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Biosorption and biomineralization of uranium(VI) by Saccharomyces cerevisiae—Crystal formation of chernikovite
Accepted Manuscript Biosorption and biomineralization of uranium(VI) by Saccharomyces cerevisiae— Crystal formation of chernikovite Xin-yan Zheng, Xiao-yu Wang, Yang-hao Shen, Xia Lu, Tie-shan Wang PII:
S0045-6535(17)30213-8
DOI:
10.1016/j.chemosphere.2017.02.035
Reference:
CHEM 18798
To appear in:
ECSN
Received Date: 4 August 2016 Revised Date:
24 January 2017
Accepted Date: 5 February 2017
Please cite this article as: Zheng, X.-y., Wang, X.-y., Shen, Y.-h., Lu, X., Wang, T.-s., Biosorption and biomineralization of uranium(VI) by Saccharomyces cerevisiae—Crystal formation of chernikovite, Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.02.035. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Fig. The mechanisms of uranium biosorption and biomineralization by live S. cerevisiae.
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Biosorption and biomineralization of uranium(VI) by Saccharomyces cerevisiae—Crystal formation of chernikovite X.Y.ZHENG, X.Y.WANG, Y.H.SHEN, X.LU, T.S.WANG* School of Nuclear Science and Technology, Lanzhou University, Lanzhou 730000, China
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Xin-yan Zheng
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Address: NO.222, Tianshui South Road, Chengguan District, Lanzhou, China
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Phone: 18394146571
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Fax number: +86 931 8913547
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Email: [email protected] Xiao-yu Wang
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Address: NO.222, Tianshui South Road, Chengguan District, Lanzhou, China
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Email: [email protected]
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Yang-hao Shen
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Address: NO.222, Tianshui South Road, Chengguan District, Lanzhou, China
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Email: [email protected]
Xia Lu
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Address: NO.222, Tianshui South Road, Chengguan District, Lanzhou, China
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Email: [email protected]
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Corresponding author: Tie-shan Wang
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Address: NO.222, Tianshui South Road, Chengguan District, Lanzhou, China
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Email: [email protected], [email protected]
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*
Corresponding author. Tel.: +86 931 8913547; fax +86 931 8913547.
E-mail addresses: [email protected], [email protected]. 1
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Biosorption and biomineralization of uranium(VI) by Saccharomyces
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cerevisiae—Crystal formation of chernikovite
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Xinyan Zheng, Xiaoyu Wang, Yanghao Shen, Xia Lu, Tieshan Wang*
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School of Nuclear Science and Technology, Lanzhou University, Lanzhou 730000, China
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Abstract: Biosorption of heavy metal elements including radionuclides by microorganisms is a promising and effective method for the remediation of the contaminated places. The responses of live Saccharomyces cerevisiae in the toxic uranium solutions during the biosorption process and the mechanism of uranium biomineralization by cells were investigated in the present study. A novel experimental phenomenon that uranium concentrations have negative correlation with pH values and positive correlation with phosphate concentrations in the supernatant was observed, indicating that hydrogen ions, phosphate ions and uranyl ions were involved in the chernikovite precipitation actively. During the biosorption process, live cells desorb deposited uranium within the equilibrium state of biosorption system was reached and the phosphorus concentration increased gradually in the supernatant. These metabolic detoxification behaviours could significantly alleviate uranium toxicity and protect the survival of the cells better in the environment. The analysis of microscopic and spectroscopic demonstrated that the precipitate on the cell surface was a type of uranium-phosphate compound in the form of a scale-like substance, and S. cerevisiae could transform the uranium precipitate into crystalline state-tetragonal chernikovite [H2(UO2)2(PO4)2•8H2O]. Key words: Biosorption; Biomineralization; Saccharomyces cerevisiae; Uranium; Phosphorus; Chernikovite
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Corresponding author. Tel.: +86 931 8913547; fax +86 931 8913547. E-mail address: [email protected] (Xinyan. Zheng), [email protected] (Tieshan Wang). 2
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1. Introduction Uranium pollution, harmful and prolonged in the natural environment, is primarily
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produced by mineral mining, smelting, nuclear fuel manufacturing and uranium research.
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Currently, uranium pollution has increased dramatically as a result of the development of the
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nuclear power industry (Kahouli, 2011). The toxic radionuclides have migrated into
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groundwater for decades and introduced a serious threat to the environment and public health
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(Willians et al., 2013). Therefore, the efficient removal of uranium and the safe disposal of
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radioactive waste have attracted increased attention worldwide. Biosorption of radionuclides
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by microorganisms, such as algae, fungi and bacteria is regarded as a promising and effective
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biotechnology that takes advantage of good selectivity, low cost, and easy implementation
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compared with traditional processing methods (Ahalya et al., 2003; Ding et al., 2014; Li et al.,
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2011).
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At present, the research trend of uranium biosorption can be divided into three types:
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looking for the best biosorbent that has high adsorption capacity and tolerance for uranyl ions
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(Marques et al., 1991); exploring the different biosorption behaviour of uranium under
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various conditions including cell activity, ion strength, pH value, contact time, biomass and
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uranium concentration (Das et al., 2002; Xia et al., 2013; Xiaoliang et al., 2012); and
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investigating the mechanisms of uranium immobilization, such as extracellular accumulation,
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cell surface sorption, and intracellular precipitation (Mingxue et al., 2010; Pereira et al.,
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2010 ). Plenty of researchers have focused on the mechanism of uranium removal (Nicolas et
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al., 2015) because the interaction between uranium and microorganisms is complicated and
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not fully understood (Wang and Chen, 2006). As a fungal biosorbent, Saccharomyces
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cerevisiae not only has more powerful resistance to toxic uranyl ions but can also survive in
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the external environmental conditions with a lower pH value than other microbes. This wide
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safe acceptance, low-cost, easily obtained, and ideal model of genetic manipulation make S.
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cerevisiae become more suitable as a biological adsorbent (Krishnan and Ayyappan, 2006).
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Especially, as the waste product from fermentation industry, S. cerevisiae is a potential
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adsorbing material (Dhankhar et al., 2011; Sakamoto et al., 2010; Strandberg et al., 1981) .
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Previous studies have observed that phosphorus has a vital role in the process of the
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uranium wastewater treatment by microbes because the phosphorus can combine with
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uranium more likely to form a different kind of uranium-phosphate minerals, such as autunite
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[Ca(UO2)2(PO4)2], uramphite [(NH4)(UO2)PO4·3H2O], chernikovite [H2(UO2)2(PO4)2·8H2O],
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and ningyoite [U2O(PO4)2·H2O] (Beazley et al., 2007; Bernier et al., 2010; Dunham et al.,
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2011; Khijniak et al., 2005; Macaskie et al., 1992, Merroun and Selenska, 2008; Rui et al.,
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2013; Sousa et al., 2013). Also the addition of phosphorus into the adsorption system could
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improve uranium biosorption capacity (Khijniak et al.,2005; Shelobolina et al., 2009; Sousa et
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al., 2013). However, a comprehensive study on the interaction between uranyl ion and
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phosphate radical during the process of biosorption is still lacking. Meanwhile, the previous
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study ignored the responses that some microorganisms could release phosphorus under the
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stress of toxic uranyl ions.
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Compared with the previous work, the present study focuses on the responses of live S.
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cerevisiae under the stress of uranyl ions and the uranium biomineralization mechanisms
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involved in the process of biosorption. The interaction between uranyl ion, pH value, and
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phosphate radical during the process of biosorption was investigated by carrying out the 4
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batch-type adsorption experiments. Even though the change in pH values (Xia et al., 2013)
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and the participation of phosphorus (Tieshan et al., 2017) during the biosorption process have
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been studied in our previous work, the synchronicity between uranyl ions, hydrogen ions, and
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phosphate ions was found for the first time. In addition, the speciation and stability of the
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uranyl ion complexes were simulated by Visual MINTEQ 3.0 under the appropriate
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phosphorus concentration and atmospheric conditions. Finally, the interaction of uranyl ions
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with cells and characteristic of uranium precipitate were analysed by scanning electron
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microscopy coupled with energy-dispersive X-ray analysis (SEM-EDX), X-ray photoelectron
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spectroscopy (XPS), and X-ray diffraction (XRD).
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2. Materials and Methods
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2.1 Culture and preparation of biomass
The S. cerevisiae were obtained from Angel Yeast Co., Ltd., China and were cultivated in
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culture medium that included yeast extract (1%), peptone (2%), and dextrose (2%). After
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activation (30 , 2h) and first generation culture (30
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second generation (30 , 30 h) from the liquid medium by centrifugation (1700*g for 5 min).
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Then, the cells were washed three times with sterilized deionized water and maintained at
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4 °C. Using the method of cell dry weight, a certain volume (2 mL) of the live cell suspension
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was placed in an electrothermal blowing dry box for 4 h at 100 °C, the dry weight was
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measured three times, averaged, and the concentration of live cells biomass solution was
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calculated. Next, the known suspension of refrigerated live cells was graded and diluted to the
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required concentration (1 g/L, 2 g/L, 3.2 g/L) for the experiments.
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2.2 Chemical reagents
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, 16h), the cells were harvested in the
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All chemicals used in the experiments were purchased as analytical purity. Uranyl stock 5
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solution used in this study was prepared by dissolving uranyl nitrate hexahydrate
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(UO2(NO3)2·6H2O) in 1 mol/L HNO3, then diluted to the desired concentration (1000 mg/L,
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100 mg/L, 10 mg/L) for experiments and was stored at pH 3.0. NaOH and HNO3 solutions
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were utilized to adjust the pH of solutions.
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2.3 Bath uranium sorption experiments
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Mingxue et al. (2010) have found that the growth of S. cerevisiae was not affected by a
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uranium concentration of 400 mg/L. Therefore, uranium concentrations used in the bath
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experiments (1 mg/L, 10 mg/L) have no effect on cell activity.
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First, 50 mL polyethylene tubes were filled with 3 mL of 0.1 mol/L NaNO3, and then a
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15 mL cell suspension that we had already prepared was added. To make cells adapt better to
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the adsorption conditions, the mixed cell suspension was pre-equilibrated by shaking for 30
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min at 30
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volume of the mixed uranium cell suspension was kept at 30 mL by adding deionized water.
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Triplicates were prepared for each condition. At the desired contact time intervals, the cells
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were separated by centrifugation at 10000 rpm for 15 min, and the uranium concentrations
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were determined using the pulsed-laser induced fluorescence method (Xia et al., 2013).
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Parallel assays without cells were conducted as controls in all experiments to ensure that
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uranium sorption on the test tube wall was negligible.
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(250 rpm). Next, 3 mL U(VI) stock solution was added to the mixture. The
To determine the dynamic changes of pH value in the biosorption process, the kinetic
investigation of pH value was conducted over a contact time range of 0-3540 min.
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To determine the dynamic changes of phosphate concentration in the biosorption process,
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the inorganic phosphate in the supernatant was measured by the molybdenum blue
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To evaluate the desorption behaviour in the biosorption process, the initial concentration of uranium was set at 10 mg/L.
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spectrophotometric method (Osmond, 1887).
To investigate the behavior of phosphate released in the biosorption process, the concentration of biomass was set at 1 g/L.
The adsorption efficiency R (%) and biosorption capacity q (mg/g) were calculated by the following formula (1), (2): R(%) = ( ρ - ρe ) / ρ ×100%
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q = ( ρ - ρe )V / CeV
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(1) (2)
where ρ and ρe (mg/L) are the initial and final concentrations of uranium in solutions,
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respectively, q (mg/g) is the biosorption capacity of uranium by the cells, V (L) is the volume
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of the solution and Ce (g/L) is the concentration of the S. cerevisiae.
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2.4 Characterization using SEM, XPS and XRD
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The uranium concentrations of 400 mg/L and 100 mg/L were set for the electronic
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microscopic and spectroscopic techniques, respectively. After the cell biomass (1.6 g/L) was
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pre-equilibrated for 1 h at 30 °C with an ionic strength of 0.01 mol/L NaNO3, 1 g/L uranium
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stock solution was added. After the adsorption process in the whole system, the centrifugal
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collected cells were washed twice with deionized water for the removal of extra uranyl ions.
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SEM sample preparation: The fresh wet pastes were fixed with more than ten times the
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volume of 2.5% glutaraldehyde (2 h, 4 °C). The biomass was washed with the deionized
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water again instead of phosphate buffer solutions, to avoid uranyl ion-phosphate precipitation.
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After being dehydrated by 5 min each time in a graded ethanol series (25%, 50%, 80%, 100% 7
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ethanol), the suspension was dropped onto a 1 cm2 size piece of aluminium foil with a plastic
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head dropper and was dried naturally in the air. Finally, we placed the sample into a freeze
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dryer for 12 h.
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XPS sample preparation: After the centrifugation, the fresh wet pastes were dropped onto
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an 1 cm2 size piece of aluminium foil directly with a plastic head dropper and dried naturally
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in the air.
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XRD sample preparation: At the desired contact time intervals (0, 1, 2, and 4 d), the cells
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were separated by centrifugation at 10000 rpm for 15 min. Next, the centrifugally collected
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cells were washed with deionized water and the concentrated suspension was dropped onto a
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microslide with a plastic head dropper and was dried naturally in the air.
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The surface morphology analysis of cells was determined by field emission scanning
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electron microscopy (FESEM-EDX, JEOL JSM-6701F JPN). The elemental composition was
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determined by X-ray photoelectron spectroscopy (XPS, Kratos Axis Ultra DLD). The
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crystallographic structure of the samples was examined by grazing-angle X-ray diffraction
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(XRD, Philips, X'pert pro) using Cu Kα radiation (λ = 0.154056 nm) over the range of 5° to
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65°(2θ) with a scan speed of 5°/min and a step size of 0.02°. The uranium compound was
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identified via the PDF-2 database of the Joint Committee on Powder Diffraction Standards
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(JCPDS ) .
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3. Results and Discussion
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3.1 Relationship between pH value, inorganic phosphate and uranyl ions in the
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supernatant during the process of biosorption
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Fig. 1 showed the changes of pH value, inorganic phosphate concentration, and uranyl
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ion concentration in the supernatant as a function of contact time at initial pH 4.50 ± 0.10 in 8
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0.01 mol/L NaNO3. As shown in Fig. 1a, the uranium concentration and the pH value of the
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control sample remained stable during the contact time. The pH value increased while the
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uranium concentration in the supernatant decreased with contact time in the biosorption
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process. The significant increase in the pH value indicated a release of hydroxyl ions (Xia et
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al., 2013) or participation of hydrogen ions during the uranium precipitation reaction. Under
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the present experimental conditions, the biosorption equilibrium was attained within 73 min
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with relatively high uranium efficiency (67.8% ± 2.2%). The pH value increased rapidly
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within the first 100 min because of the buffer function of cell suspensions. One hundred
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minutes later, when the concentration of uranium in the supernatant was rising (190 min, 0.46
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mg/L), the pH value was reduced simultaneously (190 min, 6.40). Vice versa, when the
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uranium concentration was reduced (251 min, 0.39 mg/L), the pH value was increased (251
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min, 6.78) at the same time. A similar trend was observed by Xia et al. (2013), who found that
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the pH value displays a dramatic shift before and after U(VI) biosorption by S. cerevisiae.
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Therefore, there was a negative correlation between uranium concentration and pH value in
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the supernatant during the process of biosorption, which proved that hydrogen ions may be
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involved in the active process to form the uranium precipitate.
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In addition, the relationship between inorganic phosphate and uranyl ions in the
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supernatant during biosorption was investigated (Fig. 1b). When the concentration of uranium
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in the supernatant was rising (190 min, 0.46 mg/L), the inorganic phosphate concentration
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was increasing simultaneously (190 min, 4.11 mg/L). Vice versa, when the uranium
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concentration was reduced (251 min, 0.39 mg/L), the inorganic phosphate concentration was
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also reduced (251 min, 3.46 mg/L) at the same time. Therefore, there was a positive 9
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correlation between uranyl ions and inorganic phosphate in the supernatant during the contact
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time from 50 minutes to 680 minutes (shaded), which proved that inorganic phosphate ions
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were involved in the action in the uranium precipitation process.
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No obvious positive correlation was observed between uranium and phosphorus
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concentrations within 70 min because the uranium removal was attributed primarily to such
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factors as complexation and electrostatic interaction. In addition, the concentration of the
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phosphate stayed at a relatively low level. Therefore, the first stage with fast sorption within
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70 min was not discussed in this part of this paper. After 1425 minutes, the phosphate
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concentration in the supernatant increased gradually, and that trend was different from the
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change of uranium concentration due to the release of phosphorus from the cells when S.
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cerevisiae was stimulated by uranyl ions. This specific phenomenon was investigated in
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section 3.2.2.
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All off the above data indicated that hydrogen ions and phosphate ions might be involved
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in the chernikovite precipitation. Therefore, the following chemical action equation for the
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precipitation mechanism was proposed,
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H++PO43-+UO22+ ⇋ UO2HPO4
3.2 Response of live S. cerevisiae under the stress of uranyl ions
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3.2.1 The desorption behaviour
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(3)
As Fig. 1 shows, after the biosorption system reached the equilibrium state (within 73
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min), the biosorption efficiency fluctuated significantly in the following adsorption process.
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This special behaviour was defined as the desorption process in this work. To determine the
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existence of the desorption behaviour more accurately under the stress of uranyl ions, further
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experiments in uranium biosorption by active S. cerevisiae at 10 mg/L U(VI) were explored. 10
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As shown in Fig. 2, equilibrium was attained after 373 min with an accumulation of 17.32 ±
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0.32 mgU/g dry biomass, which represents 86.6% ± 0.7% of U(VI) removal. However, during
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the process of the biosorption system reaching the equilibrium state, there were two
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significant reduction points that the adsorption efficiency was obviously lower than the trend
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values at 53 min and 313 min (black arrow in Fig.2). Similar results concerning the
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desorption behaviour of live cells have been observed by M. Vogel (2010) who investigated
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the biosorption of U(VI) by the green algae Chlorella vulgaris, indicating that living algae
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could release the bound uranium during an ongoing incubation.
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3.2.2 Phosphate release
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The behaviour of the phosphate released during U(VI) exposure over time was observed
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in section 3.1.2. To determine the trend of phosphate release during the adsorption process
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more accurately, reduce the consumption of the phosphorus during uranium-phosphate
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precipitation process, and enhance the stimulation of uranyl ions to live cells, further
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experiments of uranium biosorption by active S. cerevisiae with 10 mg/L U(VI) in 1 g/L dry
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biomass were explored.
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The change of phosphate content in the supernatant during uranium adsorption by live S.
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cerevisiae was shown in Fig. 3. The phosphorus concentration in the supernatant increased
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gradually with contact time, and it reached 30.01 ± 3.00 mg/L at 2655 min. As a result, the
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behaviour of the phosphate release, that live cells transported phosphorus from intracellular to
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extracellular (Nicolas et al., 2015), is another response when live S. cerevisiae cells remain in
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the toxic uranium solutions. Therefore, the free phosphorus and uranyl ions could form the
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uranium-phosphorus precipitate on the cell surface (section 3.4.1), which reduced the uranium
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toxicity of the solution significantly. These results agreed with other studies demonstrating
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that several species can precipitate uranium via a phosphate release mechanism (Beazley et al.,
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2007). The metabolic detoxification behaviour of live S. cerevisiae cells is called
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“self-protection” in this work.
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3.3 Uranium species in the supernatant in the presence of free phosphate
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An important factor for understanding the biosorption behaviour and the interaction of
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uranium with S. cerevisiae is the change of uranium speciation in the mixed solution which
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influences the bioavailability of the uranium. The mixed solution is composed of uranyl
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nitrate and live S. cerevisiae cells that could release phosphate during biosorption. Due to the
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change in the pH value and the phosphate concentration with contact time, the pH value of the
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solution varied from 2.0 to 10.0 and the phosphate concentration was set to 10 mg/L
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(according to Fig.3).
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Uranium speciation depends on the pH value, uranium concentration, ion strength, and
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the phosphorus concentration in the biosorption system. The phosphorus in solution could
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produce a great change for uranium species compared with the control. The dominant species
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and the changes in the uranium species with pH value can be seen in Fig 4. UO22+ dominates
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the speciation under highly acidic conditions, whereas the uranyl carbonate becomes the main
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species of uranium at higher pH values. The uranyl phosphate UO2HPO4, UO2PO4- and uranyl
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hydroxides (UO2)3(OH)5+ are formed in significant amounts during the process of biosorption
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when the pH value changes from 4.3 to 7.3.
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The uranyl phosphate is the perdominant uranium species in the solution, and the result
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is largely consistent with the equation (3). However, it is not possible to accurately calculate
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the uranium species because the composition changes result from the release of organic and
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inorganic compounds during biosorption.
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3.4 Mesoscopic and spectroscopic analysis
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3.4.1 SEM and EDX analysis
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Fig. 5 shows the SEM micrographs and EDX analysis of S. cerevisiae before and after
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biosorption. The cell surface was smooth and integral in the control sample (Fig. 5a and 5b).
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After being exposed to uranyl ions, the cell surface became rough, and a scale-like uranium
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precipitate appeared on it (Fig. 5c and 5d). In addition, Fig. 5e shows that distinct peaks of
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uranium and phosphorus were found after biosorption, which indicated that the sediment on
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the cell surface maybe a form of uranyl phosphate.
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3.4.2 XPS analysis
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To explore the interactions between uranium and S. cerevisiae on cell surface, the
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uranium deposition was analysed by XPS. The Fig. 6a shows the full spectrum of the X-ray
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photoelectron binding energy curves of S. cerevisiae before and after biosorption. After the
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adsorption, distinct peaks of uranium and phosphorous are displayed in Fig. 6a.
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Fig.6b shows the fine spectral peak of P2p at approximately 131 eV. The P2p spectra and
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U4f spectra were fitted by the XPS Peak Fit and the details are described in Table 1. As shown
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in Table 1, the binding energy of phosphorus had shifted a little (from 131.17 to 130.72 eV)
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after uranium biosorption by S. cerevisiae, which indicated that the phosphorus-containing
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compounds on the cell surface were changed after interaction with uranyl ions. In addition,
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the area of the P peak represented the phosphorus content on the cell surface, and the increase
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in the phosphorus content (from 218.47 to 1810.95) indirectly demonstrated that the
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precipitation on cell surface was a kind of uranium-phosphate compound and uranyl ions
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could stimulate the phosphorus release from the cytoplasm. The U4f spectra (Fig. 6c) showed that intense peaks at approximately 382 and 393 eV
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corresponded to the spin-orbit split in the U4f7/2 and U4f5/2 states, respectively (Kushwaha
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et al., 2010). Satellite peaks at approximately 391 and 393 eV proved that S. cerevisiae could
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reduce uranium from U(VI) to U(IV).
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3.4.3 XRD analysis
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Investigating the structural stability of uranium speciation is necessary to understand the
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fate and transport of uranium in real environments (Cerrato et al., 2013), and XRD was
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employed to determine the crystal structure of the uranium precipitation in this study. Fig. 7
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shows the diffraction spectrum of S. cerevisiae with uranium at different time intervals.
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Following the increase in contact time (0 d, 1 d, 2 d, 4 d), the diffraction peaks gradually
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appeared, and there were obvious intense crystalline peaks after the uranium exposure within
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4 days. In addition, the diffraction peaks of uranium complexes that were observed could well
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be indexed as tetragonal chernikovite [H2(UO2)2(PO4)2•8H2O] (JCPDF#08-0296). The match
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parameters with S. cerevisiae after biosorption are shown in Table 2, which includes the angle
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of the peak position, the crystal indices, the interplanar crystal spacing, and the crystalline
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dimension.
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These results indicated that S. cerevisiae could transform amorphous uranium precipitate
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into nano-crystalline, and the possible complexes of uranium with yeast phosphate would
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facilitate uranium precipitation (Kazy et al., 2009).
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3.5 Possible biosorption and biomineralization mechanisms of uranium by S. cerevisiae
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Biosorption of uranium by S. cerevisiae is a complicated interaction process. Based on
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the all abovementioned analyses, the possible uranium adsorption and biomineralization 14
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mechanisms in the biosorption process have been proposed. The process of interaction between uranyl ions and S. cerevisiae cells underwent three
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stages: fast sorption stage, relative equilibrium stage, and further sorption stage. In the fast
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sorption stage, majority of the uranyl ions were attached to the cell surface because of the
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complexation by functional groups include hydroxy, amide, and phosphate (Tsuruta, 2004)
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and electrostatic interaction. Moreover, there was a negative correlation between uranium
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concentration and pH value in the supernatant, which proved that hydrogen ions may be
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involved in the action to form the uranium precipitate. Similarly, there was a positive
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correlation between uranyl ions and inorganic phosphate in the supernatant, which proved that
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inorganic phosphate ions were involved in the action to form the uranium precipitate.
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Therefore,
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proposed
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form a chernikovite precipitate in a scale-like shape (Fig. 5) in the fast sorption and relative
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equilibrium stage. More experimental evidence proved these results: uranyl phosphate was the
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dominant uranium species in solution (Visual MINTEQ 3.0), distinct peaks of uranium and
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phosphorus were displayed after adsorption (XPS). In the further sorption stage the crystal of
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chernikovite [H2(UO2)2(PO4)2•8H2O] were formed gradually (XRD).
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hydrogen ions, phosphate ions and uranyl ions were involved in the reaction (3) to
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Live S. cerevisiae cells show a major response during the process of biosorption to
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reduce the uranium toxicity of the solution and help themselves survive well: (1) The
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behaviour of desorption. When S. cerevisiae remained in the toxic uranium solutions, the
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uranium deposit that was bound onto the cell surface would desorb before the biosorption
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system reached the equilibrium state. (2) The release of phosphorus. When S. cerevisiae 15
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remained in the toxic uranium solutions, the phosphate was gradually released from inside to
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outside of the cells and formed a uranium-phosphorus precipitate with the uranyl ions to
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reduce the uranium toxicity of the solution significantly. These responses constitute metabolic
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detoxification behaviour, known as “self-protection”, allowing them better adapt to the
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environment more efficiently.
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4. Conclusions
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This study investigated the mechanism of uranium biomineralization and the response of
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live S. cerevisiae under the stress of uranyl ions during the interaction process. The new
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phenomenon that the uranium concentration has a negative correlation with the pH value and
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a positive correlation with the phosphate concentration in the supernatant was found, which
347
indicated that hydrogen ions, phosphate ions and uranyl ions were actively involved in the
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chernikovite precipitation. The crystals of chernikovite [H2(UO2)2(PO4)2•8H2O] were formed
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gradually within 4 days. In additon, live cells could desorb uranium and gradually release
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phosphate during the biosorption process, which dramatically alleviated the uranium toxicity
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and protected their survival better in the environment. However, biosorption is a complicated
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interaction process, and the further biosorption mechanism of S. cerevisiae cells still needs to
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be studied.
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Acknowledgements
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The authors acknowledge the experimental support by the members at the College of
Chemistry, Lanzhou University for the XPS and XRD measurements support.
References Ahalya, N., Ramachandra, T.V., Kanamadi, R.D., 2003. Biosorption of Heavy Metals. Res. J. Chem. Environ. 7, 16
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71-78. Beazley, M.J., Martinez, R.J., Sobecky, P.A., Webb, S.M., Taillefert, M., 2007. Uranium biomineralization as a result of bacterial phosphatase activity: insights from bacterial isolates from a contaminated subsurface. Environ. Sci. Technol. 41, 5701–5707. Bernier-Latmani, R., Veeramani, H., Vecchia, E.D., Junier, P., Lezama-Pacheco, J.S., Suvorova, E.I., Sharp, J.O., Wigginton, N.S., Bargar, J.R.,2010. Non-uraninite products of microbial U(VI) reduction. Environ. Sci. Technol. 44, 9456–9462. Cerrato, J.M., Ashner, M.N., Alessi, D.S., Lezama-Pacheco, J.S., Bernier-Latmani, R., Bargar, J.R., Giammar, D.E., 2013. Relative reactivity of biogenic and chemogenic uraninite and biogenic noncrystalline U(IV). Environ. Sci. Technol. 47, 9756-9763. Das, S., Kedari, C., et al. 2002. Performance of immobilized Saccharomyces cerevisiae in the removal of long lived radionuclides from aqueous nitrate solutions. J. Radioanal. Nucl. Ch. 253, 235-240. Dhankhar, R., Hooda, A., Solanki, R., Sainger, P.A., 2011. Saccharomyces cerevisiae: a potential biosorbent for biosorption of uranium. Int. J. Eng. Sci. Technol. 3, 5397-5407. Ding, C.C., Feng, S., Li, X.L., Liao, J.L., Yang, Y.Y., An, Z., 2014. Mechanism of thorium biosorption by the cells of the soil fungal isolate Geotrichum sp. dwc-1. Radiochim. Acta. 102, 175-184. Dunham-Cheatham, S., Rui, X., Bunker, B., Menguy, N., Hellmann, R., Fein, J., 2011. The effects of non-metabolizing bacterial cells on the precipitation of U, Pb and Ca phosphates. Geochim. Cosmochim. Acta 75, 2828–2847. Kahouli, S., 2011. Re-examining uranium supply and demand: new insights. Energy Policy. 39, 358-376 Kazy, S.K., D'Souza, S.F., Sar, P., 2009. Uranium and thorium sequestration by a Pseudomonas sp.: mechanism and chemical characterization. J. Hazard. Mater. 163, 65-72. Khijniak, T.V., Slobodkin, A.I., Coker, V., Renshaw, J.C., Livens, F.R., Bonch-Osmolovskaya, E.A., Birkeland, N.K., Medvedeva-Lyalikova, N.N., Lloyd, J.R.,2005. Reduction of uranium(VI) phosphate during growth of the thermophilic bacterium Thermoterrabacterium ferrireducens. Appl. Environ. Microbiol. 71, 6423–6426. Krishnan, K.K., Ayyappan, S., 2006. Heavy metals remediation of water using plants ans lignocellulosic agrow astes. Rev. Envion. Contam. Tocicol. 188, 59-84. Kushwaha, S., Sreedhar, B., Padmaja, P., 2012. XPS, EXAFS, and FTIR as Tools to probe the unexpected adsorption coupled reduction of U(VI) to U(V)and U(IV) on borassus flabellifer based adsorbents. Langmuir. 28, 16038-16048. Li, L., Hu, Q., Zeng, J.H., Qi, H.Y., Zhuang, G.Q., 2011. Resistance and biosorption mechanism of silver ions by Bacillus cereus biomass. J. Environ. Sci. 23, 108-111. Macaskie, L.E., Empson, R.M. , Cheetham, A.K., Grey, C.P., Skarnulis, A.J., 1992. Uranium bioaccumulation by a Citrobacter sp. as a result of enzymatically mediated growth of polycrystalline HUO2PO4. Science. 257, 782–784. Marques, AM., Roca, X., et al., 1991. Uranium accumulation by Pseudomonas. sp. EPS -5028. Appl. Microbiol. Biotechnol. 35, 406-410. Merroun, M.L., Selenska-Pobell, S., 2008. Bacterial interactions with uranium: an environmental perspective. J. Contam. Hydrol. 102, 285–295. Mingxue, L., Faqin, D., Xiuying, Y., Wenming, Z., Liangyu, H., Xiaofeng, P., 2010. Biosorption of uranium by Saccharomyces cerevisiae and surface interactions under culture conditions. Bioresource Techno. 101, 8573-8580. Nicolas Th., Virginie Ch., Fréderic C., Magali F., Thomas V., Claire S., Virginie C., Catherine B., Laureline F., 2015. Use of combined microscopic and spectroscopic techniques to reveal interactions between
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uranium and Microbacterium sp. A9, a strain isolated from the Chernobyl exclusion zone. J. Hazard. Mater. 285, 285-293. Osmond, F., 1887. Dosage colorimétrique du phosphore, Bull. Soc. chim. (Paris). 47, 745–748. Pereira, P.H.F., Voorwald, H.J.C., Cioffi, M.O.H., Da Silva, M.L.C.P., Rego, A.M.B., Ferraria, A.M., et al., 2014. Sugarcane bagasse cellulose fibres and their hydrous niobium phosphate composites: synthesis and characterization by XPS, XRD and SEM. Cellulose. 21, 641-652. Rui, X., Kwon, M.J., O’Loughlin, E.J., Dunham-Cheatham, S., Fein, J.B., Bunker, B., Kemner, K.M., Boyanov, M.I., 2013. Bioreduction of hydrogen uranyl phosphate: mechanisms and U(IV) products, Environ. Sci. Technol. 47, 5668–5678. Sakamoto, F., Toshihiko, O., Fujii, T., Haruyuki, L., 2010. Response of Saccharomyces cerevisiae to heavy element stress: lead vs. uranium. Geomicrobiology Journal. 27, 240-244. Shelobolina, E.S., Konishi, H., Xu, H., Roden, E.E., 2009. U(VI) sequestration in hydroxyapatite produced by microbial glycerol 3-phosphate metabolism. Appl. Environ. Microbiol. 75, 5773–5778. Sousa, T. , Chung, A.P., Pereira, A., Piedadec, A.P., Morais, P.V., 2013. Aerobic uranium immobilization by Rhodanobacter A2-61 through formation of intracellular uranium – phosphate complexes. Metallomics. 5, 390–397. Strandberg, G.W., Shumate, S.E. and Parrott, J.R., Microbial, 1981. Cells as biosorbents for heavy metals accumulation of uramium by saccharomyces cereviseea and pseudomonas aetuginosa. Appl. Environ. Microb. 41, 237-245. Tieshan W., Xinyan Zh., Xiaoyu W., Xia L., Yanghao Sh., 2017. Different biosorption mechanisms of Uranium(VI) by live and heat-killed Saccharomyces cerevisiae under environmentally relevant conditions. J. Environ. Radioactiv. 167, 92-99. Tsuruta, T, 2004. Adsorption of uranium from acidic solution by microbes and effect of thorium on uranium adsorption by Streptomyces levoris. J. Biosci. Bioeng. 97, 275–277. Vogel, M., Günther, A., Rossberg, A., Li, B., Bernhard, G. , Raff, J., 2010. Biosorption of U(VI)by the green algae Chlorella vulgaris in dependence of pH value and cell activity. Sci. Total. Environ. 409, 384–395. Wang, J. and Chen C., 2006. Biosorption of heavy metals by Saccharomyces cerevisiae: a review. Biotechnol. Adv. 24, 427-51. Willians, K.H., Bargar, J.R., Lovely, D.R., 2013. Bioremediation of uranium-contaminated groundwater: a systems approach to subsurface biogeochemistry. Curr. Opin. Biotechnol. 24, 489-497. Xia, L., Xiaojiao, Z., Tieshan, W., 2013. Mechanism of uranium(VI) uptake by Saccharomyces cerevisiae underenvironmentally relevant conditions: Batch, HRTEM, and FTIR studies. J. Hazard. Meter. 262, 297–303. Xiaoliang, W., Guowen, P., Yan, Y., Yanfei, W., Tingting, H., 2012. Uranium adsorption by dry and wet immobilized Saccharomyces cerevisiae. J. Radioanal. Nucl. Chem. 291, 825–830.
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Table1. Parameters for P2p spectra of S. cerevisiae before and after biosorption FWHM
Area
P-Control
131.17
1.64
218.47
P-Uranium
130.72
1.76
1810.95
U-Uranium
380.2
1.29
761
382
1.44
391.1
1.21
392.9
1.47
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BE(eV)
5839.3 555
3877
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Table2. Parameters for XRD pattern of S. cerevisiae after biosorption (h k l)
d
XS(Å)
9.6
(001)
9.2
170
15.9
(101)
5.6
241
17.8
(110)
5.0
279
23.3
(102)
3.8
25.3
(200)
3.5
27.2
(201)
3.3
30.1
(211)
3.0
32.2
(103)
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2θ
198
346 278
216
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Fig. 1. The changes of pH value (a), phosphate concentration (b) and uranyl ion concentration in the supernatant as a function of contact of time. [Dry biomass]= 0.5 g/L, U(VI) = 1.0 mg/L, I = 0.01 mol/L NaNO3, 30 °C, 250 rpm.
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Fig. 2. Uranium bound on S.cerevisiae. [Dry biomass]= 0.5 g/L, U(VI) = 10.0 mg/L, I = 0.01 mol/L NaNO3, pH 5.40±0.10, 30 °C, 250 rpm.
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Fig. 3. Phosphate concentration in the supernatant during the biosorption process. [Dry biomass]= 1.0 g/L, U(VI) = 10.0 mg/L, I = 0.01 ml/L NaNO3, pH 5.40±0.10, 30 °C, 250 rpm.
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Fig. 4. The relative species distribution of U(VI) in presence of CO2, pCO2 = 3.16×10-4 atm. U(VI) = 10.0 mg/L, phosphate concentration = 10 mg/L, I = 0.01 mol/L NaNO3.Calculation made with Visual MINTEQ 3.0.
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Fig. 5. SEM micrographs and EDX analysis (e) of S. cerevisiae before (a) and after (c) biosorption. b, d correspond to details the square in a, c. U(VI) = 400 mg/L, I = 0.01 mol/L NaNO3, pH 5.40±0.10, and 30 °C, 250 rpm, t = 2 h.
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Fig. 6. X-ray photoelectron binding energy curves of S. cerevisiae before and after biosorption. a. full spectrum, b. P2p spectra, c. U4f sepctra. U(VI) = 100 mg/L, [Biomass] = 1.6 g/L, pH 5.40, I = 0.01 mol/L NaNO3, 30 °C, t = 2 h.
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Fig. 7. XRD pattern of the S. cerevisiae with uranium at different time intervals and comparison with reference database (vertical lines) for chernikovite (JCPDF#: 08-0296). U(VI) = 100 mg/L, [Biomass] = 1.6 g/L, pH 5.40, I = 0.01 mol/L NaNO3, 30 °C.
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Highlights
Hydrogen ions, phosphate and uranyl ions could form a chernikovite precipitate.
Uranium desorption and phosphorus release are cell responses in uranium
Tetragonal crystals of chernikovite were formed.
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solution.
S0045-6535(17)30213-8
DOI:
10.1016/j.chemosphere.2017.02.035
Reference:
CHEM 18798
To appear in:
ECSN
Received Date: 4 August 2016 Revised Date:
24 January 2017
Accepted Date: 5 February 2017
Please cite this article as: Zheng, X.-y., Wang, X.-y., Shen, Y.-h., Lu, X., Wang, T.-s., Biosorption and biomineralization of uranium(VI) by Saccharomyces cerevisiae—Crystal formation of chernikovite, Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.02.035. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Fig. The mechanisms of uranium biosorption and biomineralization by live S. cerevisiae.
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Biosorption and biomineralization of uranium(VI) by Saccharomyces cerevisiae—Crystal formation of chernikovite X.Y.ZHENG, X.Y.WANG, Y.H.SHEN, X.LU, T.S.WANG* School of Nuclear Science and Technology, Lanzhou University, Lanzhou 730000, China
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Xin-yan Zheng
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Address: NO.222, Tianshui South Road, Chengguan District, Lanzhou, China
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Phone: 18394146571
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Fax number: +86 931 8913547
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Email: [email protected] Xiao-yu Wang
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Address: NO.222, Tianshui South Road, Chengguan District, Lanzhou, China
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Email: [email protected]
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Yang-hao Shen
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Address: NO.222, Tianshui South Road, Chengguan District, Lanzhou, China
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Email: [email protected]
Xia Lu
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Address: NO.222, Tianshui South Road, Chengguan District, Lanzhou, China
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Email: [email protected]
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Corresponding author: Tie-shan Wang
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Address: NO.222, Tianshui South Road, Chengguan District, Lanzhou, China
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Email: [email protected], [email protected]
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*
Corresponding author. Tel.: +86 931 8913547; fax +86 931 8913547.
E-mail addresses: [email protected], [email protected]. 1
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Biosorption and biomineralization of uranium(VI) by Saccharomyces
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cerevisiae—Crystal formation of chernikovite
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Xinyan Zheng, Xiaoyu Wang, Yanghao Shen, Xia Lu, Tieshan Wang*
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School of Nuclear Science and Technology, Lanzhou University, Lanzhou 730000, China
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Abstract: Biosorption of heavy metal elements including radionuclides by microorganisms is a promising and effective method for the remediation of the contaminated places. The responses of live Saccharomyces cerevisiae in the toxic uranium solutions during the biosorption process and the mechanism of uranium biomineralization by cells were investigated in the present study. A novel experimental phenomenon that uranium concentrations have negative correlation with pH values and positive correlation with phosphate concentrations in the supernatant was observed, indicating that hydrogen ions, phosphate ions and uranyl ions were involved in the chernikovite precipitation actively. During the biosorption process, live cells desorb deposited uranium within the equilibrium state of biosorption system was reached and the phosphorus concentration increased gradually in the supernatant. These metabolic detoxification behaviours could significantly alleviate uranium toxicity and protect the survival of the cells better in the environment. The analysis of microscopic and spectroscopic demonstrated that the precipitate on the cell surface was a type of uranium-phosphate compound in the form of a scale-like substance, and S. cerevisiae could transform the uranium precipitate into crystalline state-tetragonal chernikovite [H2(UO2)2(PO4)2•8H2O]. Key words: Biosorption; Biomineralization; Saccharomyces cerevisiae; Uranium; Phosphorus; Chernikovite
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*
Corresponding author. Tel.: +86 931 8913547; fax +86 931 8913547. E-mail address: [email protected] (Xinyan. Zheng), [email protected] (Tieshan Wang). 2
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1. Introduction Uranium pollution, harmful and prolonged in the natural environment, is primarily
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produced by mineral mining, smelting, nuclear fuel manufacturing and uranium research.
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Currently, uranium pollution has increased dramatically as a result of the development of the
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nuclear power industry (Kahouli, 2011). The toxic radionuclides have migrated into
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groundwater for decades and introduced a serious threat to the environment and public health
52
(Willians et al., 2013). Therefore, the efficient removal of uranium and the safe disposal of
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radioactive waste have attracted increased attention worldwide. Biosorption of radionuclides
54
by microorganisms, such as algae, fungi and bacteria is regarded as a promising and effective
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biotechnology that takes advantage of good selectivity, low cost, and easy implementation
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compared with traditional processing methods (Ahalya et al., 2003; Ding et al., 2014; Li et al.,
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2011).
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At present, the research trend of uranium biosorption can be divided into three types:
59
looking for the best biosorbent that has high adsorption capacity and tolerance for uranyl ions
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(Marques et al., 1991); exploring the different biosorption behaviour of uranium under
61
various conditions including cell activity, ion strength, pH value, contact time, biomass and
62
uranium concentration (Das et al., 2002; Xia et al., 2013; Xiaoliang et al., 2012); and
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investigating the mechanisms of uranium immobilization, such as extracellular accumulation,
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cell surface sorption, and intracellular precipitation (Mingxue et al., 2010; Pereira et al.,
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2010 ). Plenty of researchers have focused on the mechanism of uranium removal (Nicolas et
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al., 2015) because the interaction between uranium and microorganisms is complicated and
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not fully understood (Wang and Chen, 2006). As a fungal biosorbent, Saccharomyces
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cerevisiae not only has more powerful resistance to toxic uranyl ions but can also survive in
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the external environmental conditions with a lower pH value than other microbes. This wide
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safe acceptance, low-cost, easily obtained, and ideal model of genetic manipulation make S.
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cerevisiae become more suitable as a biological adsorbent (Krishnan and Ayyappan, 2006).
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Especially, as the waste product from fermentation industry, S. cerevisiae is a potential
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adsorbing material (Dhankhar et al., 2011; Sakamoto et al., 2010; Strandberg et al., 1981) .
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Previous studies have observed that phosphorus has a vital role in the process of the
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uranium wastewater treatment by microbes because the phosphorus can combine with
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uranium more likely to form a different kind of uranium-phosphate minerals, such as autunite
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[Ca(UO2)2(PO4)2], uramphite [(NH4)(UO2)PO4·3H2O], chernikovite [H2(UO2)2(PO4)2·8H2O],
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and ningyoite [U2O(PO4)2·H2O] (Beazley et al., 2007; Bernier et al., 2010; Dunham et al.,
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2011; Khijniak et al., 2005; Macaskie et al., 1992, Merroun and Selenska, 2008; Rui et al.,
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2013; Sousa et al., 2013). Also the addition of phosphorus into the adsorption system could
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improve uranium biosorption capacity (Khijniak et al.,2005; Shelobolina et al., 2009; Sousa et
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al., 2013). However, a comprehensive study on the interaction between uranyl ion and
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phosphate radical during the process of biosorption is still lacking. Meanwhile, the previous
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study ignored the responses that some microorganisms could release phosphorus under the
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stress of toxic uranyl ions.
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Compared with the previous work, the present study focuses on the responses of live S.
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cerevisiae under the stress of uranyl ions and the uranium biomineralization mechanisms
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involved in the process of biosorption. The interaction between uranyl ion, pH value, and
89
phosphate radical during the process of biosorption was investigated by carrying out the 4
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batch-type adsorption experiments. Even though the change in pH values (Xia et al., 2013)
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and the participation of phosphorus (Tieshan et al., 2017) during the biosorption process have
92
been studied in our previous work, the synchronicity between uranyl ions, hydrogen ions, and
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phosphate ions was found for the first time. In addition, the speciation and stability of the
94
uranyl ion complexes were simulated by Visual MINTEQ 3.0 under the appropriate
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phosphorus concentration and atmospheric conditions. Finally, the interaction of uranyl ions
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with cells and characteristic of uranium precipitate were analysed by scanning electron
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microscopy coupled with energy-dispersive X-ray analysis (SEM-EDX), X-ray photoelectron
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spectroscopy (XPS), and X-ray diffraction (XRD).
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2. Materials and Methods
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2.1 Culture and preparation of biomass
The S. cerevisiae were obtained from Angel Yeast Co., Ltd., China and were cultivated in
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culture medium that included yeast extract (1%), peptone (2%), and dextrose (2%). After
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activation (30 , 2h) and first generation culture (30
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second generation (30 , 30 h) from the liquid medium by centrifugation (1700*g for 5 min).
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Then, the cells were washed three times with sterilized deionized water and maintained at
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4 °C. Using the method of cell dry weight, a certain volume (2 mL) of the live cell suspension
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was placed in an electrothermal blowing dry box for 4 h at 100 °C, the dry weight was
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measured three times, averaged, and the concentration of live cells biomass solution was
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calculated. Next, the known suspension of refrigerated live cells was graded and diluted to the
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required concentration (1 g/L, 2 g/L, 3.2 g/L) for the experiments.
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2.2 Chemical reagents
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, 16h), the cells were harvested in the
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All chemicals used in the experiments were purchased as analytical purity. Uranyl stock 5
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solution used in this study was prepared by dissolving uranyl nitrate hexahydrate
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(UO2(NO3)2·6H2O) in 1 mol/L HNO3, then diluted to the desired concentration (1000 mg/L,
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100 mg/L, 10 mg/L) for experiments and was stored at pH 3.0. NaOH and HNO3 solutions
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were utilized to adjust the pH of solutions.
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2.3 Bath uranium sorption experiments
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Mingxue et al. (2010) have found that the growth of S. cerevisiae was not affected by a
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uranium concentration of 400 mg/L. Therefore, uranium concentrations used in the bath
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experiments (1 mg/L, 10 mg/L) have no effect on cell activity.
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First, 50 mL polyethylene tubes were filled with 3 mL of 0.1 mol/L NaNO3, and then a
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15 mL cell suspension that we had already prepared was added. To make cells adapt better to
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the adsorption conditions, the mixed cell suspension was pre-equilibrated by shaking for 30
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min at 30
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volume of the mixed uranium cell suspension was kept at 30 mL by adding deionized water.
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Triplicates were prepared for each condition. At the desired contact time intervals, the cells
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were separated by centrifugation at 10000 rpm for 15 min, and the uranium concentrations
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were determined using the pulsed-laser induced fluorescence method (Xia et al., 2013).
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Parallel assays without cells were conducted as controls in all experiments to ensure that
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uranium sorption on the test tube wall was negligible.
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(250 rpm). Next, 3 mL U(VI) stock solution was added to the mixture. The
To determine the dynamic changes of pH value in the biosorption process, the kinetic
investigation of pH value was conducted over a contact time range of 0-3540 min.
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To determine the dynamic changes of phosphate concentration in the biosorption process,
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the inorganic phosphate in the supernatant was measured by the molybdenum blue
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To evaluate the desorption behaviour in the biosorption process, the initial concentration of uranium was set at 10 mg/L.
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spectrophotometric method (Osmond, 1887).
To investigate the behavior of phosphate released in the biosorption process, the concentration of biomass was set at 1 g/L.
The adsorption efficiency R (%) and biosorption capacity q (mg/g) were calculated by the following formula (1), (2): R(%) = ( ρ - ρe ) / ρ ×100%
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q = ( ρ - ρe )V / CeV
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(1) (2)
where ρ and ρe (mg/L) are the initial and final concentrations of uranium in solutions,
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respectively, q (mg/g) is the biosorption capacity of uranium by the cells, V (L) is the volume
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of the solution and Ce (g/L) is the concentration of the S. cerevisiae.
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2.4 Characterization using SEM, XPS and XRD
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The uranium concentrations of 400 mg/L and 100 mg/L were set for the electronic
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microscopic and spectroscopic techniques, respectively. After the cell biomass (1.6 g/L) was
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pre-equilibrated for 1 h at 30 °C with an ionic strength of 0.01 mol/L NaNO3, 1 g/L uranium
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stock solution was added. After the adsorption process in the whole system, the centrifugal
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collected cells were washed twice with deionized water for the removal of extra uranyl ions.
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SEM sample preparation: The fresh wet pastes were fixed with more than ten times the
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volume of 2.5% glutaraldehyde (2 h, 4 °C). The biomass was washed with the deionized
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water again instead of phosphate buffer solutions, to avoid uranyl ion-phosphate precipitation.
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After being dehydrated by 5 min each time in a graded ethanol series (25%, 50%, 80%, 100% 7
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ethanol), the suspension was dropped onto a 1 cm2 size piece of aluminium foil with a plastic
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head dropper and was dried naturally in the air. Finally, we placed the sample into a freeze
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dryer for 12 h.
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XPS sample preparation: After the centrifugation, the fresh wet pastes were dropped onto
161
an 1 cm2 size piece of aluminium foil directly with a plastic head dropper and dried naturally
162
in the air.
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XRD sample preparation: At the desired contact time intervals (0, 1, 2, and 4 d), the cells
164
were separated by centrifugation at 10000 rpm for 15 min. Next, the centrifugally collected
165
cells were washed with deionized water and the concentrated suspension was dropped onto a
166
microslide with a plastic head dropper and was dried naturally in the air.
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The surface morphology analysis of cells was determined by field emission scanning
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electron microscopy (FESEM-EDX, JEOL JSM-6701F JPN). The elemental composition was
169
determined by X-ray photoelectron spectroscopy (XPS, Kratos Axis Ultra DLD). The
170
crystallographic structure of the samples was examined by grazing-angle X-ray diffraction
171
(XRD, Philips, X'pert pro) using Cu Kα radiation (λ = 0.154056 nm) over the range of 5° to
172
65°(2θ) with a scan speed of 5°/min and a step size of 0.02°. The uranium compound was
173
identified via the PDF-2 database of the Joint Committee on Powder Diffraction Standards
174
(JCPDS ) .
175
3. Results and Discussion
176
3.1 Relationship between pH value, inorganic phosphate and uranyl ions in the
177
supernatant during the process of biosorption
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Fig. 1 showed the changes of pH value, inorganic phosphate concentration, and uranyl
179
ion concentration in the supernatant as a function of contact time at initial pH 4.50 ± 0.10 in 8
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0.01 mol/L NaNO3. As shown in Fig. 1a, the uranium concentration and the pH value of the
181
control sample remained stable during the contact time. The pH value increased while the
182
uranium concentration in the supernatant decreased with contact time in the biosorption
183
process. The significant increase in the pH value indicated a release of hydroxyl ions (Xia et
184
al., 2013) or participation of hydrogen ions during the uranium precipitation reaction. Under
185
the present experimental conditions, the biosorption equilibrium was attained within 73 min
186
with relatively high uranium efficiency (67.8% ± 2.2%). The pH value increased rapidly
187
within the first 100 min because of the buffer function of cell suspensions. One hundred
188
minutes later, when the concentration of uranium in the supernatant was rising (190 min, 0.46
189
mg/L), the pH value was reduced simultaneously (190 min, 6.40). Vice versa, when the
190
uranium concentration was reduced (251 min, 0.39 mg/L), the pH value was increased (251
191
min, 6.78) at the same time. A similar trend was observed by Xia et al. (2013), who found that
192
the pH value displays a dramatic shift before and after U(VI) biosorption by S. cerevisiae.
193
Therefore, there was a negative correlation between uranium concentration and pH value in
194
the supernatant during the process of biosorption, which proved that hydrogen ions may be
195
involved in the active process to form the uranium precipitate.
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In addition, the relationship between inorganic phosphate and uranyl ions in the
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197
supernatant during biosorption was investigated (Fig. 1b). When the concentration of uranium
198
in the supernatant was rising (190 min, 0.46 mg/L), the inorganic phosphate concentration
199
was increasing simultaneously (190 min, 4.11 mg/L). Vice versa, when the uranium
200
concentration was reduced (251 min, 0.39 mg/L), the inorganic phosphate concentration was
201
also reduced (251 min, 3.46 mg/L) at the same time. Therefore, there was a positive 9
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correlation between uranyl ions and inorganic phosphate in the supernatant during the contact
203
time from 50 minutes to 680 minutes (shaded), which proved that inorganic phosphate ions
204
were involved in the action in the uranium precipitation process.
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No obvious positive correlation was observed between uranium and phosphorus
206
concentrations within 70 min because the uranium removal was attributed primarily to such
207
factors as complexation and electrostatic interaction. In addition, the concentration of the
208
phosphate stayed at a relatively low level. Therefore, the first stage with fast sorption within
209
70 min was not discussed in this part of this paper. After 1425 minutes, the phosphate
210
concentration in the supernatant increased gradually, and that trend was different from the
211
change of uranium concentration due to the release of phosphorus from the cells when S.
212
cerevisiae was stimulated by uranyl ions. This specific phenomenon was investigated in
213
section 3.2.2.
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All off the above data indicated that hydrogen ions and phosphate ions might be involved
215
in the chernikovite precipitation. Therefore, the following chemical action equation for the
216
precipitation mechanism was proposed,
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H++PO43-+UO22+ ⇋ UO2HPO4
3.2 Response of live S. cerevisiae under the stress of uranyl ions
219
3.2.1 The desorption behaviour
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(3)
As Fig. 1 shows, after the biosorption system reached the equilibrium state (within 73
221
min), the biosorption efficiency fluctuated significantly in the following adsorption process.
222
This special behaviour was defined as the desorption process in this work. To determine the
223
existence of the desorption behaviour more accurately under the stress of uranyl ions, further
224
experiments in uranium biosorption by active S. cerevisiae at 10 mg/L U(VI) were explored. 10
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As shown in Fig. 2, equilibrium was attained after 373 min with an accumulation of 17.32 ±
226
0.32 mgU/g dry biomass, which represents 86.6% ± 0.7% of U(VI) removal. However, during
227
the process of the biosorption system reaching the equilibrium state, there were two
228
significant reduction points that the adsorption efficiency was obviously lower than the trend
229
values at 53 min and 313 min (black arrow in Fig.2). Similar results concerning the
230
desorption behaviour of live cells have been observed by M. Vogel (2010) who investigated
231
the biosorption of U(VI) by the green algae Chlorella vulgaris, indicating that living algae
232
could release the bound uranium during an ongoing incubation.
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3.2.2 Phosphate release
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The behaviour of the phosphate released during U(VI) exposure over time was observed
235
in section 3.1.2. To determine the trend of phosphate release during the adsorption process
236
more accurately, reduce the consumption of the phosphorus during uranium-phosphate
237
precipitation process, and enhance the stimulation of uranyl ions to live cells, further
238
experiments of uranium biosorption by active S. cerevisiae with 10 mg/L U(VI) in 1 g/L dry
239
biomass were explored.
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The change of phosphate content in the supernatant during uranium adsorption by live S.
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cerevisiae was shown in Fig. 3. The phosphorus concentration in the supernatant increased
242
gradually with contact time, and it reached 30.01 ± 3.00 mg/L at 2655 min. As a result, the
243
behaviour of the phosphate release, that live cells transported phosphorus from intracellular to
244
extracellular (Nicolas et al., 2015), is another response when live S. cerevisiae cells remain in
245
the toxic uranium solutions. Therefore, the free phosphorus and uranyl ions could form the
246
uranium-phosphorus precipitate on the cell surface (section 3.4.1), which reduced the uranium
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toxicity of the solution significantly. These results agreed with other studies demonstrating
248
that several species can precipitate uranium via a phosphate release mechanism (Beazley et al.,
249
2007). The metabolic detoxification behaviour of live S. cerevisiae cells is called
250
“self-protection” in this work.
251
3.3 Uranium species in the supernatant in the presence of free phosphate
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An important factor for understanding the biosorption behaviour and the interaction of
253
uranium with S. cerevisiae is the change of uranium speciation in the mixed solution which
254
influences the bioavailability of the uranium. The mixed solution is composed of uranyl
255
nitrate and live S. cerevisiae cells that could release phosphate during biosorption. Due to the
256
change in the pH value and the phosphate concentration with contact time, the pH value of the
257
solution varied from 2.0 to 10.0 and the phosphate concentration was set to 10 mg/L
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(according to Fig.3).
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Uranium speciation depends on the pH value, uranium concentration, ion strength, and
260
the phosphorus concentration in the biosorption system. The phosphorus in solution could
261
produce a great change for uranium species compared with the control. The dominant species
262
and the changes in the uranium species with pH value can be seen in Fig 4. UO22+ dominates
263
the speciation under highly acidic conditions, whereas the uranyl carbonate becomes the main
264
species of uranium at higher pH values. The uranyl phosphate UO2HPO4, UO2PO4- and uranyl
265
hydroxides (UO2)3(OH)5+ are formed in significant amounts during the process of biosorption
266
when the pH value changes from 4.3 to 7.3.
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The uranyl phosphate is the perdominant uranium species in the solution, and the result
268
is largely consistent with the equation (3). However, it is not possible to accurately calculate
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the uranium species because the composition changes result from the release of organic and
270
inorganic compounds during biosorption.
271
3.4 Mesoscopic and spectroscopic analysis
272
3.4.1 SEM and EDX analysis
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Fig. 5 shows the SEM micrographs and EDX analysis of S. cerevisiae before and after
274
biosorption. The cell surface was smooth and integral in the control sample (Fig. 5a and 5b).
275
After being exposed to uranyl ions, the cell surface became rough, and a scale-like uranium
276
precipitate appeared on it (Fig. 5c and 5d). In addition, Fig. 5e shows that distinct peaks of
277
uranium and phosphorus were found after biosorption, which indicated that the sediment on
278
the cell surface maybe a form of uranyl phosphate.
279
3.4.2 XPS analysis
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To explore the interactions between uranium and S. cerevisiae on cell surface, the
281
uranium deposition was analysed by XPS. The Fig. 6a shows the full spectrum of the X-ray
282
photoelectron binding energy curves of S. cerevisiae before and after biosorption. After the
283
adsorption, distinct peaks of uranium and phosphorous are displayed in Fig. 6a.
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Fig.6b shows the fine spectral peak of P2p at approximately 131 eV. The P2p spectra and
285
U4f spectra were fitted by the XPS Peak Fit and the details are described in Table 1. As shown
286
in Table 1, the binding energy of phosphorus had shifted a little (from 131.17 to 130.72 eV)
287
after uranium biosorption by S. cerevisiae, which indicated that the phosphorus-containing
288
compounds on the cell surface were changed after interaction with uranyl ions. In addition,
289
the area of the P peak represented the phosphorus content on the cell surface, and the increase
290
in the phosphorus content (from 218.47 to 1810.95) indirectly demonstrated that the
291
precipitation on cell surface was a kind of uranium-phosphate compound and uranyl ions
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292
could stimulate the phosphorus release from the cytoplasm. The U4f spectra (Fig. 6c) showed that intense peaks at approximately 382 and 393 eV
294
corresponded to the spin-orbit split in the U4f7/2 and U4f5/2 states, respectively (Kushwaha
295
et al., 2010). Satellite peaks at approximately 391 and 393 eV proved that S. cerevisiae could
296
reduce uranium from U(VI) to U(IV).
297
3.4.3 XRD analysis
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Investigating the structural stability of uranium speciation is necessary to understand the
299
fate and transport of uranium in real environments (Cerrato et al., 2013), and XRD was
300
employed to determine the crystal structure of the uranium precipitation in this study. Fig. 7
301
shows the diffraction spectrum of S. cerevisiae with uranium at different time intervals.
302
Following the increase in contact time (0 d, 1 d, 2 d, 4 d), the diffraction peaks gradually
303
appeared, and there were obvious intense crystalline peaks after the uranium exposure within
304
4 days. In addition, the diffraction peaks of uranium complexes that were observed could well
305
be indexed as tetragonal chernikovite [H2(UO2)2(PO4)2•8H2O] (JCPDF#08-0296). The match
306
parameters with S. cerevisiae after biosorption are shown in Table 2, which includes the angle
307
of the peak position, the crystal indices, the interplanar crystal spacing, and the crystalline
308
dimension.
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These results indicated that S. cerevisiae could transform amorphous uranium precipitate
310
into nano-crystalline, and the possible complexes of uranium with yeast phosphate would
311
facilitate uranium precipitation (Kazy et al., 2009).
312
3.5 Possible biosorption and biomineralization mechanisms of uranium by S. cerevisiae
313
Biosorption of uranium by S. cerevisiae is a complicated interaction process. Based on
314
the all abovementioned analyses, the possible uranium adsorption and biomineralization 14
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mechanisms in the biosorption process have been proposed. The process of interaction between uranyl ions and S. cerevisiae cells underwent three
317
stages: fast sorption stage, relative equilibrium stage, and further sorption stage. In the fast
318
sorption stage, majority of the uranyl ions were attached to the cell surface because of the
319
complexation by functional groups include hydroxy, amide, and phosphate (Tsuruta, 2004)
320
and electrostatic interaction. Moreover, there was a negative correlation between uranium
321
concentration and pH value in the supernatant, which proved that hydrogen ions may be
322
involved in the action to form the uranium precipitate. Similarly, there was a positive
323
correlation between uranyl ions and inorganic phosphate in the supernatant, which proved that
324
inorganic phosphate ions were involved in the action to form the uranium precipitate.
325
Therefore,
326
proposed
327
form a chernikovite precipitate in a scale-like shape (Fig. 5) in the fast sorption and relative
328
equilibrium stage. More experimental evidence proved these results: uranyl phosphate was the
329
dominant uranium species in solution (Visual MINTEQ 3.0), distinct peaks of uranium and
330
phosphorus were displayed after adsorption (XPS). In the further sorption stage the crystal of
331
chernikovite [H2(UO2)2(PO4)2•8H2O] were formed gradually (XRD).
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hydrogen ions, phosphate ions and uranyl ions were involved in the reaction (3) to
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Live S. cerevisiae cells show a major response during the process of biosorption to
333
reduce the uranium toxicity of the solution and help themselves survive well: (1) The
334
behaviour of desorption. When S. cerevisiae remained in the toxic uranium solutions, the
335
uranium deposit that was bound onto the cell surface would desorb before the biosorption
336
system reached the equilibrium state. (2) The release of phosphorus. When S. cerevisiae 15
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remained in the toxic uranium solutions, the phosphate was gradually released from inside to
338
outside of the cells and formed a uranium-phosphorus precipitate with the uranyl ions to
339
reduce the uranium toxicity of the solution significantly. These responses constitute metabolic
340
detoxification behaviour, known as “self-protection”, allowing them better adapt to the
341
environment more efficiently.
342
4. Conclusions
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This study investigated the mechanism of uranium biomineralization and the response of
344
live S. cerevisiae under the stress of uranyl ions during the interaction process. The new
345
phenomenon that the uranium concentration has a negative correlation with the pH value and
346
a positive correlation with the phosphate concentration in the supernatant was found, which
347
indicated that hydrogen ions, phosphate ions and uranyl ions were actively involved in the
348
chernikovite precipitation. The crystals of chernikovite [H2(UO2)2(PO4)2•8H2O] were formed
349
gradually within 4 days. In additon, live cells could desorb uranium and gradually release
350
phosphate during the biosorption process, which dramatically alleviated the uranium toxicity
351
and protected their survival better in the environment. However, biosorption is a complicated
352
interaction process, and the further biosorption mechanism of S. cerevisiae cells still needs to
353
be studied.
354
Acknowledgements
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The authors acknowledge the experimental support by the members at the College of
Chemistry, Lanzhou University for the XPS and XRD measurements support.
References Ahalya, N., Ramachandra, T.V., Kanamadi, R.D., 2003. Biosorption of Heavy Metals. Res. J. Chem. Environ. 7, 16
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71-78. Beazley, M.J., Martinez, R.J., Sobecky, P.A., Webb, S.M., Taillefert, M., 2007. Uranium biomineralization as a result of bacterial phosphatase activity: insights from bacterial isolates from a contaminated subsurface. Environ. Sci. Technol. 41, 5701–5707. Bernier-Latmani, R., Veeramani, H., Vecchia, E.D., Junier, P., Lezama-Pacheco, J.S., Suvorova, E.I., Sharp, J.O., Wigginton, N.S., Bargar, J.R.,2010. Non-uraninite products of microbial U(VI) reduction. Environ. Sci. Technol. 44, 9456–9462. Cerrato, J.M., Ashner, M.N., Alessi, D.S., Lezama-Pacheco, J.S., Bernier-Latmani, R., Bargar, J.R., Giammar, D.E., 2013. Relative reactivity of biogenic and chemogenic uraninite and biogenic noncrystalline U(IV). Environ. Sci. Technol. 47, 9756-9763. Das, S., Kedari, C., et al. 2002. Performance of immobilized Saccharomyces cerevisiae in the removal of long lived radionuclides from aqueous nitrate solutions. J. Radioanal. Nucl. Ch. 253, 235-240. Dhankhar, R., Hooda, A., Solanki, R., Sainger, P.A., 2011. Saccharomyces cerevisiae: a potential biosorbent for biosorption of uranium. Int. J. Eng. Sci. Technol. 3, 5397-5407. Ding, C.C., Feng, S., Li, X.L., Liao, J.L., Yang, Y.Y., An, Z., 2014. Mechanism of thorium biosorption by the cells of the soil fungal isolate Geotrichum sp. dwc-1. Radiochim. Acta. 102, 175-184. Dunham-Cheatham, S., Rui, X., Bunker, B., Menguy, N., Hellmann, R., Fein, J., 2011. The effects of non-metabolizing bacterial cells on the precipitation of U, Pb and Ca phosphates. Geochim. Cosmochim. Acta 75, 2828–2847. Kahouli, S., 2011. Re-examining uranium supply and demand: new insights. Energy Policy. 39, 358-376 Kazy, S.K., D'Souza, S.F., Sar, P., 2009. Uranium and thorium sequestration by a Pseudomonas sp.: mechanism and chemical characterization. J. Hazard. Mater. 163, 65-72. Khijniak, T.V., Slobodkin, A.I., Coker, V., Renshaw, J.C., Livens, F.R., Bonch-Osmolovskaya, E.A., Birkeland, N.K., Medvedeva-Lyalikova, N.N., Lloyd, J.R.,2005. Reduction of uranium(VI) phosphate during growth of the thermophilic bacterium Thermoterrabacterium ferrireducens. Appl. Environ. Microbiol. 71, 6423–6426. Krishnan, K.K., Ayyappan, S., 2006. Heavy metals remediation of water using plants ans lignocellulosic agrow astes. Rev. Envion. Contam. Tocicol. 188, 59-84. Kushwaha, S., Sreedhar, B., Padmaja, P., 2012. XPS, EXAFS, and FTIR as Tools to probe the unexpected adsorption coupled reduction of U(VI) to U(V)and U(IV) on borassus flabellifer based adsorbents. Langmuir. 28, 16038-16048. Li, L., Hu, Q., Zeng, J.H., Qi, H.Y., Zhuang, G.Q., 2011. Resistance and biosorption mechanism of silver ions by Bacillus cereus biomass. J. Environ. Sci. 23, 108-111. Macaskie, L.E., Empson, R.M. , Cheetham, A.K., Grey, C.P., Skarnulis, A.J., 1992. Uranium bioaccumulation by a Citrobacter sp. as a result of enzymatically mediated growth of polycrystalline HUO2PO4. Science. 257, 782–784. Marques, AM., Roca, X., et al., 1991. Uranium accumulation by Pseudomonas. sp. EPS -5028. Appl. Microbiol. Biotechnol. 35, 406-410. Merroun, M.L., Selenska-Pobell, S., 2008. Bacterial interactions with uranium: an environmental perspective. J. Contam. Hydrol. 102, 285–295. Mingxue, L., Faqin, D., Xiuying, Y., Wenming, Z., Liangyu, H., Xiaofeng, P., 2010. Biosorption of uranium by Saccharomyces cerevisiae and surface interactions under culture conditions. Bioresource Techno. 101, 8573-8580. Nicolas Th., Virginie Ch., Fréderic C., Magali F., Thomas V., Claire S., Virginie C., Catherine B., Laureline F., 2015. Use of combined microscopic and spectroscopic techniques to reveal interactions between
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uranium and Microbacterium sp. A9, a strain isolated from the Chernobyl exclusion zone. J. Hazard. Mater. 285, 285-293. Osmond, F., 1887. Dosage colorimétrique du phosphore, Bull. Soc. chim. (Paris). 47, 745–748. Pereira, P.H.F., Voorwald, H.J.C., Cioffi, M.O.H., Da Silva, M.L.C.P., Rego, A.M.B., Ferraria, A.M., et al., 2014. Sugarcane bagasse cellulose fibres and their hydrous niobium phosphate composites: synthesis and characterization by XPS, XRD and SEM. Cellulose. 21, 641-652. Rui, X., Kwon, M.J., O’Loughlin, E.J., Dunham-Cheatham, S., Fein, J.B., Bunker, B., Kemner, K.M., Boyanov, M.I., 2013. Bioreduction of hydrogen uranyl phosphate: mechanisms and U(IV) products, Environ. Sci. Technol. 47, 5668–5678. Sakamoto, F., Toshihiko, O., Fujii, T., Haruyuki, L., 2010. Response of Saccharomyces cerevisiae to heavy element stress: lead vs. uranium. Geomicrobiology Journal. 27, 240-244. Shelobolina, E.S., Konishi, H., Xu, H., Roden, E.E., 2009. U(VI) sequestration in hydroxyapatite produced by microbial glycerol 3-phosphate metabolism. Appl. Environ. Microbiol. 75, 5773–5778. Sousa, T. , Chung, A.P., Pereira, A., Piedadec, A.P., Morais, P.V., 2013. Aerobic uranium immobilization by Rhodanobacter A2-61 through formation of intracellular uranium – phosphate complexes. Metallomics. 5, 390–397. Strandberg, G.W., Shumate, S.E. and Parrott, J.R., Microbial, 1981. Cells as biosorbents for heavy metals accumulation of uramium by saccharomyces cereviseea and pseudomonas aetuginosa. Appl. Environ. Microb. 41, 237-245. Tieshan W., Xinyan Zh., Xiaoyu W., Xia L., Yanghao Sh., 2017. Different biosorption mechanisms of Uranium(VI) by live and heat-killed Saccharomyces cerevisiae under environmentally relevant conditions. J. Environ. Radioactiv. 167, 92-99. Tsuruta, T, 2004. Adsorption of uranium from acidic solution by microbes and effect of thorium on uranium adsorption by Streptomyces levoris. J. Biosci. Bioeng. 97, 275–277. Vogel, M., Günther, A., Rossberg, A., Li, B., Bernhard, G. , Raff, J., 2010. Biosorption of U(VI)by the green algae Chlorella vulgaris in dependence of pH value and cell activity. Sci. Total. Environ. 409, 384–395. Wang, J. and Chen C., 2006. Biosorption of heavy metals by Saccharomyces cerevisiae: a review. Biotechnol. Adv. 24, 427-51. Willians, K.H., Bargar, J.R., Lovely, D.R., 2013. Bioremediation of uranium-contaminated groundwater: a systems approach to subsurface biogeochemistry. Curr. Opin. Biotechnol. 24, 489-497. Xia, L., Xiaojiao, Z., Tieshan, W., 2013. Mechanism of uranium(VI) uptake by Saccharomyces cerevisiae underenvironmentally relevant conditions: Batch, HRTEM, and FTIR studies. J. Hazard. Meter. 262, 297–303. Xiaoliang, W., Guowen, P., Yan, Y., Yanfei, W., Tingting, H., 2012. Uranium adsorption by dry and wet immobilized Saccharomyces cerevisiae. J. Radioanal. Nucl. Chem. 291, 825–830.
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Table1. Parameters for P2p spectra of S. cerevisiae before and after biosorption FWHM
Area
P-Control
131.17
1.64
218.47
P-Uranium
130.72
1.76
1810.95
U-Uranium
380.2
1.29
761
382
1.44
391.1
1.21
392.9
1.47
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BE(eV)
5839.3 555
3877
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Table2. Parameters for XRD pattern of S. cerevisiae after biosorption (h k l)
d
XS(Å)
9.6
(001)
9.2
170
15.9
(101)
5.6
241
17.8
(110)
5.0
279
23.3
(102)
3.8
25.3
(200)
3.5
27.2
(201)
3.3
30.1
(211)
3.0
32.2
(103)
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2θ
198
346 278
216
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Fig. 1. The changes of pH value (a), phosphate concentration (b) and uranyl ion concentration in the supernatant as a function of contact of time. [Dry biomass]= 0.5 g/L, U(VI) = 1.0 mg/L, I = 0.01 mol/L NaNO3, 30 °C, 250 rpm.
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Fig. 2. Uranium bound on S.cerevisiae. [Dry biomass]= 0.5 g/L, U(VI) = 10.0 mg/L, I = 0.01 mol/L NaNO3, pH 5.40±0.10, 30 °C, 250 rpm.
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Fig. 3. Phosphate concentration in the supernatant during the biosorption process. [Dry biomass]= 1.0 g/L, U(VI) = 10.0 mg/L, I = 0.01 ml/L NaNO3, pH 5.40±0.10, 30 °C, 250 rpm.
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Fig. 4. The relative species distribution of U(VI) in presence of CO2, pCO2 = 3.16×10-4 atm. U(VI) = 10.0 mg/L, phosphate concentration = 10 mg/L, I = 0.01 mol/L NaNO3.Calculation made with Visual MINTEQ 3.0.
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Fig. 5. SEM micrographs and EDX analysis (e) of S. cerevisiae before (a) and after (c) biosorption. b, d correspond to details the square in a, c. U(VI) = 400 mg/L, I = 0.01 mol/L NaNO3, pH 5.40±0.10, and 30 °C, 250 rpm, t = 2 h.
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Fig. 6. X-ray photoelectron binding energy curves of S. cerevisiae before and after biosorption. a. full spectrum, b. P2p spectra, c. U4f sepctra. U(VI) = 100 mg/L, [Biomass] = 1.6 g/L, pH 5.40, I = 0.01 mol/L NaNO3, 30 °C, t = 2 h.
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Fig. 7. XRD pattern of the S. cerevisiae with uranium at different time intervals and comparison with reference database (vertical lines) for chernikovite (JCPDF#: 08-0296). U(VI) = 100 mg/L, [Biomass] = 1.6 g/L, pH 5.40, I = 0.01 mol/L NaNO3, 30 °C.
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Highlights
Hydrogen ions, phosphate and uranyl ions could form a chernikovite precipitate.
Uranium desorption and phosphorus release are cell responses in uranium
Tetragonal crystals of chernikovite were formed.
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solution.