Biosorption of uranium by Saccharomyces cerevisiae and surface interactions under culture conditions

Bioresource Technology 101 (2010) 8573–8580

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Biosorption of uranium by Saccharomyces cerevisiae and surface interactions under culture conditions Mingxue Liu a,c,*, Faqin Dong b, Xiuying Yan c, Wenming Zeng c, Liangyu Hou c, Xiaofeng Pang a a

School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, People’s Republic of China National Defense Key Discipline Laboratory of the Nuclear Waste and Environmental Safety of the Commission of Science, Technology and Industry for National Defense, Southwest University of Science and Technology, Mianyang 621010, People’s Republic of China c Life Science and Engineering College, Southwest University of Science and Technology, Mianyang 621010, People’s Republic of China b

a r t i c l e

i n f o

Article history: Received 24 August 2009 Received in revised form 7 May 2010 Accepted 9 June 2010

Keywords: Saccharomyces cerevisiae Biosorption Uranium Surface interaction

a b s t r a c t Few studies have focused on biosorption by microorganisms under culture conditions. To explore the biosorption of uranium by Saccharomyces cerevisiae under culture conditions, the S. cerevisiae growth curve, biosorption capacity and surface interaction under batch culture conditions were investigated in this study. The growth curve showed that uranium (<300 mg L1) did not markedly inhibit the growth of S. cerevisiae under short culture time. The maximum scavenging efficiency reached 92.4% under 6–10 h culture conditions, and the adsorption quantity of S. cerevisiae increased with initial uranium concentration. Centrifuging and drying after biosorption caused the volume reduction ratio to reach 99%. Scanning electron microscope results demonstrated that uranium interacted with yeast cell surfaces, as well as culture medium, and produced uranium precipitate on cell surfaces. Fourier transformed infrared spectra revealed that cell walls were the major sorption sites, and –OAH, –C@O and –PO2 contributed to the major binding groups. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Biosorption is becoming one of the more attractive alternative methods for the removal of radioactive ions and heavy metals from wastewater (Tuzen et al., 2008). Biosorption is not only cost effective, but it also provides an opportunity for the recycling of waste materials. Many researchers have studied the biosorption of uranium with microorganisms such as bacteria, actinomycetes, fungi and yeasts (Tsuruta, 2004). The bacterium Arthrobacter nicotianae was found to possess a high uranium adsorption ability of approximately 698 mg (2.58 mmol) uranyl ions g1 dry weight (Tsuruta, 2002). Arthrobacter and Bacillus sp. isolated from North American uranium deposits could accumulate about 2.5 mmol of uranium g1 dry weight within one hour (Tsuruta, 2007). Streptomyces levoris could adsorb about 0.38 mmol of uranium g1 dry weight from the solution in one hour (Tsuruta, 2004). Trichoderma harzianum was shown to have the maximum uranium biosorption capacity of approximately 612 mg uranium g1 dry weight (Akhtar et al., 2007). Analyses were also carried out using living and non-living

* Corresponding author at: Life Science and Engineering College, Southwest University of Science and Technology, Mianyang 621010, People’s Republic of China. Fax: +86 816 6089521. E-mail address: [email protected] (M. Liu). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.06.063

Saccharomyces cerevisiae for bioaccumulation and biosorption of uranium, respectively, from low radioactive solutions (Tsuruta, 2002; Popa et al., 2003; Liu et al., 2004; Tykva et al., 2009). The maximum degree of bioaccumulation of S. cerevisiae reached a value of nearly 8.75 mmol uranyl ions g1 dry weight (Popa et al., 2003). However, most previous adsorption experiments were carried out under batch conditions with living or dead isolated microbial cells from the culture medium or by-products, and there are few experiments carried out under culture conditions. The biosorption of radioactive nuclides under culture conditions may be more favorable to traditional biosorption methods for the following reasons: (1) compared to the separate processes, the combination of biosorption and microorganism culture takes less time and energy; (2) microorganisms may adsorb radioactive nuclides by cell metabolism under culture conditions, in addition to the adsorption by physicochemical process; (3) biosorption with microorganisms under culture condition facilitates the development of continuous industrial treatment of contaminated wastewater; and (4) biosorption can generate large volume reduction ratios. Therefore, the goals of this research were to explore the feasibility for biosorption of uranium by S. cerevisiae under culture conditions, investigate the uranium biosorption capacity of S. cerevisiae and analyze the surface interaction between S. cerevisiae and uranium.

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The volume reduction ratio (VDR) was calculated using the following formula:

2. Methods 2.1. Apparatuses

VDRð%Þ ¼

Vs  100% Vi

ð3Þ

This study used a Leica-S440 instrument coupled with energy dispersive X-ray (EDX, UK) analysis, a Spectrum One FTIR (Version BM, USA) and a TU-1900 spectrophotometer (Pgeneral, China). All measurements were carried out at the Analysis and Testing Center of the Southwest University of Science and Technology.

where Vi (mL) is the volume of the culture medium used for biosorption under culture conditions and Vs (mL) is the volume of S. cerevisiae biomass sediment dried by vacuum freeze-drying after biosorption.

2.2. Reagents

2.6. Uranium concentration measurements

All chemical reagents were of analytical grade unless otherwise stated. Doubly distilled deionized water was used throughout the study. The uranium solution was prepared by dissolving a calculated amount of uranyl acetate dihydrate (UO2(CH3CO2)22H2O) in water. ArsenazoIII stock solution (1 mmol L1) was prepared by dissolving 0.0776 g of arsenazoIII in 100 mL water. The working solutions were prepared by diluting the stock solution to appropriate volumes. The yeast culture medium was composed of glucose 5%, urea 0.1%, (NH4)2SO4 0.1%, Na2HPO4 0.05% and yeast extract 0.05% with a pH 4.5.

Timed samples (10 mL) were centrifuged (22 °C, 15 min, 4000 rpm), and the supernatant was assayed for residual uranium using a spectrophotometer at 652 nm using arsenazoIII (Yong and Macaskie, 1995).

2.3. Yeast S. cerevisiae yeast was provided by the Experiment Center of the Life Science and Engineering College, Southwest University of Science and Technology. 2.4. Growth curve of S. cerevisiae and toxicity experiments Solutions of uranium were mixed with suspensions of growing S. cerevisiae at the exponential phase and were inoculated in medium Erlenmeyer flasks, which were kept on an orbital shaker (80 rpm) at a constant temperature (30 °C). The initial uranium concentrations varied from 0 to 600 mg L1 and were set at 0, 40, 60, 100, 120,140, 160, 180, 200, 240, 300, 400 and 600 mg L1. The samples were then allowed to culture. The O.D. values of culture samples were measured by a spectrophotometer at a 560 nm wavelength every 2 h for 18 h. The experiments were run in triplicate.

2.7. SEM, EDS and FTIR spectroscopy analysis The surface characters of S. cerevisiae cells before and after uranium biosorption were analyzed using scanning electron microscopy (SEM) coupled with EDX analysis. The timed S. cerevisiae cell sample was placed on cover slip and air-dried. The samples were then fixed in a 2.5% glutaraldehyde solution for 7 h. Next, samples were dehydrated by 20 min incubations in a graded ethanol series (30%, 50%, 70% and 95% ethanol) and two incubations in 100% ethanol. Finally, the samples were air-dried. Before using, the samples were sputter-coated with 4 nm of gold particles. The elemental composition of a sample was determined using the characteristic X-ray spectrum from energy dispersive X-ray spectroscopy (EDS) by EDX analysis coupled with SEM. In this study, the detected elements were quantified from the EDS spectra using the classic ZAF model. Fourier transformed infrared spectrometry (FTIR) conducted on dried samples of S. cerevisiae before and after biosorption was recorded on KBr pellets at room temperature using a FTIR spectrometer. The sample compartments were separated though centrifuging. The precipitation was collected and was flushed with dry air to reduce any interference of H2O and CO2. The data point resolution of the spectra was 2 cm1 and 25 scans were accumulated for each spectrum. Data analysis focused on the 400– 4000 cm1 region.

2.5. Biosorption experiments Cell culture and uranium addition methods followed those described above (Section 2.4) and the pH was allowed to drift freely. The initial pH was around 4.5 and drifted to around 3.0 by the end of the culture. After a preset contact time (10 time gradients between 0 and 18 h), the cell suspensions were centrifuged (22 °C, 15 min, 4000 rpm), and the sediments were washed twice with doubly distilled deionized water. The sediments were dried with dry, hot air, and some of the sediments were dried through vacuum freeze-drying. The experiments were run in triplicate. The biosorption efficiency (R) was calculated by the following formula:

Rð%Þ ¼

q  qe  100% q

ð1Þ

The biosorption quantity of uranium was calculated according to the following equation:

q¼

ðq  qe ÞV m

ð2Þ

where q (mg g1) is the amount of uranium adsorbed per gram of dry weight, q (mg L1) is the initial concentration of uranium, qe (mg L1) is the final (or equilibrium) concentration of the uranium in solution, V (L) is the volume of solution and m (g) is the dry mass of S. cerevisiae.

3. Results and discussion 3.1. Growth curve of S. cerevisiae and toxicity of uranium The continuous industrial treatment of radioactive waste by microbes under culture conditions requires microbe survival and growth in a radioactively contaminated aqueous environment. To investigate the tolerance of yeast cells to uranium, living S. cerevisiae cells were cultivated in a culture medium containing uranium at various concentrations. The growth curve of S. cerevisiae in a culture medium containing uranium was similar to the S-type growth curve (Fig. 1); the exponential phase of growth was very short, and the time to reach the stable phase was extended compared to classical S-type growth curves. During the first 2 h of uranium addition to the medium, the uranium promoted the growth of S. cerevisiae. When the culture time exceeded 10 h, the growth speed decreased compared to control experiments. The S. cerevisiae growth under different uranium concentration culture conditions was most similar to control culture conditions, with the exception of when uranium concentration reached 600 mg L1 and S. cerevisiae growth was inhibited when cultured for 4 h. The inhibiting concentration was almost equal to the inhibiting concentration of

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1.8

1.6

OD/560nm

1.4

1.2

1

0.8

0mg/L

100mg/L

200mg/L

400mg/L

600mg/L

0.6

0.4 0

2

4

6

8

10

12

14

16

18

culture time/h Fig. 1. Growth curve of S. cerevisiae under different uranium concentrations.

uranium for Deinococcus radiodurans, which is 2.5 mmol L1 (about 670 mg L1) (Ruggiero et al., 2005). The SEM results also showed that most S. cerevisiae cells’ morphology did not change after they were cultured in 100 mg L1 and 300 mg L1 uranium conditions. These results indicated that uranium did not produce adverse effects on cell growth or morphology of S. cerevisiae. Our previous research has shown that the yeast cells can endure approximately 300 Gy (in an accumulated dose) X-ray irradiance in a cultured medium and can recover well when cultured again (unpublished data). These findings suggest that the yeast can survive and grow in a radioactively contaminated aqueous environment. However, radioactive uranium should be used to evaluate the yeast growth in such contaminated aqueous environments in further research. These data and other studies (Ruggiero et al., 2005; Khijniak et al., 2005) suggest that the biosorption of uranium by microorganisms under culture conditions is feasible. 3.2. Biosorption of uranium 3.2.1. Biosorption efficiency The data shown in Fig. 2A indicate that the curve of biosorption efficiency under different uranium concentrations was similar to the adsorption isotherm curve where biosorption efficiency increased with increased initial uranium concentration. The data showed that the S. cerevisiae cells had low uranium biosorption efficiency when the initial uranium concentration was lower than 40 mg L1. When the initial uranium concentration exceeded 160 mg L1, the biosorption efficiency reached 80–90% when the culture time was prolonged. If analyzed using the average degree, the data described that the growth of S. cerevisiae was facilitated by uranium in the initial 2 h and led to the decrease of biosorption efficiency. The growth of S. cerevisiae began to become restrained at the sixth hour while the biosorption efficiency was increasing. When the culture time reached 10 h, the biosorption efficiency began decreasing with the inhibiting growth of S. cerevisiae cells. The maximum biosorption efficiency of S. cerevisiae to uranium under culture conditions reached 92.4%. 3.2.2. Biosorption quantity The dependence of biosorption quantity on initial uranium concentration (Fig. 2B) showed that the biosorption quantity (q) in-

creased with the increase of initial uranium concentration (q) and decreased with the culture time. The maximum biosorption quantity was 102 mg g1 dry weight. When the initial uranium concentration was below 100 mg L1, the maximum biosorption quantity was lower than 25 mg g1 dry weight. When the initial uranium concentration exceeded 300 mg L1, the biosorption quantity increased greatly and the average biosorption quantity reached about 60 mg g1 dry weight. The maximum adsorption or bioaccumulation quantity of uranium by living or non-living S. cerevisiae with traditional biosorption method varies from 30 mg g1to about 2000 mg g1 dry weight (Popa et al., 2003), and the average biosorption quantity has been determined to be 100–160 mg g1 dry weight (Omar et al., 1996; Riordan et al., 1997; Liu et al., 2004). Therefore, the maximum biosorption quantity in this research was equal to the average level of biosorption quantity reported by previous studies (Omar et al., 1996; Riordan et al., 1997; Liu et al., 2004). Some previous studies have shown that the uranium removal rate was high at the beginning of biosorption and equilibrium was completely established after 30–60 min. In these studies, uranium biosorption increased with time during the 30–60 min and remained nearly constant after this period (Bayramog˘lu et al., 2006; Omar et al., 1996; Akhtar et al., 2007). In this study, the uranium biosorption was also very rapid, and the equilibrium was reached in less than 2 h. The biosorption quantity of uranium did not change greatly after 2 h for each initial uranium concentration. However, the biosorption efficiency increased slowly with culture time. This finding was due to S. cerevisiae cells growing at complete culture time and leading to the quantity increase of biomass, and accordingly the equilibrium needed to regenerate. Therefore, the biosorption under culture conditions was a dynamic change process when compared to traditional biosorption using separated biomass. According to the results of the biosorption experiments, the optimal culture time was 6–10 h for uranium scavenging under culture conditions. 3.2.3. Effect of culture medium on biosorption quantity The curve of equilibrium uranium concentration and biosorption quantity was similar to the adsorption isotherm curve when cultured for 2 h with an initial uranium concentration between 40–300 mg L1. If the initial uranium concentration exceeded 300 mg L1, and the culture time exceeded 2 h, the curve no longer

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A

100 90 80 70

R/%

60 50 40 30 0h

20

8h

2h

16h

10 0 0

100

300

200

400

500

600

ρ /mg L

-1

B

90 80 70

q/mg.g-1 dry weight

60 50 40 30 20 10

0h

2h

8h

16h

0 0

100

200

300

400

500

600

ρ /mg L-1

-10

Fig. 2. The dependence of (A) biosorption efficiency (R) and (B) biosorption quantity (q) of S. cerevisiae on initial uranium concentration (q) under culture conditions.

VDR / %

100

100mg/L

99

300mg/L 600mg/L

98 0

2

4

6

8

10

12

14

16

culture time/h Fig. 3. Volume reduction ratio (VDR) profile for biosorption of S. cerevisiae under culture conditions.

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followed the adsorption isotherm curve. For example, q = 600 mg L1, qe = 1.38 mg L1, the real q = 63.04 mg g1 and the predicted q = 26.12 mg g1. The data indicated that when the initial uranium concentration was high, the q value could not be calculated from the model. The reason for this may be that when the initial uranium concentration was high and culture time was long, the uranium could interact with the culture medium besides being adsorbed to yeast cell surfaces. These interactions may have produced an exaggerated biosorption effect, which is mentioned in the SEM results. The exaggerated biosorption effect suggests that the biosorption quantity is an apparent adsorption quantity. This effect is not based on the biosorption equilibrium because the biosorption quantity also includes the sediment weight formed by the culture medium interacting with the uranium, in addition to the uranium weight adsorbed by yeast cells. The real biosorption quantity should only include the uranium weight adsorbed by yeast cells (e.g., Langmuir model, Freundlich model). These results demonstrate that the biosorption of uranium under culture conditions is a very complex system. Many adsorption rules cannot be described as simple linear models. 3.3. Volume reduction ratio The results shown in Fig. 3 demonstrated that the volume reduction ratio (VDR) was above 99% for all initial uranium concentrations. The maximum VDR was 99.5%. The VDR decreased with culture time. This is because the S. cerevisiae cells could still grow under culture conditions, and the biomass increased and led to the decreased VDR. Incineration is a convenient means of volume reduction for the management of radioactive waste. Incineration causes waste volume and weight to be reduced by factors of 100 and 10, respectively, for combustible solid wastes (Kim et al., 2002). Therefore, the VDR would reach almost 100% if the yeast cells sediments were treated by ashing after biosorption. This analysis indicated that the biosorption under culture conditions was an ideal method for volume reduction. We therefore suggest a candidate method for the disposal of radioactive wastewater that combines biosorption by microbes under culture conditions with continuous centrifuging separation and incineration or ashing, which will be described in detail in another manuscript. 3.4. Surface interaction results 3.4.1. SEM and EDS results The SEM results (see Fig. 4) showed that the S. cerevisiae cell surface was covered with the uranium precipitate. The quantity of precipitate increased with the increase in initial uranium concentration and the interaction time. The same uranium depositions have been described in other studies. Following the bioaccumulation of uranium from the 0.1 mol L1 uranium solution, an attachment of yeast cells to the bioaccumulating material has previously been observed (Popa et al., 2003). Uranium accumulated by Bacillus sphaericus JG-A12 is located at the cell surface (Merroun et al., 2005). Surface-bound uranium in Pseudomonas fluorescens is spread over the entire cell envelope in an outer membrane-peptidoglycan-plasma membrane complex as fine-grained, platy uranium minerals (10 nm to 1 lm) (Krueger et al., 1993). Only a few S. cerevisiae cells’ surface morphology changed greatly after biosorption. Some sediment of uranium with the culture mediums could be seen on the cover slip. When the uranium stock solution was mixed with suspensions of growing S. cerevisiae inoculated in a medium to carry out the biosorption process, the yellow precipitate could be found under high uranium concentration conditions (e.g., 600 mg L1). The yellow precipitate was also found immediately after the injection of uranyl acetate into a test tube containing growth medium for culture Thermoterrabacterium

Fig. 4. Scanning electron micrographs of S. cerevisiae cells (A) before and (B) after biosorption of uranium under culture conditions (culture time = 4 h and uranium concentration = 300 mg/L).

ferrireducens (Khijniak et al., 2005). These results also support the conclusion of the biosorption of uranium and the finding that when the initial uranium concentration was high, the q value could not be calculated according to the model because the heavy metal uranium may act with the medium components (such as the proteins, peptides, polysaccharides, etc.) to produce the increased biosorption quantity. The same results have been found with unwashed yeast biomass from a local brewery (Riordan et al., 1997). The EDS results before and after adsorption (Fig. 5) showed that the content of calcium decreased and peak of natrium, and that the magnesium nearly disappeared after biosorption. A new peak of uranium appeared after biosorption. The uranium contents in yeast cell after biosorption reached 12.11% (wt%) or 0.79% (at%). The content of phosphonium increased greatly after adsorption. The ion exchange process occurred when uranyl ions in the solution were transferred from the solution to biomass and chemical bonds were formed between uranyl ions and the S. cerevisiae cell walls. This phenomenon is often found in heavy metal biosorption (Davis et al., 2003). The high silicium content may be due to the thin yeast cell thickness on the carrier of glass cover slip. 3.4.2. FTIR spectroscopy results FTIR is an important tool that identifies functional groups. The vibrancy signals before and after biosorption of uranium (Fig. 6) showed that the peak of –OH shifted from 3427.6 to 3398.6 cm1, indicating that the binding of uranium to the cell wall of yeast enhanced the bond length and the vibrancy shifted to red

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Fig. 5. The EDS results of S. cerevisiae cells (A) before and (B) after biosorption of uranium under culture conditions (culture time = 4 h and uranium concentration = 300 mg/L).

wave. The peak of symmetrical stretching vibration of the phos1 phodiester group [ts(–PO , and the vibration 2 )] was 1092.6 cm absorption band of polysaccharide skeleton was 1042.9 cm1. After absorption, the peaks shifted to 1071.9 and 1022.1 cm1, respectively. The peak at 1739.6 cm1 was the absorbing band of carboxyl groups (tC@O). The peak almost disappeared after adsorption. This showed that the carboxyl groups and metal ion were coordinated, which may have been caused by the oxygen atom of carboxyl groups coordinating with the metal ion and leading to the shitting of symmetrical stretching vibration of carboxyl groups (tC@O). This process also caused the 1739.6 cm1 band to disappear (e.g., Lin et al., 2004). The FTIR results revealed that S. cerevisiae cell wall was the major sorption site, and that –O–H, –C@O and –PO2 contributed to the major binding groups.

Most secondary uranium minerals were composed of uranyl ions. These uranium minerals presented a characteristic peak at 800–1100 cm1 because the uranyl ion group possessed the characteristic structure and the fast U-O band due to the high valence state of uranium. After adsorption, a new peak emerged at 918.1 cm1 (Fig. 6B), which was a characteristic peak of the uranyl ion group. The precipitate formed in the adsorbing course under culture conditions might have been andersonite (Na2Ca(UO2)(CO3)36H2O) or autunite (Ca[UO2PO4]26H2O) according to the FTIR data from the literature (Zhang, 1986; Popa et al., 2003). The inverse symmetry stretching vibration peaks of andersonite and autunite are 920 and 922 cm1, respectively. A Citrobacter sp. accumulated an uranyl bond via precipitation with phosphate ligand to form NH4UO2PO4 (Macaskie et al., 2000). The yellow precipitate

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Fig. 6. FTIR spectra of S. cerevisiae cells (A) before and (B) after biosorption of uranium under culture conditions (culture time = 4 h and uranium concentration = 300 mg/L).

formed immediately after the injection of uranyl acetate into the growth medium and was identified as uranium phosphate ((NH4)(UO2)(PO4)3H2O) by X-ray powder diffraction (XRD) analysis (Khijniak et al., 2005). The EDS results of P:U:Ca were 1.9:1.0:1.2(at%) in this study. Therefore, the precipitate may have been autunite. This result also indicated that yeast cells did not change the property of UAO bands after biosorption. A comparison of FTIR figures before and after adsorption (Fig. 6) showed no other great peak changes. These data also showed that the S. cerevisiae cell wall structure did not change significantly. 4. Conclusions This study suggests that S. cerevisiae had good biosorption abilities for uranium under culture conditions. The yeast surface acted as the major sorption site and could adsorb approximately 92% of the uranium from the solution under 6–10 h culture conditions

and generated 99% VDR. Therefore, the biosorption by microbes under culture conditions can be used as a potential alternative or additive process to the existing technologies in continuous industrial disposal of radioactive wastewater. However, the biosorption under culture conditions is a complex system, and there are still many questions that need to be studied for engineering applications. Acknowledgements The authors thank the National Nature Science Foundation of China and the China Academy of Engineering Physics for a NSAF grant (Grant Number: 10776027), thank the National Defense Key Discipline Laboratory of the Nuclear Waste and Environmental Safety of the Commission of Science Technology and Industry for the National Defense Foundation for funding (Grant Number: 08zxnp04) and research professor Dong Zhang, Houjun Kang from

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