033 BIOSORPTION OF STRONTIUM FROM AQUEOUS SOLUTION ...

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*Tel: 27 12 420 5894, Email: Evans. ... from aqueous solution by a growing sulphate reducing bacteria consortium. The effect of the ... Results obtained showed at all initial strontium concentrations ranging from 25-500mg/L, bulk strontium ...
033 BIOSORPTION OF STRON TIUM FROM AQUEOUS SOLUTION BY SULPHATE REDUCING BAC TERIA Nonhlanhla Ngwenya and Evans M. N. Chirwa* Department of Chemical Engineering, University of Pretoria Pretoria 0002, RSA *T el: 27 12 420 5894, Email: [email protected]

ABSTR AC T 2+

This study investigates the sorption of Sr from aqueous solution by a growing sulphate reducing bacteria consortium. The effect of the presence of strontium at varied concentrations on the grow th of sulphate reducing bacteria was also evaluated. The presence of strontium hindered the rate of sulphate reduction, and consequently sulphate reducing bacteria grow th. Results obtained showed at all initial strontium concentrations ranging from 25-500mg/L, bulk strontium removal occurred within the first 30 minutes of incubation, suggesting that removal occurred through passive processes, such as biosorption and chemical precipitation. When the sequential leaching procedure w as applied for the partitioning of strontium species in the solid fraction, it w as found that about 68% of the removed strontium w as adsorbed on the surface of the bacteria by relatively weak electrostatic interaction, while about 22% was bound to carbonates, 4% bound to oxides, 3% bound to sulphides and remainder occurring as an immobile fraction. Key Words: sulphate reducing bacteria, biosorption, complexation, metal precipitation, bioremediation.

INTRODUC TION Radioactive wastes are generated in a number of facilities and are discharged in a wide range of concentrations of radioactive materials and in a variety of physical and chemical forms. Radioactive waste is generated at various stages of the nuclear fuel cycle, which includes the mining and milling of uranium ore, fuel fabrication, and reactor operation and spent fuel reprocessing. Radioactive waste is also produced as a result of the ever-increasing use of radioisotopes in medicine, industry and agriculture, and its diverse complexity has created an uncertainty in the choice of suitable 137 90 processes for its remediation. Fission products such as cesium ( Cs) and strontium ( Sr) are the most abundant contaminants, with strontium as the major radiochemical component (1). These fission products are routinely or accidentally released along mine waste waters, and are considered as most hazardous radiotoxic elements for the environment. The proper management of radioactive waste is of major concern so as to avoid the possible 90 hydrogeologic migration of strontium ( Sr), which has a high mobility in soils and groundwater sy stems (2). Radioactive strontium has a half-life of 28 years, and due to its chemical similarity with calcium, it is easily incorporated into bone and continues to irradiate localized tissues with the eventual development of bone sarcoma and leukemia (3). Radioactive waste toxicity is due to the presence of radionuclides and fission products in the waste, and their removal is a critical element in the treatment of radioactive effluents as it decreases the radiotoxicity of the final waste by a factor of 20 to 30, making easier its disposal (4). While it has already been established that approaches that utilize microbial interactions for sequestration of environmental metal and radionuclide contamination offer practical solutions, however, their application in the treatment of radioactive wastewater is still not known. T he use of microorganisms in the treatment of radioactive wastewater is mainly governed by their survival in extreme conditions of heat, dessication and radiation. Microorganisms are ubiquitous in nature, and recent studies have shown that microorganisms can survive in irradiated environments such as the walls of a pool storing nuclear materials at a Spanish nuclear power plant (5) and in an underground granitic rock nuclear waste repository in Canada, where a 1 6 range of heterotrophic aerobic and anaerobic bacteria ranging from 10 to 10 cells/g dry weight buffer were found. 2 Among these were approximately 10 sulphate reducing bacteria (SRB) and methanogenes per gram of dry weight buffer (6). Since then there has been ongoing research on the use of microorganisms, particularly SRB to treat radioactive waste of different radiation levels. T he sulphate reducing bacteria metal remediation concept is not new, however their occurrence in radioactive environments has aroused a renewed interest in their application for radioactive waste remediation. Sulphate reducing bacteria has been shown to catalyze the reduction of toxic forms of metals and radionuclides, such as U(VI), T c(VII), Pd(II), Cr(VI), As(V) and Mo(VI) to less toxic forms (7; 8; 9; 10; 11, 12 and 13). In this study, the sorption of strontium by a mixed sulphate reducing bacteria culture and its ability to survive in an aqueous environment heavily polluted by strontium was evaluated. Results obtained in this study will help gain an insight on the potential application of sulphate reducing bacteria for the subsequent removal of fission products and

subsequently the recovery of valuable fissile material, particularly uranium, from nuclear wastewaters for reuse in nuclear reactors.

MATERIALS AND METHODS Source of microorganisms and culture conditions A mixed sulphate reducing bacteria starter culture previously isolated from a coal mine dumpsite was kindly supplied by CSIR (Pretoria). T he culture was kept at 4°C until further use. A 10% (v/v) of the starter culture was used to inoculate a 10L stock culture of sulphate reducing bacteria. The microorganisms were grown in a modified Postgate medium C (Table 1). All reagents were of analytical grade and were purchased from Merck (South Africa). This medium is devoid of iron to prevent the formation of a black iron sulphide precipitate, which affects sphetrophotometric measurements. All the components were added and then dissolved in 990 mL of distilled/deionized water. After the pH of the medium has been adjusted to pH 7.5 using concentrated NaOH, it was then autoclaved for 15 minutes at 15 psi pressure-121°C. Then under a UV hood 10 mL of a sterile solution prepared by dissolving 0.1g sodium thioglycollate and 0.1g ascorbic acid was introduced into the sterilized medium just before inoculation. The microorganisms were grown under a nitrogen atmosphere, incubated in the dark at 37°C and on an orbital shaker at 200 rpm. T he stock culture was sub-cultured every 2-4 weeks. T able 1. Composition of media used for the growth and enumeration of sulphate reducing bacteria. omponent odium lactate (60% solution w/v) east extract H4Cl a2SO4 H2PO 4 gSO 4.7H2O odium citrate.2H2O aCl 2.6H2O eSO .7H O 4

2

Modified Postgate C 6 mL 1.0g 1.0g 4.5g 0.5g 0.06 0.1g 0.06g -

Postgate C 6 mL 1.0g 1.0g 4.5g 0.5g 0.06 0.1g 0.06g 0.004g

Enumeration of sulphate reducing bacteria Sulphate reducing bacteria numbers were estimated using the three-tube most probable number (MPN) method. The inoculum was serially diluted by anaerobically transferring 1 mL volumes into rubber-sealed tubes containing 9mL Postgate medium C (T able 1) prepared as described before. The culture tubes were then incubated in the dark at 37°C in a shaker at 200rpm for up to 28 days. Blackening of the medium due to FeS formation will be regarded as a positive result. Preparation of reagents All reagents used in this study were prepared using ultrapure water from a Milli-Q water purification unit. Chemicals used were of analytical grade unless stated, and were purchased from Merck (South Africa). All glassware used was previously cleaned and soaked in 10%HNO 3 and rinsed with pure water. A standard strontium solution was prepared by dissolving 3.0429g of SrCl2.6H2O in 10mL HNO 3/ultrapure water solution (1:1) and then diluted to 1L to give a final concentration of 1000µg/mL Sr. Working standard solutions were prepared by diluting the stock solution with ultrapure water to make 2, 4, 6, 8 and 10µg/mL. Batch bioreactor set-up All experiments were conducted in duplicate. Batch experiments were conducted in 2L rubber-sealed anaerobic bioreactors filled with 1.5L Postgate medium C to allow headspace for gaseous exchange as shown in Figure 1. Prior to inoculation, a sterile solution of strontium to give final concentrations of 100, 300 and 500mg/L were added to each of the bioreactors under a UV hood. Each of the batch bioreactors were then inoculated with 5mL of 4.3×10 5 cells/gram of actively growing sulphate reducing bacteria cells harvested by centrifugation (7000g x 15 minutes). Nitrogen gas was regularly purged into the system to keep it anaerobic, and at the same time flushing out the hydrogen sulphide gas produced into a zinc acetate trap solution. T he bioreactors were operated in a fume cupboard, incubated at 37°C, and

continuously shaken at 200rpm. To monitor the growth of sulphate reducing bacteria, changes in sulphate, sulphide, strontium concentrations and pH were monitored daily over a 10 day incubation period.

Sampling port Nitrogen gas inlet

H2S outlet

Bioreactor

Shaker Figure 1. Schematic of the laboratory-scale anaerobic batch bioreactor. 10%diagram Zinc acetate solution Analytical procedures T he concentration of sulphate was determined using the turbidimetric method (14). Prior to sulphate determination samples were centrifuged (7000g x 15 minutes) to remove suspended solids. To a 5mL sample, 0.25mL of conditioning reagent (50mL glycerol, 30mL concentrated HCl, 75g NaCl, 100mL ethanol and 300mL deionized water) was added, and mixed thoroughly. After that an excess amount of finely ground BaCl2 was added and mixture stirred for 1 minute and its absorbance was measured at 420nm. Sulphate concentration was calculated from a calibration curve obtained using a similar procedure. Sulphide concentrations were measured using test kits for the Spectroquant® Nova 60 (Merck, Germany) procured from Merck, South Africa. Sample pH was measured using an Orion 3 Star series pH meter (Labotech, South Africa). To determine the distribution of strontium species in the insoluble fraction a five-step sequential extraction procedure (15) was carried out as follows: Step I: To extract exchangeable species, 8mL of 1M MgCl2 solution (pH 6) was added to 2g of the pellet obtained by centrifugation (7000g × 15 minutes). T he mixture was stirred at 25ºC for 1 hour. The mixture was then centrifuged and the supernatant was used for strontium analysis. Step II: To extract species bound to carbonates, 10mL of 0.1M sodium acetate adjusted to pH 5 using acetic acid was added to the residue obtained at step I. The mixture was stirred at 25ºC for 3 hours, and then centrifuged and the supernatant was used for strontium analysis. Step III: T o extract species bound to oxides, 10mL of 0.1M Hydroxyl ammonium chloride (pH 2) was added to the residue obtained from step II. T he mixture was also stirred at 96ºC for 3 hours, and then centrifuged and the supernatant was used for strontium analysis. Step IV: T o extract species bound to sulphide, 5mL of 30% hydrogen peroxide adjusted to pH 2 with HNO3 and 3mL 0.02M HNO3 was added to the residue obtained from step III. T he mixture was stirred for 2 hours at 85ºC. T his was followed by the addition of 5mL aliquot of 3.2M ammonium acetate (pH 2) in 20% (v/v) HNO 3 to avoid adsorption of the extracted metal into the oxidized fraction. The mixture was stirred for a further 30 minutes at 85ºC and then centrifuged and the supernatant was used for strontium analysis. Step V: To extract residual strontium species, 2mL each of H2O 2, HNO3 and Hydrofluoric acid were added to the residue obtained from step IV. The mixture was stirred at 96ºC for 3 hours, and then centrifuged and the supernatant was used for strontium analysis. 2+

For all other metal analysis experiments, total strontium (SrT ), solid-phase strontium (SrS ) and dissolved Sr (SrD) species concentrations were determined by separately. Prior to SrS and SrD determination, raw samples were centrifuged at 15 000g x 20 minutes to separate the dissolved and insoluble fractions. To determine SrD, 2.5mL of the

supernatant was dispensed into 10mL acid washed tubes to which 0.5mL of 30% H2O 2 and 0.1mL of 70% trace metal grade HNO 3 were added and heated at 60°C overnight. The resulting pellet was also subjected to the same acid digestion treatment. Following digestion or extraction, strontium concentrations were determined using atomic absorption spectrophotometer (AAS) or inductively coupled plasma atomic emission spectroscopy (ICP-AES). RESULTS AND DICSUSSION Effect of strontium on SRB growth Sulphate-reducing bacteria (SRB) derive their energy through the anaerobic reduction of sulphate to sulphide. However in the presence of strontium concentrations higher that 100mg/L, the rate of sulphate reduction is lowered (Figure 2a). In the absence of strontium (control), SRB effected a 56% reduction of the initial sulphate concentration in the reactor

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Ti me (days) Figure 2 Rates of sulphate reduction (a) and changes in pH (b), by sulphate reducing bacteria in the absence and presence of strontium at different initial concentrations.

over a ten-day period. On the other hand, the presence of strontium resulted in a lower sulphate reduction rate. There was no significant difference in the amount of strontium reduced at the different initial concentrations (30% at 100mg/L and 31% at both 300 and 500mg/L). T hese findings depict the detrimental effect of strontium at concentrations higher than 100mg/L on the growth of SRB. Since all bioreactors were inoculated with actively growing cells, higher sulphate reduction rates were observed in the first 2 days of incubation, both in the absence and presence of strontium. SRB oxidize carbon sources to yield energy, a process that also results in the generation of alkalinity in the form of bicarbonate ions. In all bioreactors the pH was maintained above 7, although there was an initial drop observed on the first day (Figure 2b). In the absence of strontium there was a slight increase in system pH from 7.2 to 7.4, and an increase from 7.24 to 7.54 in the presence of 500mg/L strontium. However, there was a slight drop in pH at strontium concentrations of 100 and 300mg/L (7.29 to 7.14 and 7.28 to 7.23, respectively). Precipitation of different initial strontium concentrations In all test experiments, there was an instant removal of strontium from solution during the first day of incubation (Figure 3). This instant removal of strontium from solution regardless of the initial concentration indicates that bulk removal was not due to an active process but a passive process, including both chemical precipitation and biosorption. Chemical precipitation being a result of either a reaction between strontium and available ligands in the media, including sulphate or the sulphide and bicarbonate ions produced. Worth noting is the fact that a reaction between strontium and sulphate results in an insoluble strontium sulphate salt. T here was a 92 and 96% strontium removal at initial concentrations of 300 and 500mg/L, respectively, and only 26% removal at an initial concentration of 100mg/L. At decreased initial strontium concentrations of less than 100mg/L (50 and 25mg/L), bulk removal occurred in the first 30 minutes of incubation (Figure 4). More strontium was removed (70%) at initial concentration of 50mg/L as compared to only 49% removal at an initial strontium concentration of 25mg/L. Decreasing the initial strontium concentrations or shortening the sampling intervals and incubation time did not yield results different from previous observations with higher concentrations. In view of the obtained results, it was clear that bulk strontium removal from solution occurs within the first 30 minutes of incubation, and higher rates of removal were observed at higher initial strontium concentrations. Distribution of solid-phase strontium species Results obtained in this study suggest that major strontium removal occurred through biosorption since about 68% of the solid phase strontium was found to be occurring in the exchangeable fraction (Figure 5). T his fraction gives an indication of the amount of strontium that is absorbed on the biomass surface by relatively weak electrostatic interaction, and can be released by ion-exchange processes (16). T his confirms earlier

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Time (da ys) Figure 3 Residual strontium concentrations in the supernatant after exposure to a growing sulphate reducing bacteria consortium.

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Fractions Figure 5 Partitioning of strontium precipitates in the solid fraction after exposure to a growing sulphate reducing bacteria consortium. findings suggesting that a passive process is responsible for bulk strontium uptake from solution. Therefore, it can be stated that in all experiments strontium was instantly removed from solution until a threshold limit was reached, as in the case of biosorption. T he elevated concentrations of strontium in this fraction may be due to the complexing ability of the organic ligands present in the surfaces of the bacteria cells. Similarly, high metal concentrations were obtained in the exchangeable fractions of both soil amended with sewage sludge and sulphide-rich tailings of a mine (17, 18). T he remainder of the solid-phase strontium was a result of a chemical precipitation due to the presence of ligands released into the media as products of both sulphate reduction and carbon oxidation. About 22% of the strontium was bound to carbonates, 4% bound to oxides and hydroxides, 3% sulphides and the remainder occurring as an immobile fraction.

CONCLUSION

T he presence of strontium has an effect on the rate of sulphate reduction by sulphate reducing bacteria. Strontium uptake from solution was metabolism-independent regardless of the starting strontium concentration. The sorption or uptake of strontium from solution onto sulphate reducing bacteria biomass occurred until the threshold limit is reached, and further metal uptake would have resulted in cell death. Most of the strontium removed from solution was bound onto biomass surface through cell surface complexation, ion exchange, adsorption, electrostatic and hydrophobic interactions and micro-precipitation, while the rest was through chemical precipitation. T hese results indicate that sulphate reducing bacteria can play a vital role in the remediation of wastewaters heavily polluted with high concentrations of fission products and

radionuclides provided the contact time is shortened such that metal uptake does not necessarily result to cell death. REFERENCES

1. JS Watson, CD Scott and BD Faison, Adsorption of Sr by immobilized microorganisms, Applied Biochemical Biotechnology 20/21, p699-709 (1989).

2. L Dewiere, D Bugai, C Grenier, V Kashparov and N Ahamdach, 90Sr migration to the geosphere from a waste burial in the Chernobyl exclusion zone, Journal of Environmental Radioactivity 40, p139-150 (2004). 3. J-P Chen, Batch and continuous adsorption of strontium by plant root tissues. Bioresource Technology 60, p185-189 (1997). 4. J-J Gautrot and P Pradel, High Level Waste and Spent Fuel: T ackling Present and Future Challenges. T he Uranium Institute. T wenty third Annual International Symposium (1998). 5. E Chicote, D Moreno, A Garcia, I Sarro, P Lorenzo and F Montero, Biofouling on the walls of a spent nuclear fuel pool with radioactive ultrapure water, Biofouling 20, p35-42 (2004). 6. S Kováč ová, J Lesyn and E Sturdik, E. Recent trends in Nuclear Waste Disposal. HEJ Manuscript. Slovakia. (2002). 7. DR Lovley, and EJ Phillips, Reduction of uranium by Desulfovibrio desulfuricans. Applied and Environmental Microbiology 58, p850-856 (1992). 8. DR Lovley, PK Widman, JC Woodward, EJP Phillips, Reduction of uranium by cytochrome c3 of Desulfovibrio vulgaris. Environmental Microbiology 59(11), p3572-3576 (1993). 9. DR Lovley and EJP Phillips, Reduction of chromate by Desulfovibrio vulgaris and its cytochrome c3. Applied and Environmental Microbiology 60, p726-728 (1994) 10. JR Lloyd, P Yong and LE Macaskie, Enzymatic recovery of elemental palladium by using sulphate reducing bacteria. Applied and Environmental Microbiology 64, p4607-4609 (1998). 11. MD T ucker, LL Barton and BM T hompson, Removal of U and Mo from water by immobilized Desulfovibrio desulfuricans in column reactors. Biotechnology and Bioengineering 60, p90-96 (1998). 12. JR Lloyd, J Ridley, T Khizniak, NN Lyalikova and LE Macaskie, Reduction of technetium by Desulfovibrio desulfuricans: biocatalyst characterization and use in a flowthrough bioreactor. Applied Envirnmental Microbiology 65, p2691-2696 (1999). 13. WL Smith and GM Gadd, Reduction and precipitation of chromate by mixed culture sulphate reducing bacterial biofilms. Journal of Applied Microbiology 88, p983–991 (2000). 14. APHA, Standard methods for the Examination of Water and Wastewater, 14th Edition. American Public Health Association. APHA-AWWA-WPC. John D. Lucas Co. Baltimore. 15. A Tessier, PGC Campbell and M Bisson, Sequential extraction procedure for the speciation of particulate trace metals, Analytical Chemistry 51(7), p844-851 (1979). 16. O Dahl, H Nurmesniemi and R Poykio, Sequential extraction partitioning of metals, sulfur, and phosphorus in bottom ash from a coal-fired power plant, International Journal of Environmental Analytical Chemistry 88(1), p61 - 73 (2008). 17. J Nyamagara, Use of sequential extraction to evaluate zinc and copper in a soil amended with sewage sludge and inorganic metal salts, Agriculture Ecosystems and Environment 69(2), p135-141 (1998). 18. E Carlsson, J T hunberg, B Ohlander and H Holmstrom, Sequential extraction of sulfide-rich tailings remediated by the application of till cover, Kristineberg mine, northern Sweden, T he Science of T he Toal Environment 299(1-3), p207-226 (2002).