Environ. Sci. Technol. 1996, 30, 2371-2375
Biodegradation of Tributyl Phosphate by Naturally Occurring Microbial Isolates and Coupling to the Removal of Uranium from Aqueous Solution RUSSELL A. P. THOMAS AND L. E. MACASKIE* School of Biological Sciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
Tributyl phosphate (TBP) is widely used as a solvent and plasticizer with a special use as a solvent for the extraction of uranium and plutonium from other radionuclides in nuclear fuel reprocessing. Although the biodegradation of alkyl phosphates by microorganisms has been noted in the literature, biodegradation of TBP is little-documented. The proposed products of the degradation are potentially useful; the liberated 1-butanol as a biomass growth substrate and the co-released inorganic phosphate moiety as a ligand that can precipitate with heavy metals to retain these as biominerals on the biomass. Microorganisms growing on TBP were isolated from industrially contaminated sites and from laboratory enrichment cultures. A mixed culture, containing Pseudomonas spp., supported the deposition of heavy metal (uranyl) phosphate and the removal of the latter from solution (biodecontamination). This general biocatalytic approach has been reported previously; these new isolates show potential for the simultaneous treatment of two classes of waste: the breakdown of one waste (TBP) could be harnessed to the treatment of another class of waste (heavy metals) This approach would represent a considerable advance over established bioprocesses for metal removal, where the cost of the addition of exogenous organophosphate substrate (phosphate donor for the metal precipitation reaction) may ultimately limit their use.
Introduction Tributyl phosphate (TBP) is a complexing agent used in uranium extraction and nuclear fuel reprocessing (1). It is also used as a solvent for other metal extractions, in aircraft hydraulics fluids, in herbicide solutions, in carbonless copying paper systems, as a surface coating composition, and in a range of other applications as a plasticizer or defoamer (2). The solvent properties of TBP facilitate * Corresponding author telephone: 0044-121-414-5889; fax: 0044121-414-6557.
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Published 1996 by the Am. Chem. Soc.
uranium and plutonium extraction from nuclear fuel reprocessing solutions (1); residual TBP can persist in lowactive waste streams at, for example, 112 µM (3). The concentration of uranium present by comparison is 42 µM (3). In a typical fuel rod storage pond, the concentrations of TBP and uranium are higher, at 300 and 142 µM, respectively (3). In summary, TBP wastes can arise from various industrial activities unrelated to metal processing or in specialized instances where co-treatment of the organophosphate solvent and the heavy metal would be required. The latter poses the dual problem of antimicrobial solvent effects together with heavy metal toxicity and was chosen as a ‘worse case’ scenario for evaluation of a possible role for biotechnological processes in waste remediation. This preliminary study aims to establish the feasibility of this approach and highlights the potential of a new mixed culture for this purpose. TBP, a phosphotriester, is chemically hydrolyzed via the intermediates dibutyl and monobutyl phosphate (DBP and MBP, respectively) to butanol (3 mol/mol) and phosphoric acid (1 mol/mol) (4). The pathway of biodegradation would probably be similar (3, 5). A report detailing the biodegradation of trimethyl phosphate proposes a similar stepwise hydrolytic removal of the methyl groups (6). The pesticide parathion (O,O-diethyl O-p-nitrophenyl phosphorothiorate), a tertiary organophosphate with a similar type of structure to TBP, can be fully hydrolyzed enzymatically via the intermediates p-nitrophenol and diethyl thiophosphate (DETP) (7, 8). DETP is analogous to DBP, but biodegradation of these phosphodiesters is little-investigated. Although the liberated butanol could serve as a utilizable carbon source, there are no reports of TBP utilization in this context. One report notes TBP biodegradation by two strains of Pseudomonas; here TBP was used as the sole phosphorus source (5). Little is known of the enzymology of TBP degradation, although the participation of phosphotriesterases, diesterases, and monoesterases is implicated. Phosphate released by the latter has been coupled to the removal of uranium from solution as cell-bound hydrogen uranyl phosphate (HUO2PO4) using immobilized cells of a Citrobacter sp. (9, 10). The supplied phosphomonoester was glycerol 2-phosphate (G2P), the hydrolysis of which gave inorganic phosphate as a precipitant ligand at a high enough concentration locally to exceed the solubility product of HUO2PO4 and to permit metal removal from dilute solution in juxtaposition to nucleation sites at the cell surface (10). The present study couples the biodegradation of TBP by new microbial isolates to the removal of uranium from aqueous solution in a flow-through system and demonstrates the potential of TBP as a feedstock for a metal waste treatment process. In addition to providing a method for the treatment of TBP-loaded wastes per se, this approach could provide a use for waste phosphotriesters as additives to wastes from metallurgical industries, which would require a organophosphate feedstock for enzymatically mediated metal phosphate biomineralization onto biomass (11, 12).
Materials and Methods Isolation of a TBP Degrading Culture. A mixed culture capable of growth on TBP as the sole carbon and phos-
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phorus source was isolated from the Mersey river at Warrington (U.K.). River water (8 mL) was added to 92 mL of minimal medium that comprised (g/L) the following: CaCl2, 0.025; MgSO4‚7H2O, 0.2; NaCl, 0.1; (NH4)2SO4, 5.0; disodium EDTA, 0.015; ZnSO4‚7H2O, 0.0066; MnCl2‚4H2O, 0.00171; FeSO4‚7H2O, 0.0015; CoCl2‚6H2O, 0.000483; CuSO4‚ 5H2O, 0.000471; NaMoO4‚2H2O, 0.000453; 3-(N-morpholino)propanesulfonic acid (MOPS buffer), 5.225. The pH was adjusted to 7 by the addition of 12.6 mL of 1 M NaOH, and 0.53 g/L of TBP (final concentration) was added as the sole utilizable carbon and phosphorus source. Control tests utilized TBP-unsupplemented media to check for ‘background’ growth on the EDTA and MOPS components as carbon sources in the presence of added inorganic phosphate. The stock TBP (BDH Chemicals, U.K.) contained no other organophosphate species, as determined by 31P nuclear magnetic resonance spectroscopy (K. M. Bonthrone, perssonal communication). No growth of the initial culture was noted after 4 weeks (aerobic shake-flask culture 30 °C). A sample (10 mL) of the suspension was further diluted by subculture into 90 mL of fresh minimal medium containing 0.53 g/L TBP. Growth now occurred and was followed turbidimetrically at OD600. The growing culture was subcultured at weekly intervals by the same method. Identification of Component Microorganisms. Component microorganisms of the culture were isolated on nutrient agar (Oxoid, U.K.) by spread-plating 0.2 mL of microbial suspension at a 10-3 dilution on a nutrient agar plate in triplicate. Single colony isolates were tested for their ability to utilize butanol, DBP, and TBP as a carbon source by the addition of a loop sample of each colony type to 10 mL of the minimal medium containing either 0.44 g/L butanol, 0.63 g/L DBP, or 0.53 g/L TBP (all BDH, England). The isolates were Gram-stained by the Huckner method using the Sigma Gram stain kit (13). Gram-negative bacteria (the major components of the culture as isolated on the nutrient agar plates) were identified further using the API 20NE kit (Biomeriuex, France). Tributyl Phosphate Utilization by Growing Cells. All growth experiments were in triplicate. A total of 10 mL of the mixed culture (OD600 of 0.35) in stationary phase (72 h) was inoculated into 90 mL of fresh medium in a 250-mL conical flask. TBP (0.53 g/L final concentration) was added as the carbon and phosphorus source. Uranyl nitrate (UO2(NO2)2‚6H2O): BDH) was added as appropriate after 13 h of aerobic growth (30 °C) to a final concentration of either 1 mM (0.50 g/L) or 100 µM (0.05 g/L). The controls were uranium- and TBP-unsupplemented cultures and TBP- and uranium-supplemented cell-free media. Upon uranium addition at the higher concentration, precipitation was indicated by an immediate increase in the OD600; as a check for further growth, protein assays were done using the bicinchoninic assay kit (Sigma). Samples (1 mL) were removed at suitable time intervals and stored at -20 °C for up to 2 weeks prior to analysis. Samples stored thus gave identical results to fresh, unstored samples. The spent medium (culture supernatant) was assayed for residual TBP and DBP by gas chromatography (610 series, ATI Unicam). The method of Kuno et al. (14) was adapted with use of a PEG 20M macrobore column (Pye Unicam). The phosphate content of the supernatant was measured by an adaptation of the method of Pierpoint (15, 16); the uranium content was analyzed using arsenazo III (16 ,17).
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X-ray Diffraction (XRD) Analysis. Cultures (90 mL) containing 1 mM or 100 µM uranyl nitrate were centrifuged after 64 h. The pellet was air dried and washed with 1.5 mL of acetone three times, filtered under vacuum, and air dried for 24 h at room temperature. The yellow solid was examined by X-ray powder diffraction analysis (XRD) as described by Yong and Macaskie (16). The spectrum acquired was compared to the reference database for HUO2PO4 (18) and to the spectrum of HUO2PO4 prepared by biomineralization onto Citrobacter biomass as described previously (16). Biomass Immobilization and Uranium Removal by Immobilized Cells in a Flow-Through Column. A 100mL inoculum of the mixed culture (OD600 of 0.35) was transferred to an air lift fermenter (volume 2.3 L) constructed in the laboratory. An additional 1.9 L of minimal medium was added with 0.53 g/L TBP as the carbon and phosphorus source. The growing culture was supplemented with a futher 0.53 g/L TBP after 48 h to give a final OD600 of 0.535. The biomass was harvested by centrifugation, washed twice in sterile isotonic saline (8.5 g/L NaCl), and stored at -20 °C. For use, a sample of frozen biomass, equivalent to 1.33 g fresh weight, was thawed and resuspended in 30 mL of sterile isotonic saline. For immobilization (entrapment) in a foam matrix, the cell suspension was mixed with 30 g of polyurethane prepolymer (Hypol 3000; Hampshire Chemicals Ltd.) and left to set at room temperature for 30 min. A control used 30 mL of isotonic saline in lieu of cells. The set foam was cut into 0.5 × 0.5 × 0.5 cm cubes, 180 of which were placed in the lumen of a 90-mL glass column (14.5 × 2.8 cm). The column was washed with isotonic saline at a flow rate of 1 mL/min for 2 h and challenged with 1.35 L of minimal medium (pH 7) with or without 0.53 g/L TBP and 0.5 g/L uranyl nitrate (1 mM). The medium was pumped through the column upward by a peristaltic pump (Watson Marlow, Falmouth, England) at a flow rate of 0.75 mL/min (0.5 bed vol/h). Samples of the influent and outflow solution (1 mL) were periodically removed and stored at -20 °C prior to analysis for TBP, phosphate, and uranium as decribed above.
Results and Discussion Identification of the Components of the Mixed Culture. No growth was observed in any TBP-unsupplemented cultures, although a mixed population gave growth in TBPsupplemented medium. Attempts to isolate the microbial components of the culture on minimal agar with TBP were unsuccessful due to the plasticizing effect of the compound in regions where the TBP had formed droplets within the agar matrix. Instead, isolations were made on nutrient agar plates. No attempt was made to isolate oligotrophic organisms, since these would be more difficult to grow on a sufficient scale for industrial use; for the latter, wellestablished species such as Pseudomonas would be preferable given the large amount of information available on this genus, its well-established record for degradation of xenobiotics, and the relatively-well established genetic systems for possible later strain improvement. Identification of the major Gram-negative isolates obtained by this method and their ability to utilize butanol, DBP, and TBP are shown in Table 1. Five Gram-positive components were also isolated but were not identified further since they comprised only a small proportion of the population by colony examination and were lost from the
TABLE 1
Characterization of Major Bacterial Components of TBP Utilizing Mixed Culture As Isolated on Nutrient Agar and Growth of Single Isolates on TBP, DBP, and Butanol in Liquid Culturea isolate no.
identification (% agreement by API20 NE test)
% of total colonies isolated
butanol
1 2 3 4 5 6 7 8
Pseudomonas sp. (low discrimination) Pseudomonas putida (very good, 99.8%) Pseudomonas vesicularis (low discrimination) Comomonas acidovorans (low discrimination) CDCgr.IV C-2 (low discrimination) Comomonas acidovorans (low discrimination) Pseudomomas sp. (genus level) no matching identification
10 10 10 10 0.15. Boldface: predominant isolate obtained. The frequency of occurrence of each isolate (% of total isolates by colony type) is approximate.
culture on further subculture. Oligotrophic organisms are often slow-growing and would be expected to be outgrown, although this was not investigated. The predominant organisms isolated and identified and those retained in the culture upon further serial subculture were pseudomonads; one isolate was unidentified by the API20 NE method but was only a minor component of the stable mixed population identified (isolate 8, Table 1). Isolate 7 (a Pseudomonas sp.) was a major component identified, comprising approximately 30% of the bacteria isolated from the culture, and was the only isolate to grow well on TBP, DBP, and butanol in subsequent shake-flask tests (Table 1). In pure culture, this organism grew on TBP with a doubling time of 8 h to a final OD600 of 1.3 with a rate of TBP consumption of 37 µmol h-1 mL-1 culture and a comparable rate of phosphate release of 32 µmol h-1 mL-1. This stoichiometry was seen also with the other isolates when examined individually in liquid culture (19). None of the isolates retained the ability to utilize TBP indefinitely. Growth and TBP utilization were maintained for eight serial subcultures, after which this ability was irreversibly lost (19). Since strain stability is an important criterion for industrial use and since the mixed culture was of a stable composition, the latter was used in further studies. Growth and TBP Utilization of the Mixed Culture. Cellfree control experiments established that the OD600, TBP, inorganic phosphate, and uranium (where added) concentrations remained unchanged throughout (Figure 1A,B). Growth experiments showed a decrease in the TBP content of the medium corresponding to an increase in OD600 (Figure 1D) and indicating utilization of TBP as a carbon and phosphorus source for growth. Only slight phosphate release was observed (Figure 1C), which was not stoichiometric with TBP consumption by the mixed culture (Figure 1D), in contrast to the observations with pure cultures tested individually (19; see above). Possibly excess phosphate was stored by a non-isolated component of the mixed culture; indeed, some microbial components of the mixed population showed accumulation of polyphosphate using the Neisser stain (20). The TBP degradation intermediate, DBP, was not detected in the medium during growth. This would be expected if TBP hydrolysis or uptake of the triester is the rate-limiting step. Intitially the medium contained approximately 0.1 mM phosphate ‘background’ (Figure 1B), which spontaneously increased to approximately 0.4 mM when the cells were added. This could be accountable as cellular phosphate release. A permeabilizing effect of TBP had been noted in
FIGURE 1. Tributyl phosphate (TBP) utilization and phosphate release by growing cultures. The mixed culture was pregrown in minimal medium supplemented with TBP as the carbon and phosphorus source as described under Materials and Methods and inoculated to an initial OD600 of 0.05 as shown into fresh TBP medium. (A) Control, uninoculated medium. Uranyl nitrate was added after 13 h. (O) OD600; (0) concentration of TBP in the cell-free medium; (4) uranyl ion concentration in the cell-free medium. (B) Control: TBPunsupplemented culture inoculated with biomass. (O) Biomass growth (OD600); ()) phosphate concentration. (C) Phosphate release (), () by the culture supplemented with 2 mM TBP. (D) Growth (O, b) and TBP utilization (0, 9) by the TBP-supplemented culture. Data are shown for two independent experiments. Open symbols: experiment I. Filled symbols: experiment II.
a previous study using Citrobacter sp. (R. E. Dick and L. E. Macaskie, unpublished), but this was discounted in the present study since the phosphate release was the same in the absence or the presence of TBP. The latter would also discount the phosphate efflux that can accompany organophosphorus compound uptake (21). In a second series of experiments, uranyl nitrate (100 µM or 1 mM) was added to the cultures after 13 h (Figure 2). This addition corresponded to a fall in the inorganic phosphate concentration in the medium (Figure 2A,C), suggesting that the uranium precipitated out rapidly and spontaneously. Uranyl nitrate (1 mM) inhibited TBP utilization by the culture after a short delay (Figure 2D). Growth was apparently promoted by the addition of 100 µM UO22+ (Figures 1D and 2B) when monitored turbidi-
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FIGURE 2. Effect of uranyl ion on growth and TBP utilization and uranyl deposition by the mixed culture. The cultures were supplemented with uranyl ion to a final concentration of 100 µM (A, B) or 1 mM (C, D) as shown (arrowed). (A) Phosphate release (), () and uranyl concentration (4, 2) in cultures supplemented with 100 µM uranyl ion. (B) Growth (O, b) and TBP utilization (0, 9) in cultures supplemented with 100 µM uranyl ion. (C) Phosphate release (), () and uranyl concentration (4, 2) in cultures supplemented with 1 mM uranyl ion. (D) Growth (O, b) and TBP utilization (0, 9) in cultures supplemented with 1 mM uranyl ion. Data are shown for two independent experiments. Open symbols: experiment I. Filled symbols: experiment II.
FIGURE 3. X-ray powder diffraction analysis of the yellow precipitate associated with the biomass harvested from the experiments shown in Figure 2. Solid line: diffraction analysis of the biomass-associated precipitate. Vertical lines: Crystallographic standard of H2(UO2)2(PO4)2‚8H2O.
metrically, but a parallel protein assay failed to show a similar increase, and it is likely that the increased turbidity was attributable to the formation of uranium phosphate precipitate. The yellow precipitate produced with 1 mM uranyl ion was identified by X-ray powder diffraction analysis (Figure 3). The observed spectrum (solid line) was in accordance with the reference spectrum of H2(UO2)2(PO4)2‚8H2O (vertical lines, Figure 3) and with the hydrogen uranyl phosphate produced by Citrobacter sp. (16) and Acinetobacter sp. (22) in previous studies of uranium phosphate biomineralization by these microorganisms. Use of Immobilized Biomass for Continuous Removal of Uranium. The above data suggest that phosphate derived from TBP hydrolysis supports the removal of uranyl ion from solution, since this was the only source of phosphate provided in the experiments. However, it is not
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FIGURE 4. TBP consumption and uranyl ion removal by immobilized biomass of the mixed culture challenged in a continuous flow system. Biomass was immobilized and retained within columns as described under Materials and Methods. Challenge solution (agitated) was passed at a flow rate of 0.75 mL/h as described, with monitoring of the inflow and outflow solutions: ()) influent TBP; (() effluent TBP; (O) influent uranyl ion; (b) effluent uranyl ion. The data are pooled from two experiments. For the uranium and TBP input solutions, the error was within 10-20%, attributable to inhomogeneity in the TBPin-water suspension, containing uranyl adduct. The reproducibility of samples taken from the exit solution was within 10%. Cell-free columns gave identical concentrations of TBP and uranyl ion in the inflow and effluent solutions, and no uranyl ion was removed by cell-supplemented columns in the absence of TBP.
clear from the above whether TBP hydrolysis was directly coupled to uranyl deposition or whether the latter occurred via phosphate leached from cellular pools. More extensive tests used immobilized biomass challenged with uranyl ion and TBP in a continuous-flow system. Columns containing cell-free ‘Hypol’ foam cubes neither degraded nor removed TBP. The concentration of this and the uranyl ion content in the influent and effluent solution were identical over 24 h of continuous flow. Similarly, biomass-filled columns removed negligible uranyl ion in the absence of TBP, discounting a major role for uranium biosorption. By comparison, columns with biomass and supplemented with TBP removed approximately 70% of the input uranyl ion and 99% of the TBP over 24 h (Figure 4). By calculation from the known biomass loading introduced into the column and the determined amount of uranyl ion removed, it was calculated that the uranyl loading on the cells was more than 100% of the biomass dry weight. Mobilized phosphate from cellular stores could not have accounted for the extensive uranyl deposition. Indeed, a strain of Acinetobacter, which accumulates polyphosphate to approximately10% of the bacterial dry weight and which couples subsequent phosphate mobilization to the stoichiometric accumulation of heavy metals (22) would have accumulated uranyl ion to only approximately 25% of the dry weight by calculation if all of the deposited phosphate was made available for metal removal. In accordance with previous studies using Citrobacter sp. (23) and in view of the uranyl toxicity to growing cells in this investigation (Figure 2), it is unlikely that the biomass was actively growing in the column during
challenge. Previous studies have shown that phosphohydrolytic activity can occur in the absence of cellular growth (growth-decoupled operation), and assuming that the phosphotriesterase and diesterase activities are comparably stable to the phosphomonoesterase activity reported previously (3, 10, 16), there is no reason why the columns should not function in the absence of biomass growth. Examination of the column exit solution showed that the precipitate did not remain within the column but partially washed-out and settled subsequently. This suggests that for industrial operation a settling step would be required. Nevertheless, this study demonstrates ‘proof of principle’ for the integrated biological treatment of two classes of waste, which is receiving increased attention with respect to realistic industrial situations.
(7) (8) (9) (10)
Acknowledgments
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The authors wish to thank Mrs. G. Basnakova for help with the XRD study, Dr. K. M. Bonthrone for 31P NMR analysis of TBP and Drs. J. R. Lloyd and A. Morby for helpful discussions. The support of the BBSRC (Studentship No. 93302170) to R.A.P.T. is acknowledged with thanks. The work was funded, in part, by the European Community (Environment: Contract EV5V-CT 93-0251).
(11) (12) (13) (14) (15) (16) (17) (18)
(20) (21)
(22)
Literature Cited (1) McKay, H. A. C. Belg. Chem. Ind. 1964, 12, 1278. (2) Jones, C. J.; Brown, D. A. In Science and Technology of Tributyl Phosphate. Miscellaneous Industrial Uses; Schulz, W. W., Navratil, J. D., Eds.; CRC Press: Boca Raton, FL, 1987; Vol. 2, p 35. (3) Macaskie, L. E. CRC Crit. Rev. Biotechnol. 1991, 11, 41. (4) Belskii, E. Russ. Chem. Rev. 1977, 46, 828. (5) Rosenberg, A.; Alexander, M. Appl. Environ. Microbiol. 1979, 37, 886. (6) Ghisalba, O.; Kuemzi, M.; Ramos-Tombo, G. M.; Schar, H. P. Chimia 1987, 41, 2206.
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Barik, S.; Sethunathan, N. J. Environ. Qual. 1978, 7, 346. Barik, S.; Sethunathan, N. J. Agric. Food Chem. 1979, 27, 1391. Macaskie, L. E.; Dean, A. C. R. Biotechnol. Lett. 1985, 7, 457. Macaskie, L. E.; Empson, R. M.; Cheetham, A. K.; Grey, C. P.; Skarnulis, A. J. Science 1992, 257, 782. Macaskie, L. E.; Hewitt, C. J.; Shearer, J. A.; Kent, C. A. Int. Biodeterior. Biodegrad. 1995, 35, 73. Roig, M. G.; Manzano, T.; Diaz, M.; Pascual, M. J.; Paterson, M.; Kennedy, J. F. Int. Biodeterior. Biodegrad. 1995, 35, 93. Collins, C. H.; Patricia, M. L. Microbial Methods; Butterworth & Co.: London, 1984; p 93. Kuno, Y.; Hina, T.; Akiyama, T.; Matsui, M. J. Chromatogr. 1991, 537, 489. Pierpoint, W. S. Biochem. J. 1957, 65, 67. Yong, P.; Macaskie, L. E. J. Chem. Technol. Biotechnol. 1994, 63, 101. Clark, P. J.; Butler, A. J.; Macaskie, L. E. Biotechnol. Tech. 1990, 4, 345. Anonymous. Powder Diffraction File Card No. 29-670. JCPDS: Swarthmore, PA, 1992. Thomas, R. A. P.; Macaskie, L. E. Bioremediation: New Scientific developments and Prospects for Future International Development. In Abstracts of the ASM/SGM Symposium; Aberdeen, Scotland, Sept 1995; p 95. Gurr, E. The Rational Use of Dyes in Biology; Leonard Hill: London, 1965. Cook, A. M. Combined carbon and phosphorus or carbon and sulphur substrates. In Mixed and Multiple Substrates and Feedstocks; Hamer, G., Egli, T., Snozzi, M., Eds.; European Federation of Biotechnology: Braunschweig Germany, 1989; p 71. Dick, R. E.; Boswell, C. D.; Macaskie, L. E. In Biohydrometallurgical Processing Vol. II; Jerez, C. A., Vargas, T., Toledo, H., Wiertz, J. V., Eds.; The University of Chile: Santiago, 1995; p 177. Yong, P.; Macaskie, L. E. Bull. Environ. Contam. Toxicol. 1995, 54, 892.
Received for review November 15, 1995. Revised manuscript received March 18, 1996. Accepted March 18, 1996.X ES950861L X
Abstract published in Advance ACS Abstracts, May 1, 1996.
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