Phosphatase production by a Citrobacter sp. growing in batch culture and use of batch cultures to investigate some limitations in the use of polyacrylamide gel-immobilized cells for product release A. J. Butler, D. S. Hallett and L. E. Macaskie Microbiology Unit, D e p a r t m e n t o f Biochemistry, University o f Oxford, Oxford, U K
Phosphatase production by a Citrobacter sp. increased during exponential growth but was transiently repressed by glucose in medium containing glycerol 2-phosphate as the sole phosphorus source. In inorganic phosphate medium, phosphatase production was enhanced by carbon starvation, but was inhibited by an excess of glucose, or in glycerol-sufficient cultures upon entry into stationary phase. This was prevented by the provision of glycerol 2-phosphate as the phosphorus source. The data are consistent with known multiple regulatory patterns for other bacterial acid phosphatases and would account for interbatch variations in phosphatase titer. Such variation can be employed to prepare batches of cells of different phosphatase titer which can be used as tools to investigate the substrate diffusional limitations involved in the use of polyacrylamide gel-immobilized cells for phosphate release.
Keywords: Citrobacter sp.; phosphatase; immobilizedcells; diffusionalconstraints Introduction Bacterial alkaline phosphatases are well understood as enzymes classically inducible under conditions of phosphate starvation. Alkaline phosphatase gene regulation and the phosphate response in Escherichia coli are well documented, l and the role of these enzymes in phosphate metabolism is widely accepted. In contrast, acid phosphatases are less well understood. Regulation of their production is controlled by more disparate factors, such as the nutrient status of the growth medium,2 and their roles have been assigned variously as, for example, hexose phosphatase 3-5 or polyphosphate hydrolases, 6'7 but detailed investigations are scant. A recent suggestion2 develops the latter concept further by postulating a role as a "safeguard" enzyme to protect the cell from temporarily acidic conditions by buffering the periplasm with phosphate released from com-
Address reprint requests to Dr. Macaskieat the MicrobiologyUnit, Department of Biochemistry, University of Oxford, South Parks Road, OxfordOXl 3QU, UK. Received 6 September 1990; revised 7 February 1991 716
pounds such as polyphosphates, but the true physiological role remains unclear. Similarly, although the genetics and regulation of alkaline phosphatase activity are relatively well understood, ~ it is only recently that detailed study of acid phosphatase has been undertaken. 2 Consequently, manipulation of acid phosphatase titer has generally relied upon physiological approaches. 8 An atypical acid-type phosphatase produced by a Citrobacter sp. has been harnessed to the removal of heavy metals from aqueous flows. 9 The heavy metalresistant, preformed enzyme functions in nongrowing ("resting") and immobilized cells in the liberation of HPO 2- from an organic phosphate " d o n o r " molecule with stoichiometric precipitation of heavy metals as cell-bound MHPO 9. Clearly, the phosphatase of this strain has industrial potential, and the present communication identifies some factors governing enzyme production by batch-grown cells. A high level of production during growth is necessary to ensure maximal metal accumulation by the cells when subsequently immobilized in fixed-bed, flow-through columnar reactors, where the efficiency of metal removal from the flow is related to the phosphatase specific activity of the immobilized cells. 9
Enzyme Microb. Technol., 1991, vol. 13, September
© 1991 Butterworth-Heinemann
Phosphatase production by Citrobacter sp.: A. J. Butler et al. Previous work has shown that metal removal was enhanced by increasing the loading of biomass per columnar reactor. 1° Fundamental work described in the present communication provides information on the control of phosphatase production by the Citrobacter sp. and also allows the formulation of a simple means to assess the degree of substrate diffusional constraint introduced under conditions of increased biomass load, using a polyacrylamide gel-immobilization system.
Experimental Organism and growth conditions The Citrobacter sp. (strain N14) was as described previously. 11 Standard minimal medium contained (g 1-1): Tris buffer, 12.0; (NH4)2SO4, 0.96; glycerol 2-phosphate (5.5 H20; B.D.H. Ltd.), 0.67; KC1, 0.62; MgSO4 • 7H20, 0.063; FeSO4 • 7H20, 0.00032, with the pH adjusted to 7.0 with 2 M HCI. The carbon source was either glucose or glycerol (0.3-3.0 g 1-1), as stated in individual experiments. In some cases, glycerol 2-phosphate was replaced by disodium hydrogen orthophosphate to an equivalent molar concentration of phosphate; this is stated where appropriate. Growth experiments were performed using 30-ml batch cultures shaken at 30°C routinely inoculated with 1 ml of overnight batch culture that had been previously maintained by daily subculture in the minimal medium. The cultures were grown to the mid-logarithmic phase of growth (A600 of 0.3-0.4; Varian series 634 spectrophotometer, corresponding to ca. 2-3 × 108 cells ml- 1). The sample size for determination of phosphatase activity (mid-logarithmic phase) was generally 10 ml; the volume of samples harvested at lower or greater cell concentrations was adjusted to give an approximately equal cell count per sample. In some cases, this necessitated the scale-up of culture volumes. For cell immobilization, large volumes (3 1) were required. Here aeration was provided by vigorous streams of sterile air, as described previously. N Harvests were generally performed in the late logarithmic or early stationary phase of growth (glycerol-glycerol 2-phosphate cultures) for maximal biomass production.
Preparation of immobilized cell columns The 3-1 batches were harvested by centrifugation, washed in isotonic saline (8.5 g 1-1 of NaCI), and immobilized in a polyacrylamide gel, as described previously. 11Each harvest (total of 5 g wet weight of cells) provided material for five replicate columns• When set, the gel was shredded through a stainless steel sieve 11 and divided into five equal parts by weight. Each aliquot was washed with several changes of saline to remove residual monomeric material, packed into glass columns (1.7 × 13 cm), and challenged with flows of composition 50 mM Tris-HC1, pH 7, supplemented with substrate (5 mM p-nitrophenyl orthophosphate; disodium salt; B.D.H. Ltd.) at flow rates controlled by an external peristaltic pump (Watson-Marlow).
Determination of phosphatase activity of resuspended and immobilized cells Samples of growing cultures were harvested by centrifugation, washed in isotonic saline, and resuspended in saline at approximately 2-3 x 109 cells ml-1. They were diluted 10-fold in M O P S - N a O H buffer (pH 7) and assayed for phosphatase activity by the liberation of p-nitrophenol from the substrate (p-nitrophenyl phosphate), as described previously. 12 Phosphatase specific activity (one milliunit) is expressed as nmol of p-nitrophenol liberated min-1 mg bacterial protein-1, with bacterial protein determined by the Lowry method and related to A600 of the bacterial suspensions, as described previously. 12 For determination of column (bioreactor) activity, samples of outflow from the columns were added to 0.2 M NaOH for visualization of the liberated p-nitrophenol versus p-nitrophenol standards similarly treated. Bioreactor efficiency is expressed in terms of the conversion factor, X = (So S)/So, where So = input concentration of substrate (here 5 mM) and S = residual concentration of substrate at time t; t is defined as the bioreactor residence time and is related to the flow rate, F, according to t = Total bioreactor volume (ml) volume occupied by solids (ml) Flow rate (ml min-1)
Variation of phosphatase specific activity during growth, and the effect of carbon source In glycerol 2-phosphate-minimal medium, the Citrobacter sp. grew with a doubling time of 90 rain, irrespective of the carbon source (glucose or glycerol; Figure la and b). The variation in phosphatase specific activity between batches previously reported 12is illustrated in Figure 1, where the activity for two cultures after 6 h was 400 and 280 milliunits, and a third experiment (not shown) where the activity was 260 milliunits. Enzyme activity was not maintained at a constant level but increased during growth, with a phosphatase specific activity doubling time of 225 min (Figure la), as compared to a cell doubling time of 90 min. A previous study 11 suggested that the potential of the cells to accumulate Cd (and by inference the phosphatase activity of the cells, although this was not tested) was carbon source related. The effect of addition of glucose to the glycerol cultures is shown in Figure lb, where to facilitate comparison between experiments using cultures of different specific activity (above), the maximum activity for the experiment (3 h from the initiation of treatment, corresponding to 6 h of growth from the original inoculation) was afforded a value of 100% with the activities of other samples expressed relative to this (Figure lb). After 3 h of treatment, the glucose-supplemented cultures expressed only 75% of the activity of the controls, but the growthdependent rate of increase was similar for each. The apparently reduced activity of the glucose cultures was
Enzyme Microb. Technol., 1991, vol. 13, September 717
3= o t~
dO0 E E
1'0 09 0-8 0"7 06 0-5
300 ~ w l -t
2 100 a_
Time (h) from inocutation
0 1 2 Time (h) from initiation of treatment
Figure 1 (a) Growth and phosphatase specific activity of Citrobacter sp. N14 in glycerol-minimal medium at 30°C supplied with glycerol 2-phosphate as the sole phosphorus source. (0) Bacterial growth; (&) phosphatase specific activity, experiment I; (I') phosphatase specific activity, experiment II. Phosphatase specific activity (one milliunit) is defined as nmol of product (pnitrophenol) liberated min -1 mg bacterial protein -~. (b) Effect of carbon source on phosphatase activity of Citrobacter sp. N14 at 30°C. The culture was inoculated at an initial biomass ml -~ as shown in legend to Figure la and grown for 3 h (2 divisions) in glycerol-minimal medium. The culture was divided into four, to which was added: (a) nothing; (b) glycerol (100 mM) to a final concentration of 10 raM; (c) glucose (100 mM)tO a final concentration of 10 mM; (d) 10% of the volume as water. The subsequent growth of all cultures was identical and pooled data are shown (O). Cultures a, b, and d gave identical results. Closed and filled symbols represent independent experiments. (&, A) Phosphatase activity of glycerol-supplemented culture; (T, V) phosphatase activity of glucose-supplemented culture. Data are expressed as percentage of maximum activity for each experiment (see text); 100% corresponded to specific activities (nmol p-nitrophenol min -~ bacterial protein -1) of 260 and 386 milliunits for experiments I and II, respectively
would comprise a significant proportion of the available carbon (see below). In the inorganic phosphate medium, carbon-limited cells ceased growth after 5 h (Figure 2a and b). Depletion of the carbon source at this point was confirmed by assay; neither residual glucose nor glycerol was detected in the appropriate culture supernatants. The carbon-sufficient cells continued to grow for a further 2-3 h (Figure 2c and d). Glucose repression was again evident, with the enzyme produced at only about 10% of the maximal level (Figure 2d) in the glucose-sufficient cultures; glucose starvation promoted an increase in enzyme production after cessation of growth (Figure 2b). With glycerol cultures (limiting, Figure 2a, or nonlimiting, Figure 2c), enzyme production was similar, reaching over 90% of the final level of 317 milliunits at a point corresponding to carbon depletion in the starved cultures (Figure 2a). Maximal activity was maintained during starvation (Figure 2a), whereas glycerol-sufficient cells lost 90% of the activity during the stationary phase (Figure 2c). Negligible enzyme activity was extruded into the medium; the activity of culture supernatants removed at 24 h was ca. 20 nmol ofp-nitrophenol min -~, equivalent to 1 mg of bacterial protein in the culture, accounting for less than 10% of the "lost" activity. It could be argued
-0-2 i~ ° ' -
-1-0 J -1.2 '
(d) ¢ . . . . 411"~ 0.2 .~
-0.4 ~ attributable to a transient glucose-mediated lag of approximately 45 min before resumption of phosphatase production.
Effect of carbon starvation The above clearly suggests a carbon-regulatory effect on phosphatase production, and indeed previous work ~3 has shown that the enzyme was overproduced fourfold under stringent carbon limitation in continuous culture. The effect of carbon starvation in batch cells was investigated (Figure 2). Here it was necessary to substitute inorganic phosphate for the glycerol 2phosphate routinely used, since the glycerol 2-phosphate phosphorus source also provided a source of potentially available glycerol upon cleavage of the organic phosphate. The amount of nascent glycerol 718
-1-2 II 10 13 23 25 .
5 Figure 2
10 1323250 Time (hours)
Effect of carbon starvation on phosphatase activity of Citrobactersp. N14 grown in inorganic phosphate medium. Cells were grown (inocula from overnight glycerol-glycerol 2-phosphate cultures: 1 ml inoculum into 30 ml experimental medium) in medium supplemented with: (a) 0.3 g 1-1 glycerol; (b) 0.3 g 1-1 glucose; (c) 2 or 3 g 1-1 glycerol; (d) 2 or 3 g 1-1 glucose. (0) Bacterial growth (As00). (&, V, O) Phosphatase specific activity (% of maximum; three experiments, where maximum activity was that observed in the glycerol-sufficient cultures at 7.5 h)
Enzyme Microb. Technol., 1991, vol. 13, September
Phosphatase p r o d u c t i o n b y C i t r o b a c t e r sp.: A. J. Butler e t al. Table 1 Calculation: Substrate diffusional constraints within the polyacrylamide gel immobilization system Given substrate conversion efficiencies Xl and X 2 for bioreactors of enzyme loading Eol and Eo2 respectively at F = constant (Figure 5 mM p-nitrophenyl phosphate; Krnapparent for p-nitrophenyl phosphate in gel-immobilized cells = 1.8 mM15:
3); S o =
F[SoX1 + Krn" In(1/(1 - X1))] 9,~5 F[SoX2+ Kin" In(1/(1 - X2))]' Situation 2: One culture of constant specific activity: biomass loading of 1 g and 2 g wet weight of cells per bioreactor Relative Eo value Value for X (Experimental data b) Eol = 1.0 X1 = 0.64 Eo2 = 2.0 )(2 = 0.77 Calculated X2 by substitution of known values for Eol, Eo2, and )('2 into the equation = 0.95 Actual value for )(2 by experiment = 0.77 Deviation from expected value = 19%
Situation 1 : Two batches of specific activity of 89 and 150 units respectively; constant biomass loading per bioreactor Relative Eo value Value for X (Experimental data a) Eol = 1.0 Xl = 0.66 Eo2 = 1.685 X2 = 0.91 Calculated X 2 by substitution of known values for Eo~, Eo2, and X1 into the equation = 0.91 Actual value for X2 by experiment = 0.91 Deviation from expected value = 0% a Data obtained from Figure 3c and d b Data obtained from Figure 3a and b
that the stationary phase cells had died and suffered autolysis, and that the enzyme was unstable in cellfree medium. However, this was discounted, since cell viability was found to remain unchanged. Furthermore, whole-cell enzyme retained 100% of the initial activity after storage for 17 days at 4°CI3; immobilized cell columns retained full metal-accumulative ability over several weeks9; and cell-free extracts of the phosphatase enzyme were stable in storage at 4°C (B. C. Jeong & L. E. Macaskie, unpublished).
Effect of phosphate source on phosphatase production by glycerol-sufficient cells The above indicates that optimal conditions for phosphatase production are late exponential phase (glycerol-sufficient cultures) or stationary phase (glycerol-limiting cultures). From the data shown in Figure 2, it is difficult to reconcile maximal biomass production with maximal phosphatase activity; both are needed for economic commercial biomass use. It was found that the use of glycerol cultures supplemented with glycerol 2-phosphate in lieu of inorganic phosphate overcame these problems. In this case, in the carbon-limiting cultures the concentration of added glycerol (0.3 g 1- ~) was 3.35 mM which was potentially increased by ca. 2 mM using the glycerol from the glycerol 2-phosphate. The increase in the available carbon (ca. 60%) was matched by a corresponding increase in the biomass content per milliliter as the culture entered the stationary phase (not shown). However, the concentration of glycerol was still limiting, as confirmed by assay, and under these conditions the phosphatase specific activity in the stationary phase was increased by ca. 55% as compared to the mid-logarithmic phase value• The situation with carbon-sufficient cells was noteworthy. Here phosphatase activity was maintained at a constant level as the cultures entered the non-carbon-limited stationary phase, in contrast to the situation with cells
grown similarly but using inorganic phosphate (cf. Fig-
ure 2c). Use of interbatch variations in phosphatase activity as a tool to illustrate diffusional limitations in polyacrylamide gel-immobilized cells Previous work has shown that phosphatase activity varies greatly between cell batches~Z; the above results suggest that the specific activity is related to the stage of growth and the nutrient (both carbon and phosphate) status of the medium. Use was made of this intrinsic variation to obtain two large-scale preparations (3 1 each) of specific activities of 89 and 150 milliunits, respectively, which were immobilized (1 g wet weight per bioreactor) as described above. In calculation the term Eo (Table 1) is loosely defined as the "total enzyme available," being the product of the number of cells lying within the substrate diffusional threshold and the phosphatase titer per cell. Here the ratio of the two specific activities was 150 : 89, and values for Eo were assigned as 1.685 and 1.0, respectively (see calculation). Flow rate-activity relationships were obtained (Figure 3c and d), from which the bioreactor efficiency or conversion value, X, at F = limiting and constant at 116 ml h-~ was calculated from the known product liberated (determined by assay; Figure 3) and comparison with the input substrate (5 mM); X = (So - S)/So; see above. Since, for a plug flow reactor EoK3 = F[SoX + Km • In(l/(1 - X))], ~4it follows that, using a ratio method (see calculation), a value for X2 can be calculated using substituted known values for Eo~, Eo2, and X1. The value for X2 obtained by experiment agreed with that predicted by calculation• This procedure was repeated by changing Eo by simply doubling the biomass load from 1 to 2 g wet weight per bioreactor (i.e. 10 g instead of 5 g wet weight of cells immobilized)• Here, since
E n z y m e M i c r o b . T e c h n o l . , 1991, v o l . 13, S e p t e m b e r
.e5 - -
2O _E 1-0
o= 0 ,- 5.0 E
5s ~ - - - - ~
8 .__. k-0 o
O~~o oooe 0
6"0 4"0 In F (mr h-I)
Figure 3 Product (p-nitrophenol) liberation by Citrobactersp. immobilized in a polyacrylamide gel. The cells were immobilized as described and challenged with flows supplemented with 5 mM p-nitrophenyl phosphate. Product liberation was determined at various flow rates as shown. The conversion factor X was calculated as described in the text from the product liberated at F = 116 ml h -1 (arrowed). Open and filled symbols denote two experiments; each point is the mean of several replicate estimations. Bioreactor compositions were as follows: (a) 1 g wet weight of cells bioreactor-1; (b) 2 g wet weight of cells bioreactor -1, prepared from the same culture (specific activity was the same in both preparations); (c) 1 g wet weight of cells bioreactor -1 at a phosphatase specific activity of 89 milliunits; (d) 1 g wet weight of ceils bioreactor-1 at a phosphatase specific activity of 150 milliunits.
the specific activity was constant, Eo varied with the biomass loading, i.e. Eo~ = 1.0 and Eo2 = 2.0. A similar comparison of calculated and actual values for Xz gave a discrepancy (underestimation) of 19% from the expected values (see calculation). Since Eo = [number of available cells][phosphatase titer/cell], it follows that this error represented the proportion of cells that were unavailable ("masked") by the greater biomass loading.
Discussion Earlier work on the production of phosphatase by Citrobacter sp. (a close relative of Escherichia coil) established that carbon-starved cells overproduced phosphatase ~3in accordance with the established regulation of the production of bacterial acid-type phosphatases by the carbon status of the medium) Traditionally, bacterial phosphatases are divided into "acid" and "al720
kaline" types; the latter are classically induced under conditions of phosphate starvation. 16However, induction of " alkaline" phosphatase by phosphate limitation does not occur in all E. coil strains, '7 while the "acid" phosphatase structural gene aph A is also induced in the absence of phosphate. 1 Although described as an "acid-type" phosphatase on the basis of the pattern of regulation by carbon supply and on the basis of responses to several diagnostic tests, the Citrobacter phosphatase has an optimum pH of around neutral in whole cells ~2or when purified (B. C. Jeong and L. E. Macaskie, unpublished). Evidence for the role of inorganic phosphate in the control of production of this enzyme had not been previously sought; in the light of the above, this was undertaken. Evidence for a dual control was obtained. Synthesis of the enzyme was repressed only transiently by glucose during exponential growth when phsophate was provided as glycerol 2-phosphate, but was marked and sustained in glucose-sufficient inorganic phosphate medium. Starvation for carbon source (glucose) overcame the phosphate-inhibitory effect. This pattern was confirmed using the nonrepressive substrate glycerol as the carbon source. Here enzyme production occurred throughout exponential growth, but as the cultures entered stationary phase with excess residual glycerol and phosphate in the medium, the activity fell to the low level associated with the glucose-repressed cultures. Similarly, starvation for glycerol overcame this effect, and enzyme production was maintained maximally into the stationary phase. These results have important implications for the production of a high yield of active biomass for subsequent immobilization. In inorganic phosphate medium, good cell yield occurred in the stationary phase under carbon-sufficient conditions, but the enzyme was produced at high levels only under conditions where growth was inhibited by the carbon supply. Substitution of glycerol 2-phosphate for inorganic phosphate overcame these problems. The complexity of control of enzyme production may account for interbatch variations in phosphatase activity reported previously '2 and confirmed in the present study. This property was exploited in the use of the Citrobacter sp. as a tool to investigate product release by polyacrylamide gel-immobilized cells. It was demonstrated that in the immobilized cells, p-nitrophenol and inorganic phosphate are produced stoichiometricaUy (L. E. Macaskie, unpublished), and inorganic phosphate had no effect on the activity of extant enzyme. 13 Estimation of the concentration of product in the column outflow at a fixed, limiting flow rate provided a measure of the efficiency of product release by the immobilized cells. Use of cells of a greater phosphatase specific activity gave a corresponding increase in the ability of the immobilized cells to liberate product, whereas attempts to achieve this quantitatively by increasing the cell loading per column were unsuccessful, probably attributable to the greater diffusional constraints associated with the use of a denser packing of cells per unit of volume of gel. For maximum bioreactor
E n z y m e M i c r o b . T e c h n o l . , 1991, v o l . 13, S e p t e m b e r
Phosphatase production by Citrobacter sp.: A. J. Butler et al. performance, it is thus advantageous to produce the cells under conditions of maximum phosphatase production per cell in order to minimize the biomass content per column and thus the substrate diffusional constraints. This is best achieved by first understanding the principles underlying the control of phosphatase production and activity by the cells; investigations into these physiological and genetic aspects are continuing.
3 4 5 6 7 8 9
Acknowledgements The authors wish to acknowledge with thanks the financial assistance of BTP Ltd. in support of this work, in association with Pembroke College, Oxford.
10 11 12
Wanner, B. L. in Phosphate Metabolism and Cellular Regulation in Microorganisms (Torriani-Gorini, A., Rothman, F. G., Silver, S., Wright, A. and Yagil, E., eds.) Am. Soc. Microbiol. Publ., 1987, pp, 12-19 Touati, E., Dassa, E., Dassa, J. and Boquet, P. L. in Phosphate Metabolism and Cellular Regulation in Microorganisms (Torriani-Gorini, A., Rothman, F, G., Silver, S., Wright, A. and Yagil, E., eds.) Am. Soc. Microbiol. Publ., 1987, pp. 31-40
14 15 16 17
Dvorak, H. F., Brockman, R. W. and Heppel, L. A. Biochemistry 1967, 6, 1743-1751 Rogers, D. and Reithel, F. J. Arch. Biochem. Biophys. 1960, 89, 87-104 Van Hofsten, B. and Porath, J. Biochim. Biophys. Acta 1962, 64, 1-12 Dassa, E. and Boquet, P. L. FEBS Lett. 1981, 135, 148-150 Dassa, E., Cahu, M., Desjoyaux-Cheral, B. and Boquet, P. L. J. Biol. Chem. 1982, 257, 6669-6676 Bolton, P. G. and Dean, A. C. R. Biochem. J. 1972, 127, 87-96 Macaskie, L. E. and Dean, A. C. R. in Adv. Biotechnol. Proc. Vol. 12 Biological Waste Treatment (Mizrahi, A., ed.) Alan R. Liss, New York, 1989, pp. 159-201 Macaskie, L. E., Wates, J. M. and Dean, A. C. R. Biotechno/. Bioeng. 1987, 30, 66-73 Macaskie, L. E. and Dean, A. C. R. J. Gen. Microbiol. 1984, 130, 53-62 Macaskie, L. E., Blackmore, J. D. and Empson, R. M. FEMS Microbio/. Lett. 1988, 55, 157-162 Hambling, S. G., Macaskie, L. E. and Dean, A. C. R. J. Gen. Microbiol. 1987, 133, 2743-2749 Fulbrook, P. D. in Industrial Enzymology. The Application o f Enzymes in Industry (Godfrey, T. and Reichelt, R., eds.) Macmillan, London, 1983, pp. 8-40 Clark, P., Butler, A. J. and Macaskie, L. E. Biotechnol. Techn. 1990, 4, 345-350 Torriani, A. Biochim. Biophys. Acta 1960, 38, 460-479 Kuo, M. H. and Blumenthal, H. J. Nature 1961, 190, 29-31
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