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EDITHA KARL-KROUPA, CLAYTON F. CALLIS, and ELI SEIFTER Monsanto Chemical Co., Dayton, Ohio
Stability of Condensed Phosphates in Very Dilute Solutions b
c
Many types of cellular materials speed up decomposition of tripolyphosphate in surface waters.
IN
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AQUEOUS solution, condensed phosphates react with water to give less condensed phosphates and eventually orthophosphate as an end product. T h e chain and ring phosphates are unique among inorganic poly acids and their salts in that they hydrolyze at an extremely slow rate. The half life of these P-0-P linkages a t neutral p H and room temperature is of the order of magnitude of years (75). Much literature (75) on the hydrolytic degradation of phosphates shows that several important factors affect the rate of such reactions. These major factors and their approximate effect on the rate are condensed in Table I (5). A number of investigators (7, 3, 6, 7, 9-72) have reported that under certain conditions the enzymatic (73) hydrolysis of chain and ring phosphates can be extremely rapid. Studies of these effects in very dilute solution have been limited by the lack of reliable analytical techniques. Recently, paper chromatography was developed into a practical and reliable method for the differential analysis of phosphate mixtures (8) and extended for use in very dilute solutions of phosphate mixtures (partper-million range). With such a tool available, observations of the instability of very dilute, nonsterile stock solutions of condensed phosphates, as well as the current interest in the fate of condensed phosphates in surface waters, prompted this study-the stability of sodium tripolyphosphate in aqueous solutions containing various common living organisms. Tripolyphosphate hydrolyzes to give 1 mole of ortho- and 1 mole of pyrophosphate (75)) and by using the paper chromatographic techniques developed for this study the complete reaction was followed-Le., the concentration of tripoly-, pyro-, and orthophosphate was measured at any stage of the reaction.
Experimental T h e hydrolysis experiments were carried out in glass-stoppered, 2-liter flasks
immersed in a water bath controlled to 24 f 1" C . The solutions were prepared by adding known amounts of stock phosphate solution to the sterile flask containing sterile water (sterilized by autoclaving). T h e organisms were then introduced and the solution made up to volume with sterile water. T h e stock phosphate solution was prepared by dissolving 11.2 grams of laboratory prepared sodium tripolyphosphate hexahydrate in 1 liter of sterilized water. The organisms selected for this investigation are given with a brief description of their treatment prior to addition to the dilute phosphate solution. Elodea (a green higher plant)-leaves were treated to remove adsorbed material by conventional rneans4.e.) washed with calcium hypochlorite for 20 minutes, rinsed in tap water, immersed in ca. 0.570 Santomerse solution, rinsed in tap water, and then finally in distilled water. Vaucherza and Ceratophyllum (higher green algae)-treatment same as Elodea. Gleocapsa (single green algae)-a pure culture grown in liquid inorganic medium, harvested by centrifugation, washed with 1% citrate and 1% tartrate solutions, and finally with distilled water. Allomyces (fungus)-cultured in organic medium containing yeast extract and glucose, filtered, washed with 1% citrate, 1% tartrate, and finally with distilled water. Escherichia coli (bacteria)-a pure culture growth in liquid medium containing yeast extract, glucose, and peptone, and harvested by slow-speed centrifugation
Table 1. Factors Affecting the Rate at Which Chain and Ring Phosphates Undergo Hydrolytic Degradation Factor Temperature PH Enzymes Colloidal gels (3) Complexing cations Concentration Ionic environment in solution (4, 16)
Approximate Effect on Rate 106-106 faster from freezing to boiling lOS-10' slower from strong acid to base As much as 106-106 faster As much as 104-106 faster Several times faster Roughly proportional Severalfold change
to avoid heating. The precipitate obtained was rejected, as it contained large amounts of precipitated minerals and organic matter. T h e supernatant was then further centrifuged, and the centrifuged cells washed with 1% citrate, 1% tartrate, and twice with distilled water to remove the bulk of the organic matter. A 100-mg. maximum of organic noncellular matter is estimated to be present in the culture used to inoculate 2 liters of dilute phosphate solution. The containers were shaken to ensure homogeneity before removing a sample for analysis. A uniform suspension of the microorganisms was possible only in the E. coli experiments; in all other cases, the weight of microorganism per unit volume of solution was not constant during the experiment. Sample volumes of 100 ml. were removed under aseptic conditions a t arbitrarily selected time intervals. T h e aliquot was then filtered through a bacteria filter, and stirred for 45 minutes with 1 gram of Dowex 50 cation exchange resin in the sodium form. T h e resin was removed by filtration and the first 25 ml. of filtrate were discarded. T h e next 10 ml. were transferred into a Petri dish, 4-inch diameter, and allowed to evaporate at room temperature. T h e dry residue was taken up with 0.4 ml. of a 0.5% solution of sodium Versenate, and applied in a band on a sheet of chromatographic paper. Details for completing the paper chromatographic analysis have been described (8). T h e procedure involves the separation of the mixture in a 21/2- to 3l/~-hour run into bands containing the ortho-, pyro-, and tripolyphosphates, which can be cut apart and measured quantitatively by a colorimetric procedure. T h e reliability of this analysis has been shown (8) to be f 0 . 1 p.p.m. phosphorus in the range of 1 to 5 p.p.m.
Results T h e distributions of the phosphates found at various arbitrarily selected time intervals are summarized in Table I1 for the control and for the solutions containing various organisms. The degradation was measured under resting conditions of the organism with the exception of the E. coli experiment, where growth was observed both from turbidity and bacterial counts. VOL. 49, NO. 12
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DECEMBER 1957
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Table II.
Hydrolysis of Sodium Tripolyphosphate with Time in Various Aqueous Cellular Suspensions (pH range, 6.9 t o 6.1; temperature, 24 I 1’ C.; initial concentrationa of total phosphorus, 5.5 P.P.m.) yo of Total Phosphorus Dry Wt. of Organism Added, Time, Ortho. Pvro. TriDolv. G . / 2 L. Soln. Hours Organism Added +2,--i% None
Sterile water
Elodea
. a .
0.155
Gleocapsa
Vaucheria
0.0 28.0 144.5 339.0 820.0 4.5 26.3 75.3 120.5
2.5 X
1.3
0.260
0.318
x
1.8 1.8 1.8 2.0 5.4 4.0 4.6 24.7 33.1 7.6 12.2 13.0 23.1 50.3 4.2 7.3 64.3 87.9 3.3 5.1 13.3 14.8 80.5 4.1 3.6 6.5 11.6 64.8 97.7 21.4 22.5 22.1 27.1 83.6
10-8
Aliomyces
10
Escherichia colib
80 X 10-6
24.3 48.0 72.8 140.3 5.5 27.3 76.0 123.8 2.5 4.8 21.5 28.0 121.0 1.7 3.5 5.3 24.8 53.5 147.0 0.5 25.0 47.3 71.8 140.3
5.8 5.7 5.4 6.1 9.3 6.0 7.8 19.2 16.8 10.4 10.6 12.8 16.2 29.6 5.8 8.6 14.2 6.2 5.2 6.0 11.4 11.8 9.3
92.4 92.5 92.8 91.9 85.4 89.9 87.5 56.0 50.0 82.0 77.1 74.1 60.8 20.1 90.0 84.1 21.5 5.9 91.4 88.8 75.2 73.3
6.8 6.3 7.6 9.4 16.6 1.5 10.0 7.4 10.1 17.6 11.0
89. I 90.0 85.9 79.0 18.6 0.7 68.6 70.1 67.7 55.2 5.4
10.2
a Total phosphorus concentration a-as followed in each experiment; deviations were within experimental error (10.1 p.p.m.) except for Ceratophyllum with an increase of 0.5 p.p.m., and Escherichia coli with a decrease of 0.5 p.p.m Average dry weight calculated from bacterial counts at beginning and end of experiment. Growth occurred as evidenced by appearance of turbidity and increase in bacterial count from 1 X 104 to 1 X 108 cells per mi.
Induction periods of approximately a day (in two cases much longer) were observed, and this may be attributed to shock to the organism Qnchanging media. Half-lives estimated by plotting the data are tabulated in Table 111. I n the control experiment, no change in the tripolyphosphate content within experimental error was observed over a period of more than 300 hours. T h e half-life of sodium tripolyphosphate found in this control experiment is the same as the value obtained in previous measurements in a 1% solution (75) within the experimental error of these dilute-solution measurements.
Discussion T h e general conclusion-all of the many varieties of cellular material tested greatly accelerate the rate of hydrolytic breakdown of tripolyphosphate-is that this is a n enzymatic catalysis that would occur with a large number of cellular materials. An acceleration as high as a thousandfold was observed. Under
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growing conditions, a n even more rapid breakdown and utilization might be expected to occur. Because of the possibility of metabolism, the possible presence of inhibitors, and the variations, it is difficult to predict accurately what rate of breakdown would be expected in a n unknown nonsterile system. T h e catalytic breakdown of tripolyand pyrophosphates in the presence of cellular material is undoubtedly dependent upon the concentration of the cellular material. In the experiments described, the quantity of cellular material responsible for the hydrolysis was extremely large compared to the quantity of phosphate. I n many practical applications of phosphates, detection of the catalytic breakdown would be impossible, because, in ordinary solutions of about 1% concentration, the relative amount of cellular material would be low and the small changes in concentration of a given phosphate undetectably small. Comparison with previously reported data (75) indicates that the catalytic
INDUSTRIAL A N D ENGINEERING CHEMISTRY
Table 111. Comparison of Rates of Degradation of Tripolyphosphate in Solutions Containing Various Organisms 24pprox- Half-Life, Hours5 imate Including Induc- induction tion period Excluding Time, tripoly- induction Organism* Hours phosphate period Control Elodea Gleocapsa Vaucheria Ceratophvllum Allomyces Escherichia coli
26 55 26 18 25 65
141 110 60 68 41 85
IO-20,000 123 66 35 59 19c 20
a Corrected for change in solid to liquid ratio during run. Dry weights added are listed in Table 11, column 2. Rate of degradation decreased considerably from reported rate after 60 hours. ~
breakdown is nonspecific for tripolyphosphate. Judging from the experimental precaution followed, it is believed that 90% or considerably more of any observed effect is attributable to the organism designated. Also, no catalytic degradation of tripolyphosphate was observed in similar experiments from the addition of kaolin, if the powdered kaolin was pretreated in a dry 110” C. oven.
Literature Cited (1) Bamann, E., Heumulleri E., Ar~atzirzeissenschajten 28, 535 (1940). ( 2 ) Bamann, E., Meisenheimer, M., Ber. 71B, 2086, 2233 (1938). ( 3 ) Ging, N. S., Sturtevant, J. M., J . Am. Chem. SOC. 76,2087 (1954). ( 4 ) Green, J., IND.EM. CHEM.42, 1542 (1950). ( 5 ) Griffith, E. J., Van Wazer, J. K.,
unpublished report.
( 6 ) Ingelman, B., Malmgren, H., Acta Chem. Scand. 1, 422 (1947); 2, 365 (1948); 3,157, 1331 (1949). (7) Jenner, H. D., Kay, H. D., J . Biol. Chem. 93, 733 (1931). ( 8 ) Karl-Kroupa, E., Anal. Chem. 28, 1091 (1956). (9) Kitsato, T., Biochem. Z . 197, 257 (1928); 201, 206 (1928). (10) Mann, T., Biochem. J . 38, 339, 345 I1 9441. Nguber;, C., Fischer, H. A., Enqrnologra 2, 241 (1938). Schaffner, A., Krumey, F., Z . physiol. Chem. 255,145 (1938). Sumner, J..B,, Myrback, K., “The Enzymes,” Academic Press, New York. 1950-51. Vol. I: chaa. 11. pp. 473-510, by J. Roche; chap: 12, pp. 511-16, by B. Ingelman. Vol. 11: part 1, chap. 46, pp. 11450, by S. P. Colowick; chap. 47, pp. 151-61, by H. M. Kalckar. (14) Van Wazer, J. R., Griffith, E. J., McCullough, J. F., J . Am. Chem. SOC. 74. 4977 11952): Anal. Chem. 26, 1755 (1954). (15) Van Wazer, J. R., Griffith, E. J., McCulloueh. J. F.. J . Am. Chem. Soc 77, 287 (1955). ’ RECEIVED for review June 11, 1956 ACCEPTED February 23, 1957 Division of Water, Sewage, and Sanitation Chemistry, 129th Meeting, ACS, Dallas, Tex., April 1956.
