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this essential difference in the methods of revivifying the zeolites is made by the use of the term “biozeolite” in referring to the normal use of activated sludge. It should be possible, however, to drop all analogies when dried sludge is regenerated with sodium chloride (cf. the Guggenheim process). The relation of the Schmutzdeclce of the slow sand filters to the biological slimes of sewage treatment is already well known and generally accepted. In each case it is now possible to refer the primary adsorption of organic matters by these gelatinous formations to zeolitic action. The use of sodium aluminate for the clarification of siliceous waters has already been interpreted as leading to the formation of a highly adsorbent aluminosilicate or zeolite. The removal of manganese (or iron) by certain classes of microorganisms may be viewed as the biological equivalent of filtration through a manganese zeolite. In this case the evidence rests on certain analyses of the gelatinous envelope which surrounds these zo6glmal formations (9, 8).
Conclusion Considering that the difference between a polluted water and a domestic sewige is one of degree and not of kind, it is not surprising that the mechanisms of purification should in each case be basically the same. Viewed as adsorption phenomena or as base-exchanging reactions, it is not reasoning by analogy to relate the clarification process in sewage treatment with the corresponding process of removing impurities, organic or inorganic, in water purification. I n each case attention should be focused on the identity of the adsorbents and not on the degree of pollution, the nature of the impurities, or the mechanical arrangements for bringing about the desired result. Some of the difficulties in visualizing this new
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concept of water purification and sewage treatment have been anticipated in earlier papers of the author.
Literature Cited (1) Ardern, E.,Brit. Assoc. Advancement Sci., 2nd Rept. on Colloid Chemistry, 1921. (2) Beythien, A., Hempel, H., and Kraft, L., 2. Untersuch. Nahr. Gsnussm., 7, 215-21 (1904). (3) Buzagh, A. v., Kolloid-Z., 49, 35-9 (1929). (4) Dallyn, F. A., and Delaporte, A. V.,Can. Engr., 50, 193-5, 213-5 (1926). ( 5 ) Dienert, F., Rev. hug., 44,113-66 (1929). (6) Genter, A. L.,Sewage Works J., 6, 689-720 (1934). (7) Gleason, G.H., and Loonam, A. C., Ibid., 5, 61-73 (1933). (8) Jackson, D. D.,Trans. A m . Micros. Soc., 23, 31-9 (1901); J. SOC.Chem. Ind., 21, 681-4 (1902). (9) Mellor, J. W., Comprehensive Treatise on Theoretical and Inorganic Chemistry, Vol. 6, p. 575,New York and London, Longmans, Green & Co., 1925. (10) Parsons, A. S., W d e r Works and Sewerage, 76, 397 (1929); Can. Engr., 58, 125 (1930). (11) Parsons. A. S., and Wilson, H., Surveyor, 72, 221-6, 252 (1927); J. Roy. Sanit. Inst., 48, 494-509 (1928). (12) Slater, C. S., and Byers, H. G., U. 6. Dept. Agr., Tech. Bull. 461 (1934). (13) Spoehr, H.A,, J. A m . Chem. SOC.,46, 1494-1602 (1924). (14) Snoehr, H.A., and Milner, H.W., Ibid., 56, 2068-74 (1934). 27, 683-6 (1936). (15) Theriault, E.J., IND.ENQ.CHIOM., (16) Theriault, E. J., Butterfield, C . T., and McNamee, P. D., J . Am. Chem. SOC.,55, 2012-24 (1933). (17) Theriault, E. J., and McNamee, P. D., IND. ENQ. CBIM., 22, 1330 (1930); Pub. Health &pt8., 46, 1301-19 (1931). (18) Wagenbds, H.H., Theriault, E. J., and Hommon, H.B., Pub. Health Bull., 132 (1923). (19) Wilson, H., Proc. Assoc. Managers Sewage Disposal Works, 1930, 171-87. R ~ C I ~ I VJuly E D 26, 1936.
Action of Amylases on Starch H.C. GORE The Fleischmann Laboratories, New York, N. Y.
OT
H E present status of the problem of the action of amylases on starch perhaps can best be shown by describing briefly the three types of diastatic activity of plant amylases which, thanks to progress made within recent years, can now be followed quantitatively. These three types of activity may be called “saccharogenesis” or attack on raw starch, “liquefying power,” and “saccharification” or Lintner activity.
Saccharogenesis In introducing the term “saccharogenesis,” Bailey (1) meant the direct attack of diastase on the raw starch of flour. This phenomenon is of the utmost importance to the baker, not only because the maltose sugar developed is the cheapest form of sugar available to him, but also because it is formed steadily throughout the proofing of the dough, thus constantly supplying the yeast with fermentable sugar for gas formation, the process continuing even after the loaf reaches the oven. The method of measurement of the saccharogenesis, or autolytic diastatic power of a flour, developed in its present form by Blish and collaborators (,@, was first worked out by Rumsey (14) in 1922. I n his original method, flour was mixed with nearly 10
parts of water a t 27” C., and the resulting suspension was kept a t 27” C. for an hour. Then a solution of sodium tungstate followed by sulfuric acid was added, and the suspension filtered. Reducing substances measured by copper reduction and calculated as maltose were then estimated in the filtrate. The method as modified by Blish (2) is operated a t 30” C. with pH control and employs a new technic for sugar estimation based on the Hagedorn-Jensen blood sugar method. Here alkaline ferricyanide is used as oxidant, and the excess is measured by titration with standard thiosulfate. This method is more rapid, more exact, and more convenient than any of the sugar methods involving copper reduction. The use of this method opens the subject of saccharogenesis of starch in a way not before possible and already has been of the greatest value to the cereal chemist. The formation of reducing substances estimated as sugar is comparatively rapid during the fist hour and falls off gradually during the remainder of the incubation period. Rumsey (14) was the first to point out the remarkable p H response. He found the maximum to be a t p H 4.7 to 4.8 with a broader range of maximum between 4.0 and 5.3. He showed also the great accelerative effect of temperature. At 0” C. autolytic diastatic activity was low, was much greater a t 35” than a t 27” C . (the standard temperature which he selected), and was still faster at 55” C.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Liquefying Power The liquefying power, or ability to destroy the viscosity of starch paste, can now be measured with excellent precision (6, 7 , 8). The technic consists of adding a measured amount of enzyme infusion to a special 5 per cent stirred potato starch, and observing the fall in viscosity at 21" C. over a measured interval. The weight of starch liquefied is then read from a curve or table. The precision is 5 per cent or better. The phenomenon is illustrated by the experiment shown in which a 5 per cent solution of freshly cooked stirred potato starch paste, kindly prepared by S. Jozsa, is mixed with a small amount of filtered malt infusion and allowed to stand for a few minutes. Two phenomena occur, rapid loss in viscosity and a peculiar whitening effect which cannot yet be explained. Ordinary flour has sufficient liquefying power to convert all of its starch in a single hour at the temperature a t which the measurements are made (21" C.) if it were present in the form of a paste and were thus freely accessible to the enzyme. In the malting of berley the liquefying power increases many fold. Starch does not occur in nature in the form of a paste, and one might ask why the liquefying power is of any particular significance, especially since in malt an enormous excess of activity seems to be present, far more than is sufficient to take care of any liquefaction usually required in mashing. The answer is that there are only a few of the activities of diastatic plant products which we can measure with a satisfactory degree of accuracy. The hope that correlations may be found in the future between the liquefying power and desired properties in the dough, loaf, or mash, is the justification for the use of this method, and preliminary results have justified this hope. Thus Landis (11) showed that a simple relationship exists between the amount of liquefying enzyme derived from malt added to the dough batch, and the increase in saccharogenic activity, the latter being independent of the Lintner value of the malt. An illustration of the probable effect of the liquefying activities of the diastases present in the dough batch is shown in work of Schulta and Landis (16). Here the effect observed was produced by modifying the starch of the dough, by the action of added diastase, during the short interval in the oven while the starch grains were swelling and the diastase was still active. The effect was observed by measuring the staling when the resulting bread was subsequently kept a t different temDeratures. The staling rate was measured bv noting the ;ate of attack on the &umb by flour diastase, using a fermentation method in which the attack on the starch of the crumb was evidenced by the volume of carbon dioxide formed. The data clearly show the marked retardation in staling rate a t the different storage temperatures ascribed to the presence in the formula of a very small amount (1 per cent based on the flour) of diastatic malt sirup; the attack on the starch which resulted in its slower reversion rate probably took place in the oven. Saccharifying Power A third type of activity of plant amylase which can now be measured with a satisfactory degree of accuracy is the saccharifying power or Lintner activity. Historically this was the first manifestation of diastase to be observed and measured. Green (4) states that the diastase of germinating barley was discovered by Kirchoff (9) in 1814. It was not until 1879, however, that Kjeldahl (IO) developed his law of proportionality, according to which the reducing substances formed upon the digestion of starch paste estimated as maltose increase directly as the time until about 40 per cent of the starch has been converted.
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To the student of enzyme kinetics this law presents an anomalous situation, since similar enzyme actions (e. g., that of invertase and the liquefying power enzyme of malt, acting on their respective substrates) are represented, not by straight lines, but by curves, the reactions beingconstantlyretarded. However, if we accept the most accurate methods of analysis applied to the study of saccharifying activity up to the present time-namely, the data published by Hanes (6) in 1932Kjeldahl's law seems to be correct, a t least during the early stages of the reaction. Thus, Hanes showed that, a t slightly above 1 per cent concentration of the soluble starch sub-' strate, straight-line relations seem to prevail. The anomaly evidently presents a difficult and important problem. In 1888, Lintner (12)made the Kjeldahl law the basis of his classical Lintner method of estimation of diastatic power. At the same time he published his method of preparation of Lintner's soluble starch still used in all malt analysis laboratories, and the Lintner scale for expression of the diastatic activity of malt. ' His method consisted of placing 10-cc. portions of 2 per cent solution of Lintner's soluble starch into a series of test tubes, about 2 cm. in diameter, and adding very small amounts of 6 per cent malt infusion, ranging say from 0.1 cc. upwards, serially in increasing quantities. After mixing and allowing to stand at room temperature for an hour, 6 cc. of Fehling solution were added to each tube, and the tubes were heated and allowed t o cool. The diastatic activity was then estimated by noting the tube in which reducing substances were present in sufficient amount just to cause all of the cupric ion to be reduced.
As in case of the liquefying power, flour apparently is amply endowed with Lintner activity. I n the case of barley malt the Lintner activity, while ample, increases only slightly during malting, and the rate of increase is far less than that of liquefying power. Comparison of Methods If we contrast the three quantitative methods of measuring diastatic activity just described, we find that in the case of both wheat flour and barley malt, the saccharogenic power, or attack on raw starch is the diastatic function which is by far the slowest, and therefore, probably most significant. The writer (3) showed that, under the conditions of the Rumsey saccharogenic diastase method, 1.2 per cent of reducing substances, reckoned as maltose, was liberated from 100 grams of flour in one hour a t 27" C.; that in the liquefying-power method starch paste was liquefied at the rate of 111 grams Der hour a t 21 " C. by 100 grams of flour: and that under the conditions of tGe Lincner method 620 grams of reducing substance, calculated as maltose, were produced by the diastase of 100 grams of flour in one hour at 21 " C., acting on a solution of soluble starch. Of these precisely measurable activities, the saccharogenic power has been much studied by the cereal chemist in the last decade on account of its prime importance in flour technology. Mangels (IS) showed clearly the extreme importance of starch resistance when he measured the rate of attack of diastatically active filtrates of different flour suspensions on commercial starch and contrasted the data thus obtained with the Rumsey values obtained when an active filtrate obtained from Marquis wheat acted on wheat starch, on starch prepared from Marquis wheat, and on starch made from durum wheat. Comparatively little difference existed between the action of the different flour filtrates on wheat starch. On the other hand, very large differences in resistance to attack by diastase were exhibited by the three starches. Thus the susceptibility of the Marquis wheat starch to diastatic attack was very slight (about one-fourth that of the commercial wheat starch) and that of the durum wheat starch was very great (over four times that of the commercial wheat starch).
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Recently Swanson (16) subjected the diastatic power of wheat and flour to an extended investigation by the new Blish method. No explanation is given for the great differences which existed between the activities of the ground wheats and the flours prepared therefrom, the former often being much more active. In the solution of such a problem the measurement of the liquefying and saccharifying powers of the ground wheat and flour may be of assistance. The development of resistance on the part of bread starch during staling is illustrated by data published by Whymper ’ (17) in 1930 showing again that the lightly cooked starch present in bread crumb develops marked resistance in the staling process. Here baked loaves were permitted to become stale and the susceptibility to attack of the starch by malt diastase was measured from day to day. Marked resistance to diastase attack developed. We still do not know to what extent the saccharogenic, liquefying, and saccharifying powers of malt and flour are due to one or several diastatic enzymes. This discussion has , been confined entirely to plant diastase, as illustrated by the amylases of flour and barley malt. It was impractical to include the extremely important amylases secreted by molds and by bacteria, and those of animal origin, because of the intricacy of the subject. Surely the factors inherent in the subject of starch resistance to diastase attack are worth studying, especially since
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the worker now has before him the possibility of linking his discoveries with the chemical and physical studies on starch now being so brilliantly conducted by others.
Literature Cited (1) Bailey, “Chemistry of Wheat Flour,” New York, Chemical Catalog Co., 1925. (2) Blish and Sandstedt, Cereal Chem., 10, 189 (1933). (3) Gore, J . Assoc. Oficial Agr. Chem.. 16,403 (1933). (4) Green, Reynolds, “Soluble Ferments and Fermentation,” Cambridge Univ. Press, 1901. (5) Hanes, Biochem. J . , 26, 1406 (1932). Johnston and Jozsa, J . Am. Chem. SOC.,57, 701 (1935). Jozsa and Gore, IND.ENG.CHISM., Anal. Ed., 2,26 (1930). Jozsa and.Johnston, Ibid., 7, 143 (1935). Xirchoff, Schweios J . , 14,389 (1815),from Green’s “Soluble Ferments and Fermentation.” Kjeldahl, series of papers from Carlsberg Lab., 1879; 2. ges. Brauw., 3,49,84,123,149,179,222 (1880). Landis, paper presented before meeting of Am. Assoc. of Cereal Chemists, Denver, Colo., June, 1935. Lintner, J. prakt. Chem.. [2]34,378 (1886); 36,481 (1888). Mangels, Cereal Chem., 3,316 (1926). Rumsey, Am. Inst. Baking, Bull. 8 (1922). Schultz and Landis, Cereal Chem., 9,305 (1932). Swanson, Ibid., 12,89 (1935). Whymper, Arlcady Rev., 7, 45 (1930). R ~ C E I V EJune D 16, 1935. Presented a6 part of the Symposium on Starch before the Division of Agricultural and Food Chemistry at the 89th Meeting of the American Chemical Society, New York, N. Y.,April 22 to 26.1935.
Oxygen Absorption Tests on ASPHALT CONSTITUENTS ROBERT R. THURSTON AND EDWIN C. KNOWLES The Texas Company, New York, N. Y. FIG. I . APPARATUS FOR OXYGEN
ABSORPTION MEASUREMENTS
@T
MANOMETE
HE process of oxidizing residua obtained from the distillation of petroleum oils with air a t elevated temperatures has been known for a long time and has been commercially practiced in the United States for nearly forty years (3). This process is relatively simple and consists in intimately contacting the residuum’with air in a suitable still a t a suitable temperature until a product of the desired consistency is obtained. The residuum so treated changes its physical characteristics and becomes harder, heavier in gravity, lower in ductility, and higher in softening point. The chemical reactions which cause these physical changes are rather complex, but it is known that the proportion of solid and liquid constituents in the asphalt as well as the nature of these constituents has a definite bearing on the characteristics of the products. The extent to which physical and chemical changes take place, and the relationship obtainable between hardness, ductility, and softening point, depend not only upon the source and characteristics of the original residuum but also upon the processing of that residuum previous to oxidation and upon the conditions of oxidation. From an economic standpoint this process is extremely important because useful asphalts can be made by it from petroleum residua not suitable for the manufacture of asphalts
