procedure of adding lerrocyanide 10 heated mash but n u t nirasuring and adjusting the concentration subsequently, as used u p to the present time, was most probably the cause of the instability previously noted (70). Using the control measures outlined in this work, high yields of citric acid have been obtained consistently with three different crop years of molasses. Potassium ferrocyanide appears to have a function in addition to the removal of interfering substances from beet molasses, since at least 10 p.p.m. were required in the mash for desirable pellet development. Although it is the general view that the ferrocyanide treatment controls mold growth indirectly by removing heavy metals (7), the need for a small excess after treatment suggested that ferrocyanide may also serve to control growth directly during the growth stage of fermentation. During the time of high acid production, ferrocyanide was not required to control growth and even relatively small amounts (above 20 p.p.m.) affected acid production adversely. Possibly ferrocyanide conditioned the mold early in the fermentation for subsequent acid production in a way independent of growth, since it has been shown that the ion affects the production of certain respiratory enzymes ( 9 ) . This aspect, however, cannot be assessed from the results. The results of this investigation are the subject of a L. S. patent application.
Acknowledgment
The author acknowledges the technical assistance of Cholette and G. W. Folkard.
N. U.
literature Cited (1) Clark, D. S., Lentz, C. P., Can. J . .Microbial. 7, 447-53 (1961). (2) Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A , Smith, F.: Anal. Chem. 28, 350-6 (1956). (3) Gerhardt, P. P., Dorrell, W. LIT., Baldwin, I. L., J . Bacterial. 52, 555-64 (1946). (4) Kolthoff, M., Pearson, E. A,, Ind. Eng. Chem., Anal. E d . 4,
147-50 (1932). (5) Marier, J. R., Boulet, M., J . Dairy Sci. 41, 1683-92 (1958). (6) Marier, J. R., Clark, D. S., Analyst 85, 574-9 (1960). (7) Martin. S. M., Can. J . Microbiol. 1, 644-52 (1955). (8) North American Cyanamid, Ltd., Montreal, Quebec, “Cyanamid Nitrogen Chemicals Digest. vol. 7, The Chemistry of the Ferrocyanides,” pp. 32, 81, 1953. (9) Ramakrishnan, C. V., Steel, R., Lentz, C. P., Arch. Biochem. Biophys. 5 5 , 270-3 (1955). (10) Steel, R., Lentz, C. P., Martin, S. M., Can. J . Microbiol. 1, 299-311 (1955). (11) Steel, R., Martin, S. M., Lentz, C. P., Ibid., 1, 150-7 (1954). RECEIVED for review July 7, 1961 ACCEPTEDDecember 4, 1961 Symposium on Physical-Chemical Aspects of Fermentation, 140th Meeting, ACS, Chicago, September 1961. Contribution from the Division of Applied Biology, National Research Council. Ottawa 2, Canada. Issued as N.R.C. No. 6673.
HYDROLYSIS OF DIALDEHYDE STARCH G hoxal and ErJythose Production in Sulfurous Acid-Barium Su@e Solutions C . A. W I L H A M , T. A. M c G U I R E , J . W . V A N C L E V E , F. H . O T E Y , A N D C L M E H L T R E T T E R , A’orthern Regional Research Laboratorv, U. S. Defiartment of Agriculture, Peoria, Ill.
. .
The commercial availability of dialdehyde starch has stimulated interest in the production of chemicals from this new polymer. Previous work has indicated that reductive hydrogenolysis yields ethylene glycol and erythritol. Reported i s a direct acid hydrolysis which produces glyoxal of known industrial value and erythrose which can b e reduced to erythritol. Hydrolysis of 20% concentrations of dialdehyde starch can be accomplished if barium sulfite i s used in conjunction with sulfurous acid. Glyoxal complexes with barium sulfite and i s partially precipitated during hydrolysis. Degradation reactions are reduced, and resultant yields of glyoxal and erythrose were 90 and 75% of theory, respectively.
concentrations of dialdehyde starch (DAS) were successfully hydrolyzed to glyoxal and erythrose ( 9 ) . In this earlier work H2S03 failed to protect aldehyde groups in DAS concentrations above 470 and reversion reactions decreased yields of both components. In the current study. the admixture of B a s 0 3 with H2S03 has increased fivefold the concentration of DAS that can be hydrolyzed, giving yields comparable to those obtained a t 4% concentration tvith H2S03 alone. The B a s 0 3 stabilization is attributed to its ability to complex with gl?-oxal ( Z ) , precipitating a portion of the complex from the hydrolysis mixture. Details presented here establish conditions for maximum conversion of high concentrations of DAS to glyoxal and erythrose, separation of these two substances, purification of glyoxal for commercial use, and purification of erythrose for eventual reduction to erythritol. ILUTE
62
I & E C P R O D U C T RESEARCH A N D D E V E L O P M E N T
Hydrolysis Procedure
Barium sulfite was prepared by converting 129 grams of BaC03 (0.595 mole) in 140 ml. of water with excess SO2 for approximately 2 hours at room temperature in an cpen Parr pressure apparatus of 1-liter capacity. Seventy grams of DAS (0.438 mole on a dry basis) were mixed with the slurry along with the final 70 ml. of water required to provide a 257, (w. w . ) DAS concentration. The apparatus was closed and chilled; then 320 to 340 grams of S O 2 were condensed in the bomb from a weighed cylinder. This mixture, after standing overnight at room temperature, was placed in a heating jacket. stirred mechanically, and brought to temperature in approximately 30 minutes. Temperature within the bomb was controlled by a Variac and measured \vith an iron-Constantan thermocouple and a Leeds & Sorthrup potentiometer. The hydrolysis was timed when the contents of the bomb reached within 1 O of the hydrolysis temperature. After hydrolysis the bomb was cooled. the SO2 was vented, and the contents of the bomb \vere adjusted to 40% ethyl
alcohol concentration to precipitate the barium glyoxalbisulfite complex. T h e complex was centrifuged out? washed, filtered, and air-dried. Both the precipitate and erythrose concentrate were analyzed for glyoxal and erythrose.
Analytical Procedures Glyoxal. T h e extent of hydrolysis of each dialdehyde unit to glyoxal was determined by reacting glyoxal with 1,2dianilinoethane as described by Wise, Mehltretter, and Van Cleve ( 7 7). "Apparent" Erythrose. Since there is no method specific for erythrose, Lambert and Seish's chromatropic acid method ( 4 ) was adapted, and total reducing sugars were expressed as "apparent" ery-throse.
Calculation of Yields. Exact hydrolysis yields were difficult to determine because not all units of DlZS produce glyoxal and erythrose. Approximately 57G of the anhydroglucose units of the original starch were not periodate-oxidized, according to methods used (7: 70), and instead yielded glucose, which was determined by the chromotropic acid method. I n addition, after periodate oxidation and hydrolysis, the 476 nonreducing end groups ( 6 ) produce glyceraldehyde and glyoxal. Both components can be determined by the methods described. Apparently only 957, of the DAS units produce glyoxal, while all yield reducing sugars. The extent of hydrolysis was the ratio of analytical values for each component to the theoretical 1 mole of erythrose and 0.95 mole of glyoxal per mole of D,AS unit.
Variables Time and Temperature. Optimum conditions for maximum yields of glyoxal and erythrose were determined by hydrolyzing a 25c0 (w./w.) concentration of DAS with 12 moles of H2SO3 and 1.65 moles of B a s 0 3 per mole of DAS unit a t a series of temperatures ranging from 90' to 130' C. Optimum hydrolysis times were established for each temperature by separate experiments in sealed tubes under conditions knoxzn to give results comparable to hydrolyses reported in this study. Maximum yields of 9576 for glyoxal and 707, for erythrose ivere maintained constant between 90' and 110' C. At the higher temperatures of 120" and 130' C . erythrose
yields decreased faster than those of glyoxal. Although the hydrolysis can be reduced to 45 minutes a t 130" C. in contrast to 9 hours a t 90" C . , the higher temperatures Lvere unsuitable because of much lower yields. Actually the hydrolysis rate with H2SO3-BaSO3 was about one third of that with H2SO3 alone. For studying other variables, DAS was hydrolyzed a t 100' C. Under these conditions, extent of conversion at various intervals of time is shown in Figure 1 . LVithin limits of accuracy of the analytical method, erythrose conversion was constant at 75Y0 after 3.5 hours. Similarh, glyoxal yields appeared to level off a t 3.5 houis, but continued hydrolysis produced glyoxal in excess of theory without any change in erythrose. Extended hydrolyses also produced other degradation products which react bvith the glyoxal reagent, as described below. These studies indicate that yields for glyoxal near 100% may be consistently 10% high. Glyoxal conversion values corrected for these discrepancies exceed maximum yields attained in the sulfurous acid hydrolysis. Optimum conditions for higher DAS concentrations are similar. Time-yield curves from sealed tube experiments either reproduce or parallel those a t the 10% concentration. Dialdehyde Starch Concentration. Optimum DAS concentration for the hydrolysis was established by the maximum yield of the more labile erythrose. Figure 2 shows that the 757, erythrose yield was maintained until the DAS concentration exceeded 20%. Under these same conditions glyoxal remained about constant until the concentration reached 4070. Barium Sulfite Concentration, The stabilizing action of B a s 0 3 may result from a twofold function. During hydrolysis a certain proportion of the glyoxal is precipitated as the barium glyoxal-bisulfite complex, limiting reversion reactions. In addition, a barium balance on the hydrolyzate indicated that a portion of the barium complexed with erythrose and probably reduced the reactivity of these aldehyde groups. Because barium forms complexes with both components, the quantity of B a s 0 3 required to produce maximum yields during hydrolysis was determined. At least 1.38 moles of barium sulfite per mole of D.4S unit were necessary for optimum glyoxal and erythrose yields. O n this basis, B a s 0 3 concentrations of
e
70t
..**
10% w / w D i a l d e h y d e Starch 12 M o l e s S O 2 'l P e r M o l e 1.65 M o l e s B a s 0 3 1 D. A . 5 . Temp. 100"~. '
-
Hydrolysis T i m e Figure 1.
-
Hr.
Relation of hydrolysis time to yield
D i a l d e h y d e S t a r c h Contn., Figure 2. to yield
Z
Relation of dialdehyde starch concentration
VOL.
1
NO.
1
MARCH 1962
63
Table 1.
Effect of Sulfur Dioxide Concentration on Hydrolysisa of Dialdehyde Starch Mole Sulfur Dioxide per Glyoxal, Apparent M o l e D A S Unit % Erythrose, 7 0
4
94.5
8
99
70.6 69.5 12 92.5 68.0 a 2570 concentration of D A S , 1.5 moles of Bas03 per mole of DAS rcpeating units, 4.5 hours at 100’ C.
1.5 moles or above should be used to assure optimum hydrolysis. Sulfur Dioxide Concentration. Although the quantity of free SO2 influences glyoxal yield considerably during H2SOa hydrolysis, in this study the SO2 concentration was not a critical factor (Table I). Other Mineral Sulfites. Sodium, calcium. zinc, and magnesium sulfites, prepared and investigated as substitutes for the more expensive barium salt, produced lower yields. Calcium sulfite was the most effective substitute, because a portion of the calcium complex of glyoxal was insoluble under reaction conditions. Maximum yields from the hydrolysis of a 20% DAS concentration with CaSOa-H2S03 were 75% glyoxal and 65% erythrose, or 15 and 10% lower, respectively, than with the barium salt. Separation of Glyoxal a n d Erythrose. Hydrolyzates using B a s 0 3 when treated with ethyl alcohol at 40% concentration or by refrigeration a t 4’ C. for 20 hours separate 87% of the “analytical” glyoxal from erythrose as the barium glyoxal complex. Model experiments with pure glyoxal indicate that 98% should normally precipitate. This difference between isolated and analytical yields arises from substances produced during extended hydrolyses that react with the glyoxal reagent. Consequently, analyses performed on extended hydrolyses may be 10% high. Regardless of the separation process, each yields an insoluble glyoxal complex, from which the glyoxal must be recovered, an erythrose solution containing glyoxal impurities, unhydrolyzed DAS, and/or other degradation products. Regeneration of Glyoxal from Barium Glyoxal Complex. An adequate method requires complete removal of glyoxal without degradation and recovery of the barium in a form suitable for recycling to the hydrolysis process. These requirements were met by substituting calcium for bariLm in the complex with subsequent recovery of barium sdfite, decomposing the formed calcium complex with sulfuric acid, and concentrating the liberated glyoxal to commercial levels. Optimum conditions for substituting calcium for barium in the complex were to heat the barium complex for 30 minutes on a steam bath with 1.5 to 2.0 moles of calcium sulfite per mole of complex and to stir for 2 hours. The barium sulfite was filtered off and purified by saturating a water dispersion with sulfur dioxide to solubilize the excess calcium sulfite. The final product was 90% barium sulfite and represented about 85yG of the available barium in the complex. The influence on the hydrolysis of regenerated barium sulfite with its slight calcium impurities is discussed under barium recovery. The calcium glvoxal-bisulfite complex was easily decomposed with sufficient H2SOI to maintain a p H between 0.5and 1.5 while steam was being passed through the slurry. Scholfield (8) established that this range of acidity was necessary for optimum stability of glvoxal. A batch process combined with concentration in vacuo may be used to attain the 30% glyoxal 64
I & E C P R O D U C T RESEARCH A N D D E V E L O P M E N T
of commerce. Some 8 to 9% of the glyoxal was destroyed during decomposition of the complex with steam and acid. No additional loss occurred in the concentration step. In addition to excess sulfuric acid? glyoxal from the calcium complex contained about 1% erythrose and 0.4 gram of C a s 0 4 per 100 ml. Such a concentrate would be much purer than glyoxal prepared industrially ( 7 ) . Barium Recovery. A barium balance on the hydrolyzate from a 20% DAS concentration and 1.65 moles of B a s 0 3 per mole of DAS units indicated that 60 to 70% of the barium was precipitated with the complex a t 40% ethanol concentration. Twenty per cent of the barium in solution was recovered by treating the erythrose concentrate with C a s 0 3 a t 100” C. for 1.5 hours. This C a s 0 3 treatment of the glyoxal complex and erythrose concentrate recovered some 65 to 70% of the barium originally added to the hydrolysis. Recovered B a s 0 3 contained some 10% CaS03, possibly sugar acids, and other impurities capable of interfering with a normal hydrolysis. When this impure B a s 0 3 ”as returned to the hydrolysis of a 2070 DAS concentration under optimum conditions, no decrease in the normal yield of either glyoxal or erythrose occurred. Extensive recycling of B a s 0 3 has not been evaluated. Reduction of Erythrose to Erythritol. Some purification of the erythrose concentrate was necessary because it contains sulfite ions that readily poison Raney-nickel hydrogenation catalysts. Making the erythrose solution alkaline with barium hydroxide precipitated the sulfite impurities completely. This purified erythrose was reduced with a commercial nickel catalyst at 180’ C. for 2 hours under a hydrogen pressure of 2200 p.s.i.g. ( 5 ) . Conversion was determined by the quantitative chromatography procedure of Dimler et al. (3) using the chromotropic acid assay ( 4 ) adapted for erythritol. Reductions vary from 88 to 100%. Initial crystallization from a concentrated hydrogenation mixture yields some 5070 of the erythritol present. Complete recovery was hampered by the presence of reduced degradation products (sorbitol and glycerol) in which erythritol is soluble, Solvent extraction procedure shows promise of increasing the recovery of erythritol. Acknowledgment
The authors appreciate the assistance of Ben Hughes, Angela Rudolphi, and Diane Graff in providing data for various phases of this work. literature Cited
(1) Bohmfolk, J. F., Jr., McNamee, R. W.,Barry, R. P., IND. ENG.CHEM.43, 786 (1951). (2) Debus, H., Ann. Chem. Liebigs 102, 23 (1857). (3) Dimler, R. J., Schaefer, W. C., $Vise, C. S., Rist, C. E., Anal. Chem. 24. 1411 (1952). f4\ Lambert. M:. Neish. A. C.. Can. J . Rescarch 28B. 8 3 (1950). (5j Otey, F.H., Sloan, ’J. W.,’Wilham, C. A., Mehltret‘ter, C. L., IND.ENG.CHEM.53, 267 (1961). (6) . , Pigman, W. W., Goepp, R. M., Jr., “Carbohydrate Chemistry,” p. 518, Academic Press, New York, 1948. (7) Rankin, J. C., Mehltretter, C. L., Anal. Chem. 28, 1012 (1956). (8) ~, Scholfield, H. E. (to General Aniline and Film Corp.), U. S. Patent 2,494,060 (Jan. 10, 1950). (9) Van Cleve, J. W., Mehltretter, C. L., Abstracts of Papers, 134th Meeting, ACS, p. 25D, 1958. (IO) Wise, C. S., Mehltretter, C. L., Anal. Chem. 30, 174 (1958). i l l \ $Vise. C. S.. Mehltretter. C. L., Van Cleve, J. W., Ibid., ‘ $1, 1241’ ( i w j . RECEIVED for review Ami1 30, 1961 ACCEPTED fuly 31; 1961 Division of Carbohydrate Chemistry, 139th Meeting, ACS, St. Louis, Mo., March 1961. Mention of firm names or commercial products does not constitute an endorsement by the U. S. Department of Agriculture.
