such as labor, amortization, taxes. and insurance. A charge of $33.00 net per barrel was used as the cost for the oleoresin and $4.00 as a processing charge in the calculations. However, in converting oleoresin to size, processing costs are higher than for preparation of rosin and turpentine because of the additional steam required for the stripping operation and for hold up time in the equipment. If $5.00 per barrel was allowed for processing, the cost of the paste sizes would increase approximately $0.002 per pound. This processing charge is well within the limits obtained from a n engineering study made by another group ( 4 ) . Sizes of these types can be prepared for about $0.08 per pound. Dry sizes can be made or approximately $0.115 per pound. These prices are to be compared with the present selling prices for sizes shown in Table IV. The economic advantages of the process are shown in comparison of the values of the size, the value of the rosin in the size if sold for cash, and value of the size if this rosin were converted to size. For conventional size these values per 1000 pounds are $77.1 1, $75.35, and $78.25, respectively. These same values for fortified size are $80.45, 971.91, and $81.53. The pine gum processors and probably the farmers as well. if this process were used, could net more if the pine oleoresin were converted to paper size rather than gum rosin. Also, savings are evident over the present practice of isolating rosin and con-
verting it to size and amount, to the expense involved in shipping and handling the rosin and size manufacturing. Acknowledgment
T h e authors wish to express their appreciation to A. G. Dreis, formerly with A. G. Dreis and Associates, Chicago, Ill., present address Newport Industries, Division of Heyden Newport Chemical Corp., Pensacola, Fla., for sizing efficiency tests reported and for his encouragement and advice. Literature Cited
(1) Am. SOC.Testing Materials, Philadelphia, Pa., “Volatile Oils in Rosin,” D 889-58, Part 8, p. 932, 1958. (2) Baird, P. K., Curran, C. E., Tappi 23, 5 (1940). ( 3 ) Bump, A. H. (to Monsanto Chemical Co.), U. S. Patent 2,383,933 (Sept. 4, 1945); Wilson, W. S.,Bump, A. H. (to Monsanto Chemical Co.), U. S. Patent 2,628,918 (Feb. 17, 1953); Hastings, R., Dreschel, E. K., Strazdins, E. S. (to .\merican Cyanamid Co.), U. S. Patents 2,771,464 (Nov. 20, 1956) and 2,791,578 (May 7, 1957). (4) Decossas, K. M., Southern Utilization Research and Develop-
ment Division, New Orleans, La., unpublished results, 1960. (5) Loeblich, V. M., Lawrence, R. V., TND. ENG. CHEM.50, 619 (1958) ; and (to U. S. Government) U. S.Patent 2,846,430 Aug. 5, 1958. RECEIVED for review August 28 1961 ACCEPTEDJanuary 2, 1962
SUBMERGED CITRIC ACID FERMENTATION
OF SUGAR BEET MOLASSES Efect of Ferrocyanide Control D, S
.
C LA R K
,
Division of Applied Biology, National Research Council, Ottawa 2, Canada
The stability of the submerged citric acid fermentation of ferrocyanide-treated beet molasses b y Aspergillus niger was improved b y precise control of the ferrocyanide treatment. In the improved procedure, the concentration of ferrocyanide in mash after the initial ferrocyanide treatment was accurately adjusted to levels required for optimum acid production. Adjustment was essential since unavoidable variations in ferrocyanide content of mash after treatment were sufficiently large to produce a marked effect on duplication of yield. With adjustment made either before inoculation or during the first stage of fermentation, high yields (above 7570 conversion in 140 hours) were obtained consistently in fermentations carried out in 2.5-liter glass tower fermentors.
has been ustd in many citric acid fermentation processes to make crude molasses substrates suitable for fermentation. Its effect has generally been attributed to the precipitation of harmful trace metals ( 3 ) ,but a few studies (7) have indicated that the chemical has a desirable toxic action on the mold itself. Although the amount of ferrocyanide to be added initially to each molasses has been determined by trial and error using mold growth and acid production as criteria, no effort appears to have been made to determine the optimum range of ferrocyanide concentration in the substrate during the different stages of fermentation or to develop methods of controlling the concentration. A study has been niade, therefore, of the optimum ferrocyanide requirement for fermentation of beet molasses by Aspergillus nzger and of the factors that affect fer. rocyanide concentration during substrate preparation. OTASSIUM FERROCYASIDE
Experimental The fermentation procedures, equipment, and methods of analysis used have been described previously ( 7 , 70, 77). Briefly, “standard” pellet-type inoculum ( 7 7) was prepared by adding 106 spores of A . nzger NRC A-1-233 to 1500 ml. of molasses substrate (mash) in 6-liter flasks and incubating the suspensions at 29’ C. for 18 to 24 hours on a rotary shaker. Fermentations were carried out a t 31’ C. in a 2.5-inchdiameter borosilicate glass tower fermentor using 2.5 liters of mash inoculated with 5 X l o 5 pellets. The mash was sparged with air for the first 24 hours of fermentation (growth stage) and with oxygen for the remaining time (acid-producing stage) using a gas flow rate of 700 cc. per minute in all tests. The tests were done in duplicate. Citric acid was determined colorimetrically as anhydrous citric acid by the method of Marier and Boulet ( 5 ) . The sugar content of mash was deVOL. l
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termined as sucrose by the colorimetric method outlined by Dubois and others (2). Mash for inoculum preparation and fermentation tests was prepared from Chatham beet molasses (Canada & Dominion Sugar Co., Chatham, Ontario, Canada; 1959 crop unless otherwise stated), diluted to 12Yo sugar concentration with tap water. The mash was adjusted to p H 6.0 and sterilized by heat (steam a t 15 p.s.i.g. for 25 to 45 minutes) in glass containers. After sterilization, the mash was treated while still hot with sufficient K4Fe(CN)6 (added as a 207, aqueous solution) to leave some of this chemical remaining in solution after cooling. Most of the ferrocyanide added reacted with substances in the mash to form a grayish-white precipitate (8). The post-sterilization p H was adjusted to 6.5. The ferrocyanide content was determined colorimetrically as Prussian blue by a previously reported method ( 6 ) and if low, adjusted to the desired level by further addition. Two methods were used to adjust the ferrocyanide concentration if it was above the desired level: The ferrocyanide could be decomposed by heating to 120' C. for 30 minutes (8) and then the desired quantity added after cooling. This heat treatment was sufficient for coInplete decomposition under the conditions used. Also, the necessary amount of ferrocyanide could be precipitated with zinc. The calculated amount of zinc [3 moles of Z n + + reacted with 2 moles of Fe(cx)6----(4)]was added as a 5% solution of ZnSOb. It was not necessary to remove the precipitate formed. Both methods gave good results, but since the latter was simpler and could be used during fermentation, it was preferred. KO phosphate or other metabolites were added to the mash (70). I n the study of factors that affect ferrocyanide concentration in mash during substrate preparation (source of molasses, sterilization conditions, and presterilization pH), molasses was diluted as described above, adjusted to the desired pH, sterilized (steam a t 15 p.s.i.g., 25 minutes, unless otherwise stated), treated while hot with K4Fe(CN)6, cooled to room temperature, and tested for its ferrocyanide content. Tests were carried out with 1500 ml. of mash in 6-liter flasks. Results A ferrocyanide concentration of 10 to 30 p.p.m. was required in the mash a t the beginning of fermentation for
optimum yields of citric acid (Figure 1). This range was affected little by molasses crop year. Tests in which the ferrocyanide concentration was measured during fermentation showed, however, that ferrocyanide was only necessary early in the fermentation (Figure 2) and that the concentration generally decreased during fermentation. LVhen the ferrocyanide concentration was maintained a t different levels throughout fermentation, although ferrocyanide was not required during the acid-producing stage, concentrations up to 20 p.p.m. had no adverse effect on yields (Figure 3). High concentrations of ferrocyanide (up to 400 p.p.m.) during the growth stage of fermentation did not affect yield significantly if they were reduced to values below 20 p,p.m. prior to the start of the acidproducing stage of fermentation (Table I ) , High concentrations could also be reduced a t later times during fermentation without affecting yield, but the time necessary to complete fermentation was lengthened (Figure 4). Appearance and growth rate of the pellets were related to ferrocyanide concentration. Pellets capable of producing optimum yields of citric acid later in fermentation mash were developed in about 21 hours in inoculum mash containing 10 to 40 p,p.m. of ferrocyanide. These pellets ranged between 0.1 to 0.2 mm. in diameter. At 5 p.p.m., the pellets matured more rapidly (18 hours) but tended to adhere to one another, foIming clumps during subsequent fermentation. Pl'ith no ferrocyanide, growth was filamentous and pellets were not formed. \\'hen the concentration was above 60 p.p.m., inoculum development was seriously retarded. In fermentation mash containing less than 10 p.p.m. of ferrocyanide, large (up to 4 mm. in diameter), soft filamentous pellets, which would produce little citric acid subsequently, were formed in 24 hou;s from "standard" inoculum, whereas in mash containing 10 to 40 p.p.m., small (about 0.8 mm. in diameter), round pellets were developed during this period. Smaller pellets were produced with increase in concentration above 40 p.p.m. (about 0.7 mm. in diameter a t 100 p.p.m. and about 0.5 at 400 p.p.m.). Since the results showed that optimum yields of citric acid could be obtained consistently only when the ferrocyanide concentration was controlled during growth of pellets and during the time of high acid production, tests were made to determine the effect of the usual mash preparation procedure
_1
0 \
200
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; a-"
CHATHAM
1957
I75
MOLASSES
f
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d
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150
125
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P z
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a
CHATHAM 1959 MOLASSES-*
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50
D V
4
25
u
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FERROCYANIDE,
80
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P P M.
Figure 1. Effect of ferrocyanide concentration in mash a t the beginning of fermentation on yield of citric acid Ferrocyanide concentration in inoculum mash, 15 p.p.rn.
60
I&EC PRODUCT RESEARCH A N D DEVELOPMENT
0
20
40
60
FERMENTATION
Figure 2. Change during fermentation
80 TIME,
100
120
140
HOURS
in ferrocyanide
concentration
Table 1.
Effect of Reducing Ferrocyanide Concentration on Citric Acid Yield“
Citric Acid Yield after 7 4 0 Hr., % ’ Wt./Vol. Fe(CN)a---Fe( CW,) ---Initial reduced reduced Fe(CN)B----, No to 75p.p.m. to 7 5 p p . m . P.P.M. change before inoc. at 24-hr. period 15 10.5 9.6 100 3.7 8.9 8.7 200 4.5 8.5 400 3.2 8.8 8.2 a Ferrocyanide concentration in inoculum mash, 75p.p.m.
...
Table II.
Effect of Sterilization, Agitation, and Cooling on Ferrocyanide Concentration Ferrocyanide Ferrocyanide Added Remaining after after Sterzlzration, Cooling, P.P.M. P.P M . b Factora Treatment 350 25 Sterilization Boiled at atm. press. 45 ‘7
...
min.
Autoclaved 15 min p 3.i.g.
on the concentration of ferrocyanide remaining in mash after the ferrocyanide treatment. Sterilization and post-sterilization conditions did not significantly affect the ferrocyanide content of prepared mash (Table 11). T h e conditions, therefcre, which have been shown to affect yield (70), do not appear to d o so by causing variations in the ferrocyanide concentration. The p H of mash before sterilization had a small but definite effect on the ultimate ferrocyanide content in that the concentration decreased about 10 p,p.m. for a decrease of 1 p H unit in the range p H 4 to 7 . T h e variation in the ferrocyanide content of mashes prepared under similar conditions from the same barrel of molasses was large (*15 p.p.m.) compared with the narrow range of concentration optimum for citric acid production. I n addition, the ferrocyanide content of mashes prepared identically from different samples of the same crop year of molasses and from different crop years varied even more (up to +150 p.p.m.). These results indicate the necessity of adjusting the ferrocyanide level in prepared mash to within the optimum concentration rance. Discussion The results show that the stability of the fermentation depended largely on precise control of the ferrocyanide concentration in the inoculum and fermentation mashes. A concentration of 10 to 40 p.p.m. was most suitable for the
, 15
350
25
350 23 Autoclaved 30 min , 1 5 p.s.i.g. 350 27 Autoclaved 45 min 15 p.s.i.g. 400 80 agitation^ No agitation 400 79 10 vigorous rotary shakes by hand 15 min. on shaker turn400 83 ing 160 r.p.m. 350 35 Coolingo Cooled rlowly a t room temp Cooled immediately in 350 38 ice bath Cooled 0 5 hr. a t room 350 36 temp., then to 20’ C. in ice bath a Testsfor each factor made with a separate sample of a well mixed supply of molasses. b Average of three tests. c After sterilization (steam, 7 5 p.s.i.g., 30 min.) and ferrocyanide addition.
production of “standard” inoculum in shake flasks within the desired incubation time (20 to 24 hours). Pellets capable of producing high yields of citric acid were developed from “standard” inoculum during the first 24 hours of fermentation in mash containing 10 to 400 p.p.m. of ferrocyanide, but optimum acid production did not occur subsequently unless the concentration was adjusted to a level below 20 p.p.m. Without adjustment during fermentation, optimum yields were obtained only if the ferrocyanide concentration a t the beginning of fermentation was between 10 to 30 p.p.m. Concentrations within this narrow range, however, are difficult to achieve consistently without measurement and adjustment, because of the relatively large variations in concentration that occur in spite of careful mash preparation. Therefore, the
i
0
>
1
I
a: =
7
t
z a
6
_I
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5 4 -
U
u
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F s ( C N l i REMOVED AFTER 100 P.P.M. ADDED
24
HR
AND
I O MIN.
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] 20
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100 120 140
FERMENTATION
TIME,
160 180 2 0 0 HOURS
Figure 4. Effect on citric acid yield of removing ferrocyanide from mash during fermentation Ferrocyanide concentration in inoculum mash, 15 p.p.m.; tation mash at 24-hour period, 1 OOp .p.m.
VOL.
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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
