Production of 2,S-Butanediol from Acid-Hvdrolvzed Starch
GEORGE E. WARD', 0. GLENN P E W 1JOHN2, AND ROBERT D. COGHILL8 Northern Regional Research Laboratory, of Agriculture, Peoria, Ill.
U.S . Department
J
T
HE fermentation process for the production of 2,Sbutanediol from carbohydrates, previously known only on the laboratory scale, has received recent attention from many laboratories. The impetus has come from the wartime urgency of producing l,&butadiene for the manufacture of Buna-type synthetic rubbers. A method for producing 1,3-butadienefrom 2,3-butanediol, based on the pyrolysis of the diacetate, had previously been disclosed by Hill and Isaacs (8). Bacteria of the Baca'llua polymyxa type convert unsaccharified starchy substrates to L2,3-butanediol (14) and ethyl alcohol, whereas bacteria of the genus Aerobuctmtransform most monoand disaccharides principally to m-2,3-butanediol (B, 14) accompanied by small quantities of d-2,3-butanediol and ethyl alcohol. Use of the latter bacteria requires that starchy substrates be converted to reducing sugar either by acid or enzymes prior to fermentation. The fermentation of refined sugar solutions and of saccharified grain mashes by Aerobacter will be discussed in detail in Iater communications. The present paper is concerned primarily with the production of 2,3-butanediol from acid-hydrolyzed refined wheat and corn starches, which have not been used previouslyaa a source for butanediol. Starch hydrolysates were studied as substrates for butanediol production primarily because it w&s believed that the recovery of butanediol would be more easily accomplished from the resulting fermented liquors than from whole grain mashes. Starches are cheaper than refined glucose but more expensive than grain on the carbohydrate basis. The object of the present investigations was to determine the proper laboratory cooking conditions for preparing fermentable mashes from starch by acid hydrolysis and to ascertain the yields of butanediol. In the course of the investigation, variations in the fermentation of starch hydrolyzates were observed, and some degree of success was attained in explaining and overcoming these variations.
J
Acid-hydrolyzed starches fermented by Aerobacter aerogenes gave 13.5 to 14.0 pounds of 2,3-butanediol per 34 pounds of pure starch (equivalent to one bushel of corn). Proper balance in trace element content was important in determining the efficiency of the fermentation; addition of manganese, cobalt, or molybdenum decreased butanediol production; addition of copper or zinc improved the fermentation of some, but not all, samples. Treatment of starch hydrolyzates by an ion-exchange agent was generally effective in increasing the butanediol yield.
utes. When the or anism was inoculated into this medium from agar slants 1mf of sterile 20%. urea solution was also added to each flask. (The urea was sterilized separately from the glucose solution to avoid excessive caramelisation of the latter.) The mais fermentation medium was prepared by cooking 3liter portions of aqueous starch sus ensions (usually of about 10% concentration) in &liter Florence gash in a laborato sterilizer at 22 to 25 pounds steam pfesure (128'. to 131' C.)%r periods up to 4 hours. H drochloric and sulfuric acids (c.P.) were used as hydrolysis catagsts. Acid concentrations of the starch slurries during hydrolysis ranged from 0.02 to 0.12 normal. Before steaming the acidic starch slurries were gelatinized in a hot water bath while being agitated; this procedure avoided lumping and was conducive to a more uniform and efficient saccharification. All production media contained, per liter besides the s a c c h d e d starch: MgSOd. 7Hz0,0.25 gram; KdsPO1, 0.60 gram; urea, 2.0 grams; and calcium carbonate 5.0 to 10.0 grams. This salt and urea composition had been found near optimal in previous studies of the fermentation of glucose media. (Extra calcium carbonate was supplied in the one instance where 0.12 N hydrochloric acid was employed for hydrolysis.) The first two mlta were added to the medium before cooking; the urea and calcium carbonate were each sterilized separately (the former as a ZOyo aqueous solution, the latter dry) and were added before inoculation. The quantit of calcium carbonate employed was sufficient to neutralize to pd5.5-0.0 the acid used for hydrolysis, and also to act aa a means of pH control during the fermentation. After addition of all the components, each 3-liter lot of medium waa MATERIALS AND METHODS inoculated with 100 ml. of an tL to 20-hour-old inoculum culture. The organism used, Aerobacter aerogenes NRRLB199, has been The fermentations were conducted in rotating aluminum drum found to be among the best butanediol-producing strains. It was fermenters (7) which were operated at 30' C., 10-12 r.p.m., 5 maintained in stock culture on agar slants of the following mepounds ga e pressure and an air flow of 50 to 100 ml. per minute, dium (in grams per liter) : which had%een found near-optimal conditionsin previous studies of butanediol production from glucose. Difco tryptone 6 Difco yeast extract 5 Except for one lot of wheat starch prepared a t this laboratory, Gluooae 1 Agar 15 all wheat and corn starches employed were commercial samples obtained from representative manufacturers. Inoculum was developed by growing the organism 8 to 20 The progress ot the fermentations was followed by anal ses for hours at 30" C. in an unagitated and unaerated medium of the reducing sugar, by the cuprous titration method of Shatfer and composition: Hartmsnn (If). Total sugar was determined by subjecting 5 ml. of the mash to the action of 25 ml. of 0.24 N hydrochloric acid Glucose 60 F ~ ~ S O.'AE?gram , 0.25 in the laboratory sterilizer for 1 hour a t 121' C.; reducin sugar ,PO4 grsm 0.60 was then determined on a portion of the neutralized hydroqyzate. C ~ C OLams ~, 6.0 This method was adopted because it was convenient for routine Distilled water to make 1 liter work produced a near-maximum quantity of reducing sugar from starch, and caused no appreciable destruction of glucose, as measThe medium was dispensed in 100-ml. portions into ZOO-ml. ured b copper reduction. Erlenmeyer flasks and sterilized by steam a t 121O C. for 30 min2,3-&~tanediolwas determined by periodic acid oxidation of an extract or distillate prepared from the culture media, ementially * Present address, Schenley Laboratories, Inc., Lawrenceburg, Ind. according to the method of Johnson (9). A correction for ace9 Deceased. toin w&s necemary. Acetoin waa determined b an un ublished * Present address, Abbot Laboratories, North Chicago, Ill. colorimetric method, based on the reduction olFolin-flu phoe1189
1190
INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 37, No. 12
fermentation of these mashes; Table I1 shows yields on the TABLEI. EFFECTOF COOKINGCONDITIONS ON BUTANEDIOL basis of pounds of butanediol per 100 pounds of starch as reYIELD FROM STARCH 2778 ceived and 34 pounds of moisture-free starch (equivalent to (Mashes 1 and 2 hydrolyaed 1.25 hours at 130° C.; mashes 3 and 4 hydroone bushel of corn). It is assumed that 0.07 gram of butanediol lvzed 4 hours s t 130° C.: distllled water used throughout) per 100 ml. of mash was derived from the glucose in the inoFerProducts culum. The acetoin and ethyl alcohol from this source are Sugar, Grams/100 hll. ?$- ~ ~ m S ’ l OM Ethyl O” FerHC1 ignored, since they were very small in quantity. The yielde menter ConcenOriginal Final Time, tanAce- 8100No. tration Free Total (free) Hr. diol toin hol of butanediol from starch 2778, especially when low acid concen32 3.72 0.20 0.33 1 0 . 1 2 N 10.04 10.36 0 . 3 0 trations were used for hydrolysis, were among the best obtained 33 3 . 7 4 0.11 0 . 2 5 2 0.12 N 10.35 10.36 0 . 3 8 47 4.23 0.21 0.07 3 0.04 N 10.25 10.30 0 . 5 2 from acid-hydrolyzed starches. 40 4.18 0.16 0.20 4 0 . 0 2 N 10.22 10.48 0 . 4 9 At the time when these good yields were being obtained from starch 2778, much poorer yields (approximately 10 pounds of butanediol per 34 pounds of starch) were being obtained from another wheat starch, designated as SS, even though i t was phomolybdic acid reagent by this compound. No correction lor interfering substances was necessary. Ethyl alcohol was deterhydrolyzed and fermented in the same manner. This starch mined by observing the refractive index of a distillate from a long had been prepared by a wet-milling process (13) from a soft packed column which held back all the 2,3-butanediol. An emwhite winter wheat grown in Michigan. Distilled water had been pirical correction was applied for diacetyl produced from the used in the manufacture of this starch, and the following peracetoin present by virtue of its reaction with alkaline copper reagent, which was added to the distillation flask. The analytical centage analyses (dry basis) show that it was of a purity a t least methods for 2,3-butanediol, acetoin, and ethyl alcohol mixtures equal to that of starch 2778: a m exacting. They require the application of several correction factors and will be published later. Starch SS Starch 2778 I n so far as possible, all fermentations were terminated as soon 0.12 0.15 Ash aa the minimum reducing sugar concentration was reached; pye98 98 Starch 0.32 vious investigations had shown that maximum 2,3-butanediol 0.19 Protein ( N X 5 . 7 ) production coincided with this point. The yields shown in the tables are expressed as percentages of the theoretical yields, baaed Typical results obtained upon fermenting untreated hydrolyzatas on reducing sugar consumed. This method of calculation largely of starch SS are shown in Table 111. Not more than 60% of the avoids deviations between various experiments due to differences fermented sugar appeared as butanediol, and almost 20% waa in medium concentration or in degree of completeness of fennentation. accounted for as ethyl alcohol. This was in marked contrast to the results obtained by the fermentation of starch 2778 (Table HYDROLYSIS CONDITIONS 11), which gave a 74 to 86% conversion of reducing sugar t o butanediol and only about 5To conversion to ethyl alcohol. A hydrolysis procedure that would yield the maximum quanHydrolysates of starch SS fermented much faster than hydrolytity of fermentable sugar was desired, so as to obtain the highest zates of starch 2778. possible yield of butanediol from a given quantity of starch. Goering (6) found that cooking starch 4 hours a t 25 pounds steam ANAEROBIC FERMENTATION pressure with 0.02 N sulfuric acid yielded hydrolyzates from which high yields of alcohol were obtained. I n the industrial Table IV presents results of a study of the anaerobic fermentaproduction of dextrose by the acid hydrolysis of cornstarch, aption of hydrolyzates of the two starches, of an unsupplementedproximately 0.025 N hydrochloric acid is employed 30 minutes glucose-nutrient salt medium, and of a glucose-nutrient salt a t about 45 pounds pressure. Accordingly, i t appeared likely medium supplemented with concentrated corn steep liquor that the use of approximately 0.02 N acid would yield fermentable (5 grams per liter). These fermentations were conducted in the mashes which would have a high dextrose equivalent and would aluminum fermenters, which were revolved but were not supnot require the addition of a large quantity of neutralizing agent plied with air. The hydrolyzate of starch 2778, like the unprior t o fermentation; thus, a high salt content was avoided supplemented glucose medium, required about 60 hours to comwhich might have an unfavorable effect on the fermentation and plete the fermentation, whereas the hydrolyzate of starch SS on recovery operations. fermented in 31 hours. Starch SS fermented about as fast To compare the effects of different laboratory cooking condianaerobically as aerobically, whereag starch 2778 fermented tions on butanediol production, mashes were prepared from much more slowly anaerobically than i t did aerobically. When starch 2778, a refined wheat starch supplied by an industrial fermented anaerobically, all mashes produced large quantities concern. This starch contained 8.19% moisture, and analyzed of ethyl alcohol; in fact, the ratio between butanediol and ethyl 98y0 starch on the dry basis, by the A.O.A.C. starch-by-diastase alcohol was approximately 2 to 1, the same as has been conmethod (1). Two mashes (350 grams of starch in 3 liters total sistently obtained in the fermentation of unsaccharified grain volume) were cooked 1.25 hours under pressure a t 130” C. with mashes by Bacillus polymyxa under anaerobic conditions. 0.12 N hydrochloric acid in distilled water, and two other similar The addition of 0.5% corn steep liquor to the glucose medium mashes were cooked 4 hours at the same temperature with 0.02 N greatly accelerated the rate of fermentation but had virtually no and 0.04 N hydrochloric acid in distilled water. After cooling, 40 effecton the ratio of products formed under these conditions. grams of sterile calcium carbonate and 20 ml. of 5 N sodium hydroxide were added to the mashes cooked with 0.12 N hvdrochloric acid. and 25 grams of sterile calcium carboFROM TABLEI I TABLE 11. YIELDSCALCULATED nate were added t o the mashes cooked Yield, Lb. 100 Lb. Staroh Yield, Lb./34 Lb. Pure Yield of Theoretical with the more dilute acid. The sodium Dry Sturch Bused oh & t c o s e ConsumAda (sa keaeired) Ferhydroxide was required to bring the pH ButaneEthyl ButaneEthyl menter ButaneEthyl diol Acetoin alcohol diol Aoetoin alcohol No. diol Aaetoin alcohol of the more acid cooks up to the range 12.3 0.7 1.1 75 4 7 1.8 2.0 1 32.5 (5.5 to 7.0) known to be desirable for the 2 5 0.8 74 12.4 0.4 2.2 2 32.7 1.0 initiation of fermentation, but no sodium 3 36.8 1.9 0.6 13.9 0.7 0.2 86 4 1 4 4 36.4 1.4 1.8 13.8 0.5 0.7 85 3 hydroxide was required for the hydrola “Glucom consumed” is assumed to equal original free sugar minus final free sugar. yzates prepared with the smaller quantities of acid. Table I gives results of
December, lW
INDUSTRIAL A N D ENGINEERING CHEMISTRY
1191
TABLE 111. BUTANEDIOL PRODUCTION FROM UNTREATED HYDROLYZATES OF STARCH SS (All maahea cooked 4 hours at l3O0 C.) Fermenter
Fermentation Time, Hr. 22 17 18
Acid and Water Used for Hydrolysis 0.03 N HCl in.distd. water 0.06 N Hi804 in t a p water 0. 06 N Hi604 in t a p water
No. 1 2
3
Gluoose Consumed, G./100 M1. 9.06 8.1 8.2
Products, Grams/100 Butanediol Acetoin 2.79 0.10 2.29 0.18 2.39 0.17
M1. Ethyl alcohol 0.86 0.06 0.77
Yield, % of Theoretical Baaed on Glucose Conaulhed ButaneEthyl diol Aoetoin alcohol 60 2.4 19.0 66 4.4 16.0 57 4.2 18.4
ss
TABLE. Iv. ANAEROBIC FERMENTATION OF GLUCOSE MEDIUMAND HYDROLYZATE$ OF STARCH 2778 AND STARCH (Distilled water used throughout; media for fermenters 1 and 2 steriliced 0.76 hour a t 121° C.: mashes for fermenters 3 and 4 cooked 4 hours a t 130' C.) Yield % of Theoretical, Based FerFermentsGlucose Products, Grams/100 M1. dn Glucoae Consumed menter Carbohydrate Acid for, tion Time, Consumed ButaneEth 1 ButaneEth 1 NO. Employed Hydrolysur Hr. G./100 M1: diol Acetoin alco~ol diol Acetoin alcogol 1 Gluoose None 63 9.9 2.79 0.02 1.29 65 0.4 26 2 Gluoose 0.6% corn steep liquor None 39 9.6 2.62 0.07 1.21 62 1.6 25 3 Starch2778 0.04 N HCl 66 10.0 2.99 0.09 1.36 68 1.8 26 4 Starch 88 O.lONHC1 31 9.9 2.68 0.08 1.28 51 1.6 25
+
TABLEV.
COMPARISON OF FERMENTATIONS OF STARCH 2778 HYDROLYZDD IN DISTILLED WATERAND
FerFermentaGlucoee Products. Grams/100 M1. menter Acid for tion Time, Consumed ButaneEthyl No. Water Hydrolysis Hr. G./lOO Mi. diol Acetoin alcohol 1 Distilled 0.08 N HtSOe 42 11.89 4.43 0.24 0.33 2 Peoria t a p 0.08 N &so4 37 12.1 3.63 0.20 0.97 3 Peoria tap 0.03 N HCI 24 8.60 2.47 0.10 0.61 0 All mashes cooked 4 hours a t 130' C. Further examples of dietilled water with at~trch2778 are given in Table I.
EFFECT O F VARIOUS FACTORS
GBOWTEI FACTORS. No difference in the proportions of ethyl alcohol and butanediol was ohservedwhen aerated glucose cultures were supplemented with biotin, nicotinic acid, paminobenzoic acid, pyridoxin, inositol, thiamine, riboflavin, and sodium pantothenate. Therefore, it was considered unlikely that these compounds were responsible for the anomalous results obtained with some starch hydrolyzates. ACID,ALKALI, ALCOHOL. Three portions of starch SSweresubjected to exhaustive extraction in the cold with 0.1 N sodium hydroxide, 0.1 N hydrochloric acid, and 65% aqueous ethyl alcohol, and the extracted starch was used for preparing fermentation mashes. Neither alcohol nor hydrochloric acid produced any improvement in this starch, but the sodium hydroxide treatment increaaed the butanediol yield to about 72% of theoretical and greatly decreased ethyl alcohol production. TRACE E~~la6erm.When Peoria tap water instead of distilled water was used in preparing mashes of starch 2778, the yield of butanediol decreased, the yield of ethyl alcohol increased, and the fermentation time was reduced (Table V). This suggested that the fermentation of starch hydrolyzates might be influenced adversely by traces of metallic elements in the Peoria water supply and also in starch SS but absent from starch 2778. Peoria tap water (4,6,10) is reported to contain the following: Fe 0.1-0.2 p.p.m. F About 0.1 p.p.m. Ca
% K Mn
72-97 37-46 About 26 About 4 About 0.12
Si01 HCOa 804 Cl
NOS
12-17 420437 5-62 14-23 1-4
Spectroscopicanalyses of the ashes of the two starches showed striking differences in relative content of inorganic elemenb. Starch 2778 contained about ten times as much lead, copper, iron, and strontium, about five times as much zinc, about twice as much calcium, about the same quantity of boron, phosphorus, aluminum, tin, silicon, nickel, and vanadium, and about half as much manganese, magnesium, molybdenum, chromium, and titanium as did starch SS. The absolute quantities of the various elements were determined only in the case of nickel, which
IN
PEORIA TAPWATERO
Yield, 7 of Theoretical Baaed on &lucose Coneuded ButaneEthyl diol Acetoin alcohol 73 4.1 6 69 3.2 16.7 67 2.4 11.7
was found to be present to the extent of 1 to 2 parts per million parts of starch. ADDEDIONS. These trace element data suggested determining the effect of added ions on the fermentation of hydrolyzates of starch 2778 (Table VI). At the ion concentrations employed, copper, fluorine, or a mixture of iron and zinc had little effect on butanediol or alcohol production; manganese (5 p.p.m.), cobalt, or a mixture of cobalt and molybdenum greatly reduced the quantity of butanediol formed. Alcohol production was stimulated by cobalt and by the cobalt-molybdenum mixture. Nickel, chromium, or fluorine (0.1 p.p.m.) increased butanediol production appreciably above the value obtained from the control culture, but not above the yield obtained in similar control cultures in other experiments. Fluorine and manganese, added together a t the concentrations in which they occur in Peoria tap water, had little effect on butanediol or alcohol production, but did appear to accelerate the fermentation appreciably. Increasing the fluorine concentration tenfold, in the presence of the same quantity of manganese, caused no appreciable change in the results obtained. Although these experiments did not fully explain the tap water effect, they showed that traces of certain metallic elements could influence the production of ethyl alcohol and have a pronounced deleterious effect on butanediol production from a starch that otherwise would give a good yield of this product. IMPROVEMENT IN YIELDS
ION-EXCHANGIU AGENTS. Removal of the injurious ions by precipitation was adjudged impossible owing to the appreciable solubility of the common compounds, particularly the hydroxides, carbonates, and sulfides, of some of the metals concerned. Ionic substitution by cation-exchange agents was therefore investigated. A hydrolyzate prepared from starch SS was adjusted to pH 5.1 and filtered to remove the slight flocculent precipitate always present at this stage. A 1-liter portion of the filtrate was then stirred for 30 minutes with 20 grams of ZeoKarb H (regenerated on the hydrogen cycle). After separation
1192
INDUSTRIAL AND ENGINEERING CHEMISTRY TABLEVI.
Fermenter
No. 1 2 3 4 S
6 7 8 9
10 11 12
Vol. 37, No. 12
OR STARCH 2778 EFFECTOB ADDEDIONSON BUTANEDIOL PRODUCTION FROM HYDROLYZATES
(All media Dreoared with distilled water and hydrolyzed 4 hours a t 130° C. with 0.03 N HCI) - . Yield, % of Theoretical Based on FermentaGlucose, Products, Grams/100 M1. Glucose Consurded tion Time, Consumed Hr. G./100 Mi. Butanediol Aoetoin Ethyl alcohol Butanediol Acetoin Ethyl alcohol
Element Added,a P.P.M. None 5 Fe 5 Zn 5 hln 0.1 c u 1 Co 1 Mo 0 . 1 Ni 0 . 1 Cr
+ +
1c o 0.1F 1.0 F 0.1 F 1.0 F
++ 0.12 Mn 0.12 Mn
26 32 27 31 27 28 26 23 30 29 20 24
7.61 8.46 8.64 8.60 8.46 7.51 7.43 7.86 8.86 8.83 8.58 8.30
2.89 2.99 2.41 3.09 2.36 3.04 3.11 2.42 3.57 3.33 2.74 3.12
0.17 0.21 0.37 0.25 0.15 0.06 0.06 0.11 0.13 0.30 0.55 0.16
0.12 0.;5 b
0.75 0.04 0.12 0.63 0.26 0.26 0.23 0.22
74 69 54 71 54 79 82 60 79 74 62 74
4.6 5.1 8.8 6.0 3.6 1.6 1.6 2.9 3.0 6.9 13.1 3.9
3.1 3.5 b
17.4 1.1 3.3 15.7 5.8 5.8 5.3 5.2
a The elemente were aupplied in the form of the following salts: FeSO4, Zns04, MnS04, CUSOI, Co(CHaCOO)r, (NHhMoO4, NiSO4, KyCrO4, and NaF. The salts were added to the starch slurries prior to gelatinization and hydrolysis. b None detected. ~~~
TABLE VII.
EFFECT
OF
TREATING HYDROLYZATES OF STARCH 8s WITH ZEO-KARBH
AND WITH
DARCO G-60
(All mashes made with distilled water and hvdrolvzed 4 hours a t 130" C. with 0.03 N HC1) Yield, % ' of Theoretical Based on FermentaGlucose Products, Grams/100 M1. Glucose Consuded Fermenter Treatment Prior to tion Time, Consumed ButaneEth 1 ButaneEthyl No. Fermentation Hr. G./100 MI: diol Acetoin alcocol diol Acetoin alcohol 1.70 0.04 0.12 59 1.5 4.2 Darco G-60 at pH 4 . 9 5.575 18 0.06 57 9.7 Darco G-60 at pH 8 . 0 1.87 0.31 2.0 19 6 . 2gn Zeo-Karb H at pH 5 . 1 L separated beb b 70 3.2 0.10 fore fermentation 2.29 24 6.32' Zeo-Karb H present during cooking and fermentation 54 0.26 0.34 7.7 22 8.69 2.43 6.1 Zeo-Karb H added a t pH 1 . 6 & present 13.8 2.9 66 2.42 0.12 0.59 23 8.38 in medium during fermentation Zeo-Karb H contacted at,pH 1 . 6 , sepa4.3 36 0.17 0.02 rated before pH was raised 1.55 0.5 43 8.13 E These mashes became diluted during the treatments; fermentation wau practically complete in all caaea. None detected.
*
of the Zeo-Karb H by filtration, the solution was supplemented with the usual nutrient salts and sterilized, then inoculated and fermented in the customary manner. The favorable results are shown in Table VII (fermenter 3). It was found necessary to separate tlie ion-exchange agent from the treated solution prior to fermentation. Treatment with Zeo-Karb H at pH 1.6 (the pH of the mash immediately after hydrolysis) gave particularly poor results. Treatment with activated carbon a t pH 8.0 was successfully employed by Bortels (8)to Eemove trace elements from nutrient media. Two 1-liter portions of the above hydrolyzate were treated with 20 grams of Darco G-60; one portion was contacted with the carbon at pH 4.9 and the other portion at pH 8.0. After removal of the carbon by filtration, the solutions were s u p plemented with the necessary nutrient salta and fermented. No improvement resulted from these treatments (Table VII). The batch tieatment with the ion-exchange agent employed in these experiments is less efficient than continuous passage of the solution through a column or tower containing the ion exchanger. I n the authors' work the latter method has been successfully employed with many samples of starch hydrolyzate liquors. The efficacy of the ion-exchange treatment for improving the fermentation characteristic8 of mashes was further demonstrated by the treatment of starch hydrolyzates prepared in a pilot plant iron cooker. Such liquors, when supplemented with the usual salts, urea, and oalcium carbonate, would not ferment, presumably because of the high iron content. However, when these hydrolyzates were subjected to treatment with Zeo-Karb H in the manner described for fermenter 3 (Table VII), fermentation proceeded a t a normal rate and normal yields of products were obtained. The fermentation characteristics of starches hydrolyzed in bronze and copper equipment have shown similar improvement after treatment with Zeo-Karb H. Comparative studies were made on samples of commercial starch-converter liquors that had been prepared continuously in bronze equipment. It was generally observed that such hydrolyzates fermented a little more slowly than hydrolyzates prepared
in glass apparatus; some samples showed a marked slowness in initiating fermentation. However, all such commercial samples were more suitable for fermentation than hydrolyzates prepared in our infrequently operated pilot-plant equipment. Starchconverter liquors that had been treated with Zeo-Karb H fermented more rapidly than untreated samples; presumably the copper content was reduced to a noninhibitory level by the ionexchange treatment. IONADDITIONS.Since considerable copper was found in the ash of starch 2778, which had given good yields of butanediol, the effect of added copper ion on the fermentation of hydrolyzates of starch SS was determined. Hydrolyzates prepared with Peoria tap water were treated with copper sulfate at concentrations to yield 0.01, 0.10, and 1.0 part of copper per million parts of medium (Table VIII). It was found that copper greatly improved the yield of butanediol and reduced the yield of ethyl alcohol. The response of hydrolyzates of twenty-four corn and wheat starches to the addition of copper (0.01 p.p.m.) was investigated to determine if it would be desirable to add this quantity of copper to all hydrolysed starch mashes to increase the yield of butanediol. None of the starches investigated showed such striking improvement as was noted for starch SS (8 to 14% increase in butanediol yield). Six snmples showed 5 to 7% increases in butanediol yield; five samples showed 2 to 4% increases; ten samples showed substantially no change; and three samples showed 6 to 15% decreases. Studies were conducted t o determine if hydrolyeates of some of these commercial starches might be improved for use in the butanediol fermentation by the addition of traces of metallic ions other than copper. Most samples showed no improvement with the limited number of ions tested. Table IX shows results obtained with cornstarch 2892, which showed poorer fermentation efficiency in tap water than in distilled water, no improvement from copper, but considerable improvement from treatment with zinc. Although many of the starches studied gave poorer yields of butanediol in tap water than in distilled water, several samples gave a good as or better yields than in tap water.
INDUSTRIAL AND ENGINEERING CHEMISTRY
December, 1945
1193
DISCUSSION
explained on the basis of inactivation or extraction of some deleterious principle. The majority of starch samples studied have given yields of butanediol equivalent to approximately 13 pounds per 34 pounds of starch. The theoretical yield is 18.9 pounds. Losses in yield are due to the incomplete conversion of starch to fermentable sugar by the hydrolysis procedure, to reversion and destruction of glucose by the acid treatment, and to the fact that not all of the fermentable sugar is converted to butanediol in the fermentation. According to information received from industrial operators, approximately 95% conversion of starch to glucose is possible in plant operation when relatively low starch concentrations (10%) are used. In the best laboratory fermentations (Table I, fermenters 3 and 4) about 86% of the fermented reducing sugar was converted to butanediol. Using 95 and 86% as the maxima to be expected for glucose formation aqd butanediol formation, respectively, a possible yield figure of (0.95 X 0.86 X 18.9 =) 15.5 pounds of butanediol per bushel of grain (34pounds of starch) is obtained. This value is considerably in excess of the best yields obtained in the present investigations. In the cookers employed, heat penetration through the unagitated maah was relatively slow, and there was evidence that the mash near the walls of the flasks was overconverted before the mash a t the center of the flaeks was sufficiently heated. Pilot-plant cooking tests, conducted by co-workers, have shown that more efficient hydrolysis can be attained in agitated cookers or continuous cookers. It appears that little trouble would be encountered in fermenting commercially produced starch hydrolyzates, particularly if the hydrolyzing equipment was operated continuously. Bronze converters maintained in continuous operation undergo less corrosion than converters operated a t infrequent intervals. Recently, considerablework has been done on the fermentation of acid-hydrolyzed wholegrain (corn and wheat) mashes by workers at the University of Wisconsin, by the research staff of Joseph E. Seagram & Sons,Inc., and by the Northern Regional Research Laboratory. Such mashes were found to yield 13 to 14 pounds of butanediol per bushel of grain processed, without especial attention being given to the metallic ion content of the mash. The ion content is apparently not determinative in whole grain mashes, possibly because of the much higher concentration of all metallic constituents and probably because of the protective action exerted by the proteinaceous constituents. (It is well known that concentrations of heavy metals, which are toxic to organisms in media low in protein, are not toxic to the same organisms in media that are rich in proteinaceous substances.) Whether starch or whole grain would be preferred for the industrial Droduction of 2.3-butanediol would be dependent upon the ability to recover the 2,3-butanediol from the fermented TABLE VI'II. EFFECTOF ADDEDCOPPER I O N ON BUTANEDIOL PRODUCTION FROM mashes, and upon the importance attached HYDROLYZATES OF STARCH SS to the recovery of high-quality by-product (All mashes made with tap water and hydrolysed 4 hours at 130' C. with 0.05 N HI SO^) feed. From the standpoint of fermepYield 7 of Theoreticd &wed on Productshl. tation only, it would appear that better Grams,100 Fermen- Glucose Glucore donsum% Fertation Conmenter Cu Added, Time, sumed Butane- AceEth 1 Butane- AceEthyl average yields could be Obtained from No. P.P.M. Hr. G./iOO MI. diol toin a l c o ~ o l diol toin alcohol acid-hydrolyzed whole grain mashes, and 1 None 7.33 1.95 0.29 0.80 61 8.1 21.4 that such mashes would require less ad2 0.01 2.69 0.14 0.23 69 3.8 6.0 1 l66 7.56 OblO 66 7.3 5 6 justment of composition. 3 0.1 17 7.53 2.54 0.27
These studies indicate that trace elements play an important role in the production of 2,3-butanediol from acid-hydrolyzed starch. The presence of certain ions, particularly those of manganese, cobalt, or molybdenum, is reflected in a decreased production of 2,3-butanediol and an increased production of ethyl alcohol under aerobic conditions, and an increased rate of fermentation under anaerobic conditions. As is usually the case with trace-element phenomena, the balance between various ions ~ p p s a r st o be a determinative factor. The present investigation has shown that mashes which are unbalanced with respect to ions can be improved, for fermentation purpo~es,by treatment with an ion-exchange agent or by addition of traces of certain metallic ions, such as copper and zinc. The latter treatment has given satisfactory resulta for individual samples studied but does not appear to be broadly applicable, since each lot of starch must be tested and prescribed for, according to ita needs. The ion-exchange treatment, on the other hand, appears t o be generally applicable; it has corrected the naturally existing unbalance of ions in starch hydrolyzates and has also removed excessive quantities of adventitious iron and copper from hydrolyzates prepared in metal equipment. Metallic ions in starch hydrolyzates may be derived from several sources. They may originate in the grain itself and not be separated from the starch during its manufacture; they may be absorbed or adsorbed from processing water by the starch granules during manufacture; they may be present in the water used for preparing the hydrolymtea; and they may arise from the corrosion of metallic equipment used for the acid hydrolysis. The precise functions of the injurious and the corrective metallic ions are not understood. Virtually nothing is known regerding the enzyme systems that catalyze the production of the fouraarbon compounds, butanediol and acetoin, or that shift the metabolism of Aerobader toward production of these compounds in preference to ethyl alcohol; i t may be that some of the metals (copper, zinc, manganese, cobalt, molybdenum) found to influence butanediol production from starch hydrolysates play m e part in these systems. Silverman (la) reported that an enayme preparation, isolated from AerobwAm aerogms and able to effect the quantitative conversion of pyruvic acid to carbon dioxide and acetoin, required the presence of cocarboxylase and either Mn++ or Mg++. The possibility of some minor organic constituent of the starches playing an important role in these enzymic processes a n n o t be overlooked; increased butanediol production from starch SS,which was treated with sodium hydroxide, might be
4
1.0
17
7.31
2.64
0.16
70
4.2
None deteated.
TABLE IX. EFFECTOF METALLIC IONS ON BUTANEDIOL PRODUCTION FROM HYDROLYZATES OF CORNSTARCH 2892 (All mashea hydrolysed 4 hours at 130° 0. with 0.033 N HCI; nnetale, pupp1ied.a~CUSOI and ZnSOd. were added to starch slurries before gelatlniration of hydrolysis) Yield. % ,"of . .Theoretical. Basad - - - nn - .Metal FermentsGlucose P?oducts, Crams/100 M1. Glucose Conaumed Fermenter Added tion Time. Conaumed ButaneEthyl ButaneEth 1 No. P.P.M: Water Hr. Q./100 MI: diol Acetoin alcohol diol Acetoin slco2(ol 18 6.6.7 2.40 0.11 1 None Distilled 0.37 71 3.4 11.0 2 None Peoria tap 19 7.60 2.13 0.22 0.62 54 5.9 16.0 3 1cu Peoria tap 19 7.37 2.04 0.I6 0.67 64 4.4 17.8 0.06 4 5 Zn Peoria tap 17 7.05 2.89 0.43 80 1.7 12.0
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1194
INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 37, No. 12
ACKNOWLEDGMENT
LITERATURE CITED
The authors gratefully acknowledge assistance received from many of their co-workers. In the Fermentation Division, 2. Louise Smith and Max D. Reeves performed the analyses for 2,3butanediol, acetoin, and ethyl alcohol; Harold P. Vind and Lucille B. Czapla made the analyses for sugar; and Robert G . Benedict maintained and supplied pure bacterial cultures. Spectroscopic analyses of the starches were made by Eugene H. Melvin and Robert J. Bertc of the Analytical and Physical Chemical Division; and chemical analyses of the starches were made by Fontaine R. Earle and his assistants, of the same division. The piloeplant cooks referred to were conducted by James M, Van Lanen, Fred W. Tanner, Jr., and Virgil E. Sohns of the Agricultural Motor Fuels Division. The strain Aerobacter aerogenes NRRLB199, which was used in the experiments, was kindly made available by C. H. Werkman of Iowa State College in 1941. The commercial starch-converter liquors were supplied by courtesy of the Corn Products Refining Company.
(1) Assoc. of Official Agr. Chem., Methods of Analysis, 5th ed., p. 369 (1940). (2) Boeseken and Cohen, Rec. tmw. chim., 47,839 (1928). (3) Bortels, Bwchem. Z.,182,301 (1927). 23,996 (1931). (4) Churchill, IND.ENG.CKEM., (5) Collins, Lamar, and Lohr, U. 9. Geol. Survey, Water Supply Puper 658,61 (1932). (6) Goering, Iowa State Coll. J. Sci., 16,65 (1941). (7) Herrick, Hellbach, and May, IND.ENO.CHBIM., 27, 681 (1935). ( 8 ) Hill and Isaace, Brit. Patent 483,939 (1938); U. S. Pateit 2,224,912(1941). (9) Johnson, IND.ENO.CHEM., ANAL.ED., 16, 626 (1944). (10)Permutit Co., personal communication. (11) S h d e r and Hartmann, J. Biol. Chem., 45,365 (1921). (12) Silverman,Iowa Stat6 Coll. J . Sci., 17,120 (1942). 36,404 (1944). (13) Slotter and Langford, IND. ENO.CHEM., (14) Ward, Pettijohn, Lockwood, tLnd Coghill, J . Am. Chem. SOC.,66. 541 (1944);
ZEIN FIBERS
PRBSENTED (by title) before the Diviiion of Agricultural and Food ChemCHEMICAL SCCIICTY in New ietw at the 108th ,Meeting of the AMERICAN
York, N.Y.
e
Preparation by Wet Spinntng C. B. CROSTON, C. D. EVANS,AND A. K. SMITH Northern Regional Research Labomtory, A new method for pmducing textile fibers from zein and the equipment used in the process are described. Zein fibere which have high tensile strength and wool-like properties are produced from alkaline dispersions of zein. The spinning dispersions are formulated to give a product of high viscosity by the use of denaturing agents, such as alcohol or urea, or by aging the solutions. The filaments are coagulated in an acid bath in which salts may or may not be used. The coagulated filaments are given a mild formaldehyde precure prior to stretching and drying. The influence of the extent of precure on the load-elongation properties of the fiber and on fiber strength is determined. Shrinkage and water resistance of the fibers are controlled by acetylation followed by a strong formaldehyde treatment.
P
ROCESSES for making protein fibers from zein have been patented by Swallen (6) and Meigs (4)and reviewed briefly by Harold (3). The Swallen process involves the use of organic solvents such as ethyl alcohol in the preparation of the spinning solution. This process also uses 20% or more of plasticizer and 2 t o 5% of formaldehyde in the spinning dispersion, and extrudes the solutions through standard spinnerettes. The extruded filaments are solidified by evaporation of the solvent (dry spinning) or by a liquid coagulant (wet spinning). Meigs described a wet spinning process for a wide variety of proteins-namely, the “globulins, prolamines, and phosphoproteins”-and includes zein among the proteins he u8es for examples. The Meigs patent contains rather startling claims for stretching the fibers 300 to 20000J0,regardless of the type of protein used. Zein has been mentioned in other patents as one of the proteins from which fibers can be spun or regenerated. However, the absence in the literature of any information on their strength or other properties makes impossible their practical evaluation or aompaxison with other fibers.
U. S . Department of Agriculture, Peoria, I l l .
Zein is the only prolamine which has attained any substantial industrial importance. Its solubility in organic solvents has determined t o a large extent its commercial utilization for f3ms and coatings, and at present its largest use is aa a replacement for shellac. Likewise, the fiber spinning methods described in the literature use organic solvents in the preparation of spinning solutions. This investigation was directed toward the development of a practical process for making zein fibers by a wet spinning method and toward evaluation of the fibers. The equipment used in this work differs from that for spinning viscose rayon and other protein fibers only in details of construction and in arrangement. Data are presented on the preparation of zein-spinning dispersions, the effect of treating the fiber with formaldehyde prior to stretching (precure), and the effect of precure on the wet and dry strengths of the fibers. The use of trditional organic solvents for zein has been temporarily abandoned in the present investigation, and an aqueous alkaline dispersion for wet spinning has been developed. The object in making the change is to avoid the extra cost which arises in recovering organic solvents. It has been knownfor some time that zein is soluble in alkaline solution, but only recently the details of this type of dispersion have been investigated and ita limitations defined by Ofelt and Evans (6). In their studies on the solubility of zein in sodium and potassium hydroxide and quaternary ammonium hydroxide, Ofelt and Evans showed that a highly concentrated zein solution can be prepared in the pH range 11.3 to 12.7. A complete description of this dispersion procedure is being prepared for publication. AQUEOUS SPINNING DISPERSION
High-viscosity zein solutions of 13 to 16.5% aoncentration me required for spinning fibers, and the upper limits in this range
