September 1950
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
activity by the method of Corman and Langlykke ( 6 ) ,as modified later a t the Northern Regional Research Laboratory. It may be necessary to alter the enzyme-substrate ratio when cultures of lower enzyme activity are used. Three conversion agents-barley malt, corn malt, and fungal culture-are compared in Table 11. The average alcohol yield obtained by conversion with 7% barley malt was 5.14 proof gallons per bushel (wet basis), tu determined from twelve fermentations. This yield appears to be equivalent to that which could be obtained b y the use of approximately 11% corn malt, according to the data of the Brazilian worker, Almeida ( 2 ) . The yield obtained by conversion with 10% of fungal culture, however, was much better than that obtained by the use of either barley or corn malt. The average yield with fungal conversion was 6.51 proof gallons per bushel, as determined from twenty-four fermentations. The plant efficiency of fermentations converted by barley malt was 74.1% as compared to 90*5% for those converted by fungal cultures. DISCUSSION
The fungal cultures employed in these experiments were prepared in a distillers’ dried solubles-corn medium; substitutes for these ingredients undoubtedly would have to be used by distilleries located in cassava producing regions. No attempt was made at this time to test substitute mediums. Experience has shown that a variety of starchy and proteinaceous materials can be used in the production of fungal cultures having high enzyme potency. Cassava stillage logically would serve as the basic ingredient of the medium. Soybean meal has proved to be a satis-
1783
factory substitute for distillers’ dried solubles and crude cwsava
or rice meal can be used in place of corn meal. SUMMARY
Cassava starch can be converted into alcohol most efficiently when submerged culture fungal enzyme preparations are used to hydrolyze the starch into fermentable sugars. Investigators who have employed acid hydrolysis report yields of 43 to 74% of the theoretical. The use of barley malt for conversion has resulted in yields of 70 to 74% of the theoretical, and the use of an equivalent amount of corn m d t resulted in lower yields. When mold bran preparations were used for conversion, yields of 80 to 85% of the theoretical were obtained. Cassava mashes converted by submerged fungal cultures, as reported in this paper, resulted in a plant efficiency of 90%. LITERATURE CITED
(1) Adams, S. L.,Balankura,B., Andreasen, A. A., and Stark, 15‘. H., IND.ENQ.CHEM., 39,1615(1947). (2) Almeida, J. R., J . de Pirucicnbu, Piracicaba, Est. S. Paulo, Brazil, 1-92 (1943). (3) Back, T. M., Stark, W. H., and Scalf, R. E., IND.END.CHEM., ANAL.ED..20.58 (1948). (4) Ranzon, J., Fulmer, E. I.,‘and Underkofler, L. A., Iowa State Coll. J.Sci,, 23,219 (1949). (6) Corman, J., and Langlykke, A. F., Cereal Chem., 23,190 (1945). (6) Sandstedt, R. M., Kneen, E., and Blish, M. J., Ibid., 16, 712 (1939). (7) Stark, W. H., Adams, S. L., Soalf, R. E., and Kolaohov, P., IND. ENQ.CHEM.,ANAL.ED.,15,443(1943). RECEIVED February 23,1950.
Rate of Secondary Fermentation of Corn Mashes Converted by A. niger S. C. PAN, A. A. ANDREASEN, A N D PAUL KOLACHOV Joseph E. Seagram & Sons, Inc., Louisville, Ky.
E
Submerged fungal cultures convert an appreciable porX T E N S I V E studies the secondary fermentation tion of starch into difficultly hydrolyzable dextrin. Exphase consists of the ferhave been made in reperimental data show that the rate of secondary fermentacent years concerning the mentation of sugars protion is dependent upon the rate at which this dextrin is duced b y the hydrolysis of use of submerged fungal hydrolyzed. The rate of secondary fermentation can be “limit dextrin” (4). cultures for the conversion increased significantly by varying a number of conditions, of grain mashes (1, 12). Three phases, similar to thereby reducing the time required for complete fermentathose just described for Practically all these studies, tion. There is a bettw correlation between the velocity f e r m e n t a t i o n of g r a i n however, have dealt with constant, K , and maltase activity of the fungal culture the yields that can be obmashes converted by barley than with the a-amylase or limit dextrinase activity. tained, while the question malt, also occur during the of rate of f e r m e n t a t i o n fermentation of corn mashea has received very little atconverted by submerged tention. Inasmuch as, in industrial application, the length of cultures of the mold AepergiElus niger. Attempts have been made timerequired for complete fermentation is of economic importance, to accelerate the rate of secondary fermentation (the third phase), a study of the rate of fermentation of corn mashes converted because it requires the greatest amount of time. Conditions for both the preparation of the submerged fungal culture and the by submerged fungal cultures was considered advbble. The fermentation of grain mashes converted by barley malt fermentation can be readily modified and many variables c ~ r consists of three overlapping phases: a lag Phase; a Primary pable of effecting a faster secondary fermentation can be inor main fermentation phase; and a secondary fermentation phase vestigated. (8, 6). The lag phase is a period during which yeast growth takes EXPERIMENTAL METHODS AND PROCEDURES place, The main fermentation phase is characterized by the Yeast and Mold Strains Used. S e w - Yeast strain sc lY, rapid fermentation of preformed sugars and is followed by a period a distillers’ Yeast, W a s employed h dl fermentation experiments. of slow fermentation-the secondary fermentation phaseAspergillis niger NRRL 337 (Northern Regional Research during which time there is a small, but significant, increase in the Laboratory) w&9 used in the preparation of the submerged alcohol content, aa shown by the data of Thorne et aE. (81). fungal culture. Other mold strains were tested and are discussed According to modern concepts of starch chemistry (7,14, f6), below.
1784
INDUSTRIAL A N D ENGINEERING CHEMISTRY
Carbohydrate Qm;/r00mI.
i-
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GLUCOSE
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FE R , DEXTRIh M E N 1I I UG MASHES
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MALT CONVERTED /, MAP" an I
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MASHES Dx!T\ NOT FERMENTED IS IN
3.0
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F U ~ G A Lc ULTURE CONVERTED MASH
.
1; -Ip
9.0
2.0
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TIME YOURS-
60
I
/z/ CARBOHYDRATES TOTAL
11.0
50
40
30
activity of the glucogenic enzyme e., enzyme(s>s litting maltose or other glucose pofymers. Because the biochemical nature of such an enzyme system requires further elucidation, we continue t o refer to the maltose-hydrolyzing ensyme(s) as maltase. Determination of Carbohydrates. The total amount of carbohydrates in a mixture of dextrin and fermentable sugars was determined as glucose after complete h drolysis with hydrochloric acid 60) by the method of Somogyi (18). The quantity of dextrin in such a mixture. wm determined by treating the sample with baker's yeast according to the procedure of S t a r k a n d Somogyi (19) and analyzing the treated Sam le for glucose after complete hyfrolysis with hydrochloric acid. s y s t em-i
SECONDARY FERMENTATION PHASE
20:
Val. 42, No. 9
1
I I l
EXPERIMENTAL RESULTS I
I
I
I
I
I
J
Differentiation of Fermentation Phases. The curves in Figure 1 illustrate the progress of fermenFigure 1. Change in Carbohydrate Concentrations during Fermentation of Malt tation of a corn mash converted and Fungal Culture-Converted Corn Mashes by bsrley malt and s u b m e r g e d culture of A . niger. After an Reparation of Submerged Fungal Culture. The mold W a s initial phase of 5 to 7 hours, the diminution of total carbopropagated at 30" C. for 48 to 60 hours either in 2-liter cone-shaped hydrate during the primary or main fermentation phase flasks, according to the description. by Adams el al. (I), or in (fermentation of Preformed sugars) was a linear function with shake flasks with 120 ml. of medlum in 750-ml. Erlenmeyer flasks. time. This rapid fermentation proceeded at a rate of 0.47 The shaker was operated a t 100 cycles per minute with a stroke length of 2 inches. A sterilized medium containin 5 grams of gram per lo()ml, per hour U P to 26 hours in mal&converted mash, distillers' dried solubles, 1 Gam of ground corn, anfO.5 gram of at which t h e 75% of the total carbohydrate had disappeared, calcium carbonate per 100 ml. was used routinely. and a t 8 rate Of 0.49 gram per 100 ml. per hour Up to 21 hours Masbg_"=d Fermentation Procedure. Corn was mashed and fermented according to the laboratory procedure.described in fungal enzyme-converted mash, at which time 60% of the by Stark et al. (10). Barley malt equal to 7% by total carbohydrate had disappeared. Following this rapid prior fungal culture equal to 10% by final volume, was used for Weight, conmary phase, the rate of carbohydrate diminution became slower. version. A bminute conversion period 6 0 0 c. was employed. less than 1 hour elapsed before the addition of yeast. Ali This slow fermentation period constituted the secondary ferfermentations were incubated a t 30" c. Immediately after the mentation phase, which required a greater time than the other introduction of yeast, a sample was taken to determine the initis! two phases. Both fermentations required 60 hours for complete total carbohydrate content after digesting for 2 hours at 55 to 60" C. to ensure complete dextrinization. .Periodic samples, fermentation. taken during fermentation, were heated in a boiling water bath for 10 minutes to stop ensymic activity and centrifuged, Total Corbohydrotes and the centrifugate was analyzed for gm/iOornl. I total carbohydrate and dextrin content. Comparative fermentatlon rates were obtained by plotting carbohydrate concentration against time, with zero hour corresponding to the time a t which yeast was inhoduced. CONVERTED MASH 10.0 Determination of Enzyme Activities. Alpha-amylase and limit dextrinase activities were determined by the methods of Sandstedt et al. (I6) and Back et al. (9), 8.0 respectively. Units of a-amylase were expressed as grams of soluble starch dextrinized in 1 hour at 30" C. Limit 6.0 dextrinase activity was expressed as per % FUNGAL CULTURE cent hydrolysis of limit dextrin in 1 hour under the conditions of the determina% FUNGAL CULTURE tion. The method of Corman and 4.0 % FUNGAL CULTURELanglykke (6), as modified later a t the Northern Regional Research Laboratory, was employed for evaluating the maltase 20 activity. The modification consists of measuring the increase in reducing power of maltose substrate after 30 and 120 I I I I I I I I 0 1 minutes of incubation. Units of maltase IO 20 30 40 !?io 60 -MALT activity were expressed as milligrams of FUNGAL CULTURE0 IO 20 30 40 SO 60 maltose hydrolyzed per hour a t 30" C. TIME HOURS and pH 4.4. According to Corman and Langlykke (6), the maltose hyFigure 2. Rate of Fermentation at Different Concentrations of Converaion Agents drolyzing power is a measure of the Ib
20
pF,!,T$&+ MAIN FERMENTATIONPHASE+
I30
40
50
60
TIME HOURS
S ~ C O N O A R Y FERMENTATION PHASE+
-
-
-
1
September 1950
INDUSTRIAL AND ENGINEERING CHEMISTRY
1785
TIME HOURS In unfermented mashes, the hyLOGARITHM O F 0 IO 20 drolysis of dextrin, formed during TOTAL C4980HYDPATC concantration primary conversion, slowed down rapidly and came almost to a standstill in 24 hours (Figure 1). Similar curves were obtained from the feiI h I menting mashes, but here a re0.5 newed rather rapid hydrolysis of the dextrin took place when the 0.4 preformed fermentable sugars had almost disappeared. The fermen0.3 tation of sugars resulting from the hydrolysis of this dextrin evidently 0.2 constituted the secondary fermentation phase. The rate of secondary fermentation apparently was governed by the rate at which this 0.0 dextrin was hydrolyzed, because the rate of fermentation of the preformed sugars was much greater. IO., OEXTRIN The fact that the mash was pracI I I I J tically free of fermentable sugars 0 IO 2 0 30 40 50 during the secondary phase (Figure TIME HOURS 1) could be accounted for by the Figure 3. Course of Secondary Conversion and Fermentation same reason. The curve (Figure 1, inset) for the fermentation of glucose (in a 1.5% yeast extract medium), which was a straighbline function throughout the entire fermendextrin was observed. When fungal culture alone waa intation period, further substantiates this reasoning. troduced, hydrolysis of the dextrin took place, but the rate of Effect of Concentration of Conversion Agents upon Rate of hydrolysis soon fell off, as illustrated in Figure 3. (A paper dealFermentation. Further data to show that the rate of secondary ing with a kinetic study of the secondary conversion is being fermentation depends upon the rate of dextrin hydrolysis, which prepared for publication.) Figure 3 shows the curve obtained in turn depends upon the enzyme concentration, is presented in when both yeast and fungal culture were introduced. A comFigure 2. The rate of primary fermentation, which was governed parison of this curve (excluding the first 5 to 7 hours of yeast by the rate of fermentation of preformed sugars, was prwtically growth) with the latter portion of the fermentation curve in unaffected by differences in the concentration of conversion Figure 1, which illustrates the secondary fermentation phase, agent. On the other hand, the rate of secondary fermentation shows that they are superimposable. Therefore, fermentation was shown to be dependent upon the concentration of conversion of the residual dextrin medium was shown to represent the agent. Fermentation was far from complete after 60 hours when secondary phase of a normal fermentation of corn mash. 4% barley malt or 5% fungal culture was used, whereas fermentaA straight line is obtained when the logarithms of the dextrin tion was complete in much less than 60 hours when 16% malt or concentrations shown on the above curve are plotted against time. 20% fungal culture was used. This shows that the rate of secondary fermentation is proporFermentatidn of Residual Dextrin Medium. In order t o study tional to the dextrin concentration presenbi.e., the over-all the secondary fermentation phase done, a medium prepared as reaction follows the course of a first-order reaction. The velocity follows was employed. TEMPERATURE OC. Corn was mashed in the usual manner and converted with 10% 25 (by volume) of submerged fungal culture for 4.5 hours at 55" to 60" C . The mash was then sterilized and fermented with 7.0 east. After fermentation the suspended solids were removed g y centrifuging and the alcohol was removed by eva oratin the supernatant liquid under reduced pressure to two t h r d s of the onginal volume. The resulting preparation ww ke t in a refrig6.0 erator. T o test the rate of fermentation in this rne$um, ali uots TEMPERATURE of the preparation were diluted, so that after the addition the conversion agent the volume waa equivalent to that of the original mash. A final volume of 32 ml. waa found convenient for tests in 200 X 25 mm. test tubes. T o avoid contamination 5.0 the medium was sterilized before use. This medium, designated aa "residual dextrin medium," contained 4.2 to 4.5 grams of dextrin per 100 ml. Experiments 4.0 in which the primary conversion was extended to 9 hours at 55O to 60" C. before sterilization and fermentation, showed very little further reduction in the dextrin content. This dextrin must, therefore, be the same as the one that is responsible for the slow 3.0 secondary fermentation of corn mashes (Figure 1). The average molecular size (or chain length) of this dextrin was rather low4 to 5 glucose units per molecule-as estimated from the ratio of 2.0 total reducing power after acid hydrolysis (8, 83) to the direct 2 3 4 5 6 7 reducing power. PH When yeast alone was introduced into the residual dextrin Figure 4. Effect of p H and T o m p e r a t w e upon R a t e medium, little or no growth took place and no diminution of the of Socondary Fermontation
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1"'
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3
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INDUSTRIAL A N D ENGINEERING CHEMISTRY
Vol. 42, No. 9
337 was used, as it has been receiving considerable study in this laboratory and elsewhere (1, 11, 12). Effect of pH and Amount of Calcium CarLimit bonate upon Rate of Secondary Fermentation. Dextrin01There wtw a marked drop in pH after autoclavFermentation, Velocity Maltase, Ya"I";;Amylase, ing if the dried solubles-corn medium was adOrganisms % Constant, Units/ d:olysis/ Units/ Tested 8 hours 24 hours K X 102 MI. MI. All. justed to an initial pH above 6; therefore, all the A . niaet mediums tested in this experiment were sterilized NRRL337 13.9 50.2 3.48 2.92 14.80 1.92 A . niger a t pH 6 and then adjusted to different pH levels NRRL330 16.4 64.5 5.35 6.90 13.15 0.79 aseptically. The mediums contained no calcium carA . oryzae 6.3 18.2 0.83 0.42 5.00 0.2 R . javanicus 4.5 27.0 1.64 0.21 0.00 0.0 bonate and the pH was adjusted twice a day with A . nigcr mutant30B 7.4 55.6 4.58 3.92 14.30 2.03 sterilized 2 N sodium hydroxide and 2 N sulfuric A . niger acid during propagation. Table I1 shows that the mutant 1OC 6.5 18.5 0.82 0.42 9.80 1.15 A . niger optimum pH for mold propagation, judged by the rnutant3OC 1 1 . 5 43.3 2.54 1.06 11.15 1.38 A . niger rate of secondary fermentation, is between 4.8and .. mutant 15E 5.5 41.5 2.98 1.85 10.70 5.6. Because the pH was in this range when calMedium. Dried solubles 5 g r a m d l 0 0 ml., corn 1.0 grani/100 rnl., CaCOa 0.5 grarn/100 cium carbonate was used in the medium, no pH ml. Propagation temperature. 32-36' C. (enzyme activities lower than usual st 30' C.) adjustment was made when calcium carbonate was Age. 48 hours. Fermentation temperature. 30" C. used. Table I1 also presents data showing the effect of using varying amounts of calcium carbonate. The data show that as the amount of calcium carbonate in the medium was increased. a-amylase activity also increased; yet the rate of secondary constant, K , represented by the slope of the straight line, would fermentation was hardly affected. Therefore, 0.5 gram per therefore give a convenient measure for the rate of secondary 100 ml. of calcium carbonate was used routinely. fermentation. In all the following experiments, the rate of secondary fermenTemperature and Age. Twc-liter, cone-shaped flasks were tation was determined by using the residual dextrin medium. employed to propagate the cultures for testing the effect of Fungal culture equivalent to 10% by final volume was introduced temperature. The cultures were incubated in water baths maintained at different temperatures ranging from 20" to 35' C. as conversion'agent, followed by a 2% yeast inoculum. The Samples were taken periodically and the rate of secondary fermenting medium was analyzed for dextrin content (as glucose, fermentation was determined. Apparently the production of the after acid hydrolysis) after 8 and 24 hours, and the value for K enzyme responsible for secondary conversion reached its maxiwas calculated according to the equation: mum in approximately 48 hours when incubated a t 30' C. dextrin concentration at 8 hours 2.303 K = - 16 (Table 111). Longer periods of cultivation failed to increase this log dextrin concentration at 24 hours enzyme activity. The propagation temperature affected the enzyme activity markedly; the optimum was found to be approxiwhere 2.303 is the factor for converting common to natural mately 25' C. Propagation at 30" and 35' C. resulted in lower logarithms and 16 is the time interval between dextrin determinarates of secondary fermentation, whereas 20' C. wm probably too tions. (Because the medium was practically free from fermentlow to ensure maximum growth and enzyme production. able sugars after 8 hours, the total carbohydrate content was Medium Ingredients. The amounts of distillers' dried solublea determined in place of dextrin.) and corn required in the medium to produce maximum enzyme Effect of pH and Temperature upon Rate of Secondary Feractivity for secondary conversion were studied. As illustrated mentation. Aliquots of the residual dextrin medium were adin Table IV, poor enzyme production resulted if either of these justed to different pH levels and sterilized. The pH of each was ingredients was omitted. The same table shows that with a measured immediately after the addition of yeast and fungal constant concentration of dried solubles ( 5 grams per 100 ml.) culture and again a t the end of 24 hours. There was a negligible the rate of secondary fermentation increased as the concentradrop in pH when the initial value was 2.8, but the pH dropped tion of corn was increased-up to 4 grams per 100 ml. Duplicate 0.7 unit when the initial value was 6.5. The optimum pH range experiments have shown that this increase is greater a t corn conwas 4.2 to 4.7, as shown in Figure 4, and because the initial pH of the medium was 4.53, no pH adjustment was necessary. A plot of K us. temperature is also given in Figure 4. It is obvious from this curve Table 11. Effect of p H and Amount of Calcium Carbonate upon Rate that the rate of secondary fermentation increased of Secondary Fermentation as the temperature increased. Therefore, a faster Limit fermentation rate resulted when elevated temperaCaCOa Velocity DextrinaAdded, G . % sCg;, Maltase. ase, % .H Amylase, tures were used instead of the conventional 30" to per sugar Units/ drolysislUnits/ 32' C. during the secondary fermentation phase. pHRange 100 M I , 8hours 24hours K X 102 M1. All. MI. Rate of Secondary Fermentation with Submerged 2.8 .. 12.0 23.7 0.89 ... ... .. 13.3 43.6 2.67 ... ... .. .. 4.1 .... Cultures of Different Organisms as Conversion 5.6-4.8 18.0 64.6 5.25 ... ... 13.7 56.4 4.28 ... ... .. 6.5-5,1 .. Agents. The conversion powers of several am7.8-6.0 .. 14.6 51.5 3.52 ... ... .. ylolytic molds, available in the authors' stock 9.1-7.8 7.0 35.7 2.34 ... .. 4.9-5.3 i:o 17.5 62.8 5.00 ... ... .. culture collection including A . niger NRRL 330, A . 2 . 90 5.4-5.1 0.0 24.8 78.0 7.41 10.50 17.3 oryzae ATCC 4814, and some ultraviolet mutants 5.5-5.4 0.2 25.9 77.3 7.38 8.80 117.85 9.70 43 . 10 40 5 . 6 5 . 4 0 . 5 2 7 . 2 7 8 . 5 7 . 3 9 1 1 . 5 5 from A . niger NRRL 337, were tested. Anon5.6-5.4 1.0 26.0 76.9 7.06 12.30 20.70 5.00 ... .. amylolytic mold, Rhizopus javanicus, was also in5.6-5.4 2.0 24.4 77.0 7.45 ... cluded. Table I shows that A . niger 330 and the Organism A . niger NRRL 337. Medium. ' Dried solubles, 5 grams/100 ml.. corn 1.0 gram/100 ml. ultraviolet mutant 30B were somewhat better Propagation temperature. pH experiment, 28-30' C. CrCOs experiment, 25-27" C. (enzyme activities higher at lower propagation temperatures). secondary conversion agents than A . niger 337; Age. 66 hours. all the other strains gave rather poor results. Fermentation temperature. 30' C. In all the following experiments, only A. niger
Table I.
Rate of Secondary Fermentation with Submerged Cultures of Different Organisms as Conversion Agents
.
I
.
September 19W
1787
besides a-amylase (1, 11), the fungal culture also exhibits enzyme activities capable of hydrolyeing limit dextrins (limit dextrinase, IS) and maltose Limit (maltase or glucogenic enzyme system, 6). In Dextrin6 order to test whether one or more of these known TemperaVelocity Maltase we, % ture Fermentation, % Constant, uNb/ ’ Hydrolysis/ Amylase, Units/ enzymes, or some other enzyme, is responsible for C,’ Hod. 8 hours 24 hour8 K X 101 MI. MI. MI. secondary conversion, the a-amylase, maltase, and 35 19 14.3 39.0 2.11 .. 35 49 17.3 56.8 4.05 2:97 ii:i2 .. limit dextrinase activities of the fungal cultures 1.48 1.24 9.65 a9.8 2.19 30 19 14.3 used in the preceding experiments were determined 15.30 1.72 3.71 65.6 5.42 18.1 30 49 3.92 15.15 1.85 63.0 4.93 (Tables I to V). 30 65 18.3 2.15 5.37 16.00 63.7 5.14 30 86 17.1 Higher calcium carbonate concentrations pro16.0 71.4 6.75 25 49 duced higher a-amylase activities, but had almost 21.3 75.0 7.15 8:SO 18:55 2134 25 65 no effect upon the rate of secondary fermenta8.2 28.8 1.60 .. 20 65 tion (Table 11). These results seem to be a t variOrganism. A. nioer NRRL 337. Medium. Dried solubles 5 grams/100 ml.. corn 1 gram/100 ml., CaCO: 0.5 gram/100 ml. ance with thmb reported by Tsuchiya et al. (22), Fermentation temperature. 30’ C. who stated that calcium carbonate did not affect a-amylme production, provided the pH was maintained above 4. Prolonaina the DroDaga- tion for more than 2 days led to a gradual increase of acentrations below 1 gram per 100 ml. With a constant concenamylase activity, while the rate of secondary fermentation tration of corn (1.0 gram per 100 ml.) the rate of secondary fershowed a uniform decrease (Table V, experiment 51G). A. mentation also increased as the concentration of dried aolubles naer NRRL 330 (Table I) produced little a-amylase, yet i t was increased up to 5 grams per 100 ml. A further increase in proved to be an efficient secondary conversion agent. These the concentration of dried solubles not only failed to increase the resulte show that a-amylase has little or no bearing on secondary rate of secondary fermentation, but tended to reduce it. Thin conversion under these conditions. stillage, when used to replace distillers’ dried solubles and water That the rate of secondary fermentation does not correlate in the medium, was found to be equivalent to 5 grams per with the limit dextrinase activity is shown in experiment 51G, 100 ml. of dried solubles on the basis of secondary fermentation Table VI where prolonged propagation of the culture caused a rates (I, 21). drop in the rate of secondary fermentation while the limit dexThe effect of other nutrient materials was tested by incortrinase activity was almost unaffected. Similar results were Doratinz them into a suboDtimal medium containing 2 grams of ist tiller^' dried solubles per 100 ml. and‘ 1 gram of corn per 100 ml. Data in Table IV show that the addition of 2 Table IV. Effect of Medium Ingredients for Propagating Fungal Cultures grams of wheat bran per 100 ml. of upon Rate of Secondary Fermentation medium gave somewhat better reaults Medium Ingredients, Fermentation, % Velocity Limit G./100 MI. Dextrinase % than the use of additional dried solubles. Dried 8 24 Constant Maltase Hydrolysis/ rr-.4myIase The results obtained when corn steep solubles Corn Others0 hours hours K X 10; Unitn/M\. MI. Uni ts/M 1.’ liquor, malt sproub, and soybean meal 5 0 ... 11.6 29.6 1.40 ... 5 0.5 ... 13.5 44.3 2.74 2:39 8.0 0:80 were used were little or no better than 5 1.0 ... 20.6 65.1 5.13 4 64 14.0 2.55 5 2.0 ... 24.8 6.86 6.05 74.8 14.7 3.30 when 5 grams per 100 ml. of dried 5.94 5 3.0 . . . 21.5 69.6 4.35 13.9 2.79 solubles were used. Animal stick liquor 5 4.0 .,. 23.6 79.2 8.14 5.26 15.1 3.48 0 1 ... 6.0 16.5 0.77 ., inhibited enzyme production at the con1 1 ... 15.3 47.3 2.99 2:s7 1:oo centration used. 2 1 ... 18.2 56.0 3.89 3.94 lo 0 1 69 5 1 ... 20.6 65.1 5.13 4.64 14 0 2.55 Effect of Prolonged Propagation. No 7.5 1 ... 20.6 67.2 5.54 4.55 13 5 2.61 10 61.8 4.75 3.40 1 ... 18.2 11 3 -2. 88 _reduction in the enzyme activity for sec5 1 21.7 62.5 4.60 4.73 19.4 2.60 ondary fermentation was observed for 1 T.S:‘ 21.1 61.5 4.56 0 .. .. .. 2 1 2C.S.L. 20.8 62.7 4.71 .. .. cultures propagated for periods up to 86 2 1 2M.8. 21.6 63.8 4.83 hours in the preceding experiment (see 1 2 W.B. 25.0 71.0 5.92 2 6 .’80 1813 2’68 2 1 2A.8.L. 8.6 29.8 1.63 1.15 10.6 0.87 Table 111); however, decreases were 2 1 18.M. 20.5 62.6 4.73 .. .. noted when 14-liter quantities of mold Organirm. A. niosr NRRL 337. Medium. All contain 0.5 gram/100 ml. CaCOa. culture were propagated for prolonged Propagation temperature. 30DC. Age. 48 hours. periods in a thin stillage-corn medium Fermentation temperature. 30’ C. (Table V). The rate of secondary ferT.8. thin stillage. C.8.L. corn steep liquor: M.S. malt sprouts: W.B. wheat bran: A.S.L. animal stick liquor; 6.M. soybean meal. mentation began to decrease after 2 days of propagation in a medium conTable V. Effect of Prolonged Propagation upon Rate of Secondary taining calcium carbonate (experiment Fermentation 51E)and after 4 days in a medium conPropagation Fermentation, % Velority Limit taining no calcium carbonate (experiExpt. Time, 8 24 Constant Maltase Dextrinase 9 a-.4mylase, NO. Days hours hours K X 101’ Units/Mi. HydrolysisjMf. Unlts/llIl ment 51G); the decrease was rather uni32 0 76.4 6.60 51E 2 11.62 19 4 4 50 form in both cases. For practical pur3 24.0 68.1 5.60 7.43 16.4 4 21.6 60.7 4.30 5.07 12.3 2.74 poses, therefore, propagation should not 5 19.0 55.2 3.68 3.38 12.7 .. be continued for more than 48 hours. 6 19.0 48.2 2.78 3.09 12.85 .. Table 111.
s
INDUSTRIAL AND ENGINEERING CHEMISTRY
Effectof Temperature and Age upon Rate of Secondary Fermontation
..
...
-
..
Correlation of Rate of Secondary Fermentation with Known Enzyme ACtivities of Fungal Cultures. Although
the enzyme system of the fungal culture involved in the hydrolysis of starch and its degradation products is not exactly known, it has been demonstrated that
51G
2 20.6 72.0 3 21.0 71.3 4 21.0 70.3 5 21.2 63.5 6. 6 19.0 48 _. Organism. A. nioer NRRL 337. Thin stillage, oorn 3 grams/lOO ml., Medium. c,n\
“IU,.
Experiments run in 14-liter quantities.
7.00 6.70 6.59 5.10 3.20
13.60 13.90 13.80 9.90 7.43
15.9 15.4 17.8 15.6 16.1
1.00 0.67 1.67 3.06 3.65
CaCO, 0.5 gram/100 ml. (expt. 51Eh no CaCOa (expt.
1788
INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 42, No. 9
scribed in the preceding paragraph. After 18.5 hours of fermentation, the temperature was Propagation Fermentation Velocity raised to 37’ C. The mash was analyzed periodFermentation* % Constant. Propagation Temperature, Temperature, ically for total carbohydrate and the fermentation c. c. 8 hours 24 hours K X 10* Medium“ was considered complete when the total carbo30 30 15.5 58.8 4.49 hydrate dropped below 0.6 gram per 100 ml. A t 30 37 26.5 77.6 7.41 the end of 42 hours the fermenting mash was analyzed for alcohol content and the yield calcu26-27 30 25.2 72.8 6.31 lated. For comparative purposes, fermentations 2773.*pcg;; of corn mashes that had been converted with 7% 2% %.B. 26-27 37 37.3 85.0b 9.00 (by grain weight) of barley malt and 10% (by Organism. A. niger NRRL 337; ,age 62 hours. Propagation made in 2-liter aeration flask. volume) of submerged fungal culture propagated D S dried solubles C., corn W.B wheat bran. in the usual distillers’ dried solubles-corn medium b Residual sugar a t ekd of 24 hours,”0.66 grain/100 ml., indicating practically complete fermentation. at 30” C. were also tested; the results are summarized in Table VII. Normal alcohol yield and residual sugar were obtained in 42 hours, as observed in 4- to b d a y cultivations in experiment 51E, Table against 52 hours for malt-converted mash and 60 hours for corn V. A . niger NRRL 330 and the mutant 30B were better secondmash converted with fungal culture prepared and used in the ary conversion agents than A . niger NRRL 337, yet their limit usual manner. Therefore, an increase in the rate of secondary dextrinase activities were almost identical (Table I). Animal fermentation substantially shortened the total time required stick liquor affected the rate of secondary fermentation adversely, for complete fermentation of a corn mash. but the limit dextrinase activity suffered far less reduction than DISCUSSION A N D CONCLUSIONS either a-amylase or maltase activity (Table IV). With only one minor discrepancy (Table 111, cultures of difAlthough the enzyme system contained in fungal cultures ferent ages incubated a t 30’ C.), the maltase activity ran parallel differs from that in barley malt, both convert an appreciable to the rate of secondary fermentation. This parallelism is portion of starch into difficultly hydrolyzable dextrins which illustrated by the data of practically every experiment presented resist further conversion until primary fermentation is complete, in Tables I to V. Comparison between maltase activity and the or until the preformed sugars have been removed. It is not velocity constant, K, are of course more consistent within an known whether the dextrin produced by fungal enzymes has the experiment than between two different experiments. The data same chemical nature as the “limit dextrin” produced by malt indicate that the rate of secondary fermentation correlates more amylases. The hydrolysis of this dextrin is undoubtedly reclosely with the maltase activity than with the other two enzyme sponsible for the slow rate of secondary fermentation. The reactivities. This appears to agree with Corman and Langlykke sults presented in this paper indicate that maltase (or the enzyme (6),who found that the alcohol yield correlates more closely which exhibits maltose hydrolyzing power) may play a more with the “glucogenic” activity-i.e., maltase activity-of the important part than a-amylase or limit dextrinase in the secondfungal cultures than with the a-amylase activity. ary conversion of this dextrin produced during primary conCombined Effect of Improved Propagation and Fermentation version by fungal enzymes. Further work is required to deterConditions. Attempts were made to combine in one experiment mine whether maltase, acting alone, would give the same results. all the beneficial effects observed thus far. The rate of secondary An examination of the literature reveals that maltase of mold fermentation was determined a t 37’ C. with the fungal culture differs from maltase of yeast in many respects (Schwimmer, 1 7 ) . propagated in a distillers’ dried solubles-corn-wheat bran medium Leibowitz pointed out that the mold maltase does not split at 26’ to 27” C. (see Table IV). The results are given in Table methyl a-glucoside, whereas yeast maltase does (9, IO). He VI, where results with fungal cultures prepared in the usual proposed the name “glucomaltase” for mold maltase, because manner are also presented for comparison. Apparently a comthis enzyme apparently requires a free aldehyde group, such as bination of high temperature for fermentation, low temperature occurs when two glucose units are linked together to form maltfor propagation of the culture, and the use of the improved ose. It does not seem unreasonable to assume that mold maltase medium resulted in a rate of secondary fermentation ( K X loa = can also hydrolyze oligosaccharides containing more than two 9 ) twice as high as the usual rate ( K X 102 = 4.49). Under these glucose units-i.e., dextrin of small molecular size such as optimal conditions, the secondary fermentation was practically that present in the residual dextrin medium. This assumption complete in 24 hours, a reduction of 16 to 20 hours (cf. not only offers a plausible explanation for the correlation between Figure 3). the rate of secondary conversion and the maltase activity of the Finally, corn mash was fermented according to the usual profungal culture, but also agrees with the view of Corman and cedure with 10% by volume of the fungal culture as conversion Langlykke that the glucogenic enzyme system hydrolyzes maltose agent, which had been propagated under the conditions deas well as other glucose polymers. The possibility cannot be excluded that the fungal culture contains an enzyme(s), other than those studied, which is responsible for the secondary conTable VII. Fermentation of Corn Mash under Improved version. Procedures Table VI.
Combined Effectof Temperature and Improved Medium O
SUMMARY
52 0.50 5.25 Barley malt 30 60 Fungal oultured 30 0.48 5.38 42 0.53 6.34 Fungal culture‘ 37 a Temperature during primar fermentation at 30” C. b Taken from curves, total cargohydratee during fermentation va. time. C On the basis of corn as received, whiah contained 9.1% moisture. Data are avera e of 6 fermentations for each conversion agent. 1 Propapatefin 5% dried aolubles and 1.0% oorn medium a t 30° C. Propagated in 2% dried aolublea, 1.0% corn, and 2.0% wheat bran medium at 26’ t o 27’ C.
Submerged fungal cultures, like barley malt, convert as appreciable portion of starch into difficultly hydrolyzable dextrin. It is believed that the slow rate at which this dextrin is hydrolyzed is responsible for the slow rate of secondary fermentation. A number of experimental conditions were found to affect the rate of secondary fermentation, as determined by using a medium containing this dextrin. High temperature for fermentation (37’ C.) and low temperature for mold propagation (25” C.) are two of the salient factors in increasing the rate of secondary fermentation. Use of more efficient mold strains or replacement
September 1950
INDUSTRIAL AND ENGINEERING CHEMISTRY
of 3% distillers’ dried solubles with 2% wheat bran in the propagation medium also shows a beneficial effect. Under the most favorable conditions thus far, the fermentation of corn mash converted with submerged fungal culture can be completed in 42 hours instead of the usual 60 hours. The secondary fermentation was found t o follow the course of a firseorder reaction. There is a better correlation between the velocity constant, K,and the maltase activity of the fungal culture than with a-amylase or limit dextrinase activity. LITERATURE CITED
Adama, S. L., Balankunr, B., Andreasen, A. A., and Stark, W. H., IND.ENQ.CHEM.,39,1615 (1947). Andreasen, A. A., Am. Brewer, 82, No. 4,30 (1949). . Back, T. M., Stark, W. H., and Scalf, R. E., Anal. Chsm.,
20, 56 (1948). (4) Back, T. M., Stark, W. H., and Vernon, C. C., IND. ENQ.CHBM., 40.80 (1948). ( 5 ) Corman, J., and Langlykke, A. F., Catsal Chem., 25, 190 (1948). (6) Foth, G., “Handbuch der Spiritus-Fabrikation,” Berlin, Paul Parey, 1929. (7) H o p k i ~R. , H., Advances in Enwmol., 6, 389 (1946). (8) Hopkina, R. H., Dolby, D. E., and Stopher, E. G., Wallsratain Labe. Cotnmuns., 5, 125 (1942).
1789
Leibowits, J., 2.phyeiol. Chem., 149, 184 (1925). Leibowits, J., and Mechlinski, P., Ibid., 154, 64 (1926). Le Menae, E. H., Corman, J., Van Lanen, J. M., and Lsnglykke, A. F., J . Bad.,54,149 (1947). Le Mense, E. H., Sohna, V. E., Corman, J., Blom, R. H., Van Lanen, J. M., and Langlykke, A. F., IND.ENQ.CHEM., 41, 100 (1949).
Lippa, J. D., Roy, D. K., Andreasen, A. A., and Kolachov, P., Abstracts of Papers, 114th Annual Meeting, AMERICAN CHEMICAL SOCIETY, p. 14A, 1948. Meyer, K. H., Advances in Colloid Soi., 1, 143 (1942). Myrblick, K., A d v a m Curbohydrate Chem., 3,251 (1948). Sandatedt, R. M., Kneen, E.,and Blish, M. J., Cereal Chem., 16,712 (1939).
Schwimmer, S., J . BioZ. Chem., 161,219 (1945). Somogyi, M., Ibid., 160,61 (1945). Stark, I. E., and Somogyi, M., Ibid., 142, 579 (1942). Stark, W. H..Adams. 8. L., Scalf. R. E.. and Kolachov. P.. IND. ENQ.CEEM.,ANAL.ED.,15, &3 (1943). Thorne, C. B., Emeraon, R. L., Olson, W. J., and Peterson, W. H., IND.ENG.CHEM., 37, 1142 (1945). (22) Tsuchiya, H. M., Corman, J., and Koepsell, H. J., Abstraota of Papers, 49th General Meeting, 80oiety of American Bacteriologists, 1949. (23) Vola, G. W., and Caldwell, M.L.,J.BioE. Chem., 171, 667 (1947). REaEZVED
February 23, 1950.
Sterile Air for Industrial Fermentations W. H. STARK Vickers-Vulcan Procam Engineering Company, Ltd., Montreal, Canada C. M. POHLER The Vulcan Copper & Supply Company, Cincinnati, Ohio
Air sterilization is a major problem in many industrial fermentations. When reciprocating compressors are installed and operated as described, sterile air is delivered by the compressor, and the need for the customary carbon or glass wool filters is eliminated. The development and use of this method is discussed as well as the economic factors to be considered in selecting air compreesion and sterilization systems over a wide capacity range.
T
HE
*
increasing w e of aerobic fermentation procesw on a commercial scale for the production of antibiotics, vitamins, enzymes, and other commodities has necessitated the develop ment of systems which will deliver sterile air at a high rate. The initial investment and the operating costs are substantial and warrant careful study. Furthermore, a poorly designed or improperly operated system may result in financial loeses of considerable magnitude. The criteria of the ideal sterile air system may be stated as follows: 1. Complete elimination of all viable microorganisms 2. A high degree of reliability 3. Ease and simplicity of operation 4. Minimum capital and operating costs Filtration through cotton, glasa wool, or carbon, and various scrubbing systems have been used. Glam wool and carbon filters are in extensive use and are perhaps the most popular methods for the production of sterile air in industry. Terjeaen and Cherry (3)reported reaently on the results of a series of careful investigations on the filtration efficiency of glam wool and slag wool. Aside from this report little published information exists, probably because of the rapid expansion of the fermentation industry. It is probable that each organization that has been faced with the problem has worked out its own solution on an empirical basis. This paper describes heat-of-compreasion systems for air
sterilization and presents a brief discussion of some of the economic factors to be considered in the selection of an air sterilization system. The experimental work was done a t Azucarera Cooperati valefayette, Arroyo, Puerto Rico, and at Joseph E. Seagram & Sons, Inc., Louisville, Ky. AIR STERILIZATION WITH HEAT OF COMPRESSION
There have been no previous reports in the literature on the use of heat of compression to sterilize air. The first known commerical installation is one that was made by Langlykke ( I ) at the Lafayette butyl alcohol plant in Puerto Rico. This unit was installed to provide sterile air for fermentor, seed tank, and line cooling. It is illustrated in Figure 1. The installation is straightforward and consists of a water-cooled reciprocating compressor designed to operate at 100 pounds per square inch gage. The water-cooled unit was an old compressor and was replaced after a short period of time with an air-cooled unit. The latter was selected in the interest of obtaining higher discharge temperatures than were possible with the water-cooled unit. The conventional air inlet filter has been replaced with a Winch pipe, approximately 55 feet high, which is packed with chain. A tee with a safety valve and a main line cutoff valve waa installed in the air discharge line as close as practicable to the cylinder head. The valves, discharge line, and the first receiver in the system were insulated to maintain high air temperatures for aa long a time M posaible. This system was operated as follows:
The unit was sterilized with steam at 100 pounds per square inch age for 30 minutes or steam at 15 pounds per square inch age for 2 hours on the downstream side of the main cutoff valve. %he cornpressor waa started and was run for a proximately 30 minutes, with the air discharged through the reIef valve. The heat so generated was relied on to sterilize the compressor and the short pipe aonnection between the cylinder head and the msin valve. As won as the steam was turned off, the main
