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
1776
RESULTS AND DISCUSSION
The data obtained are summarized in Figure 1. The most rapid biosynthesis of Chloromycetin occurs during the time of most rapid mycelial formation, suggesting that Chloromycetin is a product of relatively young cells. The same period is characterized by a rapid decrease in assayable glycerol and ammonia nitrogen. The explanation of the concomitant sharp but transitory increase in reducing substance is not clear, but possibly may reflect a temporary excess of metabolizable material derived from glycerol. The changes in total nitrogen in the filtered beer are reflected in the corresponding changes in mycelial dry weight. Autolysis of mycelium during the last 2 days of incubation doubtless accounts for much of the observed increase in ammonia nitrogen which in turn is presumably responsible for the rise in pH. SUMMARY
1. An exploratory study has been made of chemical changes occurring in the medium during growth of Streptomyces venezuelae in shaken flask cultures.
Vol. 42, No. 9
2. Data on nitrogen and glycerol utilization, reducing substance, mycelial dry weight, pH, and Chloromycetin content are reported. ACKNOWLEDGMENT
The authors are indebted to Gertrude Rodney for analyses for nitrogen and reducing substance, to Margaret Galbraith for Chloromycetin determinations and to Charles Childs for assistance with the glycerol analysis. LITERATURE CITED
(1) Ehrlich, J., Gottlieb, D., Burkholder, P. R., Anderson, L. E., and Pridham, T. G., J . Bad., 56,467 (1946). (2) Hagedorn, H. C., and Jensen, B. N., Biochem. Z.,135, 46 (1923). (3) Ma, T. S., and Zurtzaga, G., IND.ENG.CIIEM.,ANAL.ED., 14, 260 (1942). (4) Smith, R. M., Joslyn, D. A., Gruhzit, 0. M., McLean, I. W,, Jr. Penner, M. A , , Ehrlich, J., J . B a d . , 55, 425 (1946). ( 5 ) Sumner, J. B., J . Bid. Chem., 65, 393 (1925). (6) Voris, L.,Ellis, G., and Maynard, L. A,, Ibid., 133, 491 (1940).
RECEIVED January 6,1950.
Riboflavin by Fermentation with Ashbya gossypii V. F. PFEIFER, F. W. TANNER, JR.1, CHARLES VOJNOVICH, AND D. H. TRAUFLER Northern Regional Research Laboratory, Peoria, Ill.
R
IBOFLAVIN, vitamin B2, may be produced in commercial quantities by biosynthesis when nutritive mediums of proper compositions are subjected to pure culture fermentation by one of several yeastlike organisms. Currently most of the commercial riboflavin produced by aerobic fermentation is probably obtained by biosynthesis with Eremothecium ashbyiz in submerged culture
W).
capacity of 30 liters. They are 39 inches tall, 10 inches in diameter, and each is equipped with a top-entering mixer. The propeller blade is 5 inches from the dishedbottom of the vessel. An air sparger consisting of one Aloxite cylinder (No. 0 porosity) is mounted horizontally 1 inch from the bottom of the vat and 1 inch below the agitator blsde. Air from t,he service line is sterilized by passage through a column packed with glass wool. The inoculum, 1%by volume of a 24-hour culture of Ashbya gossypii NRRL Y-1056,was developed in accordance with the suggestions of Tanner, Vojnovich, and Van Lanen (9). Its production is discussed in detail in the description of the pilot plant work. Liquid inoculum was transferred aseptically to the small tanks from flasks equipped with siphons that were actuated by sterile air. Previous work showed that the complete medium was easily overcooked by batch sterilization in the fermentor with subsequent impairment of riboflavin yields. To minimize this, the corn steep liquor and the animal stick liquor were sterilized separately. After adjusting the pH of a water slurry containing the corn steep liquor and the glucose to 6.5 with sodium hydroxide, it was cooked in the vat fermentors a t 121" C. for 30 minutes by means of direct injection of steam. Cooling of the liquor was accomplished by circulating cold water through the jackets of the tanks while internal pressure was maintained with sterile air. The animal stick liquor, thoroughly dispersed in 3 to 4 volumes of tap water, was autoclaved for 30 minutes a t 121' C., cooled, and
Lperiments were conducted on semipilot plant and pilot plant scales to supply engineering data on the production of riboflavin by submerged culture of Ashbya gossypii. The following factors affecting the yield of riboflavin were investigated: sterilization methods, medium composition, aeration and agitation, fermentation temperature, and strain variation. In satisfactory fermentations, riboflavin yields of 500 to M8-y per ml. were obtained. One 1250-gallon fermentation was conducted, and the fermented liquor was processed to dried concentrate with a 969'' recovery of contained riboflavin. Operating and investment wsts for the process were approximated. Estimates indicate that riboflavin in the form of dried concentrate may be produced commercia!ly by this process at a total plant production w s t as low as 3.75 cents per gram.
Wickerham et al. (10)found that a related species, Ashbya gOSS@i, also produced large quantities of this vitamin when propagated under proper conditions. Tanner, Vojnovich, and Van Lanen (9) investigated most of the important factors influencing the biosynthesis of riboflavin in submerged aerobic cultivation by Ashbya gossypii on a laboratory scale, and established suitable mediums for commercial production employing inexpensive and readily obtainable raw materials. This paper described semipilot plant and pilot plant experiments that were conducted to obtain engineering data on the process and information on which to base approximate cost calculations. PRELIMINARY INVESTIGATIONS
Paralleling the pilot plant studies, experimental fermentations were conducted in two aluminum tanks to determine the effect of medium composition on the production of riboflavin. Each of these jacketed vats has a total capacity of 50 liters and a working 1 Present address, Chas. Pfizer C Company, Brooklyn, N. Y.
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
aseptically added to the fermentors concurrently with the inoculum. The method of analysis for riboflavin is dwcribed in the section that deals with the pilot plant investigations. Table I shows results of fermentations in the small vats. In all experiments reported, the mixer was rotated at the same speed210 r.p.m. With the mediums containing 2% glucose, the concentrations of corn steep liquor and animal stick liquor were increased gradually from 1.0 and 0.5% to 3.0 and LO%, respectively. (All concentrations of corn steep liquor and animal stick liquor mentioned throughout this paper are on an "as is" or wet basis.) The riboflavin yield fluctuated between 150 and 3367 per ml. until concentrations of 1.5% corn steep liquor and 0.8% animal stick liquor were employed, when the yield rose to 6487 per ml. With these concentrations as minima, and 3.0% corn steep liquor and 1.0% animal stick liquor as maxima, riboflavin production varied between 525 and 7 3 6 per ~ ml. With the minimum required concentration of animal stick liquor (0.8'%) and less than 1.5% corn steep liquor, poor yields were obtained, as was the case when 1.5% corn steep liquor was employed with less than 0.75% animal stick liquor. The pilot plant data show that the critical requirements of the two proteins supplement8 vary with the batches of corn steep and animal stick liquor which are employed. Several experiments, unreported, were conducted to ascertain the influence of sugar concentration on riboflavin yield. No improvement in production was observed when the concentration of glucose was increased from 2 to 2.5 and 3%. Tanner, Vojnovich, and Van Lanen (9) found that maximum yields of riboflavin were obtained in laboratory shaker cultures when the medium contained 4% glucose. When the rate of aeration was increased from 0.25 to 0.5 volume per minute per volume of medium, no significant difference in riboflavin yield was noted. PILOT P L A N T INVESTIGATIONS
Equipment. Pilot plant fermentations were conducted in a stainless steel fermentor of 300-gallon capacity and in two copper fermentors, each with a capacity of 800 gallons. The stainless steel fermentor is 13 feet tall with an internal diameter of 24 inches and is equipped with a half jacket. Agitation is provided by a pyopeller-type agitator attached to a horizontal shaft mounted 15 inches above the bottom of the fermentor. The speed of the agitator can be varied between 100 and 250 r.p.m. Temperature of the medium can be maintained within 0.5' F. of the desired point (85" F.) by circulation of water through the jacket. The temperature of the circulating water is automatically controlled. All fermentations in this tank were conducted at a volume of 200 gallons, corresponding to a liquid depth of approximately 9 feet. Sterile air for the fermentor was introduced through a set of seven Aloxite aerator stones or through a perforated pipe-cross sparger. Air was sterilized by passage through a 10-inch inside diameter packed column containing 8 feet of 10- to 24mesh activated carbon. Each copper fermentor is 5 feet tall and 5.5 feet in diameter, and is equipped with internal coils for heating or cooling. Agitation is provided by a top-entering propeller-type agitator rotating at a fixed speed of 100 r.p.m. Fermentation temperature is controlled by water circulating through the internal coils. Most fermentations in these vessels were made at a volume of 300 gallons, corresponding to a liquid depth of approximately 2 feet. Sterile air for each fermentor was supplied through a perforated pipe-cross sparger. Air was sterilized by passing it through a 16-inch inside diameter packed column containing 8 feet of 10- to 24-mesh activated carbon. Method. Medium was sterilized batchwise or continuously. With batch cooking all ingredients of the medium were put into the fermentor and heated to the desired temperature by the direct introduction of steam. After the liquor had been retained at this temperature for a definite time, it was cooled. For continuous
1772
Table I. Effect of Composition of Medium on Riboflavin Yields by A. gossypii in Semipilot Plant Equipment (Rateof aeration for a11 experiments was 0.25 volume air per minute per volume of medium) Medium Composition0 % ~~~i~~~ Animal Corn Riboflavin Fermentation stiok steep Yield, Time liquorb y/Mi. Hour; liquorb 0.5 1.0 300 140 0.6 1.0 292 136 0.5 1.5 216 67 0.5 1.5 182 67 0.5 3.0 156 137 0.5 3.0 336 96 0.8 1.0 308 137 0.8 1.0 331 96 0.8 1.5 648 160 0.8 1.5 536 87 0.8 2.2 640 139 0.8 2.2 728 114 1.0 2.0 525 98 1.0 3.0 736 184 All media contained 2 7 lucose. b "As is" basis. stick &&or and a t e y liquor used contained spprodmately 65and 47% dry substance, respectively.
sterilization and cooling, the complete medium was pumped from a storage tank a t a constant rate to a steam jet heater, where it was mixed with steam and instantaneously heated to the desired temperature. The liquid then passed through a bank of insulated pipe for the required retention time. The sterile medium was led from the cooker to a cooler, which consists of a pipe coil immersed in cold water, and thence to the fermentor. The p H of the medium was adjusted when necessary by the addition of a sterile solution of sodium hydroxide. The liquor was then inoculated with 0.50 to 0.75% by volume of a liquid culture of Ashbya gossypii, and aeration was begun. Determinations of sugar and riboflavin contents and pH of the liquor were made throughout the course of the fermentation. Precautions were taken at all times to maintain pure culture conditions. When contamination did occur, poor yields of riboflavin were obtained invariably. Stock cultures of Ashbya gossypii NRRL Y-1056 were carried on agar slannts. To prepare inoculum for pilot plant fermentstions, a loop of culture was transferred to a sterile slant and maintained at 30' C. for 24 hours. A loop of this young culture was transferred to 100 ml. of liquid medium in a 5Wml. flask. After incubation at 26' to 30' C. on a reciprocating shaker for approximately 24 hours, the contents of the flask were used to inoculate 6 liters of sterile medium in a 9-liter glass bottle. This culture was aerated by sterile air from a perforated tube a t the bottom of the bottle. It was cultured for 24 hours and then transferred to 200 or 300 gallons of Sterilized medium in a pilot plant fermentor. This quantity of culture corresponds to 0.75 and 0.5% of inoculum, respectively. Compositions of the media used in culturing inoculum are a9 follows: 500-rnl.shaker flasks
RIP" ta
pR (before sterilization)
Water to make, ml.
6.8 100
Sterilization: 15 minute? at 121' C. (15pounds per square inoh gage)
..
Sterilization: 30 minutes at 121' C. (15 pounds per square inch gage)
9-liter bottles Oluaose, % Corn steep liquor % Animal stiok liqu&r % bH (before sterilixktion) ater to make
2 0 1 .o 0.5 6.5 6 liters
Steriliaation: 45 minutes at 121' C. (15 pounds per square inch gage)
1778
INDUSTRIAL A N D ENGINEERING CHEMISTRY
Tabla 11.
NO. 17 18 84 41 41 40
COBC,
Corn Steep Liquor',
7%
Animal Stick Li uora,
%,
Tank
%
88' 88
2.0 1.9 2.1
1.9 1.9 1.9
I:! 1 .o
3 4
1.8 1.9 2.2
1.9 1.9 1.9
1.0
1/2
1.0 1.6
1/2 1/4
ss
SS 3 4
43 43 42 75
88 3
1.9 2.0 1.9 2.6
1.9 1.9 1.9 1.9
77
3
2.5
1.Q
Air*
Sterilization method Batchwiae, 250° F., 45 rnin 6 5 p H B a t c ~ w i k250' F..25 rnin., 4.4 p H Continuous, 275' F.,5 min., 6.5 p H Continuous 275O F.,5 rnin., 6.5bH Continuous, 275' F.,9 min.. 4.5 DH.all together Continuous, 275O F., 5 min., 4.5 p H , all toeethsr ~. ~ ~ . . '
64
ss
9.1
1.9
1.0
1/2
44
88
2.2
1.9
1.0
1/2
0
b
alters unknown constituents of the nutrients. When the medium is sterilized batchwise in Maximum the fermentor, it i s exposed to Riboflavin Yield, comparatively high temperRemarks 7/M1. atures for long periods, since 0 heating and cooling of the enVery ala;' 5 Very slow 88 tire batch are slow processea. In the equipment employed for 312 Stick sterilised sop408 these experiments, 200 gallons arstely 356 of liquor could be batch-sterilized in about 120 minutes, 152 Stick sterilized asp. 124 whereas by the continuous arately at 300° 192 method the medium was ... 704 heated, retained, and cooled in about 7 minutes. Since the re= ... 704 action appears to be associated with the nutrients, it might be 608 Stick sterilized 8ep656 possible to lessen the heat-sensiarately tivityof themedium by suitable alteration of its composition. Medium composition. The fermentation mediums used in these experiments were composed of commercial corn sugar, corn steep liquor, and packing house or animd stick liquor, with 0.1 to 0.2% soybean oil added aa an antifoam agent. Thew materials are readily obtainable in Iarge quantities and are relstively inexpensive. The composition of the corn steep and animal stick liquors used in these experiments is given in Table 111.
Effect of Sterilization Methods on Riboflavin Yields by A. gosoypii in Pilot Plant Equipment Glu-
Run
Continuous, 275' F.,5 min., 4.5 p H
"As is" basi?. Volume of Lur per minute per volume of medium. Stsinless stlel.
Riboflavin in the fermented liquor was determined by hydrolyzing a sample in the autoclave for 30 minutes at 15 pounds per square inch gage at a pH of 4.5 to 5.0 maintained by acetic acidsodium acetate buffer. The hydrolyzed material was subjected to fluorometric analysis on a Coleman Model 12 electronic photofluorometer. The & content of the unknown waa calculated by using a riboflavin solution prepared from U.S.P.grade riboflavin 89 a comparison standard.
Table IV. Effect of Medium Composition on Riboflavin Yields by A . gossypii in Pilot Plant Equipment
RESULTS
The following factors affecting the yield of riboflavin were investigated on a pilot plant scale: sterilization methods, medium composition, aeration and agitation, fermentation temperature, and strain variation. Sterilization methods. The following methods of medium sterilization were studied: batch cooking a t high and low pH; continuous Sterilization at high and low pH; and continuous sterilization at low pH, with separate sterilization of a portion of the nutrients to avoid undesirable side reactions. Typical remlts are given in Table 11. In most cases negligible amounts of riboflavin were produced when the medium with a p H of 6.0 to 6.5 was sterilized batohwise for 25 to 60 minutes at 250' F. Continuous sterilization at 275' F. for 5 minutes at a pH of 6.5 resulted in improved riboflavin yields, but the most consistent and mtisfactory results were obtained when the pH of the medium was 4.5, and continuous sterilization was conducted a t 275' F. with a retention time of 5 minutes. Continuous sterilization for 9 minutes a t 275" F. was also satisfactory, but sterilization for 5 minutes a t 300' F. wm uneatisfactory. Riboflavin yields were approximately equal whether the stick liquor was sterilized separately or with the other nutrients. It is believed that poor yields of riboflavin result when the medium is batch-sterilized because excessive heating destroys or Tabla 111.
Composition of Nutrients
Item Corn steep liquor A
B C D
Val. 42, No. 9
E F Packing house stick liquor, ev. compn.
Solids, %
Nitrogen, %
43.0 34.8 45.0 41.0 49.6 52.6
3.50 2.50 3.75 4.20 4.30
64.3
8.44
3.15
Run No. 59 59 51 51 47 47 55 55 53 53 51 51 54 52 44 50 48 57 57 64 66 66
67 67 62 64 62 60 60 73 75 77 69 79 71 86 90 88
89 89 87 87 94 92 95 97
Tank 4 3 3 4 3 4
a
4 3 4 3 4
ssc 88 ss
88 9s 3 4 3 3 4 3 4 4 4 3 3 4 3 3 3 3 3 3
ss
9s 88 3 4 3
4
ss
88 98 88 a "Asis" basis. b
Glucose,
Corn Steep Liquoro,
2.0 2.0 2.0 2.1 2.1 2.2 1.1 1.4 1.6 1.8 2.0 2.1 2.1 2.1 2.2 2.6 3.3 2.5 2.6 2.0 2.0 2.0 1.9 1.9 2.5 2.4 2.5 2.5 2.5 2.4 2.6 2.5 2.6 2.5 2.7 2.0 2.6 3.5 2.5 2.5 2.9 3.2 2.0 2.0 2.0 2.1
0.6 1.2 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 1.9 2.5 3.1 1.9 1.9 2.5 2.5 3.1 2.0 2.5 2.5 3.1 3.9 1.9 1.9 1.9 2.5 2.5 3.1 1.9 3.0 4.0 2.5 2.5 2.7 3.0 1.6 1.9 2.2 2.5
%
%
Animal Stick
Liquora,
%
0.3 0.6 0.9 0.9 0.6 0.6 0.9 0.9 0.9 0.9 0.9 0.9 1.0 1.0 1.0 1.0 1.0 1.2 1.6 0.9 1.2 0.9 1.2 1.6 1 .o 1.2 1.2 1.2 1.6 1.2 1.6 1.9 1.2 1.6 1.2 1.0 1.6 2.0 1.6 2.0 1.7 2.0 1.0 1.0 1.0 1.0
Volume of sir per minute per volume of medium. Shinlcss steel.
Steep A A A A
A
-4
A
i
A A A A A A A A
A A B B B B B B B B B B C C
c
C C C
D
D D D D D
D E
E E E
ivlaxi mu m Riboflavin Yield ~/ivlI;
140 260 512 552 500 530 260 420 536 520 512 552 608 568 656 576 568 848 436 368 448 520 552 336 360 418 552 336 160 560 704 704 560 680
136 292 272 240 408 496 320 432 240 416 576 448
1779
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
September 1950
Corn steep liquors vlrrled widely in their utility as medium supplements for riboflavin production, and the optimum 1 3 0 eoamount of corn steep liquor to be used in a fermentation was dependent on the bl particular batch of material used. All batches of corn steep liquor tested could be used to produce high yields of riboflavin after the optimum concentration had been experimentally determined. The optimum amount of corn steep liquor could not be correlated with its content of solids or nitrogen but had to be determined by conducting fermentations with each batch of material. 100 30Changes in pH, sugar concentration, and riboflavin content of a typical fermentation (No. 54) are shown in Figure FERMENTATION TIME, HOURS 1. Fermentation time is usually 96 to Figure 1. Changes in Medium during Fermentation 120hours. Aeration and Agitation. A series of experiments was conducted in which aeration was varied from 0.125 to 1 volume of air per minute per Amounts of each constituent were varied in an effort to find volume of fermentation medium and in which agitation was the optimum proportions for highest riboflavin yield. Results are varied by changing the speed of the agitator. Results of these exsummarized in Table IV. Continuous steriltation of the meperiments are given in Table V. dium at 275" F. and pH of 4.5 with a holding time of 5 minutes was used in each fermentation. As already noted, the liquid level in the stainless steel vessel was approximately 9 feet above the bottom in the standard 200To obtain satisfactory riboflavin yields it was necessary to maintain the concentrations of corn steep liquor and packing gallon fermentation, whereas the liquid level in each copper tank house stick liquor within narrow limits, depending on the sugar was approximately 2 feet above the bottom in the standard 300gallon fermentation. The efficiency of aeration should be better concentration employed. With a sugar content of 2.0% the medium yielded 500 to 6OOy per ml. when optimum amounta of in the stainless steel fermentor because of the greater retention corn steep liquor and animal stick liquor were used, whereaa a time of the air bubbles, and yields obtained in this vessel were medium with a sugar concentration of 2.5% and optimum noticeably higher than those procured in the copper tanks, when fermentations were conducted under similar conditions, amounts of corn steep and animal stick liquors yielded 650-700 y per ml. The highest yield in any fermentation waa 848 7 per ml. The highest riboflavin yields were obtained in experiments using When the concentrations of corn steep liquor and animal stick 0.25 volume of air and an agitator speed of 100 r.p.m. When a higher rate of aeration was employed, the fermentation time and liquor were kept constant and the sugar content of the mediums yield were both reduced. was increased, the riboflavin yield was enhanced until the optimum sugar concentration was reached, after which little change occurred. It was found Table V. Etfect of Aeration and Agitation on Riboflavin Yieldr by A. goaaypii that 2.0 to 2.5% glucose waa in Pilot Plant Equipment ample fortheproduction of high Corn Animal M.aximurn yields of riboflavin. A itator OluSteep Stick Riboflavin 8 eed Liquor", Liquor", Yield, oose, Run When the concentrations of % % Airb R.%.M: Remarks No. Tank ?/MI. % sugar and animal stick liquor 100 ..... 464 35 3 1.9 were kept constant and the corn 100 2.0 ..... 512 51 3 100 51 4 2.1 ..... 652 steep liquor concentration was 100 ..... 568 39 4 2.0 2.1 100 49 3 ..... 432 varied, the riboflavin yield in100 2.1 4 ..... 464 49 100 creased until the optimum con2.0 472 39 3 100 2.0 4 Faat fermentahon 376 35 centration was reached, after 1.9 1.0 1/4 100 70 880 2.0 which the yield decreased with 100 ..... 1.9 1.0 1/4 2.3 88 81 100 ..... 2.1 1.9 52 88 increase in the content of corn 100 1.9 ..... 2.1 88 54 100 steep liquor. For example, in 88 1.9 25 lb. pressure 648 2.1 1.0 1/4 76 25 lb: pressure, slow fermen600 100 1.9 2.1 88 1.0 1/8 74 experiments 94, 92, 95, and 97 tation 0 2.1 1.9 Very slow 320 88 1.0 1/4 a3 the concentrationsof corn steep 100 1.9 2.3 88 ..... 580 1.0 1/4 81 100 624 liquor were 1.6, 1.9, 2.2, and 1.9 2.0 88 1.0 1/4 70 250 1.9 Fast fermentaiiin 336 2.0 63 88 1.0 1/4 2.5%, respectively, and, cor100 2.2 656 44 88 100 2.1 ..... 568 respondingly, the riboflavin 88 52 100 2.1 608 ss 54 yields were 240, 416, 576, and 250 2.0 88 Fast fermenta&n 276 56 250 2.1 Fast fermentation 400 88 58 4487 per ml. This illustrates 250 Fast fermentation 880 1.8 88 61 the gradual increase in ribo..... 2.2 100 676 88 2.0 95 560 0 ..... 2.0 2.2 ss 102 flavin productionwith increased 0 ..... 2.1 2.2 1.0 3/4 536 88 100 content of corn steep liquor in 100 Strain NRRL Y-1056-N1 631 88 2.0 105 0 NRRL Strain Y-1056-N1 686 2.1 88 104 the medium, as well as the dea "As is" basis: crease in yield which occurs b Volumes of am per minute per volume of medium. when excessive amounts of e Stainless steel. corn steep liquor are employed. 90
I
I
I
I
I
I
I
I
I
I
I
g
-
f
;:8
.
.
.
32"
.
:o
.
%
I
.
.
.
INDUSTRIAL AND ENGINEERING CHEMISTRY
1780 Corn
Animal
Vol. 42, No. 9
Laboratory Culture ArAbyo gorsypii Inoculum Tank
Evaparofot Sulfuric Acid
Air Filter
Air Compressor
7 Figure 2.
le Drum Drier
v Riboflorin Concentrate
Flow Sheet for Riboflavin Production by Fermentation
When the agitator in the stainless steel fermentor was rotated
natural variants was chosen m superior to the original strain and was tested in a series of pilot plant fermentations. Results are given in Table VI. This particular strain, designated NRRL Y-1056.N1, gave consistently higher yields of riboflavin than did extent that the normal production of riboflavin waa disturbed. the standard NRRL Y-1056. The isolation of this strain, with Without mechanical agitation, more air waa required for a yield other findings of the investigations on variability and mutation, equivalent to that obtained with mechanical agitation. will be published elsewhere. The results indicate the desirability Fermentations were made in the copper fermentors at total of isolating high-yielding strains a t regular intervals in order to maintain riboflavin yields at the highest possible values. volumes of 300 and 500 gallons to determine the effect of fermentation volume on riboflavin yield. These volumea corresponded Semicommercial Fermentation.' One fermentation was conto liquid depths of approximately 2 feet and 3.25 feet, respecducted in a 4000-gallon steel fermentor 8 feet high and 8.5 feet in diameter, with conical top and bottom, and equipped with a tively. Mediums used contained 2.5% sugar, 2.5% corn steep liquor, and 1.5% animal stick liquor. Each fermentation was perforated pipe sparger for distribution of sterile air. Air wtts sterilized by passing it through a 16-inch inside diameter packed conducted with mechanical agitation and 0.25 volume of air. Riboflavin yield a t the 30@gdlon volume was 6807 per ml., and column containing 8 feet of 10- to 24mesh activated carbon, Temperature of the fermentation was controlled by water circuthe yield at the 5OO-gallon volume was 7447 per ml. This may be due to more effective aeration and agitation and indicat>esthat lated through internal coils. Fermentation volume w&s 1250 fermentations are more satisfactory when carried out with high gallons, which corresponded to a liquid depth of approximstely liquid depths. As already noted, yields in the stainless steel 3.5 feet. The fermentor was not provided with an agitator. fermentor with a liquid depth of 9 feet were always noticeably Themedium a t a pH of 4.4 was sterilized continuously a t 275' F. higher than yields in the copper tanks with liquid depth of 2 feet. with a retention time of 6 minutes. Before inoculation it conFermentation Temperature. Fermentations were made at tained 2.1% glucose, 2.2% corn steep liquor (Steep E), and 1.0% 85" and 95' F. to determine the effect of incubation temperature animal stick liquor. After adjusting the pH to 6.25 with sterile sodium hydroxide solution, the temperature of the medium waa on riboflavin yield. Sugar utilimtion was much faster in the brought to 83' F., 1% inoculum was added, and aeration was 95' F. fermentation, but riboflavin production was lowered begun using 0.375 volume during the first 24 hours of the ferdrastically. Strain Variation. Paralleling the pilot plant fermentations, a mentation, and 0.66 to 0.75 volume during the remaining 88 special study was undertaken in the laboratory by T. G. Pridham hours, Although it was realized that this was insufficient air to give a satisfactory riboflavin yield in a tank of this type, not of the fermentation division to obtain and test natural and induced variants of Ashbya gosaypii NRRL Y-1056 for riboflavin equipped with a mechanical agitator, it waa aa much as could be production. On the basis of shaker-flask experiments, one of the conveniently supplied, Temperature of the fermentor dropped to 65' F. for the 19-hour period (71-90 hours). In spite-of inadequate aeration, no agitation, Table VI. Effect of Strain Variation on Riboflavin Yield. by A . goraypii in Pilot low temperature, and low depth Plant Equipment Corn Animal MaQmum of liquid, a riboflavin yield of GluSteep Stick Riboflavin 2647 per mi. waa obtained. Run cow. Liquor', Liquor', Yield The fermented liquor waa % % % Airs R.P.M: Strain Steep r/MI: No. Tank 108 SSC 2.0 1.9 1.0 1/4 100 Y-1056 F 496 concentrated to a sirup con106 SS 2.0 1.9 1.0 1/4 100 Y-1056.N1 F 608 taining 27% solids and dried on 95 ss 2.0 2.2 100 Y-1056 E 576 SS 2.0 2.2 105 100 Y-1056.Nl E 632 a double-drum dryer operated 102 ss 2.0 2.2 1.0 1/2 0 Y-1056 E 560 with steam at 100 pounds per 104 SS 2.1 2.2 1.0 1/2 0 Y-1066. N1 E 688 '"As is" basis. square inch gage. Dried ribob Volumes of air per minute per volume of medium. flavin concentrate of the follow8 Stainless steel. ing analysia waa produced:
at 250 r.p.m. riboflavin was produced rapidly but with a lower yield than was obtained in the standard fermentation at 100 r.p.m. Possibly agitation interfered with mycelial growth to an
"E?
::: %
INDUSTRIAL AND ENGINEERING CHEMISTRY
Septsmber 1950 Nitrogen Ash Carbohydratas Fat. Mourture Riboflavin
Per Cent 8.40 19.84 8 -69 (as glucose) 0.44 2.82 1.17 (96% recovery)
A portion of the sirup was passed through a spray dryer and yielded a product of similar composition. However, it was lighter in color, lower in bulk density, and caked considerably when storedbfor 3 months, although the moisture content of the material was only 6%. This is in contrast to the drum-dried material which showed no tendency to cake during storage. DISCUSSION AND COST ESTIMATES
Analysis of the experimental results indicates that satisfactory yields of riboflavin can be obtained if fermentation variables are closely controlled. The medium should contain 2.0% glucose, 1.8 to 2.1% suitable corn steep liquor, 1.0%animal stick liquor, and a small amount of some antifoam agent. Steriliation of the medium a t pH 4.6 should be carried out continuously at 276' F. with a retention time of 5 minutes. Seed culturea should be brought up aseptically on the same type medium, and 0.6 to 1% employed for inoculation of the fermentors. Mechanical agitation should be used to keep the medium well mixed but not so strongly agitated aa to interfere with mycelial growth. Sterile air must be dispersed in the medium. Approximately 0.25 volume of air per minute per volume of fermentation medium is a satisfactory rate of aeration. After fermentation for 96 to 120 hours at 83' to 85' F., the riboflavin content of the liquor should be 500 to 6007 per ml. The fermented liquor may be evaporated to sirup in conventional evaporators and dried to a concentrate containing 2.6% riboflavin on steam-heated drum dryers. Further experiments to increase the yield of riboflavin will be conducted on a pilot plant scale to determine the effect of inocu-
1781
lum quantity and age, corn steep liquor from various sources, and use of other nutrienta and experimental teohniquea. A flow sheet for estimating production costa of the process employing conditions described here is shown in Figure 2. Preliminary cost calculations based on an annual production of 9000 kg. of riboflavin in the form of dried concentrate indicate a total plant production cost of 3.75 cents per gram. It is estimated that thii figure may be as high 88 6.6 to 7 cents per gram in a small plant producing 850 kg. per year. Calculated investment costs for the plants involved vary from $150,000 for the small plant to $400,000 for the large plant, exclusive of steam plant costs. Cultures employed in this investigation may be obtained from the culture collection of the Northern Regional Research Laboratory. I n undertakiig studies with these organisms, investigators should keep in mind that they represent pathogens isolated originally from developing cotton bolls. Hence, a11 cultures m d f e mentation residues should be sterilized before disposal. LITERATURE CITED (1) Deseive, E., Die Milohroisaenschqft, 3, 141-8 (1947). (2) Foster, J. W., British Patent 593,027 (1947). (3) Guilliermond, A., Fontaine, M., and Raffy, A,, Compt. rend., 201,1077-80 (1935). (4) Mayer, R. L., French Patent 913,165 (1946). (5) Pfiaer, Chas. & Co., Inc., British Patent 693,953 (Oot. 30, 1948). (6) Piersma, H. D., U. S. Patent 2,400,710 (1946). (7) Rudert, F. J., Ibid., 2,374,503 (1945). (8) Schopfer, W. H., Eleb. Chim. Acta, 27, 1017-32 (1944). (9) Tanner, F. W., Jr., Vojnovich, Charles, and Van Lanen, J. M., J . Bact., 58, 737-45 (1949). (10) Wickerhsm, L. J., Flickinger, M. H., and Johnaton, R. M., Arch. Biochsm., 9 , 9 5 4 (1946).
RECEIVED September 28, 1949. Report of a etudv nude under the Researoh and Marketing Act of 1948. Bureau of Agricultural and Industrial Chemistry, U. 5. Department of Agriculture.
Ethyl Alcohol from Cassava C. TEIXEIFW, A. A. ANDREASEN, AND PAUL KOLACHOV Joaeph E. Seagram dt Son., Inc., Lauiauille, Ky.
C
ASSAVA is a p l a t Cassava is a plant native to Brazil and other South American countries, but it has native to Brazil and been introduced into many other tropical regions. The plant offers an inexpensivq o t h e r S o u t h American source of starch for the production of alcohol but the lack of a suitable saccharifying countries, but it has been agent has been the main reason for its not being exploited more fully for this purpose. With acid hydrolysis, alcohol yields of 43 to 74% of the theoretical have been reported; c u l t i v a t e d rather exteneively in the weatem parts conversion with barley malt has resulted in yields of 70 to 74% of the theoretical, and of tropical Africa and India, equivalent amounts of corn malt have produced lower yields. When mold bran preparathe Malay Archipelago, tions have been used for conversion, yields of 80 to 85% of the theoretical have been o b Java, and the Philippines. tained. Results reported in this paper show it is possible to obtain plant efficienciea The plant belongs to the of 90% from cassava mashes converted with submerged fungal cultures. family Euphorbiaceae and the main species i s Manihd utilissimo. Pohl, with many varieties. roots per acre and after 2 years from 7 to 10 tons. It is usually more economical to harvest the roots after the-plants have been The cultivated varieties are herbaceous shrubs, 3 to 9 feet high. A cluster of tapering, cylindrical roots, which may attain a length allowed to grow for 2 years. up to 3 feet and a diameter of 6 to 9 inches, is produced at the Cassava starch probably is best known as tapioca or arrowroot base of the stalk (Figure 1); starch is stored in these roots. Both starch. The roots contain 26 to 32% starch and the cleaned sweet and bitter varieties are cultivated in Brazil; the root eap roots are proceased for storage by cutting them into chips which of the bitter variety contains hydrocyanic acid but the acid ia are dried. The dried chips contain approximately 75% starch dispelled when the roots are cooked. and 7% moisture, Caasava roots offer an inexpensive source of starch which has a competitive advantage over starch from other The yield of cwava, in Brazil, varies according to the length of time the plant is allowed to grow before the roots are hanreested. sources. During recent years the cultivation of cassava in & a i l haa After 1 year of growth the plants will yield from 3 to 5 tons of been and Over 9J000,000 tons were produced in 1 Present addreu, Secretaria Da AgriouItura Institub Agronomioo, Camhaving a value of $115,000,000. The two main factors which pinas-BrPsil. "**J
