EngrnTring
Production of Itaconic Acid by Aspergillus terreus in 204iter Fermentors .
Process development
G E O R G E E. N. N E L S O N , DONALD H. TRAUFLER, S I N A H E. KELLEY,
AND
LEWIS B. L O C K W O O D 1
NORTHERN REGIONAL RESEARCH LABORATORY, U. S. DEPARTMENT OF AGRICULTURE, PEORIA, ILL.
cold water was passed through the jacket to cool the liquors rapidly to 34" C.
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TACOKIC acid production by Aspeigzllus terreus has been investigated for several years at the Northern Regional Research Laboratory, since this process offers a possible new outlet for carbohydrates made available by agriculture. Three papers were published describing the surface culture studies (3-5). In shaker culture studies, it was shown that the nutrient requirements of submerged mycelia are quite different from those of surface growth ( 8 ) . The investigation of submerged cultures has been extended to a semipilot plant scale, and results on the fermentation as conducted in 20-liter fermentors are reported here. Yields of 45 to 54% by weight have been consistently obtained in 4 to 6 days, using 6% initial glucose concentration. The highest yield reported in the shaker studies was 33%.
During fermentation the temperature of the tanks was held a t about 34' C., and the acidity was adjusted to pH 1.8 to 2.0 by addition of alkali or acid as required. The air pressure was maintained a t 15 pounds per square inch. This pressure provides a significant increase in oxygen tension. The increase in pressure is possibly not critical although it has been shonn to accelerate some other fermentations. This factor has not been fully investigated. Glucose was determined by the method of Shaffer and Hartmann (6). The sugar used was a commercial grade of glucose monohydrate, but the calculations were made on the basis of the anhydrous compound. Nitrogen was determined by the microKjeldahl method. Itaconic acid was measured by the bromination method of Friedkin ( 1 ) . Paper chromatography shon ed only traces of acids other than itaconic and sulfuric in the culture liquors. h measure of the growth of the organism was obtained by filtering a 100-ml. sample, oven drying at about 95' C and weighing the mycelium.
APPARATUS AND METHODS
The stock cultures of the organism, A . terreus NRRL 1960, were grown on malt agar slants or in Kolle flasks on the same medium. Spores scraped from about 2 square cm. of the agar (roughly 500,000,000 cells) were used to inoculate 125 ml. of spore germination medium in a 300-ml. Erlenmeyer flask. This medium contained 60 grams of glucose, 5 grams of MgS04.7€€,0, 2.67 grams of ammonium sulfate, and 1.5ml. of corn steep liquor pel liter. The inoculated spore germination cultures were kept on a rotary shaker a t 30" C. for 2 or 3 days. At this age the mycelia consisted of discrete pellets 1 to 2 mm. in diameter. Ordinarily one of these flasks was used to inoculate each fermentor. The fermentors are constructed of No. 316 stainless steel, which is not corroded by the acid a t the low pH (1.8 to 2.0) maintained in this fermentation. Through the detachable cover of each tank extends a shaft carrying a six-vaned agitator, electrically driven by a V-belt. The tanks are surrounded by jackets for steam sterilization and for temperature control with water. Aeration of the culture is provided by compressed air introduced through a porous stone centered beneath the agitator. The temperature is controlled by an electric thermoregulator situated in the cooling water outlet. Samples are drawn off through a gate valve in the bottom of the fermentor, continual agitation by the propeller keeps the mycelium in suspension and ensures homogeneous samples. Air flow is regulated by a needle valve on the exhaust line; a manometer, fitted with a previously calibrated orifice, indicates the amount of exhaust gas escaping. The volume of the fermentation medium is maintained by prehumidifying the air. The humidifier is a tank nearly full of m-ater, with a layer of stainless steel filings in the bottom to disperse the air into fine bubbles. The fermentation media, ordinarily 15 liters in volume, were sterilized by either direct or indirect steam. I n the direct method, steam was passed into the jackets until the medium n a s heated to 100" C.; then steam was injected into the solution through the aerator stones until 15 pounds pressure (120' C.) was reached. In the indirect method, steam was passed into the jackets only. After 30 to 45 minutes a t 15 pounds pressure, the steam was shut off and aeration started. In both methods, 1
Present address, Takarnine Laboratory. Inc., Clifton, N.
J.
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RESULTS OF FERMENTATIOhS
The studies reported here deal with pH control, glucose concentration, nitrogen supply, magnesium sulfate concentration, effect of ferrous iron, rate of aeration, effects of interruption of aeration, size of inoculum, and corn steep liquor concentration. CONTROLOF pH. As in the previous work (a, 3, 6) close control of the pH of the medium was necessary for high acid production. Concentrated nitric acid, which was used in the shaker cultures for pH adjustment, proved unsatisfactory in the 20-liter fermentors. The pH rose again soon after the addition of nitric acid, presumably because of assimilation of the nitrate ion. Such frequent addition of nitric acid was required that the procedure became impractical. Sulfuric acid is evidently not so rapidly metabolized. A single acidification with concentrated sulfuric acid at the start of the fermentation proved sufficient. The use of ammonium sulfate in the medium was also effective in regulating the pH, since assimilation of ammonium ion by the mold released sulfuric acid. This sometimes increased the hydrogen ion concentration to such an extent ( < pH 1.7) as to inhibit the growth of the organism. To readjust the acidity to the desired range-pH 1.8 to 2.0-concentrated potassium hydroxide or ammonium hydroxide was added. GLVCOSE CONCEXTRATION. I n the shaker cultures, the optimal glucose concentration was about 6%. I n view of the potential economic advantage, higher sugar levels were tried, but without success. Not only was the fermentation rate diminished, but the efficiency of conversion of sugar to acid was decreased, Table I gives data from an experiment of this type, the glucose concentrations being about 6, 9, 12, and 16%.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
May 1952
25
TABLE I. FERMENTATION OF G L U C O S AT ~ VARYING
1167
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CONCENTRATIONS
(Other nutrients: 2 grams of MgSO&THpO,2.67 grams of (NHdaSOa, and 3.0 ml. of corn steep liquor per liter; temperature, 34O 3~ 3' C.: air flow l / t e volume per minute; air pressure, 15 pounds per square inch) Av. Fermentation
Itaconic Acid Ac- Glucose Yield cumuConof Initial labed, sumed Aci:, G./L. G./L.' G. Glucose, G./L. At 135 Hours 59 46 59 27.3 47 46 88 21.5 64 38 122 24 52 29 157 14.9 a Based on glucose consumed. Based on glucose supplied.
"2: Peak
Titer, Hr.
Itaconic Rate;--G. Acid Ac- Yield Glucose oumuof Conlated Acid, sumed/ G./L: G.b L./Hr A t Peak Titer 27.3 24.7 49.5 29.2
135 231 326 398
46 28 41 19
0.44 0.33 0.37 0.36
NITROGENSUPPLY. The principal source of nitrogen employed in these cultures was the ammonium ion, usually supplied a8 ammonium sulfate. Routine determinations instituted early in the tank studies showed a rapid utilization of nitrogen. When an initial supply of about 1.6 to 2.6 grams of ammonium sulfate per liter was provided, the fermentation proceeded satisfactorily. When the initial supply was only 0.6 gram per liter, however, practically no acid was formed, as illustrated by curve A , Figure 1. At 189 hours (point X ) 9.1 grams of ammonium nitrate (0.6 gram per liter) were added to the tank and the acid titer began to rise immediately. This shows that a considerable amount of nitrogen must be present from the start to promote high acid yields. Curve B represents a fermentation initially supplied with 2.67 grams per liter of ammonium sulfate; this level of nitrogen gave the best results. In another experiment, urea was added to a culture deficient in nitrogen, but there was no sharp increase in acid titer and the final yield was very low. The other nutrients supplied to these fermentations were 60 grams of glucose, 5.0 grams of MgS04.7Hz0, and 1.5 ml. of corn steep liquor per liter. MAGNESIUM SULFATECONCENTRATION. I n the surface cul-
0.
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80
120
Figure 1.
I X I 160 200 Time, Hours
~-
240
280
Effect of Nitrogen Supply o n Yield of Itaconic Acid
ture work ( 3 ) about 5 grams of MgSOc.7H20 per liter gave the best results, while in shaken flasks the optimum was about 0.75 gram. I n the 20-liter fermentors highest yields were obtained with 5 or 10 grams, thus paralleling the surface culture work. Table I1 shows this rather unexpected result. The slightly greater yield with 10 grams per liter is probably not significant, the difference being within the limits of experimental error. EFFECT OF IRON.I n surface cultures a small amount of iron (2 to 20 mg. per liter) stimulated acid production ( 3 , 6). Above 20 mg. per liter a t pH 2.0, the acid yield decreased ( 3 ) . When the first experiments were conducted in the tanks, accidental contamination with iron from an inlet pipe strongly inhibited growth and acid production. To determine the tolerance limit of the mold to iron under the later experimental conditions, the experiment summarized in Table 111 was set up. Iron was supplied as ferrous ammonium sulfate in amounts equivalent to 15, 30, and 60 mg. of ferrous ion per liter. The table shows increasTABLE 11, EFFECT OF MAGNESIUM SULFATE CONCENTRATION ing toxicity with increasing iron concentration. Very small inocula were found most SIZE OF INOCULUM. (Other nutrients: 2 6 grams of (NH4)zSO4 and 1.5 ml. of corn steep liquor per liter) effective in the shaker flask work ( 2 ) . At first the same proporcMgS04.7HzO Coucn., G./L.tion of inoculum was used in the 20-liter fermentors (approxi1 3 5 10 mately 200 pellets per 15-liter culture). Later, it was found that a larger inoculum (125 ml. or 0.8% by volume) together with Original glucose, grams/liter 63 56 62 57 Glucose at harvest, grams/liter 8 4 1 2 immediate acidification gave improved yields. Increasing the Age at harvest, hours 161 161 95 89 Itaconic acid a t harvest, gramdliter 24 23 31 29 inoculum size still further was ineffective, as the data in Table IV Yield, wt. %" 38 41 50 51 show. The larger inoculum in this case was a 1-liter culture Theoretical yield, % 60 61 70 73 shaken in a Fernbach flask. The other nutrients and the operata Grams itaconic acid Grams glucose supplied ing conditions were the same as in Table 111. Calculated from glucose consumed, assuming 1 mole of glucose yields RATEOF AERATION.It was found that a low rate of air flow 1 mole of itaconic acid. was adequate to maintain the fermentation. Early work indicated that as little as 0.3 liter of air per minute (1/6~ volume) gave TABLE111. EFFECTOF IRON good yields of itaconic acid. The best yields (as high as 54% in (Other ingredients: 2.67 grams of (NH&S04 5 grams of MgS04.7H10 and
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1.5 ml. of corn steep liquor per liter; aeratio; rate, l/ao volume per mihute; air pressure, 15 pounds per square inch: tem erature, 30" to 34O C.; and fermentaJion time, 158gours) --Iron 0
Original glucose, grams/liter Residual glucose, grams/liter Itaconic acidLgrsms/liter Yield wt. % Theoietical yield, % Grams itaconic acid produced.
69
8 23 39 62
Concn., Mg./L.15 30 60 57 21 16 28 62
58 32 11 19 59
55 37 7 13 54
TABLE IV.
COMPARISON OF INOCULA Amount of -Inoculum. Vol. 700.8 6.6
Initial glucose, grams/liter Gluoos? consumed, grams/liter Itaconic acid roduced, grams/liter Yield, wt. % B Age a t completion, hours Mycelial weight, grams/liter Grams acid produced. Grams sugar supplied
61 60 31 51 120 4.1
62 62 28 45 136 7.3
INDUSTRIAL AND ENGINEERING CHEMISTRY
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TABLEV.
EFFECTOF AIR FLOW IKTERRUPTIONS
(Same medium used in both fermentors: the air flow was 5 liters per minute or 1/a volume) Duration of Duration of Air Flow Air Flow Total Stoppage, Total Stoppage, Bcidity' Rlin. Aciditya Jfin. Age, Hr. Fermentor A Fermentor B 17
54 90
50
48 114
..
256 292 326 476 Finished
113 141 191 213 258 hI1. of 1 N KOH per liter.
1
...1.
..
a
..
..
138
20
178
30 15
234 266 308
'i ..
180 184
90
6 days) were obtained with 0.5 liter per minute ('/a0 volume) and this became the standard aeration rate. Increasing the air flow above this figure did not improve the yield or give faster fermentations. It is realized that aeration is affected by agitation. I n most of these experiments the agitation was gentle, with the agitator speed about 100 r.p.m. EFFECTOF INTERRUPTED AERATION. Fermentation often slowed down markedly after addition of alkali or antiform agents such as lard oil or octadecanol. At first this effect was attributed to toxic action of the addendum, but later experiments, summarized in Table V, indicate that it was due to interruption of the air flow. The total acidity listed includes some sulfuric acid; a t 17 hours practically the total titer is sulfuric acid. The interruptions were for varying periods a t the hours listed; thus fermentor B was stopped for 20 minutes a t the end of 54 hours. When the
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fermentation is allowed to proceed without interruption of air flow and without adjustment of the pH, the p H goes down to as low as 1.6 and sugar is no longer consumed. Fermentors should have provision for the addition of materials without stoppage of aeration. CORNSTEEPLIQUORCONCENTRATION. I n the shaker cultures the optimum steep liquor content was found to be 0.15% by volume (a). Results obtained with the stainless steel fermentors followed the same pattern. Tank cultures containing only the steep liquor materials carried over in the inoculum (amounting to approximately 6 mg. of steep solids per liter of the fermentation medium) were exceedingly slow and gave reduced yields. On the other hand, higher concentrations, 0.3 to 1.5% by volume, resulted in heavier mycelia (over 10 grams per liter as compared with 5 grams per liter in the best fermentations) and correspondingly decreased acid production. LITERATURE CITED
(1) Friedkin, M., IND.EXG.CHEY.,AXAL.ED.,17, 637 (1945). (2) Lockwood, L. B., a n d Nelson, (2. E. N.,Arch. Biochem., 10, 365 (1946).
(3) Lockwood, L. B., a n d Reeves, &/I. D., Ibid., 6, 455, 482 (1945). (4) Lockwood, L. B., a n d W a r d , G. E., IND.ENG.CHEX, 37, 405 (1945). (5) Moyer, A. J., a n d Coghill, R. D., A m h . Biochem., 7, 167 (1945). (6) Shaffer, P. A, a n d H a r t m a n n , A. F., J . B i d . Chem., 45, 365 (1921). RECEIVED for review August 11, 1951. AccEPrsD November 8 , 1951 Presented in part before the Division of Agricultural and Food Chemistry a t QOCIETY, Atlantic City, N. J., the 116th Meeting of the AMEHICASCHEACICAL September 1949. Taken in part from a thesis submitted by G. E. K.Nelson to the Department of Chemistry in t h e Graduate Division of Bradley University in candidacy for the degree of Master of Science.
E
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Process JACK W. RIZIKA'
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AND
WARREN M. ROHSENOW
MASSACHUSSETTS INSTITUTE OF T E C H N O L O G Y , C A M B R I D G E , M A S S .
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IK
ORDER to reduce the uncertainty of thermocouple readings the problem of estimating thermal error in thermocouples often arises. A thermocouple can measure only the temperature of its measuring junction, and to do this a great deal of care is required (6). Even though an excellent thermocouple circuit is provided to obtain an accurate measurement of the junction temperature, thermal errors may cause the junction temperature to differ greatly from the temperature of the substance being measured. In research work and industrial applications it is often necessary to know more accurately the temperature of the substance or fluid being measured. In most cases in which it is desired to measure the temperature of a fluid, part of the length of the t,hermocouple is exposed to the fluid, the thermocouple passes through a wall, and most of the thermocouple is in the surrounding environment, usually the atmosphere. Many times the effect of conducting heat down the wire and protecting tube, from the hotter to the colder sections, produces serious differences between the junction temperature and the fluid temperature. The followiiig analysis provides a means of estimating the thermal error in order to obtain a closer evaluation of the fluid temperature. 1
Present addreas, Oallatin C-35, Harvard University, Boston 63, Mass.
Assummom The following assumptions will be made in the analysis: 1. Heat flow and temperature distribution are independent of time, Le., steady state. 2. Thermocouple material is homogeneous and isotropic. 3 . There are no energy sources within the thermocouple itself. 4. Thermal conductivity, k , of the thermocouple system is uniform and constant. 5. The film coefficient of heat transfer, h, is uniform and constant over the surface of the thermocouple system. 6. Temperature of the surrounding fluid is uniform and constant. 7 . Thermocouple thickness is so small compared with its length that temperature gradients normal to the surface may be neglected. 8. Heat transferred through the tip of the thermocouple is negligible com ared with that passing through the sides. 9. The waf between the fluid and environment (atmosphere) is of negligible thickness. Therefore there is no heat exchange between the wall and the thermocouple system. ANA LYSI §
For purposes of the analysis consider the thermocouple system of length, L,shown in Figure 1. The thermocouple system, con-
