In this pilot plant new process control' applications in fermentation were studied. The following article describes them in detail
I
GEORGE J. FULD and CECIL G. DUNN Department of Food Technology, Massachusetts institute of Technology, Cambridge 39, Mass.
N e w Process Control Applications in Fermentation A P P = I c x T m x s of automatic control to fermentation processes have been limited mainly to relatively simple problems. Temperature, air flow input, back-pressure, and foaming in the fermentor can be regulated ( 6 ) . Automatic pH control has been widely used in the laboratory with fairly good success (2, 7, 8), but on a plant scale has been limited by the lack of dependable, steam-sterilizable pH electrodes until recently (9). A continuous sugar control system for baker's yeast propagation would have advantages, especially if it gave higher yields and better reproducibility in a batch process. For continuous fermentation automatic sugar control is most desirable.
When it is desired to control any variable automatically, that variable, or some directly related variable, must be measured continuously. Generally the design of a suitable instrument for this measurement is one of the most difficult features of any control problem. The possible use of several devices, such as optical rotation and specific gravity, to measure sugar concentration has been discussed ( 4 ) . Continuous infrared spectroscopic analysis might be applied. Measurement of absorption of sugar in a water solution is difficult, because of considerable interference by the water absorption itself. If a method can be developed for measuring infrared spectra of aqueous solutions or for continuous
quantitative extraction of sugar into a suitable solvent, infrared absorption analysis may have possibilities in sugar control. Another device has recently become available that might be adaptable to sugar control. The Autoanalyzer (Technicon) can measure reducing sugars continuously. The only other device commercially available that may possibly be used for sugar control is continuous refractive in. dex measurement. The errors inherent in the use of such measurements for this purpose have been investigated and are described here. A method of continuous compensation for these errors is proposed. VOL. 49, NO. 8
AUGUST 1957
1215
50-gallon pilot plant fermentor provides automatic control o f temperature, antifoam, and pH
+C
Equipment and Procedure The 50-gallon pilot plant fermentor used for these studies (5, 3E, 4E, SE, 9E-78E) has provision for automatic temperature control, automatic antifoam control, automatic pH control, and a supply of sterile air. Where possible, all controls and valves are mounted on a central panel for ready accessibility. All parts of the equipment which come into contact with microorganisms are constructed of stainless steel except in the sugar control system. Aseptic conditions are maintained, except in the sugar control system where the high cost does not warrant the use of stainless steel. -4continuous control refractometer is used for the sugar control system (7E,2E). A bypass line from the pH control circulating system supplies mash to the refractometer. Preliminary experiments showed that a cell-free stream was necessary to obtain an accurate reading with the refractometer. A continuous laboratory centrifuge provides this stream for measurements. The refractometer optical system is shown in Figure 1. A light beam is focused and collimated so that it passes through a hollow sample cell. The measured stream passes continuously through the sample cell. A variable prism, controlled by a small servo-motor, corrects the slit through which the beam passes before being focused, so that it is centered in the field of a photomultiplier tube. A revolving polarizer blacks out alternate sides of the field, seen by the photomultiplier 1800 times a minute. When intensity in the field becomes un-
source
photomultiplier I
+
I I
Figure 1 . Optical control system of Bausch & Lomb control refractometer
I
n
ground glass
condenser
thin reflector
VIEWING LENS SYSTEM SIDE ViEW AT @
collective lens
coliective
fixed polo r o i d Ly-----J
eye p i e c e
c o l,i i m a t i n g objective
7 -
objective
field appearance
1
1-
I
rotating p a la r o i d
I amici compensator hollow p r i s m
liquid
INDUSTRIAL AND ENGINEERING CHEMISTRY
variable Prism
range shift prism
telescope obi ec t ive
i l
first surface mirror
121 6
I +;t;eq'l:ective
surfoce mirror
FERMENTATION EQUIPMENT A N D DESIGN balanced, the tube causes an amplifier to drive a small motor which shifts the variable prism, correcting the slit refractive index of the flowing sample. The position of the variable prism is measured both mechanically (to give a visual indication of the index) and electrically through a Wheatstone bridge circuit which supplies the input to a recorder-controller. The actual sugar control system is shown schematically in Figure 2. The stream of yeast is passed through a continuous centrifuge (Westphalia LWA205, 5E) from the p H circulating system (from valve 171). The centrifuge is arranged with two 0.5-mm. underflow nozzles, with the peripheral nozzles blanked off. If smaller nozzles, or the peripheral nozzles, are used, the bowl becomes clogged with yeast during the fermentation. The stream containing the yeast is passed into a 2-liter aspirator bottle and pumped directly back to the fermentor by a small centrifugal pump, PU-3, SE. The overflow stream (cell-free) is also delivered to a 2-liter aspirator bottle, pumped through the refractometer by another centrifugal pump, PU-2, SE, and returned directly to the fermentor. The stream coming from the centrifuge is reasonably free of air bubbles. As the centrifugal pump keeps the liquid level low in the holdup bottle, it pulls in considerable amounts of air that become entrained in the circulating stream, causing errors with respect to the refractometer. To prevent these errors, a large test tube is used as an air trap; it must be periodically purged of the accumulated air. The presence of the air trap is undesirable, as it causes a definite holdup in the circulating stream and a changing input to the trap requires several minutes for detection in the output stream from the trap. The air trap can be elimi-
Table 1.
Semisynthetic Yeast Propagation Medium (Initial volume, 150 liters) Medium, Grams/Liter
Sugar (commercial glucose) Ammonium phosphate, monobasic Potassium phosphate, monobasic Magnesium sulfate, anhydrous Sodium citrate L-Asparagine Yeast extract (Difco) d-Biotin Calcium pantothenate Inositol Thiamine-HC1 Pyridoxine-HC1 Zinc sulfate heptahydrate Ferrous ammonium sulfate Cupric sulfate pentahydrate Antifoam (GE Antifoam 60, 30% solids)
Variable 0.93 0.19 0.11 0.67 0.17 0.17 0.000013 0.00033 0.0067 0.0027 0.00067 0.00027 0.00010 0.000017 0.06
TO FERMENTOR
I
I
-1 REFRACTOMETER I 1
IR-l
1
4
SUGAR TO FERMENTOR
pH CIR. SYSTEM
I
2 PU-I
Bottles
Figure 2.
Sugar control system
nated, if the liquid level in the aspirator bottle is maintained at a slightly higher level. Piping for conducting the streams in the system was constructed of Tygon tubing; thus aseptic conditions could not be maintained. The construction of a stainless steel system with positive displacement pumps would enable the system to be run aseptically. The refractive index of the stream is recorded by the recorder-controller, RC-2,
ZE,and a pneumatic signal is used for control. If the refractive index falls below the set point, the positioner, POS-4, 16E,opens the diaphragm valve, DRV-1, 7E, and meters in sugar solution as required from the reservoir, R-1, 7E. The reservoir may be sterilized by steam supplied through valve 128; sugar may be added manually by bypass valves 126,173, and 174. A short section of borosilicate glass pipe serves as a drip indicator.
Table II. Determination of Components (Baker's yeast propagation 150 liters in synthetic medium, run 86) Total Yeast Sugar Yield, (as Nitrogen Volume Time, Glucose), Phosphorus, (as "a), Alcohol, yo of Sample Hr. % % % Wt. Yo Total 1 24.30" 0.1075 0.447 2 0 0.750 0.0215 0.118 0.009 3 0.08 0.760 0.0232 0.118 0.007 20.0 4 1.00 0.760 0.0242 0.099 0.023 20.0 5 2.00 0.740 0.0215 0.111 0.053 25.0 6 3.00 0.700 0.0232 0.122 0.106 37.5 7 4.00 0.573 0.0268 0.136 0.213 50.0 8 5.05 0.450 0.0242 0.124 0.356 62.5 75.0 9 6.00 0.395 0.0275 0.151 0.477 10 7.00 0.280 0.0283 0.170 0.427 87.5 11 8.42 0.280 0.0300 0.167 0.680 100 12 9.00 0.117 0.0275 0.194 0.802 100 13 10.00 0.025 0.0282 0.218 0.725 100 14 11.00 0.026 0.0280 0.218 0.675 100 15 20.00 0.008 0.0282 0.199 0.705 100 a Concentrated sugar.
...
...
VOL. 49, NO. 8
...
...
Sugar Added, % of Total
...
25.2 25.2 25.2 26.2 29.3 44.5 48.3 67.6 78.2 97.2 97.5 100.0 (95.6) (87.5)
AUGUST 1957
1217
Figure 3.
Refractometer control curve for run 86
Experiments with media which contained molasses that had not been decolorized indicated that control of sugar regulation was lost during the later stages of fermentation. after approximately 37, molasses had been added to the system ( 3 ) . The molasses utilized for this work was a 50-50 mixture of blackstrap and beet molasses, “heat broken” but not decolorized. Therefore. several experiments were conducted with a semisynthetic medium that was virtually colorless (Table I). The medium which was sterilized before inoculation was similar to that described by Olson and Johnson (70); it contained hydrolyzed cornstarch (commercial glucose, Corn Products Refining Co.) as the sugar source and 0.017% of yeast extract (Difco). Observations
A typical experiment was conducted using 150 liters of medium, with 6 ounces of active dry yeast (Fleischmann) as the inoculum and an aeration rate of about 11.5 standard cubic feet per minute. Air was mixed with USP oxygen to give an oxygen concentration of approximately 48y0. This corresponds to a superficial velocity, based on air. of 8.2 feet per minute. (This was calculated back to a basis of air containing 2 1 7 , oxygen for convenience.) Analyses for sugar (as glucose), nitrogcn, phosphorus.
alcohol, and yeast yield are presented in Table I1 (3,4). The yeast yield was calculated on the basis of wet yeast obtained by centrifugation ( 7 7). The yield of yeast for this run was 2.6770 (wet yeast of 25% solids). The set point of the refractive index control curve (Figure 3). was 70. which corresponds to a refractive index of
Table 111.
1.334030. The variation of the refractive index over the 8-hour period of control was + 2 divisions on the curve in Figure 3, which corresponds to i0.000058 R . I . unit. If the variation of refractive index was purely an effect on sugar concentration, the calculated variation corresponding to this change in refractive index would be *0,07% sugar. The sugar concentration during the period of fermentation over which control was exercised showed a decrease from 0.76 to 0.287,:which can mainly be attributed to the formation of alcohol and the effect of accumulation of nonfermentable material in the commercial glucose. The accumulation of higher carbohydrate in the medium should be proportional to the total amount of sugar added to the fermentation (not the actual sugar concentration present). Because the medium is virtually colorless, the amount of sugar added at any time cannot be calculated by the color of the medium as in the molasses media studies ( 4 ) . An isotope dilution procedure was used for measuring the amount of sugar added. utilizing a nonrnetabolizable radioactive isotope, calcium-45. The calcium was precipitated as the oxalate and counted with an automatic printing Geiger counter (7). The counts per minute in the samples are proportional to the amount of sugar added to the fermentation. The amount of nonfermentable carbohydrate in the commercial glucose was calculated to be ’7.69%, and from the total amount of sugar added, the per cent of nonfermentable carbohydrate present in the fermentation at any time was calculated (Tables I11 and IV). The amount of nonfermentable carbohydrate in the commercial glucose was determined by direct analysis, after first vacuum drying
Nonfermentable Carbohydrate
(Baker’s yeast propagation 150 liters in synthetic medium. Run 86. 14.10 pciiries of Ca46 added t o sugar solutions) Actual Net Net Count Total Sugar Count,’Min. above Coned. Integrated Time, Present, in 20-ML Initial Sugar Added, Sugar Added, Sample Sample Hr. % Count 5% 70 1 24.30 1915a 2 0 0.750 124b 0 25.2 3 0.08 0.760 121 0 0 25.2 4 1.00 0.760 121 0 0 25.2 5 2.00 0.740 124 3 1.3 26.2 3.00 0.700 134 13 5.4 29.3 6 7 4.00 0.573 183 62 25.8 44.5 8 5.05 0.450 195 74 30.8 48.3 9 6.00 0.395 240 119 50.0 62.6 10 7.00 0.280 291 170 70.8 78.2 11 8.42 0.280 352 23 1 96.3 97.2 9.00 0.117 353 232 96.7 97.5 12 13 10.00 0.025 361 240 100 100.0 (95.6) 14 11.00 0.026 345 226 15 20.00 0.008 316 195 (87.5)
...
...
...
...
...
...
...
Concentrated sugar solution. Approximately 10.1 pcuries of Ca45added t o concentrate, 20.3 liters of 25% by weight commercial glucose. Expected count/min., 2022. * Approximat,ely 4.0 pcuries of Ca45added t o main medium, which contained 7.0 liters as 25% commercial glucose solution. Expected count/min., 112.
12 18 INDUSTRIAL A N D ENGINEERING CHEMISTRY
and then determining the per cent of disaccharides and monosaccharides in replicate samples by the method of Sullivan (12). I t is possible to calculate the hypothetical sugar concentration that would have been found if the nonfermentable sugar were utilizable and the alcohol formed were actually sugar. (The refractometer cannot measure the difference .) However, 1 gram of alcohol per liter would have the same effect on refractive index as about 0.38 gram of sugar. Calculations of hypothetical sugar concentrations, taking into account the effect of nonmetabolizable components and nonmetabolizable components plus alcohol produced, are shown in Table IV. The average actual sugar concentration over the period of control wqs 0.549%, with a variation of =k0.269y0 and a standard deviation of 0.288%. The average hypothetical sugar concentration, corrected for the nonmetabolizable components, is 0.670% with a variation of h0.194yo and a standard deviation of 0.15070. When corrections are made for both the alcohol and nonmetabolizable components, the average sugar concentration is 0.768%, with a variation of &0.131% and a standard deviation of 0.065%. These values of sugar concentration over the period of control are plotted in Figure 4. Discussion of Results The control system operated satisfactorily, in that a constant refractive index value was maintained throughout the fermentation. The actual sugar concentration in the medium gradually decreased. However, this does not mean that the system was not controlled; without control, the sugar added initially, to a level of about lyo,would have been completely utilized within 1 or 2 hours,
Table IV.
Sample 3 4 5 6
7 8 9 10
Correction for
FERMENTATION EQUIPMENT A N D DESIGN
Q H Y P O T H E T l C b L SUGAR C O R R E C r E O
a
ALCOHOL
O't
TIME
IN
HOURS
Figure 4. Actual sugar and hypothetical sugar concentration corrected for nonmetabolizables and for nonmetabolizables and alcohol v5. time, run 86
depending on the activity of the yeast inoculum. Some experiments of this nature have been made (3) Although a number of not too evident factors may be concerned, a major cause for decrease in available sugar was the relatively high production of alcohol during the fermentation. The high alcohol production may have been due to an inadequate sparging system, the sugar level maintained, or have been a characteristic of the medium. Although the stream from the fermentor was controlled at 30.0' i 0.2" C., there was no additional temperature control in the refractometer. This may have accounted for a random error in the control. Nonfermentable carbon compounds and ash from the sugar source will cause a decrease in the controlled sugar concentration during fermentation. A theoretical calculation of hypotheti-
Alcohol Production and Nonmetabolizable Polysaccharides
(Baker's yeast propagation 150 liters in synthetic medium. Run 86) HypothetActual ical (Corr. yo NonSugar yo Total metaboliz- for Non(as able metaboAlcohol, Sugar Time, Glucose) Added lizable) Added Hr. Wt. 70 % 0.823 0.007 25.2 0.063 0.08 0.760 0.823 0.023 0.063 25.2 0.760 1.00 0.033 0.806 26.2 0.740 0.066 2.00 0.106 29.3 0.074 0.774 3.00 0.700 44.5 0.112 0.213 0.685 4.00 0.573 0.121 0.356 0.571 48.3 5.05 0.450 62.6 0.157 0.477 0.552 6.00 0.395 78.2 0.196 0.427 0.476 7.00 0.280 0.244 0.680 0.524 97.2 8.42 0.280
11 Av. concn. sugar Variation Std. dev.
FOR N O N - M E T b O O L 1 2 b B L E S
02t
0.549 0.670 ~k0.194 f0.269 0.150 0.288 Total sugar added (as glucose) 3.015% Total carbohydrates added 3.267% Fraction solids nonmetabolizable 7.69y0
Hypothetical Sugar (Corr. for Nonmetabolizable) 0.825 0.832 0.818 0.814 0.766 0.706 0.732 0.637 0.781
0.768 f0.131 0.065
cal sugar concentrations accounts for almost the entire decrease in sugar, when the effects of compounds that might decrease the amount of controlled sugar are considered. The variation of the hypothetical sugar concentration, corrected for the effect of alcohol and nonmetabolizable fraction, gave an average value of sugar constant within 5 ~ 0 . 1 3 7for ~ 95% probability limits. The variation due to periodic cycling of the control is about =ko.0770 sugar. I t is thus considered that the hypothetical sugar concentration is constant with time within the experimental error of the procedures involved. If the errors due to nonmetabolizable fractions and alcohol production are continuously corrected by a measuring device which automatically and continuously resets the set point of the refractive index controller, more exact control may be obtained. Experiments are now in progress. There is no convenient method for continuously measuring alcohol in the presence of other compounds. However, if the circulating stream were continuously passed into a vacuum flash chamber with as small a holdup as possible under steady-state vaporization conditions, it might be possible to vaporize over 95% of the alcohol before the stream was passed through the refractometer. The following method might be applicable for correction of the nonmetabolizable fractions which tend to accumulate in the fermentor. Baker's yeast is produced with mixtures of cane and beet mdlasses, which are occasionally decolorized to some extent. If the decolorization procedure were standardized, a correlation would exist between the per cent transmittance of molasses at 420 mp and the concentration of molasses present. A relationship could then VOL. 49, NO. 8
AUGUST 1957
12 19
Processing Equipment (1E) Artisan Metal Products, Inc., Waltham, Mass., stainless steel 316 pressure vessel, 5-gallon capacity. (2E) Bausch & Lomb Optical Co., Rochester, N. Y., process control refractometer (not commercially available). “Continuous Refractometer Reference Manual,” catalog 33-45-70. Equipped with a Honeywell, Minneapolis Philadelphia, Pa., recorder Y152controller, Model P141X-93-11. Similar equipment is available from The Barnes Engineering Co., Consolidated Electrodynamics Corp., and Precision Scientific Co. (3E) Beckman Instruments, Inc., South Pasadena, Calif., pH electrode chamber, Type 9075.
-
be established between the color of the molasses and the refractive index of the nonmetabolizable (nonfermentable carbohydrates and ash components) por. tions (3, 4). The variation of such a correction factor from one batch of molasses to another is not known, but if the curves have similar shapes, determination of one or two points might determine the new relationship. As a control device for this correction, the color of the actual fermentation can be measured continuously by a flow colorimeter. As the color intensity increases, the correction required in the refractive index setting increases. By using a cascade control system, with the proper conversion cams, the set point of the refractive index controller can be reset automatically and continuously to correct for variation due to nonmerabolizable accumulation. I n a continuous fermentation, such as the continuous batch reactor type, this elaborate correction probably is not necessary. At the point where steady state conditions are reached, and control is started, the fraction of alcohol and nonmetabolizable components remaining in the system should not vary appreciably with time. Refractive index can then be used adequately to control sugar addition to the system. If some device other than a refractometer could be used for the continuous measurement of sugar, it could be inserted a t the same position as the refractive index indicator in the system. I n the present recirculating system, the dead time is 1 to 3 minutes and the time constant for this part of the loop 1 to 2 minutes. These figures are approximate and studies are being undertaken to obtain more exact data. A detailed dynamic analysis of the control loop is planned. The studies presented so far are only a preliminary to a n intensive research program. I t is hoped that dynamic analy-
1 220
(4E) Ibid., amplifier, Model W. (5E) Centrico, Inc., Englewood, N. J., centrifuge Model LWA-205 with stainless steel bowls. (6E) Cherry Burrell Corp., Chicago, Ill., centrifugal pump, Model UA. (7E) Conoflow Corp., Philadelphia, Pa., diaphragm control valve, Type AB-10, 1/2-inch stainless steel 316 construction. (8E) Eastern Industries, Inc., N e w Haven, Conn., midget centrifugal pump, Type D-11, stainless steel construction with Teflon packing. (9E) Ibid., centrifugal pump, Type E-1. (10E) Electronics Corp. of America, Cambridge, Mass., foam level probe-type 60ER1. (11E) Ibid., relay, Type 10CB1. (12E) Fischer & Porter Co., Hatboro,
sis of an entire fermentation system can be achieved from a servo-mechanical viewpoint. Once control is achieved, so that variables can be maintained constant with time, it is hoped that optimum yield studies can be analyzed statistically. With the possibility of controlling the variables, it would also be possible to vary these values with time by means of a program controller. T o obtain optimum conditions, a continuous increase or decrease of pH, temperature, or nutrient concentration with time over the batch cycle of the fermentation might be required. Studies such as this may be carried out, once successful control has been established. Summary
Continuous refractive index measurement is used for the automatic control of the sugar concentration in yeast propagation. I n colorless, or slightly colored media, a relatively constant refractive index may be maintained throughout a propagation. However, under present conditions the actual sugar concentration maintained in the medium decreases gradually with time. The amount of this decrease depends on a number of factors, among which are the quantity of alcohol produced and the accumulation of nonmetabolizable fractions associated with the carbon source. This study has emphasized some of the difficulties inherent in such a control process. A possible means of automatically correcting for this decrease i s discussed. This system when perfected should provide a useful research tool and may have practical advantages, if its expense can be justified. Acknowledgment
Several firms have made generous contributions which enabled the studies to be undertaken. Especial thanks are
INDUSTRIAL AND ENGINEERING CHEMISTRY
Pa., flowmeter, rotameter, Type 7075, Float BSU-44, 4-24-10. (13E) Foxboro Co., Foxboro, Mass., positioner Valvactor, Type A 1822. (14E) International Engineering In.., Dayton, Ohio, agitator assembly with Model S-7205 reducer. (15E) Minneapolis-Honeywell Regulator Co., Minneapolis, Minn., recorder controller Type 152 P13V-95. (16E) Moore Products Co., Philadelphia, Pa., valve positioner, Type 72LN315. (17E) Taylor Instrument Cos., Rochester, N. Y., temperature recorder controller, Type 122RN125. (18E) Weston Electrical Instrument Corp., temperature indicator, Model 2211.
extended to the Bausch & Lomb Optical Co., Rochester, N. Y . ; Beckman Instruments, Inc., Fullerton, Calif. ; the Minneapolis-Honeywell Co., Inc.. Philadelphia, P a . ; and the Vulcan Copper and Supply Co., Cincinnati, Ohio. Literature Cited (1) Bronner, F., “Effect of Food Phytates on Absorption of Radioactive Calcium in Human Beings,” Ph.D. thesis, Department of Food Technology, Massachusetts Institute of Technology, 1952. (2) Deindoerfer, F. H.,Wilker, B. L., IND. ENC.CHEMI.49,1223-6 (1957). (3) Fuld, G. J., “Investigation of Automatic Controls as Applied to Fermentation Proce~ses,~’ Sc. D. thesis, Department of Food Technology, MassachusettsInstitute of Technology, 1956. (4) Fuld, G.J., Dunn, C. G., Food Technol. 11, 15-18 (1957). (.5 .) Fuld. G. J.. Dunn. C. G.. “50-Gallon Pilot Plant Fermentor’ for Instruction Purposes.” in Dress. ( 6 ) Gaden, E. Id., Jr., Chem. Eng. 63, I
I
_N O- . 4,lSY-74 (1956). I
1 _ ^
._ ,G*C., Appl. Microbiol. 2, 2-4 (7) Lakata,
(175.4). (8) Neish. A. C., Ledinpham. G. A , , Can. J . Research‘B27, 694-704 11949). (9) Nelson, H.A.,Maxon, W. D., Elfer-
dink, J. H., IND.ENG.CHEW48. 2183-9 (1956).
(10) Olson, B. H., Johnson, M. J., J . Bacterial. 57, 235-46 (1949). (11) Singh, K.,Agarwal, D. W., Peterson. W. H., Arch. Biochen. 18, 181.-93 (1 948). (12) Sullivan, J. T., “Guide to An;!ysis of Forage Plants--Supplement, Agricultural Research Service, U. S. Department of Agriculture, TJniversity Park, Pa. (Circa. 1953). RECEIVED for review November 20, 1956 ACCEPTED June 11, 1957 Division of Agricultural and Food Chemistry, Symposium on Fermentation Process and Equipment Design, 130th Meeting, ACS, Atlantic City, N. J., September 1956. Part of Sc.D. thesis presented by G. J. Fuld in the Department of Food Technology, Massachusetts Institute of Technology, in 1956. Contribution 303 from Department of Food Technology.
