Gluconic Acid Production on E. A. GASTROCK AND N. PORGES W. S. Agricultural By-products Laboratory, Iowa State College, Ames, Iowa
P.A. WELLS A N D A. J. MOYER U. S. Department of Agriculture, Washington, D. C.
T
HE production of gluconic acid from glucose by the action of various microorganisms has beenstudied here a t intervals over a period of several years. The earlier work ( 2 , 6 )with Penicillium luteum purpurogenum was developed to semiplant scale (3) by the use of surface growths in aluminum pans measuring 109 x 109 X 5 cm. (43 X 43 x 2 inches). Fermentations with Penicillium chrysogenum (6) were more satisfactory, and the process was improved further by using submerged growths under increased air pressure and agitation in sintered-bottom washing bottles (4). The most promising results were obtained with a selected strain of Aspergillus niger (7, 9 ) . The rotary aluminum drum (1) was developed by the application and extension of the principles utilized in the work with the sintered-bottom washing bottles (4). The success of the experimental work u p to this point led to the design of the large rotary aluminum drum shown in Figure 1 and to its installation a t the Agricultural By-Products Laboratory a t Ames, Iowa ( 8 ) . The purpose of extending these investigations to pilotplant scale was to establish the applicability of the rotary fermenter to commercial practice.
The various media used throughout this study are summarized in Table I. They vary but slightlyfrom thosepreviously described (7, 9).
Peptone Potatoes Agar
cacrh _ _ _ ..
B Sporulation
30.0 0.10 0.12 None 0.225 0.25 200 20.0 4.0
50.0 0.12 0.144 0.56 None 0.20 None 1.5 None
C D Cermina- Fermentation tion 100.0 0.25 0.30 0.80 None 0.02 None None 37.5b
5.8 30
Speed, r. p. m. 30 (155) Temperature, C. 375 Time, hours
24
This solution was then transferred as inoculum through sterile hose connections to the large rotary aluminum drum (Figure 1) containing the volumes and concentrations of medium D as indicated in Table I1 for the different runs. The large drum had been prepared as follows: After thorough washing, the required amounts of water, sugar, and nutrient salts were added. Steam was passed through the heating coils until the temperature reached 84" C., as measured in the exit, with a slight flow of air through the drum. The separately sterilized calcium carbonate was then added through the handhole, and the con. tents of the drum were cooled by circulating water throu h the coils. About 1.5 hours were required for heating to 84" and 2.5 hours for cooling to 30" C. A positive pressure of sterile filtered air was maintained in the large drum during cooling to prevent any development of vacuum which might cause infiltration of contaminating materials. CALCIUM GLUCONATE RECOVERY.At the end of each run, the solution contained in the large drum was transferred through a pipe line to a number of cotton bags in order to separate the mycelial growth. Clear filtrates were produced in most cases. When recovery as calcium gluconate was intended, a suspension of calcium hydroxide to the extent of 98 per cent of the free gluconic acid present was added with vigorous agitation. Precipitation of calcium gluconate began almost immediately, but the development of a crop of firm crystals and the com letion of the crystallization usuaily required 24 to 48 hours. d o l i n g to temperatures below 20 C. was found to be efficacious. The calcium gluconate crystals were separated from the mother liquor in a centrifuge and were washed twice with cold water. The washed gluconate was removed from the centrifuge, transferred to aluminum trays, and dried below 80" C. to constant weight in a steam dryer. The mother liquors and washings were concentrated under vacuum and, after cooling, a second crop of calcium gluconate was recovered. The entire process described is presented schematically in the flow sheet (Figure 2).
OF MEDIAUSEDFOR GLUCONIC ACID TABLEI. SUMMARY PRODUCTION BY Aspergillus niger
A Culture
was divided into 7- and 10-liter portions which, with proportional amounts of calcium carbonate, were transferred aseptically to two small sterile rotary aluminum drums, similar in all respects to those previously described ( 1 ) except for their lengths (19 and 29 instead of 12 inches) (48.3 and 73.7 instead of 30.5 em.). The charge used in each case approximated one-third of the total capacity of the drum. During the germination period (7) the small drums were operated under the following conditions: Air pressure, lb./sq. in. gage (cm. Hg) Air flow, cc./liter/min.
Media, Apparatus, and Technic
Ingredient
The results of studies on the large-scale production of gluconic acid from glucose by submerged growths of Aspergillus niger are presented. The optimum conditions for acid production in the large-scale rotary drum fermentation equipment were essentially the same as those found previously for similar laboratory-scale apparatus. The necessary neutralization of the acid produced was best accomplished by adding 26 grams of calcium carbonate per liter of culture solution. The most efficient glucose concentration for gluconic acid production was between 15 and 20 per cent. Fermentations made with ungerminated fungus spores were satisfactory, although a somewhat
8.
Varies 0.156 0.188 0.388 None None None None 26.0b
Cc. per liter: None 45 40 None Beer Kind of water Distd. Distd Tap Tap a Refined corn sugar, containing 91.5 per cent of dextrose and corresponding closely to dextrose monohydrate was used in all media except as noted in the footnotes to Table 11. This &gar was described as commercial glucose in previous papers (7, 9). b Separately sterilized. I_
The organism, Aspergillus niger strain 67, was cultured on slants of medium A for 7 days at 30" C. These 7-day old cultures were used to inoculate twenty or more one-liter Erlenmeyer flasks, each containing 150 cc. of medium B and maintained at 30" C. for 7 days. The heavy crop of spores, with the fungus pellicle, was transferred aseptically to 17 liters of medium C and thoroughly macerated with a mechanical agitator. This 782
Pilot-Plant Scale-
=
=
Effect of Variables on longer period of time was required as compared to fermentations made with germinated spore preparations. The same mycelial growth could be used for several successive fermentations : this eliminated a number of preliminary operations and resulted in an over-all economy of time. The maximum charge capacity of the large-scale rotary fermenter, consistent with economy of time, was between 40 and 45 per cent of the total capacity. Under the best conditions, with 15 per cent glucose solutions, yields of gluconic acid in excess of 95 per cent, based o n the sugar present, were obtained in a 24-hour fermentation period.
Production by Submerged Mold Growths with the contents during its passage through the drum, the exit temperature closely approached the temperature of the fermenting medium.
Amount of Neutralizing Agent Previous work (4, 7 , 9) showed that it was necessary to neutralize part of the gluconic acid formed during the fermentation process and that the rate of glucose conversion, as measured by the decrease of glucose in grams per 100 cc. per hour, was more rapid in the presence of undissolved calcium carbonate than when free acid was present. Curve B, Figure 3, shows the initial lag period up to an age of approximately 4 hours, followed by a continually increasing activity to the age of 18 to 19 hours. The p H curve, D, shows a sharp drop from 5.2 to 3.5 between the ages of 17 and 20 hours. On a theoretical basis-i. e., calculated from the calcium gluconate content of the fermented solution and from the original glucose present-the calcium carbonate should have been neutralized a t a glucose content of 5.8 grams per 100 cc. This value was attained a t an age of about 17.5 hours. The activity reached a maximum about an hour after the p H value started to drop. The decrease in activity was fairly rapid thereafter and, toward the end of the fermentation, was more pronounced because of the smaller amounts of glucose present. The correlation of p H with rate of glucose utilization a t various drum speeds, glucose concentrations, etc., showed similar relations to those presented in Figure 3. I n all cases the point of greatest activity was practically coincident with the point a t which free acid developed; thereafter, the activity decreased. The p H remained at a value close to 5.5 until free acid developed. Then it dropped rapidly to 3.5 or less, indicating a p H of 5.5 to be favorable for gluconic acid production. The amount of calcium carbonate that may be used in the fermentation medium is determined by a number of factors. Under conditions of equilibrium, calcium gluconate is soluble in distilled water to the extent of approximately 4.0 grams per 100 cc. a t 30" C.; however, in the presence of other materials such as gluconic acid, this value is greater. Two and six-tenths grams of calcium carbonate per 100 cc. used for partial neutralization is equivalent to 11.67 grams of calcium gluconate monohydrate or 10.21 grams of gluconic acid per 100 cc. This amount of calcium carbonate dissolved easily, and very little tendency of the calcium gluconate to crystallize was noted during most of the fermentations. When the fermentation proceeded more slowly, the development of free acid after the disappearance of calcium carbonate was slower also. Under these conditions a tendency for the calcium gluconate to precipitate was noted. When conditions existed
AXALYTICAL DATA. Glucose and p H determinations a t intervals indicated the course of the fermentation. The total gluconic acid produced was ascertained by adding the free gluconic acid value to the soluble calcium present calculated as gluconic acid. An identification ratio, serving to indicate the production of gluconic acid solely, was obtained as follows: The fermented solution was completely neutralized with excess calcium carbonate, warmed, and filtered. A known volume of the filtered solution was concentrated, and the calcium gluconate content was determined by crystallization and recovery from an alcoholic solution. The calcium content of another portion of the solution was determined by permanganate titration and calculated to calciumgluconate. Theidentification ratio is the value for calcium gluconate determined by direct crystallization divided by the permanganate value and is equal to unity if gluconic acid alone is produced. The values given in the tables and figures are the results of individual runs. The different series represent certain trends, therefore, and must be considered as a group and not as individual runs. Temperature Control of Fermentation Medium The transformation of glucose to gluconic acid is an exothermic oxidizing reaction, but in the previous work with the small drums (7, 9) and with bottles (4) no serious rise in temperature was experienced. However, in the large drum the heat generated was sufficient to raise the temperature of its contents to a value more than 10" C. in excess of the optimum, although the temperature of the room in which the small and large drums were installed was thermostatically controlled. It was necessary, therefore, to provide a spray of cooling water to control the temperature of the large fermenter and its contents during a run. This device was made automatic by the installation of a thermoregulator in a well within the exit from the drum. The thermoregulator was arranged to operate, through a relay, a solenoid valve in the cooling water line. The exit temperature was controlled within * 1 O C. Because of the intimate association of the air 783
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INDUSTRIAL AND ENGINEERING CHEMISTRY
JULY, 1938
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tive growth, and this sugar therefore was available for gluconic acid production. The smaller quantity of mycelial growth had the technical advantages of rapid filtration of the final fermented liquor and a lower retention of final liquor by the mycelial growth. The maximum activity developed, represented by the decrease in glucose in grams per 100 cc. per hour, showed a n almost linear relationship to the rotation of the drum. This indicates that substantial increases in the speed of this reaction should be possible and that changes in the interior baffling arrangement of the drum, to give greater agitation and provision for increased speed of rotation, are subjects worthy of future study. A further advantage of higher speeds was the ability to bring the final glucose content to a value well under 1.0 gram per 100 cc. This was not brought out by the final results, for it was the practice to stop the runs when the glucose content reached or approached this value, but the general slope of the curves indicated that further fermentation would have ensued if the runs a t 9.5 and 12.8 r. p. m. had been continued. The curves at 4.0 r. p. m. and some at 6.0 r. p. m., in various series, showed the tendency to flatten out a t residual glucose values above 1.0 gram per 100 cc. With a final glucose value of 1.5 grams per 100 cc. it is impossible, even with complete conversions, to obtain a yield of calcium gluconate, on the basis of sugar present, in excess of 90 per cent. However, with a final glucose value of 0.5 gram per 100 cc., yields of over 96 per cent on the basis of sugar present were obtained without undue prolongation of the fermentation period.
SHEETOF GLUCONIC ACIDPRODUCTION FIQURE 2. FLOW 1. Tube culture 2. Sporulation flasks 3. Small rotating aluminum germination drums (17-liter volume) 4. Large rotating aluminum drum fermenter (540-liter volume) 5. Air inlet: water for fermentation solution and inoculum from 3 are also introduced here 6. Air outlet 7. Handhole: commercial dextrose, nutrients, and sterile calcium carbonate are added here 8. Bag filter 9. Aluminum tank for neutralization and crystallization; calcium hydroxide milk is added here 10. Centrifuge (stainless-steel basket, aluminum-lined curb) 11. Vacuum evaDorator for mother liauors 12. Condenser 13. Vaouum dryer 14. To calcium gluconate storage
-
that retarded the fermentation, higher speeds of rotation were conducive to the formation of calcium gluconate crystals. The presence of precipitated calcium gluconate seemed to impede the free access of the solution to the fungus growth, an effect tantamount to a decrease in rotation or agitation. This resulted in a further slowing down of the reaction.
Rate of Rotation The four runs comprising this series are presented in Figure 4 and were made a t 4.0, 6.0, 9.5, and 12.8 r. p. m. (The maximum speed possible with the large drum under the existing driving conditions was 12.8 r. p. m.). Factors other than rate of rotation were kept constant. The essential data, not shown on the curves, are given in Table IIA. The curves in Figure 4 indicate little difference in the time required for complete fermentations a t 12.8, 9.5, and 6.0 r. p. m. The fermentation rate, however, a t 4.0 r. p. m. was slower. The yield of calcium gluconate based on sugar consumed manifested a steady rise with increasing speed of the drum, which is in agreement with previous findings in the small drum studies (9, Table 11). This relationship was apparently associated with the decrease in total weight of mycelial growth. There was approximately one-third as much fungus weight a t 12.8 as at 4.0 r. p. m.; the difference, 493 grams, indicated a proportionately smaller amount of sugar utilized for vegeta-
8-RATE O f GLUCOSE DECREASE C-FREE ACID CONTENT I
1
1
1
I
I
1
1
I
I
AGE I N HOURS
I
,
1 24
1
FIGURE3. CORRELATION OF PH WITH UTILIZATIONOF GLUCOSE(15.6 GRAMS PER 100 C c . ) BY A . niger AT 6.0 R. P. M.
A previous discussion (9) indicated the possibi1ity:that degree of agitation might be expressed more adequately by means of peripheral speed than by r. p. m. when comparing fermentations in rotary drums of different diameters. The results with the large rotary drum here reported, however, when compared with previous findings with small drums (Q), show approximately the same time required for fermentations under comparable conditions in both large and small rotary drums a t the same speeds of rotation, although the peripheral speed of the larger drum was 3.9 times that of the smaller drum.
Rate of Air Flow Prior study (9) with small drums indicatedlno significant difference in fermentation period when using air a t rates of flow of 500 and 375 cc. per liter of medium per minute, but a t 250 and 125 cc. slightly longer time was required. Table IIB
VOL. 30, NO. 7
INDUSTRIAL AND ENGINEERING CHEMISTRY
786
OF VARIOUSFACTORS UPON GLUCOXIC ACID PRODUCTION IN LARGE ROTARY DRUMBY SUBMERGED GROWTHS OF TABLE 11. EFFECT Aspergillus niger UNDER AIR PRESSURE OF 30 POUNDS GAGE(155 CM. MERCURY)
Run
No.
Air Flow c c .,/ l . ,/ min.
Total Speed Volume
R. p .
m.
Liters
Refined Glucose in Corn Fermen- Fermentation Inocu- Sugar tation Media lum Charged Period Original Final
KO.
Hours
Grams/100 cc. R a t e of Rotation 2.7 14.7 0.8 15.6 0.4 14.9 1.6 15.2
A. 8 9 5 22
375 375 375 375
4.0 6.0 9.5 12.8
530 530 530 530
20 flasks 91 germinated 91 91 91
34.0 25.5 24.0 21.8
---
Calcium GluconateEquiva- EquivaYields based on: Ideqtifilent to lent to In total glucose final Glucose Glucose cation consumed present Ratio charge consumed liquor
Kg. Kg. (Germinated Spores) 105.2 87.8 100.2 105.2 105.2 102.5 105.2 94.9
KO. 83.2 95.8 100.2 93.9
B. R a t e of Air Flow 0.8 105.2 100.2 95.8 l5,6 0.9 105.2 99.6 98.5 15.2 100.1 100.1 0.7 105,2 15.5 R a t e of Rotation (Ungerminated Spores) 103.8 102.3 0.2 105.2 15.8 100.9 99.4 0.7 105.2 15.5 0.8 105.2 100 0 97.5 15.9 105.2 95.6 92.8 1.5 15.6 0.8 105.2 100.2 99.6 15.9
%
%
;Max. Glucose Fungus Decrease Weight Attained G./IOO Grams cc./hr.
94.8 96.6 97.8 98.9
79.1 91.1 95.3 89.3
0.976 1.003 1.022 0.992
730 396 237
0.60 0.88 1.12 1.40
95.6 98.9 100.0
91.1 93.7 96.0
1.003 1.014 1,005
396 306 335
0.88 0.75 0.75
98.5 98.5 97.5 97.1 99.4
97.3 94.5 92.7 88.2 94.7
0.962 1.006 0.970 0.982 0,988
2660 705 206 324
0.65 0.85 1.35 1.33 1.55
95.6 99.8 97 3 99.2
91.1 95.4 89.5 83.5
1.003 1.022 0.994 1.020
396 605 1092 2440
0.88 0.80 0.75 0.75
7 6 0 3
95.3 91.1 96.5
1.022 1.003 0,977 0.960
396 405 2721
1.12 0.88 1.08 0.45
97.5
92.7
1.35
20flasks 91 germinated 91 91
25.5 28.8 30.5
530 40 flasks un- 91 germinated 91 530 91 530 91 530 91 530
C. 63.5 38.3 30.8 30.0 30.5
530 530 530 530
20 flasks ger- 91 I18 minated 147 177
25.5 36.0 50.0 75.0
9.5 6.0 9.5 6.0
530 530 662 795
20 flasks 91 germinated 91 108 131
24.0 25.5 28.8 50.0
Volume 14.9 15.6 15.0 15.4
9.5
530
30.8
15.9
0.970
..
375
9.5
530
13.5
15.1
3.1
5
b
b
b
b
1.002
457 c
1.15
20
375
9.5
548
40 flasks un- 91 germinated Mycelium 91 from No. 18 Mycelium 98 from No. 19
23.0
15.8
0.4
b
5
h
b
b
0.995
616
0.85
5
375 375 375
9.5 9.5 9.5
530 530 530
20 flasks ger- 91 minated lOld 169d
24.0 27.0 47.0
100.2 94.9 96.9
97.7 105.2 100 9
95.3 90.6 55.9
1.022 0.974 1.004
353 300
1.12 0.85 0.70
12
11
375 250 125
6.0 6.0 6.0
25 16 18 23 27
375 375 375 375 375
4.0 6.0 9.5 12,.8
9 14 17 31
375 375 375 375
6.0 6.0 6.0 6.0
5 9 29 32
375 375 300 375
9
530 530 530
D. 15.6 20.6 25.0 30.8
E.
F. 18 19
375
G.
28 30
Concentration of Glucose 0.8 105.2 100.2 130.1 0.9 136.1 171.0 157.3 1.9 4.9 203.5 171.4 of Fermentation Medium 0.4 105.2 102.5 0.8 105.2 100.2 0.5 125.7 121.3 2.1 151.4 131.5
Re-use of Fungus Growth 0.8 105.2 100.0
Grade of Commercial Glucose Used 14.9 0.4 105.2 102.5 16.0 2.2 104.8 90.3 25.8 11.6 173.0 96.0
95.8 129.7 153.0 170.0 1C0.2 95.8 121.3 130.6 97.5
97 95 100 99
s62
a The speed of this run was 4.0 r. p. m. for 12 hours and 12.8 r. p. m. thereafter. b These values are not available as a result of unknown volumes associated with mycelia. c
d
Calculated from volume of myce!ial slurry and representative sample.
No. 80 chipped corn sugar, containing 80.1 per cent of reducmg sugars, calculated as dextrose, with a purity of 90.8 per cent.
and Figure 5 show results obtained with rates of air flow of 375, 250, and 125 cc. per liter per minute a t 6.0 r. p. m. The time required to reduce the glucose to 1 gram per 100 cc. or less increased as the air flow was decreased, but this reduction was not as great as might have been expected. Only about 1 hour more was required in the run made a t 125 cc. per liter per minute than the one at 250 cc. The maximum activity in the case of the minimum air flow resulted in a reduction of glucose a t the rate of 0.75 gram per 100 cc. per hour. Based on 530 liters (140 gallons) of fermenting solution, this would require the oxygen from approximately 23 liters (0.81 cubic foot) of air per minute. Since the total air flow was 67 liters (2.37 cubic feet) per minute, the reaction accounted for nearly 35 per cent of the air passing through the drum when the activity was greatest. The maximum activity observed in this study was a decrease of 1.55 grams of glucose per 100 cc. per hour. The oxygen demand here corresponded to an equivalent of approximately 48 liters (1.68 cubic feet) of air per minute or 24 per cent of the 200 liters (7.06 cubic feet) per minute flowing through t h e drum (run 27, Table IIC). These calculations show the effectiveness of the process in removing oxygen from air in its passage through the drum. It is believed that additional increases in the speed of the reaction would come about through a more intimate contact of the air and the fermenting medium. The use of oxygen or air enriched with oxygen is a further possibility, but it is likely also that the cost and the engineering difficulties would be increased.
Quantity and Kind of Inoculum The characteristics of a series of five runs made without pregermination of the spores in the small drums are presented in Table IIC and Figure 6. All of the fermentations listed were inoculated directly with the macerated spore-covered mats from forty one-liter flasks instead of twenty as used when the spores were pregerminated. Runs were made a t 4.0, 6.0, 9.5, and 12.8 r. p. m., respectively, and another a t a speed of 4.0 r. p. m. for 12 hours and 12.8 r . p. m. for the balance of the run. The 9.5 r . p. m., 12.8 r. p. m., and combination runs gave practically identical curves. However, a definite improvement in the maximum activity was attained in the combination run with the development of a rate of glucose consumption of 1.55 grams per 100 cc. per hour, the highest value noted during this entire study. The runs a t 4.0 and 6.0 r. p. m. were slower as to maximum activity and over-all time. A very long lag period, varying from 12 to 21 hours, was characteristic of these runs and was required, apparently, for the building up of sufficient vegetative growth and for enzymatic activation. At 9.5 r. p. m. there was a longer initial lag period than a t 6 r. p. m., probably due to a more rapid vegetative growth in the slower run. However, a t 4.0 r. p. m. there was a very long lag period, evidently due to the greatly decreased agitation resulting in an insufficient intermingling of reacting materials and thereby producing a slower reaction in spite of the increased mycelial growth. Conversely, the rapid mixing a t 12.8 r. p. m. caused a rapidreaction
INDUSTRIAL AND E,NGINEERING CHEMISTRY
JULY, 1938
787
I
dl 8 -I
8 0
AGE IN HOURS
FIGURE 4. COXVERSION OF GLUCOSE ACIDBY A . niger UNDER DIFFERENT RATESO F FERMENTER ROTATION
and a shorter lag period although the weight of growth might have been less. Comparison of the curves in Figure 6 with those in Figure 4 demonstrates a similarity between the runs made a t comparable speeds, if approximately 6 hours of the lag periods of the ungerminated runs are not considered. Those runs made with ungerminated spores had, in general, greater weights of mycelial growth. This was due probably to the longer total fermentation time as well as to the more favorable spore-volume relation and to the amount of nutrients available during the germination period (in this case, the lag period). The relation between fermentations with germinated and with ungerminated spores in the large rotary drums was similar to that noted previously in the smaller drums (7). The tendency was for the maximum activity to be higher with an ungerminated inoculum than with a pregerminated inoculum. The determination of mycelial content was made a t intervals during a number of runs near the end of this project, and, where availabIe, these results were plotted in the figures. The plotted values should be considered as indicative only; the final mycelial weights in the tables are more reliable. The following observations were made: The rate of mycelial growth proceeded slowly at the beginning of a fermentation and increased most rapidly towards the end. The greatest increase occurred after free gluconic acid was formed. Higher drum speeds and decreased rates of air flow retarded this growth. Increased myceIial growths were associated with higher sugar concentrations and were due probably to the longer fermentation periods as well as to the greater amount of available sugar. At 4 and 6 r. p. m. ungerminated spores, when used as inoculum, produced greater mycelial growths
OF GLUCOSE TO GLUCONIC FIGURE 5 . CONVERSIOX ACIDBY A . niger UNDER VARIOUS RATESOF AERATION
than germinated spores, probably because of the longer fermentation periods a t these lower speeds. Oxidative action was not proportional to vegetative growth taking place during the fermentation, for the greatest decline in activity occurred when the growth was most rapid. The amount of mycelial growth during the different steps of the process was found to be as follows: During the sporulation period the weight approximated 0.5 gram per flask or 10 grams from twenty flasks; during the germination period the 10 grams of macerated growth increased to between 60 and 144 grams, the average being close to 65 grams; during the fermentation period, the mycelial weight attained values of 200 to 3000 grams.
Concentration of Glucose The data pertaining to four runs, made a t concentrations of approximately 15, 20, 25, and 30 grams of glucose per 100 cc. in the original fermentation media, are tabulated and shown graphically (Table I I D and Figure 7 ) . The increase in fermentation time was greater proportionally than the increase in sugar percentage. To illustrate, 24 hours were required to reduce a solution of 15.6 grams per 100 cc. to one of 1.0 gram per 100 cc., and 35 hours to reduce a solution of 20.6 grams per 100 cc. to the same value. This disproportionality became greater as the initial sugar content of the fermenting medium increased. It is important to note that the final unfermented glucose content increased also with higher concentrations. The same trends were evident in work done with the small drums (7, Figure 4) and point to lower efficiencies a t the higher concentrations.
I U
2400
01 0
E ' 0 2000g
v)
s
I
s U
U
v)
l600g
s=
0
Y
s
P
U -1
1200;:-
w -1
18
95 R P M
? 800
=
400
FIGURE 6. VSE OF UNGERMINATED SPORES OF A . niger CONVERSION OF GLUCOSE TO GLUCONIC ACIDAT VARIOUSRATES O F FERMENTER ROTATION
FOR T H E
AGE IN HOURS
FIGURE7. CONVERSION OF GLUCOSE TO GLUCONIC ACID BY A . niger AT DIFFERENT SUGARCONCENTRATIONS
gallons or 33 per cent of the drum volume) were compared with run 29 a t 662 liters (175 gallons or 42 per cent) and with run 38 a t 795 liters (210 gallons or 50 per cent). At 9.5 r. p. m. an increase of 25 per cent in the volume (from 530 to 662 liters) required an increase of 26 per cent in comparable fermentation time (from 23 to 29 hours). This is equivalent to an increased over-all efficiency in that four runs a t 662 liters would produce as much calcium gluconate as five runs a t 530 liters. However, when the charge was increased to 795 liters, this 50 per cent increase in volume required a 127 per cent increase in time for comparable fermentation. Although these comparisons were not made a t the same speeds, it is felt that the optimum volume is in the neighborhood of 662 liters or 42 per cent of the drum volume. The charge of 795 liters raised the level of the solution to the axis of the rotary drum. The distance through which the films of solution were required to fall as the drum rotated was thereby reduced; as a result the
.
LABORATORY-SCALE FERMENTATION APPARATUS
Inasmuch as the preparation of the drum and its contents and the emptying procedure consumed the same amount of time in all cases, some advantage might accrue if the glucose concentration were increased above 15 grams per 100 cc. Such an increase in glucose content and consequently a higher gluconic acid percentage should increase the final recovery. In a commercial installation, a consideration of all of these factors will be necessary before a decision can be made concerning the optimum percentage of sugar to be used. The calcium carbonate content was not increased as the sugar percentage was raised because the difficulties due to crystallization of calcium gluconate then would have become more pronounced, Considerable crystallization of calcium gluconate needles was observed in the runs with both 25 and 30 grams per 100 cc. At the highest concentration the soluble calcium, calculated as gluconic acid, decreased from 11.0 grams per 100 cc. a t 24 hours to 9.8 grams a t 70 hours. Most of this decrease occurred before the run was 46 hours old, the higher gluconic acid content evidently inhibiting the precipitation near the end of the run.
AGE IN HOURS
FIGURE9. GLUCONIC ACIDPRODUCTION FROM GLUCOSE BY REPEATED USE OF A. niger GROWTH
amount of agitation, splashing, and foaming was reduced. All of these function to bring the fungus growth, the solution, and the air stream in intimate contact. The effect of the volume increase, in the case of the 795-liter charge, was similar to a marked decrease of the drum speed.
Re-use of Fungus Growth An interesting series appears in Table IIF and Figure 9. A supply of active fungus growth was on hand a t the completion of run 18 and was used to inoculate run 19. The sugar solution, nutrients, and calcium carbonate were given no heat treatment, but care was taken to keep the materials clean. The final activity of run 18 was evidenced by a decrease of 1.12 grams of glucose per 100 cc. per hour, and it was significant that this rate was resumed substantially in run 19. For 6 hours a rate of decrease of 1.15 grams of glucose per 100 cc. per hour was maintained, and then the rate dwindled to a value of approximately 0.55. This run was stopped a t an age of 13.5 hours, the glucose content having been reduced from 15 to 3 or 12 grams per 100 cc. in that time. After filtration, the same fungus growth was used in run 20, which had been prepared identically to run 19. This run began a t a rate of decrease of 0.50 gram of glucose per 100 cc. per hour, slowed down slightly, and then increased to a final value of 0.85. The dotted portions of the curves of runs 18 and 19 are the projections of runs 19 and 20, respectively, and show the continuance of the succeeding runs a t the preceding rates. The fungus weight after three fermentations was only 616 grams, and the elimination of the initial lag
_ - - - AGE - I N HOURS FIGURE8. COMPARISON OF GLUCONIC ACID PRODUCTION FROM GLUCOSE BY A . niger, UsING DIFFERENT VOLUMES
The great increase in mycelial growth a t the higher glucose concentrations was noteworthy, not only !because of its amount but also because of the extremely low gluconic acid conversion activity associated with it.
Volume of Fermentation M e d i u m Table IIE and Figure 8 show the essential data concerning runs a t different volumes; runs 5 and 9 using 530 liters (140 788
periods by such inoculation should make this procedure attractive from the commercial viewpoint. The increasing activity in run 20 may have been due to reactivation of the enzymes under the favorable p H conditions a t the beginning of the run as well as to fresh mycelial growth (7). Undesirable effects of contamination were absent, and the liquors in runs 19 and 20 were light in color and yielded an excellent grade of gluconate.
Grade of Commercial Glucose Used I n addition to the experiments with refined corn sugar, others were made using chipped sugar containing 80.1 per cent of reducing sugars with results as presented in Table IIG and Figure 10. The data on run 5 using refined corn sugar (91.5 per cent dextrose) are included for comparison. Although run 30 with an initial glucose content of 25.8 grams per 100 cc. was considered unsatisfactory, run 28, with an initial glucose content of 16 grams per 100 cc., compared favorably with run 5 especially in view of the slightly higher initial glucose content. The great difference between the runs using chipped sugar is believed to be the inhibiting effect of the nonsugar content of the chipped sugar. This yiew is supported by the low final mycelial weight (300 grams) in run 30 after 47 hours, whereas most of the runs of similar duration had mycelial growths of 1000 to 2700 grams. The inhibiting effect was not so marked in the lower concentration of chipped sugar, evidently because of the greater dilution. The unusual yields, based on sugar consumed, in excess of 100 per cent as reported in Table IIG are evidently the result of hydrolysis of any maltose present to 2 molecules of glucose.
Summary 1. A pilot-plant-scale, rotary aluminum drum fermenter was used to produce gluconic acid from glucose by submerged growths of Aspergillus niger. This was a successful adaptation of the previously developed laboratory-scale process. 2. The conversion of glucose to gluconic acid was most rapid and efficient a t the highest rotation rate available with present equipment. This was also true in the earlier studies
LARGE-SCALE FERMENTATION APPARATUS
decreased to 125 cc., the fermentation time was increased slightly. 4. Correlation of pH of the culture solution with glucose utilization showed a maximum conversion rate a t pH values above 5.0. The control of pH was accomplished satisfactorily by the addition of 2.6 grams of calcium carbonate per 100 cc. of fermentation medium. 5. Temperature control to prevent overheating due to the heat evolved in the process was found necessary in the pilot-plant scale operations. Satisfactory results were obtained by the use of a thermostatically controlled water spray on the exterior of the fermenter. 6. The optimum glucose concentration was indicated to be between 15 and 20 grams per 100 cc. Concentrations up to 30 grams per 100 cc. were fermented with decreasing efficiency. 7. The charge of the large-scale rotary fermenter was increased from 33 to 42 per cent of the total capacity with a corresponding increase in the fermentation time. A further increase to 50 per cent was unsatisfactory. In the latter case the effect was similar to a marked decrease of the drum speed, inasmuch as the degree of agitation was lessened and the fermentation rate thereby retarded. 8. A comparison of different types of inoculum on both small and large scale showed germinated spores to be superior to ungerminated spores. However, when the same mycelial growth was used for successive fermentations, the lag period was eliminated, the over-all fermentation time was decreased, and some of the preparatory operations were eliminated. 9. I n a typical fermentation with germinated spores, a charge of 91 kg. (200 pounds) of refined corn sugar in a total volume of 530 liters (140 gallons) was fermented in less than 24 hours, with yields of gluconic acid in excess of 95 per cent of the sugar present and over 97 per cent of the sugar consumed.
Literature Cited (1) Herrick, H. T., Hellbach, R., and May, 0. E., IND. ENQ.CHEM., 27, 681-3 (1935). (2) Herrick, H. T., and May, 0. E., J . B i d . Chem., 77, 185 (1928). (3) May, 0. E . , Herrick, H. T., Moyer, A. J., and Hellbach, R . , IND. ENQ.CHEM.,21, 1198 (1929). (4) May, 0. E . , Herrick, H . T., Moyer, A. J., and Wells, P. A , , Ibid.,26, 575 (1934). (5) May, 0. E . , Herrick, H . T., Thorn, C., and Church, W. B., J . B i d . Chem., 75, 417 (1927). (6) Moyer, A. J., May, 0 . E . , and Herrick, H . T., Zentr. Bakt., Parasitenk., 11, 95,311-24 (1936). (7) Moyer, A. J . , Wells, P. A., Stubbs, J . J., Herrick, H. T . , and May, 0. E., IND.ENQ. CHEM.,29, 777-81 (1937). (8) Wells, P. A., Lynch, D. F. J., Herrick, H. T., and May, 0. E., Chem. & Met. Eng., 44, 188 (1937). (9) Wells, P. A., Moyer, A. J., Stubbs, J. J . , Herrick, H . T., and May, 0. E., IN^. ENG.CHEM.,29, 653-6 (1937).
FIQURE 10. PRODUCTION OF GLUCONIC ACIDFROM CHIPPEDCORNSUGARBY A . niger
on a laboratory scale, and a correlation of both findings indicated the effect of the degree of agitation on the fermentation rate to be expressed more adequately by r. p. m. than by peripheral speed. 3. An air flow of 375 cc. per minute per liter of solution was used in most of the individual runs. When this rate was
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