Calcium Gluconate from Juice of Cull
Cider was fermented by gluconic-acidforming organisms. B. xylinus, B. oxidans, and P. divaricatum grew poorly. P. purpurogenum and P. citrinum grew well. Best yields were obtained with P. citrinum. Good yields of gluconic acid, calculated in terms of glucose used, were obtained through fermentation by P. citrinum. The yield based on total sugar used, however, was low. Fermentation by P. citrinum produced gluconic acid from sucrose and glucose solutions but not from fructose solution. This probably explains the low yield of gluconic acid based on total cider sugars destroyed.
and Surplus Apples CLIFFORD FROST, J. L. ST.JOHN, AND H.W. GERRITZ Agricultural Experiment Station, The State College of Washington, Pullman, Wash.
QT
HE success of calcium gluconate as a calcium carrier in human and animal diet and its preparation by fermentation of glucose made it seem worth while to investigate possibilities of preparing it from agricultural waste. The present trend of vigorous apple grading makes available an abundant supply of carbohydrate material from this source. Should it be found possible to convert these carbohydrates to calcium gluconate economically, a double purpose would be accomplisheda cheap supply of calcium gluconate would be available and an agricultural waste would be utilized. Numerous microorganisms produce gluconic acid as part of the end products of their metabolism. Among the mold species of penicillium, aspergillus, mucor, sterigmatocystis, and citromyces (8-5, 7-12,1.6, 15, l7,18,20-29, 32) produce gluconic acid to some extent, In the presence of calcium carbonate some species of acetic bacteria, not capable of oxidizing the CHOH group; oxidize glucose to gluconic acid. Brown (13)found that B. aceti produced gluconic acid from glucose. B. acetosum, B. racens, B. vini acetai, and B. ascedens were reported by Hermann (19)to produce gluconic acid. Takahashi and Asai (30) obtained gluconic acid in yields of 80 to 100 per cent of the theoretical from glucose solutions by the action of B. hoshigaki var. rosea and B. industriurn var. hashigaki, isolated from dried persimmons. Later the same authors (31) reported B. hashigaki var. gb.mronicurn 11 nou. sp. to produce gluconic acid in 76 per cent yields from 10 per cent glucose solutions. Bernhauer (11)obtained a French patent for the preparation of gluconic acid by the action of bacteria or fungi, including B. xylinum, B. oxidans, B. industriurn, and B. glucwonicum on sugar solutions. Investigations in the preparation of gluconic acid have so far been concerned with glucose or glucose-rich sirups. Apple-juice carbohydrates, on the contrary, consist mostly of fructose although they contain some glucose and sucrose.
B. oxidans and B. xylinum, were also used but growth was so slow that the cultures were finally discarded. Preliminary experiments showed P. divaricatum to be poorly adapted to growth on apple cider. P. purpurogenum and P. citrinum grew well and were used in the remainder of the problem. The general nature of the experiment consisted in growing the mold cultures on cider or nutrient agar slants until vigorous sporulation occurred, then transferring spores to the filtered sterilized cider where growth was desired. Experiments were conducted to determine the eftect of adding nutrient salts to the cider, on the production of calcium gluconate. The calcium gluconate was crystallized from the sugar solution by a method similar to that of Herrick and May (84):
The mycelia were removed from the solution, washed with warm water, and pressed. The washings were added to the original solution, and the whole was filtered. Calcium carbonate was added to neutralize the gluconic acid, and the excess was filtered off. The solution was boiled to break down any acid carbonate formed and refiltered. The filtrate was boiled down to about one-fourth its original volume and cooled. Ninety-five per cent alcohol was added slowly at frequent intervals with stirring until a 60 per cent alcohol solution was produced. Om standing, the calcium gluconate crystallized in characteristic cauliflower-like knob formations. These were filtered out, washed with 50 per cent alcohol, and air-dried. Purity of the calcium gluconate was determined by washing and determining calcium on the ash and by determining gluconic acid polarimetrically according to the method of Bennet-Clark (6). Yields are given in terms of calcium gluconate dihydrate. Glucose was determined by the iodometric method of Evans (16). Reducing sugars were determined by the Mumon-Walker method (1). Sucrose was determined by hydrolysis with hydrochloric acid and subsequent determination of reducing sugara (1).
Fermentation of Cider Methods
P. citrinum WITH NUTRIENT SALTS.Aliquots of cider in %liter Erlenmeyer flasks were treated with nutrient salts and autoclaved at 15 pounds per square inch (1.05 kg. per
The molds used were P. purpurogenum var. rubisclerotium, P . citrinum, and P. divaricatum. Two species of bacteria, 75
VOL. 28, NO. 1
INDUSTRIAL AND ENGINEERING CHEMISTRY
76
OF CIDER" BY Penicillium citrinum TABLE I. FERMENTATION
WITH
NUTRIENT SALTSADDED
yo of Theoretical Yield Based on:
Reducing Total Ca GluTotal Total Sugar Sugar Glucose Glucose conate sugar Glucose glucoae Vol. Remaining Remaining Remaining Time Used Obtained used used present cc. c Gram per cc. Days % Grams 0.0162 4.12 55.4 36.2 27 32.8 0.0749 0.0767 65.5 200 51.7 27 26.9 0.0186 4.46 33.3 0.0740 0.0740 64.5 250 25.2 46.5 28 0.0130 4.78 33.6 0.0710 0.0726 72.3 250 27 40.4 0.0193 6.52 77.9 45.8 0.0754 0.0754 58.9 250 44.2 80.4 29.3 0.0092 35.5 0.0734 0.0717 27 5.05 250 38.7 0.0130 4.29 52.2 37.7 0.0728 0.0731 72.3 28 200 52.2 0.0111 39.8 0.0637 0.0636 76.4 35 28.8 9.07 400 0.0143 29.1 20.2 0.0723 0.0734 28 17.3 2.85 250 69.6 Analysis of cider used (gram per 00.): reducing sugar, 0.1248; total sugar, 0.1286; gluoose, 0.0470. Incubated at room
Flask
a
.
Nutrient Salts Added Name Amount Gram NaNOa 0.1 NaNOa 0.1 0.044 MgSO4.7HnO MgSOa.7HrO 0.044 0,008 NaHnP04.HzO 0,008 NaHaPOcHnO None None temperature (23-2Se C.).
.. ..
TABLE11. FERMENTATION OF CIDER& BY P. citrinum WITHOUT ADDEDNUTRIENT SALTS
made from 14-day-old glucose agar slant cultures which had been prepared from earlier cider culCaGlu- ,-% .- of - Theoretical ~ - - - - - Yieldtures. After 3-day incubation at room temperaReducing Total Based on: Glucose Sugar sugar conate Total Total ture, flasks 1 and 2 were transferred to a 37' C. RemainRemain- Remain- Glucose Obsugar Glucose glucose cabinet and retained a t that temperature for the Flask Time ing ing ing Used tained used used present remainder of the period. The remaining flasks Days -Gram percc.% Grams were kept at room temperature. Fermentations 1 24 0.0124 0.0746 0.0760 73.4 Lost 2 24 0,0124 0.0748 0.0762 73.4 9.9 3i:4 6017 2i:S were terminated after 20 to 23 days, Results 3 31 0,0159 0.0640 0.0640 65.9 12.0 38.1 82.2 54.2 4 26 0.0102 0.0720 0,0730 78.1 11.5 36.1 66.5 52.0 are given in Table 111. 5 31 0.0162 0.0636 .... 67.4 12.5 .. 89.0 60.0 From flasks 1 and 2 kept a t 37" C., only a Total 45.9 flocculent light brown material was obtained by Analysis of cider used (gram per 00.): reducing suvar, 0.1285; total sugar 0 1302. the precipitation treatment, The precipitate reglucose 0.0466. Volume in each flask = 400 cc. No nutrient salts were added. IAcubated a t r o o 4 temperature (23-28' C . ) . mained as a film of brown eluelike material on drying. No calcium gluconate was present in either flask. Sucrose had almost completely disappeared from the fermented solution, but only about half sq. cm.) pressure for 15 minutes. The cooled solutions were inoculated with P. citrinum from slants of Bacto glucose agar. as much glucose was used by the organisms in flasks kept a t Growth covered the surface of the cider in 3 days. Sporula37" C . as in flasks kept a t room temperature. High temtion began early. The flasks were incubated a t room temperatures were not conducive to the formation of gluconic perature (23' to 28" C.). The fermentation ranged from acid from cider by P. citrinum. 27 to 35 days. Treatment and results are reported in Table I. More than 35 per cent of the glucose present was deFair yields of calcium gluconate, based on glucose used, were composed in 20 to 23 days in the flasks kept a t room temperaobtained. One of the magnesium-treated cultures gave a ture. Good yields of calcium gluconate were obtained, but, much higher yield than other cultures, but if was also much based on total glucose present, they were not as high as yields higher than its duplicate. The results in general are not sufin the earlier experiment where fermentation was continued for 31 days. ficiently consistent to warrant definite conclusions regarding the effect of nutrient salts. The quantity of calcium gluconate produced was in some P. citrinum WITHOUT NUTRIENT SALTS. Portions (400 cases more than the theoretical from the glucose used. P. cc.) of filtered cider were placed in 3-liter Erlenmeyer flasks, citrinum must, therefore, have converted cider sugar other stoppered w i t h c o t t o n , a n d s t e r i l i z e d a t 15 pounds per s q u a r e inch (1.05 kg. per sq. cm.) p r e s s u r e for 15 m i n u t e s . The cooled TABLE 111. FERMENTATION OF CIDER^ BY P . citrinum ON CIDER INOCULATED FROX CIDERCULTURES solutions were inoculated with P. citrinum from 19-day-old glucose agar slant cultures. Growth % of Theoretical Yield covered the entire surface in 3 days. Mycelia Reducing Total Ca Glu- Total Baaed on: Totat ~
v
Glucose
Sugar
Sugar
Glu-
conate sugar
Glu-
glucoae
p e n e t r a t e d the liquid to a depth of an inch Remain- Rgmain- Remain- cose Obprescoae pres(2.54 em.) or more. The flasks were incubated Flask Time Temp. 1ng 1 w ing Used tained ent used ent a t room temperature. Days C. -&am per c c . 7 % Grams I 21 37 0.0352 o.m99 0.0960 0.0962 15.9 None .. ... .. After about 20 days of growth, the mycelia 22 37 o.0375 o.1030 None started to wrinkle and sink into the cider. 3 20 Room 0.0300 0.0878 .... 28.4 2.4 l0:4 l 0 + : 3 30:5 4 20 Room 0.0288 0,0936 .... 34.3 Flasks 1 and 2 were treated for removal of cal5 21 Room 0.0252 0,0942 .... 333 129...348 322 ... 704 111 013 ... 740 109.5 95.7 38.1 6 22 Room 0.0283 0.0917 .... 94.1 30.5 cium at the end Of 24 days* The 7 22 Room 0.0282 0,0913 .... 90.4 29.2 calcium gluconate crystallized out in 36 hours. 8 23 Room 0.0276 0,0920 . . .. 33 24 .. 33 23 .. 13 11 03 .. 40 114.8 39.4 Flask 4 was treated for calcium gluconate rea Analysis of cider used (gram per cc lucose 0.0419. reducing sugar, 0.1086; total sugar, 0.1226. No nutrient salts were addecf Volkme of i 5 0 cc. was used. moval after 26-day growth, and flasks 3 and 5 were allowed to remain for 31 days. Results are given in Table 11. than glucose to gluconic acid or have hydrolyzed sucrose, Better yields of calcium gluconate based on glucose used thus producing glucose to replace glucose used. Sucrose were obtained in these experiments on cider than in the earlier had practically disappeared from the fermented solutions. experiment where different nutrient salts were added to the It was necessary to add less alcohol than a quantity equal to cider cultures, Yields increased with length of fermentation the volume of concentrated solution in order to prevent pretime up to the longest period of 31 days in this experiment. cipitation of flocculent material. Another series of flasks captaining cider with no nutrient P. citrinurn WITH AND WITHOUT DISODIUM PHOSPHATE. salts added was prepared. Portions (150 cc.) of cider were Filtered cider was measured into 3-liter Erlenmeyer flasks. placed in flasks, sterilized, and cooled. Inoculations were O
JANUARY, 1936
INDUSTRIAL AND ENGINEERING CHEMISTRY TABLEIV. FERMENTATION OF CIDER.BY P . citrinum WITH
77
ADDEDSALTS
AND WITHOUT
% of Theoretical Yield Based on: Flask
Nutrient Salts Name Amount Gram None None NaHzPOd.H*O 0:os NaHpP 0 4 . HzO 0.08 0.04 NaHzPO4.HnO 0.04 NaHzPOd.H20 None None
Reducing Sugar Remaining
..
0.0734 0.0840 0.0740 0.0732 0.0741 0.0783 0.0741 0.0741
.. ..
Analysis of cider used (gram per cc.): the others. 0
Total Sugar Remaining Gram p e r cc.-
Glucose Kemaining
Glucose Used
% 61.0 37.5 57.8 65.6 69.1 43.6 61.7 59.3
0.0168 0.0270 0.0182 0.0148 0.0133 0.0243 0.0165 0.0175
0 :osio 0.0754 0.0738 0.0744 0.0789 0.0749
....
Ca Gluconate Obtained Grama 17.7 6.9 8.4 9.0 8.7 5.a 10.8 11.6
Total sugar used
Total glucose used
2i:7 29.7 32.8 26.9 17.4 38.5
89.8 113.6 90.4 84.7 77.8 73.6 108.2 119.8
..
Total glucose present 64.8 42.6 52.2 65.6 53.8 32.1 66.8 71.1
Time Days 42 41 34 40 40 34 41 42
reducing sugar, 0.1068; total sugar, 0.1226; glucose, 0.0431. Volume was 600 co. in ffask 1 and 300 cc. in all
b
Six hundred cubic centimeters were placed in flask 1, and 300 cc. were added to each of the other flasks. An 0.08-gram portion of disodium phosphate was added to flasks 3 and 4, and 0.04 gram was added to flasks 5 and 6. No salts were added to the other flasks. The flasks were plugged with cotton, then sterilized for 15 minutes a t 15 pounds per square inch (1.05 kg. per sq. cm.) pressure, cooled, and inoculated with P. citrinum from cider cultures. As in earlier experiments, good growth was obtained in 3 days. The flasks were incubated at room temperature. Results are given in Table N. Good yields of calcium gluconate on the basis of glucose used were obtained. I n fact, the yield in some cases was too high to have come from glucose alone. It is probable that sucrose present in the cider was at least partially converted into gluconic acid by P. citrinum. As indicated in earlier experiments, the longer period of fermentation appeared to give a better yield of gluconic acid but the time interval was too short to warrant definite conclusions. On the whole, the cider to which no disodium phosphate was added gave a slightly higher yield of calcium gluconate than the treated cider. Other water-soluble and alcohol-insoluble material was present in the fermented cider which precipitated as a light brown flocculent mass when excess alcohol was added to the solution. When too much of the flocculent material to be washed out with 50 per cent alcohol was precipitated, the precipitate was redissolved in water and only sufficient alcohol was added to precipitate the calcium gluconate. P. purpurogenum var. rubisclerotium. Six 500-cc. portions of filtered cider were sterilized in 3-liter Erlenmeyer flasks and inoculated with P. purpurogenum var. rubisclerotium from 30-day-old cultures on Bacto glucose agar slants. Nutrient salts were added to the cider. The flasks were kept a t room temperature in the dark. Good growth covered the surface in 4 days. The fermentation was allowed to progress for 26 days. Sugar determinations were made and the gluconic acid was precipitated as calcium gluconate by concentrating and adding alcohol. Results are given in Table V. Large percentages of the cider sugars were decomposed by P. purpurogenum,but the yield of calcium gluconate was low. TABLE V. Flask 1
3 4 5 6 a
Nutrient Salts Name NaN03 NaHzPOd MgSOi Same as 1 without MgSOl Same as3 Same as 1 without phosphate Sameas 5
FBRMENTATION OF CIDER^ Amount Gram ::4
0.044
Sugar analysis of cider used (gram per cc.):
... ... ... ...
5
Time Days
Glucose Remaining C -
The precipitation of flocculent material with the calcium gluconate required redissolving the precipitate and recrystallizing. Some calcium gluconate was probably lost in the operation. Slightly better yields of calcium gluconate were obtained from cultures to which magnesium and phosphorus were added, but the data are too meager to warrant conclusions. Yields of gluconic acid were not as good as those obtained with P. citrinum.
Fermentation of Sugar Solutions The results of cider fermentation by both P . citrinum and P. purpurogenum indicate that only glucose or glucose and sucrose are converted into calcium gluconate by these molds. Fructose in the cider was used to a much lesser degree by both microorganisms. To determine what was taking place in the cider cultures, pure sugar solutions to which nutrient salts had been added, were inoculated with P. purpurogenum. The nutrient salt consisted of 0.05 gram NaN03, 0.004 NaH2P04, 0.0022 MgSOe-7H20, 0.009 CaCL, and 0.005 KC1 per 100 cc. of sugar solution. FERMENTATION BY P. purpurogenum var. rubisclerotium. (a) Sucrose Fermentation. A solution containing 20 grams of sucrose and the nutrient salts was inoculated with P. pu:purogenum and allowed to ferment a t room temperature for 20 days. At the end of that time it was analyzed for sugars and treated for calcium gluconate precipitation in the same manner as cider solutions. It contained 10 grams of sucrose per 100 cc. and only faint traces of reducing sugar. No calcium gluconate was present. Alcohol precipitation gave a small amount of the characteristic flocculent precipitate obtained from cider. (b) Fructose Fermentation. A fructose solution containing 7.9 grams of fructose and the salt mixture per 100 cc. was sterilized and inoculated with P. purpurogenum. The s o l p tion was fermented 22 days a t room temperature, analyzed for sugars, and treated for calcium gluconate precipitation. The fructose remaining was found to be 4.12 grams per 100 cc. No calcium gluconate was present, but a small amount of the characteristic flocculent precipitate occurring in fermented cider was obtained. BY
P . purpurogenum var. rubiscleroiium
Reducing Total Sugar Sugar Remaining Remaining Gram per cc.
Glucose Used
% of Theoretical Yield Ca Baaed on: Gluconate Total Glucose Obtalned augar used used
%
Grams
26
0.0129
0.0579
0.0608
65.04
2.435
8.54
17.09
26 26 26 26
0.0131 0.0148 0.0092 0.0116
0.0639 0.0665 0 0602 0 0620
0.0684 0.0631 0.0668
64.50 59.89 75.07 68.56
2.265 0.880 1.02 0.886
9.44 3.49 3.52 3.17
lg.03 6 69 6.18 5.88
....
glucose, 0.0369; reduclng Bugar, 0.0990; total sugar, 0.1088. Volume in each flask was 500 cc.
INDUSTRIAL AND ENGINEERING CHEMISTRY
78
( c ) Glucose Fermentation. A glucose solution containing 10 grams of glucose per 100 cc. and nutrient salts was steriliaed and cooled. The solution was inoculated with P. purpurogenum and incubated at room temperature for 22 days. At the end of that time sugar analyses were made and the calcium gluconate was precipitated, washed with 50 per cent alcohol, and weighed. The solutions contained 0.6 gram of glucose per 100 cc. A yield of 4.87 grams of air-dry calcium gluconate per 100 CC. of solution was obtained, or a yield of 46.7 per cent based on glucose used. These experiments appear to show that, although P. purpurogenum produced gluconic acid from glucose, it did not do so from fructose or sucrose. Furthermore, the sucrose was probably utilized without hydrolysis since no gluconic acid was obtained from the sucrose fermentation, and only slight traces of reducing sugars remained.
Sugar
Sugar Added
Fructose Fructose Fruotose Fructose Glucose Sucrose 6 Gram of
nesium sulfate was added. However, the yield on a duplicate fermentation was lower than that from cider to which no nutrient salt was added. Other nutrient salt cultures in the same series did not give a yield definitely higher than that obtained from a cider culture to which no nutrient salt was added. I n terms of calcium gluconate yield based on the theoretical yield possible from total sugars fermented, the effect of nutrient salts is less marked than when based on glucose used, The former basis of calculation appears most logical when sucrose is present, since P. citrinum has been shown to ferment sucrose to gluconic acid. Higher yields of calcium gluconate based on glucose used were obtained in the second series of fermentations than in the first. On the basis of total sugar used, however, the nutrient salt cultures of the first series containing added magnesium and phosphorus gave slightly higher yields. The
TABLEVI. FERMENTATIONS OF PURESUGARS BY P. citrinum % of Theoretical Yield Based on: Glucpse Fruotose ca
Time Days 0.0878 34 0.069 20 0.069 20 0.069 20 30 0.0963 0.0983 30 nutrient aalts in 100 co. volume: Gram./ffi.
VOL. 28, NO. 1
Remaining -Gram
.... .... .... .... 0.0016
Remaining
per c c . 7
Gluconate Grams
Sugar content
......
Sugar used
.. .. ..
Nutrient Salts
None None Bacto-peptone None As given" None As given" except for CaCh .... 6.29 52: 2 As given" 53: 1 As Given" 3.64 0.0020 0.0308 28.7 44.4 0.008 NaHaPOrHzO, 0.009 MgSOc7Hz0, 0.10 NaNOa, 0.06 KC1, and 0.018 CaCh. 0.072 0.021 0.208 0.0194
FERMFINTATION BY P. citrinum. Sucrose solutions, fructose solutions, and glucose solutions containing nutrient salts were prepared. They were sterilized and cooled, and then inoculated with P. citrinum. Growth covered the solutions in 4 days. The fructose solutions were allowed to ferment a t room temperature for 20 to 34 days. The glucose and sucrose solutions fermented 30 days under the same conditions. When the fermentation was discontinued the solutions were analyzed for sugars and treated for calcium gluconate precipitation. Results are given in Table VI. A good yield of calcium gluconate was obtained from sucrose fermentation amounting to 44.4 per cent of the sucrose used in the fermentation. The amount (3.28 grams) of reducing sugar remaining in solution showed that sucrose was probably hydrolyzed by P. citrinum and the glucose was then preferentially oxidized to gluconic acid. The nature of the nonglucose reducing sugar was not determined. Some fructose remained in solution but a large portion was destroyed. The results were erratic. No calcium gluconate could be obtained from the fermented fructose solutions. A flocculent precipitate separating as a thin brown mass on the bottom of the beaker was obtained on addition of alcohol to the concentrated solution. The precipitate was similar to that obtained from cider along with the calcium gluconate crystals when an excess of alcohol was added. It is probable that the flocculent material obtained from cider fermented by P. citrinum is produced from the fructose fermented. Only 0.15 gram glucose per 100 cc. remained in the solution fermented by P. citrinum. A good yield of white calcium gluconate crystals was obtained on alcoholic precipitation, amounting to 6.29 grams per 100 cc. solution or a 53.1 per cent yield based on glucose used. P. citrinum,like P. purpurogenum,did not convert fructose to gluconic acid, but, unlike P. purpurogenum, it produced a good yield of gluconic acid from sucrose. P. citrinum gave the better yield of gluconic acid but did not remove the glucose from solution as completely as P. purpurogenum.
Discussion of Results I n the first fermentation series (Table I) the highest yield of calcium gluconate was obtained from cider to which mag-
ti $:ti
cider used in both fermentations contained only traces of sucrose. The yields of calcium gluconate increased with length of fermentation period in the second series. Cider cultures of P. citrinum incubated a t 37" C. in the third series of fkrmentations showed poor growth, and no gluconic acid was formed. Apparently a temperature of 37" C. is not favorable to the production of gluconic acid by these organisms. The cultures grown at room temperature gave yields of gluconic acid above the theoretical for glucose fermented. In view of results obtained in fermentations of pure sugar nutrient salt solutions by P. citrinum, it appears that gluconic acid was produced from sucrose in the cider. This cider contained 1.4 per cent sucrose whereas ciders used in the two earlier series contained only a slight amount. Higher than theoretical yields based on glucose destroyed were again obtained in the fourth series of fermentations (Table IV). As in the previous fermentation, the sucrose was almost completely removed. The cultures to which sodium phosphate was added gave lower yields of gluconic acid, based on glucose used, than cultures to which no phosphate was added. I n series I, the reverse is true if the average of duplicates is taken. However, the difference between duplicates on cider of series I to which no nutrient salts were added was too great to warrant accepting their averages. With the possible exception of magnesium sulfate, the addition of nutrient salts to cider cultures of P. citrinum did not increase the yield of gluconic acid. I n some cases they appeared t o cause even a lowering in yield. Fermentation of cider by P. purpurogenum resuIted in decomposition of a large portion of the cider sugars but in low yields of gluconic acid. It will be necessary to experiment more fully with P. purpurogenum, however, before conclusions can be safely drawn. From pure sugar solutions containing nutrient salts, P . purpurogenum fermented large quantities of sucrose and fructose but gluconic acid was not present. Glucose was almost completely fermented from its nutrient salt solution in 22 days with the production of 4.87 grams of calcium gluconate per 100 cc. of 10 per cent glucose solution. This yield is again lower than that obtained by fermentation with P. citrinum.
JANUARY, 1936
INDUSTRIAL AND ENGINEERING CHEMISTRY
Fermentation by P. citrinum of pure sugar nutrient salt solutions of fructose, sucrose, and glucose gave good yields of gluconic acid from sucrose and glucose solutions, but no P. gluconic acid was present in the fructose solution. citrinum, like P. purpurogenum, did not ferment fructose to gluconic acid, but, unlike the latter, it fermented sucrose to gluconic acid. The presence of copper reducing material which did not reduce alkaline iodine solution indicates that P. citm'num probably hydrolyzes the sucrose to glucose and fructose, and then ferments the glucose to gluconic acid.
Literature Cited (1) Assoc. Official Agr. Chem., Methods of Analysis, 3rd ed., 1930. (2) Amelung, H., Z. physiol. Chem., 166, 209 (1927). (3) Angeletti, A.,Ann. chim. applicata, 22,59 (1932). (4) Angeletti, A., Ann. Schiapparelli, 6, 83 (1932). (5) Angeletti, A,, and Cerruti, C. F., Ann. chim. applicata, 20, 424 (1930). ( 6 ) Bennet-Clark, T. A.. Biochem. J.,28, 45 (1934). (7) Bernhauer, K.,Biochem. Z., 153,517-21 (1924). (8) Ibid., 172,296-312 (1926). (9) Zbid., 197, 136 (1928). (10)Ibid., 197, 287 (1928). (11) Bernhauer, K., French Patent 707,614 (1930). (12) Bernhauer, K., and Schulhof, L., U. S. Patent 1,849,053(1932).
79
(13) Brown, A. J., J . Chem. Soc., 49, 172-87 (1886). (14) Butkevitch. W., Biochern. Z., 154, 177-90 (1924). (15) Ibid., 182, 99-109 (1927). (16) Evans, D.I.. Ann. Botany, 42, 1 (1928). (17) Falck, R.,German Patent 553,238(1924). (18) Falck, R.,and Kapur, S. N.,Ber., 57B, 920-3 (1924). (19) Hermann, T.S.,Austrian Patent 127,373(1931). (20) Herrick, H. T., and May, 0. E. J . B i d . Chem., 77, 185 (1928). (21) Herrick, H . T., and May, 0. E., U. S. Patent 1,726,067(1929). 22, 1172 (22) May, 0.E.,and Herrick, H. T.. IND. ENQ.CHBIM., (1930). (23) May, 0.E.,Herrick, H , T., Meyer, A. J., and Hellback, R., Ibid., 21, 1198 (1929). (24) May, 0. E., Herrick, H. T., Thom, C., and Churoh, M. B., J . BioE. Chem., 75, 417 (1927). (25) Molliard, M.. Compt. rend., 178, 41 (1924). (26) Muller, I. D.,Biochem. Z., 199, 136 (1928). (27) Schreyer, R.,Ibid., 240, 295 (1931). (28) Sumiki, Y.,J . Agr. Chem. SOC.Japan, 6,17 (1930). (29) Ibid., 6,23 (1930). (30) Takahashi, T., and Asai, T., Ibid., 6, 223 (1930). (31) Ibid., 8,703-19 (1932). (32) Wehmer, C., Biochem. Z., 197, 418 (1928). RBUBIVID June 21, 1936. This paper representa one phaae of a, project on the utilization of apple waste. Published a8 Soientifio Paper No, 320, College of Agriculture and Experiment Station, State College of Washing-
ton, Pullman, Waah.
Adsorption by Activated Sludge EMERY J. THERIAULT A N D PAUL D. MCNAMEE U. S. Public Health Service, Washington, D. C.
HE necessity in sewage treatment for differentiating between clarification, a very rapid process, and the much slower processes of biological or enzymatic oxidation has been emphasized in a previous paper (11). The general conclusion drawn from the review of the various theories of sewage clarification was that in all probability the adsorptive principle or clotting agent in activated sludges was related to the gelatinous matrix itself and not to the bacteria, the protozoa, or the various hypothetical enzymes. I n this sense the gelatinous matrix of the activated sludge flocs should be regarded as an intermediary between the bacteria, etc., and the sewage, thereby avoiding a difficulty in explaining rapid clarification beyond the oxidative powers of the microorganisms. Following this general theory of sewage clarification, it was next shown (10) that the gelatinous envelop of the zooglaal masses or biological slimes of sewage treatment is unmistakably zeolitic in composition if not in actual behavior. Presumptive evidence that the sludge might actually perform as a aeolite was afforded by the colloidal nature of the material, the rapidity of action, and, especially, the absence of replaceable anions. Wagenhals, Theriault, and Hommon ( I S ) , reporting on a survey of sewage treatment plants, noted a striking decrease in alkalinity when sewage was passed through oxidizing devices coated with biological slimes. I n the extreme case the alkalinity of a sewage was reduced from the original value
Further evidence is presented in support of the theory (10)that the clarification of sewage by the so-called activated sludges depends on the presence of an aluminosilicate complex chemically the same as the zeolites of water purification. As in the case of ordinary zeolites the adsorption of ammonia and of organic matters by the sludge complex can be represented with reasonable accuracy by the Freundlich formula. In percentages, the proximate analysis of activated sludge gives: zeolite, 30; bacterial cells, 35; adsorbed matters, 20; water, 5 ; and carbon from humus, 10.
of 99 p. p. m., in terms of calcium carbonate to the highly softened value of 8 p. p. m. in the oxidizing effluent. Since the nitrate content of the effluent was only 5.2 p. p. m., the decrease could not be explained wholly as a result of acid formation during nitrification. In the light of present findings it is pertinent to interpret this decrease in titratable alkalinity in terms of base exchange, particularly as the plant in question (Fitchburg, Mass.) was by no means overloaded a t the time of the survey. Similarly, the rapid decrease in ammonia, which is observed when activated sludge is mixed with sewage, might be interpreted in terms of the well-known procedure of Folin and Bell for the removal of ammonia from urine by zeolites or the corresponding laboratory procedure for producing ammonia-free water. There were good reasons, therefore, for supposing that the gelatinous matrix of the activated sludges, biological slimes,
