gt “GLE LEO €00 PZ6l IIA ll Ploe SJuue! “| UoRejUaUsay pice a1uUeL 2£12Z2€ dod Aseaqry Aysuaaiun, 1laus0D i 1 TANNIC ACID FERMENTATION. I TANNIC ACID FERMENTATION. II EFFECT OF NUTRITION ON THE PRODUCTION OF THE ENZYME TANNASE BY LEWIS KNUDSON From THE LasporaTory oF Puant PuysioLogy, CoRNELL UNIVERSITY, Iraaca, New Yorx) FROM JOURNAL OF BIOLOGICAL CHEMISTRY Vou. XIV, No. 3, Apriz, 1913 Reprinted from Tou Journau or Brouocica Cuemisrry, Vou. XIV, No. 3, 1913. TANNIC ACID FERMENTATION. I. By LEWIS KNUDSON. (From the Laboratory of Plant Physiology, Cornell University, Ithaca, N. Y.) (Received for publication, January 30, 1913.) I, PREFACE. During 1907-1908, a preliminary investigation of the conditions of tannic fermentation was made by the writer with the purpose of improving the practical methods involved. In the course of this preliminary investigation a number of interesting observa- tions were made, justifyi further study of the conditions of the fermentation and of the relation of various organisms to the process. With the progress of the investigation other phases suggested themselves, until ultimately four distinct but correlated parts of the subject were experimentally studied. Part I, here reported, includes chiefly: (1) the toxicity of tannic acid for vari- ous fungi; (2) a comparison of the organism Aspergillus niger and Penicillium sp. in the fermentation of tannic acid; (3) the condi- tions and influence of various factors on the fermentation process. Part II is concerned primarily with the influence of nutrition on the production of the enzyme tannase, and will be reported in a subsequent paper. These investigations were begun at the suggestion of Prof. B. M. Duggar and prosecuted in his laboratory. It is a pleasure here to express my thanks for the advice, kindly criticism and assistance, which he has so generously given. II. INTRODUCTION. Chemical nature of tannic acid. Wagner! has grouped the tannins into a ‘physiological’ and a “pathological” series, the latter including, as most important, the tannin of oak galls as 1R. Wagner: Beitrage zur Kenntnis und zur quantitativen Bestimmung der Gerbsauren, Zettschr. f. anal. Chem., v, pp. 1-10, 1866. 159 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XIV, NO. 3. 160 Tannic Acid Fermentation well as the tannin of sumach and chestnut. The pathological tannins are hydrolyzed by boiling with acids or through the action of the enzyme tannase, gallic acid resulting. For other distin- guishing characteristics of the tannins Trimble’s? and Proctor’s® treatises may be consulted, and for a discussion of the diverse views held regarding their chemistry reference should be made to special papers on the subject. This paper is concerned with the fermentation of the tannin from oak galls, which is frequently termed gailotannic acid. History of tannic acid fermentation. Scheele* found in 1786 that gallic acid was present in the gall nuts. Robiquet® attributed the fermentation of the gall nuts to a ferment within the gallnut. Laroque* considered the formation of gallic acid from tannic acid to be due either to a ferment or to oxidation. He further found that various toxic substances could inhibit the fermentation. Ed. Robiquet’? showed that the tannic acid was trans- formed during the fermentation and he believed the transformation to be due to the ferment pectase which he extracted from the gall nuts. Witt- stein® stated that beer yeast aided tannin fermentation by fermenting the sugars and other products present. Van Tieghem® was the first, however, to demonstrate that tte formation of gallic acid during fermentation is due to the action of fungus organisms, and not to enzymes pre-existing in the galls, nor to oxidation by the air. He stated further that the organisms were Penicillium glaucum and a new organism which he named Aspergillus niger. He found that if the growth was submersed, the tannic acid was con- verted into gallic acid and glucose, the glucose being gradually used up, the gallic acid remaining. He stated further that if the growth was on the sur- face, sporulation and greater growth occurred and that the tannic acid was destroyed directly, the slight hydrolysis being due to submerged mycelium, the resulting glucose and gallic acid being then assimilated. °H. Trimble: The Tannins, Part I, 168 pp.; Part II, 172 pp., 1892. *H. K. Proctor: Leather Indusiry. Laboratory Book, 2nd edition, 450 pp., 1908. 4 Quoted from H. Trimble: loc. cit. 5M. Robiquet: Faits pour servir & de l’acide gallique, Ann. de chim. et de phys., 2° serie, lxiv, pp. 385-409, 1837. 6 A. Laroque: Neue Untersuchungen tiber Gallussiiure, Ann. d. Chem. u. Pharm., xxxix, pp. 87-100, 1841. 7 Ed. Robiquet: Recherches sur la fermentation gallique, Ann. de chim. et de phys., 3° serie, xxxix, pp. 453-460, 1853. 8 Wittstein: Jahresber. wiber die Fortschritte der Chemie, 1858, p. 435. * Ph. VanTieghem: Sur la fermentation gallique, Compt. rend. de V Acad. des Sci., xv, pp. 1091-95, 1867. Lewis Knudson 161 Miintz!° found that fermentation occurred through the action of Pent- cillium glaucwm. Fernbach" grew Aspergillus niger in Raulin’s solution with the sugar replaced by tannic acid, and then extracted from the organ- ism the enzyme tannase. Pottevin,! in a similar manner and at the same time, extracted the enzyme tannase from the same fungus. He noted also that the enzyme was developed when Aspergillus niger was grown on Raulin’s solution with the sugar replaced by gallic acid. He stated that the tannase acted on tannate of gelatin and also hydrolyzed methyl salicylate and ethyl salicylate. Manea#’ showed that synthetically prepared digallic acid was not split up by Aspergillus niger and Penicillium glaucum into gallic acid, and therefore concluded that the hydrolyzable tannin of the gall nut could not be a digallic acid. The latter in high concentrations was toxic to the organism. Fur- ther, Manea estimated quantitatively the digallic and tannic acid used by each organism. In a study of the fermentation process he employed pure cultures, adding a previously sterilized Raulfn’s solution rendered strongiy acid. The quicker the fermentation, the richer was the yield of gallic acid obtained. Kunz-Krause™ found an octyl gallotannoid, CssHs0O33, which through the action of a mould was transformed to gallic acid. III. METHODS. Culture solution. Throughout all the work the culture solutions used were a slight modification of Richards’® solution or of Cza- pek’s solution. These solutions are designated respectively A and B and are as follows: 10 Mintz: Ber. d. deutsch. chem. Gesellsch., 1877, p. 1773. A. Fernbach: Sur la tannase, Compt. rend. de l’Acad. des Sci., cxxxi, pp. 1214-15, 1901. 12H. Pottevin: La tannase. Diatase dedoublant l’acide gallotannique, Compt. rend. del’ Acad. des Sct., cxxxi, pp. 1215-17, 1901. 13 A. Manea: Sur les acides galiotanniques &t digalliques. These, Geneva, 1904. (Cited from Lafar: Handb. d. technische Mykologie, I, p. 663.) 144 Kunz-Krause: Fragmente zu einer Monographie d. Tannoide, Pharm. Centralbl., Halle, 1898. (Cited from Lafar: Handb. d. technische Mykologie, I, p. 662.) 15 H, M. Richards: Die Beeinflussung des Wachstums einiger Pilze durch chemische Reize, Jahrb. f. wiss. Bot., xxx, pp. 665-688, 1897. 16 Quoted from A. W. Dox: Intracellular Enzymes of Penicillium and Aspergillus, U. S. Dept. of Agric., Bureau of Animal Industry, Bulletin 120, 70 pp., 1910. 162 Tannic Acid Fermentation SoLution A. Soxution B. KNO§ seid ccincinaderienne Py 1.0 gram. MgSOz.......-+---++ +005 0.5 gram. KHiPOg.. 2... eee 0.5 gram. K,HPO,.........-----+++: 0.1 gram. MgSO4........---0 0 eee 0.25 gram. KCl...........- YS ahaeexes 0.5 gram. Distilled H,O........ «....100 ce. NaNOgiccseccs4sansacie es 2.0 gram. Distilled H.O...........-- 1000 ce. The source of carbon was cane sugar, tannic acid or gallic acid, either alone or supplementing each other, depending upon the experiment. A 10 per cent concentration of sugar was employed, as experience has shown that the better growth is secured with this concentration than with the lower concentration. This fact is developed in a subsequent table. Methods of inoculation. In all of the fermentation experiments the method of inoculation employed was that proposed by Hasse- Ibring.”” Methods of analysis. The volumetric method of Dreaper’® was used for some experiments, but for most of the work the volu- metric method proposed by Jean’ was used. Both methods have imperfections, but they are approximately accurate. In all cases analyses were checked by duplicate determinations and usually by more. Method of washing and weighing the fungus felt. For the experi- ments, the results of which are included in tables I and II, the method used in washing the felt free from gallic and tannic acid was as follows: The felt was removed by means of a bent needle and floated on distilled water, the water being renewed until it gave no furthercoloration. In order to secure the submersed growth, the solution was poured into a cylinder and the submersed growth, which now usually floated on the surface, was then removed by needles. All of the mycelium was then placed in a crucible, which had been brought to constant weight at 105°, heated for five hours at the same temperature and then weighed. This method, as well as the use of filter paper, possesses obvious disadvantages as well as being inaccurate. For most of the work, therefore, the following method was used: The Gooch 17H. H. Hasselbring: Carbon Assimilation of Penicillium, Bot. Gazette. xlv, pp. 176-193, 1908. 18W. P. Dreaper: Estimation of Tannic and Gallic Acid, Chem. News, xe, pp. 111-112, 1904. 1 F, Jean: Die Bestimmung des Tannins und der Gallussiure, Chem. Centralbl., 1900, pp. 1107-08. Lewis Knudson 163 filter was prepared in the usual manner, as employed in quantitative chem- ical analysis, and filtration was made by means of the Gooch funnel with suction. The original solution was first decanted into the Gooch crucible. The felt was then washed in the flask four or five times with distilled water at room temperature; or, if gallic acid had been precipitated, warm water was used. The washing of the felt continued until the wash water was per- fectly clear. The felt was then placed in the Gooch crucible, the flask again washed and the wash water poured into the Gooch crucible. The advan- tages of the method consist in the rapidity of the filtration and the accuracy which results from the thorough washing, which latter is important when the culture solution is to be analyzed and absolute weight of mycelium is to be obtained. It is an especially accurate method of securing all the fungous mycelium, and by exercising a little care there is no noticeable loss of spores. IV. TOXICITY OF TANNIC ACID FOR CERTAIN FUNGI. In the literature of tannic acid fermentation only two organisms are mentioned as possessing the property of effecting this fermen- tation; these are Aspergillus niger and Penicillium glaucum. In order to determine whether other organisms are capable of effecting the transformation, a considerable number of filamentous fungi were carefully tested with respect to their ability to grow in tannic acid solutions. ie As a nutrient medium a bean decoction was made by boiling 1 ‘liter of laboratory preserved beans with a liter of tap water. The ‘juice was then filtered off and diluted to 2 liters. With this decoc- tion as a solvent, four concentrations of tannic acid were made; namely, 0.25 per cent, 2 per cent, 5 per cent and 10 per cent. Test tubes were employed as culture vessels, to each of which were added 10 cc. of the solution. Small wads of filter paper were added to afford a solid substratum. The tubes were prepared in duplicate, sterilized, inoculated and kept at room temperature. They were examined at intervals and the final observations made at the end of two weeks are recorded in table ‘A. It is especially noteworthy that the 5 per cent permitted the growth of only one-third of the organisms, while in the 10 per cent solution only Aspergillus flavus; Aspergillus niger and Penicillium sp. are able to grow. A separate experiment indicated that Aspergillus oryzae could withstand 10 per cent tannic acid. An experiment was also made to determine if any of these organ- isms could utilize tannic acid as a source of carbon. Solution B with 11.6 grams of tannic acid per 100 cc. of solution was used. 164 Tannic Acid Fermentation TABLE A. CHARACTER OF GROWTH ORGANI8BM 0.25 per cent | 2 per cent 5 per cent 10 per cent 1. Penicillium brevicaule....| good good good 2. Penicillium camembertt...| good good very slight 3. Penicillium claviforme....| good good good 4, Penicillium duclaucit..... good good none 5. Penicillium granulatum...| good good good 6. Penicillium italicum...... good slight slight 7. Penicillium lilacinum.....! good slight | none 8. Penicillium purpurogenum| good none none 9. Penicillium sp............| good good good good 10. Aspergillus flavus......... good good good good 11. Aspergillus niger......... good good ‘good: good 12. Trichoderma lignorum....| good none none 13. Mucor circinelloides......| good slight | none 14. Mucor rouzit............. good slight | none 15. Mucor spinosus........... good none none 16. Polyporus sulphureus.....| good none none 17. Polyporus resinosus...... good none none 18. Fomes megaloma..........| no growth | none none 19. Chaetomium sp........... good growth] none none 20. Chaetostylon sp.......... good growth] none none 21. Stysanus sp.............. good growth’ none none 22. Cephalothecium roseum....| good | none none 23. Circinella umbellata...... good none Erlenmeyer flasks of 150 ce. capacity were employed, and in each were placed 50 cc. of the culture solution. After Sterilization these flasks were inoculated and maintained at room temperature. In addition to the above list of organisms the following were tested: Aspergillus oryzae, Nectria ipomoeae, Fusarium oxysporum, Phyco- myces nitens and Stilbella sp. Of all organisms tested only Asper- gillus niger and Penicillium sp.2° developed. Duplicate cultures of all these organisms on Chinese galls gave similar results, except in this case Aspergillus flavus produced a very slight growth. VanTieghem” found that both Aspergillus niger and Penicillium glaucum could withstand a saturated solution of tannic acid, and 20 One other species of Penicillium, as indicated in the appendix. is able to develop upon a 10 per cent tannic acid solution. 21 Loe. cit. Lewis Knudson 165 the fact that both develop in moistened gall nuts, which contain per dry weight 60 per cent of tannic acid, is evidence that for these two organisms the tannic acid is not toxic. Toxicity of tannic acid for Aspergillus flavus and Aspergillus oryzae. An experiment was conducted to determine the growth at various concentrations of tannic acid with and without 10 per cent cane sugar. In one case tannic acid (Merck’s tested reagent) was added to solution B; in the other, tannic acid + 10 per cent sugar wasused. Test-tube cultures with 15 cc. of the solutions were em- _ ployed. The results in general showed that these two fungi develop normally in the presence of 2.5 per cent tannic acid, but greater concentrations decrease the rate of germination and inhibit the growth. In the 15 per cent concentrations of tannic acid, after nine days, only one-third of the surface was felted. Up to 7.5 per cent concentration the entire surface was felted. Conclusion and discussion. The experiments on the toxicity of tannic acid indicate that of all the organisms tested, Asper- | gillus niger and Penicillium sp.“ are best adapted for the tannic i acid fermentation. These two organisms were, therefore, selected for more detailed investigation, though the other two organisms previously mentioned were also reserved for further study. Since the above experiments were made, a bulletin has appeared on the toxicity of tannin by Cook and Taubenhaus.22 The major- ity of a large number of parasitic organisms tested by them with respect to the toxicity of tannin show retardation of growth’ at from 0.1 per cent to 0.8 per cent of tannin. The few saprophytic forms tested exhibit a more marked resistance. My own experi- ments indicate also that the saprophytic forms can withstand relatively higher concentrations of tannic acid than the parasitic forms. 22 See appendix for description of this organism. 23M. T. Cook and J. J. Taubenhaus: The Relation of Parasitic Fung! to the Contents of the Cells of the Host Plant, Delaware Agric. Exp. Station, Bulletin 91, 77 pp., 1911. 166 Tannic Acid Fermentation VY. COMPARISON OF ORGANISMS AND INFLUENCE OF TIME AND GROWTH ON FERMENTATION. Comparison of organisms. Employing an infusion of gall nuts containing 10.5 per cent tannic acid, VanTieghem* found that the fermentation was completed by Aspergillus niger in six days and by Penicillium glaucum in eight days, when the temperature of incubation was 35° and the supply of oxygen was limited by keep- ing the flask stoppered. In the weaker concentrations, Penicillium glaucum fermented the tannic acid more vigorously. Since in VanTieghem’s experiments the temperature was rather high and " the conditions of growth approached anaerobic conditions, it was believed that a better comparison could be made by supplying the optimum conditions for the growth of each. In all the natural fermentations (in unsterilized solution) of the tannic acid, Asper- gillus niger always developed first and then Penicillium sp. So marked is this succession that by exposing a nutrient solution of 10 per cent tannic acid to the air a pure culture of Aspergillus niger may usually be obtained. This suggests that Aspergillus niger may possess the greater fermentative capacity, but an experi- ment was required. In the first experiment solution A and tannic acid were used, 50 cc. of the solution being placed in Erlenmeyer flasks of 150 cc. capacity. The flasks were sterilized for forty-five minutes at 115°, inoculated, and kept for two weeks at a temperature of from 18°-28°, the average being close to 24°. The felts were then removed, thoroughly washed, and the wash water added to the culture solution, the solution being then brought up to 500 cc. volume and analyzed according to Dreaper’s*® method. The figures below are for Aspergillus niger, the averages of six cultures, while for Penicillium sp) the averages of five cultures are given. TABLE I. 'TANNIC ACID) LOSS IN aki ACID DRY ORGANISM Te acenteee | ITANNIC ACID i Season sktaie has eONaGs, e grams grams grams grams gram Aspergillus niger..........| 0.228 1,244 0.200 2.752 0.848 Penicillium sp............ 0.332 1.140 1.780 1.172 0.537 Checkinsccnunteereninccny slay 1.472 2.952 24 Toc. cit. 2 Toc. cit. Lewis Knudson 167 The loss in gallic acid indicates that this substance is used by the organisms as a source of carbon, which fact agrees with the observations of VanTieghem” and Pottevin.2”? According to Van- Tieghem, when growth occurs on the surface the tannic acid is uti- lized directly without previous conversion into gallic acid. There is no evidence for this assumption. If the tannic acid is not utilized directly, and it probably is not, then Aspergillus niger is a more vigorous fermentative organism than Penicillium sp. for in the Penicillium culture more tannic acid remained and the decrease in gallic acid was only 39 percent. The larger gallic acid content of the Penicillium culture is related to the smaller amount of growth and not to the greater practical efficiency as a fermenta- tive organism, and this point is more apparent from Jater work. In table II there are given, separately, data for four of the Penicillium cultures. This table emphasizes a general relation between the amount of growth and the extent of fermentation; furthermore, the disappearance of gallic acid is correlated with increased growth. TABLE II. “TANNIC ACID GALLIC ACID NO. | PN CURE | ayxwre acim | FN CULTURE | gyrirc xc | “FUNGUS” grams grams grams grams gram Check 1.472 2.952 1 1.216 0.156 2.396 0.556 0.295 2 0.140 1.332 2.240 0.700 0.450 3 0.152 1.320 1.760 1.192 0.550 4 0.100 1.372 1.308 1.644 0.700 Culture No. 1 seems to indicate that the tannic acid transfor- mation is dependent upon the amount of growth, for only a small amount of tannic acid was transformed, but a greater growth during the same period in the other cultures resulted also in almost complete transformation. Since the most economical production of gallic acid is dependent upon the amount of growth, and growth amount is a function of time, temperature, aeration, and nutrition, then these factors should be important. Growth is emphasized because with it 26 Loc. cit. 27 Loc. cit. 168 Tannic Acid Fermentation the amount of the enzyme is probably correlated, at least with respect to Penicillium sp. Culture No. 1 shows a small decrease in tannic acid and a high decrease in gallic acid. In culture No. 2 the loss of tannic acid is greater than loss of gallic acid. The probable explanation is that Penicillium sp. utilizes first the gallic acid and then transfor- mation of the tannic acid occurs. In culture No. 1 the smaller growth has been at the expense of tannic acid. It does not seem possible to demonstrate that tannic acid is not directly utilized but it seems probable that it must be converted into gallic acid. Influence of duration and extent of growth and comparison of organ- isms. In order to determine the yield of gallic acid at different intervals, and so to note the influence of growth and duration on the fermentation, an experiment was made as follows: Solution B was used and to it were added 7.5 grams of Merck’s purified tannic acid per 100 cc. of solution. The concentration of the tannic acid was such that it came within the limits, as found by Van Tieghem,?* most favorable for Penicillium glaucum. For the investigation 50 cc. of the culture solution were placed in Erlenmeyer flasks of 150 cc. capacity. The cultures were sterilized for fifteen minutes at 5 pounds’ pressure and then inoculated according to the method described. The cultures were kept in an incubator at 31° and, at intervals, as indicated, duplicate cultures were taken for analyses. The mycelium was removed by filtering with suction through the Gooch crucible, and it was then washed with warm water to dissolve all adhering gallic acid. The solution was then brought up to 500 cc. and analyzed according to the method of Jean.*® The results of the experiment are given in Table IIT. In culture No. 8 the amount of gallic acid decrease was 0.126 gram and the tannic acid decrease only 0.069 gram, recalling the case of No. 1, table II. Penicillium sp., it seems, therefore util- izes the gallic acid first, and then the secretion of enzyme involves the transformation of the tannic acid. In all of the succeeding cultures the tannic acid decrease is greater than the increase of gallic acid, and this is to be accounted for, again, not by a direct utilization of tannic acid but by the fact that the tannic acid is first converted into gallic acid; and the constant increase of the °8 Doc. ett. 29 Loc. cit. Lewis Knudson 169 TABLE III. Organism, Aspergillus niger. TANNIC ACID] 145, op |@ALLIC ACID} Loss DRY courumn xo, | PURAMON a SE Pan Ae ooo on oA om | tcam or days grams grams grams grams gram 1 4 0.750 0.853 2.584 | +0.484 | 0.0314 2 6 1.057 0.546 2.191 | +0.091 | 0.1568 3 8 0.252 1.351 1.814 | —0.286] 0.1946 4 10 0.306 1.297 1.001 | —0.499 | 0.4688 5 12 0.160 1.443 1.460 | —0.640 | 0.4933 6 16 0.159 1.444 1.406 | —0.694 | 0.5137 7 28 0 1.603 0.898 | —1.202 | 0.6345 Check | 1.603 2.100 Organism, Penicillium sp. 8 4 1.534 | 0.069 1.974 | —0.126 | 0.0157 9 6 1.330 0.273 2.191 | +0.091 | 0.0800 10 j 8 1.296 0.305 1.769 | —0.331 | 0.1458 ¢ 11 ' 10 0.750 0.853 1.669 {| —0.481 | 0.3031 12 . 12 0.427 1.176 1.333 | —0.767 | 0.3880 13 16 0.597 1.006 1.530 | —0.670| 0.4096 14 | 28 0.106 1.497 1.277 | —0.823 | 0.4284 gallic acid prevents the utilization of the gallic acid from being made manifest. If the Aspergillus niger and Penicillium sp. cultures are com- pared, one finds that the tannic acid in the Aspergillus culture is transformed to the extent of 81 per cent by the tenth day; whereas in the Penicillium cultures this transformation, for the correspond- ing time, is only 53.3 per cent of the tannic acid. Moreover, by the fourth day the gallic acid had increased in culture No. 2 by nearly 0.5 gram, while in culture No. 8, for a corresponding time, there was a decrease of gallic acid. With this concentration and temperature, therefore, the Aspergillus niger is a morevigorous fermentative organism than Penicillium sp. If now the time factor and amount of growth be examined in their relation to the gallic acid, it is found that with the above solutions and under the specified conditions, the gallic acid de- creases after the sixth day and is utilized in the further metabolism of the organism. Z 170 Tannic Acid Fermentation Comparison of fermentative capacity in an infusion of gall nuts. For the investigation an extraction of the gall nuts was made as follows: 1800 grams of the Aleppo gall nuts were placed in one Jar and the same quantity of Chinese nuts in another. To each were added 3 liters of tap water, and the extraction was allowed to continue for five days, when the extract was filtered. To each jar were added again 2 liters of water, and after one day the ex- tracts were filtered and combined with the previous filtrates. Then the two extracts from the Chinese and Aleppo galls were mixed. After a second filtration the solution was ready for the culture vessels. For this purpose cultures were made in the usual way, using Erlenmeyer flasks of 150 cc., each, with 50 cc. of the infusion. TABLE IV. TANNIC scrp| GALL.c AcID DRY F DURATION |IN CULTURE), annie nei IN CULTURE), goaee aon WEIGHT OF CULTURE NO. SOLUTION SOLUT:ON FUNGUS days grams grams grams grams gram Check 1.637 1.797 Aspergillus niger. 0.955 1 4 0.545 1.092 2.752 : 2 6 0.218 1.419 3.218 1.421 | 0.197 3 8 0 1.637 2.500 0.703 | 0.262 4 12 0 1.637 2.527 0.730 | 0.171 5 20 0.206 1.431 2.527 0.730 | 0.205 6 56 0 1.637 2.527 0.730 | 0.148 Penicillium sp. 7 4 1.330 0.307 2.050 0.253 | 0.043 8 6 0.545 1.092 2.627 0.730 | 0.157 8 2 9 0.409 1.128 2.387 0.590 | 0.110 10 1 0.034 1.603 2.471 0.674) 0.171 11 20 0.085 1.552 2.457 0.660 | 0.117 12 56 0.152 1.415 2.247 0.450} 0.151 A comparison of the two organisms, as regards their fermenta- tive capacity, shows again that Aspergillus niger is a more efficient organism. Note especially that in four days the Aspergillus cul- tures exhibit an increase of gallic acid nearly four times as great as that in the Penicillium cultures, and all of the tannic acid had been converted in the former by the eighth day, while in the Peni- Lewis Knudson 171 cillium cultures only 70 per cent was converted in the same time. Not only does Aspergillus niger produce a more rapid fermenta- tion, but also a greater production of gallic acid is effected; for, as indicated, the maximum yield of gallic acid in the Aspergillus cultures is twice that in-the Penicillium cultures, and this in spite of the fact that a greater weight of Aspergillus niger is produced. Furthermore, in the Penicillium cultures there occurs a decrease of the gallic acid even when considerable tannic acid is still present in the culture solution. In order to understand these differences between Aspergillus niger and Penicillium sp., the composition of the infusion must first be considered and an idea of this may be obtained from con- stituents of the gall nuts. In addition to tannic acid, the gall nuts contain (Guibert**) gallic acid, chlorophyll, starch, gums, sugar, proteins and various inorganic salts and other compounds. A water extract of the gall nuts would contain in solution and suspension a certain amount of most of these substances, and the subsequent sterilization would probably result in transformations which would make certain of the organic compounds more avail- able. Aspergillus niger is an omnivorous organism in its relation to the utilization of carbon compounds. The growth of this organism for the first few days is probably at the expense of the other organic substances present, and the gallic acid, in this case, accumulates. All the facts indicate that while Aspergillus niger is utilizing the organic compounds other than tannic acid, it secretes the enzyme tannase (this point will be further developed in a separate paper), and consequently the transformation of the tannic acid goes on, and gallic acid accumulates. When the other organic compounds are exhausted, the gallic acid is utilized, and then the decrease begins. As shown previously, Penicillium sp. tends to utilize the gallic acid before it transforms the tannic acid, for gallic acid is a favorable nutrient carbon compound for this organism. Further- more, as I will show in a later paper, the presence of the other or- ganic compounds may decrease the secretion of the enzyme tan- nase by Penicillium sp.; and since the utilization of the gallic acid exceeds the formation of this substance, there results a decrease a0 Quoted from H. Trimble: loc. cit. 172 Tannic Acid Fermentation of the gallic acid despite the fact that tannic acid is present in the solution. A comparison of tables III and IV is of interest. The one experiment with a synthetic solution and the other with the infu- sion were conducted at the same time and under identical condi- tions as regards sterilization, inoculation and incubation. More- over, the tannic acid andthe gallic acid content of the culture solutions are nearly the same. In table V, only the duration of experiment, decrease of tannic acid, the loss or gain of gallic acid and the dry weights of the fungus produced, are included. TABLE V. Inrusion or GaLu Nuts: Tawnic Acip = 1.627 GRAMS Gauuic Acip = 1.797 GRAMS Sotution B + 1.603 gRams TAannic Acip + 2.100 gRams GaLtic AcIp DURATIO RY : LOSS OF | or ani OF watiakn On|) Oso... || CAINOF weicut oF TTANNIC ACID|O to t¢ agin] FUNGUS /TANNIC ACID|GALLac acrpj “Ear. days grams gram gram grams grams gram Aspergillus niger. 4 _ | 0.753 | +0.484 | 0.031 1.092 | +0.955 6 0.546 | +0.091 | 0.0156 | 1.419 | +1.421 |] 0.197 8 1.351 | —0.286| 0.194 1.637 | +0.703 | 0.262 12 1.443 | —0.640| 0.493 1.637 | +0.730} 0.171 16 1.444 | —0.694| 0.513 20 1.637 | +0.780| 0.148 Penicillium sp. 0.069 | —0.126|] 0.015 0.307 | +0.253 | 0.048 4 6 0.273 | +0.091 | 0.080 1.092 | +0.760} 0.157 8 0.305 | —0.331 | 0.145 1.128 | +0.590 |} 0.110 12 1.176 | —0.767 | 0.188 1.603 | +0.674| 0.171 16 1.016 | —0.670; 0.4096 20 1.415 | +0.450} 0.151 It is interesting to note that in the synthetic solution the gallic acid decreased after the eighth day, while in the gall nut infusion it showed at that time a marked increase, and this increase was maintained thereafter. In the synthetic solution on the eighth day there was a loss of 0.286 gram of gallic acid, while in the gall nut infusion there was a gain of 0.703 gram of gallic acid, and in Lewis Knudson 173 the latter the tannic acid was completely transformed. The differ- ence in the amount of tannic acid transformed was not sufficient, however, to account for the loss of gallic acid in the synthetic solu- tion and the marked increase in the infusion culture. Despite the greater weight of the fungus this increase of gallic acid was more in the infusion culture. A similar condition existed at the end of six days. The explanation of this point seems to be found in the fact that the infusion cultures contain various organic compounds which are utilized in place of the gallic acid; that is, elected in preference. At the end of four days probably none of the gallic acid has been assimilated. The subsequent decrease in gallic acid was due to its use after the exhaustion of more favorable organic nutrients. After the sixth day in the infusion culture there was no further growth of the organisms and no decrease in the gallic acid. The growth was less than one-half of that in the synthetic cultures, probably due to the lack of inorganic nutrients, although the presence of injurious metabolic products might also have been a factor, as the organic food supply was by no means exhausted. The conditions which obtained for the Aspergillus niger cultures apply also to those of Penicillium sp. Another point of interest brought out by the comparison is the more rapid fermentation in the infusion than in the synthetic solution. In the Aspergillus cultures the tannic acid was com- pletely transformed in the gall nut infusion by the eighth day, when the weight of the fungus was 0.262 gram. In the synthetic cul- tures transformation was not complete by the sixteenth day, when the weight of dry mycelium was 0.513 gram. In the Penicillium cultures, also, the results are comparable. In another experiment in which a gall nut infusion was used containing 2.04 grams of tannic acid and 0.56 gram of gallic acid per 50 cc., at room temperature, there was maintained for thirty days an increase of gallic acid. Practically all of the fermentation occurred before the eleventh day, and the gallic acid was protected by the other organic substances. Since the experiments with the infusion cultures indicated that the gallic acid was protected to a certain extent by the election of other organic substances, it was determined to try the addition of sugar to the culture solution with respect to its effect on the election of foods and hence on the yield of gallic acid. 174 Tannic Acid Fermentation VI. INFLUENCE OF THE ADDITION OF SUGAR. Effect of & per cent sugar. For these cultures solution A was used to which was added in the one series tannic acid alone, and to the other, tannic acid and cane sugar. Sugar was added at the rate of 5 grams per 100 cc. of solution, and the amount of tannic acid is indicated by the check. The cultures were made in liter flasks, and 500 cc. of the solution were used for each. The flask and contents were sterilized, inoculated and kept in the incubator at a temperature which varied from 27°-30°C. The results of the experiment given in table VI are in all cases obtained from dupli- cate cultures. TABLE VI. | ITANNIC ACID] rogg yy GALLIC ACTD| ~— GAIN DRY | DURATION [IN CULTURE, wwro actp|'N CULTURE) OR LOSS OF | WEIGHT OF CULTURE NO. SOLUTION SOLUTION |GALLIC ACID| FUNGUS | days grams grams grams grams grams Series I. Solution A + tannic acid. 1 | 10 4.568 8.193 | 11.095 | +6.867 1.42 2 | 20 0.203 | 12.558 2.190 | —2.038 3.66 3 | 30 | 0.050) 12.711 0.142 | —4.086 4.00 Check 12.761 | 4.228 Series II. Solution A + tannic acid + cane sugar. 4 | 10 0.913 | 11.848] 8.761 | +4.533) 6.008 5 / 20 0.812; 11.949! 3.904} —0.324) 10.35 6 30 0.101 | 12.660; 0.143 | —4.085 | 13.10 12.761 | Check 4,298 The results of the experiment seemed at first surprising. Instead of getting a protective action of the sugar with respect to the gallic acid, and thereby an increased yield of gallic acid, the opposite condition seemed to result. The yield of gallic acid was actually less at the end of ten days in the solution containing sugar than in the solution which lacked sugar, even though more of the tannic acid was transformed in the former. At the end of twenty days more gallic acid was left in the cultures of series II than in the corre- sponding culture solutions of series I, and so a certain protective action of the sugar is evident. At the end of thirty days practi- cally all of the tannic acid and gallic acid had disappeared from Lewis Knudson 175 the culture solutions. In explanation of the seeming failure of the sugar to protect the gallic acid, the dry weights.of felts produced in the corresponding cultures must be compared. The weight of fungus produced in each culture of series II was, at the end of each period, at least three times as great as the weight of the correspond- ing cultures of series I. This increased growth and the accompany- ing increased respiration were sufficient to utilize practically all of the organic nutrients supplied, usually all of the sugar and some of the gallic acid. Effect of 10 per cent sugar. Since negative results wereobtained as regards the protection of the gallic acid by 5 per cent sugar.a new series of cultures was made with 10 per cent sugar in solution B to which was added the tannic acid required. On analysis the solution showed after sterilization 4.171 grams of tannic acid and 2.198 grams of gallic acid per 50 cc. The cultures were incubated at a temperature of 28°, though it dropped occasionally, owing to an imperfect thermostat, to 25° and rose likewise to 32°. The cultures were taken down at definite intervals, the weight of the felts determined, and the analyses of the culture solutions made according to the methods previously described. The results follow in table VII. The protective action of the sugar is at once evident. Since the concentration of tannic acid here is double that in the experiments which are included in table III, and therefore a comparison of the . yields of gallic acid is not possible, yet the great increase of gallic acid and the maintenance of this increase prove that the sugar has been utilized in place of the gallic acid. Here it is obviously not true that the greater the weight the less the gallic acid. Cultures Nos. 4, 5, 6 and 7 all vary in weight, yet the amount of gallic acid in each is approximately the same, and any difference may be due to the imperfect method of analysis. Even at the expiration of thirty-five days no decrease of the gallic acid was evident. It may be concluded, therefore, and further experiments prove, that _ the 10 per cent sugar protects the gallic acid. While an increase of gallic acid is maintained in the Penicillium cultures, the increase is relatively small and is due to a slower trans- formation of the tannic acid; practically no transformation of tannic acid occurred until after the fourteenth day. Previous to this time the growth was entirely submersed, but afterwards fructi- THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XIV, NO. 3. 176 Tannic Acid Fermentation TABLE VII. een purarion | zw couTunn souu- | INOREASE OF Gal- | DRY WetauT OF NO. TIONS days grams grams gram Check 2.198 Aspergillus niger. 1 3 4.929 2.731 0.1579 2 6 5.028 2.830 0.4093 3 8 5.533 3.335 0.4135 4 14 *6 320 4.122 0.5148 5 20 *6 320 4.122 0.4470 6 25 *6§ 292 4.094 0.4820 7 35 *6 348 4.150 0.4986 Penicillium sp. 8 6 2.190 0 0.0076 9 14 2.261 0.063 0.332 10 20 3.764 1.574 0.265 11 25 3.764 1.574 lost 12 35 +3.820 1.630 0.4515 13 56 4.101 1.911 0.5570 * Transformation of tannic acid complete. ¢ Considerable tannic acid still left in culture solution. fication occurred. According to Malfitano® the secretion of the enzymes from Aspergillus occurs just after fructification, and it is possible to conclude that the lack of transformation is explain- able by non-secretion of the enzyme by the submersed growth. VanTieghem,? however, found that transformation was effected when the growth was submersed; and some experiments of the writer subsequently confirm the fermentation by submersed growth. The non-transformation is probably due to the inhibiting action of the sugar on the secretion of the enzyme tannase, but not on the formation of the enzyme, for subsequent experiments have shown that with 2 per cent tannic acid and 10 per cent sugar the enzyme is formed. The inhibition of enzyme secretion by the presence of organic nutrients has been observed by Puriewitsch.® 2G. Malfitano: La proteolyze chez |’ Aspergillus niger, Ann. d. l’Inst. Pasteur, xiv, pp. 60-81, 1900. 32 Loc. cit. 1K. Puriewitsch: Ueber die Spaltung der Glycoside durch die Schim- melpilze, Ber. d. deutsch. bot. Gesellsch., xvi, pp. 368-377, 1898. Lewis Knudson 177 The transformation of salicin or arbutin was inhibited in the pres- ence of certain amounts of glucose, cane sugar or starch. Like- wise Katz*™ found that Penicillium glaucum did not secrete the enzyme diastase when, along with 0.25 per cent soluble starch, either +. 2 per cent glucose or 15 per cent cane sugar was offered. Asper- gillus niger was not influenced noticeably in the secretion of the tannase by the presence of the 10 per cent cane sugar, while the Penicillium sp. was markedly influenced. Another noteworthy fact in regard to the Penicillium sp. is the small increase of gallic acid between the twentieth and thirty-fifth day. It seems that the enzyme secreted was so very little, or of such feeble activity or destroyed, that a very limited transforma- tion only was effected. Even at the end of fifty-six days the tannic acid was not entirely converted, and the increase of gallic acid after the end of thirty-five days was relatively small. Influence of concentration of sugar on growth. For an explana- tion of the protective action of 10 per cent sugar the following table is suggestive. Each result is the average of sixteen cultures of Aspergillus niger. TABLE VIII. eee ae COMPOSITION OF NUTRIENT SOLUTION ix couaURE Dae naa per cent grams 1 Check: 50 cc. sol. A + trace FeeCls + 2.5 gms. sugar.................. 5 ' 0.779 2 50 cc. sol. A + trace Fe2Cl; + 8.55 gms. sugar................. 17.1 1.239 3 50 cc. sol. A + trace FeCl; + 17.1 gms. sugar................. 34.2 1.513 4 50 cc. sol. A + trace Fe.Cls + 25.65 gms. sugar................ 51.3 1.590 5 50 cc. sol. A -+ trace FeCl; : 7 + 37.00 gms. sugar................ 74.0 1.308 6 50 cc. sol. A + trace Fe2Cl, + 42.50 gms. sugar............... 85.0 1.230 The table shows that the optimum sugar content is above 5 per cent. It is possible that the increased growth in the culture solution of over 10 per cent sugar is produced not by an assimi- 34 J, Katz: Die regulatorische Bildung von Diastase durch Pilze, Jahrb- f. wiss. Bot., xxxi, pp. 599-618, 1898. 178 Tannic Acid Fermentation lation of more sugar but by a stimulus, the result of high concen- tration. The table is offered here, however, only to indicate that with the nutrient solution used 5 per cent sugar is not sufficient for greatest growth. It throws light also upon the failure of the 5 per cent sugar to protect the gallic acid in the previous experi- ment. VII. ELECTION OF ORGANIC SUBSTANCES. Historical. VanTieghem® stated that the glucose formed as a result of tannic acid fermentation was utilized and the gallic acid left behind. Pas- teur demonstrated that Penicillium glaucum exhibited an election of the dextro-tartaric acid when both the dextro- and laevo-tartaric acids were present. Duclaux’s** observations revealed the fact that when Aspergillus niger was offered salts of butyric and acetic acid in a mixture, it first used the latter and then the butyric acid. Furthermore, he proved that this was not due to the better nutrient value of acetic acid, for when the acetate was offered with the tartrate (an especially good nutrient) the acetate was utilized more rapidly. The election then was not merely a matter of rela- tive food value. Pfeffer? found that under certain conditions the use of glycerin by fungi may be protected by dextrose and even better by peptone, and he showed also that the relative concentration of each had an effect upon the election. Puriewitsch** found with two organisms an election with respect to the products of amygdalin. With this substance as the source of carbon it was first transformed; then the dextrose and lastly the benzaldehyde was used. He found further that salicin was not transformed in the presence of six times its quantity of dextrose, twelve times its quantity of saccharose or fourteen to sixteen times its quantity of starch. Election of cane sugar. In order to determine definitely whether or not Aspergillus niger and Penicillium sp. elect cane sugar, when it is offered together with gallic acid, two series of cultures were made. Solution B was used, to which was added in the one case the gallic acid and 10 per cent of cane sugar, the cultures being made in Erlenmeyer flasks as before. In a similar manner cultures were made in which the gallic acid alone was offered as the source of carbon. Sterilization and inoculation were made by the usual 35 Toc. cit. 86. DuClaux: Sur la nutrition intracellulaire, Ann. de l’Inst. Pasteur, lii, pp. 97-112, 1889. a7 W,, Pfeffer: Ueber Election organischer Nahrstoffe, Jahrb. f. wiss. Bot., XxXvill, pp. 205-268, 1895. 38 Toc. cit. Lewis Knudson 179 methods, and the cultures incubated at 28° C. The results ob- tained are included in table IX. TABLE IX. GALLIC ACID ; DURATION IN CULTURE ena DRT WEIGRT ORGANISM SOLUTION OF FUNGUS days grams grams grams Series I. Solution B + 10 per cent sugar + gallic acid. Cheek secs cae ces 2.837 Aspergillus niger....... 7 2.837 none 0.3491 Penicillium sp......... 7 2.837 none 0.1109 Aspergillus niger....... 10 2.837 none 0.4589 Penicillium sp......... 10 2.837 none 0.3676 Series II. Solution B + gallic acid. CHECK a ascsacie aa torant 2.837 Aspergillus niger:...... 7 2.429 0.337 0.1000 Penicillium sp......... 7 2.837 |notdetected| 0.010 Aspergillus niger....... 10 1.474 1.363 0.3434 Penicillium sp......... 10 2.557 0.280 0.108 A glance at the table reveals the fact that both Penicillium sp. and Aspergillus niger elect cane sugar and leave behind in the culture solution the gallic acid. Cane sugar is therefore proven conclu- sively to protect the gallic acid. It is of interest to note that the addition of the sugar permits of a more rapid and more extensive growth during the ten-day period. VIII. INFLUENCE OF AERATION. Limiting supply of oxygen. VanTieghem*® stated that under aerobic conditions the tannic acid was utilized directly, and that the small amount of gallic acid formed was also assimilated. The preceding experiments and others of the writer, not here men- tioned, show that transformation of the tannic acid occurs even when all the growth is on the surface. However, if sugar is not offered with the tannic acid, the increased growth may, as shown, be at the expense of the gallic acid formed. If the tannic acid is 39 Doc. ctt. 180 Tannic Acid Fermentation offered as the only source of carbon, the yield of gallic acid is obviously dependent upon the amount of growth. Van Tieg- hem? found that 0.022 gram’s weight of mycelium transformed 48.3 grams of tannic acid in ten days at 35°. The growth is greatly diminished by the absence of oxygen, consequently the oxygen supply is a factor influencing the yield of gallic acid. In order to compare the yield under aerobic and anaerobic conditions, another experiment was performed. Solution B was used, to which was added tannic acid. Into each of six Erlenmeyers of 150 cc. capacity were placed 50 cc. of the solution. Four of the flasks were plugged with cotton, while the other two were fitted with perforated rubber stoppers. After sterilization the perfora- tions were plugged with pieces of glass rod and these, together with two of the flasks plugged with cotton, were inoculated with Asper- gillus niger. The flasks fitted with the rubber stoppers contained approximately 100 cc. of air and therefore about 20 cc. of oxygen. The cultures were incubated at 31° and at the end of twenty-eight days and forty days analyses were made. The results follow in table X. TABLE X. * TANNIC ACID LOSS OF GALLIC ACID} LOSS. Dan CULTURE BOLUTION DURATION |IN CULTURE TANNIC ACID IN CULTURE! OR GAIN IN| WEIGHT OF SOLUTION SOLUTION |GALLIC ACID} FUNGUS days grams grams grams ene Anaerobic (limited)...... 40 0 1.603 3.286 | +1.186} 0.158 Aerobic.......... 28 0 1.603 0.898 | —1 202] 0.634 Check........... 1.603 2.100 It is at once evident that the inhibition of growth due to defi- ciency of oxygen is favorable to a good yield of gallic acid. No doubt if the culture in limited oxygen supply had been analyzed sooner a greater yield of gallic acid could have been obtained. With respect to the condition in aerobic cultures it may be stated that cultures, identical with those above, showed on the fourth day a gain of 0.484 gram of gallic acid, though only half of the tannic acid had been transformed, and the weight of the mycelium pro- duced was 0.0314 gram. Comparison of methods. In order to determine more definitely the yield of the gallic acid under conditions in which the supply of 40 Loc. cit. Lewis Knudson 181 oxygen varied, as well as to compare the yields under these condi- tions with that obtained when the most favorable conditions were offered, as by the addition of sugar, the following experiment was made: Series I. Solution B + tannic acid, flask plugged with cotton and aero- bic conditions maintained. Series II. Solution B + tannic acid + 10 per cent cane sugar, otherwise like the above. Szrizes III. Solution B + tannic acid, flasks stoppered with rubber stoppers and containing therefore only 75 cc. of air or approximately 15 cc. of oxygen. Series IV. Solution B. + tannic acid, flasks stoppered with perforated rubber stoppers, fitted with glass and rubber tubing and clamps. The air was replaced by passing a stream of nitrogen (oxygen-free air) through the flasks for a period of five minutes after inoculation had been made. All the inoculations were made with spores of Aspergillus niger, according to the method described by Hasselbring.‘1 The temperature of incubation varied from 30°-35°. Erlenmeyer flasks of 125 ec. capacity were employed The results obtained are included in table XT. TABLE XI. Aspergillus niger. Duration, ten days. |TANNIC ACID GALLIC ACID DRY seates | gnowna cuampen /SCOMPUR? gaunt acto Ps ogg (@ALLie ACID “pases grams grams grams grams gram Check 4.433 3.089 I Unlimited air supply 0.409 | 4.024 | 5.618 | 2.529 | 0.3166 II Unlimited air supply 0.683 | 3.760 | 6.515 | 3.426 | 0.3341 III 75 cc. air 1.023 3.410 6.151 3.062 0.0114 IV Nitrogen 1.707 2.726 5.420 2.331 0.0013 In comparing the rate of tannic acid transformation under the different conditions it is found that in the order of rapidity of transformation they are series I to series IV. The yield of gallic acid was greatest in series II, the addition of cane sugar in this case protecting the gallic acid; somewhat less in series III; still less in series I, where the mycelium was abundant, despite the fact that it led in tHe amount of tannic acid transformed; and last in series IV. It is noteworthy that the amount of mycelium pro- 4t Doc. cit. ‘ 182 Tannic Acid Fermentation duced in this last was only 1.3 mgm., yet sufficient of the enzyme was liberated to transform a quantity of tannic acid more than 2000 times the weight of the mycelium produced. From an economic standpoint the method of series I is wasteful of gallic acid, as the organism utilizes much of this substance in its metabolism. Series II has an advantage over series III, and series III over series IV, only in the rapidity of the transformation. The small amounts of growth in series III and IV require such a slight amount of gallic acid in their metabolism that the yield of gallic acid in those series would finally be practically equal to that obtained in series II, to the cultures of which sugar had been added. This fact is borne out by the larger amounts of tannic acid left in the culture solutions of series II and series IIT. Ix. SUMMARY. 1. Tannic acid is toxic to a large number of fungi at relatively low concentrations. 2. Aspergillus niger is a more vigorous fermentative organism than Penicillium sp. 3. The fermentation was found to be more rapid in the gall nut infusion than in the synthetic solution in which tannic acid was the only source of carbon. The presence of other organic compounds in the gall nut infusion protected to a certain extent the gallic acid. 4, The addition of 5 per cent sugar did not protect the gallic acid but simply increased the growth. The addition of 10 per cent sugar protected the gall'c acid entirely. 5. When gallic acid and cane sugar to the extent of 5.5 per cent and 10 per cent, respectively, were offered together, the cane sugar was elected and the gallic acid left in the culture solution. 6. Fermentation can take place under anaerobic conditions, and 1 mgm. of mycelium is sufficient to effect the transformation of 2.706 grams of tannic acid in ten days. 7. In an approximately 15 per cent solution of tannic acid, fermentation was most rapid when the tannic acid alone served as the source of carbon, and when aerobic conditions were main- tained; yet the method of fermentation is wasteful from the stand- point of an economical yield of gallic acid. Lewis Knudson 183 8. The economical methods are (a) those in which growth occurs under aerobic conditions and the tannic acid is supplemented by cane sugar; or (b) those in which, with tannic acid alone, the sup- ply of oxygen is limited to a small amount. 9. The presence of 10 per cent cane sugar does not inhibit the secretion of the enzyme tannase by Aspergillus niger, but it does seem to inhibit to some extent the secretion of the tannase by Peni- cillium sp. 10. The enzyme is secreted into the culture solution by sub- mersed mycelium as well as by surface growth. There is no evi- dence that tannic acid is used directly; but the evidence seems to indicate that tannic acid is first transformed into gallic acid and the gallic acid then utilized. APPENDIX. Investigators previously occupied with tannic acid fermentation usually employed Aspergillus niger together with a species of Penicillium which they have designated Penicillium glaucum. As has been pointed out by Thom‘? the name Penicillium glaucum has in the past been applied to so many differ- ent species that the only idea conveyed by its use is a general concept of the genus. VanTieghem* applied the name to denote the species of Penicillium which he isolated from gall nuts, and it is probable that other students of tannic acid fermentation used the same organism. In the work of the writer a trial of a number of organisms was made and the Penicillium employed was secured from a culture labeled Penicillium olivaceum. That identification was incorrect, and then it was believed that the organism might be the one which develops on the gall nuts, but this also proved erroneous, as is indicated subsequently. In attempting to determine the Penicillium sp. used in these experiments it was found that the organism did not correspond to any of the species des- cribed by Thom.** Instead, however, of withholding publication of these investigations until the organism should be definitely determined, it was thought best to present here a brief description and certain cultural charac- teristics of the organism. It is, furthermore, the intention of the writer to make a study of the relation of the various species of Penicillium to tannic acid fermentation, and it is hoped by that time to have determined definitely the two species of Penicillium which are now known to develop in a 10 per cent solution of tannic acid. The Penicillium sp. used in these experiments possesses only a single whorl of unbranched conidia-bearing cells (sterigmata), and might therefore 42 Charles Thom: Cultural Studies of Penicillium,Bureau of Animal Industry, Bulletin 118, pp. 107, 1910. 43 Loc. cit. 44 Toc. cit. 184 Tannic Acid Fermentation be grouped with the genus Citromyces as founded ‘by Wehmer; but Thom* does not consider this a valid basis for differentiation of genera, and prefers to include this form of conidiophore under the genus Penicillium. This latter concept is here followed. ; Description of organism. Colonies on 15 per cent gelatin are, when young, of a faint green color which changes to an otter brown. On 15 per cent gela- tin + 3 per cent sugar the olive'green changes to ashy gray and then to greenish black. On bean agar the color is at first bluish green, and changes to dark olive green, and finally to a grayish color. The surface is velvety. The conidiophores arise vertically from the substratum and in length vary from 100u to 700u. The fructification averages 90 in length (it may be 200,), and its width is approximately 15u. The conidiiferous cells average 10u in length, the conidia are spherical, and 3y in diameter. A single whorl of simple conidia-bearing cells only is present as is represented by figures 1 and 2. Fig 1. Fie 2. Conidial fructification showing simple conidiferous cells (x 600). Cultural character. At room temperature 15 per cent gelatin is liquified in six days with the production of a strong ammoniacal odor; in the pres- ence of 3 per cent sugar the total liquefaction is retarded a day or more. The presence of bean juice still longer delays the liquefaction of the gelatin. On 15 per cent gelatin + 3 per cent sugar, the lower surface is colored yellow- ish to reddish brown, when grown in solution B + 10 per cent cane sugar the lower surface of the mycelial felt may be of asalmoncolor. Fruitingonsuch a solution at room temperature usually requires eight days. This organism possesses the ability to ferment tannic acid and with 10 per cent tannic acidin solution B at a temperature of 30°C., gallic acid may be precipitated in about seven days. Gall nut Penicillium. Only one other species of Penicillium has so far been found to grow on 10 per cent tannic acid, and this is the one isolated from the gall nuts. . It grows more slowly in 10 per cent tannic acid. Inthe presence of sugar it produces an intensely red color in the substratum and is a slow liquefier of gelatin. It differs from the other also in possessing more than one whorl of conidiiferous cells, and has other distinguishing features. It appears to be Penicillium rugulosum. 48 Loc. cil. Reprinted from Tae JouRNAL or Brorogicat Cuemistry, Vou. XIV, No. 3, 1913. TANNIC ACID FERMENTATION. II. EFFECT OF NUTRITION ON THE PRODUCTION OF THE ENZYME TANNASE. By LEWIS KNUDSON. (From the Laboratory of Plant Physiology, Cornell University, Ithaca, New York.) (Received for publication, January 30, 1913.) I. PREFACE, In an investigation upon tannic acid fermentation reported in the previous paper, it was found that when cane sugar. and tannic acid are offered simultaneously to either Aspergillus niger or Penicillium sp., the sugar is utilized as the source of carbon while the tannic acid is fermented, gallic acid resulting. Some of the results indicated that the rate of fermentation was influenced by the concentration of the sugar. It was deemed important, there- fore, to determine if varying the relative amounts of tannic acid and sugar inthe nutrient solution has an influence upon the amount of the enzyme tannase produced in the fungus thus grown. Since tannic acid is probably not commonly utilized in nature by Aspergillus niger and Penicillium sp. as a source of carbon, experi- ments were also made to determine if the enzyme is produced when the fungus is cultivated on nutrient solution lacking tannic acid. The writer wishes to acknowledge his indebtedness to Prof. B. M. Duggar for assistance received during the course of the investiga- tion. II], INTRODUCTION. Regulatory production of enzymes. Historical. A number of investiga- tions have been made on the regulatory formation of the enzymes, but for the most part conclusive investigations are lacking. Experimenting with two species of bacillus, Brunton and MacFayden! found that when culti- 17. Lauder Brunton and A. MacFayden: The Ferment Action of Bac- teria, Proc. Roy. Soc., xlvi, B, pp. 543-553, 1899. 185 186 Nutrition and Tannase Production vated on starch paste these developed the enzyme diastase; but if the starch were replaced by meat extract no diastase was formed. Fermi? found that of ten bacterial organisms, which developed the tryptic ferment in the presence of peptones or albumen, none developed the enzyme on a sugar- containing nutrient solution. Wortman’s? observations established the fact that the addition of tartaric acid prevented the formation of diastase in bacteria which inhabited decaying potatoes. Fermi and Montesano’s‘ investigations indicated that the presence of sugar is not absolutely neces- sary for the formation of the enzyme invertase. Various other investi- gators have studied the influence of nutrition on the formation of enzymes in bacteria. Dubourg® stated that a yeast which did not normally possess the invert- ing enzyme was capable of developing it by proper cultivation. As a cul- ture solution in the latter case, yeast water was used, to which was added 5 per cent cane sugar and 5 per cent grapesugar. The yeast, after cultivation, was thoroughly washed and then transferred to a cane sugar solution. In the latter inversion occurred. The form of yeast was not an identified strain. He reported that he was also able to develop the enzyme which fermented galactose by similar methods. Klocker,® employing the methods of Dubourg, was unable to develop invertase in Saccharomyces apiculatus or maltase in Saccharomyces marzi- anus which organisms do not normally possess these enzymes. The ability to form specific enzymes, according to Klécker, is therefore a constant char- acter of the yeast organism. Recently Harden and Norris’ ‘‘have trained” the yeast Saccharomyces Carlsberg I to ferment galactose by cultivating the yeast on hydrolyzed lac- tose in yeast water to which was added 0.15 per cent of monobasic potassium’ phosphate. Normally galactose is not fermentable by the above men- tioned yeast. According to Kohl’ the chemical nature of the solution and the aeration of the culture influence the amount or activity of the enzyme formed in the yeast organism, while the temperature of storage of the yeast also markedly affects the enzyme content. . *Claudio Fermi: Weitere Untersuchungen iiber tryptischen Enzyme der Mikroorganismen, Centralbl. f. Bact., x, pp. 401-408, 1891. 3 J. Wortman: Untersuchungen tiber das diastatische Ferment der Bac- terien, Zetischr. f. physiol. Chem., vi, p. 287, 1882. *C, Fermi and C. Montesano: Die von den Mikroben bedingte Inver- sion des Rohrzuckers, Centralbl. f. Bact., i, Abt. II, pp. 482-87, 542-56. 5 BH. Dubourg: De la fermentation des saccharides, Compt. rend. del’ Acad. des Sct., exxviii, pp. 440-42. S Alb. Klécker: Ist die Enzymebildung bei der Alcoholgdrungspilzen ein verwertbares Artmerkmal?, Centralbl. f. Bact., vi, Abt. II, pp. 241- 45, 1900. 7A, Harden and R. V. Norris: The Fermentation of Galactose by Yeast and Yeast Juice, Proc. Roy. Soc., Ixxxii, B, pp. 64549, 1910. 8F. G. Kohl: Die Hefpilze, 1908, pp. 79-81. Lewis Knudson 187 Busgen® showed that Aspergillus oryzae,on bouillon, as well as in a sugar- containing solution, formed the enzyme diastase. According to Pfeffer,!® Penicillium glaucum did not secrete diastase in the presence of 10 per cent sugar and, even when only 1.5 per cent sugar was present, the starch was only slightly attacked. Aspergillus niger behaved differently, producing dias- tase even in the presence of 30 per cent cane sugar. Employing a nutrient solution containing 0.25 per cent of soluble starch Katz! found that starch was saccharified by Penicillium glaucum. The addition of 2 per cent grape sugar or 1.5 per cent cane sugar prevented the formation of the diastase. An addition of 1.5 per cent cane sugar depressed the formation of the diastase, while an addition of 0.05 per cent had no effect. Lactose and maltose in a 3 per cent concentration decreased the rate of starch transformation, while a 10 per cent concentration still further depressed the formation of diastase. A 4 per cent addition of erythrodextrin had no effect whatsoever in protect- ing the starch. Neither did a 10 per cent addition of quinic acid, 4 per cent glycerin or 2 per cent potassium tartrate have any effect upon the secre- tion of the diastase. The addition of peptone to the solution increased the secretion of the diastase. With Aspergillus niger the growth on starch nutrient solution was slow, and five days were required for the transforma- tion of the starch. The addition of 1.5 per cent cane sugar decreased the time to two days, 15 per cent sugar increased the time of transformation by one day and 30 per cent sugar increased the time by two days. Bacterium megatherium behaved much the same as Penicillium glaucum. Dox? has shown that the carbohydrate-splitting enzymes, amylase, inulase,.raffinase, sucrase, maltase and lactase are formed in Penicillium camembertt, regardless of the carbohydrate which has served as the source of carbon in the nutrient solution. The amount of the particular enzyme could be increased, however, by cultivating the organism on the correspond- ing carbohydrate. Likewise, other enzymes are formed independently of the presence in the nutrient solution of the corresponding substance on which the enzyme acts. According to Went, the ten enzymes which he investigated in Monillia sitophila could be divided into three groups according to the influence of nutrition on their formation. The first group includes those which are formed in slight amounts regardless of the nutrition, the second group in- 9M. Biisgen: Aspergillus oryzae, Ber. d. deutsch. bot. Gesellsch., iii, pp. 66-77, 1885. 10 Quoted from R. Green: The Soluble Ferments and Fermentation, p. 32. 11 J, Katz: Die regulatorische Bildung von Diastase durch Pilze, Jahrb. f. wiss. Bot., xxxi, pp. 599-618, 1898. 12 A. W. Dox: The intracellular Enzymes of Penicillium and Aspergillus, U. 8. Dept. of Agric., Bureau of Animal Industry, Bulletin 120, 70 pp., 10. ae F. C. Went: Ueber den Einfluss der Nahrung auf die Enzymbildung durch Monillia Sitophila (Mont.), Sacc., Jahrb. f. wiss. Bot., xxxvi, pp. 611-664, 1901. 188 Nutrition and Tannase Production cludes the enzymes which are formed only under several different forms of nutrition, while the third group includes such enzymes as are formed only when the substance on which the enzyme acts is present in the culture solu- tion. Butkewitsch" also has shown that nutrition has an influence upon the secretion of the gelatin-dissolving enzyme by Aspergillus and Penicillium. III. METHODS OF EXPERIMENTATION. In all of the experiments upon the influence of nutrition on the production of the enzyme the organisms were cultivated in a syn- thetic nutrient solution, the inorganic composition being that of Czapek’s! formula. Throughout this paper the solution has been designated for convenience solution B and is as follows: Magnesium sulphate (cryst.)...........0.. 0000s cece eee ee 0.5 gram Dibasic potassium phosphate..................0.000005. 1.0 gram Potassium chloride.............0.000 00000 cece cee eee 0.5 gram Sodium nitrate........0.000 0000 c cece eee eee eee 2.0 grams Distilled awatensn, adr cargo ds eauetbe Sains ek Gade eas 1000 cc. All cultures were grown at a temperature of 28° or 30°. The fungus felt, when formed and before spore production, was removed from the nutrient solution, as it has been shown by Malfitano!® that the most abundant secretion of enzymes into the culture solu- tion takes place just after spore formation. The felt after removal was treated according to Albert and Buchner’s method for the pre- paration of ‘‘Acetondauerhefe,” as described by Dox,!” though in the method here employed the felt was not run through a hashing macbine. After the mycelium was dry, it was pulverized in a mortar and then placed in a vial until its enzymatic activity was to be determined. For determining the presence of the enzyme or the relative “4 W. Butkewitsch: Umwandlung der Eiweisstoffe durch die niederen Pilze im Zusammenhange mit einer Bedingungen ihrer Entwickelung, Jahrb. f. wiss. Bot., xxxviii, pp. 147-240, 1903. 8 Quoted from A. W. Dox: The Intracellular Enzymes of Penicillium and Aspergillus, U. S. Dept. of Agric., Bureau of Animal Industry, Bulletin 120, 70 pp. ,1910. 6G. Malfitano:. La proteolyse chez |’ Aspergillus niger, Ann. de. l’Inst. Pasteur, xiv, pp. 60-81, 1900. A.W. Dox: The Intracellular Enzymes of Penicillium and Aspergillus, loc. ctt. Lewis Knudson 189 amount of it present, the procedure was as follows: Either a 0.5 per cent, a 0.75 per cent or a 1.0 per cent solution of tannic acid was employed. To the flasks containing the solution were then added equal weights of the pulverized mycelium, the enzymatic activity of which was to be determined. There was also added, as an antiseptic, 2 per cent toluene. The flasks containing the solution and mycelium powder, being tightly stoppered, were then incubated for a definite period and then the solution analyzed for gallic acid according to Jean’s'® method. The relative increase in the gallic acid is taken as a measure of the amount of tannase pres- ent. IV. INFLUENCE OF CONCENTRATION OF SUGAR AND TANNIC ACID ON PRODUCTION OF TANNASE. Influence of concentration of tannic acid on the amount of enzyme produced. It was found in certain experiments,’ in which 10 per cent sugar plus tannic acid had been added to the nutrient solution, that the addition of sugar could not prevent the secre- tion of the enzyme tannase by Aspergillus niger, but the secretion by Penicillium sp. was markedly decreased. In order to deter- mine the minimum concentration of tannic acid which would stimulate the formation of the enzyme tannase and also what influence the concentration of tannic acid would have upon the amount, of tannase produced within the organism, the following experiment was made: Solution B was used, to which was added 10 per cent cane sugar. Liter flasks were employed, and to each were added 500 cc. of the solution. The flasks were plugged and sterilized, and when cool the tannic acid was added to each in vary- ing amounts, as is indicated in the table. For inoculation the method described by Hasselbring?” was employed, though 1 cc. of the spore-containing water was used instead of a single drop. These cultures were incubated at 28°C., and the felt was then removed and treated according to the method described. In determining the enzymatic activity of the mycelium powder 0.3 18 PF, Jean: Die Bestimmung des Tannins und der Gallussiure, Chem. Centralbl., 1900, pp. 1107-08. ; 19 |, Knudson: Tannic acid Fermentation I, this Journal, xiv, p. 159, 1918. 20H. H. Hasselbring: Carbon Assimilation of Penicillium, Bot. Gazette, xlv, pp. 176-193, 1908. 190 Nutrition and Tannase Production gram of the dried powder was added to a flask containing the tan- nic acid solution. Each flask contained 75 cc. of an approximately 0.9 per cent solution to which was added, as an antiseptic, 1 cc. of toluene. The results after one week’s incubation at 34° are given in table I. The solution contained at the beginning of incu- bation 0.555 gram tannic acid and 0.327 gram gallic acid. TABLE I. Effect of concentration of tannic acid on production of enzyme tannase; using nutrient solution B + 10 per cent sugar + tannic acid. Pertod of incuba- tion, 7 days.* = -— aapieon I ia 7 | AMOUNTOF GAIN 'TANNIC ACID] IN GALLIC ITANNIC ACID} IN GALLIC ADDED ACID ADDED AC{D a per cent gram ie per cent gram 0.00 | 0 f| O01 | © 0.01 | 0 0.10 | 0.058 0.10 | 0.059 0.49 | 0.065 : 0.80 | 0.223 | Pentcillium sp. 0.80 | 0.085 A ie 1.00 | 0.262 1.00 | 0.108 ESS 2.00 | 0.388 2.00 | 0.159 | 4.00 | 0.515 4.00 | 0.293 | 10.00 | 0.515 f| 0.525 AD \ No sugar vee SHO IRAE ao THIET GE pote ee Oe Pee tha Ob thes ten It is evident from the preceding table that there is a regulatory formation of the enzyme. There is a progressive increase in the amount of tannase with increase of tannic acid in the culture solu- tion. It is noteworthy that no tannase was produced when growth took place in a nutrient solution which lacked tannic acid. Itis somewhat remarkable that the formation of enzyme could be stimulated by 0.1 per cent of tannic acid (actually about 0.066 per cent) when there was present at the same time cane sugar in an amount more than one hundred times as great as the tannic acid. The stimulation by this small amount of tannic acid is even more surprising when previous experiments” are recalled in which the gallic acid formed from the tannic acid was protected by the cane sugar, no determinable amount of the gallic acid being assimilated. 21 UL, Knudson: loc. cit. Lewis Knudson 191 Why the increase in concentration of tannic acid should increase the amount of tannase is difficult to explain. The amount re- moved by the organism from the solution is undeterminable with the present method of analysis. Is the stimulation produced within the cell, or is it caused by contact of the tannic acid with the plasma membrane? . Tannic acid precipitates albuminous substances, and it might be possible that it reacts in this manner with the plasma membrane and this precipitation might be the stimulus for the production of the enzy- me. This explanation would not, however, include the stimulation to production of the tannase by gallic acid. A suggestive explanation for the increase of the tannase with increased concentration of the tannic acid is afforded by the work of Katz. In his experiments Katz found that diastase could be precipitated by tannic acid and rendered inactive, though when freed from the tannic acid by washing with alcohol it becomes active. Bearing in mind this precipitation by tannic acid and working on the hypothesis that if the diastase formed and secreted into the culture solution were removed from solution more diastase would be formed, he added tannic acid to the culture solution, assuming that in this way there should be an increase in the quan- tity of the diastase formed. In his experiment Katz actually found that the total quantity of enzyme formed (that secreted into the nutrient solution and that present in the fungus) was greater in the culture which contained 0.5 per cent tannic acid than in the control, the ratio of the diastatic activity being 143 to 100. The results seem to confirm his hypothesis, but there are a number of factors which suggest another explanation of the results obtained. In some previous experiments® of the writer it is noted that in the presence of 10 per cent sugar the tannic acid was fermented, and in table I, here reported, it is clear that even in the presence of only 0.1 per cent tannic acid the enzyme tannase is formed and in all probability secreted. The tannic acid of the culture solution would, therefore, be fermented and rendered inac- tive as regards its capacity to precipitate the diastase liberated, and this condition doubtless occurred in the experiment of Katz. 2 J, Katz: Die regulatorische Bildung von Diastase durch Pilze, Jahrb. f. wiss. Bot., xxxi, pp. 599-618, 1898. 23 Loc. cit. THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XIV, NO. 3. 192 Nutrition and Tannase Production While a temporary precipitate of the diastase and tannic acid may exist, it is more reasonable to assume that the increase of diastase must be ascribed to some other cause. Increased growth may possibly occur, as a result of the addition of tannic acid, and with this may be correlated an increase of diastase. A comparison of Aspergillus niger and Penicillium sp. in the formation of the enzyme tannase is of interest because it affords a partial explanation of the relatively slower transformation of the tannic acid by Penicillium sp.24 In both cases the amount of tannase produced in a 0.1 per cent concentration of tannic acid is the same. At 2 per cent concentration the amount of gallic acid, resulting from the action of the Aspergillus powder, was 3.88 times the transformation effected by the Penicillium powder. In the preceding experiment no attempt was made to determine the amount of enzyme secreted into the nutrient solution, and an experiment was required to determine this point as well as to verify the preceding results. The methods of experimentation were essentially the same as in the previous experiment except that 250 cc. Erlenmeyer flasks were employed with 100 cc. of the nutri- TABLE II. Aspergillus niger. Effect of concentration of tannic acid on the production of tannase, using solution B+ 10 per cent cane sugar + tannic acid. Average period of incubation for mycelial powder, sixty-four hours; for enzyme excreted, ninety hours. AMOUNT |WEIGHTOF sues bese anomie: eae Mapes AOTAT NG RaAeEOR manna iat) aemmaenors | avenge ont | imcauasnion pats |" aumaace LIC ACID LIC ACID per cent 4 gram gram gram gram 0 0.083 0 0 | 0 0.5 0.102 0.013 0 0.013 0.10 0.111 0.011 0 | 0.011 0.50 0.149 0.064 0.008 0.072 1.0 0.290 0 084 0 033 0.117 2.0 0.323 0.090 0.041 0.131 4.0 0.207 0.103 0.033 0.136 8.0 0.034 0.157 0 0.157 24 Toid. : Lewis Knudson 193 ent solution. The tannic acid in all cases was added after the culture solutions had been sterilized. The fungus mat was removed and treated as previously described, and for testing its tan- nase content 0.05 gram of the mycelium powder was added to 50 ce. of 1 per cent tannic acid solution, containing as an anti- septic 1 per cent toluene. The experiments were all made in dupli- cate and one series of the solutions was incubated for forty-eight hours and the other for eighty hours. After the fungus felts had been removed from the culture solution and the liquid filtered, alcohol was added to precipitate any enzymes present, the precipitate being collected on filter paper. The filter paper with whatever precipitates accompany- ing were then added to flasks containing the tannic acid solution. After ninety-six hours’ incubation these solutions were analyzed for gallic acid. The results obtained are given in table II. The results obtained verify the figures of the preceding table. With an increase in concentration of tannic acid there is a corres- ponding increase in the amount of enzyme produced. The excre- tion of enzyme into the culture solutions was evident only in the cultures having a relatively high percentage of tannic acid and at most the excretion was small. Influence of concentration of sugar. Since in the preceding exper- iments the amount of tannase produced per unit weight of the fun- gus varies with the concentration of the tannic acid, it seemed desirable to determine the effect of maintaining constant the tannic acid concentration while varying the concentration of the cane sugar. In the first experiment duplicate cultures of Aspergillus niger were made in liter flasks containing 300 cc. of solution B + 2 per cent tannic acid + varying amounts of sugar. The myce- lial mats formed were removed, treated as before described and then tested for tannase activity. In measuring the activity of the tan- nase of each culture 100 ce. of a 0.5 per cent tannic acid solution were employed to which was added 1 per cent toluene as an anti- septic. To each flask was added 0.2 gram of the powdered myce- lium. Incubation was given at 34°, and at the end of ninety hours the solutions were analyzed for gallic acid. In table III are given the results of the experiment. The results are the averages of the two tests. ae, . Tt is here clearly shown that with increased concentration of 194 2 per cent tannic acid + cane sugar. t Nutrition and Tannase Production TABLE III. Aspergillus’ niger. Effect of concentration of sugar on production of tannase; using solution Bo Period of incubation, ninety hours. AMOUNT OF CANE SUGAR ADDED INCREASE OF GALLIC | ACID AMOUNT OF CANE SUGAR ADDED INCREASE OF GALLIC ACID per cent 0 +1 +2 +8 gram 0.224 0.206 0.206 0.179 per cent +12 +16 +24 gram 0.166 0.134 0.130 sugar, the tannic acid being maintained at a constant figure, there is a decrease in the amount of the enzyme produced. In order to verify the above results another experiment was made along substantially the same lines with Aspergillus niger. To test the tannase activity of the dry mycelium 100 cc. of a 0.5 per cent tannic acid solution were used to which was added 1 per cent toluene. In-each case 0.05 gram of the powdered mycelium was employed. The cultures were made in duplicate and the determinations likewise. One set was incubated at 24°C. for four days and the second set at the same temperature for six days before analyses were made for gallic acid. In the following table there is given the composition of the nutrient solutions used and the increase in the gallic acid resulting, which increase of course is a measure of the tannase activity of the various cultures. TABLE Iv. Aspergillus niger. Effect of concentration of sugar on production of tannase; using solution B+ 2 per cent tannic acid + cane sugar. DRY WEIGHT OF INCREASE IN GAL- INCREASE IN GAL- suGar ADDED égetaica oF 2 |4 Sager omhnrok 6 pave incne uniOw ee ee per cent gram gram gram gram 32 0.158 0.179 0.179 0.179 16 0.083 0.270 0.278 0.274 12 0.074 0.233 0.270 0.251 8 0.101 0.270 0.278 0.274 4 0.093 0.270 0.270 2 0.065 0.270 0.296 0.283 0.5 0.074 0.303 0.332 0.317 0 0.035 0.303 0.368 0.335 Lewis Knudson 195 The results confirm the evidence of the preceding experiment, though the differences due to sugar concentration are not so marked. This may have been due to the longer period of incubation. Vv. INFLUENCE OF NUTRITION. Since Dox has found in Penicillium camemberti that the various enzymes are produced irrespective of the nutrition of the fungus, an experiment was made to determine definitely if the enzyme tannase could be produced in two Penicillium species when tannic acid is withheld from the culture solution. Penicillium sp., which I have previously described,® and Penicillium rugulosum were employed in the experiment. They were cultivated in 100 ec. of a nutrient solution which was composed on the one hand of solution B + 5 per cent sugar and, on the other, solution B + 5 per cent sugar + 2 per cent tannic acid. The mycelial felts, as before, were removed just before spore formation, treated as pre- viously described and pulverized. For determining the presence of the enzyme tannase 100 ce. of a 1 per cent tannic acid solution were used to which was added 2 per cent toluene as an antiseptic and 0.1 gram of the pulverized mycelium. The solutions were incubated for twenty-eight days at a temperature of 33°. They were then analyzed for increase in gallic acid. All cultures and determinations were made in triplicate. As is evident from the table tannase was formed only in the presence of tannic acid. TABLE V. GALLIC ACID, COMPOSITION OF SOLUTION PRESENT | GAIN ORGANIEM Penicillium SD ines Solution B + 5 per cent sugar] 0.287 0 Penicillium rugulosum.| Solution B -+ 5 per cent sugar 0.287 0 Penicillium sp.....-.-- Solution B + 5 per cent sugar + 2 per cent tannic acid 0.822 0.535 Penicillium rugulosum.' Solution B + 5 per cent sugar c ms + 2 per cent tannic acid 0.822 0.535 0.287 0 Experiments were next made to determine the influence of replacing the sugar by other compounds and of adding to the nutrient solution various reagents. For this experiment solution 25 Loc. cit. 196 Nutrition and Tannase Production A> was used, and as culture vessels liter flasks were employed. Into each flask were placed 365 cc. of the solution, and to it were added the sugar and other reagent, or the sugar was omitted and some other carbon compound substituted for it. The solutions were sterilized, inoculated and incubated at a temperature of 28°C. The felt, was removed just before spore production and treated in the manner previously described. To determine the presence of tannase, 0.3 gram of the powdered, dried mycelium was introduced into 100 cc. of a 0.75 per cent solu- tion of tannic acid, to which had been added as an antiseptic 2 ec. of chloroform. After incubation at 31° for one week, the solu- tions were analyzed for gallic acid. The results obtained are given in table VI. TABLE VI. Penicillium sp. COMPOSITION OF NUTRIENT SOLUTION eee Saas Solution A + 25 grams cane sugar.. : 0.202 0 Solution A + 25 grams cane sugar Be ce. iy HCI. 0.202 0 Solution A + 15 grams corn starch............ : 0.208 0 Solution A + 25 grams glycerin................. 0.202 0 Solution A + 25 grams gallic acid............... 0.370 0.128 Solution A + 25 grams tannic acid.............. 0.713 0.511 The control contained 0.202 gram gallic acid at the beginning and at the end of the incubation. The gallic acid stimulated the formation of the enzyme only one- fourth as much as did the tannic acid. Slight acidity had no effect in stimulating the production of the enzyme. Glycerin and starch, both of which are relatively poor food compounds, were supplied, and if enzymes are stimulated to formation by con- ditions approaching starvation, as suggested by Wortman,?? then the tannase should have developed; but the results were negative. An experiment similar to that above was made with Asper- gillus niger, 400 cc. of solution being used and the methods of exper- imentation the same as before. The results obtained are as fol- lows: , ° KH2PO,, 0.5 gram; KNO;, 1 gram; MgSou, 0.25 gram; Distilled water, 100 ce. 27 Loe. cit. Lewis Knudson 197 TABLE VII. « Aspergillus niger. COMPOSITION OF NUTRIENT SOLUTION CEUELGIACID: ||| AG ALEIC ACL, PRESENT INCREASE Solution A + 25 grams sugar..................... 0.209 0 Sulotion A + 25 grams sugar + 5 ce. §, HCl..... 0.202 0 Solution A + 25 grams sugar + 5 grams _ gallic BOL tie. ie ht acleadmant cen WARY ly eat, © eae mee eS, 0.202 0 Solution A + 25 grams sugar + 2 grams resorcin. 0.190 0 Solution A + 25 grams sugar + 1 grams hydro- QUINONE se sees yee sayy seeds wdinnand mccwumm nea sate 0.202 0 Solution A + 25 grams sugar + 5 grams peptone. 0.202 0 Solution A + 25 grams sugar tannic acid......... 0.691 0.489 From the above table, it is evident that in Aspergillus niger there is a very marked regulatory formation of the enzyme. Pep- tone, which stimulates the secretion of diastase, according to Katz?8 has no influence in stimulating the formation of tannase. Gallic acid of the strength used had no effect, which result is surprising in view of the results indicated in table VI and of the further fact that Pottevin?® found the enzyme tannase to be formed in Asper- gillus niger when the organism is cultivated in Raulin’s solution with the sugar replaced by gallic acid. Since pyrogallol is a de- composition product of tannic acid, it was thought that perhaps it might stimulate the formation of the enzyme, but the amounts used proved toxic in this particular experiment. Resorcin and hydroquinone were both employed because of their constitutional similarity to pyrogallol, both being dihydroxybenzenes, while the pyrogallol is a trihydroxybenzene. Negative results, however, were obtained. In a similar manner experiments were made in which 10 per cent cane sugar plus various other substances were added to solution A. For the experiment 500 cc. of the solution were used. In deter- mining the presence of tannase, 0.2 gram of the powdered mycelium was added to 75 cc. of a 0.9 per cent tannic acid solution to which had been added 2 per cent toluene as an antiseptic. The results are given in table VIII. 28 Toc. cit. ; 7 3 Za ‘ 29H. Pottevin: La tannase. Diastase dédoublant l’acide gallotanique, Compt. rend. de l’Acad. des Sci., exxxi, pp. 1215-17, 1901. 198 Nutrition and Tannase Production TABLE VIII. Aspergillus niger. COMPOSITION OF NUTRIENT SOLUTION GALLIC ACID INCREASE Solution B + 10 per cent sugar + 35 mgm ZnSO,.H.0.. None Solution B + 10 per cent sugar + 20 mgm salicylic acid. None Solution B + 10 per cent sugar + 1 gram gallic acid... None Solution B + 10 per cent sugar + 200 mgm. methyl sali- CV BOs 5 te acradntc mda watiardaciageen mina aap haeens Oe 5h batch None Solution B + 10 per cent sugar + 200 mgm. ethyl sali- CHA av eumne lacus une om wean se won RO RRS GAIT None Solution B + 10 per cent sugar + 50 mgm. methyl sali- cylate............ sodden Soci pecuGa VIS GIGS eos nce osaenta ste None Solution B + 10 per cent sugar + 50 mgm. ethyl sali- MAC Secs essa aya eublauadh Gale dns dah dndesbnsobedaaneunsiten uaNe SUES None Pottevin® states that tannase has the property of hydrolyzing methyl and ethyl salicylate. in my cultures the methyl or ethyl salicylate did not incite the development of the enzyme. The amount of salicylate present, however, was small. These concen- trations may have been too weak to stimulate the production of the enzyme but probably the hydrolysis of these two substances is due to another enzyme. Salicylic acid also was used because of its relation to gallic acid, salicylic acid being a monohydroxy- benzoic acid, while gallic acid is a trihydroxybenzoic acid. The former was necessarily used at a very low concentration, and was without effect. The zinc sulphate was used at a concentration which is stimulating to the fungus growth, as shown by Richards,?! but no formation of the tannase resulted. Since in some of the experiments tannase was not produced when the organism was grown in a solution with the carbon sup- plied as cane sugar and gallic acid, an experiment was made with the carbon supplied only as gallic acid. At the same time experi- ments were made to determine the influence of certain glucosides. Solution B was used, and 500 cc. of it placed in each of three liter flasks. These were plugged and sterilization made at 115° for ten minutes. To each of the three flasks was added gallic acid, amygdalin and salicin, respectively, and the culture solutions 30 Loc. cit. 31H. M. Richards: Die Beeinflussung des Wachstums einiger Pilze durch chemische Reize, Jahrb. f. wiss. Bot., xxx, pp. 665-88. Lewis Knudson 199 Ha inoculated. The growth in gallic acid was rapid, but not eavy. In the solutions in which the glucosides were present, growth was at first very slow, but after the transformation of the glucosides had begun, growth was rapid. While it required eight days to develop the first felts in the two glucoside solutions, the second felts (after the removal of the first) developed in two days. The felts first formed were removed and treated according to the methods previously described, and the powdered mycelium then added to 100 cc. of a 0.90 per cent solution of tannic acid which contained also 2 per cent of toluene. After incubating two days at 40°C., the solutions were analyzed for gallic acid. The composi- tion of the nutrient solutions and the gallic acid formed by the powdered mycelium are given in table IX. TABLE Ix. Aspergillus niger. COMPOSITION OF NUTRIENT SOLUTION GALLIC ACID | GALLIC AcID PRESENT INCREASE Solution B 500 cc. + 10 grams gallic acid......... 0.546 0.219 Solution B 500 ce. + 10 grams salicin............. 0.327 0 Solution B 500 cc. + 2 grams amygdalin......... 0.327 0 The control contained 0.327 gram gallic acid and 0.555 gram tannic acid. In preceding tables it has been seen that gallic acid at certain concentration in the presence of 10 per cent sugar does not stimu- late the formation of the enzyme tannase. When gallic acid is present alone as the source of carbon, the enzyme is produced. If tannic acid is a glucoside, it might be expected that the presence of another glucoside would stimulate the production of some tan- nase, but such was not the case. Nevertheless, glucosides were transformed by the organism. While the gallic acid stimulates the formation of the enzyme tannase, it does not do so as effectively as the tannic acid. VI. EFFECT OF CONCENTRATION OF GALLIC ACID ON PRODUCTION OF TANNASE. Since the amount of tannase produced is regulated by the relative concentrations of sugar and tannic acid it was deemed important to determine if the amount of the tannase produced could be in- 200 Nutrition and Tannase Production creased by increasing the concentration of the gallic acid. In the experiment, the results of which are given in table X, the usual methods were followed. Erlenmeyer flasks of 250 cc. capacity were used and 100 ce. of the nutrient solution. For determining the tannase activity of the various cultures 0.05 gram of the dried mycelium was added to 50 ce. of 1 per cent tannic acid solution and the solutions incubated for forty-eight and eighty hours respec- tively at a temperature of 30°. The culture solutions were tested for their tannase content by precipitating any enzyme present with alcohol and collecting on a filter any precipitate. The test for the enzyme was made as before. Incubation was made at a tempera- ture of 30° for ninety hours. The results in table X are the aver- ages of two cultures. TABLE X. Aspergillus niger. Effect of concentration of gallic acid on the production of tannase. Average period of incubation for mycelial powder 64 hours and for enzyme excreted 90 hours. WEIGHT OF INCREASE IN GALLIC ACID FUNGUS. AV. OF 2 CULTURES Mycelium Enzyme ex- | Total increase Av. of 2 ereted. Av. of 2) in gallic acid COMPOSITION OF NUTRIENT SOLUTION gram gram gram Solution B +10 per cent sugar + 0.1 per cent gallic acid...... 0.240 0 0 0 Solution B + 10 per cent sugar + 0.5 per cent gallic acid...... 0.442 0.024 0 0.024 Solution B + 10 per cent sugar + 1.0 per cent gallic acid...... 0.324 0.045 0 0.045 Solution B + 10 per cent sugar + 1.5 per cent gallic acid...... 0.280 0.046 0 0.046 An examination of the above table reveals the fact that the enzyme tannase is produced when the concentration of gallic acid is 0.5 per cent in the presence of 10 per cent sugar. At a concen- tration of 1.0 per cent gallic acid in the presence of 10 per cent sugar the amount of tannase produced is double that produced when only 0.5 per cent gallic acid is present. In the presence of Lewis Knudson 201 10 per cent sugar the gallic acid, so far as my previous results® show, 1s not at all absorbed from the nutrient solution. Why the increase in gallic acid should increase the amount of tannase produced is therefore difficult to explain. The writer is not pre- pared at present to offer an explanation. VII. SUMMARY. 1. There is a progressive increase of tannase in the two organ- isms Aspergillus niger and Penicillium sp. with increased concen- tration of tannic acid in Czapek’s solution containing 10 per cent sugar. 2. In a full nutrient solution containing, as a source of carbon, 2 per cent tannic acid, the addition of cane sugar decreases the quantity of the tannase produced by the organism. The higher the concentration of sugar the lower the quantity of tannase pro- duced. 3. Aspergillus niger produces more tannase or a more active tan- nase per unit weight than Penicilliwm sp. 4. The production of tannase in Aspergillus niger, Penicillium sp. and Penicillium rugulosum is stimulated by tannic acid and gallic acid, only, and the former is more effective than the latter. . 5. There is a progressive increase of tannase in Aspergillus niger with increased concentration of gallic acid in a nutrient solution containing 10 per cent sugar. . VIII. DISCUSSION. Dox*? in his excellent paper makes the following statement: ‘‘There is no evidence that enzymes not normally formed by the organism in demon- strable quantities can be developed by special methods in nutrition. The influence of adding a particular substance to the medium is, therefore, not to develop an entirely new enzyme, but to stimulate the production of an existing enzyme, which is normally formed under all conditions.” In the light of Dox’s results it seems somewhat surprising that the enzyme tannase should be formed only under special nutrition. It may be as Dox*4 32 Loc. cit. 33 A. W. Dox: The Intracellular Enzymes of Penicillium and Aspergillus, loc. ctt. a4 A. W. Dox: Enzyme Studies of Lower Fungi, Plant World, xv, pp. 40-43, 1912. 202 Nutrition and Tannase Production has stated concerning my work that the formation of the enzyme tannase is an exception to the general rule. The work of other investigators previ- ously mentioned has indicated, however, that the production of certain enzymes in other organisms is governed entirely by the character of the nutrition. Dox® has considered only the influence of external environment upon the formation of the enzymes. It is to be expected that protease, lipase, nuc- lease, inulase and perhaps some of the other enzymes would be produced _ because the substances which they transform are present in the mycelium. If the action of an enzyme is reversible and they are synthesizing agents, then the question arises: ‘“‘can the products of the decomposition induce the formation of enzyme?’ In my experiments the only substance besides tannic acid capable of inducing the formation of the tannase is gallic acid, which is a decomposition product of tannic acid. Might it be possible that all of the enzymes are produced only in response to the influence of the zymolyte or to the products of its decomposition present either in the nutrient solution or in the mycelium? There is a con- siderable amount of evidence indicating that one or the other is always pres- ent, but there is also evidence that certain enzymes are seemingly produced in the entire absence of the zymolyte or the products of its decomposition. The whole problem is a complex one and requires investigation. 3 Enzyme Studies of Lower Fungi, loc. cit. THE WAVERLY PRESS | BALTIMORE, U.S.A.