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The effect of temperature on the trace element requirements for citric acid production by aspergillus… Kitos, Paul Alan 1952

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The E f f e c t of Temperature on the Trace Element Requirements f o r C i t r i c Acid production by Aspergillus niger PAUL" ALAN KITOS A. THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIRMSNTS FOR THE DECREE OF MASTER OF SCIENCE IN AGRICULTURE i n the Department of Dairying ile accept t h i s thesis as conforming to the Standard required from candidates f o r the degree of MASTER OF SCIENCE IN AGM CULTURE Members of the Department of THE UNIVERSITY OF BRITISH COLUMBIA' May 1?$2 ABSTRACT The e f f e c t of temperature of fermentation on the production of c i t r i c acid, and on the trace e l e -ment requirements f o r the maximum production of c i t -r i c acid hy A s p e r g i l l u s niger 7-2-4 were studied. Y i e l d and e f f i c i e n c y of production of c i t r i c acid were progressively greater from 33° to 23°C« How-ever, i n order to obtain maximum y i e l d s of c i t r i c acid at temperatures lower than 30°C. i t was necess-ary to increase the l e v e l s of the trace minerals, zinc, i r o n , copper and manganese, above those found to be optimal at 30° C« The trace mineral requirement f o r maximum acid production at 25°C« was twice as great as that at 30°c< which i n turn was twice as great as that at 33°C- i n addition, fermentation at the lower tem-perature was much l e s s sensitive to excesses of the various trace element than at a higher temperature• A mineral balance which i s affected by tempera-ture of fermentation i s involved i n the n u t r i t i o n of A« niger f o r optimal c i t r i c a c i d production. Fermentation time i s longer at 23°,c« than at 30°C. However, the acid y i e l d per u n i t of carbohydrate used i s higher at the lower temperature* Trace elements other than zinc, i r o n , copper and manganese are probably essential for maximum acid production, preliminary evidence was obtained which indicated that cobalt and molybdenum were ac-t i v e i n t h i s respect. .ACKHOlfLEBGMMfS I wish to express my sincere g r a t i t u d e to Dr. J . J . R. Campbell, under whose d i r e c t i o n t h i s p r o j e c t was c a r r i e d out, f o r h i s keen i n t e r e s t , i n s p i r a t i o n and cooperation through-out t h i s i n v e s t i g a t i o n . I also wish to thank N e i l Tomlinson f o r h i s advice on many of the problems encountered i n t h i s work, and f o r many o f the techniques de-veloped i n t h i s f i e l d . I also wish to thank Dr. p. C. Truss e l l f o r h i s i n t e r e s t and cooperation, and the B r i t i s h Columbia Research Council f o r the a s s i s t a n c e granted to t h i s p r o j e c t . • TABLE OF CONTENTS Pag® introduction 1 H i s t o r i c a l 2 Experimental Methods 9 Experimental 13 mscussion 33 Conclusion 38 Bibliography 40 1. INTRODUCTION Although as early as 1935 (37) the n u t r i t i o n a l requirements f o r the growth of the mold 1 Aspergillus  niger" had been established, l i t t l e information on the n u t r i t i o n a l requirements f o r c i t r i c acid produc-t i o n by t h i s organism was available. Tomlinson, Campbell and T r u s s e l l (44, 45) were able to show that the requirements f o r growth and a c i d production are q u a l i t a t i v e l y , although not quantitatively, similar. 'This suggests that c i t r i c acid i s a normal c e l l me-t a b o l i t e that accumulates as a re s u l t of n u t r i t i o n a l imbalance, l/hether the e f f e c t s of trace elements on the physiology of A. niger can be attributed to the ions alone or i n combination or to an imbalance or complex formation of an ion or ions has not yet been established. 'Temperature of incubation i s known to cause rad-i c a l changes i n the growth of certain fungi (4). i t therefore seemed desirable to determine the e f f e c t of temperature on trace element requirements f o r growth and c i t r i c a c i d production, and to establish a quan-t i t a t i v e relationship between these two variables f o r A> niger. (2) HISTORICAL The n u t r i t i o n a l requirements f o r growth of A« niger include the elements carbon, hydrogen,, oxygen, nitrogen, sulfur, phosphorus, potassium and magnesium. Various other elements have been, suggested as being, essential for, or stimulatory to, the growth of t h i s organism. Hi e l sen (30) showed a p o s i t i v e growth response to traces of cadmium, barium, strontium, calcium, lithium, zinc, berylium, copper or manganese. Bertrand (3, 4) found that addition of traces of vanadium were stim-^ ulatory to growth i n Steinberg's (37) medium. Steinberg (40) was able to increase growth i n a non-optimal medium by small additions of titanium, colum-bium and palladium. He l a t e r showed that sodium and berylium increased the fungus growth i n the presence of suboptimal amounts of potassium and magnesium (41). Steinberg (37, 38) was able to show that added zinc, i r o n , copper, manganese, gallium and molybde-num augmented growth but that only zinc, i r o n and perhaps copper were stimulatory to a c i d accumulation under h i s conditions. Butkewitseh and Barinova (7) had previously' recognized that zinc was important while Porges (.34) likewise had recommended that not only zinc but also i r o n are essential i n acid pro-duction. In contrast, Chrzaszcz and Peyros (13) claimed that under t h e i r conditions zinc i n h i b i t e d a c i d production, but that small additions of i r o n were stimulatory' Using a raw cane sugar medium Zaldevar (3D was unable to detect s i g n i f i c a n t e f f e c t s of either zinc or i r o n on mycologi.cal c i t r i c a c i d production. Very recently, however, Chesters and Robinson (11) i l l u s t r a t e d increased acid y i e l d s from cultures p a r t i a l l y d e f i c i e n t i n z i n c A f t e r studying the ef f e c t s of numerous trace elements on c i t r i c acid production by A. niger, Bernhauer (2) was unable to detect any appreciable ef f e c t from G.01/o of t i n , aluminum, cobalt, n i c k e l , chromium, selenium, palladium, platinum, copper or ir o n . . Manganese gave inconsistent r e s u l t s while zinc, s i l v e r , tellurium, wranium, antimony and tungsten i n -h i b i t e d a c i d production, i n 1949 firkama (17) noted a 60 - 73^ decrease i n acid production, and a corres-ponding increase i n sugar u t i l i z a t i o n upon addition of i r o n to the fermentation. But, as i s true . with much of the work reported here, the amounts of trace 4. minerals used were extremely great. The r e s u l t s lend l i t t l e s ignificance to the conclusion that the element concerned has a depressive e f f e c t . Shu and Johnson (35) using a p u r i f i e d and well defined medium t r i e d to eliminate the i n h i b i t o r y ef f e c t of manganese on acid production by using an inoculum of manganese depleted spores. They con-cluded that using submerged culture technique A. niger does not require copper or manganese, but does require zinc and i r o n f o r optimal y i e l d s of c i t r i c acid (3&)« Apparently a phosphate require-ment i n excess of that required for good mycelial growth, was essential f o r maximal c i t r i c a c i d y i e l d s . Tomlinson (43), commenting on these r e s u l t s , sug-gested that t h i s may be due to the' introduction of the necessary trace elements f o r a c i d production as impurities i n the phosphate. Tomlinson went on to show that under defined conditions there i s a d e f i -n i t e requirement f o r zinc, i r o n (44), copper and manganese (45), and that one or more as yet unde-termined factors are also necessary f o r optimal c i t r i c acid y i e l d s . i n the past f i f t y years a great deal of time and e f f o r t has been devoted to a q u a l i t a t i v e study of the inorganic requirements f o r various functions of A- niger. Interpretation of the available l i t e r -ature i s dangerous due. to the number of variables that may complicate the experimental results- For example: - s t r a i n of organism used, P H of growth, energy source, available oxygen and innumerable other factors influence the r e s u l t s . One of the most important variables i s temperature of growth and fermentation. perlman (32) stated that most str a i n s of A« niger grow well between 1.5° C and 40° C> and that high y i e l d s of c i t r i c acid are most evident between 24° and 30° C- He found that l i t t l e or no a c i d accumulates at or above 37° C He a t t r i -buted the high mycelial weights and low a c i d y i e l d s at 37° C to increased metabolic a c t i v i t y at elev-ated-temperatures. However, growth i s not necessr a r i l y associated with c i t r i c a cid production (33 ) , although good y i e l d s of c i t r i c a c i d may be due to the i n a b i l i t y of the organism to u t i l i z e c i t r a t e as a metabolic substrate. Yon Loesecke ( 4 9 ) reported that Wehmer used fermentation temperatures between 13°C« and 20°C. These low temperatures warranted invest i g a t i o n but upon consultation, the o r i g i n a l t r e a t i s e did not state the temperature of fermentation (50). Only i n rare instances have such low temperatures as these been used i n phy s i o l o g i c a l studies on A» niger (3 , 29). Virtanen and pulkki (48) claim-ed that 20°C. i s the optimal temperature for c i t r i c acid accumulation. But i n view of the fa c t that when the acid was removed as rapidly as i t was form-ed without allowing the p H of the medium to r i s e , the best a c i d y i e l d s were obtained at' 3 0 °C the optimum f o r c i t r i c acid formation couj.4 not be 20°G» Using replacement cultures, Butkewitsch and Barinova (7) obtained hikjier y i e l d s at more rapid rates at 20° and 2 5 °C than at temperatures outside of t h i s range. Chatteacjee (9) also observed 25°c. to be optimal f o r c i t r i c acid accumulation. A great number of other references-to temper-ature of fermentation may be c i t e d but only a few of these are pertinent to t h i s work. SzlJ.cz used the temperature range between 18° and 28°C as part of the b a s i s f o r a patent on c i t r i c a cid production (42). Doegler and prescott had previously narrowed 7 t h i s range to 24° to 28°C. (15). Above 30° C they ob-served a decrease i n c i t r a t e accumulation coincident with an increase i n oxalate. Chrzaszcz and Peyros (13) adopted 28° to 30°C as suitable, whereas Kost-uichev and Berg (22) and l a t e r Mel'nixova et a l . (28) used 30° to 32°C as fermentation temperatures. Eisenmann and Blumenfeld (16) recommended using a temperature range between 28°c and 35° C« S i m i l a r l y Das Gupta et a l . (14) using a s t r a i n of Mucor were able to obtain f a i r y i e l d s of both c i t r i c and o x a l i c acids i n eight days at 35° - 37°C Using a s l i g h t l y d i f f e r e n t approach, Bluman (6) was unable,to induce high c i t r i c acid, production by heat shocking the spores. Working with submerged cultures (3&, 37) Shu and Johnson obtained good c i t r i c acid y i e l d s at 23°C. Tomlinson et a l . (43 f 44, 45,46), working with s t a t i o n -ary cultures and the same s t r a i n of A» niger as was used by Shu and Johnson, obtained equally good y i e l d s of c i t r i c a c i d at 30°G. I t may be'concluded from such varied reports that the most desirable temperature f o r maximum c i t r i c acid production varies to a marked de-gree with conditions of fermentation. To what extent does the temperature aff e c t condi-tions of growth and acid production? There i s only a 8. paucity of data available to a s s i s t i n any attempt to answer t h i s question, using standard conditions t Nielsen and Dresden (2?) showed that A. niger has a Q^Q of approximately J.2 between the temperatures of 7°C. and 17°C« This value i s decreased to 1.7 between 2/]°Q and-. 37° C* Thus an increase i n meta-b o l i c rate accompanies an'increase i n temperature . but the rate of increase decreases with a r i s e i n temperature. Following a preincubation period of no more than three days at 28°©.to allow f o r good mycelial development, Kovats (24) lowered the tem-perature to 20°G« to obtain maximum y i e l d of acid* Evidently temperatures as low as 2Q°Q. are riot de-l e t e r i o u s to acid production. This leads to the' p o s s i b i l i t y that c i t r i c acid production may not necessarily be related to maximum fungal develop-ment. SSPEEBIMTAL METHODS 'The organism used -throughout t h i s -work was A. niger 7*2-4, a single c e l l i s o l a t e of A.T.G.C. .A. niger No. 1015 (.31). I t was maintained as a - • s o i l stock culture* Before inoculation into f e r -mentation f l a s k s the spores were transferred s e r i -a l l y through three "four-day" cultures on 2.j» agar slants. Composition'of the sporulation medium was the same as the fermentation medium except that agar was added, trace elements were omitted, and the p H was approximately 6.0. The agar was washed with several changes of glass r e d i s t i l l e d water before use. 'The spores, used as inoculum, were transferred by l i g h t l y passing a chemically cleaned s t e r i l e glass rod over the surface of the sporulated culture. "A. spore suspension was prepared i n glass r e d i s t i l l e d water so that one agar slant provided s u f f i c i e n t spores f o r 5 mis. of inoculum. The f l a s k i n which the spore suspension was made up had a ground glass stopper. This permitted vigorous agitation i n order to produce a homogeneous suspension. Two mis. of spore suspension were used as inoculum f o r 1.00 mis. 10,. of medium, one ml. of t h i s suspension contained ap-proximately f i v e m i l l i o n spores. A l l Glassware used i n the experimental \vork was chemically cleaned hy soaking overnight i n '10?& n i t r i c acid, r i n s i n g with tap water ten times, d i s t i l l e d water twice, and glass r e d i s t i l l e d water twice. .The glassware was then f i l l e d with water r e d i s t i l l e d from glass, autoclaved at 15 l b s . pressure f o r 15 minutes, and f i n a l l y rinsed again with water r e d i s t i l l e d from glass. This procedure i s . i n accordance with the r e -commendations of Henry and Smith (20).. 230 ml. pyrex Erlenmeyer f l a s k s , covered with i n -verted 100 ml. beakers were used as fermentation vessels. The sporulati.on slants were 15 cm. x 15 mm. test tubes covered with inverted glass v i a l s . Fine granular sucrose, a product of the B.. C. Sugar Refinery, was used as carbon source, i t was p u r i f i e d f o r the fermentation medium by cation ex-change treatment through. 170 cms. of Permutit Q (645 mis. wet volume) and 60 cms. of Amberlite IMC -50 (232 mis. wet volume) at the rate of approximately 80 mis. per hour. Reagent grade chemicals were used except f o r the FeCl^ which was p u r i f i e d by cation exchange treatment. 11. The stock mineral solution was as follows (46): Sucro se 15S* K 2 H P 0 4 MgS04-7H20 0.28.96 0.06^ 0.03?* H 01 to p H 2.0 a approximately 1 ml.of 1 I H CI. Trace mineral supplements are indicated f o r each experiment. The f i n a l -volume of the medium was 100 mis. per f l a s k . . . ' Upon termination of fermentation, the contents of the f l a s k s were f i l t e r e d and the mycelial mat was wash-ed and squeezed as dry as possible by hand. The f i l -t r a t e was collected and made up to 250 mis. volume with tap water, A 23 ml. a l i q u o t xvas l i t r a t e d with 0-5 B HaOH to determine t o t a l a c i d i t y . The remainder of the f i l t r a t e was heated to. 100°C. i n an autoclave (steamed) f o r one-half hour to prevent further a c t i -v i t y . Aliquots of the supernatant were analyzed f o r re s i d u a l reducing sugar by a modified Layne-Eynon procedure (1) . Representative samples were selected f o r determination of c i t r i c acid by a pentabromace-tone procedure (.47) and f o r determination of oxa l i c a c i d by p r e c i p i t a t i o n as the calcium s a l t and t i t r a -t i o n with a standard' solution of potassium permanga-nate. 12. The mycelial mats were dried to a constant weight at 90° C- i n i n d i v i d u a l aluminum dishes. They were weighed to within 0.01 grams on a tors i o n 'bal-ance. 1> EKPERIM.MTAL In order to study the influence of temperature on the mineral requirements f o r maximal c i t r i c acid production, i t was necessary to select conditions that would serve as a bas i s f o r the i n i t i a l experi-ments. She conditions established by Tomlinson et a l . (4j>) as optimal f o r fermentation at 30°C. were adopted. The normal trace mineral l e v e l s used were as follows: Zinc as ZnS0 4. 7H20 30 jXS* f> i r o n as FeCl^ 11 jag. t> Copper as CuSCX^SHgO 4 jig. f> Manganese as MnClg. 0.3p.g.fi These l e v e l s were termed the 100?S l e v e l s . By varying a l l the mineral l e v e l s together about the established norm over, a range of Qfi to 300?°, a l l other conditions being held constant, the gross e f f e c t s of d i f f e r e n t trace mineral l e v e l s could be shown. These variable l e v e l s are recorded i n table 1. 14 Table 1* Trace Mineral Levels and the Corresponding percentage of the Established normal. percen- i Trace Elements per 100 mis. tage -Level i . Zinc Iron Copper Manganese • n •J -Tig ' • ug •ug ! • ug 23 12.5 2.73 1.0 0.075 50 25.0 5-50 2.0 0.130 15 37.5 8.23 3.0 0.223 100 50.0 11.00 4.0 0.300 123 62.3 13.75 3.0 0.375 130 75-0 16.50 6.0 0.450 175 87-5 19.23 7.0 0.525 200 100.0 22.00 8.0 0.600 225 112.5 24.75 9.0 0.675 250 125*0 27.30 10.0 0.750 300 150.0 33.0 12.0 0.900 400 200.0 44.00 16.0 1.200 The e f f e c t s of the various mineral l e v e l s re-corded i n Table 1. were studied at three d i f f e r e n t temperatures: - 25°C. and 300C- and 35°C. (JFlg ,1). 13-U G . i The Effect of Mineral Level on Citrio Aold production at ainree Different Temperatures. FIG- I MINERAL LEVELS - % 25° C-30° C-35° C 16. ihe..results c l e a r l y indicate that at ten days the y i e l d of c i t r i c acid progressively increases with de-creasing temperature over the range 35° C to 25°C. The increase i n y i e l d was accompanied hy a decrease i n s e n s i t i v i t y to the o v e r a l l trace mineral l e v e l s . Above 30°CN the requirements f o r optimal c i t r i c acid y i e l d s were f a r more s p e c i f i c than those below 30°C. I t i s d i f f i c u l t to compare the a c t i v i t i e s of an organism at one temperature, to those at another, due to the ef f e c t of temperature on a c t i v i t y rate. Pro-bably the most satisfactcr y method of comparison i s the c i t r i c acid produced per u n i t of sugar u t i l i z e d . Mathematically, t h i s represents the e f f i c i e n c y of the system i n converting sugar to acid. 'Thus, as a basis of comparison, the e f f i c i e n c y of the fermenta-tions at the point of highest acid y i e l d i s used. However, i n figure 1 the 25° C. fermentation e l i c i t e d no sharp peak i n the acid y i e l d s - Therefore the high-est mineral l e v e l producing a maximum c i t r i c acid .yield was used i n the comparison i n table 2. 17. Table 2. Comparative E f f i c i e n c y of C i t r i c Acid production at Three Temperatures: Temp. • Mineral • Sugar • C i t r i c ft E f f i c -Acid °C Level f° used g- Y i e l d g. iency. 25 200 13-17 8.96 68.0 30 100 13-90 8.02 57-8 33 30 13-28 7.42 55.9 At 23°iG. A. niger appeared to be much more e f f i -cient i n c i t r i c a cid production than at either 30°C-or 35°C Likewise i t was.more e f f i c i e n t at 30°C. than at 3i5°C However, i n order to s a t i s f y any d i s -crepancy that may arise due to a comparison of the ef f i c i e n c y at three d i f f e r e n t mineral l e v e l s , a com-parison at a single mineral l e v e l (100?£>) showed that the r e s u l t s were analagous to those i n table 2# Table 2« Temp. Sugar . C i t r i c . f> E f f i - •Mycelial °c Acid used g y i e l d g ciency weight g 23 11.76 8.89 75.7 2.03 30 13.90 8.03 37-8 2.43 33 14.31 6.58 45-9 3.03 18. The r e s u l t s at the 100f« mineral l e v e l , recorded i n table 3, emphasize the increasing e f f i c i e n c y of A s p e r g i l l u s niger i n the production of c i t r i c acid r e s u l t i n g from a decrease i n temperature. There i s also a coincident decrease i n mycelial weight with the decrease i n temperature. I t appears that the de-creasing e f f i c i e n c y i s due to the excessive amount of mycelium formed at the higher temperature. The pro-portion of sugar being converted to acid decreased, and the proportion being synthesized into mycelial structure increased with increasing temperature. Under the experimental conditions used i n t h i s work, i t has been possible to establish a r e l a t i o n -ship between fermentation temperature and the gross requirements of trace minerals f o r maximum production of c i t r i c acid, i t was shown (table 2) that f o r every 5°G, decrease i n temperature i n the range of 25° to 3 5°C, there was a doubling of the trace mineral re-quirements. This rela t i o n s h i p has only been proven v a l i d when zinc, iron, copper and manganese were s i -multaneously varied. However, i t does indicate the p o s s i b i l i t y of a s i m i l a r r e l a t i o n s h i p f o r each element when varied i n d i v i d u a l l y . The fact that A. niger i s highly e f f i c i e n t i n 1? . c i t r i c a c i d production at 23°C introduces the poss-i b i l i t y of more sa t i s f a c t o r y r e s u l t s at temperatures lower than 23°C Under s i m i l a r conditions of n u t r i -t i o n , a y i e l d of only 3-31 grams of c i t r i c acid was obtained at 22 °c. i n ten days. This represents a 64.3f° e f f i c i e n c y based on 8.22 grams of sucrose used. 'Therefore, although the y i e l d i s low, the e f f i c i e n c y of the organism i n c i t r i c acid production i s high. The mycelial ^veight at ten days i s only 1.53 grams. Thus with almost h a l f of the o r i g i n a l sugar s t i l l present, the p o s s i b i l i t y of obtaining high acid y i e l d s at low temperatures such as t h i s , i s very good. 'The technique, employed by Kovats (24), of lowering the fermentation temperature a f t e r an i n i t i a l period of mycelium formation at 28°C. may solve the problem of hastening complete fermentation at low temperatures. Another approach, although used unsuccessfully by Bluman (6) , i s to heat shock the spore inoculum, i t may also be advantageous to use pregerminated spores as inoculum i n such an experiment. These methods would tend to reduce the time required f o r mat forma-t i o n which seems to be the l i m i t i n g f a c t o r at low tem-peratures. No further work at temperatures below 25 °C was carried out. 2 0 . Although i t was shown that there i s an o v e r a l l increase i n trace mineral requirement at 25°C, over that at 30°C«, i t does not follow that a s i m i l a r e f f e c t w i l l exist for the i n d i v i d u a l trace elements. In order to discover the effect of temperature on the, i n d i v i d u a l minerals required f o r maximal c i t r i c acid production, a series of experiments were conduct-ed i n which the only variable was the l e v e l of the. element i n question. The trace minerals other than the variable were maintained at the 10Gfo l e v e l record-ed i n table 1, and the temperature was held at 23°C The r e s u l t s f o r zinc and i r o n are i l l u s t r a t e d i n f i g -ure 2, while those f o r copper and manganese are i l l u s -t rated i n figure 3« Figures 2 and 3 are sim i l a r to the 23°C. curve i n figure 1, i n that the s e n s i t i v i t y of acid produc-ti o n to high mineral l e v e l i s noticeably l e s s than that at 30° C The ef f e c t of temperature on the i r o n require-ments i s c l e a r l y shown i n figure 2. There was an i n -crease from 11 | i g. per 100 mis. at 30°C« to approxi-mately 16.5 p. g per 100 mis. at 23°C This corre-sponds to an increase of 30;?& over the established 10056 FIG.2 . The E f f e c t of ¥aried Levels of Zinc and i r o n on C i t r i c A c i d production at 25°C. FIG. 3 The E f f e c t s of Yaried Levels of Copper and Manganese on C i t r i c A c i d production at 23 c» FIG- 2 JL J 50 J00 150 200 250 300 VARIABLE LEVEL - % 22. normal. Unlike i r o n , zinc did not show a d e f i n i t e peak i n acid production, although i t did increase i t s requirement to 125f° of the established normal. An ex-cess of zinc as high as 300f« of the normal did not cause ia decrease i n acid accumulation. A. niger appar ently has a requirement f o r zinc, but i s not sensitive to excesses of t h i s m e t a l l i c ion. This i s probably true at 30°iG* also since Tomlinson et a l (44) showed that six t y times as much zinc as was used at the 'IOO9& l e v e l i n t h i s work resulted i n only a s l i g h t l y decreas ed y i e l d of c i t r i c acid. However, the r e s u l t s obtain-ed by Chesters and Robinson (11) indicate a marked s e n s i t i v i t y to zinc at as low as 20 p. g per 100 mis.* of medium at 28° C. At present there i s no tenable ex-planation f o r t h i s discrepancy. The requirements f o r both copper and manganese at 2j?°C. are increased above those at 30°C. Likewise A« niger i s sensitive to small excesses of these trace elements at the higher temperature* The optimum l e v e l of manganese under these conditions i s 0.375 p. g per 100 mis. of fermentation medium,-an increase of 0.075 n. g over that f o r the 30°C. conditions. The optimum l e v e l of copper i s 5 j i g per 100 mis.,-an increase of 1*0 P- S over that f o r the 30 °C l e v e l . 23. I f the s e n s i t i v i t y of A. niger to excesses of iron, copper and manganese, i l l u s t r a t e d i n figures 2 and 3, i s absolute, then varying the four mineral l e v e l s together should indicate a s e n s i t i v i t y equal to that of the most c r i t i c a l of these trace elements. But at 23°C. there does not appear to he such an effe c t as can he seen by comparing the 23°C. curve of figure 1 with the i r o n curve of figure 2, and the copper and manganese curves of figure 3» Therefore there must be an int e r p l a y between these traee ele-ments, or between them and other constituents of the fermentation medium, that exerts a protective e f f e c t on the a b i l i t y of the organism to cause an accumula-t i o n of c i t r i c acid. There i s very l i t t l e understand-ing of the mechanism of an interplay such as t h i s (33), and the study of i t i s outside of the scope of t h i s t r e a t i s e . According to figures 2 and 3, the following trace mineral l e v e l s appear to be most desirable f o r maximum c i t r i c acid production: 62.5 p. s per 100 mis. 16.5 u g 11 . " 5.0 u g 11 it 11 0.373 u g .1  24. 'Hie question n a t u r a l l y arises whether or not the requirements f o r eaeh of the inorganic elements w i l l d i f f e r from those established i n the experiments just reported when tested i n t h i s new basal medium. 1 i In order to answer t h i s question, i r o n was used; as an example because i t was affected more than zinc, copper or manganese when the temperature was lowered : from 30°C. to 25°C. 'Therefore zinc, copper and manganese were maintained at the newly established l e v e l s while i r o n was varied as i n table 1. To i l l u s t r a t e the effect of the new l e v e l as compared to the old l e v e l of minerals, and to bring out the effect of i r o n at the new l e v e l , the r e s u l t s are contrasted with the eff e c t of i r o n shown i n figure 2. This i s i l l u s t r a t -ed i n figure 4. At the higher l e v e l of trace minerals there i s a s i g n i f i c a n t l y greater acid y i e l d over the range stud-i e d . The curve at the 100?& l e v e l , and that at the 123$> l e v e l , are very nearly p a r a l l e l except that be-tween 11 )i g and 13* 73 ja g a stimulation occurs at the higher basic l e v e l . Apparently the higher l e v e l of trace minerals reduces the i r o n requirement from the 130?& l e v e l to between IOO96 and 1237&. The only a v a i l -24-A. FIG- 4 The Eff e c t of l a r i e d Levels of Iron at two d i f f e r e n t Levels of the Other Trace Elements (Zinc, Copper and Manganese)• FIG - 4 IRON LEVEL ° HIGH LEV EL "LOW LEVEL I I J 300 350 400 % 25. able explanation f o r t h i s phenomenon i s that there must be an interplay between the medium constituents or a spqring effect on one or more minerals on the i r o n requirement, increasing the mineral l e v e l s at 23°C« may cause an increase i n c i t r i c acid y i e l d by accelerating the metabolic a c t i v i t y of the c e l l . Table 4 presents evidence for t h i s p o s s i b i l i t y by comparing the a c t i v i t y of the mold at the two miner-al l e v e l s . Table 4. A Comparison of the Metabolic A c t i v i t i e s of A. niger at 2 3 ° C , Varying i r o n at Both the Low( .lOOjO-and Hi&h (\23f°) Mineral l e v e l s : Fe ; Mat Weight a Sucrose used a E f f i c i e n c y ?° —r a Level a. gf123f* g. 1. 100^ gf 12596 g a. • 100J4 a. 125/° a - 1. - -' 1 a. , .. ... a. a 4 «. a. •1 •a 23 a. 1.03 X 1.21 U 3.0 * 5.77 t 74.0 a. 69.0 a a. I a a. 30. a. 1.43 t. 1.63 » 6.4 1 8.06 t. 71.7 a 72.3 a. - a. 1. ' a a 100 •i 2.02 a 2.12 a 9.48* 11. 76 a. 70.7 a 75.7 - J a. a. a . . 1 a 125 it 2.12 i . 2. 17 a 11-4 M2.4? a 67-7 a. 72.8 a a. a. . . 1. a. 150 a. 2. 11 j . 2.28: a. 11.87*12.24 a. 66-5 a. 67-3 a. . . . a. a. a . . ... a a. 200 a. 2.35 i 2.42 a 12.12'13.17 it 6.2. 1 a. 68.2 a. J. a . •. a. a a. - * 300 a. i 2.41 a. ». 2.60 a a 11.34*13.00 a a. a 53-1 a. a 63.0 26. According to table 4 the fermentation cultures with the 125^ trace mineral l e v e l generally use a greater amount of sugar, produce a greater mycelial weight and produce more acid at ten days than do those at the 100,£ trace mineral l e v e l . The 100?& fermentation decreases i n e f f i c i e n c y with increases i n i r o n . However, the 125$ fermentation increases i n e f f i c i e n c y u n t i l a l e v e l of 11 ja g of i r o n per 100 mis. i s reached. The e f f i c i e n c y then declines with further additions of i r o n . Although the mycelial weight of the i l 2 5 c u l -tures i s higher than that of the cultures, there i s a greater e f f i c i e n c y of c i t r i c acid production over the entire range through which i r o n was varied. Thus greater mycelial weights do not necessarily lower the y i e l d of acid i n ten days fermentation. Tomlinson (43), using 15 grams of sucrose per 100 mis. of fermentation medium, showed that only eight days were necessary to obtain maximum y i e l d s of c i t r i c acid with A- niger. i n addition he found that the' a c i d i t y began to decrease aft e r the eleventh day. S i m i l a r l y , MacDonald (25), using glucose as a carbon source at 30°c. showed that a c i d accumulation reached i t s highest l e v e l at the ninth day of fermen-27. t a t i o n . However there were no data a v a i l a b l e on the time requirements of A« n i g e r f o r c i t r i c a c i d produc-t i o n at 23°C Pr e v i o u s work had i n d i c a t e d that at ten days i n a 25°G- fermentation, approximately two grams o f sugar remained unfermented from the f i f t e e n grams o r i g i n a l l y added. The time r e q u i r e d f o r complete u t i l i z a t i o n o f a v a i l a b l e carbohydrate should also be the time r e q u i r -ed to reach the maximum y i e l d o f c i t r i c a c i d . There-f o r e , u s i n g t h r e e d i f f e r e n t fermentation temperatures, the time r e q u i r e d f o r maximum accumulation o f c i t r i c a c i d was determined. The r e s u l t s are recorded i n t a b l e 5. Table 3 'Time Required to Obtain Maximum Y i e l d s of C i t r i c A c i d at Various Temperatures. Perm.- Maximum Temper- . Time- - Y i e l d of- Sugar -f> E f f i - - M y c e l i a l ature °c days- C i t r i c used g ciency. Weight g A c i d g 23 13 9.43 14.38 65.8 2.35 30 11 9.13 14.27 64.3 2.18 33 10 7.38 14.00 32.8 2.43 28 In a l l cases the termination of acid accumulation i s •almost concurrent with complete u t i l i z a t i o n of a v a i l -able carbohydrate. The e f f i c i e n c y of the 25°C. c u l -ture i s s t i l l greater than that of either the 30°C. or 35°C. culture, but the time required to reach the highest y i e l d i s two days longer than that f o r the 30°C culture. The r e s u l t s obtained here do not cor-roborate those of Tomlinson (43). Under conditions of t h i s experiment the c i t r i c acid accumulation at 30°C. continued u n t i l the eleventh day, but not u n t i l the thirteenth day did the a c i d i t y begin to decrease. At 33°C the fermentation was complete i n ten days, but the y i e l d of c i t r i c a c i d was r e l a t i v e l y low. The acid-i t y began to decrease by the eleventh day. The 2_5°CJ. fermentation was not complete u n t i l the thirteenth day. I t did not begin to decrease i n a c i d i t y u n t i l the six-teenth day. Because the r e s u l t s obtained from the cultures at 25°C. were being compared to si m i l a r cultures at 30°C , the ten day fermentation time was necessary. However, at ten days the acid i s s t i l l being produced rapid l y . This introduces a greater p o s s i b i l i t y of error. ' The re s u l t s are a measure of rate of acid formation rather 2 9 . than maximum a b i l i t y to form c i t r i c acid. Throughout t h i s study i t was noted that mat weight increased r a p i d l y during c i t r i c a c i d produc-t i o n ( f i g . 5 oz f i g . 6 ) ; However, Virtanen et al (48) and Kostuichev et a l (23) claimed that c i t r i c acid accumulation by A« niger occurs only on termination of nitrogen metabolism. Quantitative studies on nitrogen uptake of the organism were not undertaken i n t h i s work, but the fact that mycelial weight i n -creases continuously during acid accumulation pre-cludes the statement that c i t r i c acid i s not formed during growth. I t would be d i f f i c u l t to separate ac-t i v e c e l l metabolism from nitrogen uptake. 'The p o s s i b i l i t y that one or more trace minerals or growth factors other than those used here may b"e necessary f o r maximum y i e l d s of c i t r i c acid has been suggested by Tomlinson et a l ( 4 3 ) i and others. Two of these elements are cobalt and molybdenum. Using 0.01 jd! "g* s of cobalt per 100 mis. of medium, the y i e l d of c i t r i c acid was increased 0.5 grams over the normal, at ten days, using 0.1 jn g of molybdenum re-sulted i n an increase i n acid y i e l d of 1 * 13 grams over the normal at ten days. There i s l i t t l e doubt that these and other fact o r s are either essential or 30. PIG. 5 The E f f e c t of Fermentation Time on M y c e l i a l Weight of A. niger At Three Different Temperatures. FIG. 6 The E f f e c t o f Fermentation Tim© on C i t r i c A d d production at fare©, Different Temperatures. CITRIC ACID (grams) MYCELIAL WEIGHT(grams) _ _ _ ro ro w 11. stimulatory to maximum c i t r i c acid production and c e l l u l a r metabolism. However,the d i f f i c u l t i e s i n -volved i n studying the requirements of the organism f o r these f a c t o r s are many and great. A knowledge of the functions of each mineral would help to overcome most of the obstacles involved i n a study of trace mineral n u t r i t i o n . Much more experimental work on the trace mineral n u t r i t i o n of .A. niger than i s recorded here, was carr-i e d out but.was not reported due to the p o s s i b i l i t y of confusing the picture that has been presented. Much of the work was based on the p r i n c i p l e of establishing an optimal mineral l e v e l about which each element was varied. The mineral l e v e l s thus determined to be most sat i s f a c t o r y for maximum c i t r i c acid production were used as the basis f o r the succeeding experiment i n which the i n d i v i d u a l trace mineral levels.were again varied. Because of t h e i r apparent inconsistency, the r e s u l t s were extremely d i f f i c u l t to evaluate. However, they did indicate that one of the following p o s s i b i l i t i e s may occur: 1. A metal ion may have a sparing action on another. 2. An element i n r e l a t i v e l y high concentration may be 32. "toxic or may mask the ef f e c t of another element. U i i s approach w i l l he of l i t t l e value u n t i l - t h e e f f e c t s of these elements and t h e i r mechanism of action are known. 33-DISCUSSION Many of the cationic requirements of l i v i n g organisms are known to be coenzymes essential f o r the normal functioning of certain enzymes* Magnesium i s necessary f o r a number of enzymes concerned i n gly-c o l y s i s . Copper appears to be part of a large protein molecule having a respiratory function (21). However, a requirement f o r a metal does not necessarily mean that the metal i s a functional part of the system. Green et al . ( 1 ? ) suggested that manganese, cobalt, c a l -cium, cadmium, i r o n and zinc substitute for magnesium i n yeast carboxylase probably because the metal i s not c a t a l y t i c but merely acts as a l i n k betxveen the pro-t e i n and the prosthetic group. An explanation such as t h i s may aid i n c l a r i f y i n g the inte r p l a y of minerals observed i n the experimental work. Foster (18) l i s t e d the possible l o c i of a c t i v i t y of a number of trace elements. He noted seven enzymes that are s p e c i f i c f o r ir o n , three for copper and one for z i n c He l i s t e d 24 enzymes that require bivalent cations such as magnesium, manganese, zinc, cobalt, i r o n and calcium. He included oxalacetic acid car-boxylase and oxalosuccinic carboxylase, both of which require manganese as coenzyme and both of which are probably functional i n c i t r i c acid formation or degra-34. dation i n A» niger-I f manganese i s s p e c i f i c a l l y required i n the two enzyme systems mentioned above, a deficiency of t h i s metal would be expressed by a decrease i n a c t i v i t y or complete i n a c t i v a t i o n of the enzymes concerned. The required amount of manganese would support maximum a c t i v i t y of the enzyme. There i s evidence available which supports the theory that c i t r i c acid i s formed by way of the Krebs t r i c a r b o x y l i c acid-cycle i n A* niger" (8 , 2 6 , 2 7 ) . i f t h i s metabolic pathway does exist in-A* niger, the i n h i b i t i o n of oxalosuccinic carboxylase may cause an accumulation of the pre-cursors of oxalosuccinic acid. Therefore c i t r i c acid, due to it's s t a b i l i t y and the e q u i l i b r i a of the neigh-boring reactions, may be the product of maximum accu-mulation. The i n h i b i t i o n of oxalosuccinic carboxy-lase could be achieved by creating a deficiency of the essential coenzyme, manganese. However, mangan-ese would be necessary f o r the formation of c i t r i c a c id through the agency of oxalacetic acid carboxylase. The c r i t i c a l status of manganese i n maximum c i t r i c a c id accumulation could therefore be a r e s u l t of the need f o r s u f f i c i e n t of the cation for function of ox-ala c e t i c carboxylase but i n s u f f i c i e n t to permit the 35 functioning of oxalo succinic carboxylase. Chemical and b i o l o g i c a l , reactions can be charact-erized by a Q 1 Q. An increase i n temperature generally r e s u l t s i n an accelerated reaction rate. On the other hand, the influence of concentration of substrate on an enzyme reaction i s expressed by the Michaelis Con-stant. This i s the concentration of substrate necess-ary f o r h a l f maximum a c t i v i t y of the enzyme. 'There-fore an increase in. the concentration of the reactants may counteract the e f f e c t of decrease i n temperature. In t h i s manner the temperature could af f e c t the a c t i -v i t y of an enzyme system by suppressing the rate, or a l t e r i n g the extent of enzyme-metal complex formation. By increasing the concentration of the l i m i t i n g com-ponent, which i n . the work done here could be the cation, the e f f e c t of the lower fermentation temperature may be pa r t l y or wholly overcome, and sa t i s f a c t o r y y i e l d s at lower temperature l e v e l s may r e s u l t . A s i m i l a r expla-nation may aid i n understanding the decrease i n sensi-t i v i t y to the various trace elements that was evident when fermentation temperature was lowered from 35°C. to 2 5 ° C For many of the trace elements involved i n n u t r i -MQ& of A. niger only hypothetical mechanisms of action are known, i n a review on the r o l e of zinc i n fungi, Chesters and Molinson (12) reveal that t h i s element was shown to he essential f o r both growth and spor-u l a t i o n of A* niger. Most reports indicate that the addition of zinc r e s u l t s i n an increase i n the amount of mycelium formed per.unit of sugar u t i l i z e d . Zinc, as well as i r o n , copper, manganese, phosphorus, potas-sium and magnesium play an important r o l e i n u t i l i z a -t i o n of carbohydrate (10). Chesters and Rolinson (12) conclude that the exact r o l e of zinc i n carbohydrate u t i l i z a t i o n or acid formation i n fungi i s s t i l l unknown. A f t e r showing that Molybdenum was essential for maximal growth of A* niger, Steinberg postulated that-the locus of action of t h i s element was i n the enzyma-t i c reduction of n i t r a t e to n i t r i t e and hydroxamic ac i d (39). This p o s s i b i l i t y may be studied'using n i t r a t e as the sole nitrogen source. Comal t i s important as a nucleus i n vitamin B12 i n many organisms and presumably serves the same function i n fungi, i r o n i s essential for the prosthetic group of catalase, peroxidase, cytochromes, cytochrome o x i -dase and various other enzymes concerned with r e s p i r a -t i o n . Copper i s essential f o r the a c t i v i t y of ascorbic a c i d oxidase, tyrosine oxidase and other enzymes. 37« Although zinc probably has many l o c i of a c t i v i t y i n fungi, none have been d e f i n i t e l y established. K e i l i n and Mann ( 2 2 ) , using animal red blood c e l l s , found that zinc was essential as a constituent of carbonic anhydrase. Zinc may have a function s i m i l a r to t h i s i n molds. Assuming that organic products of fungi are meta-b o l i c intermediates or products of a side reaction normally occurring i n the organism, the n u t r i t i o n a l elements required f o r optimal growth should also be necessary for the formation of these products, one or more physiological abnormalities may cause the accumulation of an intermediate or side reaction com-pound. However, the c e l l would be normal i n other respects and would therefore have most of the n u t r i -t i o n a l requirements of the normal c e l l . In order to obtain e f f i c i e n t production of c i t r i c acid, i t may be necessary to p a r t i a l l y i n h i b i t the mycelial develop-ment to prevent conversion of carbohydrate to excess-ive s t r u c t u r a l material, i n t h i s respect a low my-c e l i a l weight may be desirable f o r a high acid y i e l d . But i t i s u n l i k e l y that the mechanism of formation of c i t r i c a c i d can be separated from other metabolic a c t i v i t i e s . 58. CONCLUSIONS 1. Decrease i n the temperature of fermentation from 30 °c. to 2J?°C. resulted i n an increase i n the trace elements necessary for maximum accumulation of c i t r i c a cid by A* niger 72-4. 2. 'The decrease i n temperature was accompanied by a decrease i n s e n s i t i v i t y of A» niger to the trace element content of the medium. 5* The e f f i c i e n c y of the fermentation was greatest at the lowest temperature. 4. A decrease i n fermentation temperature from 30°C. to 25°C. resulted i n a 25f» increase i n the require-ment f o r zinc, copper and manganese; a 50?s i n -crease i n the requirement for i r o n , and a 25^ -100fo increase i n the requirement for a l l four of these minerals when varied concurrently. j>. A change i n the o v e r a l l -trace mineral l e v e l r e-sulted i n a change i n the requirements f o r the in d i v i d u a l elements. An inter p l a y or sparing action exists between the four trace elements. 6. Growth of the mold and c i t r i c acid formation are closely related. 3?... 7. Tae time required for completion of fermentation under the conditions used i n t h i s work was ten days at 33° C , eleven days at 30°C and thirteen days at 23 °C 8. Cobalt and Molybdenum are probably necessary i n trace quantities f o r maximum y i e l d of c i t r i c acid. 40. BIBLIOGRAPHY 1. Association of O f f i c i a l A g r i c u l t u r a l Chemists 1940 O f f i c i a l and Tentative Methods of Analysis 5th Ed. p. 498. 2. Bernhauer, K. 1941 formation of Acid from Sugar by A« niger Z1 Factors determining the accumu-l a t i o n of c i t r i c acid. Biochem.Z. 309: 151 - 178. 3« Bertrand, D. 1941 importance of the Trace-Element Yanadium f o r A. niger Comp. Rend. Acad. S c i . 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