Mycological Production of Citric Acid in India V. Effect ... · Mycological Production of Citric Acid in India V. Effect of some low molecular weight alcohols ... citric acid based

Mycological Production of Citric Acid in IndiaV. Effect of some low molecular weight alcohols

Jyoti S i n g h and B. S. M e h r o t r a

Botany Department, University of Allahabad

One of the important advances in the technology of the citric acidfermentation was the observation that the use of low molecular weightalcohols such as methanol, ethanol, or isopropanol as adjuncts to themedium greatly promotes citric acid production both in surface andsubmerged cultures. This observation was first made by S a k a g u c h iand B a b a (1951) and it was later elaborated by M o y e r (1953).

One of the effects of alcohols was to increase the tolerance levelof manganese, iron and zinc considerably. The sensitivity of the fermen-tation to these trace metals usually precluded the use of crude carbo-hydrates as a carbon source unless these were first subjected to consi-derable purification. These alcohols by poisoning the fermentationcounteracted the inhibitory effect of metallic ions and other impurities,thus permitting the use of crude substrates such as cane molasses,high test molasses, ground corn, wheat flour, and wheat starch. Amongthe solvents found useful were methanol, ethanol, isopropanol. Also,the tolerance to alcohols by the moulds was found to be associatedwith pH of the medium, and amount of inoculum (M o y e r, 1953 a).

C i e g l e r and R a p e r (1957a; 1960) using a culture ofA. f o n s e c a e u s NRRL 67 were able to confirm the stimulatoryeffect of methanol as reported by M o y e r (1953) and in additioncorrelated stimulation with the presence of manganese. Also, theyreported that the effect of adding methanol to fermentation media ismore complex than merely increasing tolerance of the mould to tracemetals. T a h a and E l - Z a i n y (1959) subsequently reported thatmethanol masked the stimulatory action of zinc but also eliminatedthe inhibitory effect of iron and manganese. Additional papers relatingto the alcohol effect have been published by S p r i n g e r and G e r-n e t (1956); K a w a h a r a et al. (1956); and U s a m i and T a k e-t o m i (1961).

There are two theories concerning the role of methanol orethanol in stimulating the citric acid production. According tothe first theory, postulated by E r k a m a et al. (1949) and S t e r nand O c h o a (1951), citric acid arises by condensation of acetate withanother intermediate compound (Oxalacetate). Therefore, ethanol might

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act as a donor of acetate. But it failed to prove the role of methanolwhich has a more profound effect than ethanol. According to the se-cond theory put forward by M o y e r (1953); S p r i n g e r and G e r-n e t (1956); C i e g l e r and R a p e r (1957), the alcohols might actas metabolic poison somewhat in the same manner as claimed foraceto-naphtene (I v a n o v and M a k r i n o v a , 1941) thus altering thenormal carbohydrate metabolic pathway so that more of citric acidaccumulates.

In view of the importance of alcohols on citric acid metabolismtheir effect on citric acid production by the same six strains of A. nigergroup taken for earlier studies ( M e h r o t r a and S i n g h , 1971),was undertaken. The study includes the following aspects of the pro-blem:1. Comparatice effect of different alcohols on citric acid production.2. Effect of alcohols on the length of incubation period.3. Effect of alcohols on concentration of sugar needed for maximum

production of acid.4. Effect of the alcohol needed for maximum production of acid in

relation to the pH of the medium.

M a t e r i a l s a n d M e t h o d s

The organisms taken for the study were the same six strains ofA, niger designated M2, L3, RK2, A2, SJt and O40 as in the previousstudies. The fermentation medium employed contained: Sucrose (com-mercial market sugar), 150 g; NH4NO:I, 2.23 g; KH2PO4, 1.0 g; MgSO4 .. 7 H2O, 0.223 g; and distilled water to make 1000 ml. Market sugar wastaken as the chief carbohydrate source but the effect of alcohol wasobserved also on crude carbohydrate sources as Gur and Starch. Themethod of preparation of the medium was the same as described inprevious papers of the series ( M e h r o t r a and S i n g h , 1971; andS i n g h and M e h r o t r a , 1971). The pH of the medium was adjustedto 2.4—2.8 after sterilization. The different alcohols tried were methyl,ethyl, isopropyl and butyl. These alcohols in different concentrationswere added aseptically after about 24 hours growth of the cultures.The cultures were incubated at 28° C (+ 1) for days.

The estimations were made for titrable acidity by neutralizing2 ml aliquot with N/10 NaOH taking phenolphthalein as indicator;total residual sugar by S o m o g y ' s (1945) method; and citric acid bymethod of B e r n h a u e r (1926).

R e s u l t s

1. Comparative effect of different alcohols on citric acid productionby six strains of A. niger group with three different sources of carbo-hydrate, i. e., sucrose (market sugar). 'Gur' & starch.

244


Sydowia Annal. Mycol. Ser. 2, Vol. XXVI Plate I

EFFfCT OF SOME LOW MOLECULAR WEIGHT ALCOHOLS ON CTRIC AC 10 PRODUCTION n

BY SIX STRAINS OF ASPFRr.lLLUS NIGFR GROUP L



The effect of 0.5, 1.0, 2.5 and 2.5% methyl, ethyl, isopropyl andbutyl alcohols on the production of citric acid by six strains of A. nigergroup taking market sugar as carbohydrate source is presented in theplate I, figs. 1—24. It •was observed that methanol even at 0.5%stimulated citric acid formation markedly. The yield increased •withthe increase in methanol concentration upto 2.5 ml/culture. The opti-mum concentration of methanol for maximum production was 0.5—1.0%for L3; 0.5—2.5% for M2 and 2.5% for the rest of the strains thoughthe yield was not much effected by still higher concentration of me-thanol in the case of L3. Ethanol had stimulatory effect at 0.5% con-centration but at higher concentration it retarded acid production.Ethanol at 0.5% was equally good as methanol in the case of M2 andL3. Isopropanol at lower concentration was ineffective but at higherconcentration retarded the yield. However, a slight increase in theyield was obtained at 0.5—1.5% in the case of M2 and 2.0—2.5% in thecase of La. Butanol at 0.5% concentration slightly stimulated acidproduction but at higher concentration (1.0%) retarded the yield andat still higher concentration it proved to be toxic.

It was also seen that there was great enhancement in the acid pro-duction with 2% methanol when crude carbohydrates as Gur and starchwere used.

2. Effect of alcohols on the length of incubation period.Effect of 2.5% methanol (the most effective concentration of me-

thanol found in the earlier experiment) on all the strains and 0.5%ethanol for M2 and L3 (equally good as methanol) was observed onthe length of the incubation period required for optimum acid produc-tion. The results are presented in the Table 2. It was evident fromthe results that though the yield was higher in the case of alcoholsupplemented cultures than in 'control' (without alcohol) medium, theoptimum incubation time required to obtain the highest yield was thesame, i. e., 7—8 days. These alcohols, therefore, did not effect theoptimum fermentation period required for the highest yield.

3. Effect of alcohols on the sugar concentration required for maximumyield.

The effect of 2.5% methanol on different sugar concentration ispresented in Table 3. It was observed that when the percentage ofsugar was increased from 150 to 175 g/liter and alcohol was supplemen-ted to the culture media the strains consumed almost all of the sugarand about 70—80% yield was obtained based on the sugar consumed.At still higher sugar concentration, i. e., 20% the yield was almost thesame as at 17.5% but when the concentration of alcohol was raised to3.0—3.5% higher yields of citric acid were obtained.

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Table 2

Effect of alcohol on the lenght of incubation period

Strain AlcoholNo. concen-

trationin ml/culture

1 2

M2 Methanol2,5%

Ethanol0,5%

Li3 Methanol2,5 /o

Ethanol0,5%

RKj Methanol2,5%

Aj Methanol2,5%

SJ t Methanol2,5%

O10 Methanol2,5%

Daysofincuba-tion

3

6789

6789

6789

6168089

6789

6789

6789

6789

Totalacidityin ml/culture

4

15601810185015801960192017701800101014601770150011607188018901470195518001660144:017301730175011401670170016951300172016101700

Sugar Calciumconsumed citratein gm/culture

5

13,85515,015,015,014,95515,015,015,09,275

13,68515,015,011,27513,68515,015,012,1415,014.85515,012,46515,014,6714,875

8,514,04515,015,012,43513,6513,5715,0

in gm/culture

6

14,257515,81515,60514,2917,6116,96216,7216,9458,625

13,53515,46712,6510,282513,53516,60516,602513,40516,31514.01314.0513,515,814,87515,269,17

14,3215,0514,9712,31514,77714,33513,27

Citricacidin gm/culture

7

10,991112,191712,0311,01613k5775.13,07512,88913,0636,649

10,435611,92359,75187,9267

10,435612,8112,808810,41112,510,802610,8310,3312,1811,46711,764

7,06911,03911,611,53269,4936

11,391511,0410,23

Percentyield ofcitric acidbased onsugarconsumed

8

79,381,380,373,490,786,7885,987,171,676,280,065,0170,376,285,385,085,783,372,772,282,818,178,0179,0883,178,677,376,976,485,681,369,2

4. Effect of alcohol in relation to the pH of the medium.

The effect of 1.0% methanol, ethanol, isopropanol and butanolwas studied both on a medium with low pH, i. e., 2.4 and also onewith a higher pH, i. e., 5.0 (this is the original pH of the medium after

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Table 3

Effect of alcohol on concentration of sugar needed for maximum productionof citric acid by six strains of A. niger

Strain SugarNo. concen-

tration

1 2

M2 15,017,5

20,0

L3 15,017,5

20,0

RK2 15,017,5

20,0

A2 15,017,0

20,0

SJX 15,017,5

20,0

O40 15,017,5

20,0

Methanoladded inm l /culture

3

2,52,52,53,03,5

2,52,52,53,03,5

2,52,52,53,03,5

2,52,52,53,03,5

2,52,52,52,03,5

2,52,52,53,03,5

Totalacidityin ml/culture

4

199026002330235024601480297021002185227018902010238022202200180020002040210021601790212020302410259019601070225024302445

Sugarconsumein gm/culture

5

15,017,516,95517,3419,04515,017,0820,020,020,015,016,82519,319,3419,21015,017,518,018,52519,0515,017,518,518,8219,21515,017,516,43519,32519,77

Calcium3d citrate

in gm/culture

6

17,13518,42516,9219,0419,8813,602518,82516,65518,6119,7516,132517,2917,9517,887520,0116,516,9517,623518,252518,915,6717,03516,33518,99518,6117,03518,527516,1718,19519,5425

Citricacidin gm/culture

7

13,224714,213,0414,667915,325410,48614,512212,839514,346415,22512,436513,328813,837613,784415,55512,7113,1113,58614,0714,5712,0913,132312,59214,64314,346413,132314,27212,465414,02615,065

Percentyield ofcitricacid

8

88,182,376,985,180,069,985,164,1971,776,183,080,071,671,281,0284,875,075,575,976,680,675,068,0676,674,687,5582,775,372,676,2

autoclaving). The results are presented in table 4. It was observedthat the yield of citric acid increased on addition of 1.0% alcohol whenthe pH of the medium was very low (2.4). At 5.0 pH the yield was verymuch, retarded except in the case of methanol added to cultures. In thecase of 1.0% methanol the amount of acid produced both at higherand lower pH was almost the same but when the higher concentrationof methanol was tested there was retardation in yield at pF 5.

247


D i s c u s s i o n

The low molecular weight alcohols have been found to exert aprofound stimulatory effect on citric acid production in the presentcase. It is also evident from the results that of all the alcohols used(methanol, ethanol, isopropanol, butanol) methanol was the most ef-fective for all the six strains. M o y e r (1953 a) also found methanolto be the best alcohol for this purpose. Ethanol at 0.5% concentrationwas equally good as methanol for only two strains used in the presentstudy. These alcohols stimulated acid production only up to a certainconcentration after which they exerted an inhibitory effect on growthas well as acid production probably due to the toxicity caused by thesealcohols. Butanol was the most toxic among all the alcohols used butat lower concentration even butanol showed stimulatory effect. Higheryields of citric acid in the presence of alcohols with crude carbohydra-tes as market sugar, Gur and starch indicates that these alcohols in-crease the tolerance level of metallic impurities present in them.

The increased utilization of sugar and its greater conversion intocitric acid in the presence of alcohols is in favour of the theory proposedby Er k a m a et al. (1949), and S t e r n and O c h o a (1951) that thealcohols provide acetate to the citric acid cycle. This acetate combineswith the dicarboxylic acid formed from the higher sugar content andforms tricarboxylic acid. This theory however, fails to prove the roleof methanol as it cannot be a donor of acetate.

The report of M o y e r (1953 a) that the tolerance to these alcoholsby the mould was found to be associated with pH is also substantiatedby the present observations. The lower pH seems to minimize thetoxicity caused by these alcohols and because methanol is least toxic,high yields were obtained even at 5.0 pH in the presence of 1.0% metha-nol. When higher concentrations of methanol were added the acid pro-duction was obtained only in the case of the medium with low pH.Mo y e r (1953 b) reported that these alcohols divert mould metabolismin slightly acidic (pH 6.3) media from gluconic to citric acid productionand enhance the yield in the more acidic media usually used in thecitric acid fermentation.

The exact role of these alcohols on fermentation is still not clear.M o y e r (1953 b) has postulated in relation to citric acid productionby A. niger strains that alcohols and acidity jointly bring about al-teration in the normal carbohydrate metabolic pathway.

S u m m a r y

Effect of different concentrations of low molecular weight (me-thanol, ethanol, isopropanol and butanol) alcohols on citric acid pro-duction by six strains of A. niger, viz., M2, L3, RK2, A2, SJj, and 040

248


was studied. Methanol in 2.5% concentration for all the strains and0.5% ethanol for strain M2 and L3 were found to be the best forcitric acid production when market sugar was the carbohydrate source.Also, high yields of citric acid were obtained from crude carbohydra-tes as 'Gur' and maize starch with 2% methanol. Effect of 2.5% metha-nol for all the strains and 0.5% ethanol for strain Mä und L3 was obser-ved on the length of the incubation period also. It was found that thesealcohols do not shorten the fermentation period. Effect of 2.5% metha-nol on sugar concentration to get maximum yield was also observed.It •was concluded from the results that the acid production increasedwith higher concentration of sugar in the presence of alcohol. Butwhen a very high concentration (2.0%) of sugar was taken a higherpercentage of alcohol (3.5%) was required. It was also observed thatthe acid yield in the presence of alcohol increased only when themedium was highly acidic. Methanol (1%) showed stimulating effecteven in the case of media with high pH (5.0) but when a higher con-centration of methanol was tested the yield retarded considerably.

A c k n o w l e d g e m e n t s

This work was supported by a grant to the second author by theMinistry of Defence, Research and Development Organization, Govern-ment of India. The authors express their grateful thanks to the Direc-tor of the Organization.

Re fe r ece s

C i e g l e r , A. and K. B. R a p e r, 1957: Application of heterokaryons ofAspergillus to commercial type fermentation. App. Microbiol., 5:106—110.

— and —• 1960: Relationship of trace metals to the alcohol effect incitric acid synthesis by A. Fonsecaeus Arch. Microbiol., 37: 188—192.

E r k a m a , J. H. and B. H a g e r s t r a n d , 1949: The effect of aeration onthe formation of citric acid in surface mould cultures. Acta. Chem.Scand., 3: 858—861.

* Ivanov , N. N. and N. A. M a k r i n o v a, 1941: Doklady Akad. Nauk.SSSR, 30: 356.

K a w a h a r a , H. and K. M a t s u k u b o , 1956: Journ. Ferm. Technol., 34:153—158.

M e h r o t r a , B. S., and J y o t i S ingh , 1971: Mycological production ofcitric acid in India: I. Utilization of some cheaper and readily avail-able carbohydrate source (under publication); II. Effect of factorsother than nutritional (under publication); III. Effect of differentnitrogen compounds (under publication).

Moye r , A. J., 1953a: Effect of alcohols on mycological production ofcitric acid in surface and submerged culture. I. Nature of alcoholeffect, Appl. Microbiol., 1: 1—7.

— 1953 b: II. Fermentation of crude carbohydrate sources, Appl. Micro-biol., 1: 8—13.

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• S a k a g u c h i , K. and Bab a, 1951: (Abstract) Chemical abstract,April 10 issue.

S ingh , Jyoti and B. S. M e h r o t r a , 1971: Mycological production ofcitric acid in India: IV. Effect of different sulphur and phosphoruscompounds (under publication).

S p r i n g e r , R. and C. G e r n e t, 1956: The influence of ethanol on thesynthesis of organic acids by Aspergillus niger. Arch. Pharm., 289:48—52.

S t e r n , J. R. and S. O c h o a, 1951: Enzymatic synthesis of citric acid.I. Synthesis with soluble enzymes. J. Biol. Chem., 191: 161—172.

Taha , D. M. and E 1 - Z a i n y, 1959: The mutual influence of methanoland trace elements on the mycological production of citric acid. Arch.Microbiol., 33: 124—127.

• U s a m i , S. and N. T a k e t o m i , 19G1: Action of methanol and furfurolon citric acid fermentation. J. Chem. Soc. Japan, 64: 2072.

*) Originals not seen.

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Mycological Production of Citric Acid in India V. Effect ... · Mycological Production of Citric Acid in India V. Effect of some low molecular weight alcohols ... citric acid based