B I O S Y N T H E S I S OF C I T R I C ACID

T H E I N F L U E N C E O F H Y D R O G E N I O N C O N C E N T R A T I O N O N T H E M I C R O B I A L P R O D U C T I O N O F C I T R I C ACID BY V A R I O U S

A S P E R G I L L U S N I G E R S T R A I N S .

by

KRISHNA BAHADUR ~ ANANT NARAIN MALVIYA

Chemistry Department, University o/ Allahabad, Allahabad, India

(with 1 fig.)

(3.111.1966)

INTRODUCTION

The formation of metabolites in the microbial cultures have been greatly influenced by the hydrogen ion concentration of the medium. SORENSEN (1912) was the first to work out the role of hydrogen ion concentration in the biochemical and microbiological investi- gations. NEIStt (1949) observed that the growth as well as the me- tabolic activity of microorganisms are subject to the pH value of the culture media.

CURRIE (1917) maintained the pH range of 3--4 of his culture media by employing hydrochloric acid and showed that by pH control the formation of metabolites (citric and oxalic acid) is con- siderably varied. DOELGER & PRESCOTT (1934) observed that be- tween pH range 1.60 to 2.20 more citric acid is formed in the cul- ture medium and the yield of oxalic acid is reduced. OBSZEWSI<I reported pH value 1.2 to 2.0 as optional for the good yield of citric acid.

In most of the experiments cited above, when influence of hy- drogen ion concentration of the microbial solution on the forma- tion of citric acid were studied, either the initial pH was made up without any effort to maintain this pH during the fermentation, or the pH of the medium was examined at regular intervals and was adjusted as desired by the addition of hydrogen or hydroxyl ions. Such a s tudy cannot give a complete s tudy of the enzymatic activities at certain pH value of the medium.

360 K. BA.ttADUR ~: A. N. MALVIYA

In the present work the pH value of the microbial solutions has been controlled by the use of potassium chloride-hydrochloric acid buffer as desired. Here the influence of hydrogen ion concentration on the production of citric and oxalic acid by the four strains of Aspergillus niger has been worked out.

M~ETHOD A N D ~ A T E R I A L

A mineral nutrients solution containing 0.5 g sodium chloride, 0.5 g potassium sulphate, 0.5 g magnesium sulphate, 0.5 g calcium chloride, 0.5 g mono-sodium-di hydrogen phosphate and 2.0 g am- monium sulphate per litre was prepared. 10 ml of this mineral so- lution were taken in each culture medium contained in 250 ml co- nical flasks and to each flask 70 ml of distilled water were added. The flasks containing the culture solution were divided into four sets of four culture media each. To the media of one set 20 ml of potassium chloride and hydrochloric acid buffer solution of pH one was added. To the other three sets 20 ml of the buffer solution of pH 1.4, 1.8 and 2.2 were added. The buffer solution of KC1 and HC1 was made as follows:

(A) 0.2 M so lu t ion of KC1 (14.91 g in 1OOO ml) (13) 0.2 M HC1.

50 mI of (A) p H 1.0 1.4 1.8 2.2

x ml of (13) x 97.0 41.5 16.6

6.7

To each of the culture media 10 g of sucrose as the source of car- bon were added. The flasks were cotton plugged and were auto- claved at 15 lbs. steam pressure for th i r ty minutes. After sterili- zation the culture media were rearranged in four sets, each contain- ing culture media of pH 1.0, 1.4, 1.8 and 2.2. Each set was seeded by a trace of strain of A. niger. Following four strains were selected for the study.

St ra in No. Source

1. 363 I n d i a n Agr icu l tu ra l R e s e a r c h I n s t i t u t e New Delhi 2. 612 N a t i o n a l Chemica l Labo ra to ry , P o o n a 3. 122 4. B(Local ) I so la t ed b y Dr. I3. S, IV[EHROTRA ( B o t a n y D e p a r t -

m e n t , U n i v e r s i t y of Al lahabad)

The culture media were incubated at 30°C and were analysed after ten days.

The Aspergillus niger molds were separated from the culture so- lution by filtration through a dried and weighed filter paper. The mass of the mycelium was washed 4 or 5 times with distilled water, dried at 70--80 ° C in an air oven, cooled in a desiccator and weighed.

B I O S Y N T I t t g S I S OF C I T R I C A C I D 361

The culture solution was made up to 200 ml. Hundred ml of this solution were taken out and made neutral by the addition of ammonia. To this hot, neutral solution 2 % calcium chloride so- lution were added to completely precipitate the citric and oxalic acid, formed during fermentation, as calcium citrate and calcium oxalate respectively.

On cooling the precipitate was filtered through dry and weighed filter paper. The precipitate was washed 3 or 4 times with distilled water and was dried in air oven at 120 ° C. The dried precipitate was cooled overnight in a desiccator and weighed. The weighed precipitate of citrate and oxalate was treated with 50 % hot acetic acid solution. The precipitate of calcium oxalate remained on the filter paper while calcium citrate drained off. The calcium oxalate was washed 5 or 6 times with distilled water, dried, cooled in a desic- cator for overnight and weighed. Thus the amount of citric acid and oxalic acid was calculated.

50 ml of the remaining culture solution were taken in another flask. This was hydrolysed with 2 ml 2N HC1. After hydrolysis the excess of acid was neutralized by adding a few drops of dilute so- dium carbonate solution. The solution was made up to 100 ml and was t i trated against boiling standard Fehling solution using me- thylene blue as an indicator near the end point. Note: - (1) Only A.R. Chemicals were used.

(2) The pH of the culture medius was tested with an elec- tric pH meter.

DISCUSSION

In the case of A. niger strain 363 the yield of citric acid increases with the rise in the hydrogen ion concentration of the culture media. This increase in the production of citric acid is accompanied by a decrease in the yield of oxalic acid, increase in the growth of the mold and less consumption of the sugar. At pH value 1.0, the yield of citric acid observed is 0.0600 g while at pH value 2.2, 0.1609 g citric of acid is obtained. It is interesting here to find

O B S E R V A T I O N

TABLE I

The study o/the growth o/ mold and the production of citric and oxalic acid at di//erent hydrogen ion concentrations in the culture media o/ A. niger strain 363.

p H of t h e cu l tu re yield of citr ic acid yield of oxal ic g rowth of s u g a r lef t m e d i u m (g) acid (g) mold (g) (g)

1.0 0.0600 0.0042 0.2900 6.9785 1.4 0.0610 0.0217 0.5530 6.5600 1.8 0.0968 0.0007 0.9120 5.8570 2.2 0.1690 0.0023 0.9670 7.1304

362 K, B A H A D U R ~5 A. N . M A L V I Y A

TABLE I I

The study o I the growth o1 mold and production o/citric acid and ozalic acid at dil/erent hydrogen ion concentrations in the culture media o/ A. niger strain 612.

p H o f t h e cu l tu re yield of ci tr ic acid yield of oxal ic g r o w t h of s u g a r le f t m e d i u m (g) acid (g) mo ld (g) (g)

1.0 0.0746 0.0133 0.1698 9.1111 1.4 0.0263 nil 0.9010 7.6278 1.8 0.0181 nH 1.0730 8.00 2.2 0.0094 0.0005 0.4150 8.6315

TABLE III

The study o1 the growth of mold and production o1 citric acid and oxalic acid at di//erent hydrogen ion concentrations in the culture media o I A. niger strain 122.

p H of t h e cu l tu re yield of citr ic acid yield of oxal ic g rowth of s u g a r lef t m e d i u m (g) acid (g) mo ld (g) (g)

1.0 0.0649 0.0039 0.0830 7.1304 1.1 0.0710 nil 0.5168 9.3714 1.8 0.0443 0.0043 0.2790 8.8648 2.2 0.0445 0.0070 0.9620 6.8332

TABLE I V

The study o/the growth o/mold and production ol citric acid and o,~mlic acid at di/lerent hydrogen ion concentrations in the culture media o/ A. niger strain B

p H o f t h e cu l tu re yield of citr ic acid yield of oxal ic g rowth of suga r le f t m e d i u m (g) acid (g) mold (g) (g)

1.0 0.0289 0.0007 0.2590 7.6278 1.4 0.0379 0.0040 0.4500 8.8648 1.8 0.0123 0.0049 0.2670 9.3717 2.2 0.0182 0.0140 0.2930 7.2888

that less consumption of the substrate (sugar) gives more formation of citric acid accompanied by a good growth of Aspergillus niger mold in the microbial culture.

The metabolic activity of the strains 612, 122, and B markedly differs from the strain 363. The A. niger strain 612 gives better yield of citric acid at low pH value (1.0) of the culture medium. In this case the yield of citric acid decreases with the rise in the hydrogen ion concentration of the culture media. The decrease in the yield of citric acid is accompanied by the more consumption of the sugar. The growth of the mold is least at low pH value (1.0) of the culture media. Thus when there is less consumption of sugar the growth of the mycelium is less but the formation of metabo-

B I O S Y N T H E S I S O F C I T R I C A C I D 363

lites (citric and oxalic acid) is large in the case of A. niger strain 612. The pH value 1.4 is suitable for the good yield of citric acid in

the case of A. niger strain 122. The good yield of citric acid is ac- companied by less consumption of sugar and comparatively less growth of the mycelium and practically no formation of oxalic acid m this case.

0 2 0

~ - s t r a i n 363 J[~-strain 612 J'-1--strain 122 [7~--strain B

0.15 ._c

~t u -= 0.10 (3

g 0.05

0.00 I I I

1.0 f.4 1.8 pH Values

2.2

Fig. 1. Production of citric acid at 10 % sugar concentrat ion by different strains of A. niger.

The A. niger strain B produces better yield of citric acid at pH 1.4 of the culture media. At this pH value the growth of the mold is also higher than other hydrogen ion concentration of the culture media studied.

Thus out of all these strains the A. niger strain 363 is the best citric acid producing organism and the most suitable pH value of the culture medium for a good yield of citric acid is 2.2 (see fig.). The production of citric acid, and the growth of mold differs from A. niger strain to strain at various hydrogen ion concentration of the microbial culture.

Summary

The A. niger strain 363 is better in its citric acid producing capa- city than other strains studied. The pH value of the culture media differs from strain to strain for the good yield of citric acid. When there is good yield of citric acid the sugar consumption is less and mycelial growth is also less.

364 K. BAHADUR ~:; A. N. MALVIYA

Acknowledgements

Authors are thankful to C.S.I.R., New Delhi for financing the research and to Dr. B. S. MEHROTRA for providing the Strain B. We also thank I.A.R.I., New Delhi and N.C.L. Poona, for pro- viding A. niger strains.

References

SORErCSEN, S. P. L. (1912). tJber die Niessung und Bedeutung der Wasserstoffionen- konzentration bei biologischen Prozessen., Ergeb. Physiol., 12, 393.

NEISH, A. C., BLACKWOOD, A. C. & LEDIENGHAM, C. A. (1949). 2--3, ButanedioI Fermentation; First Int. Cong. Biochem. (Cambridge) England, 333.

CURRIE, J. N. (1917). The Citric acid Fermentat ion of Aspergillus Niger; J. biol. Chem.; 31: 15.

DOELGER, W. P. & PRESCOTT, S. C. (1934). Citric acid Fermentation; Ind. Eng. Chem., 26: 1142.

OBSZEWSKI. W. (1952). Polske. Aked. Umiejetnosai, Prace Bolnie-zolesne, 62, 16.