Effect of amino acids and vitamins on citric acid biosynthesis

Effect of amino acids and vitamins on citric acid biosynthesis

Food Biotechnology S. Bielecki, J. Tramper and J. Polak (Editors) 9 2000 Elsevier Science B.V. All rights reserved. 251 Effect o f amino acids and v...

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Food Biotechnology S. Bielecki, J. Tramper and J. Polak (Editors) 9 2000 Elsevier Science B.V. All rights reserved.

251

Effect o f amino acids and vitamins on citric acid biosynthesis W. Lesniak and W. Podgorski Food Biotechnology Department, Wroclaw University of Economics, Komandorska 118/120, 53-345 Wroclaw, Poland

It was observed that a some amino acids and vitamins have a stimulating effect on citric acid biosynthesis, particularly when it concerns Aspergillus niger mutants, indicating sensibility to deficiency of these substances in the fermentation medium. Therefore, the aim of the present experiment was to evaluate the effect of some amino acids and vitamins on submerged citric acid biosynthesis by the UV mutant Aspergillus niger B-64-5 in sucrose mineral medium. It was found that the citric acid formation was stimulated by the amino acids valine and glutamic and aspartic acid, and by the vitamins thiamine-HCl, nicotinic acid amide and ascorbic acid.

INTRODUCTION Some organic and non-organic compounds have a stimulating effect on Aspergillus niger mycelium growth and citric acid synthesis. It was observed that some amino acids and vitamins have the same effect. It applies particularly to the process of citric acid biosynthesis in synthetic medium with Aspergillus niger mutants, indicating sensibility to deficiency of these substances in the fermentation medium. It was found that Aspergillus niger strains exposed to UV rays can loose the ability of biosynthesis of some amino acids. Among them are glutamic acid, proline and ornithine which are also needed for the production of other amino acids. The basic role is for glutamic acid, which is the first organic product of nitrogen assimilation in most microorganisms. Then, as the result of glutamic acid carbon chain reconstruction with contribution of other basic amino acids, the residual amino acids are produced. According to this, it is supposed, that decrease in the rate of essential amino acids biosynthesis leads to reduction of mycelium biomass production. Experiments of other authors showed that from the amino acids in the medium, glutamic acid was used most, followed by aspartic acid and alanine, and then, used the least, leucine and isoleucine [ 1]. In Aspergillus niger mycelium some of the amino acids are assimilated completely, while others are only deaminated. Deamination of amino acids leads to synthesis of pyruvic acid, ct-ketoglutaric acid, succinic acid, fumaric acid and oxalic acid, which are intermediate products of metabolism. It was shown [1] that addition of aspartic or glutamic acid to sucrose-mineral medium increases by 8 - 38% the biomass formation and by 9 - 16% the amount of citric acid formed by the UV mutant Aspergillus niger T.1. The minimal concentration of glutamic acid

252 exerting a favourable effect on growth and acid formation is 0.05%. It is also well known that many vitamins play an important role in the enzymatic activities of the microbial metabolism, where they act as coenzymes for different reactions. In relation to vitamins, there is an opinion that their presence in media for Aspergillus niger is not necessary because of the possibility of that strain to synthesise vitamins itself. Nevertheless, it was shown that addition of small amounts of biotin to medium stimulates Aspergillus niger growth, as well as addition of pantothenic acid [2]. The citric acid biosynthesis is stimulated by addition of thiamine, pyridoxine and nicotinic acid [3]. Therefore, the present experiment was carried out to evaluate the effect of amino acids and vitamins on submerged citric acid biosynthesis by the active mutant Aspergillus niger B-64-5.

MATERIALS AND METHODS Strain: selected UV mutant Aspergillus niger B-64-5. Medium: sucrose - 150 g/1 NH4NO3 2 g/1 KH2P04 - 0 . 2 g/1 MgSO4-7H20

- 0.2 g/l

pH 2.8-3.0 Fermentation was carried out in 750 ml flasks containing 125 ml of the medium on a shaker (200 rpm). Total acidity was determined by titration of 2 ml of fermentation broth with 0.1 M NaOH against phenolphthalein. Citric acid was estimated colourometrically according to Marrier and Boulets [5]. Mycelial dry weight was determined by drying at 105~ until constant weight was attained. Reducing sugars were determined according to the Lane Eynon method as modified by Soczynski [6].

RESULTS In the first stage of experiments, the effect of different concentrations of amino acids i.e. alanine, leucine, isoleucine, glycine, proline, serine, arginine, threonine, lysine, glycine, valine and glutamic and aspartic acid, on the growth and activity of Aspergillus niger was studied, (Table 1). It was found that valine, glutamic acid and aspartic acid increased biosynthesis of citric acid by 6-8%, while glycine, serine and arginine induced an increase of 4-5%, as compared to the basic medium. In contrast, alanine, leucine and iso-leucine at a higher concentration (0.01%) decreased biosynthesis of citric acid by 24-40% (Fig. 1). The biomass of the UV B-64-5 mutant of Aspergillus niger generally decreased by about 5-10% with a concentration increase of amino acids added as compared to the basic medium (Fig.l). Only for the addition of alanine at concentration 0.002-0.004% an increase of biomass by 15% was observed. In the second stage of experiments, the basal medium was supplemented by each of the following vitamins in the proper concentration: aseorbic acid, thiamine-HC1, riboflavin,

253 pyridoxine, nicotinic acid amide, mesoinositol, calcium pantothenate, folic acid and PABA (para-amino benzoic acid) (Table 2 and Fig. 2). Table 1 Effect of amino acids on mycelial growth and citric acid biosynthesis

Amino acid

]3-alanine

L-leucine

DL-isoleucine

glycine

DL-proline

DL-serine

DL-arginine

DL-threonine

DL-lysine

DL-valine

glutamic acid

aspartic acid

Biosynthesis parameters

Addition of amino acids calculated as amino acid nitrogen [%]

[g/l], [g d.s./1]

0.0

0.001

0.002

0.004

0.006

0.010

citric acid

126

126

117

110

78

72

mycelium

12.0

11.7

13.0

13.9

11.1

10.6

citric acid

126

125

127

122

119

76

mycelium

12.0

12.8

11.2

10.6

11.2

11.1

citric acid

126

118

118

114

119

96

mycelium

12.0

11.8

11.3

10.6

12.2

11.4

citric acid

126

125

126

132

129

115

mycelium

12.7

12.2

11.7

11.4

11.8

10.9

citric acid

126

123

126

124

123

123

mycelium

12.7

10.8

11.3

11.6

11.2

11.3

citric acid

126

122

131

123

120

123

mycelium

12.7

11.4

10.6

10.9

10.8

10.8

citric acid

120

120

121

121

123

126

mycelium

13.1

12.2

11.8

11.6

12.1

12.2

citric acid

120

118

122

118

119

115

mycelium

13.1

11.3

11.7

11.3

11.6

12.2

citric acid

120

116

116

120

117

114

mycelium

13.1

12.0

11.8

11.9

12.5

13.0

citric acid

113

115

120

119

120

115

mycelium

14.2

13.3

13.0

13.1

13.0

13.1

citric acid

113

118

115

118

120

122

mycelium

14.2

13.5

14.4

14.2

14.1

13.4

citric acid

113

120

120

113

115

116

mycelium

14.2

13.6

13.1

13.1

13.2

13.1

254 Table 2 Effect of vitamins on mycelial growth and citric acid biosynthesis

Vitamin

Biosynthesis parameters 0.0

0.1

0.5

1.0

5.0

10.0

citric acid

120

120

122

126

125

124

mycelium

13.5

12.6

11.8

12.3

11.8

11.4

citric acid

120

118

117

110

67

0.0

mycelium

13.5

12.3

11.6

12.0

9.2

0.0

citric acid

120

118

118

120

120

118

mycelium

13.5

12.4

11.8

11.6

11.8

12.2

citric acid

113

114

116

114

110

104

mycelium

14.6

12.8

13.6

13.0

13.2

13.7

citric acid

106

106

110

112

110

112

mycelium

14.6

13.5

12.6

13.3

12.8

12.4

citric acid

106

114

118

109

110

105

mycelium

14.6

12.0

12.1

12.2

12.0

12.5

citric acid

113

115

115

113

112

112

mycelium

14.0

13.9

12.6

12.5

12.6

12.4

citric acid

113

112

113

113

106

105

mycelium

14.0

12.9

13.8

13.2

13.2

13.9

citric acid

113

110

109

105

110

108

mycelium

14.8

13.2

12.3

12.6

12.6

12.1

citric acid

113

108

108

110

109

108

mycelium

14.0

12.4

13.6

13.1

13.1

12.8

[g/l], [g d.s./1] thiamine-HCl

riboflavin

pyridoxine-HC1

cobalamin

ascorbic acid nicotinic acid amide mesoinositol calcium pantothenate folic acid para-aminobenzoic acid

Addition of vitamins [mg/l]

255

F i g u r e 1. E f f e c t o f a m i n o acids on citric acid p r o d u c t i o n . 1 - 13-alanine, 2 - 1-1eucine, 3 - D L - i s o - l e u c i n e , 4 - g l y c i n e , 5 - D L - p r o l i n e , 6- D L - s e r i n e , 7 D L - a r g i n i n e , 8 - D L - v a l i n e , 9 - g l u t a m i c acid, 10 - aspartic acid. C o n t r o l = 100%.

F i g u r e 2. E f f e c t o f v i t a m i n s on citric acid p r o d u c t i o n . 1 - t h i a m i n e - H C 1 , 2 - riboflavin, 3 - p y r i d o x i n e - H C 1 , 4 - c o b o l a m i n , 5 - a s c o r b i c acid, 6 nicotinic acid a m i d e , 7 - m e s o i n o s i t o l , 8 - c a l c i u m p a n t o t h e n a t e , 9 - folic acid, 1 0 - paraa m i n o - b e n z o i c acid. C o n t r o l = 100%.

256 Only thiamine-HCl, nicotinic acid amide and ascorbic acid in higher concentrations (1 - 5 mg/l) increased biosynthesis of citric acid by 5 - 10%. In contrast to these vitamins, riboflavin added in a concentration 5 mg/1 decreased citric acid yield to about 50% (Fig. 2) and at a concentration of 10 mg/1 completely stopped growth of mycelium and therefore biosynthesis of citric acid. Other studied vitamins showed negligible effects on citric acid biosynthesis and growth of mycelium. We conclude that mutant Aspergillus niger B-64-5 does not show a strong sensibility to deficiency of amino acids or vitamins in the sucrose - mineral medium. REFERENCES 1. J.D. Kasatkina, Mikrobiologija, 30 (1961) 3. 2. D.N. Lal and A.S. Srivastava, Zentralbl. Mikrobiol., 137(5) (1982) 381. 3. W. Mashhoor, R.F. Gamal, A.A. Refaat, S.A. Nasr, Annals Agric. Sci., 33(1) (1988) 153. 4. W. Lesniak, Prace Naukowe WSE Wroclaw, Technologia, 52(74) (1974) 49. 5. J.R. Mailer and M. Boulets, J. Dairy Sci., 41 (1958) 1683. 6. S. Soczynski, Przem. Spozywczy, 9 (1955) 416.