Studies on some Nutritional Factors Influencing the Production of Gluconic Acid

Zbl. Bakt. Abt. II, Bd. 131, S. 361-374 (1976)

[Faculty of Agriculture, Ain·Shams University, Shoubra, Cairo, Egypt]

Studies on some Nutritional Factors Influencing the Production of Gluconic Acid S. A. Z. Mahmoud, M. EI-Sawy, and O. O. Nom EI-Din Ibrahim Summary Investigations were carried out to formulate an economical medium that can give maximum yield of gluconic acid by the submerged culture technique, using Aspergillu8 niger NEELs. It was found that the highest yield of acid could be secured when a medium of the following ingredients was used: 13.0 % glucose syrup, 0.1 % NaNOs, 0.025 % calcium superphosphate, 0.007 % KCI, 0.025 % MgS0 4 • 7H20, and 0.5 % CaCO a. It was also found that glucose syrup, which represents an available and cheap carbohydrate source for gluconic acid production by fermentation, can replace glucose powder which is more expensive.

Zusammenfassung Untersuchungen wurden durchgefiihrt zur Formulierung eines wirtschaftlichen Mediums, das einen Hochstbetrag an Glukonsiiure unter Verwendung von A8pergillus niger NEELa bei Anwendung der Submerskultur·Technik erzielen kann. Man stellte fest, daB der hiichste Saurebetrag mit einem Medium folgender Zusammensetzung erzielt wurde: 13,0 % Glukosesirup, 0,1 % NaNO a, 0,025 % Kalziumsuperphosphat, 0,007 % KCI, 0,025 % MgS04 • 7H2 0 und 0,5 % CaCO s' Auch wurde festgestellt, daB Glukosesirup, der eine verfiigbare und billige Kohlenhydratquelle fiir die Herstellung von Glukonsaure durch Fermentierung darstellt, Glukosepulver, das kostspieliger ist, ersetzen kann.

Gluconic acid fermentation, as any other fermentation process, is greatly affected by the nutritional components of the medium. The variation in the nature and quan· tity of the nutritional requirements were studied by many investigators. TARA et a1. (1960) found that the optimum concentration of glucose for gluconic acid production in submerged culture by Aspergillus phaenicis was lO-15 %. FALK and KAPUR (1924) showed that the optimum nitrogen concentration, that secured the highest yield, was 0.036 % of nitrogen in the form of ammonium nitrate. HERRICK and MAY (1928) reported that 1 % NaNO a was good and suitable for acid production by P. pur· purogenum. Studies in this investigation were focused on the nutritional factors that favour a high yield of gluconic acid by fermentation. It was put in consideration the utili· zation of available and cheap raw materials for acid production. Such studies will shed some light on the basic informations needed for the possible industrialization of gluconic acid by fermentation in Egypt.

362

S. A. Z. MAlLlIOl"D,

;vr. ElrI-'AWY, and 0.

O. NUl'R EL·DIN IBRAHIM

Materials and Methods In this study Aspergillus niger NRRLa was used for the production of gluconic acid from different raw materials. The use of glucose syrup and cane"sugar molasses as carbon sources, corn steep liquor (C.S.L.) and cotton seed cake as nitrogen sources, and calcium superphosphate as phosphorus source were investigated. It should be pointed out that glucose monohydrate, chemically pure, was investigated as a carbon source in order to achieve a comparison between this source and the other tested carbon sources. Chemical analysis of employed materials are as follows: 1. Glucose mono· hydrate : specific rotation, (a) D20 + 52° to + 53°; loss on drying, 10 %; lead (Pb), no more than 0.001 %. 2. Glucose syrup: 78 % total sugar (as glucose), 46 % reducing sugar, 32 % non·reducing sugar, 16% moisture, 0.03% protein, 0.02% fats, 300 p.p.m. acidity, and 120 p.p.m. 802' 3. Cane· sugar molasses: 60.8 % total sugar, 16.6 % reducing sugar, 44.2 % sucrose, 27.4 % moisture, 0.12 % nitrogen, 5.09 % ashes, 5.09 % calcium, 0.297 % iron, 0.035 % manganese, and 0.013 % phosphorus. 4. Corn steep liquor (C.S.L.): 41 % moisture, 4.29 % total nitrogen, 24.37 % protein, and 12 % ash. 5. Cotton seed cake: 11 % moisture and volatile matter, 24 % protein, 5- 6 % fat and oil, 1 % ash, and 23 % fibers. 6. Calcium superphosphate: 61.1 % Ca(H ZPO t )2' 33 % moisture, 1.6 % Fe 2 0 a, and 0.14 % MgO. Czapek's agar medium (Difco 1972) was employed for propagation, maintenance, and sporulation of the moulds. The basal fermentation medium used was that suggested by MAHMOUD et al. (1973). Its composition is as follows: 20.0 g glucose powder, 0.100 g NaNO a, 0.010 g KH2 P0 4 , 0.025 g MgS04 • 7H20, 0.005 g KCI, 0.5 g CaC0 3 ; water up to 100 ml, and initial pH 6.5. The fermentation process was carried out in 250 ml flasks, each containing 50 ml of the medium. After sterilization, the flasks were inoculated with standard inoculum of spore suspension (0.5 million spores for each flask) and incubated at 30°C for 120 hours on a rotary shaker, i.e., using the submerged culture technique. Four replicates were made for each studied factor. It should be noted that the period of incubation, used in this study (120 hours), was found by MAHMOUD et al. (1976) to be a suitable period for attaining maximum yield of gluconic acid. During the fermentation period, necessary chemical analyses, namely pH, total titrable acidity (PEPPLER 1967), total and consumed sugars g/IOO ml fermented medium (ELOHIL 1933) as well as gluconic acid g/100 ml culture (TAHA et al. 1960) were done after 48, 72, 96, and 120 hours from the start of the experiment. Determination of interfering substances, as citric acid (MARIER and BOULETS 1958) and oxalic acid (EL-HABIBI 1972), were estimated in the fermentation culture, and amounts of gluconic acid were corrected, if necessary. Yield of gluconic acid was calculated as percentage of the weight of the original sugar used. ~cld=

weight of gluconic acid produced . . . X 100 WeIght of ongmal sugar

The conversion coefficient (c.c.) of gluconic acid was calculated on the base of consumed sugar. c.c.

=

weight of gluconic acid produced

---,--,---,-------- X

weight of sugar consumed

100

Results and Discussion A. Effect of some nutritional factors 1. Glucose powder

To study the effect of concentration of carbon source on gluconic acid production, glucose powder of the basal fermentation medium was added in different concentrations, namely 5, 10, 15, 20, and 23 g/100 ml medium.

1.92

4.32

6.02

0.0

4.0

4.0

3.5

4.5

10

15

20 (control) 23

~

0

Q

~

obi;

.~

S 00 0 0 ....

0:

~

~E '0 ~

0.0 0.0

120.0 2.35

40.0 0.78

25.0 0.5

25.0 0.5

E-<

.

~

~

E-<~

<1

-

Initial pH = 6.0. 1) C.C. = conversion coefficient.

2.88

3.5

5

;:l ....

~o

"'0

w.bi;

::x::

~

0

o~

.:

00

s '" '"~ g ~~ .... S. o S

~

F ermentation period in hours

48 hours

;;>.,

Sugar %

....

6.88 7.74 2.56

3.0 3.2

5.67

3.84

w.bi;

~

'"0 0: 0

S

o~

o

;:l 0

;:l~

.:

w

3.0

3.0

3.0

;;>.,

::x::

e

'" e Q)

S

0

0 0

e .

~

140.0 2.74

300.0 5.88

240.0 4 .71

3.0

3.0

3.0

0

~

9.4

7 .1

3.24

~

0 0 0 0

'a e abo....

0:

'0

'"~

~..,

5.76 325.0

6.37

13.54 610.0 11.96

10.72 480.0

E-< "

7 .52 360.0

bIl

3.0

200.0 3.92

00

4.16 165.0

"

3.0

Eo<

120.0 2.35

=' -

~~ E-i~

... 0

" E~ ~

~"3 ~ I': 0 o ~ S

'"

~ ~ S ..,

'U

~

;:l ....

00 00

.~

0:

'0 :;

~

~3

96 hours

obi;

...• ~::!

< ...

•

'" 3... g S

~

72 h ours

Table 1. Effect of glucose powder con centration on the production of gluconic acid

'U

0:::;-

94.8 88.0

12.32 525.0 10.29

3.0

740.0 14.5

650.0 12.8

83.9 44.3

99.9 72.5

100.0 85.3

8.62 104.0 86.2

4.4

><

]

~

~

"

S

15 ~ 0 8bi50

§

0:

.8

14.0

12.8

8.28 440.0

4.64 225.0

~

3 E-< ~ rZbi;E-ig

g bIl....

~

o S . '"

'0 :;

Q) ....

'"03

3.0

3.0

3.0

3.0

;;>.,

::x::

:::

§..:: s

"...,

~ ";

0 0

'" s ~ ~

~

120 h ours

w ~ w

l

Q

~

~

~

H

fA

..,o

Q

.,..

~

e

o·

;;-

~ ..,

Z

S CP

eJ.

o

'" g

(:0

ere

364

S.

A. Z.

1!AHM01 ' j),

M.

EL,SAWY,

and O. O.

NODR EL-DIN IBRAHIM

Table 1 shows that maximum yield was attained when glucose was used in 10 and 15 % concentration, being 88.2 % and 85.3 %, respectively. It was found that the conversion coefficient showed the highest value when using 10 and 15 %, being 104 and 100 %, respectively. Therefore, glucose powder in 13 % concentration was considered to be the suitable concentration to be used in the medium. These results are in agreement with TARA et al. (1960). 2. Nitrogen source Different nitrogen sources were used by many investigators for the production of gluconic acid. In this investigation, seven different organic and inorganic nitrogen sources were used to assess the most suitable one for gluconic acid production by A. niger NRRL3 . The sodium nitrate of the basal medium was replaced by equivalent amounts of nitrogen (0.165 g Njl) of each of the following nitrogen sources: ammonium sulfate, ammonium nitrate, peptone, corn steep liquor (C.S.L.), urea, and cotton seed cake. Results of Table 2 show that peptone attained the highest yield of gluconic acid in a short time (12.45 gjl00 ml culture in 96 hours). It also gave the highest amount of gluconic acid, as well as the highest conversion coefficient, at the end of the experimental period; being 96.5 and 106.5 %, respectively. This was followed by sodium nitrate and ammonium nitrate, each gave a yield of 95.7 %. In general it may be concluded that, on the basis of yield and conversion coefficient, nitrogen sources could be arranged in the following descending order: peptone > NaN0 3 = NH 4N0 3 > C.S.L. > urea> (NH4)2S04 > cotton seed cake. When comparing peptone with sodium nitrate as nitrogen source for gluconic acid production by A. niger NRRL3 , it could be seen that the former gave low increase in yield, being 0.8 %. Such insignificant increase does not compensate for the high cost of peptone. Therefore, it is recommended to use sodium nitrate as nitrogen source in the fermentation medium. To determine the proper concentration of sodium nitrate, the following levels were used: 0.00, 0.05, 0.10, 0.15, 0.20, 0,25, and 0.30 gj100 ml medium. The above mentioned concentrations were added to the basal llledium, whereas

other ingredients were kept unchanged. Results in Table 3 show that maximum yield of gluconic acid (96.2 %) was obtained when sodium nitrate was added at 0.10 %. When the sodium nitrate concentration was increased above this level, the yield gradually decreased, reaching 64.2 % at 0.30 % NaN0 3 . The same trend was also observed in the conversion coefficient, the highest conversion (105 %) was observed in treatment containing 0.1 % sodium nitrate. Positive relation can be observed between the biomass, representing fungal growth and the concentration of nitrogen source. The highest weight of the biomass was recorded in the high NaN0 3 (0.3 %) treatment, being 0.9 gj100 ml culture. It appears that increasing the nitrogen source, increased the biomass on the expense of the product. From the above experiment, it can be concluded that the optimum concentration of sodium nitrate, which secured the highest yield and conversion coefficient, was 0.1 %. These results are in line with those observed by MOLLIARD (1922 and 1924) who demonstrated that gluconic acid wasformed when the nitrogen content of the nutritive solution was comparatively low. BERNRAUER (1924) showed that thin mats, grown at a low temperature with a low supply of nitrogen, favoured the production of gluconic acid. On the other hand, media high in nitrogen, producing abundant mycelial mats and incu bated at a relatively high temperature, favoured the production of citric aeid rather than of gluconic acid.

0)

0 0

S

0

4.48 180.0 3.53

10.28 445.0 8 .72

4.48

2.36 100.0 1.96

3.0

3.0

4.0

3.0

4.0

NH4N0 3

Peptone

C.S.L.

Urea

Cotton seed cake

50.0 0.98

Initial pH = 5.7. Initial sugar = 12.8%. 1) C.C. = conversion coefficient.

1.16

4.48 195.0 3.82

3.0

(NH()zS04

50.0 0.98

3.52 120.0 2.35

3.0

bIl

NaNO a (control)

::1-

Sbii

~~ ~-3

00

'a S

"

"'5

E-! "

0)

oil

'0

<>0

:::;-.

~ 't:lB

:a g 4 3

0-

~

~"3

'"""' S "S .

0)

s ...3

't:l

W

48 hours

Fermentation period in hours

~

Nitrogen source

3.0

3.0

3.0

3.0

3.0

3.0

'" 3.0

~

'" ::I... S

::I

---

c

S c

178.0

360.0

3.52 104.0

4.6

7.2

2.04

3.49

7.06

10.56 550.0 10.78

9.31

8.96 475.0

6.86

Sbii

0

6.37

350.0

g

"

'§::I ~_

,,as -"

8'3

't:lE

7.84 325.0

8.0

::I

E-!!:

.

wbii ~

::1-

~o

"'0

<~

" S S

0-

;:: o

00

;::l!:

't:l '"

72 hours

Table 2. Effect of different nitrogen sources on gluconic a cid production

3.0

3.0

3.0

3.0

3.0

3.0

3. 0

~

\,,)

S

::;-

S

0)

Wbo~g

~

:;; g 4 3 _ E-!.!':

0"'"

"s .

;:J

~

~

8 "3B g....,

't:l

0

8.33

9.55

Sbo

::1-

"

00

as

:;

as "

.~

~ 't:lB

460.0

5.44 228.0

7.73 310.0

9.2

4.47

6.08

9 .0

11.68 635.0 12.45

10.54 550.0 10.78

8.95 425.0

9.55 487.0

96 hours

3.0

2.5

3 .0

3 .0

3.0

3.0

'" 3 .0

~

S

~

::I

8

S 0)

E-!.!': • ::I E-! "

•

'0

~ ;:;-: Q

S bii 0

::I _

'a88S

as "

'3

~

't:lB

~

't:l

>'? o 0;

625.0 10.29

99 .0 80.3

7.78 335.0

6.57

10.75 540.0 10.58

84.4 51.3

98.3 82.7'

10.88 570.0 11.17 102.6 86.4

11.68 635.0 12.45 106.5 96.5

11.52 625.0 12.25 106. 3 95.7

10.4

11.52 625. 0 12.25 106.3 95.7

::I Wbo

0-

;::


~ " S " s < ... :;;0 • ::I t:4J0 ::I

;::l.!':

15

120 hours

oT

U1

~

Cl

~

~.

Q

l:I

g

H

a,

(;l

o

oT

Q

~

o· ~

;;:

q

>::

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(0

3

en o

p

::>

§. 1

concentration~

:~.5

3.2

3.5

4.0

4.0

4.0

0 .05

0.10 (control)

0.15

0.20

0.25

0.30

::I

00

0

Cl

84.0

0 .0

E-;:: · ::I E- C.)

-<· ::I. .

•

S

~

o

4. 37

5.0

3.4

4 .:16

58.0

63.0

42.0

95.0

5.32 137.0

3.56

0.0

'"b()~ 0 m::1 -bO

" S "0

0 -

::I

§;::

so

1.14

1.23

0.82

1.86

2.69

1.65

0.0

3.0

3.0

3.0

3.0

:3.0

3.0

4 .8

~

0

c

C.)

-

Cl

b()

3.08

3.08

:U2

5.16

6.6

6.58

0.0

lZl

0.0

"

" _

4.8

169.0

169.0 3.313.0

7.1

7.1

6.8

8.2

4.86 :l.O

8.6

0.0

bo

::I CD

~o

.... 0

o

S

0

::I

0-

::I

00

9.0

3.31 3.0

Cl ....

S Z ::1-

Cl

"t

6.82 3.0

6.61 3 .0

0 .0

go ;:q C5 ~ ""

00

·2 S

oj 0

-cz o-a

Cl ....

174.0 341.0 3 .0

248.0

:148.0

337.0

""'

20 ~ s ~~ ~ ~

-

::J

E

e "s . 0

::I

~

S ::I 1""'1;::

"C 0 co ..

72 hours

go .... C5 ~ ~

00

::,.i

·2s

/Xl

-cE-a

·0

Cl

Initial pH = 5.6. Initial sugar = 10.6 %. 1) C.C. = Conversion coefficient.

4.0

""

;:q

I::::I

¥

Cl

48 h ours 96 hours

~

-

0.0

C5

~

284.0 5.57

284.0 5.57

274.0 5.37

:179.0 7.43

494.0 9.68

3.0

3.0

3.0

3.0

3.0

3 .0

4.8

~ ""

g0

00

·2 S

0

'" "

'S

-OZ

I::

469 .0 9 . 19

O~ O

E-;:: E-• ::I 0

-<• ::I...

• co

-S

.....

o

so S

Cl ....

8.65

8.65

8. 2

9.72

9.72

9.42

0.0

~o ::I~ lZlb(;

.... 0

::I " 0 o S

::I ::I;:: 00 ::I

Cl

-0

120 hours

of sodium nitrate (nitrogen source) on the production of gluconic acid

F ermentation p eriod in hours

0.00

%

NaNO a

Table 3. Effect of different

Cl

345.0

345.0

330.0

415.0

0

9.7 2

0.0

Sb(;

"

~

::I .....

.§

~

~

'" 0 0 -

'Q

-0..,

6.76

6.76

6.46

8.15

520.0 10.2

496.0

0 .0

E- "

-< S H':= . ::I

•

~

:g

o

6o

0 .0

:;;

-0

;:;

o-S2

96.~

78.8 64.2

78.8 64.2

78.0 60.3

83 .5 76.4

105.0

103.0 91.5

0.0

cj cj

o-S2

'" S

0.9

0.9

0 .85

0.81

0.66

O.:~

0.0

i:Q

B

Z

0)

... 0_

lf1

oc

b(;

o.-

so

~

~

....

I

~

t::1

""t"

l'=!

c:

z o

~

g>

~

-..-:

tr:

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;s::

s.

;t

>

;:::;

~

r-

:;n

0:>

8tudieR on Rome Nutritional Factors Influencing

367

3. Phosphorus source a) Di-sodium phosphate Di-sodium phosphate was added in the following concentration: 0.000, 0.010, 0.015, 0.020, 0.025, and 0.030 g/100 ml to the basal medium. Other ingredients of the medium were kept unchangEd. Results in Table 4 show that gradual increase in the yield of gluconic acid was obt'1ined with the increase in concentration of phosphate. Maximum yield (95.5 %) was recorded when di-sodium phosphate was used at 0.02 %. When di-sodium phosphate concentration was increased above thi'llevel, the yield of gluconic acid began to decrease. The same trend was also observed for the weight of the biomass of the fungal growth. The highest weight of the biomass (l.1 g/100 ml) was obtained when di-sodium phosphate was used at 0.02 %. It appears that there is a balance between the nitrogen and phosphorus sources, giving the best yield and growth of the organisms. The conversion coefficient, which ranged between 100109 %, was slightly affected by the varying cOll'J:mtrations of di-sodium phosphate. The reverse was observed in case of sodium nitrate (Table 3) where the conversion coefficient was affected by the concentration of the added nitrogen source. It is of interest to note that, when di-sodium phosphate-free medium was used, the amount of sugar consumed after 120 hours was 3.2 g, giving a yield of 23.5 % of gluconic acid. On the other hand, no sugar consume and no yield of gluconic acid was obtained wheil sodium nitrate-free medium was used (Table 3). This shows clearly that nitrogen source should be present in higher levels than phosphorus source in the medium. From the above data it can be concluded that the optimum concentration of disodium phosphate for gluconic acid production is 0.02 %. b) Calcium superphosphate Since the di-sodium phosphate is an expensive phosphorus source, it was found of interest to study the effect of calcium superphosphate, which is an available and cheap phosphorus source. For this purpose the 0.02 % di-sodium phosphate of the basal medium was substituted by an equivalent amount of phosphate of superphosphate which was found to be 0.025 %. Other ingredients of the medium were kept unchanged. It can be noticed from Table 5 that calcium superphosphate, when replacing di-sodium phosphate in equivalent amounts on phosphorus basis, gave higher yields of both gluconic acid and the bio-mass. This may be attributed to the presence of trace elements as contaminants in the superphosphate, beside phosphorus and calcium necessary for the organisms. For example, after 120 hours of fermentation the yield was 84.3 % and 92.0 % in case of di-sodium phosphate and calcium superphosphate, respectively. Also a slight increase in the conversion coefficient was observed in the case of calcium superphosphate. Therefore, it is recommended to use calcium superphosphate in the basal medium as phosphate source for producing higher yields of gluconic acid. Besides, calcium superphosphatc is available and cheaper th'1n di-sodium phosphate. 4. Potassium chloride (as potassium source) Potassium chloride was added to the basal medium in the following concentrations: 0.000,0.003,0.005,0.007, and 0.009 g/IOO ml medium. Other ingredients of the medium were kept unchanged. Results (Table 6) show that a remarkable increase in gluconic acid yield (52.3 %) was secured when KCI concentration in the medium was increased from 0.00 to

4.5

4.5

4.5

3.5

4.5

4.5

0.005

0.010 (control)

0.015

0.020

0.025

0.030

~

S

o

1.4

2.6

4.6

2.7

1.3

0.5

0.5

rIltiii

~.-<

t,;g

i:I 0 0-

en

@j

Cl

"0

E:=: Cl tiii

00

·S s

'0 "3 ., 0

g;:

"Oil

10.5 0.2

21.0 0.4

158.0 3.0

21.0 0.4

15.6 0.3

10.5 0.2

0.0 0.0

I'-<· :: 0

1'-<':::

~ <·· ...'"~

o o .-<

s

Initial pH = 6.0. Initial sugar = 13.6. 1) C.C. = conversion coefficient.

5.0

~

0.000

Na zHP0 4

48 hours

3.0

3.0

3.0

3.0

3.0

3.0

4.0

>l,

~

0

,......f

S

;:::.-

~

rIl

6.4

8.5

10.1

6.6

6.4

4.2

E:=: Cl bii

00

oS S

0

~

g;:

"Oil

'Z .,

147.0 2.9

242.0 4.7

426.0 8.4

163.0 3.2

158.0 3.0

195.0 3.8

21.0 0.41

H.=! bii E-< g

~~

t,; g ""1

o S . '"

0-

~

S S.E g ~"'5

"0 g;:

1.6

72 hours

Fermentation period in hours

3.0

3.0

3.0

3.0

3.0

3.0

3.5

>l,

~

Cl

o

o

S '-'0

~ "'" "3

9.1

10.9

11.7

9.6

8.5

7.2

3.2

S

0o

I!J

8.4

7.3

7.1

1.8

411.0

8.1

527.0 10.3

606.0 11.9

426.0

374.0

363.0

90.0

.-<

3.0

3.0

3.0

3.0

3.0

3.0

3.0

~

Cl 0

S

'"

S .

......

~

Cl

11.0

11.6

12.1

11.0

10.6

9.8

3.2

.,

0

'" ~ (.)

606.0 11.9

637.0 12.5

663.0 13.0

606.0 11.9

579.0 11.4

><

.8

...,...-01

~ "'0

108.0 87.5

107.7 92.0

107.4 95.5

108.0 87.5

107.5 83.8

105.0 75.7

100.0 23.5

bii d

~

3.2

Cl

E

00;:::;-~ 0 .

°SS

°

g;:

"Oil "2

527.0 10.3

163.0

~:::~~ rIlbOl'-<",

;;; g ""1

'" S

0......

i:I

S"'" 0 ~ "3 ~

Cl

"0

120 hours

., '" ~ ·S S · ' " t,;g <· ...~ 0 0 1'-<'::: ~.-< E:=: ~ rIltiii I'-<· 0~ Cl tiii >l,

...

§Eg

'"

"0

96 hours

Table 4. Effect of different levels of di·sodium phosphate on the production of gluconic acid

aJ

::

0.75

0.75

1.1

0.85

0.75

0.65

0.35

I:Q '"

o~

tJ1

S ~ 0':::

en

.bii

o o

S

~

CN

I:"'

~

~

Z

tj

I:"'

lo:j

~

o o ~

[

~

in

lo:j

is:

.ti

o

> ~ :::

is:

~

~

"X.

00

....'"

....

cr; p.

,:;;;:

II>

!"'"

.....

~

cr;

~

N

'"""

1) C.C.

I~tial

=

pH

%

3.5

3,5

~

l:r:I

;::;-

P"'""I

0 0

s

0

2.1

1.6

rn

='

bIl

~

bIl O

~

Q)

l3

52.7

41 .0

Eo<-oJ ."3 Eo< 0

~

S OS.

~ r,.) 0-

::;j ~

'" -oJE; S

"0 '"

48 hours

1.0

0.8

abo

='-

00 00

0

·S s

ce

"OE 0c) "3

~

3.0

3.0

~

Q)

6.3

4.6

234.0 4.6

3.0

3.0

Q)

='

S

0

8.5

5.8

rnbo

g>-

~g

o

0-

.:

00

Q)

s ....El ::l-

"0

96 hours

];:: l:r:I "bo ~

00

0

·s s

03

'5 "'S

~

"OE

194.0 3.0

rnbilE-io

H..:a

g "1 El

~~

~

r,.)

s

~ Q) S ....=' 00 ='"3 ... ~ ::::r 0""" o S .S

"0

72 hours

Fermentation p eriod in h ours

= 6.0. Initial sugar = 12.8 %. Conversion coefficient.

calcium superphosphate 0.025%

di·sodium phosphate 0.02 (control)

Phosphorus source in m ed ium III

3.0

455.0 8.9

~

l:r:I

3.0

s~

§;::

00

0

:1

·s s

ol

'0

"OE

~

II)

11.6

10.6

rnbo

::l-

~o

S "0

o

0-

~~ ~ g

"0

120 hours

319.0 6.3

. " II)

~ El Eo
]-" o

s o

T a ble 5. Effec t of calcium s uperphosphate (as phosphorus source) on gluconi c a cid production

Q)

03

"

~ ~~

abo

00 00

600.0 ll.8

550.0 10.8

Eo<· 0='

· ::l

...::

o

~

ce ..II)

>1:1

.~

"

='

S ::l oo±!

00

00

bo

o

so

105.0 92.0

1.06

102.0 843.0 0.78

·ss * * . ~ Eo
S

o

o

s

~.

~ ~

~

(JQ

5'

f

'"

c:>

'...,g"

"':j

~

o·

e<-

~

~

00

o

§

00

~

r:n e<-

Q;> ..

16.8

26.0

42.0

2.7

3.3

3.7

2.4

1.4

4.0

4.0

3.5

3.5

4.0

0.001

0.003

0.006 (control)

0.007

0.009

0 .5

1.7

0.8

0.5

0.3

0.2

s~

88 ~-

.:: S

., "

'c "3

Q;>

"OE

Initial pH = 6.0. Initial sugar = 12.8 %. I) C.C. = conversion coefficient.

26.0

89.0

10.2

1.4

4.0

.. 0

0.000

-]

8

S

~

~ ~ 0o S

§E

Q;>

"0

48 hours

Fermentation period in hours

~ ~ ~o ~-3 ~­ wbi5 Eo< "

KCI%

3.0

3.0

3.0

3.0

3.0

3.0

= ~

Q;>

-

5.2

8.9

6.8

4.5

3.9

2.3

~rLl~

~o

"'0

i"j

Q;>

.~

~

1:

,,-

§

"0..,

~

Q

~

....

a~

240.0 4.7

430.0 8.4

326.0 6.4

211.0 4.1

167.0 3.3

79.0 1.6

H

H,!: .


" '8 8

~ 0

~-;: 00 .-

"0

sE 8S

72 hours

S .

;::;-Q;>

3.3

9.0

481.0

9.4

638.0 12.5

11.9

8.3

3.0

421.0

7.8

6.4

554.0 10.9

324.0

6.4

167.0

~-

3.6

::j -

1Z2

a~

"0

., " '20 0'8

'c "3

10.1

3.0

Q;>

.

"03

tn :=; Eo<'!: • ~ ev Eo< "

;;0<'13

"S

~ (,,) 0""

~"3

" S .,e..,3 0 0

"0

3.0

3.0

3.0

3.0

~

96 hours

3.0

3.0

3.0

3.0

3.0

3.0

~

~

~ C)

"

.,

~

"0

y o ·

102.0 92.2

650.0 12.7 600.0 11.8

11.9 11.6

107.0 99.2

622.0 12.2

11.9

103.0 95.3

103.0 92.2

600.0 U.8

104.0 89.0

102.0 :36.7

\.5

C)

;:::;.

oi?

11.4

4.7

:s ....

abO

00 " 0

S

575.0 11.3

240.0

.,

·13

~ 'c "3

"0';:;

11.0

4.6

~ .;:::::! rnevEo
8 E-I.!: <'1 :3 bO_

~

0""

c:,)

S

"s S.

Q;>

E8 §"3 ....

OJ

"0

120 hors

Table 6. Effect of different concentrations of potassium chloride (as potassium source) on gluconic acid production

.........

~

tS

;>-

~

~

~ ......

tj

':"

~

s;Xl

~

~

O

P-

~

~

.;;

:E

;>-

J:,

r

~

~

~ <:;j

o

S::; ... :,; io:

N

~

'7'

o

-:t

C;:I

to

".:

3.5

3.5

3.5

3.6

3.5

O.oI

0.02

0.026 (control)

0.03

0.04

::l-

4.6

96.0 1.9

117.0 2.3

178.0 3.6

7.3

6.1

107.0 2 .1

40.0 0.8

10.0 0.2

Eo; '"

• ::l

9

~~ s~

.S

5.4

1.8

1.0

oo"bii

~

~

·s

~

"d ... ~
Initial pH = 5.7. Initia.l sugar = 13.0 %. 1) C.C. = Conversion coefficient.

3.5

~

~o

"'0

'" s

Eo;':::

9. "'1

s:: -'" 0

---

9 8

::l ::l'::: '" ::l

"d

8 2:

48 hours

F ermentation period in hours

0.00

MgSO, . 7H2 0 %

3.5

3.5

3.0

3.5

3.6

3.6

~

.~

~

"dE :3 ~ '"

5.5

7.4

9.0

7.5

5.6

2.4

115.0 2.3

180.0 3.5

235.0 4.6

165.0 3.2

95.0 1.9

40.0 0.8

H '"

s:: '" 9 89 S. 2: .SSo ~ ::s ~8 "'0 ::l_ §O- H'::: . ::l CSt(i ooti5

",'3

15 ~ S § ... 8....

72 hours

3.0

3.0

3.0

3.0

3 .0

3.0

~

~"3

-

o ",

.....

340.0 6.7 400.0 7.8 460.0 9.0

450.0 8.8 440.0 8.6

10.6 11.2

10.8 10.4

260.0 5.1

CSt(i

9.7

7.9

r;

'" '"

'0

~

"d..-

< ~ .~::s ~_

j

::s- ~~ ootii Eo; '"

~o

"'0

'" s

s:: -'" 0

]sz~ so

96 h ours

U.6 11.6 11.9

11.6 11.6

3.0 3.0

3.0 3.0

10.4 3.0

3.0

~

::s-

OObV

~o

"'0

'" s

g 0-

~

§':::

] ~

120 h ours

Table 7. Effect of different concentrations of magnesium sulfate (as magnesium source) on gluconic acid production

~

8.3

00

546.0 10.7

566.0 11.1

647.0 12.7

576.0 11.3

~

"ai

"d

~

92,2 82 ,3

95.6 85.3

106.7 97.6

97.4 83.9

97.4 83.9

79.8 63.8

'" '" ~ '29 §~ ali) oo

'5 "'3

2:

"dE

576.0 11.3

423.0

Eo; '"

. ::l

"'Eo;'::: 1 ~

9.

o......

9o

S

-:J ......

<:>:I

Jg

e.

::s

~

~

til

~ '" 0-

[

""

~.

Z

(0

~

::s

o'"

(p'

ct"

g.

Ul

372

S. A . Z.

MAHMOUD, M. EL·SAWY,

% (t';:ro

and O. O.

NOUR EL·DIN IBRAHIM

o

o Ol

e.mHno lUI 00 I /lil P!o'S otuoonm

o

lUI OOIi]UI Y.L·.L

o

e.m~lno

""

10

o
o

e.mHno lill OOI/lil peumsuoo .mlilns

00

o M

e.m~lno lUI OOI/lil

P!o'S oluoonm

o

t-

o

M

o

.,.;

e.m~lno

lUI 00I /1UI ·V·.L·.L e.mnno lUI 00 I /lil peUInsu oo .tl'llilns

00

.,.; o M

e.m~ln o TUI OOI/lil P!O~

otuoonm eJn~lno

lurooliIw 'V';L ';L e.m~lno lUI OOI/lil peUInSUO() J'SlilnS

o

o "" M

o IL:i o M

o

M

e.m~Tno lUI OOI/lil

pIO'S oluoonID e.m~]llo

ltuOOI /IUI 'V".L".L

~

e.mnno lUI OOI/lil peUInsuoo .mlilns

10 M

o

M

o ..c: 00 '
Studies on some N utritional Factors Influencing

373

0.001 %. This indicates the importance of potassium chloride as a source for potassium for gluconic acid production. Increasing potassium concentration showed marked response in the yield of gluconic acid, reaching the highest level (99.2 %) at 0.007 % of KCl. Further increase in potassium chloride decreased the yield of acid. The same trend was also found in the case of the conversion coefficient. Therefore it can be concluded that the optimal concentration of potassium chloride to produce maximum yield is 0.007 %. 5. Magnesium sulphate (as magnesium source) Magnesium sulphate was added to the basal medium in the following concentrations: 0.00,0.01,0.02, 0.025, 0.03, and 0.04 gj100 ml medium. Results (Table 7) show that the yield of gluconic acid increased from 63.8 to 83.8 %, due to the increase of magnesium sulfate concentration in the medium from 0.0 to 0.01 %. This shows the importance of magnesium in gluconic acid fermentation. The maximum yield (97.6 %) and conversion coefficient (106.7 %) were obtained when the magnesium sulfate concentration in the medium was 0.025 %. B. Effect of different cheap carbon sources The composition of the above tested fermentation medium, which gave the highest yield of gluconic acid, is as follows: 13 %glucose powder, 0.1 % NaN0 3 , 0.025 % calcium superphosphate, 0,007 % KCI, 0.025 % MgS0 4 • 7H 20 , and 0.5 % CaC03 • The carbon source in this medium was glucose powder in a concentration of 13.0 %. In view of the fact that such source is expensive, it was found of importance to investigate cheaper and locally produced raw materials, as glucose syrup and black -strap molasses, to replace glucose powder for gluconic acid production by A. niger NRRL 3 · An amount of 17 g of glucose syrup or 30 g of molasses was added to the above medium to give a final sugar concentration of 13.0 %. Other ingredients of the medium were kept unchanged. From Table 8 it can be noticed that the highest yield of gluconic acid, namely 75.4, 59.4, and 47.9 'Yo, were obtained when glucose powder, glucose syrup, and molaE'ses were used, respectively, as carbon sources. The same trend was also observed in case of the conversion coefficent. Glucose syrup gave a lower yield than glucose powder. This may be due to the fact that glucose powder, which contains only glucose, is easily converted by the gluconicdehydrogenase of the moulds to gluconic acid. Glucose syrup, that resulted from starch hydrolysis, contains 46 % invert sugars in the form of dextrin-maltose. Such inverted sugars, in order to be converted to gluconic acid, must first be hydrolysed by the mould enzymes (amylase and maltase) to glucose. Consequently, glucose syrup is expected to produce low yield of gluconic acid, when compared with glucose powder, during the fermentation period. Molasses, as shown in Ta ble 8, gave the lowest yield of gluconic acid. This may be due to the presence of some impurities and inhibitors that retarted the fermentation process. It has been shown by many investigators (TAHA et al. 1963 and N'Ew'EIGY 1971) that, although the Egyptian molasses are rich in sugars and minerals, essentially needed for the growth a nd activities of the industrial micro-organisms, yet the high content of these minerals and the presence of some inhibitory elements constitute a problem for industrial fermentation worthy of investigation. Comparing glucose powder with glucose syrup as carbon sources, the former gave an increase in acid amounting to 2.1 gjlOO ml culture (Table 8). Such increase in

~n4

S. A. Z.

MAHi\lOl'D.

lVI. EL-t-lAWY. and O. O. NOUR EL-DIN IBRAHIM, Studies on some

yield is not economical in view of the high cost of glucose powder. This indicates that glucose syrup, which is locally produced and cheap in price, can be used as carbon source instead of glucose powder which is an expensive import product.

References BERNHAUER, K.: Zum Problem der Saurebildung durch A. niger. (Vorlaufige Mitteilung.) Biochem· Z. 153 (1924),517 (c.f. Prescott and Dunn 1959). Difco Manual: Difco manual of dehydrated culture, media, and reagents. 3rd ed., 1972. Difco Laboratories, Detroit, Michigan, U.S.A. EL-HABIBI, A. M.: Ecological and phytochemical studies on Pro8opis stephaniana. Ph. D. Thesis, Fac. Sc., Cairo University, U.A.R, 1972. ELOHIL, J. T.: Report of the subcommittee on the development of a volumetric copper reduction method of sugar determination. Cereal Chem. 10 (1933),470-476. FALK, R., und KAPUR, S. N.: Uber Gluconsaure-Bildung durch Fadenpilze. Ber. 57B (1924), 920 (c. f. Prescott and Dunn 1959). HERRICK, H. T., and MAY, O. E.: The production of gluconic acid by the Penicillium luteum-purpurogenum, Group II. Some optimal conditions for acid formation. J. BioI. Chem. 77 (1928), 185. MAHMOUD, S. A. Z., EL-SAWY, M., and NOUR EL-DIN, 0.0.: Studies on the production of gluconic acid by fermentation (under publication, 1973. In: Egypt J. Td. Sci). MARIER, 1. R, and BOULETS, M.: Direct determination of citric acid in milk with an improved pyridine acetic acid method. J. of Dairy Science 41 (1958), 1683. MOLLIARD, M.: StlI' une nouvelle fermentation acide produite par Ie Sterigmatocystis nigra. Compt. rend. 174 (1922), 881 (c.f. Prescott and Dunn 1959). - Nouvalles raJh3rch93 sur form'1tion d'acides organiques par Ie Sterigmatacystis niger en millieux desequilbres. Compt. rencl. 178 (1924),41 (c.f. Prescott and Dunn 1959). NEWEIGY, N. A.: Citric acid fermentation. Ph. D. Thesis, Fac. Agric., Ain Shams University, U.A.R., 1971. PEPPLER, H. G.: Microbial Technology. Reinhold Pubi. Corporation, New York, Amsterdam, London 1967. PRESCOTT, S. C., and DUNN, C. G.: Industrial Microbiology. 3rd. ed. McGraw-Hill Book Company, New York, Toronto, London 1959. TAHA, E. E. D. M., CAD, A. M., and ABBAS, M. H.: Microbial production of gluconic acid by Egyptian mould. Arch. Mikrobioi. 36 (1960), 109-115. TARA, E. M., WAGER, K., and ZAKI, D. M.: The Chemical Composition of Egyptian Cane-sugar. J. Chem. (U.A.R) 2 (1963),163-180. Author's address: Prof. Dr. S. A. Z. MAHMOUD, Microbiological Dept., Fac. of Agriculture, Shoubra, Cairo (A.R.E.).