Selection of Suitable Physico-Chemical Conditions for Gluconic Acid Production by a Mutant Aureobasidium Pullulans SG 80
S. Ganguly* , S.K. Mandal, S.K. Patra
1Department of Biological Sciences, Sankrail Abhoy Charan High School (H.S.), Sankrail, Howrah, West Bengal, India
2Department of Organon of Medicine, Midnapore Homeopathic Medical College and Hospital, Midnapore (W), West Bengal, India
3Department of Physiology, Surendranath College, Kolkata, West Bengal, India
*Corresponding Author E-mail: subhadeepgangulyphysiol@rediffmail.com
ABSTRACT:
An experimental study was conducted to improve the production of gluconic acid by the mutant Aureobasidium pullulans SG 80 by optimizing different physic-chemical conditions for fermentation. After optimization of different factors, maximum production was obtained with : glucose,8%; (NH4)2SO4,1.0%; K2HPO4,0.15%; CaCO3, 0.25% , MnSO4,,15%; and MgSO4.7H2O,0.25% with maximum utilization of sugar at pH,6.5;volume of medium,30ml; cell density, 5x106 cells/ml; shaker speed ,200 rpm and temperature, 300C.
KEYWORDS: gluconic acid, Aureobasidium pullulans, fermentation, optimization,utilization
Gluconic acid and its salts are used in different food, pharmaceutical and hygienic products and textile industries1-5. In recent years its worldwide annual production is 60,000 tonns and available in market as 50% technical grade aqueous solution (by mass)5.To meet such huge demands, several technics have been adopted among which production by microbial means was proved to be the best as it is cheap, comparatively easier and produces stereo-specific isomer3,4. Thus, for last several decades several attempts have been made to increase its microbial production6-11.The relation between different Physico-Chemical factors and bacterial growth is very crucial in this context. Considering the facts, our present study was intended to examine different Physico-Chemical parameters on gluconic acid fermentation by the mutant Aureobasidium pullulans SG80 ,to optimize its Physical environment as well as to select suitable synthetic medium for this mutant to maximize the production.
MATERIALS AND METHODS:
Microorganism: A regulatory mutant Aureobasidium pullulans X36 was isolated from the soil of Sankrail , West Bengal, India, which accumulated only 13.6 gm/ml gluconic acid.
This strain was subsequently subjected for mutational treatment with UV irradiation and the maximum producer (36.3mg/ml) was isolated and maintained for further trials.
Composition of Maintenance medium:
Maintenance medium composed of: glucose, 2%; (NH4)2SO4, 0.4% ; MgSO4.7H2O,0.01%; biotin,0.1µg/ml.
Fermentation medium:
Glucose, 10%; Co(NH2)2,0.2%; K2HPO4,0.1%;MnSO4, 0.1%; MgSO4 . 7H2O, 0.15%. Fermentation was carried out at pH,7.0;volume of medium, 25ml,cell density, 3X106 cells/ml at 300C using 100 ml Erlenmayer conical flask at 150 rpm.
Estimation of gluconic acid:
Gluconic acid was estimated by hydroxamate method as described by Lien et al.(1959)12.
Estimation of residual sugar:
Residual sugar was estimated by the method of Gusakov et al.(2011)13.
RESULTS AND DISCUSSION:
1. Optimization of Physical Parameters
1.1. Optimization of initial pH
|
Initial pH |
Gluconic acid (mg/ml) |
Residual sugar (%) |
|
5.0 |
**30.0±0.618 |
**16.2±0.874 |
|
5.5 |
**33.2±0.742 |
*14.3±0.881 |
|
6.0 |
*37.6±0.661 |
12.9±0.876 |
|
⫵6.5 |
**39.2±0.42 |
**11.6±0.642 |
|
7.0(Control) |
36.3±0.669 |
13.2±0.761 |
|
7.5 |
*34.7±0.874 |
13.9±0.813 |
(Values were expressed as mean ± SEM, where n=6;* *p<0.01, *p<0.05 when compare to control, where ⫵maximum production.)
1.2. Optimization of volume of medium
|
Volume of medium (ml) |
Gluconic acid (mg/ml) |
Residual sugar (%) |
|
20 |
**35.6±0.773 |
**13.6±0.081 |
|
25(control) |
39.2±0.661 |
11.6±0.613 |
|
⫵30 |
**41.2±0.731 |
*10.1±0.614 |
|
35 |
37.3±0.673 |
**13.0±0.684 |
(Values were expressed as mean ± SEM, where n=6; **p<0.01, *p<0.05 when compare to control, where ⫵maximum production.)
1.3. Optimization of inoculum cell density
|
Cell density (cells/ml) |
Glutamic acid (mg/ml) |
Residual sugar(%) |
|
2x106 |
37.3±0.682 |
**13.0±0.661 |
|
3x106(control) |
41.2±0.661 |
**10.1±0.876 |
|
4x106 |
*42.3±0.743 |
*9.6±0.632 |
|
⫵5x106 |
**44.1±0.687 |
8.4±0.713 |
|
6x106 |
43.2±0.775 |
8.8±0.687 |
(Values were expressed as mean ± SEM, where n=6; **p<0.01,
*p<0.05 when compare to control, where ⫵maximum production.)
1.4. Optimization of Shaker speed
|
Shaker speed (rpm) |
Gluconic acid (mg/ml) |
Residual sugar(%) |
|
50 |
**37.2±0.668 |
**13.0±0.616 |
|
100 |
**41.6±0.671 |
**10.0±0.662 |
|
150(control) |
44.1±0.832 |
8.4±0.653 |
|
⫵200 |
**47.3±0.761 |
**6.8±0.611 |
|
250 |
**46.1±0.683 |
**7.3±0.668 |
(Values were expressed as mean ± SEM, where n=6; **p<0.01,
When compare to control, where ⫵maximum production.)
1.5. Optimization of temperature
|
Temperature (0C) |
Gluconic acid (mg/ml) |
Residual sugar (%) |
|
25 |
**33.2±0.743 |
**14.2±0.775 |
|
26 |
**36.1±0.586 |
**13.3±0.771 |
|
27 |
**37.1±0.881 |
**13.1±0.832 |
|
28 |
**39.6±0.661 |
**11.5±0.581 |
|
29 |
**43.2±0.732 |
**8.7±0.881 |
|
⫵30(control) |
47.3±0.631 |
6.8±0.659 |
|
31 |
**44.2±0.596 |
**8.5±0.732 |
(Values were expressed as mean ± SEM, where n=6; **p<0.01,
when compare to control, where ⫵maximum production.
2. Optimization of Chemical constituents of the medium
2.1. Selection of carbon source
|
Carbon source (s) |
Gluconic acid (mg/ml) |
Residual sugar (%) |
|
Glucose (control) |
47.3±0.685 |
6.8±0.661 |
|
Fructose |
**13.2±0.961 |
**18.6±0.745 |
|
Sucrose |
**11.6±0.771 |
**20.3±0.773 |
|
Lactose |
**16.2±0.823 |
**17.2±0.683 |
(Values were expressed as mean ± SEM, where n=6; **p<0.01,
When compare to control, where ⫵maximum production.)
2.2. Optimization of glucose concentration
|
Glucose (%) |
Gluconic acid (mg/ml) |
Residual sugar (%) |
|
6.0 |
**45.2±0.732 |
**3.2±0.582 |
|
⫵8.0 |
*49.1±0.816 |
*5.8±0.911 |
|
10.0(control) |
47.3±0.643 |
6.8±0.682 |
|
12.0 |
**46.1±0.716 |
**9.3±0.766 |
(Values were expressed as mean ± SEM, where n=6; **p<0.01 and *p<0.05 when compare to control, where ⫵maximum production.)
2.3. Selection of suitable nitrogen source
|
|
Gluonic acid(mg/ml) |
Residual sugar(%) |
|
⫵(NH4)2SO4 |
**53.3±0.668 |
**3.9±0.618 |
|
NH4Cl |
**43.6±0.736 |
8.6±0.718 |
|
CO(NH2)2(control) |
51.6±0.636 |
**5.0±0.711 |
|
(NH4)2HPO4 |
**33.1±0.716 |
**14.3±0.832 |
|
(NH4)2H2PO4 |
**30.6±0.694 |
**16.0±0.811 |
(Values were expressed as mean ± SEM, where n=6; **p<0.01,
when compare to control, where ⫵maximum production.)
2.4. Optimization of nitrogen source
|
(NH4)2SO4 [%N] |
Gluconic acid (mg/ml) |
Residual sugar (%) |
|
0.6 |
**46.8±0.881 |
*7.1±0.663 |
|
0.8(control) |
49.1±0.672 |
5.8±0.737 |
|
⫵1.0 |
**51.6±0.831 |
5.0±0.843 |
|
1.2 |
**46.2±0.691 |
*7.2±0.743 |
(Values were expressed as mean ± SEM, where n=6; **p<0.01
and *p<0.05 when compare to control, where ⫵maximum production.)
2.5 Optimization of mineral elements required
|
Mineral element(s) |
Concentration (%) |
Gluconic acid (mg/ml) |
Residual sugar (%) |
|
K2HPO4 |
0.1(control) |
53.3±0.683 |
3.9±0.461 |
|
|
0.15 |
53.7±0.719 |
3.8±0.661 |
|
|
0.2 |
53.0±0.882 |
4.1±0.732 |
|
CaC03 |
0.0(control) |
53.7±0.839 |
3.8±0.628 |
|
|
0.10 |
53.8±0.781 |
3.8±0.661 |
|
|
0.15 |
54.±0.669 |
3.6±0.713 |
|
|
0.20 |
54.2±0.832 |
3.5±0.533 |
|
|
⫵0.25 |
54.8±0.569 |
3.3±0.664 |
|
|
0.30 |
54.2±0.855 |
3.5±0.832 |
|
MNSO4 |
0.1(control) |
54.8±0.919 |
3.3±0.661 |
|
|
0.15 |
55.2±0.766 |
3.1±0.613 |
|
|
0.20 |
55.0±0.781 |
3.2±0.583 |
|
MgSO4.7H2O |
0.10 |
54.8±0.832 |
3.3±0.661 |
|
A |
0.15(control) |
55.2±0.991 |
3.1±0.736 |
|
|
0.20 |
*55.9±0.763 |
2.9±0.764 |
|
|
0.25 |
*56.2±0.771 |
2.6±0.668 |
|
|
0.30 |
*56.0±0.864 |
2.7±0.673 |
(Values were expressed as mean ± SEM, where n=6; and *p<0.05 when compare to control, where ⫵maximum production.)
Table 1.1-2.5 depicted the patterns of effect of different physico-chemical factors on gluconic acid production by the mutant Aureobasidium pullulans SG 80. Maximum production was obtained with a synthetic medium composed of: glucose, 8%; (NH4)2SO4, 1.0%; K2HPO4, 0.15%; CaCO3, 0.25%, MnSO4, 15%; and MgSO4.7H2O, 0.25% with maximum utilization of sugar at pH, 6.5; volume of medium,30ml; cell density, 5x106 cells/ml; shaker speed, 200 rpm and temperature, 300C. Anastassiadis and Rehm (2006) also reported sililar pattern of result using similar bacterial strain14. CuSO4.5H20 showed adverse effect where as KCl and KH2PO4, FeSO4.7H2O, ZnSO4.7H2O, Na2MoO4.2H2O, and CaCl2 showed no effect on production.
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Received on 28.12.2012 Modified on 10.01.2013
Accepted on 17.01.2013 © AJRC All right reserved
Asian J. Research Chem. 6(1): January 2013; Page 83-85