VEGETOS: An International Journal of Plant ResearchOnline ISSN: 2229-4473
Print ISSN: 0970-4078

Research Article, Vegetos Vol: 30 Issue: 4

Gibberellins Production Techniques by Immobilization

Ahamad S1*, Sharma SK1 and Agarwal DK2

1Directorate of Research, Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu (J and K), India

2Division of Plant Pathology, IARI, New Delhi, India

*Corresponding Author : Ahamad S
Directorate of Research, Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu (J and K), India
Tel: 7889808422
E-mail: [email protected]

Received: May 08, 2017 Accepted: August 08, 2017 Published: August 10, 2017

Citation: Ahamad S, Sharma SK, Agarwal DK (2017) Gibberellins Production Techniques by Immobilization. Vegetos 30:4.doi: 10.4172/2229-4473.1000317

Abstract

The gibberellins are one of the major groups of growth promoting hormones which play an essential role in regulation of growth and development of angiospermic plants. Gibberellins are secondary metabolites of the fungus, Gibberella fujiluroi with its imperfect state of Fusarium moniliforme. F. moniliforme gives higher yields of GA3 on a variety of media and is associated with bakane diseases of rice. The performance of immobilized growing cells of Gibbrella fujikuroi-52 was affected by the immobilization agent used, nature and age of cells, mycelial cell density and size of beads. The beads, prepared by using standardized procedures, could be used for 7 cycles without affecting productivity in semi-continuous culture. The rate of production of gibberellic acid was 0.33-65 mg/l/h. In immobilized cells, GF-52 produced highest GA3 i.e. 0.99 g/l as compared to 0.77 g from in un-immobilized cells. Immobilized cells produced 0.74 g/l as compared to 0.57 g/l in un-immobilized cells.

Keywords: GA3; Gibberellic acid; Gibberella fujikuroi; Immobilization; Fusarium monilifurme

Introduction

Gibberellins are secondary metabolites of the fungus, Gibberella fujikuroi with its imperfect state Fusarium moniliforme. Solid State Fermentation (SSF) considered as comparatively cheaper and efficient [1-3]. This process offers many economic and practical advantages like low cost of substrate, less capital investment and less plant operation. In SSF the production of GA, is found to be influenced by a number of physical and nutritional factors. By optimizing these factors, the GA3 yield can be increased [4-7]. Although the SSF technique is known to be highly suitable for fungal cultures, it is not yet exploited by the industry for the production of GA3 [4].

The gibberellins are one of the major groups of growth promoting hormones which play an essential role in regulation of growth and development of angiospermic plants. Gibberellins are secondary metabolites of the fungus, Gibberella fujiluroi with its imperfect state of Fusarium moniliforme. F. moniliforme gives higher yields of GA3 on a variety of media [3] and is associated with bakane diseases of rice. Enhanced yields can also be achieved by means of genetic manipulation of fungal strains, optimization of fermentation condition or by application of new and more efficient bio-processes [8-12]. A promising method currently being investigated is the use of immobilized cells under the conditions of semi-continuous or continuous fermentation [13-15]. Interest in immobilized whole cells system has increased in the last twenty years because of the inherent advantages derived from this method.

The first report on the production of GA, by immobilized mycelia of F. moniliforme was based on the use of n-alkanes as carbon source [14]. The yield of GA3 achieved by using immobilized cells was however, very row [14-16 ]. Kahlon and Malhotra [14] produced GA3 in shake flasks using F.monliforme fungal mycelia, entrapped in sodium alginate. The performance of immobilized growing-cells in the production of GA3 was found to be significantly affected by various factors. With regard to yield of GA3 from free as well as immobilized mycelia, there was no significant difference [4,17]. However, continuous production of GA3 from free as well as I reactor by immobilized mycelia of G.fujikuroi in calcium alginate beads increased the yield [14]. Immobilized cells also produced GA3 with high stability [18]. In recent, a number of advantages on the use of immobilized growing cells are being reported and it has been speculated that these will be applied extensively in future due to their economic nature compared to conventional fermentation techniques [19]. Hence, many studies were undertaken for GA3 production by immobilization.

Material and Methods

Mycelial cell production

Five fungal isolates namely, Gibberella fujikuroi (GF- 52), G. fujikuroi (GF-1068) Fusarium monliforme (FM-2451), F moniliorme (FM-I) and F moniliorme var. subglutinans (FMS-2193) were obtained from Indian Type culture collection (ITCC), New Delhi. FM-I was isolated from wheat kernels and maintained on potato dextrose agar (PDA) medium slants at 4°C. Isolates were grown in Czapek-Dox broth medium in shake flasks at 28 ± 1°C till the initiation of GA3 production phase, i.e. 3 days. The mycelial cells were separated by centrifugation at 5000 rpm for 20 minutes and used in the immobilization experiment. All these operations including the immobilization and storage of beads were done under aseptic conditions. The fungal mycelia were entrapped in sodium alginate beads as described by Kren et al. [20].

Immobilization Techniques

Batch culture

For immobilization, selected fungal cultures were grown in Czapek-Dox broth for 9 days. The myceliar mat was harvested after the incubation periods, washed thoroughly and homogenized in a homogenizer. The homogenates was mixed with sterile sodium alginate solution. The resultants mixture was added drop wise to the sterilized 2.5% CaCl2 solution with the help of an injection syringe. After 30 minutes, the beads were formed (1-3 mm in diameter) and were decanted, washed repeatedly with distilled water and transferred to Czapek-Dox medium for supporting cell proliferation. After 20- 30 hours incubation, the beads were transferred to GA3 production broth (Czapek-Dox). The flasks were maintained in a Psychrotherm incubator shaker at 120 rpm for 9 days. The temperature was maintained at 30 ± 2°C. At the end of the incubation period, the culture broth was decanted, filtered and GA, produced was estimated spectrophotometrically. All the experiments in the present studies were conducted in triplicates.

Semi-continuous culture

An inverted conical fluidized bioreactor of 1 lite capacity, for continuous culture of plant cell suspension, was taken filled with 500 ml of medium and 2% sodium alginate and sterilized. Immobilized beads (75 ml) were added aseptically and the fermentation was allowed to continue at 30 ± 1°C for 120 h. To effect aeration and agitation, air was introduced from the bottom of the bioreactor at 10 volume of air/volume of medium per minute (v/v/m) level. Evaporation loss was compensated by adding 5 ml distilled water at l0 hour intervals. At the end of incubation, the liquid was drained off and intact beads remaining in the bioreactor were washed twice with same volume of the sterile medium. The stabilized sodium alginate medium in 500 ml quantity was added and the fermentation was continued as a second cycle. The procedure was repeated up to the 10th cycle and GA3 formed in each cycle estimated.

Results and Discussion

Effect of immobilization on GA3 production under submerged fermentation (SmF) condition

To study the effect of immobilization of fungal cultures on GA, production, the selected culture GF-52 was immobilized in sodium alginate beads and was evaluated for GA3 production under SmF condition. From the results obtained (Table 1) it was clear that immobilized fungal cultures produced significantly higher quantities of GA3 than the un-immobilized cells. Apart from the type strain, the fungus, GF-52 produced significantly higher quantity of GA3 (0.88 g/l) followed by F.moniliforme-2451, F.moniliforme var subglutinans-2193, F.moniliforme-I and G. fujikuroi-1068 with yield of 0.70, 0.62, 0.55 and 0.53 g/l, respectively.

GA3 Yields (g/l)
Isolates/ Cultures
cells
un–immobilized immobilized cells Culture Means
GF-52 0.77 0.99 0.88
FM-2451 0.61 0.79 0.7
FMS-2193 0.54 0.7 0.62
FM-I 0.48 0.62 0.55
GF-1068 0.46 0.59 0.53
Substrate Means 0.57 0.74 -
S.Ed Treatment    Culture Culture and Treatment
0,03 0.03 0.08
0.06 0.06 N.S.

Table 1: Effect of immobilization on GA3 production under SmF techniques.

Performance of immobilized growing cells prepared by using standardized technique, in 1 litre capacity inverted conical fluidized bioreactor over 10 cycles of 120 hours each presented in Table 2. The rate of the production of GA3 in the first 8 cycles was almost constant but after that it started to decreased. Though the beads were firm and stable even after the l0 cycle, the outlet surface became rough and developing small openings. These were caused by the outgrowth of mycelial cells which ultimately got separated from the beads probably due to vigorous fluidization caused by the high rate of aeration. The pH medium remained unaltered at 5.5 throughout the operation period of 5 days of all cycles.

Cycle No*  GA3 production mg/litre Rate of production mg/litre/hour
1 72 0.60
2 78 0.65
3 76 0.63
4 72 0.60
5 76 0.63
6 67 0.55
7 69 0.57
8 76 0.63
9 62 0.11
10 41 0.33

Table 2: Semi-Continuous production of GA3 by immobilized growing cells of G.fujikuroi-52.

Nava Saucedo et al. [11] reported the same gibberellic acid production in both free and immobilized mycelium shake flask cultures was 0.384 and 0.408 mg GA3 of biomass/l/day, respectively. However, the immobilized growing cell system have not proved economical so far in the production of GA3, probably due to the involvement of any complex steps and regulatory mechanisms in converting carbon substrates into GA3. Kumar and Lonsane [15] produced GA3 in shake flasks using the mycelia of F. moniliforme entrapped in sodium alginate beads. However, the inceased due to immobilization was insignificant. The yield of GA3 achieved so far by using immobilized cells were very low i.e. 52 mg after 120 hour in sodium alginate [16]. The successful use of mobilized enzymes and immobilized whole cells in laboratory and industrial processes had created lot of interest among scientists leading to application of this technique to other products including GA3 production [17]. The results of the present study revealed that immobilized cells produce more GA3 when compared to un-immobilized cells (Table 1). Immobilized cells of GF-52 produced 0.99 g l-1 against 0.77g l-1 un-immobilized cells. Since the studies were conducted only once and the immobilized cells were not repeatedly used for GA3 production, final conclusion can be drawn only after further investigations. Kumar and Lonsane [17] reported that immobilized cells produced more GA3 with high stability. In the present investigation, G.fujikuroi-52 maintained its lead followed by F. moniliforme-2451 in GA3 yield which is again indication of the stability of the above mentioned fungi with regard to GA3 production. The diminished prospects for improvements in economics in SmF techniques [4] and the recent studies leading to 1.2-1.4 g of GA3 per kg dry mold bran (DMB) or per 1 liter crude extract as well as reduced downstream processing expenses by extending solid state fermentation techniques to the production of GA3 indicates [15-17] that the solid state fermentation, rather than immobilized growing cell system, is the key for reducing the cost of production of GA3 to a substantial level [16].

Conclusion

The results of the present study revealed that immobilized cells produce more GA3 when compared to un-immobilized cells. Immobilized cells of GF-52 produced 0.99 g l-1 against 0.77g l-1 unimmobilized cells. In the present investigation studies, G.fujikuroi-52 maintained its lead followed by F. moniliforme-2451 in GA3 yield which is again indication of the stability of the above mentioned fungi with regard to GA3 production.

Acknowledgement

Authors are thanks to Ex. Prof. A.K. Sarbhoy, Division of Plant Pathology, IARI, New Delhi for providing facilities during the course of investigations.

References

Track Your Manuscript

H5 Index

SCImago Journal & Country Rank

Share This Page