BIOTECHNOLOGY TECHNIQUES Volume 7 No.1 (January 1993) pp.15-18

THE USE OF FIBROUS NETWORK OF MATURED DRIED FRUIT OF LUFFA AEGYPTICA AS IMMOBILIZING AGENT

M. IQBAL AND S. I. ZAFAR Biotechnology and Food Research Centre

PCSIR Laboratories Complex, Lahore-54600, Pakistan

SUMMARY

A fibrous network of matured dried fruit of Luffa aegyptica is reported as a new and inexpensive material for the immobilization of algal ce/Is. Structure of the fibrous network and the technique of immob~1~vation is described. The unicellular red alga Porphyridium cruentum was used as the study organ/sm.

INTRODUCTION

A large number of reports concerning immob~1~v-ation of algae have been published in recent years. ImmobiliT.ation is useful for the maintenance of algal cultures (Tamponnet et al. 1985) and for the commercial production of biochemicals such as polysaccharides from Porphyridium (Gudin et al. 1982), glycerol from Duna1~pUa (Grizean and Navarro 1986), glycolate from Ch/orella (Day and Codd 1985), and hydrocarbons from Botryococcus (Baillez et al. 1985). Both natural and synthetic polymers have been used for the immobilization of cells. Natural polymers include metabolic products such as agar, kappa-carrageenan and alginate, all of which were observed to result in high viability of the immobilized cells (Robinson et al. 1986). Among the synthetic polymers used for immobili 7ation, acrylamide bisacrylamide and serum albumin/glutaraldehyde have been found unsuitable for immobilization because of the toxicity of the pre- polymeric materials (Weetall and Krampitz 1980, Brouers et al. 1982). Thepenier et al (1985) also noted the destruction of a large proportion of Porphyr/d/um cells when immobilized with polyurethane matrices. In view of these reports, natural materials seem to have advantage over the synthetic immobilizing agents. The authors are aware of no prior report describing the use of any structural material of biological origin such as plant fibres as an agent for immobilization of microbial or plant cells. Accordingly, fibrous network of matured dried fruit of Luffa aegyptica was investigated for the immobilization of algal cells to add to the limited variety of natural materials being used presently. This paper, therefore, describes the physical structure of the fibrous network and the technique of immobili~-ation. The unicellular red alga Porphyridium cruentum was used as the test organism.

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MATERIALS AND METHODS

Algal Strain. Porphyridium cruentum, strain IAM-R-I was from the Culture Collection of Algae and Microorganisms, Institute of Applied Microbiology, University of Tokyo, Japan. Cultures were grown axenically in i00 ml artificial sea water (ASW) medium (Jones et a/. 1963) contained in 250 mi Erlenmeyer flasks. All the cultures were shaken at i00 rev/min under c_o~tinuous illumination with cool white light at an intensity of 50 pE m --s.

Immohi]i v-~:k)n Technique. Matured dried fruit of Luffa aegyptica was peeled to obtain the placental cushion of the ovary made up of fibrous network. Seeds were removed by cutting the cushion into two halves. These halves were cut into rectangular pieces (approximately 18x20 mm and 2-3 mm width), soaked in boiling water for 30 rain, washed thoroughly with tap water, and placed in distilled water for 24 h. Distilled water was changed 3-4 times. The fibrous network pieces were ove R dried at 70°C and sterilized by autoclaving at 120°C and 1.06 kg/cm pressure for 20 min. These pieces were then soaked in ASW culture medium for 5-10 rain under aseptic conditions and were thereafter suspended in the stationary phase cultures of P, cruentum. These fibrous network pieces were removed from the culture flasks after 3-4 days. In order to determine the immobilization and viability of the Porphyrid/um cells, these pieces were placed in fresh ASW culture medium. The growth was monitored as the increase in biomass for 24 days and observations recorded periodically. For the determination of the biomass, the immobilized cells were washed with d/stilled water and oven dried at 80°C for 72 h to constant weight. For scanning electron microscopy, samples were fixed, dehydrated and dried according to the methods of Lee and Tan (1988). The dried specimens were coated with platinum and viewed on a scanning electron microscope.

RESULTS AND DISCUSSION

Matured dried fruit of L. aegyptica has a cushion-like structure made up of organized fibrous network. The fibres are of different sizes ranging from 150]/m to 80011m (Fig. la). Cut pieces of the network present a multilayered mesh with pore size 2.0 to 4.0 mm overlapping each other (Fig. ib). Scanning electron microscopy of the matured dry fruit showed that surface of each thread is irregular, rough, ridged, having numerous depressions (Fig. id).

The cells of P. cruentum were observed to be immobilized alongside the fibrous network as early as within 24 hours of incubation. However, the pieces were continued to be kept within the algal culture medium for further 3-4 days to allow more cells to be immobilized. The immobili~.ed cells were, thereafter, subcultured in fresh ASW culture medium and maintained in batch culture for 24 days. The cells of P. cruentum were observed to have completely covered the fibrous network pieces within 16-20 days (Fig. lc). Scanning electron microscopy studies showed that the immobilized

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complete, (b) rectangular network piece used for immobilization (c) immobili~.ed cells of P. cruentum on the network; and scanning electron micrograph of (d) fibrous network, (e) immobilized cells within the network.

E~ lO

x 6

l , , - • -r- 6

LU

>" / ~ IMMOBILIZED CELLS H r,- 2 D

I I I I I

0 5 10 15 20 25

T I M E ( D A Y S )

Fig. 2. Growth of free and immobilized cells of P. cruentum grown in batch culture.

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algal cells were mostly aggregated along the surface of the fibrous threads (Fig. le).

Under the experimental conditions described here, no significant difference was observed in the biomass production by the immobi]i~.ed and the free cells (Fig. 2). This is the first report of immobilization on a structural network of plant fibres which, as an immobilizing agent, is different in nature from other biological materials being of the nature of metabolites. In addition to the fibrous network of matured dry fruit of L. aegyptica being a successful immobilization agent, the plant material used is relatively cheap. In view of these practical advantages, coupled with the toxicity problems associated with synthetic immobilization agents (Weetall and Krampitz 1980, Brouers et al. 1982, Thepenier et al. 1982), the high cost of other sources of natural origin, and ease with which the technique can be applied, the fibrous network of matured dried fruits of L. aegyptica are a very useful alternate to the presently used both synthetic and natural immobl]i 7ation agents. Further studies with this natural material for the immobilization of algal/plant cells, with respect to the production of secondary metabolities, are in progress.

REFERENCES

Brouers, M., Collard, F., Jeanfils, J. and Loudeche, R. (1983). In: Photochemical, P hotoelectrochemical and P hotobiological Processes, Hall, D. O., Palz, W. and Pirrwitz, D., eds. pp. 171-210, Dordrecht, Reidel Publishing Co. B~]i~z, C, Largeau, C. and Casadevall, E. (1985). Appl. Microbiol. Biotechnol., 23, 99-105. Day, J. G. and Codd, G. A. (1985). Biotechnol. Lett., 7, 573-576. Grizeav, D. and Navarro, J. M. (1986). Biotechnol. Lett., 7, 261-264. Gudin, C., Therpenier, C. Thomas, D. and Chaumet, D. (1982). In: Solar World Forum, Hall, D. O. and Morton, J., eds. pp. 2255-2558, Oxford, Pergamon Press. Jones, R. F., Speer, H. L. and Kury, W. (1963). Physiol. Plant., 16, 636-643. Lee, Y. K. and Tan, H. M. (1988). J. Gen. Microbial., 134, 635-641. Robinson, P. K., Mak, A. L. and Trevan, M. D. (1986). Process Biochem., 21, 122-127. Tamponnet, C., Gudin, C. and Thomas, D. (1985). Physiol. Plant., 63, 277-283. Therpenier, C., Gudin, C. and Thomas, D. (1985). Biomass, 7, 225-240. Weetall, H. H. and Krampitz, L. O. (1980). J. Solid-Phase Biochem., 5, 115-121.

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