BIOTECHNOLOGY TECHNIQUES . Volume 8 No.12 (Dec.1994) p.885-888 Received 28th October

CHEMICAL STERILIZATION OF NUTRIENT MEDIA FOR PLANT CELL CULTURES USING DIETHYLPYROCARBONATE. T. Macek *1, J. Knill, T. Van~k 1, J. Bla~ek 1, M. Mackovrl2 1) Dept. of Plant Tissue Cultures, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n. 2, 166 10 Prague 6, 2) Dept. of Biochemistry and Microbiology, Faculty of Food and Biochem. Technology, ICT Prague, Technick~.3, 166 28 Prague 6, Czech Republic SUMMARY

Diethylpyrocarbonate (DPC), when added at about l g/I to culture media is able to sterilize them. DPC kills all the contaminating microorganisms, and in contact with water it decomposes to ethanol and carbon dioxide in amounts not inhibiting the growth of plant cells. The plant cells cultivated on media treated by DPC did not show changes in their basic characteristics. INTRODUCTION

The usual cultivation period of a culture of submerged plant cells takes from five to twenty days, in experiments with callus cultures is not exception 40 or even 100 days. For this reason removal or killing of all microorganisms is usually an absolute concept in plant cell cultivation. Sterilization by heat can destroy some auxins or cytokinins needed for plant cell growth, causes changes in some starting material, like caramelization of sugars, etc. To avoid the above mentioned problems a simple and convenient method has been tested in our laboratory for the sterilization of media using diethylpyrocarbonate, DPC (C2H5C)-CO-O-CO-OC2H5). The agent kills all contaminating microorganisms, and in contact with water it decomposes to ethanol and carbon dioxide in amounts not inhibiting the growth of the organism. At room temperature the decomposition takes about 10 hours, higher temperature increases the decomposition rate. We tested the method successfully with medium according to Murashige and Skoog (1962), using a producing strain of Solarium a v i c u l a r e . The cells cultivated during a half year of regular subcultivation each 7 days on media treated by DPC did not show changes in their growth characteristics and production pattern.

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METHODS:

The medium to be sterilized is mixed with up to 2 g DPC/I, and left standing at room temperature overnight, to allow decomposition of DPC. DPC is poorly soluble in water, it should be thoroughly mixed with the solution to be sterilised, and then poured to the presterilized cultivation vessel. Unless otherwise stated, all manipulations were performed in a laminar air flow cabinet, and for described experiments all glassware was presterilized to avoid any contamination from other source than the medium itself. To find the necessary concentration of DPC, liquid medium according to Murashige and Skoog (1962)i further mentioned as MS, was prepared with DPC added in different concentrations, see Table 1. Part of the flasks was closed by cap immediately after filling the medium, part was left standing open in the laboratory and part was autoclaved at 125~ for 30 minutes. Last quarter of the flasks was autoclaved only for 5 minutes at 125~ The number of contaminated flasks was checked visually after 5, 7, 11 and 25 days. The presence of contamination in medium sterilized by DPC in concentration of l g/I was tested after a two day storage at 27~ by cultivation test using selective nutrient media for bacteria, yeasts and fungi. 0.1 ml of the tested medium wasdirectly plated to Petri dishes containing peptone-glucose agar medium for growth of bacteria and Sabouraud-maltose medium (Difco) for the growth of yeasts and fungi. Each experiment was carried out in 3 parallels. To concentrate the possible contamination, 10 ml of the DPC-treated medium was filtered through Millipore sterile filters (45 urn), which were put on Petri dishes with Plate Count Agar medium (Difco) or Sabouraud agar. The Petri dishes for estimation of bacteria were incubated for 48 hours at 37~ for yeasts and fungi for 72 hours at 28oc. To follow the effect of residues of DPC, present in media at the time of inoculation, the cells were inoculated immediately after the addition of DPC into normally sterilized medium. Tested organism was a suspension culture of Solanum aviculare, strain AVI-3D (for details on this culture see Macek 1989), at high inoculum densities (3g FW/20 ml), cultivated on a roller, 5 rpm, in dark at 27~ The growth of the fine suspension was expressed as the growth value, defined as fresh weight increase per lg inoculum after 9 days of cultivation. Growth value, g.v. = (FW-FWo)/FWo, where FW is the fresh weight of the harvested cells, and FWo is the fresh weight of the inoculum. The S. aviculare cells were regularly subcultivated every 7th day on media sterilized by DPC for a half year period. Every two month the growth characteristics, the production of betulinic acid and the ability to transform exogeneous organic compounds have been tested, by methods described elsewhere (Van~k et al. 1989).

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RESULTS: The validity of the original idea was proved by experiments comparing the effect of different concentrations of DPC in medium, to find its lowest content necessary for obtaining a totally sterile solution, and the effect of residual DPC on the viability of plant cell suspension culture. Table 1. Sterilization of liquid nutrient medium MS by DPC, contamination tested visually after 5, 7, 11 and 25 days. DPC autocl, autocl, no autocl, no autocl. 30min/125~ 5min/125~ closed open 2g/I 0 0 0 100%/5d 1 g/I 0 0 0 100%/5d 0.2g/I 0 0 70%/11 d 100%/5d 0.05g/I 0 0 100%/5d 100%/5d ' 0.025 g/I 0 25%/5d 100%/5d 100%/5d 0 0 50%/5d 100%/5d 100%/5d The Petri dishes with microbial selective media showed a clear difference between those plated from media treated with DPC and without. All samples from media treated by DPC were free of contamination, while the control ones were heavily contaminated. The growth value of plant cells after inoculation of the medium containing different concentration of DPC is summarised in Table 2. Table 2: Effect of DPC concentration in medium (v/v) on the growth of plant cell suspension of Solanum aviculare AVI-3D (g.v.). DPC (% v/v) growth value

0.2% 0.1

0.1% 0.6

0.05% 0.025% 0.01% 0% 6.35 6.30 6.05 6.24

During a half year of regular subcultivation on media treated by DPC the tested culture did not show changes in its growth characteristics, its ability to produce betulinic acid (1.3% DW) or to biotransform methoxybenzylcyclohexanone, as was tested by HPLC.

DISCUSSION: The antifermentation effect of DPC was already found in 1955 in the laboratories of Bayer A.G., Lewerkusen (patent 1961), and much effort has been put into testing its ability for stabilization of wine, beer or soft drinks. It did not find larger application due to its toxicity if not allowed to decompose, and lower effectivity in drinks with high starting number of microorganisms. Results in Table 1 show, that for exhibiting the sterilising activity the lowest efficient concentration of DPC is about l g/l. For the case that waiting 12 hours for DPC decomposition is a too long time period, after one hour standing at laboratory temperature the DPC can be quickly decomposed by short autoclaving for 5 minutes at 125~

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So far as DPC is decomposed totally in water solutions, and the concentrations used for the sterilization are under 0.2 %, no harmful residual amount of DPC will be present in the medium, if used for inoculation after standing overnight at room temperature. Results in Table 2 confirm that the proposed way of sterilization does not influence negatively the growth of plant cells. This experiment also proved, that the presence of 0.05% of DPC itself in the medium was not influencing the c e l l viability, for which the definitive assessment is the active growth and division. The long-time cultivations (25 passages) on DPC-treated media did not cause detectable changes in their basic characteristics. Other benefits from using DPC can be derived from the fact, that during preparation of nutrient media it can be used also for stabilisation of the stock solutions, like it was described for the stabilisation of RNase-free saccharose in preparation of sucrose gradient for ultracentrifuges (Holtzhauer 1988). This has two practical aspects, the stock solutions are longer stable, and the starting number of microorganisms in the final medium before sterilization is lowered, too. We also experienced that the application of DPC may be possible not only for the sterilization of media for cultivation of plant cells, but also of media for microbial cultivations and animal cell cultures. In contrast to plant media these usually do contain much more proteins, and thus are more difficult to sterilize by DPC. In this cases we tested only the sterility Of the resulting solution, but not yet the effect on the organism itself. The self-sterilising property of the solution of DPC in medium can be also used for the sterilization of the whole vessel, but only in the case that contact of the liquid with all parts of the vessel can be ensured for a proper time period. REFERENCES:

Holtzhauer M, Biochemische Labormethoden, Springer Verlag Heidelberg Berlin (1988) Macek T, in: Biotechnology in Agriculture and Forestry, Y.P.S. Bajaj, ed., Vol. 7, 443-467, Springer Verlag Heidelberg Berlin New York Tokyo (1989) Murashige T, Skoog F, Biologia Plantarum 15, 473-497 (1962) Swiss Patent 3355019, 53c, 6/01 Bayer AG, Lewerkusen (1961) Van~k T, Wimmer Z, Macek T, Saman D, Svatow A, Romar~uk M, Biocatalysis, 2, 265-272 (1989) Wallhausser K H in: Biotechnology, Vol.2, Rehm H-J and Reed G, eds., 153-214, Verlag Chemie (1985) Thiswork has been supported by the grant No. 204193/0836 of the GrantAgencyof the Czech Republicand the ProjectCOST66. ACKNOWLEDGEMENT

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