Production of gibberellic acid by fed-batch solid state fermentation in an aseptic pilot-scale reactor

Process Biochemistry, Vol. 32, No. 2, pp. 141-145, 1997 Copyright © 1996 Elsevier Science Ltd Printed in Great Britain. All fights reserved 0032-9592/97 $17.00 +0.00 PIi:

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Production of gibberellic acid by fed-batch solid state fermentation in an aseptic pilot-scale reactor S. Bandelier, R. Renaud and A. Durand* INRA-Platform for Development in Biotechnology (LRSA-PPB), 17 Rue Sully, 21034 Dijon, France (Received 6 April 1996; accepted 6 July 1996)

Abstract

A new patented aseptic pilot-scale reactor (50 litre) was used and its performance validated in the case of the production of gibberellic acid (GA3) with Gibberella fujikoroL Production was carried out by fed-batch solid state fermentation on a wheat bran supplemented medium at about 50% dry matter and 28°C. After 11 days cultivation, a production of 3 g of GA3 per kg of dry matter was obtained. The results show the evolution of the different physical and biochemical variables during this process, proving the efficiency of this reactor in the case of secondary metabolite production. Copyright © 1996 Elsevier Science Ltd

4, 11]. Indeed, the SSF technique has shown a number of economic advantages over submerged fermentation processes in the production of microbial biomass and metabolites and in the valorization of agro-industrial by-products [13-15]. The main drawbacks of this include the difficulty in controlling important culture parameters (temperature, water content of the medium), the lack of reactors well adapted to such a process, the difficulty in scaling-up the production to an industrial level [16] and for some specific applications, such as the use of the fungus with a low growth rate, the difficulty in maintaining aseptic culture conditions during the process. Furthermore, no reference is mentioned in the literature concerning, at a pilot-scale level, the use of an aseptic reactor. Recently, we have described [17] the development of a pilot-scale reactor working under aseptic conditions and the results obtained during sterility tests. The present work describes the application of this reactor to the production of gibberellic acid by fed-batch solid state cultivation. After a brief description of the reactor design, the process is described and the results obtained in relation to published data are discussed.

Introduction

(;ibbereUic acid (GA3) is an important plant growth regulator of economic and industrial importance [1,2] used in agriculture (e.g. elimination of dormancy in seeds), nurseries, viticulture, tea gardens, induction of flowering, acceleration of germination in the brewery industry, etc. [3-5]. GA3 was produced originally by liquid surface fernlentation and now it is traditionally produced throughout the world by submerged fermentation using ( ;ibberella fujikoro't" or Fusarium moniliforrne [6]. The cost of GA3 production using submerged fermentation is very high, mainly due to extremely low yield and extensive downstream processing. Recently, different studies have been carried out to decrease the production cost using several approaches: (1) screening of fungi [1,2]; (2) optimization of the nutrients and culture conditions [4,7]; (3) development of new processes (immobilized cells, fed-batch culture) [8-11]; (4) minimization of the cost of the extraction procedure [12].

Materials and Methods

Another technique, solid state fermentation (SSF), has also been investigated to increase the yields of GA3 and also minimize production and extraction costs [1,

Materials The fermenter used (Fig. 1) consisted mainly of a vertical cylindrical shape reactor in stainless steel with an

*To whom correspondence should be addressed. 141

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insulating jacket. With a total capacity of 50 litres, it has a planetary agitation device for mixing. The reactor bottom includes a wire mesh to hold the solid substrate and distribute equally the thermostated air. Sterile air flows through a system which comprises a humidificatiori chamber and a heating and cooling battery. By means of different probes located in the air flow, the air is conditioned in order to regulate the temperature and water content of the solid medium. Moreover, a handling system developed allows sampling and the maintenance of aseptic conditions. In the upper part of the fermenter, a sterile system allows the addition of liquid or solid substrates and inoculum. Different software (in Borland Pascal Object) have also been developed to automatically sterilize, control and regulate the parameters and to acquire the data using a personal computer [17, 21].

Microorganism and culture medium The organism used was Gibberella fujikoro~" PPB 92 from the INRA collection. The fungus was maintained on potato dextrose agar and subcultured every month. Two media were used. For liquid inoculum preparation the culture was grown in Czapek-Dox medium at 28°C for 4 days on a rotary shaker (120 rpm).

The solid medium used for GA3 production was wheat bran wetted with a nutritive solution. 6 kg (dry matter) of commercial wheat bran (about 8"5% starch on a dry weight basis) was first autoclaved for 3 h at 120°C. After transfer into the reactor, it was mixed with 250g of soluble starch (Fluitex trademark, Roquette, France) per kg of dry wheat bran and sterilized again for 2 h at 121°C. This solid medium was wetted with a nutritive solution [11] having the following composition (in g/kg dry matter): magnesium heptahydrate, 0.1; sunflower oil, 14.32 ml; urea (sterilized by filtration), 2.15; and a mineral salts stock solution (diluted 10 times) in 0.2 N chlorohydric acid. The composition of this stock solution was (in mg/ 100 ml): zinc sulphate heptahydrate, 7; copper sulphate pentahydrate, 7; magnesium sulphate heptahydrate, 7; iron sulphate heptahydrate, 7; and concentrated chlorhydric acid, 16.9 ml. After sterilization and cooling at room temperature, this nutritive solution was inoculated with the liquid inoculum (15% v/w) and aseptically added to the solid medium in order to reach a final dry matter percentage near 50%. The feed, used at the end of 72, 96 and 120 h, was corn starch instead of soluble starch. Corn starch, in dry form, was sterilized in paper bags in an autoclave

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143

Production of gibberellic acid in a pilot-scale reactor

and added aseptically to the fermenting medium. For each feed, 500 g of corn starch and 500 g of sterile water were added under mixing.

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Culture conditions A~ described previously [17], the solid medium tempt'rature and water amount were maintained, respectively, at 28°C and 50% by means of the temperature and relative humidity regulation of the inlet a i . The airflow rate was maintained at 15 litres/min/ k~ dry matter. The medium was automatically mixed for 10 s every 2 h or immediately after the addition of corn starch and sterile water.

Results and Discussion

R~gulation of the culture variables A,, mentioned previously, two important variables (the temperature and the dry matter percentage of the solid medium) must be regulated during solid state ferm,mtation. As shown in Fig. 2, the medium temperature was maintained at 28"5+1"3°C throughout. Although the

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Analytical procedures CMture samples were harvested aseptically every day. The dry matter percentage of the culture medium was determined after drying to constant weight in an o~ en at 80°C. Biomass was estimated for these dry samples by an indirect methodology based on glucosamine determination after chitin hydrolysis [18]. A relation between the ghlcosamine amount and the dry biomass had been established previously for this strain [19], allowing the expression of the biomass in g of fungal dry biomass pt:r kg of dry solid medium. 1 g of the fresh solid medium was used to extract m ea and starch by adding 10 ml of distilled water. Aiter mixing for 10min at room temperature on a shaker followed by paper filtration, urea concentration w,ts determined with a Boehringer urea kit (Urea/ Ammonia kit N ° 542946) and starch concentration by the anthrone method. GA3 was extracted [17] from fresh culture samples at pit 2.5 with a 10% (v/v) aqueous solution of ethanol, at a solid medium to solvent ratio of 1:3. The solution w:ts mixed (100 rpm) for I h at 25°C. The extract was chrified with 0"5 ml each of zinc acetate and potassium ferocyanate, centrifuged (5000g for 5min) and filered (Seppak-Millipore). The filtrate was diluted with an equal volume of 40% (v/v) aqueous solution of mcthanol. GA3 concentration was determined by HPLC [20]. The column used was a /aBondapak C~8 reeersed phase column (Millipore) and the UV detecti~,n was at 206 nm.

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standard deviation may seem significant (compared to values obtained classically in submerged fermentation), the air conditioning system designed has given very acceptable results. Indeed, with a 50 cm deep layer, the medium temperature variations were satisfactory and no gradients were observed in the layer (results not shown). However, some improvements could be obtained by fitting the temperature regulation loop parameters, taking into account the inertia of the solid medium. As shown in Fig. 3, the inlet temperature set-point

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S. Bandelier et al.

144

was maintained at 28°C until around 50 h cultivation and was then changed progressively to 22°C at the end of the process, in order to remove the heat generated by the metabolism. These changes were made manually according to the evolution of the values indicated by the medium temperature probe. It may be possible in the future to develop a strategy via the PC to automatically alter the air inlet temperature set-point after measuring the rate at which the medium temperature is increasing. The airflow rate and the relative humidity of the input air were maintained initially at 80___2.5 litre/min and 98.0 + 2.0%, respectively. In order to improve the accuracy of the medium temperature regulation, it was also possible to increase the airflow rate and/or the mixing frequency. The evolution of the second important variable is shown in Fig. 4. The dry matter percentage of the solid medium was maintained at 49.85+0.58% dry matter, proving the efficiency of the regulation system.

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Fig. 5. Evolution of glucosaminc and GA3.

Biomass production and soluble starch consumption As it is impossible to measure the biomass directly in the solid medium, growth was estimated as glucosamine (Fig. 5). The maximum growth rate reached from 1 to 5 days cultivation was #,1 = 0.018 h 1 In 5 days, a production of 8.9 g glucosamine/kg dry matter was obtained. As mentioned previously [20], for this strain of Gibberella fujikoro't" on this medium, 1 g of glucosamine corresponds to 11.6 g of dry mycelium. The total biomass produced was therefore about 103 g/ kg dry matter. During the growth phase, 236.4g of soluble starch

per kg dry matter were consumed for growth (Fig. 6) giving a biomass yield of 43-5%.

GA3 and urea evolution As shown in Figs 5 and 6, the GA3 biosynthesis began before urea was totally consumed. From 72 to 144 h of cultivation, GA3 production rate was low (10 mg GA3/ kg dry matter/h). After 6days, when urea was exhausted, the GA3 production rate reached a constant value around 25 mg GA3/kg dry matter/h until 10 days cultivation. The maximum concentration obtained (3 g GA3/kg dry matter) after 10days cultivation, was

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Production of gibberellic acid in a pilot-scale reactor higher than the results mentioned in the literature [3, 4. 8, 11], which are around 1-1.5 g GA3/kg dry matter when similar media were used. In conclusion, one of the fundamental reasons why such processes have not been used up to now for secondary metabolite production at large scale is the necessity to maintain sterility. Indeed, during 11 days cultivation, many problems can occur (e.g. contamination, regulation of the temperature and the water amount of the solid medium). The aim of this work was to prove the efficiency of this reactor during a fed-batch solid state fermentation process by taking as a model the production of GA3. Moreover, we have also demonstrated the ability to t~ke samples and to add products during the fermentation without problems. References

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