Optimization of biomass, pellet size and polygalacturonase production by Aspergillus sojae ATCC 20235 using response surface methodology

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Abstract

A two-step optimization procedure using central composite design with four factors (concentrations of maltrin and corn steep liquor (CSL), agitation speed and inoculation ratio) was used in order to investigate the effect of these parameters on the polygalacturonase (PG) enzyme activity, mycelia growth (biomass) and morphology (pellet size) of Aspergillus sojae ATCC 20235. According to the results of response surface methodology (RSM), initial concentrations of maltrin and CSL and agitation speed were significant (p < 0.05) on both PG enzyme production and biomass formation. As a result of this optimization, maximum PG activity (13.5 U/ml) was achievable at high maltrin (120 g/l), at low CSL (0 g/l), high agitation speed (350 rpm) and high inoculation ratio (2 × 107 total spore). Similarly, maximum biomass (26 g/l) could be obtained under the same conditions with only the difference for higher level of CSL requirement. The diameter of pellets in all optimization experiments ranged between 0.05 and 0.76 cm. The second optimization step improved the PG activity by 74% and the biomass by 40%.

Introduction

Enzymes that hydrolyzes pectic substances, which contribute to the firmness and structure of plant cells, are known as pectiolytic enzymes or pectinases. Based on their mode of action, these include, polygalacturonase (PG), pectin esterase, pectin lyase and pectate lyase [1]. Pectinases are extensively used in the industrial clarification of wine and fruit juice, in tomato pulp and oil extraction, in chocolate and tea fermentation and in vegetable waste treatment. In the fruit juice extraction and clarification process, these are used together with amylases whereby a reduction of 50% in the filtration time is observed [2], [3]. Furthermore, in combination with other enzymes like cellulases, arabinases and xylanases they have shown to increase the pressing efficiency enormously [4]. Recent applications have emerged in the treatment and degumming of natural fibers used in paper and textile industry [5], [6]. For example pectinases in conjunction with amylases, lipases, cellulases and hemicellulases have been used to remove the sizing agents from cotton, in a safe and eco-friendly manner by replacing toxic soda [7]. Moreover, pectinases are used in animal feed production reducing the feed viscosity and increasing the absorption of nutrients [7]. In the industrial market they contribute to almost 25% of the global enzyme sales, where this contribution is estimated to increase further by the year 2009 [1], [8]. Therefore in order to meet this high demand, it is highly important to produce pectinase enzyme in a cost effective and productive way.

It is well documented, that several organisms are able to produce pectin degrading enzymes including plants, filamentous fungi, bacteria and yeast [9], [10], [11], [12]. For industrial purposes moulds such as Aspergillus niger, Coniotryrium diplodiela, Penicillum and Rhizopus species are preferred, because as much as 90% of the enzyme can be excreted into the culture medium [9], [13], [14], [15]. Pectinase production by filamentous fungi varies according to the type of strain, cultivation conditions (pH, temperature, aeration, agitation and incubation time) and the growth medium composition (particularly carbon and nitrogen sources) [16]. Therefore these have to be specified individually for each and every single strain of interest.

In this study, Aspergillus sojae ATCC 20235 (from here on A. sojae only), which has been mostly used in the production of a well-known Japanese food (koji), by means of solid-state fermentation [12] is considered in the production of polygalacturonase enzyme (PG), which attracts the most attention among the family of pectionolytic enzymes due to its wide use. To best of our knowledge there is no literature report on the pectinase production by this organism in submerged or solid state fermentation. Therefore, this paper will be one of the initial studies working in this field.

In our previous study [17], it was demonstrated that this organism held a considerable potential for the production of the polygalacturonase enzyme. In the corresponding study it was shown that, if this organism was subcultured in molasses agar seed media and grown in complex growth media agitated at 250 rpm at 30 °C, it would exhibit a pellet morphology that is of great interest to large scale submerged fermentations due to easier downstream processing. It was believed that with further optimization study using statistical tools such as response surface methodology (RSM), further increase in the enzyme yields could be attained. This determines the overall scope of this paper. As it is known, the application of statistical experimental design techniques in fermentation process development can result in improved product yields and reduced process variability, development time and overall costs [18]. With this perspective the concentrations of maltrin and corn steep liquor as carbon and nitrogen sources, respectively, together with inoculation ratio and agitation speed were chosen as the independent factor variables significant to large scale fermentations, using central composite design (CCD). The responses of interest were the PG enzyme activity, biomass and the pellet size as a measurement of the morphology of the culture.

The significance of this paper lays in the determination of the optimum regions for maximum PG production and biomass formation with a desired pellet morphology using low cost carbon and nitrogen sources. Therefore this paper will provide information, that can benefit the food and enzyme industry, and also be a new reference point for the microbiology area by providing knowledge regarding the growth requirement of A. sojae, which has been lacking so far in the literature.

Section snippets

Microorganism and spore production

A. sojae ATCC 20235 was purchased in the lyophilized form, from Procochem Inc., an international distributor of ATCC (American Type of Culture Collection) in Europe. The propagation of this culture was done on YME agar slant medium containing, malt extract (10 g/l), yeast extract (4 g/l), glucose (4 g/l) and agar (20 g/l), incubated at 30 °C until well sporulation (1 week). Stock cultures of these strains were prepared with 20% glycerol water and stored at −80 °C.

The spore suspensions used as

Results and discussion

Factors like carbon and nitrogen sources and their concentrations have always been of great interest to the researchers in the industry for the low cost media design especially when strains lacking information on their growth requirements, are under consideration such as the case here [22], [23]. It is also known that 30–40% of the production cost of industrial enzymes is estimated to be the cost of growth medium [24]. These sources together with factors like agitation speed and inoculation

Conclusions

To date, no reports are available in literature regarding the optimization of fermentation conditions for mycelial growth, pellet size and PG enzyme production by A. sojae. Therefore, this study will serve as a base line of the initial studies in this field. The study does not only provide novel information on the growth requirement of this organism, but also serves as an example for the application of the statistical techniques to the fungal systems by giving the end user flexibility for

Acknowledgements

Financial support of Izmir Institute of Technology through the projects IYTE 2004,04 and IYTE 2004,08 is gratefully acknowledged.

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