Aspergillus Oryzae in Solid-State and Submerged Fermentations Progress Report on a Multi-Disciplinary Project
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FEMS Yeast Research 2 (2002) 245^248 www.fems-microbiology.org Aspergillus oryzae in solid-state and submerged fermentations Progress report on a multi-disciplinary project Rob te Biesebeke a;b, George Ruijter a;e, Yovita S.P. Rahardjo a;c, Marisca J. Hoogschagen a;c, Margreet Heerikhuisen b, Ana Levin a;b, Kenneth G.A. van Driel a;d, Maarten A.I. Schutyser a;c, Jan Dijksterhuis a;d, Yang Zhu b, Frans J. Weber a;c, Willem M. de Vos a;e, Kees A.M.J.J. van den Hondel b, Arjen Rinzema a;c, Peter J. Punt a;b;Ã a Wageningen Centre for Food Sciences, P.O. Box 557, 6700 AN Wageningen, The Netherlands b TNO Nutrition and Food Research Institute, P.O. Box 360, Utrechtseweg 48, 3700 AJ Zeist, The Netherlands c Wageningen University, Food and Bioprocess Engineering group, P.O. Box 8129, 6700 EV Wageningen, The Netherlands d ATO B.V., P.O. Box 17, 6700 AA Wageningen, The Netherlands e Wageningen University, Laboratory of Microbiology, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands Received 3 September 2001; received in revised form 1 February 2002; accepted 5 March 2002 First published online 24 April 2002 Abstract We report the progress of a multi-disciplinary research project on solid-state fermentation (SSF) of the filamentous fungus Aspergillus oryzae. The molecular and physiological aspects of the fungus in submerged fermentation (SmF) and SSF are compared and we observe a number of differences correlated with the different growth conditions. First, the aerial hyphae which occur only in SSFs are mainly responsible for oxygen uptake. Second, SSF is characterised by gradients in temperature, water activity and nutrient concentration, and inside the hyphae different polyols are accumulating. Third, pelleted growth in SmF and mycelial growth in SSF show different gene expression and protein secretion patterns. With this approach we aim to expand our knowledge of mechanisms of fungal growth on solid substrates and to exploit the biotechnological applications. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Solid state; Submerged fermentation; Morphology; Polyol; Amylase; Protease; Genome-wide expression analysis; Aspergillus oryzae 1. Introduction fungus is confronted with gradients in concentration of substrates and enzymes, the presence of a substrate^air Solid-state fermentations (SSFs) are applied in many interface, and gradients in water content and temperature traditional food fermentation processes and o¡er possibil- [3]. Water is frequently a limiting factor for fungal growth ities for improved production of novel as well as existing in SSF and heat removal is one of the major problems in food products and ingredients [1]. In recent developments, large-scale SSF. These problems do not occur with SmF of the organisms are used in SSF to produce high yields of fungi. In SmF, water is abundantly present and gradients pure enzymes which are much more e⁄ciently produced in temperature, oxygen concentration and nutrients are than in submerged fermentations (SmF) [2]. small or absent. Fungi play a key role in SSF, for their hyphal develop- The focus of SSF research has been primarily on pro- ment allows them to e¡ectively colonise and penetrate the cess and fermenter design where the organism involved is solid substrate [1]. As a consequence of hyphal growth the regarded as a black box [4]. The molecular mechanisms underlying the behaviour of fungi in SSF have not been described in detail. Aspergillus oryzae is an obligate aero- * Corresponding author. Tel.: +31 (30) 6944-496; bic ¢lamentous fungus frequently used in SSF processes. Fax: +31 (30) 6944-466. We have initiated a multi-disciplinary study on molecular E-mail address: [email protected] (P.J. Punt). mechanisms of A. oryzae SSF on wheat kernels to under- 1567-1356 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S1567-1356(02)00092-2 FEMSYR 1477 5-6-02 246 R. te Biesebeke et al. / FEMS Yeast Research 2 (2002) 245^248 will be more pronounced than in fermentations with fungi that produce little aerial mycelium, such as C. minitans. Fig. 1. Conceptual representation of ¢lamentous fungal growth on a sol- 3. Substrate hydrolysis and product formation id substrate. The bold numbers correspond to the four layers indicated in the text. Upon penetration of the wheat kernel the fungal hyphae will ¢rst come across a variety of polysaccharides in the kernel coat. The core of the kernel consists largely of stand the di¡erences observed between SSF and SmF and starch and hydrolysis by fungal amylases results in release to improve the application of fungi in biotechnological of glucose, which is used as the main carbon and energy processes. In this letter we report our progress with SSF- source. Accumulation of malto-oligosaccharides is investi- grown A. oryzae considering morphology, basic metabo- gated to address the question whether these compounds lism and gene expression. play a role in induction of amylases. Currently, it is be- lieved that growth in the substrate particle is limited by availability of oxygen [11] and that amylase activity is 2. Fungal growth on a solid substrate su⁄ciently high to hydrolyse starch at a higher rate than the rate of utilisation by the fungus. Our data using Wheat kernels are an excellent substrate for use in A. oryzae growing on wheat dough are in agreement mixed solid-state bioreactors [5]. A drawback of using with this, although glucose accumulation in the substrate such a complex substrate is that its composition may varied strongly with culture conditions. For example, the vary and its pre-treatment may a¡ect the fermentation. glucose content of wheat dough with an initial water con- An important goal of pre-treatment is to give the grain tent of 1 kg per kg dry matter increased from an initial 2 g an increased water content [5]. In most wheat types di¡er- kg31 substrate to 2.7 g kg31 substrate after 48 h of culti- ence in respiration could be explained by di¡erence in vation. On the other hand, in dough with 0.7 kg water per water binding inside the kernel [6], and for some grain kg dry matter, glucose increased from 2.3 to 16 g per kg types the pre-treatment may a¡ect water availability, substrate in 48 h, which may be related to slower growth which warrants further studies. and/or higher amylase induction (see later). Temperature control is one of the main problems in In the SSF process, A. oryzae encounters nutrient gra- large-scale SSF [4,5,7], so it is important to know how dients. Oxygen is mainly or only available at the outside of temperature a¡ects the fungus. The heat production of the kernel, whereas carbon substrates are present only in- the fungus is proportional to its respiration rate [4]. There- side the kernel. Since aerial hyphae are not in direct con- fore we measured the respiration rate of A. oryzae grown tact with the substrate, they should be provided with a on wheat kernels at di¡erent temperatures. Based on this it carbon and energy source by transport of one or more is clear that the respiration has a temperature optimum carbon compounds through the hyphae. Mannitol is between 30 and 35‡C. The oxygen uptake rate (OUR) is found at a very high concentration in mycelium growing a re£ection of metabolic activity and is also related to heat on wheat kernels (Fig. 3) and would be a good candidate. production. A better understanding of oxygen consump- tion in fungal SSF might be useful to deal with heat re- moval problems encountered in SSF. In a conceptual model, we distinguish four layers in the mycelium (Fig. 1). Fig. 2 illustrates A. oryzae growing out of a wheat kernel, showing both mycelium on the surface of the ker- nel as well as abundant aerial mycelium. For Rhizopus oligosporus and Coniothyrium minitans, we found that the OUR during SSF is limited by di¡usion of oxygen in the layer of densely packed fungal hyphae [8,9]. For A. oryzae, however, we found that the aerial hyphae con- tribute up to 75% to the OUR. This was demonstrated by suppressing their formation by covering colonies of A. oryzae with a gas-permeable polycarbonate membrane [10]. Measurements with micro-electrodes in the layer of aerial hyphae (1 in Fig. 1) showed no decrease in oxygen Fig. 2. A. oryzae growing out of a wheat kernel. The picture was taken concentration, i.e. no di¡usion limitation. From these re- after 24 h of incubation with a low-temperature scanning electron mi- sults it can be concluded that cooling problems in SSF crograph at 20U ampli¢cation. The bar at the bottom represents 1 mm with A. oryzae or other aerial mycelium-producing fungi in length. FEMSYR 1477 5-6-02 R. te Biesebeke et al. / FEMS Yeast Research 2 (2002) 245^248 247 Polyhydroxy compounds (polyols), such as glycerol, man- quence analysis after isolation of protein bands from nitol, erythritol and arabitol, can be accumulated in ¢la- SDS^PAGE gels. A 50-kDa protein from both the SmF mentous fungi to a high concentration. The composition and SSF SDS^PAGE gel was identi¢ed as K-amylase. of the polyol pool is dependent on the growth condition Three SSF-speci¢c proteins of 35, 28 and 20 kDa were (e.g. in [12]) and polyols therefore apparently play a role in isolated from the SSF SDS^PAGE gel and the 28-kDa the adaptation to the environmental conditions.