G. AGGELIS⁽¹⁾, A. KAVADIA⁽¹⁾, K. PAPALOPOULOS⁽¹⁾, S. PAPANIKOLAOU⁽²⁾, I. CHEVALOT⁽²⁾, TH.TRYFONA⁽¹⁾, H. GEMA⁽¹⁾, I. KEFALOGIANNI⁽¹⁾, I. CHATZIPAVLIDIS⁽¹⁾, D. DIMOU⁽¹⁾, F. BLACHARD⁽²⁾, M. KOMAITIS⁽³⁾ and I. MARC⁽²⁾

⁽¹⁾Laboratory of General and Agricultural Microbiology

Dept. of Agricultural Biotechnology

Agricultural University of Athens, Iera odos 75, 118.55 Athens-Greece

⁽²⁾ Laboratoire des Sciences du Genie Chimique

C.N.R.S. - E.N.S.I.C./E.N.S.A.I.A., U.P.R. 6811., 13, rue du Bois de la Champelle, 54500, Vandoeuvre-Les-Nancy, France

⁽³⁾Laboratory of Food Chemistry and Analysis

Dept. of Food Science and Technology

Agricultural University of Athens, Iera odos 75, 118.55 Athens-Greece

running title: STORAGE SUBSTANCES BY MICROORGANISMS

Some microorganisms, growing under particular environmental conditions, are able to accumulate significant quantities of storage material, lipids or polysaccharides. These products are of biotechnological interest because of their composition and/or structure (Ratledge, 1987; 1993; Seviour et al., 1992).

Lipids from oleaginous fungi belonging to Zygomycetes often contain polyunsaturated fatty acids (PUFA) of the ω6 family, such as γ-linolenic acid (Aggelis et al., 1987; 1988; Sajbidor et al., 1988; Hansson and Dostalek, 1988; Lindberg and Hansson, 1991; Chen and Chang, 1996), arachidonic acid (Shinmen et al., 1989), and in some cases of the ω3 family, such as eicosapentanoic acid (Bajpai et al., 1992). These fatty acids are used in the clinical nutrition and in the cosmetics industry, and have gained the scientific interest for many years.

Production of lipids by oleaginous yeasts is also of scientific interest (Ykema et al., 1986; Brown et al., 1989; Saxena et al., 1998). Especially, in the case of some species, e.g. Yarrowia (Candida) lipolytica, the lipids produced have a fatty acid composition similar to that of the cocoa-butter (Papanikolaou, 1998; Papanikolaou et al., 2000). Thus these lipids could be used, partially at least, as a cocoa-butter substitute.

Non-oleaginous microorganisms e.g. the yeast-like fungi Auroebasidium pullulans and many free-living azotobacteria produce, in high C/N ratio media, considerable quantities of extra-polysaccharides, of biotechnological interest (Galiotou et al., 1998; Kefalogianni et al., 2000; Chatzipavlidis et al., 2000). Moreover, in the case of free-living azotobacteria the formation of extra-cellular polysaccharides is related with the rhizosphere colonization and the survival capability of these bacteria. Therefore, this property could be used for the selection of strains useful in the agriculture.

The aim of this paper was to model microbial growth and the synthesis-degradation of storage substances. The models proposed were used for the prediction of growth parameters in laboratory cultures, and for the quantitative determination of the ability of the microorganisms to transform the carbon source to storage material. Some results, concerning oleaginous and non-oleaginous microorganisms are also reported.

II. General Model for Predicting Growth and Formation-Degradation of Storage Substances by Microorganisms

In the conceptual (structured) model presented in Figure 1, it was assumed that the microbial cell contains an x mass, that actively contributes to the growth, and a p mass of storage material. It was postulated that the carbon source (substrate, S) was, simultaneously or not, transformed to x mass and to p mass. However, after the depletion of the carbon source from the culture medium, further microbial growth depends on the microbial capability to re-consume reserve material. Therefore, the process of synthesis-degradation of storage material is depended on the specific growth rate of x mass on substrate, on the specific rate of storage material synthesis, and on the specific growth rate of x mass on storage material.

Three different equations were used to model specific growth rate (Table 1). A classic monod-type equation, already used by Professor Glatz (Glatz et al., 1984), was used to model growth of Zygomycetes growing in media having nitrogen source as limited factor and glucose as carbon source (Kavadia et al., 2000). The second is a linear equation useful for batch cultures which was used to model growth of Mucor circinelloides (Aggelis and Sourdis, 1997) and Yarrowia lipolytica (Papanicolaou, 1998) on industrial fats and Aureobasidium pullulans on mixtures of glucose and pectin (Galiotou et al., 1998). Finally, a Verhulst-type equation, with the parameter x_max, that represents the carrying capacity, and without limited factor, was used to model empirically the growth of free living azotobacteria (Kefalogianni, 2000). Equations of this type are often used for modeling natural ecosystems in which the limited factor is unknown (Aggelis et al., 1998).

The equation of Pirt (Table 2) was used to model the synthesis of storage material (Pirt, 1975). Especially in the case of some bacteria, e.g. Azospirillum it was found that the specific rate of polysaccharide synthesis was related to the specific growth rate, and therefore, the synthesis of polysaccharide was a growth-associated process.

On the contrary, in the case of Aureobasidium pullulans, as well as in the case of many species of Zygomycetes, the synthesis of storage material (polysaccharide and lipids respectively) was a non growth-associated process, as the specific rate of synthesis remained constant.