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Production of polyunsaturated fatty acids by Pythium ultimum in solid-state cultivation

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Abstract

The oleaginous fungus Pythium ultimum was cultivated on various solid substrates in order to achieve fungal oil enriched in the polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid. Cultivation parameters, such as incubation temperature and time, substrate composition, and moisture were optimized, so as to obtain as much as 3.5 mg eicosapentaenoic acid and 2.6 mg arachidonic acid/g of wet substrate consisting of 28.5% pearled barley, 5.75% spent malt grains, 5.75% linseed oil, and 60% nutrient solution.

Introduction

Polyunsaturated fatty acids (PUFAs) are components of lipids, which function as cellular energy stores and constituents of cell membranes. PUFAs’ intake through the diet is essential in human development and physiology and might also prevent some diseases [1], [7]. At present, marine fish and products of animal origin are major sources of 5,8,11,14,17-eicosapentaenoic acid (EPA) and arachidonic acid (AA), which are biogenic precursors of a wide variety of biologically active eicosanoids [1], [16]. Alternative sources of such PUFAs include several algae [3], [11], marine bacteria [9], and some primitive fungi such as the Oomycetes (Pythium, Phytophtora) and the Zygomycetes genus Mortierella [5], [10], [12], [13].

Shimizu et al. [10] achieved EPA and AA yields as high as 1.88 g/l and 2.44 g/l, respectively, from submerged cultures of Mortierella alpina. Relatively low PUFA yields have been reported in the literature [5], [12] from submerged cultivation of Pythium species (EPA up to 0.38 g/l; AA up to 0.32 g/l), which could be ascribed to both low biomass production and lipid accumulation. The aim of this work was to devise a cultivation process that would allow such limitations to be overcome. As many Pythium species are phytoparasites growing on the surface of plant tissues or penetrating inside them [2], solid-state cultivation (SSC) was evaluated as an alternative method for the production of fungal oil. SSC is a process in which microorganisms grow on a moist solid substrate in the absence of free water. Cheap raw materials or byproducts of the agro-food industry are utilized as solid substrates [6], [8], thus making this process economically attractive.

Section snippets

Microorganism maintenance and inoculum preparation

Pythium ultimum MUCL 16164 was maintained and monthly subcultured on Czapek-Dox agar. Spore suspension for inoculum was prepared washing the mycelium grown on malt agar with a solution of 0.1% Tween 80 in water, so as to obtain ca. 1.105 spores/ml.

Liquid cultures (LC)

Cultivation conditions were adapted from those described by Gandhi and Weete [5]. Briefly, 2 ml of spore suspension were inoculated into 50 ml of Vogel’s medium [16] in 250-ml Erlenmeyer flasks and cultivated for 6 days at 21°C with reciprocal

Results and discussion

During the first 3 days of cultivation on solid substrate the mycelial growth appeared on the surface of substrate particles, which were penetrated by the fungus in the following days. At the end of the fermentation, the separation of the fungal biomass from the residual substrate was not feasible, nor were indirect assays (protein, glucosamine) satisfactory for the estimation of biomass production. The fungus accumulated PUFA-rich lipid in higher yield when cultivated on the cereal-based

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