The present invention relates to a variant of oleaginous yeast of the speciesTrichosporon oleaginosus.

More in particular, the present invention relates to a variant of oleaginous yeast of the speciesTrichosporon oleaginosus, characterized by mutations affecting cell wall synthesis that change the morphology thereof with respect to the wild-type strain of the same species. In particular, thanks to said mutations, cell aggregates are formed which, with respect to the wild-type strain of the same species, lower the viscosity of the culture broth, are more easily separable therefrom and thus make their recovery easier.

Said variant of oleaginous yeast of the speciesTrichosporon oleaginosusis also characterized by yields of oleaginous cellular biomass and intracellular accumulation of lipids that are similar or even higher than those of the wild-type strain.

Further, the present invention relates to a process for the production of lipids through the cultivation of said variant of oleaginous yeast of the speciesTrichosporon oleaginosus.

The lipids thus obtained can be advantageously used as synthesis intermediates, particularly in the so-called “green chemistry” sector, or in the production of biofuels such as “biodiesel” or “green diesel”, which can be used as such, or mixed with other fuels for transportation.

The production of lipids through microbiological methods is proposed as an advantageous alternative to current production methods from renewable sources. With respect to the extraction of lipids from plants, microbiological processes are more cost effective as they are more easily scalable, require less labour, and exploit the property of the microorganisms to quickly reproduce at the expense of low cost substrates, such as, for example, derivatives from the hydrolysis of lignocellulosic materials. Furthermore, they are independent from climatic factors and do not compete with the agricultural exploitation of soil for food purposes.

Particularly promising for this purpose are oleaginous yeasts, i.e. yeasts that can, under specific cultivation conditions, accumulate lipids, especially triglycerides, for over 25% of their dry weight. The use of oleaginous yeasts for the production of lipids is part of the prior art as described, for example, in: Meng X. et al, “Renewable Energy” (2009), Vol. 34, pag. 1-5; Galafassi S. et al, “Bioresource Technology” (2012), Vol. 111, pag. 398-403.

Generally, said lipids are obtained through the cultivation of oleaginous yeasts under aerobic conditions in bioreactors. Oleaginous yeasts are grown using sugar solutions that can be obtained from starchy plants or sugary fruits and are defined as “first generation sugars”, or are preferably obtained through the treatment and saccharification of non-edible lignocellulosic biomasses, and are therefore defined as “second generation sugars”. Lignocellulosic biomasses may be, for example, agricultural residues such as wheat straw or corn stalks, or plants that cannot be used for the food sector such as common reed, fibre sorghum, miscanthus or processing residues of non-edible plants such as guayule, eucalyptus, poplar, or forest residues or processing residues from the papermaking industry. Such mixtures of second generation sugars, which are said hydrolyzates, contain sugars with 5 (C5) and 6 (C6) carbon atoms. Oleaginous yeasts are naturally able to grow on C5 sugars such as xylose and C6 sugars such as glucose, producing cellular biomass and, under particular growth conditions, accumulate lipids in a high percentage with respect to the dry cellular weight.

From the cultivation of said oleaginous yeasts in the bioreactor, a culture broth is obtained including oleaginous cellular biomass to be recovered. Subsequently, the lipids accumulated within the cells must be extracted and separated from the culture broth still present and from the cell debris resulting from appropriate lysis or rupture techniques.

One of the main criticalities in relation to the production of lipids through microbiological methods relates to the fact that the volumetric productivity (i.e. the quantity of lipids to be obtained per unit of volume of culture broth) is limited and, in general, is less than 100 g/L. Consequently, the industrial application of these production processes envisages the use of significant volumes of culture broth of said microorganisms. These large volumes of culture broth must be, of course, treated to recover the resulting oleaginous cellular biomass and proceed with the extraction of the lipids, in large plants, with high set-up and process costs.

For the purpose of guaranteeing the cost effectiveness of the lipid production process, the culture of oleaginous yeasts must reach a high cell density, therefore allowing the productivity of said process to be increased in order to minimize the volume of culture broth and the number of bioreactors used. At the end of growth, the oleaginous cellular biomass obtained must be concentrated in order to reduce the volumes of material to be treated in the subsequent extraction steps of the products obtained (e.g., microbial oils, lipids) and optimize the size of the equipment and volumes of reactants necessary for recovery.

The technical solutions currently used for recovering oleaginous cellular biomass from the culture broth at the end of the growth of the aforesaid oleaginous yeasts are mainly of a physical or chemical nature.

The oleaginous yeasts used for the industrial production of lipids, especially in the case in which they reach high cell density, can lead to culture broths with high viscosity in which the oleaginous cellular biomass obtained is difficult to separate from the aqueous phase, because of its morphological characteristics and the synthesis of substances with surfactant properties. Further, the viscosity of the culture broth makes the cultivation of the oleaginous yeasts in the bioreactor more difficult and costly, because of the lower mixing and oxygenation efficiency of the culture medium. Said criticality is particularly clear in the event that the lipid production process is carried out using oleaginous yeasts that are high lipid producers such as, for example, those belonging to the genusTrichosporon.

Commonly, for separating the oleaginous cellular biomass obtained at the end of the lipid production process from the culture broth, techniques are used that imply a difference in density between the two phases of culture broth (i.e. aqueous phase and oleaginous cellular biomass) such as centrifugation, spontaneous sedimentation, hydrocyclones, filtration, filter pressing, microfiltration, ultrafiltration, sometimes continuously coupled to the bioreactor in which said process is carried out. In the event of using oleaginous yeasts belonging to the genusTrichosporon, the efficiency of said techniques is, however, very low, because the oleaginous cellular biomass obtained has a very similar density to that of the aqueous phase.

For the purpose of increasing the efficiency, flocculants can be added to the culture broth so as to create cell aggregates that, because of their dimensions, sediment more easily with respect to single cells. However, the addition of flocculants in the proportions prescribed by the prior art has not led to an improvement of the separation of the oleaginous cellular biomass from the aqueous phase. Further, the addition of flocculants can be incompatible with the subsequent lipid extraction processes, because of the high affinity of said flocculants with the solvents used for extraction. In practice, said flocculants could be extracted together with the intracellular lipids and therefore represent impurities to be removed prior to the use of the lipids themselves.

Other methodologies used for the concentration of oleaginous cellular biomass are based on filtration techniques. However, the substances with surfactant properties often produced by oleaginous yeasts hinder the permeation of the aqueous phase through the membrane used for filtration, making the process inefficient.

Methods are also known that envisage the evaporation of excess water such as, for example, heating to induce evaporation, or drying cellular biomass with techniques such as, for example, spray drying or freeze-drying: however, said methods are effective in the concentration of cellular biomass, but require high energy consumption and are not economically feasible for an industrial process for the subsequent production of synthesis intermediates, particularly in the so-called “green chemistry” sector, or in the production of biofuels.

Methods are also known that envisage thermal treatments, at neutral or acidic pH, generally performed after the culture broth has been drained from the bioreactor used in the process for the production of lipids, at the end of said process, so as to make it suitable for centrifuging/filtering and to facilitate the recovery of the oleaginous cellular biomass obtained. However, said methods also require further energy consumptions and, when operating at acidic pH, the neutralization of the acidified waste.

Further, some of the methods described above can lead to the degradation of the cell structures, comprising the characteristics of the final product to be obtained. Finally, the methods described above do not tackle the problem of the viscosity of the culture broth and the consequent increase in energy required due to the need to maintain both good stirring and good oxygenation, within the bioreactor used.

Studies have been performed in order to overcome the aforesaid drawbacks.

For example, international patent application WO 2013/155050 describes an oleaginous microorganism for the production of materials from renewable sources, wherein said microorganism comprises a genetic modification not present in the non-modified microorganism, and wherein said microorganism produces a fermentation broth having a lower viscosity than the one produced by the non-modified microorganism. The aforesaid oleaginous microorganism is said to have a reduced synthesis of exocellular polysaccharides and to be able to produce lipids with high yields.

Since the production of materials from renewable sources, particularly lipids, advantageously usable in the production of synthesis intermediates in the so-called “green chemistry” sector, or in the production of biofuels, is still of great interest, the study of new oleaginous yeasts able to produce lipids with good yields and to be more easily recoverable from the culture broth is also of great interest.

The Applicant therefore set out to solve the problem of identifying an oleaginous yeast able to produce lipids with good yields and more easily recoverable from the culture broth.

The Applicant has now identified a variant of oleaginous yeast able to produce lipids with good yields and more easily recoverable from the culture broth.

In particular, the invention concerns a variant of oleaginous yeast of the speciesTrichosporon oleaginosusfiled on May 17, 2017 according to the Budapest Treaty at the Leibniz-Institut DSMZ—Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH—InhoffenstraBe 7 B 38124 Braunschweig (Germany), deposit number DSM 32508, characterized by mutations affecting the synthesis of the cell wall that modify its morphology with respect to the wild-type strain of the same species. In particular, thanks to said mutations, cell aggregates are formed which, with respect to the wild-type strain of the same species, lower the viscosity of the culture broth, are more easily separable therefrom and thus make their recovery easier. Further, said variant of oleaginous yeast is characterized by yields of oleaginous cellular biomass and intracellular accumulation of lipids that are similar or even higher to those of the wild-type strain.

A further aspect of the present invention relates to a process for the production of lipids through the cultivation of said variant of oleaginous yeast of the speciesTrichosporon oleaginosusDSM 32508.

