FIELD OF THE INVENTION

The present invention concerns a method for the cultivation of algae, preferably microalgae, for example, but not only, the Chlorella zofmgiensis microalgae, for the production of molecules of economic interest such as for example carotenoids, omega 3, lipids and carbohydrates, using photobioreactors selectively exposed to natural or artificial light.

BACKGROUND OF THE INVENTION

The properties of algae and in particular of microalgae are known, which, since they are able to perform photosynthesis, are important for life on Earth. In fact they produce about half of the atmospheric oxygen and simultaneously use the carbon dioxide of greenhouse gases to grow photo-autotrophically.

It is also known that it is possible to accumulate to a large extent the desired products in algae/microalgae by modifying environmental factors, such as for example temperature, lighting, pH, the CO ₂ supply, salts and nutrient substances.

It is also known that for the optimal growth of algae/microalgae the sizes and geometry of the container, or photobioreactor, in which they are grown, as well as the exposure to light/irradiation and the concentration of cells inside the photobioreactor are all important.

However, known plants for the cultivation of algae are rather complex and not very economical.

One purpose of the present invention is to perfect a method for the cultivation of algae, preferably microalgae, which is simple, reliable and economical.

The Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages.

SUMMARY OF THE INVENTION

The present invention is set forth and characterized in the independent claim, while the dependent claims describe other characteristics of the invention or variants to the main inventive idea.

In accordance with the above purpose, a method according to the present invention, for growing algae, preferably microalgae, comprises a first step in which the growth of a biomass of algae takes place.

In accordance with one characteristic of the present invention, the method also comprises a second step, in which the biomass of algae is concentrated, and a third step in which in the concentrated biomass of algae the transformation and accumulation of molecules of economic interest take place.

In accordance with another characteristic of the present invention, the first step comprises a first sub-step in which the algae, for their growth, are disposed in one or more suitable photobioreactors, which are selectively exposed directly to natural light, and/or to artificial light.

In accordance with another characteristic of the present invention, during the first sub-step CO ₂ -enriched air is insufflated into each photobioreactor, in a percentage preferably comprised between about 0.1% and about 25%, even more preferably in a percentage of 5%.

In accordance with another characteristic of the present invention, during the first sub-step the inoculation of the culture takes place with an initial concentration of the biomass of algae preferably comprised between about 0.2 g/1 and about 0.4 g/1, and preferably at a temperature comprised between about 15°C to about 30°C, even more preferably 22 ± 2 °C.

In accordance with another characteristic of the present invention, during the first sub-step the cultures are kept in a growth defined as“semi-continuous”, since the algae, in a second sub-step of the first step, are collected when their concentration reaches a predefined value, preferably comprised between about 0.7 g/1 and about 1.5 g/1.

In accordance with another characteristic of the present invention, in the second step the biomass, which after the first step had a concentration preferably comprised between 0.7 g/1 and 1.5 g/l, is taken to a much higher concentration, preferably comprised between 5 g/1 and 300 g/1.

In accordance with another characteristic of the present invention, in the third step the concentrated biomass of algae is disposed in another photobioreactor, in which the conditions are optimized to have an imbalance between the availability of energy for cells, such as light, and the inability to produce biomass, because the cells are limited in their availability of carbon dioxide and/or mineral nutrients, so that the algae increase the synthesis of molecules of economic interest.

In accordance with another characteristic of the present invention, during the third step the combination is provided of several stimuli at the same time, including the addition of salt (e.g. NaCl) and glucose, which allows both to increase the yield of the molecules of economic interest, and also to reduce the time needed for stimulation.

In accordance with another characteristic of the present invention, during the third step the concentrated biomass of algae is exposed to a moderate light intensity, preferably comprised between about 20 pmoles and about 200 pmoles of m V ¹ photons, removing the CO ₂ .

BRIEF DESCRIPTION OF THE DRAWINGS

These and other characteristics of the present invention will become apparent from the following description of some embodiments, given as a non-restrictive example with reference to the attached drawings wherein:

- fig. 1 is a flow chart that schematically shows the method for the cultivation of algae, preferably microalgae, according to the present invention;

- fig. 2 is a schematic illustration of the method in fig. 1 ;

- fig. 3 is another schematic illustration of the method in fig. 1 ;

- fig. 4 is a schematic illustration of a step of the method in fig. 1.

DETAILED DESCRIPTION OF SOME EMBODIMENTS

With reference to fig. 1, 2 and 3, a method 10 according to the present invention for the cultivation of algae, preferably of microalgae, for example the Chlorella zofingiensis microalgae, essentially comprises three steps, that is, in sequence, a first step 11 in which a biomass of algae is grown, a second step 12 in which the biomass of algae is concentrated, and a third step 13 in which in the concentrated biomass of algae, molecules of economic interest are transformed and accumulated, such as for example carotenoids, omega 3, lipids and carbohydrates, which steps will be described hereafter in more detail. Here and hereafter in the description, by the term algae, we also intend to include microalgae.

We must clarify that the method 10 is not limited to the cultivation of the Chlorella zofingiensis microalgae, since potentially it can also be extended to other species of microalgae, such as for example other species of Chlorella, Haematococcus pluvialis, Dunaliella salina, Nannochloropsis sp, Isochrysis sp. and others.

The first step 11 comprises a first sub-step 11 in which the algae, for their growth, are disposed in one or more suitable photobioreactors 14 (fig. 2), which can be of any known type, for example Drechsel bottles, working on a laboratory scale, or vertical, concentric, spiralized cylinders, panels, horizontal and vertical tubes, open or closed tanks, working on larger scales, or which will be developed in the future, which are selectively exposed directly to natural or artificial light. In the first sub-step 111 the algae are kept in optimal conditions for maximum cell proliferation, which are known to those skilled in the art, to obtain maximum productivity of the biomass, with minimum nutrients.

In particular, in the photobioreactor 14 the biomass of algae is insufflated with CO ₂ enriched air, preferably in a percentage comprised between about 0.1% and about 25%, for example 5%. The inoculation of the biomass of algae occurs with an initial concentration of the biomass preferably from about 0.2 g/1 to about 0.4 g/1, preferably at a temperature comprised between about 15°C and about 30°C, more preferably at a temperature of 22 ± 2°C.

By way of non-restrictive example, for the growth of Chlorella zofingiensis, a growth medium is used, having the reagents listed in the table below, with the corresponding concentrations.

Table 1. Composition of the growth medium for Chlorella zofingiensis

Among all these reagents, the fundamental parameter is the concentration of NaNO ₃ , which preferably varies between about 0.2 g/1 and about 1.5 g/1. From the tests carried out it was found that the best results were obtained with a concentration of about 0.375 g/1 of NaNO ₃ , with which the maximum productivity of biomass was achieved with a lower investment of nutrients.

It is clear that for species of algae other than Chlorella zofingiensis, the composition values of the growth medium set forth above could change.

In these conditions the cultures are kept in a growth defined as “semi- continuous”, since the biomass, in a second sub-step 112, is collected when its concentration reaches a predefined value, preferably comprised between about 0.7 g/1 and about 1.5 g/1. Indicatively this happens every 2, 3 or 4 days.

The first step 11 also comprises a third sub-step 113 (fig. 1) in which it is periodically checked whether maximum productivity has been reached and, if it has, in a fourth sub-step 114, the biomass contained in the photobioreactor 14 is diluted (fig. 2). Once the desired concentration has been reached, part of the biomass is used for the production of molecules in the third step 13, as will be described in more detail later, the rest is used for the re-inoculation of the photobioreactor.

The second step 12 comprises a sub-step 121 (fig. 1) in which the biomass of algae is concentrated, using a suitable compaction apparatus 15, shown schematically in fig. 3, for example similar or identical to that described in the European patent EP 2.326.409.

In particular, in the second step 12 the biomass which, at exit from the first step 11, had a concentration preferably comprised between 0.7 g/1 and 1.5 g/1, is taken to a much higher concentration, preferably comprised between 50 g/1 and 300 g/1, that is from 5% to 30%.

At the end of sub-step 121, the algae thus concentrated can therefore be, alternatively, either collected in a sub-step 122, to be used directly as natural fertilizers, animal feeds, human nutraceuticals, etc., or dried and stored, to be subsequently used for the same purposes, or are transferred (flow 123) into a photobioreactor 16 (figs. 3 and 4), to be subjected to the third step 13.

It is also provided that the algae exiting from the sub-step 122 (fig. 1) can be again subjected to the first step 11, after adding salt or fresh water (flow 124).

In the third step 13, the concentrated biomass of algae is disposed in the photobioreactor 16 (figs. 3 and 4), in which the conditions are optimized to have an imbalance between the availability of energy for the cells, for example light, and the inability to produce biomass, because the cells are deprived of carbon dioxide and nutrients. In these conditions, the algae increase the synthesis of molecules of economic interest.

Fig. 4 is a schematized view of the photobioreactor 16, which comprises a container 17 made of a material that can be sterilized, safe for health and resistant to sea water, such as for example glass, polycarbonate, Plexiglass. The container 17 is provided with a first nozzle 18 for the introduction of CO ₂ , a second nozzle 19 for the injection of chemical components and a third nozzle 20 only for the gases to exit.

In the container 17 there are also at least a first probe 21 to detect the temperature and a second probe 22 to detect the pH value.

Inside the container 17 there is a stirring device 23, for example with blades, which can be driven so as to produce a gentle and continuous stirring of the algae.

Preferably the biomass is inserted into the container 17 so as to fill it completely or almost completely.

A group of lamps 24, for example LEDs, is provided to selectively illuminate the container 17 and a screening device 25 is provided to selectively interrupt the light and create dark conditions in the container 17.

In particular, after the concentration referred to in the second step 12, the biomass in the third step 13 is treated under stress conditions to stimulate the production of molecules of economic interest, such as for example, but not only, carotenoids, omega 3, lipids and carbohydrates.

The third step 13 substantially comprises a sub-step 131 (fig. 1), in which the concentrated biomass is processed in the photobioreactor 16 in small volumes, preferably comprised between about 1/10 and 1/100 of the volume of the culture in the first step 11, and a sub-step 132 in which the molecules of economic interest are extracted from the photobioreactor 16.

An important characteristic is that it is possible to carry out this treatment at high concentrations, which makes the method industrially much cheaper, with lower costs for both construction and maintenance, and with the possibility of maximizing the volumes.

The method 10 is more efficient if the first step 11 is optimized, for example as described above, but it can function, with a lower yield, even without this optimization.

Among the stimuli that can be used to induce the accumulation of molecules, some are known, such as for example, bright light, nutrient deprivation, temperature, pH, addition of salts, addition of organic substrates.

One of the characteristics of the present invention is that the combination of several stimuli at the same time, such as for example the addition of salt and glucose to the nutrient limitation, proves to be effective. This allows to increase the yield of molecules of economic interest, but also to reduce the time necessary for stimulation.

Another characteristic that can be used to induce molecule synthesis is the exposure of the concentrated biomass of algae, preferably with a density from about 5 g/1 to about 300 g/1 of biomass, to a moderate intensity of light, preferably from about 20 pmol to about 200 pmol of nfi s photons, removing CO ₂ .

The carbon dioxide can be removed by different methods, the simplest of which provides to hermetically close the container 17. In this case the available CO ₂ will be consumed very quickly.

The mixing of the concentrated biomass of algae to obtain continuous exposure to light can be guaranteed with streams of air without or added with CO ₂ or by mechanical mixing, for example by means of the stirring device 23, with pumps, or other. In this way an increase in the accumulation of molecules of economic interest is obtained.

It is clear that modifications and/or additions of steps may be made to the method as described heretofore, without departing from the field and scope of the present invention. It is also clear that, although the present invention has been described with reference to some specific examples, a person of skill in the art shall certainly be able to achieve many other equivalent forms of methods for the cultivation of algae, preferably microalgae, having the characteristics as set forth in the claims and hence all coming within the field of protection defined thereby.