Microalgae in artificially illuminated vertical photobioreactors.

Giuseppe Torzillo1,2, Giuseppe Quaranta2

1) Senior researcher at Institute of Bioeconomy, and Emeritus professor at University of Costa Rica;

2) Partner of the company Equilibrium

2) Giuseppe Quaranta CTO Equilibrium company

State of the art

Currently the massive culture of microalgae is dominated by three species: Arthrospira platensis (commercially Spirulina), Chlorella vulgaris and Dunaliella salina. These three species mainly grew in open basins, in lanes of variable length and width mixed by means of rotating blades at a speed of about 0.3 m/s.

The depth of the crops varies between 10 and 25 cm. The greater depth is usually adopted in summer to prevent excess temperatures which could lead to the death of the crop. The threshold of resistance to high temperature depends very much on the strain. For example, Arthrospira grows fine between 30 and 35°C.

 Above 35 °C growth slows down and then stops, and within 1-2 days the culture dies. Most microalgae cultures prefer temperatures between 20 and 30°C. Marine species have generally lower temperature optimums (20-25°C). Therefore, these species must be grown in bioreactors where the temperature can be controlled. The same considerations apply to the Haematococcus pluvialis species, recently produced on the market for the extraction of astaxanthin, a secondary carotenoid very useful for use as an antioxidant. The same can be said for the production of Phaeodactlilum tricornutum (diatom) for the production of fucoxanthin. These last two species are cultivated in generally tubular closed systems which allow the temperature limits to be kept within 25 °C using a cooling system. Therefore, the use of open ponds is possible for extremophile species growing in very selective media, for example Arthrospira (high salinity ~ 20 g/l salts, pH 9.5-11) and Dunaliella salina (high salinity). Chlorella vulgaris is a special case as it is perhaps the wildest of these three species and is characterized by a high growth rate which allows it to compete with other polluting microalgae. Furthermore, about half of the production takes place in heterotrophic fermenters using glucose as a source of energy and carbon. Normally, the production of the three main species is around 10 gm-2 day-1 which corresponds to a conversion efficiency of light into energy stored in the biomass of around 1/10 of the theoretical one (10% on a solar basis). Therefore this type of cultivation can be profitable only in cases where the areas destined for the production of microalgae are located in desert areas with high insolation, and in countries where the cost of land and labor are very low. Indeed, Spirulina is produced by Earthrise in the desert of California, some areas of China, especially in Mongolia, in this case using greenhouses to prevent excessive reduction of crop temperature. Currently 2/3 of the world production of Spirulina (about 20,000 tons) takes place in China. As far as Dunaliella is concerned, it is produced in the Negev desert (Israel) not far from the Red Sea, and in desert areas of Australia in open basins without any agitation except that operated by the wind. In Australia, the daily yield drops to 2-3 g/m2 of dry weight. Dunaliella is cultivated for the production of beta-carotene which is present in the biomass (about 10%). Synthesis of carotene in Dunaliella requires exposure of cells to a stressor usually high salinity and low nitrogen content in the medium. Stress conditions (deprivation of nitrogen) are necessary to favor the accumulation of astaxanthin (3-5% of dry weight) in Haematococcus pluvialis. Therefore, the production of microalgae is normally extensive and is economically feasible only in areas with low or very low cost of land, areas that are illuminated almost all year round, and where it is possible to find low-cost labor. In general, the cost of production in tanks varies between 10-20 US$ per kg of dry matter. The production of microalgae in Europe is practically non-existent if we exclude small initiatives by growers with surfaces of less than 1000 m2 of tanks. To be competitive, the annual production must exceed 40 tons. To take advantage of the economy of scale, it is necessary to reach about 10 Ha. Therefore, despite the recognized benefits of algae biomass for human health, Europe including the Mediterranean area is excluded as the cost of land is high and often in competition with traditional agriculture which is required to meet the growing needs of the growing population and to the growing tourism economy.

The vision of Equilibrium

The Equilibrium company, aware of the problems exposed above, proposes a vertical solution for the production of microalgae. Based on the use of expecially designed artificial lighting with high efficiency LEDs, carefully selected in terms of emission and intensity spectrum so as to allow the absorption by the photosynthetic pigments of photons administered, avoiding excesses of light which would lead to a reduction in photosynthetic efficiency and differentiating the spectrum according to the cultivation of cyanobacteria (Spirulina sp.), microalgae (Chlorella sp.) and diatoms (Phaeodactlilum sp.). Cultivation takes place 24/24 H. In fact, unlike plants, microalgae (in general) do not require periods of darkness to synthesize particular components. The advantages of this type of approach are briefly listed below:

1) Release of production from environmental factors. The artificially illuminated bioreactor can be installed virtually anywhere.

2) Possibility of administering a precise photon flux (commonly referred to as PFD) such as not to exceed the light saturation intensity of photosynthesis (Ek), i.e. remaining within the linear portion of the photosynthetic curve. This makes it possible to approximate the light utilization efficiency to a value close to the theoretical one (about 20% of the PAR).

3) Another innovative strength of the lighting system adopted by Equilibrium is the almost uniform distribution of light. Even in indoor cultivation systems, whether they are tubular or paneled, the light is normally administered from the outside. This involves a loss of light absorbed by the interface (glass or Plexiglas, etc.) which is estimated at around 10%. Furthermore, to favor the exposure of the cells to light, it is necessary to give the culture a turbulent motion which, however, is never capable of subjecting the cells to an ordered pattern of light-dark cycles. In the case of Equilibrium the catenaries (light strips) directly immersed in the culture are placed at such a distance as to distribute the light uniformly and sufficiently. In cultivation systems lighted from the outside it is necessary to move the cells from the center (with poor lighting or even in the dark) towards the walls, vice versa with Equilibrium it is the light, properly distributed by the LEDs which reaches the cells without having to subject them to a high and unsatisfactory agitation. However, a slight mixing carried out using compressed air is also necessary for the PBR proposed by Equilibrium, essentially to avoid cell sedimentation and to reduce the oxygen tension in the reactor.

4) With Equilibrium soil consumption is very limited. In the reactor it develops vertically therefore the foot print is very limited, incomparably lower than in open basins and conventional photobioreactors, both tubular and with panels.

5) Possibility of producing biomass or a certain product (e.g. phycocyanin, PUFAS, fucoxanthin, proteins, etc.) continuously and at an almost constant concentration.

6) Possibility of adopting a continuous production process, making the production and composition of the biomass stable.

7) Maintaining the culture at a given steady-state significantly increases the efficiency according to which the bioreactor operates, since the amount of energy dissipated is very limited.

8) Cell illumination homogeneity. This is a very important aspect as Equilibrium fully satisfies as it is capable of administering the right amount of light while avoiding dissipation (commonly referred to as non-photochemical quenching NPQ).

Point 6 deserves some further clarification. Since the beginning of microalgae culture biotechnology, which can be traced back to the post-war period, precisely with the first publication in 1953 by the Carnegie Institution of New York (Burley 1953), the lighting topic has seriously occupied many researchers. The point is that the means in the hands of a biotechnologist to make the illumination of the culture homogeneous were substantially two:

• the first, to use very diluted cultures so that the light passed through the entire culture layer, however strongly decreasing the productivity as it also depends on the concentration,

• the second adopting dense crops therefore with a high light extinction coefficient but imparting a high speed (turbulence) to the crop.

In some cases it was also suggested that the very high turbulence could make it possible to exploit the flash effect. Numerous studies including those carried out by the undersigned have shown that with Spirulina the increase in speed beyond turbulence (Reynonds > 4000 ) does not lead to measurable increases in productivity while it strongly increases energy consumption. In some cases, a decrease in productivity has even been found due to the mistreatment of especially filamentous cells (e.g. Spirulina). However, moderate agitation with air is useful to keep the cells in suspension and reduce the risk of oxygen toxicity that accumulates due to photosynthetic activity. But controversial works have also been published on this topic.

The Equilibrium approach.

The illuminated reactors proposed by Equilibrium use LEDS directly immersed in contact with the culture without the use of interfaces that isolate them from contact with the algal culture. This is possible thanks to a LED protection system with an innovative coating. This coating, of which not many details can be provided, also prevents the formation of biofilm on the LED which would greatly reduce the intensity of the light emitted. The distance of the LEDs (catenaries see below), is studied in such a way as to make possible a homogeneous lighting and with minimum waste of energy. Therefore, it is not necessary to give the cells vigorous mixing since they are constantly exposed to the optimal amount of light. However, the LEDs can be activated by pulses and therefore able to supply flashes alternating with well-studied periods of darkness in the event that it is necessary to adopt light intensities higher than saturation intensity for economic reasons.

 The experience gained in outdoor culture in tubular photobioreactors with a diameter of 5 cm, completely controlled, except for the light which is sunlight, indicates that generally the average daily productivity in summer is between 300 and 400 mg/l: Equilbium believes it exceeds 1000 mg/l/day. This is possible using 24/24 H and keeping the crop parameters constant The conversion efficiency is expected to be around 20% of the visible (PAR 400-700 nm) as doses of light are administered no more than saturation of photosynthesis eliminating the waste of energy.

 A conservative estimate indicates that a 50 m3 bioreactor (basic module) would produce an amount of dry biomass equal to about 50 kg of dry weight. So we focus on a double advantage: 1) energy saving (only light used efficiently), and 24/24 operation. The TRL of Equilbrium therefore lies between 7 and 8.

 Being a fully controlled bioreactor, it can easily be used to produce certain chemicals, for example phycocyanin through the right selection of strains. In this sense Equilibrium has easy access to a vast collection of bio-ecologically interesting microalgae, cyanobacteria and diatoms. For example, the Arthrospira collection (platensis and maxima) exceeds 50 readily available strains. An extremely important genetic heritage to economically enhance the production of various chemicals starting from the most promising. Equilibrium has a know-how on the production of microalgae of considerable importance as it has internationally renowned experts on the physiology and biotechnology of microalgae among its partners. Various species of Chlorella, Scenedesmus, Chlamydomonas, and Diatoms are also available in the collection. In general, most of the relevant species from a biotechnological point of view are available, for the production of proteins, dyes, cosmetics, for use in agriculture for the production of biostimulants, fertilizers, biopesticides and for the treatment of waste water.

Description of the PBR

The peculiarities of Equilibrium's PBRx01 photobioreactor were previously illustrated, which we summarize below:

• Independence from environmental conditions such as time, season, latitude;

• Industrializable and modular plant;

• Optimal management of light efficiency and its intensity within saturation limits;

• Almost uniform distribution of light in the entire culture volume;

• Optimal architecture to reduce cell sedimentation and oxygen tension in the reactor;

• Optimal architecture for thermal management with phase change materials (PCM);

• Incomparably lower foot print than open ponds and conventional photobioreactors;

• Continuous production with almost constant concentration and composition;

• Maintaining the culture at a certain balance.