WO2020132760A1

WO2020132760A1 - Inclined culture system and method for spore resuspension for the spore culture of red algae

Info

Publication number: WO2020132760A1
Application number: PCT/CL2018/050162
Authority: WO
Inventors: Ignacio PÉREZ MASSAD; Cristian BULBOA CONTADOR; Loretto CONTRERAS; Marcela ÁVILA
Original assignee: Universidad Andrés Bello
Priority date: 2018-12-28
Filing date: 2018-12-28
Publication date: 2020-07-02
Also published as: PE20212116A1

Abstract

The invention relates to a device and method for the culture of red algae spores, which comprises: a structure with U-shaped guide frames that supports a system of frames supporting a substrate for the settlement of spores in an inclined position, which is handled from outside the water by means of a lever connected to two horizontal bars that support the guide frames; and a spore resuspension system comprising tubes in which water, air and spores circulate, to make use of non-settled spores. The invention also relates to culture in inclined frames (supplanting the paradigm of horizontal culture, used until now because of the lack of mobility of the red algae spores) and to the controlled use of the spore resuspension system, and prevents contamination caused by handling of the culture.

Description

INCLINED CULTIVATION SYSTEM AND METHOD OF RE-SUSPENSION OF SPORES FOR SPORTS CULTIVATION OF RED ALGAE

The invention relates to a new methodology for cultivating red algae (macroalgae) spores using an innovative device. This device is made up of a recirculation system that allows the re-suspension of spores by means of air injection, installed at the bottom of a pond in order to take advantage of unsettled spores. On the recirculation system there is a structure that allows to hold, tilt and rotate support frames to place racks vertically, with a substrate for the settlement of spores, in order to facilitate the handling of the racks allowing manipulation, without contact from out of the water and automating the movements.

Previous art

Procedures and devices have been described for the cultivation of red algae of commercial interest that have isomorphic triphasic life cycles, which mostly include species of the genera Gracilaria, Gigartina Chondracanthus, Sarcothalia, Kappaphycus, Euchema, Chondrus, Callophyllis, Mazzaella , Gelidium, among others. However, the solution of the present invention is not previously disclosed.

WO2013153402 describes a method for cultivating algae in canalization ponds, manipulating the physiological state of the algae by altering one or more environmental parameters such as light intensity, light wavelength, concentration and balance of nutrients, temperature of the water, algae cell density, concentration or proportion of dissolved gases. This document, unlike the invention, does not specifically refer to culture by means of spores. CL 48664 (Application No.CL 200901696) is related to the cultivation of microalgae. It has a tubular spiral system arranged vertically to achieve a more efficient use of light on crops. The structure of the photobioreactor allows a high surface / volume ratio, flexibility in light orientation, ability to prevent the accumulation of oxygen of photosynthetic origin, CO2 retention capacity and avoid overheating of the liquid medium. This system is specific for microalgae, and does not refer to its usefulness in cultures using spores.

Document W02010077146 mentions the cultivation of macroalgae with a sheet provided with openings that allow the flow of water from one side to the other, where the surface of the sheet has a structure that retains spores and seeds. Although this document mentions the cultivation of macroalgae in general, it does not indicate the production of seedlings by means of spores, and it does not use the spore re-suspension, inclination and frame rotation mechanisms contemplated in the present invention.

The horizontal culture of spores attached to ropes is known [Alveal K., Romo H., Werlinger C., Oliveira E.C. (1997) Mass cultivation of the agar-producing alga Gracilaria chilensis (Rhodophyta) from spores. Aquaculture, 148: 77-83]. In this work, a culture was carried out by means of spores, which included from the settlement of the spores with ropes, to the harvest of biomass. However, it does not use the spore re-suspension, tilt and frame rotation mechanisms contemplated in the present invention.

On the other hand, spore settlement tests have been carried out on different substrates (positioned horizontally) in ponds with constant aeration [Glenn, EP, Moore, D., Fitzsimmons, K., & Azevedo, C. (1996) Spore culture of the edible red seaweed, Gracilaria parvispora (Rhodophyta). Aquaculture, 142 (1-2), 59-74] Spore culture studies, which have included laboratory stages, in a controlled environment, have also covered species such as Gigartina skttsbergii [Romo, H., Avila, M., Núñez, M. et al. (2006) Culture of Gigartina skottsbergii (Rhodophyta) in Southern Chile. A pilot scale approac. J. Appl Phycol 18: 307]; Gelidium rex [Rojas R, León NM, Rojas RD (1996) Practical and descriptive techniques for Gelidium rex (Gelidiales, Rhodophyta) culture. Hydrobiology 326-327: 367-370]; Sarcothalia crispata [Romo, H., Alveal, K. & Werlinger, C. (2001) Growth of the commercial carrageenophyte Sarcothalia crispata (Rhodophyta, Gigartinales) on suspended culture in central Chile. Journal of Applied Phycology 13: 227]; Chondracanthus chamissoi [Ávila M., Piel M., Cáceres J. and Alveal K. (2011) Cultivation of the red alga Chondracanthus chamissoi: sexual reproduction and seedling production in culture under controlled conditions. Journal of Applied Phycology 23 (3): 529-536]; Callophyllis varié gata [Ávila, M., Piel, M. and Alcapan (2014) Indoor and outdoor culture of Callophyllis variegata (Bory) Kützing (Gigartinales, Rhodophyta) in southem Chile. Journal of Applied Phycology, 26 (2): 769-774]; Kappaphycus sp [de Paula EJ, Pereira R ÍE. Elm M, (1999) Strain selection in Kappaphycus alvarezii var. alvarezii (Solieriaceae, Rhodophyta) using tetraspore progeny. J. Appl. Phycol., 11 (1): 111-121]. However, in none of these studies has the use of supports to vertically locate and tilt the frames been described, nor have they included a spore re-suspension system to improve settlement, and thus improve productivity.

The exploitation of natural banks of algae by fishermen and companies for their sale is a poorly regulated and unsustainable practice. Although they are currently competitors in the production (extraction) of biomass, in the future there will no longer be the same natural availability of the resource, so having a stock of broodstock under controlled conditions and the possibility of obtaining biomass efficiently through Starting from them is an important necessity.

The lack of development in cultivation technologies in algae, and above all in sporoculture, they create a very good opportunity to innovate with new developments that are capable of supplying a demand in a sustainable way, that is, avoiding the massive extraction of algae from their natural environment for cultivation or propagation. Although, for now, the extraction of algae from the natural environment is unavoidable for the different options for reproduction and / or propagation that algae offer, it seeks to reduce this depredation to a minimum, making the methodologies for obtaining and cultivating biomass much more efficient. resulting.

The device of the invention is aimed at improving the settlement, inoculation of the substrate and finally the propagation and cultivation of algae by means of spores, contrary to the cases of massive vegetative propagation used for commercial cultivation such as for example of species of the genus Gracilaria. Today in Chile, there are no commercial crops in red algae based 100% on the spore crop. In Asia there is fully industrialized spore cultivation for the production of Nori (Porphyra and / or Pyropid). However, these species have a heteromorphic life cycle, where the microscopic phase is controlled and cultivated in the laboratory, maintaining the conchocelis phase as breeding stock [Publication: Bast Félix, 2014. An Illustrated Review on Cultivation and Life History of Agronomically Important Seaplants. In Seaweed: Mineral Composition, Nutritional and Antioxidant Benefits and Agricultural Uses, Eds Vítor Hugo Pomin, 39-70. Nova Publishers, New York ISBN: 978-1-63117- 571-8]. A situation that does not occur in commercial algae with an isomorphic life cycle, for which this invention has been designed, where worldwide there are important species representing the genera; Gracilaria, Gigartina Chondracanthus, Sarcothalia, Kappaphycus, Euchema, Chondrus, Callophyllis, Mazzaella, Gelidium, among others.

The present invention is directed to companies or technological centers that carry out cultivation of red algae of the isomorphic life cycle of high commercial interest, such as for example those algae of the orders Gigartinales, Gelidiales and Gracilariales. The device of the invention proposes a technological solution that facilitates the sporoculture of the aforementioned red algae, whose cultivation nowadays is carried out in a practically artisanal way, evidencing a development of the techniques and processes involved in productive scaling and their massification.

The developed invention has been able to overturn the paradigm of compulsory horizontal cultivation for this type of algae, which arises due to the lack of flagella of spores which are unable to swim. Furthermore, the invention allows applying existing technologies (such as liquid resuspension) to improve the use of the raw material used. On the other hand, the device of the invention achieves facilities in the handling of the racks, reduction of the manipulation inside the ponds and automation of processes which contributes to an increase in efficiency and productivity in the crop, increasing the amount of substrate inoculated in the same effort, with a clear advance in matters related to sporoculture, which were completely stagnant, with ancient or highly artisan protocols.

The materials used in the present invention are affordable and low cost, the construction of the system does not require specialized machines and the device is assembled in a modular way, being able to expand the structure depending on the size of the pond in order to maximize the available space . Furthermore, all components are reusable. With the above, the device of the present invention allows it to be replicated without incurring large expenses with proven results.

The device of the invention allows to start spore-based cultures, either on a laboratory or commercial scale thanks to its capacity to adapt to spaces. The present invention also allows the control of the different parameters that could affect the cultivation stage, such as the requirements of temperature, photoperiod, nutrients and light intensity. Obtaining better quality algae in the shortest possible time.

An evolution of the materials used in the construction of the device of the invention would allow it to be used on an industrial scale. By using stronger and more durable materials, it is possible to make better use of the pond space, leaving more free surface to increase the amount of substrate available for spore settlement. In addition, investing in the quality of the components, the useful life of the system would be favored, in conjunction with reducing the probability of contamination due to the deterioration of its parts (such as the oxide of the metals present), and advancing in the automation of movements that today are done manually, leaving open the possibility of implementing and perfecting the system using more resources.

The device of the invention has the advantage of doubling the amount of substrate available for spore settling, compared to a method traditionally used in the same volume. At the same time, underwater handling for the operation and turning of the racks is avoided, reducing the introduction of polluting agents. The simple but effective design allows the system to be fully automated at this stage of cultivation, requiring operator intervention only for inoculation and tilting of both sides of the racks.

Description of the Figures

Figures 1A-B show a system used in the prior art of cultivation, which comprises a rack located in a horizontal position to be inoculated with spores, and then positioned vertically for the growth of spores. Figure 2 shows a side view of the inclined racks inside the pond, showing the projection of light beams on the faces of each frame.

Figure 3 shows a perspective view of the rack system, showing the structural base, U-shaped guide frames and tilt handling bars of the device. In addition, the frames and how they are assembled in the guide frames are appreciated.

Figure 4 presents the design of the pond used in the invention, highlighting the sloping walls at the bottom of it.

Figure 5 shows a schematic of the operation of the spore re-suspension system, indicating the flows of air, water and water with spores.

Figures 6 A-B show a top view of the spore re-suspension system installed in the pond. Figure 6A shows the spore re-suspension system positioned at the bottom of the pond, Figure 6 B shows both the spore re-suspension system and the rack system arranged together for operation.

Fas Figures 7 A-B show a side view of the spore re-suspension system and the rack system, both arranged inside the pond. Fa Figure 7 A shows the rack system with a certain angle of inclination (position established to receive the spore inoculum). Fa Figure 7B shows the rack system in vertical position, final configuration of the system for the settlement and growth of spores.

Figure 8 shows the rack system with the spore re-suspension system in operation. You can see how directed bubbles come out especially towards each frame from the vertical tubes of the re-suspension system (stage in which the frames have an angle of inclination).

Figure 9 shows a productivity graph of the spore re-suspension systems of the invention (frames) with respect to a reference spore inoculation system (Control).

Description of the Invention

The present invention relates to a device for the cultivation of red algae spores comprising a pond (1); a rack system with a substrate for spore settlement; at least one U-shaped guide frame that supports each frame in an inclined position, two horizontal lateral bars that allow mobility; a lever that allows manipulation from out of the water; a spore re-suspension system comprising pipes in which water, air and spores circulate.

The present invention refers to a new methodology for the cultivation of red algae spores by means of a device comprising a spore re-suspension system based on the concept of "airlift pump", built by pipes in which air, water and spores, mounted in a pool of water with a V-shaped bottom; a system of racks with polypropylene ropes as a substrate for the settlement of spores, structured in U-shaped guide frames that hold the racks in an inclined position, two horizontal lateral bars that allow mobility, and a lever that allows manipulation from out of the water .

The abatial frame (s) (2) have sufficient mobility to expose the two faces (2a, 2b) (one face at a time) to the surface and allow the spores to settle. The design allows the racks to be kept on an inclined plane for a better use of space in the decanting of spores and for greater light penetration (3), an air and water recirculation system is also included in order to recirculate the spores. The device allows for a more efficient algae culture with a larger spore settlement area and minimizing contamination by handling the racks (2). At the same time, the process is automated, intervening as little as possible in the delicate establishment of the spores, since it allows the racks to be easily manipulated from out of the water, being able to easily install or remove them without the need to disassemble the system.

The device can be built in light and economic material such as PVC and its dimensions can be adjusted according to the scale that you want to work for the scaling of the crop.

In the spore cultivation method, racks (2) are used in which ropes (4) are arranged as a substrate for fixing and settling the spores. The ropes (4) are wound in an orderly manner on the frames (2) constructed with a PVC tube, each one made up of a square or rectangular frame. The frame (2) is immersed in a pond (1) that contains sea water. The aqueous spore suspension is poured onto the surface of the frame (2) and the system is left to stand with lighting control, photoperiod, temperature control and nutrients. The spores attach themselves to the strings and later give rise to seedlings. After reaching two months of age, the seedlings and ropes (4) can be transferred to the sea.

In Figure 2 it can be seen that the inclined plane of the frames (2) allows efficient use of the light source (3), so that it affects all of one of the faces (2a, 2b) of the frame ( 2), without leaving spaces of shade. Figure 3 shows the structural base of the frame system comprising two horizontal lateral bars on each side, parallel to each other, comprising fixed bars (5a, 5b) and mobile bars (6a, 6b), which are attached to one series of guide frames (7) that support the frames by means of bolts (8). The bolts (8) form axes that allow a tilting movement of the guide frames (7), with the bolts (8) connecting the guide frames (7) to the fixed bars (5a, 5b) and to the mobile bars (6a). , 6b).

The fixed bars (5a, 5b) are arranged horizontally, and are connected at the ends to respective portions of descending vertical bars (9a, 9b). The descending vertical bars (9a, 9b) in turn connect with a horizontal basal bar (10). The basal horizontal bar (10) joins both descending vertical bars (9a, 9b) with each other, whereby the basal horizontal bar serves as a base to anchor the frame system structure to the bottom of the pond.

The movable bars (6a, 6b) are arranged horizontally, and are connected at one end to respective portions of upward vertical bars (ia, 11b). The ascending vertical bars (lia, 11b) in turn are connected to an upper horizontal bar (12). The upper horizontal bar (12) joins both ascending vertical bars (ia, 11b) with each other. The upper horizontal bar (12) also fulfills the function of a lever to maneuver the movement of the lateral mobile bars (6a, 6b) and with this, rotate the rotary axes of the guide frames (7) to be able to tilt the frames (2) .

Figure 2 shows a side view of a preferred embodiment of the system with the frames and frames in an inclined position.

Figure 3 shows a perspective view of a preferred embodiment of the device of the invention with the frames (2) and frames (7) in an upright position. To carry out the different experiments to validate the system, it was necessary to have a pond specially designed for this purpose. Fundamental in the design is that the pond has the ability to concentrate the decanted spores in a limited area for the proper functioning of the spore re-suspension system. With the background and requirements studied, the pond (1) that is presented in Figure 4 was designed.

The pond (1) comprises translucent faces allowing a complete vision of what is happening inside it and allows it to be illuminated by any of its exposed faces (13). The pond (1) also includes two inclined planes (18) located at the bottom (19) of the pond. These allow the accumulation of particles in a central line (20) formed by the edges of the planes, located in the middle part along the base of the pond (1), thus facilitating the removal of particles through the operation of the re-suspension.

The dimensions for the pond can vary depending on the scale on which you want to build the system. This time they were chosen according to the available space, so the size of the rack system (2) was adapted to dimensions of 40 cm wide, 40 cm high and 60 cm long, giving a total useful volume of 90 liters and enough space to hold at least 5 24 x 28 cm racks.

Ropes (4) 3 mm thick are wound on the frames, which are capable of supporting around 33 meters of rope as a settlement substrate in each frame (2). By having 5 racks, a total of 160 m of useful rope (4) inoculated per pond (1) can be obtained. The useful meters of rope achieved in the device of the invention represent nearly twice the meters of rope obtained by a pond compared to other frame systems of the prior art.

Figure 5 shows the spore re-suspension system that comprises a vertical tube (21) into which air (14) is drawn in, coming from an air hose (15) by means of a blower. Air is bubbled into the vertical tube (21) by means of a diffuser (17) located at the base of the vertical tube (21). The vertical tube (21) is partially submerged in the liquid, and its upper end protrudes above the surface of the water.

The vertical tube (21) at its lower end is connected to a horizontal lower tube (24), which is connected to a descending vertical tube (28), which in turn is connected to a central horizontal tube (29). The latter has perforations (25) through which water (26) and spores (23) enter from the central line (20) formed by the edges of the inclined planes at the bottom of the pond. To the central horizontal tube (29) an equal number of descending vertical tubes (28) converge according to the number of frames present.

The vertical tube (21) at its upper end, which protrudes from the water, has a 90 ° elbow that ends in an upper hole (22). The water and the decanted spores from the bottom of the pond enter through the perforations of the central horizontal tube (29) due to the pressure exerted by the water as a result of the vacuum generated by the air bubbled by the diffuser of the vertical tube (21). The mixture of water with spores and air bubbles (27) coming from the bottom are expelled through the upper hole (22) of the 90 degree elbow located in the upper part of the vertical tube (21).

The useful life of a spore contemplates a period of time in which it reaches a substrate and settles, beginning its development, this being one of the most influential factors in the success of propagation, but by decanting almost Immediately, all spores that do not reach a substrate on their way to the bottom lose the possibility of developing and growing on the racks. This is why a way was found to take advantage of the decanted spores again by using an air injection pump that allows the spores to be lifted by air and released on the surface of the water so that they reach the string substrate before ending their journey. towards the bottom of the pond. Figure 5 presents a diagram that shows the operation of this system specially adapted for the required purpose, having the advantage that it only requires a limited air flow to be able to operate and transfer the spores from the bottom to the water surface automatically, being energy efficient and completely avoiding manual manipulation inside the pond.

The spore re-suspension system collects the particles that are freely found in the water column, and those that are perched on the glass of the central line (20) of the pond formed by the two inclined planes (18) of the base of the pond (1). The structure of the spore re-suspension system was built so that each frame (2) receives the flow from the bottom (19) of the pond (1) at the same time, so the spore re-suspension system comprises a tube vertical (21) for each frame (2).

Figure 6 A shows a construction of an embodiment of the invention that considers a design for five folding frames, so there will also be five exit holes (22) for the spore re-suspension system directed towards the frames, to the airlift of the spores. On the other hand, figure 6 B shows an embodiment of the invention in which the complete assembly of the device of the invention is observed, comprising the tank (1) with the spore re-suspension system described in Figure 6A plus the rack system described in Figure 3.

In one embodiment of the invention, the spore re-suspension system comprises a series of five vertical tubes (21) arranged in such a way that each one connects at the upper end to respective outlet holes (22). At the lower end, the five vertical tubes (21) are connected to respective horizontal lower tubes (24). The distal end of each of the five horizontal lower tubes (24) in turn is connected to a single horizontal central tube (29) through respective vertical down tubes (28). The central tube horizontal (29) has perforations (25) that allow the entrance of water (26) and spores (23) decanted at the bottom of the pond.

In another embodiment of the invention, the device comprises six frames (2), which are inserted in corresponding six guide frames (7), in turn connected to a pair of movable bars (6a, 6b) and a pair of fixed bars (5a , 5b) that are arranged horizontally, and comprise a series of six vertical tubes (21) arranged so that each one connects at the upper end to respective outlet holes (22). At the lower end the six vertical tubes (21) are connected to respective six horizontal lower tubes (24). The distal end of each of the six horizontal lower tubes (24) in turn is connected to a single horizontal central tube (29) through respective vertical down tubes (28). The horizontal central tube (29) has perforations (25) that allow the entrance of water (26) and spores (23) decanted at the bottom of the pond.

The method of cultivating red algae spores of the invention comprises providing a rack system, for the settlement of spores within a pond filled with water; tilt the rack frames to a first side from out of the water; inoculate the pond with spores avoiding splashing the surface of the water; allow spores to adhere to the substrate; re-suspend unbound spores; moving the frames out of the water so as to tilt the frames to a second side to expose the other face of the frame; keep the frames on an inclined plane so as not to generate shadows; inoculate with spores avoiding splashing the surface; allowing spores to adhere to the string substrate on the second side of the frame; recirculate spores so that spores that did not settle will settle; position the racks vertically from out of the water; leave the culture without movement and light to allow the correct adhesion of the cells (spores); illuminate the rack for the growth of settled spores.

In the method using the device of the invention, the racks are inoculated with spores when they are inclined, then the spore re-suspension system is activated for 1 minute every 3 hours, after 12 hours the racks are tilted to the opposite side thanks to the lever provided for this purpose; it is inoculated with spores; The spore re-suspension system is activated for the same time interval, and the spores are expected to adhere, then the racks are placed vertically for cultivation.

Key aspects of cultivating red algae spores through sloped rack technology innovation are presented below. The levels of the different factors included are referring to the experience carried out to test the invention. However, each species of algae has its own requirements which can be adjusted.

As for example controlled temperature and lighting conditions were tested (i) a constant temperature was used, in this case it was 16 ±

1 ° C, (ii) the photoperiod was controlled at 12:12, that is, 12 hours of darkness and

12 hours of light. (iii) lighting was provided by means of a system of 36 W fluorescent tubes, which provide a photonic flux density of at least 30 pinol s -1 m -2 measured 1 cm below the surface of the water. And after settling, a constant air flow is maintained to increase the gas exchange with air with an approximate flow of 6 1 / min.

The spores that are inoculated in the device of the invention are obtained in a process that takes around 24 hours, the final step of which is the hydration of 100 g of fertile fronds, for this case spores of the red algae Mazzaella laminarioides were used, achieving obtain an average of 8000 cel mL ¹ .

For the process of obtaining spores, reproductive fronds of the algae from their natural environment were used. The fronds were transferred to the laboratory, cleaned one by one to then induce sporulation. For this, the fronds that presented a uniform color, structures were visually selected visible reproductive, low presence of epiphytes and a size that allows easy handling (ideally over 5 cm). The selected fronds were carefully washed with draining drinking water, to then be placed in filtered sea water at 5 pm and sterilized by UV. Then only the area with evident reproductive structures (cystocarps) was selected and cleaning was performed by brushing. To finish, the fronds were washed again with previously filtered and sterilized seawater, for subsequent partial dehydration. For dehydration, the pieces of clean fronds were spread on sheets of absorbent paper. The fronds were subjected to dehydration for a period of 3 hours. The total of dried algae was cut into squares of approximately 3 cm, their weight was obtained and they were separated into two equal portions. Both portions were left to rest for 6 h in a conventional refrigerator (4 ° C), allowing them to cool down and final dehydration.

To obtain spores, a 100 g portion of algae was used, which was placed in a plastic bag (4 L) specially designed and made for the purpose. In order to have sufficient spores for inoculation, two equal bags (or vertical columns) were hung and subjected to hydration, sampling at 2, 5, 7, 9 and 12 h of hydration, taking a small aliquot and counting the concentration of cells (spores) mL ¹ in a Neubauer counting chamber.

The methodology used to obtain spores allowed to inoculate in batches of 1 L per frame face of each pond thanks to the 4 L volumetric capacity of the vertical columns used for the hydration of the fronds. The inoculation with the spores obtained from the previous step was carried out under dim light, carefully spilling the water with spores (of known concentration) homogeneously throughout the water mirror at a distance no greater than 3 cm to avoid splashing. A batch is made for each of the sides of the frame, 12 h apart. Operation of the rack system and device for the re-suspension of spores.

The algae spore cultivation method of the invention comprises tilting the frames to a first side; inoculate the pond with spores; allow spores to adhere to the substrate; re-suspend unbound spores; tilt the frames to a second side to expose the other side of the frame; inoculate with spores; allowing spores to adhere to the string substrate on the second side of the frame; re-suspend unbound spores; after 24 hours of starting the process, vertically position the racks and allow the growth of the settled seeds.

In the algae spore cultivation method of the invention, the frame must first be placed in an inclined position for a side with an inclination of approximately 50 °, then after inoculation and after 12 h in which the re -spore suspension and the cells adhere to the substrate, the frame must be tilted to the other side and repeat the process. After 24 hours from the first inoculation, the racks should be positioned vertically for cultivation.

The re-suspension of spores works by means of a device created under the concept of "air-lift pump", where thanks to the vacuum generated by the injection of air into the lower area of the pond, the water generates pressure entering the collecting tube, dragging with it the particles drift in the water, to then be lifted by the bubbles and expelled from the upper end. This system is activated for one minute every 3 h, 3 times per frame side with a flow rate of 2 L min ¹ per frame. After one week, constant aeration per tank is activated at a total flow of 6 L min ¹ .

Results. A comparison of the results was made with those available in the scientific literature, taking as reference the experiments carried out by Alveal et al. (1997) and Glenn el al. (1998), who use the traditional method available up to now in the state of the art. Furthermore, a comparison was made of a reference experiment used as a control. Results of germination, growth rate, settlement density, efficiency and productivity of each system were obtained.

The reference experiment used as a control was subjected to the same environmental conditions as the system of the invention. In the reference experiment, a single rack was positioned horizontally at the bottom of the pond to receive the spore inoculum. The substrate capacity of this rack, for the spore settlement, was 82 linear meters of 3 mm thick polypropylene rope. At 12 o'clock he was manually turned over and inoculated again. Finally, 12 hours after the second inoculum, the frame was positioned vertically in the center of the pond so that it was completely covered with water. Once this process was completed, the reference experiment was allowed to run under the same conditions as the invention.

The results of settlement density, germination, and growth rate obtained with the device of the invention were maintained or were superior to those of the reference experiment taken as a control, so it can be stated that the device of the invention, with the spore recirculation system does not negatively affect the viability of spores, thus, it was demonstrated that the invention is conducive to culture from spores compared to traditional methods.

Although in the prior art productive extrapolations have been made in terms of final biomass obtained, there are no investigations that study the efficiency or direct productivity in a cultivation system. The present invention shows the first methodology of a sporoculture system that allows measuring variables of efficiency and productivity prior to obtaining final biomass results, allowing a more accurate projection of the future of the crop. The data found in the previous art must have been subjected to extrapolations and unit conversions to be able to contrast them with the data obtained in this investigation, to show that the system is effectively more productive than the methodologies currently used _·

Glenn et al. (1998) in their work with Gracilaria parvispora Abbott, they obtained

_two

a spore density of 3,204 esp. cm using 1000 g of reproductive algae. On the other hand, Alveal et al. (1997) in their work with G. chilensis, they managed, with only 400 g of algae, to inoculate 160 m of rope at a density of less than 400 esp. cm. The above, together with the results of the present study, allowed the elaboration of Table I, in which the factor to compare is the number of settled spores obtained per gram of algae used for sporulation, in this way they are isolated by design experience. and not by the substrate used. For this, data were collected that allowed measuring productivity and finding the best way to express it, considering the most relevant factors (such as biomass and area) that give a representative result.

From the data presented in Table I, it can be seen that the productivity obtained with the rack system of the invention is superior to that obtained in the experiments carried out by Alveal et al. (1997) by 249.8%. The productivity obtained with the rack system of the invention is superior to the experiments carried out by Glenn et al. (1998) by 3594.5% and productivity is superior to the reference system by 34.4%. This demonstrates that the device of the invention has a productive efficiency well above the sporoculture methodologies carried out in previous investigations.

Table I. Comparison of results obtained in bibliography and experimentation.

These results reaffirm the correct operation and the increase in productivity achieved with the method of obtaining spores that uses the spore culture device developed by the present invention.

Figure 9 shows a graph of productivity measured in spores by system. The bars represent the total average per treatment. The frame systems of the invention are explained in black and the bar representing the values of the control system taken as reference is shown in gray. The letter "a" at the top of the average line indicates the statistical similarity between the treatment with the device of the invention and the reference treatment. In addition, the percentage difference between the average values is represented with the delta symbol (D), observing a D of 34%. The error bars represent the distribution of each sample. When comparing the productivity measured in spores settled by pond, taking as a control a reference system that takes the best of traditional methods and comparing it with the productivity obtained in spores settled by pond, an increase in productivity of 34 was observed. % achieved with the device of the invention, compared to the reference system, as shown in figure 9. This allows us to conclude that the design of the device comprising folding frames and the spore re-suspension system of the invention.

In addition, the spore settling and germination percentage was close to 100% in the system, while growth rates of close to 4% d ^{1 were obtained} . This translated into productive efficiency well above the sporoculture methodologies carried out in previous investigations. The facilities in the handling of the racks, the reduction of the manipulation inside the ponds, the re-suspension of the spores and the automation of certain processes contribute to the increase in efficiency and productivity obtained in the culture that uses the device of the present invention, since with the present invention not only the amount of inoculated substrate is increased in the same effort, but at the same time, it expands the development to the cultivation in inclined racks, demolishing the paradigm of horizontal cultivation used until now, due to the lack of mobility of the red algae spores, and opens the development to the controlled use of the spore re-suspension system, making the cultivation of these algae more attractive to grow them on commercial scales and for new investors.

Claims

1. A device for the cultivation of red algae spores, CHARACTERIZED in that it comprises a pond (1); a rack system with a substrate for spore settlement; at least one U-shaped guide frame that supports each frame in an inclined position, two horizontal lateral bars that allow mobility; a lever that allows manipulation from out of the water; a spore re-suspension system comprising pipes in which water, air and spores circulate.

2. The device according to claim 1, CHARACTERIZED in that it comprises at least two frames (2) that are inserted in corresponding two guide frames (7) in the form of a U.

3. The device according to claim 1, CHARACTERIZED in that the pond (1) comprises translucent faces, allowing it to be illuminated by any of its faces (13), comprising two inclined planes (18) located at the bottom (19), where the inclined planes comprise a central line (20) formed by the edges of the inclined planes, located in the middle part along the base of the pond (1).

4. The device according to claim 1, CHARACTERIZED in that the frame system comprising two horizontal lateral bars on each side, parallel to each other, comprising fixed bars (5a, 5b) and mobile bars (6a, 6b), which They are attached to a series of guide frames (7) to support the frames with bolts (8).

5. The device according to claim 4, CHARACTERIZED in that the bolts (8) form axes that allow a tilting movement of the guide frames (7), with bolts (8) connecting the guide frames (7) to the fixed bars (5a, 5b) and to the mobile bars (6a, 6b).

The device according to claim 4, CHARACTERIZED in that the fixed bars (5a, 5b) are arranged horizontally, and are connected at the ends to respective portions of descending vertical bars (9a, 9b), where the descending vertical bars ( 9a, 9b) in turn are connected with a horizontal basal bar (10), where the horizontal basal bar (10) joins the two descending vertical bars (9a, 9b), thus forming the base to affirm the structure of the frame system at pond bottom.

7. The device according to claim 4, CHARACTERIZED in that the mobile bars (6a, 6b) are arranged horizontally, and are connected at one end to respective portions of vertical ascending bars (lia, 11b), which at their instead they connect to a horizontal upper bar (12), where the upper horizontal bar (12) joins both ascending vertical bars (ia, 11b) with each other and extends over both forming a lever to maneuver the movement of the rotary axes of the guide frames (7) to tilt the frames (2).

8. The device according to claim 1, CHARACTERIZED in that the spore re-suspension system comprises a vertical tube (21) that comprises a diffuser (17) located at its base through which air (14) enters through a air hose (15), the vertical tube (21) at its lower end connects with a horizontal lower tube (24) that connects to a vertical down tube (28) that in turn connects with a central horizontal tube ( 29).

9. The device according to claim 8, CHARACTERIZED in that the central horizontal tube (29) have perforations (25) through which water (26) and spores (23) enter from the central line (20) formed by the edges of the inclined planes of the pond bottom.

10. The device according to claim 9, CHARACTERIZED in that the vertical tube (21) at its upper end has a 90-degree elbow that ends in an upper hole (22) through which the mixture of water with spores is expelled and air bubbles (27).

11. The device according to any of the preceding claims

CHARACTERIZED because it comprises four frames (2), which are inserted in corresponding four guide frames (7), in turn connected to a pair of movable bars (6a, 6b) and a pair of fixed bars (5a, 5b) that are arranged horizontally

12. The device according to any of claims 1 to 10

CHARACTERIZED because it comprises five frames (2), which are inserted in corresponding five guide frames (7), in turn connected to a pair of movable bars (6a, 6b) and a pair of fixed bars (5a, 5b) that are arranged horizontally.

13. The device according to any of claims 1 to 10

CHARACTERIZED because it comprises six frames (2), which are inserted in corresponding six guide frames (7), in turn connected to a pair of mobile bars (6a, 6b) and a pair of fixed bars (5a, 5b) that are arranged horizontally.

14. A method of cultivating red algae spores CHARACTERIZED because it comprises providing a rack system for spore settlement within a pond filled with water; tilt the rack frames to a first side from out of the water; inoculate the pond with spores avoiding splashing the surface of the water; allow spores to adhere to the substrate; re-suspend unbound spores; moving the frames out of the water so as to tilt the frames to a second side to expose the other face of the frame; keep the racks on an inclined plane so that do not cast shadows; inoculate with spores avoiding splashing the surface; allowing spores to adhere to the string substrate on the second side of the frame; recirculate unattached spores; position the racks vertically from out of the water; leave motionless and dim light to allow spores to adhere; illuminate the rack for the growth of settled spores.

15. The method according to claim 14, CHARACTERIZED in that the step of re-suspending the spores comprises activating the re-suspension system for 1 minute every 3 h, 3 times per side of the frame with a flow rate of 2 L miri ¹ per each rack.

16. The method according to claim 15, CHARACTERIZED because after one week it comprises activating the constant aeration by pond at a flow rate of 6 L miri ¹ total.

17. The method according to any of claims 14 to 16, CHARACTERIZED in that it comprises immersing the frame (2) in a tank (1) containing fresh, sterile and nutrient-enriched seawater; Pour the aqueous spore suspension onto the surface of the frame (2) and leave the system at rest with lighting control, light photoperiod: darkness and with temperature and nutrient control.