Method for cultivating algae of the species dictyota kunthii from ligules

Abstract

The present invention discloses a method for cultivating algae of the species Dictyota kunthii that uses ligules obtained from donor plants. The method comprises at least the following four sequential steps: Introducing and cultivating donor algae; Introducing ligules into a hatchery separated by size; Cultivating ligules in the hatchery; and cultivating ligules in ponds. This cultivating method has a duration between 9 and 13 weeks, and allows the ligules used to be separated by gauge or size, homogenising the same and ensuring a minimal size of the ligules that pass to the fattening step or pond cultivation, to thus avoid sedimentation and death of the small ligules.

Description

[0001] CULTIVATION PROCESS OF ALGAE OF THE SPECIES Dictyota kunthii BASED ON LUGULES

[0002] TECHNICAL SECTOR

[0003] The technology is focused on algae cultivation, particularly the cultivation of Dictyota kunthii, for the extraction of various compounds for biotechnological use.

[0004] STATE OF THE ART

[0005] The chemical complexity and high bioactivity demonstrated by Dictyota species has sparked market interest in various compounds from these algae, leading to an emerging commercial interest in these species, highlighting the need to cultivate them. To achieve this, understanding the life history, reproduction, and maintenance of natural populations is key to proposing strategies for domestication and controlled propagation of these species. Species of the order Dictyotales exhibit a diplohaplontic life cycle with isomorphic sporophytic and gametophytic thalli. The life cycle presents different propagation pathways, which include sexual and asexual strategies. The gametophytic (haploid) thalli are dioecious and produce sori of oogonia and spermatangia, which release the gametes into the medium, where they fuse to form a zygote, which gives rise to sporophytic (diploid) thalli.These, through meiosis, produce spores in tetrads along the thallus, which after germinating reestablish the haploid phase. Furthermore, the fragmentation of the thallus into small units capable of reattaching, as well as the presence of perennial basal / prostrate parts, which generate new thalli, have also been mentioned as important asexual pathways for maintaining natural populations. In the case of the species Dictyota kunthii, the presence of ligules (spatulate proliferations present on the surface of the thallus) adds another alternative for asexual propagation, since it has been proposed, although not yet proven, that when they detach themselves they can generate new thalli.

[0006] In many species of the order Dictyotales, the presence of fertile gametophytes has been reported as very rare or completely absent. This is consistent with what has been described for D. kunthii (such as Glossophora kunthii) on the coasts of central Chile, where the majority of the species were found to be sporophytic and infertile thalli during an annual cycle. This suggests that in many cases, the persistence of populations of Dictyota species depends on asexual reproduction, where spore reproduction would be one of the most important propagation strategies, alternating with thallus fragmentation and its subsequent re-adhesion. The above suggests that both routes, spores and fragmentation, would be alternatives for the domestication and production of the species.This would avoid the complex sexual reproduction strategy, which involves rare male and female plants and involves processes of gamete maturation, release, and external fertilization, which requires many complex stages for a production process. However, both propagation strategies have not yet been tested for species of this genus as possible mechanisms for biomass production, so the present invention is innovative in this regard, representing the first effort to develop a process for biomass production.

[0007] While there are no commercial cultures of Dictyota species to date, there are cases of studies where both spores and thallus fragments have been cultivated in the laboratory for long periods of time. These studies have demonstrated the ability of Dictyota species to grow and reproduce in confinement, although only on a laboratory scale. Even studies on D. dichotoma have shown that the plants remain healthy and retain their typical morphology, making it possible to quickly obtain large quantities of clones through asexual reproduction via spore culture. Likewise, fragmentation appears as a possible strategy for productive Dictyota cultures, since laboratory studies have shown that the Dictyota thallus can be fragmented into different pieces, which regenerate and regrow after being injured.

[0008] Regarding the information available in Chile for the species D. kunthii, it can be indicated that they are scarce and provide first-rate biological and ecological information / observations on the life of this species. So far, we know that: (1) The growth of the thalli of this alga is favored by a long day photoperiod and high light intensity, and on the other hand, the short photoperiod induces the maturation of sporangia; (2) The thalli tolerate temperatures from 10 to 23°C; and (3) Sporophytic and infertile thalli are present on the central coast of Chile throughout the year.

[0009] The aforementioned background has been carried out solely under exploratory laboratory culture conditions, without considering a productive approach. Species of the genus Dictyota have high potential for obtaining biomolecules; however, there are no commercial crops to date, anywhere in the world. Furthermore, D. kunthii has a differentiating characteristic compared to other species of the genus: the presence of multicellular structures called ligules, which are very abundant on the thalli of this species, a unique characteristic within the genus. To date, there are no reports of cultivation of species of the genus Dictyota using ligules as propagules for productive purposes. The results of the present invention are the first and only attempt to cultivate this species from ligules.

[0010] Some similar technologies that point to algae cultivation are as follows: Patent Application WO2022243984, which primarily discloses an animal feed, but also discloses a method of cultivating an Asparagopsis species to produce biomass for the preparation of a feed additive, the cultivation method comprising incubating a volume of seawater containing propagules of the Asparagopsis species under green light.

[0011] Patent application JPH05103558 which presents a method of cultivating brown algae that uses pre-treated seawater conditioned with special nutrients, where adventitious embryos are grown on growth strips.

[0012] Invention Patent JPH0286717 shows a method for proliferating algae that comprises providing precursors, such as zoospores, spores or plumules, of wakame seaweed or seaweed, these are applied to a substrate or support for the proliferation of algae, to which an aqueous solution of a sodium alginate type polymer is applied to form an adhesion film, in addition to the support, nutritional components are administered to improve reproduction and growth.

[0013] Patent Application CN 106912366 The invention relates to a method of asexual propagation of Graciaria lemaneiformis, which comprises an initial step of cleaning and disinfection, then the body of the algae is cut into 2-4 mm sections using a scalpel, which are grown in a PES culture solution containing 0.25 mg / mL of 6-BA, with a 16h:8h photoperiod (light:dark) for 7 days.

[0014] Although solutions exist for large-scale algae cultivation, new methodologies specific to certain species are still needed, taking into account the specific problems of the species required.

[0015] BRIEF DESCRIPTION OF THE FIGURES

[0016] Figure 1: Stages of the production model defined for the cultivation of D. kunthii, using the ligule-based propagation strategy.

[0017] Figure 2: Morphological aspect ratio of ligules according to caliber 1, 2 and 3. Figure 3: Ligule separation system by caliber using different sieves.

[0018] Figure 4: Column culture of ligules in a hatchery under controlled conditions where the ligules are grouped by size.

[0019] Figure 5: Views of ponds with caliber 3 ligulae that began their growth stage in the cultivation yard. Detail of ligulae of different sizes during this cultivation stage.

[0020] Figure 6: Biomass of D. kunthii ray florets grown in a hatchery. A: Size 1 culture showing a linear growth fit. B: Size 2 culture showing an exponential growth fit.

[0021] Figure 7: Accumulated biomass of ligules under culture regime in outdoor ponds.

[0022] DISCLOSURE OF THE INVENTION

[0023] A cultivation process of the species Dictyota kunthii is presented, using ligules, which are obtained from donor plants (thalli) that have been extracted from natural meadows.

[0024] This process comprises at least four sequential stages, outlined in Figure 1, which are:

[0025] A) Entry and cultivation of donor algae

[0026] B) Entry of ligules to Hatchery separated by size

[0027] C) Ligule crops in Hatchery

[0028] D) Cultivation of ligules in ponds

[0029] The duration of this process, outlined in Figure 1, is 11 weeks, of which the hatchery stages (B and C) take a total of 4 weeks, and stage D, 7 weeks. This process can vary by 1 to 2 weeks depending on factors such as the weather or the quality of the donor algae.

[0030] The main advantages of this culture protocol are: (1) it uses, in an unprecedented way, multicellular proliferations called ligules as biological material for the production of D. kunthii biomass, (2) These ligules are unique to the D. kunthii species, and are generated abundantly throughout the year, maintaining high growth; (3) a low amount of donor algae is required to obtain a high number of ligules; and (4) The culture procedure performs a caliber or size separation of the ligules used, which allows to homogenize the size and ensure the passage of the ligules to the fattening or pond culture stage (D) when they have reached a minimum size for it. This prevents very small ligules from being transferred to the ponds where, due to their shape (without branches), they quickly precipitate, despite the aeration provided to move the water column, and are deposited at the bottom of the pond, dying.

[0031] The conditions of each stage are described below:

[0032] A) Intake and cultivation of donor algae: Algae are collected from natural meadows and transported to the laboratory or cultivation site. Due to the high number of ligules present on the algal thalli, only 1 to 2 kg of donor algae are required to initiate up to three cultivation cycles. Donor algae undergo a prior disinfection stage and are maintained in 300 L outdoor cultivation ponds. They are supplied with a continuous flow of filtered seawater at 15 pm (RUNXIN TM. F6B sand filter) and disinfected by UV light. The flow rate varies between 24 and 40 L / min, obtaining a minimum of approximately 4 replacements per hour. Permanent aeration is added to each pond. To reduce excess light, the ponds are covered with mesh (60% shade). These donor algae are kept in culture for a period of 2 to 4 months, during which time the released ligules are collected weekly.B) Entry of ligules to the Hatchery separated by size: At this stage, the ligules that are naturally released from the donor algae and that are found in the ponds are separated. To do this, all donor algae, including suspended ligules, are removed by means of filtering elements, such as meshes and sieves with different pore openings. This is followed by disinfection with 7% Lugol's solution for 3 to 5 min; then, a wash on an anti-aphid mesh with plenty of seawater, allowing the separation of ligules and donor plants. The latter are weighed and returned to their pond of origin. The ligules are calibrated by size; for this purpose, three filters have been established to obtain the different cultivation sizes (Figure 2) (Table 1). These are: gauge 1: anti-aphid mesh used in agriculture to seal the entry of mosquitoes made of HDPE; gauge 2: mesh made of polyethylene; gauge 3: ratchel mesh (Figure 3).Ligules with caliber 1 and 2 are cultivated in the laboratory and when the ligules reach caliber 3 they are sent to the cultivation yard in 300L tanks.

[0033] Table 1: Types of mesh used to obtain the different sizes of ligule culture in hatchery.

[0034] Caliber Mesh Type Ligule Size

[0035] 1 Antiaphid < 1 mm

[0036] 2 Polyethylene > lmm

[0037] 3 Ratchel > 4 mm

[0038] Separation by size is essential for this type of culture, which uses ray florets as propagules to obtain biomass. Figure 2 shows the morphological relationship of the ray florets according to the different sizes. When they reach size 3, they are larger, more complex, with abundant branches, which helps them stay in the water column, preventing them from precipitating and dying, as occurs with ray florets of sizes l and 2. C) Hatchery culture of ray florets: The objective of this stage is for the ray florets to increase their size, reaching size 3, which is when they are taken to the fattening stage in outdoor cultures. Ray florets of sizes 1 and 2 are separated and grown in independent columns with an initial culture density of 10 to 15 g / L. 1(Figure 4). All culture columns are maintained under the conditions detailed in Table 2. Seawater and nutrient solution are renewed weekly, and the sizes are checked and the calibration procedure is repeated using the mesh set described in Table 1. When the ligules reach size 3, they are transferred to the next stage of grow-out in the pond.

[0039] Table 2: Hatchery culture conditions

[0040] Conditions

[0041] Temperature 15 ±2°C

[0042] Photoperiod 12:12 (L:O)

[0043] Photon Flux Density 40-80 pmol m 2 s 1

[0044] Biostimulant Solution 0.3 - 1 mL / L

[0045] Weekly seawater replacement

[0046] Permanent aeration

[0047] Column Capacity 10 - 20 L

[0048] Initial culture density 10 - 30 gL 1

[0049] D) Ligule culture in ponds: Ligules that reach caliber 3 (> 4 mm) during their passage through the hatchery are transferred to external ponds with a capacity of 300 L (Figure 5). These are supplied with a continuous flow of filtered seawater at 15 pm (RUNXIN TM. F6B sand filter) and disinfected by UV light. The flow rate corresponds to a minimum of 4 changes per hour. Permanent aeration is added to each pond. To reduce excess light, the ponds are covered with Ratchell-type mesh (60% shade). They are kept in these conditions until harvest, according to our results for up to 7 weeks with variations of 1 to 2 weeks depending on the season. Currently, there are no other alternatives for the cultivation of D. kunthii. Other similar species, such as D. dichotoma and D. menstruaüs, do not have cultivation strategies; there are only a few reports of in vitro studies that describe the growth of these species. In these species the existence of ligules is not recorded, as occurs in D.kunthii, therefore, the invention is unique and specific to this species, being the first attempt to cultivate it in the world.

[0050] APPLICATION EXAMPLES

[0051] Example 1: Staged cultivation protocol

[0052] Entry and cultivation of donor algae

[0053] Disinfection Protocol: The cultivation protocol begins with the disinfection of surfaces and materials in the cultivation room (Hatchery). To do this, all materials were disinfected, rinsed with water and a 70% ethane solution, and then disinfected with UV light for 15 minutes. Walls, floors, and countertops were disinfected with a 5% chlorine solution, followed by a generous rinse with water. The cultivation yard ponds were disinfected with a 10% muriatic acid solution and then thoroughly rinsed with seawater using a pressure washer.

[0054] Donor algae intake: Once collected, the donor algae (approximately 1.5 kg of biomass) were transported cold (10°C) to the facilities. They were then washed with seawater to remove impurities and other organisms, and only those algae that were in good condition (whole, with no signs of degradation or necrosis) were selected. These algae were then incorporated into a 300 L backyard pond, which was supplied with filtered seawater at a constant flow, aerated, and protected by a mesh cover (60% shaded). During the period they remained in culture, the ligules that naturally detached from the donor algae were collected weekly and processed for cultivation according to caliber (size).

[0055] Entry of ligules to Hatchery separated by size

[0056] Disinfection, washing, and extraction of ligules / Donor algae and ligules were extracted weekly from the backyard pond using squid conditioned with a sieve smaller than caliber 1. The squid were passed through the pond, retaining the donor algae and all the already detached ligules on the sieve. This biological material was transferred to plastic containers conditioned with a 7% Lugol's solution, where they remained for 3 minutes for disinfection, which eliminates gametes / spores of other algae that may be present in the seawater or on the surface of donor algae or ligules. Then all the material was transferred to a container where it was washed with abundant filtered seawater (1 pm and disinfected with UV). At this stage, the donor algae are separated from the detached ligules.The donor algae were returned to the backyard pond, where the procedure was repeated after a week. All collected rayfish were of varying sizes and were then sieved using a container containing the three sizes (Figure 3). Size 3 was placed at the top of the container, size 2 in the middle, and size 1 at the bottom. All rayfish were placed on the first sieve (size 3), and a constant flow of filtered (1 pm) and sterilized (UV) seawater was added. In this way, the rayfish were separated by size, with the largest retained on the top (size 3) and the smallest on the bottom sieve (size 1). Size 3 rayfish (the minimum size for rayfish culture in backyard ponds) were returned to the backyard for cultivation in a pond with continuous water flow, aeration, and wind-protected Rachell mesh.The size 2 and size 1 ray fins remain in hatchery culture (Figure 4) until they reach size 3, when they are transferred to a backyard pond for fattening. Hatchery ray fin culture.

[0057] Gradings 1 and 2 were cultured in 10 L capacity columns (bags) with filtered (1 pm) UV-sterilized seawater, with a maximum culture density of 15 g / 10 L (Figure 4). Culture conditions were kept constant (described in Table 2). The culture bags were fitted with aeration hoses and diffusers, which were sealed with parafilm at the corners of the bags. A cut was made in the top of each bag to allow water and ray florets to enter. The bags were filled with seawater to about 5 cm before reaching the 10 L mark, and finally the ray florets (15 g) were added. The bags were labeled with the following information: size, bag number, and start date of culture. Then the biostimulant nutrient solution (5 mL per 10 liters) was added, the composition of which is presented in Table 3.Finally, the bags were sealed with a gauze plug filled with hydrophobic cotton. The ligules were removed from the cultures weekly, and their fresh weight was recorded. Additionally, the ligules were separated into different sizes using the sieve sequence (Figure 3). All ligules of sizes 1 and 2 were maintained under these culture conditions until they reached size 3, while those that reached size 3 were transferred to an outdoor culture pond (Figure 5).

[0058] Table 3: Composition of biostimulant solution. -

[0059] Example 2: Production results

[0060] Following the protocol described above considering stages 1, 2 and 3, the growth of caliber 1 and 2 ligules was evaluated in the hatchery for 5 weeks to demonstrate the effective growth of the ligules and the usefulness of the protocol that separates them by caliber. Three culture bags of 10 L each were used for each caliber. Caliber 1 ligules under hatchery culture showed a growth that varied over time, and fit a linear growth (Figure 6A). The behavior of caliber 2 ligules was different, as they already presented branches facilitating their cultivation in suspension within the column, and they showed a permanent homogeneous growth, fitting an exponential growth model (Figure 6B). However, in both cases a high growth rate of 8.6 and 10.2 (% d') was obtained. 1) for sizes 1 and 2 respectively, unlike what has recently been reported in laboratory studies for the species Dictyota menstrualis (Obando et al., 2023) where negative growth rates have been obtained due to the loss of biomass from the thalli, even in nutrient-enriched treatments. These results are only comparable with the rapid growth recorded for other species of brown algae, of the kelp type, such as Macrocystis, Lessonia, Laminaria, Ecklonia, among others (Serisawa et al., 2002; Bearham et al., 2013; Camus et al., 2017; Westermeier et al., 2006). This result demonstrates the feasibility and operational advantage of separating the ligules by size, as well as the efficiency of the hatchery stage, which allows to have a large number of ligules, under a rapid growth regime until reaching size 3. Culture of ligules in ponds

[0061] A new culture batch was started, where the ligules were raised in hatcheries and ponds. For the hatchery stage, culture bags were used for each size under the conditions described above for this stage. Once the ligules reached size 3, they were transferred to 300 L outdoor ponds (Figure 4), which are supplied with a continuous flow of filtered seawater at 3 pm (RUNXIN TM. F6B sand filter) and disinfected by UV light. The flow rate corresponds to a minimum of 4 changes per hour; therefore, no nutrients are added at this stage of culture. These ponds have permanent aeration to promote the suspension of ligules in the water column. In addition, to reduce excess light, the ponds were covered with Ratchell-type mesh (60% shade). They were maintained under these conditions until harvest, according to our results for up to 7 weeks.Figure 7 shows the biomass accumulation results of the complete process described above, starting the hatchery stage with 53 g of size 2 ligules. This figure shows that the development fits a potential model (curve equation and R. 2 ) with a daily growth rate of 6 and 5 % d 1for the hatchery and pond stages, respectively. The pond stage was initiated with caliber 3 ligules (> 4 mm), with abundant branching (Figure 2). During cultivation, the ligules changed shape, increasing the number of branches, as well as their length and width, until achieving individuals with typical characteristics of the species, with dichotomous branches, which in some cases exceeded 8 cm (maximum length). This is unprecedented and marks the first time that seedlings (juveniles) of a species of the genus Dictyota have been obtained under a culture regime. At the end of this process, a total of 3,494 g were obtained in the outdoor ponds in the 12th week of cultivation.

Claims

CLAIMS 1. Algae cultivation process of the species Dictyota kunthii, CHARACTERIZED because it uses ray florets obtained from donor plants (thalli) extracted from natural meadows, and which comprises at least the following four sequential stages: a. Entry and cultivation of donor algae; b. Entry of ray florets to the Hatchery, separated by size; c. Culture of ray florets in the Hatchery; and d. Culture of ray florets in ponds.

2. Process for cultivating algae of the species Dictyota kunthii, according to claim 1, CHARACTERIZED in that the duration of this process comprises between 9 to 13 weeks, where the hatchery stages (B and C) have a duration between 2 to 6 weeks, and stage D, 5 to 9 weeks.

3. Process for cultivating algae of the species Dictyota kunthii, according to claim 1, CHARACTERIZED because the cultivation procedure carries out a separation by caliber or size of the ligules used, homogenizing it and ensuring a minimum size of the ligules that move on to the fattening or pond cultivation stage (D).

4. Algae cultivation process of the species Dictyota kunthii, according to claim 1, CHARACTERIZED because in stage (A) of entry and cultivation of donor algae, 1 to 2 kg of donor algae are collected from natural meadows, these are subjected to a previous stage of disinfection and are maintained in outdoor cultivation ponds of 300L capacity, with a continuous flow of seawater filtered at 15pm and disinfected by UV light, with a flow rate between 24 to 40 L / min, permanent aeration to each pond and protected by mesh that provides 60% shade.

5. Process for cultivating algae of the species Dictyota kunthii, according to claim 1, CHARACTERIZED in that in step (B) the ligules are separated from the donor algae by means of filtering elements, such as meshes and sieves with different pore openings; disinfection is carried out with 7% Lugol's solution for 3 to 5 min; then washing on an anti-aphid mesh with plenty of seawater, allowing the separation of ligules and donor plants, the latter being weighed and returned to their pond of origin.

6. Process for cultivating algae of the species Dictyota kunthii, according to claim 1, CHARACTERIZED in that in stage (B) the ligules are calibrated by size, using 3 filters corresponding to anti-aphid mesh (gauge 1); mesh made of polyethylene (gauge 2) and ratchel mesh (gauge 3).

7. Process for cultivating algae of the species Dictyota kunthii, according to claim 1, CHARACTERIZED because in stage (B) the ligules with caliber 1 and 2 are cultivated in the laboratory and when the ligules reach caliber 3 they are sent to the cultivation yard in 300L tanks.

8. Process for cultivating algae of the species Dictyota kunthii, according to claim 1, CHARACTERIZED in that in stage (C) the cultivation of ligules in Hatchery, the caliber 1 and 2 ligules are separated and cultivated in independent columns with an initial culture density of 10 to 30 gL' 1 , the 10- to 20-liter culture columns are maintained under the following conditions: temperature: 15 ±2°C; photoperiod: 12:12 (L:O); photon flux density: 40-80 pmol m 2 s -1 ; 0.3 to 1 mL / L of biostimulant solution; with permanent aeration.

9. Process for cultivating algae of the species Dictyota kunthii, according to claim 1, CHARACTERIZED because in stage (C) weekly The seawater and nutrient solution are renewed and the gauges are checked and the calibration procedure is repeated using the set of meshes.

10. Process for cultivating algae of the species Dictyota kunthii, according to claim 1, CHARACTERIZED in that in stage (D) the ligules that reach caliber 3 (> 4 mm) during their passage through the hatchery are transferred to external ponds with a capacity of 300 L, which are supplied with a continuous flow of filtered seawater at 15 pm and disinfected by UV light, with a flow rate between 24 to 40 L / min, permanent aeration in each pond, maintaining these conditions until harvest, between 5 to 9 weeks.

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