ES2968529A1 - INTEGRATED MIXOTROPHIC INDUCTION BIOPROCESS FOR ASTAXANTHIN ACCUMULATION IN STRAINS OF THE GREEN MICROALGA <i>HAEMATOCOCCUS LACUSTRIS</i> - Google Patents

Abstract

The present invention relates to an integrated bioprocess for the mixotrophic induction of astaxanthin (ATX) accumulation in Haematococcus lacustris, comprising, at least, the following steps: autotrophic culture growth under standard conditions; mixotrophic induction of ATX accumulation in photobioreactor; biomass harvesting and drying under standard conditions; and ATX extraction under standard conditions. The use of this integrated bioprocess enables cultivation without sanitary, seasonal, climatic or geographical constraints.

Description

Integrated bioprocess of mixotrophic induction for the accumulation of astaxanthin in strains of the green microalgae in Haematococcus lacustris

Technical Sector

The present invention is related to the industrial area, more specifically with microalgae cultures of interest in the aquaculture industry and food industry.

State of the Art

Among the so-called fine chemicals produced by microalgae, the red carotenoid pigment astaxanthin (ATX) is in high demand thanks to its important characteristics. Synthetic ATX is used as a pigment in aquaculture and the food industry, to achieve the orange or reddish color in salmon and trout kept in captivity, which can only obtain it from their diet (Liu, 2010). Furthermore, it is essential for the normal development and reproduction of salmonids, accelerating sexual maturity and increasing fertilization, egg maturation and defense against oxidative stress (Nakano et al. 1995). However, these beneficial characteristics are lower in synthetic ATX compared to natural ATX. Natural ATX has 500 times more antioxidant capacity than a-tocopherol (vitamin E), showing the highest free radical absorption capacity (ORAC) of all carotenoids (Nguyen, 2013), arousing great interest for its use as a supplement. or functional ingredient in human nutrition. Additionally, the natural pigment has applications in the pharmaceutical and nutraceutical industries (Lorenz and Cysewsky, 2000), in the case of human consumption, it has demonstrated the ability to prevent degenerative diseases (Iwamoto et al. 2000; Guerin et al. 2003). cancer prevention (McCarty, 2012), improvement of the immune response (Kobayashi et al.

1997; Lorenz and Cysewsky, 2000; Lignell and Bottiger, 2001), prevention of eye diseases (Cort et al. 2008), skin care (Lorenz, 2002; Tominaga et al. 2012), anti-inflammatory agent (Speranza et al. 2012), and inhibition of LDL cholesterol (Choi et al. 2011). These outstanding qualities make natural ATX considered the "king of carotenoids", with renewed interest for human consumption (Nguyen, 2013). This wide variety of beneficial effects has allowed the entry of microalgal astaxanthin into the product market. nutraceuticals, with a huge variety of products with antioxidant properties that are sold in health food stores (McCoy, 1999) and in various pharmacy chains. In general, accumulated evidence has shown that ATX from the microalgae previously known as lacustris. H. pluvialis (Nakada & Ota, 2016; Guiry, 2018), is beneficial for human health by preventing various diseases

Its exceptional characteristics have created an important market for its production, which is estimated to have reached US$200 million in 2015 (Frost and Sullivan 2008), maintaining significant growth year after year. Even though most of the pigment used in the aquaculture industry is of synthetic origin, the market for human consumption is actively growing and is estimated to have reached US$60 million in 2008 (Frost and Sullivan 2008), mainly in encapsulated form or in the formulation of cosmetics, beverages and functional foods.

Naturally, the fungus lacustris (ex H. pluvialis) is the most productive organism, accumulating large amounts of astaxanthin upon encystment, reaching up to 4% of the compound based on dry weight under laboratory conditions (Ambati et al. 2014; Boussiba, 2000). However, 56 kg of biomass is required to obtain one kilo of pigment at a value of US$12,000. Although the production of ATX derived from this microalgae is more expensive compared to artificial and yeast production, it is the best quality in terms of its ORAC antioxidant capacity index, and has FDA approval for use in human consumption (Nguyen, 2013 ). Additionally, natural ATX is associated with other compounds that stabilize the pigment, preventing its oxidation and increasing its duration in storage (Holtin et al. 2009; Nguyen, 2013), however, natural ATX represents only 30% of current production. (Frost and Sullivan 2008), with low productivity in crops and inductive processes of ATX accumulation (Lorenz & Cysewski, 2000), which are traditionally autotrophic, requiring large areas of land, a lot of time and high production costs. (Nguyen, 2013), which indicates a clear need for new natural sources of ATX and above all, for new ways of cultivating and particularly, of inducing the accumulation of ATX with higher productivities, which allow sustainable commercial production to cover its growing market. It should even be considered that production costs are not too far from synthetic ATX, so improvements in the ATX induction process could make the production of natural ATX more economically viable in the near future (Li et al., 2011 ).

The production of ATX from microorganisms has been a topic of intense research in recent years (Chen et al, 1997; Ip & Chen, 2005), in comparison with other producing microorganisms, green microalgae, such as H. lacustrís and C. zofingiensis have the capacity to accumulate large amounts of ATX (Rise et al, 1994; Boussiba et al, 1999, Borowitzka, 1999; Curtain, 2000). Currently, the production of natural ATX is carried out mainly through the cultivation and induction of H. lacustries under autotrophic conditions, that is, using inorganic nutrients, light and CO2 as a carbon source (inorganic carbon), requiring large areas of land, long times (21 to 27 days) for cultivation and induction of ATX accumulation and high costs in operation. In this scenario, the existing autotrophic ATX accumulation induction processes and cultures of H. lucustrís do not satisfy the global demand for natural ATX, considering that the current ATX cultivation and induction conditions present low productivities of 0.18 g ATX m-3 d-1 with 1.8% (mass percentage of ATX with respect to biomass dry) annual average on a large plot of land of 4000 m2 for crop raceways and 25000 m2 for autotrophic induction raceways (Industrial Plant Data of Pigmentos Naturales S.A.).

Wild strains of the microalga achieve a maximum production reported in the literature of 4% under ideal laboratory conditions in low culture volumes. However, the contents obtained in larger scale processes at an industrial level rarely exceed 2%. To improve productivity, random mutagenesis has been used, achieving increases between 2.5 - 2.6% (Chen et al., 2003; Gomez et al. 2013), but with reversion problems, low biomass and accumulation of other compounds. desired (An et al. 1989). The generation of transgenics can be successful (Liu, 2010; Teng, 2002; Steinbrenner and Sandmann, 2006; Kathiresan et al.

2009) but its use is not allowed at the national level or in human nutrition, emphasizing the need for natural sources (Nguyen, 2013).

With this background exposed, it is highly necessary to develop a new bioprocess of astaxanthin accumulation via mixo or heterotrophic induction in H. lacustrís as a validated tool, with great economic potential to obtain significant increases in astaxanthin, free of health, geographic or climatic restrictions.

REFERENCES

• Borowitzka, M.A. (1999) Commercial production of microalgae: ponds, tanks, tubes and fermenters. J Biotechnol 70:313-21.

• Boussiba, S., Bing, W., Yuan, J. P., Zarka, A. and Chen, F. (1999). Changes in pigments profile in the green alga Haeamtococcus pluvialis exposed to environmental stresses. Biotechnology Letters, 21(7), 601-604.

• Boussiba, S. (2000) Carotenogenesis in the green alga Haematococcus pluvialis:

Cellular physiology and stress response. Physiol Plant 108:111-117.

• Chen, Y., Li, D., Lu, W., Xing, J., Hui, B. and Han, Y. (2003). Screening and characterization of astaxanthin-hyperproducing mutants of Haematococcus pluvialis. Biotechnology letters, 25(7), 527-529.

• Choi, H. D., Youn, Y. K. and Shin, W. G. (2011). Positive effects of astaxanthin on lipid profiles and oxidative stress in overweight subjects. Plant foods for human nutrition, 66(4), 363-369.

• Cort, A., Ozturk, N., Yargicoglu, P., Yucel, I., Unal, M., Ciftcioglu, A., ... and Aslan, M. (2008). Suppressive Effect of Astaxanthin on Retinal Injury Induced by Ocular Hypertension. Free Radical Biology and Medicine, 45, S80-S81.

• Curtain, C. (2000) The growth of Australia’s algal beta-carotene industry.

Australas Biotechnol 10:19-23.

• Ip, P. F., and Chen, F. (2005b). Production of astaxanthin by the green microalgae Chlorella zofingiensis in the dark. Process Biochemistry, 40(2), 733-738.

• Li, J., Zhu, D., Niu, J., Shen, S. and Wang, G. (2011). An economic assessment of astaxanthin production by large scale cultivation of Haematococcus pluvialis. Biotechnology Advances, 29 (6), pp. 568-574.

• Liu, J. (2010) Genetic engineering of Chlorella zofingiensis for enhanced astaxanthin biosynthesis and assessment of the algal oil for biodiesel production. Doctoral thesis, University of Hong Kong. http://hdl.handle.net/10722/131813 • McCarty, M. F. (2012) Minimizing the cancer-promotional activity of cox-2 as a central strategy in cancer prevention. Medical Hypotheses 78(1): 45-57.

• Nakada, T., and Ota, S. (2016). What is the correct name for the type of Haematococcus Flot. (Volvocales, Chlorophyceae)? Taxon, 65(2), 343-348. • Nguyen, K. (2013) Astaxanthin: A Comparative Case of Synthetic VS. Natural Production. Chemical and Biomolecular Engineering Publications and Other Works. http://trace.tennessee.edu/utk_chembiopubs/94. Official Journal of the European Communities L106, DIRECTIVE 2001/18/EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 12 March 2001, on the deliberate release into the environment of genetically modified organisms and repeating Council Directive 90/220/EEC.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Stressors selected to induce ATX production in H. strains. lacustrís.

Figure 2: Three-dimensional graph of the response surface for the productivity of ATX induction by mixotrophic route in strain 024.

Figure 3: ATX chromatograms in P3 strain cultures with autotrophic induction. Signals: Left corresponds to unhydrolyzed sample; Right corresponds to hydrolyzed sample.

Figure 4. ATX chromatograms in P3 strain cultures with mixotrophic induction. Signals: left corresponds to hydrolyzed sample and right corresponds to non-hydrolyzed sample.

Figure 5. Chromatograms of ATX in cultures of strain 024 with autotrophic induction. Signs: Left corresponds to hydrolyzed sample and right corresponds to unhydrolyzed sample.

Figure 6. Chromatograms of ATX in cultures of strain 024 with mixotrophic induction. Signals: left corresponds to hydrolyzed sample and right corresponds to unhydrolyzed sample.

Figure 7: Synthetic ATX standard 10 pg/mL together with strain 024 sample NaCl treatment.

Figure 8: Stereoisomer determination in ATX of strain P3 with autotrophic induction

Figure 9: Determination of stereoisomers in ATX of strain P3 with mixotrophic induction

Figure 10: Determination of stereoisomers in ATX of strain 024 with mixotrophic induction

Figure 11: Antioxidant capacity assays (%) of ATX from strain 024 (left) and P3 (right).

Figure 12: Antioxidant capacity assays of ATX from strain 024 (left) and P3 (right).

Figure 13: Concentration of ATX and biomass obtained from strain 024 during the induction process.

Figure 14: Productivity of optimized mixotrophic induction of ATX in FBR 3 L, 5 experiences.

Figure 15:Productivity of optimized mixotrophic induction of ATX in 200 L FBR, 3 experiences.

DESCRIPTION OF THE INVENTION

A new non-autotrophic integrated bioprocess of astaxanthin (ATX) accumulation is presented, via mixotrophic induction in Haematococcus lacustrís for the nutraceutical industry.

During the development of this technology, different stressors were used, which were selected according to their ATX induction capacity. Different carbon sources were used: acetate, glycerol and acetate-glycerol; irradiance and temperature.

The bioprocess includes at least the following stages:

a) Autotrophic culture growth phase under standard conditions: an autotrophic culture is prepared under standard conditions over a period of 10-20 days in bioreactors between 5 and 800 L at 23°C, with constant bubbling and at a light intensity of 50 pE. m-2 s -1;

b) ATX accumulation induction phase: In a photobioreactor, the culture is subjected to the following conditions: Carbon source sodium acetate-glycerol mixture at 10-50 mM; temperature of 25-28°C; irradiance at 500-700 pE m-2s-1; pH between 7 - 8; stirring at 100 - 250 rpm; duration 6 to 12 days;

c) Biomass harvesting and drying phase, under standard conditions; and d) ATX extraction phase, under standard conditions.

The integrated bioprocess allows greater accumulation of the carotenoid pigment ATX in at least two strains of H. lacustrís, which represent industrial strains and polyploid strains. Since there were no significant differences between both strains studied under these optimal induction conditions, we can indicate that this process can be used for any strain of H. lacustrís. Under these conditions, for the industrial strain, ATX productivities of 3.27 g m-3 d-1 were obtained and for the polyploid strain, ATX productivities of 2.61 g m-3 d-1 were obtained. Which means increasing ATX productivity between 15 and 18 times compared to that obtained by the industry under traditional conditions in the industry in northern Chile 0.18g m-3 d-1.

Another of the competitive characteristics of the bioprocess is the reduction of induction time, achieving high productivity between 6-12 days of induction, while, with the traditional method of the industrial process, induction is achieved in periods between 15-45 days.

The photobioreactor used can have a capacity from 250ml to 200L, without having to modify the operating conditions and without affecting the quality of the ATX obtained.

This bioprocess can be implemented without health, seasonal, climatic or geographical limitations. The advantages and competitive potential of the non-autotrophic bioprocess developed are:

- Greater ATX productivity due to the optimization of two factors: i) increase in ATX production and ii) shorter induction process time.

- Lower operating cost due to: i) reduced water consumption, ii) reduced energy costs and iii) lower labor.

- Applicable to any geographical area due to the use of closed systems.

- Lower investment cost, given the lower land requirement for its implementation.

- Does not depend on seasonal variability.

- Greater control of critical process variables (for example, temperature).

- Reduces production pollution due to the use of closed systems.

In the chemical and isomeric characterization of the ATX molecule obtained in this bioprocess with respect to autotrophic induction, no differences were found in the carotenoid profiles or in the isomeric composition of the ATX molecule.

The polyploid strain (P3) was not affected in its carotenoid composition, either by its polyploidization treatment or by the non-autotrophic inductive treatments.

The industrial strain (024) was also not affected by the non-autotrophic inductive treatments in terms of the carotenoid profile and the isomeric composition of the ATX molecule (3S,3'S),

The effect that could be verified is the increase in the ATX concentration for both strains in the non-autotrophic induction treatments, especially the mixotrophic type.

In the antioxidant capacity (AC) tests, it was possible to verify that this property is relatively affected by the ATX concentration in each test. However, the different nature of each antioxidant assay does not always demonstrate that there is a directly proportional relationship between ATX concentration and antioxidant capacity (AC).

In all the antioxidant tests carried out, starting from the mixotrophic induction treatments, there was an increase in CA with respect to autotrophic induction (NaCl), although the CA test by DPPH clearly indicated that there are no significant differences between the antioxidant treatments. induction. However, in the rest of the trials there was an important effect in increasing the antioxidant capacity of the ATX molecule in mixotrophic inductions.

APPLICATION EXAMPLES

Example 1: Evaluation of stressors that induce the accumulation of astaxanthin via a mixotrophic route.

Stressors were evaluated in two strains of H. lacustrís, the first strain called 024, corresponds to the industrial strain from the company Astax Chile SpA, and strain P3 corresponds to a polyploid from the GIBMAR UdeC laboratory. The P3 strain is deposited in the Spanish Algae Bank (BEA) under the access code BEA IDA 0064B, the deposit was made on November 21, 2017.

The 4 stressors evaluated were:

• carbon sources: sodium acetate, glycerol and a mixture of them, in a concentration range between 5-100 mM;

• temperature at a range between 25-30°C;

• irradiance at a range between 400 and 700 pE m-2 s-1; and

• UV-C radiation at a range between 10-100 mJcm-2.

After induction, they were kept in a JSOS-500, JSR shaker with a stirring of 120 rpm at 25, 30 and 35°C between 6 and 12 days.

For the evaluation of each stressor, cultures grown in bottles between 2-5 L were used that were maintained for 10 days at 23°C, with constant bubbling and at a light intensity of 50 pmol m-2 s-1. After this growth phase, we proceeded with the accumulation induction treatments. These were carried out in 250 mL Erlenmayer flasks that were inoculated with 100 mL of culture and, subsequently, each of them were subjected to different treatments to induce encystment of the H. strains. lacustrís (024 and P3J. The control was left without any stressor, and was maintained under the same conditions as the remaining treatments. Additionally, NaCl was used as an industrial control (in the industry the culture is diluted and for 4 consecutive days it is added NaCl to induce encystment), to simulate these conditions, 0.25 gL-1 was added the first 4 days until 1 gL-1 of NaCl was obtained in 100 mL of the culture. The stressors were analyzed in strains 024 and P3 twice. independently, including 3 replicas for each.

In each case, the ATX productivity of the evaluated stressors was determined. The best selected conditions are presented in Table 1.

Table 1: Conditions selected for optimization.

4 stressors were selected from Figure 1 for both strains, obtaining ATX productivity up to 13 times more than the annual average of the industry in northern Chile (0.18 gm-3 d-1), these stressors were used to the optimization stage. It is important to highlight that in both strains it coincides that the highest productivity is obtained with the same stressors.

The optimization of the mixotrophic induction parameters selected in Example 1 was carried out, which generated a greater accumulation of the carotenoid pigment ATX in the two H strains. lacustrís.The stressors selected to evaluate in this optimization process were: acetate carbon source, acetateglycerol carbon source, irradiance and temperature.

These conditions were evaluated through a multivariate optimization process for which they were organized in 6 triplicate experimental blocks (n = 3 replicates for 6 treatments; N=18).

To evaluate the interaction between the factors and optimize the bioprocess, the factorial experimental design method with 3 factors and three levels was used. The selected factors correspond to the concentration of sodium acetate added to the culture, the temperature of the reactor where the cultures are maintained between 6 and 12 days and its irradiance. The response evaluated was the productivity of ATX induction for each of the strains. Twenty-one experiments were carried out in each case. In total, 4 response surfaces were obtained, evaluating the concentration of sodium acetate, the mixture of sodium acetate with glycerol, for each strain (024 and P3).

Response surfaces were evaluated by analysis of variance (ANOVA) using MODDE 12.1 data analysis software (Umetrics, Umea, Sweden) to fit the experimental data to the second-order polynomial equation using a linear regression technique.

Cultures grown in 5 L bottles were used and maintained for 10 days at 23°C, with constant bubbling and at a light intensity of 50 pE m-2 s-1. After this growth phase, we proceeded with the accumulation induction treatments. The 6 blocks were evaluated with 3 replicates in 250 mL Erlenmayer flasks inoculated with 100 mL of culture, which were subjected to the conditions established to induce the encystment of the H. strains. lacustrís (024 and P3). In addition, an industrial control was added, diluting the culture and adding NaCl for 4 consecutive days, to induce encystment. To simulate these conditions, 0.25 gL-1 was added the first 4 days until 1 gL-1 of NaCl was obtained in 100 mL of the culture.

Samples were taken from all treatments in each block between day 6 and 12 of culture in order to determine cell density, dry weight and amount of ATX.

As an example, the optimized model to increase ATX productivity in strain 024 with the stressor sodium acetate is shown in Figure 2. The temperature and irradiance values are optimal at 27.5 °C and 637 pE m-2s-1 at a concentration of 50 mM (Table 2), the latter being the most significant factor in the model, since an increase in the concentration of sodium acetate could increase the ATX productivity values of 2.29 gm-3 d-1.

Table 2: Selected stressors optimized to increase ATX productivity, through optimized mixotrophic induction

Example 3: H. lacustris strains validated based on the ATX produced

Using analytical and functional techniques that allow determining the quality of the ATX produced (isomeric composition and antioxidant capacity) by mixotrophic route, the industrial strain and the polyploid strain were validated for the best induction process in comparison with the ATX of the base strain obtained. by autotrophic induction.

Cultures grown in 2-5 L bottles were used and maintained for 10 days at 23°C, with constant bubbling and at a light intensity of 50 pE m-2s-1. After this growth phase, we proceeded with the accumulation induction treatments. The ranges of the optimized conditions described in Table 3 were evaluated to induce encystment of the H. strains. lacustris(024 and P3), each with 4 replicates in 250 mL Erlenmeyer flasks inoculated with 100 mL of culture. Additionally, an industrial induction control described above was added.

Samples were taken from all treatments between days 6 and 12 of culture in order to determine dry weight and amount of ATX.

Table 3: Optimized stressors.

Free ATX was quantified by high-performance liquid chromatography (HPLC), via enzymatic hydrolysis of carotenoid esters, and the corresponding stereoisomers were identified.

Quantification of ATX stereoisomers by high-performance liquid chromatography (HPLC).

The prepared ATX standards were filtered and injected in triplicate. These standards correspond to concentrations of 0.75; 1.5; 3.0; 4.5; 6.0; and 7.5 pg/mL of ATX. The HPLC equipment was conditioned to determine the signal corresponding to free ATX at a retention time of 7.92 min.

The analysis conditions were determined at room temperature, with a flow rate of 1 mL/min and using a column (Luna 3pm Silica) in the normal phase under isocratic conditions of hexane/acetone 82:18 v/v with detection at 474 nm. Aliquots of 20 μl were injected with a total run time of 15 min. Regarding the evaluation of some validation parameters, the method was linear in the concentration ranges evaluated and both the detection limit and the quantification limit were well below the first point of the calibration curve. Which reflects the ability of the method to determine ATX concentrations at low concentrations.

Enzymatic hydrolysis of carotenoid esters:

In Figures 3-6, free ATX is observed next to the unhydrolyzed sample for autotrophic and mixotrophic induction in strain P3 and 024 deH. lacustrís. It is important to highlight that regardless of the strain and the treatment to which the samples were subjected, the results indicate that there is no variation in the carotenoid profile between the samples analyzed and only an increase in ATX is observed as in the rest of the samples. the pigments detected, indicating an induction of the entire ATX enH metabolic pathway. lacustrís.

Stereoisomers of astaxanthin produced in the cultures (p3 and 024) ofH. lacustrís.

In Figure 7, you can see the stereoisomer profile of synthetic astaxanthin, which includes the three optical isomers that this molecule has in a ratio 1:2:1 (3S,3'S):(3S,3R):(3R, 3'R).

This isomer conformation allows us to differentiate the synthetic astaxanthin molecule from the natural one, in this case from H. lacustríla which corresponds entirely to the isomer (3S,3'S).

In figures 7 to 10, it can be observed and confirmed that the polyploidization process, as well as the mixotrophic induction treatments, do not affect the isomeric composition of the ATX molecule, maintaining the structure (3S,3'S), which validates the obtaining ATX for use as a nutraceutical in humans.

Using different tests, the relationship between the percentage of antioxidant capacity (AC) of ATX produced by mixotrophic induction, with respect to the AC of ATX obtained from the base strain by autotrophic induction, was determined.

• Spin paramagnetic resonance (EPR)

• 2,2-diphenyl-1-picrylhydrazyl (DPPH)

• 2,2'-azino-bis-(3-ethylbenzothiazolin-6-sulfonic acid) (ABTS)

• Ferric iron reducing activity/antioxidant power (FRAP)

• Oxygen radical absorption capacity (ORAC).

In the antioxidant capacity (AC) tests, it was possible to verify that this property is relatively affected by the ATX concentration in each test. However, the different nature of each antioxidant assay does not always demonstrate that there is a directly proportional relationship between the ATX concentration and the antioxidant capacity (AC), figures 11 and 12.

In all the tests carried out, there was an increase in CA with respect to autotrophic induction (NaCl), although the CA test by DPPH clearly indicated that there are no significant differences between the mixotrophic induction treatments. However, in the rest of the trials it demonstrated an important effect in increasing the antioxidant capacity of the ATX molecule in mixotrophic inductions (acetate and mixture of sodium acetate and glycerol).

The CA was determined by different antioxidant methods, since they define different reaction mechanisms, such as the transfer of hydrogen atoms (as in the case of the ORAC test), electron transfer (such as FRAP), mixing between the transfer of hydrogen atoms and transfer of electrons (such as DPPH and ABST) or inversely by proxidant capacity (EPR).

Example 4: Optimized mixotrophic inductive bioprocess to obtain ATX at laboratory scale (3L) and pilot scale (200L).

The inductive bioprocess was validated under laboratory conditions in 3 and 200L agitated Infors bioreactors.

Cultures grown in 5 and 20 L bottles were used and maintained for 10 days at 23°C, with constant bubbling and at a light intensity of 50 pE m-2 s -1. After this growth phase, the ATX accumulation induction treatment proceeded, under the conditions described in Table 4. Subsequently, the induction was carried out, both in a 3L photobioreactor and in 200 L agitated bioreactors.

Table 4: Induction parameters used in the ATX induction process in 3 and 200L.

To evaluate the replicability of the method, in the case of the 3L photobioreactor, five independent experiments were carried out under the same operational conditions (replicas). Figure 13 shows the results of the final ATX concentration expressed in % w/w of biomass samples harvested at the end of each experiment and lyophilized. Additionally, the final concentration of biomass produced in g L-1 is indicated.

With the 5 mixotrophic induction experiences, it was determined that the average productivity obtained was 3.27 g m-3 d -1, which is equivalent to 18.2 times the average annual productivity of the industry (0.18 g m-3 d -1).

In Figure 14, you can see the 5 experiments carried out in the 3L photobioreactor and Figure 15 indicates the productivity of the mixotrophic induction bioprocess in 3 experiences in 200L agitated bioreactors. In this case, ATX productivities of 2.68 g m-3 d -1 which is equivalent to 15 times the average annual productivity of the industry (0.18 g m-3 d -1).

Claims (11)

1. A non-autotrophic integrated bioprocess of astaxanthin (ATX) accumulation in Haematococcus lacustrís CHARACTERIZED because the accumulation of ATX is via mixotrophic induction and includes at least the following stages: a) Autotrophic culture growth phase under standard conditions; b) Induction phase of ATX accumulation in a photobioreactor, the culture is subjected to the following stressors: carbon source; temperature; and irradiance; c) Biomass harvesting and drying phase, under standard conditions; and d) ATX extraction phase, under standard conditions. 2. The integrated non-autotrophic bioprocess of accumulation of astaxanthin (ATX) in Haematococcus lacustrís, according to claim 1, CHARACTERIZED because the autotrophic culture of the growth phase comprises a period of 10-20 days in bioreactors between 5 and 800 L at 23°C , with constant bubbling and at a light intensity between 50 - 100 pE m-2 s -1. 3. The integrated non-autotrophic bioprocess of accumulation of astaxanthin (ATX) in Haematococcus lacustrís, according to claim 1, CHARACTERIZED because in the induction phase of ATX accumulation the carbon source is a mixture of sodium acetate-glycerol. 4. The integrated non-autotrophic bioprocess of accumulation of astaxanthin (ATX) in Haematococcus lacustrís, according to claim 3, CHARACTERIZED because the sodium acetate-glycerol mixture is at a concentration between 10-50 mM. 5. The integrated non-autotrophic bioprocess of accumulation of astaxanthin (ATX) in Haematococcus lacustrís, according to claim 1, CHARACTERIZED because in the induction phase of ATX accumulation the temperature is 25-28°C. 6. The integrated non-autotrophic bioprocess of accumulation of astaxanthin (ATX) in Haematococcus lacustrís, according to claim 1, CHARACTERIZED because in the induction phase of ATX accumulation the irradiance is 500-700 pE m-2s-1. 7. The integrated non-autotrophic bioprocess of accumulation of astaxanthin (ATX) in Haematococcus lacustrís, according to claim 1, CHARACTERIZED because in the induction phase of ATX accumulation the culture is subjected to a pH between 7 -8 and an agitation of 100 - 250 rpm . 8. The non-autotrophic integrated bioprocess of astaxanthin (ATX) accumulation in Haematococcus lacustrís, according to claim 1, CHARACTERIZED because the induction phase of ATX accumulation lasts 6 to 12 days. 9. The integrated non-autotrophic bioprocess of accumulation of astaxanthin (ATX) in Haematococcus lacustrís, according to claim 1, CHARACTERIZED because in the induction phase of ATX accumulation the photobioreactor used has a capacity of 250ml to 200L. 10. Use of the integrated non-autotrophic bioprocess of accumulation of astaxanthin (ATX) in Haematococcus lacustrís, according to claim 1, CHARACTERIZED because its use allows obtaining between 15 and 18 times more productivity of ATX compared to that obtained by the industry under traditional conditions. 11. Use of the integrated non-autotrophic bioprocess of accumulation of astaxanthin (ATX) in Haematococcus lacustrís, according to claim 1, CHARACTERIZED because its use allows cultivation without health, seasonal, climatic or geographical limitations.