Process to stimulate population growth and yield of biomolecules in microalgae

Description

1 / 46 PROCESS FOR STIMULATING POPULATION GROWTH AND YIELD OF BIOMOLECULES IN MICROALGAE “CERTIFICATE OF ADDITION OF INVENTION of process no. BR 10 2021 019778-1, filed on 01 / 10 / 2021” FIELD OF APPLICATION

[0001] The present invention applies to the field of biotechnology. More specifically, the present invention proposes a process for stimulating population growth and yield of biomolecules in microalgae. FUNDAMENTALS OF THE INVENTION

[0002] Microalgae are a natural source of valuable products with potential applications in productive sectors such as the pharmaceutical, nutraceutical, cosmeceutical, agribusiness, and animal feed industries, among others. Products of interest include carbohydrates, proteins, fatty acids, antioxidants such as carotenoids, and phytohormones. Despite all the interest and investment in research, the viability of large-scale microalgae cultivation is still restricted to a few (robust) species due to the difficulty of maintaining the cultures, low biomass yield, and low yield of biomolecules per unit of biomass. An alternative, which is the subject of this invention, is to increase the yield of biomolecules per unit of biomass while maintaining the algal growth rate. With currently existing techniques, this is achieved at the cost of decreasing the growth rate and, therefore, decreasing biomass generation.Currently, to overcome this problem, the increase in biomolecules is achieved through two-stage cultivation, as described below. In stage 1, the algae are cultivated under ideal conditions for Petition 870250016081, dated 27 / 02 / 2025, page 6 / 69 2 / 46 achieves a high growth rate and high biomass, and in step 2 the cultivation condition is changed, subjecting the biomass to a situation of extreme stress, which induces the cells to produce the biomolecule. We propose this invention to overcome two problems regarding the production of biomolecules involving stress in algal biomass. In the present invention, the production of biomass with biomolecules of interest occurs during the exponential growth of the algal population and, therefore, during biomass generation. This is a stimulus to the cell and not stress. It is to overcome the need to separate exponential population growth from biomolecule production that we propose this invention, whose objective is to increase the yield of biomolecules during algal biomass generation, without interfering with the cell division rate.Furthermore, because the increase in biomolecules occurs in microalgae cells during an exponential growth phase, the proposed process allows for the semi-continuous or continuous collection of biomass with a high biomolecule content.

[0003] It has been known since the 1980s that increasing the yield of biomolecules requires metabolic alteration, usually achieved either by suppressing macronutrients such as nitrogen and phosphorus, or by adding some toxic agent. More recently, incident light colors have been used to induce an increase in biomolecule yield without adverse effects on cell division. Because microalgae are capable of adapting to different environmental conditions through metabolic alteration involving the synthesis of biomolecules, Petition 870250016081, dated 27 / 02 / 2025, page 7 / 69 3 / 46 By altering the cultivation conditions, the microalga responds with the synthesis of biomolecules.

[0004] For the vast majority of microalgae, biomolecule production faces the problem of low yield per unit of biomass, a factor that makes the product costly and can often make commercial production unfeasible. Trying to overcome this limitation, researchers and scientists from companies and universities are seeking ways to increase biomolecule yield, either through biomass produced or through super-concentrated biomass cultures. The information that induced stress in microalgae leads to the accumulation of biomolecules has been known since the 1980s (Lombardi and Wangersky 1991; 1995; (Chia et al., 2017). However, situations of this type of stress also result in a decrease in growth rate and fragility of cell walls and membranes because they negatively affect phospholipids (Lombardi and Wangersky 1991). This leads to a decrease in biomass and an increase in cellular fragility, potentially causing rupture of cell membranes during biomass collection and processing, which invariably leads to the loss of intracellular content, where the desired biomolecules are found. In addition to these problems, there is an alteration in the quality of biomolecules, for example, fatty acids, which instead of being polyunsaturated (healthy cells), become saturated fatty acids (stressed cells). Therefore, the great interest lies in increasing the content of biomolecules in microalgae without harming biomass generation.

[0005] However, subjecting an algal cell to a stressful situation without damaging it and maintaining it in physiological condition. Petition 870250016081, dated 27 / 02 / 2025, page 8 / 69 4 / 46 active exponential growth phase requires analytical and physiological control of biomass.

[0006] Studies in the literature that focus on the search for a synergistic effect through the combination of light color treatments are based on the depletion of major nutrients, usually nitrogen and / or phosphorus, not on the addition of micronutrients, as in the present invention. The present invention differs because it shows the increase of biomolecules both by the chemical element copper alone, and also by synergy between copper and blue light, with the biochemical manipulation of biomass being carried out during exponential growth, in which, during the process, biomass production continues normally, as in the control.

[0007] The gain in carotenoid productivity is undoubtedly a major step towards obtaining this antioxidant while maintaining crop growth. Microalgae exhibit variation in the composition of their photoreceptors, crucial for adaptation to the natural variation of light in the environment. There are studies that report differences in pigment content in response to exposure to different light qualities (Rebolledo-Oyarce et al. 2019). Although several authors have shown that different colors of light can influence carotenoid production (Han et al. 2019), the effect appears to be dependent on the species studied. The results of Suyono et al. (2015) were similar to ours, showing that the microalga Haematococcus pluvialis showed a higher yield of carotenoids when exposed to blue light.

[0008] Therefore, in order to solve the aforementioned problems, the present invention discloses a Petition 870250016081, dated 27 / 02 / 2025, page 9 / 69 5 / 46 process of both synergistic interaction of copper and blue light as well as the metal (copper) alone, to stimulate population growth and biomolecule yield in microalgae. The process consists of metabolic modification for the accumulation of biomolecules in microalgae cultures, preferably Kirchneriella contorta exposed to the synergy of copper with blue light, and Chlorolobion sp. exposed to copper alone, while maintaining the cell division capacity of the biomass, which has been termed biochemical manipulation. The great advantage of the present invention is the increase in the synthesis and accumulation of biomolecules with maintenance of population growth and, therefore, biomass productivity, which generates immense interest and industrial application.

[0009] One of the advantages of the present invention is the increase in the yield of biomolecules without any deleterious effect on the growth rate, through the induction of the hormesis effect in the microalga by exposing the cells to the micronutrient copper at a previously defined concentration, which may or may not be exposed to blue light for 48 hours after 48 hours of maintenance in white light with copper, depending on the microalgal species.

[00010] Another advantage is that the synergistic effect (copper and blue light) allows for the accumulation of carotenoids and carbohydrates during exponential growth in batch systems, and for the increase of proteins and oils, exposure to copper alone is sufficient, depending on the microalgae species.

[00011] Another advantage is that in Kirchneriella contorta an increase of about 30% was obtained in the accumulated total. Petition 870250016081, dated 27 / 02 / 2025, p. 10 / 69 6 / 46 of cellular biomolecules, and because it is a process that combines two distinct processes that, acting synergistically, result in 2x more proteins and carotenoids and 3.3x more carbohydrates, intracellular biomolecules, without compromising the biomass generated. In Chlorolobion sp., exposure to copper was sufficient to increase the total oil content in dry biomass from 20% to approximately 60% in exponentially growing cells, thus generating a completely modified biomass rich in lipids.

[00012] Additionally, the present invention offers the advantage of a synergistic effect in the production of biomolecules, whether carotenoids, proteins, carbohydrates and / or lipids, by associating copper with blue light, or using only copper, depending on the microalgal species. STATE OF THE ART

[00013] Korbee et al. (2005) exposed the macroalga Porphyra leucosticta to blue light and obtained higher protein production, but a lower growth rate. Other studies are based on the deprivation of macronutrients, such as nitrogen and / or phosphorus, as in the study by Li et al. (2019). These authors observed that in nitrogen or phosphorus deprivation, the carbohydrate content in Chlorella sp. increased, but due to a significant reduction in the growth rate.

[00014] US patent 9,683,211 describes a process for obtaining the antioxidant compound astaxanthin from intermittent red light irradiation cycles within the range of 2 to 24 hours for an unspecified total time period. The authors confirm an increase in astaxanthin production by doing so. Our invention differs because in our case the Petition 870250016081, dated 27 / 02 / 2025, page 11 / 69 7 / 46 algae is exposed to light in cycles of 12 hours of light, 12 hours of darkness, respecting the nighttime period, which is important for cellular metabolism. This results in an increase in biomolecules. In our invention, the total period is 96 hours.

[00015] The difference of the present invention in view of the Korbee et al. (2005) and US 9,683,211 documents cited above consists in the increased production of biomolecules concomitantly with the exponential growth of the K. contorta microalga population in culture.

[00016] Document BR 11 2015 001637-5 describes a method using microalgae for the high-efficiency production of astaxanthin. The method comprises steps including heterotrophic microalgae culture, dilution, light-induced culture, algal cell harvesting, and astaxanthin extraction. This method takes full advantage of the rapid growth of microalgae in the heterotrophic culture stage and the high accumulation of astaxanthin caused by light-induced culture from the large number of algal cells obtained through heterotrophic culture to significantly increase the efficiency of astaxanthin production in microalgae, thus achieving low-cost, high-efficiency, and large-scale production of astaxanthin from microalgae.The method not only provides an important technical means to address the large-scale industrial production of astaxanthin using microalgae, but also ensures a broad source of raw material for the widespread use of astaxanthin. Petition 870250016081, dated 27 / 02 / 2025, page 12 / 69 8 / 46

[00017] The difference between this document and the present invention is that the latter uses photosynthetic microalgae and not heterotrophic ones, as in the cited document. It is reported that heterotrophic growth requires organic nutrients in the medium, supporting the growth of bacteria and fungi, thus introducing a high risk of contamination of the algal culture, which does not exist in the present invention, with a completely inorganic medium. Furthermore, in the invention proposed here, being based on photoautotrophy, there is fixation of atmospheric carbon dioxide directly linked to the growth of the microalga during the process of production and accumulation of biomolecules, which does not happen in heterotrophic growth, where carbon dioxide is released into the medium.Unlike document BR 11 2015 001637-5, which involves several steps, such as heterotrophic microalgae culture, dilution, light-induced culture, algal cell harvesting, and astaxanthin pigment extraction, our invention involves a single step: the production of biomolecules during algal growth.

[00018] US patent 9,499,784 discloses processes for the production of microalgae, cyanobacteria and / or their metabolites. It discloses a process involving the use of a stimulus characterized as a reduction in pH, maintaining it between pH 5.0 and pH 6.0 for a period of at least 30 minutes, but not exceeding 2 hours, and then subjecting the culture to an increase in light irradiance to 400 pmol / m² / s applied to a microalgae or cyanobacteria culture to increase the production of one or more metabolites. This is followed by an increase in pH from pH 5-pH 6 to pH 7-pH 9. Under these conditions, the Petition 870250016081, dated 27 / 02 / 2025, p. 13 / 69 9 / 46 The algal culture is maintained for a further 48 hours. A process for the production of microalgae and / or cyanobacteria is also described, comprising an adaptation phase lasting 3 to 6 months, in which an algal / cyanobacterial culture is grown in a process water feedstock and / or under light-emitting diodes (LEDs) emitting light within the wavelength spectrum between approximately 400 nm and 700 nm, and a production phase, in which the microalgae or cyanobacteria are grown in the same process water feedstock and / or under the same light conditions used in the adaptation phase.

[00019] The present invention differs from the document in that it does not alter the pH of the culture nor does it use wastewater from the process for the cultivation of the microalgae. The stimulus to which the algae are subjected before exposure to blue light is a subliminal concentration of the element copper added to the culture medium. This subliminal concentration of the micronutrient copper can, by itself, result in an increase in fats / oils depending on the microalgae species. There is no alteration of the pH nor any increase in light intensity, which remains the same; only the color of the light changes, going from white to blue to seek a synergistic effect, or alternatively, remaining white, thus without synergy of manipulating factors.

[00020] US patent 9,683,211 discloses a method for cultivating green algae that promotes the growth of green algae that are in a green swarm state by irradiating the astaxanthin-accumulating green algae with artificial light. The green algae are cultivated in a liquid medium while maintaining a state in which the color of a Petition 870250016081, dated 27 / 02 / 2025, p. 14 / 69 10 / 46 green algae culture solution is green or brown, intermittently emitting red light while continuously emitting blue light.

[00021] The difference between this document and the present invention lies in the fact that for the present invention the algae is subjected to an initial stimulus given by copper added in subliminal concentration which can be maintained and / or after 48 h of growth under this condition under white light, the color of the light is changed from white to blue seeking a synergistic effect of copper with blue light. Unlike document US 9,683,211, our invention does not mix light of different colors and does not mix intermittent red light with continuous blue light.

[00022] Document KR101287384 discloses a method for algae production to increase algae density and lipid productivity. The method involves cultivating Botryococcus braunii under optical conditions where a mixture of 640 nm wavelength LED light [red] and 460 nm wavelength LED light is projected in a 5:1 ratio. The optical conditions for cultivating Botryococcus braunii are 410 to 426 Lux and a projection duration of 14 to 18 hours. The temperature and pH conditions for cultivating Botryococcus braunii are 20 to 24°C and 6.5 to 8.5.

[00023] The difference between this document and the present invention is that in the present invention there is no mixing of LED colors, a white LED is used for stimulation with a subliminal concentration of the element copper in the culture medium and then the white LED may or may not be replaced. Petition 870250016081, dated 27 / 02 / 2025, p. 15 / 69 11 / 46 by blue LED for biomolecule production. Furthermore, in the present invention, the pH is maintained between pH 6.8 and pH 8.0 and the temperature between 24 - 27oC.

[00024] Document JP2017158587 provides a method for producing astaxanthin more efficiently by cultivating microalgae with light irradiation during the cultivation period, using a blue LED with a peak wavelength of 420 to 500 nm and a red LED with a peak wavelength of 620 to 690 nm. The ratio of the blue LED with a peak wavelength of 420 to 500 nm to the red LED with a peak wavelength of 620 to 690 nm is preferably 1:19 to 19:1 in photon flux density. Their photon flux densities are preferably 20 μmol / m² / s or more.

[00025] The difference between this document and the present invention is that in document (JP2017158587) the white and blue LED colors are not used separately, but the blue and red LED colors are used simultaneously. However, in the present invention, the white and blue LEDs are used separately. In addition to this difference, in the present invention, the algae were subjected to an initial stimulus given by exposing the cells in the culture to a subliminal concentration of the metal copper, while document JP2017158587 uses blue and red LEDs together in a previously defined proportion.

[00026] Document US2011020914 discloses a method for improving the growth, lipid content and / or protein content of bacteria, microalgae and fungi cultivated in an aquatic growth medium. The method uses the Petition 870250016081, dated 27 / 02 / 2025, p. 16 / 69 12 / 46 Contact of the culture medium with methionine hydroxy analog (D and L isomers) and its derived metal salts, such as alkaline earth metal salts, ammonium salts, alkali metal salts, copper, zinc, cobalt, chromium, selenium, manganese, and iron salts to increase the growth and lipid and protein content in microalgae cells in aqueous medium. Alternatively, the methionine hydroxy analog can be one or more metal ligands that can bind to divalent ions with a 2+ charge, such as copper, manganese, calcium, and cobalt. It is desirable, but not necessary, that the ligand carries divalent metal ions in its composition.

[00027] The difference between this document and the present invention is that in the present invention, we do not propose the use of hydroxy methionine analog (D and L isomers) and its derived metallic salts. In document US2011020914, the ligand (hydroxy methionine analog - D and L isomers) is used together with the nutrient salts of microalgae in culture medium. There is absolutely no similarity between what is disclosed by document US2011020914 and the present invention, since a basic nutrient solution, fundamentally inorganic, is used, white and blue LEDs are used at different times, and in 96 h there is an increase in the production of biomolecules.

[00028] Document WO2012101459 refers to processes for the production of microalgae, cyanobacteria and / or their metabolites. A process is described that involves the use of a stimulus applied to a microalgae or cyanobacteria culture to increase the production of one or more metabolites. The document also mentions a... Petition 870250016081, dated 27 / 02 / 2025, p. 17 / 69 13 / 46 process for the production of microalgae and / or cyanobacteria comprising an adaptation stage in which an algae / cyanobacteria culture is grown in a process water feedstock and / or under light-emitting diodes (LEDs) emitting light within the light spectrum wavelengths between about 400 nm and 700 nm and a production phase in which the microalgae or cyanobacteria are grown in the same process water feedstock and / or under the same light conditions used in the adaptation phase.

[00029] The difference between this document and the present invention is that in the present invention, no pH of the culture was altered, this not being the stimulus applied. The stimulus given to the algae in the present invention is exposure to subliminal concentrations of copper under white LED light for the first 48 hours, which is then replaced by blue LED light for the following 48 hours. In total, the process of the present invention, whether using copper plus blue light synergy or just the micronutrient copper, involves 96 hours of cultivation and, furthermore, no process water was used, nor was any adaptation of the cells carried out.

[00030] Document WO2018056160 describes a method for producing astaxanthin comprising culturing an astaxanthin-producing microalga and irradiating it with light during the green stage culture period of the microalga with a blue LED with a peak wavelength of 420–500 nm and a red LED with a peak wavelength of 620–690 nm together, and the photon flux density ratio of the blue LED to the red LED is adjusted to 2:3 to 20: 1. Petition 870250016081, dated 27 / 02 / 2025, p. 18 / 69 14 / 46

[00031] The difference between this document and the present invention is that in the latter there is no mixing of LED light colors. In the process of the present invention, white LED light is used for 96 hours of cultivation or for the initial 48 hours of cultivation, which are then replaced by blue LED light that remains for another 48 hours of cultivation, totaling a cultivation time of 96 hours. SUMMARY OF THE INVENTION

[00032] In a comprehensive manner, in a first aspect, the present invention discloses a process for stimulating the yield of biomolecules without altering the capacity for cell division, maintaining maximum population growth in microalgae so that modified biomass is generated during the exponential growth phase. The algal species can inhabit both freshwater and marine or brackish environments, provided they are cultivated in the photoautotrophic nutritional mode.

[00033] In a second aspect, the present invention discloses a process for stimulating the yield of biomolecules in photoautotrophic microalgae of the Chlorophyta division, without negatively affecting population growth, but whose growth may or may not be stimulated. Microalgal species can inhabit both freshwater and marine or brackish environments, provided they are Chlorophyta species and are cultivated in the photoautotrophic nutritional mode.

[00034] Chlorophyta microalgae are Scenedesmus quadricauda, ​​Scenedesmus bijugus, Desmodesmus communis, Desmodesmus abundans, Desmodesmus brasiliensis, Desmodesmus denticulatus, Monoraphidium contortum, Monoraphidium Petition 870250016081, dated 27 / 02 / 2025, p. 19 / 69 15 / 46 mirabile, Monoraphidium graffithii, Monoraphidium indicum, Selenastrum capricornutum, Selenastrum bibraianum, Ankistrodesmus densus, Ankistrodesmus bernardii, Ankistrodesmus flexuosus, Ankistrodesmus fusiformis, Ankistrodesmus stipitatus, Ankistrodesmus fusiformis, Kirchneriella aperta, Kirchneriella irregulares, Kirchneriella obesa, Kirchneriella contorta, Kirchneriella pseudoaperta, Hariotina reticulata, Chlorella vulgaris, Chlorella emersonii, Botryococcus terribilis, Chlorollobion guanense, Chlorollobion lunulatum, Mougeotia sp. A preferred modality, as microalgae Kirchneriella contorta e Chlorolobion sp..

[00035] The objective of the present invention is to provide an increase in the yield of biomolecules without any deleterious effect on the growth rate, therefore without interfering with the exponential growth of the population in culture, through the induction of the hormesis effect in the microalga. This effect is achieved by exposing the cells to at least one metal (selected from the group comprising copper, zinc, molybdenum or cobalt) that acts as a micronutrient, in a previously defined concentration, and which is also bioavailable, and may or may not subsequently be subjected to exposure of said microalgae to blue LED light.

[00036] Another objective of the present invention is to provide an increase in the yield of biomolecules without any deleterious effect on the growth rate, through the induction of the hormesis effect in the microalga by exposing the cells to the micronutrient copper at a previously defined concentration for a period of 24 to 48 hours under white LED light or by Petition 870250016081, dated 27 / 02 / 2025, p. 20 / 69 16 / 46 sufficient time to allow hormesis effect to be achieved, followed or not by a second exposure of at least 48h under blue LED light. BRIEF DESCRIPTION OF THE FIGURES

[00037] To obtain a full and complete visualization of the object of this invention, the figures are presented as follows. Figure 1 shows the emission spectrum of the LEDs used.

[00038] Figure 2 shows the growth curve of Kirchneriella contorta exposed to different concentrations of copper.

[00039] Figure 3 shows the growth parameters in Kirchneriella contorta cells exposed to copper and white light (48h).

[00040] Figure 4 shows the growth parameters for Kirchneriella contorta cultures exposed to copper (96 h), white LED (48 h, white bar) and blue LED (48 h, blue bar).

[00041] Figure 5 shows results of protein, carbohydrate, and carotenoid productivity in Kirchneriella contorta cultures after 48 h of exposure to copper from biochemical manipulation and white LED light.

[00042] Figure 6 shows results of protein, carbohydrate, and carotenoid productivity in Kirchneriella contorta cultures after 96 h of copper exposure and 48 h of blue LED exposure for the copper concentrations from the biochemical manipulation.

[00043] Figure 7 presents the flowchart of the development and laboratory steps of the experiment. Petition 870250016081, dated 27 / 02 / 2025, page 21 / 69 17 / 46

[00044] Figure 8 shows the growth curves of Kirchneriella contorta during 96 hours of exposure to copper (open triangle) and 48 h to blue LED (closed triangle) for the copper concentrations of the biochemical manipulation.

[00045] Figure 9 shows the photosynthetic quantum yields of Kirchneriella contorta during 96 hours of exposure to copper (open triangle) and 48 h to blue LED (closed triangle) for the copper concentrations of the biochemical manipulation.

[00046] Figure 10 shows the yield of biomolecules (carbohydrates, proteins and lipids) in Chlorolobion sp. exposed to copper at subliminal concentration and to white, blue and red LED light.

[00047] Figure 11 shows the dry biomass yield and growth rate of Chlorolobion sp. subjected to subliminal concentration copper and light from white, blue, and red LEDs. DETAILED DESCRIPTION OF THE INVENTION

[00048] The examples shown here are intended only to illustrate one of the numerous ways of carrying out the invention, however without limiting its scope.

[00049] In general, the present invention discloses, in a first aspect, a synergistic interaction process of at least one metal (selected from the group comprising copper, zinc, molybdenum or cobalt) that acts as a micronutrient and is bioavailable and light (which may be white or blue LED light) to stimulate population growth and Petition 870250016081, dated 27 / 02 / 2025, p. 22 / 69 18 / 46 yield of biomolecules in microalgae, the process comprising the following main steps:

[00050] - Step a: INDUCTION OF THE HORMONE EFFECT. Microalgae culture is initiated under white LED light with hormesis induction in the population, given by the addition of a bioavailable micronutrient metal (copper or zinc or cobalt or molybdenum) in a specific and minute concentration, which can be within the range of 10-9 to 10-8 mol L-1, preferably between 3.0x10-9 and 6x10-9 mol L-1, for a period of 24 to 48h. It is important to emphasize that the concentration of copper to which the microalga will be exposed may vary with the species and its previous history of exposure to the element. The induction of this hormesis effect by exposure to the metal may, by itself, be sufficient to lead to an increase in biomolecules and maintenance of the population growth rate, depending on the physiology of the species.

[00051] — Step b: exposure to blue LED light. The culture under the hormesis effect induced by the added metal will be exposed to blue LED light. For this, the white LED light is replaced with a blue LED light and the culture is exposed to the blue LED light for at least 48 hours. A synergistic effect is created under these conditions.

[00052] - Both stages are performed in the same photobioreactor, and the total biochemical manipulation time is 96 hours, comprising: induction of the hormesis effect under white light, which lasts 24 to 48 hours (or 96 hours depending on the microalgae species), and after this period, the white light is replaced by blue LED light. Petition 870250016081, dated 27 / 02 / 2025, p. 23 / 69 19 / 46 for at least 48 hours, this being the minimum time required to achieve the desired effect.

[00053] In a preferred embodiment, the process aims to maintain or stimulate exponential population growth and biomolecule yield simultaneously in microalgae, preferably cultivated in the photoautotrophic nutritional mode. Algal species can inhabit both freshwater and marine or brackish environments.

[00054] In another preferred embodiment, the process aims to stimulate population growth and biomolecule yield simultaneously in photoautotrophic Chlorophyta microalgae, preferably cultivated in the photoautotrophic nutritional mode. The microalgal species can inhabit both freshwater and marine or brackish environments, provided they are Chlorophyta species and are cultivated in the photoautotrophic nutritional mode.

[00055] The Chlorophyta microalgae are Scenedesmus quadricauda, ​​Scenedesmus bijugus, Desmodesmus communis, Desmodesmus abundans, Desmodesmus brasiliensis, Desmodesmus denticulatus, Monoraphidium contortum, Monoraphidium mirabile, Monoraphidium graffithii, Monoraphidium indicum, Selenastrum capricornutum, Selenastrum bibraianum, Ankistrodesmus densus, Ankistrodesmus bernardii, Ankistrodesmus flexuosus, Ankistrodesmus fusiformis, Ankistrodesmus stipitatus, Ankistrodesmus fusiformis, Kirchneriella aperta, Kirchneriella irregulares, Kirchneriella obesa, Kirchneriella contorta, Kirchneriella pseudoaperta, Hariotina reticulata, Chlorella vulgaris, Petition 870250016081, dated 27 / 02 / 2025, p. 24 / 69 20 / 46 Chlorella emersonii, Botryococcus terribilis, Chlorollobium sp., Chlorollobion guanense, Chlorollobion lunulatum, Mougeotia sp. In a preferred embodiment, the microalgae are Kirchneriella contorta and Chlorolobion sp.

[00056] In a preferred embodiment, the present invention discloses a synergistic interaction process of copper and blue light to stimulate population growth and biomolecule yield in Chlorophyta microalgae, preferably the microalga Kirchneriella contorta, and a non-synergistic process of copper exposure preferably to the species Chlorolobion sp.

[00057] Next, the aforementioned process is described in detail in another modality.

[00058] The process to stimulate population growth and biomolecule yield in microalgae comprises steps (a) and (b), wherein: ai) initiate the cultivation of microalgae in a photobioreactor with an initial cell density ranging from 10⁴ to 10⁶ cells per mL in the exponential growth phase in a culture medium with a pH between 6.8 and 8.0 in a laminar flow hood; a.ii) expose the culture from step (a), still inside the photobioreactor, to white LED light with a luminous intensity ranging from 100 to 250 μmol photons m-2s-1 and add between 3.0x10-9 and 6x10-9 mol L-1 of free metal, subject the culture thus established to the light / dark photoperiod or with continuous white light, at pH 6.8 to 8.0 and temperature of 24 to 27 °C for 24 to 48 hours or for 96 hours; bi) In the case of synergy, change the white LED to a blue LED with a luminous intensity varying between 100 and 250 μmol. Petition 870250016081, dated 27 / 02 / 2025, page 25 / 69 21 / 46 photons m-2s-1 with controlled photoperiod, with pH ranging from 6.8 to 8.0 and temperature between 24 and 27 °C for a minimum of 48 hours.

[00059] For the process of synergistic interaction of copper and blue light to stimulate the yield of biomolecules in microalgae without negatively affecting population growth and, preferably in Kirchneriella contorta and Chlorolobion sp., exponentially growing cells were used.

[00060] In stage (a), the microalgae are from the Chlorophyta division, which are photoautotrophic.

[00061] The Chlorophyta microalgae are Scenedesmus quadricauda, ​​Scenedesmus bijugus, Desmodesmus communis, Desmodesmus abundans, Desmodesmus brasiliensis, Desmodesmus denticulatus, Monoraphidium contortum, Monoraphidium mirabile, Monoraphidium graffithii, Monoraphidium indicum, Selenastrum capricornutum, Selenastrum bibraianum, Ankistrodesmus densus, Ankistrodesmus bernardii, Ankistrodesmus flexuosus, Ankistrodesmus fusiformis, Ankistrodesmus stipitatus, Ankistrodesmus fusiformis, Kirchneriella aperta, Kirchneriella irregulares, Kirchneriella obesa, Kirchneriella contorta, Kirchneriella pseudoaperta, Hariotina reticulata, Chlorella vulgaris, Chlorella emersonii, Botryococcus terribilis, Chlorollobium sp., Chlorollobium guanense, Chlorollobium lunulatum, Mougeotia sp. More preferably, the microalgae are Kirchneriella contorta and Chlorolobion sp.

[00062] In step (a), the photobioreactor used is characterized as a flask for algal cultivation, provided that it is colorless and transparent, and may be made of glass or Petition 870250016081, dated 27 / 02 / 2025, page 26 / 69 22 / 46 plastic. In step (a), the culture medium used is a nutrient solution consisting of inorganic salts that supports algal growth and is selected from the group containing WC medium (Guillard & Lorenzen, 1972), BBM medium, BG11 medium or Chu#10 medium. Preferably, the culture medium is WC medium with pH=6.8 to 7.0 or BG11 medium. More preferably, the culture medium is WC medium with pH=7.0 if the microalga is Kirchneriella contorta and BG11 culture medium if the microalga is Chlorolobion sp.

[00063] In step (a), the laminar flow equipment used may be of the vertical flow type, the horizontal flow type, or it may even be replaced by manipulation with a Bunsen burner flame on a common bench, in the absence of a laminar flow cabinet. This measure aims to reduce the risk of biological contamination at the time of microalgae inoculation. In a preferred embodiment, the laminar flow equipment used is of the vertical flow type.

[00064] In step (b), the cultures from step (a) were maintained in a culture chamber with a light / dark photoperiod or with continuous white light. Preferably, the light / dark photoperiod is 10 / 14h, or 12 / 12h or 14 / 10h. More preferably, if the microalga is Kirchneriella contorta or Chlorolobion sp., they are exposed to white LED light with a luminous intensity of 200 to 250 μmol photons m-2s-1.

[00065] Preferably, in step (b), the free metal is selected from the group comprising copper, zinc, molybdenum or cobalt, or another micronutrient. Petition 870250016081, dated 27 / 02 / 2025, page 27 / 69 23 / 46

[00066] Preferably, in step (b), 3.8x10-9 to 4x10-9 mol L-1 of free copper are added for Kirchneriella contorta and 10-8 mol L-1 of free copper for Chlorolobion sp.

[00067] Preferably, in step (b), subliminal concentrations of other micronutrient metals may be added, respecting the free copper concentration range of 10-9 to 10-8 mol L-1.

[00068] The biochemical manipulation process with synergy was carried out in two main stages, while the non-synergy process was restricted to the first stage. In the synergistic process, the microalga is initially exposed for 48 hours to white LED light and a previously defined concentration of free or bioavailable copper (between 3x10-9 and 1x10-8 mol L-1 of free metal, preferably 3.8x10-9 of free copper for Kirchneriella contorta), and then the culture is exposed to blue light (blue LED) for at least 48 h, this being the minimum time for the desired effect. The total time of the proposed process is 96 h, after which the biomass is ready to be collected. Without synergy, the culture (preferably Chlorolobion sp. exposed to 1x10-8 mol L-1 of free copper) remains in the first stage for 72 to 96 hours and the manipulation effect by stimulation is obtained.

[00069] In step (c), considering the search for synergy, the white LED is changed to a blue LED with a luminous intensity varying between 100 and 250 μmol photons m-2s-1 with a controlled photoperiod, with pH varying between 6.8 and 8.0 and temperature between 24 and 27 °C for 48 hours. In all cases, with or without synergy, in step (c), the cultures were maintained in a culture chamber with a light / dark photoperiod or Petition 870250016081, dated 27 / 02 / 2025, page 28 / 69 24 / 46 with continuous blue light. Preferably, the light / dark photoperiod is 10 / 14h, or 12 / 12h or 14 / 10h.

[00070] Literature studies show that changes in the growth stage result in alterations in the quality of the biomass obtained and that changes in growth rate, transitioning from the exponential phase to the stationary phase, are the result of stress processes, such as nutritional deficiency, shading of the crop, and / or others. Under these conditions, depending on the stress, fragility of cell membranes also occurs (Lombardi and Wangersky, 1991), with the risk of loss of cellular content, releasing the desired intracellular biomolecules into the environment. Furthermore, there may be alterations in the quality of these biomolecules, such as saturation of polyunsaturated lipids (Chia et al., 2013a), resulting in a decrease in unsaturated fatty acids and an increase in saturated fatty acids, and alterations in amino acid composition (Chia et al., 2017).

[00071] In preferred embodiments of the process, cultures were started with 5x10⁴ microalgae cells per mL⁻¹ in WC medium or BG11 medium, but the initial cell density can vary from 10⁴ to 5x10⁶ cells per mL, provided that the light intensity within the culture is maintained (100 to 250 μmol photons m⁻² s⁻¹), preferably 200 μmol photons m⁻² s⁻¹ if the organism is Kirchneriella contorta or Chlorolobion sp. The initial cells were always obtained from a culture in the exponential growth phase at pH 6.8–8.0, temperature 24–27 °C, under illumination by White LED and luminous intensity of 200 μmol photons m-2s-1 Petition 870250016081, dated 27 / 02 / 2025, page 29 / 69 25 / 46 and exposure to the micronutrient copper at concentrations higher than the concentration used in the control.

[00072] The copper concentration that resulted in the hormesis effect in Kirchneriella contorta was 3.8x10-9 mol L-1 of free copper, while for Chlorolobion sp. it was 1x10-8 mol L-1. To ensure the effectiveness of the process, the free copper concentration of the process varies in the range between 3x10-9 and 1x10-8 mol L-1 of free copper and that all other cultivation parameters (temperature, pH, light intensity and quality) remain within the ranges described for microalgae cultivation.

[00073] Next, after the initial 48 h, the second main step of the process consisted of changing from white LED to blue wavelength (blue LED) for another 48 h in cases where synergy is required. Here again, temperature and light remained within the initial tolerance range. The maximum total time of 96 h, and no more than that, was important to ensure that the microalga Kirchneriella contorta or Chlorolobion sp. were still in the exponential growth phase, thus guaranteeing the quality of the cells obtained.

[00074] In a preferred embodiment of the present invention, the increase of biomolecules of biotechnological interest was achieved from the exposure of the cultures to copper without or in combination with chemical stress from copper supplied at low concentration together with physical stress from exposure to blue LED light. Copper was responsible for the hormesis effect, promoting a subtle change in the metabolism of the microalgae Kirchneriella contorta and Chlorolobion sp., and the blue light acted on Kirchneriella. Petition 870250016081, dated 27 / 02 / 2025, p. 30 / 69 26 / 46 contorta as a second stress factor resulting in a magnification in biomolecule productivity. For Kirchneriella contorta, it is noteworthy that for protein accumulation, the stimulus of 3.8x10⁻⁹ mol L⁻¹ of free copper alone resulted in a 1.4x increase compared to the control copper concentration (1.6x10⁻⁹ mol L⁻¹). For carotenoids and carbohydrates, there was an increase compared to the control treatment (1.6x10⁻⁹ mol L⁻¹) of 2x and 3.3x, respectively, obtained after synergism of copper (3.8x10⁻⁹ mol L⁻¹) plus blue LED after 96 h of total cultivation time. In Chlorolobion sp. under white light, it is noteworthy that, for protein accumulation, the stimulus of 1x10⁻⁸ mol L⁻¹ of free copper resulted in a 2.2x increase compared to the control copper concentration (1.6x10⁻⁹ mol L⁻¹). For lipids, there was an increase compared to the control treatment (1.6x10-9 mol L-1), of 2.6x obtained by copper stimulation (1x10-8 mol L-1) after 96 h of total culture time. Example 1 - Experiment with Kirchneriella contorta to evaluate the yield of biomolecules after 96 h of cultivation.

[00075] After 48 hours under blue LED light, totaling 96 hours of cultivation, determinations of biomass, growth rate, and biomolecules were performed. Biomass was determined by measuring the dry weight on an analytical balance, with the growth rate represented by the slope coefficient a obtained after linear regression adjustment using the equation Y = aX + b, resulting from the linearization of the natural logarithm of the number of cells per mL of culture plotted as a function of experimental time. Petition 870250016081, dated 27 / 02 / 2025, page 31 / 69 27 / 46 Table 1 - Results of the tests with K. contorta for determination of biomass, growth rate and biomolecules after 48h of exposure to copper. Free Copper (mol L-1) Biomass (g L-1) Growth Rate (d-1) Carotenoids (pg mL-1 d-1) Proteins (pg mL-1 d-1) Carbohydrates (pg mL-1 d-1) 1.64x10-9 0.026 0.97 0.067 11.65 3.23 3.24x10-9 0.022 0.86 0.061 9.70 2.76 3.86x10-9 0.023 0.94 0.067 16.60 6.43 1.40x10-8 0.023 0.94 0.067 14.10 5.21 1.75x10-8 0.019 0.97 0.037 12.34 1.95 9, 66x10—8 0.020 0.93 0.060 12.56 4.66

[00076] It was observed that increasing or decreasing copper concentrations compared to the suggested range can alter the cost-benefit of the process, such as not exerting the desired hormesis effect, in addition to altering the algal growth rate and / or the composition of biomolecules.

[00077] In the case of a reduction in the concentration of free copper, the microalga may physiologically approach the control situation, and no benefit may be obtained, whereas in the case of an increase in the concentration of free copper, the microalga tends to physiologically approach a situation of stress induced by the metal. In both cases, the microalga would be moving away from the ideal condition described in this invention, and it is not possible to predict the consequences for the composition and quality of the biomolecules produced, even if, eventually, there may be a reduction or increase in the algal growth rate. Petition 870250016081, dated 27 / 02 / 2025, p. 32 / 69 28 / 46

[00078] It is also worth noting that the value of 3.8x10-9mol L-1 of free copper, listed here as a chemical factor interacting with the physical factor (blue light) to result in the desired biochemical manipulation effect, is not fixed for all strains and / or species of microalgae and, therefore, the importance of the metal concentration screening test for defining the concentration range to be subsequently detailed regarding the hormesis effect.

[00079] In this invention, it is demonstrated that the combination of environmental situations acting on distinct metabolic sites, such as the chemical effect of copper and / or the physical effect of blue light color, led to an increase in biomolecules, maintaining the culture in exponential growth. The tests performed aimed to validate the present invention, proving that copper concentrations that do not alter the cell division process, with or without synergism with blue LED, are efficient as a biochemical manipulation tool.

[00080] Carefully planned and previously defined, the copper concentration did not cause a change in the growth rate, but possibly approached a very low magnitude of oxidative stress, insufficient to cause a metabolic change that would result in a change in the growth rate. The response of Kirchneriella contorta cells to sensitization caused by free copper at a concentration of 3.8x10-9 mol L-1 in the first 48 h, by itself, already resulted in a maximum specific protein productivity 1.4 times greater than the control, while in Chlorolobion sp. it was 2.6 times more oils. The productivity of Kirchneriella contorta compared to the control was Petition 870250016081, dated 27 / 02 / 2025, p. 33 / 69 29 / 46 11.65 pg mL-1d-1 (48 hours) while of Chlorolobion sp. The yield was 6.0 pg mL-1d-1 (96 hours), as can be seen in Figure 10. In this figure, the dry biomass yield in Chlorolobion sp. was obtained in 96 h (a); Growth rates (b). Treatments: CB control white light, CuB copper white light, CA control blue light, CuA copper blue light, CV control red light, CuV copper red light. Error bar represents the standard deviation of the mean (n=3). Equal letters above the bars indicate no significant difference (ANOVA, p> 0.05). At the end of 48 h, when the cells, then exposed to copper, were additionally exposed to blue LED for 48 h, Kirchneriella contorta, compared to the control, showed a 2x increase in the production of total carotenoids and proteins, and a 3.3x increase in the production of total carbohydrates. In the control group, the biomolecule productivity values ​​were: total carotenoids (0.082 pg mL-1d-1), proteins (4.68 pg mL-1d-1), total carbohydrates (2.34 pg mL-1d-1).The dose of copper considered to induce the hormesis effect in the microalga Chlorophyta used as a model, K. contorta, was 3.8 x 10⁻⁹ mol L⁻¹ (2.37 times higher than the control) in the culture medium, while the control concentration is 1.6 x 10⁻⁹ mol L⁻¹, a concentration used for all experiments performed using WC medium. If we consider Chlorolobion sp. stimulated by copper as a model for oil production in BG11 medium, the copper concentration was 1 x 10⁻⁸ mol L⁻¹ (2.63 times higher than the control), as can be observed in Figure 11. In this figure, the percentage of biomolecules in the dry biomass of Chlorolobion sp. refers to carbohydrates (a); proteins (b); lipids (c). Treatments: CB control na. Petition 870250016081, dated 27 / 02 / 2025, page 34 / 69 30 / 46 white light; CuB copper added in white light; CA control in blue light; CuA copper added in blue light; CV control in red light; CuV copper added in red light. Error bars represent the standard deviation from the mean (n=3). Identical letters above the bars indicate a non-significant difference (ANOVA, p<0.05). The biomolecules obtained here through the manipulation process are of biotechnological interest (Becker, 2007; Ursu et al, 2014) and have been the target of manipulation processes (Safi et al, 2014). Example 2 - Experiment to determine copper concentrations for biochemical manipulation.

[00081] For the biochemical manipulation of biomass, the objective of this invention, it was necessary to expose the cells to copper concentrations capable of causing some metabolic alteration without, however, exerting a negative effect on the growth rate. To define these concentrations, an assay was performed in which the microalgae K. contorta and Chlorolobion sp. were exposed to 30 different concentrations of the metal under white light (white LED), normally used for cultivation. The thirty (30) concentrations of copper metal tested are shown in Table 3. The assay with the 30 concentrations was performed directly in 96-well microplates exposed to culture light (white LED) and monitored daily for absorbance at 684 nm, the absorption wavelength of chlorophyll a. After this assay, 6 copper concentrations to which K. contorta and Chlorolobion sp. were exposed were defined. In the case of synergy, for 48 hours under usual culture light (white LED) and subsequently this K. culture.The contorted object was exposed to blue LEDs for an additional 48 hours. The total exposure time was then... Petition 870250016081, dated 27 / 02 / 2025, page 35 / 69 31 / 46 biochemical manipulation of 96 hours, the biomass contained higher amounts of biomolecules, being 3.3x more total carbohydrates, 2x more total carotenoids and total proteins in relation to the control for K. contorta and under LED white light for Chlorolobion sp. containing 2.6x more oils in the biomass.

[00082] Figure 1 shows the spectra emitted by the white and blue LEDs used in the experiments, which were recorded with an Ocean Optics UBS 2000 radiometer equipped with BIF-600-UV-VIS optical fiber and OIBase32 data collection software (Dunedin, FL). In Figure 1, item A shows the results for the white LED and in item B, the results for the blue LED.

[00083] Figure 2 shows the results of the assay with the 30 copper concentrations, where item A shows the exposure concentrations between 10⁻⁸ and 10⁻¹⁰, item B shows the exposure concentrations between 3 x 10⁻⁶ and 3 x 10⁻⁵, and item C shows the exposure concentrations between 4 x 10⁵ and 9 x 10⁻⁴ mol L⁻¹ of nominal copper. Figure 2 presents the growth curves, and the results confirm that with increasing copper concentration there was a reduction in the growth of K. contorta, reaching the absence of growth at the highest concentrations (Figure 2, item c). This response confirms the inhibitory effect of copper at high doses, a behavior that agrees with the literature (Lombardi and Maldonado 2011; de Abreu et al. 2014; Baracho et al. 2019). The influence of copper on microalgae growth occurs due to its participation in vital metabolic processes such as photosynthesis (Lombardi and Maldonado 2011), through which the algae produce Petition 870250016081, dated 27 / 02 / 2025, pp. 36 / 69 32 / 46 carbohydrates (Malapascua et al. 2014). Based on these results, copper concentrations were chosen that did not affect photosynthesis so that the cell could continue producing biomolecules and fixing CO2.

[00084] From these results, it was confirmed not only the importance of copper in the growth of the microalga, but also at what concentrations of the metal K. contorta would continue to grow similarly to the control. This step is important because the ideal or deleterious concentration for microalgae depends on the species used.

[00085] Based on the results presented in Figure 2, the copper concentrations (mol L-1) selected for the biochemical manipulation of the biomass were: control (1.6x109); 2.2x10-9; 3.8x10-9; 1.4x10-8; 1.7x10-8 and 9.6x10-8. The biochemical manipulation of the biomass itself was then carried out, which begins with exposing the algae to these 6 selected copper concentrations, as presented in Table 2.

[00086] Table 2 - Copper concentrations (molL-1) added to the culture medium for biochemical manipulation of Kirchneriella contorta biomass and quantification of the metal in free form. Added Copper Free Copper Control 4.0x10-8 Control 1.6x10-9 1.6x10-9 2.0x10-9 3.0x10-8 3.8x10-9 2.2x10-8 1.4x10-8 1.0x10-7 1.7x10-8 3.8x10-7 9.6x10-8 Petition 870250016081, dated 27 / 02 / 2025, page 37 / 69 33 / 46 EXAMPLE 3 - Experiment on the effect of copper on growth parameters

[00087] K. contorta cultures were exposed to 6 copper concentrations (Table 2) for 48 hours under white LED light.

[00088] Figure 3 shows the growth parameters in K. contorta cells exposed to copper and white light (48 h) representing the beginning of the process, where item A shows the maximum growth rate, item B shows the biomass yield and item C shows the cell biovolume.

[00089] As planned, the results (figure 3) show that the growth rate, dry biomass and biovolume were similar, with no statistical differences between the copper concentrations.

[00090] The maintenance of growth rate, biomass yield, and biovolume are promising results for the beginning of the biochemical manipulation of K. contorta and indicate that the differences between concentrations were subtle enough not to interfere with growth. Thus, these copper concentrations are physiologically tolerated by K. contorta. In fact, copper concentrations on the order of 10-9 and 10-8 mol L-1 were used, which are likely to be found in natural aquatic environments (Davies and Bennett 1985), and probably for this reason, the microalga was able to respond physiologically well to this range. EXAMPLE 4 - Experiment on the effect of copper and blue LED on growth parameters

[00091] The cultures already exposed to copper and white light (initial 48 hours) were then transferred from white light to Petition 870250016081, dated 27 / 02 / 2025, pp. 38 / 69 34 / 46 exposure to blue light where they remained for 48 hours. Therefore, at the end of the process, there are a total of 96 hours of exposure to copper, with the initial 48 hours under white light and the final 48 hours under blue light.

[00092] Figure 4 shows the growth parameters for K. contorta cultures exposed to copper (96 h), white LED (96 h, white bar) and blue LED (48 h, blue bar). Item A shows the growth rate in the exponential phase (d-1) and item B shows the dry biomass (g L-1). Error bars represent the standard deviation from the mean (n=3) and the asterisk above the bars indicates the statistical difference (p<0.05).

[00093] Growth rates and dry biomass at the end of exposure (96 h) showed that blue light promoted an increase in growth rate in the control and at the highest Cu2+ concentration (9.7x10-8 mol L-1) and in dry biomass at a Cu2+ concentration of 3.8x10-9 mol L-1. The highest growth rate (approximately 0.6 d-1) and dry biomass (~70 mg L-1) were obtained in exposure to blue light and at 3.8x10-9 mol L-1 of Cu2+.

[00094] These results are corroborated by other studies (Teo et al., 2014; Rebolledo-Oyarce et al., 2019) that analyze the different colors of light in the growth of microalgae as the sole stress factor. According to Teo et al. (2014), the microalgae Nannochloropsis sp. and Tetraselmis sp. were exposed to blue, red, blue-red, and white light, and showed higher growth rates in blue light. The authors obtained an increase in biomass yield of ~1.5x, but did not evaluate the effect of light exposure on carotenoid productivity. Petition 870250016081, dated 27 / 02 / 2025, pp. 39 / 69 35 / 46 proteins and carbohydrates. In the process of the present invention, in addition to presenting a greater increase in biomass (~1.7x) than Teo et al. (2014), it also resulted in an increase in the productivity of these biomolecules, as elucidated below.

[00095] According to the literature, blue LEDs, with shorter and more energetic wavelengths than red LEDs, allow for greater penetration through the culture (Vadiveloo et al. 2015; Schulze et al. 2016), improving cell division and growth rates of microalgae (Teo et al. 2014). Furthermore, according to Ruyters (1984), blue light can affect a large number of photosynthetic enzymes and participants in important processes, such as pigment synthesis and photorespiration. In addition, the literature (Schulze et al. 2016) indicates that nutrient absorption is more efficient under blue light. It is known that the use of different colors of light to biochemically alter algal biomass is the subject of several studies (Yan et al. 2013; Hultberg et al. 2014; Atta et al. 2013; Teo et al. 2014; Rebolledo-Oyarce et al. 2019), which have shown that the responses can vary with the algal species.

[00096] In this experiment, the smallest cell biovolumes occurred when exposed to blue light, concomitantly with the highest growth rates (Figure 4a). It is known that microalgae exposed to white light have higher biovolumes compared to those exposed to blue light (Schulze et al. 2016). Schulze et al. (2016) propose that light quality can affect the cell cycle of microalgae, resulting in morphological changes and, therefore, changes in biovolume. Example 5 - Analysis of the effect of the combination of copper and blue LED on the production and accumulation of biomolecules. Petition 870250016081, dated 27 / 02 / 2025, pp. 40 / 69 36 / 46

[00097] The results for the specific productivity of biomolecules in the initial 48 h are presented in figure 5, under exposure to copper and white light, and an additional 48 h under blue light, totaling 96 h, in figure 6, under blue LED.

[00098] Figure 5 shows the productivity of proteins, carbohydrates, and carotenoids (pg mL-1d-1) in K. contorta cultures. Values ​​obtained after 48 h of exposure to copper from biochemical manipulation (Table 1). Item A shows proteins, B shows carbohydrates, and C shows carotenoids. Error bars represent the standard deviation from the mean (n=3), and the asterisk above the bar indicates a statistically significant difference (p < 0.05).

[00099] Figure 6 shows the productivity of proteins, carbohydrates, and carotenoids (pg mL-1d-1) in K. contorta cultures. Item A shows proteins, B shows carbohydrates, and C shows carotenoids. The colors of the bars represent the colors of the LEDs, with the white bar representing the white LED and the blue bar representing the blue LED. The error bars represent the standard deviation from the mean (n=3), and the asterisk above the bar indicates a statistically significant difference (p < 0.05). [000100] It is noted that in the first 48 h, both proteins and carbohydrates showed increased productivity at a concentration of 3.8x10-9mol L-1Cu2+. [000101] However, it is noteworthy that for proteins, the trigger for the increase resulted in a maximum production (~16 μg mL-1d-1) as early as 48h and, therefore, collection at this stage would be ideal for the industry that aims only to produce proteins from algal biomass. Petition 870250016081, dated 27 / 02 / 2025, page 41 / 69 37 / 46 The next step, manipulation through synergism between copper and blue light, is justified for the biomolecules carbohydrates and carotenoids, as explained below. [000102] In this experiment, a synergistic effect was shown between free copper at a concentration of 3.8x10-9 mol L-1Cu2+ and the blue LED light, which together resulted in an increase in the production and accumulation of total carotenoids and total carbohydrates in Kirchneriella contorta, a freshwater Chlorophyta microalga. These results are shown in Figure 6. The production of carotenoids and proteins almost doubled compared to the white LED, while that of carbohydrates was 3.3 times greater. It should be noted that all increases in biomolecules occurred with the cells in full exponential growth phase, i.e., with maintenance of the capacity for cell division and biomass production. [000103] Considering the effect of copper (3.8x10-9 mol L-1 Cu2+), it is shown that K. contorta exposed to blue light increased the productivity of carbohydrate, protein, and carotenoid biomolecules, maintaining an exponential growth phase and generating biomass with more biomolecules. In the study by Abiusi et al. (2014), Tetraselmis suecica was exposed to different light qualities (white, red, blue, and green), but at a copper concentration equal to the control. The authors found no significant difference in growth rate or biomolecule content. On the other hand, Nannochloropsis sp. under different light qualities or colors did not show an increase in carbohydrates and proteins, but a Petition 870250016081, dated 27 / 02 / 2025, p. 42 / 69 38 / 46 reduction in growth rate compared to white light (Vadiveloo et al., 2015). [000104] It is noteworthy that in the first stage of the present experiment, exposure to copper was able to generate subtle metabolic changes, without interfering with the microalga's cell division capacity, which resulted in biochemical changes when the microalga was exposed to the blue LED, in the exponential growth phase. Thus, the present process produces biomolecules in quantity and produces biomass simultaneously. In other words, it can be said that the process of the present invention produces biomass in the exponential growth phase, biochemically modified and with a gain of biomolecules, compared to the control. [000105] Other studies with isolated light stress (without coupling to chemical stress) obtained smaller increases in biomolecule productivity than those shown in our invention. Han et al. (2019) observed a 1.25x increase in carotenoid content in Dunaliella salina when exposed to blue light. For carbohydrates, Nannochloropsis oculata and Tetraselmis chuii showed increases of approximately 1.2x and 1.3x, respectively, when exposed to red light, i.e., smaller than the increases found in the present invention. Regarding proteins, studies evaluating light quality are also described. For Spirulina sp. There was an increase of ~1.8x in protein content upon exposure to red light (Prates et al., 2020), and in Porphyra leucosticta the growth rate was compromised (2x lower than in the control) due to the protein gain (Korbee et al., 2005). Petition 870250016081, dated 27 / 02 / 2025, p. 43 / 69 39 / 46 [000106] It is concluded that the present process is capable of combining two distinct processes that, acting synergistically, resulted in 2x more proteins and carotenoids and 3.3x more carbohydrates, intracellular biomolecules, without compromising the biomass generated or the growth rate of the algal population. The results clearly show a synergistic effect on the production of biomolecules, more specifically, carotenoids and carbohydrates, when copper is associated with blue light. Particularly for K. contorta, the concentration of the metal that resulted in the highest values ​​of biomolecules and did not negatively interfere with the growth rate was 3.8x10-9 mol L-1. It should be noted that the increase in proteins obtained did not require synergy with blue light, occurring immediately, with white light being sufficient. [000107] Figure 8 shows the growth curves of Kirchneriella contorta during 96 hours of exposure to copper (open triangle) and 48 h to blue LED (closed triangle) for the copper concentrations of the biochemical manipulation. [000108] Figure 9 shows the photosynthetic quantum yields of Kirchneriella contorta during 96 hours of exposure to copper (open triangle) and 48 hours to blue LED (closed triangle) for the copper concentrations of the biochemical manipulation. Example 6 - Evaluation of the synergistic interaction process of copper and blue light to stimulate population growth and biomolecule yield in K. contorta with the addition of 30 different concentrations of added copper. [000109] Figure 7 presents the flowchart of the development and laboratory steps of the experiment for Petition 870250016081, dated 27 / 02 / 2025, page 44 / 69 40 / 46 K. contorts with the addition of 30 different concentrations of added copper. - Growing Conditions: [000110] The microalga Kirchneriella contorta was cultivated in WC medium with pH adjusted to 7.0 (Guillard & Lorenzen 1972), autoclaved (20 min, 121 °C, 1 bar; AV Phoenix Luferco, Brazil) and maintained under controlled temperature (24 ± 1°C) and light intensity (photosynthetically active radiation, PAR) conditions of 200 μmol photons m-2 s-1 and with a 12:12 h photoperiod (light:dark) for all LED colors (white — control, and blue). - Screening for copper concentrations in microplates and their effects on growth: [000111] A preliminary study (copper scan test) lasting 120 h was conducted to define the range of copper concentrations to be used in biochemical manipulation assays. The scan test was performed with 30 nominal copper concentrations (added copper), and the microalga exposed to the metal was cultured in 96-well microplates. The control consisted of WC culture medium with a copper concentration of 4x10⁻⁸ mol L⁻¹. For this test, 30 100 mL cultures (inoculum 10⁴ cells mL⁻¹) were made in polystyrene tissue culture flasks. These cultures were exposed to nominal copper concentrations between 3x10⁻⁸ and 9x10⁻⁴ mol L⁻¹, and a 300 μL aliquot was transferred to wells of a microplate (in triplicate). This microplate was incubated under controlled light and temperature conditions. The culture in the microplate wells lasted 120 hours, and during this period cell growth was monitored daily by means of Petition 870250016081, dated 27 / 02 / 2025, pp. 45 / 69 41 / 46 of the absorbance at 684 nm, 570 nm, and 750 nm. For these determinations, we used a plate reader spectrophotometer. To minimize evaporation from the cultures, the microplates were covered with paraffin film. [000112] Copper was added to the culture medium 24 h before the start of the experiments to equilibrate with the culture medium reagents, and free copper (Cu2+) was determined immediately before inoculating the cells. A standard copper solution with a concentration of 1000 mg L-1 was used to prepare standards, which, when diluted, were added to the cultures at the appropriate concentrations. [000113] For biochemical manipulation, 6 copper concentrations were defined that had little effect on growth rates in the copper concentration sweep experiment performed with 30 concentrations of the metal. This allowed for balancing the stress factor for biochemical manipulation and cultures that produce a quantity of biomass close to the control, thus seeking a gain in biomolecule yield. - Biochemical manipulation: [000114] The biochemical manipulation experiment was performed in 1000 mL capacity polystyrene tissue culture flasks with a culture volume of 900 mL, held vertically and bubbled with filtered air through 0.22 μm pore diameter membranes. The addition of copper followed the same pattern described for the concentration sweep test, as set out in the previous item. [000115] The initial inoculum was 5x104 cells mL-1 from an exponentially growing culture and Petition 870250016081, dated 27 / 02 / 2025, pp. 46 / 69 42 / 46 maximum quantum yield of approximately 0.70. The quantum yield, which is a measure of cellular health, was determined using the modulated fluorescence technique. The experiment lasted 96 h. Daily determinations regarding population growth (cell counting using a flow cytometer that counts fluorescent particles) were performed (Figure 3a). At 48 h of culture (exponential growth phase), biochemical determinations were made on the biomass. Total protein (Figure 5a), carbohydrate (Figure 5b), and carotenoid (Figure 5c) concentrations were determined. Immediately after sampling at 48 h, each of the three replicates of the copper and white light treatments gave rise to 2 cultures that were then exposed to white and blue LEDs. Thus, three replicates of each treatment in the 2 LED colors were obtained.After 48 h of exposure to white and blue LEDs, the cultures were sampled for the same biochemical determinations, corresponding, therefore, to 96 h of total experimental time and exposure to copper at the 6 concentrations of the previously determined biochemical manipulation (Table 2). -Determination of free copper ions (Cu2+): [000116] The determination of free copper 24 h after its addition to the WC medium was performed according to the procedure described in Lombardi et al. (2007). This methodology is based on a copper ion selective electrode (ISE-Cu) with calibration performed using metallic buffers. A copper selective electrode (ISE-Cu) is used as the working electrode and a double-junction reference electrode under a controlled temperature of 25 ± 1 °C. The buffer Petition 870250016081, dated 27 / 02 / 2025, pp. 47 / 69 43 / 46 metallic (copper) as described in Lombardi et al. (2007), containing sodium borate, sodium nitrate, copper standard and nitrilotriacetic acid. In general, metallic buffers are able to extend the detection limits of the ISE-Cu system to 10-13- 10-10 mol L-1. The calibration curve for the ISE-Cu system is always performed at pH 5 and was made from the serial dilution of a commercial single-element copper AAS / ICP standard (1000 mg L-1). [000117] The free copper concentrations determined for the copper concentration scan test, which was performed on microplates, are shown in Table 3. From this assay, the 6 copper concentrations as shown in Table 2 were defined. Table 3 - Concentrations of added copper and free Cu ions (mol.L-1) determined by ISE-Cu and growth rates for Kirchneriella contorta obtained after exposure to the metal. Treatment Added Copper Free Copper Growth Rate 1 4.0x10-8 4.9x10-13 0.606 2 6.0x10-8 5.2x10-13 0.559 3 9.0x10-8 0.514 4 1.2x10-7 1.4x10-13 0.493 5 1.5x10-7 0.542 6 3.0x10-7 4.9x10-13 0.518 7 6.0x10-7 0.598 8 9.0x10-7 1.5x10-12 0.519 9 1.2x10-6 0.543 Petition 870250016081, dated 27 / 02 / 2025, pp. 48 / 69 44 / 46 10 1.5x10-6 6.1x10-12 0.583 11 3, 0x10-6 0.470 12 4.5x10-6 4, 1x10-11 0.542 13 6, 0x10-6 0.512 14 7.5x10-6 4.7x10-8 0.511 15 9, 0x10-6 0.411 16 1, 0x10-5 5, 1x10-5 0.193 17 1.2x10-5 0.282 18 1.3x10-5 1.4x10-4 0.294 19 1.5x10-5 - 20 3.0x10-5 6.8x10-4 - 21 4.5x10-5 - 22 6.0x10-5 - 23 9.0x10-5 - 24 1.0x10-4 2.2x10-2 - 25 1.2x10-4 - 26 1.5x10-4 4.0x10-2 - 27 3.0x10-4 - 28 4.5x10-4 1.8x10-1 - 29 6.0x10-4 - 30 9.0x10-4 4.9x10-1 - Growth parameters: [000118] Daily, 4 mL of sample were collected for culture monitoring and determination of the growth pattern. For this, cells were counted using a flow cytometer and an optical microscope in order to visually monitor the culture and check for any changes in cell volume, as well as any contamination. The growth rate (d-1) was determined by means of a Petition 870250016081, dated 27 / 02 / 2025, pp. 49 / 69 45 / 46 linear regression, using the natural logarithm of cell density (cells mL-1) plotted as a function of experimental time (days), where the slope of the line formed represents the specific growth rate in the exponential phase of the culture. The results of the growth parameters are shown in figures 2, 3 and 4. -Biochemical determinations: [000119] At the end of 48 h of exposure to blue LED and 96 h of culture exposed to copper, total proteins (Figure 5a), total intracellular carbohydrates (Figure 5b), and carotenoids (Figure 5c) were determined. For carbohydrates, 30 mL of culture were centrifuged in a refrigerated centrifuge for 20 min at 4400 rpm. The pellets formed were stored in a -22 °C freezer for later analysis. The determination of total carbohydrates followed the methodology described in Albalasmeh et al (2013). The quantification of total proteins was performed using the Slocombe method (2013). Carotenoid concentrations were determined according to Wellburn (1994). Statistical analysis: [000120] Statistical analyses of the growth rate results showed no negative effects on the exponential growth rate for the concentrations selected for biochemical manipulation (step b) within the copper concentration range (30 concentrations). [000121] Minitab software (version 17 for Windows) was used to compare the concentrations of carbohydrates, proteins, and carotenoids (discrete variables) using ANOVA (Two-Way) and Tukey's test. The representations Petition 870250016081, dated 27 / 02 / 2025, pp. 50 / 69 46 / 46 graphs were created using the IgorPro 6.0.5 program (WaveMetrics, USA). [000122] The present invention has been disclosed in this descriptive report in terms of its preferred embodiment. However, other modifications and variations are possible from the present description, and are still within the scope of the invention disclosed herein. Example 7 - Synergy experiment with copper and blue, red, and white LED light colors with Chlorolobion sp. to evaluate the yield of biomolecules in 96 h of cultivation. Chlorolobion sp. cultures were exposed to copper according to Table 2 for 96 hours under white, blue, and red LED light. All cultivation and analysis conditions were the same as those used for Kirchneriella contorta (examples 1 to 6). Figures 10 and 11 show the results obtained with Chlorolobion sp. Figure 10 shows the results for dry biomass and population growth rate over 96 hours for all cultures exposed to copper and blue, red, and white LED colors. Figure 11 shows the percentage of biomolecules in the biomass determined in 96-hour cultures. Petition 870250016081, dated 27 / 02 / 2025, pp. 51 / 69

Claims

1 / 4 CLAIMS 1. Process for stimulating the yield of microalgae biomolecules during exponential population growth in order to generate modified biomass, rich in biomolecules, comprising the steps of: a) initiating microalgae culture in a photobioreactor under white LED light, inducing hormesis effect in the population by adding a micronutrient metal at a concentration between 10-9 and 10-8 mol L-1, preferably ranging from 3.0x10-9 to 1x10-8 mol L-1 for a period of 48h to 96h; b) exposing the culture to blue LED light for at least 48h CHARACTERIZED by the exposure of the culture to blue LED light being applied when synergy is sought or to white LED light in the absence of synergy.

2. Process, according to claim 1, CHARACTERIZED by comprising in step (a): ai) initiating the cultivation of microalgae in a photobioreactor with an initial cell density ranging from 10⁴ to 10⁶ cells per mL in the exponential growth phase in a culture medium with a pH between 6.8 and 8.0 in a laminar flow hood; a.ii) exposing the culture from step (ai), still inside the photobioreactor, to a white LED with a luminous intensity ranging from 100 to 250 μmol photons m⁻² s⁻¹ and adding between 3.0x10⁻⁹ and 6x10⁻⁹ mol L⁻¹ of free metal, manipulating with a light / dark photoperiod or with continuous white light, at pH 6.8 to 8.0 and a temperature of 24 to 27 °C for 24 to 48 hours. Petition 870250016081, dated 27 / 02 / 2025, page 52 / 69 2 / 4 3. Process according to claim 1, CHARACTERIZED by comprising in step (b): bi) changing the white LED to a blue LED with a luminous intensity ranging between 100 and 250 μmol photons m-2 s-1 with a controlled photoperiod, with a pH ranging between 6.8 and 8.0 and a temperature between 24 and 27 °C for at least 48 hours.

4. Process, according to any one of claims 1 to 3, CHARACTERIZED in that the microalgae are cultivated in a photoautotrophic nutritional mode and are from freshwater, marine or brackish environments.

5. Process, according to any one of claims 1 to 4, CHARACTERIZED in that the microalgae are of the Chlorophyta division, photoautotrophic, preferably cultivated in the photoautotrophic nutritional mode, and are from freshwater, marine or brackish environments.

6. Process, according to claim 5, CHARACTERIZED by the fact that the Chlorophyta microalgae are Chlorolobion sp., Scenedesmus quadricauda, ​​Scenedesmus bijugus, Desmodesmus communis, Desmodesmus abundans, Desmodesmus brasiliensis, Desmodesmus denticulatus, Monoraphidium contortum, Monoraphidium mirabile, Monoraphidium graffithii, Monoraphidium indicum, Selenastrum capricornutum, Selenastrum bibraianum, Ankistrodesmus densus, Ankistrodesmus bernardii, Ankistrodesmus flexuosus, Ankistrodesmus fusiformis, Ankistrodesmus stipitatus, Ankistrodesmus fusiformis, Kirchneriella aperta, Kirchneriella irregulares, Petition 870250016081, of 02 / 27 / 2025, p. 53 / 69 3 / 4 Kirchneriella obesa, Kirchneriella contorta, Kirchneriella pseudoaperta, Hariotina reticulata, Chlorella vulgaris, Chlorella emersonii, Botryococcus terribilis, Chlorollobium sp., Chlorolobion guanense, Chlorolobion lunulatum, Mougeotia sp.More preferably, the microalga is Kirchneriella contorta for a synergistic process and Chlorolobion sp. for a non-synergistic process.

7. Process, according to any one of claims 1, 2, 4, 5 or 6, CHARACTERIZED in that in step (a), the culture medium used is a nutrient solution consisting of inorganic salts and supporting algal growth, selected from the group containing WC medium (Guillard & Lorenzen, 1972), BBM medium, BG11 medium or Chu#10 medium; preferably, the culture medium is WC medium with pH=7.0 and more preferably, the culture medium is WC medium with pH=7.0 if the microalga is Kirchneriella contorta and wherein the photobioreactor used is a colorless and transparent algal culture flask, which may be made of glass or plastic.

8. Process, according to any one of claims 1, 2, 4, 5, 6 or 7, CHARACTERIZED in that in step (a), the laminar flow equipment used may be of the vertical flow type, of the horizontal flow type or may even be replaced by manipulation with a Bunsen burner flame on a common bench, in the absence of a laminar flow cabinet; preferably the laminar flow equipment used is of the vertical flow type.

9. Process, according to any one of claims 1, 3, 4, 5 or 6, CHARACTERIZED by the fact that Petition 870250016081, dated 27 / 02 / 2025, page 54 / 69 4 / 4 that in step (b), the cultures are maintained in a grow room with a light / dark photoperiod or with continuous white light; preferably, the light / dark photoperiod is 10 / 14h, or 12 / 12h or 14 / 10h.

10. Process, according to any one of claims 1, 3, 4, 5, 6 or 9, CHARACTERIZED in that in step (b), if the microalga is Kirchneriella contorta, it is exposed to a white LED with a luminous intensity of 200 μmol photons m-2 s-1.

11. Process according to any one of claims 1, 3, 4, 5, 6, 9 or 10, CHARACTERIZED in that in step (b), the free metal is selected from the group comprising copper, zinc, molybdenum or cobalt or other micronutrient.

12. Process according to any one of claims 1, 3, 4, 5, 6, 9, 10 or 11, CHARACTERIZED in that in step (b), 3.8x10-9 mol L-1 of free copper are added to Kirchneriella contorta.

13. Process, according to any one of claims 1, 3, 4, 5, 6, 9, 10, 11 or 12, CHARACTERIZED in that in step (b), the cultures were maintained in a grow room with a light / dark photoperiod or with continuous blue light; preferably, the light / dark photoperiod is 10 / 14h, or 12 / 12h or 14 / 10h. Petition 870250016081, dated 27 / 02 / 2025, pp. 55 / 69

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