Bioprocess for bioremediation and biorefinery of effluents using microalgae and low-cost illuminated bioreactors with full utilization of biomass, its uses and applications.

Description

1 / 15 DESCRIPTIVE REPORT Bioprocess for bioremediation and biorefinery of wastewater using microalgae and low-cost illuminated bioreactors with full utilization of biomass, its uses and applications. FIELD OF THE INVENTION

[001] The present invention belongs to the technical field of bioprocesses for wastewater treatment and biomass production and the full utilization of its components. More specifically, the invention relates to a bioremediation bioprocess for wastewater using low-cost green microalgae in illuminated or non-illuminated bioreactors, with subsequent full utilization of the generated biomass and its applications. The process involves the bioprospecting method for local strains of green microalgae, the cultivation of different microalgae strains under synthetic conditions, the natural selection method for the strain best adapted to the effluent, the cultivation of the microalgae in the effluent in annular photobioreactors with or without synthetic lighting (1), and subsequent stages of decantation / flocculation (2), centrifugation and drying of the biomass for the formulation of sustainable bioproducts. FUNDAMENTALS OF THE INVENTION Theoretical framework

[002] With the increase in population and agricultural production, one of the environmental problems that has been aggravated is the contamination of water bodies by effluents. This contamination occurs due to the input of nutrients from untreated or poorly treated effluents, and thus it is a problem known for a long time because it favors the eutrophication of lakes, estuaries, and coastal waters. The entry of these nutrients into Petition 870240111507, dated 12 / 31 / 2024, page 10 / 29 2 / 15 bodies of water negatively impact biodiversity, promoting the proliferation of epiphytes, harmful algae, macroalgae and epiphytes, and phytoplankton (Weiskel; Howes, 1992; Usepa, 2002; Anderson et al., 2002; Delvin; Brodie, 2023).

[003] The treatment of domestic and agro-industrial effluents is based, in most cases, on physical, chemical and biological processes. These processes aim to remove solids, nutrients and organic matter, through five sequential stages: (1) preliminary treatment or pre-treatment with physical and mechanical processes; (2) primary treatment with physical-chemical and chemical methods; (3) secondary treatment with chemical and biological processes; (4) tertiary or final treatment with physical and chemical processes; and (5) treatment of the sludge formed, which may include controlled disposal, recycling or incineration (Metcalf & Eddy, 2014).

[004] Microalgae comprise photosynthetic eukaryotic and prokaryotic microorganisms with wide taxonomic, metabolic, and biochemical diversity. Microalgae-based wastewater treatment systems demonstrate effectiveness in removing inorganic compounds such as nitrate, phosphate, heavy metals, inorganic carbon, emerging contaminants, BOD, COD, and other impurities. In industrial, agricultural, and domestic wastewater, nitrogen and phosphorus are the main causes of eutrophication, and are rapidly absorbed by microalgae. Phosphorus is translocated across the plasma membrane and incorporated into organic compounds such as ADP during phosphorylation, a process that uses energy obtained from the oxidation of respiratory substrates and photosynthesis. Nitrogen, present as ammonia, nitrite, and nitrate, is converted into organic forms by microalgae.Nitrate reductase reduces nitrate to nitrite, followed by nitrite reductase which, with the NADPH produced in photosynthesis, reduces nitrite to ammonia. The ammonia is then incorporated into amino acids such as... Petition 870240111507, dated 12 / 31 / 2024, page 11 / 29 3 / 15 glutamine, using ATP released from phosphorylation (Chalivendra, 2014; Emparan et al., 2019; Sánchez-Zurano et al., 2021)

[005] Among the units of conventional wastewater treatment systems, only facultative and oxidation lagoons use microalgae as active biological components through photosynthesis, being efficient in removing nutrients, micropollutants and producing dissolved oxygen. However, factors such as high effluent turbidity and reduced microalgal biomass growth reduce the effectiveness of pollutant removal in these processes (Kim et al., 2000; Wollmann et al., 2019; Chai et al., 2021).

[006] In mixotrophic systems, where microalgae obtain energy from both photosynthesis and available organic carbon in the environment, these organisms are used for the removal of nitrogen, phosphate, and metals with high efficiency; however, a high implementation cost and low yield of algal biomass production are observed. Some ways to overcome this barrier are: increasing the efficiency of this treatment with innovative low-cost bioreactors, using effluents without pretreatment, and using the generated biomass in industrial processes in its entirety. Microalgal biomass presents a promising renewable raw material in the development of sustainable products. Considering that these organisms have an immense metabolic diversity and thus possess portions of lipids, proteins, carbohydrates, and pigments of biotechnological interest and potential industrial application (Navarro-López et al., 2020) Background of the invention

[007] The state of the art related to the bioremediation of effluents using microalgae has already demonstrated the effectiveness of these microorganisms in removing nitrogen, phosphate, and metals. Several studies and patented processes describe the use of Petition 870240111507, dated 12 / 31 / 2024, page 12 / 29 4 / 15 different strains of microalgae in wastewater treatment systems, demonstrating their ability to absorb nitrogen and phosphate compounds, as well as heavy metals and other toxic substances. However, many of these processes require complex and costly infrastructure, involving sophisticated bioreactors and advanced monitoring technologies. Furthermore, the biomass generated in these processes is, in most cases, used for a single purpose and generates new waste, such as in biodiesel production.

[008] Among the known inventions, the use of closed photobioreactors stands out, which, although efficient in maximizing microalgae growth, have high construction and operating costs, limiting their large-scale applicability. In these closed systems, the light source is located outside the photobioreactor, and as algal multiplication progresses (increase in cell density within the photobioreactor), resistance to light penetration occurs. Thus, within days, in these systems, light penetration is low and, consequently, microalgae multiplication is reduced. Another example is open lagoon systems, which, despite being more economical, suffer from low biomass production and variability in environmental conditions, compromising the efficiency of the bioremediation process.

[009] Most existing processes also do not fully consider the complete utilization of microalgae biomass. Many methods focus only on the remediation of effluents, neglecting the potential for valorization of the by-products generated, such as the extraction of bioactive compounds, production of biostimulants, pigments and other value-added products.

[010] The technical problem not solved by the state of the art is, therefore, the need for a bioremediation process that Petition 870240111507, dated 12 / 31 / 2024, page 13 / 29 5 / 15 be simultaneously efficient, low cost, accessible for different scales of application, and generate biomass that allows its full utilization. Considering that traditional photobioreactors use external light sources, unlike the present invention where the light source is located in the center of the photobioreactor (1), allowing more light distribution. Additionally, there is a need for ecological solutions for more efficient nutrient removal in effluents.

[011] The present invention solves these problems by introducing a bioprocess that uses low-cost center-illuminated bioreactors for the cultivation of microalgae in different scaled volumes. This process not only ensures the efficient removal of nutrients and contaminants from effluents, but also integrates sedimentation, centrifugation, washing and drying steps, facilitating the full utilization of the biomass produced. The innovation lies in the combination of an economically viable and efficient bioreactor system, making the process more accessible and applicable in different contexts, from small producers to large-scale industries.

[012] The advantages of the present invention include lower implementation and operating costs, thanks to the use of a low-cost bioreactor system and accessible monitoring methods. Furthermore, the integrated approach to biomass utilization allows for the production of biostimulants and bioactive compounds, adding value to the process. The efficiency of the bioprocess in removing contaminants from effluents and producing biomass rich in proteins, lipids, and carbohydrates also represents a significant environmental benefit, aligning with the principles of the circular economy and contributing to the sustainability of industrial operations. Petition 870240111507, dated 12 / 31 / 2024, page 14 / 29 6 / 15

[013] Several patent applications have been filed with the aim of enabling the bioremediation processes described above. Among the documents available in the current art, the following can be highlighted:

[014] International patent PI 0923821-2 A2 describes a device that treats wastewater and / or generates biochemical energy through photosynthesis on building facades. The device includes: a container for cultivating algae or microorganisms in water; a system for feeding and injecting building wastewater into the culture; a mechanism for regulating temperature; a means for recovering and reusing the wastewater; and possibly adequate lighting for cultivation. This system allows the production of biofuel, organic molecules, chemical compounds and proteins, with the biofuel being usable oilseed biomass in thermoelectric power plants or transformed by pyrolysis.

[015] US Patent 2011247977 describes a device and method for treating nitrogen and phosphorus in wastewater using a microalgae culture tank and a separation membrane. The microalgae, cultivated in high concentrations directly in the wastewater, allow for the efficient removal of these nutrients while generating useful biomass in a stable manner.

[016] Brazilian patent BR 132014025044-0 E2 describes a process for the production of bioenergy and biofertilizers using agro-industrial effluents, especially residues from the sugar and ethanol sector. The process involves the anaerobic digestion of vinasse to produce biogas in an anaerobic reactor, which is then used in a thermoelectric power plant. After digestion, the vinasse serves as a substrate for the cultivation of algae in dedicated reactors. These algae fix carbon dioxide (CO2), atmospheric nitrogen, and potassium dissolved in the vinasse through the Petition 870240111507, dated 12 / 31 / 2024, page 15 / 29 7 / 15 photosynthesis, resulting in biomass rich in nitrogen and potassium, which can be used as biofertilizer. The resulting liquid phase can be reused or discarded, reducing fertigation and fertilizer acquisition costs. During the off-season, the reactors are used to cultivate algae species with a higher capacity for CO2 fixation, whose biomass is subsequently digested in the anaerobic reactor to produce biogas and bioenergy.

[017] Brazilian patent BR 102021024586-7 A2 describes a hybrid modular photobioreactor (land-water) and / or cartridges for the cultivation of free or immobilized microalgae in biopolymers, applicable to wastewater treatment and the generation of bioproducts. The invention aims at the production of biomass for bioenergy and the reduction of environmental impact. The proposed platform allows the simultaneous treatment of wastewater using microalgae, either in free form or immobilized in biopolymeric matrices.

[018] Brazilian patent BR 102013020471-4 B1 describes a bioprocess for the simultaneous conversion of hybrid effluents, including organic matter (COD), total nitrogen (TKN), and total phosphorus (PO4-3), in a single heterotrophic bioreactor. This process occurs substantially without the presence of light, using wastewater from poultry, swine, or domestic sewage processing, and results in the production of microalgal biorefinery bioproducts. Furthermore, the invention encompasses the heterotrophic bioreactor necessary for carrying out this bioprocess and the application of the generated bioproducts as industrial inputs.

[019] In view of the above, the present technology aims to fill significant gaps in current approaches to wastewater treatment, particularly with regard to the integration of processes for the full utilization of Petition 870240111507, dated 12 / 31 / 2024, page 16 / 29 8 / 15 biomass. Existing patents address the use of microalgae for the removal of nitrogen, phosphorus, and carbon, but face limitations in terms of obtaining native and efficient strains for pollutant removal and in integrating biomass utilization processes. For example, the need for multiple reactors, strain immobilization, and the reliance on chemical methods in some cases result in costly and less environmentally attractive processes. Furthermore, the reuse of biomass generated during treatment is a neglected issue, leading to new environmental liabilities. This invention aims to overcome these limitations by integrating the treatment of multiple pollutants into a single efficient system, using microalgae isolated at the effluent generation site itself, and facilitating the sustainable reuse of the resulting biomass, minimizing both operational costs and environmental impact. OBJECTIVES OF THE INVENTION

[020] The present invention has as its main objective to present a sustainable, low-cost and efficient bioprocess for the treatment of various effluents using microalgae or cyanobacteria in illuminated photobioreactors concomitant with the processing and full utilization of the algal biomass generated. BRIEF DESCRIPTION OF THE DRAWINGS

[021] FIGURE 1 illustrates the side view of the annular photobioreactor coupled by a hose system to the simple decanter. 1.1) Cylindrical photobioreactor body made of transparent polymethylmethacrylate or similar material. 1.2) Air inlet. 1.3) Central cylindrical polymethylmethacrylate tube. 1.4) LED or fluorescent tubular lamp. 1.5) Microalgae culture outlet. 1.6) Polymethylmethacrylate lid. 1.7) Base of Petition 870240111507, dated 12 / 31 / 2024, page 17 / 29 9 / 15 polymethyl methacrylate. 2) Air compressor or magnetic pump. 3) Decanter. 4) Peripheral pump. DETAILED DESCRIPTION OF THE INVENTION

[022] The present invention relates to an innovative bioremediation process for wastewater using microalgae, preferably of the class Chlorophyceae and / or cyanobacteria, and low-cost center-lit bioreactors, with full utilization of the biomass generated. This process aims to provide an efficient, economical and sustainable solution for wastewater treatment, while adding value to microalgae biomass in value-added products. The process steps are described in detail below. Obtaining and isolating microalgae strains

[023] The microalgae strains, preferably from the class Chlorophyceae or cyanobacteria, used in the process are obtained from environmental samples collected at the effluent source. These samples are initially enriched in Basal Bold Media (BBM) culture medium for a period of 15 days. After this period, 1 mL of the enriched sample is transferred to a sterile BBM medium, where it remains for another 15 days. Subsequently, a serial dilution is performed in a 16-well plate using sterile culture medium to isolate the different microalgae strains present in the sample. Generally, the microalgae observed in these steps comprise green species such as Chlorella spp., Scenedesmus spp., Chlamydomonas spp., Monoraphidium spp., Pectinodesmus spp..

[024] The isolated strains are selected from previous growth in effluent, from an inoculum of 10 to 50 percent of the total effluent volume v / v, at ambient temperature, with or without artificial lighting. Concomitantly, the strains Petition 870240111507, dated 12 / 31 / 2024, page 18 / 29 10 / 15 capable of multiplying in the effluent will be inoculated together in a new effluent to select the dominant strain, in this case the one best adapted to the effluent. At the end of a period of time, the strain that becomes dominant will be inoculated in BBM medium and stored. Staged cultivation of microalgae or cyanobacteria in effluent.

[025] The cultivation of microalgae or cyanobacteria is carried out in a phased manner, beginning with the inoculation of previously isolated strains in 2L volumes of BBM synthetic medium. After the initial adaptation phase, the microalgae are transferred to progressively larger volumes with effluent at ambient temperature (5°C to 35°C), using low-cost containers, such as: first to 20L gallons, then to 80L, in annular photobioreactors (1). These volumes can also be cultivated in water gallons, water tanks, etc., respectively, and may vary according to the available structure, provided that the central lighting compartment is maintained.

[026] The annular cylindrical polymethylmethacrylate photobioreactors (1) are fixed to a support base (1.7) of various materials and have six different parts: cylindrical polymethylmethacrylate tube (1.1); photobioreactor contents outlet (1.2), aeration inlet (1.2), cylindrical lighting tube (1.3), cylindrical lamp (1.4), contents outlet valve (1.5) and lid (1.6). Magnetic compressed air pumps (4) are used for system aeration and peripheral pumps (5) for effluent movement. Artificial lighting of the photobioreactors is provided by low-cost light sources, such as LED or fluorescent lamps, strategically positioned to maximize microalgae growth efficiency and reduce costs. The dimensions Petition 870240111507, dated 12 / 31 / 2024, page 19 / 29 11 / 15 internal and external dimensions of the annular photobioreactor can vary, provided the central illumination tube is maintained. Real-time monitoring

[027] The process monitoring is carried out in real time using low-cost sensors, which measure critical parameters such as pH, temperature, total dissolved solids, conductivity, irradiance and salinity (1.8). These sensors provide data that allow quick adjustments to the culture environment, ensuring optimal conditions for microalgae growth and the efficiency of the bioremediation process. Biomass processing

[028] When microalgae or cyanobacteria reach the stationary growth phase, half, or another fraction, of the volume is removed from the larger reservoir / photobioreactors and replaced with new effluent. The removed volume is subjected to a sedimentation process to separate the biomass from the water (3). The sedimented biomass is then transferred by pumps to a centrifugation / filtration system, where it is concentrated and dehydrated. Full utilization of biomass

[029] The resulting dry biomass is processed by boiling in water for 2 hours or another form of extraction using a liquid extraction matrix, which allows the extraction of secondary metabolites. After extraction, the remaining biomass, rich in cellulose, is isolated and can be used in various applications, such as in the production of biomaterials, fertilizers and other value-added products. Applications and benefits

[030] The bioprocess described is applicable to the treatment of industrial, textile, agro-industrial, mining and effluents. Petition 870240111507, dated 12 / 31 / 2024, page 20 / 29 12 / 15 urban wastewater treatment plants provide a sustainable and efficient solution for the removal of nutrients and contaminants. The treated water can be reused, reducing the demand for fresh water and minimizing environmental impact.

[031] In addition to wastewater treatment, the microalgae biomass produced can be used directly or after processing as an agricultural biostimulant or biofertilizer, pigments, animal feed, dyes, enzymes, oils, fibers, nanofibers, nanoparticles and other derivatives of its components. The complete use of biomass promotes the circular economy, adding value to the process and contributing to environmental sustainability. Technical and economic advantages

[032] The main advantages of this bioprocess include the low implementation and operating costs, resulting from the use of accessible bioreactors and sensors. The efficiency in removing contaminants from effluents is enhanced by the high absorption capacity of microalgae and cyanobacteria. The complete utilization of biomass allows the production of various value-added products, making the process economically viable and environmentally beneficial. Example 1: Production of agricultural biostimulants and cellulose from microalgae biomass derived from domestic wastewater treatment.

[033] From the effluent collection, microalgae isolation tests are initiated by inoculating 1 mL of effluent into BBM culture medium. After preliminary growth is observed, a serial dilution is performed to isolate clonal microalgae cultures. Tubes showing growth will be scaled up to 100 mL and then the newly inoculated microalgae will be placed in the culture medium. Petition 870240111507, dated 12 / 31 / 2024, page 21 / 29 13 / 15 isolates are inoculated into the effluent to evaluate their growth. Once the growth of a specific strain in the effluent is verified, the bioremediation bioprocess is initiated using annular photobioreactors (1) containing domestic effluent and a proportion of 10% to 50% v / v of microalgae in the exponential growth phase.

[034] The photobioreactor (1) is kept in operation during the exponential multiplication phase of the microalgae, which comprises five to 20 days of operation. After this period, a fraction of the treated effluent is removed and a new fraction is added daily. The removed fraction is decanted, centrifuged / filtered and dried in an oven / freeze-dryer, thus obtaining a powdered biomass.

[035] From the biomass obtained, an aqueous fraction is made which is used for the production of agricultural biostimulant and the residue is used for the production of cellulose. The dry biomass is boiled in water or saline solution (NaCl, TRIS, for example) for 2 hours to extract bioactive compounds such as phytohormones that act by stimulating the plant's defenses and growth. The resulting aqueous fraction is filtered and used as a biostimulant, the residue is used for the production of cellulose.

[036] Residual biomass is rich in cellulose, which can be used for the production of biofuels, fertilizers, or industrial ingredients. It has applications in the formulation of fibers, nanofibers, filters, microfilters, nanofilters, various polymers, and coatings. REFERENCES ANDERSON, DM; GLIBERT, PM; BURKHOLDER, JM Harmful algal blooms and eutrophy: nutrient sources, composition and consequences. Estuaries, v. 25, p. 704-726, 2002. Petition 870240111507, dated 12 / 31 / 2024, p. 22 / 29 14 / 15 CHAI, WS; TAN, WG, MUNAWAROH; HSH, GUPTA, VK; HO, SH; SHOW, PL Multifaceted roles of microalgae in wastewater biotreatment applications: a review. Environmental Pollution, v. 269, p. 116236, 2021. CHALIVENDRA, S. Bioremediation of wastewater using microalgae. University of Dayton, 2014. DEVLIN, Michelle; BRODIE, Jon. Nutrients and eutrophication. In: Marine pollution-monitoring, management and mitigation. Cham: Springer Nature Switzerland, 2023. p. 75-100. EMPARAN, Q. et al. Role of phycoremediation for nutrient removal from wastewaters: a review. Appl. Ecol. Environ. Res, v. 17, n. 1, p. 889-915, 2019. KIM, M.H. et al. Kinetics of removing nitrogenous and phosphorus compounds from swine waste by growth of microalga, Spirulina platensis. J. Microbiol. Biotechnol, v. 10(4): p. 455-461, 2000. METCALF & EDDY. Wastewater engineering: treatment and resource recovery. McGraw Hill Education, 5° edição, 2014. NAVARRO-LÓPEZ, E. et al. Biostimulant potential of Scenedesmus obliquus grown in brewery wastewater. Molecules, v. 25, n. 3, p. 664, 2020. SÁNCHEZ-ZURANO, A. et al. Wastewater treatment using Scenedesmus almeriensis: effect of operational conditions on the composition of the microalgae-bacteria consortia. Journal of Applied Phycology, v. 33, p. 3885-3897, 2021. USEPA. Report to congress: impacts and control of CSOs and SSOs. US EPA Office of Water, Washington, DC, USA, 2004. WEISKEL, P.K.; HOWES, B. L. Differential transport of sewagederived nitrogen and phosphorus through a coastal watershed. Petição 870240111507, de 31 / 12 / 2024, pág. 23 / 29 15 / 15 Environmental science & technology, v. 26, n. 2, p. 352-360, 1992. WOLLMANN, F. et al. Microalgae wastewater treatment: Biological and technological approaches. Engineering in Life Sciences, v. 19, n. 12, p. 860-871, 2019. Petição 870240111507, de 31 / 12 / 2024, pág. 24 / 29

Claims

1 / 3 CLAIMS 1. Wastewater bioremediation process, characterized by including the following steps: (a) Reception of wastewater containing nitrogen concentrations between 10 mg / L and 1000 mg / L, phosphorus between 5 mg / L and 500 mg / L, heavy metals at a maximum concentration of 100 mg / L and organic matter with COD between 100 mg / L and 30,000 mg / L, raw or pre-treated; (b) Cultivation of microalgae and / or cyanobacteria using the wastewater in annular cylindrical polymethylmethacrylate photobioreactors, equipped with continuous aeration by compressed air or magnetic pump, natural and / or artificial lighting, operating in continuous, semi-continuous or batch modes; (c) Maintenance of controlled environmental conditions, with temperature between 5°C and 35°C and pH between 2 and 10; (d) Removal of biomass in the stationary phase, followed by decantation or flocculation, centrifugation or filtration and drying at temperatures between 30°C and 100°C or freeze-drying.

2. The bioprocess according to Claim 1, characterized by the selection of locally isolated microalgae and / or cyanobacteria from the effluent-generating environment or nearby areas, with initial cultivation in synthetic medium, followed by progressive transfer to effluents in volumes of 100 mL, 2 L and 20 L, aiming at obtaining resistant and dominant strains, under temperatures between 5°C and 35°C, pH from 2 to 10, using natural or artificial light and aeration by compressed air. Petition 870240111507, dated 12 / 31 / 2024, page 25 / 29 2 / 3 3. Annular cylindrical photobioreactor for microalgae cultivation, characterized by: (a) Cylindrical body in polymethyl methacrylate or similar transparent material; (b) Cylindrical central lighting tube with LED and / or fluorescent lamps; (c) Continuous aeration system coupled to magnetic pumps; (d) Control valve for the outlet of treated contents.

4. The process according to Claims 1, 2 and 3, characterized by the use of the algal biomass produced for the extraction of proteins, carbohydrates, pigments, lipids and secondary metabolites, intended for industrial, pharmaceutical, agricultural and food applications.

5. The process according to Claims 1 to 4, characterized by real-time monitoring using integrated sensors to measure pH, temperature, cell density, conductivity, and salinity.

6. The process according to Claims 1 to 5, characterized by the production of biofertilizers and / or biostimulants from microlegal biomass, by boiling in water for 2 hours or liquid extraction with solvent, resulting in the main fraction for agricultural use.

7. The process according to Claim 7, characterized by the use of biomass residue for the production of secondary products.

8. The process according to Claim 1, characterized by a microalgae inoculum / effluent ratio between 5% and 50%, using species from the class Chlorophyceae and / or cyanobacteria. Petition 870240111507, dated 12 / 31 / 2024, pp. 26 / 29 3 / 3 9. Use of the bioproduct generated in accordance with Claims 7 and 8, characterized by its application as an input in feed mills, biofuel plants, fertilizer industries, cosmetics, human food and chemical industries. Petition 870240111507, dated 12 / 31 / 2024, pp. 27 / 29

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