On-site modular system for processing and converting animal waste and methods and uses thereof

Abstract

On-site modular system for processing and transforming animal waste, method and use thereof The present invention relates to an on-site modular system for processing and transforming animal waste. The present invention also relates to a method for transforming animal waste in situ using said on-site modular system. The present invention further relates to the use and the resulting advantages of a processed nutrient-rich bioabsorbable nitrogen-, phosphorus- and potassium-rich liquid microalgal culture fraction obtained with the method of the invention in the management of greenhouse gas emissions from farms and in agricultural or natural soil amendment. Furthermore, the present invention further relates to the use of a processed carbon-rich solid inactivated microalgal culture fraction obtained with the method of the invention as a scavenger of toxic substances or pollutants in animal waste farms and / or for animal feed.

Description

Technical Field

[0001] This invention relates to a system for processing and converting animal waste. In particular, it relates to a modular biotechnology platform for the in-situ recovery and conversion of animal waste, especially manure slurry, by producing microalgae and their derivatives cultured on a sequentially physically processed liquid manure slurry source. This innovation enables the acquisition of high-value-added byproducts, such as nutrients for animal feed and novel agricultural phytoremediation agents and natural soil conditioners. Furthermore, it offers significant advantages compared to existing systems, including 10-95% biofixation of nitrogen, phosphorus, and potassium in the manure slurry, prevention of 50-85% of greenhouse gas emissions from livestock farms, and the ability to remove contaminants from livestock ponds. In short, it provides an efficient and sustainable solution for the management of animal waste generated by intensive livestock farms. Background Technology

[0002] Animal feces as an environmental problem Manure slurry is a type of manure fertilizer, a byproduct composed of a mixture of feces, urine, straw, washing water, farm disinfection chemicals, and degraded plastic residues. Manure slurry is primarily produced on livestock farms. Its nutrient concentration is very low because it is mostly water, and it is mainly composed of water, organic matter, nitrogen, phosphorus, potassium, macronutrients (calcium, sulfur, and magnesium), and micronutrients (boron, iron, manganese, nickel, copper, and zinc). (1,2) Its composition is directly related to the feed consumed by the animals, as the contribution from the animal's own body is practically negligible.

[0003] The steady increase in pig farm wastewater (manure slurry) has become a serious environmental concern. The high levels of nutrients and toxic pollutants in manure slurry have significant impacts on ecosystems and public health. (3) Regulatory agencies have established complex and comprehensive administrative bodies in an attempt to control or maintain these industrial activity-related issues at acceptable levels. (4) As an illustrative example, the environmental impacts associated with pig farming affect both intensive and extensive farms, although the higher animal density in intensive farms means that the generation of manure and compost requires specific management systems for the leachate and waste products (ammonia, nitrates, greenhouse gases, and various biosecurity issues such as bacterial antibiotic resistance). (5) The foundation of the integrated manure management concept leads us to take action at the source (animal feed) and make decisions regarding storage and final disposal. In addition, we must also consider farm design and management, automated and frequent removal of manure, and the development of agricultural use plans. (6,7,8,9) A comprehensive manure management program is an operational scheme, individual or collective, that results in the continuous production of this residue adapting spatially and temporally to the needs of the crop (10). It envisions measures in three different areas: 1. Management: Management measures to reduce feed, flow rate and limit the components used in subsequent applications, such as excessive levels of water, nitrogen, phosphorus or heavy metals.

[0004] The calculation of the storage pond, whose volume must allow for a balance between the continuous production of manure slurry and its discontinuous consumption as a high-quality byproduct, along with thorough strategic planning and analysis of key points and controls, forms the backbone of a manure management program. Changes in feeding and diet modifications not only contribute to animal health and their adequate production performance but must also be used to control nitrogen content in waste.

[0005] 2. Fertilization (utilization): Fertilization plans and the utilization of manure slurry are based on knowledge of both soil, crop requirements, dosage and seasonality, as well as climate and hydrological characteristics.

[0006] Optimizing water consumption in livestock farms, particularly in better and more efficient cleaning, allows for lower manure flow rates, lower storage volume requirements, and reduced transportation costs. Collaboration between livestock breeders and farmers, and collective management of the fertilizer value of manure, must enable optimized transportation and application logistics.

[0007] 3. Treatment (methods): A strategic combination of methods to modify the characteristics of fecal slurry to meet the demand for a high-quality product.

[0008] The suitability of the treatment method will depend on each geographic region, the needs emphasized by the management plan, the expected quality of the final product obtained, and the associated economic costs. (10) Livestock farming accounts for 0.8% of nitrate emissions, 9.1% of greenhouse gas emissions, and is systematically increasing in ammonia emissions (1% annually over the period 1999–2019) (2019 data). (9) According to the European Commission, (a) nitrogen emissions cause at least EUR 70 billion in losses annually in the EU; (b) 81% of agricultural nitrogen inputs into aquatic systems are caused by livestock production; and (c) 87% of ammonia emissions from agriculture into the atmosphere are caused by livestock production. (11) Under our operating conditions, the nominal composition parameters of the main pollutants in manure slurry have been determined. (12,13) - Relevant facts to consider: problems, technologies, and limitations (design quality) To date, various strategies for controlling this animal waste have been proposed; all of them involve moving the waste from point A to point B, a distance X from the producer (farm). (3,4,10,14,15,16,17,18) Driven by the energy demands of processing plants, most of these transports are carried out by road in highly polluting specialized trucks (14,18,19); others are carried out using agricultural tractors on rural roads, and are due to traditional management and organizational issues in livestock-producing areas (1,20); a small fraction of these are due to regulatory issues that have found no solution other than official transport of the waste to avoid concentration, but force the communities processing the waste to bear the full cost of its utilization and reprocessing. (2,4,10) This cost varies depending on the transport distance and volume, and its impact varies significantly depending on the economic value of the transported nutrients. Therefore, the maximum permissible transport distance depends on the economic value of what is being transported and must be assessed on a case-by-case or phased basis. During pregnancy and lactation, the transportation cost of diluted manure slurry can be equivalent to its fertilizer value for a distance of about 5-7 km, while for manure slurry during the fattening period, this distance can exceed 15 km. (13) These distances will increase significantly if centralized or collective transportation services are used, as it allows for optimization of logistics, equipment usage time and depreciation, and always meets necessary sanitary restrictions. (10) As an illustrative example, Spain has 26 manure treatment plants owned by Ignis (6), Iberdrola (4), Naturgy (2), Cs Energy (2), and other companies (including some livestock cooperatives). These facilities are dedicated to drying manure and converting it into fertilizer to mitigate its environmental impact on the soil and aquifer. In areas with a high concentration of livestock farms, they can process approximately 2.5 MM / m³ per year. 3 The waste generated—in some areas where the water is undrinkable due to excessive nitrates—avoids emissions of 700,000 TCO2 equivalents. All the manure slurry processed at these plants is transported by tanker trucks with capacities of 12,000–25,000 L, making the move of waste to the treatment facilities a major operating (€) and environmental (fuel consumption) cost for these systems, significantly reducing their effectiveness. Despite these figures, there is still a need to optimize methods to make them feasible, safe, and effective in processing waste, recycling byproducts, and improving the welfare of pig and human populations.

[0009] Cost savings are a pillar of stability in intensive animal farming, which minimizes external fluctuations (epidemics, national, regional or community interventions, investment funds, raw material price fluctuations, etc.). (21) To date, the economic value of fattening pig manure slurry has been quantified as €5 to €7.3 / m³. 3This is equivalent to the price of NPK mineral fertilizer. (6,10,11) The cost of transporting pig manure slurry varies. A 2014 study set the cost at approximately €5.40 per tonne, plus €1.98 per kilometer for transportation. The cost for transporting liquid manure was approximately €0.018 per liter, plus €0.0031 per liter per kilometer. A 2016 survey showed that the average price of manure application per kilometer was €0.011 per liter, with an additional cost of approximately €0.0031 per liter per kilometer for transportation. A 2017 report estimated the annual cost of manure application for a 4,800-head pig farm to be approximately €33,750. (22,23,24,25) At the experimental farm where the proof-of-concept was installed (Granja deExperimentación Tecnológica SL-B02859924-, ES50164.1, Central Region of Zaragoza, Spain) (PoC farm), the reference estimate for the management and transportation costs of the same manure slurry averaged €2.92 / m³. 3 For a 500-sow integrated pig farm, the annual cost associated with transporting and spreading raw pig manure by tractor and vacuum tanker and by truck ranges from €3.4 million. -3 1 km to €11.8 m -3 Displacement of 100 km. (26) Under reduced tillage conditions, the use of mineral fertilizers was replaced by pig manure slurry, completely replacing barley and partially replacing corn, which was also economically viable in rainfed regions with continental Mediterranean climates. (2) In Spain, 58,703,600 pigs were slaughtered in 2021, producing 5,220,773 tons of meat, ranking third in the world after China and the United States. Pig farming is Aragon's most important agricultural sub-sector. It accounts for 66% of final livestock production and 39% of final agricultural production, values ​​significantly higher than the national averages (42.5% and 15.9%, respectively). In light of this, it must be added that a large portion of Aragon's grain production is used for pig feed. (27) Mediterranean climates, because they lack constant rainfall, vary greatly as they move away from the coast. Due to the low humidity of the air, they acquire elements of continental climates, exhibiting significant daily and annual temperature ranges (continental Mediterranean subtype). (28) Their range can vary from mild winters and very hot summers to cold winters and mild summers, in the latter case where frost and precipitation occur in the form of snow. This is the climate of the Mediana de Aragón region, the study area where the proposed solutions are tested. Under these climatic conditions, cultivating microalgae requires constant and guaranteed energy to maintain the essential system's operators and support structures, as well as the processing of byproducts. (28) -Cultivating biotechnology systems: scaling up standards Algal recycling has been extensively studied in production demonstrations of manure recovery from pig farms (3,14,16,20) and mixed pig leachate (15,16), but significant gaps and research trends remain that require evaluation under medium to large-scale and real-world research conditions to complete practical knowledge and enable us to develop their large-scale applicability (15,17,29) and local circular economy in relation to animal health. (30) Defining culture parameters under improved, partially improved environmental conditions is one of the research objectives we have been working on throughout the invention process.

[0010] Scale-up criteria depend on experience with a specific fermentation process. There is no single, rigorous, and safe standard for scaling up aerobic cultures. For this reason, validation designs for scale-up processes validate the applicability of data obtained during the R&D phase, generating more or less fixed experimental protocols that study up to two variables simultaneously. (31,32,33) When dealing with microalgae that are highly sensitive to several factors, more than one scale-up criterion is used. Factors such as pH, temperature, or culture time are typically set according to the technology transferred from R&D and are usually established as initial assumptions that vary in the same way across both pilot and industrial scales. (34,35,36) The principle of similarity relates to the concept of similarity in geometric, kinematic, and kinetic parameters between small-scale and large-scale processes. Essentially, the greater the similarity between the technologies applied to the bioreaction, the greater the likelihood that the process will approach 100% scalability. Geometric similarity corresponds to the requirement that the ratio between lengths in the two systems must be equal. (29,32,37) As a general rule for the amplification of biological processes involving microalgae, we have established geometric similarity criteria and amplification parts of similarity criteria in the invention process. (31,32) - A brief description of the separation of solid and liquid components in pig manure slurry Solid / liquid separation systems for sludge enable improved subsequent management, such as its transportation or subsequent separate treatment of each phase of the sludge. Figure 1 Solid / liquid separation equipment allows manure slurry to be separated into two phases: a solid component and a liquid component. The goal of implementing such a separation is to improve management, facilitate the transport of the solid component, enable individual processing of each phase, or reduce emissions from the largest volume of liquid. This liquid component can enable precision irrigation systems because it contains a lower concentration of organic matter. (6,8,38) The efficiency of a separator is quantified by two main values: 1) the flow separation efficiency (ηQ); and 2) the load separation efficiency, such as nitrogen (ηN) or phosphorus (ηP). Each of these efficiencies is defined as the portion of the mass flow that transfers from the inlet to the solids section; the remainder enters the liquid section because the principle of mass conservation must be obeyed. As reviewed in Flotats Ripoll (2019), these efficiencies and unknown data can be calculated from actual measurements in a given sludge processing system (the nomenclature indicates...). Figure 2 (13) Q 2 = Q 1 - Q 3 = 18.4 t / d η Q = 2.6 / 21 = 0.124 → 12.4% Q 1 C N1 = Q 2 C N2 + Q 3 C N3 C N3 = (Q 1 C N1 - Q 2 C N2 ) / Q 3 = 7.75 kg N / t η N =Q 3 C N3 / Q 1 C N1 = 0.274 → 27.4% In the example developed by Flotats Ripoll (2019), (13) 27.4% nitrogen was output from the slurry by separating the solids fraction, with a concentration factor of 2.2; note that the output nitrogen concentration is 2.2 times the original concentration. Obtaining high concentrations (e.g., by combining several separators in series) implies a reduction in overall separation efficiency, i.e., outputting a smaller percentage of nitrogen, but at a higher concentration, which may be of interest depending on the subsequent use of the solids fraction.

[0011] Table 1 shows the efficiency ranges given for S / L separation systems classified into three main types. (13) The efficiency of a device can be improved by using coagulants (iron or aluminum salts) or flocculants (organic polymers), although this effect is not universal and depends on the composition of the slurry. When the slurry is very thin, flocculant separation can be improved by adding air bubbles through flotation. Coagulants can be replaced by electrocoagulation systems, in which an electric current dissolves electrodes typically made of iron to achieve the same particle aggregation effect.

[0012] Generally, in terms of efficiency: centrifugation >> pressure separation >> size separation. However, a wide range of efficiencies has been found (Table 1). Therefore, efficiency depends on the composition of the slurry and, most importantly, on its storage time at the time of separation; the fresher the slurry, the higher the efficiency, especially when the slurry is separated within a few hours of excretion. This is because the saprophytic slurry infundibulum involved in the decomposition process dissolves many solid compounds from the moment they form, and once dissolved, they can no longer be separated by mechanical means. Efficiency depends more on the prior treatment of the slurry than on the quality of the separator. (9,13,39) The literature reviewed estimates that phosphorus (solid fraction) separation performance is generally higher than nitrogen (liquid fraction) separation performance. (7,9,39,40) As reported by Cárdenas Calvachi and Sánchez-Ortíz (2013), the biodegradation of nitrogenous carbon is slower than that of carbon compounds because only a portion of organic nitrogen can be mineralized or converted into inorganic nitrogen, and the remainder is used to produce bacterial proteins. (41) This means that in the liquid fraction, the N / P ratio will be higher than that present in fresh manure slurry due to the increased concentrations of inorganic, organic, and protein-derived nitrogen. This ratio will be more suitable for the needs of many crops. (6,8,38) Table 1. Efficiency ranges for different types of solid / liquid separators, expressed as percentages of indicated parameters for the transfer of nitrogen (N) and phosphorus (P) to the solids fraction. Adapted from 3tres3, Flotats Ripoll, 2019.(13)

[0013] To improve separation efficiency, Cooperl cooperative (France) developed TRAC© ( Traitement par RAclage Cooperl The system consists of a system that separates the solids in pits under the slats every few hours. (42) The scraper system runs upward on a slightly sloping pit floor; it squeezes the solids upward while the liquid is discharged downward, resulting in a solid portion on one side of the house and a liquid portion on the other. The nitrogen separation efficiency measured with this system is about 55%, an efficiency that conventional separation systems cannot achieve, with the added advantages of fewer breathing problems, lower mortality, and higher productivity due to the avoidance of decomposition processes and gas emissions from the house itself. One of the most interesting conclusions of this French cooperative’s work is that it demonstrates the feasibility of treating this liquid phase by biological processes in the case of “old” slurry (low chemical oxygen demand (COD) / nitrogen (N) ratio). The salinity (composed of a mixture of ions and various concentrations of organic matter) observed in this liquid portion, along with the high ratio of “monovalent cations” to “divalent cations”, has been identified as the main parameter affecting the activity of anaerobic ammonia-oxidizing bacteria. In their experiments, it was not possible to determine further parameters specifically and quantitatively. (42) - Ultrasonic homogenization system Cavitation during ultrasonic treatment creates an unusual environment for chemical reactions. The rapid compression of gas and vapor within the bubbles generates enormous temperatures and pressures, reaching up to 5000 °C and 1000 atm, respectively. Because the bubbles are extremely small compared to the surrounding liquid, the generated heat is rapidly dissipated (>10¹⁰ °C × s⁻¹). 1) This keeps the environmental conditions essentially constant. This combination of high temperature, high pressure, and rapid cooling creates conditions that can affect protein stability. (43,44) Furthermore, rapid collapse generates shock waves that can induce mechanical effects. At the interface between the two-phase system, collapse occurs asymmetrically, with the liquid jet traveling at speeds of hundreds of milliseconds. 1 The particles travel at high speed through the cavity. Due to the impact, small solid or liquid particles are released. In liquid-liquid systems, emulsions are formed, which are generally much more stable than conventionally formed emulsions. Solids undergo fragmentation and erosion, which increases the activated zone. Cavitation also accelerates mass transfer and reduces repassivation caused by reaction products. The overall result is easier contact between immiscible or sparingly soluble reagents. Many chemical reactions can be activated by sonication due to the high-energy conditions generated. These mechanical phenomena can lead to mechanical denaturation of proteins in the environment. (43,44) Ultrasonic treatment is a highly effective method for dispersing solids or emulsions. (45) As the measured size of solid particles or liquid droplets decreases, the total contact surface area increases, and therefore the reaction rate increases. Another effect of ultrasonic treatment is mass transfer. In heterogeneous systems, reaction products accumulate on the contact surface, preventing new reactant molecules from interacting with that surface. The mechanical shear forces induced by cavitation rays generate turbulence and transport of material back and forth across the surface of solid particles or droplets, maximizing the exposure of one reagent to another. On the other hand, the combination of high-speed liquid jets, high pressures and temperatures, and very high heating and cooling rates during cavitation bubble implosion makes ultrasonic cavitation a unique means of transferring energy to chemical reactions. (45) The key component of any ultrasonic device is the transducer, an electromechanical part responsible for generating ultrasound by converting electrical oscillations produced by the generator into mechanical vibrations. (46) Typically, transducers are made of piezoelectric ceramics. When a potential difference is applied to opposite surfaces of this material, the material expands or contracts depending on the polarity of the applied charge. These fluctuations in the material's dimensions are what generate ultrasonic vibrations. The primary purpose of applying ultrasound to chemical reactions is to improve their speed and performance and / or induce specific chemical reactivity. (46) Sound waves are purely mechanical in nature, cannot be absorbed by molecules, and must be transformed into a chemically useful form through an indirect and complex process called cavitation. (47) Like all sounds, ultrasound propagates through a series of compression and expansion waves traveling through the medium. Compression cycles bind molecules together in the medium, while expansion cycles separate them. In liquid media, ultrasound expansion cycles can generate enough negative pressure to break the cohesion of liquid molecules, causing them to separate locally and create true microcavities or bubbles. Typically, this occurs in previously contaminated areas of the solution where small particles, dissolved gases, or microbubbles are present due to previous cavitation processes. These bubbles grow over several cycles, from less than a micrometer to tens of micrometers, trapping vapor or gas from the medium. The growth of the cavity during each expansion period is slightly greater than the contraction during the compression period. Thus, over many acoustic cycles, the cavity grows until it eventually reaches a critical size at which it can effectively absorb energy from ultrasonic irradiation. At this point, the cavity can grow rapidly during the acoustic cycle, acquiring an unstable size that no longer allows it to effectively absorb energy. Without this energy, the cavity cannot be sustained, and the fluid around it will violently enter the cavity, causing it to implode.

[0014] - Spirulina obtuseae ( A. platensis Cultivation conditions for production This microorganism is a Gram-negative microalga or cyanobacterium that multiplies by about 25% per day. (48) Morphologically, it is characterized by a row of cylindrical filaments with a width between 2.5 and 16 μm, which are then separated by septa visible under an optical microscope, typically in open spirals, with a distance between the coils between 0 and 80 μm, and a relatively large diameter of 15 to 60 μm. (49) It preferentially grows in alkaline media with a pH range of 8.5 to 10, with an optimal growth pH of about 9, which also inhibits the growth of other planktonic microorganisms. Its growth temperature range is 24°C to 35°C, with an optimal temperature of 33°C.

[0015] Regarding light, due to their photosynthetic properties, they require some exposure; however, the literature describes that prolonged exposure to light inhibits the growth of microorganisms. (49) - Filtration system for partial separation of solid / liquid cultures Separating manure slurry is a well-known technique for treating and recycling manure and manure slurry. (10,50) Some of the best techniques used include filtration or high-pressure systems: • Batch Filtration: Batch filtration involves separating suspended solids from a slurry containing a certain amount of related liquid. Various methods, such as pumping, gas pressure, gravity head, or centrifugal force, are used to direct the slurry towards a filter. Solids are retained by the filter media, which allows the filtrate to pass through. The flow of the filtrate is generated by a pressure differential across the system. (10) • High-Pressure Slurry Abrasion: High-pressure slurry abrasion (HPSA) is a patented particle abrasion technology used for crushing and selectively dissociating minerals. Two slurry streams are pumped under high pressure through opposing nozzles, and collisions between solid particles suspended in the slurry streams cause the feed material to break down. (51) • Combined Mechanical Sludge Conditioning and Filtration: This approach relies on sludge analysis and testing to develop a methodological solution called combined mechanical sludge conditioning and filtration. The method involves primary filtration on a continuous basis using a candle filter, which retains particles as small as 0.5 microns and discharges the solids as concentrated sludge / sludge. The concentrated sludge is then filtered on a secondary plate and frame filter press to recover residual solvents and discharge dry solids. (52) Depending on the specific application and characteristics of the slurry, these methods offer different advantages and disadvantages. Batch filtration is suitable for a wide range of particle sizes and concentrations, while HPSA focuses on the selective dissociation of minerals. Combined mechanical slurry conditioning and filtration offers a reliable method with high-quality filtrate and minimal utility consumption.

[0016] - The theoretical methods and foundations of the proposed design solution The economic, social, and environmental costs of manure contamination are a major problem in the swine industry, which is by all means a low-profit industry. (53) For this reason, the goal of this solution is to investigate a new, inexpensive, scalable, and comprehensive technology for the in-situ reuse of manure by developing an original, managed microalgae production system that will allow for the production of high-value-added byproducts: (a) new ingredients for animal feed, and (b) phytoremediation agents for agriculture and natural soil conditioners, without the need for transportation or reprocessing outside the farm. The platform will allow for the avoidance of greenhouse gas emissions (GGE), including but not limited to ammonia (NH3), nitrous oxide (N2O), methane (CH4), carbon dioxide (CO2), and hydrogen sulfide (H2S), by significantly reducing the number of days of open-air fermentation of manure.

[0017] Microalgae, with their short shelf life, high growth rate, and high CO2 utilization efficiency, represent one of the viable renewable resource technologies for producing biomass from wastewater nutrients. (54,55) The potential to convert manure nutrients into biomass for industrial uses (food, biofertilizer, biolipids, bioenergy) has attracted increasing attention, leading to a wide range of industrial applications in the circular economy of livestock farms. (14,56,57) In particular, pig farm wastewater is considered a valuable source of water and nutrients for microalgae cultivation and has been treated through anaerobic digestion, various A / O methods, microbial fuel cells, and the cultivation of various microalgae. (3,14,16) In this context, it is crucial to improve microalgae production systems to reduce the economic (transportation, management), environmental (GGE, farmland microbial contamination), or social (time-consuming operations) costs associated with animal manure planning.

[0018] Any solution applicable to manure management must involve minimizing the barriers to handling this natural waste derived from intensive animal production, namely: 1. The transportation of manure slurry (raw material) to waste disposal sites (farmland), processing (manure digesters), and drying centers (energy conversion facilities) involves high consumption of petroleum fuels, time, and energy.

[0019] 2. Farmers need to lease and manage multiple arable land plots to properly dispose of waste according to fertilization plans, which are often outsourced to third parties and are beyond their control.

[0020] 3. High concentrations of nitrates / nitrites / toxins and / or potential zoonotic microbial communities in unprocessed manure slurry can contaminate farmland.

[0021] Document US 2021 / 0207969 A1 discloses an algae growth and cultivation system that produces algal biomass as a raw material for algae-based products. Therefore, the main objective of this prior art document is to achieve this through parallel industrial steps, additional nutrients, and the coupling and cascading of different biological systems, such as cultivation with pretreated fecal slurry. Promote the growth of algal biomass maximize In contrast, the main objective of this invention is to achieve simple and unique algal cultivation. Recycling and converting sewage slurry This invention does not require additional nutrients or the development and maintenance of several parallel biological systems. It does not require maximizing algal growth, but simply optimizing it to achieve the goal (recycling manure). Summary of the Invention

[0022] According to a first aspect, the present invention relates to a modular on-site system for processing and converting animal waste.

[0023] According to a second aspect, the present invention relates to a method for in-situ conversion of animal waste using a field modular system according to a first aspect of the invention.

[0024] According to a third aspect, the present invention relates to the use of a processed, nutrient-rich, bioavailable, nitrogen-, phosphorus-, and potassium-rich liquid microalgae culture fraction obtained by a method according to a second aspect of the invention. Attached Figure Description

[0025] Embodiments of the invention will now be further described in the following paragraphs of this specification by way of illustrative examples only, and are not intended to constitute any limitation on the scope of the invention. The following description will be better understood when read in conjunction with the accompanying drawings, in which: Figure 1 A flowchart for determining the most suitable sludge management strategy for the specific site conditions. Adapted from 3tres3, Flotats Ripoll, 2019.(13) Figure 2 A schematic diagram of a screw press, indicating the names and data used to calculate efficiency. Published by 3tres3, FlotatsRipoll, 2019. Red data represent theoretically true measurements of the analyzed manure processing system. Quantities to be estimated are those that can be estimated.

[0026] Figure 3Design of a modular biotechnology platform for processing and converting animal waste in the field. Biological and contaminant risks are categorized by gray intensity: dark (high), medium (medium), and light (low). Legend: Circle (homogenization process); triangle (solid-liquid separation process); square (storage of raw materials and products); dashed square (mobile compact unit; identical module for two phases); ellipse (culture operation); thin line (flow of liquid portion); thick line (flow of solid portion). These figures correspond to the operations described in Table 3.

[0027] Figure 4 MS 100 separator. Image courtesy of Mecàniques Segalés S.L., Gurb, Spain.

[0028] Figure 5 Bioreactor 3L (BioBundle) (left) and its Ez-Control controller (right). Note that the computer is in standby mode and has been unplugged.

[0029] Figure 6 100L bioreactor (left) and cooling / heating recirculator (right). Note that the computer is in standby mode and has been unplugged.

[0030] Figure 7 Open rectangular pool bioreactor. (A) An example of a system assembled outside the facility with a culture capacity of 1200 L. (B) An example of a system assembled inside the facility with a culture capacity of 50 L. Both examples produce *Paracasus platensis* (…). Arthrospira platensis paracas ).

[0031] Figure 8 The Mediana-de-Aragon experimental station features open-tube FBR stations (up to 1,500 L). The system is in a static state with no operation. Each tube system has a filter pump and a UV lamp. It can be turned on or off depending on the light requirements of the strain being studied.

[0032] Figure 9 Basic design of a modular biotechnology platform for the processing and conversion of animal waste at the Mediana de Aragon pig farm. The manure slurry stream is marked in red, while the homogenization and microalgae culture streams are marked in green. There are two homogenization processes, at the inlet and outlet of the demonstration unit.

[0033] Figure 10A model laboratory station with a culture capacity of <50L was used, featuring a medium-scale inoculum preparation system within the facility. A. *Paracasus tarda* was injected into flasks, and floating structures began to form in the microalgal dispersion. B. A laboratory demonstration was conducted using controlled light (12 hours light / 12 hours dark). The two flasks on the left were filled with water and culture medium (50 / 50). The two middle flasks contained EMRA-derived fecal slurry-based colloidal medium. A 28-day experiment was conducted.

[0034] Figure 11 Comparative culture of *Typha latifolia* in laboratory-processed fecal slurry via EMRA and in Zarrouk medium. Conditions: 3000 lux, 12:12 hour photoperiod (light / dark), 30°C, and manual agitation.

[0035] Figure 12 Paracasa obtusifolia was grown in FBR culture within a clean chamber equipped with two lamps inside the reactor.

[0036] Figure 13 Relative growth rates of microorganisms classified by temperature range. Red boxes indicate the vegetative growth temperature ranges reported in the literature. Adapted from Blanco et al. 2015. (58) Figure 14 Growth control of Paracasa obtusigenina inoculated in an outdoor open raft reactor.

[0037] Figure 15 (A) Control photobioreactor (FBR) (open pond), culture of *Spirometra platensis* in 650 L of synthetic sea salt medium, day 0 (D0). (B) EMRA-processed fecal slurry FBR (open pond), culture of *Spirometra platensis* (from original dilution 1:1 v / v). Conditions: light intensity 3000-4000 lux, 27°C, photoperiod 12 on / 12 off, and continuous oxygen supply.

[0038] Figure 16 Comparison of Spirulina platensis growth in a 1,000 L photobioreactor (FBR) (open pond) between Spirulina-specific culture medium (control) and EMRA-derived fecal slurry-based colloidal culture medium (from the original dilution 1:1 v / v).

[0039] Figure 17 Figure 1. Three solutions used in the proposed experiments (A) and (B): piped water from Mediana de Aragon (left), culture water with Spirulina obtusifolia (middle), and EMRA-derived fecal slurry-based colloidal medium (right).

[0040] Figure 18Seed germination, day 2 (D2). Condition 1 (A): Mediana-de-Aragon drinking water; Condition 2 (B): Culture water containing Spirogyra obtusifolia; Condition 3 (C): EMRA-derived fecal slurry-based conditioner. Germination was observed to begin under all conditions.

[0041] Figure 19 Seed germination, day 4 (D4). Condition 1 (A): Mediana-de-Aragon drinking water; Condition 2 (B): Spirogyra platensis-specific culture medium; Condition 3 (C): EMRA-derived fecal slurry-based conditioner. The seeds not only germinated but were also observed to slowly grow and begin rooting. Under condition 2 (green), seed size was confirmed to be increasing, but germination took longer.

[0042] Figure 20 Day 4. Use transparent paper to keep the seedbed moist.

[0043] Figure 21 Vegetative growth, day 15 (D15). Condition 1 (A): Drinking water from Mediana de Aragon. Repeat group 1 and repeat group 2. Seedlings continue to grow.

[0044] Figure 22 Fungal growth, day 15 (D15). Condition 2 (B): Culture water containing Spirulina obtusifolia. Repeat group 1 and repeat group 2. Fungi were observed in all wells, and no germinating seeds were observed.

[0045] Figure 23 Vegetative growth, day 15 (D15). Condition 3 (C): EMRA-derived manure-based conditioner. Repeat group 1 and repeat group 2. Seedlings continued to grow.

[0046] Figure 24 Used for analyzing chickpeas ( Cicer arietinum The number of repeats in a crop.

[0047] Figure 25 Seed samples prepared under "blank" conditions are used for nutrient analysis. Please remember that these samples have not undergone any treatment, and their use is the starting point for determining the seeds used.

[0048] Figure 26 Physicochemical analysis of the 18-day culture conditions for chickpeas.

[0049] Figure 27 Retain leaf biomass for chlorophyll analysis. Detailed Implementation

[0050] In a first aspect, the present invention relates to a modular on-site system for processing and converting animal waste, comprising: (a) A treatment unit for treating animal waste, including a solid / liquid separation unit; the treatment unit allows for the acquisition of a fecal slurry-derived liquid portion of animal waste; (b) A treatment unit for the liquid animal waste portion of the homogenization unit (a), including a high-pressure homogenization and / or ultrasonic treatment unit; such treatment allows for the production of a homogenized, nutrient-rich liquid portion; (c) A culture unit for culturing microalgae, wherein the culture medium is the product from (b), namely the homogenized, nutrient-rich liquid portion; in other words, the culture step is carried out by using the homogenized, nutrient-rich liquid portion obtained in unit (b) as the culture medium. (d) A unit for collecting and processing the microalgal cultures produced in unit (c), including a high-pressure homogenization and / or sonication unit; such collection and processing allows for the production of homogenized, nutrient-rich microalgal colloidal suspensions; (e) A unit for separating the solid / liquid products generated in unit (d), i.e., a homogenized, nutrient-rich microalgal colloidal suspension, comprising a solid / liquid separation unit and / or a microfiltration unit; such separation allows for the production of two byproducts: (1) Processed nutrient-rich, bioavailable, nitrogen-, phosphorus-, and potassium-rich liquid microalgae culture fraction, and (2) Processed carbon-rich solid inactivated microalgae culture fraction. (f) Control and monitoring unit for monitoring culture conditions and processing efficiency.

[0051] In the context of this invention, the term "on-site modular system" means a collection of mechanical, physical, and biotechnological systems and units used for processing and converting animal waste located in intensive production farms and closely associated with ponds and storage sites for organic waste.

[0052] In the context of this invention, the term "fecal-derived liquid animal waste fraction" means a homogenized (i.e., sonicated) liquid fraction of nutrient-rich biocarbon molecules broken up and separated from solid particles in an animal waste suspension from an intensive farm, which serves as a microalgae culture medium in the form of a colloidal suspension with a low microbial load.

[0053] In a preferred embodiment, the microalgae are selected from Arthrospira platensis and Chlorella vulgaris. Chlorella vulgaris (or combinations thereof). In other preferred embodiments, the microalgae is *Zygophyllum obtusifolium*.

[0054] Modular on-site systems for processing and converting animal waste (also defined herein as EMRA) have been shown to be effective in producing microalgae in Mediterranean continental climate regions. The microalgae cultivated may include, but are not limited to, Spirulina platensis, Chlorella vulgaris, and other microalgae capable of growing in nutrient-rich culture media derived from animal waste. Examples include the production of cultures rich in 10–98% Spirulina platensis, within a temperature range of 10–30 °C, a light intensity range of 1500–3000 lux, and a photoperiod range of 6–12:6–12 hours (light / dark).

[0055] The on-site modular system for processing and converting animal waste according to the invention is a promising candidate for addressing the following needs: (a) saving animal feed costs (microalgae as a new food ingredient) (converting at least 0.5% to 3% of manure slurry into feed), (b) generating new income for farmers (high-tech, nutrient-rich, bioabsorbable, nitrogen-, phosphorus-, and potassium-rich liquid phytoremediation agents, up to 75% of total manure slurry production), and (c) avoiding significant greenhouse gas emissions (composed of, but not limited to, ammonia (NH3), nitrous oxide (N2O), methane (CH4), carbon dioxide (CO2), and hydrogen sulfide (H2S), accounting for 10% to 80% of total greenhouse gas emissions).

[0056] In a particular embodiment, the on-site modular system for processing and converting animal waste consists of a fully constructed modular interconnected tubular closed conversion platform, which comprises, but is not limited to, a semi-automatic pump for liquid management and a secondary storage tank for mechanical and physical manure slurry and culture modification.

[0057] In other preferred embodiments of the system of the present invention, the solid / liquid separation units in units (a) and (e) include at least one of the following: particle size and gravity separation units (static or rotary screens, grids, drums, sedimentation), pressure separation units (screw presses, plate and frame filter presses), and centrifugal separation units. The efficiency of these units is measured in terms of mass flow rate (starting from 1 kg) and the separation of nutrient loads such as nitrogen and phosphorus (starting from 1%).

[0058] Units (b) and (d) of the system of the present invention include high-pressure homogenization and / or ultrasonic treatment units for liquid and culture portions. These two homogenization units are used to (1) improve the efficiency of animal waste processing and conversion, and (2) optimize the culture medium for microorganisms, including microalgae. The homogenization unit can be based on, but is not limited to, any device that uses stirring blades and / or sound waves to break down cell structures and release intracellular substances, which is useful for fermentation, digestion, and other organic matter conversion processes. Key ultrasonic treatment platform parameters include ultrasonic treatment amplitude (at least 80), ultrasonic treatment time (starting with a cycle or continuous flow of at least 45 seconds), number of ultrasonic treatment cycles (at least 5 cycles or continuous), and ultrasonic treatment mode (on / off). Ultrasonic treatment technology is more effective for homogenizing microalgae cultures because it improves the dispersion of solids, emulsification of liquids, mass transfer, and reaction rates.

[0059] In other preferred embodiments of the system of the present invention, the culture unit (c) includes at least one open or closed photobioreactor, optionally including an aeration unit, wherein the at least one open or closed photobioreactor includes a temperature control unit. In other preferred embodiments, the at least one open or closed photobioreactor is a tubular, flat-plate, or pool-type bioreactor. This optional aeration unit is used to control bubble agitation and gas distribution, which also improves the exchange of microalgae between light and dark areas, thereby improving light transfer to the culture. The photobioreactor may be equipped with low-speed stirring blades to avoid deposition of microalgae and fouling of the reactor walls when the culture reaches high concentrations. The photobioreactor may be equipped with pumps to control pH and nutrient concentration gradients.

[0060] In other preferred embodiments of the system of the invention, the temperature in at least one open or closed photobioreactor is between 10°C and 35°C, preferably between 21°C and 31°C. This temperature is typically controlled by a temperature control unit via coils immersed inside the culture, which allows them to operate in a isothermal manner.

[0061] In other preferred embodiments of the system of the present invention, units (d) and (e) include a filtration unit, a drying unit, and an extraction unit. These units allow for the acquisition of compounds of interest such as lipids, proteins, and carbohydrates.

[0062] In other preferred embodiments of the system of the present invention, the control and monitoring unit (f) includes at least one sensor and actuator to control culture parameters, nutrient concentration and greenhouse gas emissions.

[0063] In a second aspect, the present invention relates to a method for in-situ conversion of animal waste using a field modular system according to a first aspect of the invention, said field modular system comprising any of the embodiments disclosed herein, individually or in combination, the method comprising the following steps: (i) Processing animal waste to obtain a homogenized, nutrient-rich liquid fraction; (ii) Use the homogenized, nutrient-rich liquid fraction obtained in step (i) as a culture medium to culture microalgae in a culture unit; (iii) Collect and process the microalgae culture produced in step (ii) to obtain a homogenized, nutrient-rich microalgae-derived colloidal suspension; (iv) Separate the solid / liquid homogenized nutrient-rich microalgae-derived colloidal suspension to obtain two byproducts: a processed nutrient-rich, bioabsorbable, nitrogen-, phosphorus-, and potassium-rich liquid microalgae culture fraction and a processed carbon-rich solid inactivated microalgae culture fraction.

[0064] In a third aspect, the present invention relates to the use of processed, nutrient-rich, bioavailable, nitrogen-, phosphorus-, and potassium-rich liquid microalgae culture fractions obtained by a method according to a second aspect of the invention as animal feed, said method comprising any of the embodiments disclosed herein, alone or in combination.

[0065] As an example, the method for obtaining feed for this animal is detailed below: Step 1. Process animal waste through a solid-liquid separation unit to obtain the liquid portion of the manure slurry. The manure slurry separation unit may be interconnected with both an internal manure slurry pond outlet and an external manure slurry solid portion storage pond and homogenization tank. The goal is to separate at least 97% of the nutrient-rich liquid portion from the original sedimentary manure slurry.

[0066] Step 2. Homogenize the nutrient-rich liquid portion of the manure slurry using stirring blades, high pressure, or ultrasonic treatment. The homogenization unit can be interconnected with the solid-liquid manure slurry separation unit and the fermentation bioreactor. The goal is to significantly reduce the microbial load of the original sediment and to break down and disperse the nutrient-rich (nitrogen, phosphorus, potassium) microbial structure into the colloidal culture medium.

[0067] Step 3. Cultivate microalgae in a controlled culture unit using a nutrient-rich, nitrogen-, phosphorus-, and potassium-rich colloidal dispersion as the culture medium. The culture unit may include open or closed photobioreactors, including but not limited to tubular, plate-mount, or pool-type bioreactors, interconnected with homogenization tanks and culture solid / liquid filtration units. The goal is to convert a low-microbial-load, nutrient-rich colloidal culture medium into a nutrient-rich, bioabsorbable, nitrogen-, phosphorus-, and potassium-rich, pH-controlled liquid microalgae culture.

[0068] Starting from step 3, it is worth noting that the original liquid portion of the fecal slurry can no longer be considered animal sediment waste, but rather used as a transformation medium for microalgae cultivation. Therefore, microalgae cultured in EMRA can be used as a potential food ingredient, pending regulatory approval as a new animal feed ingredient. (59,60) Step 4a. Collect and process the microbial biomass (feed component) generated in the culture unit using a culture fractionation unit (filtration or similar). The culture filtration unit may include a microfiltration or tangential filtration tubular pump or similar, interconnected with both the culture unit and the solid biomass storage tank (fresh microalgae) and the liquid culture fraction storage tank. The objective is to separate the solid culture fraction for use as a food component for on-site animals and to store the supernatant liquid culture fraction.

[0069] In a third aspect, the present invention relates to the use of processed, nutrient-rich, bioabsorbable, nitrogen-, phosphorus-, and potassium-rich liquid microalgae culture fractions obtained by a method according to a second aspect of the invention as agricultural phytoremediation agents or natural soil conditioners, said methods comprising any of the embodiments disclosed herein, alone or in combination.

[0070] As an example, the method for obtaining this agricultural phytoremediation agent or natural soil conditioner is detailed below: Steps 1 through 3 are as previously described for animal feed.

[0071] Step 4b. Homogenize the total microbial culture using a homogenization unit (stirring blades, high pressure, or ultrasonic treatment). Following an intermediate cleaning step to remove fecal-derived debris, the same homogenization unit from Step 2 can be interconnected with the culture unit and the liquid culture storage tank. The goal is to decompose microalgae and obtain significantly lower amounts of bioactive microorganisms in the processed culture.

[0072] Step 5. Separate the liquid / solid homogenized culture using a filtration unit or similar device to obtain a colloidal culture-derived liquid fraction. The same culture filtration unit as in Step 4a may include a microfiltration or tangential filtration tubular pump or similar device, which interconnects with both the liquid culture fraction storage tank and the solid biomass storage tank (fresh cell debris) and the final product storage tank after an intermediate operation cleaning step to remove culture-derived debris. The goal is to convert a low-microalgae-load, nutrient-rich, pH-controlled colloidal culture medium into a nutrient-rich, bioavailable, nitrogen-, phosphorus-, and potassium-rich liquid microalgae-derived culture fraction for use as a phytoremediation agent.

[0073] Step 6. Store the newly formed phytoremediation agent until its use. The final product storage tank can be interconnected with a homogenization unit and a dispenser equipped with a flow meter. The goal is to efficiently store and dispense nutrient-rich, bioavailable, nitrogen-, phosphorus-, and potassium-rich liquid microalgae-derived culture fractions for use as a phytoremediation agent.

[0074] The present invention further relates to the use of processed, nutrient-rich, bioabsorbable, nitrogen-, phosphorus-, and potassium-rich liquid microalgae culture fractions obtained by a method according to a second aspect of the invention as a recycler for toxic substances or pollutants in animal waste farms, the method comprising any of the embodiments disclosed herein, alone or in combination.

[0075] The present invention further relates to the use of processed, nutrient-rich, bioabsorbable, nitrogen-, phosphorus-, and potassium-rich liquid microalgae culture fractions obtained by a method according to a second aspect of the invention as fresh compost for agriculture or natural soil amendments, said method comprising any of the embodiments disclosed herein, alone or in combination.

[0076] The present invention further relates to a system according to the first aspect of the invention, comprising any embodiment disclosed herein, individually or in combination, for reducing greenhouse gas and carbon emissions in a livestock farm, said emissions being composed of, but not limited to, ammonia (NH3), nitrous oxide (N2O), methane (CH4), carbon dioxide (CO2), and hydrogen sulfide (H2S). This reduction is achieved by applying the system of the invention during the process of emptying the internal manure slurry tank in the pigpen (with a sanitary recommended maximum storage of 20 days) and by connecting the system of the invention to an external public manure slurry pond. This system, combined with regular internal manure slurry emptying, avoids at least 100 days of open-air fermentation of the residue (the legal maximum open-air storage of manure is 120 days). (10) The systems and methods of the present invention for processing and converting animal waste offer additional environmental benefits. High-pressure homogenization and / or ultrasonic treatment and filtration reduce the microbial load in the process, preventing environmental contamination from fecal slurry-derived or cultured microorganisms. Furthermore, the cultivation of microalgae eliminates odors originating from open-air fecal fermentation (significant reduction in H2S). The system also efficiently converts waste, transforming animal waste into a nutrient source for microalgae cultivation. These waste-derived nitrogen-rich solid microalgae culture fractions (algal cytoplasm and organelle walls) can be used as animal feed ingredients, providing a circular and sustainable solution for animal waste management and feeding. Moreover, this technology helps control biosafety issues such as the spread of bacterial antibiotic resistance plasmids by removing plasmids from the propagule after ultrasonic treatment.

[0077] Note that, unless the context otherwise requires, any implementation disclosed herein may be used alone or in combination with any other implementation disclosed herein. In other words, for example, a preferred option for a defining feature may be combined with a more preferred or less preferred option for other features.

[0078] The present invention will now be described with reference to the following embodiments, which are not intended to limit the scope of the invention.

[0079] Example - Design description of the proposed solution: Proof of concept Designing a modular on-site system (EMRA) for processing and converting animal waste involves an innovative breakthrough in combining various technologies to create a new, efficient, and self-sustaining combination for producing high-value manure-derived byproducts. To this end, it must meet several coexisting quality-derived design criteria listed in Table 2. In short, we have designed a high-level, sequentially robust, and extensively tested technology platform for on-site manure processing in livestock farms. Figure 3 The general procedures for EMRA operations are listed in Table 3.

[0080] Table 2. The qualities that the proposed Aragon Modular Waste Station (EMRA) must meet for feasibility in the swine industry are derived from the design parameters.

[0081]

[0082] The mechanical components that may constitute EMRA are (the operations in Table 3): 1. Pumps for transporting and handling liquids (all operations except 3, 6 and 8).

[0083] 2. Industrial high-pressure and / or ultrasonic homogenizers (Operations 6 and 8).

[0084] 3. Separators for solid and liquid components by filtration or similar methods (Operations 2 and 7).

[0085] 4. Open or closed photobioreactors, including but not limited to tubular, flat-plate or pool-type bioreactors, for culturing microalgae that exhibit better performance under the studied conditions (Assignment Y).

[0086] 5. Primary regulation system of the obtained product (liquid culture portion) <lcf>—Plant remediation agent (Assignment 8).

[0087] 6. Composting tanks, allowing the use of solid manure slurry portion (SSF) and solid culture portion (SCF) in the fertilization calendar associated with the farm management manure slurry program (Operation X).

[0088] 7. Machinery for processing compost solids (Operation X).

[0089] 8. Ponds for microalgae harvesting and subsequent inactivation (operations 5b and 5c).

[0090] 9. Quality control unit (all operations) for controlling EMRA-derived byproducts.

[0091] Table 3. Operation sequence (#) and main objectives designed for the Aragon Modular Waste Remediation (EMRA) facility to process manure slurry via microalgae.

[0092]

[0093] Example 1. Design, development, and scale-up of a proof-of-concept for microalgae production. This invention relates to a modular biotechnology platform for the in-situ recovery and transformation of animal waste, particularly manure, by producing microalgae and their derivatives cultured on a sequentially physically processed liquid manure-derived fraction. This innovation enables the acquisition of high-value-added byproducts, such as nutrients for animal feed and novel agricultural phytoremediation agents and natural soil conditioners.

[0094] To date, no previous project has used *Paracasus platensis* grown in this fecal slurry-derived colloidal medium (txid634502)(61), nor have similar results been published in indexed journals. However, laboratory conditions (500 mL) for its basal culture in water and conventional fecal slurry have been established. As part of the project, our R+D team has established them in basal media and fresh fecal slurry. Parameters such as pretreatment of fecal slurry without adding water contribution or volume correction of watery swine residues separated from solids, illumination and aeration studies of cultures, or kinetic changes of cultures in the face of environmental stress factors (temperature, reactor on / off circulation, pH correction with or without salt, etc.) were modified. Similarly, precise engineered elements were designed and modeled at a 1:1,000 scale in the experimental production facility built in Mediana de Aragon. Studies of natural pH changes and their correction kinetics were conducted on a smaller scale (150 L) before being transferred to a main semi-closed tank and FBR (2,000 L) (Example 1). The obtained theoretical products (intracellular lipids, cytoplasmic residues, and intact microalgae) were characterized to establish starting materials for byproduct verification and were one of the main research results (Example 2).

[0095] 1.1. Solution Development and Design for Full-Scale Culture Operations In the implementation of Example 1, an EMRA carbon capture and slurry removal solution was designed and validated by scaling up a culture of *Paracasus platensis*, controlling the addition of substrate and carbon source, and monitoring the generation of byproducts. The carbon capture and slurry removal solution has been designed based on validated scale-up criteria (62, 63, 64) and studies of the mechanisms and materials used in the proof-of-concept.

[0096] A demonstration facility for implementing and modifying a new greenhouse station, incorporating a manure discharge system, has been designed and drawn up to allow for the controlled addition of substrate and carbon source to microalgae. The technical design for the cultivation operation was carried out in a closed-loop intensive farm with 180 sows and their piglets (Granjade Experimentación Tecnológica SL-B02859924-, ES50164.1, Mediana de Aragon, Central Zaragoza, Spain).

[0097] 1.2. Develop and implement the proposed EMRA proof-of-concept in Mediana de Aragon Tables 2 and 3 and Figure 3 The design elements described herein are used to design and define the implementation phase of the experimental facility. Key points for controlling and establishing the platform are outlined in the EMRA design description cited in the invention summary. Our proposed solution to the manure slurry problem is phytoremediation using microalgae, which produce chlorophyll by capturing organic and environmental carbon, treating wastewater, and generating byproducts.

[0098] 1.2.1. Equipment description for validating the use of liquid fecal slurry-derived colloidal fractions in 3 L to 1,000 L photobioreactors as microalgae culture medium.

[0099] The design selected in Table 3 uses basic mechanical components supplied by a specialized company. A mobile industrial homogenizer (a non-fixed, temporary mobile device that is decommissioned after performing homogenization tasks) is installed and connected via piping to the intermediate sedimentation tank within the Mediana de Aragon greenhouse.

[0100] The solid / liquid separation unit used is the MS 100 separator (Mecàniques Segalés SL, Gurb, Spain). Figure 4 This is a device specifically designed for pigs, functioning optimally in manure slurry with a solid content not exceeding 5%. It features a filter incorporating a specific spiral to obtain a higher percentage of dry matter (35%) in the solid portion. Depending on the homogenized starting material, it can achieve a yield of 3-20 m³. 3 The yield per H is 6-30 m 3 The power consumption range is 4 kW. It is equipped with a safety pressure regulating device that automatically stops in case the filter and screen become clogged due to compression. The internal screen achieves separation of up to 250 microns in the liquid slurry fraction (LSF). A mobile station was installed on the slurry pond in Mediana de Aragon to separate the 20 m³ used in the experiments conducted in this study. 3 The manufacturer provides an example of energy consumption for a 2000-head fattening farm, ranging from approximately 2.45 kW / H (0.02 to 0.03 € / m²). 3 ), indicating less than €100 / year.

[0101] The homogenization unit used is a UIP2000hdT-230 (2000 W) industrial ultrasonic processor (Hielscher Ultrasonics GmbH, Teltow, Germany). This equipment operates continuously by recirculating industrial liquids and slurries in protected pipes within a soundproof enclosure. It is capable of processing 65,000 L every 6 hours. For this reason, it is an ideal system for homogenizing both the liquid slurry and liquid culture portions in EMRA (Emergency Medical Respiratory Regulator). Figure 1 An ultrasonic processor was connected to the outlet of the separated liquid fecal slurry section for continuous ultrasonic treatment and direct feeding of the microalgae culture in the FBR within the greenhouse. Ultrasonic treatment was performed at a depth of 65 m. 3 The energy cost of the liquid slurry portion is less than 2 kW / H.

[0102] The filtration system consists of sequential sieves ranging from 200 to 0.45 µm, separating components via high-pressure guided microfiltration and decanting (internal system, GET, Spain). This system combines a circulating pump with a liquid filter, separating up to 97% of the liquid fraction. Issues related to filter clogging and discontinuous flow in this internal system suggest that a semi-automatic tangential microfiltration system should yield better results. The research team is currently conducting experiments to confirm this hypothesis.

[0103] 3L (BioBundle 3L tubular bioreactor & Ez-Control process controller, WP0173) Figure 5 ) and 100L (100L tubular bioreactor & DHW / AFS recirculator WP0175, Figure 6 The photobioreactor is used to produce GLP master and working seeds for selected strains and to optimize the resuscitation, recovery, and initial culture operations of the studied microorganisms. The BioBundle consists of a 3L capacity externally mounted jacketed reactor assembly made of 316L SS stainless steel and a 3-rotor flowmeter system with solenoid valves (for adding components). The BioXpert Lite control system, supplemented by ADDA cards and I / O extensions, allows for complete digitization of the fermentation process. The 100L tubular bioreactor consists of a 100L capacity externally mounted jacketed reactor assembly made of 316L SS stainless steel, featuring multiple sensors and configurations, and is connected to the DHW / AFS recirculation unit. Both systems are configured to measure and control pH, temperature, dissolved oxygen (DO), level / foam, and agitation. The 3L fermenter and the software used include sensors for pH, temperature, dissolved oxygen, and level / foam.

[0104] This equipment has some fundamental characteristics for validating techniques used in cultivation processes, such as those presented in this report: • All materials in contact with the culture are made of 316L SS (stainless steel), EPDM, silicone rubber, PTFE, or borosilicate glass.

[0105] • In tests conducted by the supplier, all of these materials were proven to be non-toxic and chemically inert.

[0106] • The 3-liter BioBundle is fully validated for cGMP applications. Ez-Control is developed and documented according to the GAMP4 industry standard.

[0107] • The system is delivered along with all appropriate documentation required to operate the system, including an operation manual, general maintenance instructions, a parts list, and selected drawings.

[0108] The research team used open-air rectangular or circular ponds to scale up the modular platform and validate the system's field management. The rectangular ponds consisted of an open cultivation system designed with an open-type polyethylene outdoor raft. For this purpose, a rectangular swimming pool was constructed using an AISI 316 stainless steel structure measuring 1 x 4 x 0.4 meters and installed in the proof-of-concept facility. This structure was clad in flexible polyethylene plastic and protected with a polyester mesh to reduce environmental pollution. An aeration system was installed at the bottom of the reactor to allow algae movement and prevent its deposition. Figure 7 ).

[0109] The circular tanks consist of a reinforced polyethylene system with a semi-automatic filter and delivery system that continuously recirculates the culture medium. Light control is achieved via a programmable on / off UV lamp (not shown in the photograph). Note that the semi-open FBR system allows for better aeration and temperature transfer. pH correction is performed manually by adding to and preparing solutions in a clean room using 1-5 L of inoculum via the equipment's delivery pump. Temperatures are maintained between 5-25°C to allow the microorganisms to gradually acclimatize to the Mediana climate. Figure 8 ).

[0110] 1.2.2. Scale-up Standards Validated on the EMRA Platform Aerobic culture scale-up presents inherent challenges in controlling and monitoring reaction conditions to obtain the desired products. (31,32,33) To achieve this goal, the culture conditions for the selected strains used in the study were thoroughly investigated from the laboratory to the pilot-scale (2L). The aim was to appropriately transfer this process to industrial scale while minimizing the risk of failure. (31,33,37) Several scale-up criteria were determined based on the type of FBR used in the project (Table 4).

[0111] Table 4. Magnification standards applicable to tubular or rectangular (raft) FBR types used in this project. Note that if a common amplification standard cannot be applied to two or more FBRs, amplification based on that standard is rejected.

[0112]

[0113] 1.2.3. Experimental design and control parameters used in the proof-of-concept experiment.

[0114] An experimental design was conducted at a 1,000 L scale under EMRA-required conditions, using the growth of *Typha latum* from *Paracas* as a model to optimize microalgae culture parameters. The optimized control parameters and experimental conditions used in the proof-of-concept experiment are shown in Table 5. All results and conclusions obtained in this embodiment during 2022 and 2023 underwent consistent robustness (at least x3 operators) and reproducibility (at least x3 times) checks. Only relevant results are included in the conclusion summary.

[0115] Table 5. Optimized control parameters and experimental conditions used in the proof-of-concept experiment. The job number and sequence (#) are taken from... Figure 3 Measurements are expressed as: liters (L), hours (H), kilograms (Kg)

[0116] 1.2.3.1. Cultivation of *Typha latifolia* from Paracas Different growth conditions of primary inoculum of *Plasmodium paracassis* were tested in the laboratory using Zarrouk liquid medium. Several review articles detail the characteristics and chemical composition of the medium. (34, 49, 65) Furthermore, in Salinity TM Additional culture experiments were conducted in liquid culture medium to optimize microalgae growth.

[0117] 1.2.3.3. Growth assessment using spectrophotometry Growth assessment of *Paracasus spirochetes* cultures was conducted by periodically removing 1 mL aliquots from the culture and measuring turbidity using a spectrophotometer at 730 nm wavelength for optical density (OD) readings and chlorophyll content at 665 nm.

[0118] 1.2.3.4. Growth assessment using an optical microscope A drop of culture was placed on a glass slide and observed under an inverted microscope with a 20x magnification objective to qualitatively evaluate the growth of *Typhonium paracasii* culture. Acceptable growth of *Typhonium paracasii* provided images of the species' distinctive green colonies and intact spiral structure. Conversely, the observation of discolored colonies indicated photoinhibition.

[0119] 1.2.3.5. Determining culture growth by dry weight For cultures larger than 50 mL, take an aliquot of sufficient volume to ensure a uniform suspension of cyanobacteria. Transfer the sample to a 50 mL conical tube and centrifuge at 7000 rpm for 30 min at room temperature. Alternatively, after drying in an oven at 100°C for 12 hours, weigh the filter paper and place it in a funnel. Filter the supernatant, and once all liquid has passed through the filter, transfer the precipitate.

[0120] Subsequently, the filter was washed 2 to 3 times with distilled water to remove trace salts from the culture medium, and when the filter was observed to be free of liquid, it was transferred to a petri dish and dried in an oven at 100°C for 12 h. Finally, the filter paper and cyanobacteria were warmed together and weighed. The mass difference between the mass of the dried filter and the mass of the filter containing *Typhonium pallida* yielded the mass of the latter in the initial aliquot volume.

[0121] 1.2.3.6. Investigate the effects of photoperiod and culture medium on the growth of the selected strains.

[0122] After optimizing the conditions for algal growth in the most optima culture medium, adjustments to lighting conditions were considered during subsequent scale-up to regulate energy consumption. Furthermore, an initial adaptability test was conducted on the manure slurry present on the farm, and alternative culture media were developed. Salinity Models were created for artificial seawater to generate primary inoculum in tanks larger than 25 L.

[0123] Evaluation of culture units of 1,2,3,7,5, 50, 100 and 2,000 L.

[0124] Once the culture and optimization conditions of *Paracasus platensis* under laboratory conditions and controlled temperatures were known, it was decided to establish a production facility in a biosafety level 2 (NBS2) laboratory with a photobioreactor range of 5 to 100 L to evaluate the yield from Salinity. ™ Starting with inoculum grown in artificial seawater, the adaptability of algae and the possibility of achieving sustainable yields through inoculation into fecal slurry at room temperature were investigated. After initial upstream inoculation and full microalgae adaptation, primary inoculum was injected into FBRs up to 2,000 L to optimize microalgae culture under different experimental conditions.

[0125] 1.2.3.8. Cryopreservation Transfer one (1) mL of culture to a cryovial with 1 mL of 10% methanol (preserve 2 units per species). Immediately place the cryovial in a cold block at -20°C, then transfer it to a freezer at -80°C and store it there for 1 hour. Afterward, transfer it to a container and / or Dewar flask containing liquid nitrogen (-196°C) for further storage.

[0126] 1.2.4. The basic implementation and location scheme of EMRA in Mediana-de-Aragon.

[0127] An implementation plan has been designed and drawn up for a laboratory demonstration of a new greenhouse station connected to a modified manure discharge system, to allow for the controlled addition of substrate and carbon source to microalgae. Essentially, a laboratory system has been designed to attach to the manure pond, the most common way for livestock farms to store this manure. A basic overview is presented in... Figure 3 A basic overview of the Mediana-de-Aragon experimental facility is available in [the document / document / etc.]. Figure 9 As shown in the diagram. The strain preparation and inoculum laboratory was designed to support the experimental production facility built at a 1:1,000 scale in Mediana de Aragon. The proof-of-concept was monitored and analyzed in March through November of 2021, 2022, and 2023 (the last year ending in September).

[0128] 1.2.5. Modular station including indoor tubular photobioreactor and its auxiliary equipment.

[0129] Both rectangular pool-type (1,200L outdoor / 50L indoor) and tubular (open pool bioreactor, 2,000L indoor) photobioreactors were installed and controlled in the experimental facility. An internal lighting circuit consisting of eighteen (18) plates was installed to ensure an appropriate lux supply to the culture FBR and culture ponds.

[0130] 1.2.6. The Mediana-de-Aragon experimental pig farm has highly polluted and challenging manure slurry.

[0131] The concentrations of aluminum, copper, manganese, selenium, and zinc in drinking water from the pipeline network reaching Mediana de Aragon and extending to the farms exceeded the permissible parameters of the water quality standards (data not shown). (66) The concentrations of silver, aluminum, arsenic, boron, barium, beryllium, cadmium, cobalt, chromium, copper, iron, lithium, manganese, molybdenum, nickel, lead, rubidium, antimony, selenium, tin, titanium, vanadium, and zinc in the GET slurry exceeded the permissible parameters of the Spanish water quality standards (data not shown). (66) Under our operating conditions, the nominal composition parameters of the manure slurry have been determined. (12,13) ​​High concentrations of various heavy metals, associated with the stability of paints and building materials, as well as trace amounts of chemical disinfectants and coating compounds, were detected in the manure slurry, which may be related to poor farm management during years of lack of maintenance. GET is a young company (24 months at the time of writing) and acquired the 50-year-old farm at the end of 2021. Since then, it has made significant efforts to restore the infrastructure, facilities, and equipment of the pig farm it acquired. The farm's condition affected employees (respiratory problems) and animals (increased mortality compared to the industry average). During its existence, the company implemented a biosecurity, maintenance, and management program approved by local and regional authorities, which successfully reversed all environmental toxicity levels in the form of volatile gases, drinking water, and water pipes in the farm buildings. Excess toxic substances detected have been significantly reduced to their actual control (data not shown). However, the presence of these toxic concentrations of heavy metals in the manure slurry storage ponds (which are practically impossible to completely remove) was carefully considered when interpreting the results of the remaining project operations.

[0132] The solid content of the slurry used in this study was particularly high (49%) compared to the average slurry content in Aragon and Catalonia (which typically has a solids content of around 30%). (13) This could be due to a combination of factors: (i) greater water loss through direct evaporation (excessive heat during the summer months during the sampling period); (ii) increased seepage losses (cracks were found in the main pond during fall renovations); and (iii) lower animal loads during the sampling season, which allowed the "old" slurry to persist longer in the pond, contributing to its natural clumping and drying (crusting). In any case, the higher solids concentration may be associated with higher nominal concentrations of matrix and contaminants in the slurry, complicating its use as a phytoremediation agent.

[0133] Nitrogen dioxide is a toxic, irritating gas and a precursor to nitrate particles. These contribute to the formation of acids in the environment. It primarily affects the respiratory system. The European directive sets an annual limit of 40 micrograms per cubic meter. -3 Under high ammonium concentration conditions, it established a non-constant equilibrium reflecting the aging of the slurry, making this sample the most representative of the existing conventional storage systems in the study area. (13) These parameter concentrations can be reduced through anaerobic and aerobic denitrification processes. Biological removal of ammonia nitrogen is applied to the liquid slurry portion and is usually carried out by combining two processes catalyzed by different microorganisms: Nitrification (N): Autotrophic aerobic microorganisms obtain carbon from CO2 or bicarbonate contained in fecal slurry, and convert NH4+ into nitrogen. 4+ Converted to nitrite (NO) 2- ) and nitrate (NO) 3- ) • Denitrification (DN): Another group of heterotrophic microorganisms convert NO into nitrogen. 2- and NO 3- It is converted into N2 gas, using organic matter as a source of carbon and energy.

[0134] According to the review of this system, system efficiency varies greatly because nitrifying bacteria are very sensitive to temperature changes. A well-functioning system, adjusted according to changes in temperature and manure composition, can achieve an average nitrogen removal rate of 60% in the form of N2, relative to the nitrogen separated in the liquid fraction in the initial solid / liquid separator. Escape as NH3 and N2O of approximately 3-7% of nitrogen is common and should be avoided. (17,50,62) GET currently produces 9,219 kg of nitrogen annually according to Aragon environmental standards. (67,68) Biological nitrogen removal technologies result in the loss of fertilizer resources at the cost of energy consumption, which violates the principles of sustainability and a circular economy. (3,63) For this reason, it is not considered the best available technology for new livestock farms (BAT). (13) Chemical oxygen demand (COD) is the amount of oxygen required to chemically oxidize organic matter into CO2 and H2O. The higher the COD, the more severe the water pollution. (66,69) This result from testing fecal slurry indicates that strong aeration is necessary to consume all organic matter, which in principle would not be applicable under our study conditions, where our aim is to convert organic matter and ambient CO2 into fatty acids and algal proteins (which are likely to be consumed or used in other methods).

[0135] The recommended conductivity for greywater used in agricultural applications should be less than 1000 μS / cm. The ideal pH for irrigation water should be between 6.5 and 8.4, therefore, in principle, Mediana slurry meets this condition (data not shown).

[0136] Regardless, the processing of Mediana farm manure slurry presents a real challenge for innovative on-site modular biotechnology platforms used to process and transform animal waste.

[0137] 1.3. Aerobic microalgal culture model ranging from 200 mL to 2,000 L: Liquid fecal slurry-derived colloidal culture in *Zygophyllum platensis*. Summary of the verification results of the culture medium experiment proof of concept The production variables for large-volume aerobic culture have been studied and identified as parameters such as temperature, agitation, aeration and oxygen supply conditions, and homogenization of the colloidal fecal slurry-derived medium. The optimal culture time has been determined to achieve the highest possible yield of Spirulina in a scale-up facility. In a conventional photoreactor ( Figure 5 and 6 ) and rectangular flat culture surface ( Figure 7 and 8 The performance of the ecological platform was examined. During the study period (spring 2022 to autumn 2023), the performance of *Chlorella vulgaris* and *Anabaena* species was assessed. Anabaena sp. ), Gardita microchlorophyll ( Nannochloropsis gaditana ) and species of the genus Scenedesmus ( Scenedesmus sp. The performance of the proof-of-concept facility under extreme weather conditions was analyzed.

[0138] Apart from *Paracasus platensis* (txid634502), (61) none of the initially selected strains successfully grew in media resuspended in Mediana de Aragon network water or in processed GET fecal slurry-derived fractions. This conclusive result was surprising, as behavioral antagonism was observed in other cases, particularly for *Chlorella vulgaris* (19,70), *Scenedesmus* species (71), or *Microchlorococcus gardita* (72), where growth consistent with pig fecal slurry and runoff was observed. The composition of the culture medium showed a very significant effect on the yield of *Chlorella vulgaris* (35,73,74). This suggests that contaminants detected in both water and fecal slurry have a significant impact on the viability of algae under these conditions. We also observed the effect of excess solids content in the fecal slurry used in the study, which can alter the culture modality of the microalgae studied. (75,76,77) In any case, the growth conditions of any microorganism must be subject to the realities observed in the field (50,57,78,79) to avoid technical failures when transferring the system to the real world. Following this empirical validation, we selected *Platytoplasma palladium* for the remaining tests. (61) In this embodiment, we selected and presented *Platytoplasma palladium* as a resistant strain for the growth and bioremediation of highly polluted challenging sludge in the Mediana de Aragon (continental Mediterranean climate).

[0139] 1.3.1. Candidate strain for EMRA: *Paracasus platensis* Following a complete set of experimental trials, optimal conditions for culturing *Typhonium obliterans* in circular or rectangular FBRs up to 100 L were determined. The best results are summarized in Table 6. Based on the scale-up elements reviewed in Activity 1.2.2 (Table 4), we established in-situ optimization criteria for larger inoculum volumes in the pre-culture and primary inoculum chambers of Mediana de Aragon. Figure 9 ).

[0140] Due to the photosynthetic nature of microalgae, they require exposure to some light; however, prolonged light exposure has been described in the literature as inhibiting microbial growth. (49) These data correlate with those observed in this project when exposure was 16 hours (lower growth) compared to 12 hours (higher growth). After optimization experiments, the culture conditions for *Paracasus platensis* were finally set as follows: 12 / 12 photoperiod, 3000 lux intensity, 12:12 hour photoperiod (light / dark), 30°C, manual aeration, and cultured in Zarrouk medium. Microalgae growth results reached 2.43–3.29 g / L. -1 Between, or in other words, 2.67 ± 0.42 g L -1 This means that the optimal yield of similar strains observed in the reviewed literature was reached and exceeded, which is 4.055 mg / L. -1 Up to 2.3 g L -1 (34,80,81,82,83) Using a specific medium for *Arthrospira* from Mediana de Aragon, containing highly contaminated drinking water and a liquid fecal slurry fraction, in cultures outside the FBR, we obtained a growth rate of *Arthrospira pallacas* of 0.06 ± 0.001 g L. -1 (Low volume) to 0.05 ± 0.02 g L -1 The high volume makes it an ideal candidate for modeling the proposed solution in highly polluted environments that significantly inhibit several microalgal cultures and cyanobacteria (Chlorella vulgaris, *Scenedesmus* species, *Anabaena* species, and *Microchlorophyllaria galbana*). The yield is within the growth range previously reported for similar strains of this microalga: 4.055 mg / L. -1 (80) to 1.5 g L -1 (81), and even 2.3 g L under specific lighting conditions. -1 (34) The optimized culture conditions and environments in which the proof-of-concept produced the strain and exhibited the best performance are listed in Table 6. These yields and conditions obtained using a controlled bioreactor model and continental inland environmental conditions reliably exceed the data obtained to date for Arachnida species through literature review, ensuring that we can scale up this strain inexpensively and sustainably for a variety of economically viable purposes.

[0141] Table 6. Optimal culture conditions and yields of *Typha pala* from Mediana de Aragon (GET) in photobioreactors smaller than 100 L.

[0142]

[0143] 1.3.2. Results of the proof-of-concept study on fecal slurry-derived colloidal culture medium conditions and on-site modular biotechnology platform. The solid slurry fraction (SSF) obtained in Mediana was particularly high (49%) compared to the average SSF in Aragon and Catalonia (which is typically around 30%). (13) This could be due to a combination of factors: (i) greater water loss through direct evaporation (excessive heat during the summer months during the sampling period); (ii) increased seepage losses (cracks were found in the main tank during the 21-year autumn renovation); and (iii) lower animal loads during the sampling season, which allowed the “old” slurry to persist longer in the pond and under the grid (the internal slats of the building), contributing to the natural caking and drying (crusting) of the slurry. In any case, the higher concentration of SSF may be related to the higher nominal concentrations of matrix and contaminants in the slurry, complicating its direct and unprocessed use as a phytoremediation agent. The separation system was also less efficient for aged slurry, particularly due to sedimentation and caking problems observed in the internal ponds of the farm, which may be related to the yield and results observed for the microalgae studied, which did not show any growth. (13) Nitrogen dioxide (NO2) is a toxic, irritating gas and a precursor to nitrate particles. In high ammonium (NH4)... 4+ Under certain concentration conditions, it establishes a non-constant equilibrium related to slurry aging; the more gas, the more aged the slurry. (13) These parameter concentrations can be reduced by anaerobic and aerobic denitrification processes. Biological removal of ammonia nitrogen was applied to the liquid slurry portion and carried out via nitrification (N) under the conditions tested with *Arthropoda pallasii*. *Arthropoda pallasii* is an autotrophic aerobic microorganism that obtains carbon from CO2 or bicarbonate contained in the slurry and converts ammonia (NH4+) into nitrogen. 4+ ) is converted into nitrite (NO) 2- ) and nitrate (NO) 3- Based on reviews of other strains of the genus *Arthrospira*, system efficiency varies considerably because nitrifying bacteria are highly sensitive to temperature changes. The release of approximately 3–7% of nitrogen as ammonia (NH3) and NO2 is common and should be avoided in the proposed model. (17, 50, 62) At the farm used as a proof-of-concept facility, 9,219 kg of N per year is currently produced. -1 This meets Aragon's environmental standards. *Paracasus spirochetes* can utilize this nitrogen to convert it into organic nitrogen that plants can absorb. (67,68) In conventional farms, the highly expanded bio-nitrogen removal technology for manure consumes energy and wastes fertilizer resources, violating the principles of sustainability and a circular economy. (3,63) For this reason, for new livestock farms, Remove Nitrogen is not considered the best available technology (BAT). (13) Our solution is in the form of amino acids and proteins (microalgae growth). fixed Fecal nitrogen, carbon from suspended solids in the liquid fecal slurry portion (colloidal culture medium) (feeding) and carbon from the environment during the process (respiration).

[0144] One of the most notable results of this project was the significant impact of fecal slurry contamination on the viability and yield of microalgae cultures. The raw, unprocessed fecal slurry exhibited a substantial increase in COD, leading to high levels of contamination. (66,69) Scale-up conditions (Table 4) were established through similarity to effectively compare different culture agitation experiments and volume-scale systems, with the following parameters: (i) oxygen transfer coefficient (kla) as constant (low volume, indoor) or ambient (large volume, indoor or outdoor); (ii) tangential stirring rate (pNDi) as constant (low volume, indoor) or inapplicable (large volume, indoor or outdoor); and (iii) aeration pumping rate (F / V) as constant, via gas (low volume, indoor) or via ambient pumping (large volume, indoor or outdoor). These parameters were set to improve overall oxygen supply to the cultures (regardless of volume) and to help eliminate or buffer partial contamination detected in the raw, unprocessed fecal slurry, which is likely the reason behind the relatively low growth we observed in raw, unprocessed fecal slurry-derived *Arthrospira* cultures. The relevance of oxygen supply is evident, as various failures in cultivation aeration led to (a) death of Spirulina or (b) the onset of a seasonal phase (data not shown) in both cases. Even so, we have been able to convert LSF-derived organic matter and ambient CO2 into fatty acids and algal proteins, which can be used as a nutrient-rich, bioavailable, nitrogen-, phosphorus-, and potassium-rich liquid bio-fertilizer.

[0145] The recommended conductivity for greywater used in agricultural applications should be less than 1000 μS / cm. The ideal pH for irrigation water should be between 6.5 and 8.4; therefore, in principle, raw, unprocessed manure slurry meets this requirement. While it was possible to adequately control the pH under all tested culture conditions, we considered this due to the reduced concentration of microorganisms in the vegetative growth phase of the liquid manure-derived colloidal medium. However, in practice, no harmful effects were observed when scaling up from the initial stages, as described in other studies. The pH conditions are listed in Table 6.

[0146] Laboratory culture of *Arthrospira* was optimized by comparing Zarrouk medium (specific to this microalga) and EMRA-processed fecal slurry. Significantly increased growth was observed in *Arthrospira* derived from EMRA-processed fecal slurry in <5L laboratory cultures with 3000 lux (LED) intensity, a 12:12 hour photoperiod, 30°C, and manual agitation. Figure 11 These parameters were used as the initial experimental conditions for scaling up the EMRA method.

[0147] 1.3.3. Key parameters for scaling-up results and control The first experimental culture of *Plasmodium palladium* was conducted in an open system in which the culture was mechanically agitated with a paddle wheel. This type of system has certain limitations: (i) because it is an open culture system, the temperature is irregular, and therefore productivity may decrease. (ii) due to the low surface-to-volume ratio, it has limited light availability, which leads to shading between microalgae and reduces cellular light flux. (iii) Biomass harvesting (filtration) is costly and disadvantageous, given the very low amount of biomass produced. (iv) because it is a non-isolated system, it is more susceptible to contamination. (v) because it is not an automated system, optimizing the resources obtained is more complex. (84) In response to this system, and considering the above-mentioned disadvantages, the use of a closed culture system (FBR) was considered a possible solution to the problems caused by the open system. One of the most important contributions of the FBR designed in this proof of concept is the direct integration of a culture lighting system with a sufficient surface-to-volume ratio. (85) A significant improvement in the growth pattern of *Plasmodium palladium* was established in the laboratory FBR, whose growth pattern in the laboratory could not be completely transferred to the fecal slurry-derived colloidal medium. One of the limiting factors for achieving high-level biomass production from *Enterocera platensis* is the availability of a suitable free-flowing reactor (FBR), therefore we have tested various models and systems (described in 1.2.1) (86). Our results show that stable growth of *Typha latifolia* was observed over time in specific cultures <5 L free of heavy metals, using a 12:12 h (light / dark) photoperiod, achieving yields of 2.43–3.29 g / L. -1 Or 2.67±0.42 g L -1 The microalgal growth results met and exceeded the best yield observed in the review literature, which was 4.055 mg L. -1 Up to 2.3 g L -1 (34,80,81,82,83) In 5-25L experimental cultures conducted on indoor tubular FBRs with EMRA-rich, low-microbial-load colloidal medium, 0.1 ± 0.06 g / L should be considered. -1 sky -1 The growth of *Arthrospira* maintained an effective pH range of 10.53 ± 0.14 (Table 7), and for 25 L outdoor conditions at 30.43 ± 1.34 °C (to the theoretical maximum of 26.7 days); and 0.0561 g·L -1 sky -1 To promote growth, maintain an effective pH range of 9.5. Figure 12 And in a controlled indoor environment of 25L under LED irradiation at 29–34°C (to the theoretical maximum of 47.6 days). These unexpected growths were observed under conditions of very intense ambient heat and relative humidity variation in the region, with irradiation higher than expected at this time of year, potentially exceeding 45°C at certain times of day. In <100L FBR, with constant natural irradiation, 0.4L min -1 Forced aeration and a controlled temperature of 26±0.5℃ yielded 0.8-0.9 g / L. -1 The growth was observed. Repeat experiments were conducted in the fall, with culture yields ranging from 0.05 to 0.07 g / L. -1 sky -1 The variation between these values ​​is far lower than the yield obtained in the summer.

[0148] Anaerobic FBR culture temperatures can be operated within three different ranges: psychrophilic (ambient temperature, below 20°C), mesophilic (temperature between 30 and 40°C), or thermophilic (temperature above 40°C). (58) By increasing the temperature, the growth rate of microorganisms increases, such as Figure 13 As shown, due to increased microbial growth, the production rate of byproducts (biogas, biomass, biochar) is higher, and the residence time in the reactor is reduced.

[0149] Table 7. Culture yield (g / L) -1 Daily growth was observed, along with initial (i) and final (f) pH conditions in an outdoor photobioreactor for summer sowing. Note that the FBR collected ambient temperature and fully captured natural light. Data were collected at an outdoor tubular FBR. Data are shown as mean ± standard deviation (σ).

[0150]

[0151] Arthrospira typically grows in extremely alkaline conditions (pH=11), making it impossible for other organisms, including animals and plants, to survive. Therefore, Arthrospira achieves its maximum productivity in alkaline pH media. By surviving under such extreme conditions, it is considered a very easy species to grow because it is remarkably tolerant of pH changes. (77,87) These results regarding light-related photoperiod and temperature are consistent with those previously obtained for *Arthrospira*, where the culture growth kinetics showed tolerance to a wide temperature range from 20°C to 40°C. Maximum growth rates, i.e., cellular production with maximum accumulation of chlorophyll and phycobiliproteins, were found at 35°C and a light intensity of 2000 lux. (88,89) Regarding growth temperature, and as mentioned above, the *Arthrospira paracasa* strains used in these experiments were grown in rather extreme environments, such as pristine, unprocessed manure slurry or highly polluted drinking water. This characteristic is reflected not only in pH but also in temperature, with a high growth range between 24 and 35°C, although optimal yields were obtained at 35°C. (88) Under our conditions, optimal yields were obtained at approximately 30°C, implying significant energy expenditure during the colder periods of the year.

[0152] Photoinhibition can cause problems in the top layer of outdoor growth tanks, where temperature distribution will depend on agitation. Experiments by Vomshak and Guy in outdoor cultivation of photoinhibition-sensitive strains (90) showed that shading the culture with nets increased the daily photosynthetic rate by up to 35%. Other studies have concluded that strong solar radiation incident on hyperboloid photobioreactor cultivation systems affects the morphology and productivity of *Arthropoda platensis*. (91) It is important to consider that the light intensity per cell varies significantly depending on the growth density. The amount of light available to a cell will depend on the shading of other cells. This means that in very dense cultivation, light can become a limiting factor. This variation in light intensity during growth makes it convenient to adjust the number of lamps as the culture grows in indoor FBRs. Agitation of the culture also helps to improve this. (87) Proper agitation of the culture medium is extremely important for the growth and development of microalgae. When requirements are met and environmental conditions are satisfactory, agitation is the most important requirement for obtaining high yields of microalgal biomass. (77) Perhaps the lack of agitation under conditions of high fecal solids density (49% with Mediana slurry under our test conditions), leading to shading and lack of lighting, coupled with relatively low aeration outdoors, reduced the growth range to a maximum of 0.1–0.9 g / L observed under our initial test conditions. -1 sky -1 .

[0153] However, these factors alone cannot explain the poor performance observed from laboratory to semi-industrial scales. The surprising results of environmental pollution in both drinking water (tap water) and raw, unprocessed sludge in Mediana de Aragon led us to consider its possible impact on the observed growth. This impact was evident in the growth arrest and inhibition of vegetative growth phases in species of Chlorella vulgaris, Anabaena, Microchlorophyllium gardita, and Scenedesmus. Microalgae have been shown to effectively remove nitrogen (90–98%), phosphorus (66–98%), zinc (16–99.7%), chromium (52–96%), and mercury (77–97%) from contaminated aquatic systems. Algal strains capable of reducing metals such as Fe, Zn, and Cd by 85%, 95%, and 99%, respectively, or even eliminating Al and Cu by 100%, can be found in the literature. (92) Under our test conditions, we selected *Platycladus paracas* as a resistant strain for growth (with drastic changes in pH, temperature, and lighting) and bioremediation of highly contaminated sludge.

[0154] In the preliminary scheme for large-scale scale-up, outdoor culture of *Typha latifolia* was considered in an open culture system consisting of open outdoor polyethylene rafts. Following the methods applied in previous tests (colloidal culture medium, preparation, analytical methods), 0.0233 g·L⁻¹ was obtained in the external pond. -1 ·sky -1 The specific culture medium for the growth of Arthrospira ( Figure 14 The temperature difference was 33°C (night) - 46°C (day). This result was compared with that obtained in a closed reactor (25°C) for inoculum preparation, which produced 0.0335 g·L⁻¹. -1 ·sky -1 The results (data not shown) indicate that we have lost our growth capacity. Clearly, the isothermal system allowed for a significant increase in algal growth, approximately 43% higher. However, this reduction in growth achieved in the outdoor raft cannot be explained solely by temperature changes, and further experiments are needed to determine the cause of this decrease relative to the central inoculum.

[0155] Growth data of *Typha obtusifolia* in a 1000L FBR using the fecal slurry-derived colloidal fraction. Figure 15 The study confirmed the ability to significantly reduce total nitrogen (4.98 times lower than uncultured slurry), ammonia (4.25 times lower), total phosphorus (6.93 times lower), COD (7.77 times lower), and potassium (1.58 times lower) after modifying the residue using the EMRA method (Table 8). This transformation correlated with several key parameters, which were optimized in a series of trials at the proof-of-concept facility (data not shown). Therefore, oxygenation of the culture medium should be permanently ensured through agitation (stirring) and aeration (bubbling) in open ponds. Oxygenation of the culture medium is even more critical than temperature or pH control. Such oxygenation triggers greater bioremediation of the slurry compared to the reduction in controlled parameters in laboratory cultures without it. As an overall conclusion, the fecal slurry-derived Spirulina platensis culture processed by EMRA, in a 1000L FBR (open pond) at a light intensity of 3000-4000 lux, 27°C, a photoperiod of 12 on / 12 off, and continuous oxygenation via bubbling, has achieved better performance in converting nitrogen, phosphorus, and potassium into proteins, phospholipids, and key biomolecules for subsequent industrial applications in our proof-of-concept facility.

[0156] Table 8. Comparison of physicochemical parameters of *Typha latifolia* after 11 days of culture in the original liquid fecal slurry medium (diluted 1:1 v / v) and EMRA-processed fecal slurry. Differences (fecal slurry - modified) were calculated to examine for decreases (+) or increases (-) in the initially measured parameters. Fractions (fecal slurry / modified) were calculated to examine the proportion of such decreases (+) or increases (-) in the modified original fecal slurry.

[0157]

[0158] Based on the optimization experiment results of Spirulina-specific high-balanced culture medium (data not shown), when the culture reaches 2.67 ± 0.42 g / L... -1 When the concentration reaches the nominal value, the culture should be considered successful. The yield of *Arthrophagus platensis* from EMRA-processed fecal slurry in an open 1,000 L FBR (circular pond) was 0.35 g / L. -1 (Table 9 and Figure 16 It should be noted that reference values ​​were obtained in a low-volume scale-up process in Zaragoza after adapting to the growth of the microalgae and defining their optimal culture conditions. In short, pH control is essential, and pH fluctuations must be avoided to a greater extent to achieve optimal growth. Regarding light, when applying direct light, it is necessary to avoid shading and light inhibition at very high densities and to establish optimal growth temperatures. Optimization of pH and light control, and the use of optimal nutrient-rich, low-microbial-load colloidal culture media in longer experiments (up to 40 days), resulted in higher viability and growth of *Arthrophagus palladium* (validation is ongoing). It is estimated that, depending on the test conditions, the maximum yield of *Arthrophagus palladium* per liter should be reached on average in approximately 26.7–47.6 days. We confirmed that, under harsh conditions, the yield of *Arthrophagus palladium* was 0.35 g / L 11 days after inoculation with 10% volume of *Arthrophagus palladium* in an FBR (open pond) with optimal available technology. -1 This consists of (a) pristine, highly contaminated drinking water, and (b) more than 58 days of unusually high temperatures experienced in Spain during the summer-autumn season, during the proof-of-concept series of trials. Several longer-term experimental trials are being investigated to optimize large-scale cultivation using the EMRA method.

[0159] Table 9. Comparison of *Typha truncatula* growth in a 1000 L FBR (open pond) between synthetic sea salt (Zarrouk-like medium) (control) and EMRA-processed fecal slurry (derived from a 1:1 v / v dilution). Differences (fecal slurry - control) were calculated to examine for a decrease (+) or increase (-) factor in the initially measured parameter. Fractions (fecal slurry / control) were calculated to examine the proportion of this decrease (+) or increase (-) in the original growth control in the Mediana-de-Aragon drinking water.

[0160]

[0161] Within this framework, the EMRA system based on *Plasmodium paracassis* culture reduces the environmental impact of pig manure slurry effluents for application; (a) it produces safe byproducts for animal feed under ideal conditions using the obtained biomass (processed carbon-rich solid inactivated microalgae culture fraction); (b) it produces high-value-added NPK:C fertilizer in the same area, odorless (using the same water, producing 34.8% more) (processed nutrient-rich, bioavailable, nitrogen-, phosphorus-, and potassium-rich liquid microalgae culture fraction); (c) it promotes strong CO2 uptake in the environment; and (d) it produces consistent yields of fatty acids, proteins and polysaccharides, industrially viable and high-value sustainable biomolecules, etc. (84) Example 2. A portion of a nutrient-rich, bioavailable, nitrogen-, phosphorus-, and potassium-rich liquid microalgae-derived culture processed by EMRA is used as a biofertilizer. The main objective of this task is to conduct basic safety, usability, and efficacy tests on microalgae crop by-products as agricultural biofertilizers, and to study the characteristics of the resulting biofertilizers.

[0162] 2.1. Experimental Design Experiments were conducted to evaluate the bioremediation capacity of a portion of EMRA-processed, nutrient-rich, bioavailable, nitrogen-, phosphorus-, and potassium-rich liquid *Spirometra tarda*-derived culture obtained in a 1000 L FBR (open pond) (Example 1). In this study, 5 L of culture was processed as described in the invention. Three experimental groups of chickpea seeds sown in cotton were tested: (a) seeds irrigated with drinking water from Mediana de Aragon (Zaragoza) (control); (b) seeds irrigated with a *Spirometra tarda* culture medium cultured in synthetic sea salt; (c) and finally, seeds irrigated with an EMRA-derived fecal slurry-based modifier produced in this invention. After 18 days of culture, germinated seeds, ungerminated seeds, and developing seedlings were collected, their chlorophyll content was estimated, and nutritional analysis was performed.

[0163] 2.2. Materials and Methods 2.2.1. Materials • Drinking water from Mediana de Aragon (Zaragoza).

[0164] ·cotton.

[0165] • A fecal slurry modifier was obtained through bioremediation of *Zygophyllum obtusifolium* in IFE-22-046 technology verification and semi-industrial scale-up studies.

[0166] • Bottles for 1L centrifugation.

[0167] Seed batch number 303411.

[0168] Germinator.

[0169] Nitrile gloves.

[0170] • Salinity™ medium (Sal Aquavitro Salinity, SALAQUA16, Acuario PlantadoSL or similar).

[0171] • Plastic film.

[0172] ·pliers.

[0173] • Disposable serum pipettes.

[0174] Pipetus.

[0175] ·sprayer.

[0176] ·50 mL tube.

[0177] • Pool control solution.

[0178] 2.2.2. Equipment · Balance scales.

[0179] • Thermal stability chamber.

[0180] Centrifuge.

[0181] ·TL-D 90 Graphica 36W / 965 d lamp.

[0182] • Branson SLPe 150 ultrasonic instrument.

[0183] 2.3. Methods 2.3.1. Preparation of Crop Irrigation Solution Once the bioremediation phase is complete, the culture needs to be treated together with the biomass of *Arthropoda platensis*. For this process, 5 L of the slurry was taken from the control tank and diluted EMRA-treated slurry and sonicated for 30 minutes at 50% amplitude using a Branson SLPe 150 sonicator. Subsequently, the liquid fraction was separated from cell debris and other solids by centrifugation at 10,000 rpm for 30 minutes at 20°C to obtain a processed, nutrient-rich, bioabsorbable, nitrogen-, phosphorus-, and potassium-rich liquid microalgae culture fraction (hereafter referred to as the "amendment"). Finally, the obtained supernatant was stored at 4°C until use.

[0184] 2.3.2. Chickpea Crop 2.3.2.1. Sowing Chickpea seeds were counted and weighed according to the following conditions for distribution in the germinator, with conditions 1, 2, and 3 being two replicates. Figure 15 ).

[0185] · "blank" condition This group consists of seeds from the same batch as those used in the other conditions, but without any type of treatment. This condition is called a "blank" because it refers to a control sample that does not contain the target substance or analyte, and this control sample is used as a reference to establish a baseline for measuring the parameters to be determined under other conditions.

[0186] · Condition 1 (Negative Control): The seeds were irrigated daily with drinking water from Mediana de Aragon (more information in section 1.2.6). This condition served as a negative control to compare the results of this treatment with those obtained from seeds receiving the improver and to rule out any effects observed solely due to the addition of water. It provides a reference to the natural variability of the system and ensures that any observed effects are attributable to the variable being measured, in this case, the application of the improver.

[0187] · Condition 2 (Control Pool) Irrigation was performed using a solution from a control pool containing the liquid portion of the *Typha obtusifolia*-specific culture medium previously obtained during a 1000 L scale-up process (Example 1). Once the solution was obtained, it was processed as described in section 2.3.1. This culture yielded 2.67 ± 0.42 g / L in the validation experiment. -1 The microalgal growth results were as follows. As discussed earlier, these results met and exceeded the best yield observed in the review literature, which was 4.055 mg L⁻¹. -1 Up to 2.3 g L -1 (34,80,81,82,83) This control group was used as a reference to determine whether the changes observed in the experimental group (sewage tank) were indeed the result of the studied variable or other uncontrolled factors.

[0188] · Condition 3 (Based on EMRA-derived fecal slurry modifier) Seed irrigation was performed using an amendment (a nutrient-rich, bioavailable, nitrogen-, phosphorus-, and potassium-rich liquid microalgae culture fraction based on EMRA-derived manure slurry processed during the 1000L scale-up process (Example 1). Subsequently, the amendment was processed as indicated in section 2.3.1.

[0189] Sowing, germination, and plant growth were carried out in a thermally stable chamber with a temperature of 25°C, a relative humidity of 75%, a light intensity of 10,000 lux, and a photoperiod of 12 / 12 hours (light / dark).

[0190] 2.3.2.2. Germination The experimental and replicate groups were monitored daily, and watering was performed daily with 2 mL of solution added to each well, depending on the conditions. When this amount was found to be insufficient to keep the cotton completely moist, irrigation was combined with spraying dechlorinated water to ensure moisture during the germination stage. The dechlorinated water was sprayed first in the morning, and watering was performed six hours later, around noon, repeating these times as often as possible.

[0191] 2.3.2.3. Collecting and preparing samples for analysis For the collection, each replicate was divided into three groups: (a) ungerminated seeds, (b) germinated seeds, and (c) developing seedlings. Each new group was then weighed together with the "target" and placed in a 50 mL tube for further analysis.

[0192] 2.3.3. Analysis of Chickpeas 2.3.3.1. Analysis of chlorophyll content in plant materials Weigh approximately 0.5 g of plant material sample on an analytical balance and transfer it to a ceramic mortar. Add 10 mL (10 mL) of a 90 / 10 v / v acetone / water mixture as the solvent for pigment extraction. Grind the mixture and transfer the resulting residue to a 50 mL conical-bottom tube using another 20 mL of the 90 / 10 v / v acetone / water mixture. Incubate the extract thus obtained in the dark at 4 °C for 24 h. After this time, allow the extract to reach room temperature and centrifuge at 7000 x g for 15 min. Filter the liquid supernatant through a filter and collect it in a 25 mL volumetric flask, then dilute to the mark. Finally, measure the optical density of the liquid extract at 665, 645, and 630 nm to verify the absence of turbidity or suspended particles. The solvent itself was used as a target.

[0193] 2.3.3.2. Nutritional Analysis Standard nutrient analysis was performed on each replicate for each condition, with ungerminated and germinated seeds and developing seedlings grouped into groups. In this analysis, moisture, protein, carbohydrates, fat, ash, sugar, saturated fatty acids, salt, and nitrogen were measured.

[0194] 2.3. Results 2.3.1. Chickpea Crop 2.3.1.1. Sowing Six (6) germinators are available, two for each condition and one for each replicate, for sowing chickpea seeds. Each replicate consists of 50 holes, and a total of 5 seeds are sown in each of the holes.

[0195] Specific criteria were established for each condition to differentiate seedbeds: Condition 1 is A, Condition 2 is B, and Condition 3 is C. Figure 17 A). Irrigation solutions for each condition are shown in... Figure 17 B in.

[0196] First, a layer of transparent paper was placed at the bottom of the seedbed to prevent water loss during irrigation and to facilitate the reabsorption of water from the bottom through capillary action. Then, cotton was placed in each hole as a water reservoir for the crop. Before sowing, 250 seeds were counted and weighed, and then 5 seeds were sown in each hole, for a total of 50 seeds per replicate. The following results were obtained through conditions and replicates: Conditional weight 1: Replication group 1 (164.92 g), Replication group 2 (159.99 g).

[0197] Conditional weight 2: Replication group 1 (164.25 g), Replication group 2 (161.92 g).

[0198] Conditional weight 3: Replication group 1 (162.41 g), Replication group 2 (160.50 g).

[0199] The conditions for cultivation are (Table 10).

[0200] Table 10. Germination conditions of chickpeas.

[0201]

[0202] 2.3.1.2 Germination On the first day of cultivation (D1), it was observed that the cotton was not fully moistened after irrigation. Therefore, it was decided to combine irrigation with spraying dechlorinated water to maintain a certain level of moisture during germination, which is an important factor at this stage. The dechlorinated water was sprayed first in the morning, and then watered again about six hours later, around noon, repeating these times as much as possible.

[0203] On the second day of the experiment (D2), changes in germination began to be observed. Condition 1 had the highest number of germinating seeds, followed by Condition 3, and lastly Condition 2. Figure 18 ).

[0204] On the fourth day (D4), it was observed that the cotton was not very wet, so it was decided to spray it first in the morning and then again two hours after irrigation to maintain humidity overnight. Meanwhile, changes in seed development were again observed. Seeds in conditions 1 and 3 began to germinate, showing a change to a greener color, indicating the emergence of other new germination stages: seedlings ( Figure 19 ).

[0205] Next, it was decided to cover the tray with cling paper. Figure 20 This allows UV light to pass through, causing moisture to pool in each seedbed and forming small pores to prevent excessive condensation.

[0206] On day 5 (D5), initial fungal growth was observed, which is typical for new humidity conditions, especially in the two wells of each replicate of condition 2.

[0207] The following day, D6, fungal spread was observed along repeat group 2 of condition 2. Meanwhile, seedlings began to develop and continued to germinate only in conditions 1 and 3. From D5 onwards, germination, fungal development, and crop development were observed under all conditions until the end of the study.

[0208] On day 15 (D15), the last day of photographic documentation, under condition 1 ( Figure 21 ) and condition 3( Figure 23 In condition 3, the size of the vegetative seedlings continues to increase. In condition 4, more fungi continue to be present in the seed stage. Figure 22 ).

[0209] 2.3.1.3. Collecting and preparing samples for analysis Finally, on day 18 of the experiment, seeds were collected for nutrient analysis. Each replicate was divided into three groups: germinating seeds, ungerminating seeds, and developing seedlings. They were weighed (Table 11) and placed in 50 mL tubes.

[0210] During sample collection, seedlings in the holes containing fungi were observed to have significantly lower root development than the remaining seedlings. At the end of the test, 7 groups were generated ( Figure 24 The "blank" group contained seeds from the same uncultured batch (252.6 g). Figure 25 ), and three other test conditions and their corresponding repeat groups.

[0211] The weights in grams by condition, replicate, and group (ungerminated, germinated, seedling, and leaf weight) are presented below (Table 11). In condition 2 (control), seedlings were irrigated with a liquid fraction of a culture derived from *Typha obtusifolia* in synthetic sea salt. Since none of the seeds germinated, the samples were not grouped, and therefore weight comparisons were not considered because no germination was achieved. This is presumably due to the high salt concentration in the culture medium, which makes seed germination more difficult. In any case, this negative control validated the test as expected.

[0212] After partial cultivation of a nutrient-rich, bioavailable, nitrogen-, phosphorus-, and potassium-rich liquid microalgae culture processed from EMRA-derived fecal slurry, an increase in chickpea seed weight of 34.8% was observed [1-(36 / 55.2)].

[0213] Table 11. Chickpea seed weights under different conditions after 18 days (D18). Condition 1 (negative): Seedlings were irrigated with drinking water from Mediana de Aragon. Condition 2 (control): Seedlings were partially irrigated with a liquid portion of a specific culture medium of *Zygophyllum obtusifolium* (negative data not shown). Condition 3 (fecal slurry): Seedlings were partially irrigated with a nutrient-rich, bioavailable, nitrogen-, phosphorus-, and potassium-rich liquid microalgae culture processed from EMRA-derived fecal slurry.

[0214]

[0215] 2.3.2. Chickpea Cultivation Yield 2.3.2.1. Nutritional Analysis Once the physicochemical analysis of the samples was performed, the following results were obtained as a percentage per 100 g of sample (Table 12). As shown in Table 12 and... Figure 26 As shown, the "blank" seeds contained the highest levels of saturated fat (2.5%), total carbohydrates (50.7%), and protein (20.3%). Comparing the three experimental conditions, condition 1 (drinking water) had the highest levels of total carbohydrates (39.6%), sugars (6.42%), and protein (16.5%). The remaining higher levels of physicochemical values ​​were found in condition 2 (specific culture medium), which had higher levels of moisture (46.2%), saturated fat (0.94%), ash (5.2%), and salt (5.7%). Condition 3 (EMRA) presented intermediate values ​​between 1 and 2. Finally, the saturated fat content was similar across the three test conditions.

[0216] No significant differences in the analyzed nutrient contents were observed during the seedling growth stages under each condition. It is important to note that, taking into account moisture content, seedlings under both conditions 1 and 3 showed very similar protein contents, with an increase relative to the control seedlings. The decrease in carbohydrate and fat contents in condition 3 may indicate plant development, as the presence of nutrients (primarily amino acids) in the environment may have accelerated the rapid depletion of cotyledonary nutrients. (93) In condition 2, no seeds germinated, and higher salt and ash values ​​were observed compared to the control and conditions 1 and 3, which inhibited seed germination. (94) Furthermore, the values ​​for carbohydrates, protein, and lipids were lower, taking into account humidity.

[0217] Table 12. Nutritional value of chickpeas from different culture media. Condition 1 (negative): Seedlings were irrigated with piped water from Mediana de Aragon. Condition 2 (control): Seedlings were partially irrigated with a liquid portion of a specific culture of *Typha obtusifolia* (negative data not shown). Condition 3 (fecal slurry): Seedlings were partially irrigated with a nutrient-rich, bioavailable, nitrogen-, phosphorus-, and potassium-rich liquid microalgae culture processed from EMRA-derived fecal slurry.

[0218]

[0219] In summary, our results show that bioremediation of chickpeas with an EMRA-based manure modifier increases seed yield (by 34.8% by weight under the same conditions), while also promoting seedling development with lower humidity and water requirements, accelerating growth, and maintaining nutrient integrity.

[0220] 2.3.2.2. Analysis of chlorophyll content in plant materials The first part of the chlorophyll content calculation is the calculation of leaf biomass by condition and replication. In this case, the calculation is only performed under conditions 1 and 3, because no germination occurred under condition 2. Leaves from each seedling were removed using tweezers and scissors and preserved in 50 mL tubes. Subsequently, the amount of leaves intended for chlorophyll analysis (0.5 g) was taken, and the remainder was added to its corresponding sample for nutrient analysis. Figure 27 ).

[0221] Following the procedure described in section 2.3.3.1, to determine chlorophyll in plant material, four samples (Table 13) corresponding to plant material obtained from chickpea seeds germinated under conditions 1 (repeated 1 and 2) and 3 (repeated 1 and 2) were weighed.

[0222] Table 13. Weights of plant material obtained from chickpea seeds germinated under conditions 1 and 3. Condition 1 (negative): Seedlings were irrigated with piped water from Mediana de Aragon. Condition 3 (manure slurry): Seedlings were partially irrigated with a nutrient-rich, bioavailable, nitrogen-, phosphorus-, and potassium-rich liquid microalgae culture processed from EMRA-derived manure slurry.

[0223]

[0224] Chlorophyll content was extracted according to the procedure described in section 2.3.3.1. Finally, the absorbance of aliquots of the processed extract was measured at different wavelengths of 630, 645, and 665 nm (Table 14).

[0225] Table 14. Optical densities of chlorophyll extracts obtained under conditions 1 and 3 in replicates 1 and 2. Condition 1 (negative): Seedlings were irrigated with piped water from Mediana de Aragon. Condition 3 (manure): Seedlings were partially irrigated with a nutrient-rich, bioavailable, nitrogen-, phosphorus-, and potassium-rich liquid microalgae culture processed from EMRA-derived manure.

[0226]

[0227] Note: A: Repeat group; C: Condition; REP: Repeat.

[0228] At this point, the following formulas are applied to the calculation of chlorophyll contents A, B, and C proposed by Parsons and Strickland (1965) (Formulas 1-3, Table 15): C a (mg / L) = 11.6 absorbance 665 – 1.31 absorbance 645 – 0.14 absorbance 630 Formula 1. Chlorophyll A concentration.

[0229] C b (mg / L) = 20.7 absorbance 645 – 4.34 absorbance 665 – 4.42 absorbance 630 Formula 2. Chlorophyll B concentration.

[0230] C c (mg / L) = 55.0 absorbance 630 – 4.64 absorbance 665 – 16.3 absorbance 645 Formula 3. Chlorophyll C concentration.

[0231] Table 15. Calculation of chlorophyll A, B and C concentrations using the Parsons and Strickland formulas.

[0232]

[0233] Note: A: Repeat group; C: Condition.

[0234] Finally, the percentage of each type of chlorophyll in each extract is calculated using the following formula: %Chlorophyll A = [[(11.6 Absorbance) 665 – 1.31 absorbance 645 – 0.14 absorbance 630 ) x 0.025L / m x 100 Formula 4. Percentage of chlorophyll A.

[0235] %Chlorophyll B = [[(20.7 absorbance) 645 – 4.34 absorbance 665 – 4.42 absorbance 630 ) x 0.025L / m x 100 Formula 5. Percentage of chlorophyll B.

[0236] %Chlorophyll C = [[(55.0 absorbance)] 630 – 4.64 absorbance 665 – 16.3 absorbance 645 ) x 0.025L / m x 100 Formula 6. Percentage of chlorophyll C.

[0237] Where m: the mass of plant material obtained from chickpea seeds germinated under conditions 1 and 3 in replicates 1 and 2. The results are presented in Table 16.

[0238] Table 16. Calculate the percentages of chlorophyll A, B, and C according to formula 4-6.

[0239]

[0240] Note: R: Repeat group; C: Condition.

[0241] No significant differences were observed in the chlorophyll A, B, or C content during the seedling growth stages measured in both the conditional and replicate groups.

[0242] 2.4. Summary of Validation Results of Bioremediation of Chickpea Seeds Using EMRA-Based Manure Modifiers want.

[0243] The steady increase in pig farm wastewater (manure slurry) has become a serious environmental problem. The high levels of nutrients and toxic pollutants in manure slurry have significant impacts on ecosystems and public health. (3) Regulatory agencies have established complex and comprehensive administrative bodies in an attempt to control or maintain these industrial activity-related problems at an acceptable level. (4) Therefore, it is necessary to develop strategies to utilize or eliminate these risks (odors, groundwater pollutants) to promote (a) more efficient, profitable, sustainable, friendly and healthy production for animals, (b) residents around farms and (c) the environment. The economic value of fattening pig manure slurry has been quantified as its equivalent value based on the price of the NPK mineral fertilizer it contains, at €5 to €7 per cubic meter. 3 Between. (6) The reference estimate for the management and transportation costs of these same sludge is €2.92 / m³. 3 (Data from GET in Mediana de Aragon, central Zaragoza, Spain). It is estimated that, under reduced tillage conditions, it is possible to replace mineral fertilizers with pig manure slurry entirely in barley and partly in maize, which is also economically viable in rainfed regions with continental Mediterranean climates. (2) Microalgae, with their short shelf life, high growth rate, and CO2 utilization efficiency, have proven to be the best ecological and renewable alternative for producing biomass from wastewater nutrients. (81) Microalgae can grow under real-world conditions (outdoors) using hog farm wastewater as the sole nutrient source, which has been treated through anaerobic digestion, various A / O methods, microbial fuel cells, and the cultivation of various microalgae. (3,14,16) Examples of this management include outdoor reactor cultivation of *Scenedesmus* species for biofuel production (71) and *Crassostrea gigas* (orange-green flexural bacteria). Chloroflexus aurantiacus (95) Green microorganisms obtain the nitrogen and phosphorus necessary for their development by using fecal slurry as a growth medium.

[0244] Arthrospira contains growth regulators and antioxidants that can enhance plant tolerance by helping plants synthesize certain components. Arthrospira has a very diverse composition; it is one of the best sources of plant protein with high biological value and good digestibility; it contains 65-70% protein, is rich in chlorophyll (a pigment with great detoxification ability), B vitamins (especially B12), vitamins C, D and E, contains minerals such as potassium, selenium, copper, manganese, magnesium, zinc and phosphorus, digestive enzymes, and 7% of its content is essential fatty acids. (48) However, it can also act as a chelating agent for cyanobacterial toxins, heavy metals, pesticides or polycyclic aromatic hydrocarbons (PAHs) growing under natural conditions in polluted environments without much problem. (48) The specific composition of these molecules causes them to aggregate in storage organelles or adhere to phospholipid membranes. Therefore, in the design devised by the researchers, sonication technology was introduced into the method to minimize the risk of high concentrations of contaminants being transferred to the liquid portion of the culture (see Figure 1 ).

[0245] In short, sonication generates alternating high-pressure and low-pressure waves in an exposed liquid. During the low-pressure cycles, the ultrasound creates tiny vacuum bubbles in the liquid, which violently collapse during the high-pressure cycles. This phenomenon is called cavitation. The implosion of cavitation bubbles generates powerful hydrodynamic shear forces. These shear forces can break fibrous cellulose material into fine particles and disrupt cell walls. This releases more intracellular material, such as starch or sugars, into the liquid without damaging stored or highly concentrated organelles. (96,97) Furthermore, the breakup of cell wall material into tiny fragments allows us to flocculate or separate them.

[0246] In treating fecal slurry as runoff, microalgae showed superior removal rates of C, N, and P (91%, 91%, and 73%, respectively) compared to bacteria (74%, 80%, and 55%, respectively). (98) This experiment explored the feasibility of such low-temperature treatment by comparing the removal efficiency of 10 parameters between two membrane anaerobic photobioreactors (operating at 10°C and 22°C with a residence time of less than one day). Ultimately, the effluent concentration was within the discharge limits, and for the reactor at 22°C, the removal efficiencies of C, N, and P reached 91%, 81%, and 82%, respectively; while for the reactor at 10°C, the removal efficiencies of C, N, and P reached 87%, 77%, and 98%, respectively.

[0247] Under our test conditions, the EMRA-based manure amendment increased chickpea crop productivity by 34.8% by seed weight and during germination compared to a standardized control of conventionally irrigated plants. These results indicate that bioremediation with an EMRA-based manure amendment benefits seedlings with lower humidity and water requirements, accelerates their growth, and maintains nutrient content without difference from the control. Furthermore, the chlorophyll content in the bioremediation group remained unchanged relative to the expected growth pattern (control group), thus seedling respiration, growth, and developmental capacity remained intact, instead starting from an accelerated and improved starting point. These results surpass those obtained in similar chickpea experiments where foliar application of Spirifert biofertilizer at two points during the vegetative growth stage increased chickpea crop yield but had no effect on N, P, K levels in the grains. In addition, the treatment altered soil phosphorus and magnesium levels. (99) The main difference of this work is that it requires the addition of two different biofertilizers [Azofert®, based on pea rhizobia ( Rhizobium leguminosarum Biofertilizers of strain 2.8 x 10¹⁰ CFU and Spirulina gel (64%) and vinasse (36%) aqueous suspensions were used to achieve synergistic or additive effects, thereby increasing chickpea yield by 21-28% in soils classified as low-activity ferrous-aluminous clay (rhodic alcylic eutric) (red aluminum base saturated). (99) Although clay soils have low assimilable phosphorus content and low base saturation, they are considered fertile soils. They are deep, stable soils with favorable physical properties. By using an EMRA-based manure amendment, we achieved a 10% increase in crop yield and a 34.8% increase in weight (34.8%) in the absence of fertile soils, which makes us very optimistic about its application in degraded or contaminated soils. In other growth models, chickpea and Spirulina platensis ( Spirulina platensis Extracts of corn plants (corn ( Zea mays (100) However, in some respects, chickpeas ( Cicer arietinum L. The extract appeared to be more effective than Spirulina platensis (another strain of this microalga) in improving maize nutritional and growth parameters, likely due to its higher content of phytochemicals that can act as signaling molecules [hormones (indole-3-acetic acid, isopentenyl adenosine, gibberellin), phenols, amino acids, reducing sugars]. (100) Perhaps this synergistic effect of hormones and transmitters associated with the EMRA-based manure amendment is the reason for the higher chickpea seed yield observed in this work. Future work should use valid data from fertile eutrophic soils to confirm our hypothesis.

[0248] Example 3. On the Ecological Benefits and Carbon Capture of EMRA In recent years, the use of macroalgae and microalgae as raw materials has been revolutionary due to their high growth rates and high yields per culture area, (81) although we have seen in our results that the liquid portion of a culture derived from EMRA-based fecal slurry from *Arthrospira paracassis* can exceed the yields obtained from similar or closely related species by 10%. (99) In other words, due to *Spirulina* bioremediation, using the same amount of water, seeds can utilize carbon and nutrients derived from EMRA modifiers to produce 34.8% more agricultural products (same nutrients, same chlorophyll). In the model studied, we fixed carbon and nitrogen from the fecal slurry, and with the same amount of water, the residue could triple agricultural yields. Furthermore, due to its high photosynthetic rate and low bioavailability, *Arthrospira* can convert solar energy into carbon-storing products and thus reduce CO2 and greenhouse gas emissions (GGE). (40) Currently, the unprofitable cost / benefit ratio of the technology required for its use limits its large-scale industrial application. In this sense, there is still room for further research into advanced technological solutions based on phototropic algae, which would allow for the removal of current economic barriers.

[0249] This project has fully or partially addressed most of the gaps in the evaluation: (a) the adaptation of selected microalgae strains (both proprietary strains and comparative strains) to the continental Mediterranean climate (dry and extreme in its cold-hot cycles); (b) the impact of the characteristics of manure slurry adapted to intensive European feed production on the productivity of the adapted strains; and (c) the demonstration of efficient production of reusable byproducts for nutritional or phytoremediation applications.

[0250] *Note: Calculations and interpretations are based on differences and comparisons with strains grown in temperate or tropical climates (currently the leading strains in microalgae culture). Stable biological systems, such as those found in Japan, allow microalgae to direct all their energy toward growth and reproduction in a semi-continuous or near-continuous manner. However, in more extreme climates, microalgae invest a significant amount of energy in survival, making their reproductive cycles more seasonal (see review citations).

[0251] EMRA-based internal circulation of Arachnium production, by applying the invention immediately during the process of emptying the internal manure pond in the pigpen (with a maximum recommended storage of 20 days) and linking the invention to an external public manure pond, will result in the necessary overall reduction in the farm's carbon footprint (the current revised calculations do not take into account the energy consumption and generation from external supply to the system). This system, combined with regular internal manure emptying, avoids at least 100 days of open-air residue fermentation (the legal maximum open-air manure storage is 120 days). (10) Removing 100 days of external pond GGE avoids up to 83% of total farm GGE.

[0252] Increasing industrialization and the urgent need to avoid non-renewable fossil fuels due to climate change have led to the search for viable alternatives to renewable resources to meet growing energy demands while reducing carbon dioxide emissions. (54) The foundation of modern global energy strategies is the search for green and cost-effective alternatives to fossil fuels. Meanwhile, microalgae cultivation has the potential to significantly reduce greenhouse gas emissions from manure-based animal farms, which consist of, but are not limited to, ammonia (NH3), nitrous oxide (N2O), methane (CH4), carbon dioxide (CO2), and hydrogen sulfide (H2S). Microalgae have been identified as a promising and sustainable alternative for wastewater treatment (urban, agricultural, and industrial) and for the production of valuable byproducts. Research into these technologies is fully consistent with the strategies outlined in the Spanish National Climate Change Adaptation Plan (101), which promotes the sustainability of food systems and the adaptation of rural areas to climate change, particularly driving the circular economy and building strategies that produce smaller climate impacts and greater resilience.

[0253] Carbon and phosphorus from the fecal slurry are converted into microalgal fatty acids (phospholipids), enabling their fixation and extraction from fecal matter. Furthermore, through the conventional respiration of Spirulina, we can convert atmospheric carbon into linear hydrocarbon chains (fatty acids) more efficiently than through simple soil eutrophication carbonization. Selected Spirulina feed on nutrients in the fecal slurry, converting them into biomass through photosynthesis, initiating a carbon capture cycle in which, in addition to nitrogen and phosphorus, they require 2 kg of CO2 to produce every kg of biomass. Our proof-of-concept experimental data indicate that, under the maximum theoretical yield of *Spirulina pallens*, the yield of Spirulina can range from 181.51 g / day. -1 Up to 14,685.73 g days -1 This results in us removing 363.02-29,371.46 g of environmental carbon per day. -1 (Combined with autotrophic consumption). The combination of the two methods achieved better results, removing up to 9.1% of inorganic carbon from the slurry and converting it into usable organic fatty acids such as palmitic acid, detected in EMRA-based slurry modifiers (data not shown). This performance surpasses that described in the literature reviewed to date, making us very optimistic about deploying EMRA as an environmental solution to improve the sustainability of swine farms, not so much for carbon sequestration itself, but for avoiding the emission of greenhouse gases (methane, nitrates, etc.) from the slurry into the atmosphere.

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Claims

1. A modular on-site system for processing and converting animal waste, comprising: (a) A treatment unit for handling animal waste, including a solid / liquid separation unit; (b) A treatment unit for homogenizing liquid animal waste, including a high-pressure homogenization and / or ultrasonic treatment unit; (c) A culture unit for culturing microalgae, wherein the culture medium is a product from (b); (d) A unit for collecting and processing the microalgal cultures produced in unit (c), including a high-pressure homogenization and / or ultrasonic treatment unit; (e) A unit for separating solid / liquid products generated in unit (d), including a solid / liquid separation unit and / or a microfiltration unit; (f) Control and monitoring unit for monitoring culture conditions and processing efficiency.

2. The system according to claim 1, wherein, The microalgae were selected from *Zygophyllum obliterans* ( Arthrospira platensis ), common Chlorella ( Chlorella vulgaris (or a combination thereof).

3. The system according to claim 2, wherein, The microalgae in question is *Zygophyllum obtusa*.

4. The system according to any one of claims 1 to 3, wherein, The solid / liquid separation units in units (a) and (e) include at least one of the following: a particle size and gravity separation unit, a pressure separation unit, and a centrifugal separation unit.

5. The system according to any one of claims 1 to 4, wherein, The culture unit (c) includes at least one open or closed photobioreactor, optionally including an aeration unit, wherein the at least one open or closed photobioreactor includes a temperature control unit.

6. The system according to claim 5, wherein, The at least one open or closed photobioreactor is a tubular, flat-plate, or pool-type bioreactor.

7. The system according to claim 5 or 6, wherein, The temperature in the at least one open or closed photobioreactor is between 10°C and 35°C.

8. The system according to any one of the preceding claims, wherein, The units (d) and (e) include a filtration unit, a drying unit, and an extraction unit.

9. The system according to any one of the preceding claims, wherein, The control and monitoring unit (f) includes at least one sensor and actuator to control culture parameters, nutrient concentration and greenhouse gas emissions.

10. A method for in-situ conversion of animal waste using a field modular system according to any one of claims 1 to 9, comprising the following steps: (i) Processing animal waste to obtain a homogenized, nutrient-rich liquid fraction; (ii) Use the homogenized, nutrient-rich liquid fraction obtained in step (i) as a culture medium to culture microalgae in a culture unit; (iii) Collect and process the microalgae culture produced in step (ii) to obtain a homogenized, nutrient-rich microalgae-derived colloidal suspension; (iv) Separate the solid / liquid homogenized nutrient-rich microalgae-derived colloidal suspension to obtain two byproducts: a processed nutrient-rich, bioabsorbable, nitrogen-, phosphorus-, and potassium-rich liquid microalgae culture fraction and a processed carbon-rich solid inactivated microalgae culture fraction.

11. The use of the processed, nutrient-rich, bioavailable, nitrogen-, phosphorus-, and potassium-rich liquid microalgae culture fraction obtained by the method according to claim 10 as an agricultural phytoremediation agent or a natural soil conditioner.

12. The use of the processed carbon-rich solid inactivated microalgae culture obtained by the method according to claim 10 as a recovery agent for toxic substances or pollutants in animal waste farms.

13. Use of the processed carbon-rich solid inactivated microalgae culture fraction obtained by the method of claim 10 as animal feed.

14. The use of the processed carbon-rich solid inactivated microalgae culture fraction obtained by the method of claim 10 as agricultural fresh compost or a natural soil conditioner.

15. The use of the system according to any one of claims 1 to 9 in a farm for reducing greenhouse gas and carbon emissions therefrom.

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