Microalgae as Biofertilizers: A Sustainable Way to Improve Soil Fertility and Plant Growth

: The intensification of agricultural production in response to the global population increase and the growing demand for food has raised significant concerns regarding environmental impacts over the past few decades. Currently, modern agriculture aims to improve the quantity and quality of crop yield, minimizing the negative effects of treatments on the environment. Recently, microalgae have found extensive application as a valuable biological resource across multiple industries, including the food sector, biofuel production, and the pharmaceutical industry. In agriculture, microalgae have been seen as a promising and sustainable alternative to agrochemicals, offering a range of benefits to improve soil fertility, optimize nutrient management, and reduce reliance on synthetic fertilizers. In general, microalgae have demonstrated efficient nutrient cycling abilities, assimilating and converting essential nutrients, such as nitrogen, phosphorus, and potassium, into forms readily available for plants. Additionally, they produce bioactive substances, including phytohormones, which have a direct impact on the physiological processes of plants and promote their growth. Microalgae can also establish beneficial interactions with other soil microorganisms, supporting the growth of beneficial bacteria and fungi, thus promoting a healthy soil microbiome. On the other hand, as photosynthetic microorganisms, microalgae harness sunlight to convert carbon dioxide (CO 2 ) into organic matter through photosynthesis. This ability allows them to sequester carbon and contribute to sustainable agriculture by reducing greenhouse gas emissions. The present work provides an overview of the potential of microalgae as biofertilizers, highlighting their unique characteristics, benefits, and main limitations for effective implementation in agriculturally sustainable practices.


Introduction
Increasing agricultural production while preserving the environment has been a huge challenge faced by the scientific community [1,2]. In recent decades, the intensification of agricultural production has increased to meet the growing demand for food, but it has also led to significant negative impacts on the global environment [3]. The excessive use of chemical fertilizers is a common strategy to ensure high crop yields and make the agricultural sector economically profitable [4]. However, this practice brings serious environmental problems, including greenhouse gas emissions, degradation of soil and water quality, eutrophication of water, and biodiversity loss [5,6]. In addition, the improper use of chemical fertilizers can have significant implications for human health [7]. The persistent application of chemical fertilizers can result in the accumulation of hazardous heavy metals (e.g., cadmium, arsenic, and lead) and nitrates in the soil [8][9][10]. This contamination not only poses a threat to soil quality but also leads to the bioaccumulation of

Chemical Fertilizers and Their Environmental and Health Implications
Soil fertility is crucial for plant development and directly influences their yield. Fertile fields are invaluable assets to farmers. However, inadequate agricultural practices can lead to soil degradation and depletion. The application of chemical fertilizers is a common strategy to enhance soil fertility and provide essential nutrients, such as nitrogen, phosphorus, and potassium, for the development of plants [21]. These synthetic products are available in various forms, including solid or liquid compositions, and represent a widely adopted practice in modern agriculture due to their ability to quickly supply plants with the nutrients required for growth and increased agricultural productivity [21]. However, the over-reliance on chemical fertilizers can result in several adverse consequences on soil health and the environment, such as changes in soil pH, pest development, acidification, and soil crust formation, causing a decrease in soil organic carbon and beneficial species, thus affecting plant growth and yield, and potentially contributing to the emission of greenhouse gases [8,[22][23][24][25][26]. In addition, one of the main challenges associated with conventional fertilizers is their tendency to promote imbalanced nutrient uptake by plants [8]. Continuous reliance on these fertilizers without proper soil management can lead to the depletion of certain nutrients and the accumulation of others, disrupting the natural nutrient cycling processes [27]. Several studies have demonstrated that inappropriate use of chemical fertilizers can disrupt the equilibrium among the macronutrients, such as nitrogen, phosphorus, and potassium, leading to a decline in crop yields [28][29][30]. Sun et al. [28] evaluate the impact of different nitrogen fertilization rates on the physiological attributes of banana leaves, including plant metabolic enzymes, soluble matter, and chlorophyll, as well as soil properties (soil organic matter, enzymes, and available nutrients), and banana crop yield. The results revealed that higher nitrogen fertilization rates (≥414 g of nitrogen per plant) led to a significant reduction in soil available phosphorus content, available potassium content, glutamine synthetase activity, and sugar and soluble protein contents, when compared to lower nitrogen application rates. These results led the authors to conclude that excessive nitrogen fertilization negatively affects soil fertility and crop yield.
The persistent application of nitrogen fertilizers can also result in the accumulation of nitrate in the soil or even in plants. In general, nitrate accumulates prominently in nonleguminous plants and green leafy vegetables, like lettuce [31,32]. However, if not absorbed directly by plants, these nitrates can be leached into waterways [4]. The consumption of nitrate-contaminated groundwater or vegetables with high nitrate content may lead to serious pathological conditions [33]. Although nitrate itself is not harmful to health, its conversion to nitric oxide, nitrite, and N-nitroso compounds by salivary enzymes and oral bacteria can present potential risks, including the development of methemoglobinemia syndrome and gastric/bladder cancer [12,32,33].
Accumulation of heavy metals in agricultural soils is another common problem due to intensive chemical fertilizers application [34]. Generally, chemical fertilizers, such as superphosphate fertilizers, can contain high levels of heavy metal contaminants, such as cadmium, cobalt, copper, lead, zinc, chromium, mercury, and nickel [9,35]. Thomas et al. [36] investigated the effect of phosphorus fertilizer on heavy metal accumulation in soil and in Amaranth caudatus. According to their results, the concentration of zinc was found to be higher not only in the amended soil but also in the plants cultivated in that soil. More recently, Wei et al. [34] analyzed the availability and accumulation of heavy metals in greenhouse soils. Their results indicated that repeated application of chemical fertilizers significantly increased the accumulation of cadmium, copper, and zinc.
Cadmium is an element that is highly toxic to plants as it inhibits the fixation of carbon, decreases chlorophyll content, and reduces photosynthetic activity [37]. This element also affects the iron and zinc uptake, resulting in leaf chlorosis [38], and interferes with the uptake and transport of calcium, phosphorous, magnesium, potassium, and manganese [39]. In humans, cadmium primarily exerts its toxic effects on the kidney. Cadmium exposure has been strongly linked to renal dysfunction and kidney damage, leading to conditions such as polyuria and proteinuria [40]. However, other health adverse effects on pulmonary [41], cardiovascular [42], and musculoskeletal [43] systems have also been reported. In addition, cadmium is recognized as a human carcinogen [44].
Other studies have shown that arsenic and lead, which are commonly found in conventional fertilizers, can accumulate in the soil and plants, thus representing a risk to human health [45,46]. Chronic exposure to arsenic has been associated with numerous adverse health effects, such as arsenicosis, cardiovascular diseases, neurological disorders, respiratory diseases, diabetes, various types of cancers, and renal and reproductive diseases [47]. Lead also presents several adverse health effects. Even at very low blood levels (10 µg dL −1 BPb-blood lead level), lead directly impacts the hematological system, restraining the synthesis of hemoglobin and reducing the lifespan of erythrocytes [48]. Lead exposure also affects the cardiovascular, renal, nervous, skeletal, and reproductive systems [48,49].

Microalgae as Biofertilizers
The use of microalgae in agriculture offers a range of advantages, not only for the environment but also for the health of the soil and, subsequently, for the crops [4]. In general, microalgae exhibit growth-enhancing properties through three distinct modes, namely biofertilizers, biostimulants, and biopesticides [50,51]. Figure 1 provides a comprehensive overview of the main activities linked to microalgae-based products in agricultural practices, highlighting their action mode and impact on crop production.
Biofertilizers are probably the most common mode of utilization of microalgae biomass [50]. The meaning of biofertilizers has evolved over the years, and currently, they can be defined as preparations containing living microorganisms that stimulate plant growth by improving nutrient availability in the plant rhizosphere [52]. In contrast to chemical fertilizers, the introduction of beneficial microorganisms into the soil through biofertilizers can help to create a more diverse and healthy soil ecosystem, promoting nutrient cycling, and improving soil structure and fertility. Table 1 provides a comparison of different types of biofertilizers and traditional fertilizers, highlighting their respective advantages, disadvantages, uses, and benefits they provide in terms of promoting plant growth and their environmental impact. Although biofertilizers containing bacteria or fungi as primary components have been extensively studied, showing good results for a wide variety of crops [53,54], the use of microalgae has gained strength due to their unique properties [4]. Microalgae are a valuable source of organic carbon when applied to the soil [3]. This is a key aspect in agriculture, considering the depletion of soil organic carbon is a significant form of degradation in croplands, leading to decreased soil quality and fertility [55]. Microalgae can assimilate organic carbon into their biomass through photosynthesis, and several strains can release exopolysaccharides (EPS), which help as a carbon source and carbon sequestrant, enhancing soil aggregation and stabilization [3,56]. In fact, cyanobacteria and microalgae have been proven to be highly efficient tools in the restoration of degraded soils affected by excess salts [57]. These organisms can produce a layer of EPS on the soil surface, which retains organic carbon, nitrogen, and phosphorus. Moreover, they add organic matter and nitrogen to the soils, contributing to soil particle aggregation, increased permeability, and aeration [58]. The EPS produced by these organisms can also serve as a reservoir for water storage during water scarcity conditions and remain metabolically active when hydrated [59].
Certain cyanobacteria, in addition to having the ability to perform photosynthesis and fix atmospheric carbon, are also capable of fixing atmospheric nitrogen [60]. The protein complex nitrogenase is responsible for the conversion of atmospheric nitrogen to ammonia, which then gives rise to various nitrogenous compounds, such as polypeptides, amino acids, vitamins, and auxin-like substances, which can be subsequently released through microbial secretion or degradation after cell death [58]. Therefore, the utilization of cyanobacteria in soils enhances nitrogen availability, which is an essential nutrient for plant growth, and their beneficial effects in agricultural soils for the production of several crops have been demonstrated. Swarnalakshmi et al. [61] reported that the application of a biofilm containing the cyanobacterium Anabaena torulosa to a wheat crop resulted in a significant increase in soil nitrogen availability. Osman et al. [62] evaluated the effect of Oscillatoria angustissima and Nostoc entophytum on the production of pea plants and observed that the inoculation of soil with these species significantly increased the germination percentage, photosynthetic pigments, and growth parameters (root depth, shoot length, dry weigh and leaf area) of this plant. According to the results, the combination of Oscillatoria angustissima suspension with two different doses of chemical fertilizer (50% and 100%) resulted in greater shoot growth and dry weights of germinated peas compared to treatments with Nostoc entophytum. On the other hand, the Nostoc entophytum treatments with different doses of chemical fertilizer (50% and 100%) showed more significant increases in leaf area and root growth compared to inoculation with Oscillatoria angustissima. Furthermore, the authors verified that this increase was correlated with a higher nitrogen fixation activity by both cyanobacteria.
Microalgae also have an influence on soil microbial populations (diversity, community composition, and activity), and have the intrinsic capability to produce a wide variety of bioactive metabolites that have a positive impact on plant growth, as well as help control pests and pathogens [3]. Among these substances, phenolic compounds, carotenoids, terpenoids, polysaccharides, free fatty acids, and phytohormones are of remarkable interest since they have already been recognized as promoters of plant growth [51]. Table 2 summarizes the role of the main classes of bioactive compounds found in microalgae in agricultural practices. Table 1. Comparison of key characteristics of chemical fertilizers and different types of biofertilizers (adapted from [4]).

Characteristics Chemical Fertilizers
Biofertilizer Fungi Bacteria Microalgae/ Cyanobacteria Formation of symbiotic bonds between plant roots and soil microorganisms. An important role in the nitrogen cycle is making nitrogen available in a form that plants can use.
Ability to promote phosphorus solubilization.
The ability for nitrogen fixation by individual strains, phosphorous solubilization, and hormone production promote plant growth. Ability to capture CO2 and reduce greenhouse emissions during the addition of organic carbon to the soil.
Gradual release of nutrients for plant uptake.
Negative environmental impacts through degradation of soil, water contamination, and induction of eutrophication. Industrial-scale production and widespread use in the agricultural sector.
Not applicable.
Applicable. Another important aspect is that the biomass produced by microalgae can be converted into nutrients that are readily accessible for utilization by other plants [63,64]. The cyanobacterium Spirulina (Arthrospira platensis and Arthrospira maxima) and the green algae Chlorella vulgaris have demonstrated significant capacity for the removal of nutrients (nitrogen and phosphorous) from effluents [65,66], making them excellent candidates for soil bioremediation purposes [64]. Based on the study developed by Chaiklahan et al. [65], Spirulina platensis demonstrated a remarkable capacity for removing nutrients from wastewater with average rates of bicarbonate, total nitrogen, and phosphorus removal being 380 mg L −1 /d, 34 mg L −1 /d, and 4 mg L −1 /d, respectively. Pooja et al. [66] also showed the potential of Chlorella vulgaris to remove nutrients from wastewater, where the concentration of nitrates and phosphates were reduced by 93% and 86%, respectively, in the final treated sewage wastewater post-microalgae harvest.
Microalgal/cyanobacterial biomass also contains other essential microelements crucial for plants' growth and development, such as potassium, magnesium, sulfur, and iron [51]. These elements are involved in redox reactions and play significant roles in plants' metabolism [51]. Some cyanobacteria can also promote the solubilization of other important nutrients, such as phosphorus (another essential nutrient for plant metabolism) and other micronutrients like zinc, copper, and iron [60,67]. Afkairin et al. [68] evaluated the capacity of Anabaena sp. to solubilize phosphorus using two different organic phosphorous sources (rock phosphate and bone meal) under laboratory conditions and compared it with a commercially available bacterial consortium (Mammoth P). According to the results, the cyanobacteria treatment solubilized more phosphorous than Mammoth P, leading the authors to conclude that this can constitute an effective strategy for improving soil phosphorous availability to plants.
In addition to its influence on soil health and promoting plant protection, the application of microalgae and cyanobacteria can stimulate plant growth and development, and this can be observed in an improvement in germination rates and plant characteristics, such as increased root length, increased number of leaves, increased leaf area, among others [51]. Several studies available in the literature have demonstrated the ability of Spirulina-and Chlorella-based fertilizers to enhance plant growth [69]. Wuang et al. [69] assessed the suitability of the biomass of Spirulina platensis as agricultural fertilizer for leafy vegetables, such as Chinese Cabbage (Brassica rapa ssp. chinensis), Arugula (Eruca sativa), Pak Choy (Brassica rapa ssp. chinensis), Bayam Red (Ameranthus gangeticus), Kai Lan (Brassica oleracea alboglabra) and White Crown (Brassica rapa ssp. chinensis, F1 hybrid). The results demonstrated the potential of Spirulina-based biofertilizers to promote plant growth, affecting various biometric parameters such as leaf number, plant height, root length, and weight, as well as improving the germination process. In comparison to the controls, the supplementation of Spirulina platensis for leaf vegetables resulted in enhanced plant growth in all the tested crops. Pak choy showed the highest increase in the number of leaves from 10.33 to 13.00 and root length from 2.33 to 7.00 cm. Bayam red and Arugula exhibited a significant increase in plant height, with Arugula increasing from 16.03 to 25.37 cm and Bayam red from 31.33 to 48.67 cm. Both vegetables also demonstrated an increase in fresh weight, with Bayam red increasing from 12.52 to 26.11 g, and Arugula from 10.67 to 12.67 g. Additionally, they displayed an increase in dry weight, with Bayam red increasing from 4.28 to 10.95 g, and Arugula from 7.48 to 9.06 g. The performance of the Spirulina-based fertilizer was comparable to that of chemical fertilizer in most plant growth parameters, and it showed favorable results for one tested species-Arugula.
Dineshkumar et al. [70] analyzed the effect of Chlorella vulgaris and Spirulina platensis at different concentrations on rice growth and productivity and determined their potential as biofertilizers to achieve maximum yield. Both types of microalgae had a positive impact on the main growth parameters of the plants, such as plant height, number of leaves, leaf area, as well as fresh and dry weight, allowing a reduction of up to 50 or 75% of the recommended nitrogen fertilizer dosage. According to the results, the plant height ranged from 51.9 cm to 63. Regarding the fresh and dry weights for Chlorella vulgaris, the values ranged from 8.744 g to 13.013 g for fresh weight and from 1.287 g to 1.915 g for dry weight. For Spirulina platensis, the values ranged from 12.87 g to 20.222 g for fresh weight and from 2.631 g to 2.976 g for dry weight. Furthermore, the authors also evaluated the biological activity and chemical properties of the soil, as well as the seed yield characteristics of the rice plants. In terms of the seed yield, the results showed notable improvements in rice yield parameters when microalgae were used. For Chlorella vulgaris, the number of seeds/pod ranged from 1.65 to 2.02, the weight of 100 seeds varied from 3.43 g to 4.03 g, the seed weight/plant ranged from 2.07 g to 4.24 g, and the seed yield/pod was 14.38 g to 19.33 g. Spirulina platensis also presented good results, with the number of seeds/pod ranged from 2.22 to 2.51, the weight of 100 seeds varied from 5.94 g to 6.42 g, the seed weight/plant range from 7.12 g to 11.57 g, and the seed yield/pod was 15.56 g to 21.78 g. Regarding the chemical properties of the soil and the biological activity, the application of microalgae resulted in increased nitrogenase and dehydrogenase activity, an overall improvement in the availability of macronutrients in the soil, and a reduction in electric conductivity and soil pH.
The symbiotic relationship between plants and microalgae has also been demonstrated in other studies [71][72][73]. Uyar and Mısmıl [71] showed the benefits of growing Mentha spicata with a culture of Chlorella vulgaris in a hydroponic system. Their findings indicated that the co-cultivation of plant and microalgae under aeration conditions had a significant influence on the increase in plant weight, due to the development of new shoots and leaves. According to the results, the average weight gain of mint seedlings where Chlorella vulgaris were co-cultivated was 0.47 g, while the average weight gain in the control group was 0.29 g. Furthermore, through the assessment of photosynthetic pigments on the plant, no signs of stress were observed during its growth. Table 3 shows some representative examples of the application of some microalgae and cyanobacteria species and the benefits they generate in crops. Increase seedling attributes and seed enzymatic activities; Improve mobilization of nutrients at the seed stage. [88] * References.
Microalgae also create endosymbiotic relationships with bacteria, allowing them to grow in various ecosystems by mutually benefiting from each other's presence. It has been demonstrated that the utilization of consortia of microalgae and other microorganisms, even in extreme environmental conditions (e.g., desert), enhances soil fertility by improving properties like water retention, pollutant removal, overall stability, and providing plants with a more favorable substrate [89].

Production and Application Techniques of Microalgae-Based Fertilizers
The contribution and impact of microalgae-based biofertilizers on soils depend on various factors, such as the method of introducing microalgae into the soil and the state of the biomass, whether it is fresh, dry, or digested [3]. Considering this, the production of biofertilizers utilizing microalgae can be a relatively straightforward process. In all cases, the initial step involves the growth and harvest of microalgae biomass.
Currently, there are two main cultivation systems for microalgae production, namely open systems and closed systems, each with its own advantages and challenges. Generally, open systems involve growing microalgae in shallow open ponds or tanks of circular or raceway type, exposed to natural sunlight [90]. Open systems offer several advantages, including ease of construction, operation, and lower costs compared to closed systems [90]. Despite being suitable for large-scale production, these systems are more vulnerable to contamination, evaporation, and fluctuations in weather conditions, which can affect the productivity and stability of the cultures [90,91]. Closed systems, on the other hand, are designed to isolate microalgae from the external environment and allow for overcoming some problems detected in open systems [90]. These systems include photobioreactors, which provide better control over culture conditions and reduce the risk of contamination. Although closed systems are more expensive to set up and maintain, they offer higher productivity, allowing the production of up to three times more biomass than open systems [90,92]. Harvesting microalgae involves separating algal biomass from the growth medium, and despite the vast amount of literature available on the topic, there is a general consensus that this step remains the major bottleneck in the commercial production of microalgae biomass [50]. This can be attributed to microalgae properties, including cell size, shape, and surface charge, which require custom solutions, thus limiting commercial scalability [93]. Currently, there are several techniques that have been developed and evaluated for harvesting microalgae biomass, which can include mechanical, chemical, biological, and electrical techniques [50,94,95]. Mechanical methods are generally the most commonly used techniques for harvesting microalgal biomass [95]. However, from an economic point of view, a combination of flocculation followed by centrifugation or pressure filtration can enhance harvesting efficiencies and reduce operation and maintenance costs with lower energy inputs (less than 0.1 kWh·kg −1 algae) [96]. Other techniques have been used to harvest and concentrate the algal biomass. Table 4 summarizes the main advantages and limitations of the most frequently used techniques for microalgal harvesting.
The last step in typical microalgae biomass production involves dewatering and downstream processing for further production of commercial products [50]. Generally, the dehydration process requires high energy consumption, being an area of great concern that acts as a limiting factor in the commercialization of microalgae products [50]. However, the selection of the appropriate method depends on the end product, namely its value and properties [50]. For the biofertilizer application, primarily as a source of macro and micronutrients, the dewatering process does not affect the stability of the biomass. However, for use as pesticidal extracts or even as biostimulants, dewatering plays a fundamental role as many compounds are heat sensitive (e.g., phycobiliproteins, antioxidants, and secondary metabolites), requiring optimization of dewatering and further downstream processes [50].  [94,95]).

Harvesting Technique Advantages Limitations
Sedimentation Simple and inexpensive method; Do not require complex equipment.
Slow process, may take a long time for complete separation; May result in lower biomass recovery; Possibility of biomass degradation.

Flotation
Suitable for large-scale operations; Low-cost method; Short operation time; Low space requirement.
Requires the use of chemical surfactants.

Filtration
Efficient separation of microalgae from the medium; Cost-effective; Can achieve high biomass recovery rates; Suitable for both large and small-scale operations; Low energy consumption (natural and pressure filter); It can be used for the separation of shear-sensitive species.
Requires regular maintenance of membranes; Membrane fouling/clogging and replacement increase operational costs.

Centrifugation
Rapid and efficient separation of microalgae; High biomass recovery rates; Applicable for almost all microalgae species.
Expensive equipment and maintenance costs; High energy consumption; Possibility of cell damage. Suitable for the recovery of high-value products.

Coagulation/ flocculation
Aggregates microalgae, making harvesting easier; It can be used in combination with other methods for improved efficiency; Cost-effective for large-scale operations; Applicable to a wide range of microalgae species.
Requires the use of chemicals for flocculant formation; Chemicals may be expensive; Recycling of culture medium is limited.

Electrical based processes
Do not require the use of chemicals; Applicable to a wide range of microalgae species.
High energy consumption and equipment costs.
In the case of biofertilizers, depending on the intended formulation, i.e., solid or liquid formulations, the microalgae production differs slightly [4]. For liquid formulations, the process is simpler, involving scaling up the microalgae culture to an industrial level and supplementing it with additives to enhance cell viability over extended periods [97]. These formulations represent one of the most affordable approaches to using microalgae as a biofertilizer and can be applied during seed germination and throughout the soil cultivation phase [4]. On the other hand, solid formulations of the biofertilizer require additional steps after microalgae biomass growth and harvest, which involve the removal of water through techniques such as air/oven drying [98], lyophilization [99], or carbonization [81]. The simplest and most cost-efficient method of obtaining a solid biofertilizer is by allowing the biomass to naturally air-dry under direct sunlight. Lyophilization is an alternative method for drying microalgae cells, but its industrial-scale implementation is highly improbable due to the expensive processing costs per sample [4]. On the other hand, hydrothermal carbonization constitutes a promising technique for treating microalgae biomass, converting it into hydrocarbon. This process involves high pressures and temperatures in a liquid medium, resulting in the production of valuable nutrients that can be found in the liquid phase after the treatment, such as organic nitrogen, nitrates, ammonium, and orthophosphate derived from polyphosphates present in the raw material [4].
Currently, microalgae-based biofertilizers can be applied through various methods. The simplest approach involves directly applying the microalgal biomass or its formulations to the soil. This method ensures the distribution of nutrients and beneficial compounds throughout the root zone, promoting plant growth and soil fertility. Microalgae extracts or suspensions can also be sprayed onto the leaves of plants. This technique, known as foliar fertilization, is a widely used crop nutrition strategy that involves supplying plants with the necessary nutrients by directly spraying an aqueous solution onto the leaves, which absorb these nutrients through their cuticles and stomata [100]. As demonstrated by Youssef et al. [100], the application of microalgae through foliar spraying and soil drenching methods represents a promising approach to enhance the growth and productivity of chia plants (Salvia hispanica L.) under alkaline stress conditions. Their results revealed that foliar application with Arthrospira platensis showed better performance in herb fresh and dry weights, inflorescence number, and leaf pigments, when compared to other strains (Chlorella vulgaris, Nostoc muscorum, and Anabaena azollae). The immersion treatments with Arthrospira platensis also showed a biostimulant effect on plants, having a positive effect on all yield metrics.
Microalgae-based biofertilizers can also be used in seed germination. Bumandalai [101] evaluated the effect of Chlorella vulgaris as a biofertilizer on the germination of cucumber and tomato seeds. In this study, seeds were germinated in a culture medium containing algal strain and grown for 3, 6, 9, and 12 days. The results showed that the Chlorella vulgaris suspension had a significant impact on seed growth, leading to improved germination compared to the control group. The most effective treatments for the root and shoot lengths of tomato and cucumber seeds were 0.17 g/L and 0.25 g/L of algal suspension, respectively.
Currently, the co-cultivation of microalgae and plants has also been explored in hydroponic systems. Barone et al. [72] and Zhang et al. [73] demonstrated the positive impact of this approach. In both studies, the hydroponic co-cultivation system proved advantageous for the growth of both microalgae and plants, leading to a biostimulant effect on crop biomass attributed to algal photosynthesis. Based on the results obtained by Barone et al. [72], the co-cultivation of microalgae with plants resulted in a substantial increase in root length, reaching approximately 130% for Chlorella vulgaris (18.5 cm) and Scenedesmus quadricauda (15.5 cm). Zhang et al. [73] also showed that the co-cultivation of Chlorella infusionum and tomato in a hydroponic system led to an increase in the algal and crop biomass productivities, reaching values of 32 ± 5 g m −3 d −1 and 54.24 ± 1.81 g dm −3 d −1 , respectively. These values were significantly higher than the algal biomass productivity (16 ± 5 g m −3 d −1 ) observed in the algal monoculture and the crop biomass productivity (33.97 ± 7.58 g dm −3 d −1 ) observed in the crop monoculture. Additionally, the authors also observed that the root respiration and exudation of the crops acted as carbon sources, promoting the growth of microalgae and increasing their biomass.

Limitations of Microalgae Based-Biofertilizers
Microalgae have shown great potential in agriculture due to their ability to provide beneficial compounds and promote sustainable farming practices. However, there are some limitations that hinder their widespread use in agricultural settings. One of the main limitations to consider is the high cost associated with the production of microalgae biomass and the extraction of the metabolites of interest [51]. In general, the production of microalgae can be a very expensive process, with relatively low biomass productivity [51,102,103]. Although cultivation is widely recognized as the primary cost contributor for algal-based products, harvesting and dewatering of microalgae biomass are equally significant factors impacting the total costs [96]. Numerous studies indicate that harvesting costs account for approximately 20 to 30% of the total production costs [94,[104][105][106][107]. Generally, the considerable capital expenditure and energy consumption are mainly attributed to the need for processing large volumes of dilute algae solutions, as well as the small size of microalgal cells [108,109]. Fasaei et al. [96] evaluated the techno-economic performance of 28 different scenarios for large-scale microalgae harvesting and dewatering. The study explored the harvesting and dewatering of algal streams with dry matter ranging from 0.05% to 15% in an open pond system. The results showed that the associated costs varied between EUR 0.3 and 2.0 ·kg −1 algae, with energy consumption ranging up to 4.5 kWh·kg −1 algae. On the other hand, in closed systems with higher dry matter content, the production costs and energy consumption significantly decreased to below EUR 0.5 ·kg −1 algae and below 0.5 kWh·kg −1 algae, respectively. The findings revealed that harvesting and dewatering accounted for 3-15% of the total production costs of algae biomass. However, the maximum capacity of the method was limited, necessitating a considerable number of units for large-scale cultivation, which increased the contributions of investments and labor to the overall costs. The authors also compare single-step harvesting and dewatering or two-step operations and concluded that the latter, such as pressure filtration followed by spiral plate technology or centrifugation, was found to be more attractive from an economic point of view. The study also considered the use of flocculation in combination with a second unit operation, which required less than 0.1 kWh·kg −1 algae. However, the cost-effectiveness of flocculation methods was balanced by additional expenses for flocculants and the relatively lower biomass recovery rate compared to mechanical concentration methods. Furthermore, the impact of flocculants on the water recycle stream and fractionation and extraction steps may limit their widespread use. Labor costs were a significant factor in all scenarios, making them a crucial consideration in the analysis. Investing in a higher degree of automation could help offset labor costs and improve overall efficiency in large-scale microalgae harvesting and dewatering processes.
The application of microalgae extracts requires the extraction of bioactive compounds, and most of the commonly used methodologies are very costly and require large quantities of organic solvents [20,51]. In this context, the costs associated with the production and processing of microalgae biomass result in a higher cost of biofertilizers, biostimulants, and biopesticides, making them less competitive compared to chemical-based alternatives. Due to these factors, applications that yield higher revenues, such as the extraction of bioactive compounds for the nutraceutical, pharmaceutical, and cosmetics industries, are generally preferred for microalgae biomass [51].
In recent years, the integration of microalgae production with wastewater treatment has been proposed as an alternative to reduce the cost of microalgae production [110]. Figure 2 shows a general scheme illustrating the potential of microalgae production for simultaneous wastewater treatment and biofertilizer production.
The recovery of nutrients from wastewater and organic wastes by microalgae can contribute to the implementation of a circular economy, where waste streams are transformed into valuable resources, promoting resource recycling, reducing waste generation, and fostering environmental sustainability. This approach not only reduces the cost but also minimizes the carbon footprint associated with traditional microalgae cultivation [111].
Although the use of microalgae biofertilizers derived from wastewater treatment offers several advantages, there are also some disadvantages to consider. Microalgae have the ability to accumulate heavy metals and other contaminants present in wastewater, and, if not properly managed, the use of microalgae biofertilizers derived from wastewater treatment can potentially introduce these contaminants into the agricultural soil, posing risks to crop growth and human health. Variable nutrient composition is another limitation of microalgae biofertilizers derived from wastewater treatment. The nutrient composition of microalgae biofertilizers can vary depending on the characteristics of the wastewater used for cultivation. This variability can pose a challenge to maintaining consistent nutrient content and ratios in biofertilizers, which can affect their effectiveness in promoting plant growth. On the other hand, scaling up microalgae cultivation for biofertilizer production from wastewater treatment can be technically and economically challenging. It requires the establishment of large-scale cultivation systems and the development of efficient harvesting and processing methods to handle the high volumes of biomass generated from wastewater treatment facilities.

Figure 2.
General scheme of biofertilizer production from microalgae biomass produced in a wastewater treatment plant (adapted from [112]). Development of efficient harvesting and processing methods to handle the high volumes of biomass generated from wastewater treatment facilities.
Finally, another important limitation that has prevented the widespread use of microalgae in agriculture is the insufficient understanding of the interactions between microalgae (and their extracts), plants, and the environment [51]. Despite numerous studies conducted in this area, the great diversity of photosynthetic organisms, along with the high number of metabolites that can be extracted from them, represent a great challenge for the deep understanding of the effects and mechanisms of microalgae on soil and plants [3,51].

Conclusions
In recent years, there has been a growing trend towards the use of biofertilizers to overcome dependence on synthetic fertilizers and non-renewable resources. Numerous studies have shown that microalgae are an important source of bioactive compounds capable of regulating several plant response mechanisms, including improving soil quality and plant nutrition, providing protection against biotic and abiotic factors, and stimulating plant growth. However, despite the benefits, the success of microalgae-based biofertilizers depends on the cost-effectiveness of biomass production and processing, as well as on the implementation of energy-efficient technologies in the production process. In addition, further research studies are necessary to assess the specific effects of individual metabolite/biomass on crops, in order to determine the most suitable substances for agricultural activities.
Funding: This research was funded by Instituto de Desenvolvimento Empresarial, IP-RAM, by the Programa Operacional "Madeira 14-20" (co-funded in 85%, by Fundo Europeu de Desenvolvimento Regional (FEDER) and 15% by Autonomous Region of Madeira Government Budget), in Priority Axis 1-"Reinforce Research, Technological Development and Innovation", which includes Investment Priority 1. b-"Promotion of investment by companies in innovation and research, the development of links and synergies between companies, Research and Development (R&D) Centers", through application M1420-01-0247-FEDER-000046 submitted to the Incentive System for the Production of Scientific Knowledge and Technology of the Autonomous Region of Madeira-PROCiência 2020, by the company "AGROFOOD TECHIS, UNIPESSOAL LDA", legal person and single registration nº 515777234.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.