Utilization of Microalgae for Urban Wastewater Treatment and Valorization of Treated Wastewater and Biomass for Biofertilizer Applications

Abstract

Rapid urbanization has substantially increased freshwater consumption and consequent wastewater generation. The produced wastewater is an abundant resource of phosphorus, nitrogen, and organics. Currently, well-established activated sludge processes are utilized in conventional wastewater treatment plants to remove organics. However, removing nitrogenous and phosphorus compounds continues to be challenging and energy-intensive for urban wastewater treatment plants. Therefore, the current study aims to understand how photosynthetic microalgae can recover phosphorus and nitrogen from urban wastewater and how wastewater-grown microalgae biomass may be used as a biofertilizer and biostimulant. Utilizing microalgae biomass treated with urban wastewater as a biofertilizer promotes plant growth in a manner similar to other organic manures and conventional fertilizers while minimizing nutrient loss to the soil. Furthermore, the microalgal recovery of nutrients from urban wastewater could have potential energy reductions of 47% and 240% for nitrogen and phosphorus, respectively. In addition to producing treated wastewater suitable for a variety of irrigation systems, microalgae biomass is a potential sustainable alternative resource that could reduce conventional inorganic fertilizer usage.

1. Introduction

Human population and food demand are predicted to grow by 60–100% by 2050, resulting in significant freshwater consumption. As a result of urbanization, industrialization, food security, and the need to support global economic growth, significant amounts of urban wastewater (UW) would be produced [1,2]. The generated UW comprises several organic, inorganic, and synthetic compounds, in addition to being an abundant resource of nitrogenous and phosphorus compounds [3,4,5,6]. Releasing the nitrogen and phosphorus-rich UW directly into the environment could result in eutrophication and the leaching of harmful pollutants into the soil and the air [7]. Therefore, wastewater treatment plants (WWTPs) are used to treat urban wastewater. Primary and secondary sludges are produced from the processing of urban wastewater. The combination of primary and secondary sludge is often further stabilized using an anaerobic digestion (AD) process. Methane is recovered as part of the proportion of organic carbon from the sludge [8]. Later, the anaerobically digested sludge is concentrated, and the liquid digestate is separated and returned to the WWTP’s treatment system, while the concentrated or thicker sludge, which is high in nitrogen and phosphorus, is dumped or landfilled in a designated disposal area [8]. WWTPs continue producing methane from wastewater sludge, even though aqueous phase reforming (APR) techniques are currently being studied to convert the oxygenated organic molecules in wastewater to hydrogen at milder operating conditions [9,10]. In addition to potentially contaminating groundwater and the soil when it is landfilled, the sludge could also be incinerated as a means of volume reduction [11]. Therefore, nitrogen and phosphorus from WWTPs continue to build up in the environment and act as a source of pollution for both air and water.

Furthermore, the concentrated sludge and supernatant that is recycled back into the wastewater treatment process contribute to the additional nitrogen and phosphorus that anaerobic digestion cannot remove. Therefore, photosynthetic microalgae could potentially be utilized to remove or lower the nitrogen and phosphorus from various streams that originate from WWTPs. In addition to being able to use atmospheric carbon dioxide (CO₂), microalgae can generate biomass and high-value compounds (pigments and lipids) through the utilization of nitrogen (N) and phosphorus (P) in urban wastewater [12]. It has previously been shown that the microalgae could be successfully cultivated in open raceway ponds (ORPs), high-rate algal ponds (HRAPs), and different geometrically designed photobioreactors (PBRs) for achieving high biomass productivity and the simultaneous removal or reduction of N and P from several types of wastewater [13]. The microalgal biomass obtained through the phytoremediation of wastewater has undergone substantial research, mainly for producing gaseous and liquid biofuel and removing some recently emerging contaminants from urban wastewater [14,15]. However, the use of microalgae biomass cultivated in UWs as a potential feedstock for the production of biofertilizers and biostimulants needs more research. Additionally, phosphate and nitrogen fertilizers, which are often utilized in the form of urea and inorganic phosphorus forms, are needed by plants and crops. According to a report, the energy required to produce urea is around 30.1 GJ per ton [16]. The CO₂ emissions for the production of N and P fertilizers comprise 2.1 tons of ammonia (NH₃) and 0.343 tons of phosphorus pentoxide (P₂O₅), respectively [17]. The typical energy requirement for urban wastewater treatment is between 0.35 and 0.65 kWh/m³ or 1.26 and 2.34 MJ/m³ [18], whereas it takes 71,000 kWh/ton or 255.6 GJ/ton of energy to produce 1 ton of phosphorous at urban wastewater treatment plants combining sludge incineration and purification [19]. Thus, a potential strategy for recovering nitrogen and phosphorus from urban wastewater treatment facilities could involve the use of microalgae as a biofertilizer and biostimulant in soil and agricultural crops and plants. Therefore, one of the objectives of this review is to assess the potential of suitable microalgal strains to remove nitrogen and phosphate from urban wastewater. The review additionally studies the possibility of utilizing microalgae biomass subjected to urban wastewater treatment as a biofertilizer or biostimulant for various agricultural crops. The comparative energy economics of nitrogen and phosphorous are also studied, followed by a discussion of the potential and challenges associated with the use of microalgae-treated urban wastewater.

2. Potential Role of Microalgae in Conventional Urban Wastewater Treatment Process

Conventional WWTPs process wastewater by dividing it into a mainstream and a side stream [20]. The side streams could originate from the initial screening process or the primary clarifier just after the initial screening of the urban wastewater. The side streams are a rich source of N and P. However, the mainstream contains diluted N and P compounds and usually goes through an activated sludge process for the removal of water-soluble organics and a fraction of nitrogen and phosphorus. Later, the stream is passed on to the final clarifier to remove most of the biosolid before being discharged as a treated wastewater effluent (Figure 1). Thus, both the main and side streams finally result in effluents that could typically contain from 0.02 to 3 gL⁻¹ N and from 0.002 to 0.4 gL⁻¹ P [21,22]. As shown in Table 1, the urban wastewater originating from WWTPs in different countries contains 21 to 75 mgL⁻¹ total nitrogen (TN), and 2.3 to 35.2 mgL⁻¹ total phosphorus (TP).

UW contains varied amounts of total nitrogen and phosphorus (Table 1), even when it is collected and treated at different times inside the same WWTP. Urban wastewater has the potential to facilitate the growth of different species of photosynthetic microalgae. However, some forms of organic carbon and total nitrogen present in UW could also be taken up by microalgae in a mixotrophic mode, resulting in a higher microalgal biomass production that could eventually be utilized to produce not only biofuels but also biofertilizers or biostimulants [23]. The mechanisms by which various microalgae species take up nutrients and organic compounds like TN, TP, and COD from urban wastewater are briefly discussed in the section that follows.

Table 1. Urban wastewater characteristics form different wastewater treatment plants (WWTPs).

Country—Source	COD (ppm)	TN (ppm)	TP (ppm)	Reference
MWWTP, Zhoushan, China	317.26	53.92	4.90	[24]
WWTP, Koramangla region, South of Bangalore, India	676	64.6	28	[25]
MWWTP Wonju, South Korea	591	56	15.8	[26]
Greek Wastewater Treatment Plants (WWTPs), Greece	528	57	11	[27]
WWTP, Verona, Italy	390.5	52.3	5.24	[28]
WWTP, El Ejido, Spain	3744	52	11	[29]
WWTP, Beijing, China	301.4	81.9	8.03	[30]
WWTP, Mikkeli, Finland	730.2	61.24	11.07	[31]
WWTP, Rishikesh, Uttarakhand, India	496	24.45	9.6	[32]
WWTP, Santa Rosa Jauregui, Queretaro, Mexico	517	86	43	[33]
WWTP, Nestle Ghana Ltd., Ghana	2388	65.6	35.2	[34]
WWTP, Saint Paul, Minnesota, USA	484.8	44.9	7.9	[23]
WWTP, Turkey	375	46	4.9	[35]

Table 1. Urban wastewater characteristics form different wastewater treatment plants (WWTPs).

Country—Source	COD (ppm)	TN (ppm)	TP (ppm)	Reference
MWWTP, Zhoushan, China	317.26	53.92	4.90	[24]
WWTP, Koramangla region, South of Bangalore, India	676	64.6	28	[25]
MWWTP Wonju, South Korea	591	56	15.8	[26]
Greek Wastewater Treatment Plants (WWTPs), Greece	528	57	11	[27]
WWTP, Verona, Italy	390.5	52.3	5.24	[28]
WWTP, El Ejido, Spain	3744	52	11	[29]
WWTP, Beijing, China	301.4	81.9	8.03	[30]
WWTP, Mikkeli, Finland	730.2	61.24	11.07	[31]
WWTP, Rishikesh, Uttarakhand, India	496	24.45	9.6	[32]
WWTP, Santa Rosa Jauregui, Queretaro, Mexico	517	86	43	[33]
WWTP, Nestle Ghana Ltd., Ghana	2388	65.6	35.2	[34]
WWTP, Saint Paul, Minnesota, USA	484.8	44.9	7.9	[23]
WWTP, Turkey	375	46	4.9	[35]

Nutrient Removal Mechanism from Urban Wastewater by Microalgae

The microalgal removal efficiency of nitrogenous and phosphate-containing components from urban wastewater is reliant on their concentrations and ratio and biotic and abiotic factors affecting microalgal growth [12,36]. Previous studies have shown that for high nitrogen and phosphate removal, as well as for maximal microalgal biomass productivity, the N/P ratio should fall between 5 and 30 [37]. As indicated in Table S1 (see Supplementary Materials), the biomass densities were found to be higher for N/P ratios between 15 and 20. The low biomass density for Scenedesmus sp. (Meyen) could be due to the low nitrogen concentration in the UW and the low light intensity for the flask culture [38]. Nevertheless, urban wastewater is usually a rich source of nutrients for microalgal cultivation.

The typical nitrogen and phosphorus contents in microalgae biomass with a biomass density of 0.5 gL⁻¹ are 0.005–0.05 mg/L and 0.0005–0.003 mg/L, respectively [39,40]. As the microalgae grow in the wastewater, the cells consume these elements to produce their biomass; therefore, the higher the microalgal biomass density, the higher the recovery rates for nitrogen and phosphorus would be. However, the growth of any microalga and its biomass concentration in the wastewater would also depend on the concentration and types of nutrients (TN, TP, etc.), among other abiotic factors.

Table 2. (a) Nitrogen recovery by various microalgae from urban wastewaters. (b) Phosphorus recovery by various microalgae from urban wastewaters.

Microalgae	Initial Nitrogen Conc. (mg/L)	Initial Biomass Density (g/L)	Operating Conditions	Final Biomass Density (g/L)	Nitrogen Removal (%)	References
(a)
Arthrospira platensis	20.81 *	0.05	- Lab-scale - Temperature: 25 °C - Light intensity: 2000 lux, 3000 lux, 4000 lux - Daily illumination time: 8 h, 12 h, 16 h - Period: 10 days	0.34	≈100	[41]
Scenedesmus obliquus (Turpin)	18.52 *	0.05	- Lab-scale - Temperature: 25 °C - Light intensity: 4000 lux, 6000 lux, 8000 lux - Daily illumination time: 8 h, 12 h, 16 h - Period: 10 days	0.36	93.8	[41]
Chlorella sorokiniana	111.6	0.99	- Photobioreactor, 50 L - Temperature: 25 °C - Light intensity: 196 μmol photons m−2 s−1 - Illumination: light/dark cycles = 12:12 h - Period: 7 days	0.7	74	[45]
Chlorella sp.	22.7	0.02	- Outdoor raceway tank, 200 L - Temperature: 9.7–24.6 °C - Maximum light intensity: 1711 μmol E/m2/s - Period: 10 days	0.49	94.9	[46]
Scenedesmus sp.	22.7	0.018	- Outdoor raceway tank, 200 L - Temperature: 9.7–24.6 °C - Maximum light intensity: 1711 μmol E/m2/s - Period: 10 days	0.46	94.3	[46]
Chlorella sp.	40.65	0.1 **	- Lab-scale - Incubation: shaker, 100 rpm rotation speed - Illumination: 200 µmol m−2 s−1 continuous cool-white, fluorescent light - Duration: 9 days - Temperature: 25 °C	0.48–2.5 **	68.4	[47]
Scenedesmus sp. LX1	15.5	-	- Lab-scale - Temperature: 25 - Humidity: 75% - Light intensity: 55–60 μmol E m−2 s−1, - Light/dark periods of 14/10 h - Duration: 15 days	0.11	98.5	[38]
Tetraselmis CTP4	27.6	0.18	- 5 L reactors - 100 μmol E m−2 s−1 - HRT 6.6 d−1	1.4	92.8	[48]
Desmodesmus communis (E. Hegewald)	34	-	- Indoor 1 L photobioreactor (PBR) - 16/8 light/dark light intensity - Temperature 18–25 °C, - Aeration rate 142 mLmin−1	1.42	>98	[49]
Chlamydomonas BERC07	-	-	- 10 L PBR - 12:12 h light/dark cycle - Temperature 28 to 30 °C.	1.24	100	[50]
(b)
Arthrospira platensis	1.92 *	0.05	- Lab-scale - Temperature: 25 °C - Light intensity: 2000, 3000, 4000 lux - Daily illumination time: 8 h, 12 h, 16 h - Period: 10 days	0.34	88.6	[41]
Scenedesmus obliquus	2.01 *	0.05	- Lab-scale - Temperature: 25 °C - Light intensity: 4000, 6000, 8000 lux - Daily illumination time: 8 h, 12 h, 16 h - Period: 10 days	0.36	99+	[41]
Chlorella sp.	1.98	0.01 **	- Lab-scale - Temperature: 27 ± 3 °C - Light intensity: 2000 lux - Period: 10 days	-	82.38	[51]
Chlorella sp.	1.51	0.01 **	- Lab-scale - Temperature: 27 ± 3 °C - Light intensity: 2000 lux - Period: 10 days	-	89.39	[51]
Chlorella sorokiniana	15.5	0.99	- Photobioreactor, 50 L - Temperature: 25 °C - Light intensity: 196 μmol E m−2 s−1 - Illumination: light/dark cycles = 12:12 h - Period: 7 days	0.7	74	[45]
Scenedesmus sp. LX1	0.5	-	- Lab-scale - Temperature: 25 °C - Humidity: 75% - Light intensity: 55–60 μmol E m−2 s−1, Light/dark periods of 14/10 h - Duration: 15 days	0.11	98	[38]
Tetraselmis CTP4	5	0.18	- 5 L reactors - 100 μmol E m−2 s−1, - HRT 6.6 d−1	1.4	72.6	[48]
Desmodesmus communis	2.1	-	- Indoor 1 L PBR - 16/8 light/dark light intensity - Temperature 18–25 °C, - Aeration rate 142 mLmin−1	1.42	100	[49]
Chlamydomonas BERC07	-	-	- 10 L PBR - 12:12 h light/dark cycle - Temperature 28 to 30 °C	1.24	94	[50]

* Calculated, ** optical density at 680 nm.

shows the potential of different microalgal strains in removing or recovering nitrogen and phosphorus from different UWs. The nitrogen and phosphorus in UW are consumed by microalgae for producing proteins, carbohydrates, and lipids containing biomass. Arthrospira platensis and Scenedesmus sp. cultivated in urban wastewater had protein contents of 42.6 and 32.6%, respectively, and carbohydrate contents of 32.4 and 22.8% [41]. In the absence of any external stress conditions, cyanobacterial species like Arthrospira platensis have been reported to produce 60% proteins; under stress or limiting conditions, proteins can be reduced, but lipid production is still lower than it is for microalgal strains like Scenedesmus sp., which tend to produce more lipids and carbohydrates [42]. Microalgal biomass acts as a source of carbon, nitrogen, phosphorus, and trace metals like iron, magnesium, zinc, and calcium [43]. Mixotrophic microalgae, such as Scenedesmus sp. and Tetraselmis sp. (H.J. Carter), assimilate the organic carbon and inorganic carbonate in the wastewater to yield biomass at concentrations ranging from 0.5 to 0.7 g/L [23,44].

By utilizing the total nitrogen, total phosphorus, and organic carbon present in urban wastewaters, microalgae have shown the potential for generating biomass rich in macro- and microelements that later could be used as biofertilizers or biostimulants for various plants or crop production. Additionally, it was reported earlier that microalgae are suitable feedstock for biofertilizer applications and contain more nitrogen, phosphorus, and potassium (NPK) than conventional organic fertilizers. The following sections briefly explain how urban wastewater-cultivated biomass can be used as a possible raw material for biostimulant and biofertilizer applications.

3. Comparative Energy Assessment for Nitrogen and Phosphorus Recovery from Urban Wastewater Using Microalgae

In 2019, the global consumption levels of nitrogen and phosphorus fertilizers were 132 tons and 71 tons (as P₂O₅), respectively [17]. Moving forward, the need for these fertilizers is expected to increase to a compound annual growth rate (CAGR) of 0.6–1.5% to meet the growing demand for food and feed [17]. By the year 2030, the volume of global wastewater generation is expected to reach 470 billion m³, with average N and P concentrations of 43.7 and 7.8 mg/L, respectively [52]. Therefore, there is a huge potential to recycle N and P from UW, which could reduce the need for manufacturing fresh fertilizers and their associated environmental emissions.

The tertiary wastewater treatment process of conventional nitrification–denitrification and phosphorus removal from urban wastewater utilizes 8 to 16 kWh per capita per year in urban WWTPs [53,54]. Activated anaerobic sludge removal from the activated aerobic digestion sludge process was reported to require 2.3 kWh of energy per kilogram of N removed, and the recently discovered anammox process, which oxidizes ammonium under anaerobic conditions, requires 0.9 kWh of energy per kg of N [55,56]. The reported specific energy consumption for high-rate algal ponds (HRAPs) with biofertilizer production was reported to be 0.08 kWh/m³ to 0.1 kWh/m³ [57,58,59,60]. Depending on the nitrogen concentration (e.g., 65 mg/L) in the wastewater and the nitrogen content in microalgal strains (5%), the energy required for the efficient removal of nitrogen from urban wastewater using HRAPs is 1.23 kWh per kg N, which is 47% less energy than that previously reported, i.e., 2.3 kWh per kg N by the conventional process in the urban wastewater treatment plant.

The energy needed for conventional phosphorous removal in urban WWTPs to directly recover phosphorus and reuse it as fertilizer in agricultural fields was reported as 71 kWh per kg P [19]. Another study reported that by utilizing HRAPs, phosphorus could be removed from urban wastewater with an energy consumption of 0.23 kWh/m³ [61]. If the TP in urban wastewater is 7.8 ppm [45], then 1 m³ of wastewater would contain 7.8 g of TP; consequently, the reported energy usage as per the above-mentioned energy consumption of 0.23 kWh/m³ would be 29.4 kWh per kilogram P for phosphorus removal using HRAPs. Nonetheless, the energy required is 2.4 times less than the earlier reported 71 kWh/kgP for conventional extraction in WWTPs and reusing it as fertilizer in agriculture. Furthermore, HRAPs will simultaneously recover N and P as microalgal biomass while treating the urban wastewater. In addition, the energy needed to recover P from incinerated wastewater treatment sludge ash ranges from 45 to 70 kWh/kg P, which is substantially more than the energy used by HRAPs to recover 1 kg of phosphorus [19].

4. Valorization of Phycoremediated Microalgae Biomass from Urban Wastewater as Fertilizer

In addition to utilizing inorganic nitrogen and phosphate, microalgae can oxidize several organic substances, such as acetate, butyric acid, glucose (C₆H₁₂O₆), and acetic acid (CH₃COOH) present in UW. Several microalgae could mixotrophically and heterotrophically use these organics to produce their biomass [62,63]. On the contrary, most of the photosynthetic microalgae use inorganic carbon (e.g., carbonate) to produce their biomass. Microalgal biomass that are produced in different types of wastewater have undergone substantial research for the generation of biofuels, high-value chemicals, fish, and animal feeds [64,65]; compared to the above-mentioned products, urban wastewater cultivated microalgae biomass as a potential biofertilizer or biostimulant need to be studied more. The following section discusses the role of microalgae as a potential biofertilizer and biostimulant.

4.1. Microalgal Soil Fertilizers

As shown in Figure 2, the N- and P-rich microalgae biomass acts as a slow-release fertilizer in soil [66,67,68]. Additionally, it has been shown that the nitrogen content released by soil microalgae does not exceed the nutrient requirements of plants, minimizing the eutrophication of ground and surface waters brought on by the overuse and loss of conventional fertilizers [69,70]. Furthermore, it has been shown that phycoremediated NPK-rich microalgae biomass substantially improved corn and baby spinach crop yields, as it possesses NPK quantities greater than those found in vermicompost, de-oiled cakes, and waste slurries from biogas plants [71]. Microalgae biomass cultivated in urban wastewater rich in N and P could be directly used as soil fertilizers, as this could reduce the dependence on conventional fertilizers [72]. Various crops have recently been cultivated in urban and peri-urban areas to enhance food security, reduce poverty, and sustainable waste management and resource recovery [73]. Phycoremediated microalgae biomass could also be used as a biofertilizer in crops cultivated in urban or peri-urban areas [74]. In addition to having high NPK concentrations (

Table 3. Potential of urban wastewater-cultivated microalgae biomass as soil fertilizer and organic fertilizer.

Wastewater-Cultivated Microalgal Strain	NPK (%) Content in Microalgal Biomass	Effect of Microalgal Biomass as Biofertilizer on Soil and Crop	References
Chlorella minutissima (Fot and Novakova)	6, 1 and 0.48	Increased soil microbes and higher NPK in soil, higher yields of baby corn and spinach	[71]
Scenedesmus sp.	7.6, 1.6 and 0.9	50% inorganic fertilizer could be replaced	[70]
Chlorella and scenedesmus consortium	7.21, 1.55, 0.75	Increased shoot, root and dry weights of Solanum lycopersicum (Tomato plants)	[77]
Chlorella sp.	5.9, 1.1, 0.42	Enhanced soil macro and micronutrients, enhanced plant growth.	[78]
Scenedesmus sp.	3.6, 0.6, 0.32
Nostoc sp.	3.8, 0.8, 0.4
Chlorella minutissima, Scenedesmus spp. Nostoc muscorum (C. Agardh)	2.7, 0.1, 0.24
Organic fertilizer	NPK (%) content in organic fertilizer	Effect of organic biofertilizer on soil and crop
Cow dung manure	1.86, 0.82, 2.1	Enhanced plant growth, pod yields of okra plant.	[79]
Piggery manure	2.16, 0.8, 2.1
Green manure	2.5, 0.5, 3
Poultry manure	2.9, 0.8, 3.7

), microalgae species have been found to contain substantial quantities of phytohormones [75,76].

In a different study, the amounts of NPK (%) in farmyard manure combined with biochar for enhancing low-fertility tropical soil were 1.5, 0.0007, and 0.16, respectively. The values for the UW microalgae biomass were substantially higher in NPK content than those for the farmyard manure, as indicated in Table 3. [80]. Another study showed that palm oil mill effluent manure had N and P values (3.5 and 1.5) that were similar to certain microalgal biomass N and P values but relatively low compared to the N and P reported in some UW-cultivated microalgal biomass. [81]. Maize yield and soil fertility in Cameroon were shown to be significantly increased by the use of palm oil effluent manure in combination with other fertilizers. The UW-cultivated microalgae biomass may not only have low freshwater footprints but also be able to improve low-fertility soils and increase the yields of a variety of crops (Table 3), [81]. As depicted in Table 3, compared to potassium in organic fertilizers, the potassium level in wastewater-cultivated microalgae biomass is lower; this could be due to the microalgae requiring less potassium relative to nitrogen and phosphorus [70,82]. Although it has been shown that microalgae possess sufficient NPK to be utilized as biofertilizers, recent findings have revealed that microalgae may adsorb microplastics in UW, and this could negatively affect microalgal growth [83]. Additionally, the adsorbed microplastic may enter the soil during biofertilizer applications, but the effects of microplastic-adsorbed microalgal biofertilizers on crop productivity are still unclear and require extensive research [84].

4.2. Phytohormones Identification in Different Microalgae and Its Effect on Higher Plants

Urban wastewater-cultivated microalgae and cyanobacterial biomass have also been reported to contain a wide range of phytohormones, such as auxins, cytokinins, abscisic and gibberellic acids, ethylenes, brassinosteroids, jasmonates, and salicylic acid (

Table 4. Potential of various microalgae (Eukaryotes) and cyanobacteria (Prokaryotic microalgae) as phytohormone producers.

Phytohormone	Microalgae	Cyanobacteria	Functions	Ref.
Auxin	Chlorella pyrenoidosa, Chlorella minutissima, Scenedesmus armatus.	Synechocystis sp. (Sauvageau), Chroococcidiopsis sp. (Getlier), Phormidium sp.(Gomont, M), Leptolyngbya sp. (Anagnostidis and Komarek), Anabaena sp. (Proskina-Lavrenko and Makarova), Oscillatoria sp. (Vaucher ex Gomot), Nostoc sp.	Plant and root growth and development, promotes flowering, cell division, and differentiation.	[88,89,90,91,92,93,94]
Cytokinins	Chlorella minutissima. Nannochloropsis oceanica (Suda and Miyashita)	Chroococcidiopsis sp., Anabaena sp., Synechocystis sp., Phormidium sp., Chlorogloeopsis sp. (A.K. Mitra), Calothrix sp. (Chlonoky), Rhodospirillum sp. Oscillatoria sp.	Promotes root and shoot branching, flowering, seed germination, nutrient mobility, delayed aging, assists in cell division and morphogenesis.	[88,91,95,96,97]
Abscisic acid	Dunaliella sp. (Masjuk), Chlamydomonas reinhardtii, Chlorella minutissima, Draparnaldia mutabilis (Roth), Nannochloropsis oceanica.	Anabaena variabilis, Nostoc muscorum, Synechococcus leopoliensis, Trichormus variabilis (Woronichin).	Stress tolerance, embryogenesis, initiating antioxidant activities.	[88,95,98,99,100,101,102,103,104]
Ethylene	Chlorella pyrenoidosa.	Calothrix sp., Synechococcus sp., Nostoc sp., Scytonema sp. (Bornet and Flahault), Anabaena sp., Cylindrospermum sp. (F.E. Fritsch).	Fruit ripening, aging, delaying shoot and root development.	[105,106,107]
Gibberellic acid	Chlamydomonas reinhardtii, Chlorella sp., Tetraselmis sp.	Phormidium foveolarum, Anabaenopsis sp., Cylindrospermum sp.	Stem, flower seed development, seed, morphogenesis and seed elongation	[88,96,108,109,110]
Brassinosteroids	Chlorella vulgaris, Chlorella minutissima, Chlorococcum elipsoideum,	-	Growth promoter, lowering stress, regulating cell division, germination, and reproductive process.	[111,112]
Jasmonic acid, jasmonates	Dunaliella, Euglena gracilis (GA klebs)	Arthrospira platensis	Seed and growth development, pollen production and stress and defense mechanism, aging mechanism.	[113,114,115]
Salicylic acid	Chlorella minutissima, Scenedesmus quadricuada (Turpin) Brebisson	-	Stress regulation, antioxidant, and enzyme activity.	[116,117,118]

) [85]. However, some microalgae species grown in urban wastewater have been reported to accumulate heavy metals [86]. As a result, the analysis of microalgal biomass before use in agriculture becomes a prerequisite, even though microalgae contain phytohormones that have been reported to have beneficial effects (Table 4) on plants [87].

Extracting phytohormones from various microalgae and delivering them as foliar extracts (Figure 3) might be beneficial for the plants [119]. Tomato plant height and the number of flowers and branches can be effectively increased by applying the extracts of Tetradesmus dimorphus (Turpin) MJ Wyne as foliar sprays at 50% concentrations ranging from 0.35 to 3.75 gL⁻¹ biomass [120].

When foliar sprays of protein hydrolysates of Arthrospira sp. were sprayed on Gardenia petunia, the foliar extract stimulated enhanced root growth and increased the flowers per plant since it contains amino acids that are known to be biostimulants [76]. Another study found that spraying lettuce with Arthrospira sp. hydrolysate enhanced spermin content and promoted growth [121]. For producing microalgal biofertilizers and other biostimulants, using freshwater and commercial fertilizers would be counterproductive compared to their cultivation in nutrient-rich wastewater [119].

5. Applications of Microalgal-Treated Wastewater (MTW)

Urban wastewater from domestic households is considered important nowadays, with some countries, such as Malta and Cyprus, utilizing approximately 90 to 60% of treated effluent water due to the scarcity of freshwater [122]. In contrast, water-scarce countries, such as Israel and Jordan, utilize more than 90% of their treated urban wastewater for irrigation [123]. In existing urban WWTPs, the generated wastewater is subjected to tertiary wastewater treatment to remove microbiological contaminants, lower nitrogenous and phosphate compounds for reduced eutrophication potential, and have less adverse effects on the environment before being discharged for irrigation, fertigation, or disposal in areas other than eutrophication-sensitive zones. The conventional tertiary treatment is energy-intensive and generates greenhouse gases (CO₂, ammonia, nitrous oxides, methane from nitrifying and denitrifying reactions, and sludge) in the removal of nitrogenous waste from UW, primarily ammonia and phosphate [124]. In this context, utilizing microalgae as a resource rather than a conventional tertiary treatment could lower the release of greenhouse gases and lead to the production of microalgae biomass, which can be used as biostimulants and fertilizers, as described in earlier sections.

Several PBRs and high-rate algal ponds (HRAPs) have been designed and implemented to lower energy costs, enhance biomass productivity, and lower eutrophying compounds [125,126].

Urban wastewater that microalgae have treated can be used as irrigation for containment areas, meadows, and terraces [126]. Escherichia coli Escherich contamination is the primary barrier to the reuse of wastewater treated by microalgae [127]. According to previous studies, there are certain approaches that can be used to minimize the presence of E. coli, such as the utilization of PBRs and ORPs, with high light penetration to attain maximum ultraviolet (UV) in the growth system to lower bacterial growth, as well as the cultivation of microalgae strains in high pH to help make wastewater unfavorable for bacterial growth [128]. The chemical and physical characteristics of wastewater treated by microalgae and prospective uses in a variety of sectors are shown in Table 5.

Although microalgae-treated wastewater has the potential to be utilized for irrigation purposes (Table 5), some requirements must be fulfilled before such treated wastewater can be used for irrigation. Some important water parameters required by the Food and Agriculture Organization (FAO) are soil bulletin water discharge criteria and FAO irrigation guidelines [129]. It was reported that increasing the microalgal biomass in urban wastewater might effectively lower the salinity because increased soil salinity and total dissolved solids (TDS) in UW could cause soil to become sodic [130]. As a result, UW that has undergone microalgal treatment is appropriate for fertigation or irrigation applications [131,132,133]. Even though the nutrient levels are reduced and the salinity is decreased, before being used for fertigation or irrigation after microalgal treatment, the presence of bacterial species, such as E. coli, intestinal nematodes, certain antibiotic-resistant bacteria, antibiotic-resistant genetic material, and several organic and toxic pharmaceutical compounds, may need to be maintained as per the prescribed regulation standards.

Table 5. Microalgae-treated urban wastewater quality and potential applications.

Microalgae Strain	Cultivation System	Water Quality Parameters	Application Area	Reference
Microalgal natural consortium	Green dune PBR	TN-14 mgL−1, turbidity—29, E. coli—671, TP—1.8 mgL−1, NH4+—0.7 mgL−1 TSS—76.1 mgL−1	Agriculture usage low/D * and usage low/E * Portugal classification areas	[126]
Chlorella vulgaris	14 L transparent cylindrical PBR	pH—8.01, TN—12 mgL−1, TDS—3852 ppm, PO4—0.4 mgL−1, E. coli—n.a., EC—6.02	Irrigation of low-quality soil for castor oil crop cultivation	[131]
Chlorella sp., Scenedesmus sp.	HRAP + wetlands	COD—73 ppm, TN—32 ppm, TP—2 ppm, TSS—24 ppm, turbidity—15 NTU	Irrigation of golf courts and municipal garden areas	[60]
Chlorella minutissima	PBR	pH—8.5, TDS—136 ppm, NH4+—2.9 mgL−1, DO—8.1 ppm, EC—0.25 dS/m	Suitable for crop irrigation	[132]
Chlorococcum sp., Oscillatoria sp., Scenedesmus sp., Phacus and Chlorella sps.	Conical open microalgal pond	pH—6.9, TDS—688, NH4+—7.6, TSS—4	Amaranthus crop irrigation	[133]
Mixed microalgal consortia	HRAP combined with DAF and solar disinfection	Suspended solids 2.1–20 ppm, turbidity—9.6 NTU, Salmonella—absent, nematodes < 1	Fit for agricultural use	[134]

* Urban wastewater classification.

Table 5. Microalgae-treated urban wastewater quality and potential applications.

Microalgae Strain	Cultivation System	Water Quality Parameters	Application Area	Reference
Microalgal natural consortium	Green dune PBR	TN-14 mgL−1, turbidity—29, E. coli—671, TP—1.8 mgL−1, NH4+—0.7 mgL−1 TSS—76.1 mgL−1	Agriculture usage low/D * and usage low/E * Portugal classification areas	[126]
Chlorella vulgaris	14 L transparent cylindrical PBR	pH—8.01, TN—12 mgL−1, TDS—3852 ppm, PO4—0.4 mgL−1, E. coli—n.a., EC—6.02	Irrigation of low-quality soil for castor oil crop cultivation	[131]
Chlorella sp., Scenedesmus sp.	HRAP + wetlands	COD—73 ppm, TN—32 ppm, TP—2 ppm, TSS—24 ppm, turbidity—15 NTU	Irrigation of golf courts and municipal garden areas	[60]
Chlorella minutissima	PBR	pH—8.5, TDS—136 ppm, NH4+—2.9 mgL−1, DO—8.1 ppm, EC—0.25 dS/m	Suitable for crop irrigation	[132]
Chlorococcum sp., Oscillatoria sp., Scenedesmus sp., Phacus and Chlorella sps.	Conical open microalgal pond	pH—6.9, TDS—688, NH4+—7.6, TSS—4	Amaranthus crop irrigation	[133]
Mixed microalgal consortia	HRAP combined with DAF and solar disinfection	Suspended solids 2.1–20 ppm, turbidity—9.6 NTU, Salmonella—absent, nematodes < 1	Fit for agricultural use	[134]

* Urban wastewater classification.

6. Life Cycle Impact Assessment (LCIA) of Microalgae Biofertilizer Production Using Urban Wastewater

Few recent LCIA studies on wastewater-cultivated microalgae biomass as biofertilizers have been published, despite this being a promising strategy for achieving sustainability through a circular bioeconomy. A recent life cycle assessment (LCA) assessed the environmental impacts of UW-cultivated biomass proposed for use as biofertilizers [59,135]. According to the LCA study, UW contains micropollutants, heavy metals, and pathogens that need to be regulated and would require social acceptance in order to function as UW-cultivated microalgal biofertilizers [59,135]. In contrast to conventional microalga cultivation, which has been shown to require more chemicals, and therefore, have higher impact values than using urban wastewater-cultivated microalgae biomass as biofertilizers, microalgae’s use of N and P from urban wastewater reduces negative environmental impacts and promotes a circular bioeconomy [59,135]. Additionally, another LCA study showed that utilizing microalgae cultivated in urban waste water in HRAPs as a biofertilizer directly was more cost-effective than using energy from biomethane cogeneration and residual digestate as a biofertilizer [59]. Additionally, the LCA study reported that, when compared to UW-cultivated microalgae biomass using HRAPs for biofertilizer applications, conventional activated sludge processes, from which the sludge is transported, incinerated, or landfilled, have significantly higher impact categories related to climate change, ozone layer depletion, fossil depletion, and photochemical oxidant formation [59].

7. Future Prospects and Challenges

• In order to lower the presence of heavy metals for biofertilizer applications, microalgal strains capable of complexing and precipitating heavy metals from UW must be developed.
• Microplastics that may contaminate soil or enter food crops may be present in certain microalgal biomass; hence, microalgal biofertilizers made from microalgae grown in UW must be thoroughly assessed for the presence of microplastics [83].
• Microalgal phytohormones need to be identified, and the phytohormone production in microalgal strains grown in UW needs to be improved to produce larger phytohormone yields, as opposed to using microalgal extracts directly on plants [5].
• Future studies should place greater emphasis on growing microalgae in HRAPs rather than PBRs. Additionally, when microalgae remove eutrophicating elements, CO₂ might be used to compensate for the carbon removed during the secondary wastewater treatment step [136].
• In case the microalgae biomass needs to be separated, self-settling strains, such as Scenedesmus obliquus and Chroococcidiopsis sp., must be used for wastewater treatment, as this would eliminate the energy-consuming preliminary dewatering process [137,138,139]. Microalgal biomass harvested using inorganic metal-based and organic coagulants could interfere with their applications as biofertilizers [12]. The self-settled strain may then be centrifuged, sundried, and used as biofertilizer; alternatively, the phytohormones may be recovered from the centrifuged biomass and used as a biostimulant for plant growth.
• Additionally, microalgal co-cultivation systems need to be integrated into existing WWTPs. This requires support from governments and research organizations.

8. Conclusions

Nitrogenous, phosphorus, and organic substances that contribute to eutrophication can be found in different amounts in the process streams of traditional wastewater treatment facilities. The N/P ratios of different urban wastewaters are suitable for cultivating different microalgal species and converting eutrophying N and P compounds into protein, carbohydrate, and lipid-rich biomass. Compared to other organic manures, the microalgae biomass from treated UW has a higher NPK content, which, when used as soil fertilizers, improves crop productivity and reduces the potential for eutrophication caused by conventional inorganic fertilizers. In addition to possessing NPK, microalgae biomass from treated UW also contains numerous kinds of phytohormones or biostimulants that can stimulate the growth of specific crop parts. Additionally, microalgal-treated UW can be used for various irrigation or fertigation activities to increase crop yield after being disinfected to reduce the microbial load and meet certain FAO and European Union discharge criteria. Finally, it can be stated that using traditional procedures to remove nitrogenous and phosphate compounds from wastewater in WWTPs is far more expensive and energy-intensive than using microalgae to remove N and P from UW in HRAPs. This review concludes with future directions for a faster and more widespread acceptance of the utilization of microalgae as a sustainable resource for treating UW and producing biofertilizers and biostimulants.