Human urine is composed of eight main ionic/non-ionic species (i.e., Na⁺, K⁺, Ca²⁺, Cl⁻, SO₄²⁻, H₂PO₄⁻/HPO₄²⁻, HCO₃⁻ and urea; Golder et al., 2007). Most nitrogen in fresh urine originates from urea, which can be hydrolyzed into free ammonia (NH₃), ammonium (NH₄⁺) and bicarbonate (HCO₃⁻) during storage. Both urea and ammonium are known to support the growth of microalgae (Tuantet, 2015). However, the free ammonia in urine can inhibit the growth of microalgae (Tuantet et al., 2013), and cell death may occur in the presence of high ammonia concentrations (Belkin and Boussiba, 1991). Piltz and Melkonian (2017) found satisfactory algal growth for all dilutions but not for undiluted urine. Thus, diluting synthetic or natural urine is essential to reduce ammonia’s toxic effect (Azov and Goldman, 1982). Cultivating microalgae in urine has two scenarios. The first scenario is a continuous culture by adding fresh urine as a daily nutrient stock (Tuantet et al., 2013). A process parameter, dilution rate (D, h⁻¹), is introduced to express the relationship between the flow of the raw urine (q, L h⁻¹) and the volume of the bioreactor (V, L). The other is a batch cultivation by employing diluted urine at a lower light intensity (Jaatinen et al., 2016), in which a parameter of dilution (or dilution ratio) is used (Tuantet et al., 2019).

Via uptake and conversion of nutrients, microalgae enable urine purification and resource recovery in biofuels, biochemicals, and bio-fertilizer (Tuantet, 2015). As for biomass production, microalgae can grow autotrophically by utilizing organic/inorganic nitrogen and CO₂/bicarbonate as carbon sources. Meanwhile, microalgae cells can directly use phosphate in urine under aerobic conditions and transform it into adenosine triphosphate (ATP) or other organic substances through assimilation and proliferation (Yang et al., 2008). Figure 2 presents the microalgae species successfully cultivated in synthetic and real human urine. Some typical species and their characteristics are summarized as follows:

Chlorella is a fast-growing microalgae species with reported maximum specific growth up to 0.25 h⁻¹ under autotrophic and light saturating conditions (Cuaresma et al., 2009), the dry weight of which is contributed by 6%–8% nitrogen and 1%–2% phosphorus. Microalgae biomass of Chlorella can be directly used as a fertilizer and/or a potential source for chemicals and biofuels (Tuantet et al., 2013). Because Chlorella is rich in protein (Adamsson, 2000), the cultivated biomass from urine can be directly fed to zooplankton and the latter can be provided to fish in the constructed food chain (Adamsson, 2000). The widely investigated Chlorella in urine media includes C. sorokiniana and C. vulgaris (Figures 2A,B). C. vulgaris has higher Omega-3 fatty acids content. These fatty acids are commonly found in green leafy vegetables and oily fish such as herring, sardines, and tuna (Revellame et al., 2021). C. vulgaris prefers ammonium to nitrate as a source of nitrogen, which benefits the growth in urine (Schuler et al., 1953). Another species of Chlorella, C. sorokiniana shows great potential for commercial production as a nutrient substitute for humans and animals (Morais Junior et al., 2020). C. sorokiniana is mesophilic, the growth of which is characterized by a relatively high optimal temperature (30°C–40°C) and a maximum specific growth rate of ~0.27 h⁻¹ (Cuaresma et al., 2009). Furthermore, C. sorokiniana can synthesize neutral oil as triacylglycerol under stress conditions (e.g., under high light intensity or nutrient deficiency). The study by Tuantet et al. (2014) demonstrated that dilution of urine by two times could achieve adequate incubation of C. sorokiniana CCAP211/8K, which contributed to >90% of total nitrogen and phosphorus removal. Of all the commercial microalgae, while Chlorella has the second largest annual production inferior to Spirulina, the price of Chlorella is significantly higher on the market (Yuan et al., 2022).

Spirulina is the benchmark of microalgal biotechnology and is currently the most (3,000 dry tons annually) commercially produced microalgae (Detrell et al., 2020). Spirulina (Arthrospira platensis; Figures 2C,D), one of the essential cyanobacteria, produces high concentrations of pigments (chlorophyll a and phycocyanin), fatty acids (i.e., γ-linolenic acid) and proteins (Gutierrez-Salmean et al., 2015). A. platensis contains a more balanced ratio of saturated to unsaturated fatty acids including Omega-3 and Omega-6 (Revellame et al., 2021). Moreover, S. platensis has a relatively high cell growth rate and requires an easy process for biomass recovery as a result of the filamentous cell structure. Studies have demonstrated that the cultivation of S. platensis is viable for waste purification or aquatic food production (Chang et al., 2013). For example, S. platensis was harvested after 7 days of incubation and percentage removal of 97.0% and 96.5% was, respectively, achieved for NH₄⁺-N and total phosphorus in the urine at a 120-dilution (Chang et al., 2013).

Scenedesmus acuminatus is also widely used in the treatment of anaerobic digestion effluents and secondary domestic wastewater (Figures 2E,F), because of its capacity to grow at a high biomass concentration of 8–11 g L⁻¹ (Posadas et al., 2015; Tao et al., 2017b). Scenedesmus is one of the high protein content species, which contains 50%–56% protein, 10%–17% carbohydrate and 12%–14% lipid (Raeisossadati et al., 2020). For example, a strain of Desmodesmus sp. QL96 isolated from Tibet, China contains 17 amino acids (including 7 essential ones; Cheng et al., 2021). The properties of Desmodesmus highlight its commercial merits in biomass cultivation from urine. Scenedesmus species have been reported to take up high concentrations of nitrogen (273 mg L⁻¹) and phosphorus (58.8 mg L⁻¹; Kim et al., 2015). Scenedesmus was grown in 0.5% diluted urine supplemented with EDTA and iron, and its maximum biomass density was about 133 mg-dry weight L⁻¹.

Microalgae are generally cultivated in open or closed systems (Song et al., 2018). Raceway ponds are traditional open systems to cultivate microalgae (Table 1). While these configurations have merits including low cost and simple operation, microalgal productivity in open systems is highly susceptible to environmental conditions. In comparison, closed systems including tubular, flat panel and bubble column photobioreactors (PBRs) are relatively costly and currently limited to small-scale microalgae cultures that generate high-value products including poly-unsaturated fatty acids, carotenoids and other chemicals for pharmaceutical and cosmetics industries (Rezvani et al., 2022). The operating cost for closed reactors relates to collecting culture fluid and microalgae cells. Recent advancements in reactor design have paved the way for more efficient enrichment and collection of microalgae (Zittelli et al., 2013; Tuantet, 2015). The architectures of PBRs for microalgae cultivation in urine are summarized in Figure 3 and Table 2.

PBRs constructed for microalgae cultivation should sustain fast reaction rates, stable operation performance and a high capacity to recover nutrients via microalgae harvesting (Yang et al., 2008). In earlier experiments, Tuantet et al. (2013) cultured Chlorella on human and artificial urine microtiter plates. Column PBRs have simple configurations and better flow conditions (Figure 3A and Table 2). However, some practical problems exist when urine is used as feed. In addition to the nutrient levels of raw urine being unsuitable for algal growth, the dark color of urine and the low light penetration would challenge the PBR setup. To overcome the limitations, on the one hand, the bioreactors are typically fed with diluted urine. Conversely, an optimized system requires shorter light paths for microalgae growth. In the study by Chatterjee et al. (2019), extremely high dilution would be necessary even for only 50% nitrogen recovery in a 0.5 m deep raceway pond. To this end, a short light-path PBR (typically in the flat-panel configuration, Figure 3B) has been developed to supply light to all cells encapsulated inside the microalgal culture and thus support dense cultivation. Continuous microalgae cultivation has been carried out in a PBR with a narrow light path (i.e., 5 mm). However, an inherent conflict remained between the nitrogen removal and photosynthetic efficiency when the PBR was used to treat urine containing 0.77–2.6 g-N·L⁻¹. Because an increase in the biomass concentration/density would shield the illumination and create a “dark zone” for a considerable part of the culture, advancements to minimize the “dark zone” in order to enhance biomass productivity are of significance in a short light-path PBR (Tuantet, 2015).

From a theoretical perspective, cultivation at a high cell density is essential to sustain high nutrient removal efficiencies in urine treatment (Tuantet et al., 2014). A PBR employing a higher hydraulic retention time (HRT) can prompt biomass growth and nutrient removal, but this may require a larger volume/footprint for reactor deployment. To address this issue, the membrane separation process has been integrated with PBR to increase the capacity and improve biomass recovery efficiency (Ma et al., 2017; Nguyen et al., 2021; i.e., membrane PBR in Figure 3C and Table 2). While membrane modules used in PBRs could be similar to those in conventional membrane bioreactors, the operating protocols may differ due to the characteristics of microalgae. For instance, the formation of a cake layer on the membrane surface in PBRs could be deemed as a means to recover the suspended cells from the dilute culture, which may pave the way for the application of dynamic membrane processes in membrane PBRs for more-efficient biomass harvesting (Ma et al., 2013b). Developing a realistic PBR with a compact structure is therefore one of the most critical interests in microalgae production in human urine (Larsen et al., 2021).

Light intensities and switch modes used for microalgae production in urine are summarized in Table 2. Essentially, light intensity influences the microalgae photosynthesis and consequently their growth rate via modulation of ATP and NADPH production and essential molecules synthesis. The study by Tuantet et al. (2013) indicated that illumination was one of the most important factors influencing algal growth and nutrients removal, which is also confirmed by the analysis in Figure 4. A biomass density of 6.6 g L⁻¹ was obtained in 2-times diluted urine at a light intensity of 1,540 μmol m⁻² s⁻¹ compared to that of 3.8 g L⁻¹ in 10-times diluted urine at 1050 μmol m⁻² s⁻¹ (Tuantet et al., 2014). Note that different illumination units (e.g., μmol m⁻² s⁻¹, W m⁻¹ and lux) have been used in the literature (Yang et al., 2008; Chang et al., 2013; Tuantet et al., 2014, 2019; Tuantet, 2015), and the photosynthetic photon flux density (μmol m⁻² s⁻¹) could be converted to lux by multiplying a factor of 54–82 (for sunlight and high-pressure sodium lamps) according to the manufacturer (for example, Apogee Instruments, Inc., UT, United States). Overall, it was suggested that increasing the light intensity up to 1,500 μmol m⁻² s⁻¹ could prompt microalgae growth and improve the nutrient removal efficiency (Tuantet et al., 2014).

Temperature change also influences the solubilization and volatilization of ammonia, thus leading to pH excursion. As shown in Table 2, a temperature between 30°C and 40°C is typically applied to the incubation because, for example, the optimal temperature for the growth of S. platensis is between 29°C and 32°C (Yang et al., 2008). Nevertheless, some microalgae can tolerate low temperatures; for example, S. acuminatus could grow in human urine even at 5°C with the recovery of N and P achieving 52% and 38%, respectively, (Chatterjee et al., 2019).

As for photobioreactor design and operation, biomass and hydraulic retention time (BRT and HRT) are two key parameters (Nguyen et al., 2021). Results in Figure 4 indicate that BRT may have a positive relationship with nutrients removal from urine. In a PBR, BRT relates to biomass accumulation and thus determines the nutrient removal rates (Akerstrom et al., 2014). A short BRT (2–5 days) may not be sufficient to sustain the rapid growth of microalgae, which consequently limits the biomass production rate even when a high nitrogen uptake rate is obtained (Luo et al., 2017; Praveen et al., 2019). Likewise, the highest TP removal of 52.1% was achieved at a BRT of 7 days during incubation (Nguyen et al., 2021). It should be noted that a longer BRT may result in the deterioration of the settling/harvesting properties of the biomass (Wang et al., 2013; Ma et al., 2018a). In addition, a longer HRT resulting from an extended BRT would decrease the productivity of a PBR to treat human urine. Conversely, a short HRT leads to higher nutrient loads but compromises the removal of N and P. To address the limitations, membrane separation processes have been integrated with PBRs (MPBR) to achieve flexible control of BRT and HRT. High nutrient loads such as 90–110 mg-N L⁻¹ day⁻¹ and 5–6 mg-P L⁻¹ day⁻¹ have been used in MPBRs (Nguyen et al., 2021). In summary, the BRT and HRT should be set given the treatment efficiency and capital cost for bioreactor deployment. According to the study by Nguyen et al. (2021), a short BRT (7 days) and an extended HRT (>2 days) are thus suggested to capture nutrients from urine while minimizing the environmental impacts effectively.

Tuantet (2015) found that the algal growth was inhibited due to the exhaustion of trace elements within 24 h, highlighting the importance of trace elements in biomass production in diluted urine. As for the effect of dilution on microalgae growth, Jaatinen et al. (2016) reported that the highest biomass densities of 0.73 and 0.60 g L⁻¹ were obtained at 1:100 dilution of urine with and without the addition of trace elements. Moreover, C. sorokiniana showed the fastest growth rate in urine diluted 20 times with trace elements added (Tuantet et al., 2013).

Magnesium (Mg²⁺), iron (mainly in the form of Fe^II) and certain trace elements are present in urine at low concentrations (Tuantet et al., 2013). When urine is collected and stored, the formation of precipitates due to an increase in pH reduces the availability of these elements. Udert et al. (2003) demonstrated that the precipitation capacity of guano stone and octa calcium phosphate in urine reached 87% when the hydrolysis rate was 11%. As a result, the magnesium content in hydrolyzed urine (0.15–0.17 mg L⁻¹) could be significantly lower than in fresh urine (25.4 ± 17.0 mg L⁻¹; Zhang et al., 2014). Mg²⁺ plays a vital role in algal metabolism because it is essential for chlorophyll production (Sydney et al., 2010). The magnesium content in Chlorella sp. ranged from 0.36% to 0.80% of dry weight, and only 40 mg L⁻¹ dry biomass could be sustained at a 0.36% magnesium content (Borowitzka and Borowitzka, 1988). Therefore, magnesium supplementation is essential to promote microalgal growth in hydrolyzed urine (Table 3; Tuantet et al., 2019). No significant difference was found between the specific growth rates of microalgae fed with hydrolyzed urine with additional Mg²⁺ (μ = 0.095–0.111 h⁻¹) as compared to synthetic and fresh urine (Tuantet et al., 2013). Moreover, iron is also one of the most crucial trace metals involved in the enzymatic reactions of photosynthesis in photosystem I (PSI) and PSII (Cao et al., 2014). An increase in the iron concentration in the medium (1.2 × 10⁻² mM) would elevate the biomass as well as lipid content of C. vulgaris (Liu Z.-Y. et al., 2008). Note that significant elements such as Cl also indispensably contribute to the photosynthesis of chlorophyll and affect the uptake of trace elements (Yang et al., 2008).

Typically, microalgae production in human urine uses the internal inorganic carbon source of bicarbonate (HCO₃⁻) and/or exogenous sparged CO₂. Additional CO₂ can prompt the growth of microalgae. As shown in Table 2, the gas concentration (v/v) of extra CO₂ ranges from 1% to 20% because an overhigh CO₂ dose may cause acidification of the media. Nevertheless, field studies have shown that microalgae cultivation by using flue gasses can withstand a high CO₂ concentration of 40% (Pires et al., 2012).

Besides, microalgae can grow under mixotrophic conditions. Under autotrophic conditions, S.platensis fed with an inorganic carbon source (with ammonium or urea as the nitrogen source) demonstrated a yellowish-green appearance with relatively low protein content. At the same time, the biomass became green and difficult to settle down after adding an organic carbon source (Chang et al., 2013). Adding an organic carbon source could alleviate the inhibition effect of ammonium. For example, by adding 100 and 200 mg L⁻¹ sodium acetate (or glucose) to the synthetic urine (Table 3), the productivity of S.platensis was improved with the nitrogen and phosphorus removal increasing from 97% and 96.5% to ~100% and 98%, respectively (Chang et al., 2013). As for treating urea in human urine, adding an organic carbon source could also facilitate the removal of ammonia via biomass production. As such, introducing waste organic carbon sources (e.g., effluents from food plants) to microalgae cultivation in human urine can provide a valuable solution to increase nutrient recovery efficiency.

Medium pH directly influences microalgae growth and determines the speciation of nutrients that may support or inhibit biomass production. As aforementioned, hydrolysis of urea in human urine produces HCO₃⁻ and ammonia while raising the solution pH (Adamsson, 2000; Zhang et al., 2013). Conversion between ammonia and ammonium is primarily determined by the solution pH (pK_a = 9.25; Zhang et al., 2018; He et al., 2022; Zhang et al., 2022), and higher pH could facilitate the transformation of NH₄⁺ to NH₃ (Figure 5). NH₃ is far more toxic than NH₄⁺ because the transport of NH₄⁺ involves the participation of transporters (Figure 5C; Källqvist and Svenson, 2003; Wang et al., 2019). Ammonia or free ammonia can reduce photosynthetic activity and directly shows toxicity to microalgae (Zhang et al., 2014). The photosynthetic rate of Scenedesmus was allegedly decreased by 50% of its maximum rate in the presence of free ammonia of 20 mg L⁻¹ under basic conditions (Azov and Goldman, 1982), though different microalgae species have different tolerance to the pH-dependent toxicity of NH₃/NH₄⁺ (Tao et al., 2017a). As mentioned above, a high pH level can lead to the precipitation of unchelated trace metals, thus inhibiting algal growth (Adamsson, 2000). To stabilize the solution pH and neutralize the alkalinity especially when human urine is directly used as the feed, sparging of CO₂ (or diluted CO₂) has been carried out. In addition to serving as an inorganic carbon source, the excessive CO₂ can buffer the pH (pK_a (H₂CO₃/HCO₃⁻) = 6.30; Ma et al., 2018b; Figure 5B) and prevent the inhibition of free ammonia on algal growth (Tuantet et al., 2014).

With the absorption of ammonium in algal growth, the pH would decrease because of the production and accumulation of H⁺, which slows down the growth rate (Azov and Goldman, 1982). In a photobioreactor, when the medium pH drops from pH 6.8 to <4 during cultivation, the microalgae would be subject to the cessation of growth or even death (Hulatt et al., 2012; Jaatinen et al., 2016). C. sorokininana has shown a high specific growth rate within the pH range from 4 to 7 (Tuantet et al., 2014). In addition, consideration should be given to the source of human urine. For example, it has been reported that gender may influence the pH because the female urine demonstrated a more narrow pH window (7.1–7.9) as compared to that (5.7 and 8.0) of the male urine (Tuantet, 2015).

When non-sterilized human urine is used as the feed, bacterial contamination of the cultivations very likely occurs, which leads to competition for the nutrients and lower conversion to algal biomass. While this problem in the early growth stage may be solved by sterilization of the inoculation medium, it was reported that a large variety of bacteria were detected from C. vulgaris cultures grown on both sterilized and non-sterilized media at an extended cultivation period (Jaatinen et al., 2016). Maintaining the culture composition in the photoautotrophic mode (i.e., in the absence of organic carbon) may inhibit the competition from bacterial growth. Microalgal cells can regain dominance in the diverse community in 2 days when the organic carbon supply ceases (Zhang et al., 2014). Nevertheless, the autotrophic nitrifiers can adapt to fill a similar ecological niche compared to the microalgae though the biomass yield of ammonia-oxidizing bacteria is low (Ma et al., 2013a, 2015). In the symbiosis, a consortium of microalgae and nitrifying bacteria can decrease the need of expensive external aeration (Sun et al., 2020; Li et al., 2022). The photosynthetic oxygenation rate can drive the nitrification in urine with a volumetric nitrification rate of 67 mg N L⁻¹ day⁻¹, and a maximum biomass-specific photooxygenation rate of 160 mg O₂ gVSS⁻¹ day⁻¹ (Muys et al., 2018).

In addition, the microalgae growth also leads to the secretion of extracellular organic matter that can be used by heterotrophic bacteria (Zhang et al., 2014). In turn, microalgae can convert nutrients into cellular components through photosynthesis and respiration. Currently, some single-cell microalgae such as Chlamydomonas, Chlorella and Phormidium are proven candidate for the formation of symbiosis suitable for waste treatment and facilitates the removal of N and P (Nguyen et al., 2020a). Inoculation of Arthrospira platensis with nitrated urine has been found to exhibit better growth and produce 62% more protein than untreated urine (Coppens et al., 2016). As for biofuel production, the biomass consisting of bacteria and microalgae may provide higher biogas production than pure microalgae (Jaatinen et al., 2016). For instance, 17%–24% higher methane yields (376–403 ml-CH₄·g-VS⁻¹) were obtained from a mixture of microalgae (C. vulgaris) and bacteria (1%–10%) than the control that only contained microalgae (Lu et al., 2013). As such, the following studies to explore (i) the algal and bacterial inter-group competition and collaboration and (ii) the impacts of co-existing bacteria on biofuel conversion are required to advance the process performance.

Source-separated urine contains about 60 ± 30% of drugs and lifestyle biomarkers consumed by humans (Monetti et al., 2022). Following intake, ~50% of pharmaceuticals do not change the chemical form, and are discharged with the intermediates as human metabolites (Lienert et al., 2007). Some human metabolites (e.g., conjugates of the antibiotic sulfamethoxazole, the anti-convulsant carbamazepine and the analgesic ibuprofen; Quinn et al., 2009; Ren et al., 2022a,b; Yang et al., 2022) have detrimental effects on the environment and may pose direct toxicity to microalgae. For instance, the anticonvulsant carbamazepine and the antidepressant fluoxetine form more toxic metabolites than their parent compounds (Verstraete et al., 1997; Jelic et al., 2015; Monetti et al., 2022). In contrast to the abundant studies of the contents of N, P and organic matter (Table 1) that influence microalgae growth and biomass production, there is little investigation of the micropollutants in urine involving the metabolism of microalgae. de Wilt et al. (2016) evaluated the efficiency of microalgae C. sorokiniana to remove six spiked pharmaceuticals (147 ± 9 μg L⁻¹ diclofenac, 317 ± 33 μg L⁻¹ ibuprofen, 337 ± 23 μg L⁻¹ paracetamol, 181 ± 62 μg L⁻¹ metoprolol, 117 ± 17 μg L⁻¹ carbamazepine and 202 ± 30 μg L⁻¹ trimethoprim). Results showed that 60%–100% of diclofenac, ibuprofen, paracetamol and metoprolol could be readily removed by photolysis and biodegradation while carbamazepine was refractory (removal <30%; de Wilt et al., 2016). While the presence of micropollutants at 100–300 μg L⁻¹ did not inhibit microalgae (C. sorokiniana) growth, the deployment of pre-treatment technologies may be required at higher concentrations to prevent the pitfalls. Specific micropollutants can be removed by activated carbon, an effective absorbent for various organic and inorganic molecules because of the large surface area, porous structure and surface-bound groups (Yin et al., 2007). Activated carbon adsorption has been applied to eliminate antibiotics, beta-blockers, and nonsteroidal anti-inflammatory drugs from urine (Udert et al., 2016). Future work is essential to (i) assess the microalgae response to micropollutants at elevated concentrations, and (ii) develop cost-effective, reliable and environmentally benign processes to polish human urine under realistic conditions.

Essentially, the accumulation of neutral lipids by Acutodesmus, Phaeodactylum, Dunaliella and Nannochloropsis requires nutrient limitation or starvation, which can inhibit microalgal growth (Sun et al., 2018). While the acquisition of favorable and stable traits can be conducted via crossbreeding for crops (Armbrust, 1999; Chepurnov et al., 2008), this is not applicable to most microalgae that have some deficiency (Dismukes et al., 2008). To address the limitation, microalgae genetic engineering is considered as an optimal approach to solve this bottleneck. Direct or indirect genetic modification has been proposed as a means to improve the growth and lipid productivity of promising microalgal strains.

Genetic engineering can facilitate lipid accumulation without affecting the algal growth (Munoz et al., 2021), by modifying the single metabolic pathways including fatty acid synthesis metabolism, Kennedy pathway, polyunsaturated fatty acid and triacylglycerol metabolisms and fatty acid catabolism. For example, the synthesis of fatty acids requires a continuous supply of acetyl-CoA. Compared with wild type strains, the total lipid contents of N. oceanica and Schizochytrium sp. were increased by 36% and 11%, respectively, by employing the overexpress of the malonyl-CoA acyl carrier protein transacylase (Chen et al., 2017). In addition, commercial production of large-scale bulk of microalgae is still not feasible (Remmers et al., 2018). Genetic engineering may pave the way for increasing the photosynthetic rate to modulate the carbon flux toward lipids while maintaining high biomass production (Ajjawi et al., 2017). This strategy to prompt the yield of microalgae lipids may eventually make microalgal derivatives an effective means of commercial biofuels.