Wastewater treatment process using immobilized microalgae

Microalgae production has a long history, and today its commercial use is becoming increasingly popular. The biomass of many species is used in various industrial sectors, including as a source of food (Severo et al. 2024a), animal feed, biofertilizers, health compounds, and chemicals. The energy sector has also explored the use of microalgal biomass to obtain renewable biofuels such as biodiesel (Dzuman et al. 2022), biohydrogen (Khosravitabar & Spetea 2024), bioethanol, and biogas (Miyawaki et al. 2021), addressing the production of these inputs in the context of biorefinery (Severo et al. 2019).

The cultivation of microalgae has been carried out in different systems, but closed photobioreactors are the most established for successful production. They provide better operational control, lower incidence of contamination, and higher biomass productivity (Razzak et al. 2023). However, two issues of concern that limit scale-up are the cost of the culture medium and the various stages of biomass processing, including mainly harvesting (Ruiz et al. 2016). For example, techniques such as flotation, flocculation, and centrifugation require substantial investments in energy and chemicals to recover biomass. It is estimated that harvesting costs can represent 20–30% of the total costs of biomass production (Morais et al. 2023).

Thus, an alternative to circumvent these issues is cell immobilization, which is an economical method to separate the biomass suspension from the liquid phase. The challenges in harvesting microalgae stem from the fact that the suspended cells are very small; normally, they are only a few micrometers in size and have a low dispersion density (<1% in the culture medium). In addition, cultivation demands substantial amounts of water and nutrients, making large-scale processes economically unfeasible and complicating traditional harvesting methods (Li et al. 2024a).

The microalgae immobilization technique is based on the concept of the ‘immobilized cell’. This refers to a living cell that is restricted, either naturally or artificially, from freely migrating from its initial location within the aqueous phases of a system (Tampion & Tampion 1987). This approach emerged more than four decades ago and has been extended to microalgae, which can be immobilized in various polymer matrices and biotechnologically exploited for many purposes, whether to produce a target metabolite or to remove pollutants (de-Bashan & Bashan 2010). Many natural (e.g., alginate, agar, agarose, cellulose, and carrageenan) (de Lira et al. 2024) and synthetic (e.g., acrylamide, polyurethane, and polyvinyl) polymers are utilized for this application (de-Bashan & Bashan 2010).

In general, the immobilization of microalgae is based on the fixation or entrapment (encapsulation) of cells in a matrix through physicochemical interactions (Moreno-Garrido 2008). The technique has the following advantages: (i) cells are easily collected by simple filtration due to the size of the spheres formed compared to free microalgae cells; (ii) higher cell concentration and preservation; (iii) improved metabolic activity; (iv) reduction of equipment maintenance costs; (v) thermal stability; (vi) biodegradability; and (vii) remarkable effect on wastewater purification, removing nitrogen and phosphate compounds, including heavy metals. Besides, the easy recovery of the cells contributed to their reusability. By being immobilized in a stable matrix, the microalgae can be easily separated from the treated water and transferred to a new batch of wastewater, maintaining their functional integrity over several cycles. The stability, reusability, and reactivation of the immobilized cells depend on the matrix material and the operational conditions, but in many cases, they can be used repeatedly, making the process more cost-effective and sustainable (de-Bashan & Bashan 2010; da Silva et al. 2022; Mariano et al. 2022; Foo et al. 2023; Han et al. 2023; Severo et al. 2024b, 2024c).

Another important factor in reducing production costs is the cultivation of microalgae combined with wastewater treatment, which is a rich source of nitrogen compounds (⁠⁠, ⁠, ⁠), phosphates (⁠ ), and other inorganic ions (⁠⁠) (Gonçalves et al. 2023). These substances are considered relevant pollutants in the aquatic environment since their concentration in the water exceeds the established limits (Morán-Valencia et al. 2023). The presence of these ions is due to the discharge of industrial, municipal, or agricultural effluents. High concentrations of compounds derived from phosphorus and nitrogen result in severe eutrophication and a reduction in the amount of dissolved oxygen in water bodies, threatening aquatic organisms, the equilibrium of ecosystems, and human health (Abdel-Raouf et al. 2012).

This paper presents the experimental results of a study in which swine manure from a biodigester was used to cultivate microalgae immobilized in sodium alginate. The objective was to investigate whether immobilized Tetradesmus obliquus cells could thrive on a substrate not previously used specifically for this type of process. Additionally, another novelty of the present study lies in alginate beads that have demonstrated considerable potential to increase the growth and nutrient removal efficiency (RE) of immobilized microalgae. To the best of the authors' knowledge, the immobilization of this species and its cultivation in this type of wastewater has not been reported in the literature. Finally, the impact of the present study was to explore the potential biomass production and the simultaneous environmental benefit due to the effect of waste treatment and the safe use of the substrate combined with a natural polymer matrix.

The microalgae used in the experimental tests was T. obliquus, which were cultivated at the Biotechnology Laboratory of the Sustainable Energy Research & Development Center (NPDEAS, Curitiba, Brazil). This strain (LGMM0001) was previously identified by rDNA sequence and micromorphological analysis, as described in detail in the work of Corrêa et al. (2017).

The stock cultures were propagated and maintained in a sterile environment containing a CHU synthetic medium rich in organic and inorganic salts (Chu 1942). The medium consisted of 10 mL. L⁻¹ of solutions NaNO₃ 25 g L⁻¹, CaCl₂·2H₂O 2.5 g L⁻¹, MgSO₄·7H₂O 7.5 g L⁻¹, K₂HPO₄ 7.5 g L⁻¹, KH₂PO₄ 17.5 g L⁻¹, NaCl 2.5 g L⁻¹, and 1 mL·L⁻¹ of solutions C₁₀H₁₄N₂Na₂O₈·2H₂O 50 g L⁻¹, KOH 31 g L⁻¹, FeSO₄·7H₂O 4.98 g L⁻¹, H₃BO₃ 11.42 g L⁻¹, ZnSO₄·7H₂O 0.0882 g L⁻¹, MnCl₂·4H₂O 0.0144 g L⁻¹, NaMoO₄·2H₂O 0.00119 g L⁻¹, CuSO₄·5H₂O 0.0157 g L⁻¹, and Co(NO₃)₂·6H₂O 0.0049 g L⁻¹. The cultures were continuously illuminated with a set of 40 W white, fluorescent lamps. The incubation conditions were as follows: temperature of 22 °C, continuous aeration of 1 L min⁻¹, and light intensity of 5,500 lux.

The biodigested swine manure was used in the experiments as a culture medium. The wastewater was obtained from a local farm (Curitiba, PR, Brazil). The samples were collected, sieved, and filtered with a multilayer fiber cloth to separate the solids from the sludge. Subsequently, the wastewater was sterilized in an autoclave and kept under refrigeration (4 °C) to prevent contamination and limit the biodegradation process by bacterial activity.

The characterization of the wastewater sample was determined according to the Standards Methods for the Examination of Water and Wastewater (APHA 2005) and is presented in Table 1.

The experiments were performed under two conditions: (1) cultivation with immobilized microalgal cells and (2) cultivation with free microalgal cells. Both treatments were carried out in Erlenmeyer flasks, which were fed with 1 L of culture medium containing different concentrations of wastewater (10, 20, and 30%) diluted in sterilized water. In addition, an initial test with 100% effluent was conducted to verify the viability of the beads in the culture medium.

Cultures were operated in batch mode and illuminated with white, fluorescent lamps (40 W), constant aeration of 1 L min⁻¹, a temperature of 22 °C, and an initial pH adjusted to 9.0.

Cell concentration and cell counting were performed every 48 h to monitor the growth of the microorganism. Residence times of 240 h (10 days) were standardized for all experiments.

The samples of immobilized microalgae cultured in wastewater were determined from the collection of 30 beads, which were dissolved using a vortex with a 4% (w/v) sodium bicarbonate solution for 2 h to resuspend the microalgal cells, and subsequent cell concentration measurements (Cao et al. 2020).

Cell counting was performed using a Neubauer chamber placed in an optical microscope (Bioval™, São Paulo, Brazil) with a 400× magnifying setting.

Additionally, samples of the liquid medium were collected to determine the removal of nutrients present in the effluent and evaluate the bioremediation potential.

The pH of the culture medium was monitored using a pH meter (TE-058, Tecnal™, Piracicaba, SP, Brazil).

All sampling was performed aseptically in a laminar flow hood. Tests were performed in triplicate, and the kinetic data refer to an average of six repetitions.

In this study, the cultivation of microalgae in wastewater media with different concentrations using both free and immobilized cells was compared concerning their capacity for biomass production and nutrient removal. The results are presented and discussed below.

Compared to free cells, immobilized cells showed superior performance, especially for the experimental condition with 10% effluent concentration, which started at 48 h, and this trend was maintained until 192 h of cultivation, indicating that the immobilized culture positively impacted cell growth compared to the free culture. In this experimental condition, the maximum DW obtained from the biomass was 2,690 mg L⁻¹, which is approximately 2.3 times higher when compared to 1152 mg L⁻¹ in the free culture. Similar behavior was observed for conditions with 20 and 30% effluent concentrations for immobilized cells, whose dry biomass weights were in the order of 2,274 and 2,360 mg L⁻¹, respectively, compared to 940.2 and 1,033 mg L⁻¹ in culture with free cells.

It is worth mentioning that preliminary tests were carried out on samples containing 100% swine manure (Supplementary Material, Figure S2). The microalgae beads were entirely dissolved in the raw wastewater by the second day of treatment. Microscopic analysis showed that the growth of free cells was inhibited by the third day, indicating non-viable cells (data not shown). Therefore, the use of 100% wastewater is not appropriate for the cultivation of microalgae immobilized in sodium alginate.

Immobilized cells grew better than free ones, as reported in other studies (Lam & Lee 2012; Cheirsilp et al. 2017; Emparan et al. 2019; Saxena et al. 2021). Ruiz-Marin et al. (2010) also observed that immobilized Chlorella vulgaris and Scenedesmus obliquus exhibited robust growth with no long lag phase in contrast to free cells, suggesting that both strains were able to quickly adapt to wastewater. According to Alfaro-Sayes et al. (2023), the dry biomass weight for the Chlorella sorokiniana culture immobilized in sodium alginate was 2,870 mg L⁻¹, compared to 2,090 mg L⁻¹ in the free culture, about 1.4 times higher. In the study by Halim & Haron (2021), the number of C. vulgaris cells immobilized in calcium alginate increased exponentially and reached a maximum concentration on the eighth day of cultivation (64 × 10⁶ cells/mL) when grown with the synthetic medium. The critical factor for the cultivation of immobilized microalgae is the efficiency of gas and mass transfer between the liquid and solid phases. Nutrients can diffuse through the structures of the semipermeable beads (alginate entrapment matrix – porous material that facilitates the diffusion of nutrients), allowing cells to undergo the cell division process (Han et al. 2023).

Regarding the different concentrations of wastewater, the one with 10% of swine manure better supported the cultivation of immobilized microalgae, and this can be attributed to the fact that lower concentrations provide an ideal amount of nutrients for the microalgae, ensuring that they grow without excessive amounts of compounds that can be harmful. In addition, reduced toxicity, nutritional balance, less microbial competition, and less accumulation of undesirable factors result from the input of lower concentrations of effluents into microalgal cultivation systems. However, the magnitude of these effects may vary depending on the specific characteristics of the wastewater and the species involved (Kube et al. 2018).

Microalgae immobilization is the traditional technique for removing nutrients from wastewater (Moreno-Garrido 2008). Biodigested swine manure contains a blend of organic (e.g., proteins, lipids, carbohydrates, and volatile fatty acids) and inorganic (e.g., ammonium, phosphates, sulfates, and various metals) compounds (de Carvalho et al. 2022). Ensuring that the treated medium meets acceptable standards by removing these compounds is a significant challenge. In this sense, the removal of COD, TN, and TP by immobilized and free microalgae cells was analyzed.

The COD parameter determines the pollutant load of the water by quantifying the oxygen required for the chemical oxidation of the compounds present in the analyzed water. It is a crucial parameter to evaluate the efficiency of microalgae in removing organic matter present in wastewater (Lacalamita et al. 2024).

The COD removals for immobilized cells cultivated with 20 and 30% of wastewater with initial concentrations of 221.69 and 266.17 mg L⁻¹ were 35 and 38%, respectively. In these conditions, the COD removals were considerably reduced. These results demonstrated that certain concentrations of organic matter affect the COD removal from the effluent by inhibiting the cellular growth of the immobilized microalgae.

In general, immobilized systems were more efficient in consuming organic carbon, which is one of the most important nutrients regulating the growth and metabolism of microalgae. The carbon content in microalgae cells is approximately 50% (Han et al. 2023). This indicates that T. obliquus was able to utilize this substrate to maintain its metabolism and growth. Additionally, COD consumption is closely related to biomass growth in microalgae cultivation because organic matter, as measured by COD, can serve as a source of carbon, energy, and nutrients for microalgae metabolism.

Comparatively, in the study by Emparan et al. (2019), a COD removal rate of 55% was observed for Nannochloropsis sp. immobilized and cultivated in 10% palm oil mill effluent, a lower result than that found in this work. Halim & Haron (2021) found COD removal from synthetic wastewater of 89 and 83% for immobilized and free cells, respectively.

Both types of cultivation (free and immobilized cells) with a concentration of 30% wastewater showed better performance and a similar profile in TN removal. With initial concentrations of 34 and 34.45 mg L⁻¹, both conditions rapidly reached final concentrations of 14.6 and 12.1 mg L⁻¹, respectively. This behavior was observed during the first 48 h of cultivation, when nutrient availability was highest, where the cells moved from a less concentrated medium to a more concentrated one. Therefore, TN concentrations decreased at a slower rate after this period. The results corroborate the biomass production profile, as shown in Figure 3. In addition, TN REs of 65 and 57% were observed for immobilized and free cultures, respectively.

In general, immobilized T. obliquus had a slightly higher percentage of TN removal compared to free cells during cultivation with the other concentrations of 10 and 20% of biodigested swine manure. The concentration of TN gradually decreased in the medium.

A similar trend has been reported in several studies on the removal of nitrogenous compounds from wastewater using immobilized microalgae (Liu et al. 2012; Soo et al. 2017; Banerjee et al. 2019; Özgür & Göncü 2022).

Nitrogen is the second most prevalent constituent of microalgae after carbon. Microalgae are able to use a variety of nitrogen forms, including nitrate (⁠⁠), nitrite (⁠⁠), ammonia (⁠⁠), urea, and organic nitrogen compounds as nutrient sources. It plays a crucial role in microalgal cultures as it is a fundamental component of proteins, nucleic acids, chlorophyll, and other compounds that are essential for microalgal growth and metabolism. Besides, nitrogen has an important role in biomass synthesis and regulation of various metabolic pathways. Therefore, evaluating the total nitrogen removal, a measure that includes all forms of nitrogen present in the medium, including organic and inorganic nitrogen, helps to understand whether there are sufficient nutrients available for the microalgae in the culture medium. This is critical for optimizing growth conditions and maximizing biomass production. In addition, TN removal in microalgae cultures is essential to ensure cultivation success, prevent eutrophication, and monitor the efficiency of wastewater treatment processes (Kumar & Bera 2020).

Phosphorous concentrations decreased sharply in the first 48 h and then stabilized. For example, in the cultivation of microalgae immobilized with 20% wastewater, the initial TP concentration of 311.4 mg L⁻¹ reached a final concentration of 1.6 mg L⁻¹; the same was observed in the cultivation of microalgae with free cells, whose values varied from 312 to 7.28 mg L⁻¹. The phosphate RE by the cells was greater than 99%.

The rapid phosphate consumption in both cultures is related to the ability of the microalgae to respond effectively to nutrient stimuli in the culture medium. According to Liu et al. (2012), when the microalga C. sorokiniana immobilized in alginate was cultured in synthetic wastewater, the phosphate concentration decreased from 10.13 to 1.82 mg L⁻¹, from 10.26 to 2.30 mg L⁻¹, and from 10.26 to 2.30 mg L⁻¹ under mixotrophic, heterotrophic, and microaerobic conditions, respectively. A slight decrease in phosphate from 11.21 to 9.45 mg L⁻¹ was observed under autotrophic conditions. Other studies have demonstrated the ability of immobilized microalgae to remove phosphorus from wastewater (Garbowski et al. 2020; Halimn & Haron 2021).

Phosphorus is one of the key elements for microalgal growth, as 1 g of P can support the production of about 1.7 kg of microalgal biomass. Microalgae are able to uptake phosphorus present in wastewater in various forms, such as inorganic phosphate (⁠⁠), which is the most common form of phosphorus found in the environment and is generally the main source of phosphorus for microalgae. They have the ability to assimilate inorganic phosphate directly from the culture medium to use it in the synthesis of essential cellular compounds. In addition, microalgae can also assimilate organic phosphates, such as phosphate esters and phospholipids, and phosphonates, which are organic compounds containing phosphorus–carbon bonds (Lavrinovičs et al. 2020).

Finally, the use of immobilized microalgae has been widely investigated in recent years as a potential strategy for wastewater treatment. However, due to the complex characteristics of these media, the performance of microalgae in removing the pollutant load varies. Table 2 summarizes several studies conducted with different types of wastewaters, processes, and microalgae species immobilized in alginate matrix.

Table 2

Nutrient and pollutant removal of different immobilized microalgae processes

Microalgae .	Type of wastewater .	Cultivation system .	RE .	Reference .
Tetradesmus obliquus	Swine manure biodigested	Erlenmeyer flasks	COD: 62% TN: 65% TP: 99%	This study
S. obliquus, C. vulgaris, and C. sorokiniana	Food industrial wastewater	Annular PBR	COD: 72.2–78.5% TN: 68.5% − 84.4% : insignificant changes	Hu et al. (2021)
C. vulgaris	Wastewater reverse osmosis concentrate (ROC)	Erlenmeyer flasks	OC: varied changes TN: 85% TP: 100%	Mohseni et al. (2021)
Synechococcus pevalekii and Dunaliella salina	Rice vinasse	Fluidized bed PBR	Nitrate: 91% TS: 29%	Colusse et al. (2021)
C. vulgaris	Wastewater from a municipal secondary effluent stream	Conical flasks	NH4+: 98% : 95%	Banerjee et al. (2019)
Desmodesmus subspicatus	Sugarcane vinasse	Erlenmeyer flasks	OC: 45% TN: 49% TP: 8%	Jesus et al. (2019)
Synechocystis (mutant strain)	Shrimp wastewater	Flat-plate PBR	⁠: 71.5%	Krasaesueb et al. (2023)
Scenedesmus sp.	Raw domestic wastewater	Erlenmeyer flasks	17β-estradiol: 85–99% TN: 86%	Wang et al. (2020)
Tetradesmus obliquus	Simulated wastewater with heavy metals	Erlenmeyer flasks	Cd2+: 99.85%	Li et al. (2024b)

Microalgae .	Type of wastewater .	Cultivation system .	RE .	Reference .
Tetradesmus obliquus	Swine manure biodigested	Erlenmeyer flasks	COD: 62% TN: 65% TP: 99%	This study
S. obliquus, C. vulgaris, and C. sorokiniana	Food industrial wastewater	Annular PBR	COD: 72.2–78.5% TN: 68.5% − 84.4% : insignificant changes	Hu et al. (2021)
C. vulgaris	Wastewater reverse osmosis concentrate (ROC)	Erlenmeyer flasks	OC: varied changes TN: 85% TP: 100%	Mohseni et al. (2021)
Synechococcus pevalekii and Dunaliella salina	Rice vinasse	Fluidized bed PBR	Nitrate: 91% TS: 29%	Colusse et al. (2021)
C. vulgaris	Wastewater from a municipal secondary effluent stream	Conical flasks	NH4+: 98% : 95%	Banerjee et al. (2019)
Desmodesmus subspicatus	Sugarcane vinasse	Erlenmeyer flasks	OC: 45% TN: 49% TP: 8%	Jesus et al. (2019)
Synechocystis (mutant strain)	Shrimp wastewater	Flat-plate PBR	⁠: 71.5%	Krasaesueb et al. (2023)
Scenedesmus sp.	Raw domestic wastewater	Erlenmeyer flasks	17β-estradiol: 85–99% TN: 86%	Wang et al. (2020)
Tetradesmus obliquus	Simulated wastewater with heavy metals	Erlenmeyer flasks	Cd2+: 99.85%	Li et al. (2024b)

COD: chemical oxygen demand; TN: total nitrogen; TP: total phosphorus; OC: organic carbon; TS: total solids.

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The study by Lee et al. (2020) evaluated alginate bead sizes of C. vulgaris and Chlamydomonas reinhardtii with sizes of 2, 3.5, and 5 mm for nutrient removal. The authors reported that the appropriate size was approximately 3.5 mm. However, with a larger size, the microalgae were not able to grow completely due to empty spaces caused by cell migration to the edge of the bead, which prevented substrate diffusion and efficient light transfer. Comparatively, in this study, the size of the bead proved to be adequate, which was also sized at 3.5 mm (as shown in Figure 1), as the cells remained in both the center and the edge of the bead for 192 h of cultivation, allowing them to effectively receive light and substrate from the medium.

The study on immobilized microalgae T. obliquus in sodium alginate beads cultivated in biodigested swine manure demonstrated remarkable growth performance and nutrient RE, surpassing free cell culture, particularly in environments with lower effluent concentrations. The research highlighted the importance of optimizing nutrient concentrations in the medium to support microalgal growth while minimizing potential adverse effects. The results highlighted the high efficiency of organic carbon, total nitrogen, and phosphate removal by immobilized microalgal cells, demonstrating their potential for nutrient bioremediation in wastewater. These results provide valuable insights into the application of microalgae-based systems for sustainable and cost-effective wastewater treatment processes. Further research in this area could explore the scaling up of these techniques for practical implementation in wastewater treatment plants.

The authors thank the Brazilian National Council for Scientific and Technological Development, CNPq (Projects 446787/2020-5 and 408073/2021-7) and the Coordination for the Improvement of Higher Education Personnel, CAPES, Ministry of Education, Brazil.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.