Introduction

Daily household and industrial activities discharge water containing carbon and nutrient compounds, such as nitrogen and phosphorus. These compounds are the main causes of eutrophication in oceans and lakes (El-Sheekh et al. 2021; Nguyen et al. 2020). Eutrophication disrupts the balance of the water ecosystem and leads to environmental pollution; therefore, it is necessary to remove nutrient compounds to avoid eutrophication (Nguyen et al. 2020). Wastewater treatment for the removal of nutrient compounds has been developed since the 1900s, and biological, physical, and chemical wastewater treatment methods have been widely used (Zahuri et al. 2024). In general biological wastewater treatment, the activated sludge method is used in which organic matter is decomposed by microorganisms (Mujtaba et al. 2017; Sikosana 2019; Wilén et al. 2018). However, the decomposition of organic matter using the activated sludge method requires a large amount of oxygen, which means that a large amount of electricity is required for aeration. The energy for aeration accounts for 60–80% of the total energy used in wastewater treatment using the activated sludge method (Chauchuat et al. 2005). Furthermore, because the activated sludge method has low nitrogen and phosphorus removal efficiency, additional treatment is often required.

Wastewater treatment with microalgae including green algae and cyanobacteria has attracted attention as an energy-saving alternative to activated sludge treatment (de Morais et al. 2023; Mujtaba et al. 2017; Wang et al. 2016). As microalgae perform photosynthesis, they do not require aeration and are expected to fix dilute CO2 in the atmosphere. In addition, they are characterized by high nitrogen and phosphorus removal capacities compared to common microorganisms. In a previous study, the model green alga Chlamydomonas reinhardtii was used for wastewater treatment in a palm oil mill with high removal rates of 100% for ammonium and nitrate, 66–89% for phosphate, and 16.7–33.2% for chemical oxygen demand (COD) (Mohd et al. 2024). In another report, when wastewater was treated with the common green algae Chlorella vulgaris, the removal rate of NH3-N was 98.7–99.8% and total phosphorus was 41.0–62.5% (Kwon et al. 2020). In addition, when wastewater was treated with the cyanobacterium Arthrospira platensis, more than 98% of the COD, phosphorus, and nitrogen were removed within 4–5 days of treatment (Hena et al. 2018). However, one problem with photosynthetic microalgae is that their growth rate is lower than that of common microorganisms in an atmosphere of dilute CO2 (Chinnasamy et al. 2009). In microalgae cultivation, high-concentration carbon dioxide aeration is performed to increase the growth ability of the microalgae.

Therefore, the co-cultivation of microalgae and heterotrophic microorganisms is attracting attention to improve the growth of microalgae without the aeration of high-concentration carbon dioxide (Naseema Rasheed et al. 2023; Ray et al. 2022). Previous studies reported that the growth rate of microalgae increases under dilute CO2 atmospheric conditions when C. reinhardtii and Saccharomyces cerevisiae are co-cultured (Karitani et al. 2024a, 2024b). Co-cultivation of C. reinhardtii and Escherichia coli promotes microalgal growth (Yamada et al. 2023). These co-culture systems of microalgae and heterotrophic microorganisms are expected to increase the growth rate of microalgae, even in wastewater treatment, and improve the efficiency of wastewater treatment using microalgae. In addition, the molecular mechanisms of the formation of mutualistic relationships in microalgae co-culture systems remain unclear. It is expected that further developments in research on microalgae co-culture systems will be achieved through omics analysis, including transcriptome analysis.

In this study, we investigated different combinations of microalgae and heterotrophic microorganisms to improve the efficiency of wastewater treatment. Specifically, three types of microalgae and five types of heterotrophic microorganisms were used in combinations for wastewater treatment, and the optimal combination for wastewater treatment was determined. Subsequently, wastewater containing higher concentrations of carbon, phosphorus, and nitrogen was treated with the optimal combination of microorganisms, and the removal efficiency was evaluated. Furthermore, to elucidate the cause of the changes in wastewater treatment efficiency with the optimal combination, changes in gene expression were assessed through transcriptome analysis.

Material and methods

Media and artificial wastewater

The BG11Y medium (1 vol% BG11 broth for microbiology (Sigma-Aldrich Japan, Tokyo, Japan), 5 g/L yeast extract (Formedium, Norfolk, UK), 0.1 vol% trace metal mix A5 + Co (Sigma-Aldrich Japan)); SOT medium (16.8 g/L NaHCO3 (Nacalai Tesque, Kyoto, Japan), 500 mg/L K2HPO4 (Nacalai Tesque), 2500 mg/L NaNO3 (Nacalai Tesque), 1000 mg/L K2SO4 (Wako Pure Chemicals), 1000 mg/L NaCl (Nacalai Tesque), 200 mg/L MgSO4·7H2O (Nacalai Tesque), 40 mg/L CaCl2·2H2O (Nacalai Tesque), 10 mg/L FeSO4·7H2O (Wako Pure Chemicals), 80 mg/L Na2EDTA·2H2O (Nacalai Tesque), 0.1 vol% trace metal mix A5 + Co, pH 9.0); yeast/peptone/glucose (YPD) medium (10 g/L yeast extract, 20 g/L peptone (Formedium), 20 g/L glucose (Nacalai Tesque)), and NBRC 802 medium (10 g/L Casein peptone (Nacalai Tesque), 2 g/L Yeast extract, 1 g/L MgSO4·7H2O, pH 7.0) were used. For the solid medium, 20 g/L of agar powder (Nacalai Tesque) was added.

The 1 × artificial wastewater (0.400 g/L D-( +)-Glucose, 0.200 g/L acetic acid (Nacalai Tesque), 0.078 g/L NH4Cl (Nacalai Tesque), 0.018 g/L KH2PO4 (Nacalai Tesque), 0.013 g/L MgSO4·7H2O, 0.043 g/L CaCl2·2H2O, 0.005 g/L FeSO4·7H2O, 0.200 g/L H3BO3 (Nacalai Tesque), 0.1 vol% Trace metal mix A5 + Co (Sigma-Aldrich Japan), pH 7.5), and 2 × artificial wastewater (0.800 g/L D-( +)-Glucose, 0.400 g/L Acetic acid, 0.156 g/L NH4Cl, 0.036 g/L KH2PO4, 0 0.013 g/L MgSO4·7H2O, 0.043 g/L CaCl2·2H2O, 0.005 g/L FeSO4·7H2O, 0.200 g/L H3BO3, 0.1 vol% Trace metal mix A5 + Co, pH 7.5) were prepared as described in a previous study (Feng et al. 2011) with some modifications. Both types of artificial wastewater were autoclaved at 121 °C for 20 min before use.

Microorganisms and pre-culture conditions

The green algae C. reinhardtii NIES-2238 (National Institute for Environmental Studies), green algae C. vulgaris NIES-1269 (National Institute for Environmental Studies), and the cyanobacterium A. platensis NIES-39 (National Institute for Environmental Studies), autotrophic microorganisms used for wastewater treatment in previous studies (Hena et al. 2018; Mohd et al. 2024; Tam et al. 1990), were used. Yeasts S. cerevisiae YPH499 (NBRC 10505; NITE Biological Resource Center), S. cerevisiae NBRC 1953 (NITE Biological Resource Center), and S. cerevisiae SH-4 (National Research Institute of Brewing), and bacteria Bacillus subtilis NBRC 13719 (NITE Biological Resource Center) and Bacillus amyloliquefaciens NBRC 14141 (NITE Biological Resource Center), heterotrophic microorganisms used for wastewater treatment in previous studies (Ghasem et al. 2002; Guo et al. 2021; Xie et al. 2013), were also used.

BG11Y medium was used for the pre-culture of C. reinhardtii NIES-2238 and C. vulgaris NIES-1269. The SOT medium was used for the pre-culture of A. platensis NIES-39. Microalgae were pre-cultivated at a constant light intensity of 60 µmol-photons/m2/s (white fluorescent light), 30 °C, and 120 rpm for 5 days. Under the condition, the microalgae reached approximately the mid-exponential growth phase.

The YPD medium was used for the pre-culture of S. cerevisiae, and NBRC 802 medium was used for the pre-culture of B. subtilis NBRC 13719 and B. amyloliquefaciens NBRC 14141. Heterotrophic microorganisms were pre-cultivated at 30 °C and 120 rpm for 3 days. Under the condition, the heterotrophic microorganisms reached approximately the mid-exponential growth phase.

Treatment of wastewater with microalgae and heterotrophic microorganisms alone and in combination

Pre-cultured microorganisms were harvested, washed once with artificial wastewater, and then suspended in 15 mL of artificial wastewater in a 50-mL flask to an OD750 of 0.1. Wastewater treatment was performed at a constant light intensity of 60 µmol-photons/m2/s (white fluorescent light), 30 °C, and 120 rpm for 18 h. Experiments were conducted in duplicate to explore an optimal combination of microalgae and heterotrophic microorganisms, and in triplicate to evaluate wastewater treatment efficiency using the optimal combination.

Analysis of wastewater

To measure TOC, wastewater was centrifuged at 10,000 × g for 1 min at 20 °C. The supernatant was diluted with ultrapure water and measured using a total organic carbon (TOC) meter (Shimadzu, Kyoto, Japan). C8H5KO4, NaHCO3, and Na2CO3 were used as standards.

For the determination of PO43− concentration, the effluent was centrifuged at 10,000 × g for 1 min at 20 °C, and the supernatant was diluted appropriately with ultrapure water. Subsequently, 2 mL of the diluted supernatant and 400 μL of potassium peroxodisulfate solution (40 g/L potassium peroxodisulfate) were mixed and heated in a pressure vessel at 126 °C for 30 min. After cooling the pressure vessel to room temperature, 900 μL of the reaction solution and 60 μL of molybdenum chromogenic solution (10 g/L hexammonium heptamolybdate tetrahydrate, 400 mg/L bis[( +)tartrate]diantimony(III)acid dipotassium trihydrate, 13.3 vol% concentrated sulfuric acid, 12 g/L ascorbic acid]) were mixed. After standing at room temperature for 15 min, the PO43− concentration was calculated by measuring Abs880 using a spectrophotometer (Shimadzu). Potassium hydrogen phosphate was used as a standard.

For the measurement of NH4+ concentration, the effluent was centrifuged at 10,000 × g for 1 min at 20 °C, and the supernatant was diluted with ultrapure water. Subsequently, 2 mL of the diluted supernatant and 1000 μL of sodium hydroxide/potassium peroxodisulfate solution (3.0 g/L NaOH, 20 g/L potassium peroxodisulfate) were mixed and heated in a pressure vessel at 126 °C for 30 min. After the pressure vessel was allowed to cool to room temperature, 900 μL of the reaction solution and 60 μL of sodium hydroxide-boron solution [61.8 g/L boric acid, 8.0 g/L NaOH] were mixed, and the NH4+ concentration was calculated by measuring Abs220 using a spectrophotometer. Potassium nitrate was used as the standard.

Transcriptome analysis

Total RNA was extracted from C. reinhardtii NIES-2238 and S. cerevisiae SH-4 cells using NucleoSpin RNA (Takara Bio, Otsu, Japan) and Quick-RNA MiniPrep Plus (Zymo Research The MGIEasy RNA Directional Library Prep Set (MGI Tech, Shenzhen, China) was used to prepare a complementary DNA library for NGS of the extracted RNA. RNA sequencing was performed using DNBSEQ-G400 (MGI Tech).

The genome sequences of C. reinhardtii CC-503 and S. cerevisiae S288c were used as reference sequences for read mapping using Geneious Prime version 2020.0.3 (Tomy Digital Biology, Tokyo, Japan). DEGs were defined as genes satisfying the following conditions: significance probability (p-value) of < 0.01 and differential expression log2 ratio of <  − 1.5 or > 1.5. RNA sequencing data were deposited in the DNA Data Bank of Japan nucleotide sequence database under the accession number DRR568808–DRR568810.

Results

Optimal combination of microalgae and heterotrophic microorganisms for wastewater treatment

Microalgae and heterotrophic microorganisms were combined for 1 × artificial wastewater treatment to determine optimal combinations. Figure 1 shows the removal rates of TOC (a), PO43− (b), and NH4+ (c) in wastewater treatment using three microalgae and five heterotrophic microorganisms alone or in combination.

Fig. 1
figure 1

Removal rates of TOC (a), PO43− (b), and NH4+ (c) from 1 × artificial wastewater using microalgae and heterotrophic microorganisms alone and in combination. Data represent the averages of two replicates, and error bars indicate standard deviation

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The TOC removal rate was 11% for C. vulgaris NIES-1269, which was the highest among the three microalgae when treated alone (Fig. 1a). When only heterotrophic microorganisms were used, S. cerevisiae SH-4 exhibited the highest removal rate (84%) among the five heterotrophic microorganisms. Among the 15 microbial combinations, the combination of C. reinhardtii NIES-2238 and S. cerevisiae SH-4 resulted in the highest TOC removal rate (82%).

C. vulgaris NIES-1269 exhibited the highest PO43− removal rate (31%) among the three microalgae when the wastewater was treated with microalgae alone (Fig. 1b). When heterotrophic microorganisms were used alone, S. cerevisiae SH-4 showed the highest removal rate (94%) among the five microorganisms. Among the 15 microbial combinations, the PO43− removal rate was the highest when C. reinhardtii NIES-2238 and C. vulgaris NIES-1269 were combined with S. cerevisiae SH-4, with 93% and 94%, respectively.

A. platensis NIES-39 exhibited the highest NH4+ removal rate (34%) among the three microalgae when wastewater was treated with microalgae alone (Fig. 1c). When only heterotrophic microorganisms were used, S. cerevisiae SH-4 exhibited the highest removal rate (66%) among the five heterotrophic microorganisms. Among the 15 microbial combinations, the highest NH4+ removal rate was achieved when C. reinhardtii NIES-2238 and C. vulgaris NIES-1269 were used in combination with S. cerevisiae SH-4 (71% and 70%, respectively).

Wastewater treatment by optimal combination

Among 15 microbial combinations, the combination of C. reinhardtii NIES-2238 and S. cerevisiae SH-4 was optimal for wastewater treatment (Fig. 1). With this combination, wastewater treatment was conducted for 18 h using 2 × artificial wastewater with increased concentrations of TOC (Fig. 2a), PO43− (Fig. 2b) and NH4+ (Fig. 2c) to evaluate the wastewater treatment performance.

Fig. 2
figure 2

Removal rates of TOC (a), PO43− (b), and NH4+ (c) from 2 × artificial wastewater with Chlamydomonas reinhardtii NIES-2238 and Saccharomyces cerevisiae SH-4 alone and in combination. Data represent the average of three replicates, and error bars indicate standard deviation. Significant differences were calculated using Student’s t-test (*; 0.05 < p < 0.10, **p < 0.05). n.s., not significant

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A similarly high TOC removal rate of over 80% was achieved when S. cerevisiae SH-4 was used alone, and when C. reinhardtii NIES-2238 and S. cerevisiae SH-4 were used in combination (Fig. 2a). The combination of C. reinhardtii NIES-2238 and S. cerevisiae SH-4 achieved the highest PO43− removal rate of 93% (Fig. 2b). This was 7.0 times higher than that achieved with C. reinhardtii NIES-2238 alone and 1.1 times higher than that achieved with S. cerevisiae SH-4 alone. The combination of C. reinhardtii NIES-2238 and S. cerevisiae SH-4 achieved the highest NH4+ removal rate of 63%, which was 1.3 times higher than that achieved with S. cerevisiae SH-4 alone (Fig. 2c). Statistical analysis confirmed that there was no significant difference in TOC removal rate between the use of S. cerevisiae SH-4 alone and the combination of C. reinhardtii NIES-2238 and S. cerevisiae SH-4, but there was a significant difference in PO43− and NH4+ removal rates. Therefore, the combined use of C. reinhardtii NIES-2238 and S. cerevisiae SH-4 in wastewater treatment significantly improved the removal rates of PO43− and NH4+ from 2 × artificial wastewater.

Comparison of gene expression in wastewater treatment alone and in combination

After 18 h of wastewater treatment with C. reinhardtii NIES-2238 or S. cerevisiae SH-4 alone or in combination, total RNA was extracted from each cell and subjected to transcriptome analysis. Volcano plots showing p-values and fold-changes for each gene of C. reinhardtii NIES-2238 and S. cerevisiae SH-4 obtained by transcriptome analysis are shown in Fig. 3a and b, respectively.

Fig. 3
figure 3

Volcano plot of genes for Chlamydomonas reinhardtii NIES-2238 (a) and Saccharomyces cerevisiae SH-4 (b)

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In C. reinhardtii NIES-2238, the expression of 1371 genes, 636 with upregulated and 735 with downregulated expression, was altered by the combined wastewater treatment (Fig. 3a, Tables S1, S2). In contrast, as shown in Fig. 3b, in S. cerevisiae SH-4, the expression of 692 genes, 305 with upregulated and 387 with downregulated expression, was altered by the combined wastewater treatment (Tables S3, S4).

Gene ontology (GO) analysis was performed on the upregulated and downregulated genes in C. reinhardtii NIES-2238 and S. cerevisiae SH-4 to identify gene functions that were significantly more frequent among the differentially expressed genes (DEGs). The 20 GO terms with the highest log10 (p-value) values obtained by GO analysis are shown in Fig. 4 (for C. reinhardtii NIES-2238) and Fig. 5 (for S. cerevisiae SH-4).

Fig. 4
figure 4

Top 20 GO terms associated with Chlamydomonas reinhardtii NIES-2238 genes whose expression was increased (a) and decreased (b) via co-cultivation

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Fig. 5
figure 5

Top 20 GO terms of Saccharomyces cerevisiae SH-4 genes whose expression was increased (a) and decreased (b) via co-cultivation

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The GO terms for the upregulated genes in C. reinhardtii NIES-2238 were mainly associated with functions related to chromatin components and nucleosomes (Fig. 4a). They were also associated with functions related mainly to the transport of molecules and ions, such as transporter activity and inorganic molecular body transmembrane transporter activity. Some GO terms for the genes downregulated in C. reinhardtii NIES-2238 were related to functions of thylakoids, plastids, chloroplasts, and photosynthetic membranes (Fig. 4b).

Some GO terms for the genes upregulated in S. cerevisiae SH-4 were associated with functions related to membrane-bound organelles, cellular processes, and signal receptor activity (Fig. 5a). Some GO terms of the genes downregulated in S. cerevisiae SH-4 were associated with functions such as cellular processes, intracellular anatomical structure, and cellular anatomical entities (Fig. 5b).

Discussion

The objective of this study is to clarify the optimal combination of microalgae and heterotrophic microorganisms to improve the efficiency of wastewater treatment by combining microalgae and heterotrophic microorganisms. When C. reinhardtii NIES-2238 and S. cerevisiae SH-4 were combined for wastewater treatment, TOC decreased from 424 to 86 mg/L (equivalent to approximately 1275 mg/L and 264 mg/L of COD, respectively [Dubber et al. 2010]) with an 80% removal rate, while total phosphorus decreased from 12 to 0.90 mg/L with a 93% removal rate of PO43−, and total nitrogen decreased from 39 to 14 mg/L with a 63% removal rate of NH4+ in 18 h (Fig. 2). Similar to the present study, a previous study treating wastewater containing similar concentrations of carbon, phosphorus, and nitrogen showed that combined wastewater treatment with the microalgae C. vulgaris and the fungal Aspergillus sp. in concentrated wastewater decreased COD from 1660 to 622 mg/L with a 63% removal rate, total phosphorus from 52.6 to 5.35 mg/L with a 90% removal rate, and total nitrogen from 97.2 to 40.0 mg/L with a 59% removal rate after 24 h (Zhou et al. 2012). In another study, wastewater treatment using a combination of microalgae (C. vulgaris) and a fungus (Ganoderma lucidum) resulted in COD, total phosphorus, and total nitrogen removal rates of 80%, 85%, and 74%, respectively (Guo et al. 2017). Therefore, the wastewater treatment efficiency in this study using safer microorganisms was comparable to or higher than that of previous studies. Interestingly, in the present study, the removal rates of TOC, PO43−, and NH4+ were similar whether 1 × artificial wastewater or 2 × artificial wastewater was used. Therefore, wastewater containing high concentrations of TOC, PO43−, and NH4+ could be treated efficiently. However, the composition of actual urban wastewater varies greatly depending on factors, such as region and season. Microalgae growth and CO2 uptake are affected by many factors, including temperature, pH, CO2 concentration, light, nutrients, and flue gas composition (Hasnain et al. 2023). Future studies should examine the combination of C. reinhardtii NIES-2238 and S. cerevisiae SH-4 established in this study, including the treatment of wastewater with various TOC, PO43−, and NH4+ concentrations and actual wastewater containing various compounds.

Among the genes with increased expression in C. reinhardtii NIES-2238, many were involved in transporting molecules and ions, such as transporter activity and inorganic molecule transmembrane transporter activity (Fig. 4a). These genes included those encoding proteins responsible for the uptake of phosphate (CHLRE_02g144750v5) and ammonium (CHLRE_03g159254v5) ions (Table S1). Therefore, the combination of C. reinhardtii NIES-2238 and S. cerevisiae SH-4 may have increased the expression of these genes, contributing to increased phosphorus and nitrogen removal rates.

The GO terms of the downregulated genes in the microalga C. reinhardtii NIES-2238 were mainly related to thylakoids, chloroplasts, and photosynthesis (Fig. 4b). Previous studies have reported that when microalgae and heterotrophic microorganisms are co-cultured, light is shielded by heterotrophic microorganisms, and the photosynthetic activity of microalgae is suppressed (Karitani et al. 2024a; Yamada et al. 2023; Wang et al. 2019). In the present study, the photosynthetic activity of microalgae could have been suppressed by the co-utilization of microorganisms for wastewater treatment. However, in this study, the combination of C. reinhardtii NIES-2238 and S. cerevisiae SH-4 did not decrease the removal rate of nutrients but rather increased it. Therefore, it is likely that the light-shielding effect of the co-utilization of these microorganisms did not adversely affect the nutrient removal efficiency. However, the effect of light irradiation conditions on nutrient removal efficiency is unclear, and future optimization of light irradiation conditions for wastewater treatment combining C. reinhardtii NIES-2238 and S. cerevisiae SH-4 is required.

The GO terms associated with upregulated yeast genes in wastewater treatment in combination with the microalga C. reinhardtii NIES-2238 were primarily linked to organelles (Fig. 5a). Among the genes in this GO term were those related to the mitochondria, a type of organelle. The mitochondria are responsible for ATP production (Dimmer et al. 2002; Frey et al., 2000). Previous studies have reported that ATP depletion within S. cerevisiae causes cell damage (Schimz et al. 1979). Therefore, when the expression of mitochondrial genes is elevated, the mitochondria are more active in ATP production, and the use of this ATP may protect yeast from cell damage. In line with this, ATP1, which is involved and essential in ATP synthesis (Zhang et al. 2017), was among the upregulated genes (Table S3). Therefore, the increased expression of these genes may have contributed to protecting yeast from cell damage and improving the efficiency of wastewater treatment. Among the yeast genes with elevated expression (TRX1 and GRX3), those that protect cells from oxidative stress and damage were identified (Table S3) (Garrido and Grant 2002; Pujol-Carrion et al. 2006). In general, toxicity to microorganisms caused by oxidative stress is an issue in wastewater treatment using microorganisms (Osundeko et al. 2013; Chan et al. 2022). Therefore, the microorganisms used must be resistant to oxidative stress. In this study, the combination of microalgae and yeast increased the expression of genes that protect cells from various types of damage and stress, which may have contributed to improved efficiency of wastewater treatment.

The GO terms of S. cerevisiae SH-4, genes that were downregulated in wastewater treatment in combination with C. reinhardtii NIES-2238 included cell process control, binding, and cell development (Fig. 5b). These functions are associated with yeast cell growth. Previous studies have reported that in co-culture, competition for microbial growth occurs and microbial growth is inhibited (de Morais et al. 2023; Karitani et al. 2024a). Therefore, S. cerevisiae SH-4 growth potential may have been reduced in wastewater treatment combined with C. reinhardtii NIES-2238 in the present study. However, in this study, the nutrient removal rate was improved by the combined wastewater treatment, indicating that S. cerevisiae SH-4 growth potential did not decrease or that the reduction in S. cerevisiae SH-4 growth potential did not have a negative effect on nutrient removal.

In conclusion, three microalgae and five heterotrophic microorganisms were combined to determine the optimal combination for wastewater treatment. The results showed that the combination of C. reinhardtii NIES-2238 and S. cerevisiae SH-4 for wastewater treatment improved phosphorus removal efficiency (93% removal and 1.1 times improvement) and nitrogen removal efficiency (63% removal and 1.3 times improvement). To the best of our knowledge, this is the first study to show that a combination of green algae and yeast improves the efficiency of wastewater treatment. Transcriptome analysis revealed that one of the main reasons for the improved wastewater treatment performance of the combination of green algae and yeast was the increased expression of genes related to the uptake of phosphate and ammonium ions in the green algae. As both the green alga C. reinhardtii and the yeast S. cerevisiae are highly safe microorganisms, the establishment of their effective combination for wastewater treatment is highly significant. Based on the findings of this study, it is expected that the combination of C. reinhardtii and S. cerevisiae will further improve the efficiency of wastewater treatment, thereby establishing this technology as a new alternative to the activated sludge method of wastewater treatment. In future research, in addition to the transcriptome analysis in this study, further exploration of the biological mechanisms of microbial interactions through metabolomic and proteomic analyses, as well as research into the deletion and overexpression of specific genes, is expected to lead to further strengthening of the mutualistic relationship. Challenges for the practical application of this technology include the development of large-scale wastewater treatment facilities capable of efficiently cultivating microalgae and the development of stable control techniques for the co-cultivation system.