Euglena gracilis CCAP 1224/5Z was purchased from the Culture Collection of Algae and Protozoa¹. The microalgal cells were grown under a continuous light at a light intensity of approximately 100 μmol/m²/s in an illuminating incubator at 26°C in EM medium [1.8 g/L NH₄Cl, 0.6 g/L KH₂PO₄, 0.6 g/L MgSO₄, 60 mg/L Urea, 0.02g/L CaCl₂, 0.48 mg/L Na₂EDTA, 2 mg/L Fe₂ (SO₄)₃, 60 μL HCl, 0.01 mg/L Vb₁, 0.0005 mg/L Vb₁₂, 20 mg/L CuSO₄⋅5H₂O, 0.4 g/L ZnSO₄⋅7H₂O, 1.3 g/L Co (NH₃)⋅H₂O, and 1.6 g/L MnCl₂⋅4H₂O] until reaching stationary phase. Subcultures of alga were done every 6 days at a ratio of 10% (Afiukwa and Ogbonna, 2007).

Vibrio natriegens 1H00025 was purchased from the Third Institute of Oceanography, MNR (Xiamen, China). The bacterial cells were grown in sterilized 2216E (CM0471) medium and incubated at 26°C with rotational shaking (120 r/min) in the dark. Subcultures of alga were done every 3 days at a ratio of 1% (Weinstock et al., 2016).

Euglena gracilis cells were harvested during the stationary phase when the OD₇₅₀ reached 3.0. The OD₇₅₀ of each initial inoculum was adjusted to 3.0 after being washed three times in EG medium (Afiukwa and Ogbonna, 2007). V. natriegens cells were harvested during the exponential phase when OD₆₀₀ reached 1.0. The OD₆₀₀ of the initial inoculums were adjusted to 1.0 after being washed three times in EG medium. The inoculation volume ratio of E. gracilis to V. natriegens was 10:1 (200 mL: 20 mL). After mixing the two inoculums, additional medium was added to reach a final volume of 1.5 L. After dilution, co-cultivation of E. gracilis and V. natriegens was performed in an illuminating incubator at 26°C in EG medium under a continuous light at a light intensity of approximately 100 μmol/m²/s, no shaking or aeration was used during experiment.

The growth of E. gracilis was calculated by measuring its cell number, total chlorophyll content, and dry weight. Paramylon content was also calculated. Cell growth was measured by counting cell number with a microscope in a 0.1 mL counting chamber. The total chlorophyll of a 1 mL sample was extracted with 80% acetone and the chlorophyll content was determined using the Arnon method (Arnon, 1949). The dry weight of the 100 mL sample was measured using the oven-drying method (Edmunds, 1966). Paramylon extraction and measurement was carried out as reported previously (Takenaka et al., 1997). Cell number and total chlorophyll were measured daily over the course of 9 days. Dry weight and paramylon content were measured every two days. Three biological replicates were used for each experiment.

The cell number of V. natriegens was measured manually with a microscope in a 0.1 mL counting chamber. Samples were first stained with crystal violet (Kannan et al., 2019) at a 9:1 ratio of sample to crystal violet for 15 minutes. Next, 100 μL of the stained sample was added into the counting chamber and completely fixed on the chamber surface after being dried in an oven.

For LC-MS/MS analysis, samples taken after six days of incubation were first centrifuged at 5,000 g for 5 min at 4°C (Thermo Heraeus Fresco17, United States) to obtain supernatant. After centrifugation, 300 μL aqueous methanol (1 μg/mL of the inner label) was plunged into 100 μL of each supernatant sample, followed by vortexing for 30 s, then 10 min on ice in an ultrasonic disruptor. Samples were then incubated at −40°C for 1 h, followed by centrifugation at 12,000 rpm for 15 min at 4°C. The resulting supernatant was then utilized for LC-MS/MS measurements (Doppler et al., 2016).

LC-MS/MS analyses were performed using a UHPLC system (1290, Agilent Technologies) with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) coupled to a Q Exactive mass spectrometer (Orbitrap MS, Thermo). The mobile phase A consisted of 0.1% formic acid in water at the positive mode and 5 mmol/L ammonium acetate in water for the negative mode, and the mobile phase B was acetonitrile. The elution gradient was set as follows: 0–1 min, 1% B; 1–8 min, 1%–99% B; 8–10 min, 99% B; 10–10.1 min, 99%–1% B; 10.1–12 min, 1% B. The flow rate was set to 0.5 mL/min, while the injection volume was set to 2 μL. A QE mass spectrometer was utilized to acquire MS/MS spectra, with the collective mode set to information-dependent acquisition (IDA) in the acquisition software (Xcalibur 4.0.27, Thermo). In this mode, the acquisition software continuously evaluates the full scan MS spectrum. The ESI source conditions were set as follows: sheath gas flow rate as 45 Arb, aux gas flow rate as 15 Arb, capillary temperature 400°C, full MS resolution as 70000, MS/MS resolution as 17500, collision energy as 20/40/60 eV in NCE mode, spray Voltage as 4.0 kV (positive) or −3.6 kV (negative), respectively (Periannan, 2003).

The cell number, chlorophyll content, dry weight, and paramylon content of E. gracilis were analyzed using a parametric two-way analysis of variance (ANOVA) with treatment (co-cultivated and axenic) as the source of variations. All data fulfilled the assumptions of the parametric test and no data transformation was needed. The statistical analysis was carried out by SPSS 17.0 for Windows.

For the metabolomic study, after the raw data profiles were preprocessed, Student’s t-tests were utilized for univariate analysis, while principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) were used for multivariate analysis. Variable importance in projection (VIP) score was combined with P-value to screen significant differential metabolites. In-house database and online databases (HMDB and Metlin) were applied in metabolite identification. Metabolites which were found to be statistically significantly different in different samples were then qualitatively analyzed based on relevant KEGG pathway and literature information (Mangalam et al., 2013).

When compared to the E. gracilis control, the co-cultivation group had significantly higher biomass and produced more paramylon. As shown in Figure 1A, the average growth rate of E. gracilis was faster when co-cultivated with V. natriegens, and the co-cultivation group not only entered the exponential phase approximately 24 h earlier than the control but also entered the stationary phase 24 h later. The average division speed of E. gracilis under the co-cultivation system was also much faster than that of the control. When the control entered the stationary phase after 6 days and the cell division slowed, the co-cultivation group continued to divide rapidly. After 9 days of cultivation, the cell number of the co-cultivation group was 23% higher than that of the control group. Interestingly, as shown in Figure 1B, the chlorophyll content of the co-cultivation group (47.73 μg/mL) in the late culture period (D9) increased by 23.75% compared with that of the control (38.57 μg/mL).

Changes in the number of V. natriegens cells in the co-cultivation system are shown in Figure 1A. V. natriegens grew slowly during D1–D3 of co-cultivation, and the number of bacteria began to increase sharply from D3 onward. On D5, the growth of V. natriegens entered into a stationary phase (7.23 x 10⁵ cells/mL), while the number of V. natriegens was 15.38 times and 1.33 times higher than that on D1 (0.47 x 10⁵ cells/mL) and D9 (5.43 x 10⁵ cells/mL), respectively. The V. natriegens cell count then began to decline, indicating that this species can exist symbiotically with E. gracilis, and its presence is correlated with higher E. gracilis growth rates.

As shown in Figures 1C, 2, both the dry weight and paramylon content increased in the two groups over time, but the dry weight and paramylon content in the co-cultivation group were significantly higher than that of the control group. On D9, dry weight and paramylon content of the co-cultivation group reached their maximum values, 1.42 and 0.76 g/L, respectively, which were approximately 15 and 12% higher than the control (1.24 and 0.68 g/L).

Metabolites with p-value < 0.05 were considered significant differential metabolites, and were the driving force of the separation shown in the models constructed previously (Table 2). When compared to the control, the co-cultivation group had significantly higher levels of methyl carbamate, ectoine, choline, methyl N-methylanthranilate, gentiatibetine, 4R-aminopentanoic acid and glu-val, while the levels of 3-butylpyridine, proline, sn-glycero-3-phosphocholine, N-butyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine, and myosmine were lower.

To investigate the correlation of significantly differential metabolites, heat maps were generated (Figure 4) using the Pearson’s correlation coefficient. Positive R values denote positive correlation while negative R values denote negative correlation. The metabolite 4R-aminopentanoic acid, for example, was positively correlated with methyl carbamate, methyl N-methylanthranilate, ectoine, glu-val, and gentiatibetine, with glu-val and gentiatibetine showing the highest R values. At the same time, this metabolite was negatively correlated with 3-butylpyridine, proline, sn-glycero-3-phosphocholine, N-butyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine and myosmine, with proline and sn-glycero-3-phosphocholine showing the lowest R values.

In addition to multivariable analysis, metabolites were mapped to KEGG metabolic pathways for enrichment analysis (Table 3). Pathway enrichment analysis showed that “Glycine, serine and threonine metabolism,” “Glycerophospholipid metabolism,” and “ABC transporters” were statistically significantly enriched.

Under the optimal microalgae-bacteria ratio and co-cultivation conditions, the growth of E. gracilis can be improved significantly by V. natriegens. In the co-cultivation group, E. gracilis entered its exponential phase earlier and the exponential phase lasted longer compared to the control. The co-cultivation group also had values of cell number, chlorophyll content, dry weight and paramylon content which were 123, 124, 115, and 112% of the control, respectively.

Taken together, these results indicate that V. natriegens had a positive influence on E. gracilis under optimum growth conditions, resulting in higher reproductive efficiency, increased biomass and higher production of bioactive materials accumulation in E. gracilis. When the diatom Thalassiosira pseudonana was co-cultivated with the bacteria Dinoroseobacter shibae at a 1:1 ratio, the metabolism of T. pseudonana was altered, but its overall growth rate was unchanged (Paul et al., 2013). When cyanobacteria Microcystis aeruginosa PCC 7806 and microalga Desmodesmus subspicatus were co-cultivated in a designated dialysis tubing, the presence of M. aeruginosa did not influence the growth of the microalga at the early logarithmic growth phase, while the microalga started to out-compete the co-cultivated bacteria during the exponential phase of growth (Omidi et al., 2019). These findings indicate that species, cultivation conditions and co-cultivation ratio can all influence the results of co-cultivation. There’re other ways to optimize the biomass and bioactive materials accumulation in microalgae, different approaches were reported depend on the different purposes (Chew et al., 2017). For instance, optimizing the algal photobioreactor (Cheah et al., 2020) or modifying algal particles (Cheng et al., 2019) for wastewater treatment, many researches were also done to explore sustainable ways to utilize algae in bioenergy production (Chia et al., 2018).

Previous studies have also shown that when E. gracilis was heterotrophically co-cultivated with V. natriegens, significant increases in biomass and paramylon content were found (Kim et al., 2019). Although this previous study found positive results from co-cultivation, it did not explore the possible mechanisms or effects on the metabolites present in the system. In the current study, we not only analyzed the metabolites of the co-cultivation culture system, but also confirmed that bacteria can live in an algae-dominant environment, which may work to exclude the presence of other unwanted bacteria.

Under the optimal microalgae-bacteria ratio and cultivation conditions of this study, a balance of oxygen, carbon dioxide and nutrient substances was established between V. natriegens and E. gracilis. Meanwhile, the cell density of V. natriegens increased as E. gracilis’ cell density increased, indicating that the dead cells of V. natriegens (which could have been providing nutrients) were not the main reason why the production of E. gracilis was increased. It is plausible that V. natriegens produced metabolites that positively influenced the growth of E. gracilis. Moreover, V. natriegens entered both the stationary and decline phases earlier than E. gracilis, indicating that V. natriegens had a shorter life cycle than E. gracilis, Therefore, subsequent addition of more bacteria could be considered if this strategy was applied in the actual production process.