The marine microalgae D. salina was purchased from Shanghai Guangyu Biological Technology Co., LTD., and maintained in petri dishes using BG11 solid medium with 3% NaCl. D. salina cells were successively transferred from petri dishes to 250 ml flasks, and then cultivated in 400 ml bubble column photobioreactors with 1% CO₂ under 120 μmol m⁻² s⁻¹ and 25°C conditions. Phenol-degrading bacteria H. mongoliensis (No. 1.7454) was purchased from the China General Microbiological Culture Collection Center and maintained in petri dishes using 2216E solid culture medium. H. mongoliensis cells were successively transferred from petri dishes to 250 ml flasks, and then incubated at 30°C on a rotary shaker (150 rpm). The cells of those microorganisms, D. salina and H. mongoliensis, were harvested during their logarithmic growth phase by centrifugation. All the harvested cells were resuspended into the simulation phenol wastewater with the required biomass density and used in the following experiments.

The stock solution of phenol (2000 mg L⁻¹) was prepared by dissolving the requisite amount of phenol in sterilized BG11 medium with 3% NaCl. The solution of a required concentration of phenol was prepared by diluting the stock solution with the sterilized BG11 medium containing 3% NaCl.

Batch studies on the phenol degradation capability of D. salina and H. mongoliensis were conducted in a set of 250 ml flasks (working volume 150 ml) with breathable sealing membranes on a rotary shaker (150 rpm) under continuous illumination (120 μmol m⁻² s⁻¹) and a constant temperature of 25°C. The evaluation for D. salina was conducted with a starting cell concentration of 0.4 g L⁻¹ and a range of phenol concentrations (i.e., 100, 200, 300, 400 and 500 mg L⁻¹). One control group without phenol addition was also conducted for comparison. For H. mongoliensis, the initial inoculation concentration was set at 0.3 g L⁻¹ to test its degradation capability for 400 and 500 mg L⁻¹ phenol, respectively. All tests were performed in triplicate.

In order to investigate the effect of H. mongoliensis on phenol degradation and the growth of D. salina, different amounts of H. mongoliensis were co-cultured with D. salina at 400 mg L⁻¹ phenol. The initial inoculation concentration of D. salina was 0.4 g L⁻¹. And 0.05, 0.1, 0.15 and 0.2 g L⁻¹ of H. mongoliensis was added to adjust the microalgae to bacteria ratio to 8:1, 4:1, 8:3 and 2:1, respectively. These experiments were conducted in a set of 250 ml flasks (working volume 150 ml) with breathable sealing membranes on a rotary shaker (150 rpm) under continuous illumination (120 μmol m⁻² s⁻¹) and a constant temperature of 25°C. The phenol concentration, biomass concentration of D. salina, Fv/Fm and pH were measured daily. All tests were performed in triplicate.

An evaluation of the influence of operating conditions (phenol concentration, pH, light intensity and temperature) on the performance of the D. salina and H. mongoliensis co-culture was conducted in 250 ml conical flasks (working volume 150 ml) with breathable sealing membranes. The culture parameters at different operating conditions are shown in Table 1. The initial phenol, D. salina and H. mongoliensis inoculation concentrations were kept at 400 mg L⁻¹, 0.4 g L⁻¹ and 0.2 g L⁻¹, respectively. The initial pH was adjusted using HCl (1 mol L⁻¹) or NaOH (1 mol L⁻¹) solution to 5.5, 7.5, 9.5 and 11.5, respectively. The light intensities of 120, 240 and 360 μmol m⁻² s⁻¹ were adjusted by changing the distances between the flasks and the LED (maximum light intensity around 2000 μmol m⁻² s⁻¹). The temperature was regulated using a constant temperature water bath and set as 19, 25, 31 and 37°C, respectively.

The best degradation performance of the co-culture system was investigated with different initial phenol concentrations under optimal conditions. The initial phenol concentrations were adjusted using stock phenol solution to 300, 400, 500 and 600 mg L⁻¹, respectively. All tests were performed in triplicate.

10 ml of sample was daily withdrawn from each flask to measure the residual phenol concentration, cell density, Fv/Fm and pH.

The pH of the sample was measured using a Five Easy pH meter (METTLER TOLEDO) immediately after the sample was harvested. The maximum quantum yield of photosystem Ⅱ was determined using 2 ml of sample. The Fv/Fm value was measured using a fluorescence monitoring system (FMS2, Lufthansa Scientific Instruments Co., Ltd. United Kingdom) after the sample had been stored in dark conditions for 30 min.

1 ml of sample was centrifuged at 6,000 rpm for 10 min to obtain the supernatant. The concentration of residual phenol in the supernatant was measured by the colorimetric assay 4-amino antipyrine method (Zhou et al., 2017). This method involved the use of 4-aminoantipyrine which reacts with phenol at an alkaline pH in presence of potassium ferricyanide to form a red colored antipyrine dye which could be measured spectrophotometrically against a suitable blank at 500 nm. The residual phenol concentrations were calculated by plotting the values against a suitable standard curve (Bera et al., 2017). The removal efficiency of phenol was calculated using Eq. 1: R E ( % ) = ( C i − C t ) / C i × 100 (1a) Where, RE (%) was the removal efficiency of phenol. Ci and Ct were the concentrations of phenol at the initial stage and after the indicated time, respectively.

The biomass concentrations of D. salina and H. mongoliensis in monocultures were determined gravimetrically. Generally, 5 ml of sample was filtered using a pre-dried and pre-weighed cellulose membrane (0.45 µm pore size), washed with deionized water, dried at 105°C until reached the constant weight, cooled in a desiccator and then weighed again. The dry weight of the blank filter was subtracted from that of the loaded filter to obtain the dry weight.

The biomass concentration of D. salina in coculture was determined indirectly by measuring the chlorophyll a and b (Chl a + b) concentrations in co-culture according to the method of Russel et al. (2020). Generally, 0.5 ml of sample was centrifuged at 13,400 rpm for 10 min and the supernatant was discarded. Chlorophyll a and b were extracted from the pellets using methanol (1.5 ml) and quantified as described in Pruvost et al. (2011). The concentrations of Chl a + b (mg L-1) were calculated using Eqs 2–4: C h l   a = [ − 8.0962 × ( O D 652 − O D 750 ) + 16.5169 × ( O D 665 − O D 750 ) ] × 3 (2) C h l   b = [ 27.4405 × ( O D 652 − O D 750 ) − 12.1688 × ( O D 665 − O D 750 ) ] × 3 (3) C h l   a + b = C h l   a + C h l   b (4) Where Chl a, Chl b and Chl a + b are the concentrations of chlorophyll a (mg L⁻¹), chlorophyll b (mg L⁻¹) and chlorophyll a and b (mg L⁻¹), respectively. OD₆₅₂, OD₆₆₅ and OD₇₅₀ are the optical densities of the extraction solution at wavelengths of 652, 665, and 750 nm, respectively.

A standard curve was prepared for measuring biomass concentrations using Chl a+b concentrations in a series of D. salina suspensions. Chl a + b concentrations and biomass concentrations of D. salina were correlated according to the following Eq. 5: Y = 0.0175 X + 0.0128   [ R 2 = 0.992 ] (5) Where, Y and X are biomass concentrations (g L⁻¹) and Chl a+b concentrations (mg L⁻¹) of D. salina, respectively.

The biomass concentration of D. salina in the co-culture was calculated using Eq. 5 after measuring the concentrations of Chl a + b in the co-culture.

The means and deviations of the three replicates of each treatment and control, at each sampling time, were calculated. Data are presented as mean ± SD. Statistical analysis was carried out using Origin (ver. 9.0) software.

In order to determine phenol degradation of D. salina and H. mongoliensis, these two microbes were incubated with different phenol concentrations. D. salina was inoculated with 0.4 g L⁻¹ biomass concentration and 100–500 mg L⁻¹ phenol in the culture medium. As shown in Figure 1A, the residual phenol concentrations decreased with increasing inoculation time across all phenol concentrations. D. salina could completely 100 mg L⁻¹ phenol within 5 days. The removal efficiency of phenol decreased to 75.5% ± 3.4%, 50.6% ± 6.3%, 30.3% ± 1.3%, 27.3% ± 1.7% with increasing phenol concentrations to 200, 300, 400 and 500 mg L⁻¹, respectively. It can be seen that D. salina showed poor degradation capacity to high phenol concentrations. As shown in Figure 1B, the biomass concentrations of D. salina across all phenol concentrations were smaller than that of the control group without phenol. D. salina almost could not grow when microalgal cells exposed to 400 and 500 mg L⁻¹ phenol concentration, which was consistent with the changes of Fv/Fm values (Figure 1C). The removal efficiency of phenol and growth of D. salina were accordance with previous reports (Das et al., 2016; Duan et al., 2017; Wang et al., 2019). Das et al. (2016) reported that the diatom BD1IITG could only degrade 39.88% and 24% of 50 and 250 mg L⁻¹ phenol, respectively, after 8d incubation. Wang et al. (2019) investigated the phenol degradation capability of marine microalgae I. galbana and found that phenol with concentrations below 100 mg L⁻¹ was completely degraded after different residence times of either 2 or 4 days. However, I. galbana cells could not grow at phenol concentrations of 225 mg L⁻¹. In general, high concentration of phenol exhibits a strong toxicity to microalgae under high salt conditions, which seriously affects the growth of microalgae and even leads to death.

In contrast to D. salina, H. mongoliensis showed higher phenol tolerance and removal efficiency under high phenol and high salt concentrations. As shown in Figure 1D, H. mongoliensis could use phenol as the only carbon and energy source to grow, and completely degrade 400 and 500 mg L⁻¹ phenol within 2 days. The results were similar to the phenol degradation capacity of the genus Halomonas (García et al., 2004; Ceylan et al., 2011; Haddadi and Shavandi, 2013; Lu et al., 2015). Haddadi and Shavandi (2013) reported that Halomonas sp. strain PH2-2 could completely degrade 1,100 mg L⁻¹ phenol within 168 h. Lu et al. (2015) Reported that Halomonas sp. strain was able to degrade more than 94% of 500 mg L⁻¹ phenol over a range of 3%–10% NaCl within 4–5 days. If H. mongoliensis are introduced into the phenol degradation system of D. salina, it can accelerate the degradation of phenol, thereby reducing the stress of phenol on D. salina and promoting the growth of D. salina. Therefore, the co-culture of H. mongoliensis and D. salina might enhance the phenol degradation efficacy and increased the biomass concentration of D. salina comparing to D. salina monoculture.

In order to study the effect of operation conditions on the performance of the co-culture system, operating parameters (inoculation ratio, pH, light intensity and temperature) were systemically investigated. The experimental results are presented and discussed below.

In order to investigate the best phenol degradation performance of D. salina and H. mongoliensis co-culture, the experiments were carried out for degradation of 300–600 mg L⁻¹ phenol under optimal operating conditions (25°C, 120 μmol m⁻² s⁻¹, pH 7.5).

As shown in Figure 6A, both 300 and 400 mg L⁻¹ concentrations of phenol were completely degraded within 5 days. The phenol removal efficiencies of D. salina monoculture were only 50.6% ± 6.3%, 30.3% ± 1.3% under 300 and 400 mg L⁻¹ phenol, respectively. It can be seen that the addition of bacteria enhanced the phenol degradation efficiency. In addition, the maximum biomass concentration of D. salina in coculture were 1.5 and 1.7 times compared to the monoculture at 5 days under 300 and 400 mg L⁻¹ phenol, respectively. This might be due to the phenol degradation by H. mongoliensis, which reduce the stress of phenol to D. salina cells. Meanwhile, phenol was degraded to CO₂ and then enhanced microalgal growth as carbon source (Maza-Márqueza et al., 2013; Ryu et al., 2017).

Under the condition of higher phenol concentration, the degradation rates of 500 mg/L and 600 mg/L phenol in the co-culture system could reach to 93.4% ± 0.3% and 80.6% ± 0.1%, respectively, which is much higher than that of D. salina monoculture. However, the biomasses and Fv/Fm values (Figures 6B,C) of D. salina in the co-culture system were relatively low. In addition, the pH of the culture medium under 500 and 600 mg L-1 phenol were higher than that of under 300 and 400 mg L-1 phenol (Figures 6D). These results indicated that high concentration of phenol produced great stress on the growth of D. salina. However, comparing to previous studies (Das et al., 2016; Wang et al., 2019), the co-culture system of D. salina and H. mongoliensis in this study showed higher phenol removal rate and efficiency. To the best of the authors’ knowledge, it was the best performance for phenol degradation by microalgae and bacteria microcosm under high salt conditions. Generally, bacteria have higher phenol tolerance and degradation capability than microalgae (Mohammadi et al., 2015; Bhattacharya et al., 2018). It can be seen that the addition of salt-resistant phenol degrading bacteria significantly improved the maximum phenol degradation concentration and phenol degradation efficiency of the co-culture system, and also increased the biomass of microalgae. In order to further increase the efficiency of the system, it is necessary to screen halotolerant microalgal strains with higher phenol tolerance and degradation efficiency to establish an efficient microalgae and bacteria microcosm, and investigate the phenol removal, the growth of microalgae and bacteria and the relationship between them, and discuss the interactions between microalgae and bacteria in the process of high concentration phenol degradation.