Water pam

During the batch cultivation period, OD₇₅₀ of microalgae cultures was measured every 2 days by Thermo Fisher Scientific Microplater Reader (Varioskan LUX, Finland) to represent the cell concentration (Shen et al., 2022 (link); Zhang et al., 2022 (link)). At the final cultivation day (Day 10), 10 mL of C. roscoffensis cultures collected by Whatman GF/F filters (0.70 µm) were rinsed twice with Milli-Q water and dried to constant weight (60°C for 48 h) to determine the biomass. Moreover, F_v/F_m was monitored every 2 days using WATER-PAM (WALZ, Germany) to investigate the influences of antibiotics on the photoinactivation.

After 10 min of dark adaptation, the chlorophyll fluorescence parameter of N. haitanensis was measured using WATER-PAM (Walz, Germany). The minimum fluorescence F₀ was obtained by measuring light at an intensity of 0.1 μmol·m^− 2·s^− 1 and a pulse frequency of 1 Hz applied for 0.8 s. Next, the maximum fluorescence Fm was measured with a saturation pulse of 4000 μmol·m^− 2·s^− 1. Subsequently, the chlorophyll fluorescence parameters Fv/Fm =  (Fm-F₀)/Fm were obtained.

Photosynthetic efficiency of microbial phototrophs was measured using a Pulse Amplitude Modulated (PAM) fluorometer (Water-PAM; Walz GmbH, Effeltrich, Germany). A 3 ml aliquot of water was transferred to a quartz cuvette and after a 10 min dark-adaptation period, minimum fluorescence (F_O) was recorded. Upon application of a saturating pulse of light (pulse duration = 0.8 s; pulse intensity > 3000 µmol photons m⁻² s⁻¹) maximum fluorescence (F_M) was determined. From these two parameters, F_V∕F_M was calculated according to the equation (F_M − F_O)∕F_M (Schreiber, 2004 ).

Samples were filtered onto 42 mm diameter Whatman GF/F filters. The filters were then extracted in 10 ml of 90% methanol for 12 hours at 4°C in the dark. Chlorophyll-a was measured on a Turner 10AU fluorometer using the acidification method [27] .
A WaterPAM (Walz GmbH, Germany) was used for the maximum quantum yield (F_v/F_m) measurements. Gain settings were between 18 and 22. All samples were dark adapted at −2°C for one hour prior to measurement. One-way ANOVA was used to compare end-point data for the control and treatment.

The microalgal cell density was daily monitored using a microscope and Hausser hemocytometer until the end of the experiment. Specific growth rate (μ) for each day was calculated according to the following equation: μ = lnCt₂–lnCt₁, where Ct₂ and Ct₁ are cell density at time point t₂ (day) and the day before t₂, respectively. To analyze the photosynthetic response of antibiotic-treated algal cells, the maximum quantum yield of PS II (F_v/F_m), and the non-photochemical quenching (NPQ) were investigated using fast chlorophyll fluorescence with a Water-PAM (Heinz Walz, Germany) as previously described (Zhang R. et al., 2017 (link)).

The Water-PAM (Walz) was used to measure the variable chlorophyll fluorescence with a Water-S stirring device capable of keeping the samples homogenous and preventing sedimentation of the cells. Light-emitting diodes supplied red measuring light (spectral peak at 650 nm), actinic light, and saturation pulses (spectral peak at 660 nm). After 20 min of dark adaption, a 2-ml sample was used to analyze fluorescence parameters Fv/Fm by measuring Fo and Fm. Light treatment strategy and stirrer operation in this study were according to the previous reports (Campbell et al., 1998 (link); Cosgrove and Borowitzka, 2006 (link)).

The photosynthetic fluorescence parameters were determined using a pulse amplitude modulated fluorometer (WATER–PAM, Walz, Effeltrich, Germany). 2ml samples were taken from the incubation bottles and kept in the dark for 15min at 20°C. The assay light levels (A-PAR) between 0 and 1,120μmol photons m⁻² s⁻¹ were applied in nine steps and 45s each in fast light response curve measurements. The instant minimal (F₀ʹ) and maximal fluorescence (F_mʹ) were, respectively, determined at the end of each A-PAR and saturating light pulse (800ms, 3,000μmol photons m⁻² s⁻¹). The effective photochemical quantum yield (Yield) was calculated as: Yield=(F_mʹ−F₀ʹ)/F_mʹ (Baker, 2008 (link)). The relative electron transport rate (rETR) was calculated as: rETR=Yield×A-PAR (Ralph and Gademann, 2005 (link)). The parameters of the photosynthesis vs. irradiance curves (P–I curves) were analyzed as follows: rETR=rETR_max×tanh (a×A-PAR/rETR_max; Jasby and Platt, 1976 (link)). The maximal relative electron transport rate (rETR_max) represents the light-saturating level of rETR, and light use efficiency (a) was derived from the slope of each electron transport rate (ETR) vs. light curve. Saturating light intensity, I_k, is calculated from the expression rETR_max/a and is characteristic for the onset of light saturation.