Field sample collection and analysis
Field samples of surface water (0-50 cm) from Lake Chaohu, Lake Zhiyin (China), Reservoir Orlík (Czechia) and Reservoir Stefanski (Poland) were collected for chemical, biological and biochemical analysis on 05th Jun. 2018, 18th Jan. 2018, 22nd Oct. 2014 and 26th Aug. 2010 respectively (Fig. S1; Table. S2). 2 L of samples were taken and transported to the laboratory immediately. Fresh phytoplankton samples were inspected to minimize difficulties in determining species on the membrane filters. SRP concentrations were determined according to Murphy and Riley [15] after filtering with cellulose acetate membrane (0.45 μm pore diameter size). ELF method was used to quality detect APase on single-cell level. Samples obtained from field were treated using ELF® 97 phosphate (ELFP, InvitrogenTM) on the basis of the protocol described in Strojsova et al. [11]. 0.5 mL incubations were started by adding the ELFP solution (final concentration 27 μM) and samples were incubated at 25 °C for 2.5 hours. Each incubation was terminated by transferring the sample to a filter holder (diameter 7 mm) with a membrane filter (Millipore; 0.22 μm pore size). The filter with retained algae were placed on a microscope slide, embedded it with the anti-fading reagent Citifluor AF1 (Citifluor, London, UK), and covered with a cover slide for microscopic inspection. ELF-labelling cells were inspected by epifluorescence microscopy (Olympus BX51FL). 20-50 (depending on the cell density) images were taken randomly on the filters. The active and inactive cells with fluorescence were recorded in each image to quantify the percentages of cells with ELF labeling.
Experiment design in laboratory culture
Three strains of A. flos-aquae FACHB 1249 (A. flos-aquae 1249 for short), A. flos-aquae FACHB1171 (A. flos-aquae 1171 for short) and A. flos-aquae FACHB 1260 (A. flos-aquae 1260 for short) were attained from Freshwater Algae Culture Collection of Institute of Hydrobiology (Wuhan, China). Prior to experiments, cells were harvested in the exponential growth period by centrifugation (3000 rpm, 10 min), rinsed 3 times with P-free BG11 medium and then cultivated in P-free BG11 medium for 48 h to consume P stored in cells to a low level and equal basis [16]. After that, the culture was inoculated in BG11 [17] medium with modified P concentrations and the modifying sets were as follows:
A culture using K2HPO4 as the sole P source was defined as +DIP treatment. In P-depleted treatment, K2HPO4 was replaced by KCl. α-glycerophosphate and β-glycerophosphate alternated K2HPO4 with the same amount of P in the treatments of +DOPα and +DOPβ. We conducted the experiments in sterile condition to avoid exotic bacterial contamination.
Initial cell density of A. flos-aquae in each culture was roughly 5×108 cells L-1. All cultures were incubated in conditions suitable for cyanobacterial growth: constant white light intensity of 30 μmol photons m-2 s-1 irradiance, 12 h of light: 12 h of dark, and 25°C.
Subsamples were collected at different intervals over 59 days for chemical and biochemical analysis, e.g. SRP and DOP concentrations, cell density, photochemical efficiency, cell P quota, quality and quantity determination of APase, P storage detection etc.
Cell density, specific growth rate and photochemical efficiency of photosystem II
Samples for cell density estimation were preserved with Lugol’s solution and counted with an Olympus BX 41 microscope (Olympus Corporation, Japan) [18]. The specific growth rate was calculated according to the following equation: μ= (ln Ct2 - ln Ct1) / (t1-t0), where Ct2 and Ct1 are cell densities at times t2 and t1, respectively [19].
Fv/Fm is one of the chlorophyll fluorescence parameters which could reflect the maximum quantum yield of the photochemistry. The value of Fv/Fm will decrease when plant was stressed by environmental conditions so that it is often used to evaluate whether the plant is subjected to conditional inhibition [20]. It was examined by Water PAM2100 (Walz, Germany) and determined according to Ting and Owens [21].
P concentrations and cell P quota
SRP concentration was determined using the same method as field investigation. Total phosphorus (TP) and total dissolved phosphorus (DTP) concentrations were measured following digestion according to the measurement reported by Beattie et al. [22]. DOP concentration was calculated as the difference between DTP and SRP concentrations.
Aliquots of 5 mL from individual cultures were harvested onto 25 mm GF/F glass fiber filters (Waterman, UK) for the measurement of TP in samples to determine the specific total P content of cells according to Vahtera, et al. [12].
Size-fractionation of APA
APA was quantified by fluorometric assay [23]. It was determined in unfiltered sample (APAT) and the filtrates through 3.0 μm and 0.45 μm membrane filters (APA<3.0μm and APA<0.45μm respectively). 150 μL of Tris–HCl (pH = 8.5) was added to 2.7 mL subsample, after pipetting aliquots of fluorogenic substrate (MUFP-phosphate, final concentration of 100 μM), fluorescence was measured immediately, after incubation at room temperature for 2 hours, fluorescence was read again. The contribution of APA to the algal (APA>3.0μm) and bacterial (APA0.45–3.0μm) fractions were calculated as follows: APA>3.0μm=APAT−APA<3.0μm, APA0.45–3.0μm=APA<3.0μm−APA<0.45μm, in which APA<0.45μm represented the dissolved phosphatase activity [24].
Fluorescence measurements and microscopic inspections
In addition to quality determine algal APase by ELF method, we detected PPB in cultures of A. flos-aquae by DAPI (4',6-diamidino-2P-depletedhenylindole) according to the procedure of Bar-Yosef et al. [14]. 0.5 mL incubations were started by adding the DAPI solution (final concentration 15 μM) and samples were incubated at room temperature in dark for 5 min to detect PPB accumulation. Incubation termination, slide embedded protocol and inspection were the same as the stain of ELF. DAPI could bind to polyphosphate and show a yellow fluorescence at 526 nm under UV excitation [25, 26].
Statistical analysis
Pearson's correlation analysis and Linear regression were carried out to analyze the relationships between SRP concentration and ELFp, APA and ELFp. The significant differences among four treatments were determined by ANOVA analysis and the differences were considered significant at P <0.05 or P < 0.01. SPSS statistical software (version 18.0, Chicago, IL, USA) was used for those analysis. Data were log transformed to fit normal distribution. The plots of regression were drawn by SigmaPlot 10.0.