Culture of cyanobacteria and bacteria
Microcystis aeruginosa FACHB-905 and FACHB-907 used in this study are purchased from the Freshwater Algae Culture Collection of Institute of Hydrobiology (FACHB), Chinese Academy of Sciences (Wuhan, China). Sterilized BG11 liquid medium or BG11 agar medium (with the agar concentration of 1.5%) is used as the main culture medium for both axenic and xenic M. aeruginosa [5,6,39]. Before being used as inoculants, cyanobacteria are cultured with 200 mL BG11 liquid medium in 500 mL Erlenmeyer ﬂasks for 7 days to reach the log phase, and the culture conditions are as follows: 2000 lux white light, light: dark = 14 h: 10 h; 25 ± 1 °C [5,6]. Axenic M. aeruginosa FACHB-905A is obtained by treating with antibiotics, lysozymes, and micropicking from M. aeruginosa FACHB-905 culture.
Bacterial strains B905-1 and B905-2 are isolated from the culture solution of the cyanobacterium M. aeruginosa FACHB-905. These two bacteria are routinely grown in TY liquid medium  at 28 ± 1 °C under aerobic conditions (with the shaking speed of 150 rpm). The cell-free filtrate of strain B905-1 is obtained by centrifuging the fermentation broth at 10,000×g for 10 min and then filtering through a 0.22 μm cellulose acetate membrane . Stock cultures are kept at 4 °C, and working cultures are obtained from stock cultures through two transfers in appropriate TY liquid medium.
Isolation and purification of axenic culture
For the isolation and puriﬁcation of axenic cultures, cyanobacterial cells are treated by the solid-liquid alternate cultivation method. The xenic cyanobacterium is diluted to different multiple from 10-1 to 10-8, and the different multiple are inoculated onto sterile Petri dishes containing BG11 agar medium, respectively . After incubating for 15 to 20 d under the culture conditions above, a single cyanobacterial colony is picked by a Pasteur pipette with the aid of a microscope, and then transferred into a test tube that containing 5 mL BG11 liquid medium. The purification result is checked as the test tube becoming green, and the testing method is as follows: 0.1 mL cyanobacterial culture from test tube is spread on the Luria-Bertani (LB) agar plate [40,42] and incubated at room temperature for 3 d or more to examine the existence of associated bacteria, the absence of associated bacteria indicates this cyanobacterial culture is axenic. After the purification, the axenic cyanobacterial colony is picked up by a Pasteur pipette, then transferred to Erlenmeyer ﬂasks with BG11 liquid medium, and incubated at 25 ± 1 °C in a 14L/10D light-dark cycle. The purification procedure for axenic M. aeruginosa FACHB-905 is illustrated in Fig. 7.
Cyanobacterial inhibition bioassay
The antibiotics (including tetracycline, cephalosporin, kanamycin, penicillin and streptomycin) and lysozyme are purchased from Wuhan Dingguo biological technology Co., LTD. The sensitivities of cyanobacterium-associated bacterium to tetracycline, cephalosporins, kanamycin, penicillins and streptomycin are evaluated by the filtering paper method with the final concentration of 100 µg mL-1 for each treatment . The sensitivities of cyanobacterium to five antibiotics are carried out by adding antibiotic with the final concentration of 100 µgmL-1 in 250 mL sterilized Erlenmeyer flasks containing 100 mL xenic M. aeruginosa FACHB-905 culture (the initial cyanobacterial cell number is 1.0 × 106 cell mL-1), and the controls (CK) are added without any antibiotic. Effects of lysozyme on the cyanobacterium and associated bacteria are performed by adding lysozyme (the lysozyme is dissolved with sterile distilled water and then ﬁltered through a 0.22 μm membrane) at ﬁnal concentrations of 0, 1.0, 2.0, 5.0, and 10.0 mg mL-1 in 250 mL sterilized Erlenmeyer flasks containing 100 mL xenic M. aeruginosa FACHB-905 culture (the initial cyanobacterial cell number is 1.0 × 106 cell mL-1). Cyanobacterium-associated bacteria and cyanobacterial cell densities are determined after incubation for 2 d.
The growth curve of axenic M. aeruginosa FACHB-905A and xenic M. aeruginosa FACHB-905 are carried out at an initial cyanobacterial cell number of 1.0 × 106 cell mL-1. Effects of bacterium-cyanobacterium ratio on the growth of four kinds of different initial axenic cyanobacterial cell concentrations in BG11 liquid medium are performed as follows: the axenic M. aeruginosa FACHB-905A is firstly added in 250 mL sterilized Erlenmeyer flasks containing 100 mL BG11 liquid medium to keep the cyanobacterial cell number of 3.0 × 102, 3.0 × 103, 3.0 × 104 and 3.0 × 105 cell mL-1, respectively, and then strain B905-1 (initial cell number is 2.73 × 107 cell mL-1) is added according to bacterium-cyanobacterium ratio of 1:10 and 1:100, the controls (CK) are without the addition of strain B905-1. For the effects of bacterium-cyanobacterium ratio on the growth of axenic M. aeruginosa FACHB-905A on BG11 agar medium, the associated bacterium (B905-1) and cyanobacterium (axenic M. aeruginosa) are mixed well in BG11 liquid medium, and the ﬁnal bacterium-cyanobacterium ratios of 1:1, 1:10 and 1:100 are performed by adding different amounts of bacterium into the 100 mL axenic M. aeruginosa culture with the initial cyanobacterial cell number of 1.0 × 104 cell mL-1. The mixed suspensions are diluted to different multiples and then plated on the BG11 agar medium, each dilution gradient is repeated for three times.
The effect of cell-free filtrate of strain B905-1 on axenic M. aeruginosa FACHB-905A is carried out by adding the cell-free filtrate (2%, v/v) into a 100 mL sterilized Erlenmeyer flask which containing initial axenic cyanobacterial cell number of 1.0 × 106 cell mL-1. The cell-free filtrate is obtained by filtrating with the 0.22 μm cellulose acetate membrane. The negative control is made by adding the same amount of TY liquid medium into 100 mL cyanobacterial culture or BG11 agar plate.
All the experiments are performed under aseptic conditions, the controls (CK) and the treatments are replicated three times, and the arithmetical means (± SD) are used as the ﬁnal results.
DNA extraction, sequencing and phylogenetic analysis
The isolated bacterial strains and axenic M. aeruginosa FACHB-905A are identiﬁed based on 16S rRNA gene sequence analysis. Cyanobacterium-associated bacteria are prepared by incubating the seed culture at 37 °C with a shaking speed of 180 rpm for 20 h in sterilized LB liquid medium; while axenic M. aeruginosa FACHB-905A is cultured for 7 days in sterilized BG11 liquid medium under the condition described in Section “Culture of cyanobacteria and bacteria”. Cyanobacterium and associated bacteria cells are collected by centrifugation at 4000 rpm for 10 min (at 4 °C). DNA is extracted from the bacterial or cyanobacterial sample using the 3S DNA Isolation Kit V2.2 (Biocolor BioScience & Technology Co., Shanghai, China). Fragments of the 16S rDNA are ampliﬁed by PCR using the primers 27F (5’-GAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-ACGGCTACCTTGTTACGACTT-3’), and the amplified fragments are sequenced by AuGCT Biotech Co., Ltd. (Beijing, China) . The BLAST procedure is used to search for sequence similarity in GenBank.
Bacteria cell density is determined by colony counting method. Samples are cultured on TY agar medium at 28 ± 1 °C for 48 h, and the colonies were counted. The cyanobacterium cell number is determined by hemocytometer using light microscopy (NIKON-YS100). The cell density or cell number of each sample is counted in triplicate, and standard error of the mean is calculated for all data. Statistical analysis is performed using Version 17.0 of SPSS for Windows (SPSS, Chicago, IL, USA) .
The generation time (G) of the cyanobacterium is calculated according to equation (1):
G = (t2-t1)/[3.322(lgX2-lgX1)] (1)
where X1 and X2 are the cyanobacterium cell number at time t1 and t2, respectively.
The inhibition efficiency is calculated according to equation (2):
Inhibition efficiency = (1 - Ct/C0) × 100% (2)
where C0 and Ct are the cyanobacterium cell number of the control and test group at time t, respectively[5,6].