Cell counting through flow cytometry
For M. alcaliphilum 20ZR - S. sp. PCC7002 pair, two 0.5 mL samples of culture broth were taken and each sample was immediately treated with 0.25 mL of 50 mM EDTA and 0.25 mL of 4% paraformaldehyde to fix the cells. After 10 minutes of fixation, the samples were centrifuged at 10,000 RPM and 0.5 ml of supernatant was removed, then each sample was treated with 0.5 mL of 0.05% Tween-20 detergent for 20 minutes (away from light) to minimize cells sticking to each other. Next, after removing Tween-20 through centrifugation, the samples were washed and re-suspended in DI water. For M.capsulatusC. sorokiniana pair, the overall procedure is similar, with the differences being that the samples were first treated with 0.2% Tween-20 detergent for 20 minutes and then treated with 200 mM EDTA and 4% paraformaldehyde for 20 minutes to fix the cells. After sample preparation, 25 µL of the re-suspended sample was counted on a Beckman Coulter Cytoflex LX cytometer with 6 active lasers and 21 channels for fluorescence detection. FlowJo Version 10.6.1 was used to analyze the data obtained from the flow cytometer. As both the cyanobacteria and microalgae used in this work are green, and both methanotrophs are white, different filter of excitation wavelengths were used to help differentiate the cells in the coculture. For M.alcaliphilum 20ZR - S. sp. PCC7002 pair, the forward scatter (FSC-H) was paired with the filter of excitation wavelength at 610nm (Y610-mCHERRY-H fluorochrome) to separate the two populations. ForM. capsulatusC. sorokiniana pair, the FSC-H was paired with the filter of excitation wavelength of 710nm (Y710-PC5.5-H) to separate the two populations.