Assembly and Quantification of Co-Cultures Combining Heterotrophic Yeast with Phototrophic Sugar-Secreting Cyanobacteria

Our research aims to understand microbial networking in synthetic consortia involving sucrose-secreting cyanobacteria and heterotrophic fungi. In the past, microbiologists usually studied pure cultures of single microorganisms. In nature, however, microbes usually occur in consortia, and therefore it's essential to also understand microbial networking in mixed cultures.

One major challenge is to find suitable growth conditions, including a medium that allows growth of all consortia members. Here we mainly focus on optimizing for the highest sucrose production and secretion by the phototrophic member, which provides the basis for growth of the heterotrophic members. For tracking growth in axenic cultures, common techniques like optical density or backscatter measurements can be used.

However, these techniques do not apply to co-cultures where we need the cell count of the individual species. In our protocol, we apply a combination of fluorescent labeling and cell counting to distinguish between different co-culture partners. In addition, we could show that similar results can be achieved by different methods ranging from simple, low cost to expensive, high-throughput ones.

To begin, pellet the cyanobacterial cells after growing the culture for three days. Resuspend the pellet in 25 milliliters of sterile CoYBG-11 medium. Centrifuge the sample at 4, 000g for 20 minutes at room temperature.

Then discard the supernatant by decanting. Once the optical density is determined, add the calculated amount of CoYBG-11 in a 250-milliliter baffled flask under sterile conditions. Afterwards, add the calculated volumes of the cyanobacterial and heterotrophic cell suspensions to achieve a final culture volume of 50 milliliters.

Use 50 microliters from the one-molar IPTG stock for a 50-milliliter culture. Then place the flask in the photoincubator at approximately 200 micromoles photons per square meter per second. Supplement the environment with 2%carbon dioxide at 30 degrees Celsius, orbital shaking at 150 RPM, and 75%humidity.

For sampling, take the flask out of the incubator and take a one-milliliter sample from the culture under sterile conditions. To prepare for the microscopic quantification, ensure that the counting chamber and cover glass are free from dust and cells. Position the cover glass correctly by sliding it onto the two support bars with a little pressure while avoiding breakage.

Once the cover glass is properly positioned, observe Newton's rings between the two glass surfaces while ensuring the cover glass does not slip. Now mix the cell suspension thoroughly and apply a few microliters to the edge of the chamber. Allow the chamber to fill completely via capillary force.

Then use an appropriate objective of a light microscope and focus on the counting chamber grid lines. Count the cells in the squares appropriate for the given cell size. For quantification by particle counting, adjust the optical density of the sample to 0.1.

Then add 10 microliters of the sample to 10 milliliters of isotonic measuring buffer. Secure the lid of the sample cup and mix the sample gently by slightly tilting the cup. After placing the sample cup in the particle counter, insert a capillary with the appropriate pore size for the utilized cells into the sample cup and close the machine's door.

Start the measurement and record in an appropriate range. Cells with different cell sizes can be discriminated at this stage. For quantification by single-cell flow cytometry, adjust the optical density of the samples to arrange suitable for cytometry measurement.

Apply the given formula to ensure the cell count remains within a range of 1, 000 to 10, 000 cells per second at a flow rate of 10 microliters per minute. Transfer 300 microliters of each sample cell suspension to an individual well in a 96-well plate. Then transfer the 96-well plate to the auto sample of the cytometer.

Open dot plots and histograms for the fluorescence and scatter channels relevant to the samples. To determine the cell concentration, record a defined sample volume using the record function. Separate the signals for photo and heterotrophic organisms based on the red autofluorescence of the phototrophic organism using the histogram of the APC-H channel.

Then use the histogram of the FITC-H channel displaying only the heterotrophic population to identify the heterotrophic cells containing green fluorescent protein. Use the histogram of the PC5.5-H channel to distinguish between heterotrophic cells containing red fluorescent protein and those without it.