Validation of E-C protocol using dynamic growth of the coculture
The E-C protocol is not applicable to the static coculture with known concentrations, as it is based on the growth stoichiometry of individual microorganisms. Therefore, in this subsection, we use coculture batch growth experiments to demonstrate and validate the E-C protocol. With the validity of the cell counting method established, the individual biomass concentration obtained from the cell counting method were used to validate the E-C protocol. Figure 3 (a) and (b) plot the total OD of the coculture over 3 days for the salt water pair and fresh water pair, respectively; and Figure 3 (c) and (d) plot the gas phase composition for each coculture pair for one inoculum ratio (1:10 for the salt water pair and 1:2 for the fresh water pair), respectively. The gas compositions for the other inoculum ratios are provided in Supplementary Material Figure S2. For the fresh water methanotroph-microalgae pair, higher inoculum concentration of the microalgae resulted in better growth of the coculture. This is because the microalgae grows much slower than the methanotroph, so the methanotroph growth is limited by O2 availability. Therefore, more microalgae in the inoculum enabled better growth of the methanotroph. For the salt water pair, higher inoculation concentration of the cyanobacteria did not have much impact on coculture growth. This is because the cyanobacteria grew much faster than the methanotroph, and the methanotroph growth is limited by mass transfer of CH4 from gas phase.
Figure 4 compares the individual biomass concentration measured through the cell counting approach and the E-C protocol for both coculture pairs, where each point represents one of the duplicates, and the error bar represents the standard deviation from three cell counting measurements for the same sample. As can be seen from these figures, the results obtained from the two approaches correlated very well, particularly at low biomass concentrations. The R2 for the linear relationship between the results from the E-C protocol and cell counting approach ranges 0.90 – 0.98, which validates the results obtained from the E-C protocol.
However, Figure 4 also shows that the agreement between the cell counting approach and the E-C protocol deteriorates at higher concentrations after coculture growth. To determine which approach performs better, we calculated the total OD for each sample using the measured individual biomass concentrations, and plotted them against the measured total OD. The results are shown in Figure 5 (a) and (b) for the salt water pair and the fresh water pair respectively. Both figures showed that the total OD calculated from the E-C protocol were almost exactly the same as the measured total OD. On the other hand, the total OD calculated from the cell counting approach showed larger deviation from the measured total OD, particularly at higher concentrations. The bar chart of the mean squared error (MSE) of predictions in the total OD based on six experimental runs (three inoculum concentrations with duplicates) are plotted in Figure 5. The error bar represents one standard deviation of MSE’s. Student’s t -test shows that the MSE’s of the cell counting is statistically significantly larger than that of the E-C protocol, with a p-value of 0.0158 for the salt water pair and 0.0030 for the fresh water pair.
Besides obtaining individual biomass concentration for each microorganism in the coculture accurately and quickly, the E-C protocol also provides estimates of individual substrate consumption rates and product excretion rates. Figure 6 (a) and (b) plot the individual consumption and production rates of O2 and CO2 respectively by M. alcaliphilum 20ZR and S. sp. PCC7002 over a three-day period for the inoculum ratio of 1:10, and Figure 6 (c) and (d) plot those values for M.capsulatusC. sorokiniana , for the inoculum ratio of 1:2.
Figure 6 shows that although for many cases very small amounts of O2 were detected in the gas phase (e.g., day 2 and 3 for the salt water pair and all 3 days for the fresh water pair), significant amount of O2 was produced by the photoautotroph, which was completely consumed in situ by the methanotroph. Similarly, Figure 6 shows that the actual amount of CO2 consumed by the photoautotroph was much larger than what was directly measured in the experiment, because the CO2 produced by the methanotroph would be preferably consumed by the photoautotroph, as it was produced in situ and did not involve the mass transfer resistance from gas to liquid.