Demonstration of the E-C protocol in characterizing coculture dynamic growth
In these experiments, the E-C protocol was applied to characterize the dynamic growth of both model coculture pairs. To validate the E-C protocol’s accuracy, the individual biomass concentration within the coculture was also measured through cell counting using flow cytometry for comparison. For each coculture pair, three different inoculum concentrations were tested with duplicates. For the M. alcaliphilum 20ZR - S. sp. PCC7002 pair, the inoculum OD ratios between the methanotroph and the cyanobacteria were 1:15, 1:10, and 1:5, with the same amount of methanotroph for all three cases. For theM. capsulatus - C. sorokiniana pair, the inoculum OD ratios were 1:3, 1:2 and 1:1, also with the same amount of methanotroph for all three cases. Before and after the inoculation, all vials were flushed with the feeding gas (80% CH4 and 20% CO2), and were put under the same light intensity (190 µmol/m2/s). The coculture growth lasted for 3 days and was sampled once daily. The vials were flushed with feeding gas to replenish the gas phase after each sampling.
Modeling Framework for the Experimental-Computational Protocol
The protocol was developed based on each organism’s growth stoichiometry, the substrate exchange relationship within the coculture as shown in Figure 1(b), and the total mass balance. Eqns. (1) and (2) show the growth stoichiometry for the methanotroph and photoautotroph, respectively.
\(CH_{4}+\left(Y_{\frac{O_{2}}{CH_{4}}}\right)_{\text{meth}}O_{2}\rightarrow\left(Y_{\frac{X}{CH_{4}}}\right)_{\text{meth}}X_{\text{meth}}+\left(Y_{\frac{\text{CO}_{2}}{CH_{4}}}\right)_{\text{meth}}CO_{2}\ \)(1)
\(CO_{2}+\left(Y_{\frac{H_{2}O}{CO_{2}}}\right)_{\text{photo}}H_{2}O\rightarrow\left(Y_{\frac{X}{CO_{2}}}\right)_{\text{photo}}X_{\text{photo}}+\left(Y_{\frac{O_{2}}{CO_{2}}}\right)_{\text{photo}}O_{2}\ \)(2)
where \(X\) denotes biomass, and the subscripts “meth” and “photo” denote methanotroph and photoautotroph, respectively; \(Y_{\frac{a}{b}}\) denotes the stoichiometric coefficients between “\(a\)” and “\(b\)”, where “\(b\)” is CH4 for methanotroph and CO2 for photoautotroph. These coefficients can be obtained from literature (Akberdin et al., 2018; Bernstein et al., 2016; Kliphuis et al., 2011). If the coculture growth medium is vastly different from what is commonly used for the single culture and could affect the microorganism’s growth stoichiometry, then experimental data of the single culture cultivated on the coculture medium should be used to estimate the coefficients. The coefficients used in this work are listed in Table 1.
As shown in Figure 1 (b), only the methanotroph within the coculture can consume CH4, therefore the amount of cell growth for methanotroph can be estimated based on the measured methane consumption (i.e., \(CH_{4}\)). Similarly, the amount of the O2required for methane consumption and the amount of CO2produced can be estimated using stoichiometric coefficients as follows.
\(\left(X\right)_{\text{meth}}=\left(Y_{\frac{X}{CH_{4}}}\right)_{\text{meth}}CH_{4}\)(3)
\(\left(O_{2}\right)_{\text{meth}}=\left(Y_{\frac{O_{2}}{CH_{4}}}\right)_{\text{meth}}CH_{4}\ \)(4)
\(\left(\text{CO}_{2}\right)_{\text{meth}}=\left(Y_{\frac{\text{CO}_{2}}{CH_{4}}}\right)_{\text{meth}}CH_{4}\)(5)
Next, based on the overall mass balance of O2 and CO2, as shown in Eqns (6) and (7), we can determine the amount of CO2 consumed and the amount of O2 produced by the photoautotroph. The subscript “gas” and “liquid” denote the measurements obtained from headspace samples and liquid samples, respectively.
\({(O_{2})}_{\text{gas}}={(O_{2})}_{\text{photo}}-\left(O_{2}\right)_{\text{meth}}\ \)(6)
\({(\text{CO}_{2})}_{\text{gas}}={(\text{CO}_{2})}_{\text{meth}}-\left(\text{CO}_{2}\right)_{\text{photo}}-\left(\text{CO}_{2}\right)_{\text{liquid}}\ \)(7)
where CO2 and O2 in the gas phase (i.e. , \({(\text{CO}_{2})}_{\text{gas}}\),\({(O_{2})}_{\text{gas}}\)) are measured through GC, and the dissolved CO2 in the liquid phase (i.e. ,\(\left(\text{CO}_{2}\right)_{\text{liquid}}\)) are measured through total carbon analyser. In Eqn (6), we neglect the contribution from dissolved O2 due to its small solubility in aqueous solutions; however, in Eqn. (7), dissolved CO2 has to be considered due to its much larger solubility in aqueous solutions, especially under high pH conditions. Although it is difficult to determine the amount of dissolved CO2 in one sample due to the carbonate (\(\text{CO}_{3}^{2-}\)) and bicarbonate (\(\text{HCO}_{3}^{-}\)) salts contained in the culture medium and the equilibrium among different forms of dissolved CO2, the change in dissolved CO2 between two sampling points can be easily determined by the difference in the total inorganic carbon content of these two samples. Therefore, based on the overall mass balances (i.e., Eqns (6) and (7)), the amount of CO2consumed and O2 produced by photoautotroph can be obtained, as shown in Eqns (8) and (9).
\({(O_{2})}_{\text{photo}}={(O_{2})}_{\text{gas}}+{(O_{2})}_{\text{meth}}\ \)(8)
\(\left(\text{CO}_{2}\right)_{\text{photo}}=\left(\text{CO}_{2}\right)_{\text{meth}}-\left(\text{CO}_{2}\right)_{\text{gas}}-\left(\text{CO}_{2}\right)_{\text{liquid}}\ \)(9)
With the amount of CO2 consumed and O2produced by the photoautotroph available, the amount of biomass produced by photoautotroph growth can be obtained through two ways using growth stoichiometry, either from CO2 consumption (Eqn. (10)) or from O2 production (Eqn. (11)).
\({(X)}_{photo-1}=\left(Y_{\frac{X}{CO_{2}}}\right)_{\text{photo}}\left(\text{CO}_{2}\right)_{\text{photo}}\)(10)
\({(X)}_{photo-2}=\left(Y_{\frac{X}{O_{2}}}\right)_{\text{photo}}\left(O_{2}\right)_{\text{photo}}\)(11)
where biomass yield with respect to O2 can be obtained as the following:
\(\left(Y_{\frac{X}{O_{2}}}\right)_{\text{photo}}=\frac{\left(Y_{\frac{X}{CO_{2}}}\right)_{\text{photo}}}{\left(Y_{\frac{O_{2}}{CO_{2}}}\right)_{\text{photo}}}\)(12)
In this work, we use the average of these two approaches to estimate photoautotroph biomass accumulation, as shown Eqn. (13).
\({(X)}_{\text{photo}}=\frac{1}{2}\left[\left(X\right)_{photo-1}+\left(X\right)_{photo-2}\right]\)(23)
Results and Discussion