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