(566c) The Effect of Cultivation History on the Growth Phenotype of a Type I Methanotroph

Methane (CH₄) is the second most abundant greenhouse gas (GHG), whose global warming potential is 72 times that of CO₂ within a 20-year period. At the same time, CH₄ is a low-cost, rich source for carbon and energy, and an essential component of the global carbon cycle. Because of its low volumetric energy density, it is desirable to convert CH₄ into other forms of liquid fuels, and microbial conversion of CH₄ at ambient temperature and pressure is an attractive alternative to the thermochemical gas-to-liquid conversion, particularly for small scale application ^[1]. Among different methanotroph species, Methylomicrobium buryatense 5GB1 has been identified as a promising biocatalyst for industrial CH₄ conversion, and recently several key results have been published on the strain, including some baseline data of M. buryatense 5GB1 on its bioreactor performance as well as transcriptomic and ¹³C labeling-based analyses have been reported recently ^[2-5].

Despite these advancements towards a better understanding of the type I methanotroph’s cellular metabolism; available understanding on the difference of methane metabolism between oxygen-limited and methane-limited growth is still lacking. In our prior work, we have attempted to examine the different growth phenotypes, i.e., oxygen-limited vs. methane-limited, using batch experiment. However, we found that changing the composition of feed gas alone cannot drive the cells into different phenotypes. Instead, cell growth rate must be controlled to achieve oxygen-limited and methane-limited phenotypes ^[6].

In this work, we use continuous cultures to achieve oxygen-limited and methane-limited growth phenotypes by controlling both the cell growth rate and the feed gas composition. Additionally, we compare the differences between the two phenotypes through yield distribution.

Totally 6 steady-state growth conditions were examined, which consist of three different cell growth rates (controlled via dilution rate) and two feed gas compositions. The feed gas that contains 14% CH₄ and 23.3% O₂ is denoted as the methane-limited condition; while the feed gas that contains 14% CH₄ and 11.7% O₂ is denoted as the oxygen-limited condition. Two independent continuous runs were performed. The conditions tested in different runs were the same 6 conditions, but these conditions were carried out in different orders to test the hypothesis that the cells’ prior growth condition has a noticeable effect on their current physiological state. Figure 1 depicts the order of the different conditions being tested in the first run (CE1) in (a) and the second run (CE2) in (b). For each growth condition, once a steady-state was achieved, it was maintained for at least three days to allow multiple data points to be collected. If a variation of larger than 10% was observed in the data, the steady-state was maintained for an additional day, after which the culture condition was switched to examine a different condition.

By adjusting dilution rate and feed gas composition, different cell growth rates can be obtained with different methane and oxygen uptake rates, enabling further examination of the differences between the methane-limited and the oxygen-limited growth phenotypes. It is interesting that both continuous runs showed that the methane limited conditions produce more organic carbon while the oxygen-limited conditions produce more biomass and less organic compounds, contrary to the common belief that oxygen-limited condition would result in higher yield for the organic compounds.

To better understand the differences between the oxygen-limited and methane-limited growth phenotypes of M. buryatense 5GB1, a modified GEM was used to conduct in silico analysis by incorporating the measurements obtained from the continuous culture. One major application of metabolic network models, particularly GEMs, is to predict different growth phenotypes (e.g., how fast cells grow, what products are excreted) in various genetic and environmental conditions. Developed by the Palsson Lab, phenotype phase plane (PhPP) analysis is a powerful tool that uses FBA with the GEM to provide a global perspective on the genotype-phenotype relationship, and to help characterize different metabolic phenotypes ^[7]. In PhPP analysis, FBA is performed along two flux constraint dimensions (for this paper methane uptake rate and oxygen uptake rate), and the FBA results generated are used to construct a 2D or 3D (if growth rate is also considered) phase diagram.

In order to conduct FBA with the modified model, growth and non-growth associated ATP maintenance values are needed. The growth associated ATP maintenance value within the biomass equation was set at 23 ATP g DCW ^-1, which is considered as the low mode ^[3]. The non-growth associated ATP maintenance (NGAM) was estimated using experimental data, as listed in Table 1 for both CE1 and CE2

Table 1 clearly shows that the methane-limited phenotype requires higher NGAM, indicating higher metabolic burden to the cells. This is consistent with the experimental results of increased production of organic compounds under methane-limited conditions. In addition, the NGAM estimated for CE1 have higher values than that for CE2, for both oxygen-limited and methane-limited phenotypes. This makes sense as in CE1, cells were driven to different phenotypes when moving to a new condition, which resulted in heavier metabolic burden by expressing the enzymes needed for the different phenotype. However, in CE2, cells stayed in the same growth phenotype and only switched once when transition from condition 6 (methane limited) to condition 5 (oxygen-limited). Less frequent changes of phenotype resulted in lower NGAM. Figure 1 (c), (d), (e), and (f) plotted the phenotype planes obtained based on the NGAM estimated for the different runs. It clearly demonstrates the effect of the cultivation history on the current phenotype: For condition 6 (methane-limited condition), in CE1, it was located in the oxygen-limited phenotype, because the two conditions tested before it were both oxygen-limited (condition 4 and 5); while in CE2, it was located in the methane-limited phenotype, because both culture conditions preceding it were methane-limited (condition 2 & 3).

References

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