We realized from all these data that lower temperature and pH lead to an efficient reaction cycle. We further sought optimized conditions by running the cycle under these conditions at pH 5, 5 °C with 0, 5, and 10 mM pyridine, decreasing the maximum anhydride concentration (Table , Figure ). At the same time, it suppressed N-acylurea (Figure ). The effectiveness of suppressing N-acylurea leveled off when the pyridine was more than 5 mM which is 10% of the fuel (Figure ). Indeed, the selectivity significantly increased from 71 % (0 mM Pyridine) to 98 % (10 mM Pyridine). Therefore, we conclude that the optimal condition for our reaction cycle is to use dicarboxylic acids as catalysts that lead to 5-membered anhydrides like aspartic acid or succinic acid derivatives. When other catalysts are used, we suggest using 10% pyridine as a fraction of the fuel at pH 5 at 5 o C. Under those optimal conditions, N-acylurea was completely suppressed in the cycle of C3 with DIC under our optimal condition (Figure , Figure ). Moreover, the reaction cycle of the N-acetyl-L-aspartate derivate with a 4-carbon side chain (n = 4) under our optimal condition resulted in a 4-times decrease in the yield of N-acylurea side product (Figure ). We tested our newly optimized condition in a case using a catalyst that could phase-separate, i.e., butyric acid (C4). Due to the extra methylene group compared to C3, the activated catalyst of C4 phase separates to form oil droplets. We ran reaction cycles by fueling 100 mM C4 with 100 mM EDC under the sub-optimal condition (pH 6 without pyridine) and optimal condition (pH 5 with 10 % pyridine as a fraction of EDC). Both reaction cycles were run at 21 °C to compare the phase separation better because phase separation is strongly temperature-dependent. The reaction cycle under the sub-optimal condition did not turn turbid, suggesting no assembly was observed. However, under the optimal condition, the reaction solution became turbid within 30 seconds after EDC was applied, and this turbidity persisted for 5 minutes, as observed on UV/Vis spectroscopy (Figure ). Confocal fluorescence microscopy confirmed that the transient assembly consisted of oil droplets (Figure ). The total volume of the droplets calculated from the confocal fluorescence microscopy peaked at 579 ± 70 µm around 2 min after EDC was added and decreased to 0 after 5 minutes. Moreover, the hydrodynamic diameter of the droplets was 1.7 ± 0.1µm on dynamic light scattering (DLS). The scattering intensity profile further confirmed that droplets emerged upon the application of EDC and decayed after EDC was depleted. The droplets' lifetime was also consistent with the values obtained on UV/Vis spectroscopy and confocal fluorescence microscopy. Taken together, using the optimal versus sub-optimal conditions can make the difference between obtaining chemically fueled functions or not.