Fig. 2
Main findings of the high-throughput cultivation protocol’s optimization. A Buffer selection—six different chemical compound combinations with buffering properties were tested. The top row in the table represents the composition of the buffer encoded by symbols (in the legend); the buffers’ pKa values are marked with these symbols on the grey arrow (target pH values marked with red arrows). Some components have two pKa values making them more suitable for covering the whole span of tested pHs (it is presumed that 1 value away from the pKa value the buffer is still in its range of buffering capacity). Initially set pH, molarity, and the experimentally defined post-culture pH (control strain, main culture medium, 72 h) are given as numbers. ‘Buffering capacity’ [M L−1], and percentage of maximal growth [%max] at 48h of cultivation (a proxy for growth impairment) are given as ‘bubble graphs’. Boxes crossed out indicate that the buffer did not meet requirements in terms of (i) component—utilization by Y. lipolytica (based on HPLC data), (ii) final pH—drop in pH > 1, (iii) buffering capacity—buffering capacity level < 10 M L−1, iv) growth—pronounced growth impairment < 40% of maximal growth. Kinetic graphs represent growth and normalized r-Prot for Y. lipolytica control strain grown in a maleic-acid buffered medium (3 pH values, 2 molarities). Grey ‘bubble plots’ represent osmolarity readouts of YPG medium buffered with the indicated maleic acid buffer, and the osmolarity level known to impose severe stress for Y. lipolytica cells. B, C Continuous variables range examination (pH and temperature)—these sections represent pH and temp as numerical, continuous parameters coded from -1 (pH 3.0 or 22 °C) to + 1 (pH 7.0 or 34 °C). Bubble plots represent growth and normalized r-Prot as % values of these parameters read under pH 5.0 or 34 °C for the control strain (48 h). Kinetic graphs represent growth and normalized r-Prot for Y. lipolytica control strain in time. D, E, G Discrete variables levels examination (Oxygen availability, nitrogen, and carbon source types and concentrations)—these sections represent OA, N, and C as categoric parameters encoded as -1 (low OA/inorganic N/glucose) or + 1 (high OA/organic N/glycerol) levels. Oxygen availability variation was implemented by the use of Duetz system sandwich covers of different headspace volume exchange rates: 0.004, 0.7, and 2.5 mL min−1. Optimization of Nitrogen source and concentration was conducted with inorganic N (AS–ammonium sulfate) or organic N (CH – casamino acid hydrolysate) substrates at concentrations of 5 or 15 g L−1. Optimization of carbon source concentrations was performed with glucose at concentrations: 20, 30, 40, and 50 g L−1, marked as C20, C30, C40, and C50, respectively—and cultivated on either one of two N sources. Consumed carbon in these cultures was calculated as residual C amount based on HPLC data from 48h of cultivation in relation to its initial load. Kinetic graphs represent growth and normalized r-Prot for Y. lipolytica control strain in time. F Geometry/volume—this section presents a comparison of two types of 24-well plates with different geometry of wells (square/round) and working volume recommended by the manufacturer (2.5/1 mL). Bubble and time-point plots are constructed as in other sections. Kinetic plots: x-axis time [h] and the y-axis OD600 (optical density at 600nm) value or sFL [FL/OD600] (specific fluorescence) for growth and normalized r-Prot, respectively. Shaded areas represent standard deviation (SD) values, based on biological duplicates or triplicates. All bubble plots are calculated in percentage to the highest or optimal variant (variants 100% are framed with black-dashed lines) in 48 h of cultivation. The percentage value is indicated within the bubble and corresponds to the bubble area. If not stated otherwise, cultures were performed in high OA, 28 °C, pH 5.0, and with glucose (20 g L−1) and ammonium sulfate (15 g L−1) as carbon and nitrogen sources in 24-well plates with JMY2810 as the control strain