Dear all,

So I'm running some experiments where I will need to record growth profile data, and collect samples for glucose, ethanol and volatiles analysis. My strategy is always to use use fresh cells from a plate (so, streaked in the afternoon to fresh plate, let grow overnight at 29 degrees, and inouclate pre-culture next day, 8 hours, start culture by dilution, calculating a certain Od next day based on the 2x time). When diluting the pre-culture, I assure that they are in the beggining of exponential phase, to avoid any kind of lag-phase. I send you a figure where there are curves using the same strain and same media, but with different final biomass concentrations. From my pre-culture, I also tested dilluting the strains to a culture or actually put them in the microplate reader. I observed that, from the same pre-cutlure, when cells are dilluted something happens, since on the microplate reader, where I just loaded the pre-culture directly, cells grow like in curve 1. However, this does not happen every time, as you can see in curve 1. On curve 1, cells were treated the same way as in curves 2 and 3, but they grew just fine. Additionally, we also measured glucose. As you see, on cultures where growth arrest occurs faster and earlier, glucose takes much longer to be consumed. The growth rates between all the curves, however, are equal, it's just the exit from exponentil growth that occurs earlier. Of curse, for the cutlures 2 and 3, the glucose concentration when cells shift from exponential growth is quite high. I always use same flasks, same amount of media per flask (1/5 of the volume of the flask), fresh cells, etc. I have no clue why are they exiting exponential growth so fast and early. Hope somebody can help me! Thank you in advance.