Samples have been collected from the bioreactor directly through the sampling septum. For each time point, a volume equivalent to ca. 300 mg dry cell weight was harvested, frozen in liquid nitrogen, and further stored at -80°C until extraction.
Growth protocol
Precultivations for chemostat were carried out on a 5-ml tube of YPD medium at 28 °C for 16 h at 100 rpm. The culture in growth phase was transferred into a 1-l Erlenmeyer flask containing 170 ml of mineral medium at pH 5.6 prepared as follows (all compounds are expressed in gl-1): (NH4)2SO4, 3.5; KH2PO4, 6; MgSO4, 2; EDTA, 0.0375; ZnSO4∙7H2O, 0.0281; MnCl2∙4H2O, 0.0025; CoCl2∙6H2O, 0.00075; CuSO4∙5H2O, 0.00075; Na2MoSO4∙2H2O, 0.00005; CaCl2∙2H2O, 0.0125; FeSO4∙7H2O, 0.00875; H3BO3, 0.0025; D-biotin, 0.00025; D-L-panthotenic acid, 0.001; nicotinic acid, 0.001; my0-inositol, 0.00625; thiamin, 0.001; pyridoxine, 0.001; para-aminobenzoic acid, 0.0002. Glucose was added to a final concentration of 18 gl-1 with chloramphenicol at 10 mgl-1. After 16 h of growth at 28°C and 100 rpm, 170 ml of the broth were used to inoculate 1.7 l of the same mineral medium in a 3-l bioreactor. Chemostat and D-Stat experiments were performed in a 3-l stirred tank bioreactor with a working volume of 1.5 l using the Biostat B. Braun Biotech International (Sartorius AG , Germany) with the acquisition software MFCS/win 2.0. The temperature was regulated at 28°C and the pH at 5.6 by the in-line addition of 5 M NaOH. The fermentation started in batch under full aerobic conditions. The Dissolved Oxygen was maintained above 20% of saturation by modulating the air flow rate or the stirring rate in order to avoid oxygen limitation. 11 h after inoculation, when the glucose was totally consumed, continuous cultivation was started. For chemostat cultivation, the bioreactor was continuously fed with the mineral medium (except (NH4)2SO4) supplemented with 23 gl-1 glucose at 0.108 lh-1. The bioreactor was fed with a second reservoir containing 60 gl-1 (NH4)2SO4 at 0.0117 lh-1 corresponding to a N/C ratio of 0.129 molN.Cmol-1. The working dilution rate was 0.08 h-1. When the steady state was reached after 5 residence times (ד= 1/D), the D-Stat was set up. The feeding rate of the mineral medium supplemented with glucose was maintained constant at 0.120 lh-1 while the (NH4)2SO4 feeding rate followed a linear and smooth decrease from 0.0117 lh-1 to 0.0003 lh-1 for 50 h (deceleration factor : 0.00015 h-2) corresponding to a decrease in the N/C ratio from 0.129 to 0.0028 molNCmol-1. All other parameters were maintained unchanged.
Extracted molecule
total RNA
Extraction protocol
Frozen samples were treated by mechanical disruption, using a bead beater (Microdismenbrator, Braun, Germany) and a tungsten bead (Ø ~ 7 mm), for 2 min at 2600 rpm. After disruption, the resulting cell powder was recovered and further processed for RNA purification using the RNEasy Midi Kit (Qiagen, The Netherlands), according to the manufacturer's instruction.
Label
Cy3
Label protocol
Samples were treated with the Low Input Quick Amp labelling kit (Agilent, USA), according to manufacturer's protocol.
Scanning was performed using a INNOSCAN 900 Microarray Scanner (INNOPSYS, France). Images were further treated using Mapix v5.5.0 (Innopsys, France).
Description
Reference sample, replicate 4 (pool of sample 22, 23 and 24)
Data processing
Raw transcriptomic data were filtered and normalized using the R software and the Limma package of the Bioconductor library. Local background estimates were corrected using the “normexp + offset” method, using an offset value of 10. A « scale » normalization method was applied to normalize background substracted data between arrays.
