title: JL-9h-16-Stuttgart
organism: Corynebacterium glutamicum ATCC 13032
description: Protocols: Throughout the triple-phase process, six sequential biomass samples were harvested in a fast sampling procedure at various growth rates and oxygen availabilities. For this purpose, approximately 2 ∙ 109 cells of cell suspension were filled into precooled ( 21 °C) 2 mL reaction tubes. Pellets were then harvested in an immediate centrifugation (≥ 13,000 rcf, 30 s; centrifuge MiniSpin®, rotor F 45 12 11 (pre-cooled to -21 °C), Eppendorf AG, Hamburg, Germany). Then, the supernatant was discarded, the cell pellets frozen in liquid N2 and stored at 70 °C. Triple-phase bioprocess. To investigate the adaptation of C. glutamicum to a transition from aerobiosis via microaerobiosis to anaerobiosis a bioprocess in a 30 L stainless steel bioreactor (Bioengineering AG, Wald, Switzerland) was conceptualized. The seed train was established as follows (cultivation condition / inoculum / incubation time): 2x TY agar plate / streaked from glycerol stock / 2 3 days; o/n1 in 5 mL 2x TY / single colony / 15-16 h; o/d in 50 mL 2x YT / complete o/n1 / 7 8 h; o/n2 in 200 mL CGXII + 40 g glucose L 1 in 2 L flasks with baffles / o/d (starting biomass ~ 0.2 g CDW L 1) / 11-12 h; main culture in 10 L CGXII* + 60 g glucose L 1 in the 30 L bioreactor / o/n2 (starting biomass ~ 0.4 g CDW L 1). The online pH and dissolved oxygen concentration (DO) were measured using standard probes (Mettler-Toledo GmbH, Gießen, Germany). Calibration was achieved ex situ at 30 °C for the DO electrode at 0 % (dH2O + sodium sulfite) and 100 % (dH2O + air gassing) and pH electrodes in buffer solutions at pH 4.0 and pH 7.0. To maintain a pH of 7.4 throughout the cultivation, 25 % ammonia solution was added automatically. Excessive foaming was avoided by adding anti-foam solution manually on demand (Struktol™ J 647, Schill+Seilacher GmbH, Hamburg, Germany). Over the entire cultivation period the agitation was set to 445 rpm using a single six blade Rushton turbine and four baffles at the reactor wall. Low aeration rates of 0.1 vvm (1 L min 1; mass flow controller 0 2 L min 1, Analyt-MTC GmbH, Müllheim, Germany) were realized by gassing through a needle inside the cultivation broth. The exhaust gas was analyzed for O2 and CO2 content (BlueSens gas sensor GmbH, Herten, Germany) and the reactor overpressure kept at 0.5 bar. After 11 h of cultivation anaerobic conditions were initiated by a stop of aeration and flushing the headspace with 10 L nitrogen gas min 1 for 15 min. 2.3.4 RNA isolation was performed on sequential biomass samples (approximately 2 ∙ 10^9 cells), harvested in a fast sampling procedure, basically using the RNeasy Mini Kit (Qiagen) including a double DNA digestion step. RNA quality and quantity was checked by using NanoDrop (Peqlab) and by Agilent RNA Nano 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies). PCR was performed to assure no DNA remained in the samples. 4 µg of total RNA with RNA integrity number (RIN) > 8.0 were used for cDNA lbrary preparation. Stable RNAs were depleted with the Ribo-Zero rRNA Removal Kit (Bacteria) according to manufacturer's instructions (Epicentre). Afterwards the remaining mRNA was purified using RNA MinElute columns (Qiagen) and checked for quality and successful rRNA depletion with the Agilent RNA Pico 6000 kit on the Agilent 2100 Bioanalyzer (Agilent Technologies). The TruSeq Stranded mRNA Library Prep Kit from Illumina was applied to prepare the cDNA libraries.