Characterization of Clostridium ljungdahlii OTA1: A Non-Autotrophic Hyper Ethanol-Producing Strain
Clostridium ljungdahlii OTA1 was grown in 160 mL serum bottles containing 50 mL of modified Reinforced Clostridial Medium with 5 g/L fructose (mRCMf) and an artificial syngas headspace (110 ml; 20% CO, 20% CO2, 10% H2, balance N2, or 80% CO, balance N2). RNA from three culture bottles at 14 h (early log phase) and 36 h (late log phase) time points (six samples total). Serum bottles were opened and 0.5 ml of culture was added to 1 ml of Qiagen RNA Protect Bacterial Reagent (Qiagen, Venlo, Limburg, Netherlands). These samples were centrifuged (10 min, 21,460 x g, 4°C); the supernatant was removed, and pellets were stored at -65°C. Batch cultures used for RNA sequencing were not sampled for fermentation analysis to save as much culture for total RNA isolation as possible. To ensure that the liquid fermentation products for the cultures used for RNA sequencing were similar to the other fermentations in this study, liquid analysis (as described above) was performed on other cultures that were simultaneously inoculated.   The Qiagen RNeasy Mini Kit (Qiagen, Venlo, Limburg, Netherlands) with on-column RNAse-free DNase (Qiagen, Venlo, Limburg, Netherlands) treatment was used in conjunction with the enzymatic lysis and Proteinase K treatment (New England Biolabs, Ipswich, Massachusetts, USA) per the supplier’s protocol to isolate total RNA. RNA quality was checked by gel electrophoresis. The Epicentre ScriptSeq Complete Kit (Bacteria) (Illumina, San Diego, California, USA) was used to isolate the mRNA as well as synthesize and amplify the cDNA with a different index for each sample (twelve total). The Qiagen RNeasy MinElute Kit (Qiagen, Venlo, Limburg, Netherlands) was used to purify the mRNA. mRNA and cDNA were analyzed for quality and concentration using an Agilent 2500 Bioanalyzer on an Agilent RNA 6000 Pico Chip and DNA High Sensitivity Chip, respectively, at the North Carolina State Genomic Sciences Lab (Raleigh, NC). Indexed cDNA was pooled and subjected to bioanalysis prior to sequencing at the BGI and Children's Hospital of Philadelphia collaborative genome facility using the 100bp Single Read protocol for one lane on the Illumina HiSeq2000. Base quality calls and demultiplexing were performed with the CASAVA 1.8.2 pipeline (Illumina, San Diego, California, USA).