Bioprocess valorization of spent sulfite liquor with Corynebacterium glutamicum on minimal medium

Published: 28 May 2024| Version 1 | DOI: 10.17632/n9jkwkrf4d.1


Fermentation data of 5 replicate batch cultivations with Corynebacterium glutamicum on low molecular weight permeate of ultra-filtered spent sulfite liquor (UF-SSL) from softwood pulping (stored at 4°C). For all experiments, C. glutamicum CR099::U pXMJ19 was used, which, in contrast to the wild-type, contains genes for improved mannose uptake and the ability to metabolize xylose integrated into the genome (denoted by ::U), as well as an empty plasmid for potential product biosynthetic genes. The valorization of UF-SSL was performed without additional prior detoxification steps such as bioling or overliming using a minimal medium based on UF-SSL. The dataset consists of on-line data recorded during cultivation (pH, T, dO2, O2,offgas, CO2,offgas, weight of reactor and feeds, gassing, ...), off-line data of 9 samples per batch taken every 3h (BM; HPLC: glc, man, gal, ara, xyl, urea; Enzyme Assay: ace, lac, glu, NH4, PO4; ICP-MS: Na, Mg, Ca, Mn, Fe, Ni, Cu, Zn) and meta data (feed composition, quantity, density...). UF-SSL (42 g/L glucose, 135 g/L mannose, 57 g/L xylose, 29 g/L galactose, 13 g/L arabinose, 6.4 g/L acetate) was diluted with water to 25% (~10 g/L initial glucose concentration), sterile filtered (0.2µm) and supplemented (0.2 mg/L biotin, 12 mg/L chloramphenicol, 5 ml/L polyproylenglycol 2000). Trace elements were not added since sufficient amounts (relative to the defined medium) were present in the UF-SSL. Due to strong precipitation when PO4 was added to UF-SSL (containing 14 g/L Ca), a separate nitrogen and phosphate (NP) feed was used instead of mixing with the medium. The NP feed (66.4 g/L urea and 71.8 g/L KH2PO4, sterile filtered) was added at a constant feed rate ( V = 4.5 mL/h until 100 ml were fed) to the 1 L UF-SSL minimal medium batch volume in order to achive a CN ratio of ~1:10 and CP ratio of ~1:4 at full addition.


Steps to reproduce

Pre-culture was started from glycerol stocks stored at -80°C by plating cells on 2TY agar plates (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, 10 g/L agar, heat sterilized, 12 mg/L chloramphenicol) and incubated at 30°C for 72h. Subsequently, 2 seeding steps were performed on liquid 2TY complex medium (without agar) for 24h (1 colony, 12.5 ml medium) and 18h (12.5 ml seed 1, 225 ml medium, 25 ml 100% UF-SSL with 100 g/L 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7, sterile filtered) at 30°C with 230 rpm shaking speed. The addition of UF-SSL in the Seed 2 step is to allow for short term adaptation of the cells to the substrate (sugar utilization genes, inhibitor detoxification genes). Prior to innoculation, Seed 2 was harvested and resuspended in 0.9 g/L NaCl (saline) solution for an inoculum of 1 g/L in 75 ml transferred via styringe to avoid transferring complex components from the preculture into the batch. Innoculum volume and medium dilution (initial cell to inhibitor ratio), freshly incubated agar plates plus precise timing of preculture plus rapid handling of cells when not in medium (cell viability) were identified as critical parameters for reproducible growth in the batch. 3L working volume glass bioreactors (Labfors 5, Infors, Germany) equipped with optical dO2 probes (Visferm DO, Hamilton, Switzerland), potentiometric pH probes (Easyferm PHI, Hamilton, Switzerland) and off-gas analyzers (BlueInOne Ferm, BlueSense, Germany) were used for cultivation. Samples were collected every 3 h and stored at 4°C until analysis using a custom sampling device. pH was maintained at 7.00 +/- 0.02 by adding 2.5 M KOH and 2.5 M H2SO4, temperature at 30°C, and dO2 above 30% by increasing the stirrer speed at a constant air flow of 0.3 vvm. Prior to analysis, samples were separated into supernatant and pellet by centrifugation (3420 RCF, 4°C, 5 min). Supernatants were analyzed by HPLC (UltiMate U3000, ThermoFisher, USA) with an RI detector (RI-100, Shodex, USA) using a Pb column (Nucleogel Sugar Pb 300mm, Macherey-Nagel, Germany) with an isocratic flow of 0.4 ml/min of ultrapure water at 79°C and automated enzymatic photometric assays (CEDEX Bio HT Analyzer, Roche, Switzerland). Pellets were analyzed for biomass concentration by resuspension in saline twice, followed by serial dilution on 96-well plates (PP black, Microplate, 96-well, F-Bottom; Grainer BIO-ONE) , followed by fluorescence measurement (EX: 280/15 nm; EM: 340/20 nm) using a plate reader (Spark, Tecan, Switzerland).


Technische Universitat Wien Fakultat fur Technische Chemie


Bioprocess Optimization, Bioprocess, Fermentation


Bio-Based Industries Joint Undertaking

No 790507