Dynamic feed blending of lignocellulosic feedstocks subject to raw material variability to maintain steady-state cultivation conditions: Data set and Code

Description

Fermentation data and process control code for dynamic bleeding of lignocellulosic feedstocks to enable steady-state processing conditions in the presence of batch-to-batch variability of renewable feedstocks. Ultra-filtered spent sulfite liquor (UF-SSL), a cheap and readily available side stream from the pulp and paper industry with a significant residual sugar concentration but subject to raw material variability, should be valorised as a primary feedstock in a continuous cultivation. Wood hydrolysate (WH) from enzymatic digestion of softwood cellulose fibres after sulfite pulping was used as a secondary, higher value feedstock with constant composition. A multiple-input multiple-output controller was used to compensate for changes in primary feedstock composition, measured in-line by FTIR spectroscopy, by dynamically adjusting UF-SSL and WH feeding, as well as bleeding and cell-free harvesting of the culture. Both feedstocks were obtained from Norway spruce (Picea abies) pulping (Borregaard AS, Sarpsborg, Norway) and stored at 4°C until use. The laboratory set-up of the process consisted of two continuously stirred tank reactor (CSTR) bioreactors (Labfors 5, Infors, Germany), one referred to as the 'feed tank' used as a holding tank for the prepared UF-SSL solutions and the second referred to as the 'cultivation tank' used for the cultivation of C. glutamicum ATCC 13032 pVWEx1-manA pEKEx3-xylAB with improved mannose uptake and additional xylose uptake compared to the wild type. The MIMO control was active for the first 120 h of the process, after which the control was switched off and default settings for all feeds were used for reference. The UF-SSL batch used had a composition of 42.0 g/L glucose, 132.8 g/L mannose and 56.5 g/L xylose, as well as other sugars at lower concentrations that were not metabolised by the strain. Sugar concentrations were varied for the purpose of the experiment by dilution with deionised water (25-75% UF-SSL) and sugar composition was varied by dissolving solid glucose, mannose or xylose powder in the diluted UF-SSL. The WH batch used for this study had a composition of 468.5 g/L glucose, 17.9 g/L mannose and 15.0 g/L xylose and was diluted to 25% before use. A nutrient solution [112.5 g/L urea, 170 g/L KH2PO4] was used to provide bioavailable nitrogen and phosphate (NP). All solutions were supplemented before use [50 mg/L kanamycin sulphate (selection for presence of pEKEx3 plasmid), 100 mg/L spectinomycin dihydrochloride (selection for presence of pVWEx1 plasmid), 0.2 mg/L biotin (essential vitamin for C. glutamicum) and 0.5 mM isopropyl-beta-d-thiogalactopyranoside (plasmid induction)] and sterile filtered [0.22 µm]. 2.5 M H2SO4 and 2.5 M KOH were used for pH control. The dataset consists of feedtank data including in-line FTIR results and cultivation data including off-line sample measurements. Matlab 2024a including the System Identification Toolbox (version 24.1) was used for control implementation.

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The feed tank [20°C, 200 rpm, 0.125 L/min headspace gassing] was equipped with a silver halide fibre-optic immersion probe with a 9.5 mm optical path length and a DiComp diamond probe tip connected by a 1.5 m fibre-optic cable to a liquid N² MCT detector of a Fiber MultiplexIR FT-IR system (ReactIR 45m, Mettler Toledo, USA). Immersion of the FTIR probe tip was ensured by stepwise refilling with prepared UF-SSL solutions at different dilutions and sugar concentrations modified by dissolving solid sugars. All other feeds were provided in glass bottles with predetermined concentrations. Cultivation conditions [30°C, pH 7, 0.5 vvm air] were kept constant and dO2 was regulated to <30% by increasing the stirrer speed (400-1200 rpm). The cultivation tank was equipped with optical dO2 probes (Visferm DO, Hamilton, Switzerland), potentiometric pH probes (Easyferm PHI, Hamilton, Switzerland) and off-gas analysers (BlueInOne Ferm, BlueSense, Germany). Cell retention was performed using 2 hollow fibre modules (Microza PSP-113, Daiso Chemical Co., LTD., Japan) made of polyolefin with a membrane area of 0.1 m², a hollow fibre inner diameter of 1.9 mm, a pore size of 0.1 µm and a loop flow rate of 1 L/min. 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 sterilised, 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 shaking at 230 rpm. Prior to inoculation, seed 2 was harvested and resuspended in 0.9 g/L NaCl (saline) solution to give an inoculum of 1 g/L in 75 ml transferred via styringe. Samples were collected every 3 h and stored at 4°C until analysis using a custom sampling device. Prior to analysis, samples were separated into supernatant and pellet by centrifugation (3420 RCF, 4°C, 5 min). Supernatants were analysed 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 analysed 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). WH feed bottle weight was not available due to vibartions interferring with balance precision at low volumetric feed rates.

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