Figure legends

Figure 1. Schematic flow diagram for the design, manufacture and experimental testing of porous monoliths for applications in bioprocess engineering. (a) chemical structures of bifunctional monomers (CEA, AETAC, MAETAC, HEMA) employed, displaying (meth)acrylate groups for polymerization (blue) and active groups (red). (b) Simulated reduced plate height vs reduced velocity (Peclet) for the gyroidal scaffold (this work) and random packing of spherical particles (extracted from Schure et al., 2004). (c,f) CAD models of hollow cylinder and gyroid structures, respectively, together their 3D printed counterparts (d,g). Experimental characterization of (e) hollow cylinders using 96 multi-well plate set-up in static (batch) mode and (h) gyroid scaffold in column for dynamic testing.
Figure 2. Adsorption isotherms of (a) bovine serum albumin (BSA) on anion exchangers (based on AETAC monomer, adapted from Simon et al., 2020), and (b) lysozyme (LYS) on cation exchangers (based on CEA monomer). Ligand densities of 0 (control), 0.57, 1.14, 1.73, 2.33 mmol/mL and of 0 (control), 0.77, 1.56, 2.36, 3.23 mmol/mL were tested for the anion and cation exchanger, respectively. Isotherms were fitted with Langmuir model (continuous lines). Dashed lines corresponds to maximum binding capacity of equivalent commercial materials (Boi et al., 2020; Staby et al., 2005). (c) Separation of BSA (16 mg/mL) and myoglobin (MYO, 6 mg/mL) on AETAC-based anion exchangers (1.6 mL column volume (CV), 1.73 mmol/mL ligand density). 500 μL injection in binding buffer (20 mM Tris, pH 8.0) followed by linear gradient from 0 to 30 % elution buffer (20 mM Tris, 1 M NaCl, pH 8.0) over 20 CV at 1.0 mL/min. First peak is flow-through of overloaded BSA and MYO, second and third peaks correspond to MYO elution at 7.4 CV (3.1 mS/cm) and BSA elution at 18.7 CV (19.0 mS/cm) in line with the electrostatic interactions established at the buffer’s pH of 8 (pIBSA = 4.8; pIMYO = 7.0). (d) Separation of BSA (16 mg/ml) and LYS (4 mg/ml) on CEA-based cation exchangers (2.5 mL CV,  3.23 mmol/mL ligand density). 300 μL injection in binding buffer (25 mM phosphate, pH 7.4) followed by linear gradient from 0 to 100 % elution buffer (25 mM phosphate, 1 M NaCl, pH 7.4) over 20 CV at 1.0 mL/min. First peak is flow-through of non-binding BSA (pIBSA = 4.8) and overloaded LYS, second peak corresponds to LYS elution at 7.6 CV (33.4 mS/cm) in line with the electrostatic interactions established at the buffer’s pH of 7.4 (pILYS = 11.4).
Figure 3. (a) Formation of the BA hydrolysis product over time using a 3D printed monolith with 7.31 mg/g immobilised trypsin. (b) BA conversion (blue) and productivity (red) obtained from continuous operation of immobilized enzyme bioreactor with Schoen gyroid bed geometry in steady state mode at different flow rates.
Figure 4. (a) SEM of MAETAC monomers revealing bacterial biofilm adhered onto 3D printed support after 3 days incubation withR. opacus culture in exponential growth phase. BT is desulfurized by R. opacus into phenolic end products, as detected using Gibbs test on perfusate samples visualized by blue colour (b) and measured at 610 nm (c).