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).