2. Materials and Methods

2.1. Composition of material formulations

All chemicals employed are listed in the SI. All fomulations included 48 % v/v cyclohexanol and 12 % v/v dodecanol as porogens, and 1% w/v omirad 819 as photoinitiator. A mixture of poly(ethylene glycol) diacrylate (PEGDA, 12 % v/v) and alkoxylated pentaerythrioltetraacrylate (SR494, 12 % v/v) as crosslinkers, and 0.125 % w/v Tinuvin 326 as photoabsorber was employed for the acrylate (AETAC and CEA) formulations. The relative concentration of AETAC and CEA was varied to adjust the ligand denisity (0, 4, 8, 12, 16 % vol), with di(ethylene glycol) ethyl ether acrylate (DEGEEA) as non-functional monomer to obtain a total monomer concentration of 16 % vol. The methacrylate formulation was composed of MAETAC (12 % v/v) and HEMA (12 % v/v) functional monomers, ethylene glycol dimethacrylate (EDMA, 16 % v/v) crosslinker and Tinuvin 326 (0.1 % w/v) photoabsorber.

2.2. Model Design, Fabrication, and Characterization

Computer-Aided Design (CAD) models of hollow cylinders and gyroidal columns were created on Fusion 360 (Autodesk, USA), exported as STL files and sliced using Netfabb 2017 (Autodesk, USA). A Solflex 350 (W2P Engineering, Austria) DLP printer was employed to fabricate all parts. Post-printing, the parts were washed three times in IPA in an ultrasonic bath (Allendale Ultrasonics, UK) and then fully cured in water with a xenon Otoflash G171 unit (NK-Optik, Germany). The parts were stored in sterile 0.1 M phosphate buffer until use. A TM4000Plus SEM microscope (Hitachi, Japan) and a Zeiss Crossbeam 550 FIB SEM (Jena, Germany) were used for SEM imaging, with samples prepared by freeze-fracturing with liquid nitrogen, dring in ethanol, followed by a final wash in HMDS before sputter coating using an Emscope SC500 (Bio-Rad, UK). Mean pore sizes and distributions were evaluated from the SEM images.

2.2. Chromatography

3D printed hollow cylinders were employed in batch experiments by inserting the cylinders into 96-well plates and reading the absorbance using a Modulus II microplate reader (Turner BioSystems, USA). Batch adsorption on the AEX material (based on the AETAC monomer) involved an initial equilibration in phosphate buffer (20 mM, pH 7.4) for a minimum of 48 h, followed by addition of a BSA solution (0–32 mg/mL) in phosphate buffer. Similarly, CEX materials (based on the CEA monomer) were equilibrated in binding buffer (20 mM phosphate, pH 7.4) before loading a LYS solution (0–4 mg/mL). Flow experiments were carried out at 1 mL/min using gyroidal columns (50% external porosity, 500 µm wall thickness) slotted into 10 mm i.d. SNAP® glass housing (Essential Life Solutions, USA) and connected to an ÄKTA™ Purifier 10 system (GE Healthcare, USA) equipped with a UV detector to record absorbance at 280 nm.

2.3. Immobilized Enzyme Bioreactor

Trypsin was immobilized on CEA supports via the EDC protocol. Briefly, the 3D printed materials were equilibrated in a 0.1 M sodium phosphate (pH 7.4) activation buffer, followed by a 35-min immersion on activation buffer containing 1:10 molar excess of EDC with respect to carboxylic groups. After extensive washing in activation buffer, coupling of the enzyme was obtained by soaking the 3D printed models in trypsin solutions (1–10 mg/mL) in 0.1 M phosphate buffer pH 7.4 for 2 hours at room temperature. Non-bound trypsin was removed by washing with 0.1 mM Tris buffer (pH 8). The amount of trypsin immobilized on the 3D printed materials was calculated as the difference of the initial and final concentration of trypsin using the BCA assay (Smith et al., 1985). A control experiment was run by adding trypsin solutions to non-activated cylinders. Similarly to chromatography runs, the activity of the immobilized trypsin was tested both in batch (hollow cylinders in multi-well plate format) and dynamic conditions (gyroids with 50% external porosity, 500 μm wall thickness, 25 mm diameter, 10 mm bed height, flow rate ranging 0.5–8 mL/min). In both cases, after equilibration in 50 mM Tris buffer pH 8, a 1 mM BAEE substrate solution in 50 mM Tris buffer pH 8 was fed to the 3D printed models, and formation of the hydrolysis product (BA) product was monitored at 253 nm.

2.4. Bacterial Biofilm Bioreactor

Biofilms of Rhodococcus opacus IEGM 248 cells were obtained by perfusing fresh cultures (exponential growth phase) for 3 days in recirculation mode (1 mL/min) through gyroidal supports (50% external porosity, 2 mm wall thickness,10 mm diameter, 40 mm height) in a glass column, followed by column washes with quarter strength Ringer’s solution to remove non-adsorbed biomass (free cells). The obtained biofilms were then grown by continuous feed (2 mL/min) of a mineral salts medium (MSM, 2.0 g/l sucrose, 7 g/l Na2HPO4, 6 g/l KH2PO4, 2 g/l NH4Cl, 0.2 g/l MgCl2·6H2O, 0.03 g/l CaCl2·2H2O, 0.001 g/l FeCl3·6H2O) spiked with 0.2 mM BT as sole sulphur source. According to the biodesulfurization reaction, BT is converted intoa phenolic compounds (principally hydroxyphenylacetaldehyde) whose presence in the perfusate was confirmed using the Gibbs test (Gibbs, 1927; W. Wang et al., 2013).