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).