NBIC Webinar Presentation

PDRA: Selva M Athi Narayanan

PIs: Dr Simone Dimartino & Prof Nick Christofi

Date: 13/02/2021

Accelerator Project

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• Improved physical interactions between bacterial cells and sulphured substrates

Immobilisation

• Biofilms on porous support materials

Biofilm bioreactor

Concepts: The Known

Concepts: The Unknown

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Perfectly ordered beds vs random beads• Ordered bed better than randomly organised immobilised phase1,2

• Immobilisation on Triply Periodic Minimal Surface (TPMS) structures

Biodesulphurisation (BDS)• Removal of Sulphur from fossile fuels

1 Fee et al., (2014). 3D printed porous media columns with fine control of column packing morphology, Journal of Chromatography A. 1333, pp. 18–24.2 Simon and Dimartino (2019). Direct 3D printing of monolithic ion exchange adsorbers, Journal of Chromatography A. 1587, pp. 119–128.

Biodesulphurisation (BDS)

• Whole cell biocatalysis

• 4S pathway – oxygen intensive process

DBT → DBTO → DBTO2 → HBPS → 2HBP (Phenol) + SO32-

BT → BTO → BTO2 → sulfinate → HPEal (Phenol) + SO32-

Catalysts 2019, 9(3), 229

Dibenzothiophene

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Dibenzothiophene (DBT)

Benzothiophene (BT)

Biocatalyst – Rhodococcus• Actinobacteria

• Big genome size (>8 Mb)• more metabolically active genes

• Mycolic acid rich cell membrane• highly hydrophobic

• Alkanotrophic• Highly tolerant to solvents

• Doubling time > 5 hours

• Some strains - Excellent BT/DBT desulphurising capabilities

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Image of a 4-day old culture of Rhodococcus captured using Keyence VHX digital microscope

3D printed bioreactors - Ideas

3D Bioprinting / Bioplotting Entrapment Bioinks / FLINK

3D printed supports Adsorption Porosity optimal for bacterial percolation

Porogen of choice?

Saha et al., 2018

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Schaffner et al., (2017). 3D printing of bacteria into functional complex materials, Science Advances. 3(12), p. eaao6804.

Saha et al., (2018). Additive Manufacturing of Catalytically Active Living Materials, ACS Applied Materials & Interfaces. 10(16), pp. 13373–13380.

Kyle, S. (2018). 3D Printing of Bacteria: The Next Frontier in Biofabrication, Trends in Biotechnology. 36(4), pp. 340–341.

Shavandi & Jalalvandi (2019). Biofabrication of Bacterial Constructs: New Three-Dimensional Biomaterials, Bioengineering. 6(2), p. 44.

Dubbin et al., (2021). Projection Microstereolithographic Microbial Bioprinting for Engineered Biofilms, Nano Letters. 21, pp. 1352–1359.

Dubbin et al., 2021

Support material

• TPMS structure – gyroid 3,4

• Monomer• BUMA – hydrophobic surface• MAETAC – positive charged surface

• 3D printing conditions• DLP printer• Unit cell (2 mm & 4 mm)• Exposure time• Choice of Vat (Flex / Tilt)

• MAETAC + HEMA as monomers

Chemical Function

Dodecanol / Cyclohexanol mix (20/80) Porogen

2-hydroxyethyl methacrylate (HEMA) Monomer[2-(Methacryloyloxy)ethyl ]trimethylammonium chloride solution (MAETAC)

Monomer

Ethylene glycol dimethacrylate (EDMA SR206) Crosslinker

Omnirad 819 Photoinitiator

Tinuvin 326 Photoabsorber

Resin composition

73 Melchels et al., (2011). The influence of the scaffold design on the distribution of adhering cells after perfusion cell seeding, Biomaterials. 32(11), pp. 2878–2884.4 Aueidda et al., (2019). Mechanical properties of 3D printed polymeric Gyroid cellular structures: Experimental and finite element study, Materials and Design. 165, p. 107597.

Work flowCulture of bacteria 3D printed gyroids

Immobilisation(Adsorption)

Perfusion through a column of gyroids

BDS activityBiomass Reactor Operation

• Gibbs test• Analytical

• SEM• Culture plate method• Staining method

• Recirculation• Assessment of efficiency

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Immobilisation on 3D printed gyroids

Syringe Columns

Non-Recirculating system (for reaction) 20 µl min-1

Recirculating system (for immobilisation) 2 ml min-1

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4 cm

long

~21

cm lo

ng

50x

1000x 4000x

50x

1000x

4000xAcelerating-voltage level: 15 kV; Detector: MIX (Backscattered-electron and secondary-electron).

Gyroid with Immobilised

bacteria

Control Gyroid

(No bacteria)

Biofilms on the gyroid surface

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4000x

4000xAcelerating-voltage level: 15 kV; Detector: MIX (Backscattered-electron and secondary-electron).

Gyroid with Immobilised

bacteria

Control Gyroid

(No bacteria)

Biofilms on the gyroid surface

8000x

8000x 11

BDS activity by immobilised bacteria

Gibbs testblue colour ≡ presence of phenol

Day 1

Day 2

Day 5

Growth on NA plates to assess the microbial load

Controls

Neg Ctrl

0.01 mM 2HBP

0.05 mM 2HBP

0.1 mM 2HBP

Test samples

4th PASS 3rd PASS

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Future

• Gyroids – Autoclavable & Solvent tolerant

• Column length• 3D printer limitation• Columns with multiple stacked gyroids• Columns in series

• Retention time

• Elemental Sulphur analysis

And many other biotransformation reactions..

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Thank you