Immobilization of functional nano-objects in living engineered bacterial biofilms for catalytic applications

Abstract

Nanoscale objects feature very large surface-area-to-volume ratios and are now understood as powerful tools for catalysis, but their nature as nanomaterials brings challenges including toxicity and nanomaterial pollution. Immobilization is considered a feasible strategy for addressing these limitations. Here, as a proof-of-concept for the immobilization of nanoscale catalysts in the extracellular matrix of bacterial biofilms, we genetically engineered amyloid monomers of the Escherichia coli curli nanofiber system that are secreted and can self-assemble and anchor nano-objects in a spatially precise manner. We demonstrated three scalable, tunable and reusable catalysis systems: biofilm-anchored gold nanoparticles to reduce nitro aromatic compounds such as the pollutant p-nitrophenol, biofilm-anchored hybrid Cd_0.9Zn_0.1S quantum dots and gold nanoparticles to degrade organic dyes and biofilm-anchored CdSeS@ZnS quantum dots in a semi-artificial photosynthesis system for hydrogen production. Our work demonstrates how the ability of biofilms to grow in scalable and complex spatial arrangements can be exploited for catalytic applications and clearly illustrates the design utility of segregating high-energy nano-objects from injury-prone cellular components by engineering anchoring points in an extracellular matrix.

Keywords: bio-inorganic hybrid system; hydrogen production; living catalysis; nanoscale catalyst immobilization; semi-artificial photosynthesis.

Figure 1

Diverse catalytic applications of tunable functional E. coli biofilms with anchored nano-objects. (a) The biofilm-anchored Au NPs enable the recyclable catalytic reduction of the toxic p-nitrophenol (PNP) into the harmless p-aminophenol (PAP). (b) The biofilm-anchored heterogeneous nanostructures (Au NPs/Cd_0.9Zn_0.1S QDs) photocatalyse the degradation of organic dyes to low-toxic products based on facile light-induced charge separation. (c) The biofilm-anchored quantum dots coupled with the engineered strain enable photo-induced hydrogen production. Electrons are transferred from QDs to hydrogenase using methyl viologen (MV) as a mediator.

Figure 2

Recyclable reduction of p-nitrophenol with E. coli biofilm-anchored Au NPs as catalysts. (a) TEM, HRTEM, HAADF STEM imaging and EDS mapping of E. coli biofilm-anchored Au NPs (5.2 nm). (b) UV-Vis spectra monitoring of the reduction of p-nitrophenol catalysed by polystyrene-substrate-attached E. coli biofilm-anchored Au NPs (5.2 nm). (c) Catalytic activity comparison of three consecutive reuse instances of E. coli biofilm-anchored Au NPs (5.2 nm) on the bottom of a 12-well plate. (d) Illustration of a simple device for recyclable catalysis as enabled by functional PP-thin-flake-substrate-attached biofilm-anchored Au NPs (5.2 nm). (e) Conversion-efficiency comparison of five consecutive reuse instances of the device shown in (d). The conversion efficiencies are 100, 99.9, 98.5, 94.8 and 81.2% for five cycles of a 16-min reaction. (f)–(h) The catalytic efficiencies of p-nitrophenol reduction with E. coli biofilm-anchored Au NPs with a diameter of 2.1 ± 0.5 nm (f), 5.2 ± 0.5 nm (g) and 7.9 ± 0.6 nm (h). The TEM images in (f)–(h) were the enlarged images in the rectangular box in Supplementary Fig. 6. Note: ‘Au NPs’ here refer to water-soluble Au NPs.

Figure 3

Accelerated photodegradation of Congo red using E. coli biofilm-anchored hybrid structures as catalysts. (a) Schematic for the degradation of Congo red using E. coli biofilm-anchored QDs as catalysts. (b) TEM (left), HAADF STEM (top right) and EDS mapping (bottom right) results of E. coli biofilm-anchored Cd_0.9Zn_0.1S QDs. (c) Photodegradation of CR with free QDs, Tc_Reciever/CsgA_His biofilms or Tc_Reciever/CsgA_His biofilm-anchored QDs as catalysts. (d) Schematic of CR degradation using E. coli biofilm-anchored Cd_0.9Zn_0.1S QDs and Au NPs as catalysts. (e) TEM (left), HAADF STEM (top right) and EDS-mapping (bottom right) results of E. coli biofilm-anchored Cd_0.9Zn_0.1S QDs and Au NPs. (f) Photodegradation of CR with Tc_Reciever/CsgA_His biofilm-anchored Au NPs, Tc_Reciever/CsgA_His biofilm-anchored QDs or Tc_Reciever/CsgA_His biofilm-anchored QDs and Au NPs as catalysts. Notes: In the schematics of (a) and (d), the red spheres represent QDs while the yellow spheres represent Au NPs. The dark-brown particles represent Congo red molecules. O₂ is the major sacrificial agent for accumulated photo-generated electrons. For the EDS-mapping results in (b) and (e), the green color represents Zn, the blue color represents Au and the red color represents Cd.

Figure 4

Biofilm-anchored quantum dots coupled with the engineered strain for photo-induced hydrogen production. (a) Schematic for the hydrogen-production process in our in vivo hybrid semi-artificial photosynthesis system. (b) TEM images of Tc_Receiver/CsgA_His biofilm-anchored QDs. The bottom image is the zoomed-in image of the white rectangle in the top image. (c) Hydrogen production over time catalysed under different experimental conditions. (d) Hydrogen production under light–dark cycles. Notes: TEOA represents triethanolamine as a sacrificial agent. MV represents methyl viologen as an electron mediator. HydA represents [FeFe] hydrogenase. BL21(DE3)/pAEFG means E. coli cells containing hydrogenase. CsgA_His/QDs refers to Tc_Receiver/CsgA_His biofilms with anchored QDs. QDs used here was CdSeS@ZnS QDs. The light source used here was an artificial blue-light source with a current of 0.3 A.