Polymer-induced biofilms for enhanced biocatalysis

Abstract

The intrinsic resilience of biofilms to environmental conditions makes them an attractive platform for biocatalysis, bioremediation, agriculture or consumer health. However, one of the main challenges in these areas is that beneficial bacteria are not necessarily good at biofilm formation. Currently, this problem is solved by genetic engineering or experimental evolution, techniques that can be costly and time consuming, require expertise in molecular biology and/or microbiology and, more importantly, are not suitable for all types of microorganisms or applications. Here we show that synthetic polymers can be used as an alternative, working as simple additives to nucleate the formation of biofilms. Using a combination of controlled radical polymerization and dynamic covalent chemistry, we prepare a set of synthetic polymers carrying mildly cationic, aromatic, heteroaromatic or aliphatic moieties. We then demonstrate that hydrophobic polymers induce clustering and promote biofilm formation in MC4100, a strain of Escherichia coli that forms biofilms poorly, with aromatic and heteroaromatic moieties leading to the best performing polymers. Moreover, we compare the effect of the polymers on MC4100 against PHL644, an E. coli strain that forms biofilms well due to a single point mutation which increases expression of the adhesin curli. In the presence of selected polymers, MC4100 can reach levels of biomass production and curli expression similar or higher than PHL644, demonstrating that synthetic polymers promote similar changes in microbial physiology than those introduced following genetic modification. Finally, we demonstrate that these polymers can be used to improve the performance of MC4100 biofilms in the biocatalytic transformation of 5-fluoroindole into 5-fluorotryptophan. Our results show that incubation with these synthetic polymers helps MC4100 match and even outperform PHL644 in this biotransformation, demonstrating that synthetic polymers can underpin the development of beneficial applications of biofilms.

Scheme 1. Schematic representation of experimental approach to polymer-induced biofilms for biocatalysis: hydrophobic polymers are prepared using an in situ screening strategy based on poly(acryloyl hydrazide) (step ①). Then, a poor biofilm former strain (MC4100) is incubated in the presence of these polymers to yield biofilms (step ②). This way, the poor biofilm former strain is able to match the performance of a good biofilm former strain (PHL644) in the biotransformation of serine and 5-fluoroindole into 5-fluorotryptophan (step ③).

Scheme 2. Synthesis of functional polymers P1-mod-aldehyde, and list of polymers prepared with aldehyde conversions.

Fig. 1. Biofilm formation as measured by crystal violet staining: fractional change in absorbance at 550 nm for E. coli PHL644 cultures (A) and E. coli MC4100 cultures (B) following incubation over 24 h in the presence of 0.05 mg mL⁻¹ of P1 (black solid bar), aldehydes (solid coloured bars) and functional polymers P1-mod-aldehyde (hollow coloured bars). Data has been normalised and represents the fractional change in absorbance at 550 nm when compared to E. coli MC4100 cultures incubated in the absence of polymers (solid line). Fractional change in absorbance at 550 nm for E. coli PHL644 cultures incubated in the absence of polymers when compared to E. coli MC4100 cultures incubated in the absence of polymers is also shown for comparison (dashed line). Not buffered indicates incubation in 100 mM aqueous NaCl. Buffered indicates incubation in 100 mM phosphate buffer at pH 7. Means ± range from at least three biological replicates are shown. Full details of polymer clog D calculations are available in the ESI.

Fig. 2. Aggregation of bacteria: (A) schematic representation of polymer-induced aggregation of bacteria, the techniques used for its characterization and the best preforming polymers identified with each technique. (B) Optical density at 600 nm for E. coli MC4100 cultures following incubation for 24 h in the absence (black) and presence of 0.5 mg mL⁻¹ of P1-mod-3InA (yellow). Optical density at 600 nm of 0.05 mg mL⁻¹ of P1-mod-2InA suspended in culture media (blue) shown for comparison. (C) Changes to optical density at 600 nm for E. coli MC4100 cultures in the presence of 0.5 mg mL⁻¹ of functional polymers P1-mod-aldehyde. (D) Size distribution of suspensions of MC4100 cultures (black) and E. coli MC4100 cultures in the presence of 0.05 mg mL⁻¹ of P1-mod-3InA (yellow), following incubation over 48 h. Size distribution of suspensions of P1-mod-3InA in culture media (blue) shown for comparison. (E) Changes in the proportion of free bacteria in suspensions of E. coli MC4100 incubated in the presence of 0.05 mg mL⁻¹ of functional polymers P1-mod-aldehyde for 48 h. Data has been normalised and represents the difference when compared to E. coli MC4100 cultures in the absence of polymers. Means ± SD from at least two biological replicates are shown.

Fig. 3. Curli expression measured using a GFP reporter strain: (A) Schematic representation of the formation of curli fibres following polymer-induced aggregation of bacteria, which leads to surface-attached biofilms. (B) Best preforming polymers identified in this assay. (C) Green fluorescence against time for E. coli MC4100 pJLC-T cultures following incubation in 100 mM phosphate buffer at pH 7 in the absence (black, n = 10) and presence of 0.05 mg mL⁻¹ of P1-mod-2AFPA (yellow, n = 8) or 2AFPA (blue, n = 3). Mean ± 95% confidence intervals are shown. (D) Total GFP fluorescence for E. coli MC4100 pJLC-T cultures in the presence of 0.05 mg mL⁻¹ of P1 (black solid box) and functional polymers P1-mod-aldehyde (hollow boxes). (E) Rate of increase of green fluorescence for E. coli MC4100 pJLC-T cultures in the absence and presence of 0.05 mg mL⁻¹ of functional polymers P1-mod-aldehyde. For box and whisker plots, median is shown as a line. Box extends from 25th to 75th percentile while whiskers go from minimum to maximum value. Fit to a straight line together with prediction bands are shown.

Fig. 4. Biocatalytic activity: (A) schematic representation of polymer-induced biofilms for biocatalysis. (B) Best preforming polymers identified in this assay. (C) Percentage of 5-fluoroindole depletion, (D) 5-fluorotryptophan appearance and (E) conversion of 5-fluoroindole to 5-fluorotryptophan for E. coli MC4100 pSTB7 cultures following 48 h of incubation in 100 mM phosphate buffer at pH 7 in the presence of 0.05 mg mL⁻¹ of P1 (black solid box) and functional polymers P1-mod-aldehyde (hollow boxes), followed by incubation with reaction buffer for another 24 h. Median is shown as a line. Box extends from 25th to 75th percentile while whiskers go from minimum to maximum value. Values for E. coli MC4100 (solid line with 25th to 75th percentile) and E. coli PHL644 cultures (dashed line) incubated in the absence of polymers shown for comparison.

Fig. 5. Correlation of relevant phenotypes with 5-fluorotryptophan production: percentage of 5-fluorotryptophan appearance as a function of biomass (as measured by crystal violet staining) (A), clustering (as measured by changes to optical density at 600 nm) (B), curli production (as measured by total green fluorescence) (C), and relative metabolic activity (as measured by resorufin fluorescence) (D), for E. coli MC4100 cultures following 48 h of incubation in 100 mM phosphate buffer at pH 7 in the absence (lower white dot) and presence of 0.05 mg mL⁻¹ of P1 (black solid dot) and functional polymers P1-mod-aldehyde (coloured dots), followed by incubation with reaction buffer for another 24 h. Where relevant, data for E. coli PHL644 cultures is shown (top white dot). Median and 25th to 75th percentiles shown for at least 4 replicates.