Hyperadherence of Pseudomonas taiwanensis VLB120ΔC increases productivity of (S)-styrene oxide formation

Abstract

The attachment strength of biofilm microbes is responsible for the adherence of the cells to surfaces and thus is a critical parameter in biofilm processes. In tubular microreactors, aqueous-air segmented flow ensures an optimal oxygen supply and prevents excessive biofilm growth. However, organisms growing in these systems depend on an adaptation phase of several days, before mature and strong biofilms can develop. This is due to strong interfacial forces. In this study, a hyperadherent mutant of Pseudomonas taiwanensis VLB120ΔCeGFP possessing an engineered cyclic diguanylate metabolism, was applied to a continuous biofilm process for the production of (S)-styrene oxide. Cells of the mutant P. taiwanensis VLB120ΔCeGFP Δ04710, showing the same specific activity as the wild type, adhered substantially stronger to the substratum. Adaptation to the high interfacial forces was not necessary in these cases. Thereby, 40% higher final product concentrations were achieved and the maximal volumetric productivity of the parent strain was significantly surpassed by P. taiwanensis VLB120ΔCeGFP Δ04710. Applying mutants with strong adhesion in biofilm-based catalysis opens the door to biological process control in future applications of catalytic biofilms using other industrially relevant strains.

Figure 1

Aggregate formation of Pseudomonas taiwanensis VLB120ΔCeGFP Δ04710 in planktonic overnight cultures. A. Autoaggregates of P. taiwanensis VLB120ΔCeGFP Δ04710 (left) in comparison with free swimming cells of P. taiwanensis VLB120ΔCeGFP (right); strains were grown in M9 minimal medium (0.5% (wt/vol) glucose). B. Adhesion of P. taiwanensis VLB120ΔCeGFP Δ04710 to the glass wall of a shaking flask.

Figure 2

Initial adherence of Pseudomonas taiwanensis VLB120ΔCeGFP Δ04710 and control strain P. taiwanensis VLB120ΔeGFP to 10 cm long silicone tubings under various flow rates. After 2 h attachment phase, the medium flow of 100 μl min⁻¹, 500 μl min⁻¹ or 5 ml min⁻¹, respectively, was started for 1 h to flush out non‐adhered cells. Afterwards, the silicone tubes were removed and the first 10 cm of each were used for quantification of the attached biomass by crystal violet (CV) staining.

Figure 3

Attached and detached biofilm biomass of Pseudomonas taiwanensis VLB120ΔCeGFP Δ04710 and the control strain P. taiwanensis VLB120ΔCeGFP grown in silicone tubes applying single‐phase aqueous flow rates of 100 μl min⁻¹ and 500 μl min⁻¹ or two‐phase aqueous‐air segmented flow of 100 μl min⁻¹ medium and 100 μl min⁻¹ air. Errors bars represent standard deviation of triplicates. A. Total biofilm dry weight (BDW) of biofilms grown in 40 cm long silicone tubings. B. Detached biofilm biomass during continuous biofilm cultivation. One data point reflects the amount of biomass in 10 ml effluent.

Figure 4

Biofilm dry weight (BDW) and viable cells in biofilms determined via the resazurin and eGFP assay. Values are given as a ratio of the quantities of the mutant Pseudomonas taiwanensis VLB120ΔCeGFP Δ04710 to the parent strain P. taiwanensis VLB120ΔCeGFP. A. Biofilms after 48 h of standard cultivation under segmented flow conditions. Errors bars represent standard deviation of triplicates. B. Biofilms after 336 h of biotransformation under segmented flow conditions. Error bars represent standard deviation of five replicates.

Figure 5

Biotransformation of styrene to (S)‐styrene oxide using Pseudomonas taiwanensis VLB120ΔCeGFP Δ04710 and control strain P. taiwanensis VLB120ΔCeGFP. The experiment is divided into three phases. Phase I represents the adaptation phase of the control strain while P. taiwanensis VLB120ΔCeGFP Δ04710 already produces significant amounts of (S)‐styrene oxide. Phase II shows the production phase of both strains. After 288 h (phase III) the air flow was increased to 4 ml min⁻¹. The volumetric productivities are averaged values of five replicates. Error bars represent standard deviation of five independent replicates.