Enhanced benzaldehyde tolerance in Zymomonas mobilis biofilms and the potential of biofilm applications in fine-chemical production

Abstract

Biotransformation plays an increasingly important role in the industrial production of fine chemicals due to its high product specificity and low energy requirement. One challenge in biotransformation is the toxicity of substrates and/or products to biocatalytic microorganisms and enzymes. Biofilms are known for their enhanced tolerance of hostile environments compared to planktonic free-living cells. Zymomonas mobilis was used in this study as a model organism to examine the potential of surface-associated biofilms for biotransformation of chemicals into value-added products. Z. mobilis formed a biofilm with a complex three-dimensional architecture comprised of microcolonies with an average thickness of 20 microm, interspersed with water channels. Microscopic analysis and metabolic activity studies revealed that Z. mobilis biofilm cells were more tolerant to the toxic substrate benzaldehyde than planktonic cells were. When exposed to 50 mM benzaldehyde for 1 h, biofilm cells exhibited an average of 45% residual metabolic activity, while planktonic cells were completely inactivated. Three hours of exposure to 30 mM benzaldehyde resulted in sixfold-higher residual metabolic activity in biofilm cells than in planktonic cells. Cells inactivated by benzaldehyde were evenly distributed throughout the biofilm, indicating that the resistance mechanism was different from mass transfer limitation. We also found that enhanced tolerance to benzaldehyde was not due to the conversion of benzaldehyde into less toxic compounds. In the presence of glucose, Z. mobilis biofilms in continuous cultures transformed 10 mM benzaldehyde into benzyl alcohol at a steady rate of 8.11 g (g dry weight)(-1) day(-1) with a 90% molar yield over a 45-h production period.

FIG. 1.

Z. mobilis biofilm morphology in flow cells. (A) Phase-contrast images showing initial attachment and subsequent biofilm development during 3 days of Z. mobilis growth at a constant flow rate of 0.15 ml min⁻¹ at room temperature. The arrows indicate microcolonies. (B) CLSM images of 3-day-old Z. mobilis biofilms stained with the BacLight LIVE/DEAD viability stain. The top and bottom images are topical and vertical views of Z. mobilis microcolonies. The white triangle in the top image indicates the position where the vertical section of the biofilm was taken. Bars, 50 μm.

FIG. 2.

Qualitative analysis of benzaldehyde toxicity: CLSM images of 24-h planktonic cultures and 3-day-old Z. mobilis biofilms stained with the BacLight LIVE/DEAD viability stain. Planktonic (A) and biofilm (B and C) cultures were examined after 1 h of exposure in the absence or presence of benzaldehyde. Live cells fluoresced green, and dead cells fluoresced red. The top and bottom images in panel B are topical and vertical views of Z. mobilis microcolonies. (C) Overall biofilm morphology. (A and B) Bars, 50 μm. (C) Bars, 200 μm.

FIG. 3.

Quantitative analysis of benzaldehyde toxicity. Residual metabolic activity was measured by the specific glucose consumption rates of 3-day-old Z. mobilis biofilms and 24-h planktonic cultures after 1 h of exposure to different benzaldehyde concentrations (A) and after exposure to 30 mM benzaldehyde for up to 3 h at 30°C (B). Samples exposed to benzaldehyde for less than 3 h were preincubated in MES-buffered saline before benzaldehyde exposure to provide a total incubation time of 3 h. One hundred percent residual activity corresponded to the specific glucose consumption rate of control cultures exposed to MES-buffered saline without benzaldehyde, which was on average 30 mmol glucose mg total protein⁻¹ h⁻¹. The error bars indicate the sample standard deviations of results from independent cultures (n ≥ 4 for panel A and n ≥ 2 for panel B).

FIG. 4.

Continuous biotransformation determined by using 3-day-old Z. mobilis biofilms in the presence of 10 mM benzaldehyde and 5 g liter⁻¹ glucose (27.8 mM) with a constant flow rate of 0.15 ml min⁻¹ at 30°C. Benzaldehyde was added at time zero. The error bars indicate the sample standard deviations of results from independent reactors (n = 3). OD 600, optical density at 600 nm.