Adhesion of acinetobacter venetianus to diesel fuel droplets studied with In situ electrochemical and molecular probes

Abstract

The adhesion of a recently described species, Acinetobacter venetianus VE-C3 (F. Di Cello, M. Pepi, F. Baldi, and R. Fani, Res. Microbiol. 148:237-249, 1997), to diesel fuel (a mixture of C12 to C28 n-alkanes) and n-hexadecane was studied and compared to that of Acinetobacter sp. strain RAG-1, which is known to excrete the emulsifying lipopolysaccharide, emulsan. Oxygen consumption rates, biomass, cell hydrophobicity, electrophoretic mobility, and zeta potential were measured for the two strains. The dropping-mercury electrode (DME) was used as an in situ adhesion sensor. In seawater, RAG-1 was hydrophobic, with an electrophoretic mobility (&mgr;) of -0.38 x 10(-8) m2 V-1 s-1 and zeta potential (zeta) of -4.9 mV, while VE-C3 was hydrophilic, with &mgr; of -0.81 x 10(-8) m2 V-1 s-1 and zeta of -10.5 mV. The microbial adhesion to hydrocarbon (MATH) test showed that RAG-1 was always hydrophobic whereas the hydrophilic VE-C3 strain became hydrophobic only after exposure to n-alkanes. Adhesion of VE-C3 cells to diesel fuel was partly due to the production of capsular polysaccharides (CPS), which were stained with the lectin concanavalin A (ConA) conjugated to fluorescein isothiocyanate and observed in situ by confocal microscopy. The emulsan from RAG-1, which was negative to ConA, was stained with Nile Red fluorochrome instead. Confocal microscope observations at different times showed that VE-C3 underwent two types of adhesion: (i) cell-to-cell interactions, preceding the cell adhesion to the n-alkane, and (ii) incorporation of nanodroplets of n-alkane into the hydrophilic CPS to form a more hydrophobic polysaccharide-n-alkane matrix surrounding the cell wall. The incorporation of n-alkanes as nanodroplets into the CPS of VE-C3 cells might ensure the partitioning of the bulk apolar phase between the aqueous medium and the outer cell membrane and thus sustain a continuous growth rate over a prolonged period.

FIG. 1

(A) Adhesion of an oil droplet and its spreading to form a film at the electrode. The electrical signal (transient current) is caused by displacement of the double-layer charge (ς₁) from the contact area A_C. (B) Adhesion of bacterial cells and formation of molecular contact with the electrode.

FIG. 2

(A) Oxygen consumption rates determined with Clark’s probe in cultures of Acinetobacter sp. strain RAG-1 (▴) and A. venetianus VE-C3 (▵) grown in mineral medium containing 2 g of diesel fuel liter⁻¹. (B) Protein determination of Acinetobacter sp. strain RAG-1 (▴) and A. venetianus VE-C3 (▵) grown in mineral medium containing 2 g of diesel fuel liter⁻¹.

FIG. 3

Colonization of diesel fuel by Acinetobacter sp. strain RAG-1. (a) Decrease in surface tension of part of a diesel fuel drop colonized by RAG-1, observed with an optical microscope in the transmission mode after a 3-h incubation at 28°C in mineral medium with diesel fuel (2 g liter⁻¹). (b) Colonization of a diesel fuel droplet (diameter, 56 μm) by RAG-1, showing bacterial adhesion at the rim and on top, observed in the transmission mode after an 8-h incubation at 28°C. (c) Repellent diesel fuel microdroplets of different sizes (from 10 to less than 0.75 μm) dispersed in the medium without attached bacteria after a 24-h incubation. (d) Same image as in panel b but observed by CLSM in the fluorescence mode, formed by 16 overlapped images scanned every 0.8 μm for a total depth of 12.8 μm. This sample was stained with Nile Red fluorochrome for the neutral lipid moiety of emulsan.

FIG. 4

Colonization of diesel fuel by A. venetianus VE-C3. (a) Light microscopy in the transmission mode, showing aggregates of cells (arrows) before adhesion to the diesel fuel surface after a 6-h incubation at 28°C in mineral medium with 2 g of diesel fuel liter⁻¹. (b) The same image as in panel a but observed by CLSM in the fluorescence mode, obtained by overlapping 20 images scanned every 0.6 μm for a total depth of 12 μm. VE-C3 was stained with ConA-FITC to show polysaccharide residues of glucose and mannose in CPS. Only a fraction of the cells seen in panel a (arrows) have a fluorescent CPS after 6 h of incubation. (c) Transmission mode, showing that the surface tension of a diesel fuel drop colonized by strain VE-C3 decreases and the bacteria at the rim and on top produce elongated and indented shapes after a 12-h incubation. (d) The same image as in panel c but in the fluorescence mode, with the ConA-FITC distribution imaged by CLSM. VE-C3 cells with CPS smear the elongated rim of the diesel fuel drop showing “polysaccharide footprints.” (f) Transmission mode, showing a diesel fuel droplet with a diameter of about 20 μm, with many smaller microdroplets embedded in a microbial aggregate. After a 28-h incubation at 28°C, VE-C3 cells are still attached to diesel fuel droplet. (g) The same image as in panel f but in fluorescence mode with the ConA-FITC distribution imaged by CLSM. The aggregate of cells and diesel fuel microdroplets is glued together by a thick polysaccharide matrix excreted by the bacteria.

FIG. 5

(A) Cell hydrophobicity of A. venetianus VE-C3 at different times, measured by the MATH test: 0 h (▵) preincubated in PCA medium and then transferred to mineral medium with 2 g of diesel fuel liter⁻¹, then incubated for 12 h (□) and 36 h (○) in mineral medium with 2 g of diesel fuel liter⁻¹. (B) MATH test for Acinetobacter sp. strain RAG-1 under the same conditions as in panel A incubated for 0 h (▴), 12 h (■), and 36 h (●).

FIG. 6

Adhesion of uninduced VE-C3 and RAG-1 cells at the electrode from their suspensions in 0.1 M NaCl solution. The percentage of surface coverage (A) and the film formation time (B) are plotted as a function of cell density.

FIG. 7

Electrochemical signals in the diesel fuel-degrading cultures of RAG-1 (curve B) and VE-C3 (curve C) after 3 days of growth. The control (curve A) is an uninoculated dispersion.