Download Adherence of bacteria to hydrocarbons: A simple method for

FEMS MicrobiologyLetters 9 (1980) 29-33
© CopyrightFederation of European MicrobiologicalSocieties
Published by Elsevier/North-HollandBiomedicalPress
29
A D H E R E N C E O F B A C T E R I A TO H Y D R O C A R B O N S : A SIMPLE M E T H O D F O R
MEASURING CELL-SURFACE HYDROPHOBICITY
M. ROSENBERG, D. GUTNICK and E. ROSENBERG
Department of Microbiology, George S. WiseFaculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel
Received 20 May 1980
Accepted 20 June 1980
1. Introduction
The hydrophobic nature of the outermost surface
of various microbial cells has been implicated in such
biological phenomena as interactions between bacteria and phagocytes [1], attachment of bacteria to
host tissue [2,3], adherence of bacteria to nonwettable solid surfaces [4,5], partitioning of bacteria
at liquid : liquid [6] and liquid : air [7,8] interfaces,
and the ability of microbial cells to grow on hydrocarbons through direct contact with the immiscible
substrate [9-12].
A number of methods for studying hydrophobic
interactions of cells have been reported in the literature. These include binding of hydrocarbon and fatty
acids to cells and cell components [12,13], measurement of the force required to remove hydrocarbonbound cells [9], partitioning of bacteria in aqueous
polymer two-phase systems [ 14,15], hydrophobic
interaction chromatography [3,15] and contact angle
measurements of dried cell layers [1,16]. No single
method adequately describes cell-surface hydrophobicity since (1) the experimental conditions employed influence the observed hydrophobic interactions to some degree and (2) various non-hydrophobic effects often interfere.
Interest in the mechanism which enables direct
contact between hydrocarbon-degrading cells and
their water-insoluble alkane substrates has led us to
develop a rapid quantitative assay for the hydrophobic interaction of ceils with liquid hydrocarbons.
method is based on the degree of adherence of
cells to various liquid hydrocarbons following a brief
period of mixing. The present report describes this
technique and its application in measuring the surface
hydrophobicity of various bacterial cells.
2. Materials and Methods
2.1. Bacteria
The following test bacteria were used: Acinetobacter calcoaceticus strain RAG-1 (ATCC 31012) was
isolated previously in this laboratory [ 17,18] ; A. calcoaceticus strain BD 413 tryp E 27 was kindly provided by Dr. E. Juni. Escherichia cell B ilvA thy,
E. coli K-12 CSH 57, E. coil J-5, Bacfflus subtilis 168
and Enterobacter aerogenes CDC 659]66 were kindly
provided by Dr. E.Z. Ron. Micrococcus lysodeikticus
ATCC 4698 was kindly pr6vided by Dr. I. Friedberg.
Locally isolated strains of Staphylococcus aureus,
Staphylococcus albus and Serratia marcescens were
kindly provided by Ruth Zack. Pseudomonas aeruginosa PAS 279 was kindly provided by Dr. J. Shapiro. The test bacteria were grown at 30°C with shaking in nutrient broth. Unless otherwise stated, bacteria were harvested at earlY logarithmic growth
phase, washed twice and resuspended in PUM buffer,
pH 7.1 : 22.2 g K2I-IPO4 • 3H20, 7.26 g KH2PO4,
1.8 g urea, 0.2 g MgSO4 • 7H20 and distilled water to
i 000 ml.
2.2. Assay procedure
To round-bottom test tubes (10 mm diameter),
containing 1.2 ml of washed cells suspended in PUM
buffer, were added various volumes of test hydrocar-
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bon (n-hexadecane, n-octane or p-xylene). Following
10 min preincubation at 30°C, the mixtures were agitated uniformly on a Thermolyne Maxi Mix (Sybron)
for 120 s. After allowing 15 min for the hydrocarbon
phase to rise completely, the aqueous phase was carefully removed with a Pasteur pipette and transferred
to a 1 ml cuvette. Light absorbance was determined
at 400 nm, using a Gilford Model 240 spectrophotometer.
3. Results
The basic experiment used to measure bacterial
hydrophobicity is shown qualitatively in Fig. 1. Hexadecane was layered onto a turbid aqueous suspension
of Acinetobacter RAG-1 (Fig. 1, left tube) and then
mixed for 120 s; on standing a clear bottom layer and
a "creamy" upper layer formed (Fig. 1, middle tube).
Microscopic examination of the upper layer revealed
an oil-in-water emulsion consisting of hexadecane
droplets covered with patches of bacteria (Fig. 2).
Decrease in absorbance of the lower aqueous phase
was used as a measure of cell surface hydrophobicity.
Fig. 1. Adherence of Acinetobacter RAG-1 to hydrocarbon.
Hexadecane was added to an aqueous suspension of Acinetobacter RAG-1 (left). After mixing for 120 s and allowing to
stand, adherent cells rose with the hydrocarbon, forming a
"creamy" upper layer and a clear aqueous phase (middle).
Addition of isopropanol to a final concentration of 5% (v/v)
results in breakage of the emulsion and release of cells back
into the aqueous phase (right).
The cell-stabilized emulsions did not break even after
several days; however, addition of 5% (v/v) isopropranol brought about an immediate coalescence of
droplets and release of cells into the aqueous phase
(Fig. 1, right tube).
Results illustrating the adherence of various bacteria to the test hydrocarbons are presented in Figs.
3 - 6 . Fig. 3 compares the affinity ofE. coli with
those of two Acinetobacter strains. E. coli B showed
no significant affinity towards hexadecane or octane
and little or no affinity towards xylene. Similar
results (not presented) were obtained with M. lysodeikticus, E. aerogenes, B. subtilis and P. aeruginosa.
While Acinetobacter RAG-1 showed high affinity
towards all three hydrocarbons, Acinetobacter BD
exhibited higher affinity for octane and xylene than
for hexadecane. More than 97% of the RAG-1 and
over 99% of the BD strain cells were removed from
the aqueous phase by 0.2 ml octane.
The affinities of two species of Staphylococci
towards the test hydrocarbons are presented in Fig. 4.
Although S. albus did not adhere to hexadecane or
xylene, S. aureus exhibited a high affinity towards all
the test hydrocarbons.
Although S. marcescens grown to early exponential phase showed relatively low affinity towards
hexadecane and xylene and none towards octane,
pink-pigmented early stationary phase cells adhered
strongly to all three hydrocarbons (Fig. 5).
Loss of oligosaccharide components on the outer
surface ofE. coli rough mutant J-5 [19] was accompanied by a significant increase in its affinity towards
Fig. 2. Phase micrograph showing RAG-1 cells adhering to
hexadecane droplet following mixing. 825X.
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Fig. 6. o, E. coli K-12 (1.558); o, E. coli J-5 (1.408).
Figs. 3-6. Aff'mityof bacteria towards hydrocarbon as a function of hydrocarbon volume. Aqueous bacterial suspensions were
mixed with varying volumes of hexadeeane, octane and xylene as described in Materials and Methods. Results are expressed as
percentage of the initial absorbanee (A 400) of the aqueous suspension as a function of hydrocarbon volume. Initial absorbance is
shown in parentheses.
each of the three test hydrocarbons, as compared
with E. coli K-12 (Fig. 6).
4. Discussion
A simple quantitative method has been described
for studying the outer cell surface of bacteria, based
on the affinity of these cells for liquid hydrocarbons.
Large differences in the aftrmities of various bacteria
for hydrocarbons have been demonstrated using this
technique.
Previous observations have suggested that the ability to adhere to bulk hydrocarbon is a characteristic
of hydrocarbon-degrading microorganisms [ 11 ]. We
have shown that S. aureus and early stationary phase
S. marcescens cells adhere to a variety of fiquid hydrocarbons despite their inability to degrade them.
Thus, the ability of bacterial cells to adhere to hydrocarbon is not restricted to hydrocarbon-degrading
bacteria. Moreover, both Acinetobacter strains
adhered strongly to octane and xylene, hydrocarbons
which they are incapable of metabolizing. This indicates that the adherence of these hydrocarbondegrading microorganisms is not limited to metabolizable hydrocarbons, and suggests that direct contact
between Acinetobacter cells and bulk hydrocarbon is
due to general hydrophobic interactions rather than
specific recognition of the substrate. P. aeruginosa
PAS 279 did not adhere significantly to the test hydrocarbons under the conditions studied, despite its
ability to grow on hexadecane. This result suggests
that affmity for hydrocarbon may vary among hydrocarbon-degrading bacteria. Further studies on the
relationship between adherence to hydrocarbon and
growth of hydrocarbon-degrading bacteria are underway.
The large increase in hydrophobicity observed in
S. marcescens with increasing age of the ceils agrees
with previous reports [8,13]. The small, but significant increase in the affmity of the rough mutant
E. coli J-5 as compared with that ofE. coli K-12 indicates the increased hydrophobicity of the rough mutant, presumably due to the increased exposure of
inner core regions of the LPS layer. Similar results
have been obtained with rough mutants of Salmonella
typhimurium [1,14].
The method described here may prove useful in
enabling the separation of certain cell mixtures, and
in separating hydrophobic cell components. In addition, this technique may provide a means of enriching
for and selecting mutants which are altered in their
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surface hydrophobicity. A search for such mutants is
currently in progress, with a view towards investigating the relationship between the metabolism o f hydrophobic substrates and modifications in cell surface
hydrophobicity.
Acknowledgements
We thank Z.L. Zosim, L. Goldstein and E.A. Bayer
for stimulating discussions and constructive criticism.
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