Download Determination of bacterial cell surface hydrophobicity of single cells

FEMS Microbiology Letters 152 (1997) 299^306
Determination of bacterial cell surface hydrophobicity of single
cells in cultures and in wastewater in situ
Anna Zita, Malte Hermansson *
Biointerface and Biofouling Laboratory, Department of General and Marine Microbiology, Goëteborg University, Medicinaregatan 9 C,
S-413 90 Goëteborg, Sweden
Received 10 February 1997; revised 24 April 1997; accepted 2 May 1997
Abstract
Bacterial cell surface hydrophobicity is one of the most important factors that influence bacterial adhesion. A new method,
microsphere adhesion to cells, for measuring bacterial cell surface hydrophobicity was developed. Microsphere adhesion to
cells is based on microscopic enumeration of hydrophobic, fluorescent microspheres attaching to the bacterial surface. Cell
surface hydrophobicity estimated by microsphere adhesion to cells correlates well with adhesion of bacteria to hydrocarbons or
hydrophobic interaction chromatography for a set of hydrophilic and hydrophobic bacteria (linear correlation coefficients, R2 ,
were 0.845 and 0.981 respectively). We also used microsphere adhesion to cells to investigate the in situ properties of individual
free-living bacteria directly in activated sludge. Results showed that the majority of the bacteria were hydrophilic, indicating
the importance of cell surface hydrophobicity for bacterial adhesion in sludge, and for the overall success of the wastewater
treatment process.
Keywords:
Bacterial hydrophobicity; Microsphere adhesion to cells; Method
1. Introduction
Bacterial cell surface hydrophobicity (CSH) is one
of the most important factors that govern bacterial
adhesion to various surfaces such as the air/water
interface [1], oil/water interface [2], biomaterials [3],
teeth [4], animal cells [5], activated sludge [6], and
di¡erent solid surfaces [7].
Techniques for measuring bacterial CSH are summarized in Rosenberg et al. [8] and include adhesion
of bacteria to hydrocarbons (BATH), hydrophobic
interaction chromatography (HIC), salting out ag* Corresponding author. Tel: +46 (31) 773 2574; Fax:
+46 (31) 773 2599; E-mail: Malte.Hermansson@gmm.gu.se
gregation (SAT), contact angle measurements
(CAM), partitioning of cells in two phase systems
(TPP), adhesion to solid surfaces and binding of
fatty acids to bacterial cells. These methods give an
average value for the bacterial population and were
designed for measuring CSH of bacterial cultures
rather than in situ properties of uncultured cells.
We have developed a test for measuring CSH of
single cells. The method, microsphere adhesion to
cells (MAC), is based on enumeration of hydrophobic £uorescent microspheres that associate with the
bacterial surface during the test. The number of microspheres per cell is determined by epi£uorescence
microscopy. MAC can be used to determine CSH of
bacteria directly in di¡erent samples without prior
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 2 1 4 - 0
A. Zita, M. Hermansson / FEMS Microbiology Letters 152 (1997) 299^306
300
cultivation. Larger, blue-stained, microspheres have
been used earlier to determine CSH of yeast cells [9].
In this work the MAC method was compared with
the BATH and HIC methods. MAC was used to
describe CSH for several di¡erent bacterial populations and also for determining the CSH of bacteria
directly in wastewater, where the surface properties
of bacteria are of great interest for understanding the
process of activated sludge £occulation.
2. Materials and methods
2.1. Bacterial strains and culture conditions
Bacterial strains (see Table 1) were grown in LB
medium (tryptone [10 g/l], NaCl [5 g/l], yeast extract
[5 g/l], pH 7.5), Caulobacter maris (ATCC 15268)
was grown in 2216E medium (bacto-peptone [5 g],
yeast extract [1 g], FePO4 [0.1 g], ¢ltered sea water [1
l], pH 7.5). All strains were grown to stationary
phase on a shaker at room temperature (22^25³C)
and subsequently harvested by centrifugation,
3600 g for 10 min at 18³C, washed once, suspended
in 0.01 M phosphate bu¡ered saline (PBS), adjusted
to 1010 cells/ml and kept in PBS overnight.
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2.2. Wastewater sampling
E¥uent water was taken from the RYA wastewater treatment plant (WWTP) in Go
ë teborg, Sweden. At this plant, the wastewater is treated by a
primary sedimentation, followed by an activated
sludge process. The aeration tanks are of the plug£ow type and are aerated by di¡used air. Prior to use
the e¥uent water samples were ¢ltered through a 1.2
Wm Nucleopore (Poretics) polycarbonate ¢lter to remove small pinpoint £ocs. Samples were treated
within 2 h.
2.3. Hydrophobic interaction chromatography
HIC was performed as described before [10].
Brie£y, bacterial solutions (about 107 cells/ml) were
run through a column with a hydrophobic gel, OctylSepharose CL-4B (Pharmacia, Sweden). The samples were eluted with 12 ml of PBS and the amount
of eluted cells was determined by epi£uorescence microscopy (Olympus microscope, magni¢cation
1250, blue excitation light) of acridine orange
stained samples that were ¢ltered onto prestained
0.2 Wm Nucleopore ¢lters. Hydrophobicity was calculated as (a b)/a 100, where a is the amount of
cells added to the column and b is the amount of
cells in the eluate. Samples were run in duplicate and
for 3^5 parallel bacterial cultures, and the results are
presented as mean values.
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3
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2.4. Bacterial adhesion to hydrocarbons
The BATH assay was performed as described before [2]. 4 ml of the bacterial suspension (5 107
cells/ml) was vigorously mixed with 1 ml of n-hexadecane for 2 min. The water and organic phase were
allowed to separate for 15 min and 0.1 ml of the
aqueous phase was sampled and the amount of cells
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Table 1
Comparison of di¡erent methods for determination of cell surface hydrophobicitya
Strain
Hydrophobicity
b
Escherichia coli K51
Pseudomonas putida
Flavobacterium breve
Escherichia coli F-18(pPKL91)
Serratia marcescens
Acinetobacter calcoaceticus
a
See Section 2 for details.
b
c
% cells retained in the gel.
% cells in the aqueous phase after partitioning.
d
% cells with 3 or more attached microspheres.
Reference
c
d
HIC (%)
BATH (%)
12 þ 12
33
þ 12
MAC(%)
5þ6
[14]
25 þ 25
27
þ 10
19 þ 13
[15]
62 þ 0
4
þ1
77 þ 9
CCUG 7320
85 þ 3
2
þ1
73 þ 10
[16]
47 þ 5
1
þ0
99 þ 0
0.3 þ 0.3
83 þ 10
CCUG 190
94 þ 1
CCUG 12804
A. Zita, M. Hermansson / FEMS Microbiology Letters 152 (1997) 299^306
301
Fig. 1. Distribution of cells with di¡erent numbers of attached hydrophobic microspheres. Graph a represents Escherichia coli K51, b represents Pseudomonas putida, c represents Flavobacterium breve, d represents Serratia marcescens, e represents E. coli F-18(pPKL91) and
f represents Acinetobacter calcoaceticus.
A. Zita, M. Hermansson / FEMS Microbiology Letters 152 (1997) 299^306
302
Fig. 2. A: Micrograph showing a hydrophobic E. coli F-18(pPKL91) with attached hydrophobic microspheres and one cell with no attached microspheres (lower left cell). B: Micrograph showing Caulobacter maris with microspheres attaching to the stalk of a cell (lower)
and the £agellum of a swarmer cell (upper). The size of a single microsphere represents 0.1 Wm.
determined by epi£uorescence microscopy, as for
HIC (see above). The hydrophobicity was expressed
as (a3b)/a 100, where a is the initial cell concen-
U
tration in the aqueous phase and b is the concentration in the aqueous phase after partitioning. Samples
were run in duplicate and for 3^5 parallel bacterial
A. Zita, M. Hermansson / FEMS Microbiology Letters 152 (1997) 299^306
cultures, and the results are presented as mean values.
2.5. Microsphere adhesion to cells
Fluorescent polystyrene microspheres (diameter
0.1 Wm, Molecular Probes, Eugene, OR, USA) with
a modi¢ed surface that rendered them hydrophobic
(sulfate-modi¢ed, FluoSpheres, cat. no. F-8847) or
hydrophilic
(carboxylate-modi¢ed,
FluoSpheres,
cat. no. L-5221) were used. The excitation/emission
wavelengths were 505/515 nm, and 490/515 nm respectively.
Microspheres were diluted in distilled water to a
10% solution (v/v) and sonicated to a uniformly dispersed solution. 10 Wl of the microsphere solution
and 10 Wl of bacterial suspension were added to 0.1
ml of distilled water in an Eppendorf tube and the
solution was vigorously mixed on a Vortex for 2
min. 3 Wl of the sphere-bacterial solution was taken
out and 3 Wl of acridine orange [0.1 g/l] was added to
visualize the bacteria. 100^500 bacterial cells were
examined by epi£uorescence microscopy for attached
microspheres. The number of spheres per bacteria
was counted (0, 1, 2, 3, 4^9, or 10). The wastewater
samples were concentrated, by centrifugation, to a
cell concentration of approximately 1 108 cells/ml.
The hydrophobicity was expressed either as the fre-
v
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303
quency of cells with a certain number of associated
microspheres, or as the percent of cells with 3 or
more microspheres (cf. [9]). A high percent corresponds to a hydrophobic cell. MAC was tested on
3^5 parallel bacterial cultures and the results are
presented as mean values.
3. Results and discussion
3.1. Distribution of cell surface hydrophobicities of
bacterial cultures as measured by MAC
Fig. 1 shows the distribution of cells with di¡erent
numbers of attached hydrophobic microspheres. The
results show very clear di¡erences in the CSH between the di¡erent strains. The method seems to
give a good resolution of both hydrophilic and hydrophobic cells. It was di¤cult to exactly enumerate
more than about 10 microspheres per cell. This limits
the resolution of CSH of very hydrophobic strains.
As for most hydrophobicity tests the results should
not be interpreted as relative values because they
depend on the test conditions. Fig. 2A shows a micrograph of a hydrophobic Escherichia coli F18(pPKL91) with high numbers of attached microspheres, but also one cell with no attached microspheres.
Table 2
Hydrophobicities of free-living bacteria in wastewater with the HIC, BATH and MAC methods and sludge characteristics for the sampling occasions
Sample
Hydrophobicity
HIC (%)
Sludge characteristics
BATH (%)
e
MAC (%)
SVa
SVIb
SSVIc
SSd
1
^
86
^
220
64
45
8
2
^
47
^
185
66
42
11
3
^
43
^
215
61
45
12
4
^
47
^
240
65
44
11
5
18
^
24
450
122
54
11
6
32
^
30
550
149
57
12
7
21
^
22
370
98
50
11
8
7
71
15
370
129
106
8
9
19
100
15
890
267
117
10
30
81
19
900
303
113
7
11
34
62
18
850
336
123
26
a
Sludge volume (ml/l).
b
c
d
e
Sludge volume index (ml/g).
Stirred sludge volume index (ml/g).
Suspended solids in the e¥uent water (mg/l).
Not determined.
5
304
A. Zita, M. Hermansson / FEMS Microbiology Letters 152 (1997) 299^306
Fig. 3. Distribution of wastewater bacteria with di¡erent numbers of attached hydrophobic microspheres.
A more detailed picture of the CSH of a population is obtained with MAC than with, for instance,
the BATH and HIC methods. For the hydrophobic
E. coli F-18 and Acinetobacter calcoaceticus about
80^90% of the cells have more than 10 microspheres,
but some cells have few or no microspheres.
Although most of the E. coli K51 cells are hydrophilic, i.e. have no microspheres, some cells in the
population are hydrophobic. The fact that populations with a predominantly hydrophilic or hydrophobic character have a fraction of cells with essentially
the opposite character is seen clearly for Pseudomonas putida and E. coli F-18, indicating some heterogeneity in a population. The reason for this heterogeneity is not known. It may be the result of
di¡erences in individual cellular physiology, but heterogeneity of a P. putida population did not decrease
when cells started to grow after addition of nutrients
(LB) (data not shown).
Adhesion of blue stained microspheres has been
used to evaluate CSH of yeast cells in pure cultures
[9], [11]. The use of £uorescent microspheres in the
present work allows the use of smaller beads that are
suitable for bacteria.
3.2. Comparisons of bacterial cell surface hydrophobicity with the HIC, BATH and MAC methods
Table 1 shows the results for the hydrophobicity
of the series of bacterial strains measured by the
HIC, BATH and MAC methods. The MAC method
correlated well with both HIC (linear correlation coe¤cient R2 = 0.845) and BATH (R2 = 0.981). Adhesion of hydrophilic microspheres to the bacteria
showed no correlation with hydrophobicity (linear
correlation coe¤cient for MAC, R2 = 0.1) (data not
shown).
HIC and BATH tests do not always give the same
CSH value when a speci¢c strain is evaluated [12],
probably because BATH evaluates the overall CSH,
whereas the HIC method more likely measures localized CSH. MAC is most likely to be sensitive to
localized CSH and may complement other CSH
methods by determining hydrophobic areas on the
cell surface.
3.3. CSH of speci¢c cellular structures
The use of the MAC method to study hydrophobicity of speci¢c areas on the cells or of surface appendages was shown for Caulobacter maris which
produces both stalked cells with holdfasts and
swarmer cells with £agella [13]. The hydrophobic
microspheres attached to the distal tips of the stalk
and along the £agella (Fig. 2B), indicating that these
parts of the cells were hydrophobic. The hydrophobic microspheres speci¢cally attach to these areas
and not to the other parts of the cell. This also shows
that possibly fragile cell surface structures remained
intact during the test. The problems with determining CSH for ¢lamentous and chain-forming bacteria
can also be avoided using MAC, since the cells are
distinguished in the microscope.
3.4. CSH of free-living wastewater bacteria in situ
The MAC method is well suited for use without
culturing of bacteria prior to the analysis, which is of
great importance when studying environmental samples, since only a small part of these bacteria can be
cultivated under laboratory conditions.
Bacterial adhesion is an important factor in the
biotechnological treatment of wastewater. Laboratory experiments have shown earlier that a high
CSH was positively correlated with adhesion of bacteria to activated sludge £ocs [6]. Using the MAC
method we could now also show in situ that cells
that were non-attached had a low CSH, indicating
A. Zita, M. Hermansson / FEMS Microbiology Letters 152 (1997) 299^306
305
Fig. 4. Micrograph showing hydrophobic microspheres attaching to bacterial ¢laments extending out from a sludge £oc. The size of a single microsphere represents 0.1
Wm.
that this may be a reason why they did not attach to
MAC can be used to examine the hydrophobicity
the sludge £ocs thus supporting the earlier results.
of mixed samples in more detail compared to con-
Only 20 þ 5% of the free-living cells had 3 or more
ventional methods. Fig. 3 shows that the wastewater
attached microspheres, which showed that the ma-
bacteria had a broad distribution of cells with di¡er-
jority of cells were indeed hydrophilic (Table 2 and
ent CSH, even though most of the cells are generally
Fig.
of
hydrophilic. The possibility of correlating morpho-
23 þ 10% and 67 þ 21%, respectively, which also in-
logical types of bacterial cells with CSH is a further
dicates that the major fraction of these bacteria was
possibility o¡ered by the MAC method as shown for
3).
HIC
and
BATH
gave
mean
values
hydrophilic (Table 2). The results for the wastewater
Caulobacter maris.
samples using all three methods simultaneously gave
di¡erences in CSH between morphological groups.
results that were similar to the hydrophilic
However, it was interesting that bacterial ¢laments
P. putida,
The WWTP samples showed no
which had a HIC value of 25% and a MAC value of
extending out from sludge £ocs were sometimes cov-
19%, except for the BATH value where the waste-
ered by microspheres indicating a strong hydropho-
water samples were even more hydrophilic than
bic character (Fig. 4).
P.
putida. Water samples were collected on 11 di¡erent
occasions which showed that the results were valid
for di¡erent running conditions at the treatment
Acknowledgments
plant, e.g. sludge settling conditions, turbidity in
the e¥uent water, etc. Some of the parameters describing
the
running
conditions
plant are shown in Table 2.
at
the
treatment
This project was ¢nancially supported by The National Board for Industrial and Technical Development (NUTEK), through the STAMP consortium in
306
A. Zita, M. Hermansson / FEMS Microbiology Letters 152 (1997) 299^306
Goëteborg and The Foundation for Strategic Research through the Marine Science and Technology
(MASTEC) Programme which are gratefully acknowledged. We would also like to thank Cecilia
Dahlberg for help with the micrographs.
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