RESEARCH ARTICLE
Protists with di¡erent feeding modes change bio¢lm morphology
Anne Böhme, Ute Risse-Buhl & Kirsten Küsel
Institute of Ecology, Friedrich Schiller University Jena, Jena, Germany
Correspondence: Kirsten Küsel, Institute of
Ecology, Friedrich Schiller University Jena,
Dornburger Straße 159, 07743 Jena,
Germany. Tel.: 149 3641 949 461; fax: 149
3641 949 462; e-mail:
[email protected]
Received 21 November 2008; revised 24 April
2009; accepted 29 April 2009.
Final version published online 10 June 2009.
DOI:10.1111/j.1574-6941.2009.00710.x
Editor: Riks Laanbroek
MICROBIOLOGY ECOLOGY
Keywords
flagellates; ciliates; amoeba; biofilm
morphology; bacterial biofilm.
Abstract
The effect of Dexiostoma (filter feeder), Vannella, Chilodonella (raptorial feeders),
Spumella, and Neobodo (direct interception feeders) on the morphology of multispecies bacterial biofilms was investigated in small flow cells. The filter feeder
Dexiostoma campylum did not alter biofilm volume and porosity but stimulated
the formation of larger microcolonies compared with ungrazed biofilms. In
contrast, the raptorial feeder Vannella sp. efficiently grazed bacteria from the
biofilm surface, leading to smaller microcolonies and lower maximal and basal
layer thickness compared with ungrazed biofilms. Microcolony formation was not
stimulated in the presence of the sessile Spumella sp. Chilodonella uncinata rasped
bacteria from the outer surface leading to mushroom-shaped microcolonies. In the
presence of C. uncinata and Spumella sp., the biofilm volume was 2.5–6.3 times
lower compared with ungrazed biofilms. However, the biofilm porosity and the
ratio of biofilm surface area to biofilm volume were 1.5–3.7 and 1.2–1.8 times
higher, respectively. Thus, exchange of nutrients and gases between the biofilm and
its surrounding fluid should also be improved in deeper biofilm layers, hence
accelerating microbial growth.
Introduction
The predominant microbial life mode in streams are biofilms,
which are aggregations of bacteria and protists embedded in a
matrix of exopolymeric substances (Geesey et al., 1978; Lock
et al., 1984; Costerton et al., 1994). In streams, biofilm
bacterial activity can be higher than the activity of suspended
bacteria (Fletcher, 1986; Romanı́ & Sabater, 1999). The
morphology of biofilms, either cultivated or in streams, is
complex and highly structured with tower- and mushroomshaped microcolonies and intersected open water channels
(Lawrence et al., 1991; Møller et al., 1997; Stoodley et al.,
1999; Battin et al., 2003a, b). Liquid flow in biofilm channels
maintains nutrient and gas exchange between the biofilm and
its surrounding fluid (de Beer et al., 1994, 1996; Stoodley
et al., 1994). Especially those channels that reach deep into the
biofilm matrix also maintain exchange processes at the basal
biofilm layer. These exchange processes are enhanced at a
high porosity (biofilm-free area at the surface) and a high
biofilm surface area in relation to the biofilm volume.
Biofilm structure and dynamics are controlled by physical
and chemical conditions of the water column (Battin et al.,
2003b; Costerton, 2007), but also by protist grazing activity
(Pederson, 1990; Arndt et al., 2003). Protists can reduce the
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bacterial biomass in biofilms (Weitere et al., 2005) and alter
biofilm morphology due to grazing (Lawrence & Snyder,
1998; Matz et al., 2004; Weitere et al., 2005; Queck et al.,
2006) and possibly motility (Jackson & Jones, 1991). Bacterial stasis is prevented by grazing protists and the bacterial
community is kept in a productive state (Johannes, 1965).
Filter feeders of the subclass Hymenostomatia (Ciliophora) that frequently occur at high abundances in stream
biofilms (Risse-Buhl & Küsel, 2009) have been studied in
detail relative to their effect on biofilm morphology. The
vagile Tetrahymena sp. (Hymenostomatia) increases microcolony abundance in early (3-day-old) biofilms (Weitere
et al., 2005; Parry et al., 2007), while grazing of late (7-dayold) biofilms causes distinct but fewer microcolonies than in
the ungrazed control (Weitere et al., 2005). Hymenostomatia utilize suspended prey more efficiently than attached
prey by producing strong feeding currents (Fenchel, 1986;
Eisenmann et al., 1998; Parry, 2004). Because surfaceassociated gulper or raptorial feeders utilize mainly attached
prey (Fenchel, 1986; Hausmann, 2002), species of this
feeding mode may influence biofilm morphology more than
the investigated filter feeders. The raptorial-feeding amoeba
Acanthamoeba polyphaga drastically reduces biofilm biomass and microcolony abundance (Weitere et al., 2005).
FEMS Microbiol Ecol 69 (2009) 158–169
159
Protists influence biofilm morphology
Table 1. Characteristics of the investigated protist species with different feeding modes and their abundance in the inoculum
Species and accession number
of CCAPw
Dexiostoma campylum
(Hymenostomatia, Ciliophora)
CCAP 1611/1
Vannella sp. (Vannellida,
Amoebozoa) CCAP 1589/18
Chilodonella uncinata
(Phyllopharyngia,
Ciliophora)
Spumella sp. (Chrysophyceae,
Stramenopiles) CCAP 955/2
Neobodo designis (Neobodonida,
Kinetoplastea)
Length (mm)
Lifestyle
Feeding modez
Food source‰
Abundance in
inoculum (cells mL1)z
35–90
Water column,
biofilm
Filter feeder
Suspended and recently
attached bacteria
505
Not studied
25–70
Biofilm
Raptorial feeder
25–70
Biofilm
Raptorial feeder
Recently attached and
embedded bacteria
Recently attached and
embedded bacteria
555
Not studied
510
337
5–15
Biofilm
5–10
Biofilm
Direct interception
feeder
Direct interception
feeder
Suspended, attached, and
embedded bacteria
Suspended, attached, and
embedded bacteria
4660
2650
Not studied
9.2 104
Taxonomic classification after the system of Adl et al. (2005) and Lynn & Small (2002).
w
Cultures of three protist species are maintained at the culture collection of algae and protozoa (CCAP) in Argyll, Scotland (http://www.ccap.ac.uk).
Protists were grouped according to their feeding mode following Hausmann (2002), Boenigk & Arndt (2002), and Hausmann et al. (2003).
‰
Data are from Parry (2004).
z
The upper and lower numbers indicate initial abundance of protists in the inoculum for experiments with VYE-medium bacteria and with stream
microorganisms, respectively (n = 1). The effect of Dexiostoma campylum and Vannella sp. on the stream microbial biofilm and the effect of Neobodo
designis on the biofilm of VYE-medium bacteria were not studied.
z
Thus, differences in protist feeding modes and motility
might affect the extent of morphological biofilm changes.
Similar to planktonic bacteria (Hahn et al., 2000; Hahn &
Höfle, 2001), biofilm bacteria are also known to develop
defense strategies against grazing protists (Matz & Kjelleberg,
2005). Bacterial cell aggregations called microcolonies and
quorum-sensing-mediated production of toxins are efficient
grazing resistance mechanisms against flagellate and amoeba
grazing, respectively (Matz et al., 2004; Weitere et al., 2005).
The effect of grazing protists on biofilm bacteria was commonly studied with single-species bacterial biofilms (Matz
et al., 2002, 2004; Weitere et al., 2005; Queck et al., 2006). In
contrast, stream biofilms are usually composed of mixed
bacterial communities. Thus, we studied the effect of protists
with different feeding modes (filter, direct interception and
raptorial feeders), which typically colonize stream biofilms
(Foissner et al., 1992; Schönborn, 1996; Franco et al., 1998;
Willkomm, 2008; Risse-Buhl & Küsel, 2009) on the threedimensional (3D) morphology of multispecies bacterial biofilms. We hypothesized that raptorial feeding ciliates stimulate
microcolony formation and increase the biofilm surface area
to biofilm volume (BSA/BV) ratio of multispecies bacterial
biofilms.
Materials and methods
Protists and bacterial cultures
One filter feeder, two direct interception feeders, and two
raptorial feeders were used in this study (Table 1). Filter and
direct interception feeders use cilia or flagella to create water
FEMS Microbiol Ecol 69 (2009) 158–169
currents that carry suspended or loosely attached prey
toward the mouth (Eisenmann et al., 1998; Boenigk &
Arndt, 2002; Hausmann et al., 2003; Parry, 2004). The
studied filter feeder Dexiostoma campylum concentrates
particles by producing strong feeding currents with ciliary
membranelles (Fenchel, 1986; Hausmann, 2002). The direct
interception feeders Spumella sp. and Neobodo designis
ingest individual bacterial cells that are transported along
the flow lines created by a flagellum (Boenigk & Arndt,
2002). The raptorial feeders Vannella sp. and Chilodonella
uncinata search for bacterial prey that are loosely associated
with and permanently attached to surfaces (Parry, 2004).
Feeding is supported by pseudopods (Vannella sp.) or by
special mouth structures, the cytopharynx (C. uncinata)
(Foissner et al., 1991; Hausmann et al., 2003; Smirnov et al.,
2007).
The amoeba Vannella sp. was isolated from biofilm
samples of the third-order Ilm stream (50144 0 5800 N,
11102 0 1400 E). Because the isolation of the ciliates D. campylum and C. uncinata from stream samples was not successful, K. Eisler (Institute of Zoology, University of Tübingen)
kindly provided a culture of D. campylum that also contained the vagile ciliate C. uncinata and the sessile flagellate
Spumella sp. Dexiostoma campylum and Spumella sp. were
isolated from the original culture. However, Spumella sp.
could not be separated from C. uncinata, and experiments
were run as two (C. uncinata and Spumella sp.)- and single
(Spumella sp.)-species treatments. Protist cultures were kept
at 20 2 1C in Volvic table water with 5 mg L1 yeast extract
[Fluka; dissolved organic carbon (DOC): 3.65 0.1 mg L1]
(VYE medium) and transferred into fresh medium every 2
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160
weeks. Protist cultures were filtered (5-mm cellulose nitrate
filter, Sartorius) 1 day before the start of the experiment to
enrich protists and to eliminate bacteria from the original
culture. Overnight, the protists in the filtered suspension
could recover and further minimize the remaining bacteria
by grazing. Fixed (Lugol’s solution) protists were enumerated in Sedgewick Rafter Chambers at 100 magnification
(Axioplan, Zeiss, Jena, Germany).
Protists were cocultivated with a multispecies bacterial
community in small flow cells. The multispecies bacterial
community developed in unsterilized VYE medium (VYEmedium bacteria) within a 3-day incubation period without
protists at 20 2 1C. No additional inoculum was used. The
bacteria were counted after fixation with formaldehyde (2%
final concentration) and staining with 4 0 6-diamidino2-phenylindole (Porter & Feig, 1980) at oil immersion
(Axioplan, Zeiss).
Effect of protists with different feeding modes
on the morphology of bacterial biofilms
Biofilms were cultivated on glass coverslips that sealed three
channels (40 4 4 mm) of a flow cell made of acryl glass
(Møller et al., 1998). A peristaltic pump (Ismatec SA,
Wertheim-Mondfeld, Germany) maintained a continuous
discharge of 110 mL min1. The flow velocity ranged between
25.7 4.2 and 182.3 64.8 mm s1 at a height of 50–500 mm
above the coverslip, respectively, indicating laminar flow
conditions with Reynolds numbers of o 1. The flow
guaranteed a continuous supply of nutrients, organic carbon
(yeast extract), and gases. Bubble traps ahead of the flow
cells entrapped destructive air bubbles. Flow channels were
sterilized with a sodium hypochloride solution (NaOCl,
0.5%) for 4 h and washed with sterilized, distilled water
overnight. Sterilized VYE medium was pumped through the
flow channels for 1 h before experiments started. Flow
channels were inoculated with a bacterial suspension of
1.1–3.0 107 cells mL1 grown in the VYE medium. Bacteria
stayed in flow channels for 2 h without flow to allow
their settlement and attachment to the glass slides.
Experiments were performed at a temperature of 23 2 1C,
and 15-h light and 9-h dark conditions. After 1 day, protists
were introduced into the flow channels by syringe (for
abundances, see Table 1) and incubated without flow for
2 h. Because additional bacteria were introduced into the
system with the protists, a corresponding concentration of
bacteria (0.1–20.7 106 bacteria mL1) was added to ungrazed biofilm treatments. Experiments were run for a total
of 5 days.
For every treatment, six separate flow channels served as
independent replicates. The whole flow cell set-up was
directly placed under an inverse light microscope (Axiovert,
Zeiss) to enumerate protists and microcolonies daily in all
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A. Böhme et al.
six separate flow channels. Concomitantly, the maximal
biofilm thickness was measured with the calibrated fine
drive of the microscope. Microcolonies were defined as
aggregations of bacterial cells with an approximately circular
base and a diameter of 4 10 mm.
The bacterial biofilm was fixed with 4% formaldehyde
solution that was slowly pumped through flow channels
with the peristaltic pump to minimize alteration of the
biofilm morphology. Biofilms of six flow channels were fixed
before inoculation of the protists to determine the spatial
arrangement of bacteria in initial biofilms. All other biofilms
were fixed after 4 days of protists’ grazing activity. After
fixation, the bacterial biofilm was stained with the nucleic
acid marker propidium iodide (0.3 mM) (Sigma-Aldrich),
which stains the bacterial and extracellular nucleic acids, for
10 min in the dark. A washing step with phosphate-buffered
saline removed excess of stain. Stained biofilms were
observed with a confocal laser scanning microscope (LSM
510, Zeiss). 3D picture-stacks (z-stacks) were taken at three
randomly chosen spots in each flow channel at a magnification of 400 (objective: Appochromat 1.2 W corr) using a
helium neon laser with a vertical resolution of 0.5 mm.
Effects of protists on the morphology of a
stream microbial biofilm
Stream bacteria were obtained by taking water samples from
the third-order Ilm stream (50144 0 5800 N, 11102 0 1400 E) in
August 2007 (abiotic parameters of the stream: temperature
16.2 1C, oxygen content 8.8 mg L1, pH 8.1, conductivity
1
1
0.5 mg L1, NO
0.23 mS cm1, PO3
4
3 11.2 mg L , NH4
1
1
o 0.001 mg L , and DOC 5.49 mg L ). Stream water
(400 mL) was filtered through a 0.45-mm cellulose
acetate filter (Sartorius) to exclude protists and metazoans.
Flow channels were washed with filtered (0.2 mm, Sartorius)
and sterilized stream water. Stream bacteria (5.8 105 cells mL1) were inoculated into the flow channels and
incubated without flow for 2 h. The flexible flagellate
N. designis (length width: 6.2 3.6 mm) probably passed
the filters through overlapping pores (Cynar et al., 1985) and
was introduced into the flow channels together with the
bacterial filtrate. The direct interception feeder N. designis is
commonly found in stream biofilms and contributes about
40% to the flagellate biofilm community in the Ilm stream
(Willkomm, 2008).
To study multiple effects of protists on stream microbial
biofilm morphology, biofilms were cocultivated with
C. uncinata and Spumella sp. (for abundances, see Table 1).
Treatments with stream bacteria and N. designis were used as
control. Chilodonella uncinata and Spumella sp. were introduced into the flow channels as described in the previous
section. The treatments with one [N. designis; G(N)],
two [N. designis and Spumella sp.; G(NS)], and three
FEMS Microbiol Ecol 69 (2009) 158–169
161
Protists influence biofilm morphology
[N. designis, Spumella sp., and C. uncinata; G(NSC)] protist
species were replicated five times.
Image analysis
3D image analyses were accomplished with the ‘DAIME’
software (Daims et al., 2006). Images were edited before the
analyses. Voxels with a brightness range between 0 and 12
and with o 5 nonzero (not black) neighbor voxels were
deleted before z-stacks were masked to give all nonzero
voxels the maximum intensity. Images were segmented (2D
or 3D) to define objects and to delete auto-fluorescent
objects or protists. The analyzed parameters included basal
layer thickness (mm), BV (mm3 cm2), and BSA (mm2 cm2).
The latter two parameters were used to calculate the BSA/BV
ratio. From a 2D image at the biofilms base, the porosity
(biofilm-free area at the surface of the coverslip)
(mm2 cm2), microcolony area (mm2), and porosity within
microcolonies in relation to microcolony area (PM/MA)
(mm2 mm2) were estimated. The basal biofilm layer was
measured with the Zeiss LSM IMAGE BROWSER (CZ Image
Browser 4.0, offline version) after a new image stack was
generated that displayed the biofilms side (xz) view.
Statistical analysis
The time series of maximal biofilm thickness measurements
and microcolony abundances were analyzed using a
repeated measures ANOVA with the time as within-subject
factor and ungrazed and grazed biofilm treatments as
between-subject factors (SPSS 15.0). A one-way ANOVA with
Tukey’s test for multiple comparisons was used to test for
differences of biofilm characteristics between ungrazed (1
and 5 days) and grazed biofilms (5 days). Data were
log10(x11) transformed for the one-way ANOVA approach
due to lack of homogeneity (Levene’s test).
Results
Effects of protists with different feeding modes
on the morphology of bacterial biofilms
Protist abundances increased over time (Fig. 1), indicating
that all protist species were able to maintain growth in the
flow cells. After a lag-phase of 3 days, the abundance of the
filter feeder D. campylum increased and reached
1.3 104 cells cm2. The abundance of Vannella sp. increased rapidly after the first day and peaked with
5.1 104 cells cm2 after 3 days. Cells that hatched out of
cysts contributed to the population only to a minor extent.
The flagellate Spumella sp. in the single-species treatment
reached 98.9 104 cells cm2. In the two-species treatment,
abundance of Spumella sp. decreased to 33.5 104 cells cm2
from day 4 to 5. Spumella sp. was about 42 times more
abundant than C. uncinata, which made up 1–5% of the
total protists abundance in the two-species treatment.
The maximal biofilm thickness significantly increased
during all experiments in both ungrazed and grazed biofilms
(Fig. 1, Table 2). Grazing by D. campylum and Vannella sp.
significantly diminished the increase in maximal biofilm
Fig. 1. Protist abundance (cells cm2) in biofilms,
maximal biofilm thickness (mm), and abundance
of microcolonies (cm2) of VYE medium bacterial
biofilms cultivated in flow cells (mean SD,
n = 6). The biofilms in six separate flow channels
were observed alive during the 5-day incubation
period and biofilm morphology was not altered.
UG, ungrazed biofilms; G(D), biofilms grazed
by Dexiostoma campylum; G(V), biofilms grazed
by Vannella sp.; G(S), biofilms grazed by
Spumella sp.; and G(CS), biofilms grazed by
Chilodonella uncinata and Spumella sp.
FEMS Microbiol Ecol 69 (2009) 158–169
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162
A. Böhme et al.
Table 2. Repeated measures ANOVA for testing the effect of different protists (treatment: with and without protists) on biofilm thickness and
microcolony abundance during 5 days of biofilm formation (time)
Biofilm thickness
df
Microcolony abundance
F
Flow cell experiment: Dexiostoma campylum
Within-subject effects
Time
4, 40
50.803
Time vs. treatment
4, 40
3.294
Between-subject effects
Treatment
1, 10
1.467
Flow cell experiment: Vannella sp.
Within-subject effects
Time
4, 40
16.711
Time vs. treatment
4, 40
7.440
Between-subject effects
Treatment
1, 10
2.831
Flow cell experiment: Chilodonella uncinata, Spumella sp.
Within-subject effects
Time
4, 60
54.660
Time vs. treatment
8, 60
3.090
Between-subject effects
Treatment
2, 15
0.595
P
df
F
P
0.001
0.020
4, 40
4, 40
29.570
3.873
0.001
0.009
0.254
1, 10
4.990
0.050
0.001
0.001
4, 40
4, 40
97.414
28.992
0.001
0.001
0.123
1, 10
57.521
0.001
0.001
0.005
4, 60
8, 60
56.980
11.826
0.001
0.001
0.564
2, 15
21.796
0.001
Significant effects (P o 0.05).
df, degree of freedom; F, F-value of the repeated measures ANOVA; P, P-value of the repeated measures ANOVA.
thickness (Table 2). The large filter feeding ciliate
D. campylum caused a 72% lower maximal biofilm thickness
compared with ungrazed biofilms at the highest protist
abundance. Feeding of the raptorial-feeding amoeba Vannella sp. caused a 20–42% lower maximal biofilm thickness
compared with ungrazed biofilms between days 3 and 5. In
the C. uncinata experiment, maximal biofilm thickness of
ungrazed and grazed biofilms of the two-species treatment
increased linearly and reached a maximum of 196.5 mm. In
the single-species treatment with the flagellate Spumella sp.,
the maximal biofilm thickness reached a plateau after 2 days
and remained at 140.0 mm.
Microcolony formation started after a lag phase of 1–3
days, simultaneously, in both grazed and ungrazed biofilms
irrespective of the protist’s feeding mode (Fig. 1). Microcolony abundance was significantly affected by the protists
(Table 2). The vagile ciliate D. campylum stimulated microcolony formation compared with ungrazed biofilms by up
to 370% (Fig. 1) and microcolony size was significantly
smaller in grazed compared with ungrazed biofilms
(F1 = 27.264, P o 0.001; Fig. 2). Distinct patches of microcolonies that were c. 20 mm apart were observed in biofilms
cocultivated with D. campylum. Compared with 5-day-old
ungrazed biofilms, significantly fewer microcolonies
(50–88%) (F1 = 108.442, P o 0.001) with a lower area
(F2 = 18.489, P o 0.01) were found in the presence of the
amoeba Vannella sp. (Fig. 2). In the single-species treatment
with the sessile Spumella sp., no stimulatory effect was
found. Thus, we assume that the increase in microcolony
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Fig. 2. Effect of protists’ grazing activity on abundance and size of
microcolonies of VYE medium bacterial biofilms cultivated in flow cells
(mean SD, n = 6). Circles, ungrazed biofilms (UG); squares, biofilms
grazed by Dexiostoma campylum [G(D), black symbols], by Vannella sp.
[G(V), grey symbols], by Spumella sp. [G(S), white symbols], and by
Chilodonella uncinata and Spumella sp. [G(CS), white symbols]. Arrows
indicate changes from ungrazed to grazed biofilms.
formation of up to 400% in the treatment with C. uncinata
and Spumella sp. was due to the presence of the vagile ciliate
C. uncinata. Similarly, grazing activity of C. uncinata
appeared to account for microcolonies with a significantly
larger basal area (F2 = 4.836, P o 0.05) (Fig. 2, Supporting
Information, Table S1).
FEMS Microbiol Ecol 69 (2009) 158–169
163
Protists influence biofilm morphology
Fig. 3. Effect of protists’ grazing activity on
biofilm volume and biofilm surface area of a
VYE-medium bacterial biofilm cultivated in flow
cells (mean SD, n = 6). UG, ungrazed biofilms
after 1 day (open symbol) and after 5 days (closed
symbol) of incubation; G(D), biofilms grazed by
Dexiostoma campylum; G(V), biofilms grazed
by Vannella sp.; G(CS), biofilms grazed by
Chilodonella uncinata and Spumella sp.; G(S),
biofilms grazed by Spumella sp. Significant
differences between treatments (P o 0.05) are
displayed by different letters (a, b, c).
The CSLM images visualized the spatial arrangement of
bacteria as well as extracellular nucleic acids that were
stained with the fluorescence marker propidium iodide. In
streams, the cellular fraction forms the major part of
biofilms with 60–85% of biofilm dry weight (Aguilera
et al., 2008). Other types of exopolysaccharides that might
also contribute to biofilm morphology of flow cell biofilms
were not visualized in the CLSM pictures. However, the
combination with light microcopy observations revealed
that biofilm volume significantly increased from days 1 to 5
(Fig. 3) in all ungrazed biofilms of VYE medium bacteria
due to a higher maximal biofilm thickness, more microcolonies (Fig. 1), and a lower porosity (Fig. 4). The BSA/BV
ratio in 1- and 5-day-old ungrazed biofilms of the D.
campylum and Vannella sp. experiments were similar
(P 4 0.05). In the C. uncinata and Spumella sp. experiment,
the BSA/BV ratio was lower in 5- than in 1-day-old ungrazed
biofilms (Fig. 4).
CLSM images showed that raptorial and interception
feeders had a stronger effect on the spatial arrangement of
surface-associated bacteria than the filter feeder. Vannella
sp., Spumella sp., the combination of C. uncinata and
Spumella sp. but not D. campylum significantly reduced the
biofilm volume and biofilm surface area (Figs 3 and 4, Table
S1). Dexiostoma campylum was not grazing on the basal
layer of the biofilms and the porosity was similar to 5-dayold ungrazed biofilms. The biofilm volume was 2.5, 6.3, and
2.6 times lower in biofilms cocultivated with Vannella sp.,
Spumella sp., and the combination of C. uncinata and
Spumella sp. compared with 5-day-old ungrazed biofilms,
respectively, but similar to 1-day-old biofilms. The BSA/BV
ratio was 1.2, 1.8, and 1.3 times greater in biofilms grazed by
Vannella sp., Spumella sp., and the combination of
C. uncinata and Spumella sp. compared with 1- and 5-dayold ungrazed biofilms, respectively (Fig. 4). In addition, the
porosity of grazed biofilms cocultivated with Vannella sp.
and with the combination of C. uncinata and Spumella sp.
was similar to 1-day-old ungrazed biofilms, but significantly
higher compared with 5-day-old ungrazed biofilms (Fig. 4).
The thickness of the basal biofilm layer was reduced due to
FEMS Microbiol Ecol 69 (2009) 158–169
the grazing activity of Vannella sp., Spumella sp., and the
combination of C. uncinata and Spumella sp. by 22.7%,
36.5%, and 30.5%, respectively. The porosity within microcolonies was significantly lower in biofilms grazed by D.
campylum (F1 = 14.041, P o 0.001), indicating that more
bacteria made up the central part of a microcolony (Fig. 4,
Table S1). Vannella sp. had no effect (F1 = 0.620, P = 0.449)
on porosity within microcolonies. The grazing activity of
Spumella sp. caused a significantly higher porosity within
microcolonies (F2 = 14.721, P o 0.001) compared with
5-day-old ungrazed biofilms (Figs 4 and 5).
Effects of protists on the morphology of a
stream microbial biofilm
The abundance of N. designis reached 14.6 104 cells cm2
in the single-species treatment, but was lower in both other
treatments (Table 3). Spumella sp. made up 14.9% and 1.5%
of the total protist abundance in the two- and three-species
treatments, respectively. Abundance of C. uncinata reached
127.5 cells cm2, contributing only 0.2% to the community
in the three-species treatment.
Stream microbial biofilms were 1.2–2.4 times thicker,
with a 1.1–1.7 times higher porosity, and a 1.6–6.3 times
lower biofilm volume compared with CLSM images of
1-day-old biofilms of VYE-medium bacteria. The biofilm
volume of grazed stream microbial biofilms was 2.8–4.5
times higher in 4- compared with 1-day-old biofilms (Table
3). Porosity was lowest in the two-species treatments where
biofilm volume and maximal biofilm thickness peaked and
abundance of protists was lowest. Most microcolonies were
found in the single-species treatment with the highly
abundant vagile N. designis. The maximal biofilm thickness
of 1-day-old biofilms was similar to 4-day-old biofilms
grazed by three protist species. Despite the high abundance
of N. designis in the single-species treatment, the maximal
biofilm thickness was significantly higher in 4- than in
1-day-old biofilms. The basal layer thickness of grazed
biofilms was significantly lower in the three-species treatment compared with single- and two-species treatments.
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164
A. Böhme et al.
Fig. 4. Effect of protists’ grazing activity on BSA/
BV ratio, porosity, basal layer thickness, and
porosity of microcolonies to microcolony area
ratio (PM/MA ratio) of a VYE medium bacterial
biofilm cultivated in flow cells (mean SD, n = 6).
No mc, no microcolonies were observed in 1-dayold ungrazed biofilms; UG, ungrazed biofilms;
G(D), biofilms grazed by Dexiostoma campylum;
G(V), biofilms grazed by Vannella sp.; G(CS),
biofilms grazed by Chilodonella uncinata and
Spumella sp.; G(S), biofilms grazed by Spumella
sp. Significant differences between treatments
(P o 0.05) are displayed by different letters
(a, b, c).
Discussion
Microcolonies as grazing resistance strategy
against protistan grazers
In VYE-medium bacterial biofilms, the filter-feeding ciliate
D. campylum stimulated the formation of microcolonies
with a small basal area in distinct patches. The filter-feeding
ciliate Euplotes sp. locally clears patches and increases spatial
heterogeneity of bacterial biofilms (Lawrence & Snyder,
1998). Euplotes sp. cells regularly return to the cleared
biofilm patches probably due to released chemical cues.
Cleared patches were not observed in biofilms cocultivated
with D. campylum in the present study. However, uniformly
distributed microcolonies might have developed in patches
that were regularly visited by D. campylum cells.
Despite increased numbers of microcolonies, abundance
of D. campylum and C. uncinata increased during the 4 days
of grazing. Doubling times of both ciliates range between 11
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c
and 27 h under culture conditions of 20 1C (Laybourn &
Stewart 1974; Finlay, 1977) and between 72 and 144 h at
5–10 1C in streams (Schönborn, 1981). Thus, food limitation due to formation of microcolonies as a resistance
strategy against grazing in flow channel biofilms seemed to
be negligible.
The raptorial-feeding amoeba Vannella sp. reduced
microcolony abundance, microcolony size, and also maximal and basal biofilm layer thickness. The interesting
question is whether protists can also use, besides bacterial
cells temporarily attached, cells that are permanently attached to surfaces and microcolonies embedded in mucus.
Amoebas mainly maintain growth by feeding on biofilmassociated organisms (Pickup et al., 2007). Similar to
Vannella sp., Acanthamoeba castellanii and A. polyphaga
deplete the majority of biofilm bacteria and microcolonies
(Huws et al., 2005; Weitere et al., 2005). Microcolonies of
Pseudomonas aeruginosa are described to kill Acanthamoeba
via a conserved virulence pathway (Matz et al., 2008).
FEMS Microbiol Ecol 69 (2009) 158–169
165
Protists influence biofilm morphology
Because Vannella sp. was always highly abundant, this
virulence pathway seemed to play a minor role as a grazing
resistance strategy of VYE-medium bacterial biofilms.
Microcolony formation of the multispecies bacterial
biofilm was not affected by the sessile interception feeding
flagellate Spumella sp. In single-species biofilms, microcolonies are efficient defense strategies against grazing by vagile
flagellates (Matz et al., 2004; Weitere et al., 2005; Queck
et al., 2006). The vagile flagellate Bodo saltans induces
microcolony formation in the early stages of biofilm devel-
opment of Serratia marcescens (Queck et al., 2006). The
authors suggested that microcolony formation does not
seem to be mediated by the release of extracellular compounds or signal molecules, but by the action of the
flagellate’s flagella. In contrast, the flagella motion of the
sessile flagellate Spumella sp. seemed not to be sufficient to
stimulate microcolony formation in this study. Thus, movement of protists on the biofilm surface and not only the
flagella motion might be important to stimulate microcolony
formation.
In the presence of the direct interception feeder Spumella
sp., microcolonies had a smaller basal area and higher
PM/MA ratio compared with microcolonies of the ungrazed
control. The microcolony-forming and exopolysaccharideproducing morphotype of Pseudomonas putida is not grazed
by the interception feeder Ochromonas sp., indicating that
the production of exopolymeric substances is a grazing
resistance strategy (Matz et al., 2002). Deeply embedded
bacteria forming the central part of microcolonies are thus
protected against protists’ grazing activity. Spumella sp.
captures bacterial cells that are carried along the flow lines
of its flagellum (Boenigk & Arndt, 2002). Although not
directly observed, the high PM/MA ratio displayed in the
CLSM pictures indicated that Spumella sp. seemed to utilize
bacterial cells from the interior of microcolonies with a
flagellum that is 1.5–2 times longer than the flagellate’s
body. We hypothesize that the movement of the flagellum of
Spumella sp. loosen bacterial cells from the biofilm and even
out of microcolonies.
Fig. 5. CLSM images (xz axis) of the VYE medium bacterial biofilms
grazed by the raptorial-feeding ciliate Chilodonella uncinata and the
interception-feeding flagellate Spumella sp. (a) ungrazed biofilms at t = 1
day, (b) ungrazed biofilms at t = 5 days, (c) biofilms grazed by Spumella
sp., and (d) biofilms grazed by C. uncinata and Spumella sp. Scale
bar = 20 mm.
Grazing protists alter mass transfer of nutrients
into biofilms
Protists shape the biofilm morphology due to their grazing
activity and mobility (Jackson & Jones, 1991; Lawrence &
Snyder, 1998; Matz et al., 2004; Weitere et al., 2005).
Table 3. Effects of grazing of one, two, and three different protist species on characteristics of a stream microbial biofilm in flow cells [mean (SD), n = 5]
G t=1
Abundance of protists
Neobodo designis (cm2)
Spumella sp. (cm2)
Chilodonella uncinata (cm2)
Morphological biofilm characteristics
Biofilm volume (106 mm3 mm2)
Porosity (106 mm2 mm2)
Microcolony abundance (cm2)
Maximal biofilm thickness (mm)
Basal layer thickness (mm)
0.2 (0.4) 104
0.0 (0.0)
0.0 (0.0)
0.42 (0.05)a
0.92 (0.01)a
0.0 (0.0)
111.0 (3.0)a
4.06 (0.30)a
G(N) t = 4
G(NS) t = 4
14.6 (1.9) 104
0.0 (0.0)
0.0 (0.0)
1.18 (0.28)b
0.81 (0.04)ab
28.5 (9.1)a
160.3 (10.2)c
8.88 (039)b
2.3 (0.6) 104
0.4 (0.03) 104
0.0 (0.0)
1.91 (0.33)b
0.73 (0.05)b
2.8 (0.8)b
192.0 (14.3)bc
8.29 (0.51)b
G(NSC) t = 4
7.0 (1.7) 104
0.1 (0.03) 104
127.5 (45.4)
1.17 (0.09)b
0.76 (0.02)b
0.9 (0.5)b
145.3 (14.0)ab
6.49 (0.37)c
No microcolonies were present in 1-day-old biofilms. Thus, this value was not included in the statistical analysis.
Different italic letters show results of the one-way ANOVA and indicate significant differences between treatments (P o 0.05).
G, stream microbial biofilms after 1 day; G(N), stream microbial biofilms after 4 days; G(NS), stream microbial biofilms additionally grazed by Spumella
sp. for 3 days; G(NSC), stream microbial biofilms additionally grazed by Spumella sp. and Chilodonella uncinata for 3 days.
FEMS Microbiol Ecol 69 (2009) 158–169
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166
Changes in biofilm morphology and spatial arrangement of
surface-associated bacteria differed apparently due to the
feeding mode of protists. Biofilm volume and porosity was
not altered in biofilms cocultivated with D. campylum. Filter
feeders preferably utilize suspended rather than attached
bacteria (Eisenmann et al., 1998), because they concentrate
food particles by producing strong feeding currents (Fenchel, 1986; Hausmann, 2002). The filter-feeding ciliate Tetrahymena sp. is also able to reduce microcolony abundance
and biofilm biomass, indicating that biofilm bacteria can be
used as food source (Weitere et al., 2005). Because bacterivorous protists can maintain growth by alternatively utilizing yeast extract of the medium or detritus (Broers et al.,
1991; Scherwass et al., 2005), growth of D. campylum might
have also been dependent on the utilization of yeast extract
in the medium rather than on biofilm bacteria alone.
Because of cell mobility or the strong feeding currents of
500 mm s1 near the mouth (Fenchel, 1986), filter feeders
might slough biofilm fragments that are used as a food
source (Huws et al., 2005; Parry et al., 2007). In our
experiment, about 30% of the cells of D. campylum were
found within microcolonies or biofilm foldings where they
could slough fragments from the upper biofilm layer or
from the microcolony periphery. Sloughing might explain
the lower maximal biofilm thickness and smaller microcolonies. The low porosity within microcolonies (PM/MA
ratio) indicated that bacterial growth could also be maintained within microcolonies. Released nutrients of protists
(Zubkov & Sleigh, 1999) and the produced feeding currents
of filter feeders might enhance oxygen and nutrient transport into biofilms (Glud & Fenchel, 1999; Vopel et al., 2005).
Thus, vagile filter feeders that can reach high abundances in
biofilms at slow flowing stream sites (Risse-Buhl & Küsel,
2009) might promote nutrient transport into biofilms
grown at slow flow velocities.
Despite the stimulated formation of microcolonies,
C. uncinata, in combination with Spumella sp., also reduced
the biofilm volume. The raptorial-feeding ciliate C. uncinata
actively searches for bacteria (and small flagellates) and takes
up individual cells with its cytopharyngeal basket (a cylinder
of microtubules) (Foissner et al., 1991; Hausmann, 2002).
Feeding C. uncinata seemed to rasp single bacterial cells
from the biofilm surface by forward and backward movements (see Video S1), which might have caused the development of mushroom-shaped microcolonies (Fig. 5).
Mushroom-shaped microcolonies provide optimal diffusion
paths between the biofilm and its surrounding fluid due to a
high BSA/BV ratio (Picioreanu et al., 1998a, b; Costerton,
2007). Hence, the transport of nutrients into deeper layers of
biofilms is enhanced (Massoldeya et al., 1995). Thus, grazing
raptorial and interception feeders tested in this study
seemed to enhance the exchange of nutrients and gasses
between the biofilm and the VYE medium due to the high
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A. Böhme et al.
porosity and BSA/BV ratio of the biofilm. The better
exchange can also accelerate bacterial growth.
In stream microbial biofilms, maximal thickness increased over time irrespective of the species that were
grazing on these biofilms more than the exploitation of the
free surface area, which is indicated by the high porosity.
Only minor differences were observed between treatments
with one, two, and three grazing protist species. The
different protist feeding modes present on these biofilms
might have caused a selection for different types of bacteria,
such as loosely associated or permanently attached to a
surface. Because of the high abundance of N. designis in all
treatments, this flagellate appeared to be mainly responsible
for the altered spatial arrangement of surface-associated
bacteria. Neobodo designis moved along linear pathways that
resulted in the furrowed basal layer. Hence, exchange of
nutrients between the surrounding fluid and thicker biofilms were enhanced due to the network of channels and
voids penetrating the biofilm.
Conclusion
We demonstrated that single protist species as well as a
combination of protist species are capable of altering biofilm
morphology, affecting spatial arrangement of bacterial cells,
and removing biofilm fragments. Biofilm function that is
closely linked to biofilm structure and spatial arrangement
of bacterial cells is crucial for stream ecosystem processes
(Battin et al., 2003a). The stimulated formation of microcolonies and the high surface area of biofilms grazed by
raptorial and direct interception feeders, but also the
channel network observed in stream microbial biofilms,
yielded a rougher biofilm surface compared with ungrazed
biofilms. Considering biofilms as microbial landscapes, the
dispersal of drifting microorganisms is altered by changing
surface characteristics (Battin et al., 2007). Especially at fastflowing stream sites, more cells will stick to rough biofilms
because of lower shear forces experienced by cells shielded
from the main current and a greater surface area for
adsorption compared with smooth biofilms (Characklis,
1984). Protists’ grazing activity leading to a rough biofilm
surface, especially by raptorial feeders, might reduce the
dispersal distance and thus organism drift. At these fastflowing sites, biofilm processes are enhanced and uptake of
dissolved nutrients and carbon is improved (Battin et al.,
2003b). At slow flow velocities, internal processes might be
more important to promote biofilm function. Mass transfer
of nutrients down to the biofilms’ base at slow flow
velocities might be enhanced either (1) due to the production of strong feeding currents by vagile filter feeders or (2)
due to the grazing activity of raptorial and interception
feeders that raised the BSA/BV ratio and the porosity.
FEMS Microbiol Ecol 69 (2009) 158–169
167
Protists influence biofilm morphology
Acknowledgements
The study was funded by a grant from the German Science
Foundation (GRK 266/3). J. Bolz and P. Zipfel provided the
CLSM facilities. The authors sincerely thank A. Scherwass,
W. Schönborn, C. Augspurger, S. Kröwer, and M. Reiche for
helpful discussions, K. Eisler and G. Dürr for providing
some of the protists cultures, M. Willkomm for determining
the flagellate species, M. Hupfer for providing the peristaltic
pump, J. Schmidt for performing the stream microbial
biofilm experiment, and M. Richter, A. Hartmann, S.
Poltermann, and A. Güllmar for assistance with the handling of the CLSM.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. Results of the one-way ANOVA for testing the effect
of protists on morphological characteristics of a VYE
medium bacterial biofilm.
FEMS Microbiol Ecol 69 (2009) 158–169
Video S1. Cells of Chilodonella uncinata grazing on the
periphery of a microcolony of a VYE-medium bacterial biofilm
(200 magnification; one graduation mark = 5 mm).
Please note: Wiley-Blackwell is not responsible for the
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should be directed to the corresponding author for the article.
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