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Applied and Environmental Microbiology, September 2001, p. 4286-4292, Vol. 67, No. 9
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.9.4286-4292.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Interception of Small Particles by Flocculent
Structures, Sessile Ciliates, and the Basic Layer of a Wastewater
Biofilm
Heinrich
Eisenmann,1,*
Ioanna
Letsiou,2
Anette
Feuchtinger,2
Wolfgang
Beisker,1
Ernst
Mannweiler,2
Peter
Hutzler,2 and
Patrik
Arnz3
Flow Cytometry Group1
and Institute of Pathology,2 National
Research Center for Environment and Health, D-85764
Neuherberg/Munich, and Institute of Water Quality and Waste
Management, Technical University of Munich, D-85784
Garching,3 Germany
Received 23 January 2001/Accepted 12 June 2001
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ABSTRACT |
We investigated attachment processes of hydrophobic and
hydrophilic particles (diameter = 1 µm) to mature biofilms
grown on clay marbles in a sequencing batch biofilm reactor. During a
treatment cycle with filtered wastewater containing different
fluorescent beads, the progression of particle density in various
biofilm compartments (carrier biofilm, basic biofilm layer, biofilm
flocs, and sessile ciliates) was determined by flow cytometry, confocal laser scanning microscopy and automated image analysis. Particles were
almost completely removed from wastewater by typical processes of
particle retention: up to 58% of particles attached to clay marbles, up to 15% were associated with suspended flocs, and up to
10% were ingested by sessile ciliates. Ingestion of particles by
ciliates was exceptionally high immediately after wastewater addition
(1,200 particles grazer
1 h1) and continued
until approximately 14% of the water had been cleared by ciliate
filter feeding. Most probably, ciliate bioturbation increases particle sorption to the basic biofilm. Backwashing of the
reactor detached pieces of biofilm and thus released approximately 50%
of the particles into rinsing water. Clay marbles in the upper part of
the reactor were more efficiently abraded than in the lower part. No
indications for selective attachment of the applied hydrophobic and
hydrophilic beads were found. As a consequence of interception
patterns, organisms at elevated biofilm structures are probably major
profiteers of wastewater particles; among them, ciliates may be of
major importance because of their highly active digestive food vacuoles.
| |
INTRODUCTION |
Microbial activity in
biofilms and physical processes such as adsorption are responsible for
wastewater clearance in biofilters. These filters comprise mature
biofilms with highly dynamic microbial populations, for which
particulate material constitutes an important carbon resource
(22, 28). Consequently, the interception of particles by
the biofilm is essential for the performance of bioreactors (44). Since particles tend to accumulate hazardous
pollutants at their surface, their removal from wastewater is all the
more important (26). Processes of particle attachment and
detachment are therefore of major interest in biofilm research
(29, 35, 37, 43).
Recently, sequencing batch technology has been applied to biofilters
(47). Wastewater is periodically loaded in a fixed bed
reactor and circulated in a closed system with intermittent aeration.
In contrast, other reactor types are operated in a continuous mode, in
which wastewater permanently passes through the system. Particle
retention is a fundamental factor in these technologies, since it
determines biofilm function (35, 44). To achieve a better
assessment of reactor types, particle retention should be specifically
examined for each application.
Passive attachment of percolating particles in a porous matrix depends
on physical mechanisms such as van der Waals forces or hydrophobic
interactions (4, 32). Particle attachment to a mature
biofilm is further influenced by biofilm texture, heterogeneity,
surface charge, and the activity of organisms in various biofilm
compartments (13, 35, 43). In mature biofilms, compartments can be divided into a surface layer, comprising metazoa, ciliates, ciliate stalks, flocculent filaments, and adjacent bacteria, and a basic layer, which is embedded in a polysaccharide matrix with
channels and crevices (9, 10, 46). Each of these biofilm structures can be assumed to attract particles of different size, form,
or hydrophobicity, thus leading to a specific pattern of particles in
the biofilm.
For instance, sessile ciliates, which typically dominate the
surface of mature biofilms, can accumulate particles of a certain size
by grazing (11, 16). Moreover, they can spread particles from the bulk water to the biofilm through acceleration in grazing swirls, whereas their stalks might serve as a major attachment site
(3, 40, 45). Until now, protozoan activity within biofilms
has been investigated with the focus on particle detachment, i.e.,
bacterial grazing (14, 24, 31, 41). In mature biofilms, however, grazing activity of sessile ciliates can be assumed to enrich
particulate organic resources within the biofilm. Information on the
importance of this process is necessary, compared to passive attachment
of particles.
Microscopical tools for the quantification of particle sorption by
biofilms must provide rapid counts of a variety of particles. Flow
cytometry and confocal laser scanning microscopy (CLSM) allow extremely
fast enumeration of differently fluorescing particles if adequate
software is available to analyze large data sets and perform accurate
image analysis. In the present study, we have developed these
techniques to evaluate the attachment of two kinds of fluorescing beads
(hydrophilic and hydrophobic) to mature biofilms grown in a sequencing
batch biofilm reactor.
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MATERIALS AND METHODS |
Reactor operation.
A bench-scale submerged biofilter
(height = 63 cm; diameter = 19 cm) was routinely operated for
the treatment of municipal wastewater. The reactor was sequentially
charged with 17 liters of wastewater; an additional 5.5 liters remained
in tubes or was associated with the biofilm during exchange. Sintered
clay marbles with diameters of 4 to 8 mm were used as a support
material for the biofilm. They comprised a total surface area of 11.5 m2 and an interstitial volume of 17 liters. In
order to achieve elimination of organic pollution (dissolved and
suspended solids) as well as nitrogen and phosphorus removal, the
reactor was operated in the sequencing batch reactor mode (1,
47). For this purpose, a cycle consisting of different treatment
phases was regularly applied, namely, a filling phase of 15 min and an
unaerated recirculation phase of 3 h, followed by an aeration and
recirculation phase of up to 24 h. Recirculation was accomplished
by pumping 300 liters h1 from top to bottom of
the biofilter, resulting in an upwards flow velocity of 2.1 mm
s1. Aeration of 200 liters
h1 resulted in dissolved oxygen concentrations
of approximately 6 mg of O2
liter1. To avoid clogging, excess biomass was
hydraulically removed by backwashing once a week with water and
pressurized air. During backwashing 36 liters of water passed through
the reactor bed within 150 s.
Microbead characteristics.
Fluorescent beads (diameter = 1 µm) were added to 17 liters of membrane-filtered (0.45-µm pore
size) municipal wastewater, which was pumped to the reactor at the
beginning of a sequencing batch cycle. Two types of beads were evenly
added to a final particle concentration of 3.23 × 107 beads per ml: green fluorescing hydrophobic
beads (latex [maximum excitation and emission wavelengths, 458 and 540 nm, respectively]; catalog no. 17154; Polysciences, Eppelheim,
Germany) and red fluorescing hydrophilic beads (carboxylated
polystyrene [maximum excitation and emission wavelengths, 625 and 645 nm, respectively]; catalog no. F8816; Molecular Probes, Leiden, The
Netherlands). Different hydrophobicity was demonstrated by
shaking of an ultrasonically treated aqueous suspension of beads in
n-hexadecane or n-octane (modified after the
method of Rosenberg et al. [38]), which removed more
latex beads from the aqueous phase (69 or 97%, respectively) than
carboxylated polystyrene beads (47 or 76%, respectively).
Sampling.
Before sampling, water circulation was stopped and
the reactor was turned cautiously into a horizontal position. To avoid disturbance by movement of clay marbles, the bed was fixed by a holed
plate, fastened at the top. At each sampling time (0, 20, 45, 75, 210, 330, and 450 min and 24 h after reactor loading) two samples were
taken from lateral ports at the lower region (height = 16 cm) and
at the upper region (height = 52 cm) of the biofilter. Twenty clay
marbles and 9 ml of interstitial water per sample were collected using
tweezers or syringes, respectively. Each marble was softly panned in 40 ml of water so that loose biofilm flocs adjacent to the marble surface
were detached and quantitatively separated. Bulk water from the reactor
supernatant liquid was drawn from the reactor recirculation tube. Two
final samples were taken subsequent to reactor washing after 24.1 h. All samples were immediately fixed with buffered paraformaldehyde (4.4% [wt/vol] in [per liter] 7.60 g of NaCl, 1.25 g of
Na2HPO4, and 0.41 g of
NaH2PO4; pH 7.2), stored at
4°C, and analyzed within 6 weeks.
Microbead detection and quantification.
Bead densities were
determined from the bulk biofilm at clay marbles (carrier biofilm) and
its basic layer (basic biofilm), from sessile ciliates of the biofilm
surface layer, and from separated biofilm flocs. In addition, beads
were quantified in the biofilters' supernatant and pore water.
Flow cytometry was applied to determine the density of
fluorescing particles attached to 10 clay marbles, associated with the
flocs separated from these marbles, and in bulk water from the
biofilter supernatant and pore water. In all samples, particles were
detached and suspended by shaking (1 min) and strong sonication in a
Branson (Dietzenbach, Germany) Sonifier (model B12; resonator tip, 0.5 in.; 450 W, 40% power; amplitude, ~75 µm; time, 2 min), adequately diluted, and quantitatively introduced into a FACS Star Plus
cytometer (Becton Dickinson, Paramus, N.J.) for 3 to 10 min at a rate
of 500 to 1,200 particles per s. Green and red fluorescence excitation
was performed by two lasers (argon, 488 nm; helium-neon, 633 nm) in a
single focus. Using a long-path filter (<515 nm) combined with a
band-pass blocking filter centered around 633 nm (Kaiser Optical
Systems, Ann Arbor, Mich.), fluorescence of both bead types could be
determined without interference by the HeNe laser. Both bead types were
much brighter than the background and could be monitored on one
fluorescence channel. For a clear discrimination of red fluorescing
beads, about 30% of the emission light was directed by a mirror to a
second fluorescence channel equipped with a long-path filter (>645 nm;
Schott, Mainz, Germany). Data were analyzed by DAS 4.4 software
(2).
Particle density in the basic biofilm layer and in food vacuoles
of sessile ciliates was estimated by automated analysis of
CLSM images.
Image acquisition was performed with a Zeiss (Jena,
Germany) 510 confocal laser scanning microscope. An argon laser
with a 488-nm
wavelength activated the fluorescence of green beads
(band-pass
fluorescence filter, 505 to 550 nm), light from a helium-neon
laser
with 543 nm excited the red beads (long-path fluorescence
filter, 560 nm). To take an image, a clay marble was placed in
a
water-filled plastic dish with a bottom coverslip (Matek, Ashland,
Mass.) and observed around its point of support with an inversion
microscope (Fig.
1a). Images from the
marble surface were recorded
at various microscopical resolutions to
quantify beads in the
basic biofilm, the number of sessile ciliate
grazers, and their
ingested beads. Three image analysis programs were
therefore created
with Zeiss KS400 software.
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FIG. 1.
(a) Clay marble, partially masked by biofilm flocs, with
investigated biofilm zones schematized. The red dot marks the area of
support at a glass slide during microscopical observation with an
inverse CLSM; dotted lines depict the line of sight. Yellow letters
correspond to exemplary images below: (b) ciliate grazers at a defined
marble area (inner circle) discernible by their fluorescing food
vacuoles (two-dimensional image); (c) basic biofilm (single layer of a
three-dimensional CLSM image with beads fluorescing in the green and
red channel); (d) ciliate grazers (white) with densely packed beads in
food vacuoles (yellow circles) (single layer of a three-dimensional
CLSM image). Printed images were scaled down and modified by a photo
editing program (Adobe Photoshop) to illustrate the quality of the
originals.
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Fluorescent beads in the basic biofilm were imaged with a 20×
objective featuring a numerical aperture (NA) of 0.5 (Fig.
1c).
From
three to six marbles per sample, a biofilm area of 0.46 by
0.46 mm
2 was documented, which was embedded in a
viscous matrix and free
from flocculent structures or ciliates. Biofilm
depth was displayed
by 25 to 75 images stacked at an optical distance
of 2 µm. The
three-dimensional, smallest units of an image (voxels)
had a horizontal
density of 512 by 512, so that each voxel had a volume
of 1.62
µm
3. The brightness of beads surpassed
background fluorescence of
the biofilm by far. The volumes of red and
green fluorescing beads
were determined by automated image analysis
using a constant threshold.
Summarized bead volumes from each CLSM
image were averaged for
every sample, resulting in a general mean
coefficient of variation
(CV) of 0.57. For evaluation of bead numbers,
the volume values
were converted by division through the screen volume
of a single
bead, i.e., the volume of a bead measured by CLSM. The
screen
volumes (11.0 µm
3 for green beads and
12.5 µm
3 for red beads;
n > 100) had been determined
separately.
The number of grazing ciliates was discernible by their
fluorescing food vacuoles and estimated on two-dimensional surface
images of six to seven clay marbles per sample (Fig.
1b). The
objective
lens was 1.25× (NA, 0.035) for the sampling times 15
to 210 min due to
a documented area of about 30 mm
2. No single
beads were discernible at this resolution, but food
vacuoles from
neighboring grazers were sometimes merged to one
object, which led to
an underestimation of grazer numbers. A circular
area of the marble
surface was defined using automated image analysis,
and fluorescing
objects with diameters of more than 10 µm were
counted as ciliate
grazers. The threshold was automatically adapted
to regional variations
of the background by a dynamic filter,
which improved the segmentation
of grazers (KS400, manual for
imaging system release 3.0; Carl Zeiss
Vision, Munich, Germany]).
Direct microscopic controls at
higher resolutions verified that
more than 90% of the counted objects
were sessile ciliates. However,
high bead densities covered the biofilm
5 h after bead exposure
and caused a disturbing background
fluorescence. Therefore, grazers
were identified with a greater
objective (4×; NA, 0.1) in subsequent
samples, taking into account a
10-fold-smaller imaged area. The
overall CV per sample was 0.46 for the
smaller and 0.73 for the
greater
objective.
Particle volumes of red and green fluorescing beads in ciliate
food vacuoles were determined from three-dimensional images
taken with
a 63× objective (NA, 1.2, in water). We recorded the
particle
content of 8 to 28 grazers per sample, from at least
three marbles. The
horizontal image size was 0.084 mm
2, with a
z stack of 30 to 100 images at an optical distance
of
0.5 µm. Thus, the voxel volume was 0.042 µm
3. The volumes of red and green, mostly
agglomerated beads in each
ciliate were measured by automated image
analysis and averaged
for each sample, with a mean CV of 0.71 per
sample.
To discriminate between loosely distributed beads (optically
singular) and densely packed beads (i.e., optically clustered)
in a
ciliate, the following basic steps were performed by automated
image
analysis (Fig.
1d). To define every single ciliate, fluorescing
objects
were strongly diluted until the beads within each ciliate
were merged
together. Contours of the resulting volume arbitrarily
defined the
ciliates' bodies. Closely arranged ciliates, which
occasionally had
been merged too, were manually divided. From
the original image,
objects were radially smoothed and eliminated
if <100 voxels, i.e.,
singular beads, were excluded (KS400 manual).
The residual clustered
beads were enclosed and defined as food
vacuole content. Image
superimposition of ciliate and vacuole
contours revealed beads inside
the food vacuoles of each ciliate.
Since these beads were packed in
clusters, their volumes were
converted to numbers by subtraction of
pore space (40% at closely
cubic package according to Busch et al.
[
6]) and subsequent
division through the geometric
volume of a single bead (0.51 µm
3). The volumes
of single beads outside of the vacuoles were converted
by division
through the screen volume of a single bead. This volume
(3.4 µm
3 for green beads and 4.1 µm
3 for red beads) was derived from separate
measurements of single
beads (
n >
90).
Calculation of ciliate clearance.
The clearance of
wastewater by ciliate feeding was estimated from water volumes
represented by ingested beads. Assuming equal concentration
(c) of beads in bulk water, one ingested bead was equivalent
to a certain volume (Vb = 1/c)
of cleared water that increased with diminishing bead concentration.
For each sampling interval (i = 1, 2, ...8),
the cleared water volume was calculated from the water volume
represented by one bead in the middle of the interval
[(Vbi = 2/(ci
1 + ci)],
multiplied by the newly ingested beads per grazer at the end of the
interval (
Gi), and the extrapolated
number of grazers in the reactor (Ni). The
total volume (Vcl) of cleared water
can therefore be determined as follows:
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RESULTS |
Attachment to biofilm compartments.
After
wastewater loading, the particle concentration in wastewater
declined exponentially within 7.5 h, almost to 1% of the initial
concentration (Fig. 2). This was apparent
from an increasing transparency of the supernatant. Particle density in
the biofilters' pore water was always slightly higher, preferably in
the lower part of the reactor (Table 1).
The overall amount of floc-associated particles was already high after
20 min (7% of total), with a clear majority in the lower part of the
reactor. It doubled within the next 7 h but declined to 2% of
loaded particles in the following 17 h. The particle concentration
at the carrier biofilm (including ciliates, ciliate stalks, flocculent
structures, and basic biofilm) increased rapidly to a level of ca.
37,000 particles mm2 after 5.5 h. This
density was generally greater in the upper segment of the reactor, with
the maximum difference (46%) to the lower segment reached after
24 h. Particle density at the basic biofilm layer paralleled that
of the carrier biofilm at a level of 30 to 50% and remained relatively
stable after 45 min at up to 10,700 particles
mm2. More than 90% of loaded particles could
be detected in the first hour of the experiment, only 40% could be
detected after 24 h, but 74% could again be detected after
reactor backwashing.
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FIG. 2.
Percentage of particles (diameter = 1 µm)
recovered from several compartments of a sequencing batch biofilm
reactor after addition of particle-containing wastewater. The reactor
was back washed 24 h after wastewater charge. Symbols:  , bulk
wastewater;  , total biofilm including flocs;  , biofilm on carrier
material;  , basic biofilm layer; ×, sessile ciliate grazers.
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TABLE 1.
Mean particle concentrations and percentages of
hydrophobic and hydrophilic particles in wastewater and biofilm
compartments of a sequencing batch biofilm reactor
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Reactor backwashing.
Backwashing detached pieces of biofilm,
so that particle densities at the marble biofilm and its basic layer
substantially declined. Consequently, 45% of loaded particles were
released into bulk water, mainly into the outflowing
rinsing fraction. The majority of attached particles switched
from the upper to the lower segment of the reactor. In parallel,
floc-associated particles increased about fourfold in number and
accumulated almost exclusively in the lower region.
Ciliate grazing.
The sessile ciliate community in the
bioreactor was dominated by the peritrich ciliate genus
Epistylis, which made up to more than 99% of the sessile
community and about 87% of the total ciliate community (J. Fried,
personal communication). These ciliates reacted to wastewater addition
with immediate grazing activity, exerting maximum grazing rates within
the first 20 min (ca. 1,200 particles h1; Fig.
3a2 and a3). The maximum number of
ingested particles per grazer (ca. 1,000 particles) was reached after
3.5 h, concurring with the maximum number of grazers (5.1 grazers
mm2; Table 1). Their individual clearance rate
for this interval was 20 nl ciliate1
h1, corresponding with filtration of
12% of wastewater by the ciliate community. Altogether, about 14% of
wastewater was filtrated through ciliate feeding. After 3.5 h, the
number of grazers as well as the ingested particles decreased rapidly.
Vacuoles with conglomerated particles started to dilate, so that higher
amounts of loosely distributed particles in ciliate cells were measured
(Fig. 3b2 and b3). Until 24 h, ciliates still populated the
surface biofilm but contained no fluorescing particles (Fig. 3c2).
Instead, ciliate stalks and other large flocculent structures of the
biofilm were densely covered by particles (Fig. 3c1). Neither for
ciliates nor for the basic biofilm did we find indications for sites or periods of selective attachment of the applied hydrophilic and hydrophobic particles.
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FIG. 3.
Hydrophilic (red) and hydrophobic (green) fluorescing
particles in sessile ciliates of a mature biofilm from a sequenced
batch biofilm reactor that was charged with particle-containing
wastewater at 15 min (a), 7.5 h (b) and 24 h (c) after
wastewater addition. Columns show increasing grazer densities (a1 and
b1) and particle accumulation at elevated biofilm structures (c1);
sessile ciliate grazer colonies with compact (a2), dilated (b2), and
empty (c2) vacuoles; horizontal and lateral projections of
three-dimensional CLSM images of ciliates, and immediately after prey
acquisition (a3), during vacuole processing (b3), and during particle
egestion (c3). Bars in columns 1 to 3 represent 1 mm, 25 µm, and 10 µm, respectively. Printed images were scaled down and modified by a
photo editing program to illustrate the quality of the originals.
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DISCUSSION |
Particle retention in bioreactors.
It was found that particle
attachment to a mature biofilm is triggered by three different
processes: passive interception by biofilm flocs, sorption of
particles to the basic biofilm, and grazing activity of sessile
ciliates at the surface biofilm. All processes contribute considerably
to rapid removal of particles from wastewater in a biofilter operated
in the sequencing batch mode.
At the end of the treatment cycle almost total clearance of
1-µm-diameter particles was accomplished. Okabe et al.
(
35)
exposed particles of the same diameter in a rotating
disk reactor
and found that one-third of particles remained in bulk
water.
Studies on a rotating annular reactor or an airlift suspension
reactor also showed lower adsorption efficiencies of biofilms
in the
diurnal time frame (
13,
43,
44). Reactors in these
studies
worked in a continuous mode, which entails lower and more
constant particle concentrations than the batch mode. Biofilms
in
batch reactors seem to have a higher sorption capacity and
thus
might have a higher ability to cope with shock loading conditions.
The
high clearance efficiency of particles in the sequencing batch
reactor
can be explained by lower porosity of the filter bed and
frequent
recirculation of wastewater, which contribute to a high
contact
frequency of carriers and particles (
23,
32).
Adsorption to biofilm compartments.
In the biofilter, the
adsorption of particles was different for each biofilm compartment.
Immediately after addition of wastewater to the reactor, biofilm flocs
intercepted substantial amounts of particles, since they had primary
and ambient contact with the fluid-transported particles. The loosely
attached flocs intercepted particles preferentially in the lower part
of the reactor, whereas in the upper part, particles adsorbed more
efficiently to clay marbles. Presumably, flocs masked the biofilm
carriers in the lower segment (Fig. 1a), so that the underlying carrier
biofilm had less contact with particles. Floc-associated particles
declined after several hours, as flocs seemed to assemble more closely with the carrier biofilm and integrate into the surface layer.
Firmly attached flocculent structures of the carrier biofilm, mostly
formed by sessile ciliate colonies, protruded beyond
the basic biofilm
up to a millimeter and obviously intercepted
great amounts of particles
through oscillation in the streaming
liquid (
42). They
account for the difference between particle
densities in the carrier
biofilm and its basic layer compartment.
However, the observed pattern
of reduced particle attachment to
indentations might be modified at
higher flow velocities, which
can lead to more intense percolation of
biofilm crevices (
12).
Neither biofilm compartment showed
selective attachment of the
applied hydrophobic and hydrophilic beads.
Nevertheless, selective
sorption of more different particles could
indicate specific features
of biofilm surfaces (
5,
48).
Reactor backwashing.
After 1 day of operation, flocs and
particles were transported to the upper region of the biofilter, where
particle densities increased. In this reactor segment, backwashing led
to most efficient abrasion of the biofilm, because of more frequent
collisions of clay marbles. Numerous flocs with sorbed particles were
detached and accumulated in the lower reactor segment. The different
efficiency of backwashing might lead to an unfavorable, inhomogeneous
biomass distribution. A complete fluidization of the filter bed during washing should therefore be ensured (33).
Until backwashing, the total amount of detected particles continuously
decreased. Nondetected particles might have been trapped
in unsampled
pockets of the reactor system (e.g., by wall effects).
Alternatively,
the detection of fluorescent beads in the biofilm
could have been
ineffective for bead clusters, which increased
with proceeding particle
adsorption.
CLSM and image analysis.
Automated analysis of CLSM images
provides a detailed and fast spatial exploration of biofilms (27,
30). However, clustered particles, as they typically occur in
mature biofilms, have to be enumerated by estimate conversions of
object volumes. This would demand a precise optical screening of single
particles. We found that the volume of a single bead determined by CLSM
was several times greater than its geometric volume, which can be explained by the limited vertical resolution in the CLSM technique and
the extreme brightness of beads (20). To avoid substantial underestimation, we therefore preferred a geometric conversion of bead
clusters in food vacuoles. Another limitation was a shading effect
within densely filled food vacuoles. Beads from the upper part of
vacuoles blocked light from underlying beads, so that the vacuoles were
discerned as hemispheres. Despite these restrictions, automated CLSM
image analysis allowed more-precise and unbiased quantification of
selective ingestion and vacuole dilation. Alternative direct
microscopic quantification of ingested beads would involve more-laborious and inaccurate estimates of grazer density, vacuole numbers, and vacuole diameters (14).
Grazing rates and particle processing by sessile ciliates.
Grazing rates and particle release by epilithic sessile ciliates were
quantitatively investigated for the first time in this study. Sessile
grazers intercepted the newly available batch of particulate resources
with very high ingestion rates (1,200 particles ciliate1 h1 within 20 min), which are in the upper range compared to other ciliate feeding
types (7, 15, 21, 25, 36). Carrias et al. (8)
report even higher grazing rates for epibiotic peritriches on a pelagic
diatom, but they employed very small particles (0.5 µm), which
occupy less space in food vacuoles. The present data are the first in
situ grazing rates of sessile ciliates in a wastewater treatment
system, a system where they generally dominate the surface layer of
mature biofilms (11, 19, 36).
Grazing behavior was obviously pulsed by wastewater addition to
the bioreactor. The maximum rate of ingestion occurred
immediately
after particle addition, when suspended particles
were most available.
Because of their high initial grazing rates, we
assume that many
ciliates were satiated after 3 to 4 h and then
started vacuole
processing, which would fit with cyclic regeneration of
prey reported
for other ciliate species (
34,
39).
Possibly, vacuole processing
was powered by recurring oxygen
availability. Still unsatiated
grazers probably tried to compensate for
the decreasing availability
of suspended particles with higher
clearance rates, until particle
concentration fell below the economical
limit. In this situation,
the energetic costs for clearance activity
exceeded the potential
nutritive gain of ingested particles. The period
before ingestion
peak (at 3.5 h) was therefore the most intense in
wastewater filtering
by grazers, especially since they had also
reached their maximum
number. The subsequent start of vacuole
processing can thus be
explained by satiation or by low availability of
particles. The
cyclic regeneration of ingested material typical for
ciliates
was finally synchronized by the sequencing batch
operation.
Importance of ciliate grazing activity.
Compared to other
biofilm compartments, the interception of particles by ciliates was
relatively low. Particle ingestion by sessile ciliates might reach a
magnitude of 10% of the loaded batch. Considering particle
degradation, ciliates may have a major impact because of their
extremely active digestive food vacuoles (17, 18, 34).
Ingested wastewater particles are probably most efficiently degraded,
and released digestion products will trigger microbial activity in the
bioreactor (36).
About 14% of wastewater was cleared through ciliate filter feeding,
and even more was moved along the ciliates' bodies. Water
swirls that
are generated by grazing activity can reach an extension
of 400 µm
(
3,
16,
40). This substantial bioturbation probably
affects the liquid boundary layer at the biofilm surface and weakens
its barrier effect for dissolved nutrients (
49).
Conclusions.
Small particles can accumulate very rapidly in a
mature biofilm due to protozoan grazing activity, passive interception
by flocculent structures, and sorption to the basic biofilm. In batch biofilm reactors, the retention of particles by these processes is
highly effective. Thus, if wastewater particles are nutritious, they
are obviously an essential energy resource for the biofilm community. A
major profit can then be assumed for sessile ciliates, bacteria at
ciliate stalks, and bacteria at other elevated biofilm structures. Future experiments and models should address the
versatile function of ciliates in mature biofilms and the nutritional
value of wastewater particles in bioreactors.
| |
ACKNOWLEDGMENTS |
We are grateful to Johannes Fried for determination of ciliates
and to Eberhard Morgenroth and Stefan Wuertz for helpful comments on
the manuscript.
This work was supported by the Deutsche Forschungsgemeinschaft through
its Research Center (SFB411) on Fundamental Studies of Aerobic
Biological Wastewater Treatment, Munich, Germany.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Flow Cytometry
Group, National Research Center for Environment and Health,
Ingolstädter Landstr. 1, D-85764 Neuherberg, Germany. Phone:
49-89-3187-3426. Fax: 49-89-3187-3349. E-mail:
eisenmann{at}gsf.de.
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Applied and Environmental Microbiology, September 2001, p. 4286-4292, Vol. 67, No. 9
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.9.4286-4292.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
