Vol.

57: 161–173, 2009 AQUATIC MICROBIAL ECOLOGY Printed November 2009

doi: 10.3354/ame01341 Aquat Microb Ecol Published online October 6, 2009

OPEN
ACCESS

Effects of suspended matter quality and virus

abundance on microbial parameters: experimental
evidence from a large European river
Lisa Kernegger, Irene Zweimüller, Peter Peduzzi*
Department of Freshwater Ecology, Faculty of Life Sciences, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria

ABSTRACT: In riverine water, both suspended particulate material and viruses are prominent eco-
logical factors. The existence of various particle types and differences in viral abundance impose
variability in microenvironments. Particulates and their microbial surrounding may interact in sev-
eral ways, this interaction being strongly dependent on particle quality and the abundance of organ-
isms involved. In laboratory experiments, we used different suspended matter types (fresh and aged
mineral sediment and leaf litter, river snow) that typically occur in riverine environments as model
particles. We investigated the effects of particle quality and different ambient viral abundances (× 1,
× 2 enrichments, and inactivated viruses) on several microbial parameters (changes in bacterial and
viral abundances, bacterial production, specific bacterial production) of both the free-living and par-
ticle-attached fractions using water from a floodplain system of the Danube River (Austria). Both ses-
ton quality and variable viral abundances in the bulk water influenced some microbial parameters.
The average abundance of bacteria and viruses was significantly higher on organic than on inorganic
particles and on aged particles (for both sediment and leaf litter). Changes in bacterial abundance
during the course of the experiments were also influenced by particle quality, with, for example, aged
sediment favoring increasing abundances. Virus:bacterium ratios (VBR) were significantly higher on
organic than on inorganic particles, but significantly lower on suspended particles than in the plank-
tonic fraction. Typically, bacterial secondary production (overall and cell-specific) was higher on par-
ticles than in bulk water. Bacterial productivity in the ambient water was negatively affected by the
abundance of planktonic viruses but positively affected by that of attached viruses. These findings
from experimental systems may foster in situ studies of particle-rich environments.

KEY WORDS: Virus · Particles · Suspended matter · River · River snow

Resale or republication not permitted without written consent of the publisher

INTRODUCTION larger to finer size through physical and biological

maceration, although some processes like formation of
The presence of suspended particulate matter and its fecal pellets or aggregation may also increase particle
variable quality in various aquatic environments can size (Johnson et al. 1986, Ward et al. 1990). The non-
considerably affect the microbial community (Simon et living matrix of particles and aggregates can harbor a
al. 2002, Peduzzi & Luef 2008). Due to the multiple rich microbial community. Aggregates tend to be
sources of particles in inland waters, their environmen- amorphous, porous structures exhibiting high nutrient
tal significance may vary strongly between and even concentrations, a diverse microbial assemblage and
within particular systems (compare e.g. Hopkinson et polymer glycoconjugate distribution, as well as distinc-
al. 1998). In riverine systems, auto- and allochthonous tive physical and chemical gradients (Neu 2000, Simon
sources yield a composite of inorganic and organic sus- et al. 2002, Luef et al. 2009). When they are large
pended materials (Hein et al. 2003). Such particles may enough to be seen macroscopically in riverine environ-
be subjected to substantial alteration in size and chem- ments, the term ‘river snow’ is used for such aggre-
ical composition. They are usually converted from gates in analogy to marine snow (Neu 2000). It can be

*Corresponding author. Email: [email protected] © Inter-Research 2009 · www.int-res.com

162 Aquat Microb Ecol 57: 161–173, 2009

assumed that particle–microbe interactions strongly MATERIALS AND METHODS

depend on particle quality, and the type and abun-
dance of organisms involved. Sampling and incubation. All water samples for the
Viruses are abundant in all natural aquatic environ- experiments were taken from a floodplain section of the
ments, with abundances varying from <104 ml–1 to Danube River, downstream of Vienna at Regelsbrunn, in
>108 ml–1 (Wommack & Colwell 2000). Moreover, the Danube National Park at river kilometer 1895. In
their abundance is generally one order of magnitude Austria, the Danube River is a 9th order river, and the
higher than that of bacterioplankton (Weinbauer reach downstream of Vienna represents one of the last
2004). They can therefore significantly affect bacte- remnants of semi-natural river floodplain systems in cen-
rio- and phytoplankton (Proctor & Fuhrman 1990, tral Europe, although it is strongly impacted by regula-
Suttle et al. 1990). Viruses and suspended particulate tion. This running water system carries various particle
material may interact in several ways that can be loads depending on hydrological and biological condi-
ecologically relevant in aquatic environments. In a tions. The area has been the site of intense research ac-
previous study using thin sectioning and transmission tivities in recent years (e.g. Hein et al. 1999, Besemer et
electron microscopy (TEM), we found up to 9 × 1010 al. 2005, Luef et al. 2007, Peduzzi & Luef 2008, Peduzzi et
virus particles cm– 3 in marine snow of the northern al. 2008). Sampling of water for the experiments was
Adriatic Sea; these viruses were able to influence the conducted during a period of moderate water level fluc-
process of algal floc formation (Peduzzi &Weinbauer tuations. Experiments were carried out in the laboratory
1993a). Although suspended matter is often a promi- using rotating tanks (10 l, translucent acrylic glass,
nent factor in inland waters, only limited data on the washed with 10% HCl, tap water and finally rinsed with
abundance of viruses associated with suspended par- Milli-Q water) on a rolling table at 2 rpm.
ticulate material in freshwater exist (Luef et al. 2007, Processing of bacterial and viral concentrates. For
Peduzzi & Luef 2008, Peduzzi & Luef 2009). Riverine each experiment, water was taken from the sampling
systems display highly variable particle loads that are site at Regelsbrunn and treated as follows (Fig. 1): the
mainly triggered by hydrology. In the Danube River water was prefiltered through a 3 µm mesh, then
where suspended matter is often a prominent factor, through a GF/C (Gelman Sciences fiber filter type A/C
a variable and occasionally high proportion of viruses glass, nominal pore size 1.2 µm, 142 mm diameter) and
(between 0.43 and 35.06%) was found to be associ- finally through a 0.22 µm GV Millipore membrane filter
ated with particulate material (Luef et al. 2007). A (142 mm diameter). A bacterial concentrate was pro-
recent study reports up to 5.4 × 109 viruses cm– 3 duced from 40 l of 1.2 µm prefiltered water, which was
lectin-stained suspended material in the Danube as again filtered onto a 0.22 µm GV Millipore membrane fil-
analyzed by confocal laser scanning microscopy (Luef ter (47 mm diameter). Bacteria were kept in suspension
et al. 2009). However, interactions between sus- by gentle stirring with a glass rod, taking care that a
pended matter and natural viral assemblages are residual volume remained in the filter funnel. After de-
poorly documented. canting, the used filters were rinsed in the bacterial con-
We performed laboratory experiments to (1) test the centrate to recover most of the cells. This yielded a bac-
effect of particle quality on the dynamics of prokary- terial concentrate of ~3.4 × 107 cells ml–1. To produce a
otic and viral abundance, both on the particles and in viral concentrate, ultrafiltration (Millipore Pro Flux M12)
the surrounding water, and (2) assess how different was used. A 100 l volume of 0.22 µm prefiltered water
ambient viral abundances are involved in this interac- was ultrafiltered as described by Suttle et al. (1991). The
tion. The experiments were conducted with water from 2 obtained fractions are the retentate (viral concentrate)
a floodplain of the Danube River (Austria), using 3 dif- and the permeate (virus-free water; Weinbauer 2004). A
ferent suspended matter types (mineral sediment, leaf 30 kDa cut-off cartridge efficiently concentrated the
litter and river snow) that typically occur in this envi- virus size fraction (Suttle et al. 1991). The resulting viral
ronment as model particles. We also differentiated the concentrate contained ~1.8 × 109 viral particles ml–1.
particulate material as ‘organic or inorganic’ and ‘fresh Processing of particles. Three different particle
or aged’. We hypothesized that (1) growth conditions types (inorganic sediment, leaf litter and river snow)
for prokaryotes are generally better on particulate were used. Sediment and leaf litter particles were used
material, (2) organic material should be more attractive in 2 ways: as ‘fresh’ particles to represent freshly intro-
to microorganisms (bacteria and viruses), and (3) aged duced material into the aquatic environment (e.g. dur-
particles are more densely populated by microorgan- ing spates; in this case, particles were free of microor-
isms than freshly produced material. We further ganisms at the beginning of the experiment); and as
hypothesized that (4) differences in the abundance of ‘aged’ particles that were covered with an already
free-living and attached viruses affect the productivity formed aquatic biofilm. River snow was used as it was
of prokaryotic plankton. (see below).
 Kernegger et al.: Particles and viruses in river water 163

1 mm mesh. For the respective experiment, 220 mg of

these dried leaf particles (<1 mm particle size) was
added into each rolling tank. To produce leaf litter with
biofilm, dried leaf particles were placed in an aquar-
ium and processed similarly as the aged mineral sedi-
ment.
River snow: Unprocessed sample water was added
into rolling tanks. The tanks were kept rotating under
artificial light (daylight spectrum, 300 µmol m–2 s–1) at
2 rpm for 4 d to form suspended river snow particles as
described by Shanks & Edmondson (1989). For the
respective set of experimental incubations, the river
snow particles were carefully transferred into clean
rolling tanks containing experimental water (see next
section).
Experimental setup in rolling tanks. For each exper-
imental setup with different particulate material, 3 sets
of rolling tanks were prepared and incubated for 60 h
(Fig. 1). The 3 sets of rolling tanks had the same initial
bacterial abundance and equal density of the respec-
tive particle type but different viral concentrations.
Although all analyses were carried out in duplicate or
triplicate, it was not possible to replicate batch cultures
for each treatment due to the complexity of the experi-
mental protocol (cf. Auguet et al. 2009). For each set of
rolling tanks, microorganism-free water (30 kDa per-
Fig. 1. Preparation (filtration and concentration) of samples meate) was placed in a large jar. Approximately 1.5 l of
for culture experiments in rotating tanks. Different particulate bacterial concentrate as well as particles were added
materials were added (see ‘Materials and methods’ for
and gently stirred to obtain a homogeneous suspension
details); treatments were enriched with 2 different virus
abundances and with inactivated viruses of bacteria and particles. This suspension was split and
added into each tank of the 3 experimental treatments.
The viral concentrate was added separately; the 3 dif-
ferent treatments received different viral concentra-
Sediment: Sediment was taken from the sampling tions (see below). Aged particles were transferred from
site. It was cleaned of organic matter in a muffle the aquarium to the rolling tanks as follows: water from
furnace at 480°C for 6 h. For the experiment that was the aquarium was poured through a 3 µm mesh to col-
initially without particle-associated microorganisms lect the particles, which were then resuspended in
(fresh sediment treatment), 25 g of this mineral sedi- microorganism-free water. To ensure that each tank
ment was transferred into each rolling tank. received approximately the same amount of particles,
To produce aged sediment, the cleaned sediment this particle slurry was poured into graduated flasks,
was placed into an aquarium with 30 l of sample water stirred to keep particles suspended and equal aliquots
that had been GF/C (Gelman Sciences fiber filter type were then distributed into the rolling tanks. Particles
A/C glass, 1.2 µm) filtered. The aquarium was previ- without initial colonization were weighed and equal
ously rinsed with 10% HCl, tap water, Milli-Q water amounts of dried particles were transferred directly
and filtered sample water (in that order). Air was into each tank.
pumped into the aquarium to keep sediment in sus- The preparation of the river snow treatment differed
pension. After 6 d (a period reflecting typical retention from that of the other 4 particle types. River snow par-
times of water at the source location of the Danube ticles were carefully pipetted from the tank where they
River floodplain; Hein et al. 1999), this aged sediment had been produced into a large Petri dish. Almost all
was transferred into the experimental rolling tanks water in the Petri dish was carefully removed while
(see below). ensuring that particles did not dry up, before transfer-
Leaf litter: Leaf litter was collected from the shore at ring river snow particles into the tanks in approxi-
the sampling site. It was rinsed twice with tap water mately equal amounts.
and once with deionized water. After drying at 60°C Virus-free treatment: To investigate whether viruses
for 48 h, leaves were crushed and sieved through a have any impact at all, a ‘virus-free’ (i.e. with inacti-
164 Aquat Microb Ecol 57: 161–173, 2009

vated viruses) incubation was also conducted with total number of bacteria and viruses. To estimate a
each particle type as a control. Microwave treatment potential bias in virus counting due to extracellular
was applied to inactivate heat-labile viruses (Proctor & DNA interference, the effect of DNase treatment on
Fuhrman 1992). A 200 ml volume of the viral concen- samples was tested (Danovaro et al. 2001). The number
trate was treated at the highest power output of a of viruses obtained from DNase-treated samples did
microwave oven (500 W) for 3 min at 30 s intervals, not differ significantly from those obtained from
with the sample being iced down between microwave untreated samples (p > 0.60, Mann-Whitney U -test, n =
exposures to prevent boiling. This procedure has also 88). All counts were performed as soon as possible
been used in other studies (e.g. Weinbauer & Suttle (within 10 h) to minimize potential loss of viruses dur-
1996, Auguet et al. 2009) and was found to successfully ing storage.
inactivate viruses (Auguet et al. 2009). In the literature, attached microorganism abundance
Treatments with different viral concentrations: To is often given in cells ml–1. Since we used various par-
cover the range of viral abundances typically occurring ticle types in different amounts, we decided to express
at the sampling location, 2 different viral abundances microorganism abundance in (g dry wt)–1 of suspended
(‘low virus’ and ‘virus-rich’) were used in these experi- matter. Water samples from the tanks were filtered
ments. A 100 ml volume of the untreated viral concen- through precombusted and preweighed (W0) glass
trate was added by gentle stirring (Fig. 1). The initial microfiber filters (APFF Millipore GF/F, 47 mm diame-
viral concentration obtained in this low viral concen- ter). The filters were dried at 60°C for 24 h and
tration treatment was half the viral concentration of the weighed again (W1). The dry weight of the particulate
‘virus-rich’ treatment. The latter was obtained by matter (DW) was determined as W1 – W0.
transferring 200 ml of the viral concentrate into the Bacterial secondary production (BSP) and specific
tank. BSP. BSP was measured using the thymidine ([methyl-
3
Bacterial and viral abundance measurements. The H] TdR) incubation method and calculated using the
technique for enumerating microorganisms that was conversion factor described by Berger et al. (1995).
applied in the present study most likely also stains Measurements were performed in triplicate, and
archaea. However, since the frequently used term duplicate formaldehyde-killed samples served as con-
‘prokaryotes’ does not refer to a monophyletic group trols. For extraction, the samples were first transferred
(Pace 2006), we decided to retain the term bacteria onto 3.0 µm membrane filters (Millipore SSWP, 47 mm
(although some archaea may have also been counted) diameter) and filtered into acid-washed and rinsed
throughout the text. glass tubes; this retains the particulate material and
Samples were taken every 12 h (T0 to T5), fixed with the associated microorganisms on the filter. These fil-
formaldehyde (2% final concentration) and processed ters were washed with ice-cold trichloroacetic acid
immediately for enumeration. From these samples, (TCA) and transferred into scintillation vials for further
10 ml were filtered onto 3 µm Isopore membrane filters processing. The collected filtrate was then filtered
(TSTP, Millipore) to retain the particle-associated again onto 0.45 µm membrane filters (Millipore HAWP,
microbial fraction. Filters with particles were fixed (2% 47 mm diameter), and the filters were extracted as
formaldehyde final concentration) in 4.5 ml of 0.02 µm explained above for 3.0 µm filters. This method yields
filtered water and processed immediately. To enumer- direct estimates of BSP of both bacterial fractions — the
ate bacteria and viruses on particles, 500 µl of sodium particle-associated (> 3 µm) and the free-living (0.45 to
tetrapyrophosphate (5 mM final concentration) was 3 µm) fraction (Peduzzi & Schiemer 2008). The specific
added to the samples. During incubation for 1 h, the (cell–1) BSP (sBSP) was calculated by dividing bacterial
samples were gently shaken at 20°C. After treatment production with the corresponding bacterial abun-
with sodium tetrapyrophosphate, the samples were dance.
sonicated 3 × for 1 min, with pulsing at 40 W using a B. Statistical analyses. For all statistical analyses, we
Braun Diessel Biotech Sonifier (Labsonic U) adopted used SPSS 12.0 for Windows. Initial viral abundances
from Danovaro et al. (2001). To stain and count fixed were characterized by the initial abundances (T0) of
samples, 1 to 3 ml were filtered onto Whatman free-living and attached viruses (log10 transformed
AnoDisc filters (0.02 µm, 25 mm diameter). Free-living abundance values+1).
(directly filtered from the original samples) and parti- The outcome of the experiments was characterized
cle-attached (after sonication) bacteria as well as by the changes in bacterial abundance and productiv-
viruses were stained with SYBR Gold and enumerated ity during the course of the experiment (abundance,
within 10 h after sampling (Chen et al. 2001). Microor- sBSP and BSP of both free-living and attached bacte-
ganisms were enumerated under an epifluorescence ria). These variables were calculated as follows: for
microscope (Nikon E 800) at 1250 × magnification. On bacterial parameters, endpoint values were calculated
each filter, 20 fields were counted to determine the (Tfinal = mean of values at T3 to T5, i.e. the saturation
 Kernegger et al.: Particles and viruses in river water 165

phase of the growth curve); the difference between the Mineral sediment
starting value at T0 and the endpoint value was
expressed as a proportion of the initial value for para- Bacterial abundance in the water column increased
meters associated with free-living bacteria (abundance in all experiments with fresh and aged suspended mat-
changes, changes in specific and total production; in ter, except in the virus-rich treatment using aged sedi-
contrast to simple ratios, the resulting indices give ment (Fig. 2a). The abundance of bacteria attached to
increasing and decreasing values equal to weight); for mineral sediment also increased in all treatments
variables associated with attached bacteria, the pro- (Fig. 2b). As expected, viral abundance in the water in
portional change values described above were multi- the virus-free treatment remained below the detection
plied by 100 (by 1000 for sBSP) and the absolute value limit (Fig. 2c). Viral abundance in the water decreased
was log10 transformed (this multiplication transforms in the 2 treatments containing viruses and fresh sedi-
values to >1, thus avoiding disproportional effects due ment. It did not change significantly in the incubations
to negative log values); each log transformed value with aged sediment in the virus-rich treatment, but
was multiplied by +1 for increasing and –1 for decreas- increased in the low virus treatment. Viral abundance
ing change (Δ) values. on aged sediment particles increased in all treatments,
The different treatment of variables for attached whereas it remained more or less unchanged over the
bacteria was necessary because variables associated course of the experiment on fresh sediment particles
with attached bacteria changed over several orders of (Fig. 2d).
magnitude during the experiments (which was not the
case for free-living bacteria); then, resulting indices
had similar ranges as the other variables. Leaf litter
A principal component analysis (PCA) was per-
formed on variables describing the outcome of the Bacterial abundance in the water column increased
experiments. The number of factors was determined in all experiments with aged and fresh leaf litter as sus-
by the Kaiser-criterion. Factors were varimax-rotated pended matter, except for the virus-rich treatment
to facilitate the interpretation of the factor loadings. using aged material, where free-living bacteria in the
A 2-step regression analysis was carried out. In step water remained at a constant level (Fig. 3a). Bacterial
one, the effect of particle quality on initial viral abun- abundance on particles increased in all experiments
dances (after mixing the components in the experi- using aged and fresh leaf litter as particles (Fig. 3b).
mental tanks) and the outcome of the experiment Again, with our method, no viruses could be detected
derived from the PCA-axes described above was eval- in the water in the initially virus-free treatments
uated; this involved 5 regressions with dummy coded (Fig. 3c). Viral abundance in the water decreased in
particle qualities (4 variables for the particle qualities the treatments with fresh leaf litter at both viral abun-
‘aged sediment’, ‘fresh’ and ‘aged’ leaves, and river dances. In the treatments with aged leaf litter, free-
snow; 1 = present, 0 = absent). Fresh mineral sediment living viral abundance did not change under either
served as the reference. In a second step, the effect of abundance levels. Viral abundance on fresh and aged
particle quality on initial viral abundances and the out- leaf litter increased from the beginning to the end of
come of the experiment was removed by using the the experiments in all incubations, but fluctuated
residuals of the regressions described above. Three strongly in the final phase of the experiment (Fig. 3d).
regressions were calculated, with the residuals of
PCA1 to 3 as the dependent variable and the residuals
of viral (free-living and attached) abundances as the River snow
independent variable.
During the course of the experiments using river
snow as suspended matter, bacterial abundance in
RESULTS the surrounding water increased in the virus-free
treatment. In the virus-rich and low viral abundance
Effects of particle quality treatments, bacterial numbers in the ambient water
remained constant (Fig. 4a). Bacterial abundance on
All bacterial and viral abundances at T0 and Tfinal river snow increased in all treatments (Fig. 4b). No
from all experimental treatments are presented in viruses were detected in the water of the initially
Figs. 2 to 4. The changes in the measured parameters virus-free treatment; a slight increase or roughly
that were observed during the experiments with dif- constant abundance was observed in the other 2
ferent types of suspended matter will now be dis- virus treatments (Fig. 4c). Viral abundance on river
cussed. snow tended to increase but, as with leaf litter,
166 Aquat Microb Ecol 57: 161–173, 2009

Sediment Leaf litter

25 6
A A
Bacteriafree x 106 ml–1

Bacteriafree x 106 ml–1

T0 fresh sediment 5
20 Tfinal fresh sediment
T0 aged sediment
Tfinal aged sediment 4
15
3
10
2

5 1

0 0
5 8
B B
4

Bacteriaattached x 1011
Bacteriaattached x 1010

3 6

(g dry wt)–1
2
(g dry wt)–1

1 4
0.1

0 0
14 4
C C T0 fresh leaf litter
12 Tfinal fresh leaf litter
Virusesfree x 107 ml–1

Virusesfree x 107 ml–1

3 T0aged leaf litter

10 Tfinal aged leaf litter

8
2
6

4 1
2

0 0
16 40
14 D D
12
Virusesattached x 1010
Virusesattached x 109

10
8 30
(g dry wt)–1
(g dry wt)–1

6
4
2
20
1

10

0 0
Virus-free Low virus Virus-rich Virus-free Low virus Virus-rich

Fig. 2. Changes in the abundance (± SE) of (A) free-living and

(B) attached bacteria, and (C) free-living and (D) attached Fig. 3. Changes in the abundance (± SE) of (A) free-living and
viruses in incubations with mineral sediments (fresh and (B) attached bacteria, and (C) free-living and (D) attached
aged) under different planktonic virus concentrations. Note viruses in incubations with leaf litter (fresh and aged) under
different scale for values <1 for attached viruses and bacteria. different planktonic virus concentrations. T0: beginning of
T0: beginning of the experiment; Tfinal: average of the mea- the experiment; Tfinal: average of the measurements at the
surements at the 36th, 48th and 60th hour of the experiment 36th, 48th and 60th hour of the experiment
 Kernegger et al.: Particles and viruses in river water 167

River snow 30
6 Low virus treatment
25 Virus-rich treatment
A

Virus:bacterium ratio
T0 river snow
Bacteriafree x 106 ml–1

5 Tfinal river snow

20
4
15
3

10
2

1 5

0 0
50 In the ambient water On particles
B Fig. 5. Virus:bacterium ratios in the ambient water and on
Bacteriaattached x 1011

40 suspended particles at 2 different virus abundance treat-

ments. Data presented as box plots, with the boundary of the
(g dry wt)–1

box closest to 0 indicating the 25th percentile, the line within

30 the box marking the median, and the boundary of the box far-
thest from 0 indicating the 75th percentile. Error bars: 90th
and 10th percentiles
20

10 fluctuated strongly in the final phase of the experi-

ment (Fig. 4d).
0 In all experimental incubations, BSP fluctuated as
30 listed in Table 1. Both the overall (ml–1 experimental
C water) as well as the specific (cell–1) BSP were signifi-
25
cantly higher on particles (BSP: p < 0.001, n = 180;
ml–1

20 sBSP: p < 0.05, n = 180; Mann-Whitney U-test) than in

106

bulk water. Moreover, VBR was variable during the

15 incubations (Table 2). Generally (pooling data from all
Virusesfree x

incubations), the VBR was significantly lower (p <

10 0.001, n = 180; Mann-Whitney U-test) on suspended
matter than in bulk water (Fig. 5).
5 For further comparison, we analyzed potential
effects of organic (leaf detritus, river snow) versus inor-
0 ganic (sediment) particles as well as fresh versus aged
12
suspended material (combined sediment and leaf lit-
D
10 ter). For this comparison, we included data on both
Virusesattached x 1011

BSP and VBR.

(g dry wt)–1

6 Organic vs. inorganic particles

4 The average abundance of bacteria and viruses (for

both: p < 0.001, n = 89) as well as the VBR (p < 0.01, n =
2
89; Mann-Whitney U-test) were significantly higher on
organic than on inorganic particles. Even in the water,
0
Virus-free Low virus Virus-rich the abundance of bacteria was significantly higher (p <
0.001, n = 89; Mann-Whitney U-test) when organic par-
Fig. 4. Changes in the abundance (± SE) of (A) free-living and ticles were present. Productivity was less affected and
(B) attached bacteria, and (C) free-living and (D) attached the effect contrasted with that on abundances: sBSP of
viruses in incubations with river snow under different plank-
tonic virus concentrations. T0: beginning of the experiment;
attached bacteria was significantly higher on inorganic
Tfinal: average of the measurements at the 36th, 48th and particles (p < 0.001, n = 89; Mann-Whitney U-test); the
60th hour of the experiment overall BSP was not affected. The productivity of bacte-
168 Aquat Microb Ecol 57: 161–173, 2009

Table 1. Bacterial secondary production (BSP) and specific BSP (sBSP) for the free-living and attached fractions in experiments
with different particulate materials

BSPfree-living (µg C l–1 h–1) BSPattached (µg C l–1 h–1) sBSPfree-living (fg C cell–1 h–1) sBSPattached (fg C cell–1 h–1)
N Range Average N Range Average N Range Average N Range Average

Fresh sediment 18 0.14 – 1.22 0.69 18 0.23 – 2.22 1.42 18 0.08 – 1.02 0.32 18 0.28 – 3.48 1.50
Aged sediment 18 0.24 – 2.47 0.92 18 0.24 – 3.76 1.93 18 0.03 – 0.16 0.06 18 0.03 – 0.37 0.16
Fresh leaves 17 0.04 – 1.40 0.45 17 0.03 – 5.68 2.28 17 0.03 – 0.45 0.16 17 0.02 – 2.41 0.60
Aged leaves 18 0.48 – 1.26 0.92 18 0.72 – 3.04 1.80 18 0.09 – 0.42 0.23 18 0.02 – 0.06 0.04
River snow 18 0.13 – 0.61 0.35 18 0.26 – 1.55 0.85 18 0.01 – 0.11 0.05 18 0.07 – 0.29 0.13

Table 2. Virus:bacterium ratios (VBR) for the free-living and attached particles (p < 0.001, n = 89; Mann-Whitney
fractions in experiments with different particulate materials U-test). In contrast to the organic versus in-
organic comparison, neither the abun-
VBRfree-living VBRattached dance and the productivity of bacteria in
N Range Average N Range Average the water, nor the BSP and the VBR of
attached and freeliving microorganisms
Fresh sediment 12 2.50 – 27.9 9.47 18 0.00 – 1.40 0.72
Aged sediment 12 1.00 – 18.7 6.27 18 0.11 – 5.74 2.27 were significantly different between fresh
Fresh leaves 11 2.72 – 29.8 13.7 16 0.00 – 4.07 1.19 versus aged particles.
Aged leaves 12 2.67 – 9.76 5.51 18 0.35 – 2.47 1.05
River snow 12 3.13 – 13.7 6.48 18 0.82 – 3.07 1.55
Combined effects of particle quality and
viral abundance
Table 3. Principal component analysis (PCA). Communalities, percentage of
explained variance and factor loadings. sBSPfree: specific bacterial sec- The PCA, which was performed mainly
ondary production (BSP) of free-living bacteria; BSPfree: BSP of free-living
to determine uncorrelated variables that
bacteria; sBSPatt: specific BSP of attached bacteria; BSPatt: BSP of attached
describe the reaction of microorganisms
bacteria; bacatt: abundance of attached bacteria; bacfree: abundance of free-
living bacteria; Δ prod bacfree: changes in the productivity of free-living bac-
during the experiments, produced the fol-
teria; Δ abund bacfree: changes in the abundance of free-living bacteria; Δlowing results (Table 3): the 6 variables en-
abund bacatt: changes in the abundance of attached bacteria. Values in bold
tered into the PCA (changes in abundance,
indicate high factor loading for respective PCA-axis
sBSP and overall BSP for both free-living
and attached bacteria) were transformed
Factor loadings
into 3 PCA-axes without much loss of infor-
PCA1 PCA2 PCA3
Communality Δ prod bacfree Δ abund bacfree Δ abund bacatt mation (in total, 91.6% of the variance in
the data was explained; Table 3). The first
sBSPfree 0.947 0.970 –0.0090 0.068 axis (PCA1: Δ prod bacteriafree) was domi-
BSPfree 0.930 0.904 0.235 0.248 nated by the change in the productivity
sBSPatt 0.953 0.462 0.833 –0.2140
BSPatt 0.801 0.531 0.443 0.576
of free-living bacteria (both sBSP and
bacatt 0.966 0.155 0.033 0.970 BSP), while the second (PCA2: Δ abund
bacfree 0.900 –0.1270 0.828 0.445 bacteriafree) was strongly correlated with
the change in the abundance of free-living
Explained variance (%) 52.4 21.6 17.7
bacteria and to a certain extent with the
sBSP of attached bacteria. The third axis
(PCA3: Δ abund bacteriaatt) was dominated
ria in the water was not significantly influenced by the by the change in the abundance of attached bacteria.
presence of both organic and inorganic particles. BSP of attached bacteria could not be attributed to one of
the axes because the factor loadings were quite similar
for all 3 axes.
Fresh vs. aged particles

The average abundance of attached bacteria and Effect of particle quality on microbial parameters
viruses was significantly higher on aged particles (for
both: p < 0.001, n = 89; Mann-Whitney U-test). The sBSP Initial abundances (after mixing of the incubated
of attached bacteria was significantly higher on fresh components) of attached viruses were highly depen-
 Kernegger et al.: Particles and viruses in river water 169

Table 4. Effect of particle quality on the microbial community: initial viral abundance and changes during the experiments. Ini-
tial abund viratt: initial abundance of attached viruses; initial abund virfree: initial abundance of free-living viruses; PCA1, 2, 3:
principal component axes 1, 2, 3 (see Table 3 and text); Δ prod bacfree: changes in the productivity of free-living bacteria; Δ abund
bacfree: changes in the abundance of free-living bacteria; Δ abund bacatt: changes in the abundance of attached bacteria;
summary: parameters describing regression fit; int/sl: intercept (fresh sediment)/slope (other particle qualities). Values in bold
indicate significant (p < 0.05) parameters

Initial abund Initial abund PCA1 - Δ prod PCA2 - Δ abund PCA3 - Δ abund
viratt virfree bacfree bacfree bacatt
Reg. coefficients Int/sl p-value Int/sl p-value Int/sl p-value Int/sl p-value Int/sl p-value

Fresh sediment 0.438 0.439 0.984 0.066 1.231 0.028 0.279 0.662 –0.703 0.025
Aged sediment 1.789 0.042 –0.082 0.905 –1.093 0.139 0.351 0.697 2.177 0.000
Fresh leaf litter 1.990 0.027 0.145 0.834 –1.413 0.064 –0.390 0.667 0.894 0.040
Aged leaf litter 2.895 0.004 –0.191 0.783 –2.037 0.013 –0.725 0.428 –0.405 0.310
River snow 3.694 0.001 –0.257 0.712 –1.611 0.039 –0.634 0.487 0.847 0.049

Summary: r2 p-value r2 p-value r2 p-value r2 p-value r2 p-value

0.722 0.008 0.042 0.967 0.506 0.104 0.174 0.719 0.847 0.000

dent on particle quality (r2 = 0.72) Table 5. Effect of viral abundance on changes in the microbial community dur-
(Table 4): river snow and aged leaf lit- ing the experiment after elimination of particle quality effects. Res: residuals
from regression with particle quality; PCA1, 2, 3: principal component axes 1, 2,
ter had much higher viral densities 3 (see Table 3 and text); summary: parameters describing regression fit; viratt:
than fresh sediment. Since the initial attached viruses; virfree: free-living viruses. Values in bold are significant at
abundance of planktonic viruses was p < 0.05 after Bonferroni correction
experimentally manipulated, it could
not be explained by particle quality. Res PCA1 Res PCA2 Res PCA3
To reduce the number of variables Reg. coefficients Slope p-value Slope p-value Slope p-value
and avoid problems stemming from
Res viratt 0.820 0.003 –0.557 0.126 0.141 0.436
the colinearity of the variables, we
Res virfree –0.752 0.013 –0.175 0.659 –0.052 0.799
used the 3 PCA-axes described above
to represent the changes in the abun- Summary: r2 p-value r2 p-value r2 p-value
dance and productivity parameters of
attached and free-living bacteria. 0.508 0.010 0.342 0.066 0.056 0.687
Abundance changes in attached bac-
teria during the experiment (PCA3)
were clearly influenced by particle quality (r2 = 0.85): (residuals from PCA2 and PCA3), but they clearly
aged sediment favored increasing abundances. Abun- influenced the bacterial productivity in the ambient
dance changes of free-living bacteria (PCA2) could not water (residuals from PCA1). The initial abundance of
be attributed to particle quality, although productivity free-living viruses had a highly significant negative
changes of free-living bacteria (PCA1) showed a rela- influence on bacterial productivity in the ambient
tively high, but not significant correlation with particle water, while that of attached viruses had a highly sig-
quality (r2 = 0.51). nificant positive influence, i.e. free-living viruses
Based on the hypotheses formulated in the introduc- reduced bacterial productivity in the ambient water,
tion, we applied a second series of regression analyses whereas viruses attached on particles enhanced it
(see ‘Materials and methods’ for details of calculations) (Table 5).
as discussed in the next section. Fig. 6 summarizes the results of the analysis: changes
in the parameters of microbes attached to particles
could largely be explained by particle quality (r2 >
Effects of initial viral abundance 0.70), whereas viral abundances, both on the particles
and in the water, did not explain the changes in abun-
When the effect of particle quality was eliminated by dance of the attached microbial community. In contrast,
using the residuals of the regressions, the initial viral productivity changes of bacteria in the water were af-
abundances did not significantly explain the changes fected by viral abundances, whereas an effect of parti-
in bacterial abundance in the water and on particles cle quality on this parameter cannot be confirmed.
170 Aquat Microb Ecol 57: 161–173, 2009

Fig. 6. Summary of 2-step regression analysis: on the left side are the effects of particle quality on the outcome of the experiments
(represented by 3 PCA axes), on the right side is the influence of virus abundance, when particle quality is not considered (using
residuals) (see ‘Materials and methods’, ‘Statistical analyis’ for details). Percentages printed in bold indicate significant regres-
sions. Numbers in ellipses refer to the percentage of the variance explained by the second regression series alone (e.g. 51%
for the regression of residual PCA1 vs. virus abundances)

DISCUSSION was significantly higher than on inorganic and fresh

particles. Abundances of bacteria on particles followed
Throughout our experiments, the bacterial and viral the same pattern, although the VBR was higher only
abundances on particles (when calculated as cells or on organic particles. In natural aquatic environments,
viruses ml–1) were comparable with values reported particle quality apparently influences microbial colo-
earlier for the Danube River and the Danube flood- nization (Luef et al. 2007, Peduzzi & Luef 2008, Rie-
plains (Luef et al. 2007). Such data are clearly influ- mann & Grossart 2008). The aged and organic particles
enced by the variable abundance of particles. Unfortu- in our experiments were likely a more favorable
nately, no bacterial or viral abundance estimates based microenvironment (at least initially). This could be due
on g dry weight of suspended particles could be found to an already formed biofilm, adsorbed organic mater-
for the Danube in the literature. However, the load of ial, or the presence of an organic matrix, which may
suspended matter transported in the Danube varies stimulate prokaryote production and increase infection
considerably, and high discharge rates are typically (cf. Weinbauer et al. in press). There is evidence that
associated with higher particle loads. the higher numbers of particle-associated prokaryotes
In general, suspended matter quality determined on aged particles typically exhibit higher hydrolytic
viral and bacterial abundance on particles. We found activity; even the high metabolic activities of hetero-
that viral abundance on organic and on aged particles trophic eukaryotes result in elevated dissolved organic
 Kernegger et al.: Particles and viruses in river water 171

matter (DOM) and inorganic nutrient concentrations in tial phase of colonization. Grossart & Ploug (2000) also
the pore water or at the interface of particulate matter found reduced growth efficiency of bacteria on aged
compared to the surrounding water (Simon et al. 2002). compared to fresh aggregates.
This can result in higher host abundance and higher The productivity changes during the experiments
viral production rates. Further, Peduzzi & Luef (2008) revealed that the sBSP and BSP in the ambient water
found a significant interrelationship between the cell–1 followed similar patterns as indicated by their high
productivity of bacteria and viral abundance in a flood- correlation with PCA1, whereas the sBSP and BSP of
plain rich in aged particles with abundant organic con- attached bacteria could not be allocated to the same
stituents. All these considerations may explain why axis, i.e. did not have the same trends. This is in accor-
fresh and inorganic particles harbored fewer bacteria dance with the findings described for the pooled data.
and viruses in our experiments. On the other hand, we detected no significant effect of
The generally lower VBRs on particles relative to the particle quality on any of the productivity change vari-
bulk water from our experiments are in agreement ables.
with values from various particle environments. In From a physical standpoint, viruses can be regarded
freshwater sediments, values are typically lower; ratios as charged hydrophilic colloids with some ability to
of up to 13 have been found on floating particles in adsorb to the surfaces of suspended particulate matter.
river systems (Luef et al. 2007, Peduzzi & Luef 2009). In From the older literature, there are indications that as
lake snow, VBRs varied between 0.3 and 8.5 with an much as 99% of the viruses present in coastal marine
average of 4.7 (Simon et al. 2002). Potential reasons for waters are adsorbed to naturally occurring colloidal
this observation are extensively discussed in Wein- and particulate matter (Berg 1973). Chattopadhyay &
bauer et al. (in press). Puls (2000) have shown that van der Walls forces can
Suspended matter quality is obviously also a strong govern the process of sorption of selected bacterio-
determining factor for changes in microbial abun- phages on clays. Moreover, the attraction between a
dance. During the present experiments, the abun- viral particle and any interface appears to be strongly
dance of attached bacteria increased strongly on aged influenced by the virus’s amphipathicity, which is
sediment or on organic material such as leaves and the result of localized hydrophobic and hydrophilic
river snow. In the literature, growth rates of bacteria on regions on the surfaces of the capsid protein, as well as
organic particles mainly concern bacteria associated by the ionic strength of the suspending medium
with marine snow and apparently vary greatly; rates (Thompson & Yates 1999). This implies that even phys-
may vary with the age and state of decomposition of ical forces can be an important determining factor in
the marine snow (Alldredge et al. 1986, Alldredge & the colonization process. In our experiments, the dif-
Gotschalk 1990). In our study, even bacterial abun- ferent types of suspended particulates apparently har-
dance in the ambient water was influenced by particle bored a certain fraction of the viruses, even shortly
quality: in the treatments with organic particles, bacte- after the mixing of the components in the incubations.
rial abundance was on average much higher than in However, the abundance of viruses on particles dif-
treatments containing inorganic particles. Thus, our fered, depending on the quality of the material. In an
experiments demonstrate that favorable growth condi- environmental study of the Danube floodplain area,
tions for bacteria were provided in association with we found that, as particles with significant organic
particulate material, which agrees with several other constituents increase in abundance, more viruses are
studies in various aquatic environments (Grossart et al. attached and fewer are freely suspended (Peduzzi &
2007, Peduzzi & Schiemer 2008, Besemer et al. 2009). Luef 2008). Therefore, such particles apparently have
Attached bacteria were also found to be more active a significant potential to remove virus-like particles
(on a cell–1 basis) in a study conducted in the same (VLPs) from the water column.
Danube floodplain system where our water samples Even after eliminating the effect of particle quality in
were collected. There, the sBSP was on average 9 × our statistical analysis, variable viral abundance (both
higher than that of free-living forms (Luef et al. 2007, attached and free-living) affected the change in bacte-
Peduzzi & Luef 2008). Moreover, in an isolated back- rial productivity in the ambient water: the abundance
water of the Danube River, we found a positive relation of planktonic viruses had a negative effect whereas
between sBSP and viral abundance (Peduzzi & Luef that of attached viruses had a positive effect. Other
2008). Interestingly, the sBSP (i.e. the cell–1 productiv- studies also report a negative effect of abundant free-
ity) of attached bacteria was higher in our experiments living viruses on the productivity of bacteria sharing
on inorganic and fresh versus organic and aged parti- the same microhabitat (compare Peduzzi & Weinbauer
cles. One potential explanation is that the cell–1 activity 1993b, Peduzzi & Luef 2009). On one hand, high viral
has reached a saturation phase on already colonized abundance on particles may enhance nutrient release
particles compared to ‘pioneering’ bacteria in the ini- due to lysis of attached hosts or lysozymes. On the
172 Aquat Microb Ecol 57: 161–173, 2009

other hand, suspended particles may act as adsorbing fully stimulate further investigations, including in situ
agents that remove viruses from the ambient water, ones, on suspended matter and viral particles in river-
thus lowering the probability of new infections. The ine systems. Future studies should collect information
few studies that simultaneously measured viral abun- on virus-mediated bacterial mortality to better under-
dance and bacterioplankton production found signifi- stand whether attached viruses correspond to plank-
cant correlations (Heldal & Bratbak 1991, Maranger & tonic forms that were previously adsorbed, or if they
Bird 1995). A direct indication that increased viral pro- were produced locally from infected attached bacteria.
duction coincides with elevated levels of BSP was Exploring this question will require additional esti-
noted by Steward et al. (1996). Their study reported a mates of viral production in ambient water and on par-
significant correlation between the frequency of visi- ticulate material. Focusing on such topics will provide
bly infected cells and bacterial production rates. further key insights into the structure and function of
Accordingly, an abundant virioplankton community is aquatic systems.
probably dependent on an active bacterioplankton
host community (Wommack & Colwell 2000). A recent Acknowledgements. We thank M. G. Weinbauer for valuable
study by Riemann & Grossart (2008) speculates that, in discussions, B. Luef and M. Agis for all kinds of help in the
environments rich in particulate matter, elevated lytic laboratory, and M. Maschek for drawing Fig. 1. The Austrian
production of phages occurs due to increased activities Science Fund supported our research (grants P14721 and
17798 to P.P.).
of particle-associated bacteria.
The results presented here supplement the scarce
observations that particle-associated viruses exhibit LITERATURE CITED
some dependency on the type and quality of the mate-
rial (Farnell-Jackson & Ward 2003, Luef et al. 2007, ➤ Alldredge AL, Gotschalk C (1990) The relative contribution of
marine snow of different origins to biological processes in
Peduzzi & Luef 2008). In this context, the effect of sus- coastal waters. Cont Shelf Res 10:41–58
pended particles can be ecologically significant. For Alldredge AL, Cole JJ, Caron DA (1986) Production of hetero-
example, a study conducted in the Gulf of Mexico trophic bacteria inhabiting macroscopic organic aggre-
gates (marine snow) from surface waters. Limnol
reports that free-living viruses may bind irreversibly to Oceanogr 31:68–78
aggregates, thereby losing their infectivity (Suttle & ➤ Auguet JC, Montanié H, Hartmann HJ, Lebaron P, Casa-
Chen 1992). There is also evidence, however, that viral mayor EO, Catala P, Delmas D (2009) Potential effect of
association with colloidal and particulate material can freshwater virus on the structure and activity of bacterial
communities in the Marennes-Oléron bay (France).
prolong their survival (Kapuscinski & Michell 1980),
Microb Ecol 57:295–306
and phage production and transduction frequency Berg G (1973) Removal of viruses from sewage, effluents and
may even increase in the presence of particulate mat- waters. Present and future trends. Bull WHO 49:461–469
ter (Kokjohn et al. 1991, Ripp & Miller 1995). Based on ➤ Berger B, Hoch B, Kavka G, Herndl GJ (1995) Bacterial
our own results, we argue that not only the availability metabolism in the River Danube: parameters influencing
bacterial production. Freshw Biol 34:601–616
but also the quality of the suspended particles will be
➤ Besemer K, Moeseneder MM, Arrieta JM, Herndl GJ, Peduzzi
crucial for the survival and/or proliferation of viruses in P (2005) Complexity of bacterial communities in a river-
aquatic environments. floodplain system (Danube, Austria). Appl Environ Micro-
In conclusion, the present laboratory experiments biol 71:609–620
supported our main hypotheses: both seston quality ➤ Besemer K, Agis M, Eichberger B, Luef B, Preiner S, Peduzzi
P (2009) Sources and composition of organic matter as
and variable viral abundances in the bulk water con- substrate for bacterial growth in a large European river-
siderably influence microbial parameters. This sug- floodplain system (Danube, Austria). Org Geochem 40:
gests a potential effect of variable particle quality and 321–331
viral abundance on microbial parameters in natural ➤ Chattopadhyay S, Puls RW (2000) Forces dictating colloidal
interactions between viruses and soil. Chemosphere 41:
aquatic environments. We cautiously use the term 1279–1286
‘potential’ because experimental results have to be ➤ Chen F, Lu JR, Binder B, Liu YC, Hodson RE (2001) Appli-
translated with care to natural conditions. Enclosure cation of digital image analysis and flow cytometry to
experiments may be biased due to unknown effects enumerate marine viruses stained with SYBR Gold. Appl
Environ Microbiol 67:539–545
stemming from e.g. confinement, handling or the walls
of the containers. However, since the volume of our ➤ Danovaro R, Dell’Anno A, Trucco A, Serresi M, Vanucci S
(2001) Determination of virus abundance in marine sedi-
tanks was relatively large (10 l), the jars were kept ment. Appl Environ Microbiol 67:1384–1387
rotating to keep all material in suspension, and all ➤ Farnell-Jackson EA, Ward AK (2003) Seasonal patterns of
treatments were run at identical initial conditions viruses, bacteria and dissolved organic carbon in a river-
ine wetland. Freshw Biol 48:841–851
(except for the intended different treatments), our sta- Grossart HP, Ploug H (2000) Bacterial production and growth
tistically significant results should be relevant for in efficiencies: direct measurements on riverine aggregates.
situ processes. These experimental results will hope- Limnol Oceanogr 45:436–445
 Kernegger et al.: Particles and viruses in river water 173

➤ Grossart HP, Tang KW, Kørboe T, Ploug H (2007) Comparison cles as bioactive agent in unicellular plankton community
of cell-specific activity between free-living and attached successions. J Plankton Res 15:1375–1386
bacteria using isolates and natural assemblages. FEMS ➤ Peduzzi P, Aspetsberger F, Hein T, Huber F, Kargl-Wagner S,
Microbiol Lett 266:194–200 Luef B, Tachkova Y (2008) Dissolved organic matter
➤ Hein T, Heiler G, Pennetzdorfer D, Riedler P, Schagerl M, (DOM) and bacterial growth in floodplains of the Danube
Schiemer F (1999) The Danube restoration project: func- River under varying hydrological connectivity. Fund Appl
tional aspects and planktonic productivity in the flood- Limnol/Arch Hydrobiol 171:49–61
plain system. Regul Rivers Res Manage 15:259–270 ➤ Proctor LM, Fuhrman JA (1990) Viral mortality of marine bac-
➤ Hein T, Baranyi C, Herndl GJ, Wanek W, Schiemer F (2003) teria and cyanobacteria. Nature 343:60–62
Allochthonous and autochthonous particulate organic ➤ Proctor LM, Fuhrman JA (1992) Mortality of marine bacteria
matter in floodplains of the River Danube: the importance in response to enrichments of the virus size fraction from
of hydrological connectivity. Freshw Biol 48:220–232 seawater. Mar Ecol Prog Ser 87:283–293
➤ Heldal M, Bratbak G (1991) Production and decay of Riemann L, Grossart HP (2008) Elevated lytic phage produc-
viruses in aquatic environments. Mar Ecol Prog Ser 72: tion as a consequence of particle colonization by a marine
205–212 Flavobacterium (Cellulophaga sp.). Microb Ecol 56:
➤ Hopkinson CS, Buffam I, Hobbie J, Vallino J and others (1998) 505–512
Terrestrial inputs of organic matter to coastal ecosystems: ➤ Ripp S, Miller RV (1995) Effects of suspended particulates
an intercomparison of chemical characteristics and bio- on the frequency of transduction among Pseudomonas
availability. Biogeochemistry 43:211–234 aeruginosa in a freshwater environment. Appl Environ
➤ Johnson BD, Zhou X, Wangersky PJ (1986) Surface coagula- Microbiol 61:1214–1219
tion in sea water. Neth J Sea Res 20:201–210 ➤ Shanks AL, Edmondson EW (1989) Laboratory-made artificial
➤ Kapuscinski RB, Michell R (1980) Processes controlling virus marine snow: a biological model of the real thing. Mar Biol
inactivation in coastal waters. Water Res 14:363–371 101:463–470
Kokjohn TA, Sayler GS, Miller RV (1991) Attachment and ➤ Simon M, Grossart HP, Schweitzer B, Ploug H (2002) Micro-
replication of Pseudomonas aeruginosa bacteriophages bial ecology of organic aggregates in aquatic ecosystems.
under conditions simulating aquatic environments. J Gen Aquat Microb Ecol 28:175–211
Microbiol 137:661–666 ➤ Steward GF, Smith DC, Azam F (1996) Abundance and pro-
➤ Luef B, Aspetsberger F, Hein T, Huber F, Peduzzi P (2007) duction of bacteria and viruses in the Bering and Chukchi
Impact of hydrology on free-living and particle-associated Seas. Mar Ecol Prog Ser 131:287–300
microorganisms in a river floodplain system (Danube, ➤ Suttle CA, Chen F (1992) Mechanisms and rates of decay of
Austria). Freshw Biol 52:1043–1057 marine viruses in seawater. Appl Environ Microbiol 58:
➤ Luef B, Neu TR, Peduzzi P (2009) Imaging and quantifying 3721–3729
virus fluorescence signals on aquatic aggregates: a new ➤ Suttle CA, Chan AM, Cottrell MT (1990) Infection of phyto-
method and its implications for aquatic microbial ecology. plankton by viruses and reduction of primary productivity.
FEMS Microbiol Ecol 68:372–380 Nature 347:467–469
➤ Maranger R, Bird DF (1995) Viral abundance in aquatic sys- ➤ Suttle CA, Chan AM, Cottrell MT (1991) Use of ultrafiltration
tems: a comparison between marine and fresh waters. Mar to isolate viruses from seawater which are pathogens of
Ecol Prog Ser 121:217–226 marine phytoplankton. Appl Environ Microbiol 57:
➤ Neu TR (2000) In situ cell and glycoconjugate distribution in 721–726
river snow studied by confocal laser scanning microscopy. ➤ Thompson SS, Yates MV (1999) Bacteriophage inactivation at
Aquat Microb Ecol 21:85–95 the air–water–solid interface in dynamic batch systems.
➤ Pace NR (2006) Time for a change. Nature 441:289 Appl Environ Microbiol 65:1186–1190
➤ Peduzzi P, Luef B (2008) Viruses, bacteria and suspended par- Ward GM, Ward AK, Dahm CN, Aumen NG (1990) Origin
ticles in a backwater and main channel site of the Danube and formation of organic and inorganic particles in
(Austria). Aquat Sci 70:186–194 aquatic systems. In: Wotton RS (ed) The biology of parti-
Peduzzi P, Luef B (2009) Viruses. In: Likens GE (ed) Encyclo- cles in aquatic systems. CRC Press, Boca Raton, FL,
pedia of inland waters. Vol 3. Elsevier, Oxford, p 279–294 p 27–56
Peduzzi P, Schiemer F (2008) Bacterial dynamics, nutrients Weinbauer MG (2004) Ecology of prokaryotic viruses. FEMS
and organic matter in the water column of tropical fresh- Microbiol Ecol 28:127–182
water reservoirs of Sri Lanka. In: Schiemer F, Simon D, ➤ Weinbauer MG, Suttle CA (1996) Potential significance of
Amarasinghe U, Moreau J (eds) Aquatic ecosystems and lysogeny to bacteriophage production and bacterial mor-
development: comparative asian perspectives. Bakhuysen tality in coastal waters of the Gulf of Mexico. Appl Environ
Publishers, p 135–152 Microbiol 62:4374–4380
Peduzzi P, Weinbauer MG (1993a) Effect of concentrating the Weinbauer MG, Bettarel I, Cattaneo R, Luef B and others (in
virus-rich 2–200 nm size fraction of seawater on the for- press) Viral interactions with organic and inorganic parti-
mation of algal flocs (marine snow). Limnol Oceanogr 38: cles in the water column: implications for aquatic micro-
1562–1565 bial ecology. Aquat Microb Ecol doi:10.3354/ame01363
➤ Peduzzi P, Weinbauer MG (1993b) The submicron size frac- ➤ Wommack KE, Colwell RR (2000) Virioplankon: viruses in
tion of seawater containing high numbers of virus parti- aquatic ecosystems. Microbiol Mol Biol Rev 64:69–114

Editorial responsibility: Gunnar Bratbak, Submitted: December 5, 2008; Accepted: June 23, 2009
Bergen, Norway Proofs received from author(s): September 18, 2009