JOURNAL OF GEOPHYSICAL RESEARCH, VOL. 112, G01002, doi:10.

1029/2006JG000235, 2007

Effects of overlying velocity on periphyton structure and

denitrification
Shai Arnon,1,2 Aaron I. Packman,1 Christopher G. Peterson,3 and Kimberly A. Gray1
Received 25 May 2006; revised 25 August 2006; accepted 6 September 2006; published 11 January 2007.

[1] The effects of overlying flow conditions on periphyton structure and denitrification
were measured in three laboratory mesocosms (120 cm long and 60 cm wide) under
average velocities of 0.05, 0.5 and 5 cm/s. Periphyton was cultivated on polyethylene
benthic nets overlaying a thin layer of sand. The mesocosms were operated
continuously for four months, leading to prolific growth of periphyton on the benthic nets
and in the underlying sediments. Periphyton structural characteristics were quantified in
terms of algal/bacterial biomass, algal species composition, and microbial
enumeration. Confocal microscopy was used to investigate the spatial organization of the
periphyton. The denitrification potential of the microbial community in each mesocosm
was evaluated using the acetylene inhibition method. Different benthic microbial
communities developed under the three flow conditions, while the total microbial biomass
accrual increased monotonically with increasing overlying velocity. Denitrification
potential also increased with overlying velocity when evaluated on both a per unit area and
per unit biomass basis. The periphytic community that developed under the fastest
velocity and was characterized by the largest fractional biovolume of diatoms
(predominantly Achnanthidium minutissimum), promoted establishment of a consortium
of bacterial denitrifiers more physiologically active than those that developed under slower
overlying velocities. These results demonstrate that hydrodynamic transport conditions
play a key role in structuring benthic microbial communities, and the structural differences
that develop under different flow conditions also regulate benthic microbial processing of
substances delivered from the water column. Understanding the response of microbial
communities to physical conditions is essential for evaluating nutrients dynamics, as well
as for the development of management strategies aimed at mitigating effects of excess
nitrogen by increasing denitrification in shallow aquatic systems.
Citation: Arnon, S., A. I. Packman, C. G. Peterson, and K. A. Gray (2007), Effects of overlying velocity on periphyton structure and
denitrification, J. Geophys. Res., 112, G01002, doi:10.1029/2006JG000235.

1. Introduction ities reduce the thickness of the diffusive boundary layer at

[2] Flow conditions are an important regulator of spatial
and temporal changes in the composition of benthic micro-
bial communities, as well as of the energy flux and nutrient
cycling associated with those communities [Wetzel, 1983;
Mulholland et al., 1994; Alexander et al., 2000; Battin et
al., 2003]. The specific links between hydrodynamic pro-
cesses, the structural attributes of benthic microbial com-
munities, and their broader ecosystem function are only
beginning to be elucidated [Battin et al., 2003; Larned et
al., 2004; Singer et al., 2005]. Increasing overlying veloc-
1
Department of Civil and Environmental Engineering, Northwestern
University, Evanston, Illinois, USA.
2
Now at Department of Environmental Hydrology and Microbiology,
Zuckerberg Institute for Water Research, Blaustein Institutes for Desert
Research, Ben-Gurion University of the Negev, Sede Boqer, Israel.
3
Department of Natural Science, Loyola University, Chicago, Illinois,
USA.
Copyright 2007 by the American Geophysical Union.
0148-0227/07/2006JG000235
biofilm surfaces, thus enhancing mass transfer of dissolved
nutrients and stimulating metabolic processes such as
nutrient uptake, photosynthesis, respiration, and reproduc-
tion [McIntire, 1966; Horner et al., 1990; Jørgensen and
Des Marais, 1990; Kühl et al., 1996]. The positive effect of
increasing velocity persists until nutrient saturation occurs,
and this threshold varies considerably with environmental
and biofilm-specific attributes [Bothwell, 1989; Horner et
al., 1990]. Above velocities of 10 –15 cm/s, hydrodynamic
shear stress also begins to influence biofilm architecture,
taxonomic composition, and nutrient cycling [Keithan and
Lowe, 1985; Bergey et al., 1995; Biggs et al., 1998a;
Hondzo and Wang, 2002]. Under slow flow conditions
(laminar or transitional), shear forces are minimal, flow
variation produces differences in nutrient supply rates, and
thus biofilm structure and function should be strongly
influenced by biotic interactions among biofilm residents
[Peterson and Stevenson, 1990; McCormick and Stevenson,
1991]. Little is known, however, of the effects of variation
in overlying velocities on the structural and functional

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G01002 ARNON ET AL: FLOW, PERIPHYTON, AND DENITRIFICATION
attributes of periphytic biofilms under the slow flow con-
ditions that are common in most wetlands, lakes, and slack
water areas of streams, such as pools and off-channel
backwater areas.
[3] Periphyton encompasses a broad spectrum of biogeo-
chemical processes involved in nutrient and energy cycling
in shallow freshwater systems. Here we focus on denitrifi-
cation, an anaerobic bacterial respiration process that con-
verts nitrate to nitrite, ultimately yielding the gaseous
compounds nitrous oxide and nitrogen gas [Tiedje, 1988].
Therefore variation in the efficiency of this process has
important implications for nitrogen cycling in freshwaters,
and particularly for the protection or restoration of aquatic
ecosystems subject to excess nutrients [Alexander et al.,
2002; Böhlke et al., 2004; Richardson et al., 2004]. Under
anoxic conditions, the rate of denitrification is influenced by
nitrate concentrations, organic carbon content, temperature,
pH, and the velocity of the surrounding flow [Tiedje, 1988;
Kadlec and Knight, 1996; Eriksson, 2001; Hill and
Cardaci, 2004]. Flow conditions should affect denitrifica-
tion mainly by regulating the delivery of nitrate, oxygen and
organic carbon to denitrifying bacteria. In addition, because
the taxonomic content of bacterial consortia associated with
microalgae varies among algal species [Schäfer et al., 2002;
Grossart et al., 2005], presumably as a function of species-
specific variation in the chemical nature of algal organic
exudates [Haack and McFeters, 1982; Myklestad, 1995; van
Hannen et al., 1999], the flow regime should influence
denitrification activity by influencing the microbial com-
munity composition.
[4] In aquatic ecosystems that are nutrient-rich, periphytic
communities of high biomass can develop on the surfaces of
rocks (epilithon) and vegetation (epiphyton). While periph-
yton communities also develop on the finer sediments often
associated with wetland benthos, such as sand (epipsam-
mon) and silts (epipelon), the greater instability of these
substrata can preclude development of thick periphyton
communities of high biomass [Eriksson, 2001; Bastviken
et al., 2003], unless the sediments are stabilized by algal
mucilages [Madsen et al., 1993; Smith and Underwood,
1998]. As such, in slow-flowing habitats like wetlands, the
potential for development of periphyton assemblages of
high biomass and denitrification capacity can be limited
by amount of stable surface area available for periphytic
growth. Although denitrifying microbes also reside within
sediments, slow flow conditions limit mass transfer of
nitrate to these microorganisms, especially to those deep
within the sediment profile. By increasing the availability of
surfaces capable of supporting periphytic growth while
reducing mass transfer constraints, it may be possible to
increase denitrification potential and thus increase the
capacity of nutrient removal in constructed aquatic systems
such as wetlands and agricultural ditches. For example,
polyethylene benthic net that is used in constructed wet-
lands to stabilize sediments and to prevent fish foraging
[Hey, 1994] was recently found to support thick periphytic
growth with high denitrification potential [Sirivedhin and
Gray, 2006]. Moreover, denitrification rates in benthic-net
communities did not vary seasonally as is commonly the
case in sedimentary and epiphytic microbial communities
[Groffman and Hanson, 1997; Toet et al., 2003; Richardson
et al., 2004; Ishida, 2005; Sirivedhin and Gray, 2006].
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G01002
[5] In this study, we cultivated periphyton assemblages
on polyethylene benthic nets in laboratory mesocosms to
test the hypothesis that variation of flow conditions within
the laminar-to-transitional range plays a significant role in
controlling the total biomass, community composition, and
denitrification potential of the benthic microbial community.
2. Materials and Methods
2.1. Experimental Approach
[6] The effects of overlying flow conditions on periphy-
ton structure and denitrification activity were studied using
three recirculating laboratory mesocosms. Periphyton was
cultivated at three overlying velocities, 0.05, 0.5 and 5 cm/s,
on polyethylene benthic net [Hey, 1994] placed on top of a
sand bed. These flow conditions are representative of both
wetlands and a variety of slow-flow environments that can
be found along streams and rivers (off-channel dead zones,
backwater lakes, riparian wetlands, etc.) After four months
of cultivation under the three flow conditions, differences in
periphyton biomass and structure were evaluated by ash free
dry mass (AFDM), chlorophyll a (Chl a), bacterial abun-
dance, algal taxonomic structure, and structural imaging via
confocal laser scanning microscopy (CLSM). Denitrifica-
tion potentials (DNP) of periphyton from each flow regime
were quantified and analyzed to assess linkage to periphy-
ton structure.
2.2. Experimental Setup and Biofilm Cultivation
[7] The mesocosms consisted of three identical Plexiglas
channels (243 cm long, 63 cm wide, and 44 cm high)
connected to a 2 m3 water reservoir as illustrated in Figure 1.
The sampling area within each mesocosm was packed with
four cm of clean natural silica sand (Ottawa #12 Flint silica
sand, mean diameter 486 mm) to form a flat surface, and
was isolated from inlet and outlet sections by barriers that
controlled water column depth at 10 cm. A layer of
polyethylene benthic net, 4 mm thick with 7
7 mm mesh
size, was laid over the sand surface. Water was recirculated
within the experimental system using a centrifugal pump at
flow rate of 350 L/min, and distributed among the three
mesocosms at three different flow rates with excess water
recycled back into a common reservoir to ensure that the
microbial communities in the mesocosms were not isolated
from one another. Flow rates of 1.89, 18.9 and 189 L/min
(±10%) were used, yielding average overlying velocities of
0.05, 0.5 and 5 cm/s and Reynolds numbers of 32, 320 and
3200, respectively, spanning the laminar to transitional (or
weakly turbulent) flow range [Chaudhry, 1993].
[8] The mesocosms were each seeded with 2 L of dense
homogenized algal-bacterial slurries, derived from sedi-
ments collected during November 2004 from Mill Creek
near Wadsworth, Illinois. After allowing 24 hr for settling
under stagnant water conditions, lights were turned on and
flow was initiated in each mesocosm and maintained con-
stant (0.05, 0.5 or 5 cm/s) for four months to support biofilm
growth. Water temperature was maintained at 15 ± 2°C
and irradiance was supplied at 140 ± 10 mEin/m2 s via a
400 W metal halide lights (Hubbell), at light:dark cycle of
8:16 hr. Nutrients were supplied every three weeks to yield
the following concentrations (mg/L); 1 Ca(NO3)24H2O,
0.62 KH2PO4, 1.25 MgSO47H2O, 0.79 NaHCO3, 0.1125
G01002 ARNON ET AL: FLOW, PERIPHYTON, AND DENITRIFICATION
Figure 1. Schematic illustration of the experimental setup.
(a) Overview of the parallel-flow mesocosms. (b) Structure
of each mesocosm.
EDTAFeNa, 0.1125 EDTANa 2 , 0.124 H 3 BO 3 , 0.695
MnCl24H2O, 0.05 (NH4)6Mo7O244H2O, 0.002 Cyanoco-
balamin, 0.002 Thiamine HCl, 2.85, 0.002 Biotin and
2.85 NaSiO39H2O [Beakes et al., 1988]. Nitrate-nitrogen
(NO3-N) concentrations were maintained between 6 and
8 mg/L (using KNO3, measured according to method
4500-B [American Public Health Association, 1998]). Addi-
tions of KNO3 were accompanied by KH2PO4 additions to
maintain the N:P ratio at 16:1. Dissolved organic carbon
(DOC) was maintained at 14±4 mg/L by adding water-
extractable DOC from commercial peat in the beginning of
the experiment, and by diluting the reservoir with deionized
water when DOC increased owing to autotrophic production
(measured with Dohrmann Apollo 9000 Automated TOC
Analyzer).
2.3. Sampling Procedure and Microbial Biomass
Measurements
[9] Samples of the benthic microbial community were
taken from the mesh located in the center of the mesocosms,
excluding the 5 cm adjacent to sidewalls and the 15 cm next
to the inlet and outlet, to minimize boundary effects (see
Figure 1b). Periphyton samples were collected from
10 pieces of net, each with an area of 80– 130 cm2. The
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G01002
total area sampled represented approximately 75% of the
total available benthic surface area. These samples provided
a good assessment of within-mesocosm variability.
[10] Each net sample was divided into 2 equal parts, one
for DNP measurements and the other to quantify periphyton
structure and taxonomic content. Biomass scraped from
these latter samples was homogenized (by sonication and
vortexing) and subsampled for AFDM, Chl a, algal identi-
fication, and bacterial counts. Subsamples for algal identi-
fication and bacterial counts were preserved with 0.5%
glutaraldehyde. AFDM and Chl a were analyzed using
standard methods [American Public Health Association,
1998], while bacterial numbers were counted by epifluor-
escence microscopy (Zeiss, Axiophot) after staining with 40,
6-diamidino-2-phenylindole (DAPI). Briefly, a 10 mL
diluted sample was stained at a concentration of 2.5 mg
DAPI/mL and incubated in the dark for 15 min. Samples
were then vacuum filtered (<30 mm Hg) onto a 25-mm
black polycarbonate membrane filter with a pore size of
0.2 mm (Millipore), overlaid on a 0.45-mm-pore-size, 25-mm-
diameter nitrocellulose backing filter. The filters were
transferred to a microscope slide and a glass cover slip
was applied with a drop of immersion oil. Bacterial cell
counts were made at 400x magnification in randomly
selected fields over a wide area of the filter until a minimum
of either 200 cells or 40 fields was reached [Kepner and
Pratt, 1994]. Aliquots from subsamples for quantifying algal
taxonomic content were mounted in Taft’s syrup medium
[Stevenson, 1984] and at least 500 cells were identified via
light microscopy at 1000
magnification. Biovolume for
each taxon was calculated by taking the average of dimen-
sional measurements from at least 20 individuals and apply-
ing formulae of Hillenbrand et al. [1999]. Counts and taxon-
specific biovolume measures were then used to calculate the
relative contribution of each algal taxon to total algal
biovolume (i.e., relative biovolume).
2.4. Confocal Laser Scanning Microscopy
[11] Benthic net samples (2
2 cm, in duplicate) were
removed from the mesocosms to observe structural patterns
in the periphyton after 45, 56 and 75 days of growth. The
samples were placed into small chambers filled with filtered
(0.2 mm) water from the mesocosms. SYTO 9 (Molecular
Probes) was added to the chambers at a concentration of
20 mg/mL for 15 minutes at room temperature to stain
periphyton components for observation with confocal laser
scanning microscopy. Excess stain was flushed with filtered
(0.2 mm) water. Samples were than analyzed with a Zeiss
LSM 510 confocal microscope, using three fluorescence
channels: green (SYTO 9, excitation 488 nm, emission
500 – 550 nm), red (algal auto-fluorescence, excitation
543 nm, emission 560 nm) and far-red (algal auto-fluorescence,
excitation 633 nm, emission 653 – 717 nm).
2.5. Denitrification Potential
[12] Denitrification rates were determined with the acet-
ylene inhibition method [Yoshinari and Knowles, 1976;
Tiedje, 1988]. Benthic net samples (40 – 75 cm2) with
attached periphyton were added to 125 mL gas-tight jars
(I-CHEM septa-jarTM) filled with 70 mL of nutrient solution
to provide enriched conditions for denitrification. The
nutrient solution contained 40 mg/L NO3-N as KNO3,
G01002 ARNON ET AL: FLOW, PERIPHYTON, AND DENITRIFICATION
Figure 2. Effect of overlying velocity on microbial
biomass in 120-day-old periphyton as indicated by
(a) AFDM, (b) Chl a and (c) bacterial abundance. Variability
within each mesocosm was calculated on the basis of 9– 10
independent samples, and is indicated by the Box-Whisker
presentation; the central line indicates the median value, the
upper and lower boundaries of the box indicates the 25th
and 75th percentile values, and error bars indicate the 10th
and 90th percentile values. The results clearly show that
total benthic microbial biomass accrual increased with
increasing overlying velocity.
100 mg/L carbon as dextrose, and 225 mg/L chloramphen-
icol to inhibit microbial growth. The incubation started
immediately after the jars were flushed with N2 for three
minutes and acetylene was added (10% v/v). During the
incubation, the jars were agitated gently at 150 rpm, under
dark condition at room temperature (23 ± 1°C). Headspace
samples were taken every three hours during a 24-hour
incubation period, and N2O concentrations were measured
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G01002
immediately using a gas chromatograph (Hewlett Packard
5890) equipped with 63Ni electron capture detector at an
operating temperature of 320°C. A stainless steel Porapak Q
(80/100 mesh) column (Agilent) was used to separate the
gases at 60°C with N2 as a carrier gas (16 – 18 mL/min).
Denitrification rates were calculated from linear regression
of N2O accumulation in the headspace, after the concen-
trations were corrected for solubility using the Bunsen
coefficient [Venterink et al., 2003].
2.6. Statistical Analysis
[13] The variability of AFDM, Chl a, bacterial abun-
dance, and DNP was evaluated by obtaining multiple
samples of the periphyton growing in each mesocosm.
These results will be reported using Box-Whisker plots
showing the median and the 10th, 25th, 75th, and 90th
percentiles. The effects of overlying velocity on AFDM,
Chl a, bacterial abundance, taxon-specific relative algal
biovolume, and DNP were analyzed using one-way
ANOVA followed by Tukey’s post-hoc test. Algal taxo-
nomic structure was also analyzed via ordination by Multi-
Dimension Scaling (MDS) using Primer Version 5 [Clarke
and Gorely, 2001]. Taxa included in this analysis were those
that comprised an average of at least 3.0% of algal bio-
volume in at least one of the three flow regimes.
3. Results and Discussion
3.1. Effect of Flow Conditions on Periphyton Structure
[14] The effect of the overlying flow conditions on
periphyton growth was evaluated using multiple measures
of the benthic microbial community: AFDM, Chl a content,
and bacterial abundance. All three measures of periphyton
biomass were positively correlated with the velocity of the
overlying flow, as shown in Figure 2. Stronger correlations
were found for bacterial abundance (r2 = 0.88, p < 0.001)
and Chl a (r2 = 0.83, p < 0.001), than for AFDM (r2 = 0.3,
p < 0.05). The average AFDM under the fastest velocity
(5 cm/s) was 1.18 times higher than that of the intermediate
velocity (0.5 cm/s) and 1.96 times higher than that of the
slowest velocity (0.05 cm/s), though significant differences
at p < 0.05 were found only between the fastest and the
slowest velocities (Figure 2a). Chl a differed significantly
among velocity treatments (p < 0.05), with the average Chl
a concentration being 2.55 and 7.14 times higher under the
fastest velocity than under the intermediate and slow veloc-
ity, respectively (Figure 2b). Bacterial abundance was
significantly different only between the fast velocity and
the two slower velocities (p < 0.05), with the value under
the fastest velocity being 3.7 and 5.28 times higher than
under the intermediate and slow velocities, respectively
(Figure 2c).
[15] The observation that increasing velocities in the
laminar-to-transitional range support elevated levels of
biomass complements results of previous studies that
focused on the effects of overlying flows on periphyton
biomass under faster flow conditions [e.g., Horner et al.,
1990; Biggs et al., 1998a; Nikora et al., 1998; Hondzo and
Wang, 2002]. These studies demonstrated that shear forces
limit or reduce biomass accrual only under turbulent con-
ditions, typically at 10– 15 cm/s or more, although the exact
flow condition that limits biomass accrual varies with
G01002 ARNON ET AL: FLOW, PERIPHYTON, AND DENITRIFICATION G01002

Figure 3. Periphyton structures observed under the three different flow conditions at different spatial
scales. (a, b, c) Photographs of a 120-day-old periphyton grown at velocities of 0.05, 0.5 and 5 cm/s,
respectively. (d, e, f ) Structure of a 75-day-old periphyton resolved using three-channels CLSM (xy plane)
under the same flow conditions. Algal auto-fluorescence was detected in the red (yielding red colors in
the CLSM images) and far red (blue), and their overlay appears as pink. The third channel resolves SYTO 9,
a general nucleic acid stain (green), while the overlay of all three channels appears white. Arrows show
the orientation of the benthic nets, which is identical for all images. The flow direction is from right to
left. CLSM images of periphyton in the two slow velocities, Figures 3d and 3e, show no obvious spatial
orientation or structure of resident microorganisms, whereas quasi-hexagonal structures developed at the
fastest velocity (Figure 3f ).

substratum characteristics and the physical structure, taxo-
nomic content, and physiological condition of the periphy-
ton community [Stevenson, 1983; Biggs et al., 1998a].
Flow-related increases in periphyton biomass are normally
attributed to the increase in mass transfer from the bulk
water to periphyton, which increases with overlying veloc-
ity because of compression of the hydrodynamic boundary
layer [Jørgensen and Des Marais, 1990; Biggs et al., 1998a].
We observed no evidence of major sloughing, supporting
the interpretation that shear forces had minimal, if any,
effect on biomass accrual under the flow treatments used
here (Re = 32, 320, and 3200).
[16] Biomass accrual is influenced by internal mass
transfer processes within the biofilm in addition to external
mass transfer processes. Biofilms have a heterogeneous
internal structure, including both interstitial voids through
which advective flow can occur and dense cell aggregates
that impede diffusion [de Beer et al., 1994]. Resistance to
transport within biofilms varies with biofilm structure
[Ohashi and Harada, 1994; Beyenal and Lewandowski,
2002], which itself is influenced by a myriad of physical,
chemical, and biological factors, including flow regime and
species composition. Beyenal and Lewandowski [2002]
suggested that increasing velocities produce a denser bio-
film structure, which will reduce diffusion and therefore
tend to decrease internal mass transfer of nutrients at higher
velocities. However, it is not always observed that increas-
ing velocities produce denser biofilms, as shown by Battin
et al. [2003] and Peterson and Grimm [1992] for periphytic
communities. Gantzer et al. [1991] noted that biomass in
epilithic biofilms was more sensitive to changes in flow at
low shear velocities than at higher shear velocities, and
surmised that this reflected greater mass-transport limitation
under the slower velocities. Thus unique combinations of
external and internal mass transfer processes are expected to
control biomass accrual in a given microbial community at
different velocities.
[17] Photographs and CLSM imaging revealed that over-
lying velocity affected the physical structure of the periph-
yton that developed in the mesocosms (Figure 3). The
benthic nets provided a uniform substrate that supported
relatively homogeneous macroscopic growth of periphyton
in the slower flow treatments, as shown in Figure 3a and 3b,
while more distinct patterns were observed in the fastest
flow (Figure 3c). In all flow treatments, submerged benthic-
net surfaces were completely covered with continuous, thick
biofilms. CLSM images of periphyton in the two slow
velocities, Figure 3d and 3e, showed no obvious spatial
orientation or structure of resident microorganisms, whereas
quasi-hexagonal structures developed at the fastest velocity,
as shown in Figure 3f. The quasi-hexagonal structures were
ubiquitous and observed in all samples examined between
days 45 and 75 (total of 6 samples). Similar quasi-hexagonal
structures have been reported in biofilms grown in stream-
side flumes, wastewater treatment reactors, and marine
environments, as discussed by Battin et al. [2003, and
references therein]. The mechanisms for the formation of
these structures are still not known, but their presence
suggests that physical forces exerted by the flow at 5 cm/s,
while not of adequate intensity to limit biomass accrual, did

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Figure 4. Relative biovolume of the most common algal taxa in 120-day-old periphyton communities
developed under different velocities. Taxon identity is provided to the left of bar component to which it
applies. Tukey post-hoc test results for each taxon are provided on the right (p < 0.05). S indicates slow-
velocity condition (0.05 cm/s), M medium (0.5 cm/s), and F fast (5 cm/s). The results show that
periphytic community growing under the fastest velocity was characterized by the largest fractional
biovolume of diatoms whereas the fractional biovolume in the slower velocities was dominated by green
algae.

influence the periphyton architecture. Interestingly, the
periphyton structures in this study were observed within
the same range of velocities as those observed by Battin et
al. [2003]. Here the quasi-hexagonal microstructures were
generally parallel to the elements of the benthic net, as
shown in Figure 3.
[18] The structure of periphyton communities differed
among flow regimes in both taxonomic content and physical
attributes, as shown in Figures 3 and 4. As is common in
late-successional periphyton assemblages [Steinman et al.,
1989; Peterson and Grimm, 1992; Johnson et al., 1997],
algal species richness in 120-day communities was low in
all flow regimes, with just 12 taxa identified collectively
from all samples. Of these, 4 comprised the bulk of the algal
biovolume (Figure 4), and 6 met our criteria for inclusion
in MDS ordination (Figure 5). Ordination revealed clear
separation in taxonomic structure of assemblages developed
at 5 cm/s from those developed under slower velocities.
Under the fastest flow regime, there was significantly greater
contribution of the monoraphid diatom Achnanthidium
minutissiumum, and the small-celled, loosely filamentous
green alga, Stichococcus sp., and a reduced contribution of
two forms of the green alga Oocystis (one bright green with
distinct chloroplasts, one duller with less definitive cellular
contents). Given the similar size and shape of the two
observed forms of Oocystis, it is plausible that they repre-
sent cells of the same species in different states of physio-
logical condition, with increased prevalence of the
presumably ‘‘less healthy’’ duller form under slower flow
conditions because of the decreased supply of nutrients to
the periphyton community. Dominance of A. minutissimum
in communities developed in the highest flow velocity may
have stemmed from better nutrient supply rates associated
with this flow regime, as this diatom often responds
favorably to increased nutrient availability [Fairchild et
al., 1985; McCormick and Stevenson, 1989; Biggs et al.,
1998b]. Variation in algal community structure among flow
regimes was likely instrumental in establishing different
consortia of bacterial denitrifiers, as discussed below.
3.2. Effect of Flow Conditions on Denitrification
[19] Denitrification potential (DNP) rates, calculated per
unit area of benthic net, differed significantly among all
velocity treatments (p < 0.001) as shown in Figure 6a, with
sequential increases of about an order of magnitude for each
order-of-magnitude increase in velocity (r2 = 0.86, p < 0.001).
The DNP rates associated with the sand underlying benthic
nets were below detection limits (at least 1 order of
magnitude below the smallest DNP rate observed on the
benthic net). While DNP is expected to increase with
biomass, which also increased with increasing velocity, an
additional increase in DNP was evident when rates were
normalized to various measures of periphyton biomass, as
illustrated in Figure 6b. An increase with velocity was evident
for DNP normalized by AFDM and Chl a (p < 0.05). The
trends in DNP rates normalized by bacterial abundance
were less clear under the fastest and intermediate flow

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could limit denitrification during the illuminated part of the

Figure 5. Multi-dimensional scaling ordination of 120-
day-old periphyton samples developed under different flow
conditions in laboratory mesocosms. Data used in the
ordination were relative biovolume of the six most common
algal taxa encountered in samples, based on criteria
described in text. Ordination revealed clear separation in
taxonomic structure of assemblages developed at 5 cm/s
from those developed under slower velocities.
day, it was shown that anoxic conditions suitable for
denitrification occur in periphytic communities, even under
light conditions [Christensen et al., 1990; Lorenzen et al.,
1998]. Together, these attributes point to periphyton as a
potentially superior environment for denitrification.
[21] Here we observed that faster flow conditions fa-
cilitated a shift in microalgal community structure from
dominance by the Chlorophyte Oocystis in slower-flow
treatments to dominance by the diatom Achnanthidium
minutissimum. We attribute the high DNP rates observed
under the fastest flow condition (5 cm/s) to a combination of
the increased biomass resulting from enhanced nutrient
delivery to the periphytic microbial community and the
increased prevalence of diatoms, in this case almost exclu-
sively A. minutissimum, in the resulting microbial assem-
blage. For example, Figure 7 illustrates a positive relationship
between DNP rates and fractional biovolume of diatoms,

velocities (p = 0.064), though DNP was slightly higher in

the faster velocity than in the intermediate velocity. As
mentioned previously, periphyton developed in the three
flow conditions differed significantly in biomass measures
(Figure 2) and algal taxonomic structure (Figures 4 and 5).
Analysis of a subset of our samples with Terminal Restric-
tion Fragment Length Polymorphism (T-RFLP), and subse-
quent ordination by MDS, showed clear separation of the
fastest-velocity mesocosm from those in lower-velocity
mesocosms, which indicates significant differences in tax-
onomic representation within bacterial consortia grown
under different flow conditions [Ishida, 2005]. Thus, within
120-day-old periphyton assemblages developed under an
overlying velocity of 5 cm/s, some combination of attrib-
utes, likely biomass and algal species representation, pro-
moted establishment of a consortium of bacterial denitrifiers
more physiologically active than those associated with
periphyton established under slower flows.
[20] As a potential site for denitrification, periphytic
communities have two main advantages over microbial
communities residing within sediments. Denitrifying bacteria
within periphyton enjoy close proximity to microalgae as a
potential carbon source, and are less likely to be nutrient- Figure 6. Denitrification rates in periphyton. (a) Results
limited due to mass-transfer constraints. Both of these are expressed per unit area. (b) Results from Figure 6a, divided
likely explanatory factors behind Romani and Sabater’s by the weight of biomass (AFDM and Chl a) or number of
[2000] observation that periphyton supported significantly cells (bacterial abundance), and normalized to the highest
higher bacterial exoenzyme activity than biofilms in the value. Variability within each mesocosm was calculated on
same system that were primarily heterotrophic. While sedi- the basis of 4 – 10 independent samples, and is indicated
ments often support the anoxic conditions required for by the Box-Whisker presentation; the central line indicates
denitrification, a sustained supply of nitrate to instigate this the median value, the upper and lower boundaries of the
process, and an organic carbon source to fuel bacterial box indicates the 25th and 75th percentile values, and error
metabolism, is often lacking [Holmes et al., 1996]. In bars indicate the 10th and 90th percentile values. Missing
periphyton, in contrast, nitrate is typically the predominant whiskers are due to a low number of samples for the
form of inorganic nitrogen available, and dissolved organic 0.05 cm s1 treatment. Denitrification potential increased
carbon is regularly exuded from resident microorganisms. with overlying velocity, when compared per unit area and
Despite the fact that oxygen produced by photosynthesis also when normalized to biomass content.

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G01002 ARNON ET AL: FLOW, PERIPHYTON, AND DENITRIFICATION
Figure 7. Denitrification potential rates plotted versus the
relative biovolume of diatoms shows a positive trend, which
demonstrates a possible link between algal community and
denitrification (r2 = 0.583).
demonstrating a possible link between algal community
composition and denitrification. This general relationship
is probably more complex since the quality and quantity of
dissolved organic carbon produced by algal communities
varies with microalgal taxonomic composition [McFeters et
al., 1978; Battin et al., 2003; Kostel, 2006]. Such variation
is manifested at the species level, with observations that
diatom species, some within the same genus, produce
organic exudates that differ chemically [Myklestad, 1995;
Smith and Underwood, 2000; Hamels et al., 2004] and
support three-dimensional organic layers over their silica
frustules that differ the density and type of amphoteric
active sites [Gelabert et al., 2004]. There is also good
evidence that bacterial taxa vary in ability to exploit
different algal-derived carbon sources. For example, both
Grossart et al. [2005] and Schäfer et al. [2002] used Direct-
Gradient-Gel Electrophoresis (DGGE) to illustrate that
different diatom species support distinctive and stable
consortia of accompanying bacterial communities associate
with their cell surfaces. Finally, the quantity and quality of
carbon available to denitrifying bacteria have been shown to
affect denitrification activity [e.g., Hill and Cardaci, 2004].
Field data from three different wetlands revealed that
periphyton established on the same type of benthic net
employed here showed significant positive relationships
between DNP rates and the relative abundance of the diatom
genera Achnanthidium and Nitzschia, but not other diatoms
or nondiatom algae [Ishida, 2005]. Sirivedhin and Gray
[2006] observed similar processes in a constructed wetland
and found periphytic communities resident on benthic nets
produced higher DNP rates than microbial communities
residing in the underlying sediments. In addition, the
consistently high bulk nitrate removal, despite seasonal
and temperature variation, was attributed to the periphyton
community supported on the benthic nets. These authors
hypothesized that superior DNP rates in the periphyton
arose from the close coupling between the consortium of
microbial denitrifiers and algal component of the periphy-
ton, which provides a favorable and highly degradable
carbon source for denitrifiers. Our results support Sirivedhin
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G01002
and Gray’s hypothesis, and further illustrate that hydrody-
namic transport conditions play a critical role in these
ecological relationships.
[22] This study clearly demonstrates that flow conditions
shape the structure and ecological function of benthic
microbial communities. Such connections have been dem-
onstrated previously for faster flow conditions, showing the
effect of shear stress on biofilm structure and mass transfer
processes on its ecological function. Our results show that
flow variations can influence periphyton structure and its
functional attributes even under the slow flow conditions
characteristic of wetlands, river backwaters, and other
similar aquatic habitats. While we demonstrate that flow
history shapes the structure of the microbial community,
including both the physical structure and the microbial
community assemblage, further study is needed to explicitly
link this behavior to the effects of flow conditions on mass
transfer of nutrients and other biologically important solutes
to cells resident in the benthic biofilm. A better understand-
ing of these relationships will improve assessment of the
benthic habitat characteristics favorable for different micro-
bial populations and enhance our ability not only to manage
but also to predict the effect of excess nutrients in aquatic
ecosystems.
[23] Acknowledgments. We thank Martina Hausner for CLSM sup-
port, and Lisa Marx and Gabriel Singer for laboratory assistance and useful
discussions. This work was supported by grant CSREES-2002-01199 from
the United States Department of Agriculture (to K. A. G and A. I. P).
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S. Arnon, Department of Environmental Hydrology and Microbiology,
Zuckerberg Institute for Water Research, Blaustein Institutes for Desert
Research, Ben-Gurion University of the Negev, Sede Boqer 84990, Israel.
(sarnon@bgu.ac.il)
K. A. Gray, and A. I. Packman, Department of Civil and Environmental
Engineering, Northwestern University, 2145 Sheridan Road, Evanston, IL
60208-3109, USA (k-gray@northwestern.edu; a-packman@northwestern.
edu)
C. G. Peterson, Department of Natural Science, Loyola University, 6430
N. Kenmore Avenue, Chicago, IL 60626, USA. (cpeters@luc.edu)

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