Freshwater Biology (1991) 25, 437-450

Biofilm development on leaf and wood surfaces in a boreal river

S. W. GOLLADAY* and R. L. SINSABAUGH Department of Biology, Clarkson

University, Potsdam, NY 13699-5805, U.S.A.

SUMMARY. 1. Biofilms are organic layers that develop on submerged

surfaces. They are composed of micro-organisms, exoenzymes, and de-
tritus particles enclosed within a gelatinous matrix. While much is
known about mineral surface biofilms, those developing on organic sur-
faces have not been extensively studied. We examined the influences
of current velocity and substratum composition on biofilm development
in a fourth-order North American boreal river.
2. Arrays of white birch ice-cream sticks and sugar maple leaves were
placed at fast and slow current sites. Samples were collected periodically,
analysed for mass loss, and assayed for microbial biomass (ATP. ergos-
terol, chlorophyll a) and exoenzyme activity associated with hgnocellulose
degradation (exo- and endocellulase. ^-glucosidase, phenol oxidase,
peroxidase).
3. Biofilms developed rapidly on both surfaces. On leaves, biomass
peaked within 30 days of exposure. On wood, ATP and chlorophyll a
concentrations peaked within 30-70 days, whereas ergosterol increased
throughout the study (161 days). On leaves, current velocity had little
influence on biofilm development, although breakdown rates were greater
at the fast flow site. On wood, ATP and chlorophyll a concentrations
were greater at the fast flow site, whereas ergosterol concentrations and
breakdown rates were similar at both sites. Microbial biomass was con-
sistently greater on wood than leaves. Exoenzyme activity developed
rapidly on both surfaces; current velocity had little influence on activity.
Except for p-glucosidase, activities were greater on wood than leaves.
4. Our results suggest that fungi are an important structuring element
of organic surface biofilms and the physical stability of the substratum
strongly influences biofilm development. Leaf surfaces are susceptible to
softening and fragmentation, truncating biofiim development. In contrast,
abrasion of wood surfaces removes senescent material exposing fresh sub-
stratum for colonization. Thus, wood surfaces with their greater physical
stability, permit the development of more extensive biofilms. Wood
surfaces may represent an overlooked but important site of metabolic
activity in streams.
• Correspondenee: Dr S. W. Golladay. University of Oklahoma Biological Station HC-71 Box 205
Kingston. OK 73439, U.S.A.

437
438 S, W, Golladay and R. L. Sinsabaugh
Introduction
Biofilms are the organic layers that form on
submerged surfaces in streams or other aquatic
environments. They are composed of micro-
organisms (bacteria, algae, fungi, protozoa, and
micrometazoa), exoenzymes, and detritus par-
ticles enmeshed in a gelatinous poiysaccharide
matrix (glycocalyx) (Geesey et aL, 1978; Lock
etal., 1984). Because of their ubiquitous nature,
biofilms are probably the site of considerable
metabolic activity in many stream ecosystems.
Many aquatic micro-organisms, particularly
bacteria, are metabolically most active when
associated with surfaces {Lock et al., 1984).
As biofilms develop, micro-organisms secrete
a poiysaccharide matrix which may promote
retention of organic matter and exoenzymes.
and act as an ion exchange medium (Geesey et
aL, 1978; Lock et al., 1984). As biofilm devel-
opment continues, a complex and vertically
stratified community may form, with aerobic
autotrophic and heterotrophic micro-organisms
dominant near the surface and micro-organisms
tolerant of lower oxygen concentrations near the
base (Costerton et al.. 1987). The depth of bio-
films and extent of stratification are probably
strongly determined by flow regime, disturb-
ance history, and foraging activities of meio- and
macrofauna (e.g. Locketal., 1984; Pringle. 1985;
Power & Stewart. 1987; Steinman etal.. 1987;
Pringle et al., 1988; Grimm & Fisher, 1989).
Although biofilms develop on all submerged
surfaces, those developing on mineral substrata
(epilithic) have been most intensively investi-
gated. Epilithic biofilms are frequently domi-
nated by bacteria, cyanobacteria. and algae
(Geesey e/fl/.. 1978; Lock f/a/., 1984). Epilithic
biofilms developing on well-lit surface.s may be
dominated by autotrophs, with their hetero-
trophic components relying on algal exudates
and senescent cells as a source of nutrition
(Geesey et al., 1978; Rounick & Winterbourn
1983). Those developing in heavily shaded
streams or on the undersides of rocks tend
towards heterotrophy, relying on uptake of dis-
solved and particulate organic matter as a nu-
tritional source (Rounick & Winterbourn, 1983;
Lock era/., 1984).
Dissolved organic matter (DOM) appears to
be particularly important in promoting epili-
thon development and activity. Bott, Kaplan &
Kuserk (1984) estimated that DOM supported
roughly 55% of the epilithic bacterial biomass
in a woodland spring seep. Epilithic bacterial
cells appear to prefer low molecular weight,
labile DOM sources, which are relatively easy
to transport across cell membranes (Kaplan &
Bott, 1983). However, the abundance of these
compounds is temporally variable, often present
only as short-lived pulses following periods of
rainfall and senescence of riparian vegetation
(Kaplan & Bott, 1985). The temporal varia-
bility of DOM poses a problem for epilithic
communities. Micro-organisms must maintain
metabolic activity in order to utilize DOM during
transient periods of availability, but also must
conserve resources or rely on alternate, more
refractory compounds during periods of low
DOM availability (Kaplan & Bott, 1985). Thus,
the structure and function of epilithic biofilms
must be strongly influenced by the availability
of DOM or alternate sources of nutrition.
Biofilm development on organic substrata has
been less intensively studied, although a variety
of micro-organisms, especially fungi, colonize
leaf and wood surfaces (e.g. Willoughby &
Archer. 1973; Suberkropp & Klug. 1974; 1976;
Lamore & Goos, 1978; Chamier & Dixon, 1982;
Shearer& Von Bodman, 1983; Chamier. Dixon
& Archer. 1984). From a biofilm perspective,
organic surfaces represent a metabolic substrate
as well as a surface for colonization. Thus,
microbial communities that develop on organic
surfaces may be less dependent on external
energy sources. The purpose of this investigation
was to examine biofilm development on organic
surfaces in a stream. We hypothesized that fungi
would be an important structuring element of
organic surface biofilms because they are excel-
lent colonizers of organic matter. We also hypo-
thesized that the extent of biohim development
on organic surfaces would be limited by the
physical stability of the substrate. As wood
surfaces have a greater physical stability than
leaves they are potentially the site of greater
biofilm development. We evaluated these hypo-
theses by measuring structural and functional
indicators of biofilm development on leaf and
wood surfaces.
SKe description
This work was conducted in the St. Regis River,
a fourth-order stream, which originates in the
Adirondack Mountains of New York (Franklin

County, elevation 610 m) and flows northward
into the St. Lawrence River (Huntingdon
County, elevation 46m). The St. Regis River is
a brown-water stream (OD 254=0.14-0.48),
with dissolved/colloidal organic carbon concen-
trations ranging from 5 to 19mgP'. As it flows
downstream from the Adirondack Mountains it
traverses calcareous bedrock, raising the pH
slightly above neutral (7.4—7.6).
Our study site was located on the West Branch
of the St. Regis in the town of Stockholm
(44= 42' 51"N 74" 54'W). The channel was c.
30-50m wide with an average gradient of 1:240
and an elevation of 116 m. The substratum is
composed of exposed bedrock, boulders, and
cobbles. A small island near the west bank
creates a slow-flowing side channel 6-8m wide.
Discharge is regulated by an impoundment
13.3 km upstream from the study area. Diversion
of water for power generation causes wide and
irregular discharge fluctuations during the sum-
mer months. In the main channel, mean depth
ranges from 0.25 to 1.0 m with average velocities
of O.I-l.Om s " . In the side channel, mean
depth ranges from 0.5 to 1.0 m with an average
velocity ranging from 0.01 to 0.1m s~'. The
annual range of temperature at the study site
was 0-25''C.
Methods
Sample arrays
Biofilm development was examined on two
organic substrata, white birch (Betulapapyrifera
Marsh.) ice-cream sticks (11.5x2.0x0.2 cm) and
sugar maple (Acer saccharurn Marsh.) leaves.
Ice-cream sticks were purchased from a com-
mercial supplier. Leaves were collected at abscis-
sion from a single tree on the flood plain of the
river. Our sampling units were 8—lOg of air-
dried substrata enclosed in a 0.5-cm mesh nylon
bag (18x30cm). Bags enclosing sticks were
partitioned in eight sections with each section
containing a single stick. Leaves were placed
inside unpartltioned bags.
On 30 May 1989, arrays of sampling units
were tethered to chains securely staked to the
substrata in the main channel (high velocity
site) and side channel (low velocity site). Leaf
packs were collected after 24 h, then weekly for
1 month, then bi-weekly until breakdown was
complete (mid-August). Stick packs were col-
Biofilms on organic surfaces 439
lected after 24 h. bi-weekly for 3 months, then
monthly through November 1989.
On each sampling date, six units of leaves
and/or sticks were removed from each site,
placed on ice. and transported to the laboratory.
The contents of three were rinsed with tap-
water, dried in paper bags (50^C. 48 h) and
weighed to determine mass loss. The contents
of the remaining packs were carefully removed
from their enclosing bags and kept moist in
basins of river water. Leaves were punched into
2-cm diam. discs, and sticks were cut into halves.
Leaf discs or stick halves were then u.sed in
analytical procedures to determine the extent of
biofilm development. All extractions and assays
were usually completed within 24-36 h of sample
collection.
Laboratory protocols
The ATP content of leaf and wood biofilms
was determined using a modified cold extraction
procedure similar to that of Lee et al. (1971).
ATP is considered an indicator of total living
community biomass. Subsamples were weighed
wet and placed into 5 ml of of 5mM MgSO^/
2.5 mM EDTA buffer. ATP was extracted by
adding 5 ml of 1.2 N H2SO4. After 10 min. sam-
ples were removed and the pH of the extract
was adjusted to 7.5-7.75 with 5 N NaOH. Ex-
tracts were then frozen for storage (Anonymous.
1985). ATP concentrations were measured using
the luciferin—luciferase reaction and an LKB
1250 luminometer. Counting efficiencies were
estimated for each sample by the addition of
internal standards.
The ergosterol content of biofilms. an indicator
of fungal biomass, was measured following the
procedures outlined by Newell. Arsuffi & Fallon
(1988). Subsamples were weighed wet, placed
into 50 ml of methanol. and refluxed for 2h.
After filtering the undigested material, extracts
were saponified by the addition of 5 ml of 4%
KOH in ethanol and refluxed for 0.5 h. Extracts
were cooled and then transferred to 125 ml separ-
ation funnels containing 10 ml of distilled water.
Each sample was extracted three times with
pentane (10, 5. 5 ml). Pentane extracts were
combined and evaporated in a fume hood. The
residue was redissoived in 1.0 ml of HPLC-
grade methanol. Ergosterol wasquantified using
a reverse-phase HPLC system configured as
follows: solvent = methanol; flow rate = 2 ml
440 S. W. Golladay and R. L. Sinsabaugh
min"'; column = Altex Ultrasphere ODS
4.6mmxl5cm; absorbance detector = 282nm
with a range of 0.500; quantification = Waters
745B integrator with attenuation at 16; ergosterol
retention time = 4.1 min; replication = three
per sample; standard = 25tig m l ' solution of
ergosterol and methanol.
Chlorophyll a concentrations, with corrections
for phaeophytin, were determined spectropho-
tometrically (Anonymous. 1985). Chlorophyll a
is an indicator of algal biomass. Samples were
extracted in 25 ml of 90% MgCO.i-saturated
aqueous acetone. The suspensions were covered
and placed in the dark for 24 h at 8°C. An ab-
sorption spectrum from 5(X) to 750 nm was gen-
erated for each extract using a Gilford Response
spectrophotometer, with chlorophyll a concen-
tration estimated at an absorbance of 664 nm.
Extracts were then acidified and rescanned to
determine phaeophytin concentration, which was
estimated at an absorbance of 665 nm.
Leaves and wood were also analysed for sev-
eral classes of exoenzymes prominent during
the decomposition of lignocellulose. Samples
for exoenzyme assays were homogenized in
100ml of 50mM acetate buffer (pH 5) using a
Brinkmann poiytron. All assays were conducted
at 2rC with four replicates and two controls
per sample. Enzyme activities were expressed
as Mmole of substrate converted h ' cm "^.
Exocellulase activity was estimated by the
generation of reducing sugar from microcrystal-
line cellulose (MCC). Two ml of homogenate.
2 ml of 2% MCC in acetate buffer, and a drop
of toluene (microbial inhibitor) were tumbled
in a platelet mixer for 18 h in a capped 5-ml
polyethylene test tube. After incubation, the
tubeswere centrifuged and 1.0 ml of supernatant
was withdrawn for colorimetrie quantification
of reducing sugar using the method of Nelson
(1944). Controls were prepared by mixing 2 ml
of homogenate and 2 ml of MCC, followed by
immediate centrifugation and recovery of the
supernatant. The rate of reducing sugar gener-
ation from MCC is linked to exocellulase activity
because exocellulase can bind to crystalline cel-
lulose and hydrolyse cellobiose and glucose from
non-reducing ends of the polymer. However,
this assay is not specific for exocellulase activity
because hydrolysis of crystalline cellulose re-
quires the interaction of both exocellulase and
endocellulase.
Endocellulase activity was estimated viscome-
trically using the method of Almin & Eriksson
(1967). One ml of homogenate was mixed with
2 ml of 1% carboxymethyl cellulose in a 5-ml
test-tube. The tubes were capped and tumbled
for a variable time (0.25-2 h) depending on ac-
tivity. After incubation, the tubes were centri-
fuged and viscosity estimated by the fall velocity
of the fluid in a 0.1-m! glass pipette. Activity
was calculated in relative units based on the
difference between initial and final viscosity.
P-glucosidase activity was determined using
p-nitrophenyl-B-D-g!ucopyranoside dissolved in
acetate buffer as a substrate. Two ml of homoge-
nate and 2 ml of substrate (initial substrate con-
centration was lOmM) were placed in 5-ml
test-tubes which were capped and tumbled for
l - 3 h . After incubation, the samples were cen-
trifuged and 2 ml of supernatant was transferred
from each into a test-tube containing 0.2 ml of
1.0M NaOH. Eight ml of water were added to
each tube, and absorbance was measured at
410nm. Duplicate controls containing 2ml of
homogenate plus 2 ml of buffer and 2 ml buffer
plus 2 ml of substrate were processed in parallel.
Phenol oxidase activity was estimated by
incubating 2 ml of homogenate with 2 ml of
lOmM L-3,4-dihydroxyphenylalanine (i.-dopa)
for lh with continuous tumbling. Following
centrifugation, the absorbance of supernatant
was measured at 460nm. Controls were pre-
pared by mixing 2 ml of homogenate with 2 ml
of acetate buffer. Peroxidase activity was assayed
in parallel by mixing 2 ml of homogenate. 2 ml
of L-dopa. and 0.2ml of 0.3% H2O2. Controls
contained 2 ml of homogenate, 2 ml of buffer,
and 0.2 ml of H2O2. Peroxidase activity was
calculated as the difference between substrate
oxidation rates in the presence and absence
of peroxide. A more detailed description of
enzyme assay procedures has been reported
(Sinsabaugh & Linkins, 1990).
ResuKs
Breakdown rates
Breakdown rates (-k) calculated from re-
gressions In (% AFDM remaining) versus time
for each substratum and exposure site were
significantly different from zero (Table 1) (linear
regression. P<O.(X)1) (Webster & Benfield.
1986). At both sites, a comparison of the slopes
of regression lines (-k) indicated that leaves

TABLE 1. Breakdown rales (~k) of leaves and wood. Tjo values are esti-
mates of time until 50% mass loss based on regressions of ln(% remaining)
versus limc
Substrate Site Breakdown
rate
(day-')
Leaves Slow 0.0237
Fast 0.0484
Wood Slow 0.0016
Fast O.fX)19
decayed significantly faster than sticks (analysis
of covariance, P<0.001) (Sokal & Rohlf, 1969).
Leaves placed in the fast current site lost weight
more rapidly than those al the slow current site
(analysis of eovariance, P<0.001). There was
no difference between sites for stick breakdown
rates (analysis of covariance, P>0,05), Times
until 50% mass loss (^50) for leaves were esti-
mated to be 12 and 21 days at the fast and slow
sites, respectively. For sticks, T$Q values were
estimated to be 364 and 435 days at the fast and
slow sites, respectively.
Structural indicators of biofilm development
Biofilms developed rapidly on leaves and
wood at both fast and slow current sites (Fig.
1). Leaf ergosterol standing stock peaked eariier
at the fast current site than at the slow current
site, suggesting slightly faster colonization and
decline by fungal populations. However, with
the exception of day 21. there were no signifi-
cant differences between ergosterol standing
stocks at the fast and slow sites (two-sample t-
tests by day, P>0.05).
Ergosterol standing stocks on wood generally
increased throughout the study. With the ex-
ception of the last sample date (day 161) there
were no significant differences in ergosterol
standing stocks at the fast and slow current sites
(two-sample Mests by day. P>0.05). Ergosterol
standing stocks on wood equalled or exceeded
those observed on leaves by day 14 at the fast
site and day 42 at the slow current site. When
averaged over the entire study, ergosterol stand-
ing stocks on wood were seven times greater
than leaves at the fast site and six times greater
at the slow site.
Chlorophyll a standing stocks were relatively
variable on both substrata at both current sites.
Biofilms on organic surfaces 441
SE 7-50 r n
(days)
0.0022 21 0.94 8
0.0066 12 0.91 6
0.0001 435 0.96 8
0.0002 364 0.94 S
On leaves, algal colonization proceeded some-
what faster at the fast current site (Fig. 1);
however, there were no significant differences
in chlorophyll a standing stocks between sites
(two-sample /-tests by day, P>0.05).
On wood, chlorophyll a standing stocks were
consistently greater at the fast current site com-
pared to the slow site, with statistically signifi-
cant differences on days 28 and 42 (two-sample
/-tests by day, P<0.05). At both fast and slow
sites chlorophyll a standing stocks on wood
exceeded those observed on leaves after 14
days of exposure. When averaged over the
entire study, chlorophyll a standing stocks on
wood were thirty-six times greater than those
on leaves at the fast site and ten times greater at
the slow site.
ATP standing stocks exhibited patterns of
development similar to the other biomass indi-
cators (Fig. 1). On leaves, mean ATP concen-
trations peaked within 14 days of exposure. From
day 14 onward, mean ATP standing stocks were
greater on leaves exposed at the slow current
site, differences were significant on days 21. 28.
and 42 (two-sample /-tests by day, P<0.05).
At both sites, ATP concentrations on wood
peaked at day 63 then declined to relatively
constant concentrations. Mean ATP standing
stocks suggested a consistent pattern of greater
microbial community development on wood at
the fastflowsite; however, there were no signifi-
cant differences in individual comparisons by
date (two-sample /-tests by day, P>0.05). At
both fast and slow fiow sites, ATP standing
stocks on wood exceeded those on leaves by
day 14 of the study. When averaged over the
entire study, ATP standing stocks on wood
were seven times greater than on leaves at the
fast flow site and four times greater at the slow
fiow site.
442 S. W. Golladay and R. L. Sinsabaugh
0.75
(0)
k\
Leoves
0.000
25 50 75 100 125 150
FIG. I. Mean (±1 SE) standing stocks of microbial biomass indicators oo leal (a.c,e) and wood (b,d,0
surfaces at fast (•) and slow (o) velocity sites in the St. Regis River.
Functional indicators of biofilm development,
cellulolytic enzymes
On both leaves and wood, exoenzyme activi-
ties increased rapidly (Figs 2 & 3). On leaves,
exocellulase activities peaked by day 7. There
were no significant differences among sites (two-
sample r-tests by day. /'>0.05). On wood, exo-
celtulase activities generally increased through
day 161 with no consistent differences between
fast and stow flow sites. Exocellulase activity
developed more rapidly on leaves than wood
at both sites. However, by day 28 activities
measured on leaves and wood were comparable
and. when averaged over the entire study, exo-
cellulase activity on wood was 2 - 3 times greater
than that measured on leaves.
Endocellulase activity on leaves peaked be-
2.5
(d)
2.0
1.5
1.0
0.5
0.0
0.25
0.20
O.i5
0.10
0.05
0.00
100 125 150 175
Days
tween 14 and 21 days with no significant differ-
ences in activity between sites (two-sample
Mests by day. P>0.05). Endocellulase activity
on wood was highly variable, although general
increases were observed at both siies over the
study. Endocellulase activity appeared to develop
more rapidly on wood at the fast flow site; by
day 28 activity was significantly greater than at
the slow site (two-sample /-tests by day, /'<0.05).
By day 63, endocellulase activity had declined
at the fast fiow site and activities at the slow
flow site were significantly greater (two-sample
/-tests by day, P<0.05). For days 91-161 there
were no significant differences between endocel-
lulase activities measured on wood at the fast
and slow flow sites (two-sample /-tests by day.
P>0.05). By day 28, endoceltulase activities on
wood at both sites exceeded those on leaves;
 BiofUms on organic surfaces 443

0 25 50 75 100 125 150 25 50 75 100 125 150 175

Doys
FIG. 2. Mean ( i l SE) activities of cclluiolytic exoenzymes on leaf (a.c.e) and wood (b,d.O surfaces at fast
(•) and slow (o) current vclocily sites in the St. Regis River.

activities on wood were thirty-eight times higher
than leaves at the slow flow site and thirty-six
times higher at the fast fiow site.
On leaves, p-glucosidase activity appeared to
develop more rapidly at the slow flow site; on
day 7 activity was significantly greater than that
measured at the fast fiow site (two-sample /-test
by day, P<().05). However, by day 14 p-gluco-
sidase activity on leaves at the fast flow site had
exceeded that measured at the slow flow site
(two-sample r-test by day, P<Q.O5). From day
21 to the end of the study there was no difference
in activity on leaves at the fast and slow current
sites. On wood, ^-glucosidase activities generally
increased over the duration of the study at both
the fast and slow current sites. Activities were
significantly greater on wood at the slow flow
site on days 63 and 161 (two-sample /-tests by
day. /*<0.05). (i-glucosidase activities on leaves
generally exceeded those measured on wood.
When averaged over the study, the wood/leaf
activity ratio was 0.6 at the slow current site and
0.5 at the fast current site.
Ligninolytic enzymes
Phenol oxidase activity on leaves peaked
within 21 days of exposure at both sites (Fig. 3).
There were no consistent differences in phenol
oxidase activities between sites. On wood, phenol
oxidase activity generally increased throughout
the study at both sites. From day 63 to day 161,
activity was significantly greater on leaves at
the slow fiow site (two-sample /-tests by day,
P<0.05). Concentrations on wood generally
exceeded those on leaves by day 14 at the fast
444 S. W. Golladay and R. L. Sinsabaugh
0.05r
IV
0.00
0 25 50 75 100 125 150
Days
FIG. 3. Mean (±1 SE) activities of ligninolytic exoenzymes on leaf (a,c) and wood (b,d) surfaces al fast (•)
and slow (o) current velocity sites in the St. Regis River.
site and by day 28 at the slow site. When
averaged over the study, phenol oxidase activities
on wood were seven times greater than on
leaves at both sites.
On leaves, peroxidase activities generally
increa.sed throughout the study at both sites.
Peroxidase activity on leaves at the slow site
was significantly greater than the fast site on
day 28 (two-sample /-tests by day. /'<0.05).
Peroxidase activities on wood increased through
day 91 at the slow fiow site and day 126 at the
fast flow site. From day 61 onward, average
activities were generally greater on wood at the
slow flow site, differences were significant on
day 91 (two-sample /-test by day, P<d.O5).
By day 14, peroxida.se activities on wood were
greater than on leaves at both sites. When
averaged over the study, peroxidase activities
on wood at the fast flow site were eleven times
greater than leaves and five times greater at the
slow flow site.
Correlations among indicators of biofilm
development
Correlation analysis was performed on both
structural and functional indicators of biofilm
0 25 50 75 100 125 150 175
development. For leaves, twelve of the thirty-
six possible correlations among indicators were
statistically significant at the fast flow site, and
seventeen of the thirty-six at the slow fiow site
(Pearson coefficient of correlation, P<0.05).
Correlation analysis revealed several general
patterns of biofilm development on leaves at
both fast and slow current sites. Exoenzymes
involved in cellulose degradation were mutually
correlated at both sites (range of r=0.48-0.80
fast, « = 18; r=0.42-0.77 slow. H=24) reflecting
relatively early peaks in cellulolytic enzyme
activities on leaves (Fig. 2). At both sites the
strongest correlations were between endocellu-
lase and l^-glucosidase activities and the weakest
were between exocellulase and the other cellu-
lolytic enzymes.
Ligninolytic enzyme activities were correlated
with exposure time on leaves at both fast and
slow current sites (fast r=0,59 and 0.82. n= 18;
slow ;-=0.41 and 0.72, /i=24; phenol oxidase
and peroxidase. respectively). At both sites,
correlations of phenol oxidase and time were
relatively weak, but correlations of peroxidase
activity with time were stronger. Although both
exoen2yme activities tended to peak towards
the end of the study (Fig. 3). they were not
mutually correlated at either site.

At both sites, there was a negative correlation
between exocellulase activity and peroxidase
activity (fast r=-0.82, n=18. slow r=-0.72.
0=24). At each site, exocellulase activity peaked
very early, whereas peroxidase activity was much
slower to develop.
On leaves, structural indicators of biofilm
development (ATP, ergosterol, and chlorophyll)
were not consistently correlated with either ex-
posure time, exoenzyme activities, or each other.
The only consistent pattern observed at both
sites was a tendency for ergosterol to correlate
with endocellulase and p-glucosidase activity
(fast r=0.65 and 0.52, «-18. slow r=0.73 and
0.73, n=24; for endocellulase and p-glucosidase,
respectively). At both sites, ergosterol. endocel-
lulase. and [i-glucosidase peaked during the
middle portions of the study (Figs I & 2).
Correlation analysis of biofilm indicators on
wood were statistically significant for twenty-
two of thirty-six comparisons at the fast flow
site and twenty-five of thirty-six comparisons at
the slow flow site (Pearson coefficient of corre-
lation. P<0.05). Several general patterns of
biofilm development on wood emerge from this
analysis. At both current sites, exocellulase,
|3-glucosidase, and phenol oxidase activities
were strongly correlated with exposure time
(fast, range r=0.83-0.95, /i=24; slow, range
r=0.84-0.96, n=24). and the three enzymes
were mutually correlated (fast, range r=0.77-
0.88, n=24; slow, range r=0.82-0.90, /j=24).
Correlations between the other exoenzymes and
exposure time were weaker (fast, range r =
0.43-0.63, «=24; slow, range r=0.59-0.8O).
On wood at both sites, ergosterol was the
only structural indicator of biofilm develop-
ment that consistently correlated with either
exposure time (fast r=0.85, /i=24; slow r=^0.95)
or exoenzyme activities. Correlations were ob-
served between ergosterol and exocellulase,
(i-glueosidase, and phenol oxidase activities (fast,
range r=0.71-0.83, /i=24; slow, range r=0.S2~
0.%, M=24). Weaker correlations were observed
at both sites between ergosteroi and endocel-
lulase and peroxidase activities. This analysis
reflects the general observation that ergosterol
standing stocks and exoenzyme activities in-
creased consistently over the course of the study
(Figs 1-3). There was little tendency for the
other biomass indicators (chlorophyll or ATP)
to covary with exposure time, exoenzyme ac-
tivities, or each other.
Biofilms on organic surfaces 445
Discussion
The leaf breakdown rates reported in this study
are among the fastest reported in the literature
(e.g. Webster & Benfieid. 1986). We attribute
rapid processing to the combined effects of
relatively high stream temperature, microbial
processing, and physical abrasion. A positive
relationship between mass loss and stream tem-
perature has been observed in numerous studies
and it is generally assumed that warm conditions
promote microbia! community development
(reviewed by Webster & Benfield, 1986; Findlay
& Arsuffi, 1989). In this study, stream tempera-
ture was 20—25°C during the period when leaves
were being exposed, stimulating rapid biofilm
development (Figs 1-3). Within 21 days of
exposure, leaves were noticably softened, pre-
sumably due to the effects of exoenzyme activity.
Following softening, leaves were rapidly skel-
etonized especially at the fast current site.
There are a number of avenues for mass loss
during leaf processing in streams, including
leaching of dissolved organic matter (DOM),
biological assimilation or conversion to fine par-
ticulate organic matter (FPOM). and physical
abrasion. In the St. Regis River, physical abra-
sion appears to be an extremely important factor
determining leaf breakdown rates and probably
accounts for observed differences between sites.
Several observations support this conclusion.
Leaching losses generally are greatest within 24 h
of exposure in streams (Petersen & Cummins.
1974; Webster & Benfield. 1986). In this study,
leaves lost 12-14% of original AFDM during
the first 24 h of exposure at both sites. Thus,
while leaching represents a significant avenue of
mass loss, it probably does not contribute to
differences between sites.
Biological processing also contributes to mass
loss from decomposing leaves. Our measures
of biofilm development suggest that microbial
communities on leaves are structurally and func-
tionally similar at fast and slow current sites
in the St. Regis River. Thus, differences in
microbial processing probably do not account
for the differences in breakdown rates between
fast and slow current sites. However, there is
little doubt that microbial activity results in
significant loss of leaf mass both through direct
assimilation and conversion to FPOM or DOM
(Findlay & Arsuffi, 1989).
446 S. W. GoUadav and R. L. Sinsabaugh
Invertebrate feeding activities also contribute
to mass loss from leaves (Wallace. Webster &
Cuffney, 1982; Webster, 1983). Surveys of the
invertebrate fauna of the St. Regis River have
revealed that shredding invertebrates, (those
feeding on whole leaves) are rare (Golladay et
aL. 1989b). The most abundant macroinvert-
ebrate group colonizing leaves were larval
Chironomidae, generally classified as collector/
gatherers or predators. Their activities probably
contribute to decomposition primarily through
their influence on microbial biofilms; retreat
building has been correlated with the rate of bio-
film development (Sinsabaugh & Linkins, 1988).
The wood breakdown rates observed in the St.
Regis River are also among the fastest reported
in the literature (e.g. Golladay & Webster.
1988). Wood processing rates are influenced
by chemical composition (e.g. Melillo et al.,
1983) and exposure method (e.g. Golladay &
Webster. 1988). White birch is considered a
chemically labile wood type (low percentage
lignin, low lignin/nitrogen ratio, Melillo et al..
1983). Exposure method influences decompo-
sition because slow oxygen diffusion rates gen-
erally restrict decomposer activity to surface
layers of submerged wood (Triska & Cromack,
1980; Aumene/a/.. 1983; Harmon c/a/.. 1986).
Thus, breakdown rates are strongly influenced
by shape of wood, i.e. surface area to volume
ratios, making it difficult to compare results of
studiesusingdifferent sized pieces of wood. The
ice-cream sticks tised in this study had a relatively
large surface area compared to their volume, and
breakdown rates we observed (0.0016-0.0019
day"') were comparable to those reported by
Melillo et al. (1983) for a variety of wood chips
(range 0.0007-0.0033 day"').
The slow processing rates of wood, compared
to leaves or other vascular plant material, are
generally attributed to its more refractory chemi-
cal composition and complex molecular arrange-
ment (e.g. Webster & Benfield. 1986). The
breakdown rates observed in this study generally
support that conclusion. We did not observe
extensive surfatx softening, nor signs of abrasion
and fragmentation during the first 63 days of
exposure even though stream temperatures
were relatively high (20-25°C) and biofilm de-
velopment was occurring. On day 63. mass loss
from sticks at both sites averaged approximately
8%. During this same period mass loss from
leaves exceeded 80% of original AFDM. How-
ever, by day 63, extensive biofilms had developed
on sticks and rate of mass loss began to acceler-
ate. During the next 30 days sticks at the fast
site lost an additional 12% and sticks at the slow
site lost an additional 9% of original AFDM.
During this period stream temperatures remained
relatively high and wood surfaces became notice-
ably softened and abraded. Abrasion was more
evident on sticks collected from the fast current
site, and probably contributed to the slightly
faster breakdown rates measured there. Visual
examination also revealed a greater abundance
of detrital material accumulated on slow site
sticks, while algae (diatoms and filamentous
chlorophytes) were more abundant on the fast
site sticks. Functionally, the biofilms at both
sites were quite similar.
As with leaves, the most abundant macroin-
vertebrate colonizere were larval Chironomidae.
However, during the middle portions of the study
larval Elmidae (Coleoptera) became increas-
ingly abundant. We observed shallow grooves
in the softened wood surfaces that were often
occupied by elmids and were produced by their
feeding activity. Natural wood collected from
the St. Regis River is often extensively grooved
and elmid larvae are generally abundant col-
onists (Golladay et aL, 1989b). Similar patterns
of wood colonization and xylophagy have been
observed in Pacific north-western streams in
North America (Anderson, Steedman & Dudley.
1984; Steedan & Anderson. 1985). Thus, macro-
invertebrate feeding, as well as microbial pro-
cessing and physical abrasion, is probably a
significant avenue of mass loss for wood debris
in the St, Regis River.
Our results suggest that fungi, as indicated by
ergosterol standing stocks, are important in
the early development of biofilms on organic
surfaces. Many studies have documented the
importance of fungi, particularly aquatic hy-
phomycetes, as important early colonizers of
decaying leaf litter (Suberkropp & Klug, 1974,
1976; Chamier & Dixon, 1982; Chamier et al..
1984). As a group they are excellent penetrators,
elaborating pectinases and cellulases that en-
able them extensively to colonize leaf matrices
(Suberkropp & Klug, 1974. 1976; Chamier et
al,, 1984). Witliin3 weeks of exposure in the St.
Regis River, we observed peaks in exo-. endocel-
lulase, and |3-glucosidase activity on leaves,
those exoenzymes required for the complete
degradation and assimilation of cellulose.

Fungal colonization of leaves is probably a
random process originating from spores en-
trained in the water column (Chamier & Dixon,
1982; Chamier et al., 1984). As colonization
proceeds, aquatic hyphomycetes appear to form
'consortia' of a few mutually compatible species
(Shearer & Lane. 1983; Chamier et al.. 1984).
It has been hypothesized that fungal species
assemble because they have complementary
enzymes that permit efficient degradation of
structural polysaccharides (Chamier e/a/.. 1984).
However, other studies have suggested that
hyphomycete species have broadly overlapping
enzymological capabilities and their actual dis-
tribution is regulated by tolerance for physico-
chemical factors (temperature, nutrient), and
feeding by invertebrate grazers (Suberkropp.
Arsuffi & Anderson, 1983; Rossi 1985). Fungal
consortia may also competitively exclude other
fungi and some bacteria from leaf surfaces
(Chamier et aL, 1984). In a particular stream,
the same consortia of fungi appear to colonize all
litter types (Chamier & Dixon, 1982; Shearer &
Lane 1983). However, their rate of establishment
is influenced by leaf chemistry and fungal colon-
ization proceeds relatively slowly on refractory
leaf species (e.g. oak, Quercus spp.) (Suberkropp
& Klug, 1976; Chamier & Dixon 1982).
As leaf decomposition continues, funga! popu-
lations generally decrease in abundance and
bacterial populations increase (Suberkropp &
Klug, 1974, 1976; Findlay & Arsuffi, 1989).
Bacteria, while sometimes capable of degrading
lignoceilulose (e.g. Benner, Moran & Hodson,
1986), have a limited ability to penetrate leaf
cuticles and tend to be limited to leaf surfaces
(Suberkropp & Klug. 1974. 1976). Bacterial
biomass is generally less abundant on leaf sur-
laces than fungal biomass but has a faster turn-
over (Findlay & Arsuffi, 1989). Because of their
penetrating ability and size, fungal hyphae are
important in promoting the physical development
of leaf surface biofilms. whereas bacterial cells
probably contribute primarily to biofilm function.
Decreases in fungal abundance during the
later stages of leaf decomposition have been
associated with fragmentation and a diminished
planar surface area available for hyphal growth
(Suberkropp & Klug, 1974, 1976; Chamier et
aL, 1984). Fungal populations appear to be
limited by the physical stability and longevity of
the substratum, or perhaps simply the substratum
size. Presumably as leaf decomposition occurs,
Biofilms on organic surfaces 447
fragmentation reduces the suitability of leaf
material as a fungal substrate. In the St. Regis
River, we observed a decline in ergosterol and
ATP standing stocks that corresponded with
softening and fragmentation of leaf material.
This suggested that leaf surface biofilms were
dominated by fungal biomass during the early
stages of development and by other micro-
organisms as decomposition proceeded.
Leaf litter changes chemically as well as physi-
cally during decompo.sition. Relatively labile
chemical constituents, i.e. pectin, hemicellulose.
and cellulose, decrease in relative abundance,
whereas more refractory compounds, primarily
lignin represent a greater proportion of the re-
maining mass (Triska. Sedell & Buckley. 1975;
Suberkropp. Godshalk & Klug, 1976). During
the latter stages of leaf decomposition in the
St. Regis River we observed declines in cellu-
lolytic exoenzyme activity and increases in lig-
nolytic enzyme activity that probably reflected
the increasingly refractory composition of
decaying leaves. It appears that leaf surface
biofilms respond to both the changing physical
and chemical nature of their substrate.
The development of wood surface biofilms
has also been less intensively studied than those
developing on leaf or mineral surfaces. Yet, in
streams where it has been measured, wood
often comprises a majority of the organic matter
pool (e.g. Naiman & Sedell, 1979; Golladay et
al., 1989a). Like other surfaces, wood is col-
onized by micro-organisms following submerg-
ence. Fungi, including aquatic hyphomycetes
are important colonists and, with time, extensive
fungal assemblages develop on submerged wood
(e.g. Willoughby & Archer. 1973; Lamore &
Goos, 1978; Shearer & Von Bodman, 1983).
As with leaves, spore deposition is probably a
random process but with time relatively stable as-
semblages of a few common fungal species de-
velop (e.g. Sanders & Anderson. 1979; Shearer
& Von Bodman. 1983). Fungi isolated from
wood appear to produce a broad range of ligno-
cellulose degrading enzymes (Zare-Maivan &
Shearer, 1988).
As epixylic biofilm development continues,
exoenzyme activities increase and wood surfaces
begin to soften. Our study indicates that, while
colonization by fungi and other micro-organisms
occurs at comparable rates on both leaf and
wood surfaces, softening of the latter occurs
more gradually reflecting their more complex
448 5. W. Golladay and R. L. Sinsabaugh
molecular structure (Harada & Cote, 1985). As
softening continues, surface layers are removed
through the combined effects of physical and
biological activity. Epixyiic biofilm development
is unique in that removal of highly degraded
surface layers gradually exposes fresh material
beneath. Thus, the chemical composition of the
substrate remains relatively uniform throughout
decomposition, promoting a gradual accumu-
lation of a broad spectrum of lignocellulose
degrading exoenzymes. This is in contrast to
leaf surfaces, where the biofilm community and
abundance of exoenzymes change in response
to relatively rapid chemical and physical changes
in the substratum.
Subsurface layers of epixylic biofilms also act
as a refuge or anchor point for micro-organisms,
particularly fungi. Firm anchorage increases
the resistance to complete biofilm removal by
scouring or other disturbance while promoting
the accumulation of a relatively thick, temporally
stable, biofilm. In the St. Regis River, fungi are
an important structuring component of epixylic
biofilms and we observed relatively strong cor-
relations between indicators of fungal biomass
and indicators of biofilm function. Conceptually,
epixyfie biofilms can be thought of as a dynamic
biotic and abiotic assemblage, constantly pen-
etrating into fresh wood surfaces at the base,
and constantly eroding accumulated material
from the surface. This is in contrast to leaf
surface biofilms whose development is limited
by the ephemeral nature of the substratum, and
epilithic biofilms whose development is limited
by inert impenetrable substratum.
A final factor increasing the complexity of
epixylic biofilms is the activity of meio- and
macrofauna. Colonizing organisms can promote
biofilm development through retreat building
(e.g. Pringle. 1985; Sinsabaugh & Linkins. 1988)
or remove biofiims through feeding activity.
The net result of invertebrate activity is to
create a complex three-dimensional pattern on
wood surfaces composed of biofilm patches of
various ages. In the St. Regis River, natural
wood is often extensively grooved by invert-
ebrate activity. When viewed from a habitat
perspective, wood surfaces gradually become a
mosaic of microhabitat types providing coloniz-
ation sites for a variety of organisms with dif-
fering preferences or tolerances for current
exposure, light exposure, dissolved oxygen,
nutrient concentrations, and food.
Our results indicate that fungi are extremely
important in the development of biofilms on
both leaf and wood surfaces in streams. How-
ever, the physical instability of leaf litter makes
it susceptible to fragmentation, truncating biofilm
development. In contrast, on wood surfaces
fragmentation and abrasion removes senescent
surface layers making fresh material available
for hyphal growth. Thus wood surfaces, with
their greater physical stability, permit the de-
velopment of much more extensive biofilms.
Wood surfaces represent an overlooked but
potentially important site of metabolic activity
in streams.
Our observations, along with othere on the
microbial ecology of decomposition, have im-
portanl implications for the study of biofilm
development on organic substrata in aquatic
ecosystems. It would appear that biofilm devel-
opment is predictable with the rate influenced
by chemical nature of the substratum. Organic
surface biofilms are likely to be dominated by a
few fungal species whose abundance in turn, is
influenced by physical factors (i.e. temperature,
nutrients) (Suberkropp & Klug, 1974. 1976;
Suberkropp et aL, 1983), feeding activities of
invertebrates (Suberkropp et al., 1983; Rossi,
1985) and individual physiologies (e.g. mutual
tolerance, competitive ability, enzymatic pro-
duction) (Chamier e/a/.. 1984). Biofilm longevity
will be determined by the physical stability of
the substrata and magnitude of external disturb-
ances. Finally, emergent properties of biofilms
(e.g. respiration rates, decomposition rates,
nutrient immobilization) represent the cumulat-
ive individual physiologies modified by mutual
interaction within the biofilm matrix.
Acknowledgments
Financial support for this project was provided
by the School of Science at Clarkson University.
The senior author wishes to thank Dr A. E.
Linkins for providing laboratory space in which
to conduct this research. We thank Lisa
Raybum, Deborah Repert, and Tim Weiland
for assistance with laboratory analyses. Coral
McCallister of the University of Oklahoma
drafted the figures. Finally, we thank Dr A. G.
Hildrew and two anonymous reviewers for
many helpful comments on the first draft of this
paper.

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(Manuscript accepted 13 December 1990)