2013 - Romaní, Amalfitano, Artigas, Fazi, Sabater, Timoner, Ylla, Zoppini

Hydrobiologia (2013) 719:43–58
DOI 10.1007/s10750-012-1302-y
MEDITERRANEAN CLIMATE STREAMS
Review Paper
Microbial biofilm structure and organic matter use
in mediterranean streams
Anna M. Romanı́ • Stefano Amalfitano •
Joan Artigas • Stefano Fazi • Sergi Sabater •
Xisca Timoner • Irene Ylla • Annamaria Zoppini
Received: 26 January 2012 / Accepted: 31 August 2012 / Published online: 12 October 2012
Ó Springer Science+Business Media B.V. 2012
Abstract River and stream biofilms in mediterranean fluvial ecosystems face both extreme seasonality
as well as arrhythmic fluctuations. The hydrological
extremes (droughts and floods) impose direct changes
in water availability but also in the quantity and quality
of organic matter and nutrients that sustain the
microbial growth. This review analyzes how these
ecological pulses might determine unique properties
of biofilms developing in mediterranean streams. The
paper brings together data from heterotrophic and
autotrophic community structure, and extracellular
enzyme activities in biofilms in mediterranean
Guest editors: N. Bonada & V. H. Resh / Streams in
Mediterranean climate regions: lessons learned from the last
decade
A. M. Romanı́ (&) S. Sabater X. Timoner I. Ylla
Institute of Aquatic Ecology, University of Girona,
Campus de Montilivi, 17071 Girona, Spain
e-mail: [email protected]
S. Amalfitano S. Fazi A. Zoppini
Water Research Institute (IRSA), National Research
Council (CNR), Via Salaria km 29.3-CP10,
00015 Monterotondo, Italy
J. Artigas
Irstea, 3 Quai Chauveau CP 220, 69336 Lyon, France
S. Sabater X. Timoner
Catalan Institute for Water Research (ICRA), Emili Grahit
101, Edifici H2O Parc Cientı́fic i Tecnològic de la
Universitat de Girona, 17003 Girona, Spain
streams. Mediterranean stream biofilms show higher
use of peptides during the favorable period for epilithic
algae development (spring), and preferential use of
cellulose and hemicellulose in autumn as a response to
allochthonous input. The drying process causes the
reduction in bacterial production and chlorophyll
biomass, but the rapid recovery of both autotrophs
and heterotrophs with rewetting indicates their adaptability to fluctuations. Bacteria surviving the drought
are mainly associated with sediment and leaf litter
which serve as ‘‘humid refuges’’. Some algae and
cyanobacteria show resistant strategies to cope with
the drought stress. The resistance to these fluctuations is strongly linked to the streambed characteristics
(e.g., sediment grain size, organic matter accumulation, nutrient content).
Keywords Biofilm Extracellular enzymes Drought resistance Bacteria Algae Mediterranean river
Introduction
River and stream biofilms are microbial assemblages
of autotrophic and heterotrophic microorganisms that
are attached to organic and inorganic surfaces (rocks,
cobbles, sediment grains, leaf litter, wood). Bacteria,
algae, fungi, and protozoa developing in a biofilm
share a unique three-dimensional structure usually
embedded in polymeric materials (polysaccharides
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and peptides; Romanı́ et al., 2004a; Romanı́, 2010). As
will be discussed in this article, biofilms play a key
role in river biogeochemical cycles (organic and
inorganic matter cycling).
Microbial biofilms dwelling in streams and rivers
face an ensemble of environmental factors that vary
spatially and temporally (light regime, nutrient and
dissolved organic matter (DOM) availability, water
chemistry, and flow; Roberts et al., 2004; Stanley et al.,
2004; Artigas et al., 2009). In mediterranean streams
(med-streams), water flow has huge fluctuations from
basal conditions to drought and floods. Frequency and
severity of droughts strongly modulate river ecology
(Acuña et al., 2005) and biogeochemistry (Vázquez
et al., 2011). During the aquatic-habitat-shrinking
period, water warms up, increases in nutrient and
dissolved substances, and pH also may show larger
shifts (Ylla et al., 2010; Vázquez et al., 2011). During
flood events, large quantities of organic material and
nutrients are transported downstream (Bernal et al.,
2005; Vázquez et al., 2007; Tzoraky et al., 2007). At
the scale of the microbial biofilm, such fluctuations
become extremely arrhythmic and highly dependent
on the specific streambed substrata characteristics.
This has been both described for bacterial heterotrophs
and autototrophs that make use of summer drought
‘‘humid refuges’’ and result in high heterogeneity of
the biofilm structure and ecological functioning in and
at the surface of the river bed.
In general, biofilm structure and function is sensitive
to changes in nutrient content, temperature, oxygen,
flow velocity, and light, which determine not only the
biofilm community composition, but also its thickness,
the relative abundance of autotrophs/heterotrophs, and
ultimately its function (Gao et al., 2005; Besemer et al.,
2009a, b; Ylla et al., 2009; Dı́az Villanueva et al.,
2011). Among temperate forested streams, the seasonal
dynamics of allochthonous and autochthonous organic
matter inputs followed by the consequent biofilm
microbial use and decomposition is quite common
(Jones & Smock, 1991; Sinsabaugh & Follstad Shah,
2010). However, in med-streams, the unpredictable
drying and flooding episodes exert an additional and
critical abiotic control on microbial metabolism. In
temporary ecosystems, the processing and transformation rates of available DOM are higher in wet than in
dry periods, and changes in river sediment microbial
activity and biomass occur in response to drying and
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reinundation (Tzoraky et al., 2007; Amalfitano et al.,
2008; McIntyre et al., 2009). When river flow becomes
fragmented in pools, a decrease in the use of peptides
but increase in polysaccharide decomposition by the
microbial biofilm has been reported, indicating a
decrease in available fresh C and N organic matter
sources as drought progresses (Ylla et al., 2010).
Summer periods, moreover, are characterized by drops
in biodegradable dissolved organic C and N concentrations (Ylla et al., 2010). During the wet to drought
transition, a decrease in bacterial C production as well
as a decrease in benthic chlorophyll content have
been shown (Amalfitano et al., 2008; Ylla et al., 2010).
During this period, processing rates of deposited
material are likely to differ from those in flowing
water, and periodic hypoxia in pools causes microbial
metabolism to fluctuate between aerobic and anaerobic
pathways (Chan et al., 2005). Thus, the resistance and
recovery of the microbial autotrophs and heterotrophs
in biofilms are relevant for the maintenance of the river
function.
The objective of this review is to synthetize the
knowledge of biofilm structure and functioning in medstreams. For this purpose, the microbial biofilm structure and organic matter use in med-stream biofilms are
analyzed from the perspective of the main ecological
pulses (see Fig. 1). First, autochthonous and allochthonous organic matter inputs are summarized. Second,
the seasonality of extracellular enzyme activities and
activity ratios is synthesized and analyzed with empirical data collected from a collection of published studies
on med-streams. Third, the specific bacterial community changes in sediment and the drought resistance of
autotrophs as well as their relevance as refuges for
further colonization during rewetting are further discussed. Finally, a comparison of med- and non-medstream biofilms is made in order to highlight potential
unique characteristics of such microbial communities.
The revision is mainly focussed on stream biofilms of
the Mediterranean Basin because little information is
available from other mediterranean regions.
Seasonal sources of autochthonous
versus allochthonous organic matter
DOM comprises most of the carbon in streams and
rivers (Volk et al., 1997), which is continuously
Hydrobiologia (2013) 719:43–58
45
Fig. 1 Outline of seasonal changes in energy inputs for biofilm
functioning in a Mediterranean Basin stream. The extracellular
enzymes shown in the diagram are those corresponding to the
highest capabilities measured for each period. The relevance of
the different microbial components (alga, bacteria, fungi) are
also shown
supplied both from terrestrial input (allochthonous
material) as well as from in-stream processes (autochthonous material).
Allochthonous DOM sources mainly come from
terrestrial plant materials (leachates from surrounding
soils, grasses, and inputs from riparian trees; Graça,
1993). This allochthonous material is more recalcitrant, more resistant to biological degradation (Joffre
et al., 2001), and encompasses heterogeneous refractory organic substances of high molecular weight
(McDonald et al., 2004) and of low quality (Kaplan &
Newbold, 1995) than autochthonous material. Specifically, in med-streams, among typically deciduous
riparian vegetation, sclerophyllous-type vegetation is
present, having hard leaves with thick cuticles and
strong lignification, which could determine a greater
recalcitrance of the allochthonous material. As in
temperate regions, allochthonous inputs are generally
seasonally pulsed, where the bulk of litter fall occurs
in a short pulse in autumn (Fisher & Likens, 1973),
with additional material entering the streams by lateral
movement over the remainder of the year (Benfield,
1997). Alternatively, in med-streams, there are more
protracted periods of litter fall, with peaks occurring at
different times of the year in the Northern and
Southern Hemispheres. In the Northern Hemisphere,
the presence of autumn–winter or summer deciduous
trees can explain the autumn peak and the extended
period of litter production (Sabater et al., 2001;
Artigas et al., 2004). In severe drought, the summer
litter fall is extended from mid-summer to midautumn, which has been related to hydric stress
(Acuña et al., 2007). In the Southern Hemisphere,
the prolonged litter fall results from evergreen
phenology whereby new leaf growth is balanced by
almost simultaneous leaf fall (Gasith & Resh, 1999).
Autochthonous DOM sources are produced by the
autotrophic organisms (algae, cyanobacteria, mosses,
phytoplankton, macrophytes) as a result of photosynthetic processes (extracellular release), as well as from
predatory grazing, cell death and senescence, and viral
lysis. This DOM is of low molecular weight and is the
most labile and bioavailable in the stream pool (Bott
et al., 1984; Bertilsson & Jones, 2003). It consists of a
large array of biomolecules, including lipids, carbohydrates, polysaccharides, amino acids, proteins,
waxes, and resins (Piccolo, 2001). This organic C
source may be available mostly during episodes of
high primary production when exudates are produced,
and within biofilms where exudates and products of
cell lysis become concentrated. During springtime
biofilm blooms, stream DOC concentrations increase
as much as 37%, apparently as a result of extracellular
release by algae (Kaplan & Bott, 1989). In medstreams there are two light, leafless periods that are
favorable for primary production. The first one, and
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usually the most relevant, is in spring when the leaves
have not yet sprouted on the trees and there are clear
mild days. The second one is in autumn, when leaves
start to fall and stream water is still warm, although if
strong floods occur this second potential primary
production period may be short or non-existent
(Ylla et al., 2007).
This seasonal shift between inputs of allochthonous
and autochthonous DOM (elevated primary production in spring and DOM and POM inputs in autumn)
marks two important metabolically active periods in
med-stream biofilms (Fig. 1). The first period is characterized by a greater development of algal biomass
and the use of peptides and polysaccharides by the
biofilm (as shown by increases in b-glucosidase and
peptidase extracellular enzyme activities; Artigas
et al., 2009) providing C and N sources for microbial
heterotrophs. In autumn, the benthic heterotrophs
(bacteria and fungi) became more relevant and there is
a greater importance of plant material (cellulose,
hemicellulose, lignin) in sustaining stream system
functioning as indicated by the increase of cellulolytic
and ligninolytic enzyme activities (Artigas et al.,
2009). In winter, microbial activities decrease as a
consequence of decreases in water temperature but
biofilms still exhibit high ligninolytic activity (phenol
oxidase and peroxidase activities) as in the late
decomposition of autumn plant material (Artigas
et al., 2004).
Organic matter and biofilm metabolism
during drought and rewetting episodes
Although direct measurements of biofilm microbial
metabolism during drought are scarce, the recovery
after rewetting suggests that summer humid refuges are
relevant for river functioning. When flow resumes and
pools reconnect, the material is transported downstream, with the first water flush highly loaded in
dissolved organic carbon and nutrients (Larned et al.,
2010). This pulse of biodegradable high molecular
weight organic matter is rapidly processed probably as a
result of the activity of microbial heterotrophs (Romanı́
et al., 2006). During rewetting, the biofilm metabolism
is responding rapidly specially in those microenvironments where humidity has been maintained during
drought, such as in humid sediment patches and leaf
litter (Amalfitano et al., 2008; Ylla et al., 2010). During
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Hydrobiologia (2013) 719:43–58
the rewetting period, the majority of extracellular
enzyme activities rapidly increase, indicating that these
enzymes are important mechanisms for communities to
recover after droughts (Romanı́ & Sabater, 1997;
Zoppini & Marxsen, 2011). Flooding episodes also
induce changes in the metabolic profile of microbial
communities, probably as a result of the increase of
allochthonous organic carbon and nutrients in the
flowing water (Ylla et al., 2010). Zoppini et al. (2010)
observed that b-glucosidase and lipase activities contribute substantially in metabolizing allochthonous
materials during these periods.
This general trend of fluctuations in biofilm microbial activity during drought and rewetting episodes is
highly variable among different benthic substrata (fine
or coarse sediment, cobbles, rocks, leaves, Fig. 1). The
drought process amplifies the patchiness of metabolic
hot spots as shown by the relationships between
physical and chemical properties of sediments and the
microbial extracellular enzyme activities from five
temporary rivers of the Mediterranean Basin [Mulargia, Tagliamento and Candelaro (Italy), Krathis
(Greece), Pardiela (Portugal)] (Zoppini et al., 2010;
Zoppini & Marxsen, 2011; Casella, 2012). These
sediments were characterized by their grain-size
distribution, the concentrations of total organic carbon (TOC) and total nitrogen (TN, CHN analyzer),
and ash-free dry weight (AFDW, subtracting the ash
weight from the dry weight). These measurements
were associated to the spectrofluorometric analyses of
extracellular enzyme activities determined using artificial substrates (Zoppini et al., 2010). A canonical
correspondence analysis (CCA; ter Braak & Verdonschot, 1995) was used to examine the relationships
occurring between extracellular enzyme activities and
physical–chemical properties of the analyzed sediments (Fig. 2). For this analysis, all data were logtransformed and elaborated by the PAST software
package (PAlaeontological STatistics, ver. 2.05). The
obtained Axis 1 explained 71% of variation of the
extracellular enzyme activities, discriminating between
fine and coarse sediments. The Axis 2 explained
about 28% of the variation and discriminating between
dry and wet conditions. Peptidase and phosphatase
activities showed the highest contributions to total
extracellular enzyme activities. Phosphatase activity
is likely to be preferentially associated with sandy
sediments (0.05–0.5 mm), while peptidase activity to
coarse-grained sediments (2–0.5 mm).
Hydrobiologia (2013) 719:43–58
Fig. 2 Canonical correspondence analysis (CCA) performed
with log-transformed data of extracellular enzyme activities and
physical–chemical properties of sediments collected from five
temporary rivers of the Mediterranean Basin [Candelaro—Italy,
Mulargia—Italy, Tagliamento—Italy, Kratis—Greece, Pardiela—Portugal (Zoppini et al., 2010; Zoppini & Marxsen, 2011;
Casella, 2012)]. The arrowhead length is proportionally related to
the vector length. The dot size is proportionally related to the
percentage occurrence of each enzyme in the whole dataset. %WC
water content, %AFDW ash-free dry weight, TN total nitrogen,
TOC total organic carbon in sand, coarse, and fine sediments
Peptidase discriminates environments with relatively low contribution of biofilm organic matter
(AFDW, ash-free dry weight) and TN. The interpretation of the peptidase activity pattern is complex
because it is an amphibolic enzyme potentially
important in both carbon degradation and nitrogen
acquisition. The predominance of peptidase in coarsegrained sediments has been observed in nutrient-poor
habitats, where benthic microbial communities may
rely on high peptidase activity to obtain sufficient
amounts of organic nitrogen (Rusch et al., 2003).
Peptidase activity has been also linked to the algal–
bacterial relationship and the bacterial use of algal
released peptides (Ylla et al., 2009), and thus a higher
peptidase activity is often measured in coarse sand or
cobbles where algal biomass develops (Artigas et al.,
2009). Phosphatase activity is higher in sandy substrates with relatively higher TOC/AFDW ratios and
lower water content. Moreover, this activity is likely to
be scarcely affected by a progressive decrease in
sediment–water content (Zoppini & Marxsen, 2011).
47
During the drought, a relevant phosphatase activity is
maintained, especially in sediment biofilms (Timoner
et al., 2012), probably indicating a significant phosphorus limitation for energy and cell maintenance by
the microbial communities surviving the drought.
b-glucosidase and lipase activities are associated
with fine-wet sediments, with relatively lower TOC/
AFDW ratio where nutrients are available, as suggested by higher concentration of TN. High contribution of AFDW also allows us to infer the presence of a
variety of organic substances probably rich in N and P.
The interdependence observed between community
respiration rates and the activity associated with lipase
and b-glucosidase relies on the specificity of these
enzymes to hydrolyze energy-rich compounds. The
products of these enzyme activities (e.g., glycerol, fatty
acids, and glucose) represent readily usable sources of
energy fueling catabolic metabolism (Zoppini et al.,
2010).
Seasonality of enzyme activity ratios
The heterotrophic biofilm capacity might be further
analyzed from the perspective of the ‘‘ecoenzymatic
stoichiometry’’ theory developed by Sinsabaugh et al.
(2009) when the analysis of enzymes responsible for
C, N and P decomposition compounds is included.
Sinsabaugh et al. (2009) suggested that an equilibrium
between C:N:P activity ratios of 1:1:1 indicates an
equilibrium between the elemental composition of
available organic matter, microbial biomass and nutrient
assimilation, and growth of microorganisms in river
sediments. Similarly, Sala et al. (2001) proposed that
C:N:P activity ratios calculations are sensitive tools for
detecting nutrient limitation in marine microbial communities. Long-term extracellular enzyme activities
data collected in med-streams and rivers from Spain
[Llobregat (Ricart et al., 2010), Ter (Romanı́ & Sabater,
1999), Fuirosos (Artigas et al., 2009; Ylla et al., 2010;
Sabater et al., 2011), Solana (Romanı́ & Sabater, 1998),
and Riera Major (Romanı́ & Sabater, 2001)] and from
Italy [Mulargia and Candelaro (Zoppini et al., 2010;
Zoppini & Marxsen, 2011; Casella, 2012)] were used to
analyze the activity ratio between C:N, C:P, and N:P
organic matter use in different types of biofilms (Fig. 3).
In all these studies, the biofilm extracellular enzymes
were measured by incubating fresh biofilm samples
(between 3 and 5 replicates per site) with artificial
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Hydrobiologia (2013) 719:43–58
Fig. 3 Relationship (in logarithms) between b-glucosidase and c
peptidase (A), b-glucosidase and phosphatase (B), and peptidase
and phosphatase (C) organic matter acquisition extracellular
enzyme activities in microbial biofilms from Mediterranean
Basin rivers. The solid line indicates a 1:1 relationship. Data
include monthly or seasonal values obtained from the different
streambed substrata available in each river (biofilms on cobbles
and rocks, sand or leaves) including minimum 1 year of study.
Data for the three relationships were obtained from four temporary rivers of the Mediterranean Basin [Llobregat—Spain
(Ricart et al., 2010), Fuirosos—Spain (Artigas et al., 2009; Ylla
et al., 2010; Sabater et al., 2011), Mulargia and Candelaro—
Italy (Zoppini et al., 2010; Zoppini & Marxsen, 2011; Casella,
2012)]. Additional data of enzymatic activity from three
Spanish temporary rivers [Ter (Romanı́ & Sabater, 1999),
Solana (Romanı́ & Sabater, 1998), Riera Major (Romanı́ &
Sabater, 2001)] were added on the b-glucosidase: phosphatase
relationship (B). Differences between seasons (Spring, Summer,
Autumn and Winter) are highlighted. The specific regression
slopes and coefficients of variation for each relationship are
included in Table 1
substrates followed by spectrofluorometric analysis
(Romanı́ & Sabater, 2001). The relationships between
C:N, C:P, and N:P acquiring enzymes were calculated
after logarithmic transformation of the data and
performing linear regression analyses in order to
calculate the slope and coefficient of variation (Sigma
Plot Ver. 11.0, Systat Software, Inc.; CA, USA). In the
analyzed med-stream biofilms, the equilibrium was
observed for peptidase:phosphatase activity ratios
(N:P, slope = 1:1), but not for the b-glucosidase:peptidase (C:N) and b-glucosidase:phosphatase (C:P)
activity ratios (Fig. 3; Table 1). Overall, the b-glucosidase:peptidase and b-glucosidase:phosphatase ratios
were below the 1:1 equilibrium, indicating a greater
potential of med-stream biofilms to acquire N and P
than C. Specifically, the shifts of enzyme activity
ratios were more pronounced during spring and
winter (ranging 0.6–0.7:1), suggesting that the greatest
N and P limitation occurred during these periods.
Conversely, the highest variability in enzyme activity
ratios was observed during the summertime (C:N and
C:P variation coefficients ranging from 44 to 49%,
Table 1). This variability in organic matter acquisition
may be explained by the important changes in organic
matter availability during drought episodes (Ylla et al.,
2010). As an example, an unbalanced C:N activity
ratio (1.7–2:1) was observed during the summer–
autumn episode of 2006 in the Fuirosos stream,
indicating a greater acquisition of organic C than N
under dry conditions (Fig. 3A, white and dark-gray
symbols far above the 1:1 line). Here, the increased
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water stress in the riparian forest causing late summer
litter fall and the POM accumulation in pools may be
responsible for the enhanced organic C acquisition.
The above results suggest that in med-rivers, the
heterotrophic use of organic compounds is unbalanced
by the greater use of phosphorus and peptides,
probably indicating a limitation of nitrogen and
phosphorus at least during some periods of the year.
The stoichiometry analysis also shows that the relationship between enzyme capabilities can drastically
Hydrobiologia (2013) 719:43–58
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Table 1 Enzyme activity ratio calculations for b-glucosidase:
peptidase (Glu: Pep), b-glucosidase: phosphatase (Glu: Phos)
and peptidase: phosphatase (Pep: Phos) organic matter acquisition activities in various temporary rivers of the Mediterranean region [Llobregat—Spain (Ricart et al., 2010), Fuirosos—
Glu:Pep
Spain (Artigas et al., 2009; Ylla et al., 2010; Sabater et al.,
2011), Mulargia and Candelaro—Italy (Zoppini et al., 2010;
Zoppini & Marxsen, 2011; Casella, 2012), Ter (Romanı́ &
Sabater, 1999), Solana (Romanı́ & Sabater, 1998), Riera
Major, (Romanı́ & Sabater, 2001)]
Glu:Phos
Pep:Phos
Slope
CV
Slope
CV
Slope
CV
All data
0.8:1
42.6
0.8:1
42.3
1:1
17.4
Spring
0.6:1
31.7
0.7:1
37.8
1:1
15
Summer
0.9:1
44.5
0.9:1
49.4
1:1
16.5
Autumn
0.9:1
36
0.8:1
33
1:1
20.2
Winter
0.7:1
24.5
0.7:1
29.7
1:1
11.7
Regression slopes and coefficients of variation (italics) for each enzyme activity ratio were represented for the whole year (all) and
between seasons (spring, summer, autumn, winter)
change between seasons and substrata, such as the
much higher use of polysaccharides (high b-glucosidase, acquiring a C source) than peptidase in a
temporal med-stream (the Fuirosos) during the summer period (Fig. 3). This behavior not only underlines
the mediterranean characteristic of ‘‘fluctuations’’ but
also the adaptation of the microbial biofilm communities to the available organic matter sources. Thus,
organic matter supply in med-streams is strongly
pulsed and this finally determines the organic matter
use in biofilms.
Resistance to drought: the response
of the heterotrophic component of microbial
biofilms
The maintenance of the stream functioning in a medstream affected by severe drought episodes is linked to
the survival of the heterotrophic component of
microbial biofilms. The resistance and survival to
drought and rewetting episodes could determine the
development of a specific distinctive bacterial community. In med-streams, sediment and leaf material
Table 2 Quantitative occurrence of major bacterial taxa retrieved within the benthic substrates based on fluorescence in situ
hybridization
Proteobacteria (ALF968, BET42a, GAM42a), Bacteroidetes (e.g., CF319a) and Gram-positive bacteria (e.g., HGC69a, LGC354abc).
Values are averages from 38 samples collected from 8 Mediterranean streams [Cremera—Italy (Amalfitano & Fazi, 2008; Bandiera
2006); Mulargia—Italy, Tagliamento—Italy, Kratis—Greece, Pardiela—Portugal (Amalfitano et al., 2008; Zoppini et al., 2010);
Albegna, Ente and Fiora—Italy (Fazi et al., 2005)]
1 9 105cells/g, s 1 9 106cells/g,
1 9 107cells/g, d 1 9 108 cells/g, m 1 9 109 cells/g
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1,0
%AFDW
<0.2mm
Firmicutes
Actinobacteria
%WC
0,5
Axis 2 (30%)
are where the moist conditions might be more easily
maintained, providing a refuge in which aquatic
bacterial communities could survive in contrast to
the more harsh conditions in cobble biofilms (Ylla
et al., 2010).
The response to water stress of microbial communities in soil (Van Gestel et al., 1992; Steenwerth et al.,
2003; Fierer et al., 2003; Wu & Brookes, 2005;
Williams, 2006) or freshwater sediment (Amalfitano
et al., 2008; Fazi et al., 2008; Marxsen et al., 2010) has
been mainly studied in microcosm experiments. Some
studies showed changes in microbial diversity in
natural streambed sediments subject to hydrological
fluctuations (Rees et al., 2006; Zoppini et al., 2010).
Using a qualitative DNA-based approach, some
authors (Lueders & Friedrich, 2000; Marxsen et al.,
2010) showed community changes in sediments in
response to rewetting, while Fierer et al. (2003)
reported that the drying and rewetting cycle affects the
microbial community structure, mainly in soils that
underwent moisture stress infrequently.
A strong relation between bacterial community and
substratum characteristics (i.e., water and organic
matter content) is evident when comparing quantitative data from eight temporary med-streams (Table 2).
The highest bacterial abundance was found on organic
matter-rich allochthonous leaf accumulations and, as
expended, the lowest values on intra-sediment biofilms under dry conditions. The detritus accumulated
in stagnant waters showed a relatively high organic
matter content, mainly originating from allochthonous
inputs at an advanced stage of decomposition and by
in situ production. This substratum in pools supports
an abundant microbial community, thus acting as a
‘‘humid refuge’’ for aquatic microbes.
From a wide range of freshwater ecosystems, fluorescence in situ hybridization (FISH) studies suggested
that there were some crucial differences among bacterial
assemblages associated with different sediment and
detritus types, even at a broad phylogenetic level (Fazi
et al., 2005; Gao et al., 2005; Marxsen et al., 2010). In
med-streams multiple factors affect the bacterial community composition (Fazi et al., 2005, 2008; Zoppini
et al., 2010), and it is evident that different bacterial
domains show different distribution patterns (Table 2).
Following the protocol optimized by Fazi et al. (2007),
bacterial community composition was assessed by FISH
in sediments from five selected med-streams [Cremera—
Italy (Bandiera, 2006; Amalfitano and Fazi, 2008);
Hydrobiologia (2013) 719:43–58
Cytophaga-Flavobacteria
Alphaproteobacteria
>2.0mm
0,0
0.2-0.5mm
Gammaproteobacteria
Betaproteobacteria
-0,5
0.5-2.0mm
-1,0
-1,0
-0,5
0,0
0,5
1,0
1,5
Axis 1 (52%)
Fig. 4 Canonical correspondence analysis (CCA) performed
with log-transformed data of major bacterial taxa and the grain
size classes, water, and organic matter content of the sediments
collected from five temporary rivers of the Mediterranean Basin
[Cremera—Italy (Amalfitano and Fazi, 2008; Bandiera 2006);
Mulargia—Italy, Tagliamento—Italy, Kratis—Greece, Pardiela—Portugal (Amalfitano et al., 2008; Zoppini et al., 2010)].
%WC water content, %AFDW ash-free dry weight, TN total
nitrogen, TOC total organic carbon; \0.2 mm: ultra-fine grain
size; 0.2–0.5 mm: fine grain size; 0.5–2.0 mm medium grain
size; [2.0 mm: coarse grain size
Mulargia—Italy, Tagliamento—Italy, Kratis—Greece,
Pardiela—Portugal (Amalfitano et al., 2008; Zoppini
et al., 2010)]. A CCA was used to examine the preferential associations of major bacterial taxa with the
grain size classes, and water and organic matter content
of the sediments. The number of cells per taxon was
logarithmically transformed (ter Braak & Verdonschot,
1995). Differences in environmental factors explained
about 52 and 30% of the variation in the bacterial
community for Axis 1 and Axis 2, respectively. The
results show that in the sediments of the analyzed medstreams, the spatial and temporal dynamics of microbial
phylogenetic groups are affected by grain size distribution (Fig. 4), and thus, regulated by the frequency
and intensity of the water intermittency. The mechanisms by which the stream flow dictates the sediment
grain size patterns are fundamental to organic matter
and nutrient dynamics and, thus, to bacterial community structuring in intra-sediment biofilms (Santmire &
Leff, 2007). The grain size distribution depends largely
upon the interlocking of individual particles of different
Hydrobiologia (2013) 719:43–58
size, the near-bed flow characteristics, the longitudinal
slope, and the prevailing sediment transport conditions
(Hendrick et al., 2010). In med-regions, the strong
hydrologic seasonality and the drought severity determine the degree of disruption of the longitudinal fluvial
continuum (Butturini et al., 2003), representing additional determinants of the sediment accumulation processes on the streambed (Wittenberg, 2002).
Overall, the Alpha, Beta, and Gamma subclasses of
Proteobacteria accounted for the major fraction of the
domain bacteria that colonize the benthic substrates.
The presence of a large fraction of Betaproteobacteria
was reported at sites with high benthic organic matter
content (Wobus et al., 2003; Gao et al., 2005). This
could be attributed to their high efficiency in oxidizing
ammonia and degrading organic compounds in association with specific enzymatic activities (Araya et al.,
2003; Kirchman et al., 2004; Zoppini et al., 2010).
Moreover, they are reported to be an active component
of freshwater microbial communities in sediments
exposed to hydrological fluctuations (Amalfitano
et al., 2008; Besemer et al., 2009b; Marxsen et al.,
2010; Zoppini et al., 2010) and in biofilms during the
early succession stages (Brümmer et al., 2003; Lupini
et al., 2011).
The Alphaproteobacteria generally showed a lower
abundance than the Beta subclass, but in some cases,
they are reported to lead the transformation processes
of labile organic materials and to out-compete other
groups in mature biofilms, being highly efficient in
nutrient assimilation and substrate mineralization
(Santmire & Leff, 2007). The members of Gammaproteobacteria are reported to be relatively abundant
in presence of a high nutrient concentration and
when extra organic matter input and humic substances
become available (Wobus et al., 2003; Gao et al., 2005;
Zoppini et al., 2010). According to previous reports,
the increase in bacteria from the Cytophaga-Flavobacteria (CF) group in substrates highly loaded with
organic carbon may be a general feature of microbial
communities in freshwater habitats (Salcher et al.,
2010). Some members of the CF cluster are specialized
for the degradation of complex macromolecules
(Kirchman, 2002; Tamaki et al., 2003). Accordingly,
they constitute a significant proportion of the microbial
communities on substrates characterized by recalcitrant and high molecular weight DOM and on
suspended particles either in stream or river biofilms
(Simon et al., 2002; O’Sullivan et al., 2004).
51
The quantitative occurrence of Gram-positive bacteria in stream bed substrates is under-investigated.
Generally, the members of the polyphyletic groups of
Actinobacteria (cells with a high DNA content of
guanine and cytosine) and Firmicutes (cells with a low
DNA content of guanine and cytosine) are at least one
or even two order of magnitude lower than those of the
taxa above described. In some local cases, the Actinobacteria appear to be associated with high abundance
of cells belonging to the Betaproteobacteria (Salcher
et al., 2010). Moreover, they could constitute an important fraction of the community in dry sediments,
suggesting a high molecular and metabolic resistance
to desiccation (Amalfitano et al., 2008). Many members of the Firmicutes (e.g., Bacillus spp.) are known to
be spore-forming and, thus, were reported as the first
colonizers of the water column after sediment desiccation (Fazi et al., 2008).
Resistance to drought: the response of autotrophic
component of microbial biofilms
The river biofilms developing on the upper side of
rocks and cobbles and the surface sediment support a
great proportion of autotrophic biomass (up to
80–90% of whole biofilm biomass; Romanı́, 2010).
In med-streams, drought requires specific structural
and physiological adaptations from autotrophic microorganisms to survive, as well as particular recolonization abilities once the drought is over. Factors to be
considered start from the cellular level but eventually
involve the whole response of autotrophs to the reach
and segment scale during drought.
Periods of low or non-flow lead to water loss for
primary producers, and desiccation stress (Mosisch,
2001). Several studies point out the resistance of the
autotrophic community to drought (Romanı́ & Sabater,
1997; Robson & Matthews, 2004; Caramujo et al.,
2008; Robson et al., 2008). This is based on structural
properties (higher EPS—extracellular polymeric substances—content, species succession, resistance structures), but also on physiological properties (light-shade
adaptability, photosynthetic activity after rewetting).
Some groups and species are better adapted than
others (Kawecka, 2003; Robson & Matthews, 2004;
Ledger et al., 2008) to drought. Some have cellular or
other supraorganismic structures that provide shelter
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52
to desiccation. Cyanobacteria and green algae are
better adapted to withstand drought than are diatoms.
Some Cyanobacteria in calcareous environments
deposit carbonates, while others produce stromatolitic-like mats that provide protection to the cells
living inside, as well as a high porosity prone to
rewetting soon after rains occur (Sabater et al., 2000).
These structures show quick autotrophic (and heterotrophic) response after rewetting (Romanı́ & Sabater,
1997). Among the common genera building (and
dwelling) on those structures are Rivularia, Phormidium, Homoeothrix, or Schizothrix. Other crust-forming species also resist drought rather well. For
example, the green alga Gongrosira persists after
long desiccation, but it has been observed experimentally (Ledger et al., 2008) that its numbers may largely
decrease after drought.
Algal communities experience succession in the
process from low flow to drought. In a reach scale, there
is transition from diatom mats to encrusting forms as
dryness progresses (Romanı́ & Sabater, 1998; Ledger
et al., 2008). Diatoms are less resistant than other
groups to desiccation. Still, some produce thick mucilaginous masses that may act as protectors of the living
cells, at least during shorter droughts. Among these, the
genera Cymbella and Gomphonema include several
mucilage-forming species, which occur in med-streams
during late spring and summer. Other diatoms build
inner plates to resist desiccation and osmotic variations
related to alternate drought and rewet. These are
organisms which also subsist in subaerial environments
(Sabater, 2009). Navicula contenta, Fragilaria construens, Achnanthes lanceolata, and Denticula tenuis
persist under these conditions, and are common in
med-rivers (Tornés & Sabater, 2010). Other algae also
possess thick cell walls that protect the cell from
desiccation. The red alga Hildenbrandia rivularis
produces permanent crusts mostly in poorly lit areas
of oligotrophic streams that resist long desiccation and
return to activity after the flow returns.
Some filamentous green algae (Spirogyra, Zygnema,
and other Zygnematales and Oedogoniales) produce
zygospores through sexual reproduction that are thickwalled and become dormant to survive adverse
conditions such as drought. These filamentous forms
are adapted to shrinking water conditions. Zygnematales grow unattached to the substrata (except a few,
such as Spirogyra fluviatilis; Margalef, 1983) and
form large masses in slow-moving areas, and are
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Hydrobiologia (2013) 719:43–58
well-adapted to excess light. During the development
of drought conditions, cells may become yellowish, and
although some may have impaired their photosynthetic
apparatus, the mass itself is able to persist and adapts to
use nutrients efficiently (Borchardt, 1994). During
these conditions, masses may be able to affect nearby
water temperature, as well as pH, and therefore exert
influence on local biogeochemistry. Surprisingly some
of these assumptions remain unmeasured. It is also
remarkable that within these masses multiple species
co-exist and hybridization is common. Oedogonium
and Zygnematales are polyploid, and therefore able to
face the potential genetic drift that derives from
multiple hybridization (Margalef, 1983). Finally, some
other green algae (Cladophora or Stigeoclonium)
possess differentiated basal cells (rizoids) that not only
secure attachment, but also persist after the plant has
completed its cycle (Margalef, 1983). This helps to
explain its widespread occurrence in drought-prone
med-rivers after water recovery.
There is a reduction in chlorophyll a with the flow
drawback (Ylla et al., 2010). Streambed materials
remain exposed to high temperatures and high incident
light conditions particularly in open areas that affect
the photosynthetic apparatus. Algal cells are metabolically inactive during dry conditions, and the return of
photosynthetic activity is only known for some groups
of organisms. The return may be fast in stromatolitic
biofilms, where photosynthesis was noticeable 1 h after
rewetting in a recovery experiment (Romanı́ & Sabater,
1997). Also, terrestrial cyanobacteria recover their
photosynthetic efficiencies extremely fast, from minutes
to a few hours (Scherer et al., 1984). However, the
photosynthetic recovery of epilithic diatoms or green
algae to complete drought is unknown. Adaptations to
progressive drought conditions are better known. Lower
photosynthetic efficiencies during low flow in a medstream were associated with the carotenoid accumulation in sun-adapted biofilms (Guasch & Sabater, 1995),
which provide photooxidative protection to photoinhibition at summer ambient irradiances (Scherer et al.,
1988; Quesada et al., 1995; Dieser et al., 2010). UVprotective pigments such as carotenoids, xanthophylls,
and scytonemin accumulate in microbial mats
(Fernández-Valiente et al., 2007) and soil crust biofilms
(Belnap et al., 2007). It may be that physiological
adjustments of autotrophic cells to cope with high
temperatures and exposure to extreme solar radiation
also occur under drought episodes. These physiological
Hydrobiologia (2013) 719:43–58
adjustments may allow the cells to allocate more energy
to processes other than protecting the cell from light and
temperature, and makes it adaptive to better withstand
desiccation stress (Fleming & Castenholz, 2007) and
facilitate physiological recovery.
Recolonization ability of autotrophs is related to two
main factors. The first is related to the speed at which
drought occurs. The rapidity of drying matters, because
slow drying enables longer permanence of refuges that
allows more possibilities for the organisms to survive
(time for structural and physiological adaptations), as
well as for regrowth (Robson et al., 2008). The severity
of drought is essential in defining the existence or
otherwise of these refuges, and this depends on the
specific location of the site (altitude or topography,
local climate, relevance of water abstraction).
The second is the dominant type of substrata, i.e.,
the prevalence of sediments, wood, or cobbles in the
streambed that have different potentials to maintain
humidity and enable host recolonization. Sediments
are the most favorable habitat under complete desiccation conditions. Areas underneath cobbles may also
allow for humid conditions, where cells can survive.
With sufficient humidity, these areas may host live
cells or resting stages, which are essential to recolonization. Perennial pools, or small areas of superficial
flow, provide a refuge from which to restart colonization. Perennial pools may guarantee the sufficient
inoculum to restart colonization downstream. Density
of algae for regrowth is positively related to the water
permanence in the stream (Robson & Matthews,
2004). However, Robson et al. (2008) showed that
the main refuge source is the substrata type, and that
pools are not contributing significantly to the overall
recolonization. The algal flora developing on pools
does not match that colonizing flowing surface waters,
and additional sources (suitable substrata) are required
to complete the recolonization.
On top of these factors, algae show different
recolonization abilities. Diatoms quickly colonize
open spaces that are opened by disturbances. At the
reach scale, areas temporarily rewet and again dried
provide unsuitable substrata for autotrophs. However,
some diatoms may use these areas to initiate pioneer
communities (Tornés & Sabater, 2010). The frequency
of habitat emersion and autotrophs development is
patch-specific, and may lead to high spatial differences
in algal diversity and community composition within
short transactional distances (Tornés & Sabater, 2010).
53
Finally, the higher capacity of algae to recover its
metabolism as a result of physiological adjustments
produces high productivity immediately after rewetting
(Dodds et al., 1996; Romanı́ & Sabater, 1997), despite
lower concentrations of chlorophyll a. The temporal
extent of intermittency may substantially reduce algal
productivity resulting from the loss of drought refuges
(Robson & Matthews, 2004) and severe damage to algal
cells that may hinder the recovery. This fact may limit the
supply of both autochthonous carbon (Dodds et al., 1996)
and allochthonous carbon, because it has been demonstrated that algal photosynthesis enhances heterotrophic
metabolism, stimulating extracellular enzyme activities
(Francoeur & Wetzel, 2003; Romanı́ et al., 2004b;
Francoeur et al., 2006). Therefore, the whole ecosystem
metabolism may be affected. Adaptation to windows of
opportunity for regrowth is essential; production pulses
may supply food webs with high quality carbon
(Artigas et al., 2009).
Comparing stream biofilms from mediterranean
to non-mediterranean climate regions
The data and literature reviewed in this manuscript as
well as some specific studies focusing on the comparison between stream biofilms from mediterranean and
non-mediterranean regions highlight some distinctive
structural and functional features of mediterranean
ones. When comparing the changes during biofilm
formation in a med-stream in contrast to a Central
European stream (third-order forested streams, Artigas et al., 2012), a faster colonization by algae and
bacteria was measured at the med-stream although
reaching similar biomass values at the mature biofilm.
Also, med-river biofilms tend to have a greater content
of extracellular polymeric substances (Artigas et al.,
2012), which might be linked to the adaptation to
drought and the development of species producing
mucilage (cyanobacteria, diatoms). Chlorophyll density was more fluctuating in a forested med-stream
(with higher values in spring-early summer) while it
was more constant over the year in an Atlantic stream
(Sabater et al., 2008). For the bacterial community, it
is suggested that the hydrologic seasonality of medrivers, which change the physico-chemical features of
benthic substata, indirectly affects the occurrence and
the abundance of the major bacterial groups, and is
mainly linked to water and organic matter availability.
123
54
When rewetted streambed sediments from two low
order streams in distinct climate regions (a Central
European and a Mediterranean), a lower bacterial
biomass and lower bacterial production were measured at the med-stream (Marxsen et al., 2010). In the
same study, bacterial diversity (as measured by TGGE
analysis) was similar for both streams in dry sediments
but it was lower in Mediterranean sediments after
rewetting. Those results suggested that bacterial
community composition is more fluctuating in med
than in non-med-streams.
Under more extreme hydrologic conditions than medstreams, desert streams show some similarities in biofilm
structure and functioning. Zeglin et al. (2011) found that
changes in bacterial community composition and diversity were mainly affected by sediment water content and
conductivity. In microbial mats from a desert stream,
flooding resulted in the replacement of over 74% of the
microbial community (bacteria and algae; Abed et al.,
2011). It could be hypothesized that in desert streams the
microbial community is replaced after flooding in a
much higher degree than that occurring in med-streams
because of the lack of persistent humid refuges.
In the case of algal biomass, it is generally described
that during drought there is a decrease in organic inputs
in contrast to inorganics enhancing autotrophy, as
observed in a Central New Mexico stream (Dahm et al.,
2003). However, in med-streams this increase in
autotrophy is not clear and likely depends on the
development of the riparian vegetation and the leaf fall
that may occur in intense summer drought. In a forested
med-stream, a decrease in chlorophyll content on the
biofilm developing on sand and cobbles was measured
during the wet to drought process (Ylla et al., 2010) as
well as an increase in the heterotrophic biomass (fungi
and bacteria, Artigas et al., 2009). When comparing
seasonal data of three med-rivers to nine Atlantic rivers
(from 1 to 4 order size, including open and forested
and eutrophic and oligotrophic ecosystems) a higher
bacterial to algal biomass ratio was shown for the
Mediterranean biofilms (Romanı́ & Sabater, 2000)
suggesting a higher heterotrophy (Fig. 1).
The biofilm use of the available organic matter
also shows specific features when compared to nonmed-rivers as shown by the results of biofilm extracellular enzyme activities. From the comparison of
seasonal data from med- to Atlantic rivers (Romanı́ &
Sabater, 2000), a significant higher polysaccharide
decomposition capacity was measured for the med-
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Hydrobiologia (2013) 719:43–58
river biofilms (higher b-glucosidase and b-xylosidase), suggesting an effect of temperature and/or of a
different microbial community colonizing the substrata. However, a specific study of the effect of
drought/rewetting showed a lower polysaccharide
decomposition capacity (as well as peptide) at the
med-stream after rewetting, which was not observed in
the non-Mediterranean stream. This result was linked
to a more intense drying in the med-stream (Marxsen
et al., 2010).
Conclusions, trends, and perspectives
Hydrology appears as one of the main factors affecting
the biofilm structure and functioning. In the case of the
biofilm autotrophs, the structural and physiological
adaptations to drought are well known; much less is
known about the effect of such physiological changes
on the water quality or the effect of floods and biofilm
recolonization. In the case of the biofilm heterotrophs,
although some data are provided here about the
bacterial community composition, there are few
studies analysing the bacterial community during
drought and rewetting episodes. The processes taking
place in the sediment during the drought may be also
very relevant for the resistance and recovery during
rewetting. Linking both the autotrophs and heterotrophs there is a need for research at different times but
especially on different spatial scales that take into
account the effect of patchiness. The specific conditions at different but short time and spatial scales may
have a great effect on biofilm structure and functioning. Experiments linking laboratory and field work
including such patchiness are needed.
Finally, although one may consider that medstream biofilms are adapted to drought and floods,
and efficiently make use of the available organic
matter pulses, the question about how efficient these
communities actually are in decomposing organic
matter still remains unanswered. Further research in
analyzing the increasing frequency and intensity of
drought/flooding episodes, and their impact on biofilm
extracellular enzyme activities and biofilm architecture, could shed some light on understanding biofilm
processes. The fluctuations in microbial biomass and
in enzyme activities suggest that efficiencies are also
fluctuating, because in the rewetting there is a biomass
decrease but some activities are increased.
Hydrobiologia (2013) 719:43–58
Acknowledgments This study was funded by the projects
CGL2011-30151-C02-01, and SCARCE (Consolider-Ingenio
2010 CSD2009-00065) of the Spanish Ministry of Economy and
Competitiveness; the Italy–Spain Exchange Project CNR-CSIC
2006IT0010; and the EU project MIRAGE (FP7-ENV-2007-1
n.211732). We also thank the comments from two anonymous
reviewers as well as editors’ suggestions.
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