Leaf-litter leachate concentration promotes

Science of the Total Environment 599–600 (2017) 1677–1684

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Leaf-litter leachate concentration promotes heterotrophy in freshwater biofilms: Understanding consequences of water scarcity Aingeru Martínez a,⁎, John Stephen Kominoski b, Aitor Larrañaga a a b

Laboratory of Stream Ecology, Department of Plant Biology and Ecology, University of the Basque Country, P.O. Box 644, 48080 Bilbao, Spain Department of Biological Sciences and Southeast Environmental Research Center, Florida International University, 11200 SW 8th Street, Miami, FL 33199, USA

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Water scarcity increases the dissolved organic matter concentration in fresh waters. • Biofilm metabolism at different concentrations of Eucalyptus leachates was assessed. • The increase in leachates concentration enhanced respiration and reduced production. • Heterotrophy increases under water scarcity via dissolved organic matter concentration.

a r t i c l e

i n f o

Article history: Received 15 March 2017 Received in revised form 4 May 2017 Accepted 4 May 2017 Available online xxxx Editor: D. Barcelo Keywords: Biofilm functioning Colonization stage Dissolved organic matter Eucalyptus grandis Freshwater ecosystem

a b s t r a c t Climate change is increasing overall temporal variability in precipitation resulting in a seasonal water availability, both increasing periods of flooding and water scarcity. During low water availability periods, the concentration of leachates from riparian vegetation increases, subsequently increasing dissolved organic matter (DOM). Moreover, shifts in riparian vegetation by land use changes impact the quantity and quality of DOM. Our objective was to test effects of increasing DOM concentrations from Eucalyptus grandis (one of the most cultivated tree species in the world) leachates on the metabolism (respiration, R; gross primary productivity, GPP) and extracellular enzyme activities (EEAs) of freshwater biofilms. To test effects of DOM concentrations on freshwater biofilm functions, we incubated commercial cellulose sponges in a freshwater pond to allow biofilm colonization, and then exposed biofilms to five different concentrations of leaf-litter leachates of E. grandis for five days. To test if responses to DOM concentrations varied with colonization stage of biofilms, we measured treatment effects on biofilms colonizing standard substrates after one, two, three and four weeks of colonization. Increases in leachates concentrations enhanced biofilm heterotrophy, increasing R rates and decreasing GPP. Leachate concentrations did not affect biofilm EEAs, and changes in biofilm metabolism were not explained by treatment-induced changes in biofilm biomass or stoichiometry. We detected the lowest production:respiration ratios, i.e. more heterotrophic assemblages, with the most concentrated leachate solution and the most advanced biofilm colonization stages. Shifts in quantity of dissolved organic matter in freshwaters may further influence ecosystem metabolism and carbon processing. © 2017 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: Lab. of Stream Ecology, Dept. of Plant Biology and Ecology, University of the Basque Country, P.O. Box 644, 48080 Bilbao, Spain. E-mail address: [email protected] (A. Martínez).

http://dx.doi.org/10.1016/j.scitotenv.2017.05.043 0048-9697/© 2017 Elsevier B.V. All rights reserved.

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A. Martínez et al. / Science of the Total Environment 599–600 (2017) 1677–1684

1. Introduction Dissolved organic matter (DOM) is a mixture of organic compounds that plays a key role in aquatic ecosystem biogeochemistry (Tank et al., 2010). The amount and composition of DOM depend on different transformation processes such as biodegradation, photodegradation, or flocculation (Sachse et al., 2005). Variation in DOM is also attributed to organic matter sources, both allochthonous, which result from a combination of inputs from the adjacent terrestrial ecosystems, and autochthonous, from inside production (Webster and Meyer, 1997). Dissolved compounds in fresh waters are derived from allochthonous inputs when the hydrological connectivity with terrestrial ecosystems is high, but the reduction of this connectivity increases the relevance of autochthonous DOM sources (von Schiller et al., 2015). Such loss of connectivity is expected to be enhanced as consequence of the forecasted increases in water temperatures and changes in precipitation regimes by the end of the 21st century (IPCC, 2013), increasing the frequency of low-water level periods in lentic systems and streams turning into a fragmented and heterogeneous landscape dominated by slow running waters and isolated pools. Under these conditions, where tree leaves from surrounding vegetation usually are accumulated due to lack of advection (Acuña et al., 2005; von Schiller et al., 2011), leachates of this material (autochthonous source) represent the main DOM source in forested freshwater ecosystems (Casas-Ruiz et al., 2016). Moreover, in these scenarios, the reduction of dilution capacity due to water scarcity (Sabater and Tockner, 2009) leads to an increase in the leachates concentration. Increases of water scarcity, disease and pests, and agricultural intensifcation are changing the composition of riparian plant species globally (Kominoski et al., 2013). These changes are altering the quantity and quality of leaf-litter inputs into fresh waters (Kominoski et al., 2013), which determine the quantity and quality of leachates (Wallace et al., 2008). These shifts may contribute to differences in overall utilization of leachates by aquatic biofilms and alter the ecosystem functioning. Aquatic biofilms are assemblages of autotrophic and heterotrophic microorganisms embedded in a polysaccharide matrix (Romaní, 2010) that play a key role in biogeochemical cycling of DOM (Sabater et al., 2002) and transferring C and nutrients to higher trophic levels (Allan and Castillo, 2007). Labile DOM can stimulate biofilm growth, respiration and primary production (Ylla et al., 2012; Lovatt et al., 2014). In contrast, recalcitrant leachates (e.g., polyphenols, terpenoids, oils) can have inhibitory or toxic effects on aquatic biofilms (Friberg and Winterbourn, 1996; Kominoski et al., 2007; Schlief and Mutz, 2007; Ylla et al., 2012). The quantity and quality of DOM also regulate the synthesis of extracellular enzymes, which allow heterotrophic microbes to degrade complex organic compounds into smaller molecules for assimilation. Phosphatase activities are low when environmental P concentrations are high (Hill et al., 2012; Williams et al., 2012), and C hydrolytic enzyme activities are elevated when organic C is abundant (Hoppe et al., 1988). Understanding how the quantity and quality of DOM constrain the metabolism of biofilm communities is critical to comprehend the biogeochemical processing of C and nutrients in freshwater ecosystems. In areas in which running waters dry out to pools on summer or in areas in which pools are abundant per se the understanding of the interaction between DOM and the metabolism of the biofilm can be decisive to have a more complete model of their biogeochemical cycles. Eucalyptus is one of the most widespread exotic plant species due to plantations for wood and pulp purpose (Brown, 2000). Among the species of this genus, Eucalyptus grandis Hill ex Maiden is one of the most planted species in the world, mainly in subtropical and tropical regions (Burgess, 1988; Eldridge et al., 1994). As other Eucalyptus species, leaves of E. grandis contain high concentrations of recalcitrant chemical compounds as polyphenols, terpenoids and oils (Espinosa-Garcia et al., 2008; Tonin et al., 2014; Bachega et al., 2016) that are readily leached as DOM (Wallace et al., 2008). Moreover, the peak litter fall of

Eucalyptus often coincides with periods of seasonal low flow or water level in the tropics (Cizungu et al., 2014) and the temperate areas (Graça et al., 2002), resulting in high concentrations of C and nutrient subsidies, as well as inhibitory, recalcitrant compounds. We used Eucalyptus as a model to predict the effect of increasing recalcitrant leachates in biofilm activity. Our objective was to test effects of increasing concentrations of leaf-litter leachates on the functioning of freshwater biofilms, simulating different scenarios derived from water scarcity levels. Specifically, we tested how overall biofilm growth, metabolism [respiration (R) and gross primary production (GPP)], and extracellular enzyme activities (EEAs) involved in assimilation of simple polysaccharides and peptides (β-glucosidase and leucine aminopeptidase), complex polysaccharides (cellulase), recalcitrant C compounds (polyphenol oxidase) and phosphate (acid phosphatase and alkaline phosphatase) responded to increased concentrations of C, N and P-rich Eucalyptus leaf-litter leachates. We focused on functional measures instead of taxonomic measures to assess integrated effects on ecosystem processes. For this, we incubated biofilm at five different concentrations (one as control) of E. grandis leachates. We predicted that the increase in leachates would negatively affect growth and metabolism of biofilm and that the sensitivity of biofilms to Eucalyptus DOM concentrations would decline with increases in biofilm growth and colonization time, as the reliance of microbial biofilms on water column C and nutrient subsidies declines with microbial mat development (Pringle, 1990; Jenkinson and Lappin-Scott, 2001). Therefore, to evaluate if responses to leaf-litter leachate concentrations varied with colonization stage of microbial biofilms, we tested for effects of leachate concentrations after 1 to 4 weeks of colonization. 2. Materials and methods 2.1. Study site and experimental design In March 2014, we incubated next to each other four mesh bags containing 28 pieces (2.5 cm × 2.5 cm) of air-dried commercial cellulose sponges (0.65 ± 0.06 g for each sponge, 18.22 ± 1.37 g for each bag) in a groundwater-fed limestone pond at Florida International University (25° 45′ 29″ N, 80° 22′ 25″ W; Miami, USA) to allow colonization by biofilms. Cellulose sponge standard substrates (Sponge Cloth, The Coburn Company Inc) were used instead of natural substrates, like leaves or rocks, because they are easy to manipulate, have a high surface area to promote microbial colonization, and present lower variability in physical and chemical structure than natural substrates. In addition, cellulose provides a carbon source for the biota (mean ± SE: 30.02 ± 0.22%C; n = 8), but were very poor in nutrients (0.075 ± 0.002%N, 0.0015 ± 0.0002%P). Although microorganisms can feed on sponges, this material seems suitable for assessing the effects of increasing recalcitrant compounds and nutrients concentrations contained in Eucalyptus leachates, since the effects of changes in quality and quantity of dissolved organic matter have been proved in microbial assemblages colonizing substrates with much higher nutrients content (see Canhoto et al., 2013). After one, two, three and four weeks of colonization, one of the four bags was retrieved from the pond. Three of the 28 sponge pieces were used to quantify biofilm growth, elemental mass, R and GPP (see below the details for each measurement) after incubation in the pond prior to treatment with different leaf-litter leachate concentrations. The remaining 25 sponges were incubated individually in 35-mL glass vials with five different concentrations (four + control) of leaf-litter leachates from E. grandis (5 replicates for each concentration). Each treatment consisted of 15 mL of filtered (0.7-μm-pore size glass fiber filters, Whatman GF/F) pond water and 15 mL solution of different leachates concentrations. A control consisted of incubating colonized sponges in 30 mL of filtered pond water. To obtain the different leachate


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concentrations 15, 30, 45 and 60 g of air-dried green E. grandis leaves were submerged in 3 L of deionized water for four days. We chose this range of low-to-high leaf-litter leachate concentration to include concentrations reported by Canhoto and Laranjeira (2007) in isolated pools from Eucalyptus globulus Labill plantations. Leachate concentrations were made at the beginning of the experiment and stored in the dark at 4 °C and used throughout the experiment. We used green leaves to minimize differences in foliar chemistry that is inherent among senescent leaf litter. Microcosm vials were placed in racks and incubated in an outdoor water bath for 5 d under ambient light and temperature conditions at Florida International University. The minimal variability in climatic conditions in Miami for the entire incubation period (NOAA Online Weather Data; mean temperature 22.9 ± 0.7 °C, accumulated precipitation 66.3 mm) allowed us to accurately compare results among the four different biofilm colonization stages. To protect the vials from the exterior but to allow the gas exchange, a non-sealed cap was placed on top of each vial and all vials were shaken twice every day to avoid oxygen depletion.

2.5. Biofilm extracellular enzyme activities

2.2. Biofilm metabolism

2.6. Statistical analyses

All sampled sponges (both pre-incubated at the pond and incubated in the laboratory) were divided in two parts; half was placed in a dark bottle and half in a light bottle (Wheaton BOD bottles) with 300 mL of filtered (0.7 μm), oxygen saturated pond water to measure substratespecific microbial respiration (R) and gross primary productivity (GPP) under lab conditions (photosynthetically active radiation of 9 μmol m−2 s−1), respectively. Dissolved oxygen (DO) concentrations were recorded every 3 min for 30 min with DO sensors (YSI ProOBOD, Yellow Springs, Ohio). We used changes in DO in light and dark bottles without added sponges as control. We measured R and GPP as changes in the slope of DO concentrations over time for treatment incubations minus the slope of the control. Rates of R and GPP were corrected for container volume and sponge ash-free dry mass (AFDM; see below), expressed as mg O2 g AFDM−1 h−1.

For field data we used %AFDM, metabolism and elemental composition as response variables and constructed linear models testing effects of pre-treatment incubation (presence/absence) and/or pre-treatment incubation time (weeks). Linear and exponential models were tested in these models and fits with the highest R2 values using nontransformed data were selected. For the laboratory experiment, we calculated changes (Δ) in biofilm biomass, metabolism, and elemental content and molar ratios from pre-treatment to treatment incubations for each biofilm colonization stage. We developed linear models testing effects of leaf concentrations and biofilm pre-treatment incubation time (week) on the magnitude of changes in biofilm parameters. We tested for differences in biofilm extracellular enzymatic activities across leaf concentrations using linear models. Both linear and second-order polynomial regressions were used to test if leaf concentrations were shown just a subsidy (linear regression) effect or a subsidy/stress response with maximum values in intermediate concentrations of the leachate (second order polynomial regressions) (Odum et al., 1979). Data were transformed as needed to achieve homoscedascity and normality of the residuals. All statistical analyses were performed in R (version 3.2.5; R Development Core Team, 2016).

2.3. Biofilm biomass The two halves of cellulose sponges used for metabolism measurement were oven-dried (60 °C for 3 d) and one half of each sponge was combusted (550 °C for 4 h) to obtain AFDM. Total AFDM of sponges was estimated from dry-to-AFDM weight conversions from the combusted half. Initial AFDM of all sponges was estimated from dryto-AFDM weight conversions of non-incubated sponges (n = 8). In addition, we filtered incubation solutions from metabolism trials and treatment microcosms onto ashed, pre-weighed 0.7-μm filters. Filters were oven dried (60 °C for 3 d) and combusted (550 °C for 4 h) to calculate the AFDM of particulate sponge material lost during incubations. The remaining half of cellulose sponges were ground to a fine powder with a ball mill (8000D Spex CertiPrep, Metuchen, New Jersey) prior to determining elemental composition and stoichiometry. At week 4, the half sponges were newly cut into two pieces; one used for nutrient determination and the other one to measure microbial extracellular enzyme activities (EEAs; see below). 2.4. Biofilm elemental mass and stoichiometry We measured percent C and nitrogen (N) of cellulose sponges as a percentage of dry mass, using a Carlo Erba 1500 N CHN Analyzer (Carlo Erba, Milan, Italy). We quantified percent phosphorus (P) from combusted (550 °C for 4 h), acid-extracted cellulose sponges spectrophotometrically (ascorbic acid method; APHA, 2005). Cellulose P concentrations were expressed as a percentage of AFDM. All stoichiometry data were calculated as molar ratios.

Biofilm EEAs were quantified only from cellulose sponges under experimental conditions after 4 weeks of pond incubation. Following Saiya-Cork et al. (2002), sub-samples of cellulose sponges exposed to each leaf-litter-leachate treatment were blended with 60 mL of 50 mM acetate buffer (pH 5). The potential activities of acid phosphatase (AP), alkaline phosphatase (ALP), β-Glucosidase (BG), cellulase (CE) and leucine aminopeptidase (LA) were assayed fluorometrically (365 nm excitation, 450 nm emissions) using 4-Methylumbelliferyl phosphate, 4-nitrophenyl phosphate disodium, 4-Methylumbelliferyl β-D-glucopyranoside, 4-Methylumbelliferyl β-D-cellobioside and L-Leucine-7-amido-4-methylcoumarin hydrochloride, respectively. Polyphenol oxidase (PPO) activity were assayed colorimetrically (absorbance 460 nm) using L-dihydroxyphenylalanine (DOPA). Assays were conducted in 96-well microplates, and fluorescence was read with a Synergy H1 microplate reader (BioTek, Winooski, Vermont, USA). Appropriate blanks and controls were used to account for autofluorescence and quenching.

3. Results 3.1. Pre-treatment conditions and biofilm colonization Dissolved nutrient concentrations in pond water during the incubation period were 19.19 ± 0.27 μmol L− 1 for total dissolved nitrogen (TDN), 0.10 ± 0.01 μmol L− 1 for total dissolved phosphorous (TDP) and 456.60 ± 8.04 μmol L−1 for total organic carbon (TOC). Percent AFDM and C of cellulose sponges increased substantially after 1 week of incubation in the pond (Fig. 1, Table 2). However, substrate C:N ratio during this initial colonization period was constant (Fig. 1, Table 2). Throughout the 4-week incubation in the pond, percent AFDM increased linearly, and total N and P of substrates increased exponentially (Fig. 1, Table 2). Incubation reduced the percentage of C and C:P ratio of the substrates linearly and the C:N and N:P exponentially (Fig. 1, Table 2). Microbial respiration rates and GPP did not change with substrate incubation time (Fig. 1, Table 2). 3.2. Leachates concentration treatments Each treatment differed in total dissolved elemental concentrations (Table 1). Concentrations of TOC, TDN, and TDP were very closely related, and all increased with increasing leaf leachates concentrations.

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Fig. 1. Temporal changes in response variables of freshwater biofilms during pre-treatment incubations. Only significant lines are displayed with confidence intervals. Data of T0 (initial time point prior to incubation) is joined with the line when non-significant effects of the incubation were detected (see Table 2). %AFDM: % ash free dry mass, R: respiration, GPP: gross primary production.

Increases in TDN (3×), TOC (50×), and TDP (N650×) were measured above background pond water concentrations (leaf concentration = 0) for the highest leachate concentration treatment (Table 1). In microcosms with the most concentrated solution the amount of nutrients in the leachates represented 5.44% for C, 6.53% for N and 96.02% for P of the total content of those elements in the entire microcosm. 3.3. Biofilm biomass During incubations in microcosms in Eucalyptus leachates, mass (% AFDM) of most cellulose sponges (81.0%) increased (values of change in % AFDM from pre-treatment to the treatment N 0) (Fig. 2, Table 3). The magnitude of the increase was time-dependent, the higher the

Table 1 Total dissolved nitrogen (TDN), total dissolved phosphorous (TDP) and total organic carbon (TOC) in water (mean ± SE) for the different leaf concentration. R2 values after Pearson correlation among the four descriptors of the quality of the leachates are shown below. Leaf concentration (g L−1)

TDN (μmol l−1)

TDP (μmol l−1)

TOC (μmol l−1)

0 5 10 15 20

24.26 ± 1.17 29.15 ± 0.88 43.23 ± 0.85 64.08 ± 1.79 81.13 ± 1.77

0.38 ± 0.07 20.69 ± 5.85 62.44 ± 11.05 123.52 ± 13.80 252.69 ± 21.32

618.03 ± 25.58 7092.63 ± 385.09 13,485.42 ± 709.53 20,911.67 ± 777.80 31,153.33 ± 914.83

Leaf conc. TDN TDP

0.96

0.90 0.95

0.99 0.98 0.95

colonization stage in pond the lower the increase in biomass under treatments. Nevertheless, leachate concentration did not influence the Δ %AFDM (Fig. 2, Table 3) in the laboratory.

Table 2 Results of linear models testing effects of pre-treatment incubation (presence/absence) and/or pre-treatment incubation time (weeks) on changes in biofilm biomass (% ash-free dry mass, AFDM), metabolism (microbial community respiration, R; gross primary productivity, GPP), and elemental (carbon, C; nitrogen, N; phosphorus, P) mass (%) and molar ratios (C:N, C:P, N:P). Presence/absence effects of pre-treatment incubation on R and GPP were not tested, as these variables were only measured in incubated standard substrates. Significant P values (b0.05) are highlighted in bold. Data were transformed (natural logarithm, Ln) where necessary to meet assumptions of homoscedasticity. For molar ratios, log-transformation occurred before ratios were calculated. Variable

Source of variation

df

F

P

%AFDM (Ln)

Pre-treatment incubation Pre-treatment incubation time (weeks) Pre-treatment incubation time (weeks) Pre-treatment incubation time (weeks) Pre-treatment incubation Pre-treatment incubation time (weeks) Pre-treatment incubation Pre-treatment incubation time (weeks) Pre-treatment incubation Pre-treatment incubation time (weeks) Pre-treatment incubation Pre-treatment incubation time (weeks) Pre-treatment incubation Pre-treatment incubation time (weeks) Pre-treatment incubation Pre-treatment incubation time (weeks)

1,17 1,17 1,9 1,9 1,17 1,17 1,17 1,17 1,15 1,15 1,17 1,17 1,15 1,15 1,15 1,15

258.47 1338.13 1.75 0.06 612.41 566.32 0.21 197.55 0.30 239.15 12.87 111.96 1.11 78.08 0.08 59.74

b0.001 b0.001 0.218 0.817 b0.001 b0.001 0.654 b0.001 0.592 b0.001 b0.001 b0.001 0.308 b0.001 0.785 b0.001

R GPP %C (Ln) %N (Ln) %P (Ln) C:N (Ln) C:P N:P (Ln)


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Fig. 2. Change (Δ) in response variables of freshwater biofilms after 5-day incubations of standard substrates to different concentrations of Eucalyptus leaves. Colonization stage (weeks) are identified as follows: one, circles; two, squares; three, pyramids; and four, crosses. Lines are drawn only when the sources of variations were significant: significant effect of leachate concentration is indicated with a sloping line and significant effect of pre-treatment incubation time with parallel lines. No significant interaction between leachate concentration and pretreatment incubation time was observed (see statistics in Table 3). Thinner to thicker lines are used to represent week 1 through 4 of pre-treatment incubation. Data points have been displaced from the nominal 0, 5, 10, 15 and 20 values for the sake of clarity. %AFDM: % ash free dry mass, R: respiration, GPP: gross primary production.

In general, the concentration of C, N and P in sponges increased (85.0, 63.0 and 89.6% of the cases increased, respectively) after the incubation in microcosms (Fig. 2). The change of %C did not differ depending on the colonization stage, but was also positively influenced by leachate concentration (Fig. 2, Table 3). The variation of %N was not related to colonization stage or leachate concentration. Meanwhile, the colonization stage determined positively the change of %P, although no significant effect of the leachate concentration was observed (Fig. 2, Table 3). Incubation of the sponges in microcosms resulted in a reduction of the C:N, C:P and N:P molar ratios (66.0, 91.3 and 87.0% of the cases decreased, respectively), although the reductions were smallest for the sponges incubated for the longest in the field (Fig. 2, Table 3). However, as for variations in AFDM, stoichiometrical changes were not explained by Eucalyptus leachate concentration (Fig. 2, Table 3).

a reduction in the use of oxygen of 0.06 mg O2 g AFDM−1 h−1 in control treatment to an increase of oxygen consumption of 3.9 mg O2 g AFDM−1 h−1 at the highest concentrations of Eucalyptus leachates. Changes in GPP during incubations in microcosms also varied as a function of pre-treatment incubation time and leaf concentration gradient (Fig. 2, Table 3), with 20.2% of the sponges showing an increase and 79.8% a reduction. Declines in GPP during incubations in microcosms were highest for substrates pre-incubated for 4 weeks and incubated the highest concentration of E. grandis leachates (Fig. 2). Productivity to respiration (P:R) ratios associated with substrates did not change as a function of pre-treatment incubation time but declined with increasing Eucalyptus leachate concentrations during incubations in microcosms (F1,97 = 13.12, P b 0.001, Fig. 2). Incremental increases in leachate concentrations reduced variation in P:R among samples, with an overall 33% reduction of the variance in P:R for substrates exposed to leachates versus control (Fig. 3).

3.5. Biofilm metabolism

3.6. Biofilm extracellular enzyme activities

Microbial respiration rates generally increased after incubations in microcosms (75.8% of the cases increased), and the increments were highest for substrates pre-incubated in the field for the longest time (Fig. 2, Table 3). Respiration showed the clearest relationship with concentration of E. grandis leachates in the water, mean values ranging from

No effect of E. grandis leaf concentration on microbial EEAs was measured (Fig. 4, Table 4). Among the six EEAs measured, cellulase activities (CE) had a weak positive correlation with respiration, whereby CE explained 21% of the variance in microbial respiration rates (y = 2.75× + 2.94, R2 = 0.21, P = 0.021).

3.4. Biofilm elemental mass and stoichiometry

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Table 3 Results of linear models testing effects of leaf concentrations and biofilm pre-treatment incubation time (week) on changes in biofilm biomass (% ash-free dry mass, AFDM), metabolism (microbial community respiration, R; gross primary productivity, GPP), and elemental (carbon, C; nitrogen, N; phosphorus, P) mass (%) and molar ratios (C:N, C:P, N:P) following 5-day incubations in DOM treatments. Significant P values (b0.05) are highlighted in bold. Significant P values (b0.05) are highlighted in bold. Data were transformed (natural logarithm, Ln) where necessary to meet assumptions of homoscedasticity. For molar ratios, log-transformation occurred before ratios were calculated. Variable

Source of variation

df

F

P

Δ %AFDM (Ln)

Leaf concentration Week Leaf concentration × week Leaf concentration Week Leaf concentration × week Leaf concentration Week Leaf concentration × week Leaf concentration Week Leaf concentration × week Leaf concentration Week Leaf concentration × week Leaf concentration Week Leaf concentration × week Leaf concentration Week Leaf concentration × week Leaf concentration Week Leaf concentration × week Leaf concentration Week Leaf concentration × week

1,96 1,96 1,96 1,95 1,95 1,95 1,95 1,95 1,95 1,95 1,95 1,95 1,96 1,96 1,96 1,65 1,65 1,65 1,95 1,95 1,95 1,65 1,65 1,65 1,65 1,65 1,65

2.21 64.66 0.02 128.48 18.26 3.00 6.38 7.49 2.45 5.04 1.88 0.24 0.59 0.71 0.08 0.00 9.08 0.02 1.78 20.34 0.43 3.68 36.13 1.56 1.44 4.14 0.28

0.14 b0.01 0.89 b0.01 b0.01 0.45 0.01 0.01 0.12 0.03 0.17 0.63 0.44 0.40 0.78 0.98 b0.01 0.90 0.19 b0.01 0.51 0.06 b0.01 0.22 0.24 0.05 0.60

ΔR

Δ GPP

Δ %C (Ln)

Δ %N (Ln)

Δ %P (Ln)

Δ C:N (Ln)

Δ C:P

Δ N:P (Ln)

4. Discussion Increases in concentrations of leachates enhanced net heterotrophy of freshwater biofilms with linear increases in microbial R and decreases in GPP. Although large increases in the C and nutrient concentrations were measured with increases in leaf-litter leachate concentrations, functional changes in biofilm metabolism were not explained by treatment-induced changes in biofilm biomass or stoichiometry.

Fig. 3. Productivity to respiration ratios of freshwater biofilms under different concentrations of Eucalyptus leaves. Pre-treatment incubation weeks are identified as follows: one, circles; two, squares; three, pyramids; and four, crosses. Average, the 95% confidence interval, and the 95th quantile regression are shown.

Moreover, contrary to our prediction, biofilms at more advanced colonization stages responded more strongly to changes in concentrations of leaf-litter leachates. However, later-stage colonization of microbial biofilms can coincide with increases in algal biomass and in subsidies available to heterotrophic microbes (Sekar et al., 2004; Pohlon et al., 2010). Increases in leaf-litter leachates can have strong inhibitory effects on autotrophic responses (Lovatt et al., 2014) which may explain our observations of strong effects of leachate concentrations in latercolonization biofilm assemblages. As expected, our experimental leachate treatments differed in C and nutrient concentrations. Higher levels of isolated N and P or the combination of these two macronutrients enhance metabolic rates and growth of microbial autotrophs (Larned, 2010). Labile leachates are more easily incorporable by biofilms and reduce competition for nutrients between autotrophic and heterotrophic components of biofilms (Bechtold et al., 2012). This lower-concentration saturating response may explain the lack of a positive autotrophic response at higher concentrations of dissolved N and P in our treatment leachates. The highly recalcitrant compounds present in leaves of E. grandis (Espinosa-Garcia et al., 2008; Tonin et al., 2014; Bachega et al., 2016) can have inhibitory or toxic effects on both respiration and primary productivity of microbial biofilms (Schlief and Mutz, 2007; Ylla et al., 2012; Canhoto et al., 2013). Nevertheless, the abundance of polyphenols reported in E. grandis leaves (Espinosa-Garcia et al., 2008; Tonin et al., 2014; Bachega et al., 2016) target photosynthesis (Sun et al., 2006; Shao et al., 2009; Pohlon et al., 2010; Bährs et al., 2013), as the oxidation of these compounds leads to the formation of radicals that interfere with electron transfer chain in the Photo System II (Oettmeier et al., 1988; Laue et al., 2014). Similarly, Lovatt et al. (2014) observed that labile leaf-litter leachates had stronger inhibitory effects on autotrophic than stimulatory effects on heterotrophic assemblages of the biofilm. Our results show that along gradients in leachate concentration, biofilm R increased and GPP decreased, suggesting that E. grandis leachates promoted more heterotrophic biofilms. In addition to changing dissolved chemical properties of water, leachates can change water color and restrict light penetration, which can limit GPP and DO and promote heterotrophy. In other places, leachates of Eucalyptus species have decreased water pH below 5 (Canhoto and Laranjeira, 2007; Canhoto et al., 2013), which can enhance algal biomass (France and Welbourn, 1992) and inhibit microbial respiration (Dangles et al., 2004). Although we did not measure water pH directly, the high buffering capacity of karstic aquatic ecosystems like South Florida wetlands (Chen et al., 2010) likely explains why we did not detect an enhancement of GPP or inhibition of microbial respiration. Eucalyptus leachates promoted heterotrophic over autotrophic activities, but without changes in biofilm structure or elemental content. Moreover, variation in P:R ratios decreased with increasing leachate concentrations. Enhanced heterotrophy should increase C losses and should also be mirrored by changes in biomass and stoichiometry of biofilms. However, we did not observe differences in biofilm biomass or C:nutrient stoichiometry incubated in our different treatments. A possible explanation is that the short incubations (5 days) were sufficient to alter biolfilm metabolism but insufficient to observe these effects incorporated into the biofilm biomass. In addition, enhanced growth of heterotrophic microbes may have consumed algal biomass, thereby causing declines in GPP and increases in R but no changes in total biofilm AFDM. Activities of heterotrophic microbes (e.g., EEAs) are stimulated when algal cell lyse and release labile DOM (Romaní and Sabater, 2000), but we did not detect such changes in biofilm C and nutrient-based EEAs. Forecasted shifts in temperature and precipitation regimes (IPCC, 2013) lead to the promotion of water scarcity periods, stimulating the loss of hydrological connectivity with terrestrial ecosystems and the reduction of dilution capacity, which promote the quantity of leaf leachates (Sabater and Tockner, 2009) and their role as the main DOM source (Casas-Ruiz et al., 2016). Under this scenario, which may be


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Fig. 4. Biofilm extracellular enzymatic activities (AP, acid phosphatase; ALP, alkaline phosphatase; LA, leucine aminopeptidase; BG, β-Glucosidase; CE, cellulase; and PPO, polyphenol oxidase) at the colonized substrata under different leaf concentrations (see statistics in Table 4).

extrapolated to small lentic systems, promoted by human-made weirs and impoundments worldwide, and isolated pools in streams, increased concentrations of leachates may affect longer-term ecosystem functioning. Due to the key role of biofilm in biogeochemical cycling and the trophic base of freshwater food webs (Allan and Castillo, 2007), biofilm metabolism alteration could modify the transfer of energy and nutrients to higher trophic levels, potentially affecting secondary production of an array of consumers. Changes in autotrophic and heterotrophic components of biofilms could alter the priming effect of more recalcitrant compounds both dissolved (Rier et al., 2014) and particulate (Danger et al., 2013; Kuehn et al., 2014). Thus, declines in autotrophs could also slow down organic matter processing in fresh waters and the role of these systems in C and nutrient cycling (Battin et al., 2009; Cheever et al., 2012).

5. Conclusion Our results indicate that increases in concentrations of Eucalyptus leaf-litter leachates increased R and decreased GPP of freshwater biofilms, enhancing net heterotrophy. Due to the key role that biofilms

Table 4 Results of linear models (linear and second-order polynomial regressions) testing effects of leaf concentrations on microbial extracellular enzyme activities. AP: acid phosphatase, ALP: alkaline phosphatase, LA: leucine aminopeptidase, BG: β-glucosidase, CE: cellulase, PPO: polyphenol oxidase. Variable

AP ALP LA BG CE PPO

Linear

Polynomial

df

F

P

df

F

P

1,23 1,23 1,23 1,23 1,23 1,22

0.82 0.02 0.47 0.02 2.62 0.00

0.38 0.89 0.50 0.89 0.12 0.96

1,22 1,22 1,22 1,22 1,22 1,21

0.71 0.08 1.04 1.74 0.02 0.36

0.41 0.78 0.32 0.20 0.90 0.55

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