Microb Ecol (2012) 64:593604 DOI 10.

1007/s00248-012-0062-6

MICROBIOLOGY OF AQUATIC SYSTEMS

Labile and Recalcitrant Organic Matter Utilization by River Biofilm Under Increasing Water Temperature
Irene Ylla & Anna M. Roman & Sergi Sabater

Received: 26 November 2011 / Accepted: 21 April 2012 / Published online: 9 May 2012 # Springer Science+Business Media, LLC 2012

Abstract Microbial biofilms in rivers contribute to the decomposition of the available organic matter which typically shows changes in composition and bioavailability due to their origin, seasonality, and watershed characteristics. In the context of global warming, enhanced biofilm organic matter decomposition would be expected but this effect could be specific when either a labile or a recalcitrant organic matter source would be available. A laboratory experiment was performed to mimic the effect of the predicted increase in river water temperature (+4 C above an ambient temperature) on the microbial biofilm under differential organic matter sources. The biofilm microbial community responded to higher water temperature by increasing bacterial cell number, respiratory activity (electron transport system) and microbial extracellular enzymes (extracellular enzyme activity). At higher temperature, the phenol oxidase enzyme explained a large fraction of respiratory activity variation suggesting an enhanced microbial use of degradation products from humic substances. The decomposition of hemicellulose (-xylosidase activity) seemed to be also favored by warmer conditions. However, at ambient temperature, the enzymes highly responsible for respiration activity variation were -glucosidase and leuaminopeptidase, suggesting an enhanced microbial use of polysaccharides and peptides degradation products. The
I. Ylla (*) : A. M. Roman : S. Sabater Institute of Aquatic Ecology, University of Girona, Campus Montilivi, 17071 Girona, Spain e-mail: irene.ylla@gmail.com S. Sabater Catalan Institute for Water Research (ICRA), Emili Grahit, 101, 17003 Girona, Spain

addition of labile dissolved organic carbon (DOC; dipeptide plus cellobiose) caused a further augmentation of heterotrophic biomass and respiratory activity. The changes in the fluorescence index and the ratio Abs250/total DOC indicated that higher temperature accelerated the rates of DOC degradation. The experiment showed that the more bioavailable organic matter was rapidly cycled irrespective of higher temperature while degradation of recalcitrant substances was enhanced by warming. Thus, pulses of carbon at higher water temperature might have consequences for DOC processing.

Introduction Biofilms covering the streambed are the main processors of organic matter [56]. In freshwaters, dissolved organic matter (DOM) makes the larger proportion of the organic matter pool. DOM quantity and quality varies with its source; DOM released from autochthonous production (derived from within the aquatic ecosystem) is the most labile and bioavailable in the stream DOM pool. It consists of a large array of biomolecules, including lipids, carbohydrates, polysaccharides, amino acids, proteins, waxes, and resins [52]. In contrast, allochthonous DOM (terrestrially derived) is more recalcitrant, more resistant to biological degradation [8, 9], and encompasses heterogeneous refractory organic substances of high molecular weight [45]. Altogether, DOM in rivers usually includes minor amounts of biodegradable material that are rapidly recycled and large amounts of refractory residues that are partially degraded (photochemically or microbiologically) [11]. Current climate models predict that mean annual temperature will increase by 3.5 C in the air and 2.24.3 C in stream waters by 2,100 [33]. It is widely accepted that

594

I. Ylla et al.

global warming broadly affects and will further affect ecosystems [39, 66]. Increasing water temperature will probably result in increasing the metabolism of organic carbon [1, 53]. Gudasz et al. [30] observed that the mineralization of organic carbon in lake sediments was positively related with temperature, implying that warmer temperatures could lead to higher mineralization and a decrease of organic carbon burial. Also, Jenkinson et al. [37] postulated that one effect of global warming will be to accelerate the decomposition of soil organic matter. In streams, the microbial biofilm is a hotspot for organic matter use and recycling which is not only affected by the nature of the available organic matter source [4, 12] but also by the temperature conditions [19]. Temperature increases the respiration rates of algae, bacteria, and invertebrates [6, 7]. However, the detailed temperature response pattern of different organic substrates to be microbially decomposed remains obscure. In this study, we tested if the predicted increase in stream water temperature could differentially affect the decomposition of labile and recalcitrant material by the microbial biofilm communities. Because the recalcitrance of a molecule is defined by the activation energy required to break its chemical bonds [61], higher temperature and activation energy can promote the degradation of recalcitrant organic substrates. The Arrhenius equation indicates that the higher the activation energy for a reaction, the higher its temperature dependence. Thus, elevated temperature will theoretically accelerate the breakdown of recalcitrant compounds to a greater degree than the breakdown of labile compounds. The temperature sensitivity of organic carbon decomposition has been extensively studied in soils but the temperature sensitivity of soil organic carbon decomposition remains controversial [16, 54]. Some studies have indicated that the decomposition of older, more recalcitrant soil organic matter is less temperature sensitive [28], equally temperature sensitive [14, 20], or more temperature sensitive [13, 40] than labile soil organic matter decomposition. Relatively little is known about the potential responses of the OM degradation mediated by biofilms at higher water temperatures. While Sand-Jensen et al. [58] did not observe differences in dissolved organic carbon (DOC) degradation related to temperature in a small stream, observations in the laboratory showed that changes in water temperature affected biofilm structure and determined its DOM microbial use [19]. In river ecosystems, microbial biofilms respond to a wide array of carbon inputs, even those that might be considered recalcitrant. Microorganisms have a key role in the degradation of DOM [47, 62]. Bacterial extracellular enzymes convert high molecular weight compounds to lower molecular weight compounds that are easily assimilated by heterotrophic microbes [15]. Because the synthesis of extracellular enzymes is regulated by the organic molecules

available, they provide a powerful tool to understand the potential microbial use of DOC [55]. In this study, the analyzed enzyme activities covered the degradation of labile compounds such as simple polysaccharides and peptides (glucosidase and leucine-aminopeptidase), simple but less common polysaccharides (-xylosidase, involved in the last step of hemicellulose decomposition) and recalcitrant compounds such as lignin (phenol oxidase). The high population density within the biofilm, the expression of extracellular enzymes, and even cell-to-cell signals contribute to the response of biofilms to pulsed inputs [23]. The outcome of biofilm DOM processing under higher temperature may depend on the carbon source. In the present laboratory experiment, we simulated two extreme available DOM quality sources by the addition of high quality labile DOC (cellobiose and a dipeptide) and recalcitrant DOC (humic material). The experiment evaluated the effects of a 4 C increase in temperature on biofilm respiration and OM degradation capacity. Specifically, we aimed to determine whether a temperature increase could differentially affect the degradation of labile versus recalcitrant DOC by microbial biofilms. We hypothesized that the recalcitrant OM would be more intensively used at the higher water temperature while the use of labile carbon would be barely affected by the temperature increase.

Methods Experimental Procedure The designed experiment included two treatments with contrasting DOC quality added (labile and recalcitrant) plus a control (where no extra C was added) at two different water temperatures (14 and 18 C). Six treatments were defined: elevated temperature-labile DOC, elevated temperaturerecalcitrant DOC, elevated temperature-control, ambient temperature-labile DOC, ambient temperature-recalcitrant DOC, and ambient temperature-control. Three replicate microcosms were used per treatment. Each microcosm consisted of a glass jar (19 cm in diameter and 9 cm high) with 4045 glass tiles (1 cm2 each tile) attached to the jar s bottom. The microcosms were filled with 2 L of artificial stream water. To mix the water body inside each microcosm, water was recirculated by means of a submersible pump (Hydor, Pico 300, 230 V 50 Hz, 4.5 W). The artificial stream water was obtained by dissolving pure salts (12 mg L1 Na2SO4, 20 mg L1 Na2SiO3, 30 mg L1 CaCl2, 1 mg L1 KCl, 2 mg L1 MgSO4, and 20 mg L1 NaHCO3) in MilliQ water to reproduce the chemical composition of a typical headwater forested stream (the Fuirosos [64]), from where the biofilm inoculum was collected.

Effects of warming on DOC use

595

Colonization Period The glass tiles in the jars were colonized with epilithic biofilms previously scraped from rocks collected from the Fuirosos stream during May, when the mean stream water temperature was of 14 C. After homogenizing the biofilm inoculum (thus each inoculum comes from the same batch), biofilm aliquots (10 mL with ca. 35 g chlorophyll) were added to each microcosm once a week (during 3 weeks). Colonized biofilms were maintained inside two incubators (SCLAB-PGA500) with a constant daynight light cycle (12 h/12 h) at 14/11 C day/night temperatures, respectively. Ammonium phosphate (30 g L1) and ammonium nitrate (750 g L1) were added to all treatments every 34 days during the whole experiment (including the colonization period and experimental phase) to prevent phosphorous and nitrogen depletion. During the colonization period the water from the microcosms was replaced every 34 days, and no DOC was added in the microcosms. Experimental Stage When biofilms were 3 weeks old, the experimental temperatures and DOC conditions were applied to the respective microcosms. The two temperature treatments consisted of 14/11 C day/night temperatures, respectively for the ambient temperature treatment and 18/15 C day/night temperatures, respectively for the elevated temperature treatment. The reference 14 C corresponded to the mean water temperature in the Fuirosos stream in autumn and spring [4], while 18 C correspond to a 4 C water stream temperature increase. The DOC treatments respectively received one daily pulse of 10 mg C L1 of labile DOC (labile treatment) or 10 mg C L1 of recalcitrant DOC (recalcitrant treatment), during three consecutive days. A total of 30 mg C L1 DOC was received by the microcosms after these 3 days. No extra DOC was added to the control treatment. The labile DOC was made up as a mixture of cellobiose (5 mg C L1, from Sigma-Aldrich) and the dipeptide leucine-proline (5 mg C L1, from Sigma Aldrich). The recalcitrant DOC enrichment consisted of pure humic substances isolated from the Swannee River (from the International humic substance society, IHSS, ref. 2S101H). No further water replacements were done after the DOC pulses so that potential changes in DOC quality and quantity could be monitored after the pulses. Nutrient additions and the daynight light cycle were maintained like during the colonization period. Once the experimental temperature conditions and DOC pulses were applied, biofilms in the microcosms were sampled on day 1 (1 day after the third DOC pulse) and later on days 3, 6, 10, and 17. Glass tiles were collected at random from each microcosm. Changes in DOC quality (by means of fluorescence index (FI) and absorbance ratio) as well as

community respiration and biofilm extracellular enzyme activities were analyzed. Biofilm chlorophyll and bacterial numbers were also measured. On the last sampling date, primary production and respiration were analyzed.

Physicochemical Parameters Water temperature in each microcosm was recorded every 5 min by a submerged temperature data logger (ACR SmartButton reader) to register any possible fluctuation from the temperature established in each incubator. Water nutrient content, oxygen (Hach DO meter) and conductivity (conductivity meter, WTW) were monitored twice a week during biofilm colonization and at each sampling date during the experiment. Water aliquots from each microcosm were collected for analysis of inorganic nutrients and DOC after filtration (0.2 m pore-diameter nylon filter, Whatman). Nitrate was analyzed by ion chromatography (Metrohm 761 compact IC), ammonia was determined following Hach [32], and phosphate was analyzed spectrophotometrically, as described by Murphy and Riley [50]. All DOC samples were acidified with 2M HCl (2 %) and stored at 4 C until analysis. DOC was determined with a Shimadzu TOC 5050A.

DOC Quality Fluorescence Index Filtered water samples (Whatman GF/F filters) were collected from each microcosm on each sampling day and were adjusted to pH 2 with concentrated hydrochloric acid. Fluorescence was measured at 370 nm excitation and 450 and 500 nm emission and blank corrected (Milli-Q water acidified with HCl to pH 2). FI was calculated as the ratio of emission intensity (450 nm/500 nm) at 370 nm excitation. This ratio is indicative of the origin of the OM, more terrestrially or microbially derived. Higher FI values are indicative of microbially derived material (lower aromaticity) and lower FI values are linked with more terrestrially derived sources (higher aromaticity) [46, 63].

DOC Absorbance Water samples were collected from each microcosm at each sampling day and absorbance measured immediately. Water absorbance at 250 nm (Shimadzu UV-1800) was used to calculate the ratio Abs250/total DOC to indicate the proportion of humic material in the total DOC [24, 49]. Samples from the recalcitrant DOC treatments were diluted (1/2) with Milli-Q water.

596

I. Ylla et al.

Extracellular Enzyme Activities The biofilm extracellular enzyme activities of -D-1,4-glucosidase (EC 3.2.1.21) and -xylosidase (EC 3.2.1.37) were determined spectrofluorometrically using fluorescent methylumbelliferone (MUF; from Sigma-Aldrich) linked artificial substrates (4-MUF--D-glucopyranoside and 4-MUF-7-D-xyloside, respectively). Leucine-aminopeptidase activity (EC 3.4.11.1) was analyzed by the fluorescent-linked artificial substrate L-leucine-7-amido-4-methylcoumarin hydrochloride (from Sigma-Aldrich). Saturation curves (MichaelisMenten fitting) were previously performed to determine substrate saturation concentrations for the measure of potential extracellular enzyme activities, and a final concentration of 300 M was added to each glass tile. Preparation of MUF substrates required a previous dissolution of substrates in hydroximethylether (final solution in the assay of 0.1 %) to facilitate their dissolution in water. Concentrated substrate solutions (10 mM) were prepared with autoclaved ultrapure water and kept in the freezer (20 C) until use. After the corresponding substrate addition to the glass tiles (one for each microcosm and sampling date), samples were incubated in the dark at the respective treatment temperatures (14 and 18 C) and under continuous shaking for 1 h. Blanks (which contain filter-sterilized microcosms water) and standards of MUF and AMC were also incubated. At the end of the incubation, glycine buffer (pH 10.4) was added (1/1 vol./vol.), and fluorescence was measured at 365/ 455 nm excitation/emission for MUF and at 364/445 nm excitation/emission for AMC (spectrofluorimeter Kontron SFM 25). The intensity of fluorescence of the blanks was subtracted from all samples to correct for hydrolysis of the substrate or fluorescent substances in the water solution. Units are expressed as nmol MUF or AMC cm2 h1. Phenol oxidase activity (EC 1.14.18.1) was measured following the method outlined by Sinsabaugh et al. [60] by using L -3,4 dihydroxyphenylalanine (from SigmaAldrich) at a final concentration of 5 mM with acetate buffer. Incubations lasted 1 h. Samples were also incubated at the respective treatment temperatures (14 or 18 C) on agitation and in darkness. At the end of incubations, the absorbance of the liquid phase was measured at 460 nm (Shimadzu UV-1800). Units are expressed as mol DIQC cm2 h1. Respiratory Activity and Primary Production

two to three tetrazolium chloride from Sigma-Aldrich) into INT formazan. One tile for each microcosm plus 4 mL of water from each corresponding microcosm and two blanks were incubated in 0.02 % INT solution. Incubations were performed in a shaker at the respective treatment temperatures (14 and 18 C) for 8 h in the dark. After this, INTformazan was extracted with methanol for a minimum of 1 h at 4 C in the dark followed by sonication (2 min, Selecta, 40-W power; [10]). The extracts were filtered (GF/F filters, Whatman) and their absorbance measured spectrophotometrically at 480 nm (Shimadzu UV-1800). A stock solution of 30 g mL1 INT formazan (Sigma-Aldrich) in methanol was used to prepare a standard curve. Primary Production Net primary production (NPP) and community respiration were measured on the last sampling day of the experiment after differences in oxygen concentration (oxygen balance method) were determined in each microcosm. Oxygen concentrations were determined with a Hach DO meter probe. Three glass tiles from each microcosm were removed and incubated in Winkler vials for 1.5 h. A first incubation in the early morning under dark conditions was performed to measure community respiration. Three consecutive incubations with the same glass tiles but under light conditions were performed to determine NPP. Biofilm Structure Bacterial Numbers Live and dead bacteria were counted using the Live/Dead Baclight bacterial viability kit (Invitrogen Molecular probes) [27]. Bacterial numbers were estimated after sonication of each glass tile (one per microcosm on days 1 and 17) for 90 s using a sonication bath (Selecta) operating at 40 W and 40 kHz. After appropriate dilution (ten times) with sterile water, a 1:1 mixture of SYTO 9 and propidium iodide was added to the samples and incubated for 15 min. Samples were then filtered (0.2 m pore-diameter black polycarbonate filters, Nucleopore, Whatman) and at least 20 fields were randomly counted in each slide (Nikon E600 epifluorescence microscope). The fraction of live bacteria was calculated as the abundance of live cells divided by the total counts obtained with the Live/Dead method. Chlorophyll a

Community Respiration The electron transport system (ETS) activity in each microcosm on each sampling day was assayed after the reduction of the electron transport acceptor iodonitrotetrazolium (INT; Chlorophyll a concentration on the glass tiles (one replicate for each microcosm from the first and last sampling date) was measured after extraction in 90 % acetone for 12 h in the dark at 4 C. To ensure complete extraction of

Effects of warming on DOC use

597

chlorophyll, samples were further sonicated for 2 min in a Selecta sonication bath operating at 40 W and 40 kHz and previously protected from light. Extracts were filtered through 1.4 m mesh fiberglass filters (GF/C Whatman) and chlorophyll a concentration was determined spectrophotometrically using a Lambda UV/VIS spectrophotometer (Hitachi), following Jeffrey and Humphrey [36]. Statistical Analyses Differences in DOC concentration, FI, Abs250/total DOC, extracellular enzyme activities and community respiration (ETS) between the different treatments over the five sampling days were analyzed using a two-way repeated measures analysis of variance (RM-ANOVA). This analysis was used to test for the single source effects and the interactions between two factors: temperature (low and high) and DOC (labile, recalcitrant, and control). Probabilities within groups (day temperature, day DOC, and day temperature DOC) were corrected for sphericity using the Greenhouse Geisser correction. All probabilities were adjusted using the DunnSidak correction. All variables were log (x +1) transformed. If significant differences were detected among treatments, Tukeys HSD post hoc tests were performed to determine which treatments differed. Variability in bacterial number, chlorophyll a (both from days 1 and 17), and primary production and respiration (from day 17) were analyzed by ANOVAs (two way for primary production and respiration and three way for bacteria and chlorophyll). This analysis was used to test for the single source effects of the studied factors and their interactions: temperature and DOC source. In the case of the bacterial number and chlorophyll a, the comparison between the two sampling dates was also included (time effect). The four variables were also log (x +1) transformed. The relationship between ETS and enzymatic activities were analyzed by forward multiple linear regression analysis in order to determine the enzymes which better explain the variation in the biofilm respiratory activity. The SPSS software package for Windows (Ver. 14.0.1, SPSS Inc. 19892005) was used for all statistical analyses.

temperature treatments; 140.8 and 110.3 C, day/night, respectively at ambient temperature treatments; n 0 1,836). The decrease in nutrient content achieved 53 g PO4 L1 (corresponding to a 75 % reduction with respect to the initial nutrient concentration), 11.25 g NNH4 L1 (corresponding to a 56 % reduction with respect to the initial nutrient concentration) and 36056 g NNO3 L1 (corresponding to a 60 % reduction with respect to the initial nutrient concentration) in each 3- to 4-day period, but the periodic additions of N and P reestablished the initial nutrient conditions. Changes in DOC Content and Quality For the whole experiment and considering all DOC treatments (control, labile DOC, and recalcitrant DOC), DOC content was lower at elevated temperature (Fig. 1a; Table 1), and temperature effect showed no differences between DOC treatments (not significant temperature DOC interaction; Table 1). However, DOC evolved differently between DOC quality treatments. The initial pulse concentration (30 mg C L1) in the labile treatments rapidly decreased while in the recalcitrant-amended DOC treatments, there was a slight DOC increase throughout the experiment (Fig. 1a). DOC concentrations in the control treatment increased from 4 0.4 to 161.0 mg C L1 (Fig. 1a; Table 1). The FI was significantly lower in the recalcitrant treatment than in the labile and control treatments (Fig. 1b; Table 1). Temperature slightly increased the index values in the recalcitrant treatment throughout the experiment (temperature DOC day interactions; Table 1) but did not affect the other two treatments. The Abs250/total DOC index was much higher in the recalcitrant than in the labile and control treatments (Fig. 1c; Table 1). The absorbance index slightly decreased in the recalcitrant microcosms with higher temperature (temperature DOC interaction, Table 1). Biofilm Community Respiration, Primary Production, and Extracellular Enzyme Activities Community respiration (ETS) of the biofilms increased with temperature and the highest values were measured in the labile DOC treatments (temperature and DOC effects, Table 1; Fig. 2; Tukeys test, p <0.009). At the ambient temperature, changes in ETS over time were affected by DOC treatment, especially from days 10 to 17; steeply increasing with the labile DOC; slightly increasing in the control treatment; and maintained at lower levels with the recalcitrant DOC. In contrast, at the elevated temperature, community respiration was further enhanced at the recalcitrant and control treatments and its time evolution was similar in the three DOC treatments (Fig. 2; Table 1, temperature DOC day interaction).

Results Chemical Conditions at the Microcosms Conductivity increased in all microcosms (from 125 to 225 S cm1) throughout the experiment, while dissolved oxygen remained steady (10.50.9 mg L1). After the onset of the experimental temperature conditions, temperature remained as established in the set up in all microcosms (18 0.8 and 15 1.1 C, day/night respectively at elevated

598

c) b)
Fluorescence index DOC (mg L )

Abs250/ total DOC (m2 gC-1)

-1

a)

1.2 10 20 30 0

1.4

1.6

1.8

2.0

40

50

D ay 1 D ay 3

AT-C AT-L AT-R

D ay 6

ET-C ET-L ET-R

D ay 10

Table 1 Repeated measures ANOVA results considering the effects of temperature (elevated or ambient) and DOC source (control, labile, or recalcitrant) on DOC quality as well as on biofilm metabolic parameters
Fluorescence index 0.838 0.05 F1, 12 0 10.123 0.020 F2,
12 0 9.513

Figure 1 a DOC concentration, b fluorescence index, and c ratio Abs250/total DOC on the different sampling days under each set of conditions (ambient temperature-control (AT-C), ambient temperaturelabile DOC (AT-L), ambient temperature-recalcitrant DOC (AT-R), elevated temperature-control ( ET-C ), elevated temperature-labile DOC (ET-L), and elevated temperature-recalcitrant DOC (ET-R)). Values are meansstandard errors (vertical bars). For better clarity, half of the error bars were shown (n 0 3)
D ay 17

The net primary production (NPP) and community respiration were measured on the last sampling day. NPP decreased at elevated temperature (ANOVA, temperature effect p <0.001), and this was especially relevant in DOCamended treatments (labile and recalcitrant DOC additions; ANOVA, DOC effect, p 0 0.026; Fig. 3). NPP was lower in the recalcitrant than in the labile and control treatments (Tukeys test, p 0 0.007). Community respiration measured with the oxygen balance (dark bottles) was enhanced at elevated temperature in the control and recalcitrant treatments but not in the labile treatment (ANOVA, temperature effect, p <0.001 and DOC effect, p <0.001; Fig. 3).
Abs250/total DOC 0.564 F1, 12 0 2.950 <0.001 F2, 12 0 4381.6 <0.001 F1.6, 20.1 0 28.854 0.027 F2, 12 0 9.285 0.976 F1.6, 20.1 0 0.871 <0.001 F3.3, 20.1 0 20.689 0.510 F3.3, 20.1 0 2.352 ETS -glucosidase 0.013 F1, 12 0 15.113 0.726 F2, 12 0 2.070 <0.001 F2.9, 34.8 0 58.627 0.848 F2, 12 0 1.631 0.883 F2.9, 34.8 0 1.383 0.174 F5.8, F5.8,
34.8 0 2.769

Source of variation

DOC

Peptidase <0.001 F1, 12 0 47.045 <0.001 F2, 12 0 28.073 <0.001 F2.8, 34.5 0 9.104 0.936 F2, 12 0 1.237 0.652 F2.8, 34.5 0 1.961 0.356 F5.7, 34.5 0 2.274 <0.001 F2.8, 34.3 0 76.214 0.442 F2, 12 0 3.140 0.156 F2.8, 34.3 0 3.644 0.264 F5.7, 34.3 0 2.501

-xylosidase 0.581 F1, 12 0 2.856 0.264 F2, 12 0 4.156 <0.001 F2.6, 31.8 0 23.145 0.998 F2, 12 0 0.511 <0.001 F2.6, 31.8 0 14.323 0.006 F5.3, 31.8 0 5.187

Phenol oxidase 0.999 F1, F2,

12 0 0.153

Temperature F1, <0.001 F2, <0.001 F3.1, 0.584 F2, <0.001 F3.1, 0.020 F6.2, 0.006 F6.2,
37.6 0 4.799 37.6 0 4.126 37.6 0 11.096 12 0 2.569 37.6 0 27.872 12 0 898.509 12 0 1.610

<0.001

F1, 12 0 36.679

DOC

<0.001

0.275
12 0 4.057

F2, 12 0 1143.1

Day

<0.001

<0.001 F2.8, F2,

33.9 0 15.323

F3.3, 40.6 0 143.717

Temperature DOC

0.192

0.997
12 0 0.600

F2, 12 0 4.764

Temperature day

0.341

0.994 F2.8, F5.6, 0.013

34.8 0 4.649 33.9 0 0.739

F3.3, 40.6 0 2.612

DOC day

<0.001

0.067
33.9 0 3.466

F6.7, 40.6 0 136.906

Temperature DOC day

0.291

0.054 F5.7, 34.5 0 3.545

0.013 F5.7, 34.3 0 4.515

0.31 F5.3, 31.8 0 2.437

0.999

I. Ylla et al.

F6.7, 40.6 0 2.281

F5.6,

33.9 0 0.709

Probability within groups was corrected for sphericity by the GreenhouseGeisser correction, and p values were adjusted by the DunnSidak correction. Values of <0.05 are indicated in boldface type and values <0.1 are indicated in italics

Effects of warming on DOC use Figure 2 Temporal changes in community respiration (electron transport system) on epilithic biofilms (growing on the artificial glass substrata). Values are meansstandard errors (vertical bars). For better clarity, half of the error bars were shown (n 0 3)

599

AMBIENT TEMPERATURE
3.5

ELEVATED TEMPERATURE
Community respiration

Community respiration

g formazan cm-2 h-1

3.0

2.5

2.0
Control Labile DOC Recalcitrant DOC

1.5

Control Labile DOC Recalctrant DOC

1.0
10 1 6 10 17 D ay D ay D ay D ay D ay D ay ay D ay D ay D D ay 17 1 3 6 3

Higher temperature significantly increased -glucosidase and leu-aminopeptidase activities (temperature effects, Table 1; Fig. 4). Time evolution of -xylosidase was also significantly affected by temperature (temperature day interaction, Table 1), showing higher activity values from day 10 and until the end of the experiment in elevated temperature treatments. No significant effect of temperature was found for phenol oxidase activity (Table 1). DOC addition affected the temporal pattern of xylosidase and phenol oxidase activities (DOC day interaction, Table 1). Especially from days 10 to 17, xylosidase activity increased with labile and recalcitrant materials while phenol oxidase activity increased only when recalcitrant material was added. DOC quality as a single factor only affected leu-aminopeptidase activity, which was significantly lower in the recalcitrant treatment (Table 1; Fig. 4; Tukeys test, p <0.001). Relationships Between Community Respiration and Enzymatic Activities

Bacterial Number and Chlorophyll Dynamics The total number of bacteria (including live and dead cells) increased in all treatments (including the control) over time (ANOVA, time effect, p <0.001, Table 3). The maximum bacterial abundance was achieved at day 17 in the elevated temperature-labile treatment (Table 3), but the highest proportion of live bacteria occurred at the first sampling day in the elevated temperature-labile (503.6 %) and ambient temperature-labile (441.4 %) treatments. The percentage of living bacteria in the other treatments and days ranged from 36 to 40 %. Chlorophyll a ranged between 3.5 and 4.8 g cm2 at the beginning of the experiment. The addition of a recalcitrant DOC source enhanced chlorophyll density to 7 g cm2 but remained similar in the control and labile treatments (ANOVA, DOC day interaction, p <0.001; Table 3). Temperature effects on chlorophyll density were not significant.

Discussion In order to estimate the extracellular enzyme activities which best predict the changes in the community respiration, a forward linear multiple regression analysis was performed, considering all the enzymatic activities (independent variables) as potential contributors to community respiration (ETS). The analysis run considering all the treatments, selected leu-aminopeptidase activity as the most strongly correlated variable (p 0 0.002), followed by phenol oxidase activity ( p 0 0.049). The remaining variables (-glucosidase and -xylosidase) were of little statistical importance (p 0 0.760 and 0.385, respectively) and have not been considered in the following equation (Table 2). When just the ambient temperature treatments were considered then the significant extracellular enzyme selected by the linear regression were -glucosidase and leuaminopeptidase while in elevated temperature treatments phenol oxidase alone explained 67.8 % of ETS variation (Table 2). In this study, an interactive positive effect of DOC availability and increased temperature on biofilm microbial decomposition activity is suggested, especially when recalcitrant DOC is available. The greater effect of increasing temperature to the biofilm use of recalcitrant compounds (humic substances) is shown by the results of the biofilm community respiration. While the biofilm fed with labile carbon showed an enhanced community respiration irrespectively to temperature, the biofilm fed with humic substances showed an increase of community respiration only at higher temperature (Fig. 2). At the same time, the extracellular enzyme activities explain a large fraction of the biofilm respiratory activity variation and their respective contribution differs depending on the temperature indicating that a different organic matter source is been respired (linear regressions, Table 2). At higher temperature, the phenol oxidase enzyme activity explains a large fraction of

600 Figure 3 Net primary production (NPP) and community respiration (Resp.) per unit of chlorophyll for each set of treatments. Each bar represents the mean (standard errors) of three consecutive incubations performed with the same glass tiles along the last day of the experiment (day 17)

I. Ylla et al.

AMBIENT TEMPERATURE
1.0

ELEVATED TEMPERATURE

mg O2 mg Chl h-1

0.5

-1

0.0

-0.5 NPP Resp.

-1.0

O C

Co nt ro l

Co nt ro

DO

tra nt D

DO

La bi le

La bil e

respiratory activity variation. This enzyme is involved in the degradation of lignin and humic substances by oxidizing phenols at the expenses of oxygen [59] and, as expected, it was enhanced when humic substances were available. The relationship between phenol oxidase and respiratory activity suggest an enhanced degradation of humic substances at increased temperatures, although the time evolution of this enzyme did not show a significant effect of temperature. Significant effects of temperature on phenol oxidase were measured in peatlands [21]. The decomposition of hemicellulose seems to be also favored by the warmer conditions as shown by the increases in -xylosidase activity. Although the contribution of -xylosidase biofilm activity to biofilm heterotrophic metabolism is low, this enzyme was also positively affected by temperature after DOC additions. This underlies the suggestion that more recalcitrant compounds are more sensible to temperature increases. The -xylosidase enzyme hydrolyses simple polysaccharides such as xylobiose or xylooligosaccharides to form D-xylose as the end product [43] which are found in plant cell walls such as hemicelluloses. On the contrary, at ambient temperature, the two enzymes highly responsible for respiration activity variation were -glucosidase and leu-aminopeptidase. In streambed sediments, a significant correlation was found between community respiration and the activities of -glucosidase and leu-aminopeptidase which was linked to the specificity of these enzymes to hydrolyze energy-rich compounds [70]. The -glucosidase is involved in cellulose decomposition, specifically degrading cellobiose and other relatively small oligomers containing -D-glucose linkages by splitting off the terminal -D-glucose residue [18]. The product of -glucosidase (i.e., glucose) represents readily usable source of energy fuelling catabolic metabolism, while leuaminopeptidase (providing amino acids) is an enzyme potentially important in both carbon degradation and nitrogen

acquisition. Although these two enzymes were enhanced by temperature, they might contribute to a larger fraction of the respiration activity at ambient temperature, when the degradation of more recalcitrant compounds would be lower. The biofilm metabolic responses were accompanied by changes in the DOC quantity and quality. Biofilm metabolism is highly responsible for changes in the flowing water DOC [56], such as the significant consumption of labile DOC measured in the present experiment. However, DOC consumption was not observed after the pulse of humic substances but a slight increase in DOC concentration. The dynamics of DOC changes respond both to uptake and release mechanisms within the biofilm (bacterial mineralization, algal release, and cell lysis); thus, under the control and recalcitrant conditions, a net release of DOC was measured while a net consumption of DOC was observed for the labile treatment. In spite of this, no net DOC consumption occurred when humics were added, subtle but significant changes of DOC quality parameters were measured. In the recalcitrant treatments, the progressive increase of the FI and the decrease of the absorbance ratio (Abs 250/total DOC) respectively highlight that humic substances were losing aromaticity [46] and that the amount of phenolic compounds in total DOC was diminishing [17, 24]. The patterns of both indices indicated that transformation of material was occurring after the reception of the recalcitrant DOC. The two indices showed a slightly more noticeable behavior (higher FI, lower absorbance ratio) at higher temperature, which might indicate that temperature promoted the degradation of humic compounds. The biofilm metabolic results and the DOC quantity and quality variations suggest a differential effect of increasing temperature when a different DOC quality is available. At warming conditions, when a labile organic matter source is available, it might be preferentially and rapidly used, as indicated by the significant decrease in DOC content, and so their effects quickly vanished, but the

Re ca lci tra

Re ca lc i

nt D

O C

Effects of warming on DOC use Figure 4 Temporal changes in extracellular enzymatic activities (-glucosidase, leuaminopeptidase, -xylosidase, and phenol oxidase) at the colonized substrata of each treatment. Values are meansstandard errors (vertical bars). For better clarity, half of the error bars were shown (n 0 3)

601

AMBIENT TEMPERATURE
12 10 8 6 4 2 200 180 nmol AMC cm-2 h-1 160 140 120 100 80 60 40 20 4 nmol MUF cm-2 h-1

ELEVATED TEMPERATURE
-glucosidase
Control Labile DOC Recalcitrant DOC

-glucosidase
Control Labile DOC Recalcitrant DOC

nmol MUF cm-2 h-1

Peptidase

Pepitdase

Xylosidase

Xylosidase

Phenol-oxidase
mol DIQC cm-2 h-1 0.04 0.03 0.02 0.01 0.00
1 3 6

Phenol-oxidase

10

17

ay

ay

ay

ay

ay

ay

D ay

D ay

10

D ay

substrate is providing N and C for bacterial growth. In fact, a significant increase in bacterial density was measured at the labile treatment and at elevated temperature conditions. On the other hand, under warming conditions and when a recalcitrant organic matter source is available, respiration is enhanced and there is a greater use and transformation of the available organic matter. In freshwater lakes, it has been shown that autochthonous sources (more labile) are preferentially utilized by bacterioplankton [42] but that processing of allochthonous

organic carbon by bacteria is a major component of CO2 production through bacterial respiration [44]. Increased utilization of recalcitrant OM with higher temperature has been measured in soils and tied to microbial activation [3]. Increasing utilization of old carbon under high temperatures has been associated with changes in microbial community composition and the related expression of enzyme activities [65]. Shifts in community composition (especially bacterial) could contribute in our experiment to

D ay

17

602

I. Ylla et al.

Table 2 Forward linear multiple regression analysis considering all the enzymatic activities (independent variables) as potential contributors to community respiration ETS) Treatment considered Ambient and elevated temperature Ambient temperature Elevated temperature Linear multiple regression equation ETS 0 0.51 leu-pep+0.31 phox ETS 0 0.47 -gluc+0.47 leu-pep ETS 0 0.67 phox p values <0.001 0.002 0.005 r 0.688 0.810 0.678

The corresponding enzymatic activities abbreviations are: leu-pep leu-aminopeptidase, phox phenol oxidase, -gluc -glucosidase

the responses in community metabolism (enzyme activities and respiration rates). Findlay et al. [22] show that bacterial communities may diverge when exposed to labile versus recalcitrant DOM sources. In biofilms grown in bioreactors, the bacterial community dissimilates whether the bioreactors were fed either with humic substances or labile organic matter [51]. The addition of humic material could cause indirect effects on the biofilm microbiota. Dissolved recalcitrant substances alter water color (making it brownish-orange) and attenuate both visible and UV light [35]. Algal communities may respond to the lower light availability with increased chlorophyll a concentrations [29]. In our experiment, the chlorophyll a content was higher in the recalcitrant treatment although it was least productive, suggesting a higher chlorophyll a per cell content but not increasing biomass. Potential depression effect of humic matter on primary productivity has been also described in lakes [34]. The lowest peptidase activity in the recalcitrant treatment could not only be related with the low photosynthesis in this treatment [25, 68] but also to the inhibitory effect of humic matter on certain metabolic processes by the complexation and inactivation of bacterial enzymes [26, 48]. The addition of high quality labile DOC in this laboratory experiment simulates the autochthonous carbon availability when primary productivity reaches relevant pulses in natural aquatic ecosystems; recalcitrant DOC addition simulates the

allochthonous organic matter inputs such as those from riparian vegetation. In temperate stream ecosystems, autochthonous and allochthonous DOC inputs fluctuate seasonally showing higher peaks in spring and autumn, respectively [5, 69]. This seasonality becomes more extreme in intermittent river systems [57]. However, the DOC composition used in the experiment is structurally much more simple than the natural DOC composition (especially for the labile one), and the concentrations used are in the upper range of those found in natural systems [38]. Although allochthonous and autochthonous DOC inputs vary seasonally the final mixed DOC in streams uses to contain a high content of humic substances [2, 67]. Since the recalcitrant and labile sources are mixed together, potential relevant priming effects might occur [31] modulating the microbial response. Our results hint that the composition of the carbon supply pulses and warmer water temperatures (4 C increase) influence how DOC materials are processed by the biofilm. Labile DOC pulses trigger the response of heterotrophic biofilms that rapidly use those substances. At warming conditions, the increasing decomposition capacity of polysaccharides and peptides is not translated into greater respiration activity but increasing microbial biomass. Biofilm microbial use of inputs of terrestrial material in streams, such as recalcitrant humic substances, might be most affected by temperature increases that can accelerate their decomposition affecting the aquatic food web and promoting an efficient recycling of

Table 3 Total bacterial numbers and chlorophyll a concentration in the colonized glass substrata under each temperature (ambient and elevated) and DOC treatment (control, labile, and recalcitrant) Ambient temperature Control Total bacteria (107 bact. cm2) Day 1 1.320.09 Day 17 1.740.07 (31 %) Chl-a (g chl cm2) Day 1 3.430.43 Day 17 3.690.56 (7.6 %) Labile DOC Recalcitrant DOC Elevated temperature Control Labile DOC Recalcitrant DOC

1.110.1 1.800.2 (62 %) 3.650.2 4.130.5 (13 %)

1.070.06 1.850.03 (72 %) 3.620.41 6.861.79 (89.5 %)

1.370.09 2.020.3 (47.5 %) 4.260.29 4.320.86 (1.5 %)

1.160.03 2.360.4 (103 %) 4.290.44 4.070.03 (5 %)

1.650.2 2.030.2 (23 %) 4.820.13 7.350.5 (52 %)

Values are meansstandard errors, n 0 3. The percentage of change in day 17 with respect to day 1 is also indicated in parenthesis

Effects of warming on DOC use

603 15. Chrst RJ, Overbeck J (1990) Substrate ectoenzyme interaction: significance of beta-glucosidase activity for glucose-metabolism by aquatic bacteria. Arch Hydrobiol 34:9398 16. Davidson EA, Janssens IA (2006) Temperature sensitivity of soil carbon decomposition and feedbacks to climate change. Nature 440(7081):165173 17. De Haan H (1993) Solar UV-light penetration and photodegradation of humic substances in peaty lake water. Limnol Oceanogr 38 (5):10721076 18. Deshpande V, Eriksson KE (1988) 1,4-beta-glucosidases of Sporotrichum pulverulentum. Methods Enzymol 160:415424 19. Daz V, Font J, Schwartz T, Roman AM (2011) Biofilm formation at warming temperature: acceleration of microbial colonization and microbial interactive effects. Biofouling 27(1):5971 20. Fang CM, Smith P, Moncrieff JB, Smith JU (2005) Similar response of labile and resistant soil organic matter pools to changes in temperature. Nature 433(7021):5759 21. Fenner N, Freeman C, Lock MA, Harmens H, Reynolds B, Sparks T (2007) Interactions between elevated CO2 and warming could amplify DOC exports from peatland catchments. Environ Sci Technol 41:31463152 22. Findlay S, Sinsabaugh RL, Sobczak WV, Hoostal M (2003) Metabolic and structural response of hyporheic microbial communities to variations in supply of dissolved organic matter. Limnol Oceanogr 48(4):16081617 23. Fischer H (2003) The role of biofilms in the uptake and transformation of dissolved organic matter. In: Findlay SEG, Sinsabaugh RL (eds) Aquatic ecosystems: interactivity of dissolved organic matter. Academic, San Diego, pp 285313 24. Fischer H, Mille-Lindblom C, Zwirnmann E, Tranvik LJ (2006) Contribution of fungi and bacteria to the formation of dissolved organic carbon from decaying common reed (Phragmites australis). Arch Hydrobiol 166(1):7997 25. Francoeur SN, Wetzel RG (2003) Regulation of periphytic leucineaminopeptidase activity. Aquat Microb Ecol 31(3):249258 26. Freeman C, Lock MA, Marxsen J, Jones SE (1990) Inhibitory effects of high molecular weight dissolved organic matter on metabolic processes in contrasted rivers and streams. Freshw Biol 24:159166 27. Freese HM, Karsten U, Schumannn R (2006) Bacterial abundance, activity, and viability in the eutrophic river Warnow, Northeast Germany. Microb Ecol 51:117127 28. Giardina CP, Ryan MG (2000) Evidence that decomposition rates of organic carbon in mineral soil do not vary with temperature. Nature 404(6780):858861 29. Guasch H, Sabater S (1995) Seasonal variations in photosynthesis irradiance responses by biofilms in Mediterranean streams. J Phycol 31(5):727735 30. Gudasz C, Bastviken D, Steger K, Premke K, Sobek S, Tranvik LJ (2010) Temperature-controlled organic carbon mineralization in lake sediments. Nature 466:479481 31. Guenet B, Danger M, Abbadie L, Lacroix G (2010) Priming effect: bridging the gap between terrestrial and aquatic ecology. Ecology 91(10):28502861 32. Hach (1992) Water analysis handbook, 2nd edn. Hach, Loveland, CO 33. IPCC (2007) Summary for policymakers. Cambridge University Press, Cambridge 34. Jackson TA, Hecky RE (1980) Depression of primary productivity by humic matter in lake and reservoir waters of the boreal forest zone. Can J Fish Aquat Sci 37(12):23002317 35. Jansson M, Blomqvist P, Jonsson A, Bergstrom AK (1996) Nutrient limitation of bacterioplankton, autotrophic and mixotrophic phytoplankton, and heterotrophic nanoflagellates in Lake Ortrasket. Limnol Oceanogr 41(7):15521559 36. Jeffrey SW, Humphrey GF (1975) New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher-plants,

terrestrial organic carbon back to the atmosphere (faster C cycling), which will further enhance the warming trend. In anthropized fluvial systems, similar effects on the microbial decomposition of the recalcitrant and complex organic matter compounds that flow from waste water treatment plant outlets [41] might be expected, although these effects can be hidden by multiple stress situations (i.e., pollutants).
Acknowledgments This study was funded by projects CGL200765549/BOS, CGL2008-05618-C02/BOS, CGL2011-30151-C02-01, and SCARCE (Consolider-Ingenio CSD2009-00065) of the Spanish Ministry of Economy and Competitiveness. We thank Juanita Mora for her help in the laboratory analysis and Helmut Fischer for his advice on DOC quality interpretation. We also thank four anonymous reviewers for their suggestions.

References
1. Acua V, Tockner K (2010) The effects of alterations in temperature and flow regime on organic carbon dynamics in Mediterranean river networks. Glob Chang Biol 16:26382650 2. Amon RMW, Benner R (1996) Bacterial utilization of different size classes of dissolved organic matter. Limnol Oceanogr 41 (1):4151 3. Andrews JA, Matamala R, Westover KM, Schlesinger WH (2000) Temperature effects on the diversity of soil heterotrophs and the delta C-13 of soil-respired CO2. Soil Biol Biochem 32(5):699706 4. Artigas J, Roman AM, Gaudes A, Muoz I, Sabater S (2009) Organic matter availability structures microbial biomass and activity in a Mediterranean stream. Freshw Biol 54(10):20252036 5. Artigas J, Romani AM, Sabater S (2008) Relating nutrient molar ratios of microbial attached communities to organic matter utilization in a forested stream. Fundam Appl Limnol 173(3):255264 6. Baulch HM, Schindler DW, Turner MA, Findlay DL, Paterson MJ, Vinebrooke RD (2005) Effects of warming on benthic communities in a boreal lake: implications of climate change. Limnol Oceanogr 50(5):13771392 7. Berggren M, Laudon H, Jonsson A, Jansson M (2010) Nutrient constraints on metabolism affect the temperature regulation of aquatic bacterial growth efficiency. Microb Ecol 60:894902 8. Bertilsson S, Tranvik LJ (2000) Photochemical transformation of dissolved organic matter in lakes. Limnol Oceanogr 45 (4):753762 9. Bianchi TS, Filley T, Dria K, Hatcher PG (2004) Temporal variability in sources of dissolved organic carbon in the lower Mississippi River. Geochim Cosmochim Acta 68(5):959967 10. Blenkinsopp SA, Lock MA (1990) The measurement of electrontransport system activity in river biofilms. Water Res 24(4):441445 11. Cabaniss SE, Madey G, Leff L, Maurice PA, Wetzel R (2005) A stochastic model for the synthesis and degradation of natural organic matter. Part I. Data structures and reaction kinetics. Biogeochemistry 76(2):319347 12. Claret C (1998) Hyporrheic biofilm development on artificial substrate, as a tool for assessing trophic status of aquatic systems: first results. Ann Limnol -Int J Lim 34(2):119128 13. Conant RT, Drijber RA, Haddix ML, Parton WJ, Paul EA, Plante AF, Six J, Steinweg JM (2008) Sensitivity of organic matter decomposition to warming varies with its quality. Glob Chang Biol 14(4):868877 14. Conen F, Leifeld J, Seth B, Alewell C (2006) Warming mineralises young and old soil carbon equally. Biogeosciences 3(4):515519

604 algae and natural phytoplankton. Biochem Physiol Pflanz 167 (2):191194 Jenkinson DS, Adams DE, Wild A (1991) Model estimates of CO2 emissions from soil in response to global warming. Nature 351 (6324):304306 Kaplan LA, Newbold JD (2003) The role of monomers in stream ecosystem metabolism. In: Findlay SEG, Sinsabaugh RL (eds) Aquatic ecosystems: interactivity of dissolved organic matter. Academic, San Diego, pp 99119 Kathol M, Norf H, Arndt H, Weitere M (2009) Effects of temperature increase on the grazing of planktonic bacteria by biofilm-dwelling consumers. Aquat Microb Ecol 55(1):6579 Knorr W, Prentice IC, House JI, Holland EA (2005) Long-term sensitivity of soil carbon turnover to warming. Nature 433 (7023):298301 Krasner SW, Westerhoff P, Chen B, Rittmann BE, Nam S-N, Amy G (2009) Impact of wastewater treatment processes on organic carbon, organic nitrogen, and DBP precursors in effluent organic matter. Environ Sci Technol 43(8):29112918 Kritzberg ES, Cole JJ, Pace ML, Granli W, Bade DL (2004) Autochthonous versus allochthonous carbon sources of bacteria: results from whole-lake 13C addition experiments. Limnol Oceanogr 49(2):588596 Lachke AH (1988) 1,4-beta-D-xylan xylohydrolase of Sclerotium rolfsii. Methods Enzymol 160:679684 McCallister SL, del Giorgio PA (2998) Direct measurement of the 13C signature of carbon respired by bacteria in lakes: linkages to potential carbon sources, ecosystem baseline metabolism, and CO2 fluxes. Limnol Oceanogr 53(4):12041216 McDonald S, Bishop AG, Prenzler PD, Robards K (2004) Analytical chemistry of freshwater humic substances. Anal Chim Acta 527(2):105124 McKnight DM, Boyer EW, Westerhoff PK, Doran PT, Kulbe T, Andersen DT (2001) Spectrofluorometric characterization of dissolved organic matter for indication of precursor organic material and aromaticity. Limnol Oceanogr 46(1):3848 Meyer JL (1994) The microbial loop in flowing waters. Microb Ecol 28(2):195199 Meyer JL, Edwards RT, Risley R (1987) Bacterial growth on dissolved organic carbon from a blackwater river. Microb Ecol 13:1329 Morris DP, Hargreaves BR (1997) The role of photochemical degradation of dissolved organic carbon in regulating the UV transparency of three lakes on the Pocono Plateau. Limnol Oceanogr 42(2):239249 Murphy J, Riley JP (1962) A modified single solution for the determination of phosphate in natural waters. Anal Chim Acta 27:3136 Peter H, Ylla I, Gudasz C, Roman AM, Sabater S, Tranvik L (2011) Multifunctionality and diversity in bacterial biofilms. PLoS One 6(8):e23225 Piccolo A (2001) The supramolecular structure of humic substances. Soil Sci 166(11):810832 Porcal P, Koprivnjak JF, Molot LA, Dillon PJ (2009) Humic substances-part 7: the biogeochemistry of dissolved organic carbon and its interactions with climate change. Environ Sci Pollut Res 16(6):714726 Reichstein M, Katterer T, Andren O, Ciais P, Schulze ED, Cramer W, Papale D, Valentini R (2005) Temperature sensitivity of

I. Ylla et al. decomposition in relation to soil organic matter pools: critique and outlook. Biogeosciences 2(4):317321 Roman AM, Artigas J, Ylla I (2012) Extracellular enzymes in aquatic biofilms: microbial interactions versus water quality effects in the use of organic matter. In: Lear G, Lewis GD (eds) Microbial biofilms: current research and applications. Caister Academic Press, New Zealand Roman AM, Guasch H, Muoz I, Ruana J, Vilalta E, Schwartz T, Emtiazi F, Sabater S (2004) Biofilm structure and function and possible implications for riverine DOC dynamics. Microb Ecol 47 (4):316328 Roman AM, Vzquez E, Butturini A (2006) Microbial availability and size fractionation of dissolved organic carbon after drought in an intermittent stream: biogeochemical link across the streamriparian interface. Microb Ecol 52(3):501512 Sand-Jensen K, Pedersen NL, Sondergaard M (2007) Bacterial metabolism in small temperate streams under contemporary and future climates. Freshw Biol 52:23402353 Sinsabaugh RL (2010) Phenol oxidase, peroxidase and organic matter dynamics of soil. Soil Biol Biochem 42:391404 Sinsabaugh RL, Osgood MP, Findlay S (1994) Enzymatic models for estimating decomposition rates of particulate detritus. J N Am Bentholl Soc 13(2):160169 Thornley JHM, Cannell MGR (2001) Soil carbon storage response to temperature: an hypothesis. Ann Bot 87(5):591598 Tranvik LJ (1988) Availability of dissolved organic carbon for planktonic bacteria in oligotrophic lakes of differing humic content. Microb Ecol 16(3):311322 Vzquez E, Amalfitano S, Fazi S, Butturini A (2011) Dissolved organic matter composition in a fragmented Mediterranean fluvial system under severe drought conditions. Biogeochemistry 102:50 72 Vzquez E, Roman AM, Sabater F, Butturini A (2007) Effects of the dry-wet hydrological shift on dissolved organic carbon dynamics and fate across stream-riparian interface in a Mediterranean catchment. Ecosystems 10(2):239251 Waldrop MP, Firestone MK (2004) Altered utilization patterns of young and old soil C by microorganisms caused by temperature shifts and N additions. Biogeochemistry 67(2):235248 Walther GR, Post E, Convey P, Menzel A, Parmesan C, Beebee TJC, Fromentin JM, Hoegh-Guldberg O, Bairlein F (2002) Ecological responses to recent climate change. Nature 416(6879):389 395 Wetzel RG, Hatcher PG, Bianchi TS (1995) Natural photolysis by ultraviolet irradiance of recalcitrant dissolved organic matter to simple substrates for rapid bacterial metabolism. Limnol Oceanogr 40(8):13691380 Ylla I, Borrego C, Roman AM, Sabater S (2009) Availability of glucose and light modulates the structure and function of a microbial biofilm. FEMS Microbiol Ecol 69(1):2742 Ylla I, Sanpera-Calbet I, Vzquez E, Roman A, Muoz I, Butturini A, Sabater S (2010) Organic matter availability during pre- and postdrought periods in a Mediterranean stream. Hydrobiologia 657:217 232 Zoppini AM, Amalfitano S, Fazi S, Puddu A (2010) Dynamics of a benthic microbial community in a riverine environment subject to hydrological fluctuations (Mulargia River, Italy). Hydrobiologia 657:3751

37.

55.

38.

56.

39.

57.

40.

41.

58.

59. 60.

42.

43. 44.

61. 62.

63.

45.

46.

64.

47. 48. 49.

65.

66.

67.

50.

51.

68.

52. 53.

69.

70.

54.