Leachates from helophyte leaf-litter enhance nitrogen removal from

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Leachates from helophyte leaf-litter enhance nitrogen removal from wastewater treatment plant effluents Miquel Ribot, Joaquin Cochero, Timothy N. Vaessen, Susana Bernal, Elliot Bastias, Esperança Gacia, Albert Sorolla, Francesc Sabater, and Eugènia Martí Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b07218 • Publication Date (Web): 04 Jun 2019 Downloaded from http://pubs.acs.org on June 4, 2019

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Environmental Science & Technology

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Leachates from helophyte leaf-litter enhance nitrogen removal from

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wastewater treatment plant effluents

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Miquel Ribot1*, Joaquín Cochero2, Timothy N. Vaessen1, Susana Bernal1,3, Elliot

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Bastias1, Esperança Gacia1, Albert Sorolla4, Francesc Sabater3 and Eugènia Martí1.

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1 Integrative

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CSIC), Blanes, Girona, Spain. E-mail: [email protected], [email protected],

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[email protected], [email protected], [email protected],

Freshwater Ecology Group, Centre d’Estudis Avançats de Blanes (CEAB-

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[email protected].

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2 ILPLA

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[email protected]

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3 Departament

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Universitat de Barcelona. Av. Diagonal 643, 08028, Barcelona, Spain. E-mail:

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[email protected].

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4 Naturalea,

– Instituto de Limnología Dr. Raúl A. Ringuelet, La Plata, Argentina. E-mail:

de Biologia Evolutiva, Ecologia i Ciències Ambientals (BEECA),

Castellar del Vallés, Spain. E-mail: [email protected].

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*corresponding author

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M. Ribot and J. Cochero contributed equally to this work.

1 ACS Paragon Plus Environment


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GRAPHICAL ABSTRACT

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ABSTRACT

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Bioengineering techniques are currently used to restore degraded habitats in human-

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altered streams. Aquatic plants used in these techniques can additionally contribute to

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reducing excess nitrogen (N) from point sources via assimilation. Moreover, leachates

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from plant leaf-litter can serve as an additional source of labile dissolved organic matter

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(DOM), which can promote aerobic respiration and N removal via denitrification. We

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tested the influence of leaf-litter leachates from Iris pseudacorus and Phragmites

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australis on the structure and activity of freshwater biofilms grown in flumes fed by

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effluent from a wastewater treatment plant (WWTP). The responses of the epilithic

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biofilm to the inputs of leaf-litter leachates were compared to those measured using a

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brewery by-product rich in sugars and to the WWTP effluent water (i.e., control). All

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DOM sources significantly enhanced aerobic respiration and denitrification of the

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biofilm when compared to the controls, with increases in total microbial abundance but

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not in denitrifier abundance. The results suggest that metabolic activity of biofilms may

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be limited by bioavailability of DOM in WWTP effluent; and leaf-litter leachates of

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helophytes used in bioengineering techniques could alleviate this limitation by

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enhancing microbial N and C uptake in the receiving stream.



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INTRODUCTION

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Inputs from wastewater treatment plant (WWTP) effluents can cause

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environmental problems such as eutrophication of the receiving water body1,2, since

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they are relevant sources of dissolved organic matter (DOM)1,3 and dissolved inorganic

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nitrogen (DIN)1,4, particularly in Mediterranean streams where dilution capacity is low

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due to water scarcity5. Solutes from WWTP effluents can be metabolized to some extent

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by receiving streams4,6,7 due to the presence of microbial assemblages developed on

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streambed substrata (i.e., biofilms) which are often considered major drivers of nutrient

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processing8,9. DOM can be metabolized by heterotrophic microorganisms either

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aerobically (i.e., aerobic respiration) or under anoxic conditions via denitrification,

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where NO3- is used as electron acceptor, producing nitrogenous gasses10,11. Previous

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studies carried out in recipient streams indicate that WWTP effluents may enhance

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ecosystem respiration17, while denitrification tends to be low6,12. However, under

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specific environmental conditions (i.e., low flow) WWTP effluents may enhance

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denitrification in the recipient stream13.

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Availability of DOM and nitrate (NO3-) is relevant for denitrification. However,

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the relative role of these solutes on the limitation of denitrification depends on the

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catchment land use and stream typology. In streams with low anthropogenic pressure,

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denitrification is often limited by NO3- availability14,15. In contrast, in WWTP-

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influenced streams, denitrification is likely limited by DOM concentration16,17 and

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quality1819. The latest is because the aeration in secondary wastewater treatment process

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is aimed to reduce biochemical oxygen demand, and thus, DOM20. In addition, DOM in

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WWTP effluents tends to be enriched in refractory substances21,22, such us humic-like

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compounds which have high molecular weight and aromaticity, which tend to be less

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bioavailable than amino acids and carbohydrates23. Therefore, despite WWTP effluent


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inputs being a source of DOM to the stream, denitrification and the metabolic activity of

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microbial assemblages in recipient streams could be limited by DOM quality.

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Aquatic plants, such as helophytes, have traditionally been used in

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bioengineering techniques to reduce water nutrients loads because they take up

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inorganic compounds to meet their nutrient demand24,25. In this sense, helophytes have

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been used in a wide variety of wastewater treatment systems, such as urban and

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agricultural WWTP effluents26,27, urban storm water runoff28,29 or mine spills30.

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However, during plant senescence, leaf litter can release OM and nutrients through

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leaching and decomposition, which can increase DOM and DIN in the water

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column31,32. Even though N availability may transiently increase in the water column,

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plant leachates could have a positive effect on N removal by releasing high quality

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DOM, which fuels heterotrophic activity of microbial assemblages33,34.

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Here, we assessed the influence of the bioavailability of plant leachates and

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other DOM sources on the biofilm capacity to remove nitrate. We incubated biofilms

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developed in flumes fed by a WWTP effluent with 4 DOM sources: raw water from the

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WWTP effluent (i.e., control), leaf-litter leachates from Iris pseudacorus and

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Phragmites australis, and a brewery by-product rich in sugars (i.e., labile DOM

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source35). To explore the effect of the 4 DOM sources on biofilms, we measured: i) the

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abundance of total bacterial and denitrifying bacteria, ii) denitrification enzyme activity

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(DEA), and iii) aerobic respiration (AR). We expected higher bacterial abundance and

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higher DEA and AR in incubations amended with the different DOM sources with

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respect to control if microbial assemblages in biofilms were limited by DOM

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bioavailability from the WWTP effluent. We also expected similar responses between

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incubations amended with leachates from helophytes and with the brewery by-product if

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leachates are labile DOM sources. Previous research in the same flumes has shown that



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flumes planted with helophytes have a higher capacity to remove both DOC and DIN

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when compared to unvegetated flumes during the vegetative period36. Therefore, the

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present study seeks to expand the current knowledge on the effects of helophytes used

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in bioengineering practices on nutrient removal by exploring whether these two

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helophyte species can further promote DOM and NO3- removal during senescence.

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MATERIAL AND METHODS

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Biofilm sampling and leachate production

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We collected gravels (10 – 20 mm diameter) colonized by biofilm from nine artificial

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flumes located at the Urban River Lab (URL) experimental facility

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(http://www.urbanriverlab.com). Flumes are directly fed with treated wastewater from

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the effluent of the WWTP from Montornès del Vallès (20 km N of Barcelona, Spain). In

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all flumes, water inflow (5 L min-1) was maintained on subsurface flowpaths. Gravels in

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flumes were colonized by biofilm in 3 different experimental settings: flumes with only

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gravel (“Flume_Unveg”; n = 3), flumes with gravel and Iris pseudacorus (“Flume_Iris”;

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n = 3), and flumes with gravel and Phragmites australis (“Flume_Phragm”; n = 3). Iris

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pseudachorus and Phragmites australis in the flumes had a mean (± standard deviation)

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biomass standing stock of 2.7 kg (±0.9 kg) and 7.6 kg (±1.4 kg) of dry-weight m-2,

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respectively. We collected gravel (5-10 cm depth) at three random locations along the

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flumes and we mixed it to produce a representative composite sample from each flume.

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Samples were transported to the laboratory in a cooler and kept in the refrigerator until

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next day when we conducted the incubations.

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Leaf-litter leachates from I. pseudacorus (DOM_Iris) and from P. australis

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(DOM_Phragm) were obtained by submerging 100 g of air-dried leaves in 1.5 L of

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distilled water for 48h at laboratory ambient temperature (~23 ºC). A by-product of the


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beer brewing process was used as a reference source of highly labile DOM because it

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contains a high proportion of sugars35 (DOM_Brew). During the maceration, malt and

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cereal grains are incubated in water at high temperature (i.e., 60-70ºC) to hydrolyze

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starch into sugars (i.e., maltose and dextrin) by amylases. After the maceration, grains

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are washed and the low-density fraction that does not meet the minimum sugar

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concentration for fermentation is discarded. This fraction was the by-product used here.

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Unamended water from the WWTP effluent was used as a control treatment

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(DOM_Control).

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The concentrations of NO3-, nitrite (NO2-), ammonium (NH4+), soluble reactive

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phosphorous (SRP), total organic carbon (TOC) and dissolved organic carbon (DOC)

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from the leaf-litter leachates, from the brewery by-product, and from the WWTP

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effluent water were measured using Spectroquant© commercial kits on a

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Spectroquant© Nova 60 portable spectrometer (Merck kGaA, Darmstadt, Germany).

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Concentrations are shown in Table S1.

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Experimental procedure

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In the laboratory, we set the incubations by placing 100 g of colonized gravel into 250

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mL glass bottles and adding 150 mL of effluent water. After 12 h of acclimation, 3 sub-

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sets of incubation bottles (9 bottles each: gravel from 3 types of flumes x 3 replicates

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each) were amended with each type of DOM source (i.e., WWTP effluent water, leaf-

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litter leachates of Iris and Phragmites, and the brewery by-product). The amendments

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were targeted to produce an increase of 4 mg L-1 of DOC above the background

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concentration of the WWTP effluent water. Therefore, the concentration of N and P

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slightly varied for the different treatments of DOM sources (see Table S2). The

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experimental design (explained further in “Data Analysis”) included a total of 36



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incubation bottles, and was supplemented with three additional bottles filled with 150

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ml of MilliQ water and no gravels that served as blanks. Incubations lasted 18 h and

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were conducted twice: one to assess the abundance of total and denitrifying bacteria of

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the biofilms and to measure potential DEA, and a second time to measure aerobic

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respiration.

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Abundance of total and denitrifying bacteria

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To characterize the bacterial abundance in the epilithic biofilms of the different

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treatments and assess if it changed during the incubations, we used six pieces of gravel

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from each bottle before and after the DEA incubations. Biofilms were detached from the

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gravel surface by submersing the gravel into Tween 20 detergent, followed by

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sonication37. Supernatant was filtered through a 0.2 µm polycarbonate membrane. Total

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DNA was extracted from the filters directly after filtration using a DNA extraction kit

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(PowerSoil® DNA Isolation Kit, Mo Bio). Presence and quantification of total bacteria

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in biofilms was based on 16S rRNA gene copy numbers. Presence and quantification of

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denitrifying bacteria was based on nirS and nirK gene copy numbers, since they are

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common marker genes to trace denitrifying bacteria38. Gene copy numbers were

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obtained with quantitative real-time polymerase chain reaction (qPCR) amplification.

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The primers used for 16S were 341F and 534R39 and the primers used for nirk and nirS

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were F1aCu and R3Cu38 and Cd3aF and R3VCd40, respectively. Each sample was run in

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triplicate with standard curves ranging from 10-3 ng mL-1 to 10-7 ng mL-1 DNA of nirS,

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nirK and 16S genes, respectively. The results of the qPCR analysis were expressed in

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copies of genes per colonized surface area (i.e., copies cm-2). The surface area was

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estimated covering the gravel pieces tightly with aluminum foil and using a weight-to-

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area relationship.


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To explore the effect of the DOM treatments on the bacterial abundances we calculated the relative change (RC) over the incubation period as follows:

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𝑅𝐶 (𝑡𝑖, 𝑡𝑓) =

𝐴𝑏𝑓 𝐴𝑏𝑖

―1

(1)

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where Abi and Abf are the bacterial gene abundances at the beginning (ti) and at

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end (tf) of the incubation period (18 h), respectively. Positive values (RC > 0) indicate

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an increase in gene copy numbers, whereas negative values (RC < 0) indicate a decrease

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over the incubation period.

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Denitrifying enzyme activity assays

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The potential DEA of the epilithic biofilms was measured following the acetylene

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(C2H2) block technique41. This technique uses C2H2 to block the transformation of

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nitrous oxide (N2O) to nitrogen gas (N2); thus, the accumulation of N2O in the

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headspace of the bottles during the incubation period is used to estimate denitrification

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rates. Water in the incubation bottles was first made anoxic by pumping helium for 10

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minutes. Bottles were then sealed tight with septum-fitted screw-top lids, and 10 mL of

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acetylene (C2H2) were added to each bottle with a syringe. Bottles were gently shaken

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for several minutes to ensure that C2H2 mixed well with the water, and were incubated

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in the dark at ambient laboratory temperature (~23 ºC). Gas samples from the headspace

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were collected in 10 mL vacutainers using a double needle, after 10 min and 18 h of the

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C2H2 addition. After collecting the initial gas sample, 10mL of C2H2 were added to each

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bottle to maintain the gas volume constant and avoid pressure changes. The analysis of

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N2O concentration was conducted in the Serveis Científico-Tècnics of the University of

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Vic on an Agilent 7890A gas chromatography system (Agilent Technologies, Santa

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Clara, USA).

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Potential rates of DEA (in mg N2O g AFDM-1 h-1) were calculated as follows: 9 ACS Paragon Plus Environment


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𝑀𝑓 ― 𝑀𝑖

DEA = 𝑡 𝑥 𝑏𝑖𝑜𝑚𝑎𝑠𝑠

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(2)

where Mf and Mi are the N2O mass in the incubation bottle at the end and at the

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beginning of the incubation, respectively, t is the incubation time (17.8 h), and biomass

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is the biofilm biomass in the gravel measured as ash free dry mass (AFDM, in g). We

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estimated Mf and Mi by measuring the volume of the headspace of each bottle, the N2O

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concentration at the end and beginning of the incubation, respectively; and the volume

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of water corrected for N2O solubility in the liquid phase with an appropriate

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temperature-dependent Bunsen coefficient42. We assumed linearity in the accumulation

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of N2O in the headspace over the incubation time based on results from previous

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studies43,44,45. The AFDM associated with the gravel in each bottle was estimated as the

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difference between the dry mass (dried at 60 ºC for 24 h) and the weight after being

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ashed (at 550 ºC for 4 h in a muffle furnace).

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We used a mass balance approach to calculate NO3- loss in the incubation bottles. To do

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so, we compared the initial NO3- mass in the water with the accumulated N2O mass in

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the headspace, and we assumed that denitrification was the major responsible for that

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transformation given that incubations were conducted under anaerobic conditions.

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Aerobic respiration activity assays

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The aerobic respiration activity of the epilithic biofilms was measured using a second

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set of incubation bottles, prepared as described above. The Resazurin (Raz)-Resorufin

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(Rru) system was used as a hydrometabolic tracer46. Raz is a metabolically active

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compound that reacts irreversibly to Rru under mildly reducing conditions46; and thus,

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transformation of Raz to Rru can be used a proxy of aerobic respiration47. After the

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DOM sources were added, the incubation bottles were spiked with a Raz standing stock

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solution to achieve an initial Raz concentration of 100 µg L-1. A 5 mL water sample was 10 ACS Paragon Plus Environment

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collected 10 minutes after the Raz addition and another sample after 18 hours of

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incubation. All samples were immediately filtered through Whatman GF/F glass fiber

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filters (0.7 μm pore size), placed in acid-washed glass scintillation vials and stored on

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ice in the dark until we ran the analysis (i.e., within 0; Figure 1A). In biofilms from vegetated flumes, only

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the treatment of DOM_Brew in Flume_Iris showed a significant increase in total

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bacterial abundance (p-value = 0.002; Figure 1). ƞ2 for DOM treatments was larger than

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those of either the Flume setting factor or the interaction term.

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Changes in gene abundance of the denitrifying bacteria during the incubations differed

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substantially between nirK and nirS (Figure 1, B and C). The nirK gene was

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significantly more abundant in Flume_Iris, (Table 1, RC nirK p0.05). Based on ƞ2, both the interaction term and the DOM source factor had

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moderate effects on the variance of the gene abundance, though there were no

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significant differences in nirS among treatments.

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Potential rates of denitrification

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There were significant differences on the DEA rates related to both factors, DOM

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source and Flume setting (Table 1). The DOM source had a larger effect (ƞ2= 0.66) on

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the variability of the DEA rates than the Flume setting (ƞ2= 0.22). Biofilms with DOM

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additions (DOM_Iris, DOM_Phragm and DOM_Brew) had higher DEA rates (Fig. 2;

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Tukey test p-value < 0.05) than those incubated under unamended conditions

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(DOM_Control). Regarding the Flume setting factor, significant differences were only

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found between biofilms from Flume_Unveg and from Flume_Iris (Figure 3; Table 1).

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After the incubations, NO3- load in the bottles decreased by 4.2 ± 0.5% (mean ±

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standard deviation), 22.6 ± 4.1%, 27.2 ± 1.2% and 40.2 ± 10.4% for the DOM_Control,

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DOM_Iris, DOM_Phragmites and DOM_Brew treatments, respectively.

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Rates of aerobic respiration

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The flume setting and the DOM source influenced AR rates (Table 1; p

Leachates from helophyte leaf-litter enhance nitrogen removal from

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