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Plant-microbe interactions drive denitrification rates, dissolved nitrogen removal, and the abundance of denitrification genes in stormwater control measures Natalie R Morse, Emily G.I. Payne, Rebekah Henry, Belinda E. Hatt, Gayani Chandrasena, James P. Shapleigh, Perran Louis Miall Cook, Scott Coutts, Jon Hathaway, Michael Todd Walter, and David Mccarthy Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02133 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on July 30, 2018

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Environmental Science & Technology

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Title: Plant-microbe interactions drive denitrification rates, dissolved nitrogen removal, and the abundance of denitrification genes in stormwater control measures

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Author List:

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Natalie Morsea*; Emily Payneb; Rebekah Henryb; Belinda Hattc; Gayani Chandrasenad; James Shapleighe; Perran Cookf; Scott Couttsg; Jon Hathawayh; M.Todd Waltera; David McCarthyb

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a

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*corresponding author: Natalie Morse, [email protected], 111 Wing Drive, Riley-Robb Hall B62, Cornell University, Ithaca, NY 14850, phone:734-771-7744, fax: 607-255-4449

Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York 14850, United States

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b

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c Jacobs, Melbourne, Victoria, Australia

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d SMEC Australia, North Sydney, New South Wales, Australia

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e

Department of Microbiology, Cornell University, Ithaca, New York 14850, United States

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f

Water Studies Centre, School of Chemistry, Monash University, Clayton, Victoria, Australia

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g

MICROMON, Monash University, Clayton, Victoria, Australia

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h

Environmental and Public Health Microbiology Laboratory (EPHM Lab), Department of Civil Engineering, Monash University, Clayton, Victoria, Australia

Department of Civil and Environmental Engineering, University of Tennessee, Knoxville, Tennessee 37996, United States

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KEWWORDS: stormwater, nitrogen, microbial, metagenomics, plant-microbe interactions

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Nitrogen

Microbes

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ABSTRACT

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The microbial community and function along with nitrate/nitrite (NOx) removal rates, and

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nitrogen (N) partitioning into “uptake”, “denitrification” and “remaining” via isotope tracers, were

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studied in soil bioretention mesocolumns (8 unique plant species). Total denitrification gene reads

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per million (rpm) were positively correlated with % denitrified (r = 0.69), but negatively correlated

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with total NOx removal following simulated rain events (r = -0.79). This is likely due to plant-

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microbe interactions. Plant species with greater root volume, plant and microbial assimilation %,

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and NOx removal %, had lower denitrification genes and rates. This implies that although

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microorganisms have access to N, advantageous functions, like denitrification, may not increase.

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At the conclusion of the 1.5-year experiment, the microbial community was strongly influenced

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by plant species within the Top zone dominated by plant roots, and the presence or absence of a

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saturated zone influenced the microbial community within the Bottom zone. Leptospermum

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continentale was an outlier from the other plants and had much lower denitrification gene rpm

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(average 228) compared to the other species (range: 277 to 413). The antimicrobial properties and

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large root volume of Leptospermum continentale likely caused this denitrification gene depression.

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1.0

Introduction

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Humans have disrupted the global nitrogen (N) cycle by applying fertilizers, burning fossil

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fuels, and disturbing the landscape1. The main environmental concerns with excess N are water

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quality degradation (e.g., eutrophication, habitat loss, etc.) and increased nitrous oxide (N2O)

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emissions associated with global climate change2–4. High N deposition from fossil fuel

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combustion, fertilizer applications, septic systems, and leaky sanitary sewers contribute to urban

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N pollution5–8. As urbanization is only expected to increase, there is a strong need to mitigate N

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pollution from urban areas 9,10.

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Bioretention, or biofilters, are popular green infrastructure (GI) practices employed

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throughout the world to reduce urban pollutant loads and provide hydrological benefits to

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downstream waterways11–13. However, the N treatment performance within these systems is highly

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variable14. Plant uptake and microbial denitrification are two mechanisms to internally treat N

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within bioretention cells.

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Denitrification is a stepwise, anaerobic microbial process reducing nitrate to N2, which is

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catalyzed by several enzymes: nap, nar, nirS, nirK, nor, and nosZ15. Denitrification rates are

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generally highest under anoxic conditions16,17. Thus, bioretention cells with anaerobic saturated

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zones (SZ) overlaid by aerobic zones have been suggested as a strategy to reduce N water

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pollution18. Lab studies reported that the inclusion of a SZ enhanced NO3- removal18–20. However,

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to date, field studies on bioretention with SZ have shown variable results. Some studies have

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reported that a SZ did not markedly improve N treatment over conventional bioretention design21–

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loads, despite significantly reducing total outflow N loads.

, and Passeport et al. (2009) reported that two bioretention cells with SZ failed to reduce NOx

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While the impacts to N removal from design considerations like SZ have been considered,

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little research has explored the microbial or plant-microbe effects on N cycling within bioretention.

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denitrification microbial mechanisms are not reported. Plant-microbe interactions alter the

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microbial community and subsequent denitrification process26. Chen et al. (2013) reported that the

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abundance of denitrification genes (qPCR) in the soils was correlated with inundation time in a

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bioretention cell, but they did not examine design features or the plant-microbe interactions. Morse

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et al. (2017) reported that denitrification genes (metagenomics) were greater within often saturated

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stormwater basins than dry basins, although plant effects were not reported. Bettez and Groffman

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(2013) reported that denitrification potential was greater in stormwater systems than nearby

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riparian zones, although microbial communities or DNA profiling was not conducted. LeFevere et

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al (2012) noted that naphthalene concentrations were positively correlated with naphthalene

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degrading genes in bioretention mesocolumns, and that planted columns had greater naphthalene

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removal. A similar study examining the interaction of microbes, plants, and N removal ought to

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be conducted in bioretention systems. Ruiz-Rueda et al. (2009) reported that plant species affected

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nosZ diversity in constructed wetlands, although N treatment performance was not reported. The

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plant-microbial effects on N cycling and removal within bioretention cells is important, but largely

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unquantified. Work is needed to determine how our designs and plant selections influence the

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microbial community and particularly denitrification, especially in engineered systems designed

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to remove N.

offers a review on general plant effects in bioretention pollutant removal, although specific

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The objectives of this work were to determine whether: (1) plant species altered the

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microbial community and the denitrification process, (2) more abundant denitrification genes

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translated to better N removal from simulated rain events, and (3) a SZ enhanced the relative


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abundance of functional genes responsible for denitrification, or altered the microbial community

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at large within bioretention. By using

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quantified the microbial community structure, abundance of denitrification genes, and N uptake

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pathways and overall N removal rates. A better understanding of the interplay between the various

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physical and biological factors should allow us to optimize bioretention system design to favor the

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conditions suitable for denitrifier proliferation and, hence, long-term, permanent N removal.

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2.0

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N tracers, DNA metagenomics, and 16S profiling, we

Methods

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This research builds on an extensive mesocolumn study conducted over a 1.5-year period

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in 201420,31. The water quality monitoring and N portioning, via isotope tracers was completed in

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2014. Additional analysis was performed on these systems in 2016 to examine the microbial

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population within the columns. Soil samples collected in 2014 from the columns were frozen at -

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80ºC until DNA extraction in 2016; samples were likely unaffected by storage conditions32. By

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pairing previously completed monitoring results with the microbial analysis herein, we aimed to

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provide a comprehensive view of how these systems process N.

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2.1

Experimental Mesocolumns

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This study examines the effects of eight Australian plant species commonly used in

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stormwater management and one soil-only control on microbial communities and denitrification

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within mesocolumns. The plant species studied in these experiments were: Allocasurina littoralis

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(ALL), Buffalo sp. (BUF), Carex appressa (CAR), Dianella tasmanica (DIA), Gahnia siberiana

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(GAH), Hypocalmma angustifolium (HYP), Leptospermum continentale (LEP), and Juncus krassii

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(JUN).



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The mesocolumn construction and experimental methods are comprehensively described

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elsewhere20. Single-plant bioretention columns were grown in a greenhouse for a 6-month

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establishment period and a 1-year monitoring period. Columns were 600 mm high, with a “Top”

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300-mm zone consisting of loamy sand and a “Bottom” 300 mm zone consisting of sand and a

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gravel drainage area. Columns with a saturated zone (SZ) kept the lower 300 mm saturated with

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an elevated outlet and “Non-saturated zone” (NS) columns allowed free drainage at the bottom of

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the columns (Figure S1). Table 1 shows the column design layout with the plant species and one

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soil-only control; there were 27 SZ and 27 NS columns.

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Columns were dosed with semi-synthetic stormwater as described33: 0.99 mg L-1

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(NO3/NO2)-N, 0.41 mg L-1 NH4-N, 0.38 mg L-1 particulate organic N, 0.44 mg L-1 dissolved

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organic N, 0.36 mg L-1 total phosphorus (TP), and 150 mg L-1 total suspended solids. A dosing

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volume (3.7 L) twice-weekly was set to reflect a bioretention cell sized to 2.5% of the drainage

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area, with annual 540mm average precipitation observed for Melbourne (Australia). Outflow

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concentrations of TN (APHA 4500 N), TP (APHA 4500 P), total dissolved phosphorus (APHA

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4500 P) and NO3/NO2 (NOx; APHA 4500 NO3) were measured monthly at the National

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Association of Testing Authorities, Australia (NATA) lab (Water Studies Centre, Monash

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University, Australia). At the conclusion of the experiment, root characteristics were destructively

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sampled. Total root length (cm), volume (cm3), mass (g), and surface area (cm2) were determined

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for each column by scanning 3 subsamples (EPSON Flatbed Scanner EPSON Expression

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10000XL 1.8 V3.49) and then analyzing them with WinRHIZO software (v. 2009c, Regent

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Instruments Canada Inc.). The ratio of the sub-sample dry mass to total dry mass was used to

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calculate total root length, volume, and surface area for the entire root system.

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Table 1. Column design and soil DNA sampling layout. Configuration indicates whether the column had a non-saturated (NS) bottom zone or a saturated (SZ) bottom zone. Soil samples collected for DNA extraction were taken from the bottom (BOT) or top (TOP) zones. Numbers before “BOT” or “TOP” indicate the number of samples collected within that treatment.

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Plant Species

Abbreviation

Allocasurina littoralis

ALL

Buffalo

BUF

Carex appressa

CAR

Dianella tasmanica

DIA

Gahnia siberiana

GAH

Hypocalmma angustifolium

HYP

Juncus krassii

JUN

Leptospermum contintale

LEP

Soil Only Control

Soil

2.2

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Configuration NS SZ NS SZ NS SZ NS SZ NS SZ NS SZ NS SZ NS SZ NS SZ

Column Replicates 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

Sampled for Metagenomics 0 0 1 BOT; 2 TOP 2 BOT; 3 TOP 3 TOP 2 BOT; 3 TOP 0 3 BOT; 2 TOP 0 0 0 0 0 3 BOT; 3 TOP 2 TOP 3 BOT; 3 TOP 3 TOP 2 BOT; 3 TOP

Sampled for 16S 0 3 BOT; 3 TOP 3 BOT; 3 TOP 2 BOT; 3 TOP 3 BOT; 3 TOP 2 BOT; 2 TOP 3 BOT; 3 TOP 2 BOT; 3TOP 0 3 BOT; 3 TOP 0 3 BOT; 3 TOP 0 3 BOT; 3 TOP 3 BOT; 3 TOP 3 BOT; 3 TOP 3 BOT; 3 TOP 3 BOT; 3 TOP

Isotope Tracers 15

N isotope tracers were used to partition incoming N among plant and microbial

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assimilation (“assimilation”), microbial denitrification, and soil porewater. Full details are

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described in

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during two trials. Prior to tracer addition and thereafter every 6 hours, samples were collected from

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inflow solution and porewater and analyzed for NH4+ and NO3-. Dissolved N2O, 28N2, 29N2, and

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30

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concentrations were analyzed by gas chromatography using a NCA GSL2 elemental analyzer

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coupled to a Hydra 20-22 isotope ratio mass-spectrometer (IRMS; Sercon Ltd., UK).

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. Briefly, a 1:1 Na14NO3:Na15NO3 nutrient solution was applied to the columns

N2 were also analyzed from the samples to determine denitrification rates. N2O and N2



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Linear regression of

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N2 and

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N2 production calculated

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N denitrification and

15

N

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denitrification rates, respectively. The total amount of 15N denitrified was calculated by integrating

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the rate of denitrification over the 12-hour monitoring period. The amount of 15NO3- remaining in

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the pore water was calculated based on the final 15NO3- concentration at 12 hours. The proportion

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of 15NO3 assimilated was calculated as the difference between 15N denitrified and 15N remaining

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in the pore water.

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2.3

Soil Metagenomics and 16S

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Each soil sample analyzed for DNA was a composite sample where three samples were

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collected throughout the soil column zone, Top (TOP) or Bottom (BOT), and pooled. These

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samples were collected from the columns at the end of the 1.5-year experiment. Soils were frozen

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at -20ºC prior to DNA extraction. Total DNA was extracted from each sample using the Mo-Bio

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PowerSoil® DNA Isolation Kit as per manufacturer’s instructions. The effluent was frozen at -

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80ºC until sequencing. Prior to sequencing, DNA extract concentrations were quantified using a

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Quant-It hsDNA assay kit (ThermoFisher).

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Metagenomic and 16S rRNA sequencing was conducted at Micromon (Monash University,

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Australia). Metagenomic sequencing libraries were prepared using the Nextera XT library

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preparation kit (Illumina) according to the manufacturer’s instruction. A total of 1ng of DNA was

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used as input, and the libraries were purified with 0.6V of AxyPrep Mag PCR Cleanup beads

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(Axygen). All library types were quantitated using a Qubit fluorimeter and Quant-IT DNA HS kit

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(Invitrogen) as per the manufacturer’s instructions, and sized using an Bioanalyzer 2100 and DNA

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HS kits (Agilent) according to the manufacturer’s instructions. The libraries were denatured and

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diluted for sequencing using a NextSeq 500 (metagenomic libraries) and NextSeq 500 Mid-Output


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V2 (150cycles) kit or a MiSeq and MiSeq V2 (600 cycle) Reagent Kit (16S amplicon libraries)

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according the manufacturer’s instructions.

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Soil samples used for DNA sequencing were selected to cover a range of plant species

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types (only 5 plant species and the control were sampled for metagenomics, while all 8 species

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were sampled for 16S), and also to coincide with the isotope tracer study previously conducted 31.

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Forty-nine samples were sequenced for DNA metagenomics based on quantification results (Table

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1; each sample was from an individual replicate column), thus some treatments did not have the

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full n=3 as DNA was not successfully extracted from each column. Approximately 10M 150 base

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pair (bp) single-strand reads were sequenced for each sample (49 samples x 10M reads = 490M

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reads total). Raw data can be accessed via the European Sequencing Archive (Study:

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PRJEB24343). Next, DIAMOND (double index alignment of next generation sequencing data), a

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high throughput alignment program comparing DNA sequence reads to reference sequences, was

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performed with the sample DNA sequences against a custom, manually curated database of

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reference proteins critical for N cycling. This is analogous, but faster, than BLASTx34. DIAMOND

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returned a matrix of matches for each sequence, within each sample; results were filtered where

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percent sequence identity (pid) >50, and assigned read length >25 amino acids. Once functional

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gene count reads were obtained, they were normalized by total reads per sample and multiplied by

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one million to obtain reads per million reads (rpm), which is a relative abundance term.

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Denitrification was the process of interest for complete N removal herein. Therefore,

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analyses focused on functional genes associated with denitrification: nitrate reductase (nap and

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nar), nitrite reductase (nirK and nirS), nitric oxide reductase (cNor and qNor), and nitrous oxide

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reductase (nosZ). The sum of all these genes is herein referred to as ‘total denitrification genes’,



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which are expressed as rpm. While all these genes are indeed part of the denitrification pathway,

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nap and nar are also used in other dissimilatory nitrate reduction processes.

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Taxonomic assignment with the metagenomics sequences was conducted via kmer

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mapping and matched with known genes from the JGI/IMG database; additional details are given

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in the Supplementary Materials. The percentage of reads that could be assigned to a gene varied,

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but ranged from 50-70%. The name of the source of the gene sequences were extracted and then

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used to query NCBI to receive complete taxonomic description of the source.

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16S rRNA amplicon sequencing was conducted within all plant treatments and the soil-

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only control. Samples were selected to coincide with the DNA metagenomics samples, and also

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include additional plant treatments to increase statistical power among treatments. Eighty-four

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samples were sent for 16S sequencing, although 3 samples failed to amplify, therefore a total of

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81 samples are included in this analysis. 16S rRNA amplicon libraries were prepared by

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amplifying

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(TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG)

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reverse

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(GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC)

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primers, 5 uL of genomic DNA, 2x HiFi HotStart ReadyMix (KAPA) made up to volume with

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UltraPure water (Invitrogen). The PCR program considerations are given in Supplementary

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Materials. Approximately 40,000 16S sequences were completed per sample. Quality filtering and

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OTU picking procedures have been described elsewhere (Henry, 2016; Schang et al. 2016). The

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assembled reads were analyzed using the QIIME 1.8.0 closed-reference OTU picking workflow

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with UCLUST for de novo OTU picking and the GreenGenes 13_8 release 35. Data for 16S samples

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are

available

the

on

V3+4

the

region

Short

Read

in

50

Archive,

µL

reactions:

project

reference

1µM

forward and

PRJNA309092 8

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(ncbi.nlm.nih.gov/bioproject/PRJNA309092). Following removal of singletons (18% of OTUs)

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and doubletons (9% of OTUs), and rarefraction without replacement to 30,000 reads per sample,

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8,122 OTUs remained in the 81 samples. Summarization of relevant OTUs included only those

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with a relative abundance ≥0.2%. All other analyses used the entire rarefied dataset. Alpha and

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Beta diversity assessments were done with rarefied data including singletons and doubletons. The

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phyloseq package in R was used for 16S analysis.

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2.4

Statistical Analyses

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We used R software (version 3.1.1; R Development Core Team, 2014), and a criterion of

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95% confidence (a=0.05) was applied for all statistical analyses. Nitrogen treatment and isotope

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tracer results through the columns approximately 3 months prior to soil DNA collection were used

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to compare against DNA data.

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For metagenomics results, we used analysis of variance (ANOVA) to test if plant species

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significantly affected denitrification gene rpm between the plant treatments. Tukey honest

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significant differences (HSD) post-hoc tests determined what plant species, if any, differed

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significantly from each other. The influence of a SZ on total denitrification genes rpm was

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assessed with a mixed effects model. Total denitrification gene rpm was the dependent variable,

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sample location (Top vs Bottom zone) was the independent fixed effect, and plant species was the

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random effect variable. This controlled for random variation from species to species, while

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allowing an overall effect of SZ to be determined. Spearman correlations were calculated for total

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denitrification gene rpm, % denitrified, % assimilated, and % NOx removal to determine

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relationships among these variables.

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ANOVA was used to compare observed and Chao1 α-diversity differences between

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groups. Principal coordinate analysis (PCoA) from pairwise Unifrac and weighted Unifrac 9 ACS Paragon Plus Environment


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distances for the 16S data were created to visualize differences between groups, and analysis of

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similarity (ANOISM) tested differences between groups.

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3.0

Results and Discussion

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3.1

Soil Metagenomic Denitrification Genes

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Total denitrification genes rpm within the SZ columns were significantly and positively

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correlated with % denitrified N (r = 0.69) (Table 2). Table 2 illustrates the correlations between

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the columns; all values are the measured value for the entire column; no TOP or BOT distinction

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as processes like denitrification were quantified via flow through the entire column, and not limited

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to either zone. However, the total denitrification reads are the sum of TOP and BOT gene reads

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for that column. This indicated that the denitrification genes rpm corresponded to increased

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denitrification function, thus supporting the use of total denitrification genes rpm as a biological

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marker for actual denitrification. The % denitrified in NS columns could not be determined, as the

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tracer method required sampling from porewater, which was unavailable in the NS columns.

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Table 2. Spearman correlation values for total denitrification gene rpm, % NOx reductions, and partitioning of N as % denitrified and % assimilated determined via 15N tracer. Correlations with total denitrification genes were calculated using DNA results from the sum of both Top + Bottom zones of SZ columns (n=17). Root length, surface area, volume and mass are all total values from the column. Values in bold are significant (p

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