Effects of Facilitated Bacterial Dispersal on the Degradation and

(4) Therefore, the PAH may escape bacterial degradation and build up poorly .... phase, at equilibrium can be determined experimentally and calculated...
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Effects of facilitated bacterial dispersal on the degradation and emission of a desorbing contaminant Sally Otto, Thomas Banitz, Martin Thullner, Hauke Harms, and Lukas Y. Wick Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00567 • Publication Date (Web): 19 May 2016 Downloaded from http://pubs.acs.org on May 25, 2016

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Effects of facilitated bacterial dispersal on the degradation and emission of a

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desorbing contaminant

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Sally Otto1, Thomas Banitz2, Martin Thullner1, Hauke Harms1,3, Lukas Y. Wick1*

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Microbiology, Permoserstr. 15, 04318 Leipzig, Germany

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UFZ - Helmholtz Centre for Environmental Research, Department of Environmental

UFZ - Helmholtz Centre for Environmental Research, Department of Ecological

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Modelling, Permoserstr. 15, 04318 Leipzig, Germany

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Deutscher Platz 5e, 04103 Leipzig

German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig,

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Running title: Effects of facilitated bacterial dispersal

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Intended for: Environmental Science and Technology

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*

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UFZ. Department of Environmental Microbiology; Permoserstr. 15; 04318 Leipzig,

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Germany. phone: +49 341 235 1316, fax: +49 341 235 1351, e-mail: [email protected].

Corresponding author: Mailing address: Helmholtz Centre for Environmental Research -

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ABSTRACT ART

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ABSTRACT

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The quantitative relationship between a compound’s availability for biological removal

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and eco-toxicity is a key issue for retrospective risk assessment and remediation

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frameworks. Here, we investigated the impact of facilitated bacterial dispersal on a model

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soil-atmosphere interface on the release, degradation and outgassing of a semi-volatile

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contaminant. We designed a laboratory microcosm with passive dosing of phenanthrene

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(PHE) to a model soil-atmosphere interface (agar surface) in the presence and absence of

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glass fibers known to facilitate the dispersal of PHE-degrading Pseudomonas fluorescens

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LP6a. We observed that glass fibers (used as a model to mimic a fungal hyphal network)

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resulted in (i) increased bacterial surface coverage, (ii) effective degradation of matrix-

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bound PHE, and (iii) substantially reduced PHE emission to locations beyond the

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contamination zone even at low bacterial surface coverage. Our data suggest that bacterial

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dispersal networks such as mycelia promote the optimized spatial arrangement of

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microbial populations to allow for effective contaminant degradation and reduction of

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potential hazard to organisms beyond a contaminated zone.

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KEYWORDS: Bacterial dispersal network, biodegradation, Pseudomonas fluorescens

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LP6a, PAH, phenanthrene, passive dosing

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INTRODUCTION

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Various environmental chemicals (contaminants) serve as substrates for microorganisms,

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while simultaneously exerting toxic effects on other organisms. Both processes are

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controlled by the chemicals’ bioavailability for the one or the other effect1. It is clear that

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the degradation of a chemical by the first group of organisms reduces the exposure for the

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second and it is generally agreed that metabolic utilization may be considered as a

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detoxification mechanism. However, these degrading organisms rely on the chemical and

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should strive to maintain an appropriate level of a chemical’s availability2. The

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quantitative relationship between the availability for biological removal and ecotoxicity is

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a key issue for any attempt to weigh extant risks against prospects of remediation3. In soil

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many organic contaminants, such as semi-volatile contaminants like polycyclic aromatic

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hydrocarbons (PAH) tend to sorb to the solid soil matrix4. Thereby the PAH may escape

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bacterial degradation and build up poorly bioavailable reservoirs5-7. Driven by

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disequilibria however, bound PAH may be released again to aqueous or gaseous soil

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phases where they may become available to soil organisms. Depending on the receiving

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organisms, this may cause toxic effects8,

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specialized bacteria able to metabolize PAH as a source of carbon and energy2. Any

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presence of PAH-degrading bacteria in the vicinity of PAH reservoirs might thus reduce

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further transport and eventual outgassing of PAH to the soil air. Moreover, PAH

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degradation, through its effect on the sorption equilibrium, would drive the desorption

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flux thus accelerating the attenuation of PAH. Efficient degradation thereby ideally

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requires a homogeneous distribution of degrading bacteria.

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The distribution of specialized bacteria and their degradation potential in the soil however

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shows a distinct spatial variability10, 11, leading to regions comprising several millimeters

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up to a few centimeters, which are devoid of degrading activity12, 13. In case of limited

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bacterial mobility patchy distribution of catabolically active microbial biomass may occur

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and/or result in biodegradation as e.g. by

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and the effectiveness of such ‘bacterial filters’ may be limited14-16. However, the effects

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of these small scale spatial heterogeneities on degradation efficiency have hardly been

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evaluated so far. Starting out from the earlier discovery that fungal mycelia facilitate

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bacterial dispersal by providing continuous paths for bacterial motility even through

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water-unsaturated soil15-18, we here hypothesize that mycelial dispersal networks may

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help distributing PAH-degrading bacteria in a way that area-covering PAH degradation

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occurs and transport of PAH including outgassing is minimized. A dense dispersal

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network thus would facilitate the formation of homogenously distributed degrader

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biomass, thus preventing contaminant outgassing from a contaminant hot spot.

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In this study, the effects of bacterial distribution in presence and absence of glass fibers

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(suitably mimicking dispersal along a fungal mycelium15,

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constantly desorbing phenanthrene (PHE) were investigated. In particular we studied (i)

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bacterial dispersal on the model surface in presence and absence of dispersal networks,

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(ii) the effects of the bacterial distribution on PHE degradation, and (iii) the role of

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bacterial distribution on PHE gas phase emission.

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) on biodegradation of

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MATERIAL AND METHODS

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Organisms and culture conditions. PHE degrading soil-borne Pseudomonas fluorescens

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LP6a20, 21 was grown at room temperature on a rotary shaker at 150 rpm in Erlenmeyer

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flasks containing 200 mL of minimal medium (MM) supplemented with 1.5 mg L-1 PHE

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crystals as the sole carbon and energy source. For microcosm experiments, 72 h old

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cultures were harvested in the exponential growth phase and washed twice in 50 mM

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potassium phosphate buffer (PB) at pH 7.2 and centrifuged at 1,000 x g for 10 min. The

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pellet was re-suspended in MM to reach an OD578 of 8 (≈ 1010 cells mL-1). Cell numbers

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were counted with a MoFlo flow cytometer (DakoCytomation, U.S.) as described

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elsewhere22. For all measurements, the instrument was adjusted using fluorescent 1-µm ACS Paragon Plus Environment

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beads. Cell counting was performed with 488 nm light of 400 mW to analyze the forward

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light scatter (FSC) and the side light scatter (SSC). Measurements of cells and reference

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microbeads were performed in triplicates.

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Sorbent. Polydimethylsiloxane (PDMS) was used as sorbent for PHE. Its qualities as

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passive dosing system are described elsewhere23. PDMS O-rings obtained from Altec

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(Order no. ORS-0793-57. Cornwall, UK) with an outer diameter of 90.7 mm, an inner

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diameter of 79.3 mm, a mass of 9.12 g (C.V. 0.5 %, n = 10) and a volume of 6.81 mL

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were used for passive dosing in biodegradation experiments. For use in the microcosm

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system, PDMS rings were cut into 1 cm long pieces with a mass of 0.356 g (C.V. 1.5 %,

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n = 20). The pieces were cleaned as described elsewhere23. Methanol was used as the

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loading solvent. PDMS pieces (57 per batch) were submerged in 75 mL of saturated PHE

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solution in methanol which had been diluted with methanol to a final volume of 500 mL

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in a closed 1-L bottle and incubated at room temperature for at least 3 days to obtain

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partitioning equilibrium. The solution was decanted, surfaces of PDMS pieces were

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wiped using lint-free tissue, and residual methanol was removed by three sequential

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washes with 200 mL of Milli-Q water. The PHE loading therefore led to a final mean

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concentration of 596 µg PHE g-1PDMS in the PDMS. For an individual experiment with a

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higher PHE load (≈ 8000 µg PHE g-1PDMS) here, PDMS pieces were submerged in 500 mL

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of saturated PHE methanol solution and treated as described above. A lower PHE load in

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the PDMS (314 µg PHE g-1PDMS) for a coverage experiment (cf. below) were adjusted by

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using 25 mL of saturated PHE methanol solution which had been diluted with methanol

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to a final volume of 500 mL.

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Calculation of the partitioning of PHE. The distribution of PHE between PDMS and

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water, i.e. agar as aqueous phase, at equilibrium can be determined experimentally and

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calculated using the dimensionless partitioning ratio:

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: =

()

(1)

()

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Here cPDMS and cwater are the PHE concentration in PDMS and water, respectively and

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: = 8404 (± 315) is the dimensionless partitioning ratio and was obtained

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experimentally in this study (cf. the SI). PHE vapor-phase concentrations were not

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directly measurable and thus calculated assuming equilibrium between the gas phase and

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the agar phase using a dimensionless partitioning ratio of " : = cair / cwater =

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0.001424. Hence, the percentage fraction of the total amount of PHE in the closed abiotic

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system, #$% present at the equilibrium in the agar can be calculated by:

#$% =

1 ( ( 1 + " : ∙ ( " + : ∙ (  

(" (= 120 *+)

,

∙ 100 (2)

( (= 2 *+, "- . / + 1 *+, 012 )

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Here,

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( (= 0.76 *+), are the respective volumes. PHE concentrations in individual phases

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were calculated assuming equilibrium between the three-phases in the abiotic system and

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by using the total PHE load in the microcosm and used for the comparison between the

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experimental data in the abiotic system and the theoretically calculated value.

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Degradation and dispersal experiments. Degradation and dispersal experiments were

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conducted in microcosms with or without glass fibers as dispersal networks (Fig. 1). The

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PHE loaded PDMS allows a continuous and controlled release of the only carbon sources

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into the agar of the microcosm system as model soil atmosphere interface. The basic body

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of a microcosm consists of a glass vat (length: 3.5 cm, height: 1.0 cm, width: 1.0 cm)

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containing PHE-loaded PDMS, agar as the direct environment of the bacteria and a

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dispersal network (glass from P-D Glasseiden GmbH, Germany). Three PHE-loaded

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PDMS pieces (596 µg PHE/g PDMS) were placed at the bottom of the vat and covered with 2

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mL of minimal medium agar (MMA, 0.6% agar (w/v)). Glass fibers (approx. 300 pcs,

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length: 3.2 cm with a diameter of ≈ 8 µm) were cleaned by heating to 600 °C for 5 h.

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They were placed in parallel along the transect A-D at a distance of ≈ 0.25 cm from the

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left border of the vat in zone A and covered the whole width of the vat (Fig. 1). The

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microcosms were incubated for 18 h to allow the PHE partition between PDMS and agar.

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Then, the microcosms were inoculated with 2 µL of bacterial suspension at 0.1 cm

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distance from the glass fibers on the left-hand third of zone A (zone A, Fig. 1) to avoid

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capillary flow along them and incubated for 24 h, 96 h, 168 h (highest PHE load, cf.

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section Sorbent above), 0 h, 72 h, 120 h (medium PHE load) or 72 h (lowest PHE load),

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respectively. An agar cube (1 cm3 MMA, 2 % agar w/v) was placed at a distance of 0.3

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cm to the vat thus leaving the glass vat and the agar cube separated by an air gap (Fig. 1).

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The agar cube was used as a recipient of outgassing PHE. All microcosm setups were

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incubated in sealed standard glass Petri dishes at 25 °C and stored in a 30 L desiccator to

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minimize gaseous losses of PHE. Identical setups without glass fibers, either with or

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without bacteria were used in two independent control experiments.

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The agar cube was removed 2, 27, and 120 h after inoculation and analyzed for its PHE

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concentration by gas chromatography coupled with mass spectrometry (GC-MS, cf.

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below). As illustrated in Fig. 1, the glass vat was divided in four zones of interest (A, B,

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C and D). The surfaces of the zones were harvested individually with a spatula starting

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from zone D to a depth of about 1 mm. Due to the agar concentration used the agar allows

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for efficient bacterial dispersal on its surface only, yet restricts bacterial motility within

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the agar. The removed agar layer was then suspended in 3 mL PBS, ultravortexed for 1

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min, sonicated (with 35 kHz for 2 x 30 s with a break of 1 min)15, fixed with 2 mL 10%

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(v/v) sodium azide (NaN3) and finally analyzed for total cell number by flow cytometry

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(flow cytometry, cf. above). When glass fibers were present, they were cut at zone

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boundaries with a sharp scissor prior to sampling. The surface area of zone A was

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0.5 cm2, surface areas of zones B, C and D were 1 cm2, each. Thereafter, the remaining

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agar was removed and weighed. The PDMS pieces were removed before the PHE was

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extracted and analyzed.

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Bacterial surface coverage experiments. To estimate effects of bacterial surface

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coverage on PHE degradation and outgassing from microcosms, various initial cell

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concentrations

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(314 µg PHE/g PDMS). Suspensions of P. fluorescens LP6a calculated to result in surface

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coverages of 9%, 37% or 64%, assuming a cross-sectional area of 1.5 µm2 per cell, were

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adjusted by flow cytometry in PBS buffer and inoculated in three different regimes

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(‘heterogeneous’ or ‘homogeneous’ distribution without glass fibers or placement on

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glass fibers) on the agar surface of the microcosm system (see scheme in SI, Fig. S1).

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Microcosms were prepared and incubated for 72 h as described above but filled with 2

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mL MMA with 2 % agar to restrict bacterial movement. Heterogeneous distribution was

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achieved by spot inoculation of 3.33 µl cell suspension at each of three positions

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separated by 1 cm with the first spot placed at 0.75 cm from the left border of the glass

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vat. For homogeneous distribution, a 10 µL cell suspension was inoculated on the agar

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surface and dispersed carefully with a glass stick. For the third regime, glass fibers

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(approx. 300 pcs, length: 3.5 cm) were placed on the entire agar surface and spot

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inoculated with 10 µL cell suspension in the center of the agar surface. All setups were

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installed in triplicates along with controls without bacteria. Microcosms were harvested

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and analyzed as described above, but for the quantification of cell numbers, the whole

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agar surface was removed with a sterile spatula to a depth of about 1 mm.

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Extraction and analysis of PHE. Triplicate microcosms were harvested and their PHE

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concentration analyzed after a certain time of incubation (cf. section Degradation and

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dispersal experiments above). Briefly, agar samples were mixed during 10 min with

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approximately equal amounts of activated (5 h at 600 °C) Na2SO4 and then extracted with

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either 20 mL (agar cube) or 25 mL (agar from the vat) of a 1:3 mixture of acetone and n-

were

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hexane containing per-deuterated PHE (PHE-d10, 10 mg mL-1 99.5 %, Dr. Ehrenstorfer,

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Germany) and acenaphthylene (ACN-d10, 10 mg mL-1 99.5 %, Dr. Ehrenstorfer,

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Germany) as extraction and injection standard, respectively25. PHE (98 % HPLC),

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methanol (99.8 %), acetone (99.8 %) and n-hexane (97.5 %) were obtained from Fluka,

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Merck, or Roth (all Germany). PHE in PDMS was extracted overnight with 8 mL of the

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solvent mixture per piece. PHE was quantified in an HP 7890 Series GC as described

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elsewhere26.

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Statistical analysis. Results of the replicate series for systems with or without transport

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networks were compared to each other and to abiotic systems, using a two-sided

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Student’s t-test to test for significant differences (p-value = 0.05).

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RESULTS

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Distribution and abundance of bacteria in the presence of a dispersal networks.

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Dispersal and growth of P. fluorescens LP6a in the presence and absence of glass fibers

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were monitored as illustrated by the mean bacterial coverage in the four zones of the

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microcosms (Fig. 2). In the absence of glass fibers (Fig. 2 left), the coverage in the

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inoculation zone A rose from initially 180 % to 870 % (i.e. formed presumed multiple

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layers of bacteria within the top ≈ 1 mm scraping depth) within 120 h, while in all other

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zones coverage increased very slowly only and remained below 33 % until the end of the

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experiment. With glass fibers (Fig. 2 right) the calculated average coverage in zone A

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reached 620 % within 120 h. In all other zones, coverages remained below 100 % but

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were at least twofold higher than in the absence of glass fibers. These differences were

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clearly significant in zones B and C after 120 h, where dense coverage (55 – 68 %) was

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found in zones B to D when glass fibers were present.

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Influence of bacterial distribution on PHE degradation. Significantly higher PHE

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with the network-free setups (15 %; Fig. 3). Likewise 36 % (72 h) and 34 % (120 h)

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lower agar-dissolved PHE concentrations were measured in the agar of the vat in

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presence of the glass fiber networks compared to the setups without glass fibers

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(percentages relate to the theoretically calculated PHE concentration for the abiotic

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system; SI, Fig. S2).

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Influence of bacterial dispersal and abundance on PHE emission. Outgassing PHE

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was passively captured by an agar cube placed in the microcosms and analyzed over time

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to assess the PHE emission from the main body of the microcosm (Fig. 4). The partition

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equilibrium of PHE was obtained after 72 h incubation in the abiotic control (Fig. 4A). In

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agreement with PHE degradation data, the presence of LP6a bacteria reduced PHE

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outgassing by 77 % (72 h) and 88 % (120 h) relative to the calculated value. Although the

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microbial biomass in the presence of glass fibers was similar to the network-free scenario,

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no PHE accumulation in the agar cube, and hence, very limited PHE outgassing was

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detected when bacteria dispersed along glass fibers (Fig. 4A). Efficient bacterial dispersal

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along the glass fibers likewise resulted in reduced PHE emission from a PDMS

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containing a 13-fold higher PHE load (Fig. 4B). Here, ca. five- (96 h) and eight-fold

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(168 h) decreased PHE amounts were captured in the agar cubes when bacteria were able

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to disperse along glass fibers compared to the setup without glass fibers.

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Influence of cell coverage and distribution on PHE degradation. In order to further

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assess the effects of bacterial biomass and distribution on PHE degradation, in the

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bacterial surface coverage experiments nine regimes differing in their biomass

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distribution (‘heterogeneous’, ‘homogeneous’, ‘glass fibers’) and initial biomass (‘mean

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coverage’; cf. Materials and Methods) were analyzed for their effects on PHE outgassing,

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PHE degradation, and the abundance of strain LP6a after 72 h (SI, Fig. S1).

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Heterogeneous inoculation reduced PHE outgassing marginally irrespective of the

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homogeneously applied to the agar surface or when glass fibers allowed their dispersal

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the outgassing of PHE was significantly decreased. Reduced outgassing went along with

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similarly efficient PHE degradation for the variants with 37% and 64% initial mean

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coverage respectively, likewise irrespective of the distribution (Fig. 5 bottom). Also here,

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no significant differences between the homogeneous distribution and the setup with glass

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fibers were observed. Interestingly, the initial biomass concentration only had marginal

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effects on the extent of PHE degradation: After 72 h of incubation, the total cell

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concentrations on the agar surface were quasi similar in all regimes independent of their

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initial abundance and distribution (SI, Fig. S3).

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DISCUSSION

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Influence of dispersal on biomass distribution and PHE biodegradation. Our study

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demonstrates that facilitated dispersal and optimized spatial arrangement of microbial

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populations enhances the degradation of constantly desorbing PHE. Efficient

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biodegradation of PHE simultaneously reduced the emission of this semi-volatile

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chemical already at surprisingly low rates of bacterial surface coverage (Fig. 4). Control

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experiments, in which biomass was ab initio inoculated at different degrees of

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homogeneity indicated that the effect of dispersal networks arises indeed from its

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influence on the cell distribution, which controls the substrate availability to individual

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cells and, consequently, their degradation rates. This can be seen from distinct

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degradation performances of differently distributed bacterial inocula despite identical

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substrate provision (Fig. 5 bottom). An important observation is that distribution of

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biomass exerted substantially different pressures on the partition equilibrium of the

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chemical; whereby a more homogeneously distributed biomass was driving PHE

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desorption by reducing PHE concentrations in the vat’s aqueous phase

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based system proved well-suited for mimicking prolonged PHE release as it is observed ACS Paragon Plus Environment

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. The PDMS-

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in PAH contaminated soil. Fungal mycelia known to facilitate bacterial dispersal15, 28, 29

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were substituted by arrangements of glass fibers for better control and longevity of the

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network as well as to prevent possible metabolic and physiological interactions between

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fungi and bacteria18. Despite the model character of this setup, we believe that the

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combination of a slow substrate release system and a receiving gas phase, separated by a

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functional system for bacterial dispersal in which bacteria upon inoculation can disperse

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and dynamically develop a biological sink for the released substrate, mimics several

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relevant features of a contaminated top soil. Our data experimentally support previous

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modelling data16,

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biodegradation rate of chemicals. Although the overall flux of PHE from the source is

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increased, aqueous concentrations of PHE available for both, degradation (SI, Fig. S2)

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and/or possible toxic exposure to non-degrader organisms are reduced. This effect is

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directly seen as reduced outgassing from the system (Fig. 4) which subsequently leads to

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a lower availability of the chemical to organisms beyond the contaminated zone. The

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higher biomass concentration behind the point of inoculation developing in microcosms

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with glass fibers indicates the positive effect of dispersal networks on the degradation

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capacity of the system in spite of the same initial substrate content. The positive effect of

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dispersal became evident despite of similar biomass production in presence of glass fibers

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in the bacterial surface coverage experiments (SI, Fig. S3), where reduced PHE loads in

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the PDMS (cf. lines 120ff) may have limited PHE bioavailability and have led to variable

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PHE allocation for bacterial growth and dispersal. Differing bacterial cell yields due to

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variable resource allocation (i.e. the division of resource uptake into fractions allocated to

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different energy-demanding processes) has been described for high metabolic costs for

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motility or flagellar synthesis 32, 33-

30, 31

, showing that network-based bacterial dispersal improves the

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Implications for the management of soil contamination. Many regulators have started

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to consider bioavailability within their retrospective risk assessment and remediation

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frameworks of organic chemicals. Such decision-making may rather depend on

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appropriate description of hazard based on the bioavailability rather than on the total-

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extractable concentration of a contaminant. Hence improved information on the emergent

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interactions between microbial dispersal, biodegradation and exposure of contaminants is

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needed to ensure appropriate protection of the environment and public health. Our results

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suggest that the presence of natural bacterial dispersal networks (e.g. provided by fungal

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mycelia or plant roots) may play an important role for the biodegradation of continuously

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released contaminants. The data furthermore show that the spatial arrangement of

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degrader biomass reflects the supply and transport characteristics of the degraded

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compounds34 provided that bacterial motility is sufficiently fast as e.g., due to dispersal

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networks. These effects of continuous structures or other biological vectors for the

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degradation capacity of bacteria in soil should also be kept in mind when designing

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bioremediation schemes3, 35, 36. Invasive management techniques such as ploughing may

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thus be detrimental for fungal helper functions37, 38. Enhanced dispersal and homogeneous

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distribution of degrader biomass may also be important for highly mobile (e.g. highly

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water soluble or quickly desorbing) contaminants, as such chemicals might easily escape

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patchy degrader populations19. Finally, our findings also may also apply for assessing the

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fate of biodegradable gases of environmental concern, such as e.g. methane evolving

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from soil and sediments39, 40.

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Acknowledgements. This work has been performed in the frame of the Helmholtz

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Association Research Programme ‘Chemicals in the Environment’ (CITE) and was

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supported by the research program Chemical Active Transport (CAT) and the Helmholtz

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Impulse and Networking Fund through the Helmholtz Interdisciplinary Graduate School ACS Paragon Plus Environment

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for Environmental Research (HIGRADE). We thank Kai-Uwe Goss for valuable

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discussions and Birgit Würz, Rita Remer and Jana Reichenbach for skilled technical help.

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We are grateful to Thomas Hübschmann for help on this work.

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Supporting Information Available

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This information is available free of charge via the Internet at http://pubs.acs.org

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REFERENCES

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(1) Ehlers, L. J.; Luthy, R. G. Contaminant bioavailability in soil and sediment. Environ. Sci. Technol. 2003, 37 (15), 295a-302a; DOI 10.1021/es032524fs. (2) Johnsen, A. R.; Wick, L. Y.; Harms, H. Principles of microbial PAH-degradation in soil. Environ. Pollut. 2005, 133 (1), 71-84; DOI 10.1016/j.envpol.2004.04.015s. (3) Ortega-Calvo, J. J.; Harmsen, J.; Parsons, J. R.; Semple, K. T.; Aitken, M. D.; Ajao, C.; Eadsforth, C.; Galay-Burgos, M.; Naidu, R.; Oliver, R.; Peijnenburg, W. J. G. M.; Rombke, J.; Streck, G.; Versonnen, B. From Bioavailability Science to Regulation of Organic Chemicals. Environ. Sci. Technol. 2015, 49 (17), 10255-10264; DOI 10.1021/acs.est.5b02412s. (4) Luthy, R. G.; Aiken, G. R.; Brusseau, M. L.; Cunningham, S. D.; Gschwend, P. M.; Pignatello, J. J.; Reinhard, M.; Traina, S. J.; Weber, W. J.; Westall, J. C. Sequestration of hydrophobic organic contaminants by geosorbents. Environ. Sci. Technol. 1997, 31 (12), 3341-3347; DOI 10.1021/es970512ms. (5) Semple, K. T.; Doick, K. J.; Jones, K. C.; Burauel, P.; Craven, A.; Harms, H. Defining bioavailability and bioaccessibility of contaminated soil and sediment is complicated. Environ. Sci. Technol. 2004, 38 (12), 228a-231a; DOI 10.1021/es040548ws. (6) Weissenfels, W. D.; Klewer, H. J.; Langhoff, J. Adsorption of Polycyclic AromaticHydrocarbons (Pahs) by Soil Particles - Influence on Biodegradability and Biotoxicity. Appl. Microbiol. Biotechnol. 1992, 36 (5), 689-696; DOI 10.1007/BF00183251s. (7) Means, J. C.; Wood, S. G.; Hassett, J. J.; Banwart, W. L. Sorption of Polynuclear Aromatic-Hydrocarbons by Sediments and Soils. Environ. Sci. Technol. 1980, 14 (12), 1524-1528; DOI 10.1021/Es60172a005s. (8) Phoothiwut, S.; Junyapoon, S. Size distribution of atmospheric particulates and particulate-bound polycyclic aromatic hydrocarbons and characteristics of PAHs during haze period in Lampang Province, Northern Thailand. Air Qual Atmos Health. 2013, 6 (2), 397-405; DOI 10.1007/s11869-012-0194-3s. (9) Wincent, E.; Jonsson, M. E.; Bottai, M.; Lundstedt, S.; Dreij, K. Aryl Hydrocarbon Receptor Activation and Developmental Toxicity in Zebrafish in Response to Soil Extracts Containing Unsubstituted and Oxygenated PAHs. Environ. Sci. Technol. 2015, 49 (6), 3869-3877; DOI 10.1021/es505588ss. (10) Vinther, F. P.; Brinch, U. C.; Elsgaard, L.; Fredslund, L.; Iversen, B. V.; Torp, S.; Jacobsen, C. S. Field-scale variation in microbial activity and soil properties in relation to mineralization and sorption of pesticides in a sandy soil. J. Environ. Qual. 2008, 37 (5), 1710-1718; DOI 10.2134/jeq2006.0201s. (11) Badawi, N.; Johnsen, A. R.; Sorensen, J.; Aamand, J. Centimeter-Scale Spatial Variability in 2-Methyl-4-Chlorophenoxyacetic Acid Mineralization Increases with Depth in Agricultural Soil. J. Environ. Qual. 2013, 42 (3), 683-689; DOI 10.2134/jeq2012.0397s. (12) Gonod, L. V.; Chadoeuf, J.; Chenu, C. Spatial distribution of microbial 2,4dichlorophenoxy acetic acid mineralization from field to microhabitat scales. Soil Sci. Soc. Am. J. 2006, 70 (1), 64-71; DOI 10.2136/sssaj2004.0034s. (13) Pallud, C.; Dechesne, A.; Gaudet, J. P.; Debouzie, D.; Grundmann, G. L. Modification of spatial distribution of 2,4-dichloro-phenoxyacetic acid degrader microhabitats during growth in soil columns. Appl. Environ. Microbiol. 2004, 70 (5), 2709-2716; DOI 10.1128/Aem.70.5.2709-2716.2004s. (14) Semple, K. T.; Doick, K. J.; Wick, L. Y.; Harms, H. Microbial interactions with organic contaminants in soil: Definitions, processes and measurement. Environ. Pollut. 2007, 150 (1), 166-176; DOI 10.1016/j.envpol.2007.07.023s.

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380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428

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(15) Kohlmeier, S.; Smits, T. H. M.; Ford, R. M.; Keel, C.; Harms, H.; Wick, L. Y. Taking the fungal highway: Mobilization of pollutant-degrading bacteria by fungi. Environ. Sci. Technol. 2005, 39 (12), 4640-4646; DOI 10.1021/Es047979zs. (16) Harms, H.; Wick, L. Y. Dispersing pollutant-degrading bacteria in contaminated soil without touching it. Eng. Life Sci. 2006, 6 (3), 252-260; DOI 10.1002/elsc.200620122s. (17) Wick, L. Y.; Remer, R.; Würz, B.; Reichenbach, J.; Braun, S.; Scharfer, F.; Harms, H. Effect of fungal hyphae on the access of bacteria to phenanthrene in soil. Environ. Sci. Technol. 2007, 41 (2), 500-505; DOI 10.1021/Es061407ss. (18) Pion, M.; Spangenberg, J. E.; Simon, A.; Bindschedler, S.; Flury, C.; Chatelain, A.; Bshary, R.; Job, D.; Junier, P. Bacterial farming by the fungus Morchella crassipes. Proc Biol Sci. 2013, 280 (1773), 2242; DOI 10.1098/rspb.2013.2242s. (19) Worrich, A.; König, S.; Miltner, A.; Banitz, T.; Centler, F.; Frank, K.; Thullner, M.; Harms, H.; Kästner, M.; Wick, L. Y. Mycelia-like Networks Increase Bacterial Dispersal, Growth and Biodegradation in a Model Ecosystem at Varying Water Potentials. Appl. Environ. Microbiol. 2016, DOI 10.1128/AEM.03901-15 s. (20) Foght, J.; Semple, K.; Westlake, D. W. S.; Blenkinsopp, S.; Sergy, G.; Wang, Z.; Fingas, M. Development of a standard bacterial consortium for laboratory efficacy testing of commercial freshwater oil spill bioremediation agents. J. Ind. Microbiol. Biotechnol. 1998, 21 (6), 322-330. (21) Bugg, T.; Foght, J. M.; Pickard, M. A.; Gray, M. R. Uptake and active efflux of polycyclic aromatic hydrocarbons by Pseudomonas fluorescens LP6a. Appl. Environ. Microbiol. 2000, 66 (12), 5387-5392. (22) Hübschmann, T.; Vogt, C.; Till, S.; Rohwerder, T.; Sand, W.; Harms, H.; Muller, S. Detection of sulfur microparticles in bacterial cultures by flow cytometry. Eng. Life Sci. 2007, 7 (4), 403-407; DOI 10.1002/elsc.200720195s. (23) Smith, K. E.; Rein, A.; Trapp, S.; Mayer, P.; Karlson, U. G. Dynamic passive dosing for studying the biotransformation of hydrophobic organic chemicals: microbial degradation as an example. Environ. Sci. Technol. 2012, 46 (9), 4852-4860; DOI 10.1021/es204050us. (24) Schwarzenbach, R. P., Gschwend, P. M., Imboden, D. M. Environmental Organic Chemistry; New Jersey, Canada, 2003. (25) Schamfuss, S.; Neu, T. R.; van der Meer, J. R.; Tecon, R.; Harms, H.; Wick, L. Y. Impact of Mycelia on the Accessibility of Fluorene to PAH-Degrading Bacteria. Environ. Sci. Technol. 2013, 47 (13), 6908-6915; DOI 10.1021/Es304378ds. (26) Gros, J.; Nabi, D.; Würz, B.; Wick, L. Y.; Brussaard, C. P. D.; Huisman, J.; van der Meer, J. R.; Reddy, C. M.; Arey, J. S. First Day of an Oil Spill on the Open Sea: Early Mass Transfers of Hydrocarbons to Air and Water. Environ. Sci. Technol. 2014, 48 (16), 9400-9411; DOI 10.1021/es502437es. (27) Dechesne, A.; Badawi, N.; Aamand, J.; Smets, B. F. Fine scale spatial variability of microbial pesticide degradation in soil: scales, controlling factors, and implications. Frontiers in Microbiology. 2014, 5 (667), DOI 10.3389/fmicb.2014.00667s. (28) Harms, H.; Schlosser, D.; Wick, L. Y. Untapped potential: exploiting fungi in bioremediation of hazardous chemicals. Nat. Rev. Microbiol. 2011, 9 (3), 177-192; DOI 10.1038/Nrmicro2519s. (29) Furuno, S.; Pazolt, K.; Rabe, C.; Neu, T. R.; Harms, H.; Wick, L. Y. Fungal mycelia allow chemotactic dispersal of polycyclic aromatic hydrocarbon-degrading bacteria in water-unsaturated systems. Environ Microbiol. 2010, 12 (6), 1391-1398; DOI 10.1111/j.1462-2920.2009.02022.xs.

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(30) Banitz, T.; Wick, L. Y.; Fetzer, I.; Frank, K.; Harms, H.; Johst, K. Dispersal networks for enhancing bacterial degradation in heterogeneous environments. Environ. Pollut. 2011, 159 (10), 2781-2788; DOI 10.1016/j.envpol.2011.05.008s. (31) Banitz, T.; Fetzer, I.; Johst, K.; Wick, L. Y.; Harms, H.; Frank, K. Assessing biodegradation benefits from dispersal networks. Ecol. Model. 2011, 222 (14), 25522560; DOI 10.1016/j.ecolmodel.2010.07.005s. (32) Banitz, T.; Johst, K.; Wick, L. Y.; Fetzer, I.; Harms, H.; Frank, K. The Relevance of Conditional Dispersal for Bacterial Colony Growth and Biodegradation. Microb. Ecol. 2011, DOI 10.1007/s00248-011-9927-3s. (33) Harshey, R. M. Bacterial motility on a surface: Many ways to a common goal. Annu. Rev. Microbiol. 2003, 57, 249-273; DOI 10.1146/annurev.micro.57.030502.091014s. (34) Dechesne, A.; Or, D.; Smets, B. F. Limited diffusive fluxes of substrate facilitate coexistence of two competing bacterial strains. FEMS Microbiol. Ecol. 2008, 64 (1), 18; DOI 10.1111/j.1574-6941.2008.00446.xs. (35) Liu, J.; Liu, S.; Sun, K.; Sheng, Y. H.; Gu, Y. J.; Gao, Y. Z. Colonization on Root Surface by a Phenanthrene-Degrading Endophytic Bacterium and Its Application for Reducing Plant Phenanthrene Contamination. Plos One. 2014, 9 (9), e108249; DOI 10.1371/journal.pone.0108249s. (36) Ortega-Calvo, J. J.; Tejeda-Agredano, M. C.; Jimenez-Sanchez, C.; Congiu, E.; Sungthong, R.; Niqui-Arroyo, J. L.; Cantos, M. Is it possible to increase bioavailability but not environmental risk of PAHs in bioremediation? J. Hazard. Mater. 2013, 261, 733-745; DOI 10.1016/j.jhazmat.2013.03.042s. (37) Oehl, F.; Sieverding, E.; Mader, P.; Dubois, D.; Ineichen, K.; Boller, T.; Wiemken, A. Impact of long-term conventional and organic farming on the diversity of arbuscular mycorrhizal fungi. Oecologia. 2004, 138 (4), 574-583; DOI 10.1007/s00442-003-1458-2s. (38) Gosling, P.; Hodge, A.; Goodlass, G.; Bending, G. D. Arbuscular mycorrhizal fungi and organic farming. Agric. Ecosyst. Environ. 2006, 113 (1-4), 17-35; 10.1016/j.agee.2005.09.009s. (39) Serrano-Silva, N.; Sarria-Guzman, Y.; Dendooven, L.; Luna-Guido, M. Methanogenesis and Methanotrophy in Soil: A Review. Pedosphere. 2014, 24 (3), 291-307. (40) Nazaries, L.; Murrell, J. C.; Millard, P.; Baggs, L.; Singh, B. K. Methane, microbes and models: fundamental understanding of the soil methane cycle for future predictions. Environ Microbiol. 2013, 15 (9), 2395-2417; 10.1111/1462-2920.12149s.

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FIGURE LEGENDS

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Figure 1: Experimental setup used to assess effects of bacterial dispersal on PHE

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degradation and outgassing. A-D denote surface zones that were separately analyzed for

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bacterial biomass distribution. White lines represent the glass fibers used as model

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dispersal networks and red cylinders represent PDMS pieces used as passive dosing

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systems for continuous PHE release. An adjacent agar cube served as passive sampler for

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PHE outgassing from the vat.

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Figure 2: Calculated coverage of the agar surface of microcosms by P. fluorescens LP6a

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bacteria in % (A, B, C, D) in the absence (left) and presence (right) of glass fiber

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dispersal networks. Hundred % indicates nominal full coverage with a monolayer of

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bacteria. Values are expressed by shades of grey. Data represent averages of triplicate

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experiments. The mean measured cell concentration at a zone of interest in presence of

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glass fibers showed a statistically significant difference from the system without glass

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fibers (*) at a p-value of 0.05.

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Figure 3: PHE concentration in the PDMS in the absence and presence of glass fibers.

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Data represent averages and standard deviations of triplicate experiments for the setup

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with and without glass fibers. The abiotic control was performed in two independent

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triplicate experiments. The mean measured PHE concentration in the PDMS in presence

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of glass fibers shows a statistically significant difference from the system without glass

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fibers (a) at a p-value of 0.05.

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Figure 4: PHE concentration in the agar cube next to the glass vat from microcosms with

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PDMS containing a low PHE load (Fig 4A; system incubated for 120 h) or a high PHE

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load (Fig 4B; system incubated for 168 h). Bars represent the abiotic control (dark grey),

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the setup without glass fibers (grey) and the setup with glass fibers (grey with hatching).

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Solid line depicts calculated PHE concentrations at equilibrium in the abiotic system,

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with the dashed lines showing the 95 % confidence intervals. Data represent averages and

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standard deviations of triplicate experiments. The mean measured concentration of PHE

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in the agar cube shows statistically significant difference from the abiotic control (a) and

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a statistically significant difference from the system without glass fibers (b) at a p-value

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of 0.05.

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Figure 5: PHE concentration in the agar cube next to the vat (top) and in the PDMS

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(bottom) with the ‘heterogeneous’, ‘homogeneous’ and ‘glass fibers’ distribution regimes

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of P. fluorescens LP6a biomass after 72 h of incubation, respectively. The solid line

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depicts the mean PHE concentration of the abiotic microcosms. The dashed line depicts

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calculated PHE concentrations at equilibrium in the agar cube in the abiotic system (cf.

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equation 2; calculation of the partitioning of PHE). The mean measured concentration of

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PHE in the agar cube under different distribution regimes shows a statistically significant

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difference from the abiotic control (a) and a statistically significant difference from the

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system with a heterogeneous bacterial distribution (b) at a p-value of 0.05, respectively.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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