Effects of Facilitated Bacterial Dispersal on the Degradation and

May 19, 2016 - *Phone: +49 341 235 1316; fax: +49 341 235 451316; e-mail: [email protected]. ... in (i) increased bacterial surface coverage, (ii) eff...
0 downloads 3 Views 771KB Size
Subscriber access provided by UCL Library Services

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25

Environmental Science & Technology

1

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

2

desorbing contaminant

3 4

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

5 6 7

1

8

Microbiology, Permoserstr. 15, 04318 Leipzig, Germany

9

2

UFZ - Helmholtz Centre for Environmental Research, Department of Environmental

UFZ - Helmholtz Centre for Environmental Research, Department of Ecological

10

Modelling, Permoserstr. 15, 04318 Leipzig, Germany

11

3

12

Deutscher Platz 5e, 04103 Leipzig

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

13 14

Running title: Effects of facilitated bacterial dispersal

15

Intended for: Environmental Science and Technology

16 17 18

*

19

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

20

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

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

21

ACS Paragon Plus Environment

1

Environmental Science & Technology

22

Page 2 of 25

ABSTRACT ART

23

24

ACS Paragon Plus Environment

2

Page 3 of 25

Environmental Science & Technology

25

ABSTRACT

26

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

27

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

28

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

29

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

30

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

31

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

32

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

33

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

34

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

35

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

36

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

37

dispersal networks such as mycelia promote the optimized spatial arrangement of

38

microbial populations to allow for effective contaminant degradation and reduction of

39

potential hazard to organisms beyond a contaminated zone.

40 41 42 43

KEYWORDS: Bacterial dispersal network, biodegradation, Pseudomonas fluorescens

44

LP6a, PAH, phenanthrene, passive dosing

ACS Paragon Plus Environment

3

Environmental Science & Technology

Page 4 of 25

45

INTRODUCTION

46

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

47

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

48

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

49

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

50

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

51

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

52

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

53

quantitative relationship between the availability for biological removal and ecotoxicity is

54

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

55

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

56

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

57

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

58

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

59

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

60

organisms, this may cause toxic effects8,

61

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

62

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

63

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

64

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

65

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

66

requires a homogeneous distribution of degrading bacteria.

67

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

68

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

69

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

70

bacterial mobility patchy distribution of catabolically active microbial biomass may occur

9

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

ACS Paragon Plus Environment

4

Page 5 of 25

Environmental Science & Technology

71

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

72

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

73

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

74

bacterial dispersal by providing continuous paths for bacterial motility even through

75

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

76

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

77

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

78

network thus would facilitate the formation of homogenously distributed degrader

79

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

80

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

81

(suitably mimicking dispersal along a fungal mycelium15,

82

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

83

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

84

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

85

bacterial distribution on PHE gas phase emission.

19

) on biodegradation of

86 87

MATERIAL AND METHODS

88

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

89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

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

5

Environmental Science & Technology

Page 6 of 25

97

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

98

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

99

microbeads were performed in triplicates.

100

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

101

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

102

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

103

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

104

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

105

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

106

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

107

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

108

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

109

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

110

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

111

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

112

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

113

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

114

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

115

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

116

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

117

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

118

to a final volume of 500 mL.

119

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

120

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

121

calculated using the dimensionless partitioning ratio:

ACS Paragon Plus Environment

6

Page 7 of 25

122

Environmental Science & Technology

: =

()

(1)

()

123

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

124

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

125

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

126

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

127

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

128

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

129

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

#$% =

1 ( ( 1 + " : ∙ ( " + : ∙ (  

(" (= 120 *+)

,

∙ 100 (2)

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

130

Here,

131

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

132

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

133

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

134

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

135

Degradation and dispersal experiments. Degradation and dispersal experiments were

136

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

137

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

ACS Paragon Plus Environment

,

and

7

Environmental Science & Technology

Page 8 of 25

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

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

153

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

154

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

155

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

156

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

157

without bacteria were used in two independent control experiments.

158

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

159

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

160

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

161

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

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

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

ACS Paragon Plus Environment

8

Page 9 of 25

Environmental Science & Technology

170

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

171

extracted and analyzed.

172

Bacterial surface coverage experiments. To estimate effects of bacterial surface

173

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

174

concentrations

175

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

176

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

177

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

178

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

179

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

180

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

181

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

182

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

183

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

184

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

185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

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

193

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

194

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

195

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

were

distributed

on

agar

containing

ACS Paragon Plus Environment

PHE

loaded

PDMS

9

Environmental Science & Technology

Page 10 of 25

196

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

197

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

198

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

199

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

200

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

201

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

202

elsewhere26.

203

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

204

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

205

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

206 207

RESULTS

208

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

209

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

210

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

211

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

212

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

213

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

214

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

215

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

216

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

217

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

218

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

219

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

220

Influence of bacterial distribution on PHE degradation. Significantly higher PHE

221

removal from the PDMS pieces was detected in the presence of glass fibers (45 %) than ACS Paragon Plus Environment

10

Page 11 of 25

Environmental Science & Technology

222

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

223

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

224

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

225

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

226

system; SI, Fig. S2).

227

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

228

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

229

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

230

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

231

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

232

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

233

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

234

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

235

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

236

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

237

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

238

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

239

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

240

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

241

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

242

bacterial surface coverage experiments nine regimes differing in their biomass

243

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

244

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

245

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

246

Heterogeneous inoculation reduced PHE outgassing marginally irrespective of the

247

amount of inoculated biomass (Fig. 5 top). In contrast, when the bacteria were ACS Paragon Plus Environment

11

Environmental Science & Technology

Page 12 of 25

248

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

249

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

250

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

251

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

252

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

253

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

254

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

255

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

256

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

257 258

DISCUSSION

259

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

260

demonstrates that facilitated dispersal and optimized spatial arrangement of microbial

261

populations enhances the degradation of constantly desorbing PHE. Efficient

262

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

263

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

264

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

265

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

266

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

267

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

268

degradation performances of differently distributed bacterial inocula despite identical

269

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

270

biomass exerted substantially different pressures on the partition equilibrium of the

271

chemical; whereby a more homogeneously distributed biomass was driving PHE

272

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

273

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

27

. The PDMS-

12

Page 13 of 25

Environmental Science & Technology

274

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

275

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

276

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

277

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

278

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

279

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

280

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

281

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

282

modelling data16,

283

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

284

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

285

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

286

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

287

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

288

higher biomass concentration behind the point of inoculation developing in microcosms

289

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

290

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

291

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

292

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

293

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

294

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

295

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

296

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

297

motility or flagellar synthesis 32, 33-

30, 31

, showing that network-based bacterial dispersal improves the

298

ACS Paragon Plus Environment

13

Environmental Science & Technology

Page 14 of 25

299

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

300

to consider bioavailability within their retrospective risk assessment and remediation

301

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

302

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

303

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

304

interactions between microbial dispersal, biodegradation and exposure of contaminants is

305

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

306

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

307

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

308

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

309

degrader biomass reflects the supply and transport characteristics of the degraded

310

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

311

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

312

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

313

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

314

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

315

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

316

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

317

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

318

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

319

from soil and sediments39, 40.

320 321

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

322

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

323

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

324

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

14

Page 15 of 25

Environmental Science & Technology

325

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

326

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

327

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

328 329

Supporting Information Available

330

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

ACS Paragon Plus Environment

15

Environmental Science & Technology

Page 16 of 25

331

REFERENCES

332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379

(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.

ACS Paragon Plus Environment

16

Page 17 of 25

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

Environmental Science & Technology

(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.

ACS Paragon Plus Environment

17

Environmental Science & Technology

429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464

Page 18 of 25

(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.

ACS Paragon Plus Environment

18

Page 19 of 25

Environmental Science & Technology

465

FIGURE LEGENDS

466

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

467

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

468

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

469

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

470

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

471

PHE outgassing from the vat.

472

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

473

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

474

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

475

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

476

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

477

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

478

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

479

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

480

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

481

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

482

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

483

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

484

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

485

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

486

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

487

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

488

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

489

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

ACS Paragon Plus Environment

19

Environmental Science & Technology

Page 20 of 25

490

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

491

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

492

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

493

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

494

of 0.05.

495

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

496

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

497

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

498

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

499

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

500

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

501

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

502

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

503

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

ACS Paragon Plus Environment

20

Page 21 of 25

504

Environmental Science & Technology

FIGURES

505 506

Figure 1

ACS Paragon Plus Environment

21

Environmental Science & Technology

Page 22 of 25

507

508

Figure 2

ACS Paragon Plus Environment

22

Page 23 of 25

Environmental Science & Technology

509 510

Figure 3

ACS Paragon Plus Environment

23

Environmental Science & Technology

Page 24 of 25

511 512

Figure 4

ACS Paragon Plus Environment

24

Page 25 of 25

Environmental Science & Technology

513 514

Figure 5

515

ACS Paragon Plus Environment

25