A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Summary

A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

Abstract

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.

Introduction

Soil is densely populated by a wide range of microorganisms^1,2 such as bacteria. However, conditions in this habitat are challenging, especially in terms of water availability³. Bacteria permanently need to search for optimal conditions in heterogeneous environments⁴, but the absence of continuous water films is resulting in restricted mobility⁵ hindering them to spread freely. Also, diffusion rates of solutes (e.g., nutrients) are lowered under unsaturated conditions⁶. Thus, bacteria and nutrients are often physically separated and nutrient accessibility is limited³. As a consequence, a transp....

Protocol

1. Preparation of Dishes, Slides and Incubation Chambers

1. Prepare the following material for each microcosm: one big plastic Petri dish bottom (d = 10 cm), one modified (see step 1.2) small plastic Petri dish bottom (d = 5 cm) with lids and one counting chamber slide with three cavities.
2. Take the desired number of Petri dish bottom parts (d = 5 cm). Remove part of the brim with a saw to exactly fit a slide (26 mm edge length). To sterilize the system, soak Petri dish bottoms and lids in 70% ethanol O/N and dry them for at least 2 hr in a flow cabinet under UV-light. Store the Petri dishes in a tightly sealed plastic container, which had also been ....

Results

The results presented here have already been published earlier¹⁵. Please refer to the article for detailed mechanistic and environmental discussion.

After image recording via CLSM, a maximum intensity projection can be conducted using the respective microscope software or ImageJ to gain a first visual impression of the sample and the controls (Figure 2). Later, the data sets may be projected differently in order to show meaningful features by specific visualization .......

Discussion

The presented microcosm setup proved suitable to study bioavailability of spatially separated chemicals to degrading organisms after uptake and transport by mycelia. Potential gas-phase transport of partially volatile compounds is prevented and bacterial bioreporter cells can be visualized without elaborate sample preparation and thus with minimal disturbance of the sensitive system. At the same time, chemical analysis of the sample can be easily conducted allowing for a good control of the gained results and for quantif.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Confocal Microscope	Leica		TCS SP5X, LAS AF - Version 2.6.1; or equivalent CLSM
GC HP 7890 Series GC and Agilent 5975C MSD	Agilent		an equivalent GC/MS may be used
GC capillary column J&W 121-5522	Agilent
Cork borer	Fisher Scientific	12863952	or any other
Cover slips	Marienfeld	107222	High performance, No.1.5H
GC/MS insterts	WICOM	WIC 47080
GC/MS vials 2 ml	WICOM	WIC 41150
Lids / septa for screw cap vials	DIONEX	49463 / 049464
Lids for GC/MS vials	WICOM	WIC 43948/B
Objective Slides	Menzel		ordinary
PDMS coated glass fibers	Polymicro Technologies, Inc.		V (PDMS) = 13.55 ± 0.02 µl m-1
Petri Dishes small / big	Greiner	633-102 / 628-102
Screw cap vials 40 ml	DIONEX	48783	other glass vials may be used
Screw cap vials 60 ml	DIONEX	48784	other glass vials may be used
Acenaphthylene d08	Dr. Ehrenstorfer	C 20510100
Acetone	Carl Roth	9372.2
Activated carbon	Sigma-Aldrich	242276-1kg
Agarose	Carl Roth	2267.4
Fluorene	Fluka	46880
Kanamycin sulfate	Carl Roth	T832.2	50 mg L-1
Methanol	Carl Roth	P7171
Minimal Medium: 100 ml Solution 1 + 25 ml Solution 2 + 5 ml Solution 3 ad. 1,000 ml aqua dest
Solution 1
Ammonium sulfate	Carl Roth	3746.1	5 g L-1
Magnesium chloride x 6 H2O	Carl Roth	2189.1	1 g L-1
Calcium nitrate x 4 H2O	Carl Roth	P740.1	0.5 g L-1
Solution 2
Disodium phosphate	Carl Roth	P030.1	55.83 g L-1
Monopotassium phosphate	Carl Roth	3904.1	20 g L-1
Solution 3 (pH 6.0)
Disodium EDTA	MERCK	1084180250	0.8 g L-1
Iron(II) chloride x 4 H2O	MERCK	1038610250	0.3 g L-1
Cobalt(II) chloride x 6 H2O	Carl Roth	T889.3	4 mg L-1
Manganese(II) chloride x 1 H2O	Carl Roth	4320.2	10 mg L-1
Copper(II) sulfate	Carl Roth	P023.1	1 mg L-1
Sodium molybdate x 2 H2O	Carl Roth	0274.1	3 mg L-1
Zinc chloride	MERCK	1088160250	2 mg L-1
Lithium chloride	Carl Roth	P007.1	0.5 mg L-1
Tin(II) chloride x 2 H2O	Carl Roth	4473.1	0.5 mg L-1
Boric acid	Riedel-de-Haen	11606	1 mg L-1
Potassium bromide	Carl Roth	A137.1	2 mg L-1
Potassium iodide	Carl Roth	6750.1	2 mg L-1
Barium chloride	Carl Roth	4453.1	0.5 mg L-1
MMA			Minimal medium + agarose 0.2%
Phenanthrene d10	Dr. Ehrenstorfer	C 20920100
Potato Dextrose Agar: 24 g L-1 broth + bacto-agar 1.5%; pH 6.8
Potato Dextrose broth	Difco/ Beckton Dickinson	254920
Bacto-agar	Difco/ Beckton Dickinson	214040
Sodium acetate x 3 hydr.	Carl Roth	6779.1
Sodium sulfate	MERCK	1066495000
Toluene	MERCK	1083252500
mTY medium: 3 g L-1 yeast extract, 5 g L-1 bacto tryptone and 50 mM NaCl
Yeast extract	Merck	1037530500
Tryptone	Serva	4864702
Sodium chloride	Carl Roth	3957.1
ImageJ with logi tool plugin			http://rsb.info.nih.gov/ij/download.html and http://downloads.openmicroscopy.org/bio-formats/4.4.10
Pythium ultimum strain 67-1			Obtained from the lab of Dr. Christoph Keel; Department of Fundamental Microbiology, University of Lausanne, Switzerland
Burkholderia sartisoli RP037-mChe			Obtained from the lab of Prof. Jan Roelof van der Meer; Department of Fundamental Microbiology, University of Lausanne, Switzerland