MICROBIOLOGICAL AND MOLECULAR TOOLS FOR SOIL...
Transcript of MICROBIOLOGICAL AND MOLECULAR TOOLS FOR SOIL...
Kristian K. Brandt, Mette H. Nicolaisen, Ole Nybroe, Jan Sørensen, Chris Rensing Section of Genetics and Microbiology Department of Plant and Environmental Sciences University of Copenhagen
MICROBIOLOGICAL AND MOLECULAR TOOLS FOR
SOIL REMEDIATION
1. Detailed characterization of biodegradation by in situ studies of the abundance, expression, and diversity of key catabolic genes in indigenous bacterial degraders
2. Designed reporter bacteria as toxicity and bioavailability sensors to monitor soil quality and contaminant bioavailability during soil clean-up operations
3. Pollution-induced community tolerance (PICT) and reverse PICT as sensitive and ecologically relevant soil quality indicators
Three proposed concepts and tools for microbe-assisted soil remediation
Case 1
Surveying abundance, expression and diversity of key catabolic genes
Catabolic gene (DNA) → Transcript (mRNA) → Protein (enzyme)
Surveying catabolic genes encoding key enzymes for biodegradation: Proof-of-concept with phenoxy acid model pesticides
2,4-D / MCPA
(R)-dichlorprop
(S)-dichlorprop
Downstream analysis Real-time PCR DGGE DNA sequencing
30 g sieved soil
(R,S)-dichlorprop herbicide
Trace amounts 14C-dichlorprop
NaOH trap
RNA/DNA co-extraction
Scintillation count Microcosms
Experimental set-up
Transcriptional analysis of rdpA ,sdpA and
tfdA genes from indigenous dichlorprop
degrader population
Direct substrate induction of rdpA and sdpA genes Expression of rdpA/sdpA is linked to the mineralization of dichlorprop tfdA genes also induced by dichlorprop
Paulin et al. (2011) Environ Microbiol 13: 1513–1523
Degrader community diversity studied by DGGE
analysis of catabolic genes in soil rdpA
sdpA
tfdA
Paulin et al. (2011) Environ Microbiol 13: 1513–1523
Identification of bacterial degraders based on - Sequencing of catabolic genes - Sequencing of actively
transcribed genes (mRNA)
D. Acidovorans MC1 Variovorax paradoxus S110
Class III
S. herbicidovorans MH
Current activities: Optimization of in situ biodegradation of hydrocarbons using alkB
gene (alkane degradation) as functional gene marker
• Alkane degrader community composition is affected by nutrient addition as determined by the use of alkB gene as functional gene marker
Bioaugmentation of hydrocarbon-
contaminated soils in biopiles
using patented system for
delivery of degrader organisms
(Cleanfield Denmark)
Anja Sandal Grønskov
Case 2
Designed reporter bacteria as toxicity and bioavailability sensors
Whole-cell bacterial biosensors (bioreporters) for quantification of bioavailable toxicants
Response to Cu by two recombinant Pseudomonas fluorescens DF57 biosensor strains
[CuSO4] µM
0.01 0.1 1 10
Bio
lum
inescence
(rela
tive lig
ht units)
0
200
400
600
800
1000
1200
1400
Bio
lum
inescence
(% o
f contr
ol)
0
20
40
60
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120Strain Cu15
Strain 40E7
“lights off”
“lights on”
Analytical range
• A are anions: E.g. carbonate, phosphate, sulfide. • L are ligands: E.g. chloride, citrate, DOC (e.g. root
exudates). • Bio-available fraction: Cu2+ and some CuL ?
Cu2+ Cu CuL (aq)
CuA (s)
sorption
precipitation
complexation
Bioavailable fraction ?
Cu speciation and bioavailability in soil
Particle
Cu-EDTA complexes are NOT bioavailable
Impact of chemical speciation on bioavailability
Cu bioavailability in Cu-EDTA solutions
Nybroe et al. (2008) Environ Toxicol Chem 27: 2246–2252
Current activities: Spatial heterogeneity of bioavailable Cu as determined by bioluminescent bioreporter before and after soil remediation by soil washing technologies
See Brandt et al., 2008 (Environ Sci Technol 42:3102-3108) for details on bioreporter assay methodology.
Case 3
Pollution-induced community tolerance (PICT) and reverse PICT
Experimental field site (Hygum): Long-term (+85 yr) Cu gradient ranging from background Cu levels to high levels of Cu contamination (>3000 ppm)
High Cu soil
Pollution-induced community tolerance (PICT) constitutes an ecologically relevant soil quality indicator
Cu dose−response curves for the nonadapted bacterial community extracted from control soil (triangles) and the corresponding Cu-adapted bacterial community extracted from high-Cu soil (circles) as measured by the leucine incorporation method (left panel, n = 2) and the bromodeoxyuridine incorporation method (right panel, n = 3). Curves represent best fits using nonlinear logistic models.
Berg et al. Environ. Sci. Technol. 2010, 44, 8724-8728. DOI: 10.1021/es101798r Copyright © 2010 American Chemical Society
The pollution-induced community tolerance (PICT) concept: Cu exposure selects for Cu tolerance in soil bacterial communities
OTU, ≥97% similarity level 6538 random 16S rRNA gene sequences per sample
Current activites: • Reverse PICT: Does the bacterial community retain Cu
sensitivity after soil clean-up?
Current activites: • New PICT detection assays for organic priority pollutants and
for toxicant mixtures (metals and hydrocarbons) • Identifying toxic chemicals in field contaminated soils using
biosensors, PICT, comprehensive chemical profiling, and multivariate statistics
• WANTED: Complexly polluted field sites (metals plus hydrocarbons) that includes non-contaminated control soil
Jakub Modrzynski
Conclusions and perspectives
1. Catabolic gene approach holds promise for evaluation and optimization of biodegradation potential (activity, composition, and diversity of specific degrader community)
2. Several whole-cell bacterial biosensors are ready for environmental application (measures bioavailability or toxicity)
3. Pollution-induced community tolerance (PICT) indicates causal relationships between chemical exposure and effect, and reverse PICT holds promise for ecological validation of soil remediation
Pathways by which metal resistant N-fixing rhizobia can act as plant growth promoting bacteria in metal-impacted soils
Hao et al. (in revision) Int. J. Phytorem. Chris Rensing
Thanks for your attention
Villum Foundation
[3H]leucine incorporation assay for phenotypic community-level resistance profiling
DNA
Ribosomes
Synthesis of radiolabelled
proteins containing
incorporated [3H]leucine
Inner membrane Outer
membrane
[3H]leucine incorporation (3 hour incubation)
Protein synthesis
Biomass production
Bacterial growth
[3H]bromodeoxyuridine (BrdU) incorporation assay for phenotypic community-level resistance profiling
DNA
* *
Synthesis of radiolabelled
DNA containing
incorporated [3H]BrdU
Inner membrane Outer
membrane
* *
[3H]BrdU incorporation (3 hour incubation)
DNA synthesis
Cell division
Bacterial growth
Ribosomes
Experimental set-up
Downstream analysis Real-time PCR
RNA/DNA co-extraction
Addition of sorption site
competitor
water
• Frozen in liquid N2
• Freeze dried overnight
100 ppm
1000 ppm
10 000 ppm
Scintillation count Microcosms
Clay content
pH
Phosphate
The pollution-induced community tolerance (PICT) concept constitutes an ecologically relevant soil quality indicator
• PICT detection assays must be short (few hours) to eliminate selection during the PICT detection phase
Figure modified from Demoling & Bååth (2008)