Laboratory of Microbial Ecology and Technology (LabMET)
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Transcript of Laboratory of Microbial Ecology and Technology (LabMET)
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Laboratory of Microbial Ecology and Technology (LabMET)
Research topics and expertise
Tom Van de Wiele, PhD
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Strategic Research
Microbial communities Quorum sensing Electron shuttling Horizontal gene transfer Metabolomics
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Applied Research
Environmental Microbiology Wastewater
Microbial fuel cells Biodegradation Anaerobic treatment Nitrogen removal strategies Minimizing wast sludge
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Applied Research
Environmental Microbiology Soil / Sediments
Bioprecipitation of catalytic particles Anaerobic removal of organochlorine contaminants
Soil and river sludge clean-up Pesticide degradation and ecotoxicology
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Applied Research
Environmental Microbiology Solid Wastes
Solid waste treatment De-icing
Buildings and structures Biologically mediated CaCO3 formation Microbial induced corrosion
Air Indoor air pollution Biotrickling filtration
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Applied Research
Gastrointestinal Microbiology Functional foods
Pro- / pre- / synbiotics Bioactivation of food components Rumen microbiology
Risk assessment Environmental contaminants Toxic food processing metabolites
Phage therapy
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Applied Research
Foodchain Microbiology Drinking water
Hygienisation Water recycling Pathogen abatement
Aquaculture systems Habitat research
Epiphytes on grain Space station life cycles Deep sea methane oxidation
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Tools and instruments
Reactor Technology Microbial Fuel cells Activated sludge systems Upflow Anaerobic Sludge Bed reactors Membrane reactors Rotating disc reactors Simulator of the Human Intestinal Microbial Ecosystem (SHIME)
Dialysis reactors
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Tools and instruments
Molecular Analysis PCR DGGE FISH Realtime PCR Cloning Flow Cytometry
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Tools and instruments
Microbial analysis Epifluorescence and light microscopy Growth kinetics Microbial isolations and enrichments Metabolic activity Bioassays Biodegradation assays
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Tools and instruments
Physico – chemical analysis Gas chromatography HPLC Ion chromatography Spectrophotometry Atom absorption BOD, COD, TSS, VSS, NOX, TOC,…
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Gastrointestinal microbial ecology
Microbial biotransformation of
environmental and food compounds
in the gut and the consequences for biological activity assessment
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Colonmicrobiota and health
Colon ascendens, colon transversum, colon descendens
Water- and salt resorption Microbiota
500 species, 1014 CFU/mL ± stabile community
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Colonmicrobiota and health
Further digestion
Production of SCFA as energy source for colonocytes
Immunostimulation
Production of vitamins (K en B12)
Colonization resistance against pathogens
Formation of health-promoting components from food
Health effects:Health effects:
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Colon microbiota and health
Health effects:Health effects:
Colonization by pathogens Formation of toxins Putrefaction Formation of (geno-)toxic
compounds from food (contaminants) Recent (!): microbiota stimulate
fat uptake and synthesis
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Colon microbiota and health
A microbial community in balance
BacteroidesBacteroides
EubacteriumEubacterium
BifidobacteriumBifidobacterium
AnaërobeAnaërobe GG++ CocciCocci
ClostridiumClostridium
LactobacillusLactobacillus
MethanogenenMethanogenen
EscherichiaEscherichia colicoli
SulfaatreduceerdersSulfaatreduceerders FusobacteriumFusobacterium
EnterobacteriaceaeEnterobacteriaceae
ProteusProteus
P.aeroginosaP.aeroginosa
VeillonellaVeillonella
StaphylococcusStaphylococcus
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BacteroidesBacteroides
EubacteriumEubacterium
BifidobacteriumBifidobacterium
AnaërobeAnaërobe GG++ CocciCocci
ClostridiumClostridium
LactobacillusLactobacillus
MethanogenenMethanogenen
EscherichiaEscherichia colicoli
SulfaatreduceerdersSulfaatreduceerders FusobacteriumFusobacterium
EnterobacteriaceaeEnterobacteriaceae
ProteusProteus
P.aeroginosaP.aeroginosa
VeillonellaVeillonella
StaphylococcusStaphylococcus
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(Gibson & Robertfroid ,1995)(Gibson & Robertfroid ,1995)
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SHIME-Tec: gastrointestinal in vitro technology
Simulator of the Human Intestinal Microbial Ecosystem
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Oral exposure to bioactive compounds
Food: Soy and hop isoflavones Heterocyclic aromatic amines from grilled meat
... Environment:
Soil ingestion Inhalation of dust and subsequent ingestion
...
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Case 1. Microbial conversion of phytoestrogens Soy phytoestrogens:
Daidzine daidzein equol (microbial action)
Equol has beneficial health effects
Microbial consortium applicable as probiotic
K. Decroos et al. (2005)
Hop phytoestrogens: Isoxanthohumol hoppein (8-prenylnaringenin or 8-PN)
Carried out by colon microbiota Importance for hop supplements, beer industry…
S. Possemiers et al. (2006)
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Hop phytoestrogens
Beer: Isoxanthohumol: mg/L range 8-prenylnaringenin: traces
Menohop: food supplement for relief of menopausal symptoms 100 g 8-PN / d (1 tablet / d) IX: mg/L range
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8-PN production in vitro
• isoxanthohumol addition to SHIME
• no conversion in ascending colon
• conversion in transverse and especially descending colon
• estrogenic activity as observed with estrogen bioassay
• moderate in transverse colon
• high in descending colon
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In vitro - in vivo comparison
• SHIME run with fecal microbiota from
• woman A: high 8-PN producer
• woman B: moderate 8-PN producer
• woman C: low 8-PN producer
• In vivo: urinary excretion of 8-PN corresponds to in vitro incubation
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Biological activity assessment
Uptake of food supplement: IX intake of 1 mg/d 8-PN producing intestinal microbiota:
8-PN exposure of more than 500 g/d Range of biological activity
Risk assessment process needs to incorporate bioactivation by intestinal bacteria
Complexity: interindividual variability
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Case 2. Oral exposure to PAHPolycyclic Aromatic Hydrocarbons
Ingestion of contaminated soil Industrial and urban areas Atmospheric deposition of PAH: 50 g.ha-1.yr-1
Oral uptake Adults: 50 mg.d-1
Children: 200 mg.d-1
Occasionally: 1-20 g.d-1
HUMAN HEALTH RISK ASSESSMENT Focus on intestinal absorption and bioactivation by human enzymes
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Current knowledge on PAH bioactivation
1. PAH release from
soil / nutrition
2. Intestinal absorption
Intestine or liver cells
3. Gene expression
Cytoplasm AhR
Nucleus
mRNA
Arnt
Translate proteins
DRE
4. Possible bioactivation to toxic compounds
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What happens to non-absorbed PAHs ?
Are colon microbiota capable of biotransforming PAHs?
Are microbial PAH metabolites bioactive?
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Experimental set-up
Incubate PAH in samples from SHIME reactor
Screen for PAH metabolites Estrogen receptor bioassay: estrogenicity
LC-ESI-MS: hydroxy-PAH Pure PAH compounds PAH contaminated soil samples
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Yeast Estrogen test
Human estrogen receptor in yeast cell Estrogen responsive elements in plasmid Reporter gene lacZ
Nucleus
Estrogen
plasmid
hER
PGKpromoter
ERE
Lac-Z
CPRGYellow
CPRGRed
-Galactosidase
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SHIME: colon microbiota activate PAHs
0,00
0,50
1,00
1,50
2,00
2,50
3,00
naphthalene phenanthrene pyrene benzo(a)pyrene
nM
EE
2 e
qu
iva
len
ce
Stomach Small intestine Colon Inactivated colon
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Chemical analysis
LC-ESI-MS: hydroxylation of PAHs 1-OH pyrene: 4.3 µg/L 7-OH B(a)P: 1.9 µg/L
EE2 7-OH B(a)P
OH
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Urban playground soil sample: 50 ppm PAH
0
5
10
15
20
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stomach small intestine colon
µg
PA
H/L
rel
ease
d
% E
E2
equ
ival
ence
PAH release estrogenicity
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Biological activity assessment
PAH exposure Adult: 5 g PAH/d Child:50 g PAH/d
Colon microbiota convert PAH to pseudo-estrogenic metabolites
Hydroxylation under anaerobic conditions? Enterococcus faecalis Mucosa associated bacteria: micro-aerophilic conditions
Relevant biological activity in vivo ?
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Chemopreventive effect from prebiotics Prebiotic inulin: add to SHIME reactor Evaluate inulin as chemopreventive agent Start-up, inulin treatment (2.5 g/d) Incubate SHIME suspension with 40 µM B(a)P Monitor PAH bioactivation with yeast estrogen bioassay
Relate to prebiotic effects Metabolic analysis PCR-DGGE-sequencing Real-time PCR quantification Bifidobacterium sp.
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SCFA: colon ascendens
26% increase ** Towards propionic and butyric acid
Reversible effect
Start-up
Treat-ment
Con-trol
% AA 57 37 48
% PA 19 33 19
% BA 21 27 29 Acetic acid
Propionic acid
Butyric acid
Other acids
Total SCFA
0
10
20
30
40
50
60
µm
ol/g
Start-up Treatment Control
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Pearson correlation [0.0%-100.0%]
100
500
Case-study: inuline (Resultaten)
1. Bifidobacterium sp.
Start-up and control samples
Inulin treatment samples
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INULINE: stimulatie van de INULINE: stimulatie van de BIFIDOBACTERIABIFIDOBACTERIA
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3. Bifidobacterium longum (95% sim.)
2. Bifidobacterium infantis (96% sim.)
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PCR-DGGE van PCR-DGGE van bifidobacteriabifidobacteria
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Take home messages
Metabolic potency from gut microbiota Higher than currently anticipated Consider this process for risk assessment Interindividual variability ! Identification of responsible bacteria and process conditions needed
Modulation of biological activation through dietary factors, microbial community composition...
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Contact information
LabMET – Ghent UniversityCoupure Links 653B-9000 Gent
http://labMET.ugent.be/http://www.shimetec.be
+32/9/264.59.76