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Laboratory of Microbial Ecology and Technology (LabMET)

Research topics and expertise

Tom Van de Wiele, PhD

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Mission

Microbial Ecology

Strategic research

Applied research

Technology

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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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Twin SHIME : parallel treatment and control

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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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Large interindividual variability

Batch incubation of IX with fecal microbiota from 51 women

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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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Ascending colon: inhibitory effect

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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