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 3

Applied Research  Environmental Microbiology  Wastewater  Microbial fuel cells   Biodegradation Anaerobic treatment   Nitrogen removal strategies Minimizing wast sludge 4

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 5

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 6

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 7

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 8

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 9

Tools and instruments  Molecular Analysis       PCR DGGE FISH Realtime PCR Cloning Flow Cytometry 10

Tools and instruments  Microbial analysis       Epifluorescence and light microscopy Growth kinetics Microbial isolations and enrichments Metabolic activity Bioassays Biodegradation assays 11

Tools and instruments  Physico – chemical analysis       Gas chromatography HPLC Ion chromatography Spectrophotometry Atom absorption BOD, COD, TSS, VSS, NOX, TOC,… 12

Gastrointestinal microbial ecology

Microbial biotransformation of environmental and food compounds in the gut and the consequences for biological activity assessment 13

Colonmicrobiota and health    Colon ascendens, colon transversum, colon descendens Water- and salt resorption Microbiota   500 species, 10 14 CFU/mL ± stabile community 14

Colonmicrobiota and health Health effects:       Further digestion Production of SCFA as energy source for colonocytes Immunostimulation Production of vitamins (K en B 12 ) Colonization resistance against pathogens Formation of health-promoting components from food 15

Colon microbiota and health Health effects:      Colonization by pathogens Formation of toxins Putrefaction Formation of (geno-)toxic compounds from food (contaminants) Recent (!): microbiota stimulate fat uptake and synthesis 16

Colon microbiota and health A microbial community in balance

G +

(Gibson & Robertfroid ,1995) 17

SHIME-Tec: gastrointestinal

in vitro

technology Simulator of the Human Intestinal Microbial Ecosystem 18

Twin SHIME : parallel treatment and control 19

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  (microbial action)  equol 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) 21

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 22

Large interindividual variability Batch incubation of IX with fecal microbiota from 51 women 23

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 24

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 25

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 26

Case 2. Oral exposure to PAH  Polycyclic 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 27

Current knowledge on PAH bioactivation

3. Gene expression Cytoplasm AhR 1. PAH release from soil / nutrition 2. Intestinal absorption

Translate proteins

Arnt mRNA Nucleus DRE Intestine or liver cells

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 30

Yeast Estrogen test  Human estrogen receptor in yeast cell  Estrogen responsive elements in plasmid  Reporter gene

lacZ

Estrogen ERE PGK promoter

Lac

-Z plasmid Nucleus hER CPRG Yellow CPRG Red  -Galactosidase 31

SHIME: colon microbiota activate PAHs

Stomach Small intestine Colon Inactivated colon 3,00 2,50 2,00 1,50 1,00 0,50 0,00 naphthalene phenanthrene pyrene benzo(a)pyrene

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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 OH 7-OH B(a)P 33

Urban playground soil sample: 50 ppm PAH

PAH release estrogenicity 25 20 15 10 5 0 stomach small intestine colon

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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 ? 35

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 37

SCFA: colon ascendens    26% increase ** Towards propionic and butyric acid Reversible effect % AA % PA % BA Start up 57 19 21 Treat ment 37 33 27 Con trol 48 19 29

colon ascendens 60 Start-up Treatment Control 50 40 30 20 10 0 Acetic acid Propionic acid yric acid Ot her ac ids To tal S CF A

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Case-study: inuline (Resultaten)  PCR-DGGE van bifidobacteria

3 2 1 Inulin treatment samples 1. Bifidobacterium sp.

2. Bifidobacterium infantis (96% sim.) 3. Bifidobacterium longum (95% sim.)

Start-up and control samples INULINE: stimulatie van de BIFIDOBACTERIA

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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 University Coupure Links 653 B-9000 Gent http://labMET.ugent.be/ http://www.shimetec.be

+32/9/264.59.76

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