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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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
Health effects

• 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

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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

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Colon microbiota and health

• A microbial community in balance

(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 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

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Current knowledge on PAH bioactivation
3. Gene expression
1. PAH release from soil / nutrition
2. Intestinal absorption
Intestine or liver cells
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

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SHIME colon microbiota activate PAHs
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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

OH
EE2 7-OH B(a)P
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Urban playground soil sample 50 ppm PAH
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Biological activity assessment

• PAH exposure
• Adult 5 ?g PAH/d Child50 ?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
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Case-study inuline (Resultaten)

• PCR-DGGE van bifidobacteria

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1
3
1. Bifidobacterium sp.
2. Bifidobacterium infantis (96 sim.)
Inulin treatment samples
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