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JEPPE LUND NIELSEN
CENTER FOR MICROBIAL COMMUNITIESDEPARTMENT OF BIOTECHNOLOGY, CHEMISTRY AND ENVIRONMENTAL ENGINEERING
AALBORG UNIVERSITY
SEARCHING FOR NEW FUNCTIONAL GENES
INVOLVED IN DEGRADATION OF
MICROPOLLUTANTS
Aalborg West WWTP
0
5
10
15
20
25
30
35
1996 2002 2010
EnergyconsumptionEnergyproduction
Mio
kW
h
100th anniversary…
Future challenges: • High effluent water quality (micropollutants)• Reliable stable plants• Low energy and ressource consumption• Utilization of valuable materials
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
First Pasveersloot ditch built 1953 (Dreisschor, NL)
Ecosystem Design
Improve xenobiotic
degradation
”Marker genes”
Assess potential in
pilot/full scale systems
Improved degradation of micropollutants
Identifydegraders
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Aalborg East
Aabybro
HirtshalsAalborg West
Triclosanremoval
hrs
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Plant design and operation predicts removal and number of cells involved…
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Enumerations by FISH and relative abundance by amplicon sequences
Ecophysiology by single cellActivity measures,
i.e. MAR-FISH, SIP and various staining
techniques
Core groups found in 50Danishtreatmentplants ?
Identifying the “tourists”
Combined with FISH/Card-FISH
QuantitativeMAR
MAR-positive
EE2 utilising cells EUBmix
Estimated biovolume fraction
involved in
EE2-degradation ~ 0,1 ‰ (~105 cells/ml)
Uptake of of 3H-EE2
Principle of MAR-FISH
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Uptake of of 3H-EE2
IsolationDNA
extractionSequencing +
Assembly
Genome
Gene sequence acquisition in the genomic era
Total DNA extraction
Sequencing + Assembly
Assembly of individualgenomes
IdentityMetabolic pathways
(Functional potential / Blueprint)Genome
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Complex community
Genomes
The pre-genome era as the ‘dark ages’ (Ian Dunham, )
First 1000 microbial genomes comprise ca. 3,3mio genes
Transitive catastropheWhen a mistake is inheritedfrom gene to gene
COMBREX (COMputational Bridges to EXperiments; http://combrex.bu.edu/)
Abundance of mRNA and proteins in S. cerevisiea (Futcher et al., 1999)
mRNA content in a cell ~42,000 (S. cerevisiea) and many are non-coding! Prokaryotes has less…
(Aebersold et al., 2013)
Proteins carry out the job…
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Quantitative Proteomics pipeline
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Quantification of proteins combined with metabolomics
(+/- micropollutant) provide insight into the proteins
affected by the micropollutant!
Full or partial biochemical pathway established for degradation of: • p-cresol (effect on nitrifiers) • Gemfibrozil• Ibuprofen• Estradiol• Bisphenol A• Triclosan• Chrome
Ibuprofen
Extracellular solute binding protein family 1
Cell Membrane
Cytosol
ABC 2 Type transporter
A B C
A - AMP-dependent synthase and ligase
B – Dioxygenase containing Rieske (2Fe-2S) iron sulphur domain
C - Enoyl-CoA hydratase/isomerase
Established model of Ibuprofen degradation
Proposed pathway for ibuprofen degradation
D E
D & E in same operon?
MAR-FISH to confirmUptake of 14C-Ibuprofen
qPCR for quantification of Ibuprofen-degrading genesand their expression
Better removal of micropollutants by introducing a polishing of effluent
BIOSEP™ MBR
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Phylogeny to describe diversity
Development of microflora in polishing step for complete removal of:
CarbamazepineDiclofenacIbuprofenNaproxen
Triclosan,…
CENTER FOR MICROBIAL COMMUNITIES | AALBORG UNIVERSITY
Thank you for your attention!
and collaborations: K. Bester (AU)H. Gough (U. Washington)M. Von Bergen (Leipzig)