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

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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)