Gilda Carvalho, Cláudia Galinha, Teresa Crespo and Maria Reis Microbial characterisation of activated sludge in membrane bioreactors: a tool for biofouling assessment Gilda Carvalho, Cláudia Galinha, Teresa Crespo and Maria Reis

Microbiology as an ally for engineering Wastewater treatment systems: complex, mixed biological systems Microbial characterisation helps understanding: Microbial community and state Interactions between different microoorganisms Operational conditions for the consortia to fullfil objectives of the process Interactions and competition Process optimisation

Membrane bioreactors and biofouling Fouling caused by biological activity EPS Type of microorganisms enriched in the system Physiological state of the microorganisms Level of fouling caused by EPS

Classical and modern microbiology Culture-dependent methods Limited to isolated organisms (1%) Molecular biology Fluorescence in situ hybridisation (FISH) Denaturing gradient gel electrophoresis (DGGE) Cloning and sequencing

Fluorescence in situ hybridisation 23S rRNA 16S rRNA

FISH

FISH: hybridisation process

FISH: hybridisation process (cont.)

FISH results All Bacteria (EUB Mix) PAO (PAO Mix)

FISH quantification (with CLSM) FISH quantification with image analysis (30-40 images) Relative abundance Population dynamics

Nutrient removal activated sludge Acetate-SBR Propionate-SBR Before acclim. After acclim. All Bacteria (EUB Mix) PAO (PAO Mix) A B C D 64% 37% 89% 76% 10 mm

Methylene Blue staining (poly-P granules) FISH PAO (PAO Mix) FISH PAO (PAO Mix) 10 mm Methylene Blue staining (poly-P granules) 10 mm 10 mm Cocci 10 mm 10 mm Rods

DGGE Amplified DNA of a mixed culture (16S rDNA) Run in electrophoresis gel with a gradient of denaturant conditions (urea and formamide) Result: each different DNA sequence displays a band Fingerprinting

Biomass collected from a MBR DGGE results SRT 10d SRT 30d Population diversity Compare fingerprints at different reactor operation conditions to detect bands of interest Microbial identification Microbial identification: mention this again later Biomass collected from a MBR Jinsong et al., 2006

Clone library of 16S rDNA Plasmid with antibiotic resistance DNA extraction & purification PCR amplification with primers 27f and 1492r Plasmid with antibiotic resistance Recombinant DNA LIGATION PCR product purification Bacteria selection on X-gal medium + antibiotic Bacteria containing DNA inserts are white CLONE LIBRARY CLONING Competent E. coli cells

PCR amplification with primers SP6 and T7 Sequencing PCR amplification with primers SP6 and T7 Clone SEQUENCING

Sequenced 16S rDNA Sequence analysis: Comparison with published sequences (BLAST) Identify microorganisms present in the mixed culture

Sequenced 16S rDNA (cont.) DGGE bands can be excised and sequenced too Design new FISH probes using the ARB software Biomass DNA extraction, amplification Cloning, sequencing FISH probes

Conclusions FISH: detect and quantify probe-targeted microorganims in mixed cultures Chemical staining: physiological state / specific activity DGGE: diversity and fingerprinting Clone libraries: to separate DNA from mixed cultures Cloned DNA or DGGE bands can be sequenced: microbial identification Improved knowledge on microbial community, interactions/competition and state Correlated with process performance for optimisation of the operational conditions

Process understanding and optimisation FISH MBR process data DGGE Clone library/ sequencing