1. 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

2. 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

3. 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

4. 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

5. Fluorescence in situ hybridisation
23S rRNA
16S rRNA

6. FISH

7. FISH: hybridisation process

8. FISH: hybridisation process (cont.)

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

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

11. 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

12. 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

13. 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

14. 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

15. 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

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

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

18. 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

19. 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

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