Abstract

Filamentous microorganisms cause bulking sludge which results in major problems in industrial waste water treatment plants. However there is a lack of identification tools for these organisms. This project aims to map the filamentous biodiversity through screening a large number (> 100) industrial water systems and developing molecular techniques for identification and quantification. Filaments are micromanipulated from activated sludge and species specific 16S rRNA sequences are derived via PCR or reverse transcriptase PCR. Probes and primers for respectively fluorescent in situ hybridisation, PCR and multi-plex quantitative PCR are designed to monitor filament population composition and dynamics. Filament ecophysiology is studied both in pure culture and in situ using microautoradiography. This study will result in a methodology to study biodiversity and dynamics of filamentous microbial populations in industrial wastewater treatment

Objectives

• Describe the biodiversity of filamentous microorganisms in industrial water systems.
• Determine filament identity with both morphological and 16S RNA phylogenetic analysis; Develop species-specific detection, identifiation and quantification methods for filamentous microorganisms.
• Determine ecophysiological potential and in situ expression of physiological activity of filamentous microorganisms.
• Determine symbiotic relationships between filaments and floc formers in activated sludge.
• Develop and apply tools for monitoring filament population dynamics in industrial wastewater treatment activated sludge.

The ultimate aim of the project is to monitor the dynamics of filamentous populations in industrial wastewater treatment plants using the methods and methodologies that have been developed to analyse filamentous microorganism populations in this project.

Description of the work

Samples are taken in a large number (> 100) industrial wastewater treatment plants and other industrial water systems containing biofouling. Filaments are isolated through micromanipulation and dilution isolation. DNA is derived from pure cultures or after reverse transcriptase PCR on extremely low (10-50) numbers of micromanipulated filaments. Additionally a clone library is made from mixtures of filament containing sludges in a mixed full cycle RNA approach to obtain sequence data from filaments. Phylogenetic analyses are performed to classify organisms and develop specific probes and primers for respectively fluorescent in situ hybridisation and PCR analysis. Multiplex real time Q-PCR is developed for simultaneous quantification of different types of filaments and serve as a quick screening method. Filament physiology is studied in pure cultures with different substrates and under varying redox conditions. These studies are also performed in situ in activated sludge with a combination of FISH and microautoradiography with radio labelled substrates to define in situ physiological activity. Cell surface properties and symbiontic relations between filaments and floc forming bacteria are studied in defined mixed cultures and in situ to reach a better understanding of filament ecophysiology. All methods are applied to monitor filament population biodiversity and dynamics in a 6-9 month monitoring programme in a limited number of industrial wastewater treatment plants. Specific filament abundance is correlated with process data to identify operation conditions that may prevent bulking sludge.

Deliverables

• Species specific molecular detection methods (FISH, PCR) for filamentous micro-organisms.
• Biodiversity and phylogeny of filamentous microorganisms in industrial water systems.
• Ecophysiology of filamentous microorganisms in situ and in pure culture.
• Production of an identification manual for filamentous microorganisms in industrial water systems.
• Dynamics of filamentous microorganisms in industrial water systems.

Contacts

Coordinator

• Jaap van der WAARDE / Bioclear BV NETHERLANDS

Participant

• Marc LEMAITRE / Eurogentec S.A. BELGIUM
• Hermie HARMSEN / Groningen State University Medical Microbiology / NETHERLANDS
• Dick EIKELBOOM / TNO-MEP NETHERLANDS
• University of Aalborg DENMARK
• Claudia BEIMFOHR / Vermicon Engineering & Microbiology AG GERMANY
• Valter TANDOI / Water Research Institute, CNR ITALY