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Microbial characterisation of polyhydroxyalkanoates storing populations selected under different operating conditions using a cell-sorting RT-PCR approach


The identity of polyhydroxyalkanoates (PHA) storing bacteria selected under aerobic dynamic feeding conditions, using propionate as carbon source (reactor P), was determined by applying reverse transcriptase–polymerase chain reaction (RT-PCR) on micromanipulated cells and confirmed by fluorescence in situ hybridisation (FISH). Four genera, Amaricoccus, Azoarcus, Thauera and Paraccoccus were detected, the latter only rarely present. All the biomass was involved in PHA storage as shown by Nile Blue staining. By quantitative FISH, their specific amount was determined in this and two other systems using acetate as the carbon substrate (sequencing batch reactor [SBR] A and A1). SBR A and reactor P had the same sludge retention time (SRT, 10 days), while reactor A1 was operated with the SRT of 1 day and the double organic loading rate (OLR). Systems fed with acetate (41.1 ± 2.2 and 49.4 ± 1.4% total Bacteria, for A and A1, respectively) became enriched in Thauera independently on the SRT and OLR, while it was only present in a minor amount when propionate was used as a substrate (1.9 ± 0.2% total Bacteria). Amaricoccus was present in both reactors operated at 10 days SRT, favoured in the one fed with propionate (61.4 ± 1.9% total bacteria), and almost completely removed at the SRT of 1 day. Azoarcus cells were found in all the analysed systems (3.9 ± 0.3, 23.3 ± 1.5 and 45.9 ± 1.5 for P, A and A1, respectively), while Paracoccus was scarcely present.

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This research was developed within the agreement for Scientific and Technological Cooperation between the Gabinete de Relações Internacionais da Ciência e do Ensino Superior—GRICES (Portugal) and National Research Council—CNR (Italy). The authors acknowledge the financial support of the Fundação para a Ciência e Tecnologia (FCT) through the project POCI/BIO/55789/2004 and EU Integrated Project Contract no. 026515. Luísa S. Serafim acknowledges Fundação para a Ciência e Tecnologia for grant SFRH/BPD/14663/2003.

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Correspondence to Maria A. M. Reis.

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Lemos, P.C., Levantesi, C., Serafim, L.S. et al. Microbial characterisation of polyhydroxyalkanoates storing populations selected under different operating conditions using a cell-sorting RT-PCR approach. Appl Microbiol Biotechnol 78, 351–360 (2008).

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  • PHA production
  • Aerobic dynamic feeding
  • Mixed cultures
  • RT-PCR
  • FISH
  • Micromanipulation