Characterization of PHB storage in activated sludge extended filamentous bacteria by automated colour image analysis

The storage of poly-β-hydroxybutyrate (PHB) in extended filamentous bacteria from activated sludge was monitored by Sudan Black staining: PHB granules were blue in the reddish filaments counterstained by safranin. By quantitative image analysis of colour images grabbed on an optical microscope, the distribution of the PHB loading of the extended filaments was estimated by determination of the proportion of blue pixels of their skeleton. The method was applied for different feed compositions to demonstrate its ability to monitor the PHB synthesis and storage capacity of filamentous bacteria in mixed cultures. Fast PHB storage, within a few hours, could be observed with acetate-based feeding solutions but the storage rate decreased with more complex feeds (meat extract based feed, wastewater).

[1]  J G Holt,et al.  Nile blue A as a fluorescent stain for poly-beta-hydroxybutyrate , 1982, Applied and environmental microbiology.

[2]  Marie-Noëlle Pons,et al.  Gram-staining characterisation of activated sludge filamentous bacteria by automated colour analysis , 2004, Biotechnology Letters.

[3]  G. Daigger,et al.  Manual on the causes and control of activated sludge bulking and foaming , 1992 .

[4]  Mark C M van Loosdrecht,et al.  Effect of feeding pattern and storage on the sludge settleability under aerobic conditions. , 2003, Water research.

[5]  T. Mino,et al.  Isolation, characterization and identification of polyhydroxyalkanoate-accumulating bacteria from activated sludge. , 2000, Journal of bioscience and bioengineering.

[6]  W. Page,et al.  Formation of poly(hydroxybutyrate-co-hydroxyvalerate) by Azotobacter vinelandii UWD , 1992, Applied and environmental microbiology.

[7]  Hiroyasu Satoh,et al.  Production of polyhydroxyalkanoates (PHA) by activated sludge treating municipal wastewater: effect of pH, sludge retention time (SRT), and acetate concentration in influent. , 2003, Water research.

[8]  Maria A M Reis,et al.  Methods for detection and visualization of intracellular polymers stored by polyphosphate-accumulating microorganisms. , 2002, Journal of microbiological methods.

[9]  G. Odham,et al.  Combined Determination of Poly-β-Hydroxyalkanoic and Cellular Fatty Acids in Starved Marine Bacteria and Sewage Sludge by Gas Chromatography with Flame Ionization or Mass Spectrometry Detection , 1986, Applied and environmental microbiology.

[10]  Tomonori Matsuo,et al.  Uptake of Organic Substrates and Accumulation of Polyhydroxyalkanoates Linked with Glycolysis of Intracellular Carbohydrates under Anaerobic Conditions in the Biological Excess Phosphate Removal Processes , 1992 .

[11]  D. Dionisi,et al.  Effect of periodic feeding on substrate uptake and storage rates by a pure culture of Thiothrix (CT3 strain). , 2007, Water research.

[12]  J. May,et al.  Production of poly-\-hydroxybutyrate in Acinetobacter spp. isolated from activated sludge , 1993, Applied Microbiology and Biotechnology.

[13]  S. K. Rawal,et al.  Synthesis of PHB by recombinant E. coli harboring an approximately 5 kb genomic DNA fragment from Streptomyces aureofaciens NRRL 2209. , 2002, International journal of biological macromolecules.

[14]  W. Gujer,et al.  Intracellular carbon flow in phosphorus accumulating organisms from activated sludge systems , 1997 .

[15]  H. Hamana,et al.  Biosynthesis of poly-3-hydroxybutyrate by Sphaerotilus natans , 1995, Applied Microbiology and Biotechnology.

[16]  Geert Boeije,et al.  Chemical fate prediction for use in geo-referenced environmental exposure assessment , 1999 .

[17]  J. J. Heijnen,et al.  Poly-β-hydroxybutyrate metabolism in dynamically fed mixed microbial cultures , 2002 .

[18]  G. Eggink,et al.  Gas-chromatographic analysis of poly(3-hydroxyalkanoates) in bacteria , 1994 .

[19]  J. Crespo,et al.  Effect of carbon source on the formation of polyhydroxyalkanoates (PHA) by a phosphate-accumulating mixed culture , 1998 .

[20]  Mauro Majone,et al.  A bulking sludge with high storage response selected under intermittent feeding , 1998 .

[21]  K. Nickerson.  Purification of Poly-β-Hydroxybutyrate by Density Gradient Centrifugation in Sodium Bromide , 1982, Applied and environmental microbiology.

[22]  Marie-Noëlle Pons,et al.  Characterisation of activated sludge by automated image analysis , 2001 .

[23]  K. Sudesh,et al.  Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters , 2000 .

[24]  C. M. Hooijmans,et al.  Biological phosphate removal processes , 1997, Applied Microbiology and Biotechnology.

[25]  C. M. Hooijmans,et al.  IMPACT OF EXCESSIVE AERATION ON BIOLOGICAL PHOSPHORUS REMOVAL FROM WASTEWATER , 1998 .

[26]  R. M. Lafferty,et al.  A rapid gas chromatographic method for the determination of poly-β-hydroxybutyric acid in microbial biomass , 1978, European journal of applied microbiology and biotechnology.

[27]  M. Pons,et al.  Physiological investigations by image analysis. , 1998, Journal of biotechnology.

[28]  Eugénio C. Ferreira,et al.  Automated monitoring of activated sludge using image analysis , 2000 .

[29]  Roberto Ramadori,et al.  Effect of periodic feeding in sequencing batch reactor on substrate uptake and storage rates by a pure culture of Amaricoccus kaplicensis. , 2003, Water Research.

[30]  M. Pons,et al.  Endogenous processes during long-term starvation in activated sludge performing enhanced biological phosphorus removal. , 2006, Water research.

[31]  S. K. Rawal,et al.  Poly(3-hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli harbouring Streptomyces aureofaciens PHB biosynthesis genes: effect of various carbon and nitrogen sources. , 2003, Microbiological research.

[32]  M. Loosdrecht,et al.  Substrate flux into storage and growth in relation to activated sludge modeling , 1999 .