Leonor Guedes da Silva

Analysis of metabolic functionality and regulation of storage accumulating microorganisms under dynamic environments

Natural habitats of microorganisms are dynamic environments with non-continuous supply of carbon and energy sources. These dynamics change the selective pressure and in contrast to batch or chemostat cultivation conditions, storage metabolism can increase competitiveness. Most prominent in bacteria is the accumulation of PHA (Jiang et al. 2011) but some organisms also invest in the accumulation of glycogen and/or polyPi in anaerobic/aerobic cultivation conditions (Van Loosdrecht, Pot, and Heijnen 1997) .

Microorganisms with storage metabolism are very relevant for wastewater treatment plants, especially in enhanced biological phosphorus removal, EBPR. This process uses a mixed culture enriched in phosphate-accumulating organisms (PAOs), which takes up VFAs (volatile fatty acids) and phosphate and converts these into PHA, glycogen and polyPi in an anaerobic-feast/aerobic-famine dynamic regime.

However, the EBPR process can become unstable and mainly due to PAOs’ direct competitors: Glycogen-accumulating organisms (GAOs) (Oehmen et al. 2010). Both are able to take up VFA anaerobically, store carbon as PHA and use the accumulated intracellular storage products for growth aerobically. The main difference is that GAOs are not able to store polyPi as PAOs do. Interestingly, small differences in the process regime (Feast/famine and aerobic/anaerobic) lead to an enrichment of PAO or GAO’s. However, while the overall metabolic processes can be well described, the intermediate metabolism and the regulation of the complex metabolic switches between storage accumulation and growth and their influence on the PAO-GAO competition are still poorly understood.

Approach

In this project, novel system biology tools developed for pure culture systems are applied to unravel PAOs (and GAOs) exquisite metabolism and its regulation. By combining intracellular metabolomics, fluxomics and genome-scale metabolic modelling, we aim to clear the decades-old controversy around the metabolism of both organisms and derive hypothesis for the metabolic/post-transcriptional regulation of these systems.

Techniques

  • DGGE and FISH as culture characterization methods
  • Intracellular metabolites measurement (Taymaz-Nikerel et al. 2009)
  • Quasi-steady state 13C flux analysis (Wiechert 2001)
  • Dynamic 13C flux analysis (Nöh, Wahl, and Wiechert 2006; de Jonge et al. 2014; Abate, Hillen, and Aljoscha Wahl 2012)
  • Integration of –omics data (Lourenço et al. 2011)

  • Abate, Alessandro, Robert C. Hillen, and S. Aljoscha Wahl. 2012. “Piecewise Affine Approximations of Fluxes and Enzyme Kinetics from in Vivo 13C Labeling Experiments.” International Journal of Robust and Nonlinear Control 22 (10): 1120–39. DOI
  • De Jonge, Lodewijk, Nicolaas A A Buijs, Joseph J Heijnen, Walter M van Gulik, Alessandro Abate, and S Aljoscha Wahl. 2014. “Flux Response of Glycolysis and Storage Metabolism during Rapid Feast/famine Conditions in Penicillium Chrysogenum Using Dynamic (13)C Labeling.” Biotechnology Journal 9: 372–85. DOI
  • Jiang, Yang, Yu Dimitry, Robbert Kleerebezem, Gerard Muyzer, and Mark van Loosdrecht. 2011. “Plasticicumulans Acidivorans Gen. Nov., Sp. Nov., a Polyhydroxyalkanoate-Accumulating Gammaproteobacterium from a Sequencing-Batch Bioreactor.” International Journal of Systematic and Evolutionary Microbiology 61 (9): 2314–19. DOI
  • Lourenço, Anália, Sónia Carneiro, José P Pinto, Miguel Rocha, Eugénio C Ferreira, and Isabel Rocha. 2011. “A Study of the Short and Long-Term Regulation of E. Coli Metabolic Pathways.” Journal of Integrative Bioinformatics 8 (3): 183. DOI
  • Nöh, Katharina, S. Aljoscha Wahl, and Wolfgang Wiechert. 2006. “Computational Tools for Isotopically Instationary 13C Labeling Experiments under Metabolic Steady State Conditions.” Metabolic Engineering 8 (6): 554–77. DOI
  • Oehmen, Adrian, Carlos M. Lopez-Vazquez, G. Carvalho, M. a M Reis, and M. C M van Loosdrecht. 2010. “Modelling the Population Dynamics and Metabolic Diversity of Organisms Relevant in Anaerobic/anoxic/aerobic Enhanced Biological Phosphorus Removal Processes.” Water Research 44 (15). Elsevier Ltd: 4473–86. DOI
  • Taymaz-Nikerel, Hilal, Marjan de Mey, Cor Ras, Angela ten Pierick, Reza M. Seifar, Jan C. van Dam, Joseph J. Heijnen, and Walter M. van Gulik. 2009. “Development and Application of a Differential Method for Reliable Metabolome Analysis in Escherichia Coli.” Analytical Biochemistry 386 (1). Elsevier Inc.: 9–19. DOI
  • Van Loosdrecht, M. C M, M. a. Pot, and J. J. Heijnen. 1997. “Importance of Bacterial Storage Polymers in Bioprocesses.” Water Science and Technology. DOI
  • Wiechert, W. 2001. “13C Metabolic Flux Analysis.” Metabolic Engineering 3 (3): 195–206. DOI