The aim of this experimental workpackage is to understand mechanisms of nutrient and light-driven allocation and the fate of fixed carbon in phytoplankton cultures.We will work in the laboratory, with axenic (if possible) monocultures of selected species under well-defined conditions.
We plan to combine advanced physiological methods (variable fluorescence - ETR, oxygen evolution using MIMS, carbon fixation) with biochemical analysis of cell composition (metabolomics, FTIR) and quantitative genomics and proteomics to understand the processes that allocate photosynthetic energy into biomass.
Leading co-ordinator: David J.Suggett (UTS, Sydney, Australia)
Leading co-ordinator: Kim Halsey (OSU, Corvallis, Oregon, USA)
Local co-ordinator: Ondrej Prasil (Algatech, Trebon, Czechia)
Number of participants: 10 - 20
Field experiments: None
Lab experiments: Centre Algatech, Institute of Microbiology in Třeboň
Data management: Centre Algatech, Institute of Microbiology in Třeboň
We will grow 2-3 selected model species of single-cell phytoplankton with contrasting physiologies (e.g. prokaryotic cyanobacterium like Crocosphaera, lipid accumulating eustigmatophyte Nannochloropsis and starch accumulating green alga Chlorella). The growth will be limited by light or nutrients, so that variation of growth rates will be achieved. Using an array of methods, we will then quantitatively estimate how cells with different growth rates modulate the photosynthesis-driven energy fluxes (ETR through Photosystems, O2 evolution in Photosystem II and consumption within electron chain, gross C-fixation, N-fixation in diazotroph) and sizes and dynamics of metabolites (mostly carbon and nitrogen) storage pools. In addition, we will collect samples for analysis of gene and protein expression.
Programmable bioreactors (PSI Company) for cell growth under turbidostat or chemostat conditions, with online detection of variable fluorescence, oxygen concentration, pH and cell density). Standard cultivation systems for algal cultures. Cell counter. Flow cytometer with cell sorter. Several bench-top fluorometers for active variable fluorescence measurements (FRR, PAM). Instrument for Spectrally Resolved measurements of Fluorescence Induction. Fluorescence Kinetic Microscopes for measurements at single cell levels. Advanced confocal microscope. Dual PAM for additional measurements of variable fluorescence, Photosystem 1 activity, NADPH dynamics and transmembrane fields. Several Clark electrodes, optodes and MIMS apparatus for measuring gas exchang in algal suspension. Thermoluminescence apparatus. Spectrometer with integrating sphere, several spectrofluorometers for measuring emission and excitation spectra (also for 77K). Licensed lab for isotope work, including phtosynthetron, scintillation counter and HPLC with scintillation detector.
Standard equipment for biochemical extractions and purifications. Calibrated HPLC for pigment analysis. Dedicated GC for nitrogenaseacitivity. Elemental (C/N) analyzer. FTIR spectrometer for semiquantitative analysis of biomass composition. High precision GC and LC MS for proteomic and metabolomic work,including fatty acids and small organic molecules.
Clean room for RNA isolation, standard equipment for PCR, including q-PCR.