Phosphorus uptake by microalgae from P-rich media Alexei Solovchenko*, Šárka Moudříková**, Peter Mojzeš**, Tabea Mettler-Altmann***, Andres Sadowsky***, Bärbel Ackermann****, Alexandr Lukyanov*, Ladislav Nedbal**** *Faculty of Biology, Moscow State University, Moscow, Russia;
[email protected] **Institute of Physics, Faculty of Mathematics and Physics, Charles University, Prague, Czech Republic ***Cluster of Excellence in Plant Sciences (CEPLAS) and Institute of Plant Biochemistry, Heinrich Heine University, Dusseldorf, Germany ****Institute of Bio- and Geosciences/Plant Sciences (IBG-2), Forschungszentrum Jülich, Germany
INTRODUCTION Large amounts of phosphorus (P), a major non-renewable plant nutrient, are lost annually with wastewater. Microalgae are a promising vehicle for recycling P from wastewater (Figs. 1, 2) into biofertilizers, since they are naturally equipped to take up much more P than is necessary to support their growth, expressing a behaviour known as ‘luxury uptake’ of P [1]. Figure 1 – An image from the Envisat satellite (Full Resolution, MERIS_FR) showing Lake Erie, USA in 2011. P from farms, sewage, and industry provoked a harmful algae bloom that threatened public health due to hepatotoxin microcystin. The figure demonstrates the negative environmental impact of algae grown on P in runoff waters as well the positive potential of algae to take up large quantities of P if handled properly. (Source: [2])
Advantages of microalgal capture of P include simultaneous removal of different nitrogen species, destruction of organic pollutants, suppression of pathogenic microflora, capture of the greenhouse gasses CO2 and NOx, as well as co-generation of P-enriched biomass suitable for conversion into slow-release P biofertilizer [2].
Figure 2 – A schematic representation of potential algaebased interventions in key P-flows in the anthroposphere. The flows are represented relative to the annual quota of P-rock mining - 100%, 18 Mt(P) y–1. On the source side of the anthroposphere, the annual P-fertilizer application represents 78% and grazing 67% of the mining quota. The black arrows indicate losses of P occurring in all steps from mining (yellow), processing in the food and feed chains (green), industrial use (gray), and waste chains (brown). The losses occur in forms that may not be accessible to sequestration by algae because of involved chemical or physical forms or because of low concentration levels. In contrast, the P-rich waste streams (red) that can be considered for phosphate sequestration by algae are massive, capped as high as 64% of the annual mining quota. (Source: [2])
RESULTS and DISCUSSION Phosphorus-starved cells of all organisms exhibited a bi-phasic kinetics of Pi uptake. Within the first 2–4 h after Pi replenishment in the medium, the highest rate of Pi uptake was recorded; this rate depended on the external Pi concentration. A transient accumulation of PolyP was detected during this period using confocal Raman microscopy (Fig. 3) and confirmed by an enzymatic assay using heterologously expressed exopolyphosphatase from yeast (see Methods). The highest PolyP was detected starting from approximately 2 h after Pi addition. At this phase, the cells accumulated > 5 %DW of P.
Figure 4 – Detection of polyphosphates in C. vulgaris CCALA256 pre-starved (time 0 in the graph) and re-fed with inorganic phosphate (8 mM KH2PO4). Error bars show variability among individual cells.
The second phase coincided with the beginning of exponential growth (starting from ca. 1 d after Pi replenishment) when the cultures displayed a sustained rate of Pi uptake which was 5–10 times lower than that recorded at the first phase. The Pi uptake at the second phase was proportional to the cell division rate (Fig. 5). Little, if any, PolyP accumulation was detected during this phase although the presence of assorted P-metabolites like ATP and sugar phosphates were evident from 31P NMR measurements suggesting a high level of metabolic activity (not shown). The onset of stationary phase (slowdown of cell division rate) because of light limitation on the background of excess of Pi in the medium brought about the appearance of PolyP in the cells and a decline in Pi uptake rate from the medium. The P content of the microalgal biomass did not exceed 2% DW at this stage.
However, the use of microalgae is limited by insufficient knowledge of P uptake mechanisms under eutrophic conditions. In the present work we followed the relationships between cell division rate, inorganic phosphate (Pi) uptake rate from a Prich medium and polyphosphate (PolyP) formation in the P-starving and P-sufficient cells of three green microalgae strains as a function of external Pi concentration and P nutrition. Figure 5 – Relationships between the specific growth rate, µ, and Pi uptake rate in the studied organisms at different stages of their growth under P-replete conditions. Phase I— the phase of rapid Pi uptake; Phase II—the phase of slow Pi uptake.
METHODS • Organisms: the chlorophytes Chlorella vulgaris CCALA 256, IPPAS C-1 and Parachlorella kessleri CCALA 251 • Cultivation conditions: complete or P-free Tamiya medium (+P and –P, respectively), a greenhouse and a V-bag bioreactor (NOVAgreen®, Fig. 2) or in +P or –P Trebon medium [3] in PSI FMT150 bioreactors (Fig. 3). The cultures were grown under combined illumination comprised by natural solar illumination and/or continuous artificial illumination, bubbling with CO2 : air (1 : 25, v/v; 1 v v–1 min–1.
Figure 3 – Algal cultures in a NOVAgreen® photobioreactor
CONCLUSIONS
To obtain the cultures with depleted internal P reserves, the cells were pelleted by centrifugation (5 min, 3000 g), washed with the P-free medium, re-suspended in the same medium and incubated under the conditions described above in a Pfree medium until cessation of the cell division. At the beginning of the experiment, Pi was added to the final concentration of 400 μM.
The analysis of the kinetics of Pi uptake and PolyP formation and catabolism inside the microalgal cells suggests that: - The most rapid P uptake and its transient storage in PolyP is displayed by P prestarved cells during the post-starvation lag period.
• Measurements: The culture growth was monitored via cell number, Chl content or cell dry weight, DW [3]. The residual Pi and nitrate contents were checked daily with standard cuvette tests LCK 380 and LCK 350 (Hach Lange, Germany). To detect polyphosphate presence and distribution in the cell we employed Raman measurements [3] and an enzymatic assay using heterologously expressed exopolyphosphatase from yeast (Werner et al., 2007).
It is likely that the rate of PolyP accumulation in microalgal cells is defined predominantly by the rates of Pi uptake (which, in turn, depends on the external Pi concentration and the P nutrition history of the cell) and Pi expenditure for biosynthesis of cell building blocks (sugarphosphates, nucleic acids, phospholipids etc.).
- Polyphosphate-enriched cells were observed when cell division is slow, e.g. at the stationary phase. - Vigorously dividing cultures characterized by a rapid accumulation of biomass are not likely to accumulate large amounts of PolyP.
Figure 2 – a С. vulgaris culture in a FMT 150 photobioreactor
- Development of economically viable process combining the efficient P biocapture with a PolyP enrichment is a non-trivial task requiring a multi-stage ‘feast and famine’ approach including (i) the P removal phase (by pre-starved cultures) and (ii) regeneration of P-hungry culture.
[1] Cembella, A. et al. (1982) The utilization of inorganic and organic phosphorous compounds as nutrients by eukaryotic microalgae: a multidisciplinary perspective: I. Crit Rev Microbiol. 10, 317-391. [2] Solovchenko, A. et al. (2016) Phosphorus from wastewater to crops: an alternative path involving microalgae, Biotechnology Advances, 34, 550-564. [3] Moudříková, Š. et al. (2016) Raman and fluorescence microscopy sensing energy-transducing and energy-storing structures in microalgae, Algal Research, 16, 224-232. [4] Pal, D. et al. (2011) The effect of light, salinity, and nitrogen availability on lipid production by Nannochloropsis sp., Applied Microbiology and Biotechnology, 90, 1429-1441. [5] Werner, T.P. et al. (2007) Inorganic polyphosphate occurs in the cell wall of Chlamydomonas reinhardtii and accumulates during cytokinesis, BMC Plant Biology, 7, 51. AS and AL acknowledge the financial support of Russian Science Foundation (grant 14-50-00029).
