On the use of selective environments in microalgal cultivation

This thesis deals with selective environments in microalgal cultivation. As explained in Chapter 1 microalgae have changed the course of life on Earth dramatically by performing oxygenic photosynthesis. In oxygenic photosynthesis electrons from water are used to reduce carbon dioxide to carbohydrates or lipids using solar energy. As a waste product oxygen is produced. The production of carbohydrates and particularly lipids by microalgae attracts currently considerable scientific interest as microalgal lipids can be converted to yield biodiesel. As the CO2 emitted upon combustion of this biodiesel has recently been withdrawn from the atmosphere by the microalgae, no net increase in atmospheric CO2 level takes place. Microalgae offer advantages over other lipid production platforms as microalgae are able to reach high intracellular lipid contents and need little freshwater and arable land. Chapter 2 describes the advantages and limitations of the application of a selective environment to obtain a certain functionality in a system. A selective environment aims to give a competitive advantage to a microorganism displaying the desired functionality. By rewarding microalgae for displaying a certain functionality it becomes in the interest of microalgae themselves to display this characteristic. The best reward in nature is an increased chance of survival. A selective environment therefore tries to couple the desired characteristic to an increased chance of survival. Microalgal cultivation based on selective environment fundamentally differs from cultivation of pure cultures. Maintaining the desired culture is the goal of the latter, whereas a microalgal cultivation process based on selective environments aims to maintain a functionality in a system. The species, or multiple species, displaying the desired functionality are expected to differ at different geographical places, with changing climate conditions and over time. Under any condition the species that thrives under these specific conditions by displaying the desired characteristic is enriched. Interesting microalgal functionalities from an industrial point of view include a high carbohydrate and lipid productivity. Both of these storage compounds are produced by microalgae to endure dynamic growth conditions. By limiting the presence of the essential microalgal nutrient nitrogen (in the form of NH4 + or NO3 –) to the dark phase solely an environment is created in which production of storage compounds in the light period is an advantageous strategy. Production of these compounds in the light period will allow microalgae to metabolise the available nitrogen in the dark period by supplying carbon skeletons and energy in the dark. Chapter 3 shows that such an environment enriches carbohydrate producing microalgae from a natural inoculum. Chapter 4 shows that both the moment of nitrogen addition as the amount of nitrogen dosed per microalgae had significant influence on the metabolic behaviour of marine microalgal cultures enriched using the procedure described in Chapter 3. Carbohydrate and lipid productivity proved maximal if ammonium was supplied at the start of the dark period rather than the light period, irrespective of the amount of nitrogen dosed per microalgae. Increasing the amount of nitrogen dosed per microalgae, by increasing the volume exchange ratio from 33 to 50 percent per cycle, induced a decrease in storage compound production if ammonium was supplied in the light period whereas the storage compound productivity was comparable when ammonium was supplied in the dark period. Chapter 5 shows that the enriched microalgal community was highly dependent on an environmental parameters as the presence of silicate. If silicate was present at non-limiting concentrations the enriched culture was dominated by diatoms, whereas green algae were dominant if silicate was absent. Both cultures showed however the same functionality of producing large amounts of carbohydrates in the light period to be able to consume the supplied nitrogen source in the dark period. These results, together with the data obtained under marine conditions, showed that carbohydrate production can be achieved under various conditions, as long as a carbon fixation in the light period is uncoupled from nitrogen uptake in the dark. Diatoms have interesting characteristics for large-scale microalgal cultivation. These include a relative easy solid-liquid separation after cultivation, increased resistance to predators and the possibility to synthesize lipids under silicate limitation. Supplying NH4 + in a pulse, either at the start of the light or the dark period under non-limiting silicate levels, enriched a culture fully dominated by the diatom Nitzschia palea from a natural inoculum, as described in Chapter 6. The metabolic behaviour of the enriched culture was highly influenced by the moment of nitrogen addition. Biomass was the main photosynthetic product in the light period if nitrogen was dosed at the start of the light period, whereas carbohydrates were the main photosynthetic product if nitrogen was dosed at the start of the dark period. Subjecting the enriched cultures to prolonged periods of nitrogen or silicate limitation induced different metabolic responses. Cell numbers increased four times and carbohydrates were the main storage compounds under nitrogen limitation, while cell division abruptly ceased and lipids were the preferred storage compound under silicate limitation. In all experiments carried out in this thesis carbohydrates were the preferred microalgal storage compounds. Uncoupling of carbon fixation in the light from nitrogen uptake in the dark enriched under variable conditions (freshwater, marine, under high silicate concentrations) carbohydrate producing microalgae from a natural inoculum. Intracellular carbohydrate levels typically increased from10 to 50 % of organic dry weight in the light period. Although no liquid and gas flows leaving and entering the systems were sterilised and despite regular cleaning of the systems the enriched cultures were highly stable in time. This shows that if carbohydrate productivity is aimed for a proper selective environment has been identified and tested. A better understanding of the ecological role of lipids and carbohydrates in microalgae will help creating selective environment for lipid production. Besides drawing general conclusions, Chapter 7 elaborates more on possible strategies to enrich lipid producing microalgae. The strategy advocated in this thesis, rewarding a microalga for displaying a functionality by coupling it to an increased chance of survival by imposing a selective environment, will prove a valuable tool if the ecological role of lipids is better understood.