Microalgal photosynthesis under flashing light

Microalgae are promising organisms for a biobased economy as a sustainable source of food, feed and fuel. High-density microalgae production could become cost effective in closed photobioreactors (PBR). Therefore, design and optimization of closed PBRs is a topic of ongoing research in both academic and industrial environment.<br /> Mixing in dense algae cultures causes light/dark (L/D) cycles of different magnitudes exposing algae to flashing light. It is often said that due to a flashing light effect, productivity of a PBR can be increased. In this thesis the flashing light effect is systematically investigated and the result is a mechanistic model that can predict microalgae growth under different flashing light regimes.<br /> The review of existing literature about L/D cycle experiments in Chapter 2 provides the theoretical background of the flashing light effect (L/D cycles) and discusses possibilities to improve PBR productivity by its application. It is concluded that PBR performance can be optimized by maximizing photosynthetic rate and biomass yield on light energy based on increased or controlled mixing and, thus, L/D cycling. It is unlikely to achieve maximal enhancement based on L/D cycles because of the fast mixing required: specific growth rate measurements in well-controlled, lab-scale PBRs suggest a minimal flash frequency of 14 Hz - 24 Hz combined with short flash times (&lt; 20 ms) to achieve a maximal enhancement.<br /> The application of flashing light alone in an artificially illuminated PBR has a limited effect on PBR performance, consequently, continuous (sun) light should be preferred. Further optimization strategies can be developed based on mechanistic models that describe the influence of L/D cycles on algae productivity as will be shown later in this thesis (Chapter 5).<br /> In Chapter 3, photosynthetic efficiency and growth of the green microalga Chlamydomonas reinhardtii were measured using LED light to simulate light/dark cycles ranging from 5 to 100 Hz at a light/dark ratio of 0.1 and a flash photon flux density (PFD) of 1000 &micro;mol m-2 s-1.<br /> Light flashing at 100 Hz yielded the same photosynthetic efficiency and specific growth rate as cultivation under continuous illumination with the same time-averaged PFD, which is called full light integration. The efficiency and growth rate decreased with decreasing flash frequency. At all frequencies, the rate of linear electron transport during the flash was higher than during maximal growth under continuous light, suggesting storage of reducing equivalents during the flash, which are available during the dark period. In this way the dark reaction of photosynthesis can continue during the dark time of an L/D cycle. This is a possible explanation for the mechanism behind the flashing light effect.<br /> Another parameter that describes an L/D cycle besides frequency, is the duty cycle, it determines the time fraction algae spend in the light. In Chapter 4 the influence of different duty cycles on oxygen yield on absorbed light energy and photosynthetic oxygen evolution was investigated. Net oxygen evolution of Chlamydomonas reinhardtii was measured for four duty cycles (0.05, 0.1, 0.2 and 0.5) in a biological oxygen monitor. Over-saturating light flashes were applied in a square-wave fashion with four flash frequencies (5, 10, 50, 100 Hz). Algae were pre-cultivated in a turbidostat and acclimated to a low photon flux density (PFD).<br /> A photosynthesis-irradiance curve was measured under continuous illumination and used to calculate the net oxygen yield, which was maximal between a PFD of 100 and 200 &micro;mol m-2 s-1. Net oxygen yield under flashing light was proven to be duty cycle dependent: the highest yield was observed at a duty cycle of 0.1 (i.e. a time-averaged PFD of 115 &micro;mol m-2 s-1). At lower duty cycles maintenance respiration reduced net oxygen yield. At higher duty cycles photon absorption rate exceeded the maximal photon utilization rate and, as a result, surplus light energy was dissipated as heat, which lead to a reduction in net oxygen yield. This behavior was identical with the observation under continuous light.<br /> Understanding photosynthetic growth in dynamic light regimes is crucial to develop models that can predict PBR productivities under continuous and flashing light. Therefore, the objective of Chapter 5 was to develop and validate a mechanistic model that describes photosynthetic net oxygen evolution under flashing light based on biomass specific light absorption rate and light dissipation rate of excess absorbed light.<br /> The model describes photosynthetic oxygen evolution based on the availability of reducing equivalents (electrons), which result from the light reactions. Electrons are accumulated during the flash and serve as a pool for carbon dioxide fixation during the dark, which leads to partial or full light integration. Both, electron consumption rate and energy dissipation rate are based on a Monod-type kinetic.<br /> The underlying assumption of an electron pool seems correct and its filling and emptying is depending on the flash time. In general, with increase in flash time the energy dissipation rate increased as well. And, simulations showed that if the dark time between flashes is not sufficiently long then the pool will not be completely empty and is responsible for a high energy dissipation rate. The measured oxygen production rates were described well, but the description of the energy dissipation rate will need further investigation.<br /> Not only L/D cycles but also fluctuating light that algae experience while moving through the light gradient will influence PBR productivity. In Chapter 6 the combined effect of L/D cycles and fluctuating light on biomass yield on light energy was studied.<br /> For this purpose we used controlled, short light path, laboratory, turbidostat-operated PBRs equipped with a LED light source for square-wave L/D cycles with frequencies from 1 Hz to 100 Hz. Biomass density was adjusted that the PFD leaving the PBR was equal to the compensation point of photosynthesis (10 &micro;mol m-2 s-1).<br /> Algae were acclimated to a sub-saturating incident PFD of 220 &mu;mol m-2 s-1 for continuous light. Using a duty cycle of 0.5, we observed that L/D cycles of 1 Hz and 10 Hz resulted on average in a 10 % lower biomass yield, but L/D cycles of 100 Hz resulted on average in a 35 % higher biomass yield than the yield obtained in continuous light.<br /> The results show that the interaction of L/D cycle frequency, culture density and incident PFD lead to certain PBR productivity. Hence, appropriate L/D cycle frequency setting by mixing and dark zone setting by biomass concentration can optimize PBR productivity. And, reduce the effect that a dark zone exposure impinges on biomass yield in microalgae cultivation.<br /> The last chapter is a general discussion (Chapter 7) that places the results of the thesis into context for PBR operation. It is discussed that L/D cycle frequencies of 1-10 Hz, which can be achieved in practice, have a minor impact on biomass yield and volumetric productivity. But, the PBR can be operated with a dark zone and a major gain in biomass concentration can be achieved. However, the size of the dark zone is limited by the incident PFD. The incident PFD times the relative size of the photic zone should be in the same range where the optimal yield under continuous light can be found based on a P-I curve measurement. The photic zone is then defined as the volume with a PFD above the compensation point divided by the total volume.<br /> With simulations based on the dynamic model we could show that light integration can be explained by an electron storage pool that fills during the flash and is used during the dark. Furthermore, the dynamic model could be used to predict PBR productivities based on real fluctuating light regimes observed in photobioreactors.<br /> &nbsp