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. 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. 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 ( 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). 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 µmol m-2 s-1. 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. 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). 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 µmol 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 µmol 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. 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. 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. 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. 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. 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 µmol m-2 s-1). Algae were acclimated to a sub-saturating incident PFD of 220 μmol 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. 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. 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. 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. </p

Adjusted Light and Dark Cycles Can Optimize Photosynthetic Efficiency in Algae Growing in Photobioreactors

Biofuels from algae are highly interesting as renewable energy sources to replace, at least partially, fossil fuels, but great research efforts are still needed to optimize growth parameters to develop competitive large-scale cultivation systems. One factor with a seminal influence on productivity is light availability. Light energy fully supports algal growth, but it leads to oxidative stress if illumination is in excess. In this work, the influence of light intensity on the growth and lipid productivity of Nannochloropsis salina was investigated in a flat-bed photobioreactor designed to minimize cells self-shading. The influence of various light intensities was studied with both continuous illumination and alternation of light and dark cycles at various frequencies, which mimic illumination variations in a photobioreactor due to mixing. Results show that Nannochloropsis can efficiently exploit even very intense light, provided that dark cycles occur to allow for re-oxidation of the electron transporters of the photosynthetic apparatus. If alternation of light and dark is not optimal, algae undergo radiation damage and photosynthetic productivity is greatly reduced. Our results demonstrate that, in a photobioreactor for the cultivation of algae, optimizing mixing is essential in order to ensure that the algae exploit light energy efficiently

Perfusion system in high-density cell culture for higher yields in vaccine production

Product yields of biologicals such as monoclonal antibodies, recombinant proteins and vaccines produced in mammalian cell culture have to be improved constantly to cope with increasing demands and process economics. As an example we investigate an influenza vaccine production process with adherently growing MDCK cells [Genzel et al., 2004]. The process consists of a cell growth phase and a virus replication phase. We want to achieve high-density cell cultures and finally higher virus yields in microcarrier systems. Main focus is on batch-to-batch reproducibility and scale-up. To quantitatively analyze this process various mathematical models are being developed to describe phenomena such as attachment, proliferation and contact inhibition of cells. In the past, we achieved higher cell numbers in small scale bioreactors using different control strategies for perfusion systems during cell growth [Bock et al., 2005]. A perfusion mode with cell retention by filtration allowed a continuous medium exchange preventing substrate limitations, and metabolite inhibitions. So far, the virus titers (HA) did not increase in the same magnitude as expected from cell numbers. Similar results were obtained by Genzel et al, 2005 in an influenza vaccine process in a wave® bioreactor system and by Pohlscheidt et al., 2005 in a parapoxvirus vaccine production process. Furthermore, Pohlscheidt et al. demonstrated a successful increase in virus titers by using a 20 kDa dialysis system or an Expanded-Volume-Batch during virus replication. Therefore, a perfusion system might also be successfully applied during influenza virus replication. In addition, the removal of virus particles from the culture broth might increase the virus yield (HA) by avoiding unspecific degradation of virions by enzymes released from lysed cells. Here, we present results concerning the influence of perfusion rates during virus replication on virus yields. Bock et al., 2005. Closed loop control of perfusion systems in high-density cell culture. Proceedings of the 19th meeting of ESACT in Harrogate, United Kingdom Genzel et al., 2005. Serum-free influenza production with MDCK cells in wave-bioreactor and 5L-stirred tank bioreactor. Proceedings of the 19th meeting of ESACT in Harrogate, United Kingdom Pohlscheidt et al., 2005. Strategies for large scale production of Parapoxvirus Ovis by micro-carrier cell culture. Proceedings of the 19th meeting of ESACT in Harrogate, United Kingdo

Photosynthetic efficiency of Chlamydomonas reinhardtii in flashing light

Efficient light to biomass conversion in photobioreactors is crucial for economically feasible microalgae production processes. It has been suggested that photosynthesis is enhanced in short light path photobioreactors by mixing-induced flashing light regimes. In this study, 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 intensity of 1000¿µmol¿m-2¿s-1. Light flashing at 100¿Hz yielded the same photosynthetic efficiency and specific growth rate as cultivation under continuous illumination with the same time-averaged light intensity (i.e., 100¿µmol¿m-2¿s-1). The efficiency and growth rate decreased with decreasing flash frequency. Even at 5¿Hz flashing, the rate of linear electron transport during the flash was still 2.5 times 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 a light/dark cycle. Understanding photosynthetic growth in dynamic light regimes is crucial for model development to predict microalgal photobioreactor productivities. Biotechnol. Bioeng. 2011;108: 2905–2913. © 2011 Wiley Periodicals, In

Photosynthetic efficiency and oxygen evolution of Chlamydomonas reinhardtii under continuous and flashing light

As a result of mixing and light attenuation in a photobioreactor (PBR), microalgae experience light/dark (L/D) cycles that can enhance PBR efficiency. One parameter which characterizes L/D cycles is the duty cycle; it determines the time fraction algae spend in the light. The objective of this study was to determine the influence of different duty cycles on oxygen yield on absorbed light energy and photosynthetic oxygen evolution. 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 (BOM). Oversaturating light flashes were applied in a square-wave fashion with four flash frequencies (5, 10, 50, and 100 Hz). Algae were precultivated in a turbidostat and acclimated to a low photon flux density (PFD). A photosynthesis-irradiance (PI) curve was measured under continuous illumination and used to calculate the net oxygen yield, which was maximal between a PFD of 100 and 200 µmol m(-2)¿s(-1). Net oxygen yield under flashing light was duty cycle-dependent: the highest yield was observed at a duty cycle of 0.1 (i.e., time-averaged PFD of 115 µmol 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 which led to a reduction in net oxygen yield. This behavior was identical with the observation under continuous light. Based on these data, the optimal balance between oxygen yield and production rate can be determined to maximize PBR productivity

Photosynthetic efficiency of Chlorella sorokiniana in a turbulently mixed short light-path photobioreactor

To be able to study the effect of mixing as well as any other parameter on productivity of algal cultures, we designed a lab-scale photobioreactor in which a short light path (SLP) of (12 mm) is combined with controlled mixing and aeration. Mixing is provided by rotating an inner tube in the cylindrical cultivation vessel creating Taylor vortex flow and as such mixing can be uncoupled from aeration. Gas exchange is monitored on-line to gain insight in growth and productivity. The maximal productivity, hence photosynthetic efficiency, of Chlorella sorokiniana cultures at high light intensities (1,500 µmol m-1 s-1) was investigated in this Taylor vortex flow SLP photobioreactor. We performed duplicate batch experiments at three different mixing rates: 70, 110, and 140 rpm, all in the turbulent Taylor vortex flow regime. For the mixing rate of 140 rpm, we calculated a quantum requirement for oxygen evolution of 21.2 mol PAR photons per mol O2 and a yield of biomass on light energy of 0.8 g biomass per mol PAR photons. The maximal photosynthetic efficiency was found at relatively low biomass densities (2.3 g L-1) at which light was just attenuated before reaching the rear of the culture. When increasing the mixing rate twofold, we only found a small increase in productivity. On the basis of these results, we conclude that the maximal productivity and photosynthetic efficiency for C. sorokiniana can be found at that biomass concentration where no significant dark zone can develop and that the influence of mixing-induced light/dark fluctuations is margina

Heat transfer mechanisms of a vapour bubble growing at a wall in saturated upward flow

The aim of this study is to provide a better insight into the heat transfer mechanisms involved in single bubble growth in forced convection. In a set-up with vertical upflow of demineralized water under saturation conditions special bubble generators (BGs) were embedded at various positions in the plane wall. Power to a BG, local mean wall temperature and high-speed camera recordings from two viewing angles were measured synchronously. An accurate contour analysis is applied to reconstruct the instantaneous three-dimensional bubble volume. Interface topology changes of a vapour bubble growing at a plane wall have been found to be dictated by the rapid growth and by fluctuations in pressure, velocity and temperature in the approaching fluid flow. The camera images have shown a clear dry spot under the bubbles on the heater surface. A micro-layer under the bubble is experimentally shown to exist when the bubble pins to the wall surface and is therefore dependent on roughness and homogeneity of the wall. The ratio of heat extracted from the wall to the total heat required for evaporation was found to be around 30 % at most and to be independent of the bulk liquid flow rate and heat provided by the wall. When the bulk liquid is locally superheated this ratio was found to decrease to 20 %. Heat transfer to the bubble is also initially controlled by diffusion and is unaffected by the convection of the bulk liquid