High-density algal photobioreactors using light-emitting diodes

Lack of high-density algal photobioreactors (PBR) has been a limitation in exploiting the biotechnological potential of algae. Recent developments of highly efficient light-emitting diodes (LED using gallium aluminum arsenide chips) have made the development of a small LED-based PBR possible. We have calculated theoretical values of gas mass transfer requirements and light-intensity requirement to support high-density algal cultures for the 680 nm monochromatic red light from LED as a light source. A prototype PBR has been designed based on these calculations. A cell concentration of more than 2 x 10(9) cells/mL (more than 6.6% v%sol;v), cell doubling times as low as 12 h, and an oxygen production rate as high as 10 mmol oxygen/L culture/h were achieved using on-line ultrafiltration to periodically provide fresh medium. (c) 1994 John Wiley & Sons, Inc.     

Effects of cell density,light intensity and mixing on Undaria pinnatifida gametophyte activity in a photobioreactor

An on-line controlled 7 l sterilizable photobioreactor was used for the optimisation of a culture of gametophytes of Undaria pinnatifida. The gametophytes, which had been stored for three years in a culture cabinet at 16 degrees C, could rapidly grow in the photobioreactor under controlled conditions. The rate of increase of dissolved oxygen and pH were used to monitor the photosynthetic activity. Optimal gametophytes density changed varying the light intensity. The optimal cell densities were 3.24 and 3.45 g FW l(-1) when the cultures were exposed to 61.7 and 82.3 microE m(-2) s(-1), respectively. The optimal cell density was higher under a high photon flux density (PFD) than under low PFD. On the other hand, the optimal light intensities were different for different cell density cultures. The light saturation point was higher at high cell density cultures than at low cell density cultures. The optimal rotational speed was 150 rpm for high cell density culture in the photobioreactor.     

Optimization of algal photobioreactors using flashing lights

It has been reported that flashing light enhances microalgal biomass productivity and overall photosynthetic efficiency. The algal growth kinetics and oxygen production rates under flashing light with various flashing frequencies (5 Hz-37 kHz) were compared with those under equivalent continuous light in photobioreactors. A positive flashing light effect was observed with flashing frequencies over 1 kHz. The oxygen production rate under conditions of flashing light was slightly higher than that under continuous light. The cells under the high frequency flashing light were also observed to be healthier than those under continuous light, particularly at higher cell concentrations. When 37 kHz flashing light was applied to an LED-based photobioreactor, the cell concentration was higher than that obtained under continuous light by about 20%. Flashing light may be a reasonable solution to overcome mutual shading, particularly in high-density algal cultures.     

Enclosed outdoor photobioreactors: light regime,photosynthetic efficiency,scale-up,and future prospects

Enclosed outdoor photobioreactors need to be developed and designed for large-scale production of phototrophic microorganisms. Both light regime and photosynthetic efficiency were analyzed in characteristic examples of state-of-the-art pilot-scale photobioreactors. In this study it is shown that productivity of photobioreactors is determined by the light regime inside the bioreactors. In addition to light regime, oxygen accumulation and shear stress limit productivity in certain designs. In short light-path systems, high efficiencies, 10% to 20% based on photosynthetic active radiation (PAR 400 to 700 nm), can be reached at high biomass concentrations (>5 kg [dry weight] m(-3)). It is demonstrated, however, that these and other photobioreactor designs are poorly scalable (maximal unit size 0.1 to 10 m(3)), and/or not applicable for cultivation of monocultures. This is why a new photobioreactor design is proposed in which light capture is physically separated from photoautotrophic cultivation. This system can possibly be scaled to larger unit sizes, 10 to >100 m(3), and the reactor liquid as a whole is mixed and aerated. It is deduced that high photosynthetic efficiencies, 15% on a PAR-basis, can be achieved. Future designs from optical engineers should be used to collect, concentrate, and transport sunlight, followed by redistribution in a large-scale photobioreactor.     

On-line characterization of a hybridoma cell culture process

The on-line determination of the physiological state of a cell culture process requires reliable on-line measurements of various parameters and calculations of specific rates from these measurements. The cell concentration of a hybridoma culture was estimated on-line by measuring optical density (OD) with a laser turbidity probe. The oxygen uptake rate (OUR) was determined by monitoring dynamically dissolved oxygen concentration profiles and closing oxygen balances in the culture. The base addition for neutralizing lactate produced by cells was also monitored on-line via a balance. Using OD and OUR measurements, the specific growth and specific oxygen consumption rates were determined on-line. By combining predetermined stoichiometric relationships among oxygen and glucose consumption and lactate production, the specific glucose consumption and lactate production rates were also calculated on-line. Using these on-line measurements and calculations, the hybridoma culture process was characterized on-line by identifying the physiological states. They will also facilitate the implementation of nutrient feeding strategies for fed-batch and perfusion cultures. (c) 1994 John Wiley & Sons, Inc.     

Use of photoacclimation in the design of a novel photobioreactor to achieve high yields in algal mass cultivation

The concept of a completely new and novel photobioreactor consisting of various compartments each with a specific light regime is described. This is in response to the debate and development which have taken place in recent years concerning photobioreactor design and closed systems. It is well known that algae can photo-acclimate to various light intensities. At the extremes, they can be high light (HL) or low light (LL) acclimated. Both HL and LL acclimated algae typically have very specific characteristics indicating the plasticity of the organisms, which have developed specific strategies during evolution to cope with continuous and dynamic light fields. Not only are these considerations important in photobioreactor design, but also for the production of certain biocompounds, whose synthesis has specific light requirements. In the continuous flow photobioreactor described here, algal cells acclimated to different light conditions together permit utilization of the entire light gradient found in an optically dense medium, such as in a high-density culture. Compared to a single compartment vertical flat-plate photobioreactor, the multicompartment reactor yielded a 37% higher productivity rate. This is a significant improvement in photobioreactor performance.     

On-line estimation of the biomass activity during animal-cell cultivations

A software sensor was developed to determine the volumetric biomass activity of animal cell cultivations on-line. It was based on the on-line estimation of the ATP-production rate from the oxygen uptake and the lactic-acid production rate. The sensor was verified for a batch culture of Vero cells, and a batch and a continuous culture of hybridoma cells. For the hybridoma cells, the sensor showed a good correlation with the biomass concentration. However, this was not the case for the Vero cells. As soon as glutamine was exhausted, the biomass activity stabilized, whereas the amount of biomass almost doubled. Because the sensor developed responds to nutrient limitations much faster than becomes visible through cell density measurements, and because the volumetric biomass activity can be related to the volumetric consumption rates and production rates of important metabolites, it shows excellent possibilities for control purposes. (c) 1996 John Wiley & Sons, Inc.     

A new photobioreactor for continuous marennin production with a marine diatom: influence of the light intensity and the immobilised-cell matrix (alginate beads or agar layer)

In oyster ponds, the marine diatom Haslea ostrearia synthesises and excretes a hydrosoluble pigment of commercial interest called marennin. During the benthic stage, when algal cells are naturally immobilised in their own polysaccharides, marennin production is higher. To optimise this production, axenic cultures of H. ostrearia were immobilised in a polysaccharidic matrix (alginate or agar) and introduced into a new photobioreactor device for continuous marennin production. Solute diffusion was improved using an alginate beads monolayer, leading to higher levels of cell growth (a 2-fold higher cell concentration) and marennin productivity (7.57-8.80 mg day(-1) l(-1)). An increase in the light intensity (from 3.0 to 8.5x10(16) quanta cm(-2) s(-1)) led to an earlier and 1.3-fold higher production of marennin. However, the higher light intensity led to a higher rate of cell death [0.29 instead of 0.40 ng chlorophyll a (10(6) cells)(-1)]. Due to the secondary nature of marennin metabolism, it would be necessary to alternate between culture conditions favouring cell growth (moderate light intensity and no limiting nitrate supply) and those promoting marennin production (high light intensity and limiting nitrate supply).     

Monitoring of recombinant protein production using bioluminescence in a semiautomated fermentation process

On-line optimization of fermentation processes can be greatly aided by the availability of information on the physiological state of the cell. The goal of our "BioLux" research project was to design a recombinant cell capable of intracellular monitoring of product synthesis and to use it as part of an automated fermentation system. A recombinant plasmid was constructed containing an inducible promoter that controls the gene coding for a model protein and the genes necessary for bioluminescence. The cells were cultured in microfermenters equipped with an on-line turbidity sensor and a specially designed on-line light sensor capable of continuous measurement of bioluminescence. Initial studies were done under simple culture conditions, and a linear correlation between luminescence and protein production was obtained. Such specially designed recombinant bioluminescent cells can potentially be applied for model-based inference of intracellular product formation, as well as for optimization and control of recombinant fermentation processes.     

光径及氮浓度对缺刻缘绿藻生长、油脂和花生四烯酸积累的影响

以缺刻缘绿藻(Parietochloris incisa)为实验材料, 采用BG-11培养基, 分别在2种氮浓度和3种不同光径(LP)的柱状和平板光生物反应器中进行培养, 并探究其生长、油脂和花生四烯酸(AA)的积累规律。结果显示: 在两种光生物反应器中, 光径越小, 越有利于缺刻缘绿藻的生长。其中, 最大生物量均在17.6 mmol/L氮浓度时获得, 分别为5.09 g/L(2.5 cm-柱状)和2.98 g/L(3.0 cm-平板); 而最高油脂和AA绝对含量则均在1.0 mmol/L氮浓度和最大光径处获得, 分别为39.23%、13.21%(6.0 cm-柱状)和40.74%、11.33%(5.0 cm-平板); 另外, 两种光生物反应器中的最大油脂单位体积产率分别可以达到216.39 mg/(L·d)(17.6 mmol/L; 2.5 cm-柱状)和135.93 mg/(L·d)(1.0 mmol/L; 1.5 cm-平板); 而最高的AA单位体积产率均在1.0 mmol/L低氮条件, 最大光径处达到最大, 分别为21.65 mg/(L·d)(6.0 cm-柱状)和19.42 mg/(L·d)(5.0 cm-平板)。因此, 根据实际生产需要, 在1.0 mmol/L低氮条件下, 选择6.0 cm光径的柱状光生物反应器或5.0 cm光径的平板光生物反应器, 培养缺刻缘绿藻生产AA, 能有效降低生产成本。     

Growth monitoring of a photosynthetic micro-organism (Spirulina platensis) by pressure measurement

An on-line measurement technique for estimating biomass production rate in a photosynthetic micro-organism culture was developed and tested experimentally. The technique is based on monitoring O₂ production from the increase in pressure inside a closed photobioreactor. The data obtained by this method correlated with the direct measurement of the biomass concentration. A material balance on the components in the system allows the validity domain of the method to be defined. The method was applied to batch cultures of the cyanobacterium, Spirulina platensis, in a cylindrical photobioreactor validating existing physiological and light energy models.     

Optimal temperature and photoperiod for the cultivation of Agardhiella subulata microplantlets in a bubble-column photobioreactor

The optimal temperature and illumination photoperiod requirements for the phototrophic growth of a novel microplantlet suspension culture derived from the macrophytic marine red alga Agardhiella subulata were determined. The optimal growth temperature was 24 degrees C. The effects of illumination light-dark (LD) photoperiod (hour of light:hours of darkness within a 24 h cycle) on biomass production was studied within a bubble-column photobioreactor. The 4.5 cm diameter photobioreactor was maintained at near-saturation conditions with respect to light flux (38 mciromol photons m(-2) s(-1)), nutrient medium delivery (20% nutrient replacement per day), and CO(2) delivery (0.35 mmol CO(2) L(-1) h(-1)) so that the cumulative effects of photodamage on the cell density versus time curve at photoperiods approaching continuous light could be observed. Biomass production was maximized at 16:8 LD, where biomass densities exceeding 3.6 g dry cell mass L(-1) were achieved after 60 days in culture. Biomass production was proportional to photoperiod at low fractional photoperiods (< or =10:14 LD), but high fractional photoperiods approaching continuous light (> or = 20:4 LD) shut down biomass production. Biomass production versus time profiles under resource-saturated cultivation conditions were adequately described by a cumulative photodamage growth model, which coupled reversible photodamage processes to the specific growth rate. Under light-saturated growth conditions, the rate constant for photodamage was kd = 1.17 +/- 0.28 day(-1) (+/-1.0 SE), and the rate constant for photodamage repair was kr = 5.12 +/- 0.95 day(-1) (+/-1.0 SE) at 24 degrees C.     

On-line determination of glucose and lactate concentrations in animal cell culture based on fibre optic detection of oxygen in flow-injection analysis.

A flow-injection analysis (FIA) system based on fibre optic detection of oxygen consumption using immobilized glucose oxidase (GOD) and lactate oxidase (LOD) is described for the on-line monitoring of glucose and lactate concentrations in animal cell cultures. The consumption of oxygen was determined via dynamic quenching by molecular oxygen of the fluorescence of an indicator. GOD and LOD were immobilized on controlled pore glass (CPG) in enzyme reactors which were directly linked to a specially designed fibre optic flow-through cell covering the oxygen optrode. The system is linear for 0-30 mM glucose, with an r.s.d. of 5% at 30 mM (five measurements) and for 0-30 mM lactate, with an r.s.d. of 5% at 30 mM (five measurements). The enzyme reactors used were stable for more than 4 weeks in continuous operation, and it was possible to analyse up to 20 samples per hour. The system has been successfully applied to the on-line monitoring of glucose and lactate concentrations of an animal cell culture designed for the production of recombinant human antithrombine III (AT-III). Results of the on-line measurement obtained by the FIA system were compared with the off-line results obtained by a glucose and lactate analyser from Yellow Springs Instrument Company (YSI).     

Short-term evaluation of the photosynthetic activity of an alkaliphilic microalgae consortium in a novel tubular closed photobioreactor

A novel lab-scale tubular closed photobioreactor was developed and used for the assessment of the photosynthetic activity of an alkaliphilic microalgae mixed consortium under non-substrate limitation (i.e., bicarbonate excess), controlled irradiance, and mixing conditions. Two prominent haloalkaliphilic strains were identified as members of the consortium: Halospirulina sp. and Picochlorum sp. The photobioreactor (vol?=?0.5 L) consists of two interconnected U-shaped borosilicate glass tubes (internal diameter 2 cm) reaching a surface/volume ratio of 200 m² m^?3. This configuration specifically addressed the issue of the homogeneous light distribution among the microalgae suspended cells cultured by using fixed equidistant cool white light LEDs nearby the surface of the glass tubes. A soft homogeneous pneumatic mixing (i.e., airlift) was implemented in the culture fostering Reynolds numbers around 3000. The photosynthetic activity of the microalgae consortium was evaluated during different short-term kinetic assays by fitting the dynamics of the dissolved oxygen concentration to an oxygenic kinetic model. The photobioreactor operated in a closed loop allowed to control the produced oxygen by the extraction of the cumulated gas in the headspace. The use of this novel photobioreactor allowed the photosynthetic activity of microalgae suspended cells to be assessed, where the dissolved oxygen concentration and irradiance were the main parameters affecting the oxygenic rates under alkaline pH.     

Continuous medium perfusion leads to long-term cell viability and oxygen production in high-density photobioreactors

Summary A light-emitting diode-based photobioreactor (LED-based PBR) operated in a continuous perfusion mode with a perfusion rate of 3 to 6 reactor volumes a day supports high-density algal cultures, of cell concentrations up to 4·10⁹ cells/mL, or 25 g/L. The oxygen production rate at its peak was 13 to 15 mmol/(L·h). Continuous medium perfusion allowed for long-term stable oxygen production, while oxygen production in batch mode ceased when stationary phase was reached.     

Sustained hydrogen photoproduction by Chlamydomonas reinhardtii: Effects of culture parameters

The green alga, Chlamydomonas reinhardtii, is capable of sustained H(2) photoproduction when grown under sulfur-deprived conditions. This phenomenon is a result of the partial deactivation of photosynthetic O(2)-evolution activity in response to sulfur deprivation. At these reduced rates of water-oxidation, oxidative respiration under continuous illumination can establish an anaerobic environment in the culture. After 10-15 hours of anaerobiosis, sulfur-deprived algal cells induce a reversible hydrogenase and start to evolve H(2) gas in the light. Using a computer-monitored photobioreactor system, we investigated the behavior of sulfur-deprived algae and found that: (1) the cultures transition through five consecutive phases: an aerobic phase, an O(2)-consumption phase, an anaerobic phase, a H(2)-production phase and a termination phase; (2) synchronization of cell division during pre-growth with 14:10 h light:dark cycles leads to earlier establishment of anaerobiosis in the cultures and to earlier onset of the H(2)-production phase; (3) re-addition of small quantities of sulfate (12.5-50 microM MgSO(4), final concentration) to either synchronized or unsynchronized cell suspensions results in an initial increase in culture density, a higher initial specific rate of H(2) production, an increase in the length of the H(2)-production phase, and an increase in the total amount of H(2) produced; and (4) increases in the culture optical density in the presence of 50 microM sulfate result in a decrease in the initial specific rates of H(2) production and in an earlier start of the H(2)-production phase with unsynchronized cells. We suggest that the effects of sulfur re-addition on H(2) production, up to an optimal concentration, are due to an increase in the residual water-oxidation activity of the algal cells. We also demonstrate that, in principle, cells synchronized by growth under light:dark cycles can be used in an outdoor H(2)-production system without loss of efficiency compared to cultures that up until now have been pre-grown under continuous light conditions.     

Sequential heterotrophic/autotrophic cultivation – An efficient method of producing Chlorella biomass for health food and animal feed

Sequential heterotrophic/autotrophic cultivation method was investigated for production of high concentration of Chlorella biomass with high cellular protein and chlorophyll contents. By using autotrophic growth medium, which contains glucose as organic carbon source, for heterotrophic culture, the protein and chlorophyll contents of the cells could be increased by simply illuminating the culture broth and aerating with CO₂-enriched air at the end of the heterotrophic culture. A system was then constructed for continuous sequential heterotrophic/autotrophic production of algal biomass. The system was composed of the conventional mini-jar fermentor for the heterotrophic phase and a tubular photobioreactor for the autotrophic phase. The exhaust gas from the heterotrophic phase was used for aeration of the autotrophic phase in order to reduce the CO₂ emission into the atmosphere. With this system, it was possible to produce high Chlorella biomass concentration (14 g L-¹) containing 60.1% protein and 3.6% chlorophyll continuously for more than 640 h. During the steady state, about 27% of the CO₂ produced in the heterotrophic phase was re-utilized in the autotrophic phase. When the tubular photobioreactor was replaced with a 3.5-L internally illuminated photobioreactor, the productivity increased from 2 g L-¹ d-¹ to 4 g L-¹ d-¹. However, the chlorophyll content of the cells was lower due to the lower light supply coefficient of the photobioreactor. This revised version was published online in August 2006 with corrections to the Cover Date.     

Optimization of biomass, vitamins, and carotenoid yield on light energy in a flat-panel reactor using the A-stat technique

Acceleration-stat (A-stat) cultivations in which the dilution rate is continuously changed at a constant acceleration rate, leading to different average light intensities inside the photobioreactor, can supply more information and reduce experimental time compared with chemostat cultivations. The A-stat was used to optimize the biomass and product yield of continuous cultures of the microalgae D. tertiolecta in a flat-panel reactor. In this study, four different accelerations were studied, a pseudo steady state was maintained at acceleration rates of 0.00016 and 0.00029 h(-2) and results were similar to those of the chemostat. An increase in the acceleration rate led to an increase in the deviation between results obtained in the A-stat and in the chemostats. We concluded that it is advantageous to use the A-stat instead of chemostats to determine culture characteristics and optimize a specific photobioreactor. The effect of average light intensity inside the photobioreactor on the production of vitamins C and E, lutein, and beta-carotene was studied using the A-stat. The highest concentrations of these products were 3.48 +/- 0.46, 0.33 +/- 0.06, 5.65 +/- 0.24, and 2.36 +/- 0.38 mg g(-1), respectively. These results were obtained at different average light intensities, showing the importance of optimizing each product on light intensity.     

Intermittent light irradiation with second- or hour-scale periods controls anthocyanin production by strawberry cells*

Anthocyanin production by strawberry cells depends not only on light intensity but also on the light/dark cycle operation with hour- or second-scale periods. These findings are useful for designing and operating photobioreactors for enhanced anthocyanin production. Intermittent illumination with a second-scale period produces the same amount of anthocyanin as continuous light, suggesting that the light intensity distribution within a photobioreactor does not cause suppressed production. In the hour-scale cycle, continuous light operation enhanced anthocyanin production more than the light/dark cycle process.     

Analysis of light regime in continuous light distributions in photobioreactors

Maximum photobioreactor (PBR) efficiency is a must in applications such as the obtention of microalgae-derived fuels. Improving PBR performance requires a better understanding of the "light regime", the varying irradiance that microalgal cells moving in a dense culture are exposed to. We propose a definition of light regime that can be used consistently to describe the continuously varying light patterns in PBRs as well as in light/dark cycles. Equivalent continuous and light/dark regimes have been experimentally compared and the results show that continuous variations are not well represented by light/dark cycles, as had been widely accepted. It has been shown that a correct light regime allows obtaining photosynthetic rates higher than the corresponding to continuous light, the so-called "flashing light effect" and that this is possible in commercial PBRs. A correct PBR operation could result in photosynthetic efficiency close to the optimum eight quanta per O(2).