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Cyanobacteria and microalgae present in the aquatic or terrestrial environment may be emitted into the air and transported along with air masses over long distances. As a result of staying in the atmosphere, these organisms may develop a greater tolerance to stressful factors, but this topic is still relatively unknown. The main aim was to show an autecological characteristic of some airborne microalgae and cyanobacteria strains by a factorial laboratory experiment approach, including changes in irradiance, temperature, and salinity conditions. The additional purpose of this work was also to present part of the Culture Collection of Baltic Algae (CCBA) collection, which consists of airborne algae (AA) isolated from the atmospheric air of the southern Baltic Sea region. Altogether, 61 strains of airborne cyanobacteria and microalgae from the southern Baltic Sea region were isolated from May 2018 to August 2020. Selected microorganisms were tested in controlled laboratory conditions to identify their response to different irradiance (10-190 µmol photons m -2 s -1 ), temperature (13-23 °C), and salinity conditions (0-36 PSU). The highest numbers of cells (above 30 × 10 5 cell mL -1 ) were recorded for cyanobacterium Nostoc sp., and for diatoms Nitzschia sp., Amphora sp., and Halamphora sp. We found that for cyanobacterium Nostoc sp. as well as for green alga Coccomyxa sp. the maximum cell concentrations were recorded at the salinity of 0 PSU. Moreover, cyanobacteria Planktolyngbya contorta , Pseudanabaena catenata , Leptolyngbya foveolarum , Gloeocapsa sp., and Rivularia sp. were able to grow only at a salinity of 0 PSU. On the other hand, in the range of 16-24 PSU, the highest cell numbers of examined diatoms have been identified. Our research provided that deposited airborne microalgae and cyanobacteria showed full colonization potential. The present experiment suggests that the adaptive abilities of microorganisms, in particular those producing toxins, may contribute to the spread in the future. Thus, it may increase human exposure to their negative health effects. Any distinctive adaptations of the genera give them an additional competitive advantage and a greater chance for territorial expansion.

The presence, biosynthesis and functional role of sterols in the green microalga Haematococcus pluvialis remain poorly understood. In this work we studied the effect of high-light (HL) stress on sterol synthesis in H. pluvialis UTEX 2505 cells. HL stress induced the synthesis of sterols in parallel with that of triacylglycerides (TAG), giving rise to the synthesis of cholesterol over that of phytosterols. Blockage of the carotenogenic 1-deoxy-D-xylulose 5-phosphate (MEP) pathway is shown to be involved in HL-induced sterol synthesis. In addition, high irradiance exposure induced MEP- and fatty acid (FA)-biosynthetic transcripts. The pharmacological inhibition of these pathways suggests a possible feedback regulation of sterol and FA homeostasis. Finally, both lipid classes proved crucial to the adequate photosynthetic performance of H. pluvialis grown under HL intensity stress. Our findings reveal new insights into H. pluvialis lipid metabolism that contribute to the development of value-added bioproducts from microalgae., Competing Interests: Declaration of competing interest All authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Accumulation of high-value products in microalgae is not conducive with rapid cell growth, which is the potential conflict in microalgal production. Overcoming such conflict faces numerous challenges in comprehensively understanding cell behavior and metabolism. Here, we show a fully integrated interaction between cell behavior, carbon partitioning, carbon availability and path rate of central carbon metabolism, and have practically overcome the production conflict of Chromochloris zofingiensis. We demonstrate that elevated carbon availability and active path rate of precursors are determinants for product biosynthesis, and the former exhibits a superior potential. As protein content reaches a threshold value to confer survival advantages, carbon availability becomes the major limiting factor for product biosynthesis and cell reproduction. Based on integrated interaction, regulating the C/N balance by feeding carbon source under excess light increases content of high-value products without inhibiting cell growth. Our findings provide a new orientation to achieve great productivity improvements in microalgal production.

Cell disruption and protein solubilization protocols for the relative quantification of individual subunits in photosystems were developed for photosynthetic organisms including cyanobacterium Synechocystis sp. PCC 6803, green-algae Chlamydomonas reinhardtii, and seed plant Arabidopsis thaliana. The optimal methods for the disruption of Chlamydomonas, Synechocystis, and Arabidopsis cells were sonication, microbeads (Φ approximately 0.1 mm), and large beads (Φ = 5.0 mm), respectively. Extraction of the total proteins exceeded 90% using each optimal cell disruption method. Solubilization efficiency of membrane proteins was improved by the phase transfer surfactant (PTS) method. Ninety seven and 114 proteins from Chlamydomonas and Synechocystis, respectively, including membrane proteins such as photosystem proteins, ATP synthase, and NADH dehydrogenase, were successfully analyzed by nano-liquid chromatography tandem mass spectrometry. These results also indicated the improved efficiency of solubilization and trypsin digestion using PTS buffer. The results of the relative quantitative evaluation of photosystem subunits in Chlamydomonas and Synechocystis grown under high-light conditions were consistent with those of previous studies. Thus, the optimal cell disruption and PTS methods allow for comprehensive relative quantitative proteome analysis of photosynthetic organisms. Additionally, NdhD1 and NdhF1, which are NDH-1 subunit homologs, were increased under high-light conditions, suggesting that the NDH-1L complex, including NdhD1 and NdhF1, is increased under high-light conditions. The relative quantitative proteome analysis of individual subunits indicates the diverse functions of NDH-1 protein., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Background: Photosynthetic microalgae have been in the spotlight of biotechnological production (biofuels, lipids, etc), however, current barriers in mass cultivation of microalgae are limiting its successful industrialization. Therefore, a mathematical model integrating both the biological and hydrodynamical parts of the cultivation process may improve our understanding of relevant phenomena, leading to further optimization of the microalgae cultivation., Results: We introduce a unified multidisciplinary simulation tool for microalgae culture systems, particularly the photobioreactors. Our approach describes changes of cell growth determined by dynamics of heterogeneous environmental conditions such as irradiation and mixing of the culture. Presented framework consists of (i) a simplified model of microalgae growth in a culture system (the advection-diffusion-reaction system within a phenomenological model of photosynthesis and photoinhibition), (ii) the fluid dynamics (Navier-Stokes equations), and (iii) the irradiance field description (Beer-Lambert law). To validate the method, a simple case study leading to hydrodynamically induced fluctuating light conditions was chosen. The integration of computational fluid dynamics (ANSYS Fluent) revealed the inner property of the system, the flashing light enhancement phenomenon, known from experiments., Conclusion: Our physically accurate model of microalgae culture naturally exhibits features of real system, can be applied to any geometry of microalgae mass cultivation and thus is suitable for biotechnological applications.

Lutein is a high-value bioproduct synthesized by microalga Desmodesmus sp. It has great potential for the food, cosmetics, and pharmaceutical industries. However, in order to enhance its productivity and to fulfil its ever-increasing global market demand, it is vital to construct accurate models capable of simulating the entire behavior of the complicated dynamics of the underlying biosystem. To this aim, in this study two highly robust artificial neural networks (ANNs) are designed for the first time. Contrary to conventional ANNs, these networks model the rate of change of the dynamic system, which makes them highly relevant in practice. Different strategies are incorporated into the current research to guarantee the accuracy of the constructed models, which include determining the optimal network structure through a hyper-parameter selection framework, generating significant amounts of artificial data sets by embedding random noise of appropriate size, and rescaling model inputs through standardization. Based on experimental verification, the high accuracy and great predictive power of the current models for long-term dynamic bioprocess simulation in both real-time and offline frameworks are thoroughly demonstrated. This research, therefore, paves the way to significantly facilitate the future investigation of lutein bioproduction process control and optimization. In addition, the model construction strategy developed in this research has great potential to be directly applied to other bioprocesses. Biotechnol. Bioeng. 2017;114: 2518-2527. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

The desert microalga Chlorella ohadii was reported to grow at extreme light intensities with minimal photoinhibition, tolerate frequent de/re-hydrations, yet minimally employs antenna-based non-photochemical quenching for photoprotection. Here we investigate the molecular mechanisms by measuring Photosystem II charge separation yield (chlorophyll variable fluorescence, Fv/Fm) and flash-induced O 2 yield to measure the contributions from both linear (PSII-LEF) and cyclic (PSII-CEF) electron flow within PSII. Cells grow increasingly faster at higher light intensities (μE/m 2 /s) from low (20) to high (200) to extreme (2000) by escalating photoprotection via shifting from PSII-LEF to PSII-CEF. This shifts PSII charge separation from plastoquinone reduction (PSII-LEF) to plastoquinol oxidation (PSII-CEF), here postulated to enable proton gradient and ATP generation that powers photoprotection. Low light-grown cells have unusually small antennae (332 Chl/PSII), use mainly PSII-LEF (95%) and convert 40% of PSII charge separations into O 2 (a high O 2 quantum yield of 0.06mol/mol PSII/flash). High light-grown cells have smaller antenna and lower PSII-LEF (63%). Extreme light-grown cells have only 42 Chl/PSII (no LHCII antenna), minimal PSII-LEF (10%), and grow faster than any known phototroph (doubling time 1.3h). Adding a synthetic quinone in excess to supplement the PQ pool fully uncouples PSII-CEF from its natural regulation and produces maximum PSII-LEF. Upon dark adaptation PSII-LEF rapidly reverts to PSII-CEF, a transient protection mechanism to conserve water and minimize the cost of antenna biosynthesis. The capacity of the electron acceptor pool (plastoquinone pool), and the characteristic times for exchange of (PQH 2 ) B with PQ pool and reoxidation of (PQH 2 ) pool were determined., (Copyright © 2017. Published by Elsevier B.V.)

A density in algal suspension causes a significant change in the intensity and spectral composition of light reaching individual cells. Measurements of chlorophyll fluorescence allow us to observe any general changes in the bioenergetic status of photosynthesis. The aim of the study was to determine the effect of cultivation density on the PSII photochemical efficiency of three species of algae (Chlorella vulgaris, Botryococcus braunii and Chlorella emersonii), each with a different rate of growth - high, medium and low - respectively. The cell density of algae in suspension differentiated through the cultivation time (2, 4, and 8days) and the spectral composition of light. The results showed that the density of cultivation led to change in the photosynthetic apparatus of algae. The differences described between each day of cultivation (2, 4, and 8) in the kinetics of chlorophyll a fluorescence intensity in cells of the algal strains under study probably resulted from the different phases of growth of these cultures. In addition the results showed the beneficial effect of far red light on the photosynthetic apparatus and the growth of biomass in investigated algal strains., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Mathematical modeling is becoming ever more important to assess the potential, guide the design, and enable the efficient operation and control of industrial-scale microalgae culture systems (MCS). The development of overall, inherently multiphysics, models involves coupling separate submodels of (i) the intrinsic biological properties, including growth, decay, and biosynthesis as well as the effect of light and temperature on these processes, and (ii) the physical properties, such as the hydrodynamics, light attenuation, and temperature in the culture medium. When considering high-density microalgae culture, in particular, the coupling between biology and physics becomes critical. This chapter reviews existing models, with a particular focus on the Droop model, which is a precursor model, and it highlights the structure common to many microalgae growth models. It summarizes the main developments and difficulties towards multiphysics models of MCS as well as applications of these models for monitoring, control, and optimization purposes.

Mathematical modeling and the development of predictive dynamic models are of paramount importance for the optimization, state estimation, and control of bioprocesses. This study is dedicated to the identification of a simple model of microalgae growth under substrate limitation, i.e., Droop model, and describes the design and instrumentation of a lab-scale flat-plate photobioreactor, the associated on-line and off-line instrumentation, the collection of experimental data, and the parameter identification procedure. In particular, a dedicated methodology for parameter identification is discussed, including the determination of an initial parameter set using an analytical procedure, the selection of a cost function, the evaluation of confidence intervals as well as direct and cross-validation tests.

Microalgae are capable of acclimating to changes in light and ultraviolet radiation (UVR, 280-400 nm). However, little is known about how the ecologically important coccolithophore Emiliania huxleyi responds to UVR when acclimated to different light regimes. Here, we grew E. huxleyi under indoor constant light or fluctuating sunlight with or without UVR, and investigated its growth, photosynthetic performance and pigmentation. Under the indoor constant light regime, the specific growth rate (μ) was highest, while fluctuating outdoor solar radiation significantly decreased the growth rate. Addition of UVR further decreased the growth rate. The repair rate of photosystem II (PSII), as reflected in changes in PSII quantum yield, showed an inverse correlation with growth rate. Cells grown under the indoor constant light regime exhibited the lowest repair rate, while cells from the outdoor fluctuating light regimes significantly increased their repair rate. Addition of UVR increased both the repair rate and intracellular UV-absorbing compounds. This increased repair capability, at the cost of decreased growth rate, persisted after the cells were transferred back to the indoor again, suggesting an enhanced allocation of energy and resources for repair of photosynthetic machinery damage by solar UVR which persisted for a period after transfer from solar UVR., (© 2014 The American Society of Photobiology.)

The combined use of high rate algal ponds (HRAPs) for wastewater treatment and commercial algal production is considered to be an economically viable option. However, microalgal photosynthesis and biomass productivity is constrained in HRAPs due to light limitation. This paper investigates how the light climate in the HRAP can be modified through changes in pond depth, hydraulic retention time (HRT) and light/dark turnover rate and how this impacts light absorption and utilisation by the microalgae. Wastewater treatment HRAPs were operated at three different pond depth and HRT during autumn. Light absorption by the microalgae was most affected by HRT, significantly decreasing with increasing HRT, due to increased internal self-shading. Photosynthetic performance (as defined by Pmax, Ek and α), significantly increased with increasing pond depth and decreasing HRT. Despite this, increasing pond depth and/or HRT, resulted in decreased pond light climate and overall integrated water column net oxygen production. However, increased light/dark turnover was able to compensate for this decrease, bringing the net oxygen production in line with shallower ponds operated at shorter HRT. On overcast days, modelled daily net photosynthesis significantly increased with increased light/dark turnover, however, on clear days such increased turnover did not enhance photosynthesis. This study has showed that light absorption and photosynthetic performance of wastewater microalgae can be modified through changes to pond depth, HRT and light/dark turnover., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Microalgae is largely used in aquaculture as feed. More recently, these microorganisms have been considered as an important feedstock for biodiesel production. However, the concentration of produced biomass represents a large parcel of production costs. In this study, we have evaluated the influence of natural pH variation of culture medium, caused by photosynthetic activity, on the flocculation of the marine microalgae Nannochloropsis oculata. Experiments were conducted with the same culture with different pH values (8.5 and 9.6), obtained after exposing the cells to different light conditions. For each pH value, different treatments were composed by adding 0, 5, 10, and 30 mM of NaOH and the flocculant Flopam® (FO4800 SH) at concentrations of 0, 0.5, 1, and 5 ppm. Higher flocculation efficiencies were obtained for the culture with pH 9.6 in comparison to 8.5 for the same NaOH and Flopam concentrations. Lower concentrations of base and flocculant were needed for flocculating the culture in higher pH, representing an economy of 20 % in the costs of crop harvesting.

Under specific conditions, flashing light enhances the photosynthesis rate in comparison to continuous illumination. Here we show that a combination of flashing light and continuous background light with the same integrated photon dose as continuous or flashing light alone can be used to significantly enhance photosynthesis and increase microalgae growth. To test this hypothesis, the green microalga Dunaliella salina was exposed to three different light regimes: continuous light, flashing light, and concomitant application of both. Algal growth was compared under three different integrated light quantities; low, intermediate, and moderately high. Under the combined light regime, there was a substantial increase in all algal growth parameters, with an enhanced photosynthesis rate, within 3days. Our strategy demonstrates a hitherto undescribed significant increase in photosynthesis and algal growth rates, which is beyond the increase by flashing light alone., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

We recently evaluated the relationship between abiotic environmental stresses and lutein biosynthesis in the green microalga Dunaliella salina and suggested a rational design of stress-driven adaptive evolution experiments for carotenoids production in microalgae. Here, we summarize our recent findings regarding the biotechnological production of carotenoids from microalgae and outline emerging technology in this field. Carotenoid metabolic pathways are characterized in several representative algal species as they pave the way for biotechnology development. The adaptive evolution strategy is highlighted in connection with enhanced growth rate and carotenoid metabolism. In addition, available genetic modification tools are described, with emphasis on model species. A brief discussion on the role of lights as limiting factors in carotenoid production in microalgae is also included. Overall, our analysis suggests that light-driven metabolism and the photosynthetic efficiency of microalgae in photobioreactors are the main bottlenecks in enhancing biotechnological potential of carotenoid production from microalgae.

This study demonstrates the simultaneous production of bioelectricity and added-value pigments in a Photosynthetic Alga Microbial Fuel Cell (PAMFC). A PAMFC was operated using Chlorella vulgaris in the cathode compartment and a bacterial consortium in the anode. The system was studied at two different light intensities and the maximum power produced was 62.7 mW/m(2) with a light intensity of 96 μE/(m(2)s). The results showed that increasing light intensity from 26 to 96 μE/(m(2)s) leads to an increase of about 6-folds in the power produced. Additionally, the pigments produced by the microalga were analysed and the results showed that the light intensity and PAMFC operation potentiated the carotenogenesis in the cathode compartment. The demonstrated possibility of producing added-value microalgae biomass in microbial fuel cell cathodes will increase the economic feasibility of these bioelectrochemical systems, allowing the development of energy efficient systems for wastewater treatment and carbon fixation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

National interest and environmental advocates encourage alternatives to petroleum-based products. Besides biofuels, many other valuable chemicals used in every-day life are petroleum derivatives or require petroleum for their production. A plausible alternative to production using petroleum for chemical production is to harvest the abundant carbon dioxide resources in the environment to produce valuable hydrocarbons. Currently, efforts are being made to utilize a natural biological system, photosynthetic microorganisms, to perform this task. Photosynthetic microorganisms are attractive to use for biochemical production because they utilize economical resources for survival: sunlight and carbon dioxide. This review examines the various compounds produced by photosynthetic microorganisms., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In this paper, the influence of culture conditions (irradiance, temperature, pH, and dissolved oxygen) on the photosynthesis rate of Scenedesmus almeriensis cultures is analyzed. Short-run experiments were performed to study cell response to variations in culture conditions, which take place in changing environments such as outdoor photobioreactors. Experiments were performed by subjecting diluted samples of cells to different levels of irradiance, temperature, pH, and dissolved oxygen concentration. Results demonstrate the existence of photoinhibition phenomena at irradiances higher than 1,000 μE/m(2) s; in addition to reduced photosynthesis rates at inadequate temperatures or pH-the optimal values being 35 °C and 8, respectively. Moreover, photosynthesis rate reduction at dissolved oxygen concentrations above 20 mg/l is demonstrated. Data have been used to develop an integrated model based on considering the simultaneous influence of irradiance, temperature, pH, and dissolved oxygen. The model fits the experimental results in the range of culture conditions tested, and it was validated using data obtained by the simultaneous variation of two of the modified variables. Furthermore, the model fits experimental results obtained from an outdoor culture of S. almeriensis performed in an open raceway reactor. Results demonstrate that photosynthetic efficiency is modified as a function of culture conditions, and can be used to determine the proximity of culture conditions to optimal values. Optimal conditions found (T = 35 °C, pH = 8, dissolved oxygen concentration <20 mg/l) allows to maximize the use of light by the cells. The developed model is a powerful tool for the optimal design and management of microalgae-based processes, especially outdoors, where the cultures are subject to daily culture condition variations.

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.

The effect of light conditions on the growth of green algae Chlorella vulgaris and cyanobacteria Gloeothece membranacea was investigated by filtering different wavelengths of visible light and comparing against a model daylight source as a control. Luminescent acrylic sheets containing violet, green, orange or red dyes illuminated by a solar simulator produced the desired wavelengths of light for this study. From the experimental results the highest specific growth rate for C. vulgaris was achieved using the orange range whereas violet light promoted the growth of G. membranacea. Red light exhibited the least efficiency in conversion of light energy into biomass in both strains of microalgae. Photosynthetic pigment formation was examined and maximum chlorophyll-a production in C. vulgaris was obtained by red light illumination. Green light yielded the best chlorophyll-a production in G. membranacea. The proposed illumination strategy offers improved microalgae growth without resorting to artificial light sources, reducing energy use and costs of cultivation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In this study, an extension and actualization of a dynamic model of photosynthesis, previously published (Camacho Rubio et al., 2003), is presented. This model uses the concept of Photosynthetic Unit (PSU). In it, the processes of excited PSU net disappearance rate, photoinhibition and damaged PSU repair have been redefined in the context of photochemical and non-photochemical quenching. The phenomenon of photoacclimation in microalgae has been extensively incorporated into this model, significantly improving its prediction capabilities. A significant number of previously reported experimental results are successfully interpreted by the novel formulation: 1. Photosynthetic response of cells photoacclimated to different constant continuous irradiances (photoacclimated cells); 2. Short-term photosynthetic response of cells non-photoacclimated to different constant continuous irradiances (non-photoacclimated cells); 3. Kinetics of the photoacclimation response; 4. Photosynthetic response under intermittent light. It is expected that this model will contribute notably to the simulation of industrial algal mass cultures., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

The metabolic flexibility of some photosynthetic microalgae enables them to survive periods of anaerobiosis in the light by developing a particular photofermentative metabolism. The latter entails compounds of the photosynthetic electron transfer chain and an oxygen-sensitive hydrogenase in order to reoxidize reducing equivalents and to generate ATP for maintaining basal metabolic function. This pathway results in the photo-evolution of hydrogen gas by the algae. A decade ago, Melis and coworkers managed to reproduce such a condition in a laboratory context by depletion of sulfur in the algal culture media, making the photo-evolution by the algae sustainable for several days (Melis et al. in Plant Physiol 122:127-136, 2000). This observation boosted research in algal H(2) evolution. A feature, which due to its transient nature was long time considered as a curiosity of algal photosynthesis suddenly became a phenomenon with biotechnological potential. Although the Melis procedure has not been developed into a biotechnological process of renewable H(2) generation so far, it has been a useful tool for studying microalgal metabolic and photosynthetic flexibility and a possible step stone for future H(2) production procedures. Ten years later most of the critical steps and limitations of H(2) production by this protocol have been studied from different angles particularly with the model organism Chlamydomonas reinhardtii, by introducing various changes in culture conditions and making use of mutants issued from different screens or by reverse genomic approaches. A synthesis of these observations with the most important conclusions driven from recent studies will be presented in this review.