Modeling microalgae cultivation productivities in different geographic locations - estimation method for idealized photobioreactors

Microalgae can be used to produce versatile high-value fuels, such as methane, biodiesel, ethanol, or hydrogen gas. One of the most important factors that influence the economics of microalgae cultivation is the primary production of biomass per unit area. This is determined by productivity rates during cultivation, which are influenced by the local climate conditions (solar irradiation, temperature). To compare locations in different climate regions for microalgae cultivation, a mathematical model for an idealized closed photobioreactor was developed. The applied growth kinetics were based on theoretical maximum photon-conversion efficiencies (for the conversion of solar energy to chemical energy in the form of biomass). Known or estimated temperature effects for different algal strains were incorporated. The model was used to calculate hourly average areal productivity rates as well as annual primary production values under local conditions at seven example locations. Here, hourly weather data (solar irradiance and air temperature) were taken into account. According to these model calculations, maximum annual yields were achieved in regions with high irradiation and temperature patterns in or near the optimum range of the specific algal strain (here, desert and equatorial humid climates). The developed model can be used as a tool to assess and compare individual locations for microalgae cultivation.     

Metabolic engineering of Cyanobacteria and microalgae for enhanced production of biofuels and high‐value products

A lot of research has been performed on Cyanobacteria and microalgae with the aim to produce numerous biotechnological products. However, native strains have a few shortcomings, like limitations in cultivation, harvesting and product extraction, which prevents reaching optimal production value at lowest costs. Such limitations require the intervention of genetic engineering to produce strains with superior properties. Promising advancements in the cultivation of Cyanobacteria and microalgae have been achieved by improving photosynthetic efficiency through increasing RuBisCO activity and truncation of light‐harvesting antennae. Genetic engineering has also contributed to final product extraction by inducing autolysis and product secretory systems, to enable direct product recovery without going through costly extraction steps. In this review, we summarize the different enzymes and pathways that have been targeted thus far for improving cultivation aspects, harvesting and product extraction in Cyanobacteria and microalgae. With synthetic biology advancements, genetically engineered strains can be generated to resolve demanding process issues and achieve economic practicality. This comprehensive overview of gene modifications will be useful to researchers in the field to employ on their strains to increase their yields and improve the economic feasibility of the production process.     

Microalgal biotechnology: Carotenoid production by the green algae<Emphasis Type="Italic">Dunaliella salina</Emphasis>

Unicellular green algae of the genusDunaliella thrive in extreme environmental conditions such as high salinity, low pH, high irradiance and subzero temperatures. Species ofDunaliella are well known in the alga biotechnological industry and are employed widely for the production of valuable biochemicals, such as carotenoids. Some strains ofDunaliella are cultivated commercially in large outdoor ponds and are harvested to produce dry algal meals, such as polyunsaturated fatty acids and oils for the health food industry, and coloring agents for the food and cosmetic industries. During the past decade, the advances in molecular biology and biochemistry of microalgae, along with the advances in biotechnology of microalgal mass cultivation, enabled this microalga to become a staple of commercial exploitation. In particular, the advent of molecular biology and mutagenesis inDunaliella has permitted enhancements in the carotenoids content of this green alga, making it more attractive for biotechnological applications. Accordingly, the present review summarizes the recent developments and advances in biotechnology of carotenoid production inDunaliella.     

Isolation of a new strain of Picochlorum sp and characterization of its potential biotechnological applications

Selection of new autochthon strains is necessary, and for the moment the best strategy, to find microalgae well adapted to the local climatological conditions able to simultaneously produce several compounds of biotechnological interest and grow at high rates. We describe the isolation and characterization of a new microalgal strain isolated from the marshlands of the Odiel River in the Southwest of Spain. The new microalga belongs to the genus Picochlorum, as deduced from the analysis of its 18S rRNA encoding gene, is able to grow at a high growth rate and thrive with adverse conditions. It has an appreciable constitutive level of lutein (3.5 mg g(-1) DW) and zeaxanthin (0.4 mg g(-1) DW) which is increased to 1.8 mg g(-1) DW at high light intensities. This strain is also characterized by a very low level of linolenic acid (3.8% of total fatty acids) and no polyunsaturated fatty acids with four or more double bonds. Although the total lipid content is not particularly high, 23% of the dry weight, its fatty acid profile makes of Picochlorum sp HM1 a promising candidate for biodiesel production, and the high content in the carotenoids lutein and zeaxanthin indicates that the microalga could also be a good source for natural eye vitamin supplements, which could be obtained as co-products.     

Microalgae for high-value compounds and biofuels production: A review with focus on cultivation under stress conditions

Microalgal biomass as feedstock for biofuel production is an attracting alternative to terrestrial plant utilization for biofuels production. However, today the microalgal cultivation systems for energy production purposes seem not yet to be economically feasible. Microalgae, though cultivated under stress conditions, such as nutrient starvation, high salinity, high temperature etc. accumulate considerable amounts (up to 60–65% of dry weight) of lipids or carbohydrates along with several secondary metabolites. Especially some of the latter are valuable compounds with an enormous range of industrial applications. The simultaneous production of lipids or carbohydrates for biofuel production and of secondary metabolites in a biorefinery concept might allow the microalgal production to be economically feasible. This paper aims to provide a review on the available literature about the cultivation of microalgae for the accumulation of high-value compounds along with lipids or carbohydrates focusing on stress cultivation conditions.     

Engineering solutions for open microalgae mass cultivation and realistic indoor simulation of outdoor environments

Microalgae could become an important renewable source for chemicals, food, and energy if process costs can be reduced. In the past 60 years, relevant factors in open outdoor mass cultivation of microalgae were identified and elaborate solutions regarding bioprocesses and bioreactors were developed. An overview of these solutions is presented. Since the cost of most microalgal products from current mass cultivation systems is still prohibitively high, further development is required. The application of complex computational techniques for cost-effective process and reactor development will become more important if experimental validation of simulation results can easily be achieved. Due to difficulties inherent to outdoor experimentation, it can be useful to conduct validation experiments indoors. Considerations and approaches for realistic indoor reproduction of the most important environmental conditions in microalgae cultivation experiments—light, temperature, and substance concentrations, are discussed.     

Best practices in heterotrophic high-cell-density microalgal processes: achievements,potential and possible limitations

Microalgae of numerous heterotrophic genera (obligate or facultative) exhibit considerable metabolic versatility and flexibility but are currently underexploited in the biotechnological manufacturing of known plant-derived compounds, novel high-value biomolecules or enriched biomass. Highly efficient production of microalgal biomass without the need for light is now feasible in inexpensive, well-defined mineral medium, typically supplemented with glucose. Cell densities of more than 100 g l⁻¹ cell dry weight have been achieved with Chlorella, Crypthecodinium and Galdieria species while controlling the addition of organic sources of carbon and energy in fedbatch mode. The ability of microalgae to adapt their metabolism to varying culture conditions provides opportunities to modify, control and thereby maximise the formation of targeted compounds with non-recombinant microalgae. This review outlines the critical aspects of cultivation technology and current best practices in the heterotrophic high-cell-density cultivation of microalgae. The primary topics include (1) the characteristics of microalgae that make them suitable for heterotrophic cultivation, (2) the appropriate chemical composition of mineral growth media, (3) the different strategies for fedbatch cultivations and (4) the principles behind the customisation of biomass composition. The review confirms that, although fundamental knowledge is now available, the development of efficient, economically feasible large-scale bioprocesses remains an obstacle to the commercialisation of this promising technology.     

Profiling whole microalgal cells by high-resolution magic angle spinning (HR-MAS) magnetic resonance spectroscopy

Microalgae are a rich source of high value compounds such as carbohydrates, lipids, proteins and bioactive compounds. In particular, microalgae have been identified as a potentially important resource for carbon-capture and as a feedstock for green biofuels. Successful cultivation of microalgae can occur under a variety of nutrient and environmental conditions with each condition producing a unique distribution of compounds. In order to steer the cultivation towards a particular distribution of compounds, rapid and accurate methods for compound identification are required. Current methods for determining the absolute quantity of each component are time consuming and arduous making cultivation optimization impractical. High-resolution magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy offers a robust and rapid screening method capable of ascertaining the absolute quantity of each component with minimal sample manipulation. Sample preparation consists of harvested, centrifuged and freeze-dried whole-cell Nannochloropsis granulata from large-scale photobioreactors being accurately weighed and rehydrated with deuterium oxide and placed in an HR-MAS rotor. One-dimensional HR-MAS NMR spectra were recorded under quantitative conditions to determine the lipid and carbohydrate profile of the microalgae. The total time per sample for preparation, data acquisition and analysis was approximately 1 h. Changes in resonance profiles corresponding to varying proportions of saturated and polyunsaturated fatty acids were correlated to the time of harvest. In addition, standard two dimensional experiments were used to identify the major carbohydrate components. HR-MAS NMR spectroscopy has been used to profile the lipid and carbohydrate content of N. granulata and we have begun to establish methodologies for quality analysis/quality control for cultivation of various microalgal strains.     

Adaptation of growth and photosynthesis to certain temperature regimes is an indicator for the geographical distribution of Cosmarium strains (Zygnematophyceae,Streptophyta)

In contrast to the many investigations on possible relationships between climate and geographical distributions in macroalgae, there are almost no similar studies regarding microalgae. In this study we consider the potential influence of temperature on patterns of distribution of six Cosmarium strains isolated from various climate zones that have been cultured long-term (> 15 years) in a relatively low temperature–low light regime. Growth and photosynthetic parameters, obtained from PAM fluorometry, were used to estimate the physiological characteristics of the strains during and after various temperature treatments. Acclimation to constant temperature and light conditions tended to affect photosynthetic parameters more than algal growth characteristics. However, all of the Cosmarium strains demonstrated physiological responses that were consistent with their source location under both low and high temperature conditions, confirming that such responses are genetically preserved. The Cosmarium strains displayed photosynthetic capacities and levels of the onset of saturation that repeatedly exceeded values recorded for other microalgae and seaweeds, indicating that these desmid strains are adapted to high light. This observation, as well as the relatively high growth temperature optima for all of the Cosmarium strains, provides some support for Coesel's hypothesis on the origin of desmids in the tropical zone. Interestingly, the Cosmarium strains used in this study demonstrated not only adaptive characteristics in accordance with the temperature prevailing at their sampling sites, but also with regard to their evolutionary origin.     

A novel genetic engineering platform for the effective management of biological contaminants for the production of microalgae

Microalgal cultivation that takes advantage of solar energy is one of the most cost‐effective systems for the biotechnological production of biofuels, and a range of high value products, including pharmaceuticals, fertilizers and feed. However, one of the main constraints for the cultivation of microalgae is the potential contamination with biological pollutants, such as bacteria, fungi, zooplankton or other undesirable microalgae. In closed bioreactors, the control of contamination requires the sterilization of the media, containers and all materials, which increases the cost of production, whereas open pond systems severely limits the number of species that can be cultivated under extreme environmental conditions to prevent contaminations. Here, we report the metabolic engineering of Chlamydomonas reinhardtii to use phosphite as its sole phosphorus source by expressing the ptxD gene from Pseudomonas stutzeri WM88, which encodes a phosphite oxidoreductase able to oxidize phosphite into phosphate using NAD as a cofactor. Engineered C. reinhardtii lines are capable of becoming the dominant species in a mixed culture when fertilized with phosphite as a sole phosphorus source. Our results represent a new platform for the production of microalgae, potentially useful for both closed photobioreactors and open pond systems without the need for using sterile conditions nor antibiotics or herbicides to prevent contamination with biological pollutants.     

Biological constraints in algal biotechnology

In the past decade, considerable progress has been made in developing the appropriate biotechnology for microalgal mass cultivation aimed at establishing a new agro-industry. This review points out the main biological constraints affecting algal biotechnology outdoors and the requirements for making this biotechnology economically viable. One of them is the availability of a wide variety of algal species and improved strains that favorably respond to varying environmental conditions existing outdoors. It is thus just a matter of time and effort before a new methodology like genetic engineering can and will be applied in this field as well. The study of stress physiology and adaptation of microalgae has also an important application in further development of the biotechnology for mass culturing of microalgae. In outdoor cultures, cells are exposed to severe changes in light and temperature much faster than the time scale required for the cells to acclimate. A better understanding of those parameters and the ability to rapidly monitor those conditions will provide the growers with a better knowledge on how to optimize growth and productivity. Induction of accumulation of high value products is associated with stress conditions. Understanding the physiological response may help in providing a better production system for the desired product and, at a later stage, give an insight of the potential for genetic modification of desired strains. The potential use of microalgae as part of a biological system for bioremediation/detoxification and wastewater treatment is also associated with growing the cells under stress conditions. Important developments in monitoring and feedback control of the culture behavior through application of on-line chlorophyll fluorescence technique are in progress. Understanding the process associated with those unique environmental conditions may help in choosing the right culture conditions as well as selecting strains in order to improve the efficiency of the biological process.     

Development of thin-film photo-bioreactor and its application to outdoor culture of microalgae

Photosynthetic microalgae have received much attention as a microbial source of diverse useful biomaterials through CO₂ fixation and various types of photo-bioreactors have been developed for efficient microalgal cultivation. Herein, we developed a novel thin-film photo-bioreactor, which was made of cast polypropylene film, considering outdoor mass cultivation. To develop optimal design of photo-bioreactor, we tested performance of three shapes of thin-film photo-bioreactors (flat, horizontal and vertical tubular shapes) and various parts in the bioreactor. Collectively, vertical tubular bioreactor with H/D ratio 6:1 and cylindrical stainless steel spargers showed the most outstanding performance. Furthermore, the photo-bioreactor was successfully applied to the cultivation of other microalgae such as Chlamydomonas reinhardtii and Chlorella vulgaris. The scalability of photo-bioreactor was confirmed by gradually increasing culture volume from 4 to 25 L and the biomass productivity of each reactor was quite consistent (0.05–0.07 g/L/day) during the cultivation of H. pluvialis under indoor and outdoor conditions. Especially, we also achieved dry cell weight of 4.64 g/L and astaxanthin yield of 218.16 mg/L through long-term cultivation (100 days) under outdoor condition in 15 L photo-bioreactor using Haematococcus pluvialis, which means that the astaxanthin yield from outdoor cultivation is equal or superior to that obtained from controlled indoor condition. Therefore, these results indicate that we can apply this approach to development of optimal photo-bioreactor for the large-scale culture of microalgae and production of useful biomaterials under outdoor condition.     

Heterotrophic production of eicosapentaenoic acid by microalgae

Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid that plays an important role in the regulation of biological functions and prevention and treatment of a number of human diseases such as heart and inflammatory diseases. As fish oil fails to meet the increasing demand for purified EPA, alternative sources are being sought. Microalgae contain large quantities of high-quality EPA and they are considered a potential source of this important fatty acid. Some microalgae can be grown heterotrophically on cheap organic substrate without light. This mode of cultivation can be well controlled and provides the possibility to maximize EPA production on a large scale. Numerous strategies have been investigated for commercial production of EPA by microalgae. These include screening of high EPA-yielding microalgal strains, improvement of strains by genetic manipulation, optimization of culture conditions, and development of efficient cultivation systems. This paper reviews recent advances in heterotrophic production of EPA by microalgae with an emphasis on the use of diatoms as producing organisms.     

Hydrodynamic characteristics and microalgae cultivation in a novel flat‐plate photobioreactor

Flat‐plate photobioreactors (FPPBRs) are widely reported for cultivation of microalgae. In this work, a novel FPPBR mounted with inclined baffles was developed, which can make the fluid produce a “spirality” flow. The flow field and cell trajectory in the photobioreactor were investigated by using computational fluid dynamics. In addition, the cell trajectory was analyzed using a Fast Fourier transformation. The influence of height of the baffles, the angle α between the inclined baffle and fluid inlet flow direction (z), and the fluid inlet velocity on the frequency of flashing light effect and pressure drop were examined to optimize the structure parameters of the inclined baffles and operating conditions of the photobioreactor. The results showed that with inclined baffles built‐in, significant swirl flow could be generated in the FPPBR. In this way, the flashing light effect for microalgal cell could also be achieved and the photosynthesis efficiency of microalgae could be promoted. In outdoor cultivation of freshwater Chlorella sp., the maximum biomass productivity of Chlorella sp. cultivated in the photobioreactor with inclined baffles was 29.94% higher than that of the photobioreactor without inclined baffles. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

MICROALGAE AND CYANOBACTERIA: FOOD FOR THOUGHT1

In non‐Western civilizations, cyanobacteria have been part of the human diet for centuries. Today, microalgae and cyanobacteria are either produced in controlled cultivation processes or harvested from the natural habitats and marketed as food supplements around the world. Cyanobacteria produce a vast array of different biologically active compounds, some of which are expected to be used in drug development. The fact that some of the active components from cyanobacteria potentially have anticancer, antimicrobial, antiviral, anti‐inflammatory, and other effects is being used for marketing purposes. However, introduction of these products in the form of whole biomass for alimentary purposes raises concerns regarding the potential toxicity and long‐term effects on human health. Here, we review data on the use of cyanobacteria and microalgae in human nutrition and searched for available information on legislature that regulates the use of these products. We have found that, although the quality control of these products is most often self‐regulated by the manufacturers, different governmental agencies are introducing strict regulations for placing novel products, such as algae and cyanobacteria, on the market. The existing regulations require these products to be tested for the presence of toxins, such as microcystin; however, other, sometimes novel, toxins remain undetected, and their long‐term effects on human health remain unknown.     

Ultrahigh-cell-density heterotrophic cultivation of the unicellular green microalga Scenedesmus acuminatus and application of the cells to photoautotrophic culture enhance biomass and lipid production

Although production of biodiesels from microalgae is proved to be technically feasible, a commercially viable system has yet to emerge. High-cell-density fermentation of microalgae can be coupled with photoautotrophic cultivation to produce oils. In this study, by optimizing culturing conditions and employing a sophisticated substrate feed control strategy, ultrahigh-cell-density of 286 and 283.5 g/L was achieved for the unicellular alga Scenedesmus acuminatus grown in 7.5-L bench-scale and 1,000-L pilot-scale fermenters, respectively. The outdoor scale-up experiments indicated that heterotrophically grown S. acuminatus cells are more productive in terms of both biomass and lipid accumulation when they are inoculated in photobioreactors for lipid production as compared to the cells originally grown under photoautotrophic conditions. Technoeconomic analysis based on the pilot-scale data indicated that the cost of heterotrophic cultivation of microalgae for biomass production is comparable with that of the open-pond system and much lower than that of tubular PBR, if the biomass yield was higher than 200 g/L. This study demonstrated the economic viability of heterotrophic cultivation on large-scale microalgal inocula production, but ultrahigh-productivity fermentation is a prerequisite. Moreover, the advantages of the combined heterotrophic and photoautotrophic cultivation of microalgae for biofuels production were also verified in the pilot-scale.     

Mutation of microalgae from antifouling sensitivity to antifouling resistance allows phytoplankton dispersal through ships’ biofouling

Marine ecosystems are affected by introduced species including microalgae. We propose that biofouling on ships’ hulls is a potentially important mechanism for microalgae dispersal worldwide. Biofouling samples, for phytoplankton composition analysis, were collected in Spanish Mediterranean ports from the hulls of ships that had completed oceanic journeys from other Mediterranean ports, and long journeys from the Atlantic and Indian Oceans. Samples representing the local population of phytoplankton either in the water column or attached to the biofouling of locally-based ship-hulls were used as controls. A broad variety of microalgae species (including toxic dinoflagellates), which were not present in the local phytoplankton populations were found on the biofouling film of the ships that had been on distant journeys. In spite of the presence of the antifouling paints containing toxic compounds, microalgae were able to rapidly adapt to these non-favourable conditions. Consequently, our study shows that ships’ biofouling seems to be a powerful vector for microalgae dispersal at a global scale due to the capacity of microalgae to attach to the biofouling film and to cope by adaptation mechanisms with antifouling compounds.     

A screening model to predict microalgae biomass growth in photobioreactors and raceway ponds

A microalgae biomass growth model was developed for screening novel strains for their potential to exhibit high biomass productivities under nutrient‐replete conditions in photobioreactors or outdoor ponds. Growth is modeled by first estimating the light attenuation by biomass according to Beer‐Lambert's Law, and then calculating the specific growth rate in discretized culture volume slices that receive declining light intensities due to attenuation. The model uses only two physical and two species‐specific biological input parameters, all of which are relatively easy to determine: incident light intensity, culture depth, as well as the biomass light absorption coefficient and the specific growth rate as a function of light intensity. Roux bottle culture experiments were performed with Nannochloropsis salina at constant temperature (23°C) at six different incident light intensities (10, 25, 50, 100, 250, and 850 µmol/m² s) to determine both the specific growth rate under non‐shading conditions and the biomass light absorption coefficient as a function of light intensity. The model was successful in predicting the biomass growth rate in these Roux bottle batch cultures during the light‐limited linear phase at different incident light intensities. Model predictions were moderately sensitive to minor variations in the values of input parameters. The model was also successful in predicting the growth performance of Chlorella sp. cultured in LED‐lighted 800 L raceway ponds operated in batch mode at constant temperature (30°C) and constant light intensity (1,650 µmol/m² s). Measurements of oxygen concentrations as a function of time demonstrated that following exposure to darkness, it takes at least 5 s for cells to initiate dark respiration. As a result, biomass loss due to dark respiration in the aphotic zone of a culture is unlikely to occur in highly mixed small‐scale photobioreactors where cells move rapidly in and out of the light. By contrast, as supported also by the growth model, biomass loss due to dark respiration occurs in the dark zones of the relatively less well‐mixed pond cultures. In addition to screening novel microalgae strains for high biomass productivities, the model can also be used for optimizing the pond design and operation. Additional research is needed to validate the biomass growth model for other microalgae species and for the more realistic case of fluctuating temperatures and light intensities observed in outdoor pond cultures. Biotechnol. Bioeng. 2013; 110: 1583–1594. © 2012 Wiley Periodicals, Inc.     

Microalgae from the Selenastraceae as emerging candidates for biodiesel production: a mini review

Over the years, microalgae have been identified to be a potential source of commercially important products such as pigments, polysaccharides, polyunsaturated fatty acids and in particular, biofuels. Current demands for sustainable fuel sources and bioproducts has led to an extensive search for promising strains of microalgae for large scale cultivation. Prospective strains identified for these purposes were among others, mainly from the genera Hematococcus, Dunaliella, Botryococcus, Chlorella, Scenedesmus and Nannochloropsis. Recently, microalgae from the Selenastraceae emerged as potential candidates for biodiesel production. Strains from the Selenastraceae such as Monoraphidium sp. FXY-10, M. contortum SAG 47.80, Ankistrodesmus sp. SP2-15 and M. minutum were high biomass and lipid producers when cultivated under optimal conditions. A number of Selenastraceae strains were also reported to be suitable for cultivation in wastewater. This review highlights recent reports on potential strains from the Selenastraceae for biodiesel production and contrasts their biomass productivity, lipid productivity as well as fatty acid profile. Cultivation strategies employed to enhance their biomass and lipid productivity as well as to reduce feedstock cost are also discussed in this paper.     

Pure human urine is a good fertiliser for cucumbers

Human urine obtained from separating toilets was tested as a fertiliser for cultivation of outdoor cucumber (Cucumis sativus L.) in a Nordic climate. The urine used contained high amounts of nitrogen with some phosphorus and potassium, but numbers of enteric microorganisms were low even though urine had not been preserved before sampling. The cucumber yield after urine fertilisation was similar or slightly better than the yield obtained from control rows fertilised with commercial mineral fertiliser. None of the cucumbers contained any enteric microorganisms (coliforms, enterococci, coliphages and clostridia). In the taste assessment, 11 out of 20 persons could recognise which cucumber of three cucumbers was different but they did not prefer one over the other cucumber samples, since all of them were assessed as equally good.