New Biotechnology Volume 00, Number 00 April 2015
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Research Paper
Developing diatoms for value-added products: challenges and opportunities Weiqi Fu1,2,3,4, Kristine Wichuk1 and Sigurður Brynjo´lfsson1,4 Q1 1
Center for Systems Biology, University of Iceland, Reykjavı´k 101, Iceland 2 Q2 3 Division of Science and Math, New York University Abu Dhabi, United Arab Emirates Center for Genomics and Systems Biology (CGSB), New York University Abu Dhabi Institute, Abu Dhabi, United Arab Emirates 4 Q3 Faculty of Industrial Engineering, Mechanical Engineering and Computer Science, School of Engineering and Natural Sciences, University of Iceland, Reykjavı´k 101, Iceland
As a major primary producer in marine environments, diatoms have been considered as promising feedstocks for their applications in functional foods, bioactive pharmaceuticals, and cosmetics. This review focuses on the biotechnology potential of diatoms for value-added products like carotenoids. The impact of abiotic environmental stresses, such as intensity and quality of incident light, nutrient deficiency and silicon depletion, on diatoms has been examined to determine key factors that affect the growth performance and the accumulation of valuable compounds. Previous studies suggested that adaptive evolution could be an efficient method to improve the diatom productivity of valuable compounds. Light emitting diode (LED)-based photobioreactors were introduced and proposed as a promising new technology for producing quality products from diatoms. Currently available molecular biology tools were also summarized and discussed in relation to their application in the production of carotenoids and other valuable products. Taken together, systems biology and synthetic biology approaches have the potential to address the challenges faced while working toward the industrial application of diatoms. Introduction Q4 Marine diatoms are a major group of microalgae, which have been deemed as attractive sources for developing sustainable products such as fine chemicals, plastics and biofuels, a solution for energy crisis and for attempting to address the environmental issues globally [1,2]. The continuing threat of global climate change, caused mainly by the consumption of fossil fuels, calls for displacing existing oil-based industries with innovative and sustainable solutions [3]. The high production of lipids in many species of diatoms has attracted great attention to the potential use of diatoms as a source for biofuels [4]. It is clear that developing bio-based energy supplies, such as diatom biofuels, is also highly strategic as a response to the global energy crisis and the diminishing supply of fossil fuels. The concept of using diatoms for Corresponding author: Fu, W. ([email protected], [email protected]) http://dx.doi.org/10.1016/j.nbt.2015.03.016 1871-6784/ß 2015 Published by Elsevier B.V.
producing biofuels is advantageous in terms of being sustainable and a clean technology. However, biofuels derived from diatoms are not cost-competitive at present and may not be economically feasible in the foreseeable future [5]. Diatoms have also recently been explored as sources of bioactive compounds such as carotenoids due to their economic potential [6]. Carotenoids, based on their chemical structures, can be divided into two categories: (1) the carotenes, such as b-carotene; and (2) the xanthophylls, such as fucoxanthin [3]. It has been reported that many carotenoid species such as fucoxanthin, diadinoxanthin, diatoxanthin and b-carotene are present in the model species Phaeodactylum tricornutum [7]. Among them, b-carotene is a major primary source of vitamin A which is necessary for functions of the retina and has an effect on many tissue types through its action as a regulator of gene expression [8]. In addition, b-carotene also helps protect skin against photoaging due to its antioxidant www.elsevier.com/locate/nbt
Please cite this article in press as: Fu, W. et al., Developing diatoms for value-added products: challenges and opportunities, New Biotechnol. (2015), http://dx.doi.org/10.1016/ j.nbt.2015.03.016
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New Biotechnology Volume 00, Number 00 April 2015
exposure, and heat shock [16,17]. And these abiotic environmental stresses have significant influences on the physiology of diatoms [17]. In this section, representative stress factors including nitrogen, phosphorus and iron deprivation, silicon depletion and quality and intensity of light supply are discussed in relation to biotechnological development of diatoms.
Nutrients (N, P, Fe) deficiency
Research Paper FIGURE 1
Model of market value for algae-based product lines.
activity [9]. It has also been suggested that fucoxanthin has potential health promoting effects in humans, including anticancer, anti-obesity, and anti-diabetic effects as well as anti-malarial activity [10]. The demand for naturally synthesized carotenoids including fucoxanthin in the global market has been increasing dramatically [11]. At present, fucoxanthin is mainly produced from edible brown seaweeds, which contains less than 1.0 mg/g dry weight [12]. Compared to seaweeds, diatoms like Phaeodactylum are usually rich in fucoxanthin under controlled conditions and low in iodine content, which makes them great candidates for fucoxanthin production [13]. Currently, carotenoids have been widely used in food and feed supplement industry and cosmetics for their coloring prosperity [11]. In addition, the benefits of carotenoids to health have been brought to light and confirmed in many studies and their use as nutraceuticals is growing significantly [11]. Diatoms are diverse and have the potential for use in many different products in the global market [14]. A simple and informative market model, depicting the promising aspects of diatomderived products at three levels, is shown in Fig. 1. Although numerous studies have been performed to exploit diatom resources, it still remains largely unknown whether and how diatom-derived value-added products can be developed on the basis of socio-economic considerations. In this paper, we review the recent advances in developing diatoms for enhanced growth performance and productivity of valuable products. In addition, the development of modern photobioreactors and culture methods for diatoms are summarized. The research efforts toward biotechnology and commercialization of diatoms are highlighted and discussed. Future directions for engineering diatoms and their applications are proposed.
Diatoms were reported to have a higher lipid content than other algal classes [5]. Production of polyunsaturated fatty acids (PUFAs) or neutral lipids was stimulated and optimized under nutrient deficiency of N and P [18]. Nitrogen significantly influences algae productivity in ocean ecosystems [17]. Nitrogen deprivation is a classical approach to induce the accumulation of lipids in algae in order to produce biodiesels [3]. It was found that redox regulation of metabolism in response to N stress provided a mechanism of acclimation to rapid fluctuations in diatoms in marine environments [17]. Phosphorus is also an important nutrient to diatoms [19]. Under P stress conditions, triacylglycerols, which are direct precursors for biodiesels, were found to increase 1.65-fold in the model diatom P. tricornutum [19]. Oil bodies are the cellular organelles in algae that store neutral lipids as energy [2]. Upon shift to P stress in the stationary phase of P. tricornutum culture, the numbers and sizes of oil bodies increased more than in control cells [19]. Iron deprivation limited growth and productivity in diatoms at low light; this was attributed to an increasing Fe requirement for the biosynthesis of Fe-rich proteins [20]. In addition, Fe availability can significantly affect the expression level of silicon transporters and their efficiency in cell membrane and alter silicification in diatoms [21]. It was identified that in the coastal diatom Thalassiosira pseudonana (TpDSP1), a death-specific protein that is localized in the plastid enhances growth performance by increasing the efficiency of photosynthetic fixation of carbon dioxide during acute iron limitation at subsaturating lights [20].
Silicon (Si) deficiency Silicon is a major limiting micronutrient/element for diatom growth due to its key role in cell wall structure [22]. Dissolved silicate and particulate biogenic silicate are biogeochemically cycled in marine environments by diatoms. Diatom silica obtained in a production system can also be regarded as a marketable product [5]. It has been found that rapid accumulation of triacylglycerol can be induced in most diatoms under Si starvation, which alters their morphology; however, photosynthesis-related gene expression and protein content were not affected as under N limitation [5]. It was observed that diatom cells under Si limitation fixed more carbon dioxide into biomass toward lipids and less toward carbohydrate production, though the mechanisms are not yet understood clearly [5]. It is believed that diatoms have the potential for production of sufficient lipids in replacement of petrol-based oils as well as value-added products in the future.
Light stress
Diatoms in response to abiotic stresses Environmental stress is the key factor that affects productivity of microalgae [15]. Naturally, many environmental stresses are abiotic, such as light stress, salinity, nutrient deficiency, drought, UV 2
Due to the importance of light in the diatom photobiology and physiology field, researchers have tried to explore the mechanism and molecular basis of how marine diatoms respond to light variability and availability [23]. Specific light adaptation and light
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acclimation in diatoms under natural environments have been studied, and the use of light sensors and regulators has been proposed for controlling diatom growth and relevant responses in oceans [23]. However, understanding of light-driven processes is still in its infancy under defined light conditions. With the development of light-emitting diode (LED) technology and LED-based photobioreactors, it is possible to study diatoms in response to light stress under different patterns of light intensity and wavelengths accurately [24]. Here, some of the recent advances in diatoms with regard to photoprotection and light-induced metabolism are described. Nonenzymatic antioxidants such as carotenoids have crucial roles in antioxidant defenses through scavenging reactive oxygen species (ROS) and inhibiting their generation in algae and higher plants [3]. In green algae, it was shown that manipulating light supply, via changing intensities and light quality, enhanced the accumulation of major carotenoids like b-carotene and lutein [15]. It is thought that high light acclimation in diatom P. tricornutum is triggered by the redox state of the plastoquinone pool, similar to green algae and land plants [25]. Photooxidative stress resulting from short exposure to high light caused an increase of diatoxanthin; the xanthophyll cycle and the content of the major carotenoid species fucoxanthin was not affected by such light treatments in diatoms [26]. It has also been reported that blue light is essential to the photoacclimation to high light intensities in the diatom Phaeodactylum [24]. Compared to green algae, diatoms might accumulate more carotenoids in response to changes of light quality due to their lack of chlorophyll b, which primarily absorbs blue light, and to their low content of chlorophyll c, present as an accessory chlorophyll species in the light harvesting antennae for photosystem II [7]. As LED-based photobioreactors provide the opportunity to researchers to design and precisely control light intensities and spectra, it is now possible to study the interactions between light wavelengths and pigmentation [3]. For instance, it was found that blue light helped to enhance the accumulation of carotenoids in diatoms [27]. More efforts will be required to identify the complicated networks bridging diatom metabolism and environmental light conditions.
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of green algae in photobioreactors with a long light path since green lights can travel deeper into the culture with lower photon absorption on the surface [30]. To produce value-added products from diatoms, it is advantageous to utilize a LED-based photobioreactor system based on its ability to cultivate diatoms at high density [27]. However, the photolimitation caused by mutual shading in highdensity culture is ubiquitous in both small and large tubular photobioreactors [27]. The radial mixing time is usually as long as 30 s and the unfavorable light/dark cycle to algal culture and the consequent limitation on light availability may downgrade algal growth rate [27]. On the other hand, oversaturated lights supplied on the surface of photobioreactors may result in photodamage to diatom cells. The light energy dissipation and loss without conversion into diatom biomass are due to: (1) incomplete incorporation due to light reflection and scattering; (2) heat (non-photochemical quenching); (3) chlorophyll fluorescence; and (4) energy for diatom maintenance. Therefore, photolimitation and photoinhibition, which exist simultaneously in tubular photobioreactors, largely restrain the biomass yield on light energy in diatom culture [27]. Adaptive laboratory evolution (ALE) has been widely utilized for developing new biological and phenotypic functions and also for strain improvement in synthetic biology for bacteria and green algae [7,31]; the process of ALE is shown schematically in Fig. 2. Diatoms have a different evolutionary history compared to green microalgae. They are thought to have evolved from an ancient secondary endosymbiosis between heterotrophic and autotrophic eukaryotes [32]. Diatoms containing unique pigments in the light harvesting complex are capable of generating large amounts of specialty compounds in response to fluctuations in their environment, especially with regard to changes in illumination. It is believed that ALE will also improve the growth and productivity of diatom P. tricornutum, as was observed for green algae [27], although the Phaeodactylum cells differ metabolically, physiologically and evolutionarily from green algae [32]. Appropriate ALE
LED-based photobioreactors and adaptive evolution The prices of LED-based lighting systems have significantly decreased over decades. Emerging LED technology has the potential to achieve high conversion efficiency from electricity to light, while providing a narrow light spectrum [28]. The use of LEDs marks great advancements over existing indoor agricultural lighting in this context [29,30]. Although LED lamps are still more expensive than fluorescent lamps (FLs) on average, this initial CAPEX cost of LEDs can be offset partly by their longer useful lifetimes and better energy efficiency compared to FLs [30]. LEDs allow the control of the spectral output and the adjustment of light intensity and frequency to simulate the optimal conditions for diatom cultures. It can be expected that appropriate light conditions, for example, light intensity and spectrum for diatom growth determined with LEDs can be applied to outdoor photobioreactors using natural lights or combined natural and artificial lights. LED technology is therefore ideally suited for the exploitation of diatoms. It has been reported that a higher proportion of high frequency green wavelengths might be beneficial for high-density cultures
FIGURE 2
Schematic process of adaptive laboratory evolution to enhance productivity of targeted carotenoid products. The levels of carotenoids in Phaeodactylum cells are predicted based on our previous studies [3,27].
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TABLE 1
Photosynthetic efficiencies of algae and higher plants Different organisms
Illumination conditions
Photosynthetic efficiency (%)
Notes Theoretical maxima [40]
Green higher plants
Sunlight
13.0
C4 plants
Sunlight
6.0
Highest reported [41]
Green alga Chlorella vulgaris
Red LED irradiance
9.0
Highest reported [42]
Diatom P. tricornutum
Red LED irradiance
2.0
Recent study [27]
Diatom P. tricornutum
Combined red and blue (50:50) LED irradiance
3.3
Recent study [27]
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experiments can be designed to develop P. tricornutum strains with increased biomass yield on light energy as well as improved carotenoid productivity. For instance, the effects of light quality and intensity may be evaluated with LED-based photobioreactors prior to ALE. Combination of blue and red lights has yielded high productivity of carotenoids such as b-carotene and lutein in the green alga Dunaliella salina [3]. Abiotic environmental stresses such as mixed red and blue lights and silicon limitation may be combined for future stress-driven ALE experiments to yield high levels of carotenoid productivity in diatoms [15].
Systems biology and genetic engineering of diatoms The publication of the annotated P. tricornutum genome sequence [33] enables the reconstruction of the P. tricornutum metabolic network. Using this network reconstruction, it is possible to predict the metabolic capabilities of P. tricornutum from its genotype, perform in silico knock-ins of heterologous pathways and knockouts, and pinpoint bottlenecks [34–36]. Systems biology approaches seem to be promising in engineering of diatoms for value-added products. Diatoms possess all the advantages of photosynthetically-driven eukaryotic systems, but lack many of the disadvantages of plant-based expression systems, that is, they have high growth rates, are easy to grow and do not need extra organic carbon sources for cultivation. In addition to classical genetic approaches like mutation breeding, molecular biology tools of gene manipulation in diatoms have been developed recently [37]. Microprojectile bombardment as well as electroporation methods have been applied successfully to introduce foreign DNA into Phaeodactylum cells [37,38]. Shuttle vector pPha-NR with inducible nitrate reductase promoter system (GenBank: JN180663) has been constructed for controllable expression of foreign genes. Using both meganucleases and TALE nucleases to modify the genome of diatom P. tricornutum, high frequencies of targeted mutagenesis in the genome were attained and an enhanced lipid producing strain was obtained, resulting in a 45-fold increase in triacylglycerol accumulation [4]. It has also been suggested that genetic approaches including both classical mutagenesis and synthetic biology are the most cost-effective way to improve
production traits and explore the potential of microalgae as a biotechnological resource [39]. Therefore, it is feasible to genetically engineer P. tricornutum for enhanced production of valuable carotenoids.
Perspectives and conclusions The photosynthetic efficiencies for algae and land plants under different conditions are shown in Table 1. It is obvious that the photosynthetic efficiency in diatoms is still low. It is believed that the light can be captured and utilized by photosystem II efficiently by redesigning light-harvesting complexes with additional and controllable gene expression in diatoms like P. tricornutum. LED technology combined with cutting-edge synthetic biology approaches has the potential to improve growth performance and productivity significantly in diatoms. In particular, controllable modules may be designed with heterologous expression of enhanced green fluorescence proteins (eGFPs) as a light absorber and emitter to facilitate the existing light harvesting system in P. tricornutum cells. LED illumination with different light spectra can then be utilized to excite eGFPs and drive efficient growth. LEDaided synthetic biology may be promising for efficient production of carotenoids by optimizing the light harvesting system in diatom culture for all sizes of photobioreactors. In summary, combing advanced LED illumination with systems biology and synthetic biology approaches in diatoms may develop photosynthetic cell factories for value-added products from diatoms in a novel way in biotechnology.
Author contribution Persons designated as authors qualify for authorship.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgements This work was supported by the Icelandic Technology Development Fund (Rannis) and additional support for W.F. was provided by New York University Abu Dhabi Faculty Research Funds AD060.
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Research Paper
Developing diatoms for value-added products: challenges and opportunities Weiqi Fu1,2,3,4, Kristine Wichuk1 and Sigurður Brynjo´lfsson1,4 Q1 1
Center for Systems Biology, University of Iceland, Reykjavı´k 101, Iceland 2 Q2 3 Division of Science and Math, New York University Abu Dhabi, United Arab Emirates Center for Genomics and Systems Biology (CGSB), New York University Abu Dhabi Institute, Abu Dhabi, United Arab Emirates 4 Q3 Faculty of Industrial Engineering, Mechanical Engineering and Computer Science, School of Engineering and Natural Sciences, University of Iceland, Reykjavı´k 101, Iceland
As a major primary producer in marine environments, diatoms have been considered as promising feedstocks for their applications in functional foods, bioactive pharmaceuticals, and cosmetics. This review focuses on the biotechnology potential of diatoms for value-added products like carotenoids. The impact of abiotic environmental stresses, such as intensity and quality of incident light, nutrient deficiency and silicon depletion, on diatoms has been examined to determine key factors that affect the growth performance and the accumulation of valuable compounds. Previous studies suggested that adaptive evolution could be an efficient method to improve the diatom productivity of valuable compounds. Light emitting diode (LED)-based photobioreactors were introduced and proposed as a promising new technology for producing quality products from diatoms. Currently available molecular biology tools were also summarized and discussed in relation to their application in the production of carotenoids and other valuable products. Taken together, systems biology and synthetic biology approaches have the potential to address the challenges faced while working toward the industrial application of diatoms. Introduction Q4 Marine diatoms are a major group of microalgae, which have been deemed as attractive sources for developing sustainable products such as fine chemicals, plastics and biofuels, a solution for energy crisis and for attempting to address the environmental issues globally [1,2]. The continuing threat of global climate change, caused mainly by the consumption of fossil fuels, calls for displacing existing oil-based industries with innovative and sustainable solutions [3]. The high production of lipids in many species of diatoms has attracted great attention to the potential use of diatoms as a source for biofuels [4]. It is clear that developing bio-based energy supplies, such as diatom biofuels, is also highly strategic as a response to the global energy crisis and the diminishing supply of fossil fuels. The concept of using diatoms for Corresponding author: Fu, W. ([email protected], [email protected]) http://dx.doi.org/10.1016/j.nbt.2015.03.016 1871-6784/ß 2015 Published by Elsevier B.V.
producing biofuels is advantageous in terms of being sustainable and a clean technology. However, biofuels derived from diatoms are not cost-competitive at present and may not be economically feasible in the foreseeable future [5]. Diatoms have also recently been explored as sources of bioactive compounds such as carotenoids due to their economic potential [6]. Carotenoids, based on their chemical structures, can be divided into two categories: (1) the carotenes, such as b-carotene; and (2) the xanthophylls, such as fucoxanthin [3]. It has been reported that many carotenoid species such as fucoxanthin, diadinoxanthin, diatoxanthin and b-carotene are present in the model species Phaeodactylum tricornutum [7]. Among them, b-carotene is a major primary source of vitamin A which is necessary for functions of the retina and has an effect on many tissue types through its action as a regulator of gene expression [8]. In addition, b-carotene also helps protect skin against photoaging due to its antioxidant www.elsevier.com/locate/nbt
Please cite this article in press as: Fu, W. et al., Developing diatoms for value-added products: challenges and opportunities, New Biotechnol. (2015), http://dx.doi.org/10.1016/ j.nbt.2015.03.016
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exposure, and heat shock [16,17]. And these abiotic environmental stresses have significant influences on the physiology of diatoms [17]. In this section, representative stress factors including nitrogen, phosphorus and iron deprivation, silicon depletion and quality and intensity of light supply are discussed in relation to biotechnological development of diatoms.
Nutrients (N, P, Fe) deficiency
Research Paper FIGURE 1
Model of market value for algae-based product lines.
activity [9]. It has also been suggested that fucoxanthin has potential health promoting effects in humans, including anticancer, anti-obesity, and anti-diabetic effects as well as anti-malarial activity [10]. The demand for naturally synthesized carotenoids including fucoxanthin in the global market has been increasing dramatically [11]. At present, fucoxanthin is mainly produced from edible brown seaweeds, which contains less than 1.0 mg/g dry weight [12]. Compared to seaweeds, diatoms like Phaeodactylum are usually rich in fucoxanthin under controlled conditions and low in iodine content, which makes them great candidates for fucoxanthin production [13]. Currently, carotenoids have been widely used in food and feed supplement industry and cosmetics for their coloring prosperity [11]. In addition, the benefits of carotenoids to health have been brought to light and confirmed in many studies and their use as nutraceuticals is growing significantly [11]. Diatoms are diverse and have the potential for use in many different products in the global market [14]. A simple and informative market model, depicting the promising aspects of diatomderived products at three levels, is shown in Fig. 1. Although numerous studies have been performed to exploit diatom resources, it still remains largely unknown whether and how diatom-derived value-added products can be developed on the basis of socio-economic considerations. In this paper, we review the recent advances in developing diatoms for enhanced growth performance and productivity of valuable products. In addition, the development of modern photobioreactors and culture methods for diatoms are summarized. The research efforts toward biotechnology and commercialization of diatoms are highlighted and discussed. Future directions for engineering diatoms and their applications are proposed.
Diatoms were reported to have a higher lipid content than other algal classes [5]. Production of polyunsaturated fatty acids (PUFAs) or neutral lipids was stimulated and optimized under nutrient deficiency of N and P [18]. Nitrogen significantly influences algae productivity in ocean ecosystems [17]. Nitrogen deprivation is a classical approach to induce the accumulation of lipids in algae in order to produce biodiesels [3]. It was found that redox regulation of metabolism in response to N stress provided a mechanism of acclimation to rapid fluctuations in diatoms in marine environments [17]. Phosphorus is also an important nutrient to diatoms [19]. Under P stress conditions, triacylglycerols, which are direct precursors for biodiesels, were found to increase 1.65-fold in the model diatom P. tricornutum [19]. Oil bodies are the cellular organelles in algae that store neutral lipids as energy [2]. Upon shift to P stress in the stationary phase of P. tricornutum culture, the numbers and sizes of oil bodies increased more than in control cells [19]. Iron deprivation limited growth and productivity in diatoms at low light; this was attributed to an increasing Fe requirement for the biosynthesis of Fe-rich proteins [20]. In addition, Fe availability can significantly affect the expression level of silicon transporters and their efficiency in cell membrane and alter silicification in diatoms [21]. It was identified that in the coastal diatom Thalassiosira pseudonana (TpDSP1), a death-specific protein that is localized in the plastid enhances growth performance by increasing the efficiency of photosynthetic fixation of carbon dioxide during acute iron limitation at subsaturating lights [20].
Silicon (Si) deficiency Silicon is a major limiting micronutrient/element for diatom growth due to its key role in cell wall structure [22]. Dissolved silicate and particulate biogenic silicate are biogeochemically cycled in marine environments by diatoms. Diatom silica obtained in a production system can also be regarded as a marketable product [5]. It has been found that rapid accumulation of triacylglycerol can be induced in most diatoms under Si starvation, which alters their morphology; however, photosynthesis-related gene expression and protein content were not affected as under N limitation [5]. It was observed that diatom cells under Si limitation fixed more carbon dioxide into biomass toward lipids and less toward carbohydrate production, though the mechanisms are not yet understood clearly [5]. It is believed that diatoms have the potential for production of sufficient lipids in replacement of petrol-based oils as well as value-added products in the future.
Light stress
Diatoms in response to abiotic stresses Environmental stress is the key factor that affects productivity of microalgae [15]. Naturally, many environmental stresses are abiotic, such as light stress, salinity, nutrient deficiency, drought, UV 2
Due to the importance of light in the diatom photobiology and physiology field, researchers have tried to explore the mechanism and molecular basis of how marine diatoms respond to light variability and availability [23]. Specific light adaptation and light
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acclimation in diatoms under natural environments have been studied, and the use of light sensors and regulators has been proposed for controlling diatom growth and relevant responses in oceans [23]. However, understanding of light-driven processes is still in its infancy under defined light conditions. With the development of light-emitting diode (LED) technology and LED-based photobioreactors, it is possible to study diatoms in response to light stress under different patterns of light intensity and wavelengths accurately [24]. Here, some of the recent advances in diatoms with regard to photoprotection and light-induced metabolism are described. Nonenzymatic antioxidants such as carotenoids have crucial roles in antioxidant defenses through scavenging reactive oxygen species (ROS) and inhibiting their generation in algae and higher plants [3]. In green algae, it was shown that manipulating light supply, via changing intensities and light quality, enhanced the accumulation of major carotenoids like b-carotene and lutein [15]. It is thought that high light acclimation in diatom P. tricornutum is triggered by the redox state of the plastoquinone pool, similar to green algae and land plants [25]. Photooxidative stress resulting from short exposure to high light caused an increase of diatoxanthin; the xanthophyll cycle and the content of the major carotenoid species fucoxanthin was not affected by such light treatments in diatoms [26]. It has also been reported that blue light is essential to the photoacclimation to high light intensities in the diatom Phaeodactylum [24]. Compared to green algae, diatoms might accumulate more carotenoids in response to changes of light quality due to their lack of chlorophyll b, which primarily absorbs blue light, and to their low content of chlorophyll c, present as an accessory chlorophyll species in the light harvesting antennae for photosystem II [7]. As LED-based photobioreactors provide the opportunity to researchers to design and precisely control light intensities and spectra, it is now possible to study the interactions between light wavelengths and pigmentation [3]. For instance, it was found that blue light helped to enhance the accumulation of carotenoids in diatoms [27]. More efforts will be required to identify the complicated networks bridging diatom metabolism and environmental light conditions.
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of green algae in photobioreactors with a long light path since green lights can travel deeper into the culture with lower photon absorption on the surface [30]. To produce value-added products from diatoms, it is advantageous to utilize a LED-based photobioreactor system based on its ability to cultivate diatoms at high density [27]. However, the photolimitation caused by mutual shading in highdensity culture is ubiquitous in both small and large tubular photobioreactors [27]. The radial mixing time is usually as long as 30 s and the unfavorable light/dark cycle to algal culture and the consequent limitation on light availability may downgrade algal growth rate [27]. On the other hand, oversaturated lights supplied on the surface of photobioreactors may result in photodamage to diatom cells. The light energy dissipation and loss without conversion into diatom biomass are due to: (1) incomplete incorporation due to light reflection and scattering; (2) heat (non-photochemical quenching); (3) chlorophyll fluorescence; and (4) energy for diatom maintenance. Therefore, photolimitation and photoinhibition, which exist simultaneously in tubular photobioreactors, largely restrain the biomass yield on light energy in diatom culture [27]. Adaptive laboratory evolution (ALE) has been widely utilized for developing new biological and phenotypic functions and also for strain improvement in synthetic biology for bacteria and green algae [7,31]; the process of ALE is shown schematically in Fig. 2. Diatoms have a different evolutionary history compared to green microalgae. They are thought to have evolved from an ancient secondary endosymbiosis between heterotrophic and autotrophic eukaryotes [32]. Diatoms containing unique pigments in the light harvesting complex are capable of generating large amounts of specialty compounds in response to fluctuations in their environment, especially with regard to changes in illumination. It is believed that ALE will also improve the growth and productivity of diatom P. tricornutum, as was observed for green algae [27], although the Phaeodactylum cells differ metabolically, physiologically and evolutionarily from green algae [32]. Appropriate ALE
LED-based photobioreactors and adaptive evolution The prices of LED-based lighting systems have significantly decreased over decades. Emerging LED technology has the potential to achieve high conversion efficiency from electricity to light, while providing a narrow light spectrum [28]. The use of LEDs marks great advancements over existing indoor agricultural lighting in this context [29,30]. Although LED lamps are still more expensive than fluorescent lamps (FLs) on average, this initial CAPEX cost of LEDs can be offset partly by their longer useful lifetimes and better energy efficiency compared to FLs [30]. LEDs allow the control of the spectral output and the adjustment of light intensity and frequency to simulate the optimal conditions for diatom cultures. It can be expected that appropriate light conditions, for example, light intensity and spectrum for diatom growth determined with LEDs can be applied to outdoor photobioreactors using natural lights or combined natural and artificial lights. LED technology is therefore ideally suited for the exploitation of diatoms. It has been reported that a higher proportion of high frequency green wavelengths might be beneficial for high-density cultures
FIGURE 2
Schematic process of adaptive laboratory evolution to enhance productivity of targeted carotenoid products. The levels of carotenoids in Phaeodactylum cells are predicted based on our previous studies [3,27].
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TABLE 1
Photosynthetic efficiencies of algae and higher plants Different organisms
Illumination conditions
Photosynthetic efficiency (%)
Notes Theoretical maxima [40]
Green higher plants
Sunlight
13.0
C4 plants
Sunlight
6.0
Highest reported [41]
Green alga Chlorella vulgaris
Red LED irradiance
9.0
Highest reported [42]
Diatom P. tricornutum
Red LED irradiance
2.0
Recent study [27]
Diatom P. tricornutum
Combined red and blue (50:50) LED irradiance
3.3
Recent study [27]
Research Paper
experiments can be designed to develop P. tricornutum strains with increased biomass yield on light energy as well as improved carotenoid productivity. For instance, the effects of light quality and intensity may be evaluated with LED-based photobioreactors prior to ALE. Combination of blue and red lights has yielded high productivity of carotenoids such as b-carotene and lutein in the green alga Dunaliella salina [3]. Abiotic environmental stresses such as mixed red and blue lights and silicon limitation may be combined for future stress-driven ALE experiments to yield high levels of carotenoid productivity in diatoms [15].
Systems biology and genetic engineering of diatoms The publication of the annotated P. tricornutum genome sequence [33] enables the reconstruction of the P. tricornutum metabolic network. Using this network reconstruction, it is possible to predict the metabolic capabilities of P. tricornutum from its genotype, perform in silico knock-ins of heterologous pathways and knockouts, and pinpoint bottlenecks [34–36]. Systems biology approaches seem to be promising in engineering of diatoms for value-added products. Diatoms possess all the advantages of photosynthetically-driven eukaryotic systems, but lack many of the disadvantages of plant-based expression systems, that is, they have high growth rates, are easy to grow and do not need extra organic carbon sources for cultivation. In addition to classical genetic approaches like mutation breeding, molecular biology tools of gene manipulation in diatoms have been developed recently [37]. Microprojectile bombardment as well as electroporation methods have been applied successfully to introduce foreign DNA into Phaeodactylum cells [37,38]. Shuttle vector pPha-NR with inducible nitrate reductase promoter system (GenBank: JN180663) has been constructed for controllable expression of foreign genes. Using both meganucleases and TALE nucleases to modify the genome of diatom P. tricornutum, high frequencies of targeted mutagenesis in the genome were attained and an enhanced lipid producing strain was obtained, resulting in a 45-fold increase in triacylglycerol accumulation [4]. It has also been suggested that genetic approaches including both classical mutagenesis and synthetic biology are the most cost-effective way to improve
production traits and explore the potential of microalgae as a biotechnological resource [39]. Therefore, it is feasible to genetically engineer P. tricornutum for enhanced production of valuable carotenoids.
Perspectives and conclusions The photosynthetic efficiencies for algae and land plants under different conditions are shown in Table 1. It is obvious that the photosynthetic efficiency in diatoms is still low. It is believed that the light can be captured and utilized by photosystem II efficiently by redesigning light-harvesting complexes with additional and controllable gene expression in diatoms like P. tricornutum. LED technology combined with cutting-edge synthetic biology approaches has the potential to improve growth performance and productivity significantly in diatoms. In particular, controllable modules may be designed with heterologous expression of enhanced green fluorescence proteins (eGFPs) as a light absorber and emitter to facilitate the existing light harvesting system in P. tricornutum cells. LED illumination with different light spectra can then be utilized to excite eGFPs and drive efficient growth. LEDaided synthetic biology may be promising for efficient production of carotenoids by optimizing the light harvesting system in diatom culture for all sizes of photobioreactors. In summary, combing advanced LED illumination with systems biology and synthetic biology approaches in diatoms may develop photosynthetic cell factories for value-added products from diatoms in a novel way in biotechnology.
Author contribution Persons designated as authors qualify for authorship.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgements This work was supported by the Icelandic Technology Development Fund (Rannis) and additional support for W.F. was provided by New York University Abu Dhabi Faculty Research Funds AD060.
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New Biotechnology Volume 00, Number 00 April 2015
