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Biotechnological production of value-added carotenoids from microalgae: Emerging technology and prospects
Bioengineered
Abstract
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.
... Microalgae have been demonstrated to be an alternative source of L likely to become competitive for commercial exploitation (Fernandez-Sevilla, Acien Fernandez, & Molina, 2010;Fernandez-Sevilla, Fernandez, & Grima, 2012). In green microalgae (Chlorophyceae), L is a major carotenoid protecting cellular components from damage by reactive oxygen species or other stressors (Fu et al., 2014). This class of microalgae is potentially suitable for large-scale carotenoid or FA production due to the efficient growth of its members in photobioreactors (J. A. ...
... These stressful environmental conditions include nutrient limitations; certain temperatures, salinities, and light intensity and quality; and the addition of chemicals such as H2O2, Fe 2 +, and other redox agents to the media (Cordero et al., 2011;J. A. Del Campo et al., 2000;Fu et al., 2013;Fu et al., 2014;Pulz, 2001;S+ínchez et al., 2008;Wichuk, Brynjolfsson, & Fu, 2014). However, large-scale studies such as that of Del Campo et al. (2001) exhibited an exploitation methodology without implementing a 'stimulation step'. ...
... These stressful environmental conditions include nutrient limitations; certain temperatures, salinities, and light intensity and quality; and the addition of chemicals such as H2O2, Fe 2 +, and other redox agents to the media (Cordero et al., 2011;J. A. Del Campo et al., 2000;Fu et al., 2013;Fu et al., 2014;Pulz, 2001;S+ínchez et al., 2008;Wichuk, Brynjolfsson, & Fu, 2014). However, large-scale studies such as that of Del Campo et al. (2001) exhibited an exploitation methodology without implementing a 'stimulation step'. ...
This dissertation presents a novel technology aimed at improving the economic feasibility of sourcing metabolites of commercial interest from microalgae. This technology consists of a continuous two-phase algae-crustacean bioreactor where a selected microalgae species is cultured to produce biomass with specific characteristics in the first phase of the reactor. This microalgae biomass is continuously supplied to an interconnected zooplankton culture, which grows while bio-accumulating the algal metabolites or, in some cases, bio-converting them into more valuable compounds. To close the loop, zooplankton waste is used by microalgae as nutrients for growth. The zooplankton biomass produced, which is rich in the desired compound(s), can be manually harvested, avoiding expensive techniques such as centrifugation. Bio-accumulation and bio-conversion concepts have been tested at laboratory and pilot scales with different species configurations. Initially, the creation of a laboratory-scale prototype demonstrated the possibility of producing zooplankton oil with lutein concentrations greater than 1 mg per g oil. Afterwards, a pilot plant was developed to achieve a more detailed understanding of the process and assess productivity. The pilot plant permitted the study of this technology using different biological and environmental setups, which allowed the study of the bio-accumulation of lutein and eicosapentaenoic acid and the bio-conversion of carotenoids and fatty acids into astaxanthin and docosahexaenoic acid. This prototype exhibited a maximum productivity of 3 kg fresh zooplankton biomass per month and m 3. Despite the fact that the proposed culture system presents a reduced productivity in comparison to that of pure microalgae reactors, the implementation of this technology is demonstrated throughout this thesis to possibly represent significant reductions in costs associated with biomass production and processing when compared to the costs of currently used models. Furthermore, the application of this technology may be valuable in different areas such as wastewater bio-remediation, food production and aquaculture.
... Because of the negative impacts of increased protein and energy in the diet on health and fertility (McEvoy et al., 1997;Dawuda et al., 2002) and the high cost of ration, it is important to search for non-traditional supplements with beneficial character-istics for body. Microalgae possesses high concentration of proteins, lipids and vitamins, sulfated polysaccharides, several amounts of polyunsaturated fatty acids (Gouveia et al., 2008;Ghasemi et al., 2011;Wichuk et al., 2014;Cuellar-Bermudez et al., 2015;Gong and Bassi;. Pulz and Gross (2004) showed that polysaccharides is important as therapeutic where it support immune system and has potential role to fight virus infection (Abdel-Daim et al., 2015). ...
... Nutritional supply, D. salina, may be used as an alternative to hormonal treatments to enhance reproductive performance. D. salina has high levels of beta-carotene, glycerol, protein and other fine chemicals (Gouveia et al., 2008;Ghasemi et al., 2011;Wichuk et al., 2014;Cuellar-Bermudez et al., 2015;Gong and Bassi, 2016). ...
... January 2018 | Volume 6 | Issue 1 | Page 37 one. D. salina has high levels of beta-carotene, glycerol, protein and other fine chemicals (Gouveia et al., 2008;Ghasemi et al., 2011;Wichuk et al., 2014;Cuellar-Bermudez et al., 2015;Gong and Bassi;, which might improve the quality of oocyte and support further embryonic development. Conclusion of our work (Senosy et al., 2017) indicated that D. salina supplemented to Boer goats (10 g/day/head) accelerated ovarian follicle development and significantly, increased follicle numbers (small, medium and large follicles).The improvement of oocyte quality in D. salina group compared to control one might be related to high levels of beta-carotene and elevation of plasma metabolites and hormones levels. ...
The present study investigated the developmental competence of germinal vesicle (GV) oocytes and the resulting embryos upon dietary Dunaliella salina (DS) supplementation. Quality of GV oocytes and maturation, timing of zygotes cleavage to two-cell stage, quality of embryos and offspring number and weight were investigated of mice supplemented with DS (100g/kg ration; N=30) compared to control (N=30).Females were injected with 7.5 I.U. of equine chorionic gonadotropin (PMSG) followed by 7.5 I.U. of human chorionic gonadotropin (hCG) after 48h and mated with fertile male. Immature GV oocytes were harvested from ovaries after 48h of PMSG injection for investigating quality of oocyte, timing of breakdown of germinal vesicle (GVBD) and extrusion of first polar bodies and maturation rate (%).Zygotes were harvested from uterine tubes 29-30hafterhCG injection and followed for first cleavage whereas quality of blastocysts were evaluated after collection from uterine horn at96h of hCG. Improvement of oocyte quality has been indicated in DS group whereas GVBD and maturation rate were not differed between DS and control groups. Although D. salina did not change timing of first cleavage of zygotes, blastocysts' quality were significantly (P<0.05) increased in DS group. Furthermore, offspring number (9.9 ± 0.26 & 8.2 ± 0.30) and weight (12.01 ± 0.31 & 9.84 ± 0.37) at birth were increased significantly (P<0.05) in the DS group compared to control one. In conclusion, supplementation of D. salina could increase reproductive performance through improvement quality of germinal vesicle oocytes and preimplantation embryos.
... For instance, a recent comprehensive analysis indicated that more fossil energy was consumed than bioenergy produced in most scenarios using a theoretical open raceway pond facility for outdoor cultivation of microalgae (20). Among many factors that affect algal growth rates, photolimitation from unfavorable light-dark cycles caused by insufficient mixing and photoinhibition due to oversaturation of light on the surface, both of which exist simultaneously in dense cultures of microalgae, act to limit photosynthetic efficiency and reduce overall biomass productivity (21). In diatoms, a key mechanism for the abatement of photoinduced stress is nonphotochemical quenching (NPQ), in which energy-dependent quenching (qE) is the most important part of NPQ in diatoms, because there is no state-transition quenching (qT) and photoinhibitory quenching (qI) is little (22). ...
... Improving photosynthesis is of considerable interest for biotechnological applications, aquaculture, and agriculture (34). However, for the production of biofuels, biomass, and bioactive compounds from microalgae, one of the major limitations is low photosynthetic efficiency at full sunlight or high light intensities for dense cultures (21). Researchers in the field have proposed some solutions to address the issue of low photosynthetic efficiency by applying flashing lights, redesigning vertical PBRs, and introducing rapid mixing in the cultivation systems for potential production at industrial scales (35). ...
... A significant amount of the excess light energy is dissipated as heat or chlorophyll fluorescence that cannot be used by algal cells for photosynthesis (36). In a dense culture of algal suspensions, the algal cells at the surface, or the side exposed to the light sources, receive more light energy than they can use for the downstream carbon dioxide reduction; the excess energy is lost through heat dissipation and chlorophyll fluorescence (21). The algae below the surface or on the side opposite to the light sources in a PBR cannot obtain sufficient light energy due to mutual shading in algal cells (21). ...
Diatoms, considered as one of the most diverse and largest groups of algae, can provide the means to reach a sustainable production of petrochemical substitutes and bioactive compounds. However, a prerequisite to achieving this goal is to increase the solar-to-biomass conversion efficiency of photosynthesis, which generally remains less than 5% for most photosynthetic organisms. We have developed and implemented a rapid and effective approach, herein referred to as intracellular spectral recompositioning (ISR) of light, which, through absorption of excess blue light and its intracellular emission in the green spectral band, can improve light utilization. We demonstrate that ISR can be used chemogenically, by using lipophilic fluorophores, or biogenically, through the expression of an enhanced green fluorescent protein (eGFP) in the model diatom Phaeodactylum tricornutum. Engineered P. tricornutum cells expressing eGFP achieved 28% higher efficiency in photosynthesis than the parental strain, along with an increased effective quantum yield and reduced nonphotochemical quenching (NPQ) induction levels under high-light conditions. Further, pond simulator experiments demonstrated that eGFP transformants could outperform their wild-type parental strain by 50% in biomass production rate under simulated outdoor sunlight conditions. Transcriptome analysis identified up-regulation of major photosynthesis genes in the engineered strain in comparison with the wild type, along with down-regulation of NPQ genes involved in light stress response. Our findings provide a proof of concept for a strategy of developing more efficient photosynthetic cell factories to produce algae-based biofuels and bioactive products.
... Adaptive laboratory evolution is another cost-effective approach that does not r quire previous knowledge of the genetics of the microalgal strain under investigation (Fi ure 1B). Furthermore, it also avoids the introduction of foreign genetic material into t target cells; thus, the improved strains pose no regulatory issues due to the long biosafe record of such technologies ( Figure 1B) [48,49]. In adaptive laboratory evolution expe ments, cell cultures are subjected to a continuous selective pressure over a long period time [25,40,41,50]. ...
... Adaptive laboratory evolution is another cost-effective approach that does not require previous knowledge of the genetics of the microalgal strain under investigation ( Figure 1B). Furthermore, it also avoids the introduction of foreign genetic material into the target cells; thus, the improved strains pose no regulatory issues due to the long biosafety record of such technologies ( Figure 1B) [48,49]. In adaptive laboratory evolution experiments, cell cultures are subjected to a continuous selective pressure over a long period of time [25,40,41,50]. ...
Microalgae have become a promising novel and sustainable feedstock for meeting the rising demand for food and feed. However, microalgae-based products are currently hindered by high production costs. One major reason for this is that commonly cultivated wildtype strains do not possess the robustness and productivity required for successful industrial production. Several strain improvement technologies have been developed towards creating more stress tolerant and productive strains. While classical methods of forward genetics have been extensively used to determine gene function of randomly generated mutants, reverse genetics has been explored to generate specific mutations and target phenotypes. Site-directed mutagenesis can be accomplished by employing different gene editing tools, which enable the generation of tailor-made genotypes. Nevertheless, strategies promoting the selection of randomly generated mutants avoid the introduction of foreign genetic material. In this paper, we review different microalgal strain improvement approaches and their applications, with a primary focus on random mutagenesis. Current challenges hampering strain improvement, selection, and commercialization will be discussed. The combination of these approaches with high-throughput technologies, such as fluorescence-activated cell sorting, as tools to select the most promising mutants, will also be discussed.
... To showcase this software in action, this work presents two case studies focusing on two different proteins of interest, namely basic region/leucine zipper motif 1 (bZIP1) transcription factor from Chlorella sp. HS2, found in all species of genus Chlorella, and lycopene β-cyclase (LCYB) as found across all the species in the taxa Ochrophyta (20)(21)(22). These proteins were selected because of their relevance to stress responses in algae and land plants; a desirable phenotype in the context of bioengineering algae for enhancing biofuel and bioproduct yield (23,24). ...
Background: Microalgae constitute a prominent feedstock for producing biofuels and biochemicals by virtue of their prolific reproduction, high bioproduct accumulation, and the ability to grow in brackish and saline water. However, naturally-occurring wild type algal strains are rarely optimal for industrial use. Bioengineering of algae is necessary to generate superior performing strains that can address production challenges in industrial settings, particularly the bioenergy and bioproduct sectors. One of the crucial steps in this process is deciding on a bioengineering target: namely, which gene/protein to differentially express. These targets are often orthologs which are defined as genes/proteins originating in a common ancestor in divergent species. Although bioinformatics tools for the identification of protein orthologs already exist, processing the output from such tools is non-trivial, especially for a researcher with little or no bioinformatics experience.
Results: The present study introduces AlgaeOrtho, a user-friendly tool that builds upon the SonicParanoid orthology inference tool and the PhycoCosm database from JGI (Joint Genome Institute) to help researchers identify orthologs of their proteins of interest in multiple diverse algal species. This tool includes an application with a user interface, to upload an ortholog protein group file (created using SonicParanoid), and a query file that includes their protein sequence(s) of interest in the FASTA format. The output generates a table of the putative orthologs of their protein of interest, a heatmap showing sequence similarity (%), and a tree of the putative protein orthologs. Notably, the tool would be instrumental in identifying novel bioengineering targets in different algal strains, including targets in not-fully-annotated algal species, since it does not depend on existing protein annotations.
Conclusions: We tested AlgaeOrtho using two case studies, for which orthologs of proteins relevant to bioengineering targets were identified from a range of algal species, demonstrating its ease of use and utility for bioengineering researchers. This tool is unique in the protein ortholog identification space as it can visualize putative orthologs, as desired by the user, across several algal species.
... The green unicellular algae Chlorella vulgaris serves as a source of valuable metabolites for the food industry [28][29][30], agriculture [22][23][24][25][26][27][28][29][30][31][32][33][34], cosmetics [35][36][37], and biodiesel production [38][39][40][41][42]. There is a significant demand for chemical stimulants that promote the growth of this algae, as well as the accumulation of lipids [43][44][45], proteins [46,47], carbohydrates [48][49][50], and pigments [51] such as chlorophylls [52,53] and carotenoids [54][55][56][57][58]. We selected several synthesized compounds with favorable characteristics, such as high synthesis yield and improved solubility in polar solvents. ...
1H-Pyrrole-2,3-diones, fused at [e]-side with a heterocycle, are suitable platforms for the synthesis of various angular polycyclic alkaloid-like spiroheterocycles. Recently discovered sulfur-containing [e]-fused 1H-pyrrole-2,3-diones (aroylpyrrolobenzothiazinetriones) tend to exhibit unusual reactivity. Based on these peculiar representatives of [e]-fused 1H-pyrrole-2,3-diones, we have developed an approach to an unprecedented 6/5/5/5-tetracyclic alkaloid-like spiroheterocyclic system of benzo[d]pyrrolo[3′,4′:2,3]pyrrolo[2,1-b]thiazole via their reaction with Schiff bases and carbodiimides. The experimental results have been supplemented with DFT computational studies. The synthesized alkaloid-like 6/5/5/5-tetracyclic compounds have been tested for their biotechnological potential as growth stimulants in the green algae Chlorella vulgaris.
... However, some carotenoids are characteristics of specific microalgae groups. Diatoms, such as the genus Phaeodactylum, can produce the neoxanthin-related xanthophylls diatoxanthin, dinoxanthin, diadinoxanthin, and fucoxanthin, whereas some green algae can synthetize α-carotene, ε-carotene, δ-carotene or astaxanthin (Mulders et al., 2014;Wichuk et al., 2014). Furthermore, Doppler et al. (Doppler et al., 2021) isolated a novel strain of the microalga Tetraedron minimum in Iceland from a terrestrial habitat. ...
Given their multifaceted roles, carotenoids have garnered significant scientific interest, resulting in a comprehensive and intricate body of literature that occasionally presents conflicting findings concerning the proper characterization, quantification, and bioavailability of these compounds. Nevertheless, it is undeniable that the pursuit of novel carotenoids remains a crucial endeavor, as their diverse properties, functionalities and potential health benefits make them invaluable natural resources in agri-food and health promotion through the diet. In this framework, particular attention is given to ketocarotenoids, viz., astaxanthin (one of them) stands out for its possible multifunctional role as an antioxidant, anticancer, and antimicrobial agent. It has been widely explored in the market and utilized in different applications such as nutraceuticals, food additives, among others. Adonirubin and adonixanthin can be naturally found in plants and microorganisms. Due to the increasing significance of natural-based products and the remarkable opportunity to introduce these ketocarotenoids to the market, this review aims to provide an expert overview of the pros and cons associated with adonirubin and adonixanthin.
... Culture conditions suitable for cell growth are not usually conducive to the accumulation of secondary metabolites (Tafreshi and Shariati 2009). Carotenoid production in industry has been achieved by a two-step method, with D. salina cultivated in the best conditions for growth to produce more biomass (Wichuk et al. 2014), and then progressively switched to the best conditions for accumulating carotenoids with the highest carotenoid output. Red light is often employed during the growth period to boost biological yield since studies have shown that red light is more favorable to the growth of D. salina (Pereira and Otero 2019). ...
Dunaliella salina is a high-quality industrial effector for carotenoid production. The mechanism by which red light regulates carotenoid synthesis is still unclear. In this study, a transcription factor of DsGATA1 with a distinct structure was discovered in D. salina. The recognition motif of DsGATA1 was comparable to that of plant and fungal GATA, despite its evolutionary proximity to animal-derived GATA. The expression of DsGATA1 in D. salina was still noticeably decreased when exposed to red light. Analysis of physiological and biochemical transcriptomic data from overexpressed, interfering, and wild-type strains of DsGATA1 revealed that DsGATA1 acts as a global regulator of D. salina carotenoid synthesis. The upregulated genes in the CBP pathway by DsGATA1 were involved in its regulation of the synthesis of carotenoids. DsGATA1 also enhanced carotenoid accumulation under red light by affecting N metabolism. DsGATA1 was found to directly bind to the promoter of nitrate reductase to activate its expression, promoting D. salina nitrate uptake and accelerating biomass accumulation. DsGATA1 affected the expression of the genes encoding GOGAT, GDH, and ammonia transporter proteins. Moreover, our study revealed that the regulation of N metabolism by DsGATA1 led to the production of NO molecules that inhibited carotenoid synthesis. However, DsGATA1 significantly enhanced carotenoid synthesis by NO scavenger removal of NO. The D. salina carotenoid accumulation under red light was elevated by 46% in the presence of overexpression of DsGATA1 and NO scavenger. Nevertheless, our results indicated that DsGATA1 could be an important target for engineering carotenoid production.
Key points
• DsGATA1 with a distinct structure and recognition motif was found in D. salina
• DsGATA1 enhanced carotenoid production and biomass in D. salina under red light
• DsGATA1 is involved in the regulation of N metabolism and carotenoid synthesis
... Mikroalglerin gıda ve yem uygulamalarında, farmasötik biyoaktif bileşiklerde ve biyoyakıt üretiminde kullanılmak üzere kaynak materyal olarak umut verici olduğu yaygın olarak kabul edilen bir gerçektir. Tipik olarak birincil üreticiler olarak görev yapan bu organizmalar, hem tatlı su hem de deniz ekosistemlerindeki fitoplankton topluluklarında ağırlıklı olarak bulunur [4]. Mikroalgal biyoteknoloji çeşitli ticari kullanım alanlarında uygulama alanı bulmaktadır. ...
Potential and Applications of Microalgae-Mediated Microbial Fuel Cells
Abstract
In an age where energy resources are becoming increasingly limited, the search for sustainable and renewable
energy production has come to the fore. Microbial fuel cells emerged as a result of this search and have gained
importance in recent years. These cells have the ability to convert organic substances directly into electricity
through the biochemical reactions of microorganisms. In this study, horse manure wastewater and dry microalgae
biomass were used as substrates in the anode compartment, and Chlamydomonas sp. microalgae were used as an
electron acceptor in the cathode compartment. Graphite felt electrodes were used in both chambers. As a result of
the experiments carried out in this system, a maximum power density of 29.93 mW/m2 and an open circuit voltage
of 599.6 mV were recorded. At the same time, 49.35% COD removal was achieved. This study highlights the energy production potential of a dual-chamber microbial fuel cell using wastewater and microalgae. The use of Chlamydomonas sp. microalgae in the cathode region shows how the oxygen produced as a result of photosynthetic activity can be used in electron transfer. These results show that treating wastewater in sustainable ways and at the same time using biological organisms such as microalgae in energy production is a promising
strategy in the environmental and energy field. This method contributes to environmental sustainability by combining waste management and renewable energy production.
... From the last few decades, the importance of microalgae is expanding due to its beneficial products, including proteins, lipids and pigments which are highly recommended for human consumption in food and medicine [1]. Microalgae is a major part of phytoplankton community found in almost all marine and freshwater ecosystem [2]. It also exhibits a rich source of natural antioxidant properties. ...
Modern society is facing problems in producing reliable renewable energy source. A mixed process of carotenoid extraction and producing biofuels from algal biomass offers an attractive application to promote renewable energy due to its accessible mass production. Algae, a simple aquatic plant, with extraordinary biological features is capable to be used in different ways. Algae is studied as an important feedstock for carotenoids to be used in food industries, cosmetics and pharmaceutical industries and biofuels. Considering the nutraceutical properties and high value pigments of microalgae, this survey focuses on different carotenoids and micro algal species. Detailed study of carotenoid synthesis and some of the food products in which carotenoids are present is also interpreted. Furthermore different pigment extraction methods and enlightenment of future aspects of solvent selection from sustainable belongings is addressed in this paper.
... Culture conditions suitable for cell growth are not usually conducive to the accumulation of secondary metabolites [4]. Carotenoid production in industry has been achieved by a two-step method, with D. salina cultivated in the best conditions for growth to produce more biomass [5], and then progressively switched to the best conditions for accumulating carotenoids with the highest carotenoid output. Red light is often employed during the growth period to boost biological yield since studies have shown that red light is more favorable to the growth of D. salina [6]. ...
Background
Dunaliella salina is a high-quality industrial effector for carotenoid production. Although the accumulation of carotenoids in D. salina increases under red light conditions, the content of carotenoids in the algal cell decreases. The mechanism by which red light regulates carotenoid synthesis is still unclear.
Results
In this study, a transcription factor of DsGATA1 with a distinct structure was discovered in D. salina. The recognition motif of DsGATA1 was comparable to that of plant and fungal GATA, despite its evolutionary proximity to animal-derived GATA. The expression of DsGATA1 in D. salina was still noticeably decreased when exposed to red light. Analysis of physiological and biochemical transcriptomic data from overexpressed, interfering and wild-type strains of DsGATA1 revealed that DsGATA1 acts as a global regulator of D. salina carotenoid synthesis. The upregulated genes in the CBP pathway by DsGATA1 were involved in its regulation of the synthesis of carotenoids. DsGATA1 also enhanced carotenoid accumulation under red light by affecting N metabolism. DsGATA1 was found to directly bind to the promoter of nitrate reductase to activate its expression, promoting D. salina nitrate uptake and accelerating biomass accumulation. DsGATA1 affected the expression of the genes encoding GOGAT, GDH and ammonia transporter proteins. Moreover, our study revealed that the regulation of N metabolism by DsGATA1 led to the production of NO molecules that inhibited carotenoid synthesis. However, DsGATA1 significantly enhanced carotenoid synthesis by NO scavenger removal of NO. The D. salina carotenoid accumulation under red light was elevated by 46% in the presence of overexpression of DsGATA1 and NO scavengers.
Conclusion
It was found that a transcription factor of DsGATA1 from D. salina has a distinct structure and recognition motif. The novel gene encoding DsGATA1 enhanced the production of carotenoids under red light and endowed D. salina with high algal biomass. The regulation of terpenoid metabolism by DsGATA1 is different from that reported for GATA factors. DsGATA1 yet enhanced the production of NO in D. salina. Nevertheless, our results indicated that DsGATA1 could be an important target for engineering carotenoid production.
... In future research, scientists should take a holistic approach to optimize microalgae culture by combining new strategies for metabolic engineering, strain selection, photobioreactor design and process optimization to produce low-cost, high-yield and environmentally friendly methods [10]. ...
This scientometric review and bibliometric analysis aimed to characterize trends in scientific research related to algae, photobioreactors and astaxanthin. Scientific articles published between 1995 and 2020 in the Web of Science and Scopus bibliographic databases were analyzed. The article presents the number of scientific articles in particular years and according to the publication type (e.g., articles, reviews and books). The most productive authors were selected in terms of the number of publications, the number of citations, the impact factor, affiliated research units and individual countries. Based on the number of keyword occurrences and a content analysis of 367 publications, seven leading areas of scientific interest (clusters) were identified: (1) techno-economic profitability of biofuels, bioenergy and pigment production in microalgae biorefineries, (2) the impact of the construction of photobioreactors and process parameters on the efficiency of microalgae cultivation, (3) strategies for increasing the amount of obtained lipids and obtaining biodiesel in Chlorella microalgae cultivation, (4) the production of astaxanthin on an industrial scale using Haematococcus microalgae, (5) the productivity of biomass and the use of alternative carbon sources in microalgae culture, (6) the effect of light and carbon dioxide conversion on biomass yield and (7) heterotrophy. Analysis revealed that topics closely related to bioenergy production and biofuels played a dominant role in scientific research. This publication indicates the directions and topics for future scientific research that should be carried out to successfully implement economically viable technology based on microalgae on an industrial scale.
... Moreover, D. salina supplementation to growing Red Tilapia was found to accelerate fecundity (Al-Madani; personal communication). Hence, D. salina nutritional supplementation might be used as an alternative to hormonal treatment to enhance reproductive performance because of its high level of beta-carotene, glycerol, protein and other fine chemicals (Wichuk et al., 2014;Cuellar-Bermudez et al., 2015;Gong and Bassi, 2016). Further studies are still required of other microalgae species for exploring their effects on ovarian follicle development, viability of the resulting oocytes and embryos. ...
... These deleterious impacts resulted from increased carbon emissions by humans, particularly through greenhouse gases (Arora et al. 2018). Therefore, there is a need to find alternative clean energy sources, and transition to a bio-based economy with a low carbon footprint (Wichuk et al. 2014;Kusmayadi et al. 2021;Rajak et al. 2022). ...
Since the last few decennia microalgal biomass is of industrial significance for achieving sustainable biofuels pharmaceuticals, nutraceuticals, and functional foods. However, the major bottleneck that needs to be addressed for achieving sustainability is the low biomass productivity of microalgae. In this context, we employed a statistical approach, response surface methodology (RSM), for medium optimization to enhance the biomass of the marine strain Nannochloropsis oculata UTEX 2164 since N. oculata has good growth rate and lipid content along with other biochemical constituents. We investigated the role of macronutrients such as nitrogen (NaNO3—sodium nitrate as the source), phosphorus (NaH2PO4—monosodium phosphate as the source), and carbon (NaHCO3—sodium bicarbonate as the source) in F/2 medium as major factors that obviously control biomass production. The medium optimization was undertaken first to enhance algal biomass and the preliminary data analysis accounted for these quantifiable variables using the one-factor-at-a-time (OFAT) method with varying nitrogen, phosphorus, and carbon concentrations predicting their effect on the overall biomass yield. These findings were refined for the RSM-based experiments using a central composite design (CCD) and later evaluated to demonstrate the combined effect of nitrogen (N) and phosphorous (P) interactions by measuring biomass yields. Our data demonstrate that after nine days of culture with a 21.08 mg L⁻¹ of N-NO3⁻ concentration, the maximum biomass concentration achieved was 576 mg L⁻¹, compared to 460 mg L⁻¹ in the control. Overall, the employed statistical modeling achieved 25% DCW more biomass concentration than the control, with a coefficient of variance (CV) of 8.22%. Thus this study paves the way to further utilize this alga or the refined medium composition to acquire higher cell biomass in other algae.
... Photosynthesis in algae is facilitated by pigments, which are brightly colored chemical compounds. Carotenoids, chlorophylls, and phycobilin are the three primary types of microalgal photosynthetic pigments [127,128]. Microalgae pigments are eye-catching natural colors that include high-value components with health-promoting qualities that include antioxidants, vitamin precursors, neuroprotective, and • Although shrimp survival, digestive enzyme activity, and fatty acid content were not affected, their specific growth rate was much higher than in the control group. ...
Sustainable management of natural resources is critical to food security. The shrimp feed and fishery sector is expanding rapidly, necessitating the development of alternative sustainable components. Several factors necessitate the exploration of a new source of environmentally friendly and nutrient-rich fish feed ingredients. Microalgal biomass has the potential to support the growth of fish and shrimp aquaculture for global food security in the bio-economy. Algal biorefineries must valorize the whole crop to develop a viable microalgae-based economy. Microalgae have the potential to replace fish meal and fish oil in aquaculture and ensure sustainability standards. Microalgae biomasses provide essential amino acids, valuable triglycer-ides such as lipids, vitamins, and pigments, making them suitable as nutritional supplements in livestock feed formulations. Fish and microalgae have similar nutritional profiles, and digestibility is a critical aspect of the aquafeed formulation. A highly digestible feed reduces production costs, feed waste, and the risk of eutrophication. Due to low input costs, low carbon footprint, waste-water treatment benefits, and carbon credits from industrial CO 2 conversion, microalgae-based fish and shrimp feeds have the potential to provide significant economic benefits. However, several challenges must be addressed before microalgal biomass and bioproducts may be used as fish feeds, including heavy metal bioaccumulation, poor algal biomass digestion, and antinu-trient effects. Knowledge of biochemical composition is limited and diverse, and information on nutritional value is scattered or contradictory. This review article presents alternative approaches that could be used in aquaculture to make microalgal biomass a viable alternative to fish meal. ARTICLE HISTORY
... The unsaturated long-chain fatty acids extracted from microalgae exhibit important health benefits including potential anti-inflammatory and anti-carcinogenic effect on humans [80,81]. Apart from the three major fractions, microalgae contain pigments such as chlorophylls, carotenoids, phycocyanin and astaxanthin [82], which are beneficial in pharmaceutical and cosmetic industry [83,84]. Despite encasing numerous beneficial and high-value components, the current extraction of bioactive components from microalgal biomass only focused on a single product extraction. ...
Microalgae have been explored as a sustainable alternative to fuel and feed on natural resources. Microalgae possess numerous advantages over their renewable counterparts such as soybean and palm oil. It does not compete with agricultural land or freshwater for food crop production, making it a potential biofuel source. However, commercialisation of microalgae biodiesel is yet to make a presence in the billion-dollar biofuel industry due to the bottlenecks. These include rigid microalgae cell wall, low biomass concentration in the harvested culture and high downstream costs. Therefore, a fossil fuel-derived concept of refinery can be introduced to microalgae to overcome as aforementioned challenges. This project aims to focus on the algae downstream process for biorefinery applications. First, a novel biocomponent extraction method, named sugaring-out assisted liquid biphasic electric flotation (LBEF) system, for protein separation from Chlorella vulgaris was developed. High yield of proteins (69.66±0.86 %) was extracted from microalgae with a rapid and single-step process. Following this, a multiphase integrated system that focused on the extraction of two or more biomolecules in microalgae was introduced. This system focused on simultaneous component extraction rather than conventional cascade approach. The system were incorporated in two different studies. First study aimed to extract two biomolecules (protein and lipid), whereas second study focused on a concurrent three biomolecules extraction approach. The parameters of this system such as volume ratio of ammonium sulphate and t-butanol, flotation air flowrate, flotation time, ultrasound pulse settings and pH were optimised to achieve a high recovery of biomolecules. Highest yield of protein, lipids and carbohydrates were observed at 96.59±8.15 %, 61.02±0.91 % and 52.69±1.90 %, respectively. Control run without flotation technique resulted in lower yield of proteins, lipids and carbohydrates at 25.33±3.50 %, 52.96±4.59 % and 32.44±0.29 %, respectively. Whereas, control run without flotation and cell-disruption technique had lowest yield of proteins, lipids and carbohydrates at 16.73±1.26 %, 51.13±6.27 % and 26.21±0.23 %, respectively. Besides, a large-scale set-up up to 10-15 times was tested out. Recycling ability of the chemicals involved in the extraction were presented. Up to 90 % of the alcohols and salt used in the experiment were recycled. Lastly, the extracted proteins from the multiphase integrated system were purified and its application in wound healing of human keratinocyte cells was investigated. Proteins were adsorbed on a gelatine-glutaraldehyde membrane. This membrane system was used to observe the wound healing of keratinocytes. The biocompatibility, cell adhesion, proliferation and wound scratch of human keratinocyte cells were studied and presented. Overall, multiphase integrated system presented in this project serves as a successful demonstration of microalgae biorefinery concept. The improved yield of biomolecules provide potential applications of microalgae in biofuel, food and medicine field industry. Future studies should focus on analysis of life-cycle cost and optimising the operational cost required for this whole biorefinery set up. The project presented in this thesis offers a platform for future biorefinery research and possible commercial large-scale utilisation.
... The cultivation systems more often used for D. salina cultivation are large unstirred open ponds or paddlewheel stirred raceways. A two-step cultivation is commonly applied: the first stage, a "green" phase, where growth is done under optimal conditions; a second stage, the "orange" phase, where the culture is submitted to stress factors and the carotenoid production is enhanced (Wichuk et al., 2014). ...
Microalgae industrial production is viewed as a solution for alternative production of nutraceuticals, cosmetics, biofertilizers, and biopolymers. Throughout the years, several technological advances have been implemented, increasing the competitiveness of microalgae industry. However, online monitoring and real‐time process control of a microalgae production factory still require further development. In this mini‐review, non‐destructive tools for online monitoring of cellular agriculture applications are described. Still, the focus is on the use of fluorescence spectroscopy to monitor several parameters (cell concentration, pigments, and lipids) in the microalgae industry. The development presented makes it the most promising solution for monitoring up‐and downstream processes, different biological parameters simultaneously, and different microalgae species. The improvements needed for industrial application of this technology are also discussed.
... Hence, the β-carotene extraction from natural resources is experiencing an exponential evolution in several fields of industrial production. In fact, it has a highly added value in preventing many diseases, in cosmetics and as natural colorant in food (Bogacz-Radomska & Harasym, 2018;Wichuk et al., 2014). In addition, some forms of β-carotene, like the 9-cis β-carotene, are difficult to synthesize chemically and overpriced (estimated €500,000/g). ...
Recently, β-carotene has gained tremendous importance as a bioactive molecule due to the growing awareness of the harmful effects of synthetic products. β-carotene is a high-value natural pigment that has the highest demand in the global carotenoid market owing to its proven antioxidant properties relevant for several diseases. To date, Dunaliella salina is the most important producer of natural β-carotene and is the subject of important industrial efforts. However, the extraction of β-carotene remains challenging since all the proposed techniques present a risk of product contamination or loss of quality due to solvent residuals and to a low yields. The purpose of this study was to set up a green, ecological and innovative process of extraction of the two major β-carotene isomers from the halophilic microalgae Dunaliella salina. Based on molecular modeling, docking, and drug design, we conceived and synthesized two chimeric peptides (PP2, PP3) targeting specifically the two major isomers: all-trans or 9-cis β-carotene. The experimental protocol used in this study demonstrated the ability and the efficacy of those two peptides to cross the cell membrane and bind with high affinity to β-carotene isomers and exclude them towards the extracellular medium while preserving the integrity of living cells.
Interestingly, the tested peptides (PP2, PP3) exhibit significant β-carotene extraction yields 58% and 34% respectively from the total of the β-carotene in microalgae cells. In addition to its simplicity, this process is fast, independent of the source of the β-carotene, and selective. These results would allow us to set up a green, ecological, and very profitable process of extraction from microalgae containing high amounts of enough β-carotene. Our innovative approach is highly promising for the extraction of Dunaliella salina biomass on an industrial scale.
... Chlorophyll constitutes a primary component of algal biomass, possesses antioxidant properties, and is mostly utilized as coloring agents. Carotenoids are natural liposoluble pigments found in algae, consisting of hydrocarbons and their oxygenated derivatives (Wichuk et al., 2014). The hydrocarbon carotenoids are named as carotenes, whereas the oxygenated derivatives are known as xanthophylls (Hu et al., 2018). ...
Microalgae have gained attention as feedstock for value-added chemicals production. The extraction potential of four different organic solvents such as methanol, chloroform, dimethyl sulphoxide (DMSO) and acetone was assessed for obtaining chlorophyll (Chl a and Chl b), and total carotenoids over 48 h at every 4 h intervals utilizing mixed microalgal consortium. Maximum of 4.62 μg/ml of Chl a, 4.78 μg/ml of Chl b and 1.76 μg/ml of total carotenoids were obtained with DMSO, methanol and 80% acetone after 16 h, 40 h and 36 h respectively. Maximum astaxanthin yield of 0.82 μg/ml was obtained with DMSO after 20 min, which was found to decline on further increase in extraction time. Supplementing the solvent extraction process with microwave and ultrasound increased the pigment yield. The study would facilitate utilization of natural microalgal pigments as an eco-friendly and economical alternative for synthetic colorants used in the feed and agricultural industries.
... While large parts of the required demand are manufactured by chemical synthesis, microbial production of natural β-carotene is gaining importance. Aside from the use of microalgae and other microbes (Dufossé, 2017;Qiang et al., 2019;Wichuk et al., 2014;Yuan and Alper, 2019), both, S. cerevisiae and E. coli-based host systems have been established, reaching titers at gram scale with the prokaryote after engineering precursor biosynthesis and optimizing fermentation conditions Yang and Guo, 2014). We have now demonstrated that R. capsulatus can accumulate 30 mg/L (15 mg/g DCW) β-carotene, which is identical to the recently described yield that could be gained in the closely related R. sphaeroides (Qiang et al., 2019), but notably without bioprocess optimization. ...
Terpenes constitute one of the largest groups of secondary metabolites that are used, for example, as food-additives, fragrances or pharmaceuticals. Due to the formation of an intracytoplasmic membrane system and an efficient intrinsic tetraterpene pathway, the phototrophic α-proteobacterium Rhodobacter capsulatus offers favorable properties for the production of hydrophobic terpenes. However, research efforts have largely focused on sesquiterpene production. Recently, we have developed modular tools allowing to engineer the biosynthesis of terpene precursors. These tools were now applied to boost the biosynthesis of the diterpene casbene, the triterpene squalene and the tetraterpene β-carotene in R. capsulatus SB1003. Selected enzymes of the intrinsic isoprenoid pathway and the heterologous mevalonate (MVA) pathway were co-expressed together with the respective terpene synthases in various combinations. Remarkably, co-expression of genes ispA, idi and dxs enhanced the synthesis of casbene and β-carotene. In contrast, co-expression of precursor biosynthetic genes with the squalene synthase from Arabidopsis thaliana reduced squalene titers. Therefore, we further employed four alternative pro- and eukaryotic squalene synthases. Here, the synthase from Methylococcus capsulatus enabled highest product levels of 90 mg/L squalene upon co-expression with ispA. In summary, we demonstrate the applicability of R. capsulatus for the heterologous production of diverse terpene classes and provide relevant insights for further development of such platforms.
... Although several studies have addressed the effect of different culture conditions on β-carotene content in D. salina, its accumulation leads to reduced growth rates, including extreme temperatures, high salinity, and nitrogen limitation (Xu and Harvey 2019). So, molecular approaches have shown great potentials to enhance the accumulation of bioproducts, such as metabolic engineering, transcriptional engineering, and gene disruption strategies (Wichuk et al. 2014;Zhang et al. 2018). ...
Dunaliella salina ( D. salina ) has been exploited as a novel expression system for the field of genetic engineering. However, owing to the low or inconsistent expression of target proteins, it has been greatly restricted to practical production of recombinant proteins. Since the accurate gene editing function of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system, β-carotene hydroxylase gene was chosen as an example to explore D. salina application with the purpose of improving expression level of foreign genes. In this paper, based on pKSE401 backbone, three CRISPR/Cas9 binary vectors were constructed to targeting exon 1 and 3 of the β-carotene hydroxylase of D. salina CCAP19/18 (Dschyb). D. salina mutants were obtained by salt gradient transformation method, and the expression of Dschyb gene were identified through real-time fluorescent quantitative PCR. Moreover, carotenoids content was analyzed by high-performance liquid chromatography at different time points after high intensity treatment. Compared with wild type strains, the β-carotene levels of mutants showed a significant increase, nearly up to 1.4 μg/ml, and the levels of zeaxanthin decreased to various degrees in mutants. All the results provide a compelling evidence for targeted gene editing in D. salina . This study gave a first successful gene editing of D. salina which has a very important practical significance for increasing carotene yield and meeting realistic industry demand. Furthermore, it provides an approach to overcome the current obstacles of D. salina , and then gives a strong tool to facilitates the development and application of D. salina system.
... Although several studies have addressed the effect of different culture conditions on β-carotene content in D. salina, its accumulation leads to reduced growth rates, including extreme temperatures, high salinity, and nitrogen limitation (Xu et al. 2019). So, molecular approaches have shown great potentials to enhance the accumulation of bio-products, such as metabolic engineering, transcriptional engineering, and gene disruption strategies (Wichuk et al. 2014;Zhang et al. 2018). ...
Dunaliella salina ( D. salina ) has been exploited as a novel expression system for the field of genetic engineering. However, owing to the low or inconsistent expression of target proteins, it has been greatly restricted to practical production of recombinant proteins. Since the accurate gene editing function of CRISPR/Cas system, β-carotene hydroxylase gene was chosen as an example to explore D. salina application with the purpose of improving expression level of foreign genes. In this paper, based on pKSE401 backbone, three CRISPR/Cas9 binary vectors were constructed to targeting exon 1 and 3 of the β-carotene hydroxylase of D. salina CCAP19/18 (Dschyb). D. salina mutants were obtained by salt gradient transformation method, and the expression of Dschyb gene were identified through real-time fluorescent quantitative PCR. Moreover, carotenoids content was analyzed by high-performance liquid chromatography at different time points after high intensity treatment. Compared with wild type strains, the β-carotene levels of mutants showed a significant increase, nearly up to 1.4 μg/ml, and the levels of zeaxanthin decreased to various degrees in mutants. All the results provide a compelling evidence for targeted gene editing in D. salina . This study gave a first successful gene editing of D. salina which has a very important practical significance for increasing carotene yield and meeting realistic industry demand. Furthermore, it provides an approach to overcome the current obstacles of D. salina , and then gives a strong tool to facilitates the development and application of D. salina system.
... The survival rate and productive value of energy was not differed (P >0.05) among control and D. salina treated groups in this study. Algae of D. salina has high levels of beta-carotene, glycerol, protein and other fine chemicals (Gouveia et al., 2008;Ghasemi et al., 2011;Wichuk et al., 2014;Cuellar-Bermudez et al., 2015;Gong and Bassi, 2016). Chemical composition of D. salina algae indicated 8.47% moisture, 54.17% crude protein, 0.80% fiber, 11.42% total lipid, 18.47% ether extract and 6.67% ash (Mohammed 2018 b). ...
... 161 D. bardawil has the highest recorded βcarotene content (80 mg g −1 dw), 40 while recombinant C. zofingiensis and Saccharomyces cerevisiae reported less than 6 mg g −1 dw. 163 In another study, a new synthetic PSY gene was designed by combination of PSY genes from D. salina, C. reinhardtii and Mariella zofingiensis, and cloned into Scenedesmus sp. CPC2. ...
... The survival rate and productive value of energy was not differed (P >0.05) among control and D. salina treated groups in this study. Algae of D. salina has high levels of beta-carotene, glycerol, protein and other fine chemicals (Gouveia et al., 2008;Ghasemi et al., 2011;Wichuk et al., 2014;Cuellar-Bermudez et al., 2015;Gong and Bassi, 2016). Chemical composition of D. salina algae indicated 8.47% moisture, 54.17% crude protein, 0.80% fiber, 11.42% total lipid, 18.47% ether extract and 6.67% ash (Mohammed 2018 b). ...
... Apart from the three major fractions, microalgae contains various pigments such as chlorophylls, carotenoids, phycocyanin and astaxanthin [28]. These are employed in the pharmaceutical and cosmetic industry [29,30]. It is evident that microalgae encase numerous beneficial and high-value components. ...
Microalgae biomass contains various useful bio-active components. Microalgae derived biodiesel has been researched for almost two decades. However, sole biodiesel extraction from microalgae is time-consuming and is not economically feasible due to competitive fossil fuel prices. Microalgae also contains proteins and carbohydrates in abundance. Microalgae are likewise utilized to extract high-value products such as pigments, anti-oxidants and long-chain polyunsaturated fatty acids which are useful in cosmetic, pharmaceutical and nutraceutical industry. These compounds can be extracted simultaneously or sequentially after biodiesel extraction to reduce the total expenditure involved in the process. This approach of bio-refinery is necessary to promote microalgae in the commercial market. Researchers have been keen on utilizing the bio-refinery approach to exploit the valuable components encased by microalgae. Apart from all the beneficial components housed by microalgae, they also help in reducing the anthropogenic CO2 levels of the atmosphere while utilizing saline or wastewater. These benefits enable microalgae as a potential source for bio-refinery approach. Although life-cycle analysis and economic assessment do not favor the use of microalgae biomass feedstock to produce biofuel and co-products with the existing techniques, this review still aims to highlight the beneficial components of microalgae and their importance to humans. In addition, this article also focuses on current and future aspects of improving the feasibility of bio-processing for microalgae bio-refinery.
... In this context, microalgae offers several advantages due to their higher photosynthetic efficiencies, accounting for ~40% of atmospheric CO 2 assimilation, thus reducing global warming, as well as an ability to thrive in non-arable/off-shore areas and mimic any stress conditions by altering their cellular pathways (Brennan and Owende 2010, Markou and Nerantzis 2013, Li et al. 2015, Jutur, Nesamma, and Shaikh 2016. The potential use of microalgae in any industrial biorefinery will be a step forward for the production of a low or/and zero carbon footprint technology (Wichuk, Brynjolfsson, andFu 2014, Hariskos andPosten 2014). ...
... Currently, D. salina biomass production has become an attractive opportunity for companies selling green products whose varieties and demands have increased in recent years on the large shopping centres, cosmetics stores and pharmacies in large cities (Benemann, 2013;Raja et al., 2008;Spolaore et al., 2006). For this reason, in order to obtain higher yields in algal biomass rich in carotenoids is developing studies that include design and implementation of closed (for example, photobioreactors) or open ''raceways" systems, genetic improvement of strains, metabolomics research to obtain new molecules, cultivation physiology associated with the overproduction of metabolites, many scientific and technological efforts have been carried out (Lamers et al., 2008;Zhu and Jiang, 2008;Kleinegris et al., 2011;Wichuk et al., 2014;Andersen and Kawachi, 2005;Gallego et al., 2013). ...
The objective was evaluate the carotenogenic activity of Dunaliella salina isolated from the artificial salt flats of municipality of Manaure (Department of La Guajira, Colombia). Two experimental testings were designed, in triplicate, to induce the reversibility of the cell tonality depending on the culture conditions. In the first test (A), to induce the reversibility from green to red tonality in D. salina cells, these were cultured in J/1 medium at a concentration of 4.0 M NaCl, 390 µmol m−2 s−1, 0.50 mM KNO3. In the second test (B), to induce the reversibility from red to green cell tonality, the cultures were maintained in J/1 medium 1 M NaCl, 190 µmol m−2 s−1, 5.0 mM KNO3 and pH 8.2. The population growth was evaluated by cell count and the pigment content was performed by spectrophotometric techniques. It was found that in both tests the culture conditions influenced the population growth and the pigments production of D. salina. There was a significant difference between the mean values of total carotenoids in the test A with 9.67 ± 0.19 μg/ml and second test with 1.54 ± 0.08 μg/ml at a significance level of p
... Though microalgal group makes up the major source of carotenoids, metabolic engineering in carotenoids biosynthesis pathway in these microorganisms is impeded by the lack of strategies for genetic transformation of commercially important microalgal species (Wichuk, Brynjolfsson, and Fu 2014). Even sometimes, the successful transformation does not guarantee long-term stability. ...
The versatile use of biopigments in food, feed, cosmetic, pharmaceutical and analytical industries emphasized to find different and renewable sources of biopigments. Microalgae, including cyanobacteria, are becoming a potential candidate for pigment production as these have fast-growing ability, high pigment content, highly variable and also have “Generally recognized as safe” status. These algal groups are known to produce different metabolites that include hormones, vitamins, biopolythene and biochemicals. We discuss here the potential use of microalgal biopigments in our daily life as well as in food and cosmetic industries. Pigment like carotenoids has many health benefits such as antioxidant, anti-inflammatory properties and also provide photo-protection against UV radiation. This review details the effect of various abiotic and biotic factors such as temperature, light, nutrition on maximizing the pigment content in the microalgal cell. This review also highlights the potential of microalgae, whether in present native or engineered strain including the many metabolic strategies which are used or can be used to produce a higher amount of these valuable biopigments. Additionally, future challenges in the context of pigment production have also been discussed.
... To achieve the economic viability for the carotenoid production from microalgal platforms, increasing the productivity of biomass is a prerequisite. In this regard, lightdriven metabolism and the low photosynthetic efficiency of microalgae in photobioreactors are the main hurdles [64]. To improve the efficiency of sunlight-to-biomass conversion in the microalgal biofactories that generally occur with a significantly lower efficiency (35%-80%), several strategies have been successfully attempted [65], which include (1) engineering of the light-harvesting antenna to reduce non-photochemical quenching losses and light attenuation; (2) engineering photosynthetic electron transport with increased numbers or activity of Cytb6f and plastoquinone complexes present in the photosynthesis apparatus in order to overcome kinetic constraints that result in energy losses; (3) engineering of the energy/reductant sink capacity of Calvin-Benson cycle; and (4) engineering of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the construction of synthetic chloroplastic photorespiratory bypasses have been found to be beneficial in terms of achieving higher biomass productivity. ...
Carotenoids are a diverse group of isoprenoid pigments that play crucial roles in plants, animals, and microorganisms, including body pigmentation, bio-communication, precursors for vitamin A, and potent antioxidant activities. With their potent antioxidant activities, carotenoids are emerging as molecules of vital importance in protecting against chronic degenerative disease, such as aging, cancer, cataract, cardiovascular, and neurodegenerative diseases. Due to countless functions in the cellular system, carotenoids are extensively used in dietary supplements, food colorants, aquaculture and poultry feed, nutraceuticals, and cosmetics. Moreover, the emerging demand for carotenoids in these vast areas has triggered their industrial-scale production. Currently, 80%–90% of carotenoids are produced synthetically by chemical synthesis. However, the demand for naturally produced carotenoids is increasing due to the health concern of synthetic counterparts. This article presents a review of the industrial production of carotenoids utilizing a number of diverse microbes, including microalgae, bacteria, and fungi, some of which have been genetically engineered to improve production titers.
... Light supply and delivery are limiting factors when scaling up PBRs for photoautotrophic cultivation. The extra light/dark cycles due to mixing effects in large PBRs are unfavorable to algal growth (Wichuk et al., 2014). They largely decrease the biomass productivity and the biomass concentration and this greatly increases the operating and downstream processing costs (Liang et al., 2009). ...
... The neoxanthin-related xanthophylls diatoxanthin, dinoxanthin, diadinoxanthin, and fucoxanthin are only found in diatoms such as those of the genus Phaeodactylum, while α-carotene, εcarotene, δ-carotene or astaxanthin are only found in green algae ( Fig. 6) [148,151]. Furthermore, stressing conditions such as nitrogen deprivation, strong solar irradiation, drought or hypersalinity cause some microalgae to accumulate large amounts of carotenoids that provide an intense reddish pigmentation to the stressed cells [152]. In this case, however, the carotenoids are not accumulated in the thylakoid membranes but in oily droplets or lipid vesicles located either in chloroplasts (in thylakoids) or in the cytoplasm (as oleosomes). ...
Carotenoids are lipophilic isoprenoid compounds synthesized by all photosynthetic organisms and some non-photosynthetic bacteria and fungi. With some notable exceptions, animals (including humans) do not produce carotenoids de novo but take them in their diets. In photosynthetic systems carotenoids are essential for photoprotection against excess light and contribute to light harvesting, but perhaps they are best known for their properties as natural pigments in the yellow to red range. Carotenoids can be associated to fatty acids, sugars, proteins, or other compounds that can change their physical and chemical properties and influence their biological roles. Furthermore, oxidative cleavage of carotenoids produces smaller molecules such as apocarotenoids, some of which are important pigments and volatile (aroma) compounds. Enzymatic breakage of carotenoids can also produce biologically active molecules in both plants (hormones, retrograde signals) and animals (retinoids). Both carotenoids and their enzymatic cleavage products are associated with other processes positively impacting human health. Carotenoids are widely used in the industry as food ingredients, feed additives, and supplements. This review, contributed by scientists of complementary disciplines related to carotenoid research, covers recent advances and provides a perspective on future directions on the subjects of carotenoid metabolism, biotechnology, and nutritional and health benefits.
... These unusual pigments accumulate in select diatoms species and these pigments have great potential applications in cosmetics and food supplements. Pigments from diatoms showed a major role in photo-protection (Wichuk et al., 2014;Fu et al., 2015;Kuczynska et al., 2015). Benefits of carotenoid pigments from algae are presented in Table 7. ...
Microalgae are rich source of various bioactive molecules such as carotenoids, lipids, fatty acids, hydrocarbons, proteins, carbohydrates, amino acids, etc. and in recent year's carotenoids from algae gained commercial recognition in the global market for food and cosmeceutical applications. However, the production of carotenoids from algae is not yet fully cost effective to compete with synthetic ones. In this context the present review examines the technologies/methods in relation to mass production of algae, cell harvesting for extraction of carotenoids, optimizing extraction methods etc. Research studies from different microalgal species such as Spirulina platensis, Haematococcus pluvialis, Dunaliella salina, Chlorella sps., Nannochloropsis sps., Scenedesmus sps., Chlorococcum sps., Botryococcus braunii and Diatoms in relation to carotenoid content, chemical structure, extraction and processing of carotenoids are discussed. Further these carotenoid pigments, are useful in various health applications and their use in food, feed, nutraceutical, pharmaceutical and cosmeceutical industries was briefly tocuhced upon. The commercial value of algal carotenoids has also been discussed in this review. Possible recommendations for future research studies are proposed.
... Many microorganisms, such as Acetobacter and Pseudomonas bacteria Candida and Rhodotorula (carotenogenic yeast), are considered almost as phenol analyzer (Walker et al., 2014;Wichuk et al., 2014). Phenol metabolism in Rhodotorula sp. was examined and results showed that phenol hydroxylated before splitting into catechol in R. rubra and then oxidized into cis, cis-muconic acid (Boşça and Sanin, 2015). ...
The substitution of synthetic food dyes with natural colorants continues to be assiduously pursued. The current list of natural carotenoid colorants consists of plant-derived annatto (bixin and norbixin), paprika (capsanthin and capsorubin), saffron (crocin), tomato and gac fruit lycopene, marigold lutein, and red palm oil (α- and β-carotene), along with microalgal Dunaliella β-carotene and Haematococcus astaxanthin and fungal Blakeslea trispora β-carotene and lycopene. Potential microalgal sources are being sought, especially in relation to lutein, for which commercial plant sources are lacking. Research efforts, manifested in numerous reviews and research papers published in the last decade, have been directed to green extraction, microencapsulation/nanoencapsulation, and valorization of processing by-products. Extraction is shifting from conventional extraction with organic solvents to supercritical CO2 extraction and different types of assisted extraction. Initially intended for the stabilization of the highly degradable carotenoids, additional benefits of encapsulation have been demonstrated, especially the improvement of carotenoid solubility and bioavailability. Instead of searching for new higher plant sources, enormous effort has been directed to the utilization of by-products of the fruit and vegetable processing industry, with the application of biorefinery and circular economy concepts. Amidst enormous research activities, however, the gap between research and industrial implementation remains wide.
As the global population continues to grow, the demand for dietary protein is rapidly increasing, necessitating the exploration of sustainable and nutritious protein sources. Algae has emerged as a promising food source due to their high value ingredients such as proteins, as well as for their environmental sustainability and abundance. However, knowledge gaps surrounding dietary recommendations and food applications restrict algae’s utilization as a viable protein source. This review aims to address these gaps by assessing the suitability of both microalgae and macroalgae as alternative/complementary protein sources and exploring their potential applications in food products. The first section examines the potential suitability of algae as a major food source by analyzing the composition and bioavailability of key components in algal biomass, including proteins, lipids, dietary fiber, and micronutrients. Secondly, the biological effects of algae, particularly their impact on metabolic health are investigated with an emphasis on available clinical evidence. While evidence reveals protective effects of algae on glucose and lipid homeostasis as well as anti-inflammatory properties, further research is required to understand the longer-term impact of consuming algal protein, protein isolates, and concentrates on metabolic health, including protein metabolism. The review then explores the potential of algal proteins in food applications, including ways to overcome their sensory limitations, such as their dark pigmentation, taste, and odor, in order to improve consumer acceptance. To maximize algae’s potential as a valuable protein source in the food sector, future research should prioritize the production of more acceptable algal biomass and explore new advances in food sciences and technology for improved consumer acceptance. Overall, this paper supports the potential utility of algae as a sustainable and healthy ingredient source for widespread use in future food production.
Microalgae are photosynthetic unicellular organisms that live in almost all environments on earth. They occur as a single-celled or colonial form in chain and filaments. In nature, they are important sink and source of carbon, supporting the food chain in all ecosystems in the world. They are also an important component to balance the nutrient cycle particularly in aquatic ecosystems. Microalgae are taxonomically classified based on their structural morphology, nutritional requirements, motility, biochemistry composition, etc. Despite the use of microalgae in biofuel production, microalgae are widely cultivated on a commercial scale in recent years due to their rich content of natural bioactive compounds including polysaccharides, lipids, pigments, proteins, vitamins, minerals, and antioxidants. These microalgal bioactive compounds are widely applied in various industries, ranging from human and animal food, cosmeceutical, pharmaceutical to other bio-based products. The biological functions and the application of the high commercial value products sourced from microalgae were discussed in this chapter.
Cyanobacteria like other microalgal species are considered a key element in the new bioenergy concepts. Nevertheless, despite their enormous potential, this is still not enough to compete with natural fossil fuels for the production of microalgae as biofuels, with technical–economic competitiveness to other commercial technologies. Financial problems connected with the steps of the upstream and downstream processes must be overcome. Recent techno-economic analyses and life cycle assessments of microalgae-based production systems have suggested that the only most possible way for scaling up the cyanobacteria biomass technology passes through complete and optimal utilization of the cell components in an integrated biorefinery setup. This chapter provides a comprehensive analysis of the present CO2 biofixation approaches and technologies using cyanobacteria under the strategy of biorefinery with cells treatment. Herein are discussed various cultivation techniques to maximize desirable products of cyanobacteria biorefinery. Advanced methods for metabolites isolation are analyzed to ensure the stable quantity and quality of cyanobacteria-based metabolites. Optimal biomass production in advanced closed photobioreactors raises many scientific and engineering problems, which occur when the scale increased. Scale-up is the last and most difficult technological step where all hypotheses are checked. Biorefinery deals with this problem in each stage of setup. The complexity and sustainability of this approach are the foremost concerns of specialists analyzing trends in the environmental, technological, and economic dynamic changes.
Microalgae are photosynthetic unicellular organisms that live in almost all environments on earth. They occur as a single-celled or colonial form in chain and filaments. In nature, they are important sink and source of carbon, supporting the food chain in all ecosystems in the world. They are also an important component to balance the nutrient cycle particularly in aquatic ecosystems. Microalgae are taxonomically classified based on their structural morphology, nutritional requirements; motility; biochemistry composition; etc. Despite the use of microalgae in biofuel production, microalgae are widely cultivated on a commercial scale in recent years due to their rich content of natural bioactive compounds including polysaccharides, lipids, pigments, proteins, vitamins, minerals, and antioxidants. These microalgal bioactive compounds are widely applied in various industries, ranging from human and animal food, cosmeceutical, pharmaceutical to other bio-based products. The biological functions and the application of the high commercial value sourced from microalgae were discussed in this chapter.
Salt stress is an abiotic stress to plants in especially saline lakes. Dunaliella, a halophilic microalga distributed throughout salt lakes and seas, can respond to different salinity stresses by regulating the expression of some genes. However, these genes and their function and biological processes involved remain unclear. Profiling these salt-stress-related genes in a high-salt-tolerant Dunaliella species will help clarify the salt tolerance machinery of Dunaliella. Three D. salina_YC salt-stress groups were tested under low (0.51 mol/L), moderate (1.03 mol/L), and high (3.42 mol/L) NaCl concentrations and one control group under very low (0.05 mol/L) NaCl concentration and 3 transcriptome results that were deep sequenced and de novo assembled were obtained per group. Twelve high-quality RNA-seq libraries with 46 585 upregulated and 47 805 downregulated unigenes were found. Relative to the control, 188 common differentially expressed genes (DEGs) were screened and divided into four clusters in expression pattern. Fifteen of them annotated in the significant enriched Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were validated via qPCR. Their qPCR-based relative expression patterns were similar to their RNA-seq-based patterns. Two significant DEGs, the geranylgeranyl diphosphate synthase coding gene (1 876 bp cDNA) and diacylglycerol O-acyltransferase coding gene (2 968 bp cDNA), were cloned and analyzed in silico. The total lipid content, superoxide dismutase specific activity, and beta, carotene content of D. salina_YC increased gradually with increasing salinity. In addition, the expression of 11 validated genes involved in fatty acid biosynthesis/degradation, active oxygen or carotenoid metabolisms showed significant changes. In addition, algal photochemical efficiency was diminished with increasing salinity, as well as the expression of 4 photosynthesis-related genes. These results could help clarify the molecular mechanisms underlying D. salina responses to the Yuncheng Salt Lake environment and lay a foundation for further utilization of this algal resource.
Carotenoids are synthesized de novo by algae, photosynthetic plants and fungi. They are isoprenoid molecules that are responsible for various colors of fruits and vegetables such as yellow, orange and some red variants. Carotenoids are lipophilic compounds. These compounds can be classified into two categories – carotenes and xanthophylls. A variety of macroalgae and microalgae are rich in carotenoids and they help in the absorption of sunlight. These carotenoids are also used as food pigments industrially in dairy products, beverages, among others as feed additives. They also find application in cosmetics and in pharmaceuticals given the growing demand for natural products in these sectors. Some carotenoids also provide provitamin A. Producing carotenoids from algae brings with it several advantages. For instance, it is easy to produce, cost‐effective, and environmentally friendly. Their extraction with higher yields is easier and raw materials are not scarce or have seasonal constraints. Of late, there has been an increase in interest in dietary carotenoids due to their ability to reduce incidence of some chronic diseases that involve free radicals as well as for their antioxidant properties. It is likely that carotenoids protect cells from oxidative stress by quenching singlet oxygen damage through a variety of mechanisms. As such carotenoids that have been derived from algae could be a strong natural resource in the search for good functional ingredients. In this chapter, we discuss the structure of various algal carotenoids, their distribution and potential applications for human health.
The chemistry of natural products has been achieved through the improvement of physical and chemical techniques for the isolation and purification of secondary metabolism products such as carotenoids. Carotenoids, also called tetraterpenoids, produce the particular pigmentation found in nature such as red, yellow, and orange colors. These pigments have gained attention due their physiological and ecological roles. Several challenges in carotenoid analysis have promoted news techniques of extraction and purification considering chemical and physical properties of these compounds. In this chapter, the phytochemistry, classification, and biosynthesis of carotenoids were addressed, including their recent analytical advances. Throughout this chapter, their chemistry is also presented in detail and intrinsically related to the several health benefits and different industrial applications. Thus, the need to further develop the identification and quantification methods of such molecules as well as novel and green analytical techniques was emphasized.
Carotenoids are metabolites produced by microalgae that perform as accessory pigments in photosynthesis and as photoprotective molecules. In order to increase the accumulation of carotenoids
in the microalga Scenedesmus dimorphus, salinity (NaCl and KNO3) and blue LED light were evaluated as stress factors when reaching the stationary phase of culture growth in such microalga. After
evaluation, the best of the two salts studied was KNO3 0.6M, since this culture reached the highest concentration of total carotenoids (0.3357±0.02 μg carotenoids mL-1 ), in contrast to 0.1791±0.01 µg
mL-1 with NaCl 0.6 M. Subsequently, the process was scaled in an adapted container of 4 L, entering
the stationary phase on day 12. On day 14 the KNO3 was added at 0.6 M; this experiment lasted 20
days of cell count, obtaining on the last day a total of 0,7395±0,05 μg of carotenoids mL-1
by means of
spectrophotometry. Afterwards, drying of the culture with salinity was carried out, obtaining a dry weight of 5.7 g, with a productivity of 0.0927 g L-1 d -1. Finally, through HPLC chromatography, the lipid
profile of the culture was analyzed, resulting in 21.1% MUFAS, 35.9% SFAS, and 43% PUFAS (34% omega-6 and 8.9% omega-3). Based on the above, it was determined that salinity is a possible factor to
increase in a greater proportion the concentration of lipids than that of carotenoids, specifically in a ratio of 1:2,500 carotenoids and lipids, respectively.
Keywords: Microalga, biomass, carotenoids, lipids, salinity.
Los carotenoides son metabolitos producidos por microalgas que cumplen la función de pigmentos accesorios en la fotosíntesis y de moléculas fotoprotectoras. Con el fin de incrementar la acumulación de los carotenoides en la microalga Scenedesmus dimorphus, se evaluó la salinidad (NaCl y KNO3) y la luz LED azul como factores de estrés en la fase estacionaria del crecimiento del cultivo de esta alga. De las dos sales estudiadas la mejor fue KNO3 0,6M, puesto que el cultivo alcanzó la mayor concentración
de carotenoides totales (0,3357 ± 0,02 µg de carotenoides mL-1) en contraste con 0,1791 ± 0,01 µg mL-1 del cultivo NaCl 0,6 M. Posteriormente, se escaló el proceso en un recipiente adaptado de 4 L, alcanzando fase estacionaria en el día 12 y adicionando el KNO3 0,6M el día 14; en el día 20 (final del cultivo) se obtuvo mediante espectrofotometría un total de 0,7395±0,05 µg de carotenoides mL-1.
Seguido, se realizó el secado del cultivo con salinidad, obteniendo un peso seco de 5,7 g y una productividad de 0,0927 g L-1d-1. Finalmente, a través de cromatografía HPLC, se analizó el perfil lipídico del cultivo, obteniendo 21,1 % de MUFAS, 35,9 % de SFAS y 43 % de PUFAS (omega-6 de 34 % y omega-3 de 8,9 %). Con base en estos resultados, se determinó que la salinidad es un posible factor para incrementar en mayor proporción la concentración de lípidos que de carotenoides, específicamente en una proporción de 1:2.500 de carotenoides y lípidos, respectivamente.
The increasing market demands of β-carotene as colorant, antioxidant and vitamin precursor, requires novel biotechnological production platforms. Yarrowia lipolytica, is an industrial organism unable to naturally synthesize carotenoids but with the ability to produce high amounts of the precursor Acetyl-CoA. We first found that a lipid overproducer strain was capable of producing more β-carotene than a wild type after expressing the heterologous pathway. Thereafter, we developed a combinatorial synthetic biology approach base on Golden Gate DNA assembly to screen the optimum promoter-gene pairs for each transcriptional unit expressed. The best strain reached a production titer of 1.5 g/L and a maximum yield of 0.048 g/g of glucose in flask. β-carotene production was further increased in controlled conditions using a fed-batch fermentation. A total production of β-carotene of 6.5 g/L and 90 mg/g DCW with a concomitant production of 42.6 g/L of lipids was achieved. Such high titers suggest that engineered Y. lipolytica is a competitive producer organism of β-carotene. This article is protected by copyright. All rights reserved
β-carotene is a valuable pigment abundant in some microalgal species but the low β-carotene productivity of microalgae has become the major obstacles against its commercialization. This work aims to improve the productivity of algae-based β-carotene via genetic engineering approaches. First, a synthetic psy gene construct (891 bp) encoding 297 amino acids was expressed in Scenedesmus sp. CPC2 host to enhance the β-carotene production. The synthetic psy gene was designed by considering the highest consensus of amino acids (i.e., 62% identity) from Chlamydomonas reinhardtii, Dunaliella salina and Mariella zofingiensis. The original β-carotene content in wild type Scenedesmus sp. CPC2 is 10.8 mg/g-cell when grown on BG11 medium under 2% CO2 aeration, 150 μmol/m²/s light intensity and 25°C. After transformation of the psy gene into the microalgal host, the β-carotene content of the best recombinant strain (i.e., transformant CPC2-4) significantly increased to over 30 mg/g-cell. The optimal production of β-carotene with the CPC2-4 recombinant strain was achieved when the strain was grown on BG11 medium amended with 0.075 g of MgSO4, giving approximately 3-fold higher β-carotene content than that of the wild-type strain. The best cellular β-carotene content obtained (i.e., 31.8 mg/g) is superior to most algae-based β-carotene production performance reported in the literature.
Microalgae make up the largest and likely most diverse group of photosynthetic organisms in freshwater and marine systems. As new technologies are emerging for the study of bioactive compounds from microalgae, this group is drawing attention as a promising source of natural products that have wide applications in the food and pharmaceutical industries. Algae-derived bioactive compounds are attractive resources for drug screening, given their tremendous structural diversity and biological availability. In this chapter, we first discuss medicinally important products, such as carotenoids, including β-carotene, fucoxanthin, astaxanthin, and lutein, as well as essential fatty acids that originate in microalgae. We then briefly introduce screening assays for antioxidant, antimicrobial, antiviral, anticancer, and immunomodulatory effects, and explore biosynthesis of natural products, which have been widely used in food and cosmetics for their antioxidant effects and nutritional value, and we discuss the potential use of fucoxanthin and its derivatives as anticancer agents. In addition, we describe health benefits of the essential fatty acids eicosapentaenoic acid and docosahexaenoic acid. Further, this chapter emphasizes that microalgae provide a rich source of compounds for therapeutic drug screening and describes examples of screening assays for detection of biological activities of algae-derived compounds.
The unicellular green alga Chlorella zofingiensis has been proposed as a promising producer of natural astaxanthin, a commercially important ketocarotenoid. But the genetic toolbox for this alga is not available. In the present study, an efficient transformation system was established for C. zofingiensis. The transformation system utilized a modified norflurazon-resistant phytoene desaturase (PDS-L516F, with an leucine-phenylalanine change at position 516) as the selectable marker. Three promoters from endogenous PDS, nitrate reductase (NIT), and ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RBCS) genes were tested, with the RBCS promoter demonstrating the highest transformation efficiency. Inclusion of the first intron of the PDS gene further enhanced the efficiency by 91 %. Both particle bombardment and electroporation methods were examined, and the latter gave a fourfold higher transformation efficiency. The introduction of PDS-L516F, which exhibited a 33 % higher desaturation activity than the unaltered enzyme, enabled C. zofingiensis to produce 32.1 % more total carotenoids (TCs) and 54.1 % more astaxanthin. The enhanced accumulation of astaxanthin in transformants was revealed to be related to the increase in the transcripts of PDS, β-carotenoid ketolase (BKT), and hydroxylase (CHYb) genes. Our study clearly shows that the modified PDS gene is a dominant selectable marker for the transformation of C. zofingiensis and possibly for the genetic engineering of the carotenoid biosynthetic pathway. In addition, the engineered C. zofingiensis might serve as an improved source of natural astaxanthin.
In spite of attractive attributes, diatoms are underrepresented in research and literature related to the development of microalgal biofuels. Diatoms are highly diverse and have substantial evolutionarily-based differences in cellular organization and metabolic processes relative to chlorophytes. Diatoms have tremendous ecological success, with typically higher productivity than other algal classes, which may relate to cellular factors discussed in this review. Diatoms can accumulate lipid equivalently or to a greater extent than other algal classes, and can rapidly induce triacylglycerol under Si limitation, avoiding the detrimental effects on photosynthesis, gene expression and protein content associated with N limitation. Diatoms have been grown on production scales for aquaculture for decades, produce value-added products and are amenable to omic and genetic manipulation approaches. In this article, we highlight beneficial attributes and address potential concerns of diatoms as biofuels research and production organisms, and encourage a greater emphasis on their development in the biofuels arena.
A highly efficient nuclear transformation method was established for the pennate diatom Phaeodactylum tricornutum using an electroporation system that drives multi-sequence pulses to introduce foreign DNAs into the cells. By optimizing pulse conditions, the diatom cells can be transformed without removing rigid silica-based cell walls, and high transformation efficiency (about 4,500 per 10(8) cells) is achieved.
There is a particularly high interest to derive carotenoids such as β-carotene and lutein from higher plants and algae for the global market. It is well known that β-carotene can be overproduced in the green microalga Dunaliella salina in response to stressful light conditions. However, little is known about the effects of light quality on carotenoid metabolism, e.g., narrow spectrum red light. In this study, we present UPLC-UV-MS data from D. salina consistent with the pathway proposed for carotenoid metabolism in the green microalga Chlamydomonas reinhardtii. We have studied the effect of red light-emitting diode (LED) lighting on growth rate and biomass yield and identified the optimal photon flux for D. salina growth. We found that the major carotenoids changed in parallel to the chlorophyll b content and that red light photon stress alone at high level was not capable of upregulating carotenoid accumulation presumably due to serious photodamage. We have found that combining red LED (75 %) with blue LED (25 %) allowed growth at a higher total photon flux. Additional blue light instead of red light led to increased β-carotene and lutein accumulation, and the application of long-term iterative stress (adaptive laboratory evolution) yielded strains of D. salina with increased accumulation of carotenoids under combined blue and red light.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-012-4502-5) contains supplementary material, which is available to authorized users.
Background
Although there are many different expression systems for recombinant production of pharmaceutical proteins, many of these suffer from drawbacks such as yield, cost, complexity of purification, and possible contamination with human pathogens. Microalgae have enormous potential for diverse biotechnological applications and currently attract much attention in the biofuel sector. Still underestimated, though, is the idea of using microalgae as solar-fueled expression system for the production of recombinant proteins.
Results
In this study, we show for the first time that completely assembled and functional human IgG antibodies can not only be expressed to high levels in algal systems, but also secreted very efficiently into the culture medium. We engineered the diatom Phaeodactylum tricornutum to synthesize and secrete a human IgG antibody against the Hepatitis B Virus surface protein. As the diatom P. tricornutum is not known to naturally secrete many endogenous proteins, the secreted antibodies are already very pure making extensive purification steps redundant and production extremely cost efficient.
Conclusions
Microalgae combine rapid growth rates with all the advantages of eukaryotic expression systems, and offer great potential for solar-powered, low cost production of pharmaceutical proteins.
Dunaliella bardawil Ben-Amotz & Avron, but not most other Dunaliella species, has a unique property of being able to accumulate, in addition to glycerol, large amounts of β-carotene when cultivated under appropriate conditions. These include high light intensity, a high sodium chloride concentration, nitrate deficiency and extreme temperatures. Under conditions of maximal carotene accumulation D. bardawil contains at least 8% of its dry weight as β-carotene while D. salina grown under similar conditions contains only about 0.3%. Electron micrographs of D. bardawil grown under conditions of high β-carotene accumulation show many β-carotene containing globules located in the interthylakoid spaces of the chloroplast. The same algae grown under conditions where β-carotene does not accumulate, contain few to no β-carotene globules. The β-carotene-rich globules were released from the algae into an aqueous medium by a two-stage osmotic shock technique and further purified by centrifugal ion on 10% sucrose. The isolated purified globules were shown by electron microscopy to be free of significant contamination and composed of membrane-free osmiophilic droplets with an average diameter of 150 nm. Reversed phase high performance liquid chromatography of a total pigment extract of the cells revealed the presence of β-carotene as the major pigment, together with chlorophylls a and b, α-carotene and the xanthophylls lutein, neoxauthin and zeaxanthin. β-Carotene accounted for essentially all the pigment in the purified globules. Analysis of the algal and globule β-carotene fractions by HPLC showed that the β-carotene was composed of approximately equal amounts of all-trans β-carotene and of its 9-cis isomer. Intact D. bardawil cells contained on a dry weight basis about 30% glycerol, 30% protein, 18% lipid, 11% carbohydrate, 9%β-carotene and 1% chlorophyll. The β-carotene globules were composed of practically only neutral lipids, more than half of which was β-carotene. It is suggested that the β-carotene globules may serve to protect D. bardawil against injury by the high intensity irradiation to which this alga is usually exposed in nature.
Chlorella sorokiniana has been selected for lutein production, after a screening of thirteen species of microalgae, since it showed both a high content in this carotenoid and a high growth rate. The effects of several nutritional and environmental factors on cell growth and lutein accumulation have been studied. Maximal specific growth rate and lutein content were attained at 690 μmol photons m(-2) s(-1), 28 °C, 2 mM NaCl, 40 mM nitrate and under mixotrophic conditions. In general, optimal conditions for the growth of this strain also lead to maximal lutein productivity. High lutein yielding mutants of C. sorokiniana have been obtained by random mutagenesis, using N-methyl-N'-nitro-nitrosoguanidine (MNNG) as a mutagen and selecting mutants by their resistance to the inhibitors of the carotenogenic pathway nicotine and norflurazon. Among the mutants resistant to the herbicides, those exhibiting both high content in lutein and high growth rate were chosen. Several mutants exhibited higher contents in this carotenoid than the wild type, showing, in addition, either a similar or higher growth rate than the latter strain. The mutant MR-16 exhibited a 2.0-fold higher volumetric lutein content than that of the wild type, attaining values of 42.0 mg L(-1) and mutants DMR-5 and DMR-8 attained a lutein cellular content of 7.0 mg g(-1) dry weight. The high lutein yield exhibited by C. sorokiniana makes this microalga an excellent candidate for the production of this commercially interesting pigment.
For photosynthesis, phototrophic organisms necessarily synthesize not only chlorophylls but also carotenoids. Many kinds of carotenoids are found in algae and, recently, taxonomic studies of algae have been developed. In this review, the relationship between the distribution of carotenoids and the phylogeny of oxygenic phototrophs in sea and fresh water, including cyanobacteria, red algae, brown algae and green algae, is summarized. These phototrophs contain division- or class-specific carotenoids, such as fucoxanthin, peridinin and siphonaxanthin. The distribution of α-carotene and its derivatives, such as lutein, loroxanthin and siphonaxanthin, are limited to divisions of Rhodophyta (macrophytic type), Cryptophyta, Euglenophyta, Chlorarachniophyta and Chlorophyta. In addition, carotenogenesis pathways are discussed based on the chemical structures of carotenoids and known characteristics of carotenogenesis enzymes in other organisms; genes and enzymes for carotenogenesis in algae are not yet known. Most carotenoids bind to membrane-bound pigment-protein complexes, such as reaction center, light-harvesting and cytochrome b(6)f complexes. Water-soluble peridinin-chlorophyll a-protein (PCP) and orange carotenoid protein (OCP) are also established. Some functions of carotenoids in photosynthesis are also briefly summarized.
There are currently intensive global research efforts aimed at increasing and modifying the accumulation of lipids, alcohols, hydrocarbons, polysaccharides, and other energy storage compounds in photosynthetic organisms, yeast, and bacteria through genetic engineering. Many improvements have been realized, including increased lipid and carbohydrate production, improved H(2) yields, and the diversion of central metabolic intermediates into fungible biofuels. Photosynthetic microorganisms are attracting considerable interest within these efforts due to their relatively high photosynthetic conversion efficiencies, diverse metabolic capabilities, superior growth rates, and ability to store or secrete energy-rich hydrocarbons. Relative to cyanobacteria, eukaryotic microalgae possess several unique metabolic attributes of relevance to biofuel production, including the accumulation of significant quantities of triacylglycerol; the synthesis of storage starch (amylopectin and amylose), which is similar to that found in higher plants; and the ability to efficiently couple photosynthetic electron transport to H(2) production. Although the application of genetic engineering to improve energy production phenotypes in eukaryotic microalgae is in its infancy, significant advances in the development of genetic manipulation tools have recently been achieved with microalgal model systems and are being used to manipulate central carbon metabolism in these organisms. It is likely that many of these advances can be extended to industrially relevant organisms. This review is focused on potential avenues of genetic engineering that may be undertaken in order to improve microalgae as a biofuel platform for the production of biohydrogen, starch-derived alcohols, diesel fuel surrogates, and/or alkanes.
In the study of metabolic networks, optimization techniques are often used to predict flux distributions, and hence, metabolic phenotype. Flux balance analysis in particular has been successful in predicting metabolic phenotypes. However, an inherent limitation of a stoichiometric approach such as flux balance analysis is that it can predict only flux distributions that result in maximal yields. Hence, previous attempts to use FBA to predict metabolic fluxes in Lactobacillus plantarum failed, as this lactic acid bacterium produces lactate, even under glucose-limited chemostat conditions, where FBA predicted mixed acid fermentation as an alternative pathway leading to a higher yield. In this study we tested, however, whether long-term adaptation on an unusual and poor carbon source (for this bacterium) would select for mutants with optimal biomass yields. We have therefore adapted Lactobacillus plantarum to grow well on glycerol as its main growth substrate. After prolonged serial dilutions, the growth yield and corresponding fluxes were compared to in silico predictions. Surprisingly, the organism still produced mainly lactate, which was corroborated by FBA to indeed be optimal. To understand these results, constraint-based elementary flux mode analysis was developed that predicted 3 out of 2669 possible flux modes to be optimal under the experimental conditions. These optimal pathways corresponded very closely to the experimentally observed fluxes and explained lactate formation as the result of competition for oxygen by the other flux modes. Hence, these results provide thorough understanding of adaptive evolution, allowing in silico predictions of the resulting flux states, provided that the selective growth conditions favor yield optimization as the winning strategy.
Dunaliella salina has been exploited as a new type of bioreactor due to its unique advantages. However, this bioreactor application was restricted for absence of a high-efficiency and stable transformation method at present. In the present study, the cells of D. salina were transformed by glass beads. The results of histochemical staining revealed that the GUS gene was successfully expressed in the positive transformants, and PCR and PCR-Southern blot analysis further demonstrated that the bar gene was integrated into the D. salina genome. Moreover, the three transformation methods, including glass beads, bombardment particle and electroporation, were compared for screening a high-efficiency transformation method for gene engineering of D. salina. The results showed that transformation efficiency of the glass beads was the highest, approximately 10(2) transformants/microg DNA. It is concluded that the established glass beads method has been demonstrated to be an optimal transformation way for D. salina.
Haematococcus pluvialis under stress conditions overproduces the valuable red ketocarotenoid astaxanthin. Two proposed strategies for commercial production are under current analysis. One separates in time the production of biomass (optimal growth, green stage) and pigment (permanent stress, red stage), while the other uses an approach based on continuous culture under limiting stress at steady state. The productivities, efficiencies and yields for the pigment accumulation in each case have been compared and analyzed in terms of the algal basic physiology. The two-stage system indoors yields a richer astaxanthin product (4% of dry biomass) with a final astaxanthin productivity of 11.5 mg L(-1) day(-1), is more readily upscalable and amenable to outdoors production. Furthermore, each stage can be optimized for green biomass growth and red pigment accumulation by adjusting independently the respective ratio of effective irradiance to cell density. We conclude that the two-stage system performs better (by a factor of 2.5-5) than the one-stage system, and the former is best fit in an efficient mass production setup.
To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially β-carotene production, carotenogenic genes from the carotenoid-producing
yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these genes from an episomal expression vector resulted in unstable strains, the genes were integrated
into genomic DNA to yield stable, carotenoid-producing S. cerevisiae cells. Furthermore, carotenoid production levels were higher in strains containing integrated carotenogenic genes. Overexpression
of crtYB (which encodes a bifunctional phytoene synthase and lycopene cyclase) and crtI (phytoene desaturase) from X. dendrorhous was sufficient to enable carotenoid production. Carotenoid production levels were increased by additional overexpression
of a homologous geranylgeranyl diphosphate (GGPP) synthase from S. cerevisiae that is encoded by BTS1. Combined overexpression of crtE (heterologous GGPP synthase) from X. dendrorhous with crtYB and crtI and introduction of an additional copy of a truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (tHMG1) into carotenoid-producing cells resulted in a successive increase in carotenoid production levels. The strains mentioned
produced high levels of intermediates of the carotenogenic pathway and comparable low levels of the preferred end product
β-carotene, as determined by high-performance liquid chromatography. We finally succeeded in constructing an S. cerevisiae strain capable of producing high levels of β-carotene, up to 5.9 mg/g (dry weight), which was accomplished by the introduction
of an additional copy of crtI and tHMG1 into carotenoid-producing yeast cells. This transformant is promising for further development toward the biotechnological
production of β-carotene by S. cerevisiae.
To investigate the potential of double-stranded RNA interferencing with gene expression in Dunaliella salina, a plasmid pBIRNAI-Dsa was constructed to express hairpin RNA (hpRNA) containing sequences homologous to phytoene desaturase gene (pds), a key gene in carotenoid biosynthesis, and transformed into D. salina by electroporation. The relative transcription level of pds in pBIRNAI-Dsa-treated cells to nontreated cells was quantitated and the gene silencing efficiency by RNAi was evaluated via real-time polymerase chain reaction (PCR). The transcriptions of pds of the pBIRNAI-Dsa-treated group changed compared to those of the control group, and the 2(-delta deltaC)(T) was lowest on the 7th day, corresponding to 0.281265-fold of the relative pds control transcript; a relatively strong gene inhibition effect was therefore deduced. The transcript of pds may be modulated in a wide range, and a reduced transcription even to 28% of the normal level may be tolerated for its survival. This study shows that dsRNA-mediated genetic interference can induce sequence-specific inhibition of gene expression and pBIRNAI-Dsa can be used for transient suppression of gene expression in D. salina. The aim of this study was to exploit dsRNA-mediated gene silencing and to provide a foundation for gene function research in D. salina.
Recent years have witnessed a rising trend in exploring microalgae for valuable carotenoid products as the demand for lutein and many other carotenoids in global markets has increased significantly. In green microalgae lutein is a major carotenoid protecting cellular components from damage incurred by reactive oxygen species under stress conditions. In this study, we investigated the effects of abiotic stressors on lutein accumulation in a strain of the marine microalga D. salina which had been selected for growth under stress conditions of combined blue and red lights by adaptive laboratory evolution.
Nitrate concentration, salinity and light quality were selected as three representative influencing factors and their impact on lutein production in batch cultures of D. salina was evaluated using response surface analysis. D. salina was found to be more tolerant to hyper-osmotic stress than to hypo-osmotic stress which caused serious cell damage and death in a high proportion of cells while hyper-osmotic stress increased the average cell size of D. salina only slightly. Two models were developed to explain how lutein productivity depends on the stress factors and for predicting the optimal conditions for lutein productivity. Among the three stress variables for lutein production, stronger interactions were found between nitrate concentration and salinity than between light quality and the other two. The predicted optimal conditions for lutein production were close to the original conditions used for adaptive evolution of D. salina. This suggests that the conditions imposed during adaptive evolution may have selected for the growth optima arrived at.
This study shows that systematic evaluation of the relationship between abiotic environmental stresses and lutein biosynthesis can help to decipher the key parameters in obtaining high levels of lutein productivity in D. salina. This study may benefit future stress-driven adaptive laboratory evolution experiments and a strategy of applying stress in a step-wise manner can be suggested for a rational design of experiments.
Adaptive laboratory evolution (ALE) is an effective method to improve microalgal strains. The growth phenotypes of three strains (cc4324, cc4326 and cc4334) of green microalgae Chlamydomonas reinhardtii were enhanced by ALE. As a result, endpoint strains exhibited higher growth rates. Upon the utilisation of ALE strategy, the biomass concentrations of the endpoint strains of cc4324, cc4326 and cc4334 became 1.17, 1.33 and 1.48 times of those of the starting strains. The total lipid content of the original strains was increased gradually from 32% to 36.67% in the endpoint strain cc4326 and abruptly increased from 24.27% to 44.67% in the endpoint strain cc4334 by nitrogen starvation. Slight growth impairment was also observed in low-starch mutants exposed to nitrogen starvation stress. However, this impairment was quickly resolved after nitrogen was replenished. These findings demonstrated that the biomass concentration and lipid productivity of low-starch mutants can be enhanced by ALE.
The holophilic alga Dunaliella parva produces glycerol as a major product of photosynthetic ¹⁴ CO 2 incorporation and accumulates very large amounts of intracellular glycerol. A method was adopted for the determination of the cell water space based on the distribution of ¹⁴ C sorbitol and ³ H 2 O between the cells and the medium. Using these measurements the internal concentration of glycerol was found to be isoomotic with that of the medium over a broad range of 0.6 to 2.1 m NaCl. When the extracellular salt concentration of an algal suspension was increased or decreased, the intracellular water content immediately varied so as to keep an osmotic equilibrium between the cells and the medium. During the following 90 min under metabolic conditions, glycerol content changed until a new level was reached. Since no leakage of intracellular glycerol was observed above 0.6 m NaCl, these alterations in glycerol content are interpreted as due to metabolic formation and degradation of intracellular glycerol. Determination of the glycerol sensitivity of enzymic and photosynthetic reactions of cell‐free preparations from D. parva showed a broad range of tolerance to high concentrations of glycerol.
These results indicate that osmoregulation in Dunaliella depends on the synthesis or degradation of intracellular glycerol in response to the external salt concentration. A proposed scheme of glycerol synthesis in Dunaliella is suggested.
Green microalgae have recently drawn attention as promising organisms for biofuel production; however, the question is whether they can grow sufficient biomass relative to limiting input factors to be economically feasible. We have explored this question by determining how much biomass the green microalga Chlorella vulgaris can produce in photobioreactors based on highly efficient light-emitting diodes (LEDs). First, growth results were improved under the less expensive light of 660 nm LEDs, developing them in the laboratory to meet the performance levels of the traditional but more expensive 680 nm LEDs by adaptive laboratory evolution (ALE). We then optimized several other key parameters, including input superficial gas velocity, CO(2) concentration, light distribution, and growth media in reference to nutrient stoichiometry. Biomass density thereby rose to approximately 20 g dry-cell-weight (gDCW) per liter (L). Since the light supply was recognized as a limiting factor, illumination was augmented by optimization at systematic level, providing for a biomass productivity of up to 2.11 gDCW/L/day, with a light yield of 0.81 gDCW/Einstein. These figures, which represent the best results ever reported, point to new dimensions in the photoautotrophic performance of microalgal cultures.
Use of bubble columns as photobioreactors requires a quantitative knowledge of radial mixing in these columns. A complete model of liquid-phase dispersion was used to simultaneously characterize axial and radial mixing in a relatively large (0.06 m3, 2.3 m tall, 0.193 m in diameter) bubble column photobioreactor. Axial and radial dispersion coefficients and mixing times were determined in tap water and sea water for superficial aeration velocities of up to . The measured axial dispersion coefficients (Dz) were generally consistent with the predictions of the well established correlations, thus validating the complete dispersion model used in the analysis. The Dz values ranged from ∼150 to and were highly reproducible. There was evidence that the existing literature data on Dz in bubble columns are slightly underestimated, as consistent underestimation was found to be a characteristic of the widely used dispersion model that disregards radial dispersion. The value of the radial dispersion coefficient was typically about 1% of the Dz value under any given condition. Except at incipient aeration, the radial dispersion coefficient was not as sensitive to the magnitude of the aeration rate as was the axial dispersion coefficient. The mixing time data were generally consistent with the existing correlations.
Unicellular algae are characterized by an extreme flexibility with respect to their responses to environmental constraints. This flexibility probably explains why microalgae show a very high biomass yield, constitute one of the major contributors to primary productivity in the oceans and are considered a promising choice for biotechnological applications. Flexibility results from a combination of several factors including fast changes in the light-harvesting apparatus and a strong interaction between different metabolic processes (e.g. respiration and photosynthesis), which all take place within the same cell. Microalgae are also capable of modifying their photosynthetic electron flow capacity, by changing its maximum rate and/or by diverting photogenerated electrons towards different sinks depending on their growth status. In this review, we will focus on the occurrence and regulation of alternative electron flows in unicellular algae and compare data obtained in these systems with those available in vascular plants. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.
Microalgae are considered one of the most promising feedstocks for biofuels. The productivity of these photosynthetic microorganisms
in converting carbon dioxide into carbon-rich lipids, only a step or two away from biodiesel, greatly exceeds that of agricultural
oleaginous crops, without competing for arable land. Worldwide, research and demonstration programs are being carried out
to develop the technology needed to expand algal lipid production from a craft to a major industrial process. Although microalgae
are not yet produced at large scale for bulk applications, recent advances—particularly in the methods of systems biology,
genetic engineering, and biorefining—present opportunities to develop this process in a sustainable and economical way within
the next 10 to 15 years.
The photosynthetic microalga Haematococcus pluvialis, a potential source of astaxanthin, was cultivated under illumination with LEDs emitting red (λmax=625nm), green (λmax=525nm), blue (λmax=470nm), blue-purple (λmax=410nm) and purple (λmax=380nm) light and a fluorescent lamp, and the effects of wavelength on cell growth and astaxanthin accumulation were studied. LEDs emitting light of short wavelengths (380–470nm) were found to induce astaxanthin accumulation of up to 5–6% per dry cell, although the induction caused the suppression of cell growth. From these results, we proposed a new strategy of cultivating H. pluvialis under illumination with red LEDs without inducing a high level of astaxanthin accumulation, and then switching to illumination with blue LEDs at a high light intensity to induce a high level of astaxanthin accumulation.
Chlorella vulgaris is a popular food supplement in Asia and is currently marketed as a nutritional supplement. However, available scientific studies do not support its effectiveness for preventing or treating any disease in humans. Because Chlorella contains numerous nutrients, including antioxidants, it is thought to exert antioxidative functions by scavenging free radicals created by various environmental factors such as smoking. The purpose of this study was to investigate whether 6 wk of Chlorella supplementation to smokers is protective against oxidative damage in a randomized, double-blinded, placebo-controlled trial.
Fifty-two smokers, aged 20-65 y, were given 6.3g of Chlorella or placebo every day for 6 wk. Blood samples were drawn at the beginning and after the supplementation. Plasma antioxidant vitamin levels and lipid peroxidation levels were measured. As a marker of oxidative stress, lymphocyte DNA damage was measured.
Chlorella supplementation increased plasma vitamin C (44.4%), alpha-tocopherol (15.7%), and erythrocyte catalase and superoxide dismutase activities. Although 6 wk of Chlorella supplementation resulted in a significant decrease in lymphocyte DNA damage, as measured by comet assay, placebo supplementation also decreased the measured amount of lymphocyte DNA damage.
Chlorella supplementation resulted in the conservation of plasma antioxidant nutrient status and improvement in erythrocyte antioxidant enzyme activities in subjects. Therefore, our results are supportive of an antioxidant role for Chlorella and indicate that Chlorella is an important whole-food supplement that should be included as a key component of a healthy diet.
Marine diatoms rose to prominence about 100 million years ago and today generate most of the organic matter that serves as food for life in the sea. They exist in a dilute world where compounds essential for growth are recycled and shared, and they greatly influence global climate, atmospheric carbon dioxide concentration and marine ecosystem function. How these essential organisms will respond to the rapidly changing conditions in today's oceans is critical for the health of the environment and is being uncovered by studies of their genomes.
Carotenoids are ubiquitous and essential components of photosynthetic tissues in plants, algae and cyanobacteria. They participate in the light harvesting process and prevent photooxidative damage of the photosynthetic apparatus. Although de-etiolation and growth under different light conditions were reported to have pronounced effects on carotenoid contents in higher plants and algae, very little is known about the light regulation of carotenogenesis on a molecular level. In the present study, we chose the unicellular green alga Chlamydomonas reinhardtii to investigate the regulation of carotenoid biosynthesis genes in response to light. The carotenoid genes phytoene synthase and phytoene desaturase were selected for gene expression studies. Both phytoene synthase and phytoene desaturase revealed a fast up-regulation in response to light, which seemed to be due to transcriptional control. Only blue light was effective whereas illumination with red light did not lead to elevated transcript levels of phytoene synthase and phytoene desaturase. The inhibition of photosynthesis did not abolish the light induction of carotenoid genes. Comparison with published results showed that the carotenoid genes are simultaneously expressed with other genes involved in chlorophyll biosynthesis and light harvesting. This simultaneous expression may represent one mechanism for the coordinated biosynthesis of carotenoids, chlorophylls and the proteins of the photosynthetic apparatus.
The biotechnology of microalgae has gained considerable importance in recent decades. Applications range from simple biomass production for food and feed to valuable products for ecological applications. For most of these applications, the market is still developing and the biotechnological use of microalgae will extend into new areas. Considering the enormous biodiversity of microalgae and recent developments in genetic engineering, this group of organisms represents one of the most promising sources for new products and applications. With the development of sophisticated culture and screening techniques, microalgal biotechnology can already meet the high demands of both the food and pharmaceutical industries.
Annotated genome sequences can be used to reconstruct whole-cell metabolic networks. These metabolic networks can be modelled and analysed (computed) to study complex biological functions. In particular, constraints-based in silico models have been used to calculate optimal growth rates on common carbon substrates, and the results were found to be consistent with experimental data under many but not all conditions. Optimal biological functions are acquired through an evolutionary process. Thus, incorrect predictions of in silico models based on optimal performance criteria may be due to incomplete adaptive evolution under the conditions examined. Escherichia coli K-12 MG1655 grows sub-optimally on glycerol as the sole carbon source. Here we show that when placed under growth selection pressure, the growth rate of E. coli on glycerol reproducibly evolved over 40 days, or about 700 generations, from a sub-optimal value to the optimal growth rate predicted from a whole-cell in silico model. These results open the possibility of using adaptive evolution of entire metabolic networks to realize metabolic states that have been determined a priori based on in silico analysis.
Organisms adapt to environmental changes through the fixation of mutations that enhance reproductive success. A recent study by Dekel and Alon demonstrated that Escherichia coli adapts to different growth conditions by fine-tuning protein levels, as predicted by a simple cost-benefit model. A study by Fong et al. showed that independent evolutionary trajectories lead to similar adaptive endpoints. Initial mutations on the path to adaptation altered the mRNA levels of numerous genes. Subsequent optimization through compensatory mutations restored the expression of most genes to baseline levels, except for a small set that retained differential levels of expression. These studies clarify how adaptation could occur by the alteration of gene expression.
To mitigate some of the potentially deleterious environmental and agricultural consequences associated with current land-based-biofuel feedstocks, we propose the use of biofuels derived from aquatic microbial oxygenic photoautotrophs (AMOPs), more commonly known as cyanobacteria, algae, and diatoms. Herein we review their demonstrated productivity in mass culturing and aspects of their physiology that are particularly attractive for integration into renewable biofuel applications. Compared with terrestrial crops, AMOPs are inherently more efficient solar collectors, use less or no land, can be converted to liquid fuels using simpler technologies than cellulose, and offer secondary uses that fossil fuels do not provide. AMOPs pose a new set of technological challenges if they are to contribute as biofuel feedstocks.
An outlook on microalgal biofuels PMID:20705853; http://dx.doi.org/10.1126/science
- Rh Wijffels
- Barbosa
Wijffels RH, Barbosa MJ. An outlook on microalgal biofuels. Science 2010; 329:796-9; PMID:20705853; http://dx.doi.org/10.1126/science.1189003
Regulation of electron transport in microalgae PMID:21167125; http://dx.doi. org/10.1016/j.bbabio
- P Cardol
- G Forti
- Finazzi
Cardol P, Forti G, Finazzi G. Regulation of electron transport in microalgae. Biochim Biophys Acta 2011; 1807:912-8; PMID:21167125; http://dx.doi. org/10.1016/j.bbabio.2010.12.004
The place of diatoms in the biofuels industry http://dx.doi. org/10.4155/bfs.11.157 13 Astaxanthin production by Haematococcus pluvialis under illumination with LEDs://dx.doi.org/10
- M Hildebrand
- Ak Davis
- Smith
- Sr
- Jc Traller
- Abbriano
- T Katsuda
- A Lababpour
- K Shimahara
- Katoh
Hildebrand M, Davis AK, Smith SR, Traller JC, Abbriano R. The place of diatoms in the biofuels industry. Biofuels 2012; 3:221-40; http://dx.doi. org/10.4155/bfs.11.157 13. Katsuda T, Lababpour A, Shimahara K, Katoh S. Astaxanthin production by Haematococcus pluvialis under illumination with LEDs. Enzyme Microb Technol 2004; 35:81-6; http://dx.doi.org/10.1016/j. enzmictec.2004.03.016