1933353
Accelerated triacylglycerol production and altered fatty acid composition in oleaginous microalga Neochloris oleoabundans by overexpression of diacylglycerol acyltransferase 2
BackgroundMicroalgae are promising sources of lipid triacylglycerol (TAG) for biodiesel production. However, to date, microalgal biodiesel is technically feasible, but not yet economically viable. Increasing TAG content and productivity are important to achieve economic viability of microalgal biodiesel. To increase TAG content, oleaginous microalga Neochloris oleoabundans was genetically engineered with an endogenous key enzyme diacylglycerol acyltransferase 2 (NeoDGAT2) responsible for TAG biosynthesis.ResultsThe integration of NeoDGAT2 expression cassettes in N. oleoabundans transformant was confirmed by PCR. The neutral lipid accumulation in the transformant detected by Nile red staining was accelerated and 1.9-fold higher than in wild type; the lipid bodies in the transformant visualized under fluorescence microscope were also larger. The NeoDGAT2 transcript was two-fold higher in the transformant than wild type. Remarkably higher lipid accumulation was found in the transformant than wild type: total lipid content increased 1.6-to 2.3-fold up to 74.5 ± 4.0% dry cell weight (DCW) and total lipid productivity increased 1.6- to 3.2-fold up to 14.6 ± 2.0 mg/L/day; while TAG content increased 1.8- to 3.2-fold up to 46.1 ± 1.6% DCW and TAG productivity increased 1.6- to 4.3-fold up to 8.9 ± 1.3 mg/L/day. A significantly altered fatty acid composition was detected in the transformant compared to wild type; the levels of saturated fatty acid C16:0 increased double to 49%, whereas C18:0 was reduced triple to 6%. Long-term stability was observed in the transformant continuously maintained in solid medium over 100 generations in a period of about 4 years.ConclusionsOur results demonstrate the increased TAG content and productivity in N. oleoabundans by NeoDGAT2 overexpression that may offer the first step towards making microalgae an economically feasible source for biodiesel production. The strategy for genetically improved microalga presented in this study can be applied to other microalgal species possessing desired characteristics for industrial biofuel production.
18234512
Biodiesel from microalgae.
Continued use of petroleum sourced fuels is now widely recognized as unsustainable because of depleting supplies and the contribution of these fuels to the accumulation of carbon dioxide in the environment. Renewable, carbon neutral, transport fuels are necessary for environmental and economic sustainability. Biodiesel derived from oil crops is a potential renewable and carbon neutral alternative to petroleum fuels. Unfortunately, biodiesel from oil crops, waste cooking oil and animal fat cannot realistically satisfy even a small fraction of the existing demand for transport fuels. As demonstrated here, microalgae appear to be the only source of renewable biodiesel that is capable of meeting the global demand for transport fuels. Like plants, microalgae use sunlight to produce oils but they do so more efficiently than crop plants. Oil productivity of many microalgae greatly exceeds the oil productivity of the best producing oil crops. Approaches for making microalgal biodiesel economically competitive with petrodiesel are discussed.
10323847
The promise and challenges of microalgal‐derived biofuels
Microalgae offer great promise to contribute a significant portion of the renewable fuels that will be required by the Renewable Fuels Standard described in the 2007 Energy Independence and Security Act of the United States. Algal biofuels would be based mainly on the high lipid content of the algal cell and thus would be an ideal feedstock for high energy density transportation fuels, such as biodiesel as well as green diesel, green jet fuel and green gasoline. A comprehensive research and development program for the development of algal biofuels was initiated by the US Department of Energy (DoE) more than 30 years ago, and although great progress was made, the program was discontinued in 1996, because of decreasing federal budgets and low petroleum costs. Interest in algal biofuels has been growing recently due to increased concern over peak oil, energy security, greenhouse gas emissions, and the potential for other biofuel feedstocks to compete for limited agricultural resources. The high productivity of algae suggests that much of the US transportation fuel needs can be met by algal biofuels at a production cost competitive with the cost of petroleum seen during the early part of 2008. Development of algal biomass production technology, however, remains in its infancy. This perspective provides a brief overview of past algal research sponsored by the DoE, the potential of microalgal biofuels and a discussion of the technical and economic barriers that need to be overcome before production of microalgal-derived diesel-fuel substitutes can become a large-scale commercial reality. Published in 2009 by John Wiley & Sons, Ltd.
15481966
Microalgae for biodiesel production and other applications: A review
Sustainable production of renewable energy is being hotly debated globally since it is increasingly understood that first generation biofuels, primarily produced from food crops and mostly oil seeds are limited in their ability to achieve targets for biofuel production, climate change mitigation and economic growth. These concerns have increased the interest in developing second generation biofuels produced from non-food feedstocks such as microalgae, which potentially offer greatest opportunities in the longer term. This paper reviews the current status of microalgae use for biodiesel production, including their cultivation, harvesting, and processing. The microalgae species most used for biodiesel production are presented and their main advantages described in comparison with other available biodiesel feedstocks. The various aspects associated with the design of microalgae production units are described, giving an overview of the current state of development of algae cultivation systems (photo-bioreactors and open ponds). Other potential applications and products from microalgae are also presented such as for biological sequestration of CO2, wastewater treatment, in human health, as food additive, and for aquaculture.
27125322
Genetic Engineering of Algae for Enhanced Biofuel Production
ABSTRACT 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 H2 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 H2 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.
206526311
An Outlook on Microalgal Biofuels
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.
4766016
Metabolic regulation of triacylglycerol accumulation in the green algae: identification of potential targets for engineering to improve oil yield
Summary The great need for more sustainable alternatives to fossil fuels has increased our research interests in algal biofuels. Microalgal cells, characterized by high photosynthetic efficiency and rapid cell division, are an excellent source of neutral lipids as potential fuel stocks. Various stress factors, especially nutrient‐starvation conditions, induce an increased formation of lipid bodies filled with triacylglycerol in these cells. Here we review our knowledge base on glycerolipid synthesis in the green algae with an emphasis on recent studies on carbon flux, redistribution of lipids under nutrient‐limiting conditions and its regulation. We discuss the contributions and limitations of classical and novel approaches used to elucidate the algal triacylglycerol biosynthetic pathway and its regulatory network in green algae. Also discussed are gaps in knowledge and suggestions for much needed research both on the biology of triacylglycerol accumulation and possible avenues to engineer improved algal strains.
205741989
A look at diacylglycerol acyltransferases (DGATs) in algae.
Triacylglycerols (TAGs) from algae are considered to be a potentially viable source of biodiesel and thereby renewable energy, but at the moment very little is known about the biosynthetic pathway in these organisms. Here we compare what is currently known in eukaryotic algal species, in particular the characteristics of algal diacylglycerol acyltransferase (DGAT), the last enzyme of de novo TAG biosynthesis. Several studies in plants and mammals have shown that there are two DGAT isoforms, DGAT1 and DGAT2, which catalyse the same reaction but have no clear sequence similarities. Instead, they have differences in functionality and spatial and temporal expression patterns. Bioinformatic searches of sequenced algal genomes reveal that most algae have multiple copies of putative DGAT2s, whereas other eukaryotes have single genes. Investigating whether these putative isoforms are indeed functional and whether they confer significantly different phenotypes to algal cells will be vital for future efforts to genetically modify algae for biofuel production.
12001181
Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight.
We recently reported the cloning and characterization of an Arabidopsis (ecotype Columbia) diacylglycerol acyltransferase cDNA (Zou et al., 1999) and found that in Arabidopsis mutant line AS11, an ethyl methanesulfonate-induced mutation at a locus on chromosome II designated as Tag1 consists of a 147-bp insertion in the DNA, which results in a repeat of the 81-bp exon 2 in the Tag1 cDNA. This insertion mutation is correlated with an altered seed fatty acid composition, reduced diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) activity, reduced seed triacylglycerol content, and delayed seed development in the AS11 mutant. The effect of the insertion mutation on microsomal acyl-coenzyme A-dependent DGAT is examined with respect to DGAT activity and its substrate specificity in the AS11 mutant relative to wild type. We demonstrate that transformation of mutant AS11 with a single copy of the wild-type Tag1 DGAT cDNA can complement the fatty acid and reduced oil phenotype of mutant AS11. More importantly, we show for the first time that seed-specific over-expression of the DGAT cDNA in wild-type Arabidopsis enhances oil deposition and average seed weight, which are correlated with DGAT transcript levels. The DGAT activity in developing seed of transgenic lines was enhanced by 10% to 70%. Thus, the current study confirms the important role of DGAT in regulating the quantity of seed triacylglycerols and the sink size in developing seeds.
4051260
Diacylglycerol acyltransferase: A key mediator of plant triacylglycerol synthesis
Many plants deposit TAG in seeds and fruits as the major form of storage lipid. TAG production is of tremendous socioeconomic value in food, nutraceutical, and industrial applications, and thus numerous conventional and molecular genetic strategies have been explored in attempts to increase TAG content and modify the FA composition of plant seed oils. Much research has focused on the acyl-CoA-dependent reaction catalyzed by diacylglycerol acyltransferase (DGAT), which is an integral endoplasmic reticulum protein and has also been shown to be present in oil bodies and plastids. DGAT enzymes exhibit diverse biochemical properties among different plant species, many of which are summarized here. In addition to catalyzing a critical step in TAG biosynthesis, there is evidence that DGAT has roles in lipid metabolism associated with germination and leaf senescence. TAG can also be formed in plants via two different acyl-CoA-independent pathways, catalyzed by phospholipid: diacylglycerol acyltransferase and diacylglycerol transacylase. The current understanding of the terminal step in TAG formation in plants and the development of molecular genetic approaches aimed at altering TAG yield and FA composition of TAG are discussed.
18685785
Diacylglycerol acyltransferase type 2 cDNA from the oleaginous microalga Neochloris oleoabundans: cloning and functional characterization
Currently, biodiesel production from microalgae is technically, but not yet economically, feasible. Increasing microalgal triacylglycerol (TAG) content via genetic engineering could be a potential approach to improve the efficiency of biodiesel production. In all organisms studied so far, diacylglycerol acyltransferase (DGAT) is the primary enzyme of TAG biosynthesis. In this study, we cloned the cDNA encoding DGAT type 2 (NeoDGAT2) from the oleaginous microalga Neochloris oleoabundans. The open reading frame of NeoDGAT2 cDNA was 978 bp encoding a protein of 325 amino acids. Although N. oleoabundans has been classified in the class Chlorophyceae, NeoDGAT2 was closely related to Treboxiophycean DGAT2 as it shared 80 % amino acid identity with CvDGAT2-2 of Chlorella variabilis, but distantly related to Chlorophycean DGAT2 as it shared only 34 % identity with CrDGAT2A (DGTT4) of Chlamydomonas reinhardtii. As suggested by hydropathy analysis, NeoDGAT2 might contain 4 to 5 transmembrane domains. We tested whether NeoDGAT2 cDNA encoded a protein with DGAT activity by functional complementation assay in yeast Saccharomyces cerevisiae mutant H1246. NeoDGAT2 was able to compensate the endogenous DGAT2 activity of mutant H1246 and restore the lipid body formation and TAG synthesis. The successful cloning of cDNA encoding a protein with DGAT activity provides a candidate for genetic manipulation in microalgae to increase TAG content for biodiesel production.
207701544
Identification and characterization of PtDGAT2B, an acyltransferase of the DGAT2 acyl‐Coenzyme A: Diacylglycerol acyltransferase family in the diatom Phaeodactylum tricornutum
Diacylglycerol acyltransferase (DGAT) plays a pivotal role in triacylglycerol (TAG) formation in some oleaginous organisms. We describe here the identification of a type 2 DGAT (PtDGAT2B) in the diatom Phaeodactylum tricornutum that contains four putative type 2 acyl‐CoA:DGATs, sharing little sequence similarity with each other. TAG synthesis and lipid body formation could be completely restored in a Saccharomyces cerevisiae TAG‐deficient quadruple mutant by expressing PtDGAT2B. Up‐regulation of PtDGAT2B precedes the accumulation of TAG. Functional analysis of enzyme activity in vivo demonstrated that expression of PtDGAT2B can increase the proportion of unsaturated C16 and C18 fatty acids in yeast TAG.
30932125
Functional study of diacylglycerol acyltransferase type 2 family in Chlamydomonas reinhardtii
Algal triacylglycerol biosynthesis is of increasing interest for potential biodiesel production. A model microalga, Chlamydomonas, has multiple isoforms of diacylglycerol acyltransferase type 2 (DGTT) catalyzing the final step of triacylglycerol biosynthesis; however, the functions of the isoforms are poorly understood. Here, we performed heterologous complementation assay of Chlamydomonas DGTT1 to 4 in a yeast mutant defective in triacylglycerol biosynthesis. DGTT1, 2 and 3 but not 4 complemented the phenotype, including triacylglycerol levels. Interestingly, complementation by DGTT2 increased triacylglycerol content by 9‐fold.
5650993
Enhancement of extraplastidic oil synthesis in Chlamydomonas reinhardtii using a type-2 diacylglycerol acyltransferase with a phosphorus starvation–inducible promoter
When cultivated under stress conditions, many plants and algae accumulate oil. The unicellular green microalga Chlamydomonas reinhardtii accumulates neutral lipids (triacylglycerols; TAGs) during nutrient stress conditions. Temporal changes in TAG levels in nitrogen (N)- and phosphorus (P)-starved cells were examined to compare the effects of nutrient depletion on TAG accumulation in C. reinhardtii. TAG accumulation and fatty acid composition were substantially changed depending on the cultivation stage before nutrient starvation. Profiles of TAG accumulation also differed between N and P starvation. Logarithmic-growth-phase cells diluted into fresh medium showed substantial TAG accumulation with both N and P deprivation. N deprivation induced formation of oil droplets concomitant with the breakdown of thylakoid membranes. In contrast, P deprivation substantially induced accumulation of oil droplets in the cytosol and maintaining thylakoid membranes. As a consequence, P limitation accumulated more TAG both per cell and per culture medium under these conditions. To enhance oil accumulation under P deprivation, we constructed a P deprivation-dependent overexpressor of a Chlamydomonas type-2 diacylglycerol acyl-CoA acyltransferase (DGTT4) using a sulphoquinovosyldiacylglycerol 2 (SQD2) promoter, which was up-regulated during P starvation. The transformant strongly enhanced TAG accumulation with a slight increase in 18 : 1 content, which is a preferred substrate of DGTT4. These results demonstrated enhanced TAG accumulation using a P starvation–inducible promoter.
33119046
Altered lipid composition and enhanced lipid production in green microalga by introduction of brassica diacylglycerol acyltransferase 2.
Higher lipid biosynthesis and accumulation are important to achieve economic viability of biofuel production via microalgae. To enhance lipid content, Chlamydomonas reinhardtii was genetically engineered with a key enzyme diacylglycerol acyltransferase (BnDGAT2) from Brassica napus, responsible for neutral lipid biosynthesis. The transformed colonies harbouring aph7 gene, screened on hygromycin-supplemented medium, achieved transformation frequency of ~120 ± 10 colonies/1 × 10(6) cells. Transgene integration and expression were confirmed by PCR, Southern blots, staining lipid droplets, proteins and spectro-fluorometric analysis of Nile red-stained cells. The neutral lipid is a major class (over 80% of total lipids) and most significant requirement for biodiesel production; this was remarkably higher in the transformed alga than the untransformed control. The levels of saturated fatty acids in the transformed alga decreased to about 7% while unsaturated fatty acids increased proportionately when compared to wild type cells. Polyunsaturated fatty acids, especially α-linolenic acid, an essential omega-3 fatty acid, were enhanced up to 12% in the transformed line. Nile red staining confirmed formation of a large number of lipid globules in the transformed alga. Evaluation of long-term stability and vitality of the transgenic alga revealed that cryopreservation produced significantly higher quantity of lipid than those maintained continuously over 128 generations on solid medium. The overexpression of BnDGAT2 significantly altered the fatty acids profile in the transformed alga. Results of this study offer a valuable strategy of genetic manipulation for enhancing polyunsaturated fatty acids and neutral lipids for biofuel production in algae.
5103842
A type 2 diacylglycerol acyltransferase accelerates the triacylglycerol biosynthesis in heterokont oleaginous microalga Nannochloropsis oceanica.
Oleaginous microalgae have received a considerable attention as potential biofuel feedstock. However, lack of industry-suitable strain with lipid rich biomass limits its commercial applications. Targeted engineering of lipogenic pathways represents a promising strategy to enhance the efficacy of microalgal oil production. In this study, a type 2 diacylglycerol acyltransferase (DGAT), a rate-limiting enzyme in triacylglycerol (TAG) biosynthesis, was identified and overexpressed in heterokont oleaginous microalga Nannochloropsis oceanica for the first time. Overexpression of DGAT2 in Nannochloropsis increased the relative transcript abundance by 3.48-fold in engineered microalgae cells. TAG biosynthesis was subsequently accelerated by DGAT2 overexpression and neutral lipid content was significantly elevated by 69% in engineered microalgae. The fatty acid profile determined by GC-MS revealed that fatty acid composition was altered in engineered microalgae. Saturated fatty acids and polyunsaturated fatty acids were found to be increased whereas monounsaturated fatty acids content decreased. Furthermore, DGAT2 overexpression did not show negative impact on algal growth parameters. The present investigation showed that the identified DGAT2 would be a potential candidate for enhancing TAG biosynthesis and might facilitate the development of promising oleaginous strains with industrial potential.
7733154
Improvement of Neutral Lipid and Polyunsaturated Fatty Acid Biosynthesis by Overexpressing a Type 2 Diacylglycerol Acyltransferase in Marine Diatom Phaeodactylum tricornutum
Microalgae have been emerging as an important source for the production of bioactive compounds. Marine diatoms can store high amounts of lipid and grow quite quickly. However, the genetic and biochemical characteristics of fatty acid biosynthesis in diatoms remain unclear. Glycerophospholipids are integral as structural and functional components of cellular membranes, as well as precursors of various lipid mediators. In addition, diacylglycerol acyltransferase (DGAT) is a key enzyme that catalyzes the last step of triacylglyceride (TAG) biosynthesis. However, a comprehensive sequence-structure and functional analysis of DGAT in diatoms is lacking. In this study, an isoform of diacylglycerol acyltransferase type 2 of the marine diatom Phaeodactylum tricornutum was characterized. Surprisingly, DGAT2 overexpression in P. tricornutum stimulated more oil bodies, and the neutral lipid content increased by 35%. The fatty acid composition showed a significant increase in the proportion of polyunsaturated fatty acids; in particular, EPA was increased by 76.2%. Moreover, the growth rate of transgenic microalgae remained similar, thereby maintaining a high biomass. Our results suggest that increased DGAT2 expression could alter fatty acid profile in the diatom, and the results thus represent a valuable strategy for polyunsaturated fatty acid production by genetic manipulation.
33372487
Taxonomic status of the species of the green algal genusNeochloris
Komárek has recently reviewed the various species assigned to the green algal genusNeochlorisStarr (Chlorococcales, Chlorococcaceae) and removed those with uninucleate vegetative cells to a new genus,Ettlia.Watanabe & Floyd, unaware ofKomárek's work, also reviewed the species ofNeochloris and distributed them among three genera—Neochloris, Chlorococcopsis gen. nov., andParietochloris gen. nov.—on the basis of details of the covering of the zoospore and the arrangement of the basal bodies of the flagellar apparatus. This paper reconciles these two treatments and makes additional recommendations at the ranks of genus, family, order, and class.
6205404
Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans
Microalgal lipids are the oils of future for sustainable biodiesel production. However, relatively high production costs due to low lipid productivity have been one of the major obstacles impeding their commercial production. We studied the effects of nitrogen sources and their concentrations on cell growth and lipid accumulation of Neochloris oleoabundans, one of the most promising oil-rich microalgal species. While the highest lipid cell content of 0.40 g/g was obtained at the lowest sodium nitrate concentration (3 mM), a remarkable lipid productivity of 0.133 g l−1 day−1 was achieved at 5 mM with a lipid cell content of 0.34 g/g and a biomass productivity of 0.40 g l−1 day−1. The highest biomass productivity was obtained at 10 mM sodium nitrate, with a biomass concentration of 3.2 g/l and a biomass productivity of 0.63 g l−1 day−1. It was observed that cell growth continued after the exhaustion of external nitrogen pool, hypothetically supported by the consumption of intracellular nitrogen pools such as chlorophyll molecules. The relationship among nitrate depletion, cell growth, lipid cell content, and cell chlorophyll content are discussed.
21820296
Neochloris oleabundans UTEX #1185: a suitable renewable lipid source for biofuel production
Energy crises, global warming, and climatic changes call for technological and commercial advances in manufacturing high-quality transportation fuels from unconventional feedstocks. Microalgae is one of the most promising sources of biofuels due to the high yields attained per unit area and because it does not displace food crops. Neochloris oleabundans (Neo) microalga is an important promising microbial source of single-cell oil (SCO). Different experimental growth and lipid production conditions were evaluated and compared by using optical density (540 nm), dry-weight determination, and flow cytometry (FC). Best Neo average biomass productivity was obtained at 30°C under conditions of nitrogen-sufficiency and CO2 supplementation (N+/30°C/CO2), with an average doubling time of 1.4 days. The second and third highest productivities occurred with N-sufficient cultures without CO2 supplementation at 26°C (N+/26°C) and at 30°C (N+/30°C), with doubling times of 1.7 and 2.2 days, respectively. Microbial lipid production was monitored by flow cytometry using Nile red (NR), a lipophilic fluorochrome that possesses several advantageous characteristics for in situ screening near real time (at line). Results showed maximum lipid content (56%) after 6 days of nitrogen depletion under nitrogen starvation without CO2 supplementation (N−/30°C), followed by N−/30°C/CO2 and N−/26°C conditions with 52% lipid content, after 5 and 6 days of N starvation, respectively. The adequate fatty acid profile and iodine value of Neo lipids reinforced this microalga as a good source of SCO, in particular for use as biodiesel.
452823
Microalgae as a raw material for biofuels production
Biofuels demand is unquestionable in order to reduce gaseous emissions (fossil CO2, nitrogen and sulfur oxides) and their purported greenhouse, climatic changes and global warming effects, to face the frequent oil supply crises, as a way to help non-fossil fuel producer countries to reduce energy dependence, contributing to security of supply, promoting environmental sustainability and meeting the EU target of at least of 10% biofuels in the transport sector by 2020. Biodiesel is usually produced from oleaginous crops, such as rapeseed, soybean, sunflower and palm. However, the use of microalgae can be a suitable alternative feedstock for next generation biofuels because certain species contain high amounts of oil, which could be extracted, processed and refined into transportation fuels, using currently available technology; they have fast growth rate, permit the use of non-arable land and non-potable water, use far less water and do not displace food crops cultures; their production is not seasonal and they can be harvested daily. The screening of microalgae (Chlorella vulgaris, Spirulinamaxima, Nannochloropsis sp., Neochloris oleabundans, Scenedesmus obliquus and Dunaliella tertiolecta) was done in order to choose the best one(s), in terms of quantity and quality as oil source for biofuel production. Neochloris oleabundans (fresh water microalga) and Nannochloropsis sp. (marine microalga) proved to be suitable as raw materials for biofuel production, due to their high oil content (29.0 and 28.7%, respectively). Both microalgae, when grown under nitrogen shortage, show a great increase (~50%) in oil quantity. If the purpose is to produce biodiesel only from one species, Scenedesmus obliquus presents the most adequate fatty acid profile, namely in terms of linolenic and other polyunsaturated fatty acids. However, the microalgae Neochloris oleabundans, Nannochloropsis sp. and Dunaliella tertiolecta can also be used if associated with other microalgal oils and/or vegetable oils.
85024233
Lipid composition of the nitrogen starved green alga Neochloris oleoabundans
Neochloris oleoabundans has been cultivated in mineral medium deficient in nitrogen. The yield of lipids was 35–54% of cell dry weight. Triglycerides comprised 80% of the total lipids. Aliphatic hydrocarbons, sterols, pigments, glycolipids and phospholipids comprised the remaining lipid fraction. Saturated, monounsaturated and diunsaturated octodecanoic acid represented approximately one-half of the total fatty acids.
44826345
Stable nuclear transformation of the oleaginous microalga Neochloris oleoabundans by electroporation
Biodiesel from microalgae is technically feasible, but not yet economically viable. A potential approach to improve microalgae as an economically viable biodiesel feedstock is to increase microalgal lipid content via genetic engineering. Genetic manipulation of microalgae requires the accessibility to stable nuclear transformation. In this study, we describe a strategy for developing a stable nuclear transformation system of the oleaginous microalga Neochloris oleoabundans using electroporation. The hygromycin B-resistant gene Hyg3, which was used as a positively selectable marker, consisted of aph7” gene encoding aminoglycoside phosphotransferase of Streptomyces hygroscopicus and intron1 of Chlamydomonas reinhardtii rbcS2 gene, under the control of C. reinhardtii HSP70A-RBCS2 hybrid promoter. The transformation frequency was 5.2 × 10−4 transformants mg−1 DNA. The transformants showed stable hygromycin B-resistant phenotype for at least 6 months in the absence of the antibiotic selection. Co-transformation frequency of unselectable green fluorescent protein gene (Gfp) adapted to C. reinhardtii codon usage (ChGfp) and selectable Hyg3 gene was 2.6 × 10−4 transformants mg−1 DNA; up to 90 % of the transformants exhibited green fluorescent protein (GFP) activity. The ChGfp and Hyg3 gene were integrated into the nuclear genome of N. oleoabundans. The GFP fluorescence signal of the transformants under confocal laser scanning microscope was visible. The successful stable nuclear transformation system not only provides a basis for molecular genetics study, but also enables subsequent genetic engineering in the microalga to increase lipid content for biodiesel production. The strategy for developing the stable nuclear transformation system presented in this study may be applicable to other microalgal species without sequenced genome.
84574175
Fluorometric determination of the neutral lipid content of microalgal cells using Nile Red
The fluorophore Nile Red (9-diethylamino-5H-benzo[α]phenoxazine-5-one) has been used to determine neutral lipid in microalgal cells. Cellular fluorescence of stained cells and gravimetrically or chromatographically determined lipid were linearly correlated when Nile Red was excited at 488 – 525 nm and the fluorescent emision measured at 570 – 600 nm. Nile Red is a vital stain which allowed flow cytometric sorting of live microalgal populations based on their lipid content.
13210003
Lipid productivity as a key characteristic for choosing algal species for biodiesel production
Microalgae are a promising alternative source of lipid for biodiesel production. One of the most important decisions is the choice of species to use. High lipid productivity is a key desirable characteristic of a species for biodiesel production. This paper reviews information available in the literature on microalgal growth rates, lipid content and lipid productivities for 55 species of microalgae, including 17 Chlorophyta, 11 Bacillariophyta and five Cyanobacteria as well as other taxa. The data available in the literature are far from complete and rigorous comparison across experiments carried out under different conditions is not possible. However, the collated information provides a framework for decision-making and a starting point for further investigation of species selection. Shortcomings in the current dataset are highlighted. The importance of lipid productivity as a selection parameter over lipid content and growth rate individually is demonstrated.
19561234
The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas.
The Chlamydomonas reinhardtii HSP70A promoter can be induced by both heat shock and light. Several characteristics of this promoter suggest its usefulness as a tool for improved transgene expression in this alga. (i) It may by itself confer high inducibility to a transgene. Fusion of the HSP70A promoter to reporter genes HSP70B or ARS yields high levels of transgene product that, as shown for ARS, may accumulate when repeated cycles of heat shock induction are applied. (ii) It activates other promoters. Using HSP70B as a reporter gene, we show that the HSP70A promoter serves as a transcriptional activator when placed upstream of the promoters RBCS2, beta 2 TUB and HSP70B. Activation of these promoters was observed both under basal conditions and upon light induction. In addition, transformation rates obtained for the eubacterial resistance gene aadA were significantly increased, when expression of this gene was controlled by the HSP70A-RBCS2 promoter fusion as compared to the RBCS2 promoter alone.
23801204
Repeated consensus sequence and pseudopromoters in the four coordinately regulated tubulin genes of Chlamydomonas reinhardi.
The 5' coding and promoter regions of the four coordinately regulated tubulin genes of Chlamydomonas reinhardi have been mapped and sequenced. DNA sequencing data shows that the predicted N-terminal amino acid sequences of Chlamydomonas alpha- and beta-tubulins closely match that of tubulins of other eucaryotes. Within the alpha 1- and alpha 2-tubulin gene set and the beta 1- and beta 2-tubulin gene set, both nucleotide sequence and intron placement are highly conserved. Transcription initiation sites have been located by primer extension analysis at 140, 141, 159, and 132 base pairs upstream of the translation initiator codon for the alpha 1-, alpha 2-, beta 1-, and beta 2-tubulin genes, respectively. Among the structures with potential regulatory significance, the most striking is a 16-base-pair consensus sequence [GCTC(G/C)AAGGC(G/T)(G/C)--(C/A)(C/A)G] which is found in multiple copies immediately upstream of the TATA box in each of the four genes. An unexpected discovery is the presence of pseudopromoter regions in two of the transcribed tubulin genes. One pseudopromoter region is located 400 base pairs upstream of the authentic alpha 2-tubulin gene promoter, whereas the other is located within the transcribed 5' noncoding region of the beta 1-tubulin gene.
24571190
A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii.
The use of Chlamydomonas reinhardtii as a model system has been hindered by difficulties encountered in expressing foreign genes. We have synthesised a gene encoding green fluorescent protein (GFP) adapted to the codon usage of C. reinhardtii (cgfp). After verifying the gene was functional in Escherichia coli, the cgfp was fused in frame to the phleomycin resistance gene ble from Streptoalloteichus hindustanus and expressed in C. reinhardtii under control of the rbcS2 promoter and intron sequences. The GFP-fluorescence was seen only in the nucleus demonstrating the nuclear accumulation of the Ble-GFP fusion protein. The cgfp was also fused to the chlamyopsin gene, cop, and expressed in C. reinhardtii under control of the cop promoter. The eyespot became fluorescent indicating that the opsin-GFP fusion protein was correctly directed into the eyespot along with the endogenous unmodified opsin. We conclude that cgfp provides a useful tool to visualize protein synthesis and localisation in vivo in C. reinhardtii and possibly in related green algal species.
39990147
An engineered Streptomyces hygroscopicus aph 7" gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii.
We have developed a positively selectable marker for the green alga Chlamydomonas reinhardtii using the Streptomyces hygroscopicus aminoglycoside phosphotransferase gene (aph7"). Its expression is controlled by C. reinhardtii regulatory elements, namely, the beta2-tubulin gene promoter in combination with the first intron and the 3' untranslated region of the small subunit of ribulose bisphosphate carboxylase, rbcS2. C. reinhardtii cell-wall deficient and wild-type strains were transformed at rates up to 5 x 10(-5) with two constructs, pHyg3 and pHyg4 (intron-less). Transformants selected on plates with 10 microg/ml hygromycin B exhibited diverse levels of resistance of up to 200 microg/ml that were stably maintained for at least seven months; they contained two to five copies of the construct integrated in their genomes. Transcription of the chimeric aph7" gene, correct splicing of the rbcS2 intron, and polyadenylation of the transcripts have been verified by sequencing of RT-PCR products. Average co-transformation rates using pHyg3 and a second selectable plasmid were about 11%. This advocates the hygromycin-resistance plasmid, pHyg3, as a new versatile tool for the transformation of a broad range of C. reinhardtii strains without the sustained need for using auxotrophic mutants as recipients.
8556800
Altered Lipid Composition and Enhanced Nutritional Value of Arabidopsis Leaves following Introduction of an Algal Diacylglycerol Acyltransferase 2[C][W]
With the aim of improving the energy density of plant vegetative tissues, this work uses algal diacylglycerol acyltransferase type two enzymes to alter acyl carbon partitioning in Arabidopsis vegetative tissues, increasing acyl-CoA–dependent triacylglycerol synthesis and thereby increasing the nutritional value of leaves. Enhancement of acyl-CoA–dependent triacylglycerol (TAG) synthesis in vegetative tissues is widely discussed as a potential avenue to increase the energy density of crops. Here, we report the identification and characterization of Chlamydomonas reinhardtii diacylglycerol acyltransferase type two (DGTT) enzymes and use DGTT2 to alter acyl carbon partitioning in plant vegetative tissues. This enzyme can accept a broad range of acyl-CoA substrates, allowing us to interrogate different acyl pools in transgenic plants. Expression of DGTT2 in Arabidopsis thaliana increased leaf TAG content, with some molecular species containing very-long-chain fatty acids. The acyl compositions of sphingolipids and surface waxes were altered, and cutin was decreased. The increased carbon partitioning into TAGs in the leaves of DGTT2-expressing lines had little effect on transcripts of the sphingolipid/wax/cutin pathway, suggesting that the supply of acyl groups for the assembly of these lipids is not transcriptionally adjusted. Caterpillars of the generalist herbivore Spodoptera exigua reared on transgenic plants gained more weight. Thus, the nutritional value and/or energy density of the transgenic lines was increased by ectopic expression of DGTT2 and acyl groups were diverted from different pools into TAGs, demonstrating the interconnectivity of acyl metabolism in leaves.
22903012
Influence of fatty acid composition of raw materials on biodiesel properties.
The aim of this work was the study of the influence of the raw material composition on biodiesel quality, using a transesterification reaction. Thus, ten refined vegetable oils were transesterificated using potassium methoxide as catalyst and standard reaction conditions (reaction time, 1h; weight of catalyst, 1 wt.% of initial oil weight; molar ratio methanol/oil, 6/1; reaction temperature, 60 degrees C). Biodiesel quality was tested according to the standard [UNE-EN 14214, 2003. Automotive fuels. Fatty acid methyl esters (FAME) for diesel engines. Requirements and test methods]. Some critical parameters like oxidation stability, cetane number, iodine value and cold filter plugging point were correlated with the methyl ester composition of each biodiesel, according to two parameters: degree of unsaturation and long chain saturated factor. Finally, a triangular graph based on the composition in monounsaturated, polyunsaturated and saturated methyl esters was built in order to predict the critical parameters of European standard for whatever biodiesel, known its composition.
58897724
Technical aspects of biodiesel production by transesterification—a review
Biodiesel is gaining more and more importance as an attractive fuel due to the depleting fossil fuel resources. Chemically biodiesel is monoalkyl esters of long chain fatty acids derived from renewable feed stock like vegetable oils and animal fats. It is produced by transesterification in which, oil or fat is reacted with a monohydric alcohol in presence of a catalyst. The process of transesterification is affected by the mode of reaction condition, molar ratio of alcohol to oil, type of alcohol, type and amount of catalysts, reaction time and temperature and purity of reactants. In the present paper various methods of preparation of biodiesel with different combination of oil and catalysts have been described. The technical tools and processes for monitoring the transesterification reactions like TLC, GC, HPLC, GPC, 1H NMR and NIR have also been summarized. In addition, fuel properties and specifications provided by different countries are discussed.
98011642
Cetane numbers of branched and straight-chain fatty esters determined in an ignition quality tester
The cetane number, a widely used diesel fuel quality parameter related to the ignition delay time (and combustion quality) of a fuel, has been applied to alternative diesel fuels such as biodiesel and its components. In this work, the cetane numbers of 29 samples of straight-chain and branched C1 –C 4 esters as well as 2-ethylhexyl esters of various common fatty acids were determined. The cetane numbers of these esters are not significantly affected by branching in the alcohol moiety. Therefore, branched esters, which improve the cold-flow properties of biodiesel, can be employed without greatly influencing ignition properties compared to the more common methyl esters. Unsaturation in the fatty acid chain was again the most significant factor causing lower cetane numbers. Cetane numbers were determined in an ignition quality tester (IQT) which is a newly developed, automated rapid method using only small amounts of material. The IQT is as applicable to biodiesel and its components as previous cetane-testing methods. Published by Elsevier Science Ltd.
35319135
Identification of plant genetic loci involved in a posttranscriptional mechanism for meiotically reversible transgene silencing.
Numerous reports describe phenomena of transgene silencing in plants, yet the underlying genetic and molecular mechanisms are poorly understood. We observed that regeneration of Arabidopsis thaliana plants transgenic for the rolB gene of Agrobacterium rhizogenes results in a selection for transgene silencing. Transgene silencing could be monitored in this system by reversion of the visible RolB phenotype. We report a phenotypic, molecular, and genetic characterization of a meiotically reversible transgene silencing phenomenon observed in a rolB transgenic line. In this line, the rolB gene is expressed strongly and uniformly in seedlings, but in the course of further development, the rolB gene is silenced erratically at a frequency that depends on the dosage of rolB. The silenced state is mitotically stable, while complete resetting of rolB gene expression occurs in seedlings of the following generation. The silencing of rolB correlates with a dramatic reduction of steady-state rolB transcripts, while rolB nuclear run-off transcripts are only moderately reduced. Therefore, rolB gene silencing seems to act predominantly at the posttranscriptional level. The process of rolB gene silencing was found to be affected by two extragenic modifier loci that influence both the frequency and the timing of rolB gene silencing during plant development. These genetic data demonstrate a direct involvement of defined plant genes in this form of gene silencing.
22640213
RNA degradation and models for post-transcriptional gene silencing
Post-transcriptional gene silencing (PTGS) is a form of stable but potentially reversible epigenetic modification, which frequently occurs in transgenic plants. The interaction in trans of genes with similar transcribed sequences results in sequence-specific degradation of RNAs derived from the genes involved. Highly expressed single-copy loci, transcribed inverted repeats, and poorly transcribed complex loci can act as sources of signals that trigger PTGS. In some cases, mobile, sequence-specific silencing signals can move from cell to cell or even over long distances in the plant. Several current models hold that silencing signals are `aberrant' RNAs (aRNA), which differ in some way from normal mRNAs. The most likely candidates are small antisense RNAs (asRNA) and double-stranded RNAs (dsRNA). Direct evidence that these or other aRNAs found in silent tissues can induce PTGS is still lacking. Most current models assume that silencing signals interact with target RNAs in a sequence-specific fashion. This results in degradation, usually in the cytoplasm, by exonucleolytic as well as endonucleolytic pathways, which are not necessarily PTGS-specific. Biochemical-switch models hold that the silent state is maintained by a positive auto-regulatory loop. One possibility is that concentrations of hypothetical silencing signals above a critical threshold trigger their own production by self-replication, by degradation of target RNAs, or by a combination of both mechanisms. These models can account for the stability, reversibility and multiplicity of silent states; the strong influence of transcription rate of target genes on the incidence and stability of silencing, and the amplification and systemic propagation of motile silencing signals.
34967853
Biofuels from algae: challenges and potential
Algae biofuels may provide a viable alternative to fossil fuels; however, this technology must overcome a number of hurdles before it can compete in the fuel market and be broadly deployed. These challenges include strain identification and improvement, both in terms of oil productivity and crop protection, nutrient and resource allocation and use, and the production of co-products to improve the economics of the entire system. Although there is much excitement about the potential of algae biofuels, much work is still required in the field. In this article, we attempt to elucidate the major challenges to economic algal biofuels at scale, and improve the focus of the scientific community to address these challenges and move algal biofuels from promise to reality.
19644102
Investigation of biomass and lipids production with Neochloris oleoabundans in photobioreactor.
The fresh water microalga Neochloris oleoabundans was investigated for its ability to accumulate lipids and especially triacylglycerols (TAG). A systematic study was conducted, from the determination of the growth medium to its characterization in an airlift photobioreactor. Without nutrient limitation, a maximal biomass areal productivity of 16.5 g m(-2) day(-1) was found. Effects of nitrogen starvation to induce lipids accumulation was next investigated. Due to initial N. oleoabundans total lipids high content (23% of dry weight), highest productivity was obtained without mineral limitation with a maximal total lipids productivity of 3.8 g m(-2) day(-1). Regarding TAG, an almost similar productivity was found whatever the protocol was: continuous production without mineral limitation (0.5 g m(-2) day(-1)) or batch production with either sudden or progressive nitrogen deprivation (0.7 g m(-2) day(-1)). The decrease in growth rate reduces the benefit of the important lipids and TAG accumulation as obtained in nitrogen starvation (37% and 18% of dry weight, respectively).
7311923
A rapid method of total lipid extraction and purification.
Lipid decomposition studies in frozen fish have led to the development of a simple and rapid method for the extraction and purification of lipids from biological materials. The entire procedure can be carried out in approximately 10 minutes; it is efficient, reproducible, and free from deleterious manipulations. The wet tissue is homogenized with a mixture of chloroform and methanol in such proportions that a miscible system is formed with the water in the tissue. Dilution with chloroform and water separates the homogenate into two layers, the chloroform layer containing all the lipids and the methanolic layer containing all the non-lipids. A purified lipid extract is obtained merely by isolating the chloroform layer. The method has been applied to fish muscle and may easily be adapted to use with other tissues.