Enhancement of BODIPY505/515 lipid fluorescence method for applications in biofuel-directed microalgae production

This paper describes a microalgal cell lipid fluorescence enhancement method using BODIPY(505/515), which can be used to screen for lipids in wild-type microalgae and to monitor lipid content within microalgae production processes to determine optimal harvesting time. The study was based on four microalgae species (Dunaliella teteriolecta, Tetraselmis suecica, Nannochloropsis oculata, and Nannochloris atomus) selected because of their inherent high lipid content. An extended analysis was carried out with N. oculata due to the depressed fluorescence observed when compared with the other experimental strains. BODIPY(505/515) lipid fluorescence was determined for two solvent pre-treatment methods (DMSO and glycerol) and four staining condition parameters (analysis time, staining temperature, dye concentration, and algal cell concentration). It was found that lipid fluorescence of thick cell-walled microalgae, such as N. oculata, is significantly enhanced by both the pre-treatment methods and staining condition parameters, thereby significantly enhancing lipid fluorescence by ca. 800 times the base autofluorescence. The lipid fluorescence enhancement method provides a quick and simple index for in vivo Flow Cytometry quantification of total lipid contents for purposes of species screening or whole culture monitoring in biofuel-directed microalgae production.     

Chlamydomonas starchless mutant defective in ADP-glucose pyrophosphorylase hyper-accumulates triacylglycerol

Many microalgae and plants have the ability to synthesize large amounts of triacylglycerol (TAG) that can be used to produce biofuels. Presently, TAG-based biofuel production is limited by the feedstock supply. Metabolic engineering of lipid synthesis pathways to overproduce TAGs in oleaginous microalgae and oil crop plants has achieved only modest success. We demonstrate that inactivation of ADP-glucose pyrophosphorylase in a Chlamydomonas starchless mutant led to a 10-fold increase in TAG, suggesting that shunting of photosynthetic carbon partitioning from starch to TAG synthesis may represent a more effective strategy than direct manipulation of the lipid synthesis pathway to overproduce TAG.     

Patterns of variation in the lipid class and fatty acid composition of Nannochloropsis oculata (Eustigmatophyceae) during batch culture

Changes in the lipid and fatty acyl compositions of the marine microalga Nannochloropsis oculata Droop were examined during a batch culture growth cycle. During the early phase of batch culture the cellular proportion of triacylglycerols (TAG) increased. This was in addition to the increases in TAG observed in many microalgal species in the stationary-phase. Concomitant increases in the relative proportions of both saturated and monounsaturated fatty acids and decreases in the proportion of polyunsaturated fatty acids in total lipid were also associated with this phase. The separated individual lipid classes were found to have characteristic fatty acyl compositions. The relative proportion of lipid per cell, the relative proportions of the individual lipid classes and the fatty acyl compositions of the individual classes were all subject to variability during the growth cycle. The changing total lipid fatty acyl composition of N. oculata was found to be determined by the proportion of the total lipid present as TAG. The data suggest that the changes observed in the fatty acyl composition of N. oculata are a result of the partitioning of photosynthetically fixed carbon between polar and neutral lipid class biosynthesis and fatty acyl desaturation and elongation pathways. The effect of such a partitioning of carbon is discussed in relation to the effects of environmental variables and growth phase upon the balance of lipid class and polyunsaturated fatty acids (PUFA) synthesis in marine microalgae.     

Isolation and evaluation of oil-producing microalgae from subtropical coastal and brackish waters

Microalgae have been widely reported as a promising source of biofuels, mainly based on their high areal productivity of biomass and lipids as triacylglycerides and the possibility for cultivation on non-arable land. The isolation and selection of suitable strains that are robust and display high growth and lipid accumulation rates is an important prerequisite for their successful cultivation as a bioenergy source, a process that can be compared to the initial selection and domestication of agricultural crops. We developed standard protocols for the isolation and cultivation for a range of marine and brackish microalgae. By comparing growth rates and lipid productivity, we assessed the potential of subtropical coastal and brackish microalgae for the production of biodiesel and other oil-based bioproducts. This study identified Nannochloropsis sp., Dunaniella salina and new isolates of Chlorella sp. and Tetraselmis sp. as suitable candidates for a multiple-product algae crop. We conclude that subtropical coastal microalgae display a variety of fatty acid profiles that offer a wide scope for several oil-based bioproducts, including biodiesel and omega-3 fatty acids. A biorefinery approach for microalgae would make economical production more feasible but challenges remain for efficient harvesting and extraction processes for some species.     

基于模糊综合评价的产生物柴油微藻藻种筛选

产生物柴油微藻大规模培养对微藻藻种的性能要求较高。从丰富的藻种资源中筛选到高品质的藻种一直是个亟待解决的问题。通过研究3株产油微藻,从系统工程的角度综合整个微藻生物柴油的技术工艺,建立了以生长速率、含油率、油脂组成等18种指标的二级评价体系,采用二级模糊综合评价的模糊数学方法对产生物柴油微藻的性能进行综和分析、筛选。最终确定供评价的三株微藻二级模糊综合评价集：小球藻LICME001［0.360 0.315 0.192 0.069 0.064］,微绿球藻LICME002［0.277 0.331 0.236 0.104 0.052］和葡萄藻LICME003［0.325 0.371 0.232 0.071 0.060］。根据最大隶属度法则分析得：小球藻LICM001株产生物柴油微藻品质为优等级别,适合产生物柴油的技术工艺要求;微绿球藻LICME002和葡萄藻LICME003为良等级别的产生物柴油藻种。     

A homolog of Arabidopsis SDP1 lipase in Nannochloropsis is involved in degradation of de novo-synthesized triacylglycerols in the endoplasmic reticulum

Organisms of the microalgal genus Nannochloropsis produce high levels of triacylglycerols (TAGs), an efficient raw material for biofuels. A complete understanding of the TAG-breakdown pathway is critical for improving the productivity of TAGs to meet future needs. Among a number of lipases annotated as TAG lipase in the genomes of every organism, Arabidopsis SUGAR-DEPENDENT 1 (AtSDP1) lipases are characterized as a type of crucial TAG lipase in plants, similar to ScTgl3–5 in Saccharomyces cerevisiae. Homologs of the AtSDP1 TAG lipases are universally found in the genomes of plants, fungi, and algae. Here we identified two homologs of AtSDP1 TAG lipases in the oleaginous microalga species Nannochloropsis oceanica, NoTGL1 and NoTGL2. We generated single- and double-knockout strains for these lipases by homologous recombination. Whereas overall TAG content in the NoTGL2 single-knockout mutant was identical to that of wild type, the NoTGL1 knockout showed a two-fold increase in TAG content per cell in early log phase under nutrient-sufficient conditions without affecting growth. Homologs of AtSDP1 in S. cerevisiae are localized to the surface of lipid droplets, and AtSDP1 is transported from peroxisomes to the surface of lipid droplets. In contrast, NoTGL1 localized to the endoplasmic reticulum in both Nannochloropsis and yeast. We suggest that homologs of AtSDP1 lipases in Nannochloropsis modulate de novo TAG biosynthesis in the endoplasmic reticulum, unlike the roles of these lipases in other organisms. These results provide important insights into the mechanisms of TAG metabolism catalyzed by homologs of AtSDP1 lipase, which are highly conserved across species.     

Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances

Microalgae represent an exceptionally diverse but highly specialized group of micro-organisms adapted to various ecological habitats. Many microalgae have the ability to produce substantial amounts (e.g. 20–50% dry cell weight) of triacylglycerols (TAG) as a storage lipid under photo-oxidative stress or other adverse environmental conditions. Fatty acids, the building blocks for TAGs and all other cellular lipids, are synthesized in the chloroplast using a single set of enzymes, of which acetyl CoA carboxylase (ACCase) is key in regulating fatty acid synthesis rates. However, the expression of genes involved in fatty acid synthesis is poorly understood in microalgae. Synthesis and sequestration of TAG into cytosolic lipid bodies appear to be a protective mechanism by which algal cells cope with stress conditions, but little is known about regulation of TAG formation at the molecular and cellular level. While the concept of using microalgae as an alternative and renewable source of lipid-rich biomass feedstock for biofuels has been explored over the past few decades, a scalable, commercially viable system has yet to emerge. Today, the production of algal oil is primarily confined to high-value specialty oils with nutritional value, rather than commodity oils for biofuel. This review provides a brief summary of the current knowledge on oleaginous algae and their fatty acid and TAG biosynthesis, algal model systems and genomic approaches to a better understanding of TAG production, and a historical perspective and path forward for microalgae-based biofuel research and commercialization.     

A lipid droplet protein of Nannochloropsis with functions partially analogous to plant oleosins

As our understanding of the dynamics of lipid droplets (LDs) in animal, plant, and fungal cells is rapidly evolving, still little is known about the formation and turnover of these organelles in microalgae. Yet with the growing importance of algal feedstock for the production of biofuels and high-value lipids, there is a need to understand the mechanisms of LD dynamics in microalgae. Thus, we investigated the proteins associated with LDs of the emerging heterokont model alga Nannochloropsis sp. and discovered an abundant hydrophobic lipid droplet surface protein (LDSP) with unique primary sequence but structural similarities to other LD proteins. LDSP abundance in Nannochloropsis cells closely tracked the amount of triacylglycerols during conditions of oil accumulation and degradation. Functional characterization of LDSP in an Arabidopsis (Arabidopsis thaliana) OLEOSIN1-deficient mutant allowed a separation of its physical and structural properties in its interaction with LDs from its physiological or biochemical activities. Although LDSP presence in Arabidopsis predictably affected LD size, it could not reverse the physiological impact of OLEOSIN deficiency on triacylglycerol hydrolysis during germination.     

Lipidomic analysis can distinguish between two morphologically similar strains of Nannochloropsis oceanica

The two morphologically similar microalgae NMBluh014 and NMBluh‐X belong to two different strains of Nannochloropsis oceanica. They possess obviously different feeding effects on bivalves, but are indistinguishable by 18S rRNA and morphological features. In this work, lipidomic analysis followed by principal component analysis and orthogonal projections to latent structures discriminant analysis provided a clear distinction between these strains. Metabolites that definitively contribute to the classification were selected as potential biomarkers. The most important difference in polar lipids were sulfoquinovosyldiacylglycerol (containing 18:1/16:0 and 18:3/16:0) and monogalactosyldiacylglycerol (containing 18:3/16:3 and 20:5/14:0), which were detected only in NMBluh‐X. Additionally, an exhaustive qualitative and quantitative profiling of the neutral lipid triacylglycerol (TAG) in the two strains was carried out. The predominant species of TAG containing 16:1/16:1/16:1 acyl groups was detected only in NMBluh‐X with a content of ~93.67 ± 11.85 nmol · mg^?1 dry algae at the onset of stationary phase. Meanwhile, TAG containing 16:0/16:0/16:0 was the main TAG in NMBluh014 with a content of 40.25 ± 3.92 nmol · mg^?1. These results provided the most straightforward evidence for differentiating the two species. The metabolomic profiling indicated that NMBluh‐X underwent significant chemical and physiological changes during the growth process, whereas NMBluh014 did not show such noticeable time‐dependent metabolite change. This study is the first using Ultra Performance Liquid Chromatography coupled with Electrospray ionization‐Quadrupole‐Time of Flight Mass Spectrometry (UPLC‐Q‐TOF‐MS) for lipidomic profiling with multivariate statistical analysis to explore lipidomic differences of plesiomorphous microalgae. Our results demonstrate that lipidomic profiling is a valid chemotaxonomic tool in the study of microalgal systematics.     

Leveraging algal omics to reveal potential targets for augmenting TAG accumulation

Ongoing global efforts to commercialize microalgal biofuels have expedited the use of multi-omics techniques to gain insights into lipid biosynthetic pathways. Functional genomics analyses have recently been employed to complement existing sequence-level omics studies, shedding light on the dynamics of lipid synthesis and its interplay with other cellular metabolic pathways, thus revealing possible targets for metabolic engineering. Here, we review the current status of algal omics studies to reveal potential targets to augment TAG accumulation in various microalgae. This review specifically aims to examine and catalog systems level data related to stress-induced TAG accumulation in oleaginous microalgae and inform future metabolic engineering strategies to develop strains with enhanced bioproductivity, which could pave a path for sustainable green energy.     

微藻三酰甘油合成途径及其最新研究进展

三酰甘油(triacylglycerols,TAGs)是动物、植物、微生物和微藻细胞主要的储藏性脂类,它可应用于食品、轻工业和生物燃料等方面,是一种新型可再生能源——生物柴油生产的重要原料。与高等油料作物相比,微藻具有光合作用效率高、生长速度快、油脂产量高、不占用农业耕地和适应多种生长环境等优势,是一种潜在的新型生物柴油生产原料。然而,目前人们对有机体,尤其是微藻细胞内TAG合成与积累的分子机制及细胞的代谢调控机制还知之甚少。对TAG合成的一系列重要过程,包括脂肪酸的合成,TAG生物合成的主要途径和旁路途径,以及与TAG合成相关的关键酶和重要基因等进行了综述,特别对微藻细胞中与TAG合成相关的关键基因的最新研究进展进行了总结,旨在更好地了解油脂代谢的调控途径,为最大限度地供应生物柴油的生产原料提供理论基础。     

Triacylglycerol accumulates exclusively outside the chloroplast in short-term nitrogen-deprived Chlamydomonas reinhardtii

In microalgae, triacylglycerol (TAG) biosynthesis occurs by parallel pathways involving both the chloroplast and endoplasmic reticulum. A better understanding of contribution of each pathway to TAG assembly facilitates enhanced TAG production via rational genetic engineering of microalgae. Here, using a UPLC-MS(/MS) coupled with TLC-GC-based lipidomic platform, the early response of the major glycerolipids to nitrogen stress was analyzed at both the cellular and chloroplastidic levels in the model green alga Chlamydomonas reinhardtii. Subcellular lipidomic analysis demonstrated that TAG was accumulated exclusively outside the chloroplast, and remained unaltered inside the chloroplast after 4?h of nitrogen starvation. This study ascertained the existence of the glycolipid, digalactosyldiacylglycerol (DGDG), outside the chloroplast and the betaine lipid, diacylglycerol-N,N,N-trimethylhomoserine (DGTS), inside the chloroplast. The newly synthesized DGDG and DGTS prominently increased at the extra-chloroplastidic compartments and served as the major precursors for TAG biosynthesis. In particular, DGDG contributed to the extra-chloroplastidic TAG assembly in form of diacylglycerol (DAG) and DGTS in form of acyl groups. The chloroplastidic membrane lipid, monogalactosyldiacylglycerol (MGDG), was proposed to primarily offer DAG for TAG formation outside the chloroplast. This study provides valuable insights into the subcellular glycerolipidomics and unveils the acyl flux into the extra-chloroplastidic TAG in microalgae.     

Physiological role of neutral lipid accumulation in eukaryotic microalgae under stresses

In the studies of lipid metabolism of unicellular photoautotrophic eukaryotes (microalgae), the main attention is commonly paid to polar membrane lipids and their fatty acid (FA) composition, whereas neutral lipids, represented predominantly by triacylglycerols (TAG), are insufficiently studied. As was reported recently, the role of these compounds in microalgae is not limited to their storage function. It was found that TAG are frequently involved in adaptation to environmental conditions. This review summarizes experimental data obtained so far allowing to distinguish at least three aspects of TAG adaptive function in microalgae. First, these compounds are the source of long-chain FA, the building blocks for membranes necessary for rearrangements of the photosynthetic apparatus. Second, TAG biosynthesis consumes excessive photoassimilates preventing photooxidative injuries under stresses which reduce cell capacity of photosynthesis product utilization. Third, TAG deposited as cytoplasmic oil bodies form a depot for secondary carotenoids in carotenogenic microalgae producing an optical screen protecting the cell against photodamage by excessive PAR.     

Changes in the lipid composition and maximisation of the polyunsaturated fatty acid content of three microalgae grown in mass culture

Three species of microalgae were grown in mass culture to investigate the influence of culture technique and growth phase on the production of 20:5(n?3) and 22:6(n?3). These polyunsaturated fatty acids (PUFA) are considered to be essential in many marine animals diets for high growth and survival rates. The species of microalgae examined wereNannochloropsis oculata, Pavlova lutheri andIsochrysis sp. (clone T.Iso). All batch cultures (logarithmic and stationary phase) and semi-continuous cultures (logarithmic phase) examined contained high levels of the long-chain (n?3) PUFA, but production could be maximised by harvesting at specific times and growth phases. Maximum cellular content (pg cell^-1) of long-chain PUFA was found in logarithmic phase batch cultures ofN. oculata and in stationary phase cultures ofP. lutheri. The cellular content of PUFA in cultures ofIsochrysis sp. did not change significantly with culture technique or growth phase. Alternatively, stationary phase cultures of all three species showed increased proportions (%) and cellular contents of triacylglycerols, and saturated and monounsaturated fatty acids with correspondingly decreased proportions of polar lipids and most PUFA relative to logarithmic phase cultures. The exception was the proportion and cellular content of 22:6(n?3) inP. lutheri which increased with triacylglycerol content. The mass of long-chain (n?3) PUFA per volume of culture was significantly higher in stationary phase cultures due to the higher cell counts per volume. These findings indicate that the opportunity exists to maximise PUFA production by microalgae with the potential to improve animal growth and reduce production costs in mariculture operations and may be of use in the large scale culture and harvesting of microalgae for the biotechnology industry.     

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

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

Polyunsaturated fatty acid profiles of whole body phospholipids and triacylglycerols in anadromous and landlocked Atlantic salmon (Salmo salar L.) fry

We compared the fatty acid compositions and gains of whole body triacylglycerols (TAG) and phospholipids (PL) in anadromous and landlocked Atlantic salmon (Salmo salar) fry, of the same age, fed the same commercial marine oil-rich diet over a 42-day feeding trial. The landlocked strain exhibited significantly (P<0.05) higher growth rate and feed efficiency, due principally to a higher fat retention, particularly of monounsaturated and saturated fatty acids (SFA). n-3 and n-6 long-chain polyunsaturated fatty acid (PUFA) gains and retentions were significantly higher (P<0.05) in the landlocked fry. Great similarities were found in the fatty acid profiles of whole body TAG of both strains. However, marked genotypic differences were observed in the PUFA profiles of whole body PL fractions. The total PUFA, n-3 PUFA and docosahexaenoic acid (DHA) level in PL was significantly higher (P<0.05) while the SFA level, and the PUFA C18/C20 and eicosapentaenoic acid/arachidonic acid ratios were significantly lower (P<0.05) in the anadromous fry than in landlocked fry. Our results indicate that the level of DHA in salmon PL is under strong genetic control and that the capacity for incorporation, and possibly for the conversion of dietary n-3 and n-6 PUFA, is higher in the landlocked strain.     

Novel bacterial isolate from Permian groundwater, capable of aggregating potential biofuel-producing microalga Nannochloropsis oceanica IMET1

Increasing petroleum costs and climate change have resulted in microalgae receiving attention as potential biofuel producers. Little information is available on the diversity and functions of bacterial communities associated with biofuel-producing algae. A potential biofuel-producing microalgal strain, Nannochloropsis oceanica IMET1, was grown in Permian groundwater. Changes in the bacterial community structure at three temperatures were monitored by two culture-independent methods, and culturable bacteria were characterized. After 9 days of incubation, N. oceanica IMET1 began to aggregate and precipitate in cultures grown at 30°C, whereas cells remained uniformly distributed at 15°C and 25°C. The bacterial communities in cultures at 30°C changed markedly. Some bacteria isolated only at 30°C were tested for their potential for aggregating microalgae. A novel bacterium designated HW001 showed a remarkable ability to aggregate N. oceanica IMET1, causing microalgal cells to aggregate after 3 days of incubation, while the total lipid content of the microalgal cells was not affected. Direct interaction of HW001 and N. oceanica is necessary for aggregation. HW001 can also aggregate the microalgae N. oceanica CT-1, Tetraselmis suecica, and T. chuii as well as the cyanobacterium Synechococcus WH8007. 16S rRNA gene sequence comparisons indicated the great novelty of this strain, which exhibited only 89% sequence similarity with any previously cultured bacteria. Specific primers targeted to HW001 revealed that the strain originated from the Permian groundwater. This study of the bacterial communities associated with potential biofuel-producing microalgae addresses a little-investigated area of microalgal biofuel research and provides a novel approach to harvest biofuel-producing microalgae by using the novel bacterium strain HW001.