High-throughput metabolic screening of microalgae genetic variation in response to nutrient limitation (original) (raw)
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Research Paper, 2015
Fourier Transform Infrared (FT-IR) spectroscopy is a robust method for macromolecular analysis and differentiation of microorganisms. However, most studies are performed in controlled conditions and it is unclear whether this tool is appropriate for the identification of eukaryotic microalgae species from variable environments. In order to address this, nine closely-related species of marine and freshwater microalgae were grown under controlled (non-stressed) and variable (non-stressed and stressed) conditions, including nutrient-stressed and wastewater-stressed conditions. Following optimization of data processing methods, FT-IR spectra from all species and conditions were compared. The substantial metabolic changes that were caused by nutrient starvation restricted the ability of FT-IR spectroscopy to differentiate the microalgal species grown under variable conditions efficiently. Comparison of unsupervised and supervised multivariate data analysis methods found that principal component-discriminant function analysis was able best to differentiate between some species under controlled conditions but still gave poor differentiation under variable environmental conditions.
The Analyst, 2014
The metabolic profiling of various microalga species and their genetic variants, grown under varied environmental conditions, has become critical to accelerate the exploration of phytoplankton biodiversity and biology. The accumulation of valuable metabolites, such as glycerolipids, is also sought in microalgae for biotechnological applications ranging from food, feed, medicine, cosmetics to bioenergy and green chemistry. In this report we describe the direct analysis of metabolites and glycerolipids in small cell populations of the green alga Chlamydomonas reinhardtii, using laser ablation electrospray ionization (LAESI) mass spectrometry (MS) coupled with ion mobility separation (IMS). These microorganisms are capable of redirecting energy storage pathways from starch to neutral lipids depending on environmental conditions and nutrient availability. Metabolite and lipid production was monitored in wild type (WT), and genetically modified C. reinhardtii strains with an impaired starch pathway. Lipids, such as triacylglycerols (TAG) and diacylglyceryl-N,N,Ntrimethylhomoserine (DGTS) were monitored over time under altered light conditions. More than 200 ions related to metabolites, e.g., arginine, cysteine, serine, palmitate, chlorophyll a, chlorophyll b, etc., were detected. The lipid profiles at different light intensities for strains with impaired starch pathway (Sta1 and Sta6) contained 26 glycerolipids, such as DGTS, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), as well as 33 TAG species. Results were obtained over a 72-hour time period in high and low light conditions for the WT species and the two mutants. Our results indicate that LAESI-IMS-MS can be utilized for the rapid analysis of increased TAG production at elevated light intensities. Compared to WT, the Sta6 strain showed 2.5-times higher lipid production at 72 hours under high light conditions. The results demonstrate our ability to rapidly observe numerous changes in metabolite and lipid levels in microalgae populations. These capabilities are expected to facilitate the exploration of genetically altered microalgae strains for biofuel production.
High-Throughput Metabolic Profiling for Model Refinements of Microalgae
Journal of Visualized Experiments
Metabolic models are reconstructed based on an organism's available genome annotation and provide predictive tools to study metabolic processes at a systemslevel. Genome-scale metabolic models may include gaps as well as reactions that are unverified experimentally. Reconstructed models of newly isolated microalgal species will result in weaknesses due to these gaps, as there is usually sparse biochemical evidence available for the metabolism of such isolates. The phenotype microarray (PM) technology is an effective, high-throughput method that functionally determines cellular metabolic activities in response to a wide array of entry metabolites. Combining the high throughput phenotypic assays with metabolic modeling can allow existing metabolic network models to be rapidly reconstructed or optimized by providing biochemical evidence to support and expand genomic evidence. This work will show the use of PM assays for the study of microalgae by using the green microalgal model species Chlamydomonas reinhardtii as an example. Experimental evidence for over 254 reactions obtained by PM was used in this study to expand and refine a genome-scale C. reinhardtii metabolic network model, iRC1080, by approximately 25 percent. The protocol created here can be used as a basis for functionally profiling the metabolism of other microalgae, including known microalgae mutants and new isolates. network models can guide the rational designs for the rapid development of optimization strategies 1 , 2 , 3 , 4. Although approximately 160 microalgal species have been sequenced 5 , there are, to our knowledge, only 44 algal
Journal of Applied Phycology, 2014
An economically feasible microalgal lipid industry heavily relies on the selection of suitable strains. Because microalgae lipid content increases under a range of adverse conditions (e.g. nutrient deprivation, high light intensity), photosynthetic activity is usually strongly reduced. As a consequence, lipid productivity rapidly declines overtime, after reaching a maximum within the first days of cultivation. The microalgae Chlorella vulgaris, Chlorococcum littorale, Nannochloropsis oculata, Nannochloropsis sp., Neochloris oleoabundans, Stichococcus bacillaris and Tetraselmis suecica were compared on fatty acid content and productivity, and also on photosynthetic activity under nitrogen (N) starvation. Cultures in N-replete conditions were used as reference. Photosystem II (PSII) maximum efficiency was followed during the experiment, as proxy for the change in photosynthetic activity of the cells. Strains with a high capacity for both lipid accumulation as well as high photosynthetic activity under N starvation exhibited a high lipid productivity over time. Among the tested strains, Nannochloropsis sp. showed highest fatty acid content (45 % w/w) and productivity (238 mg L −1 day −1) as well as PSII maximum efficiency, demonstrating to be the most suitable strain, of those tested, for lipid production. This study highlights that for microalgae, maintaining a high photosynthetic efficiency during stress is the key to maintain high fatty acid productivities overtime and should be considered when selecting strains for microalgal lipid production.
Algal Research, 2016
Neochloris oleoabundans is an oleaginous microalga of biotechnological and commercial interest. A proteomic study was performed to compare the abundance of proteins under two different culture conditions: preferential lipid accumulation and preferential carbohydrate accumulation. When N. oleoabundans was cultivated under long periods of nitrogen starvation, the acyl carrier protein and the protein biotin carboxylase from the lipid synthesis pathway were found to be upregulated. The central metabolism pathways were also found to be highly activated to redirect the carbon flow toward pyruvate dehydrogenase and ATP synthesis. Pyruvate dehydrogenase was upregulated to supply the precursors for lipid production. Furthermore, in the pentose phosphate pathway, specifically glucose 6-phosphate dehydrogenase and 6phosphogluconate dehydrogenase were upregulated to supply reducing power in the form of NADPH for lipid synthesis and inorganic nitrogen assimilation. Carbohydrate synthesis-related enzymes that channel carbon to starch and sugar synthesis, such as UDP-glucose pyrophosphorylase and starch synthase, were upregulated when short durations of nitrogen limitation were applied during N. oleoabundans cultivation. However, ADP-glucose pyrophosphorylase was upregulated under preferential lipid accumulation conditions, indicating that under prolonged nitrogen starvation conditions, this enzyme potentially hydrolyzes starch chains to channel the carbon flow to lipid synthesis, which indicates a dual function of this protein.
2018
Microalgae are fast growing organisms that can be used as feedstock for the production of biofuels. The metabolism of microalgae can be manipulated by exposing them to different environmental conditions for favoring the accumulation of lipids, carbohydrates or proteins. For example, a change in growth conditions can cause the accumulation of large amounts of lipids, representing an opportunity for biodiesel production. Monitoring changes in the composition of microalgal cells is therefore important in assessing new growth conditions. However, at present, most techniques are time consuming, invasive and expensive. Here we have used FTIR microscopy to quantify lipid, protein, and starch accumulation in Neochloris minuta cells grown in the presence and absence of nitrogen. Under nitrogen deprivation the cellular lipid composition increases by a factor of 2.4, the cellular protein concentration decreases by ~60% while the starch concentration is unaltered. These estimates of biochemical...
3 Biotech, 2018
Biomass and lipid yield under nutrient depleted and supplemented conditions of N and Mg in two freshwater isolates, Chlorella sp. NC-MKM and Scenedesmus acutus NC-M2 from Meghalaya were investigated for biodiesel production. Both the strains, Chlorella sp. NC-MKM and Scenedesmus acutus NC-M2 are oleaginous in nature having lipid content of 40.2 ± 1.4 and 37.3 ± 2.6% DCW, respectively. The significant increase (92.8%) in lipid content was found in N-depleted condition while an increase (46.65%) in biomass yield was observed under Mg-supplemented condition in Chlorella sp. NC-MKM. Studying the interactive effects of nutrient depletion and supplementation, combination of N-depleted and Mg-supplemented condition was selected for further investigation to check enhanced lipid yield in Chlorella sp. NC-MKM. The results showed a significant increase in biomass yield, lipid yield and lipid content (30, 66.8, and 28.66%, respectively). Under this condition, accumulation of neutral lipid was also enhanced (47.17% M2 gated cells) compared to control (21.37% M2 gated cells). Further, FAMEs revealed that the relative percentage of saturated and mono-unsaturated fatty acids increased (66.16%) in Chlorella sp. NC-MKM compared to control that improves biodiesel properties.
Effect of nutrient supply status on biomass composition of eukaryotic green microalgae
Journal of Applied Phycology, 2013
In eukaryotic green microalgae, manipulation of metabolic pathways by altering the culture medium and/or culture conditions represents a powerful tool for physiological control and is usually more practicable than metabolic or genetic engineering. Strategies for nutrient-induced shifts in biomass composition are generally cost-efficient, environmentally friendly, applicable on a large scale and flexible for various industrially attractive microalgae species. In addition, processes, such as nutrient limitation/deprivation, can be readily scheduled and optimised to achieve high levels of productivity for the desired target compound(s). These strategies are currently used in microalgae to achieve overproduction of metabolites such as lipids, polysaccharides and pigments. This paper presents an overview of the species and strain-specific responses of eukaryotic, green microalgal cells that are triggered by variations in selected macronutrient and micronutrient availability. Individual and mutually associated physiological responses to nutrient supply status are described at the molecular level as well as discussed from the perspective of potential biotechnological applications.