Microalgae are currently emerging as one of the most promising alternative sources for the next generation of food, feed, cosmetics and renewable energy in the form of biofuel. Microalgae constitute a diverse group of microorganisms with advantages like fast and efficient growth. In addition, they do not compete for arable land and offer very high lipid yield potential. Major challenges for the development of this resource are to select lipid-rich strains using high-throughput staining for neutral lipid content in microalgae species. For this purpose, the fluorescent dyes most commonly used to quantify lipids are Nile red and BODIPY 505/515. Their fluorescent staining for lipids offers a rapid and inexpensive analysis tool to measure neutral lipid content, avoiding time-consuming and costly gravimetric analysis. This review collates and presents recent advances in algal lipid staining and focuses on Nile red and BODIPY 505/515 staining characteristics. The available literature addresses the limitations of fluorescent dyes under certain conditions, such as spectral properties, dye concentrations, cell concentrations, temperature and incubation duration. Moreover, the overall conclusion of the present review study gives limitations on the use of fluorochrome for screening of lipid-rich microalgae species and suggests improved protocols for staining recalcitrant microalgae and recommendations for the staining quantification.

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The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae

In order to realize the potential of microalgal biodiesel there is a need for substantial impetus involving interventions to radically improve lipid yields upstream. Nutrient stress and alteration to cultivation conditions are commonly used lipid enhancement strategies in microalgae. The main bottleneck of applying conventional strategies is their scalability as some of these strategies incur additional cost and energy. Novel lipid enhancement strategies have emerged to research forefront to overcome these challenges. In this review, the latest trends in microalgal lipid enhancement strategies, possible solutions and future directions are critically discussed. Advanced strategies such as combined nutrient and cultivation condition stress, microalgae–bacteria interactions, use of phytohormones EDTA and chemical additives, improving light conditions using LED, dyes and paints, and gene expression analysis are described. Molecular approaches such as metabolic and genetic engineering are emerging as the potential lipid enhancing strategies. Recent advancements in gene expression studies, genetic and metabolic engineering have shown promising results in enhancing lipid productivity in microalgae; however environmental risk and long term viability are still major challenges.

https://openscholar.dut.ac.za/bitstream/10321/2245/1/Singh_RSER_Vol55_Pg1-16_2016.pdf

Trends and novel strategies for enhancing lipid accumulation and quality in microalgae

Sustainability of the four generations of biofuels – A review

The risk of onset of cancer is influenced by poorly controlled chronic inflammatory processes. Inflammatory diseases related to cancer development include inflammatory bowel disease, which can lead to colon cancer, or actinic keratosis, associated with chronic exposure to ultraviolet light, which can progress to squamous cell carcinoma. Chronic inflammatory states expose these patients to a number of signals with tumorigenic effects, including nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPK) activation, pro-inflammatory cytokines and prostaglandins release and ROS production. In addition, the participation of inflammasomes, autophagy and sirtuins has been demonstrated in pathological processes such as inflammation and cancer. Chemoprevention consists in the use of drugs, vitamins, or nutritional supplements to reduce the risk of developing or having a recurrence of cancer. Numerous in vitro and animal studies have established the potential colon and skin cancer chemopreventive properties of substances from marine environment, including microalgae species and their products (carotenoids, fatty acids, glycolipids, polysaccharides and proteins). This review summarizes the main mechanisms of actions of these compounds in the chemoprevention of these cancers. These actions include suppression of cell proliferation, induction of apoptosis, stimulation of antimetastatic and antiangiogenic responses and increased antioxidant and anti-inflammatory activity.

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Bioactive Compounds Isolated from Microalgae in Chronic Inflammation and Cancer.

Microalgae serve as a promising source for the production of biofuels and bio-based chemicals. They are superior to terrestrial plants as feedstock in many aspects and their biomass is naturally rich in lipids, carbohydrates, proteins, pigments, and other valuable compounds. Due to the relatively slow growth rate and high cultivation cost of microalgae, to screen efficient and robust microalgal strains as well as genetic modifications of the available strains for further improvement are of urgent demand in the development of microalgae-based biorefinery. In genetic engineering of microalgae, transformation and selection methods are the key steps to accomplish the target gene modification. However, determination of the preferable type and dosage of antibiotics used for transformant selection is usually time-consuming and microalgal-strain-dependent. Therefore, more powerful and efficient techniques should be developed to meet this need. In this review, the conventional and emerging genome-editing tools (e.g., CRISPR-Cas9, TALEN, and ZFN) used in editing the genomes of nuclear, mitochondria, and chloroplast of microalgae are thoroughly surveyed. Although all the techniques mentioned above demonstrate their abilities to perform gene editing and desired phenotype screening, there still need to overcome higher production cost and lower biomass productivity, to achieve efficient production of the desired products in microalgal biorefineries.

Recent Developments on Genetic Engineering of Microalgae for Biofuels and Bio-Based Chemicals.

Summary 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 9 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 a-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.

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Altered lipid composition and enhanced lipid production in green microalga by introduction of brassica diacylglycerol acyltransferase 2

The marine based algal biofuel may be the best possible options for future energy, particularly for India where freshwater and land resources are limited. We have characterized a novel marine microalga, isolated from an Indian Ocean using genotypic and biochemical studies, which contain high lipids (48%) on dry wt. basis, among marine-microalgae reported from the Indian subcontinent. The classification of new species based on DNA barcodes studies using 16S rRNA and 23S rRNA has indicated its genetic similarity to green algae Parachlorella kessleri. Subsequently, FAME analysis has showed that it contain about 43%, saturated fatty acids, which makes new algal species highly suitable for further exploration and commercial production of the biodiesel.

DNA barcode and lipid analysis of new marine algae potential for biofuel

India's energy-mix comprises both non-renewable (coal, lignite, petroleum and natural gas) and renewable energy sources (wind, solar, small hydro, biomass, cogeneration bagasse etc). Information on reserves of non-renewable sources of energy and the potential for generation of renewable energy sources is a pre- requisite for assessing the country's potential for meeting its future energy needs. The changes in the reserves over time indicate the research and development going into the discovery of new reserves and the pace of their exploitation. The use of biomass aptly fits into the need for using renewable sources to drive our economic development. Algae have emerged as one of the most promising sources for biodiesel production. It can be inferred that they under transesterification reaction can be converted to fatty acid methyl ester (FAME). Such an approach can contribute to solve major problems of air pollution resulting from CO evolution and future crisis due 2 to a shortage of energy sources. In this study we used Chlamydomonas reinhardtii for biodiesel production and compared the amount of biodiesel produced with the biodiesel from waste vegetable oil. The study was undertaken to know the proper transesterification, amount of biodiesel production (ester) and physical properties of biodiesel produced.

Irshad Ahmad

Papers

Altered lipid composition and enhanced lipid production in green microalga by introduction of brassica diacylglycerol acyltransferase 2

DNA barcode and lipid analysis of new marine algae potential for biofuel

Biodiesel: a potential solution for energy requirements of India.