Plant triacylglycerols as feedstocks for the production of biofuels

Triacylglycerols produced by plants are one of the most energy-rich and abundant forms of reduced carbon available from nature. Given their chemical similarities, plant oils represent a logical substitute for conventional diesel, a non-renewable energy source. However, as plant oils are too viscous for use in modern diesel engines, they are converted to fatty acid esters. The resulting fuel is commonly referred to as biodiesel, and offers many advantages over conventional diesel. Chief among these is that biodiesel is derived from renewable sources. In addition, the production and subsequent consumption of biodiesel results in less greenhouse gas emission compared to conventional diesel. However, the widespread adoption of biodiesel faces a number of challenges. The biggest of these is a limited supply of biodiesel feedstocks. Thus, plant oil production needs to be greatly increased for biodiesel to replace a major proportion of the current and future fuel needs of the world. An increased understanding of how plants synthesize fatty acids and triacylglycerols will ultimately allow the development of novel energy crops. For example, knowledge of the regulation of oil synthesis has suggested ways to produce triacylglycerols in abundant non-seed tissues. Additionally, biodiesel has poor cold-temperature performance and low oxidative stability. Improving the fuel characteristics of biodiesel can be achieved by altering the fatty acid composition. In this regard, the generation of transgenic soybean lines with high oleic acid content represents one way in which plant biotechnology has already contributed to the improvement of biodiesel.     

Differential effects of nitrogen and sulfur deprivation on growth and biodiesel feedstock production of Chlamydomonas reinhardtii

Biodiesel production from microalgae is a promising approach for energy production; however, high cost of its process limits the use of microalgal biodiesel. Increasing the levels of triacylglycerol (TAG) levels, which is used as a biodiesel feedstock, in microalgae has been achieved mainly by nitrogen starvation. In this study, we compared effects of sulfur (S) and nitrogen (N) starvation on TAG accumulation and related parameters in wild-type Chlamydomonas reinhardtii CC-124 mt(-) and CC-125 mt(+) strains. Cell division was interrupted, protein and chlorophyll levels rapidly declined while cell volume, total neutral lipid, carotenoid, and carbohydrate content increased in response to nutrient starvation. Cytosolic lipid droplets in microalgae under nutrient starvation were monitored by three-dimensional confocal laser imaging of live cells. Infrared spectroscopy results showed that relative TAG, oligosaccharide and polysaccharide levels increased rapidly in response to nutrient starvation, especially S starvation. Both strains exhibited similar levels of regulation responses under mineral deficiency, however, the degree of their responses were significantly different, which emphasizes the importance of mating type on the physiological response of algae. Neutral lipid, TAG, and carbohydrate levels reached their peak values following 4 days of N or S starvation. Therefore, 4 days of N or S starvation provides an excellent way of increasing TAG content. Although increase in these parameters was followed by a subsequent decline in N-starved strains after 4 days, this decline was not observed in S-starved ones, which shows that S starvation is a better way of increasing TAG production of C. reinhardtii than N starvation.     

Advances in culture and genetic modification approaches to lipid biosynthesis for biofuel production and in silico analysis of enzymatic dominions in proteins related to lipid biosynthesis in algae

Biofuels have been shown to be a promising and highly attractive alternative for minimizing the use of fossil fuels, and microalgae have positioned themselves as potential candidates for production of lipids and other substances of commercial interest. We briefly review recent advances made in microalgae culture conditions and genetic manipulation for improving lipid yields for biofuel production – with both approaches showing similar yields of triacylglycerides, indicating that more work is required for improving lipid yield and accumulation in algae. Aiming at gaining knowledge of algae genetic manipulation and exploring future use of this information for modifying the lipid biosynthesis pathway, we investigated whether some characteristics of enzymes involved in lipid biosynthesis could relate to lipid yield and accumulation in algae. We made an in silico analysis of amino acid sequence of enzymatic domains and modeled tertiary structure of three proteins involved in the biosynthesis of lipids in microalgae: acetyl‐CoA carboxylase, Acyl‐CoA: diacylglycerol acyltransferase, and glycerol‐3‐phosphate acyltransferase. Our results suggest that, based on primary amino acid sequences and tertiary structure of proteins shared by certain algae, it is likely that these proteins may relate to lipid yield and accumulation, which makes bioinformatics a powerful tool for in silico study of proteins and for selecting genes involved in lipid biosynthesis that could be useful for heterologous transformation in algae with the long term objective of improving their yield, accumulation, and fatty acid composition by genetic engineering.     

Macroalgae in biofuel production

The conversion processes of macroalgae for biofuels can be divided into thermochemical (dry) and microbiological (wet) processes. The chemical composition of macroalgae together with the pre‐treatment method, conversion conditions, and the characteristics of the microbes involved (wet processes) determine the yield and the properties of the biofuel produced. Macroalgae are often rich in carbohydrates, and therefore well suited for biogas, biobutanol and bioethanol productions. The content of triacylglycerols (TAGs) is the best indicator for the suitability of the alga for biodiesel production. TAGs have a high conversion rate to biodiesel, high percentage of fatty acids, and they lack phosphorus, sulfur and nitrogen. Macroalgae can have high metal concentrations, which can have an impact on conversion processes: metals may inhibit or catalyse the processes. High sulfur (especially in green algae) and nitrogen contents are also characteristic to macroalgae, and may be problematic in the production of biogas (NH₃‐toxicity) and the use of the oil and biodiesel (high concentrations of H₂S and NO_x‐compounds). Macroalgae have proven to be suitable material for conversion processes, but further optimization of the processes is needed. At present, macroalgae are not economically, or in many cases not even environmentally, sustainable material when the whole production chain is considered. In this review we summarize information on the chemical composition of macroalgae in a prospect of biofuel production, and the current situation in the field of macroalgal‐based biofuel production.     

FATTY ACID AND LIPID COMPOSITION OF THE ACIDOPHILIC GREEN ALGA CHLAMYDOMONAS SP.1

The lipid and fatty acid compositions of Chlamydomonas sp. isolated from a volcanic acidic lake and C. reinhardtii were compared, and the effects of pH of the medium on lipid and fatty acid components of Chlamydomonas sp. were studied. The fatty acids in polar lipids from Chlamydomonas sp. were more saturated than those of C. reinhardtii. The relative percentage of triacylglycerol to the total lipid content in Chlamydomonas sp. grown in medium at pH 1 was higher than that in other cells grown at higher pH. A probable explanation might be that Chlamydomonas sp. has two low pH adaptation mechanisms. One mechanism is the saturation of fatty acids in membrane lipids to decrease membrane lipid fluidity, and the other is the accumulation of triacylglycerol, as a storage lipid, to prevent the osmotic imbalance caused by high concentrations of H₂SO₄.     

Strategies to enhance production of microalgal biomass and lipids for biofuel feedstock

ABSTRACT

Microalgae have enormous potential as feedstock for biofuel production compared with other sources, due to their high areal productivity, relatively low environmental impact, and low impact on food security. However, high production costs are the major limitation for commercialization of algal biofuels. Strategies to maximize biomass and lipid production are crucial for improving the economics of using microalgae for biofuels. Selection of suitable algal strains, preferably from indigenous habitats, and further improvement of those ‘platform strains’ using mutagenesis and genetic engineering approaches are desirable. Conventional approaches to improve biomass and lipid productivity of microalgae mainly involve manipulation of nutritional (e.g. nitrogen and phosphorus) and environmental (e.g. temperature, light and salinity) factors. Approaches such as the addition of phytohormones, genetic and metabolic engineering, and co-cultivation of microalgae with yeasts and bacteria are more recent strategies to enhance biomass and lipid productivity of microalgae. Improvement in culture systems and the use of a hybrid system (i.e. a combination of open ponds and photobioreactors) is another strategy to optimize algal biomass and lipid production. In addition, the use of low-cost substrates such as agri-industrial wastewater for the cultivation of microalgae will be a smart strategy to reduce production costs. Such systems not only generate high algal biomass and lipid productivity, but are also useful for bioremediation of wastewater and bioremoval of waste CO₂. The aim of this review is to highlight the advances in the use of various strategies to enhance production of algal biomass and lipids for biofuel feedstock.     

Modifying plants for biofuel and biomaterial production

The productivity of plants as biofuel or biomaterial crops is established by both the yield of plant biomass per unit area of land and the efficiency of conversion of the biomass to biofuel. Higher yielding biofuel crops with increased conversion efficiencies allow production on a smaller land footprint minimizing competition with agriculture for food production and biodiversity conservation. Plants have traditionally been domesticated for food, fibre and feed applications. However, utilization for biofuels may require the breeding of novel phenotypes, or new species entirely. Genomics approaches support genetic selection strategies to deliver significant genetic improvement of plants as sources of biomass for biofuel manufacture. Genetic modification of plants provides a further range of options for improving the composition of biomass and for plant modifications to assist the fabrication of biofuels. The relative carbohydrate and lignin content influences the deconstruction of plant cell walls to biofuels. Key options for facilitating the deconstruction leading to higher monomeric sugar release from plants include increasing cellulose content, reducing cellulose crystallinity, and/or altering the amount or composition of noncellulosic polysaccharides or lignin. Modification of chemical linkages within and between these biomass components may improve the ease of deconstruction. Expression of enzymes in the plant may provide a cost‐effective option for biochemical conversion to biofuel.     

Microalgal lipids biochemistry and biotechnological perspectives

In the last few years, there has been an intense interest in using microalgal lipids in food, chemical and pharmaceutical industries and cosmetology, while a noteworthy research has been performed focusing on all aspects of microalgal lipid production. This includes basic research on the pathways of solar energy conversion and on lipid biosynthesis and catabolism, and applied research dealing with the various biological and technical bottlenecks of the lipid production process. In here, we review the current knowledge in microalgal lipids with respect to their metabolism and various biotechnological applications, and we discuss potential future perspectives.     

Tailoring lignin biosynthesis for efficient and sustainable biofuel production

Increased global interest in a bio‐based economy has reinvigorated the research on the cell wall structure and composition in plants. In particular, the study of plant lignification has become a central focus, with respect to its intractability and negative impact on the utilization of the cell wall biomass for producing biofuels and bio‐based chemicals. Striking progress has been achieved in the last few years both on our fundamental understanding of lignin biosynthesis, deposition and assembly, and on the interplay of lignin synthesis with the plant growth and development. With the knowledge gleaned from basic studies, researchers are now able to invent and develop elegant biotechnological strategies to sophisticatedly manipulate the quantity and structure of lignin and thus to create economically viable bioenergy feedstocks. These concerted efforts open an avenue for the commercial production of cost‐competitive biofuel to meet our energy needs.     

Regulation and remodeling of intermediate metabolite and membrane lipid during NaCl-induced stress in freshwater microalga Micractinium sp. XJ-2 for biofuel production

Microalgae can accumulate a large fraction of reduced carbon as lipids under NaCl stress. This study investigated the mechanism of carbon allocation and reduction and triacylglycerol (TAG) accumulation in microalgae under NaCl-induced stress. Micractinium sp. XJ-2 was exposed to NaCl stress and cells were subjected to physiological, biochemical, and metabolic analyses to elucidate the stress-responsive mechanism. Lipid increased with NaCl concentration after 0-12 hr, then stabilized after 12–48 hr, and finally decreased after 48–72 hr, whereas TAG increased (0–48 hr) and then decreased (48–72 hr). Under NaCl-induced stress at lower concentrations, TAG accumulation, at first, mainly shown to rely on the carbon fixation through photosynthetic fixation, carbohydrate degradation, and membrane lipids remodeling. Moreover, carbon compounds generated by the degradation of some amino acids were reallocated and enhanced fatty acid synthesis. The remodeling of the membrane lipids of NaCl-induced microalgae relied on the following processes: (a) Increase in the amount of phospholipids and reduction in the amount of glycolipids and (b) extension of long-chain fatty acids. This study enhances our understanding of TAG production under NaCl stress and thus will provide a theoretical basis for the industrial application of NaCl-induced in the microalgal biodiesel industry.     

Outlook in the application of Chlamydomonas reinhardtii chloroplast as a platform for recombinant protein production

Microalgae, also called microphytes, are a vast group of microscopic photosynthetic organisms living in aquatic ecosystems. Microalgae have attracted the attention of biotechnology industry as a platform for extracting natural products with high commercial value. During last decades, microalgae have been also used as cost-effective and easily scalable platform for the production of recombinant proteins with medical and industrial applications. Most progress in this field has been made with Chlamydomonas reinhardtii as a model organism mainly because of its simple life cycle, well-established genetics and ease of cultivation. However, due to the scarcity of existing infrastructure for commercial production and processing together with relatively low product yields, no recombinant products from C. reinhardtii have gained approval for commercial production and most of them are still in research and development. In this review, we focus on the chloroplast of C. reinhardtii as an algal recombinant expression platform and compare its advantages and disadvantages to other currently used expression systems. We then discuss the strategies for engineering the chloroplast of C. reinhardtii to produce recombinant cells and present a comprehensive overview of works that have used this platform for the expression of high-value products.     

Production of Chlorella vulgaris as a source of essential fatty acids in a tubular photobioreactor continuously fed with air enriched with CO2 at different concentrations

To reduce CO₂ emissions and simultaneously produce biomass rich in essential fatty acids, Chlorella vulgaris CCAP 211 was continuously grown in a tubular photobioreactor using air alone or air enriched with CO₂ as the sole carbon source. While on one hand, nitrogen‐limited conditions strongly affected biomass growth, conversely, they almost doubled its lipid fraction. Under these conditions using air enriched with 0, 2, 4, 8, and 16% (v/v) CO₂, the maximum biomass concentration was 1.4, 5.8, 6.6, 6.8, and 6.4 g_DB L^?1 on a dry basis, the CO₂ consumption rate 62, 380, 391, 433, and 430 L^?1 day^?1, and the lipid productivity 3.7, 23.7, 24.8, 29.5, and 24.4 mg L^?1 day^?1, respectively. C. vulgaris was able to grow effectively using CO₂‐enriched air, but its chlorophyll a (3.0–3.5 g 100g_DB^?1), chlorophyll b (2.6–3.0 g 100g_DB^?1), and lipid contents (10.7–12.0 g 100g_DB^?1) were not significantly influenced by the presence of CO₂ in the air. Most of the fatty acids in C. vulgaris biomass were of the saturated series, mainly myristic, palmitic, and stearic acids, but a portion of no less than 45% consisted of unsaturated fatty acids, and about 80% of these were high added‐value essential fatty acids belonging to the ω3 and ω6 series. These results highlight that C. vulgaris biomass could be of great importance for human health when used as food additive or for functional food production. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:916–922, 2014     

Chlorella vulgaris as a lipid source: Cultivation on air and seawater‐simulating medium in a helicoidal photobioreactor

The freshwater microalga Chlorella vulgaris was cultured batchwise on the seawater‐simulating Schlösser medium either in a 1.1‐L‐working volume helicoidal photobioreactor (HeP) or Erlenmeyer flask (EF) as control and continuously supplying air as CO₂ source. In these systems, maximum biomass concentration reached 1.65 ± 0.17 g L^?1 and 1.25 ± 0.06 g L^?1, and maximum cell productivity 197.6 ± 20.4 mg L^?1 day^?1 and 160.8 ± 12.2 mg L^?1 day^?1, respectively. Compared to the Bold's Basal medium, commonly employed to cultivate this microorganism on a bench‐scale, the Schlösser medium ensured significant increases in all the growth parameters, namely maximum cell concentration (268% in EF and 126% in HeP), maximum biomass productivity (554% in EF and 72% in HeP), average specific growth rate (67% in EF and 42% in HeP), and maximum specific growth rate (233% in EF and 22% in HeP). The lipid fraction of biomass collected at the end of runs was analyzed in terms of both lipid content and fatty acid profile. It was found that the seawater‐simulating medium, despite of a 56–63% reduction of the overall biomass lipid content compared to the Bold's Basal one, led in HeP to significant increases in both the glycerides‐to‐total lipid ratio and polyunsaturated fatty acid content compared to the other conditions taken as an average. These results as a whole suggest that the HeP configuration could be a successful alternative to the present means to cultivate C. vulgaris as a lipid source. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:279–284, 2016     

Characterization of triacylglycerols from overwintering prepupae of the alfalfa pollinator Megachile rotundata (Hymenoptera: Megachilidae)

Alfalfa leafcutting bees, Megachile rotundata (F.), overwinter as prepupae. The internal lipids were extracted from prepupae that had been wintered at 4 degrees C for 7 months. Megachile rotundata prepupae possessed copious quantities of internal lipids (20% of the fresh weight) that were extracted with CHCl3/methanol (2:1). Transmission electron microscopy revealed that lipids were stored within very large intracellular vacuoles. Separation by silica chromatography revealed that 88% of the internal lipids were triacylglycerols. Ester derivatives of fatty acids from triacylglycerol components were analyzed by gas chromatography-mass spectrometry and 15 fatty acid constituents were identified. The majority (76%) of the triacylglycerol fatty acids were unsaturated fatty acids. The major triacylglycerol fatty acid constituent (30%) was the C16 monounsaturated fatty acid, palmitoleic acid (16:1, hexadec-9-enoic acid), with substantial amounts of linolenic acid (18:3, octadec-9,12,15-trienoic acid, 15%), palmitic acid (16:0, hexadecanoic acid, 14%) and oleic acid (18:1, octadec-9-enoic acid, 13%). Palmitoleic acid as the major fatty acid of an insect is an unusual occurrence as well as the presence of the 16-carbon polyunsaturated fatty acids, 16:2 and 16:3. The major intact triacylglycerol components were separated and identified by high performance liquid chromatography-mass spectrometry. A complex mixture of approximately 40 triacylglycerol components were identified and major components included palmitoyl palmitoleoyl oleoyl glycerol, palmitoyl palmitoleoyl palmitoleoyl glycerol, myristoyl palmitoleoyl palmitoleoyl glycerol, myristoleoyl palmitoyl palmitoleoyl glycerol, and palmitoyl palmitoleoyl linolenoyl glycerol. The function of these internal lipids and their relevance to winter survival and post-wintering development of M. rotundata is discussed.     

Chemical synthesis and NMR characterization of structured polyunsaturated triacylglycerols

The chemical synthesis of pure triacylglycerol (TAG) regioisomers, that contain long chain polyunsaturated fatty acids, such as arachidonic acid (AA) or docosahexaenoic acid (DHA), and saturated fatty acids, such as lauric acid (La) or palmitic acid (P), at defined positions, is described. A single step methodology using (benzotriazol-1-yloxy)-tripyrrolidinophosphonium hexafluorophosphate (PyBOP), an activator of carboxyl group commonly used in peptide synthesis and occasionally used in carboxylic acid esterification, has been developed for structured TAG synthesis. Identification of the fatty acyl chains for each TAG species was confirmed by atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) and fatty acid positional distribution was determined by (1)H and (13)C NMR spectra. The generic described procedures can be applied to a large variety of substrates and was used for the production of specific triacylglycerols of defined molecular structures, with high regioisomeric purity. Combination of MS and NMR was shown to be an efficient tool for structural analysis of TAG. In particular, some NMR signals were demonstrated to be regioisomer specific, allowing rapid positional analysis of LC-PUFA containing TAG.     

Chemistry and liquid chromatography methods for the analyses of primary oxidation products of triacylglycerols

Abstract

Triacylglycerols (TAGs) are one of the major components of the cells in higher biological systems, which can act as an energy reservoir in the living cells. The unsaturated fatty acid moiety is the key site of oxidation and formation of oxidation compounds. The TAG free radical generates several primary oxidation compounds. These include hydroperoxides, hydroxides, epidioxides, hydroperoxy epidioxides, hydroxyl epidioxides, and epoxides. The presence of these oxidized TAGs in the cell increases the chances of several detrimental processes. For this purpose, several liquid chromatography (LC) methods were reported in their analyses. This review is therefore focused on the chemistry, oxidation, extraction, and the LC methods reported in the analyses of oxidized TAGs. The studies on thin-layer chromatography were mostly focused on the total oxidized TAGs separation and employ hexane as major solvent. High-performance LC (HPLC) methods were discussed in details along with their merits and demerits. It was found that most of the HPLC methods employed isocratic elution with methanol and acetonitrile as major solvents with an ultraviolet detector. The coupling of HPLC with mass spectrometry (MS) highly increases the efficiency of analysis as well as enables reliable structural elucidation. The use of MS was found to be helpful in studying the oxidation chemistry of TAGs and needs to be extended to the complex biological systems.