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Algal bioprospecting in ecosystems leads to exploring native microalgae and the competency evaluation of economically producing lipids as biofuel or nutritional applications. In this study, ten microalgae species were screened from the saline water lake. Chlorella vulgaris, Chlorella sorokiniana, Chlamydomonas raudensis, Chlamydomonas hedleyi, Dunaliella salina, Picochlorum bazangan sp. nov., Tetraselmis bazangan sp. nov., Haematococcus lacustris, Nannochloropsis oceanic, and Scenedesmus rubescens were isolated and identified using 18SrDNA and tufA markers. Biodiesel potentials were assayed by the determination of biomass productivity, biochemical components, fatty acid profile, and biodiesel properties. The results showed that the maximum biomass yield (1.22 g/L) belonged to C. vulgaris. The highest protein, carbohydrate, chlorophyll, and carotenoid content were recorded in C. vulgaris, C. raudensis, C. sorokiniana, and D. salina, respectively. N. oceanica accumulated high lipid content and omega-3 fractions (31.09%). However, C. hedleyi had the highest lipid productivity (11.64 g/L/day) compared to other microalgae. The best species for biodiesel production was C. vulgaris, with a specific growth rate of 0.36 day−1, lipid productivity of 7.45 g/L/day, and C16-C18 fatty acid profile of 78.3%. The microalgae C. vulgaris had appropriate biodiesel properties of low viscosity (4.49), high cetane number (55.38), and relatively low cloud point (4.98). Another choice was N. oceanic, with high lipid productivity, cetane number (59.79), oxidative stability (56.43), and low iodine value (47.11). Microalgae T. bazangan sp. nov. had a cetane number (55.24), low cloud point (4.71), and C16-C18 fatty acid profile of 82.34%. Accordingly, C. vulgaris, T. bazangan sp. nov., and N. oceanic can be considered potential species for biodiesel. [ABSTRACT FROM AUTHOR]

Microalgae have been recognized as promising competitors for bioenergy production, because of their high lipid content and quick biomass development. In this work, freshwater microalgae Scenedesmus dimorphus was cultivated in a synthetic wastewater media with the object of investigating the bioenergy potential by physicochemical characterization. Batch experiments were performed photoautotrophically for biomass generation supplemented with different levels of CO2 concentration. Maximum biomass production was in the limit of 373 mg/L to 587 mg/L under different CO2 feed concentrations. Cultivation of microalgae along with CO2 supplementation found a substantial impact on buildup of lipid in microalgae biomass. Maximum 29% and 32% lipid content were achieved for 2% and 6% CO2 enrichment respectively, under nitrogen-depleted conditions. Fourier transform infrared spectroscopy, thermogravimetric analysis, and carbon, hydrogen, nitrogen, sulfur elemental analyzer instrumental tools were used to perform physicochemical characterization on biomass in order to determine its proximate analysis, ultimate analysis, higher heating value (HHV), and macromolecular composition, The higher heating value (HHV) of Scenedesmus dimorphus biomass achieved 33.26 MJ/kg with almost zero sulfur content. This HHV value is significantly higher than that of other microalgae sp. Moreover, this HHV value is also even higher than that of bituminous coal (24.3 MJ/kg) and other types of lignocellulosic biomass. Scenedesmus dimorphus biomass indicates that it can be a potential fuel feedstock for environmentally friendly bioenergy production. [ABSTRACT FROM AUTHOR]

Background: Metabolic engineering for hyperaccumulation of lipids in vegetative tissues is a novel strategy for enhancing energy density and biofuel production from biomass crops. Energycane is a prime feedstock for this approach due to its high biomass production and resilience under marginal conditions. DIACYLGLYCEROL ACYLTRANSFERASE (DGAT) catalyzes the last and only committed step in the biosynthesis of triacylglycerol (TAG) and can be a rate-limiting enzyme for the production of TAG. Results: In this study, we explored the effect of intron-mediated enhancement (IME) on the expression of DGAT1 and resulting accumulation of TAG and total fatty acid (TFA) in leaf and stem tissues of energycane. To maximize lipid accumulation these evaluations were carried out by co-expressing the lipogenic transcription factor WRINKLED1 (WRI1) and the TAG protect factor oleosin (OLE1). Including an intron in the codon-optimized TmDGAT1 elevated the accumulation of its transcript in leaves by seven times on average based on 5 transgenic lines for each construct. Plants with WRI1 (W), DGAT1 with intron (Di), and OLE1 (O) expression (WDiO) accumulated TAG up to a 3.85% of leaf dry weight (DW), a 192-fold increase compared to non-modified energycane (WT) and a 3.8-fold increase compared to the highest accumulation under the intron-less gene combination (WDO). This corresponded to TFA accumulation of up to 8.4% of leaf dry weight, a 2.8-fold or 6.1-fold increase compared to WDO or WT, respectively. Co-expression of WDiO resulted in stem accumulations of TAG up to 1.14% of DW or TFA up to 2.08% of DW that exceeded WT by 57-fold or 12-fold and WDO more than twofold, respectively. Constitutive expression of these lipogenic "push pull and protect" factors correlated with biomass reduction. Conclusions: Intron-mediated enhancement (IME) of the expression of DGAT resulted in a step change in lipid accumulation of energycane and confirmed that under our experimental conditions it is rate limiting for lipid accumulation. IME should be applied to other lipogenic factors and metabolic engineering strategies. The findings from this study may be valuable in developing a high biomass feedstock for commercial production of lipids and advanced biofuels. [ABSTRACT FROM AUTHOR]

Background: Biofuel research that aims to optimize growth conditions in microalgae is critically important. Chlamydomonas reinhardtii is a green microalga that offers advantages for biofuel production research. This study compares the effects of nitrogen-, sulfur-, and nitrogen and sulfur- deprivations on the C. reinhardtii starchless mutant cc5373-sta6. Specifically, it compares growth, lipid body accumulation, and expression levels of acetyl-CoA carboxylase (ACC) and phosphoenolpyruvate carboxylase (PEPC). Results: Among nutrient-deprived cells, TAP-S cells showed significantly higher total chlorophyll, cell density, and protein content at day 6 (p < 0.05). Confocal analysis showed a significantly higher number of lipid bodies in cells subjected to nutrient deprivation than in the control over the course of six days; N deprivation for six days significantly increased the size of lipid bodies (p < 0.01). In comparison with the control, significantly higher ACC expression was observed after 8 and 24 h of NS deprivation and only after 24 h with N deprivation. On the other hand, ACC and PEPC expression at 8 and 24 h of S deprivation was not significantly different from that in the control. A significantly lower PEPC expression was observed after 8 h of N and NS deprivation (p < 0.01), but a significantly higher PEPC expression was observed after 24 h (p < 0.01). Conclusions: Based on our findings, it would be optimum to cultivate cc5373-sta6 cells in nutrient deprived conditions (-N, -S or –NS) for four days; whereby there is cell growth, and both a high number of lipid bodies and a larger size of lipid bodies produced. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate biomass generation and determine the total lipid content of a microalgae isolated from a lagoon of the Ecuadorian Amazon. The morphological identification of the isolated microalgae was done through bibliographic comparisons and molecular identification by PCR followed by DNA sequencing and phylogenetic analysis. A 10-day batch culture was operated in 1-L glass bottles, and nutrient removal was determined daily. After harvesting, biomass was centrifuged to remove most of the water content. Half of the obtained biomass was kept wet and the other half was dried for further analysis. Cell growth, optical density, and dry weight were measured during cultivation time. In addition, total lipid and free fatty acid (FFA) percentages were evaluated. Removal of chemical oxygen demand (COD), nitrates (NO3−), and phosphates (PO43−) was 33.12%, 67.77%, and 76.73%, respectively. The biomass concentration was 2.19 ± 0.12 g/L with a productivity of 0.22 g/L per day. The best extraction percentages of total lipids and FFA were 18.43% and 61.46%, respectively. Although the results of lipids and FFA do not reach the required limits, the productivity of biomass and lipids is comparable with species that are cultivated for biodiesel production. [ABSTRACT FROM AUTHOR]

The study aimed to apply gamma radiation as a cell pretreatment method for lipid extraction and for obtaining astaxanthin from residual biomass of Haematococcus pluvialis. Factor 1 of the two-factor experimental design was represented by cell pretreatment methods: biomass with chloroform:methanol under ultrasound (BCMU), biomass with chloroform:methanol under γ radiation (BCMR), dry biomass under γ radiation (DBR), and control (without pretreatment). Factor 2 considered the vegetative and cystic phases. Cultivation was performed in a mixotrophic system, and biomass was collected in both phases, centrifuged, lyophilized, and submitted to cell pretreatment and lipid extraction. Lipid content and FAMEs were evaluated comparing pretreatment methods and life cycle phases. Total lipid content was higher with the BCMR method in the vegetative (18% DW) and cystic (14% DW) phases. Gamma radiation combined with organic solvent was more efficient for increasing lipid yield, and DBR had a lipid yield similar to BCMU. FAME content differed between phases and pretreatments for most fatty acids, mainly C16:0, C16:1, C18:1n9c, C18:2n6c, C18:2n6t, and C18:3n3. The predominance of saturated or low unsaturated fatty acids makes H. pluvialis, in both phases, suitable for biofuel production. The preservation of astaxanthin from residual cystic biomass was observed when submitted to the DBR method, with a concentration similar to the raw biomass (1.5% DW). Gamma radiation in dry biomass has an antioxidant effect. Therefore, the lipid extraction method preceded by gamma irradiation was efficient for vegetative and cystic cells of H. pluvialis and contributed to the preservation of astaxanthin from residual cystic biomass. [ABSTRACT FROM AUTHOR]

Industrial crops are becoming increasingly relevant as raw materials for different goods and services. Although there are many oilseed crops with industrial purposes, data about their production in Chile are scarce because the definition used by the Office of Agricultural Studies and Policies places agro‐industrial and industrial crops in the same category. Here, we review the available information about 12 industrial oleaginous crops at different levels of development (four native, two adventive and six introduced crops). Most of these crops have been evaluated for or are related to biodiesel production, despite the lack of commercial biodiesel production in Chile. Jojoba was the first industrial oilseed produced commercially in Chile; however, every year its production area decreases, and currently it does not exceed 100 ha. Sunflower and rapeseed, which are the first generation of raw materials for biodiesel production, have been poorly studied in Chile despite the large number of studies available on them worldwide. Camelina and castor bean have recently been studied; the former is produced in the south, while the latter is produced on the coastline in northern Chile. Further research is needed to improve the production and adaptation of these oilseed crops to climatic conditions in Chile; moreover, a new market must be opened to produce and commercialize their oils. Oilseed species native to Chile have not been extensively studied, and they represent an interesting option suitable for production under Chilean climatic conditions; this is particularly true for guindilla and chañar, because both can grow under different abiotic stresses. © 2022 Society of Chemical Industry and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The reuse of the culture media after biomass growth can be a cost-effective strategy for large-scale microalgae production, especially when the target product is biofuel. Therefore, this study aimed to investigate the effect of recycling the microalgae Tetradesmus obliquus culture medium, focusing on the improved production of biomass and lipids, biodiesel properties, and cost reduction. The experiments were carried out in duplicate and three sequential steps with successive recycling (50% of the clarified medium and 50% of the standard). It was found that cells from recycled cultures had a similar overall performance as those from the fresh medium (standard). From this verification, the main findings of this research pointed out that (i) the consumption of the nutrients was efficient, with values in the order of 99% nitrate, 20% phosphate, 64% total iron, and 9% potassium; (ii) the average contents of biomass and lipids in the recycled medium increased around 20% to 24%, respectively, concerning the standard; (iii) the polyunsaturated fatty acids pool present in the microalgal oil dropped from 20.71 to 9.78% in the recycled culture, which represents a high-quality feedstock for biofuel; and (iv) the biodiesel properties evaluated, including iodine and saponification values, were 48 g I2 100 g−1 and 254 mg KOH/goil−1, respectively. Finally, this recyclability bioprocess can reduce overall costs by about 34%, thus improving the economic viability of microalgae production. [ABSTRACT FROM AUTHOR]

Lipid droplets are cytoplasmic organelles that store lipids for energy and membrane synthesis. The oleaginous yeast Lipomyces starkeyi is one of the most promising lipid producers and has attracted attention as a biofuel source. It is known that the expansion of lipid droplets is enhanced under nutrient-poor conditions. Therefore, we prepared a novel nitrogen-depleted medium (N medium) in which to culture L. starkeyi cells. Lipid accumulation was rapidly induced, and this was reversed by the addition of ammonium. In this condition, cell proliferation stopped, and cells with giant lipid droplets were arrested in G1 phase. We investigated whether cell cycle arrest at a specific phase is required for lipid accumulation. Lipid accumulation was repressed in hydroxyurea-synchronized S phase cells and was increased in nocodazole-arrested G2/M phase cells. Moreover, the enrichment of G1 phase cells seen upon rapamycin treatment induced massive lipid accumulation. From these results, we conclude that L. starkeyi cells store lipids from G2/M phase and then arrest cell proliferation in the subsequent G1 phase, where lipid accumulation is enhanced. Cell cycle control is an attractive approach for biofuel production. [ABSTRACT FROM AUTHOR]

This scientometric review and bibliometric analysis aimed to characterize trends in scientific research related to algae, photobioreactors and astaxanthin. Scientific articles published between 1995 and 2020 in the Web of Science and Scopus bibliographic databases were analyzed. The article presents the number of scientific articles in particular years and according to the publication type (e.g., articles, reviews and books). The most productive authors were selected in terms of the number of publications, the number of citations, the impact factor, affiliated research units and individual countries. Based on the number of keyword occurrences and a content analysis of 367 publications, seven leading areas of scientific interest (clusters) were identified: (1) techno-economic profitability of biofuels, bioenergy and pigment production in microalgae biorefineries, (2) the impact of the construction of photobioreactors and process parameters on the efficiency of microalgae cultivation, (3) strategies for increasing the amount of obtained lipids and obtaining biodiesel in Chlorella microalgae cultivation, (4) the production of astaxanthin on an industrial scale using Haematococcus microalgae, (5) the productivity of biomass and the use of alternative carbon sources in microalgae culture, (6) the effect of light and carbon dioxide conversion on biomass yield and (7) heterotrophy. Analysis revealed that topics closely related to bioenergy production and biofuels played a dominant role in scientific research. This publication indicates the directions and topics for future scientific research that should be carried out to successfully implement economically viable technology based on microalgae on an industrial scale. [ABSTRACT FROM AUTHOR]

Microalgae are photosynthetic organisms functioning as the green bio-factories for various pharmaceutical and biofuel products. To date, numerous attempts have been carried out to manipulate culture conditions to maximize the production of the desired metabolites. Because light is the energy source of microalgae for their growth and metabolites biosynthesis, it has been one of the most investigated variables emphasized on the deep understanding of how microalgae respond towards light changes as an external stimulus. This review discusses the effects of different light sources, light intensities, light wavelengths and length of photoperiod on various microalgae species, especially in terms of biomass and lipid productivity. Additionally, the relationship between photoregulation processes and lipid productivity of microalgae are also deliberated. The current available approaches of microalgae mass cultivation, including different types of open and closed systems are recapitulated with the intention to highlight the significant insights for the design of future photoreactors. [ABSTRACT FROM AUTHOR]

Freshwater deficiency and growing requests for renewable energy have led to increased concern about water and energy security. Therefore, the present study attempted to evaluate the feasibility of using wastewater (WW) as a culture medium for microalgal cultivation with the aim of nutrient removal and biodiesel production. The green microalga Asterarcys quadricellulare (Chlorophyta) was isolated from a wastewater treatment plant in Menoufia, Egypt, and grown for 24 days in Bold's Basal Medium as a control and at different concentrations of secondary treated WW in distilled water (25%, 50%, 75%, and 100%). The results of 75% WW treatment recorded 96.6%, 98.4%, and 89.9% removal efficiency for nitrate, ammonia, and total phosphorus, respectively. Also, it revealed high biomass yield and biomass productivity (1.44 g L−1 and 69.0 mg L−1 day−1, respectively). In addition, it showed higher lipid production (360.6 mg L−1) and lipid productivity (17.2 mg L−1 day−1). Asterarcys quadricellulare fatty acid profile estimation revealed elevated proportions of oleic (54.92%), palmitic (20.54%), and linoleic acids (13.9%). Most of the properties of biodiesel derived from the studied microalga are consistent with those recommended by the international standards. The current study concluded that A. quadricellulare could be used for wastewater bioremediation and biomass production with high potential as biodiesel feedstock. [ABSTRACT FROM AUTHOR]

Oleaginous microorganisms and the thermal decomposition of their recovered lipids are new attractive research fields for the sustainable production of biofuels from actual biomass. In this work, the thermal decomposition of lipids from C. vulgaris and from L. starkeyi was investigated using a thermogravimetric analyzer coupled to a mass spectrometer (TGA-MS) under non-isothermal conditions. Thermal decomposition was examined using the one-step reaction model. Integral, differential, and advanced isoconversional methods were implemented to analyze the conversion-dependent activation energy. The Friedman method resulted in an accurate and reliable estimation of the kinetic parameters. The activation energy showed a strong dependence on conversion from 123.5 to 246 kJ mol-1 for lipids from C. vulgaris and from 80.3 to 299.9 kJ mol-1 for lipids from L. starkeyi. The generalized master plot approach evidenced that the thermal decomposition of lipids from C. vulgaris proceeds via three-dimensional diffusion (D3—Jander's equation). Also, the kinetic analysis indicated that Valensi's equation (D2), controlled by a two-dimensional diffusion model, is the most likely mechanism for the thermal decomposition of lipids from L. starkeyi. The rate parameters were evaluated via a kinetic compensation plot. Ion temperature evolution was investigated by TGA-MS revealing many of the functional groups evolved in the range of 350–500 °C as C1–C7 aliphatic hydrocarbons, aromatic hydrocarbons, aldehydes, carboxylic acids, and furan derivatives. All these aspects favor the lipid biosynthesis from microbial biomass to potential lipid-based building blocks. [ABSTRACT FROM AUTHOR]

Microalgal biodiesel is technically viable to power diesel engines. This third-generation biofuel has great performance of combustion and can reduce emission of greenhouse gases into the atmosphere. However, its production is currently very expensive, relative to petrochemical diesel. Development and implementation of strategies to optimize productivity and quality of biomass and thus ensure this future-proof biofuel is economically feasible to fabricate and competitive with diesel oil on an industrial scale is challenging. This meta-analytic overview documents the diversity of fastest-growing, oil-accumulating microalgae; performance of cultures and systems; strategies for inducing lipids; and quality and economics of microalgal biodiesel. The microalgae, Neochloris oleoabundans, Scenedesmus obliquus, Desmodesmus sp., and Micractinium sp., are hyperaccumulators of lipids. The genre, Micractinium sp., is a thermophile, and thus it can resist hydrothermal streams. Complementarily, it is the fastest to grow and the most productive in both biomass and biodiesel. This should be of great importance to roll-out sustainable, high-performance algal systems in marginal lands in tropical zones, where the heat often makes the planning and management of projects difficult and expensive. Heat shock, nutritional starvation, and photoperiod are the most effective algae-specific strategies for inducing mechanisms for lipogenesis in Ankistrodesmus dimorphus, Chlorella vulgaris and Leptolyngbya sp., Cylindrotheca closterium and N. oleoabundans, and Amphora subtropica and Dunaliella sp., respectively. Rhodococcus sp. produces a significant amount of biomass at very low cost, relative to diesel oil. The concept of synergistically co-culturing microalgae with this photosynthetically active bacterium may be an option to save the producer the expenditures and uncertainties of third-generation biodiesel. [ABSTRACT FROM AUTHOR]

This research aims to evaluate the effect of molasses as a source of organic carbon on the growth and lipid profile of Dunaliella salina cultivated under different light regimes to determine its strategy and potential use to produce biodiesel. Two sources of nutrients (F/2 medium, F/2; molasses, M) and two light conditions (12 h light: 12 h darkness, 12L:12D) conformed four treatments (F/2 12L:12D, F/2 D, M 12L:12D, and MD) that were investigated with three replicates each. The concentration of microalgae produced by the groups under light conditions was more than double that of those grown in darkness. The highest biomass produced (46.91 ± 6.47 g L-1) was obtained in the F/2 medium with 12L:12D treatment, while the lowest (18.99 ± 0.78 g L-1) was in the M 12L:12D group. Regardless of the culture conditions, the lipids were mainly composed of the C16:0 and C18 fatty acids. The use of molasses as a culture medium produced the highest amount of C18:1, regardless of the light regime. Molasses promoted the highest production of fatty acids methyl esters. Depending on the culture medium, D. salina cells showed different percentages of saturated (SFA), monounsaturated (MFA), and polyunsaturated (PUFA) fatty acids. The highest amount of saturated fatty acids was obtained in F/2 12L:12D. The highest amount of polyunsaturated fatty acids was presented in the mixotrophic culture (molasses and light). The cultures grown with molasses were rich in monounsaturated fatty acids. An inexpensive industrial by-product, Cane molasses could be a low-cost feedstock for biodiesel in cultivating D. salina. [ABSTRACT FROM AUTHOR]

The bioenergy crops such as energycane, miscanthus, and sorghum are being genetically modified using state of the art synthetic biotechnology techniques to accumulate energy‐rich molecules such as triacylglycerides (TAGs) in their vegetative cells to enhance their utility for biofuel production. During the initial genetic developmental phase, many hundreds of transgenic phenotypes are produced. The efficiency of the production pipeline requires early and minimally destructive determination of oil content in individuals. Current screening methods require time‐intensive sample preparation and extraction with chemical solvents for each plant tissue. A rapid screen will also be needed for developing industrial extraction as these crops become available. In the present study, we have devised a proton relaxation nuclear magnetic resonance (1H‐NMR) method for single‐step, non‐invasive, and chemical‐free characterization of in‐situ lipids in untreated and pretreated lignocellulosic biomass. The systematic evaluation of NMR relaxation time distribution provided insight into the proton environment associated with the lipids in the biomass. It resolved two distinct lipid‐associated subpopulations of proton nuclei that characterize total in‐situ lipids into bound and free oil based on their "molecular tumbling" rate. The T1T2 correlation spectra also facilitated the resolution of the influence of various pretreatment procedures on the chemical composition of molecular and local 1H population in each sample. Furthermore, we show that hydrothermally pretreated biomass is suitable for direct NMR analysis unlike dilute acid and alkaline pretreated biomass which needs an additional step for neutralization. [ABSTRACT FROM AUTHOR]

In recent years, research initiatives on renewable bioenergy or biofuels have been gaining momentum, not only due to fast depletion of finite reserves of fossil fuels but also because of the associated concerns for the environment and future energy security. In the last few decades, interest is growing concerning microalgae as the third-generation biofuel feedstock. The CO2 fixation ability and conversion of it into value-added compounds, devoid of challenging food and feed crops, make these photosynthetic microorganisms an optimistic producer of biofuel from an environmental point of view. Microalgal-derived fuels are currently being considered as clean, renewable, and promising sustainable biofuel. Therefore, most research targets to obtain strains with the highest lipid productivity and a high growth rate at the lowest cultivation costs. Different methods and strategies to attain higher biomass and lipid accumulation in microalgae have been extensively reported in the previous research, but there are fewer inclusive reports that summarize the conventional methods with the modern techniques for lipid enhancement and biodiesel production from microalgae. Therefore, the current review focuses on the latest techniques and advances in different cultivation conditions, the effect of different abiotic and heavy metal stress, and the role of nanoparticles (NPs) in the stimulation of lipid accumulation in microalgae. Techniques such as genetic engineering, where particular genes associated with lipid metabolism, are modified to boost lipid synthesis within the microalgae, the contribution of "Omics" in metabolic pathway studies. Further, the contribution of CRISPR/Cas9 system technique to the production of microalgae biofuel is also briefly described. [ABSTRACT FROM AUTHOR]

Efficient biodiesel production from biomass is essential for the green-oriented energy transition. The current incongruity between fermentation temperature and microbial thermotolerance is one of the key factors restricting biodiesel production, and it is difficult to solve this problem relying only on conventional microbial characterization and breeding methods. We propose a strategy to solve the corn stover cellulose utilization problem by developing thermotolerant biodiesel-production yeast strains. Candida tropicalis was initially improved by adaptive laboratory evolution and then mutated by atmospheric and room-temperature plasma mutagenesis to obtain a thermotolerant strain capable of growing at temperatures up to 45 °C. Two rounds of fluorescence-activated cell sorting were used to select high lipid content cells, which resulted in a 16.8% increase in lipid accumulation capacity compared to the original cells. The simultaneous saccharification fermentation of the generated strain with pretreated corn stover at 44 °C yielded a lipid generation efficiency of 86.2 ± 2 mg/g. This study provides a new method and an alternative strain for the selection and breeding of high-thermotolerance and high biodiesel-production yeasts and offers a feasible strategy for lignocellulosic fermentation for biodiesel production at elevated temperatures using the new industrial strain. [Display omitted] • A new strategy for lignocellulosic fermentation for biodiesel production. • Provides a new method and alternative strain, addressing the temperature asymmetry between microbial and enzyme catalytic (enzymatic) activity during fermentation. • Developing thermotolerant biodiesel-production yeast strains capable of growing at temperatures up to 45 °C. [ABSTRACT FROM AUTHOR]

Oleaginous algae have the ability to synthesize a high level of triacylglycerol (TAG) and are considered as the next‐generation feedstock for biofuel production. Manipulating algal lipid biosynthetic pathways has potential to overproduce TAG and represents a feasible way toward bringing down algae‐derived biofuels production cost. Here we dissected functional roles and engineering potential of six diacylglycerol acyltransferase (DGAT) genes from the marine alga Phaeodactylum tricornutum. PtDGAT1, PtDGAT2B, and PtDGAT3, residing at the chloroplast endoplasmic reticulum (ER) readily for utilizing both chloroplast‐ and ER‐derived diacylglycerol, were transcriptionally correlated with TAG accumulation. Heterologous expression in yeast, in vitro assay and overexpression in P. tricornutum all supported that PtDGAT1 surpassed the other five PtDGATs in synthesizing TAG. Compared to wild type P. tricornutum, the PtDGAT1‐overexpressing strain produced more than doubled TAG and total lipids, which reached 57% and 73% of dry weight, respectively, record‐high levels ever achieved in this alga. Our results demonstrated the strategy of screening proper engineering targets and manipulating a single gene to pull carbon flux to lipids for TAG hyper‐accumulation without growth compromise. The engineered alga with a great trait improvement may serve as a potent lipid producer for production uses. [ABSTRACT FROM AUTHOR]

The biomass of Mucor circinelloides, a dimorphic oleaginous filamentous fungus, has a significant nutritional value and can be used for single cell oil production. Metal ions are micronutrients supporting fungal growth and metabolic activity of cellular processes. We investigated the effect of 140 different substrates, with varying amounts of metal and phosphate ions concentration, on the growth, cell chemistry, lipid accumulation, and lipid profile of M. circinelloides. A high-throughput set-up consisting of a Duetz microcultivation system coupled to Fourier transform infrared spectroscopy was utilized. Lipids were extracted by a modified Lewis method and analyzed using gas chromatography. It was observed that Mg and Zn ions were essential for the growth and metabolic activity of M. circinelloides. An increase in Fe ion concentration inhibited fungal growth, while higher concentrations of Cu, Co, and Zn ions enhanced the growth and lipid accumulation. Lack of Ca and Cu ions, as well as higher amounts of Zn and Mn ions, enhanced lipid accumulation in M. circinelloides. Generally, the fatty acid profile of M. circinelloides lipids was quite consistent, irrespective of media composition. Increasing the amount of Ca ions enhanced polyphosphates accumulation, while lack of it showed fall in polyphosphate. [ABSTRACT FROM AUTHOR]

• Influence of phytohormones on algal biomass and lipid accumulation was investigated. • Auxin and cytokinin exihibited a synergystic effect on biomass and lipid yield. • Phytohormones influenced the activities of antioxidant enzymes SOD, CAT and APX. • Phytohormones modified the FAME contents that were desired for high-grade biofuels. Microalgae are considered as a promising resource for biodiesel. Nevertheless, their commercial exploitation necessitates considerable impetus towards the development of approaches for increased biomass and lipid production. The present work elucidates the impact of exogenously supplemented auxins, i.e., indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and indole-3-propionic acid (IPA) and cytokinins, i.e., benzylaminopurine (BAP) and thidiazuron (TDZ) on biomass, lipid content and fatty acid profile of Desmodesmus sp. JS07. Among auxins, IBA improved the biomass and lipid content up to 1.96 ± 0.11 g/L and 34.88 ± 3.87 %, respectively while BAP among cytokinins increased the biomass and lipid content up to 1.88 ± 0.061 g/L and 31.84 ± 1.33 % respectively. Further, the cumulative impact of IBA (10 mg/L) and BAP (5 mg/L) resulted in their synergistic effect by stimulating biomass and lipid content up to 2.34 ± 0.032 g/L and 42.43 ± 1.88 % respectively. Auxins stimulated the superoxide dismutase activity, and cytokinins increased the enzymes (catalase and ascorbate peroxidase), scavenging reactive oxidative species, thereby regulating ROS homeostasis in microalgae. A significant alteration in the fatty acid profile owing to the type and dosage of phytohormones was detected. Hence, the strategy employing phytohormones could prove to be a meaningful approach for biofuel production. [ABSTRACT FROM AUTHOR]

Developing effective, energy-efficient methods to extract lipids from microalgae remains a challenge in bioenergy. This study assesses electric pulse (EP) pretreatments to enhance lipid extraction from Chlorella protothecoides. In tandem with solvent extraction, we applied 10, 50, 100, 200, and 300 60 kV/cm, 60 ns duration EPs or 3, 17, 33, 67, and 100 60 kV/cm, 300 ns duration EPs to match the energy density u applied to the sample for each EP duration. Lipid recovery increased with increasing u for each EP duration until reaching a maximum for 17 300 ns EPs (the second lowest u) and 100 60 ns EPs (the third lowest u) to achieve a 13.3% and 16.1% increase in lipid extraction, respectively. Lipid extraction declined at higher u due to increased cell lysis. The largest net energy released calculated from the difference between the energy available from the extracted lipids and the applied energy of the EPs occurred at the lowest u for each EP duration. Applying trains of 100 μs EPs induced qualitatively similar behavior. These results suggest that a more detailed parametric study may optimize EP parameters for net energy release for specific microalgae and solvents. [ABSTRACT FROM AUTHOR]

Lipomyces starkeyi has been regarded as one of the model oleaginous yeasts. However, in this study, Lipomyces kononenkoae BF1S57 strain was found to be able to yield 60.06% (w/v) lipids (11.3 g/L lipids) from 90.0 g/L glucose, its cell mass was 18.8 ± 0.3 g/L within 96 h of a 10-L fermentation. Furthermore, the genome of L. kononenkoae has two copies of the genes encoding the β-subunit and α-subunit of fatty acid synthase, respectively. This may be related to high lipid contents in its cells. Over 94% of the fatty acids of the extracted lipids produced by L. kononenkoae BF1S57 strain was C 16:0 , C 16:1 , C 18:0 , C 18:1 and C 18:2 fatty acids. The properties of the prepared biodiesels in this study totally met the requirements of US biodiesel ASTM D6751 and EU biodiesel EN 14214. All these results demonstrated that the produced cellular lipids could be used as raw materials for biofuel production. • L. kononenkoae BF1S57 strain yielded 60.06% (w/v) lipids within 96 h of the 10-L fermentation. • Over 94% of the fatty acids of the extracted lipids was C 16:0 , C 16:1 , C 18:0 , C 18:1 and C 18:2 fatty acids. • The prepared fatty acid methyl esters could be used as biodiesels. [ABSTRACT FROM AUTHOR]

Wastewater treatment, along with the simultaneous production of valuable chemical compounds, including lipids by microalgae is a challenging but attractive study. Towards this goal, the candidate microalgae were selected from culture collections or isolated from wastewater in this study. The initial screening test using microalgae revealed that various eukaryotic as well as prokaryotic microalgae showed steady growth in municipal wastewater samples. Among them, Tetraselmis sp. NKG400013 and Parachlorella kessleri NKG021201 from culture collections, and Chloroidium saccharophilum NKH13 from the wastewater sample exhibited high biomass productivity. Furthermore, P. kessleri NKG021201 and C. saccharophilum NKH13 showed high lipid productivity (56 ± 1 mg/L/day for NKG021201, 35 ± 10 mg/L/day for NKH13). During this cultivation, 99% of nitrogen and 82% of phosphorous compounds were removed from the wastewater sample by the strain NKG021201. Analysis of fatty acid compositions of P. kessleri NKG021201 and C. saccharophilum NKH13 revealed that lipids derived from these microalgae were suitable for the application of biodiesel fuels, indicating that these microalgae were promising for wastewater treatment and lipid production. [ABSTRACT FROM AUTHOR]

Background: Nannochloropsis gaditana is a photosynthetic unicellular microalgae considered one of the most interesting marine algae to produce biofuels and food additive due to its rapid growth rate and high lipid accumulation. Although microalgae are attractive platforms for solar energy bioconversion, the overall efficiency of photosynthesis is reduced due to the steep light gradient in photobioreactors. Moreover, accumulation of lipids in microalgae for biofuels production is usually induced in a two-phase cultivation process by nutrient starvation, with additional time and costs associated. In this work, a biotechnological approach was directed for the isolation of strains with improved light penetration in photobioreactor combined with increased lipids productivity. Results: Mutants of Nannochloropsis gaditana were obtained by chemical mutagenesis and screened for having both a reduced chlorophyll content per cell and increased affinity for Nile red, a fluorescent dye which binds to cellular lipid fraction. Accordingly, one mutant, called e8, was selected and characterized for having a 30% reduction of chlorophyll content per cell and an almost 80% increase of lipid productivity compared to WT in nutrient-replete conditions, with C16:0 and C18:0 fatty acids being more than doubled in the mutant. Whole-genome sequencing revealed mutations in 234 genes in e8 mutant among which there is a non-conservative mutation in the dgd1 synthase gene. This gene encodes for an enzyme involved in the biosynthesis of DGDG, one of the major lipids found in the thylakoid membrane and it is thus involved in chloroplast biogenesis. Lipid biosynthesis is strongly influenced by light availability in several microalgae species, including Nannochloropsis gaditana: reduced chlorophyll content per cell and more homogenous irradiance in photobioreactor is at the base for the increased lipid productivity observed in the e8 mutant. Conclusions: The results herein obtained presents a promising strategy to produce algal biomass enriched in lipid fraction to be used for biofuel and biodiesel production in a single cultivation process, without the additional complexity of the nutrient starvation phase. Genome sequencing and identification of the mutations introduced in e8 mutant suggest possible genes responsible for the observed phenotypes, identifying putative target for future complementation and biotechnological application. [ABSTRACT FROM AUTHOR]

To elucidate the potential of Chlorococcum oleofaciens KF584224.1, the effects of different ferric ion concentrations (0, 10−7, 10−6, 10−5, 10−4 mol L−1) on growth, lipid accumulation, and fatty acid profile has been studied. The quantity and quality of extracted lipid was also analyzed by gravimetric and gas chromatographic methods, respectively, to evaluate their suitability according to the international standards for biodiesel (ASTM D6751 and EN 14214). An increase in ferric ion concentration up to 10−6 mol L−1 was associated with an increased specific growth rate by 1.83 folds and expanded exponential growth rate by 10 days compared with that in 0 mol L−1 ferric ion concentration. The highest biomass productivity (310.17 ± 2.19 mg L−1 d−1) and lipid productivity (138.81 ± 3.57 mg L−1 d−1) were achieved in culture medium supplemented with 10−6 and 10−5 mol L−1 ferric ion. The proportion of PUFAs and SFAs tends to decrease and increase by enhancing the ferric ion concentration, respectively. Although the quality of biodiesel produced form C. oleofaciens was found to be in accordance with the specifications in the American (ASTM D6751) and European (EN 14214) biodiesel standards, ferric ion supplementation enhanced the lipid characteristics obtained from C. oleofaciens for biodiesel production. • C. oleofaciens grew up 2.19-fold faster at 10−6 mol L−1 ferric ion concentration. • Maximum lipid productivity was obtained under 10−5 mol L−1 ferric ion concentration. • Enhancing in ferric ion supply played a positive role in SFAs accumulation. • The produced FAMEs met the biodiesel specifications of international standards. • Ferric ion concentration up to 10−5 mol L−1 improved the biodiesel quality. [ABSTRACT FROM AUTHOR]

Studies on microalgae species and strain selection have demonstrated the potential of a broad range of algal species suitable for large-scale mass production, but provided little data on the reliability and productivity of the cultures under outdoor conditions over a long period. Long-term studies as part of the development of large-scale microalgae mass production are essential at small scale to minimise expenditure in scaling-up and large-scale operations. The current study describes the long-term semi-continuous culture of 4 wild-type isolates of euryhaline Tetraselmis sp. grown in 1 m2 raceway ponds outdoors in 7% (w/w NaCl) saline water enriched with N and P without any additional carbon source under the environmental conditions in Perth, Western Australia. The long-term investigation indicated that Tetraselmis MUR-231 was not able to grow outdoors, while Tetraselmis strains MUR-167, MUR-230 and MUR-233 grew outdoors over long periods. Tetraselmis MUR-167 was cultured for over 16 months outdoors before terminated due to cell clump formation and heavy biofilm development on the pond walls, whereas Tetraselmis strains MUR-230 and MUR-233 were cultured in semi-continuous culture over 20 months. The latter two strains grew at high solar irradiance with daily maximum values of 58890 W m−2 and temperatures ranging from about 40 to 0 °C. Both strains had a similar average specific growth rate of 0.19 day−1 over the experimental period. Maximum biomass productivities were 9.11 g m−2 day−1 and 9.99 g m−2 day−1 for Tetraselmis MUR-230 and MUR-233, respectively. The maximum total lipid productivity of 4.30 g m−2 day−1 of Tetraselmis MUR-230 was slightly higher than Tetraselmis MUR-233 (4.05 g m−2 day−1). These results clearly indicate the potential of these two Tetraselmis strains for commercial application, such as a high lipid yielding feedstock under minimal culture management conditions in arid and semi-arid coastal regions such as northern Western Australia. [ABSTRACT FROM AUTHOR]

Microalgae based biofuel is an attractive alternative energy source due to its rapid growth rate and high lipid accumulation efficiency. In this study, we screened high lipid content microalga with the favourable fatty acid composition suitable for biodiesel production. Totally twelve different microalgal species were isolated from freshwater habitats. The isolates were identified as Micractinium sp., Chlorella sorokiniana, Scenedesmus bajacalifornicus, Desmodesmus sp., Scenedesmus obliquus, Coelastrum proboscideum, Chlamydomonas moewusii, Chlamydomonas debaryana, Chlamydomonas dorsoventralis, Coelastrum sp., and Ankistrodesmus sp. based on morphological features and ITS region similarity. Among the isolates, highest lipid content (33±0.07%) and lipid productivity (0.27±0.06 g L-1) were obtained from Ankistrodesmus sp. Intracellular lipid droplets of Ankistrodesmus sp. were observed through Nile red staining. The lipid content was enhanced up to 45% under the nitrogen deficient (5 mg L-1) BG-11 medium. Ankistrodesmus sp. fatty acid profile shows the presence of palmitic (16.39%), stearic (15.67%), oleic (25.66%), linolenic (21.62%), and alpha-linoleic acids (14.34%). The oleic acid was the dominant fatty acid 25.66% in the nitrogen deficient condition. [ABSTRACT FROM AUTHOR]

This section presents a review of the scientific literature published in 2018 on topics relating to sustainable bioenergy from biofuel residues and waste. This review is divided into the following sections: Feedstocks, Bioethanol, Biodiesel, Biohydrogen, Hydrogen, Biofuel Residues, Microalgae, and Lignocelluloses. [ABSTRACT FROM AUTHOR]

• Passive lipid extraction techniques that skip conventional cell lysis step for microalgal biofuel production. • Energy consumption by passive methods are compared with physical methods. • In-situ transesterification, saponification-acidification, and switchable solvents have low energy consumption. • Passive methods suitable for lipid-based biorefinery is presented. The conventional method of lipid extraction mandatorily includes cell lysis which is considered an essential process for the enhancement of extraction efficiency. However, cell lysis method especially those incorporating mechanical and physical methods such as homogenization, sonication, microwave techniques, etc., are energy intensive and drastically escalates the biofuel production cost. Therefore, the development of an alternate route of extraction skipping the conventional mechanical cell lysis step remains a challenge to be accomplished. In this context, we discuss the reports involving passive techniques that are able to extract lipid from microalgae without significant cell wall shearing. This includes methods such as in-situ transesterification, direct saponification, supercritical fluid extraction, organic solvent extraction, etc. Moreover, the review discusses the employment of passive lipid extraction methods in a biorefinery set-up, outlining the various bioproducts that could be generated along with lipid. The review concludes with the analysis of the economics of microalgal lipid extraction using passive disruption methods and compares with the processes incorporating cell lysis steps. [ABSTRACT FROM AUTHOR]

Discovery of high-yielding microalgae rich in valuable lipids, proteins, pigments, or carbohydrates is essential to the growth of nutraceutical and biofuel industries. Therefore, bioprospecting of microalgae from diverse environmental conditions has become highly significant to meet the demand of such bioresources globally. In this context, bioprospecting of three species of microalgae isolated from bloomed freshwaters of Kerala, India, was carried out; morphological and molecular characterizations revealed two of the algae as new species to science and named accordingly. The industrial potentials of these algae (Fasciculochloris boldii (MH992105.1), Sphaerocystis antoni-kadavilaii Ray & Prasanth sp. nov. (MK005299.1), and Chlorella zachariaii Ray & Prasanth sp. nov. (MH930447.1)) were assessed in terms of biomass yield and biochemical profile. The alga Sphaerocystis antoni-kadavilaii with a biomass yield of 176.8 ± 2.0% increase L−1 day−1, lipid productivity of 4.4 ± 0.3 mg L−1 day−1, the protein content of 46.1%, is found promising, in comparison with the known species such as Spirulina sp. and Chlorella sp. It is high in chlorophyll a, and total carotenoid content as well. The alga Fasciculochloris boldii showed high lipid content of 26.8 ± 1.1% and the alga Chlorella zachariaii showed 30.1% of omega-3 fractions in its total fatty acids. The Fourier transform infrared spectroscopy confirmed biodiesel feasibility of lipids from all these algae. However, the alga Fasciculochloris boldii with high proportions of saturated fatty acids in its lipid (71.2%) can be considered as a potential species for biodiesel as the biodiesel from the same also shows the required cetane number (63.3) and low iodine value (38.9). [ABSTRACT FROM AUTHOR]

Switchable solvents (SS), also termed smart solvents, have the unique ability to change their polarity/hydrophilicity through a protonation reaction using CO 2. Recently, a novel method employing the use of SS for lipid extraction in microalgae has shown promising results. In this study the efficiency of the tertiary amine N, N-dimethyl cyclohexylamine (DMCHA) to extract lipids from the eukaryotic microalgae Chlorella vulgaris and Haematococcus pluvialis , was investigated using traditional hexane as a control under wet conditions. The results revealed that high lipid yields using DMCHA can be achieved in both species, however the extraction procedure is significantly more complex compared with hexane. This study found that lipid extraction of H. pluvialis , using DMCHA to be an efficient solvent, achieving a maximum lipid yield of 64.84%. Furthermore, DMCHA achieved a maximum lipid yield of 63.85% with C. vulgaris. The practicalities of DMCHA for lipid recovery are considerably more complex at larger volumes compared with hexane. • DMCHA is biocompatible with Haematococcus pluvialis for lipid extraction. • Switchable solvents form globules with neutral lipids through Van-der Waal forces. • Microalgae cell wall rigidity reduces lipid extraction efficiency. • Switchable hydrophobic solvents are a more environmentally friendly alternative. • H. pluvialis and C. vulgaris maximum lipid recovery using DMCHA of 64.84% and 63.85%. [ABSTRACT FROM AUTHOR]

[Display omitted] • Studied effect of bacterial quorum sensing precursors (QSP) on algal lipidomic. • QSP promoted biomass (1.96–4.2X) and lipid productivity (2–4.5X) of Chlorella sp. • N-(3-Oxododecanoyl)-L-homoserine lactone increased biomass and neutral lipids. • N-(3-Hydroxyoctanoyl)-DL-homoserine lactone increased biomass and phospholipids. • QSP mediated oxidative stress coping mechanism and lipid accumulation of alga. Quorum sensing precursors of bacteria interact with microalgae in cultures and indirectly affect the algal physiology but their profound effect on the lipid and biomass in relation to improving the biofuel competence is one of the potential unexplored areas. The knowledge on the effect of phycospheric bacterial quorum sensing (QS) precursors on the lipid metabolism of microalgae under axenic and xenic conditions is highly restricted. This study endeavored to investigate an independent effect of QS precursors on the selected potential alga Chlorella sp. and unraveled the hidden prospect of QS for improvement of the lipids and identified the differences between the xenic and axenic conditions in the presence and absence of the QS precursors. Results revealed that N-(3-Oxododecanoyl)-L-homoserine lactone and N-(3-Hydroxyoctanoyl)-DL-homoserine lactone changed lipid composition, increased lipid productivity (2–4.5- folds) and biomass productivity (1.96–4.2- folds) under both xenic and axenic conditions of Chlorella sp. Further, selected QS precursors ameliorated fatty acid composition of the biomass that affected the resultant biodiesel properties at the end. N-(3-Hydroxyoctanoyl)-DL-homoserine promoted synthesis of PUFA and phospholipids with higher C18:2n6 compare to control. In contrast, N-(3-Oxododecanoyl)-L-homoserine lactone promoted SFA and neutral lipids with higher C18:00. Upon investigation of the stress physiology of Chlorella sp., it was revealed that the above QS precursors mediate the antioxidant levels and accumulate lipids, while also encourage biomass amelioration. [ABSTRACT FROM AUTHOR]

[Display omitted] • Sodium oleate (NaOL) enhances both biomass and lipid productivity in algae. • Optimum NaOL augmented with light irradiance governed the metabolite accumulation. • Total carotenoid and lipid content were significantly increased. • NaOL ameliorated the antioxidant potential of microalgae during the stationary phase. • NaOL orchestrated the metabolite overproduction in a dose-dependent manner. Microalgal metabolites have emerged as potential commodity components due to their notable beneficial nutritional and economic properties, and thus, their overproduction is of paramount importance to combat the existing ecological and dietary issues. However, the production titer of these metabolites by the algal systems at optimal cultivation conditions is inadequate to meet the burgeoning global demand for those metabolites. Therefore, a pressing demand remains to concurrently co-produce valuable metabolites without compromising cellular biomass production. Here, we report an effective strategy that simultaneously overproduces lipids, biomass and carotenoids in Chlorella vulgaris by providing sodium oleate (NaOL) in the cultivation medium. Supplement of sodium oleate at the optimal concentration (50 µg/mL) at increased light intensity enhances cellular growth rate in a dose-dependent manner. Total lipid content was quantified, which showed that lipid content was significantly increased in the NaOL-treated cells by 2.08-fold than that of the control cells. Interestingly, spectrophotometric determination of total carotenoids showed that carotenoid content was increased by 2.48-fold in the T5 (50 µg/mL) NaOL treated cells compared to the control. Interestingly antioxidant analysis showed that NaOL provision alleviated the antioxidant potential of the algal cells. Together, this report exemplifies the potential strategy to preclude the existing intricacies of co-producing metabolites in C. vulgaris by supplementing NaOL, thereby highlighting the viable chemical engineering approach for the biological production of commercially essential metabolites in microalgae. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Biofuel production by supercritical ethyl acetate was studied in fixed-bed reactor. • Synthesized biofuel properties nearly met the biodiesel standards. • The excess of triacetin content greatly increased density of biofuel. • Thermal stress strongly affected on cold flow properties of biofuel. • The utilization of alumina balls contributed to cleanliness of biofuel. Abstract This study aimed to investigate the effects of temperature, ethyl acetate to palm oil molar ratio, residence time, and packing alumina balls on triglyceride conversion, ethyl ester, and acetin contents. The complete triglyceride conversion was observed at 1.35 ± 0.05 times of the transition temperature. The packed alumina enhanced the ester content up to 20%; but it had no effect on the acetin content. Furthermore, this study also intended to determine the effects of the acetin content and thermal cracking on 19 fuel properties of lipid-based biofuel samples synthesized by supercritical ethyl acetate. The optimal condition was found at 380 °C, 20 MPa and ethyl acetate to oil molar ratio of 30:1. First, the density and kinematic viscosity of the biofuels were slightly higher than the maximum values specified by standards because of the presence of triacetin and polymerized products. Second, packing alumina balls increase the mixing intensity and thermal conductivity inside the tubular reactor resulted in higher contents of sulfated ash and carbon residue. Third, the acid values of the biofuel samples were considerably higher than the standard value of biodiesel. However, copper strip corrosion testing resulted in slight tarnishing (level 1A). The corrosiveness of fuels mainly originates from their sulfur compound content, neither biofuel sample was classified as a corrosive material. Furthermore, the high acid values of the samples interfered with the testing of oxidation stability. Finally, beneficial effects of triacetin on cold flow properties were revealed in this study. The cloud point and cold filter plugging point of lipid-based biofuels strongly depended on the thermal stress of the reaction conditions rather than the triacetin concentration. [ABSTRACT FROM AUTHOR]

Highlights • An efficient way of genome editing using CRISPR-Cas9 in microalgae. • Knockout of a key effector gene in Lands cycle have increased in TAG accumulation. • Increase in overall lipid productivity of the green alga, Chlamydomonas reinhardtii. • Upstream process for reducing the production cost of the microalgal biofuel. Abstract Biofuel derived from microalgae have several advantages over other oleaginous crops, however, still needs to be improved with its cost aspect and can be achieved by developing of a strain with improved lipid productivity. In this study, the CRISPR-Cas9 system was incorporated to carry out a target-specific knockout of the phospholipase A 2 gene in Chlamydomonas reinhardtii. The targeted gene encodes a key enzyme in the Lands cycle. As a result, the mutants showed a characteristic of increased diacylglycerol pool, followed by a higher accumulation of triacylglycerol without being significantly compensated with the cell growth. As a result, the overall lipid productivities of phospholipase A 2 knockout mutants have increased by up to 64.25% (to 80.92 g L−1 d−1). This study can provide crucial information for the biodiesel industry. [ABSTRACT FROM AUTHOR]

Commercial production of biofuel from oleaginous microalgae is often impeded by their slow growth rate than other fast-growing algal species. A promising strategy is to genetically engineer the fast-growing algae to accumulate lipids by expressing key lipogenic genes from oleaginous microalgae. However, lacking of strong expression cassette to transform most of the algal species and potential metabolic target to engineer lipid metabolism has hindered its biotechnological applications. In this study, we engineered the oxidative pentose phosphate pathway (PPP) of green microalga Chlorella pyrenoidosa for lipid enhancement by expressing a glucose-6-phosphate dehydrogenase (G6PD) from oleaginous diatom Phaeodactylum tricornutum. Molecular characterization of transformed lines revealed that heterologous PtG6PD was transcribed and expressed successfully. Interestingly, subcellular localization analyses revealed that PtG6PD was targeted to chloroplasts of C. pyrenoidosa. PtG6PD expression remarkably elevated NADPH content and consequently enhanced the lipid content without affecting growth rate. Collectively, this report represents a promising candidate to engineer lipid biosynthesis in heterologous hosts with notable commercial significance, and it highlights the potential role of plastidial PPP in supplying lipogenic NADPH in microalgae. [ABSTRACT FROM AUTHOR]

As a potential feedstock for biofuel production, a high-cell-density continuous culture for the lipid production by Cryptococcus albidus was investigated in this study. The influences of dilution rates in the single-stage continuous cultures were explored first. To reach a high-cell-density culture, a single-stage continuous culture coupled with a membrane cell recycling system was carried out at a constant dilution rate of 0.36/h with varied bleeding ratios. The maximum lipid productivity of 0.69 g/L/h was achieved with the highest bleeding ratio of 0.4. To reach a better lipid yield and content, a two-stage continuous cultivation was performed by adjusting the C/N ratio in two different stages. Finally, a lipid yield of 0.32 g/g and lipid content of 56.4% were obtained. This two-stage continuous cultivation, which provided a higher lipid production performance, shows a great potential for an industrial-scale biotechnological production of microbial lipids and biofuel production. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Microalgae incubation after PEF-treatment improves water-soluble components release. • Microalgae incubation after PEF-treatment improves lipid extraction yields. • With 20 h of incubation, energy of PEF-treatment can be reduced to 0.25 MJ/kg DW. • Incubation is also efficient at 4 °C but less than at 25 °C. Abstract Pulsed Electric Field (PEF) pre-treatment, applied on fresh microalgae Auxenochlorella protothecoides, induces spontaneous release of a substantial water fraction and enables subsequent lipid extraction using ethanol-hexane blends. In this study, fresh microalgae suspensions were treated with PEF and incubated under inert conditions. Incubation promotes the release of ions and carbohydrates and increases the yields of subsequent lipid extraction thus enabling a considerable reduction of PEF-treatment energy. With a 20 h incubation period at 25 °C, almost total lipid extraction is achieved with a specific PEF-treatment energy of only 0.25 MJ/kg DW. Incubation on ice remains beneficial but less efficient than at 25 °C. Additionally, incubating microalgae cells in suspension at 100 g DW /L or in a dense paste, was almost equally efficient. Correlation between the different results suggests that spontaneous release of ions and carbohydrates facilitates more successful lipid extraction. A direct causality between the two phenomena remains to be demonstrated. [ABSTRACT FROM AUTHOR]

We report the cultivation of Chlorella sorokiniana str. SLA-04 in media containing trace amounts of Ca and Mg. The differences in productivities of biomass, lipids and carbohydrates were assessed relative to cultures grown in standard media (BG-11) that contain approximately 8 × higher concentration of Ca and 30 × higher concentration of Mg. Culture performance in N-limited standard media was also investigated. In addition to growth and accumulation of storage products (lipid and carbohydrate), we measured the utilization of N, Ca and Mg and monitored changes in cell size and photosynthetic activity. Our results showed that limitation of Ca or Mg did not inhibit cell replication and culture growth. On the contrary, Ca-limited (Ca L ) cultures had ∼30% higher biomass productivity relative to the control with excessive nutrients possibly due to improvement in cell wall flexibility and cell division. We also observed that Ca L and Mg-limited (Mg L ) cultures had nearly 3-fold higher lipid concentration (measured as fatty acid methyl ester) and 50% higher carbohydrate concentration than the nutrient excess control cultures. Simultaneous culture growth and lipid accumulation in Ca L and Mg L cultures suggest that de novo synthesis was the primary mechanism for lipid accumulation in Ca/Mg-limited media. Overall, our study demonstrates that micro nutrient optimization, in addition to optimization of macronutrients, could significantly improve microalgal biorefinery yields. [ABSTRACT FROM AUTHOR]

This section presents a review of the scientific literature published in 2017 on topics relating to sustainable bioenergy from biofuel residues and waste. This review is divided into the following sections: Feedstocks, Bioethanol, Biodiesel, Biohydrogen, Hydrogen, Biofuel Residues, Microalgae, Lignocelluloses and other topics. [ABSTRACT FROM AUTHOR]

Acetate and ammonium were used as organic carbon and nitrogen sources, respectively, during mixotrophic cultivation of Chlorella pyrenoidosa . Cell growth, content of neutral lipid (NL), productivity of biomass and total lipid, and fatty acid profiles were investigated. Results showed that C. pyrenoidosa could endure high concentrations of NH 4 + -N (100–200 mg/L) and immediately entered logarithmic growth, when the culture media contained 2.0–10.0 g/L NaAc. The 2.0–10.0 g/L NaAc in the media also resulted in the NL content of 1.87–3.05 mg/10 9 cells, much higher than 0.5 mg/10 9 cells of the controls. The maximum productivities of biomass and total lipid were achieved under 50 and 10 mg/L NH 4 + -N respectively when the 2.0 g/L NaAc was dosed. The fatty acids were mainly composed of C16:0, C16:1, C18:0, and C18:1 under the mixotrophic cultivation, with the higher saturation compared to the controls. [ABSTRACT FROM AUTHOR]

Since the current patterns of energy consumption are mostly dependent on fossil fuel, multiple issues related to energy resources, economic stability, and environmental pollution have arisen. Biodiesel is one of the potential alternative energy resources that can achieve sustainability. However, commercializing biodiesel is restricted with the high production costs that may not compete with the conventional diesel fuel since 70 – 95% of the costs are accounted for the raw materials. Azolla macroalgae is a potential source for fatty acids that can be a feedstock for commercial biodiesel. The current investigation took Azolla filiculoides as a feedstock to synthesize biodiesel. Soxhlet apparatus was the method of choice to extract the lipids which were subjected to the optimized transesterification reactions using response surface methodology (RSM). Four independent variables, which are oil to methanol molar ratio, catalyst loading, reaction temperature, and reaction time, were analyzed to optimize the highest response of biodiesel yield. 29 runs were suggested by the model. Actual experiments were conducted to validate the model. The obtained results were close to the predicted values with a reasonable error 0.029%. The best optimum value was 81.90% that was obtained at oil to methanol molar ratio 1:6, catalyst loading 1.75 wt%, reaction temperature 60 ◦C, and reaction time 120 min. The characteristics of the yielded fuel met the biodiesel standards of MS2008:2008, ASTM D6751, and EN 14214. Thus, Azolla filiculoides macroalgae are one of the promising feedstocks to produce commercial and sustainable biodiesel fuel. • Macroalgae Azolla filiculoides was utilized as a feedstock for biodiesel production. • 19.02% of lipids was extracted by Soxhlet. • The highest obtained biodiesel yield was 81.90%. • The physicochemical properties revealed compatibility with the biodiesel standards. [ABSTRACT FROM AUTHOR]

SUMMARY: Microalgae are considered a very promising alternative for biofuel production. Several strategies were developed to modulate and improve algae metabolites production to meet the requirements for biodiesel production. Most previous research evidenced that the increase of the lipid content is accompanied by a decrease of the biomass production, which increases the cost of the downstream processing. Hence, the challenge is to find special culture conditions that increase the lipid and the biomass productivities simultaneously. In the present work, we developed a strategy for the improvement of biomass and lipid productivities in a novel local microalga isolate, Chlorocystis sp. QUCCCM14, which was not previously known as a promising strain. Indeed, culturing QUCCCM14 using f/2 medium with 10× NaH2PO4 (0.15 g L−1 NaNO3 and 5.6 mg L−1 NaH2PO4) resulted in an improvement of 3.178 folds the lipid productivity reaching 56.121 mg L−1 day−1 and enhanced the biomass productivity reaching 141.363 mg L−1 day−1, simultaneously. Comparative analyses of the FAME profiles demonstrated that fed‐batch culture with phosphate or nitrate separately leads to a high production of the omega 3 fatty acids (Linolenic acid), whereas fed‐batch culture with phosphate and nitrate simultaneously increased the production of fatty acids suitable for biodiesel production. [ABSTRACT FROM AUTHOR]

In the present scenario of depleting oil reservoir, microbial oil has gained much attention over plant and animal based sources. Among different microorganisms, yeast strains are considered superior source for oil production. The cost of oil produced by yeast could further be lowered using cheaper agro-waste and biomass as substrate. This review focuses on key topics which will help in gaining better understanding to enhance lipid production using yeast strains. The effects of oleaginous yeast co-culturing with microalgae, different cheap carbon sources of biomass, and types of yeast species on oil production were highlighted in the review. An overview of mechanisms of oil production from biomass, viz. pretreatment of biomass, fermentation and oil recovery are also provided. Constraints encountered during the oleogenesis or microbial oil accumulation and their probable solutions along with a section on different by-products obtained during oleo-genesis are also discussed. [ABSTRACT FROM AUTHOR]

Background: In the wake of rising energy demands, microalgae have emerged as potential sources of sustainable and renewable carbon-neutral fuels, such as bio-hydrogen and bio-oil. Purpose: For rational metabolic engineering, the elucidation of metabolic pathways in fine detail and their manipulation according to requirements is the key to exploiting the use of microalgae. Emergence of site-specific nucleases have revolutionized applied research leading to biotechnological gains. Genome engineering as well as modulation of the endogenous genome with high precision using CRISPR systems is being gradually employed in microalgal research. Further, to optimize and produce better algal platforms, use of systems biology network analysis and integration of omics data is required. This review discusses two important approaches: systems biology and gene editing strategies used on microalgal systems with a focus on biofuel production and sustainable solutions. It also emphasizes that the integration of such systems would contribute and compliment applied research on microalgae. Conclusions: Recent advances in microalgae are discussed, including systems biology, gene editing approaches in lipid bio-synthesis, and antenna engineering. Lastly, it has been attempted here to showcase how CRISPR/Cas systems are a better editing tool than existing techniques that can be utilized for gene modulation and engineering during biofuel production. [ABSTRACT FROM AUTHOR]

Abstract: As promising alternatives to fossil‐derived oils, microbial lipids are important as industrial feedstocks for biofuels and oleochemicals. Our broad aim is to increase lipid content in oleaginous yeast through expression of lipid accumulation genes and use Saccharomyces cerevisiae to functionally assess genes obtained from oil‐producing plants and microalgae. Lipid accumulation genes DGAT (diacylglycerol acyltransferase), PDAT (phospholipid: diacylglycerol acyltransferase), and ROD1 (phosphatidylcholine: diacylglycerol choline‐phosphotransferase) were separately expressed in yeast and lipid production measured by fluorescence, solvent extraction, thin layer chromatography, and gas chromatography (GC) of fatty acid methyl esters. Expression of DGAT1 from Arabidopsis thaliana effectively increased total fatty acids by 1.81‐fold above control, and ROD1 led to increased unsaturated fatty acid content of yeast lipid. The functional assessment approach enabled the fast selection of candidate genes for metabolic engineering of yeast for production of lipid feedstocks. [ABSTRACT FROM AUTHOR]

For the disintegration and extraction of microalgae to produce lipids and biofuels, a novel processing technology was investigated. The utilization of a hydrothermal treatment was tested on four different microalgae species (Scenedesmus rubescens, Chlorella vulgaris, Nannochloropsis oculata and Arthorspira platensis (Spirulina)) to determine whether it has an advantage in comparison to other disintegration methods for lipid extraction. It was shown, that hydrothermal treatment is a reasonable opportunity to utilize microalgae without drying and increase the lipid yield of an algae extraction process. For three of the four microalgae species, the extraction yield with a prior hydrothermal treatment elevated the lipid yield up to six times in comparison to direct extraction. Only Scenedesmus rubescens showed a different behaviour. Reason can be found in the different cell wall of the species. The investigation of the differences in cell wall composition of the used species indicate that the existence of algaenan as a cell wall compound plays a major role in stability. [ABSTRACT FROM AUTHOR]

Heterotrophic microalgae, capable of converting organic carbons to biofuel, as well as assimilating nutrients, have a great prospective in wastewater treatment. Meanwhile, the knowledge about heterotrophic microalgae is still far less than the autotrophic conterpart. Hence, in this study, 20 heterotrophic microalgal strains were isolated from a domestic wastewater treatment plant, and identified according to morphology and partial 18S and 23S rRNA gene sequences. Further, their biological traits were assessed in terms of N, P, TOC removal efficiencies, growth parameters, self-settleability and lipids production, expressed through a comprehensive selection index. By such, the optimal strains were chosen and applied back to treat the real wastewater, with or without pretreatment of sterilization. An organic-adaptable strain, i.e., Botryococcus sp. NJD-1, was ultimately recommended to achieve the concurrent biofuel production (up to 61.7% lipid content) and pollutants removal (up to 64.5%, 89.8% and 67.9% for N, P and TOC) in pristine wastewater. [ABSTRACT FROM AUTHOR]

There is a growing interest worldwide for the production of renewable oil without mobilizing agriculture lands; fast and reliable methods are needed to identify highly oleaginous microorganisms of potential industrial interest. The aim of this study was to demonstrate the relevance of attenuated total reflection (ATR) spectroscopy to achieve this goal. To do so, the total lipid content of lyophilized samples of five Streptomyces strains with varying lipid content was assessed with two classical quantitative but time-consuming methods, gas chromatography-mass spectrometry (GCMS) and ATR Fourier transform infrared (ATR FT-IR) spectroscopy in transmission mode with KBr pellets and the fast ATR method, often questioned for its lack of reliability. A linear correlation between these three methods was demonstrated allowing the establishment of equations to convert ATR values expressed as CO/amide I ratio, into micrograms of lipid per milligram of biomass. The ATR method proved to be as reliable and quantitative as the classical GC-MS and FT-IR in transmission mode methods but faster and more reproducible than the latter since it involves far less manipulation for sample preparation than the two others. Attenuated total reflection could be regarded as an efficient fast screening method to identify natural or genetically modified oleaginous microorganisms by the scientific community working in the field of bio-lipids. [ABSTRACT FROM AUTHOR]