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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]

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]

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]

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]

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]

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]

[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]

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]

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]

Use of oleaginous microorganisms as 'micro-factories' for accumulation of single cell oils for biofuel production has increased significantly to mitigate growing energy demands, resulting in efforts to upgrade industrial waste, such as second-generation lignocellulosic residues, into potential feedstocks. Dilute-acid pretreatment (DAP) is commonly used to alter the physicochemical properties of lignocellulosic materials and is typically coupled with simultaneous saccharification and fermentation (SSF) for conversion of sugars into ethanol. The resulting DAP residues are usually processed as a waste stream, e.g. burned for power, but this provides minimal value. Alternatively, these wastes can be utilized as feedstock to generate lipids, which can be converted to biofuel. DAP-SSF residues were generated from pine, poplar, and switchgrass. High performance liquid chromatography revealed less than 0.13% monomeric sugars in the dry residue. Fourier transform infrared spectroscopy was indicative of the presence of lignin and polysaccharides. Gel permeation chromatography suggested the bacterial strains preferred molecules with molecular weight ~ 400-500 g/mol. DAP-SSF residues were used as the sole carbon source for lipid production by Rhodococcus opacus DSM 1069 and PD630 in batch fermentations. Depending on the strain of Rhodococcus employed, 9-11 lipids for PD630 and DSM 1069 were observed, at a final concentration of ~ 15 mg/L fatty acid methyl esters (FAME) detected. Though the DAP-SSF substrate resulted in low FAME titers, novel analysis of solid-state fermentations was investigated, which determined that DAP-SSF residues could be a viable feedstock for lipid generation. [ABSTRACT FROM AUTHOR]

Diatoms are responsible for up to 40% of the carbon fixation in our oceans. The fixed carbon is moved through carbon metabolism towards the synthesis of organic molecules that are consumed through interlocking foodwebs, and this process is strongly impacted by the abiotic environment. However, it has become evident that diatoms can be used as 'platform' organisms for the production of high valuable bio-products such as lipids, pigments and carbohydrates where stress conditions can be used to direct carbon metabolism towards the commercial production of these compounds. In the first section of this review, some aspects of carbon metabolism in diatoms and how it is impacted by environmental factors are briefly described. The second section is focused on the biosynthesis of lipids and in particular omega-3 long-chain polyunsaturated fatty acids and how low temperature stress impacts on the production of these compounds. In a third section, we review the recent advances in bioengineering for lipid production. Finally, we discuss new perspectives for designing strains for the sustainable production of high-value lipids. [ABSTRACT FROM AUTHOR]

Almost all oleaginous microorganisms are available for biodiesel production, and for the mechanism of oil accumulation, which is what makes a microbial approach economically competitive. This study investigated the potential that the yeast Candida lipolytica UCP0988, in an anamorphous state, has to produce simultaneously a bioemulsifier and to accumulate lipids using inexpensive and alternative substrates. Cultivation was carried out using waste soybean oil and corn steep liquor in accordance with 22 experimental designs with 1% inoculums (107 cells/mL). The bioemulsifier was produced in the cell-free metabolic liquid in the late exponential phase (96 h), at Assay 4 (corn steep liquor 5% and waste soybean oil 8%), with 6.704 UEA, IE24 of 96.66%, and showed an anionic profile. The emulsion formed consisted of compact small and stable droplets (size 0.2-5 μm), stable at all temperatures, at pH 2 and 4, and 2% salinity, and showed an ability to remove 93.74% of diesel oil from sand. The displacement oil (ODA) showed 45.34 cm2 of dispersion (central point of the factorial design). The biomass obtained from Assay 4 was able to accumulate lipids of 0.425 g/g biomass (corresponding to 42.5%), which consisted of Palmitic acid (28.4%), Stearic acid (7.7%), Oleic acid (42.8%), Linoleic acid (19.0%), and γ-Linolenic acid (2.1%). The results showed the ability of C. lipopytica to produce both bioemulsifier and biodiesel using the metabolic conversion of waste soybean oil and corn steep liquor, which are economic renewable sources. [ABSTRACT FROM AUTHOR]

Co-hydroprocessing of petroleum fractions with residual lipids is a promising technology for hybrid fuels production. The aim of this study is the identification of an effective operating window for co-hydroprocessing of Heavy Atmospheric Gas Oil (HAGO) with Waste Cooking Oil (WCO) within the nominal operating limits of a refinery. In particular, two Liquid Hourly Space Velocities or LHSVs (0.66 and 1 h −1 ) and three hydrogen-to-oil or H 2 /Oil ratios (300, 400 and 500 L H 2 /L feed) were studied for a HAGO/WCO 90/10 V/V blended feedstock over a commercial hydrotreating NiMo/Al 2 O 3 catalyst. The two parameters were evaluated with respect to the attained diesel content and quality of the produced hybrid fuels. All experiments were performed at a constant pressure of 5.6 MPa and temperature of 350 °C. The results have shown that higher LHSV is preferable in terms of the hydrogen consumption affecting the conversion cost. As far as H 2 /Oil is concerned, high H 2 /Oil ratios are more effective, rendering higher conversion, diesel content and heteroatom removal. [ABSTRACT FROM AUTHOR]

Biological lipids derived from oleaginous microorganisms are promising precursors for renewable biofuel productions. Direct lipid extraction from wet cell-biomass is favored because it eliminates the need for costly dehydration. However, the development of a practical and scalable process for extracting lipids from wet cell-biomass is far from ready to be commercialized, instead, requiring intensive research and development to understand the lipid accessibility, mechanisms in mass transfer and establish robust lipid extraction approaches that are practical for industrial applications. This paper aims to present a critical review on lipid recovery in the context of biofuel productions with special attention to cell disruption and lipid mass transfer to support extraction from wet biomass. [ABSTRACT FROM AUTHOR]

Botryococcus terribilis is a peculiar species that directs a higher portion of the consumed CO 2 to metabolite biosynthesis. It results in a low growth rate associated with high oil productivity - compared to other species. In addition to its oils (mainly composed of lipids and hydrocarbons), this microalgae is also a promising source of proteins, carbohydrates, and pigments. The biosynthesis of these products depends on the cultivation conditions. Moreover, the extraction method applied has a direct influence on the quality of the products and their possible applications. Botryococcus terribilis metabolites can be used as fertilizers, biomaterials, pharmaceuticals, cosmetics, food supply, and in a range of biofuels (biodiesel, bioethanol, and biogas). However, biorefinery with this microalgae still needs to overcome economic and technical hurdles to reach a competitive market value. This work analyzed the evolution of the studies with the Botryococcus terribilis , approaching it main metabolites and relating it to the cultivation conditions, extraction methods, and the final products that can be obtained. For the first time, the studies with Botryococcus terribilis were summarized, creating a guide for further research on this microalgae by presenting the recommended conditions for its cultivation and extraction, its possible applications, the information missing in the literature, and the prospects for scale-up. • Approach the microalgae from metabolism to prospects for large-scale cultivation. • Summarize the conditions and results obtained with Botryococcus terribilis. • Botryococcus hydrocarbons are promising source of biofuels and biomaterials. • Botryococcus extracellular metabolites can be continuously extracted. • Recommendations and opportunities for further research with Botryococcus terribilis. [ABSTRACT FROM AUTHOR]

In this study, five Chlorella species ( Chlorella vulgaris, Chlorella minutissima, Chlorella pyrenoidosa, Chlorella sp.1 and Chlorella sp.2) were grown in various nutrient medium including BG-11, BBM, Fog's medium and MN medium for the evolution of biomass and lipid production potential. Among the tested medium, BG-11 was found most economical and efficient medium for all Chlorella species. To see the impact of organic carbon sources on lipid production potential, all microalgae species were also cultured in selected medium (BG-11) with different organic carbon sources like glucose, glycerol, sodium acetate, and sucrose under mixotrophic condition. The results showed that all Chlorella species performs better under mixotrophic condition, but Chlorella vulgaris achieved maximum lipid productivity (3.5 folds higher) in glycerol supplemented culture medium than control medium among all species. [ABSTRACT FROM AUTHOR]

Currently microalgae are considered as an efficient biological producer of oil. Present study was conducted to explore the lipid yield percentage of three microalgal strains Chlorella sp., Oedogonium sp. and Spirogyra sp. Bligh and Dyer, Soxhlet, Folch and Microwave assisted extraction methods were employed for oil extraction. It was observed that Bligh and Dyer method with Chloroform-methanol (1:2, v/v) gave maximum extraction yield (15.46 ± 0.240%) for Chlorella sp. whereas Folch method with chloroform-methanol (2:1, v/v) gave more lipid yield as compared to Bligh and Dyer method i.e 5.53 ± 0.240% for Spirogyra sp., 13.67 ± 0.240% and 19.69 ± 0.667% for Oedogonium sp. and Chlorella sp. respectively. Soxhlet extraction resulted in less (14.5± 0.45) percentage yield as compared to Bligh and Dyer and Folch method (16.7± 0.24). Microwave assisted extraction with chloroform- methanol resulted in highest lipid yield (30%) with Chlorella sp. indicating that it contained the maximum lipid content among all the three species. [ABSTRACT FROM AUTHOR]

Sustainable mass production of algal biofuels requires a reduction in nutrient demand and efficient conversion into fuels of all biomass including lipid-extracted algal residues (LEA). This study evaluated methane production, nutrient recovery and recycling from untreated and enzymatically pretreated Nannochloropsis LEA using semi-continuous anaerobic digestion (AD). Additionally, this process was compared to methane generation from whole Nannochloropsis alga (WA) and thermally pretreated WA. The methane production from untreated LEA and WA reached up to 0.22 L and 0.24 L per gram of biomass volatile solids (VS), respectively, corresponding to only 36–38% of the theoretical potential. Additionally, observed VS reduction was only 40–50% confirming biomass recalcitrance to biodegradation. While enzymatic treatment hydrolyzed up to 65% of the LEA polysaccharides, the methane production increased by only 15%. Alternatively, WA thermal pretreatment at 150–170 °C enhanced methane production up to 40%. Overall, an integrated process of lipid conversion into biodiesel coupled with LEA conversion into methane generates nearly 40% more energy compared to methane production from WA, and about 100% more energy than from biodiesel alone. Additionally, the AD effluent contained up to 60–70% of the LEA phosphorus content, 30–50% of the nitrogen, sulfur, calcium and boron, 20% of the iron and cobalt, and 10% of manganese, zinc and copper, which can partially replace chemical fertilizers during algal cultivation. Consequently, supplementation of Nannochloropsis cultures with 5% AD effluent was optimal for a high algal growth rate. Therefore, coupling biodiesel and methane production provides significant energy advantages along with sustainability and economic benefits from nutrient recycling. [ABSTRACT FROM AUTHOR]

Abstract: Because some algae can be grown photoautotrophically, heterotrophically, and/or mixotrophically, they show promise for lipid production as biofuel feedstock with different conditions. In this study, we analyzed microalga Chlorella sorokiniana for biomass and lipid production under three culture modes. The best growth performance was obtained under mixotrophic conditions compared with photoautotrophic and heterotrophic conditions. With the addition of 4 g L−1 glucose, the specific growth rate (3.40 d−1) and maximum biomass dry weight (DW, 3.55 g L−1) in the mixotrophic culture were 1.8- and 2.4-fold of those in the heterotrophic culture, and 5.4- and 5.2-fold of those under the photoautotrophic culture. Most significantly, the biomass yield based on consumed glucose reached values of up to 0.82 g g−1, which was only 0.34 g g−1 in the corresponding heterotrophic condition. Moreover, the lipid content in mixotrophic algae also substantially increased. At a light intensity of 50 μmol m−2 s−1, a lipid content of 45% was achieved compared to only 13% in the heterotrophic culture. The PSII inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) had little effect on the maximum DW and lipid content in the heterotrophic culture, but caused a significant decline in the mixotrophic culture. Mixotrophic cultures supplied with air grew as well as cultures supplied with 1% CO2. Mixotrophy also changed the temperature profiles for growth, increasing the optimum from 25 °C to 37 °C. These results indicate that C. sorokiniana may be an ideal candidate for mixotrophic cultivation that offers great potential in the production of renewable biomass for bioenergy applications. [Copyright &y& Elsevier]

Background Microalgae are a promising platform for producing neutral lipids, to be used in the application for biofuels or commodities in the feed and food industry. A very promising candidate is the oleaginous green microalga Scenedesmus obliquus, because it accumulates up to 45% w/w triacylglycerol (TAG) under nitrogen starvation. Under these conditions, starch is accumulated as well. Starch can amount up to 38% w/w under nitrogen starvation, which is a substantial part of the total carbon captured. When aiming for optimized TAG production, blocking the formation of starch could potentially increase carbon allocation towards TAG. In an attempt to increase TAG content, productivity and yield, starchless mutants of this high potential strain were generated using UV mutagenesis. Previous studies in Chlamydomonas reinhardtii have shown that blocking the starch synthesis yields higher TAG contents, although these TAG contents do not surpass those of oleaginous microalgae yet. So far no starchless mutants in oleaginous green microalgae have been isolated that result in higher TAG productivities. Results Five starchless mutants have been isolated successfully from over 3,500 mutants. The effect of the mutation on biomass and total fatty acid (TFA) and TAG productivity under nitrogenreplete and nitrogen-depleted conditions was studied. All five starchless mutants showed a decreased or completely absent starch content. In parallel, an increased TAG accumulation rate was observed for the starchless mutants and no substantial decrease in biomass productivity was perceived. The most promising mutant showed an increase in TFA productivity of 41% at 4 days after nitrogen depletion, reached a TAG content of 49.4% (% of dry weight) and had no substantial change in biomass productivity compared to the wild type. Conclusions The improved S. obliquus TAG production strains are the first starchless mutants in an oleaginous green microalga that show enhanced TAG content under photoautotrophic conditions. These results can pave the way towards a more feasible microalgae-driven TAG production platform. [ABSTRACT FROM AUTHOR]

The paper describes the genome sequence, gene annotation, and transcriptional profiling of the microalga Nannochloropsis gaditana B-31. The study identifies possible targets of genetic modification that could improve the yield of the strain for biodiesel production. The genome resource is available online at www.nannochloropsis.org with multiple tools for information retrieval.Nannochloropsis is rapidly emerging as a model organism for the study of biofuel production in microalgae. Here, we report a high-quality genomic assembly of Nannochloropsis gaditana, consisting of large contigs, up to 500 kbp long, and scaffolds that in most cases span the entire length of the chromosomes. We identified 10646 complete genes and characterized possible alternative transcripts. The annotation of the predicted genes and the analysis of cellular processes revealed traits relevant for the genetic improvement of this organism such as genes involved in DNA recombination, RNA silencing, and cell wall synthesis. We also analyzed the modification of the transcriptional profile in nitrogen deficiency—a condition known to stimulate lipid accumulation. While the content of lipids increased, we did not detect major changes in expression of the genes involved in their biosynthesis. At the same time, we observed a very significant down-regulation of mitochondrial gene expression, suggesting that part of the Acetyl-CoA and NAD(P)H, normally oxidized through the mitochondrial respiration, would be made available for fatty acids synthesis, increasing the flux through the lipid biosynthetic pathway. Finally, we released an information resource of the genomic data of N. gaditana, available online at www.nannochloropsis.org. [ABSTRACT FROM PUBLISHER]

Highlights: [•] Haematococcus pluvialis was cultivated in domestic secondary effluent. [•] The maximal biomass productivity of 27.8mgL−1 d−1 was achieved. [•] Nitrogen and phosphorus in the secondary effluent both were removed efficiently. [•] The maximal lipid accumulation rate of 12.0mgL−1 d−1 was obtained. [•] Haematococcus pluvialis outperformed most microalgal species in literatures. [ABSTRACT FROM AUTHOR]

There is a growing interest for the mixotrophic cultivation of microalgae using sustainable natural carbon sources. Chitin from shrimp waste was hydrolyzed using chitinase from Trichoderma asperellum and was exploited for the mixotrophic cultivation of microalgae. Chitin saccharification was optimized using Box-Behnken design to produce 50.41% (w/w) of reducing sugars using chitin, 0.83% (w/v), chitinase, 2.92 (v/v), temperature, 40 °C and time, 4.48 h. The biomass productivity of Tetradesmus obliquus and Aphanocapsa sp. was promoted to 39.1 and 40.57 mg L−1 day−1 in the presence of 0.5 g L−1 of N -acetylglucosamine oligomers (GlcNAc), which was estimated to be ∼1.9 and ∼2-folds higher than the autotrophic conditions, respectively. Modified Logistic kinetic model confirmed higher growth rates and shortened lag-time of the mixotrophic cultures. The lipid productivity of both microalgae species was >1.5-times higher than the autotrophic conditions. Nitrate removal from the culture medium was found to be an effective strategy to increase both lipid content and lipid productivity for T. obliquus , but this strategy was not effective for Aphanocapsa sp. Fatty acid analysis revealed an increase of saturated fatty acids under mixotrophic cultivation for both microalgae species. Additionally, several biodiesel evaluation parameters indicated superior characteristics for the mixotrophic culture compared to the autotrophic conditions. Accordingly, mixotrophic growth of microalgae using GlcNAc as a new, sustainable carbon and nitrogen source opens a new avenue for biofuel production with an effective waste conversion into valuable commodities. • Enzymatic production of reducing sugars from shrimp chitin was optimized to yield 50.41% (w/w). • Algal lipid productivity was enhanced >1.5 fold using chitin hydrolysate. • Nitrate removal enhanced lipid production of Tetradesmus obliquus. • Biodiesel from algal lipids obtained by mixotrophy had improved characteristics. [ABSTRACT FROM AUTHOR]

Microalgae have been considered as a promising biomass for biofuel production, but freshwater resource consumption during the scaled-up cultivation are still a challenge. Obtaining robust marine strains capable of producing triacylglycerols and high value-added metabolites are critical for overcoming the limitations of water resources and economical feasibility. In this study, a marine microalga with lipid and astaxanthin accumulation capability was isolated from Bohai Bay, China. The alga was named as Coelastrum sp. HA-1 based on its morphological and molecular identification. The major characteristics of HA-1 and the effects of nitrogen on its lipid and astaxanthin accumulations were investigated. Results indicated that the highest biomass, lipid and astaxanthin yields achieved were 50.9 g m-2 day-1, 18.0 g m-2 day-1 and 168.9 mg m-2 day-1, respectively, after cultivation for 24 days. The fatty acids of HA-1, identified in their majority as oleic acid (56.6%) and palmitic acid (25.9%), are desirable biofuel feedstocks. In addition, this alga can be harvested with simple sedimentation, achieving 98.2% removal efficiency after settling for 24 h. These results suggest that Coelastrum sp. HA-1 has several desirable key features that make it a potential candidate for biofuel production. [ABSTRACT FROM AUTHOR]

Two different strains of microalgae, one raphidophyte and one dinoflagellate, were tested under different abiotic conditions with the goal of enhancing lipid production. Whereas aeration was crucial for biomass production, nitrogen deficiency and temperature were found to be the main abiotic parameters inducing the high-level cellular accumulation of neutral lipids. Net neutral lipid production and especially triacylglycerol (TAG) per cell were higher in microalgae (>200% in Alexandrium minutum, and 30% in Heterosigma akashiwo) under treatment conditions (25°C; 330 μM NaNO) than under control conditions (20°C; 880 μM NaNO). For both algal species, oil production (free fatty acids plus TAG fraction) was also higher under treatment conditions (57 mg L in A. minutum and 323 mg L in H. akashiwo). Despite the increased production and accumulation of lipids in microalgae, the different conditions did not significantly change the fatty acids profiles of the species analyzed. These profiles consisted of saturated fatty acids (SAFA) and polyunsaturated fatty acids (PUFA) in significant proportions. However, during the stationary phase, the concentrations per cell of some PUFAs, especially arachidonic acid (C20:4n6), were higher in treated than in control algae. These results suggest that the adjustment of abiotic parameters is a suitable and one of the cheapest alternatives to obtain sufficient quantities of microalgal biomass, with high oil content and minimal changes in the fatty acid profile of the strains under consideration. [ABSTRACT FROM AUTHOR]

A huge amount of a mixture of unreacted fatty acids and mono/di/triglycerides, namely bio-heavy oil, is generated as a waste from biodiesel industry. Considering that the lipid (i.e., triglycerides) accounts for 80% of the total biodiesel production cost, valorization of bio-heavy oil into biodiesel can be a viable route for enhancing the economic/environmental benefits. Nonetheless, the high contents of free fatty acids and impurities have restricted practical valorization of bio-heavy oil into biodiesel because conventional acid/base catalyzed transesterification suffers from the presence of the impurities. To overcome the technical challenge, this study suggested a direct valorization platform for the conversion of bio-heavy oil into biodiesel via thermally induced transesterification. Prior to biodiesel production, the properties of bio-heavy oil, such as acid value (105 mg KOH g−1 bio-heavy oil), its elemental compositions and thermal stability were characterized. Acid-catalyzed transesterification with a H 2 SO 4 catalyst showed the 31.1 wt% of biodiesel yield after 24 h of reaction at 60 °C. However, thermally induced transesterification exhibited 59.3 wt% of biodiesel yield after 1 min of reaction at 400 °C using a porous medium (SiO 2) with no presence of a catalyst. The porous SiO 2 (pore size: 6 nm) provided confined spaces for lipids and methanol, allowing the rapid transesterification reactions between them at high temperature. The biodiesel yield from thermally induced transesterification was proportionate to reaction temperature by 360 °C, but it decreased at > 400 °C due to the chemical bond scissions of unsaturated hydrocarbons. C 6-22 fatty acid methyl esters (FAMEs) were produced from thermally induced (non-catalytic) transesterifications, and weight fractions of each FAMEs were constant, regardless of the reaction conditions at ≤ 400 °C. All experimental findings offer a new recycle platform for BHO into biodiesel. [Display omitted] • Bio-heavy oil was converted into biodiesel via thermally induced reaction. • Thermally induced reaction doubled biodiesel yield (59%) than acid catalyzed one. • Thermally induced transesterification was tolerant to high contents of impurities. [ABSTRACT FROM AUTHOR]

Advancement of microalgae for use as biofuel feedstock depends on bioprospecting novel strains and then applying classical strain improvement techniques to enhance desirable traits. Here we successfully demonstrate this approach using two novel strains of green algae, identified as Desmodesmus armatus and Chlorella vulgaris, to improve biomass and lipid yields. UV irradiation was utilized to generate random mutations and the resulting populations were analyzed via flow cytometry to quantify lipid fluorescence while enriching for high lipid variants using fluorescence activated cell sorting. Secondary screening was performed via in situ fluorescence staining to identify the single highest lipid producing mutants from each strain. This approach produced a Desmodesmus armatus mutant that exhibited both increased biomass productivity and higher lipid content, which doubled lipid yield from the culture. The resulting Chlorella vulgaris mutant exhibited an increase in growth only, resulting in a 33% increase in lipid yield. • Generated novel UV-mutants of Desmodesmus armatus and Chlorella vulgaris. • Mutants displayed increased biomass and fatty acid productivities and yields. • Characterized growth and lipid composition of wild type and mutant strains. • Nile Red fluorescence did not consistently correlate with lipid content in mutants. [ABSTRACT FROM AUTHOR]

The application of microalgae biomass of Chlorella sorokiniana as environmentally friendly biosorbents for removing potentially toxic elements (PTE) from water and as a source of biofuel has been thoroughly studied. In this paper, we investigate its physicochemical properties infrared spectroscopy (IR spectra), microstructure, adsorption properties); we have managed to isolate the lipid complex, which amounted to 20% of dry biomass. Studies of the lipid complex showed that 80.02% of lipids are unsaturated fatty acids (C18:1, C18:2, C18:3). Additionally, we have investigated the efficiency of using the residual biomass obtained after lipid extraction for water purification from rare-earth metals (REM) and PTE. To increase the sorption properties of residual biomass, its thermal modification was carried out and sorption materials based on heat-treated residual biomass and chitosan were created. The physicochemical and mechanical properties of the obtained sorption materials were studied. The total sorption capacity was 31.9 mg/g for REM and 349.7 mg/g for PTE. Moreover, we propose a new method for the disposal of spent sorbents as additional fuel. Spent sorbents can be considered to be biofuel in terms of energy content (20.7 MJ*kg−1). The results of this study provide the basis for increased use of microalgae. [ABSTRACT FROM AUTHOR]

The procedures for the extraction and separation of lipids and nutraceutics from microalgae using classic solvents have been frequently used over the years. However, these production methods usually require expensive and toxic solvents. Based on our studies involving the use of eco-sustainable methodologies and alternative solvents, we selected ethanol (EtOH) and cyclopentyl methyl ether (CPME) for extracting bio-oil and lipids from algae. Different percentages of EtOH in CPME favor the production of an oil rich in saturated fatty acids (SFA), useful to biofuel production or rich in bioactive compounds. The proposed method for obtaining an extract rich in saturated or unsaturated fatty acids from dry algal biomass is disclosed as eco-friendly and allows a good extraction yield. The method is compared both in extracted oil percentage yield and in extracted fatty acids selectivity to extraction by supercritical carbon dioxide (SC-CO2). [ABSTRACT FROM AUTHOR]

A two-stage cultivation method involving the initial growth in optimized conditions for biomass production followed by those for lipid production in oleaginous brackish diatom Navicula phyllepta MACC8 resulted in a proportional increase of lipid concentration along with biomass production. The diatom was further subjected to stress conditions by altering the nutrient components such as nitrate, phosphate, silicate, and temperature. Silicon deprivation resulted in the highest lipid percentage of 28.78% of weight at the end of the 18th day of the second stage. A significant increase in lipid content was observed on the complete removal of the nutrients silicon and urea one at a time, while the biomass showed a considerable reduction. The application of multiple nutrient stress conditions had a profound influence on the increased rate of lipid production. A combination of phosphate deprivation, silicate limitation and temperature reduction resulted in a significant increase in lipid percentage of 32.13% at the cost of reduced biomass (1.1 g L−1), whereas phosphate deprivation, urea limitation, and temperature reduction resulted in lipid percentage of 27.58% with a biomass of 1.44 g L−1 at the end of the second stage. Further, the results were supported by Nile red staining, FTIR, fatty acid profile and oxidative stress marker analyses. The changes in biochemical composition and oxidative stress parameters within the various stress conditions demonstrated the profound influence of the selected stress factors on the biodiesel productivity of the diatom, besides its stress tolerance. A two-phase culturing system, with multifactor stress application, especially nitrogen limitation along with phosphate starvation and temperature stress, would be the suitable method for gaining maximum biomass productivity and lipid content in diatom Navicula phyllepta MACC8 towards biofuel production. [ABSTRACT FROM AUTHOR]

Background: The extraction of lipids from microalgae requires a pretreatment process to break the cell wall and subsequent extraction processes to obtain the lipids for biofuels production. The multistep operation tends to incur high costs and are energy intensive due to longer process operations. This research work applies the combination of radicals from hydrogen peroxide with an organic solvent as a chemical pretreatment method for disrupting the cell wall of microalgae and simultaneously extracting lipids from the biomass in a one-step biphasic solution. Result: Several parameters which can affect the biphasic system were analyzed: contact time, volume of solvent, volume ratio, type of organic solvent, biomass amount and concentration of solvents, to extract the highest amount of lipids from microalgae. The results were optimized and up to 83.5% of lipid recovery yield and 94.6% of enhancement was successfully achieved. The results obtain from GC-FID were similar to the analysis of triglyceride lipid standard. Conclusion: The profound hybrid biphasic system shows great potential to radically disrupt the cell wall of microalgae and instantaneously extract lipids in a single-step approach. The lipids extracted were tested to for its comparability to biodiesel performance. [ABSTRACT FROM AUTHOR]

The use of fossil fuels has been strongly related to critical problems currently affecting society, such as: global warming, global greenhouse effects and pollution. These problems have affected the homeostasis of living organisms worldwide at an alarming rate. Due to this, it is imperative to look for alternatives to the use of fossil fuels and one of the relevant substitutes are biofuels. There are different types of biofuels (categories and generations) that have been previously explored, but recently, the use of microalgae has been strongly considered for the production of biofuels since they present a series of advantages over other biofuel production sources: (a) they don't need arable land to grow and therefore do not compete with food crops (like biofuels produced from corn, sugar cane and other plants) and; (b) they exhibit rapid biomass production containing high oil contents, at least 15 to 20 times higher than land based oleaginous crops. Hence, these unicellular photosynthetic microorganisms have received great attention from researches to use them in the large-scale production of biofuels. However, one disadvantage of using microalgae is the high economic cost due to the low-yields of lipid content in the microalgae biomass. Thus, development of different methods to enhance microalgae biomass, as well as lipid content in the microalgae cells, would lead to the development of a sustainable low-cost process to produce biofuels. Within the last 10 years, many studies have reported different methods and strategies to induce lipid production to obtain higher lipid accumulation in the biomass of microalgae cells; however, there is not a comprehensive review in the literature that highlights, compares and discusses these strategies. Here, we review these strategies which include modulating light intensity in cultures, controlling and varying CO2 levels and temperature, inducing nutrient starvation in the culture, the implementation of stress by incorporating heavy metal or inducing a high salinity condition, and the use of metabolic and genetic engineering techniques coupled with nanotechnology. [ABSTRACT FROM AUTHOR]

Background: Efficient and economically viable production of biofuels from lignocellulosic biomass is dependent on mechanical and chemical pretreatment and enzymatic hydrolysis of plant material. These processing steps yield simple sugars as well as plant-derived and process-added organic acids, sugar-derived dehydration products, aldehydes, phenolics and other compounds that inhibit the growth of many microorganisms. Lipomyces starkeyi is an oleaginous yeast capable of robust growth on a variety of sugars and lipid accumulation on pretreated lignocellulosic substrates making it attractive as an industrial producer of biofuels. Here, we examined gene expression during batch growth and lipid accumulation in a 20-L bioreactor with either a blend of pure glucose and xylose or pretreated corn stover (PCS) that had been enzymatically hydrolyzed as the carbon sources. Results: We monitored sugar and ammonium utilization as well as biomass accumulation and found that growth of L. starkeyi is inhibited with PCS hydrolysate as the carbon source. Both acetic acid and furfural are present at concentrations toxic to L. starkeyi in PCS hydrolysate. We quantified gene expression at seven time-points for each carbon source during batch growth and found that gene expression is similar at physiologically equivalent points. Analysis of promoter regions revealed that gene expression during the transition to lipid accumulation is regulated by carbon and nitrogen catabolite repression, regardless of carbon source and is associated with decreased expression of the translation machinery and suppression of the cell cycle. We identified 73 differentially expressed genes during growth phase in the bioreactor that may be involved in detoxification of corn stover hydrolysate. Conclusions: Growth of L. starkeyi is inhibited by compounds present in PCS hydrolysate. Here, we monitored key metabolites to establish physiologically equivalent comparisons during a batch bioreactor run comparing PCS hydrolysate and purified sugars. L. starkeyi's response to PCS hydrolysate is primarily at the beginning of the run during growth phase when inhibitory compounds are presumably at their highest concentration and inducing the general detoxification response by L. starkeyi. Differentially expressed genes identified herein during growth phase will aid in the improvement of industrial strains capable of robust growth on substrates containing various growth inhibitory compounds. [ABSTRACT FROM AUTHOR]

Microalgae utilized for experiments are often not produced by the researchers that are doing experiments with them. The microalgae are made storable through thermal or freeze-drying by the producer. In an industrial scaled process, because of efficiency reasons, microalgae would not be dried, but processed directly. With that, the question is, if drying already could change the composition or structure that much, that a process scaled up from laboratory to productive scale with fresh microalgae would be less efficient or even would not work at all. The effect of freeze drying on the extraction behavior for the species Scenedesmus rubescens was investigated. It was obtained in freeze-dried condition and again was delivered in fresh state. The utilized microalgae were extracted with n-hexane, without and with different pretreatments (acidic hydrolysis and hydrothermal carbonization) to examine the differences in the yields. In conclusion, it was demonstrated that freeze drying harms the cell wall and therefore this process already influences the quantity of extracted lipids. Depending on the harshness of the treatment process for cell wall disruption this might influence the extracted yield when the algae are not freeze-dried. The quality of the extracted lipids does not change when freeze-dried. [ABSTRACT FROM AUTHOR]

Background: The regulation of genes related to lipid metabolism by genetic engineering is an important way to increase the accumulation of lipids in microalgae. DNA binding with one finger (DOF) is a plant-specific transcription factor in higher plants, where it regulates carbon and nitrogen metabolic pathways by regulating key genes involved in these pathways. Overexpression of DOF can increase lipid production in plants; however, it is not clear whether overexpression of DOF can increase lipids in microalgae. Results: In this study, we cloned a DOF transcription factor, crDOF, from Chlamydomonas reinhardtii. The sequence of this transcription factor is 1875 bp and encodes a peptide of 624 amino acids with a conserved DOF domain. Overexpression of crDOF in C. reinhardtii significantly increased the intracellular lipid content. The content of total fatty acids in the transgenic algae line Tranc-crDOF-12 was 126.01 μg/mg (dry weight), which was 23.24% higher than that of the wild type. Additionally, the content of unsaturated fatty acids in the transgenic Tranc-crDOF-12 line increased significantly. Fluorescence quantitative PCR analysis showed that in the transgenic line Tranc-crDOF-12, the expression levels of BCC1, FAT1, SQD1, MGD1, DGD1 and PGP1 genes were significantly upregulated, while the expression levels of ACP1, ACS1, CIS1 and SQD2 were downregulated. Conclusions: Our results confirm that crDOF increases intracellular lipids in C. reinhardtii by regulating key genes involved in lipid metabolism. According to these findings, we propose that enhancing the lipid content in microalgae by overexpressing DOF may be achieved in other industrial strains of microalgae and be employed for the industrial production of biodiesel. [ABSTRACT FROM AUTHOR]

Background: Several abundant proteins have been identified in lipid body of an alkenone-producing marine haptophyte alga Tisochrysis lutea. The gene expression patterns of these proteins were investigated to better understand their roles in alkenone biosynthesis. For this purpose, T. lutea was first cultured in nitrogen-sufficient medium for biomass production and then shifted to nitrogen-deprived medium to induce lipid body formation. Results: There were remarkable increases in the volume of alkenone body (AB) and alkenone content in the alga after they were exposed to nitrogen depletion medium. Relative mRNA levels of the genes coding for the identified proteins V-ATPase subunit VA, V-ATPase subunit Vd, hypothetical protein EMIHUDRAFT_465,517, coccolith scale associated protein-1, cycloartenol-c-24-methyltransferase 1-like and SPFH domain-containing protein were investigated over the culture period. RT-PCR data showed that the expression of all these genes except the gene coding for SPFH domain-containing protein was up-regulated during the transition period from nitrogen-sufficient to nitrogen-deficient medium. Among them, the expression of the coccolith scale associated protein-1 gene was up-regulated 50–650 folds. These up-regulations were consistent with the increased alkenone production in nitrogen-deprived medium, suggesting that these proteins are involved in alkenone biosynthesis in T. lutea. Conclusions: Expression analysis of the lipoprotein genes suggests that five out of the six genes are up-regulated and are therefore likely to code for the identified lipoproteins associated with alkenone biosynthesis in T. lutea. These data would help better understand alkenone metabolism and engineer for improved biofuel production in T. lutea. [ABSTRACT FROM AUTHOR]

Background: Amelioration of biofuel feedstock of microalgae using sustainable means through synthetic ecology is a promising strategy. The co-cultivation model (Tetraselmis striata and Pelagibaca bermudensis) was evaluated for the robust biofuel production under varying stressors as well as with the selected two-stage cultivation modes. In addition, the role of metabolic exudates including the quorum-sensing precursors was assessed. Results: The co-cultivation model innovated in this study supported the biomass production of T. striata in a saline/marine medium at a broad range of pH, salinity, and temperature/light conditions, as well as nutrient limitation with a growth promotion of 1.2–3.6-fold. Hence, this developed model could contribute to abiotic stress mitigation of T. striata. The quorum-sensing precursor dynamics of the growth promoting bacteria P. bermudensis exhibited unique pattern under varying stressors as revealed through targeted metabolomics (using liquid chromatography–mass spectrometry, LC–MS). P. bermudensis and its metabolic exudates mutually promoted the growth of T. striata, which elevated the lipid productivity. Interestingly, hydroxy alkyl quinolones independently showed growth inhibition of T. striata on elevated concentration. Among two-stage cultivation modes (low pH, elevated salinity, and nitrate limitation), specifically, nitrate limitation induced a 1.5 times higher lipid content (30–31%) than control in both axenic and co-cultivated conditions. Conclusion: Pelagibaca bermudensis is established as a potential growth promoting native phycospheric bacteria for robust biomass generation of T. striata in varying environment, and two-stage cultivation using nitrate limitation strategically maximized the biofuel precursors for both axenic and co-cultivation conditions (T and T-PB, respectively). Optimum metabolic exudate of P. bermudensis which act as a growth substrate to T. striata surpasses the antagonistic effect of excessive hydroxy alkyl quinolones [HHQ, 4-hydroxy-2-alkylquinolines and PQS (pseudomonas quorum signal), 2-heptyl-3-hydroxy-4(1H)-quinolone]. [ABSTRACT FROM AUTHOR]

Background: Microalgae, being a phototroph, grow in the presence of light, and utilizing photons in narrow and specific range of wavelengths. There have been numerous attempts to take advantage of this trait of wavelength-dependent growth for the purpose of increasing biomass productivity. One potential option involves wavelength conversion of sunlight. In the present study, three fluorescent dyes with blue, red, and green emission spectra were employed with the aim of improving sunlight utilization efficiency and thus enhancing biomass and lipid productivity of Nannochloropsis gaditana. Results: When DPA and R101 were used to enrich blue and red spectra, biomass productivity of Nannochloropsis gaditana was increased by 35.1 and 40.3%, respectively. The maximum quantum yield values were higher than 0.6 at the early stage of growth for the cultures grown under DPA- and R101-modified solar radiation. Chlorophyll a content was also 57.0 and 32.3% higher than the control at the early growth stage under DPA- and R101-modified solar radiation, respectively. This stimulation of photosynthetic activity at the early growth stage correlated well with rapid growth under DPA- and R101-modified light during the first 4 days of cultivation. Lipid productivity consequently increased by 26.9 (DPA) and 39.4% (R101) after 10 days of cultivation. An immediate effect on lipid induction was observed in cultures under modified light, which exhibited 19.1% improvement in lipid content at the cost of some degree of impaired growth. Conclusion: Fluorescent dyes with the capability of enriching wavelengths of light favored by the algal photosystem could indeed be an effective means of promoting growth of Nannochloropsis gaditana. This strategy would be particularly powerful for mass cultivation where sunlight is the only economically viable option for illumination. [ABSTRACT FROM AUTHOR]

The majority of the fuels we use in our vehicles and other equipment on a daily basis are nonrenewable. As a result, the world is going to eventually run out of supplies of fuels. In addition, the current fuel sources are environmentally unfriendly, releasing greenhouse gases into the atmosphere. Many alternatives to using natural reservoirs as sources of fuels have been exploited, among which, the use of algal feedstock as a source of biofuel has received enormous attention. Algal biofuel was studied for a long time, and has recently gained popularity again due to the global demand for transportation fuels, the greenhouse gas effects, and the energy security risks. Funded by the NSF, at Eastern, we are cultivating algae on an outdoor pilot unit--Algal Turf Scrubber®--in dairy manure effluent for wastewater treatment and sustainable biofuel production. Algae offer an inexhaustible source of energy, nutrients and raw materials. When microalgae are grown and harvested, the biomass obtained contains lipids, which can be converted into biodiesels through chemical transformations. One of the main difficulties in implementing the use of algae for biofuel on a wider scale, is the inefficient methods of lipid extraction, and the current methods used do not exhaustively extract all the lipids, and are costly, thereby making the wide scale production of algae not economically feasible compared to the current natural fuels being used. In this project I investigated ways to maximize the amount of lipids extracted and develop simple extraction procedures that do not require a lot of energy to successfully extract the lipids required for the production of the biofuels. I used Gas Chromatography - Mass Spectrometry to characterize the extracted lipids. In addition, I will also investigate how to utilize the remaining material after the extraction is completed. [ABSTRACT FROM AUTHOR]