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Background: Raw materials composed of easily assimilated monosaccharides have been employed as carbon source for production of microbial lipids. Nevertheless, agro-industrial wastes rich in galactose-based carbohydrates have not been introduced as feedstocks for oleaginous yeasts. Results: In this study, Aureobasidium namibiae A12 was found to efficiently accumulate lipid from soy molasses and whey powder containing galactose-based carbohydrates, with lipid productions of 5.30 g/L and 5.23 g/L, respectively. Over 80% of the fatty acids was C16:0, C18:0, C18:1, and C18:2. All kinds of single sugar components in the two byproducts were readily converted into lipids, with yields ranging between 0.116 g/g and 0.138 g/g. Three α-galactosidases and five β-galactosidases in the strain were cloned and analyzed. Changes of transcriptional levels indicated GalB and GalC were key α-galactosidases, and GalG was key β-galactosidase. In 10 L fermentor, lipid production from SM and WP achieved 6.45 g/L and 6.13 g/L, respectively. β-galactosidase was responsible for lactose hydrolysis; sucrase and α-galactosidase both contributed to the efficient hydrolysis of raffinose and stachyose in a cooperation manner. Conclusions: This is a new way to produce lipids from raw materials containing galactose-based carbohydrates. This finding revealed the significance of sucrase in the direct hydrolysis of galactose-based carbohydrates in raw materials for the first time and facilitated the understanding of the efficient utilization of galactose-based carbohydrates to manufacture lipid or other chemicals in bioprocess. [ABSTRACT FROM AUTHOR]

Background: Microbial oils, generated from lignocellulosic material, have great potential as renewable and sustainable alternatives to fossil-based fuels and chemicals. By unravelling the diversity of lipid accumulation physiology in different oleaginous yeasts grown on the various carbon sources present in lignocellulose hydrolysate (LH), new targets for optimisation of lipid accumulation can be identified. Monitoring lipid formation over time is essential for understanding lipid accumulation physiology. This study investigated lipid accumulation in a variety of oleaginous ascomycetous and basidiomycetous strains grown in glucose and xylose and followed lipid formation kinetics of selected strains in wheat straw hydrolysate (WSH). Results: Twenty-nine oleaginous yeast strains were tested for their ability to utilise glucose and xylose, the main sugars present in WSH. Evaluation of sugar consumption and lipid accumulation revealed marked differences in xylose utilisation capacity between the yeast strains, even between those belonging to the same species. Five different promising strains, belonging to the species Lipomyces starkeyi, Rhodotorula glutinis, Rhodotorula babjevae and Rhodotorula toruloides, were grown on undiluted wheat straw hydrolysate and lipid accumulation was followed over time, using Fourier transform-infrared (FTIR) spectroscopy. All five strains were able to grow on undiluted WSH and to accumulate lipids, but to different extents and with different productivities. R. babjevae DVBPG 8058 was the best-performing strain, accumulating 64.8% of cell dry weight (CDW) as lipids. It reached a culture density of 28 g/L CDW in batch cultivation, resulting in a lipid content of 18.1 g/L and yield of 0.24 g lipids per g carbon source. This strain formed lipids from the major carbon sources in hydrolysate, glucose, acetate and xylose. R. glutinis CBS 2367 also consumed these carbon sources, but when assimilating xylose it consumed intracellular lipids simultaneously. Rhodotorula strains contained a higher proportion of polyunsaturated fatty acids than the two tested Lipomyces starkeyi strains. Conclusions: There is considerable metabolic diversity among oleaginous yeasts, even between closely related species and strains, especially when converting xylose to biomass and lipids. Monitoring the kinetics of lipid accumulation and identifying the molecular basis of this diversity are keys to selecting suitable strains for high lipid production from lignocellulose. [ABSTRACT FROM AUTHOR]

Background: Lipids are important precursors in the biofuel and oleochemical industries. Yarrowia lipolytica is among the most extensively studied oleaginous microorganisms and has been a focus of metabolic engineering to improve lipid production. Yield improvement, through rewiring of the central carbon metabolism of Y. lipolytica from glucose to the lipid precursor acetyl-CoA, is a key strategy for achieving commercial success in this organism. Results: Building on YB-392, a Y. lipolytica isolate known for stable non-hyphal growth and low citrate production with demonstrated potential for high lipid accumulation, we assembled a heterologous pathway that redirects carbon flux from glucose through the pentose phosphate pathway (PPP) to acetyl-CoA. We used phosphofructokinase (Pfk) deletion to block glycolysis and expressed two non-native enzymes, phosphoketolase (Xpk) and phosphotransacetylase (Pta), to convert PPP-produced xylulose-5-P to acetyl-CoA. Introduction of the pathway in a pfk deletion strain that is unable to grow and accumulate lipid from glucose in defined media ensured maximal redirection of carbon flux through Xpk/Pta. Expression of Xpk and Pta restored growth and lipid production from glucose. In 1-L bioreactors, the engineered strains recorded improved lipid yield and cell-specific productivity by up to 19 and 78%, respectively. Conclusions: Yields and cell-specific productivities are important bioprocess parameters for large-scale lipid fermentations. Improving these parameters by engineering the Xpk/Pta pathway is an important step towards developing Y. lipolytica as an industrially preferred microbial biocatalyst for lipid production. [ABSTRACT FROM AUTHOR]

Background: The unicellular alga Haematococcus pluvialis has achieved considerable interests for its capacity to accumulate large amounts of triacylglycerol and astaxanthin under various environmental stresses. To our knowledge, studies focusing on transcriptome research of H. pluvialis under exogenous hormones together with physical stresses are rare. In the present study, the change patterns at transcriptome level were analyzed to distinguish the multiple defensive systems of astaxanthin and fatty acid metabolism against exogenous salicylic acid and high light (SAHL) stresses. Results: Based on RNA-seq data, a total of 112,463 unigenes and 61,191 genes were annotated in six databases, including NR, KEGG, Swiss-Prot, PFAM, COG and GO. Analysis of differentially expressed genes (DEGs) in KEGG identified many transcripts that associated with the biosynthesis of primary and secondary metabolites, photosynthesis, and immune system responses. Furthermore, 705 unigenes predicted as putative transcription factors (TFs) were identified, and the most abundant TFs families were likely to be associated with the biosynthesis of astaxanthin and fatty acid in H. pluvialis upon exposure to SAHL stresses. Additionally, majority of the fifteen key genes involved in astaxanthin and fatty acid biosynthesis pathways presented the same expression pattern, resulting in increased accumulation of astaxanthin and fatty acids in single celled H. pluvialis, in which astaxanthin content increased from 0.56 ± 0.05 mg·L−1 at stage Control to 0.89 ± 0.12 mg·L−1 at stage SAHL_48. And positive correlations were observed among these studied genes by Pearson Correlation (PC) analysis, indicating the coordination between astaxanthin and fatty acid biosynthesis. In addition, protein–protein interaction (PPI) network analysis also demonstrated that this coordination might be at transcriptional level. Conclusion: The results in this study provided valuable information to illustrate the molecular mechanisms of coordinate relations between astaxanthin and fatty acid biosynthesis. And salicylic acid might play a role in self-protection processes of cells, helping adaption of H. pluvialis to high light stress. [ABSTRACT FROM AUTHOR]

Background: Crude glycerol (CG) and hemicellulose hydrolysate (HH) are low—value side-products of biodiesel transesterification and pulp—and paper industry or lignocellulosic ethanol production, respectively, which can be converted to microbial lipids by oleaginous yeasts. This study aimed to test the ability of oleaginous yeasts to utilise CG and HH and mixtures of them as carbon source. Results: Eleven out of 27 tested strains of oleaginous yeast species were able to grow in plate tests on CG as sole carbon source. Among them, only one ascomycetous strain, belonging to Lipomyces starkeyi, was identified, the other 10 strains were Rhodotorula spec. When yeasts were cultivated in mixed CG/ HH medium, we observed an activation of glycerol conversion in the Rhodotorula strains, but not in L. starkeyi. Two strains—Rhodotorula toruloides CBS 14 and Rhodotorula glutinis CBS 3044 were further tested in controlled fermentations in bioreactors in different mixtures of CG and HH. The highest measured average biomass and lipid concentration were achieved with R. toruloides in 10% HH medium mixed with 55 g/L CG—19.4 g/L and 10.6 g/L, respectively, with a lipid yield of 0.25 g lipids per consumed g of carbon source. Fatty acid composition was similar to other R. toruloides strains and comparable to that of vegetable oils. Conclusions: There were big strain differences in the ability to convert CG to lipids, as only few of the tested strains were able to grow. Lipid production rates and yields showed that mixing GC and HH have a stimulating effect on lipid accumulation in R. toruloides and R. glutinis resulting in shortened fermentation time to reach maximum lipid concentration, which provides a new perspective on converting these low-value compounds to microbial lipids. [ABSTRACT FROM AUTHOR]

Background: Recent studies of lipid extraction from microalgae have focused primarily on dewatered or dried samples, and the processes are simple with high lipid yield. Yet, the dewatering with drying step is energy intensive, which makes the energy input during the lipid production more than energy output from obtained lipid. Thus, exploring an extraction technique for just a thickened sample without the dewatering, drying and auxiliary operation (such as cell disruption) is very significant. Whereas lipid extraction from the thickened microalgae is complicated by the high water content involved, and traditional solvent, hence, cannot work well. Dimethyl ether (DME), a green solvent, featuring a high affinity for both water and organic compounds with an ability to penetrate the cell walls has the potential to achieve this goal. Results: This study investigated an energy-saving method for lipid extraction using DME as the solvent with an entrainer solution (ethanol and acetone) for flocculation-thickened microalgae. Extraction efficiency was evaluated in terms of extraction time, DME dosage, entrainer dosage, and ethanol:acetone ratio. Optimal extraction occurred after 30 min using 4.2 mL DME per 1 mL microalgae, with an entrainer dosage of 8% at 1:2 ethanol:acetone. Raw lipid yields and its lipid component (represented by fatty acid methyl ester) contents were compared against those of common extraction methods (Bligh and Dryer, and Soxhlet). Thermal gravimetry/differential thermal analysis, Fourier-transform infrared spectroscopy, and C/H/N elemental analyses were used to examine differences in lipids extracted using each of the evaluated methods. Considering influence of trace metals on biodiesel utilization, inductively coupled plasma mass spectrometry and inductively coupled plasma atomic emission spectroscopy analyses were used to quantify trace metals in the extracted raw lipids, which revealed relatively high concentrations of Mg, Na, K, and Fe. Conclusions: Our DME-based method recovered 26.4% of total raw lipids and 54.4% of total fatty acid methyl esters at first extraction with remnants being recovered by a 2nd extraction. In additional, the DME-based approach was more economical than other methods, because it enabled simultaneous dewatering with lipid extraction and no cell disruption was required. The trace metals of raw lipids indicated a purification demand in subsequent refining process. [ABSTRACT FROM AUTHOR]

Background: Heterotrophic cultivation of microalgae has been proposed as a viable alternative method for novel high-value biomolecules, enriched biomass, and biofuel production because of their allowance of high cell density levels, as well as simple production technology. Tetradesmus bernardii, a newly isolated high-yielding oleaginous microalga under photoautotrophic conditions, is able to grow heterotrophically, meaning that it can consume organic carbon sources in dark condition. We investigated the effect of different carbon/nitrogen (C/N) ratios on the growth and lipid accumulation of T. bernardii in heterotrophic batch culture under two nitrogen sources (NaNO3 and CO(NH2)2). In addition, we conducted time-resolved transcriptome analysis to reveal the metabolic mechanism of T. bernardii in heterotrophic culture. Results: T. bernardii can accumulate high biomass concentrations in heterotrophic batch culture where the highest biomass of 46.09 g/L was achieved at 100 g/L glucose concentration. The rate of glucose to biomass exceeded 55% when the glucose concentration was less than 80 g/L, and the C/N ratio was 44 at urea treatment. The culture was beneficial to lipid accumulation at a C/N ratio between 110 and 130. NaNO3 used as a nitrogen source enhanced the lipid content more than urea, and the highest lipid content was 45% of dry weight. We performed RNA-seq to analyze the time-resolved transcriptome of T. bernardii. As the nitrogen was consumed in the medium, nitrogen metabolism-related genes were significantly up-regulated to speed up the N metabolic cycle. As chloroplasts were destroyed in the dark, the metabolism of cells was transferred from chloroplasts to cytoplasm. However, storage of carbohydrate in chloroplast remained active, mainly the synthesis of starch, and the precursor of starch synthesis in heterotrophic culture may largely come from the absorption of organic carbon source (glucose). With regard to lipid metabolism, the related genes of fatty acid synthesis in low nitrogen concentration increased gradually with the extension of cultivation time. Conclusion: T. bernardii exhibited rapid growth and high lipid accumulation in heterotrophic culture. It may be a potential candidate for biomass and biofuel production. Transcriptome analysis showed that multilevel regulation ensured the conversion from carbon to the synthesis of carbohydrate and lipid. [ABSTRACT FROM AUTHOR]

Background: Converting wastewater sludge to lipid is considered as one of the best strategies of sludge management. The current problem of lipid production from wastewater sludge is the low yield (0.10–0.16 g lipid/g dry sludge) due to the low availability of easily uptaken materials (such as soluble monosaccharide and oligosaccharide) in sludge to oleaginous microorganism (Rhodotorula glutinis, Trichosporon oleaginosus, Lipomyces starkeyi). Pretreatments are efficient methods to improve sludge bioavailability. This study is aimed to achieve high lipid production from sludge and high sludge reduction. Results: In this study, it was observed that the soluble chemical oxygen demand (SCOD) had significantly increased after different pretreatment. The SCOD in the supernatant was increased from 32.64 to 180.25 mg/L, 924.16 mg/L, 1029.89 mg/L and 3708.31 mg/L after acidic (pH 2 for 2 h), alkaline (pH 12 for 2 h), microwave irradiation (15 min with 5 min interval), and ultrasonication (30 min at 450 W and 20 kHz frequency with 5 s on and 2 s off mode) pretreatment, respectively. Pretreatments have also increased the release of total nitrogen (TN) and total phosphorus (TP) from solids. The sludge after different pretreatments were used as a medium for lipid production, and the highest lipid content (36.67% g/g) was obtained in the fermentation with ultrasonication pretreatment sludge, and the sludge reduction was 63.10%. For other pretreatments, the lipid content and sludge reduction were 18.42% and 32.63% in acid pretreatment case, 21.08% and 36.44% in alkaline pretreatment case, and 26.31% and 43.03% in microwave pretreatment case, respectively. Conclusion: It was found that ultrasonication pretreatment was the most efficient way to increase the sludge biodegradability (SCOD) and to release TN and TP from solid phase to liquid phase. Pretreated sludge for lipid production achieved significant improvement in lipid yield and sludge reduction. Lipids produced from pretreated sludge were transesterified to biodiesel and the analysis showed that biodiesel had a similar composition as commercial biodiesel. The study reveals that pretreatment on sludge is a promising method for enhancing biological sludge management efficiency. [ABSTRACT FROM AUTHOR]

Background: Biotechnologically produced microbial lipids are of interest as potential alternatives for crude and plant oils. Their lipid profile is similar to plant oils and can therefore be a substitute for the production of biofuels, additives for food and cosmetics industry as well as building blocks for oleochemicals. Commercial microbial lipids production, however, is still not profitable and research on process optimization and cost reduction is required. This study reports on the process optimization using glucose or xylose with the unconventional oleaginous yeast Saitozyma podzolica DSM 27192 aiming to reduce the applied carbon source amount without sacrificing lipid productivity. Results: By optimizing the process parameters temperature and pH, lipid productivity was enhanced by 40%. Thereupon, by establishing a two-phase strategy with an initial batch phase and a subsequent fed-batch phase for lipid production in which a constant sugar concentration of about 10 g/L was maintained, resulted in saving of ~ 41% of total glucose and ~ 26% of total xylose. By performing the automated continuous sugar feed the total sugar uptake was improved to ~ 91% for glucose and ~ 92% for xylose and thus, prevented waste of unused carbon source in the cultivation medium. In addition, reduced glucose cultivation resulted in to 28% higher cell growth and 19% increase of lipid titer. By using xylose, the by-product xylonic acid was identified for the first time as by-product of S. podzolica. Conclusions: These findings provide a broad view of different cultivation process strategies with subsequent comparison and evaluation for lipid production with S. podzolica. Additionally, new biotechnological characteristics of this yeast were highlighted regarding the ability to produce valuable organic acids from sustainable and renewable sugars. [ABSTRACT FROM AUTHOR]

Background: Lupeol exhibits novel physiological and pharmacological activities, such as anticancer and immunity-enhancing activities. However, cytotoxicity remains a challenge for triterpenoid overproduction in microbial cell factories. As lipophilic and relatively small molecular compounds, triterpenes are generally secreted into the extracellular space. The effect of increasing triterpene efflux on the synthesis capacity remains unknown. Results: In this study, we developed a strategy to enhance triterpene efflux through manipulation of lipid components in Y. lipolytica by overexpressing the enzyme Δ9-fatty acid desaturase (OLE1) and disturbing phosphatidic acid phosphatase (PAH1) and diacylglycerol kinase (DGK1). By this strategy combined with two-phase fermentation, the highest lupeol production reported to date was achieved, where the titer in the organic phase reached 381.67 mg/L and the total production was 411.72 mg/L in shake flasks, exhibiting a 33.20-fold improvement over the initial strain. Lipid manipulation led to a twofold increase in the unsaturated fatty acid (UFA) content, up to 61–73%, and an exceptionally elongated cell morphology, which might have been caused by enhanced membrane phospholipid biosynthesis flux. Both phenotypes accelerated the export of toxic products to the extracellular space and ultimately stimulated the capacity for triterpenoid synthesis, which was proven by the 5.11-fold higher ratio of extra/intracellular lupeol concentrations, 2.79-fold higher biomass accumulation and 2.56-fold higher lupeol productivity per unit OD in the modified strains. This strategy was also highly efficient for the biosynthesis of other triterpenes and sesquiterpenes, including α-amyrin, β-amyrin, longifolene, longipinene and longicyclene. Conclusions: In conclusion, we successfully created a high-yield lupeol-producing strain via lipid manipulation. We demonstrated that the enhancement of lupeol efflux and synthesis capacity was induced by the increased UFA content and elongated cell morphology. Our study provides a novel strategy to promote the biosynthesis of valuable but toxic products in microbial cell factories. [ABSTRACT FROM AUTHOR]

Background: The development of microalgal strains for enhanced biomass and biofuel production has received increased attention. Moreover, strain development via metabolic engineering for commercial production is being considered as the most efficient strategy. Pyruvate is an essential metabolite in the cells and plays an essential role in amino acid biosynthesis and de novo fatty acid biosynthesis in plastids. Although pyruvate can be a valuable target for metabolic engineering, its transporters have rarely been studied in microalgae. In this study, we aimed to identify the plastidial pyruvate transporter of Phaeodactylum tricornutum and utilize it for strain development. Results: We identified putative pyruvate transporter localized in the plastid membrane of Phaeodactylum tricornutum. Transformants overexpressing the pyruvate transporter were generated to increase the influx of pyruvate into plastids. Overexpression of a plastidial pyruvate transporter in P. tricornutum resulted in enhanced biomass (13.6% to 21.9%), lipid contents (11% to 30%), and growth (3.3% to 8.0%) compared to those of wild type during one-stage cultivation. Conclusion: To regulate the pyruvate influx and its metabolism in plastids, we generated transformants overexpressing the putative plastidial pyruvate transporter in P. tricornutum. They showed that its overexpression for compartmentalizing pyruvate in plastids could be an attractive strategy for the effective production of biomass and lipids with better growth, via enhanced pyruvate metabolism in plastids. [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]

Background: In this study, renewable tea waste hydrolysate was used as a sole carbon source for carotenoids and lipid production. A novel Rhodosporidium toruloides mutant strain, RM18, was isolated through atmospheric and room-temperature plasma mutagenesis and continuous domestication in tea waste hydrolysate from R. toruloides ACCC20341. Results: RM18 produced a larger biomass and more carotenoids and α-linolenic acid compared with the control strain cultured in tea waste hydrolysate. The highest yields of torularhodin (481.92 μg/g DCW) and torulene (501 μg/g DCW) from RM18 cultured in tea waste hydrolysate were 12.86- and 1.5-fold higher, respectively, than that of the control strain. In addition, α-linolenic acid production from RM18 in TWH accounted for 5.5% of total lipids, which was 1.58 times more than that of the control strain. Transcriptomic profiling indicated that enhanced central metabolism and terpene biosynthesis led to improved carotenoids production, whereas aromatic amino acid synthesis and DNA damage checkpoint and sensing were probably relevant to tea waste hydrolysate tolerance. Conclusion: Tea waste is suitable for the hydrolysis of microbial cell culture mediums. The R. toruloides mutant RM18 showed considerable carotenoids and lipid production cultured in tea waste hydrolysate, which makes it viable for industrial applications. [ABSTRACT FROM AUTHOR]

Background: The necessity to develop high lipid-producing microalgae is emphasized for the commercialization of microalgal biomass, which is environmentally friendly and sustainable. Nannochloropsis are one of the best industrial microalgae and have been widely studied for their lipids, including high-value polyunsaturated fatty acids (PUFAs). Many reports on the genetic and biological engineering of Nannochloropsis to improve their growth and lipid contents have been published. Results: We performed insertional mutagenesis in Nannochloropsis salina, and screened mutants with high lipid contents using fluorescence-activated cell sorting (FACS). We isolated a mutant, Mut68, which showed improved growth and a concomitant increase in lipid contents. Mut68 exhibited 53% faster growth rate and 34% higher fatty acid methyl ester (FAME) contents after incubation for 8 days, resulting in a 75% increase in FAME productivity compared to that in the wild type (WT). By sequencing the whole genome, we identified the disrupted gene in Mut68 that encoded trehalose-6-phosphate (T6P) synthase (TPS). TPS is composed of two domains: TPS domain and T6P phosphatase (TPP) domain, which catalyze the initial formation of T6P and dephosphorylation to trehalose, respectively. Mut68 was disrupted at the TPP domain in the C-terminal half, which was confirmed by metabolic analyses revealing a great reduction in the trehalose content in Mut68. Consistent with the unaffected N-terminal TPS domain, Mut68 showed moderate increase in T6P that is known for regulation of sugar metabolism, growth, and lipid biosynthesis. Interestingly, the metabolic analyses also revealed a significant increase in stress-related amino acids, including proline and glutamine, which may further contribute to the Mut68 phenotypes. Conclusion: We have successfully isolated an insertional mutant showing improved growth and lipid production. Moreover, we identified the disrupted gene encoding TPS. Consistent with the disrupted TPP domain, metabolic analyses revealed a moderate increase in T6P and greatly reduced trehalose. Herein, we provide an excellent proof of concept that the selection of insertional mutations via FACS can be employed for the isolation of mutants with improved growth and lipid production. In addition, trehalose and genes encoding TPS will provide novel targets for chemical and genetic engineering, in other microalgae and organisms as well as Nannochloropsis. [ABSTRACT FROM AUTHOR]

Background: Lignocellulosic biomass has been commonly regarded as a potential feedstock for the production of biofuels and biochemicals. High sugar yields and the complete bioconversion of all the lignocellulosic sugars into valuable products are attractive for the utilization of lignocelluloses. It is essential to pretreat and hydrolyze lignocelluloses at high solids loadings during industrial processes, which is more economical and environmentally friendly as capital cost, energy consumption, and water usage can be reduced. However, oligosaccharides are inevitably released during the high solids loading enzymatic hydrolysis and they are more recalcitrant than monosaccharides for microorganisms. Results: A fed-batch enzymatic hydrolysis of corn stover pretreated by the sodium hydroxide–methanol solution (SMs) at high solids loading was demonstrated to reach the high concentrations and yields of fermentable sugars. Glucose, xylose, cello-oligosaccharides, and xylo-oligosaccharides achieved 146.7 g/L, 58.7 g/L, 15.6 g/L, and 24.7 g/L, respectively, when the fed-batch hydrolysis was started at 12% (w/v) solids loading, and 7% fresh substrate and a standardized blend of cellulase, β-glucosidase, and hemicellulase were fed consecutively at 3, 6, 24, and 48 h to achieve a final solids loading of 40% (w/v). The total conversion of glucan and xylan reached 89.5% and 88.5%, respectively, when the oligosaccharides were taken into account. Then, a fed-batch culture on the hydrolysates was investigated for lipid production by Cutaneotrichosporon oleaginosum. Biomass, lipid content, and lipid yield were 50.7 g/L, 61.7%, and 0.18 g/g, respectively. The overall consumptions of cello-oligosaccharides and xylo-oligosaccharides reached 74.1% and 68.2%, respectively. Conclusions: High sugars concentrations and yields were achieved when the enzyme blend was supplemented simultaneously with the substrate at each time point of feeding during the fed-batch enzymatic hydrolysis. Oligosaccharides were co-utilized with monosaccharides during the fed-batch culture of C. oleaginosum. These results provide a promising strategy to hydrolyze alkaline organosolv-pretreated corn stover into fermentable sugars with high concentrations and yields for microbial lipid production. [ABSTRACT FROM AUTHOR]

Background: Cultivation of microalgae in wastewater could significantly contribute to wastewater treatment, biodiesel production, and thus the transition to renewable energy. However, more information on effects of environmental factors, including light intensity, on their growth and composition (particularly fatty acid contents) is required. Therefore, we investigated the biomass and fatty acid production of four microalgal species, isolated in the Northern hemisphere and grown at three light intensities (50, 150 and 300 μE m−2 s−1). Results: Increases in light intensities resulted in higher biomass of all four species and, importantly, raised fatty acid contents of both Desmodesmus sp. and Scenedesmus obliquus. Fourier-transform IR spectrometry analysis showed that the increases in fatty acid content were associated with reductions in protein, but not carbohydrate, contents. Assessment of fatty acid composition revealed that increasing light intensity led to higher and lower contents of oleic (18:1) and linolenic (18:3) acids, respectively. The microalgae consumed more than 75% of the nitrogen and phosphorus present in the wastewater used as growth medium. Conclusion: The results show the importance of optimizing light intensities to improve fatty acid production by microalgae and their quality as sources of biodiesel. In addition, increase in fatty acid content is associated with decrease in protein content. [ABSTRACT FROM AUTHOR]

Background: Yarrowia lipolytica, a non-traditional oil yeast, has been widely used as a platform for lipid production. However, the production of other chemicals such as terpenoids in engineered Y. lipolytica is still low. α-Farnesene, a sesquiterpene, can be used in medicine, bioenergy and other fields, and has very high economic value. Here, we used α-farnesene as an example to explore the potential of Y. lipolytica for terpenoid production. Results: We constructed libraries of strains overexpressing mevalonate pathway and α-farnesene synthase genes by non-homologous end-joining (NHEJ) mediated integration into the Y. lipolytica chromosome. First, a mevalonate overproduction strain was selected by overexpressing relevant genes and changing the cofactor specificity. Based on this strain, the downstream α-farnesene synthesis pathway was overexpressed by iterative integration. Culture conditions were also optimized. A strain that produced 25.55 g/L α-farnesene was obtained. This is the highest terpenoid titer reported in Y. lipolytica. Conclusions: Yarrowia lipolytica is a potentially valuable species for terpenoid production, and NHEJ-mediated modular integration is effective for expression library construction and screening of high-producer strains. [ABSTRACT FROM AUTHOR]

Background: Chromochloris zofingiensis is emerging as an industrially relevant alga given its robust growth for the production of lipids and astaxanthin, a value-added carotenoid with broad applications. Nevertheless, poor understanding of astaxanthin synthesis has limited engineering of this alga for rational improvements. Results: To reveal the molecular mechanism underlying astaxanthin accumulation in C. zofingiensis, here we conducted an integrated analysis by combining the time-resolved transcriptomes and carotenoid profiling in response to nitrogen deprivation (ND). A global response was triggered for C. zofingiensis to cope with the ND stress. Albeit the little variation in total carotenoid content, individual carotenoids responded differentially to ND: the primary carotenoids particularly lutein and β-carotene decreased, while the secondary carotenoids increased considerably, with astaxanthin and canthaxanthin being the most increased ones. The carotenogenesis pathways were reconstructed: ND had little effect on the carbon flux to carotenoid precursors, but stimulated astaxanthin biosynthesis while repressing lutein biosynthesis, thereby diverting the carotenoid flux from primary carotenoids to secondary carotenoids particularly astaxanthin. Comparison between C. zofingiensis and Haematococcus pluvialis revealed the distinctive mechanism of astaxanthin synthesis in C. zofingiensis. Furthermore, potential bottlenecks in astaxanthin synthesis were identified and possible engineering strategies were proposed for the alga. Conclusions: Collectively, these findings shed light on distinctive mechanism of carotenogenesis for astaxanthin biosynthesis in C. zofingiensis, identify key functional enzymes and regulators with engineering potential and will benefit rational manipulation of this alga for improving nutritional traits. [ABSTRACT FROM AUTHOR]

In this study, confocal Raman microspectroscopy was used to detect lipids in microalgae rapidly and non-destructively. Microalgae cells were cultured under nitrogen deficiency. The accumulation of lipids in Scenedesmus obliquus was observed by Nile red staining, and the total amount of lipids accumulated in the cells was measured by gravimetric method. The signals from different microalgae cells were collected by confocal Raman microspectroscopy to establish a prediction model of intracellular lipid content, and surface scanning signals for drawing pseudo color images of lipids distribution. The images can show the location of pyrenoid and lipid accumulation in cells. Analyze Raman spectrum data and build PCA-LDA model using four different bands (full bands, pigments, lipids, and mixed features). Models of full bands or pigment characteristic bands were capable of identifying S. obliquus cells under different nitrogen stress culture time. The prediction accuracy of model of lipid characteristic bands is relatively low. The correlation between the fatty acid content measured by the gravimetric method and the integral Raman intensity of the oil characteristic peak (1445 cm-1) measured by Raman spectroscopy was analyzed. There was significant correlation (R² = 0.83), which means that Raman spectroscopy is applicable to semi-quantitative detection of microalgal lipid contetn. [ABSTRACT FROM AUTHOR]

Background: Biomass-derived jet fuel is an alternative jet fuel (AJF) showing promise of reducing the dependence on fossil fuel and greenhouse gas emissions. Hydroprocessed esters and fatty acids (HEFA) concept is also known as one of the pathways for producing bio jet fuel. HEFA fuel was approved by the American Society for Testing and Materials in 2011, and can be blended up to 50% with conventional jet fuel. Since then, several HEFA economic and life-cycle assessments have been published in literature. However, there have been limited analyses on feedstock availability, composition, and their impact on hydrocarbon yield (particularly jet blendstock yield) and overall process economics. Results: This study examines over 20 oil feedstocks, their geographic distribution and production levels, oil yield, prices, and chemical composition. The results of our compositional analysis indicate that most oils contain mainly C16 and C18 fatty acids except pennycress, yellow grease, and mustard, which contain higher values and thus would require hydrocracking to improve jet fuel production. Coconut oil has a large content of shorter carbon fatty acids, making it a good feedstock candidate for renewable gasoline instead of jet substitutes' production. Techno-economic analysis (TEA) was performed for five selected oil feedstocks--camelina, pennycress, jatropha, castor bean, and yellow grease--using the HEFA process concept. Conclusion: The resource analysis indicates that oil crops currently grown in the United States (namely soybean) have relatively low oil yield when compared to oil crops grown in other parts of the world, such as palm, coconut, and jatropha. Also, non-terrestrial oil sources, such as animal fats and greases, have relatively lower prices than terrestrial oil crops. The minimum jet fuel selling price for these five resources ranges between $3.8 and $11.0 per gallon. The results of our TEA and resource studies indicate the key cost drivers for a biorefinery converting oil to jet hydrocarbons are as follows: oil price, conversion plant capacity, fatty acid profile, addition of hydrocracker, and type of hydroprocessing catalysts. [ABSTRACT FROM AUTHOR]

Background: The sustainability of microbial lipids production from traditional carbon sources, such as glucose or glycerol, is problematic given the high price of raw materials. Considerable efforts have been directed to minimize the cost and find new alternative carbon sources. Volatile fatty acids (VFAs) are especially attractive raw materials, because they can be produced from a variety of organic wastes fermentation. Therefore, the use of volatile fatty acids as carbon sources seems to be a feasible strategy for cost-effective microbial lipid production. Results: Lipid accumulation in Y. lipolytica using synthetic and food waste-derived VFAs as substrates was systematically compared and evaluated in batch cultures. The highest lipid content obtained with acetic, butyric, and propionic acids reached 31.62 ± 0.91, 28.36 ± 0.74, and 28.91 ± 0.66%, respectively. High concentrations of VFA inhibited cell growth in the following order: butyric acid > propionic acid > acetic acid. Within a 30-day experimental period, Y. lipolytica could adapt up to 20 g/L acetic acid, whereas the corresponding concentration of propionic acid and butyric acid were 10 and 5 g/L, respectively. Cultures on a VFA mixture showed that the utilization of different types of VFA by Y. lipolytica was not synchronized but rather performed in a step-wise manner. Although yeast fermentation is an exothermic process, and the addition of VFA will directly affect the pH of the system by increasing environmental acidity, cultures at a cultivation temperature of 38 °C and uncontrolled pH demonstrated that Y. lipolytica had high tolerance in the high temperature and acidic environment when a low concentration (2.5 g/L) of either synthetic or food wastederived VFA was used. However, batch cultures fed with food fermentate yielded lower lipid content (18.23 ± 1.12%) and lipid productivity (0.12 ± 0.02 g/L/day). The lipid composition obtained with synthetic and food waste-derived VFA was similar to commercial biodiesel feedstock. Conclusions: This work demonstrated the feasibility of utilizing synthetic and food waste-derived VFA for lipid production by Y. lipolytica. The good adaptability of Y. lipolytica to the high temperature and acidic environment further illustrated its considerable potential for practical application. [ABSTRACT FROM AUTHOR]

Background: Oleaginous microalgae are promising production hosts for the sustainable generation of lipid-based bioproducts and as bioenergy carriers such as biodiesel. Transcriptomics of the lipid accumulation phase, triggered efficiently by nitrogen starvation, is a valuable approach for the identification of gene targets for metabolic engineering. Results: An explorative analysis of the detailed transcriptional response to different stages of nitrogen availability was performed in the oleaginous green alga Monoraphidium neglectum. Transcript data were correlated with metabolic data for cellular contents of starch and of different lipid fractions. A pronounced transcriptional down-regulation of photosynthesis became apparent in response to nitrogen starvation, whereas glucose catabolism was found to be up-regulated. An in-depth reconstruction and analysis of the pathways for glycerolipid, central carbon, and starch metabolism revealed that distinct transcriptional changes were generally found only for specific steps within a metabolic pathway. In addition to pathway analyses, the transcript data were also used to refine the current genome annotation. The transcriptome data were integrated into a database and complemented with data for other microalgae which were also subjected to nitrogen starvation. It is available at https://tdbmn.cebitec.uni-bielefeld.de. Conclusions: Based on the transcriptional responses to different stages of nitrogen availability, a model for triacylglycerol and lipid hyperaccumulation is proposed, which involves transcriptional induction of thioesterases, differential regulation of lipases, and a re-routing of the central carbon metabolism. Over-expression of distinct thioesterases was identified to be a potential strategy to increase the oleaginous phenotype of M. neglectum, and furthermore specific lipases were identified as potential targets for future metabolic engineering approaches. [ABSTRACT FROM AUTHOR]

Background: Simultaneous wastewater treatment and lipid production by oleaginous microalgae show great potential to alleviate energy shortage and environmental pollution, because they exhibit tremendous advantages over traditional activated sludge. Currently, most research on wastewater treatment by microalgal are carried out at optimized temperature conditions (25-35 °C), but no information about simultaneous wastewater treatment and lipid production by microalgae at low temperatures has been reported. Microalgal growth and metabolism will be inhibited at low temperature conditions, and satisfactory wastewater treatment performance will be not obtained. Therefore, it is critical to domesticate and screen superior microalgal strains with low temperature adaptability, which is of great importance for wastewater treatment and biodiesel production. Results: In this work, simultaneous wastewater treatment and lipid production were achieved by a microalgal mutant Scenedesmus sp. Z-4 at the low temperature conditions (4, 10, and 15 °C). The results showed that algal growth was inhibited at 4, 10, and 15 °C compared to that at the optimal temperature of 25 °C. However, decreased temperature had no significant effect on the total cellular lipid content of algae. Importantly, lipid productivity at 10 °C was compromised by more net energy output relevant to biodiesel production, which demonstrated that the low temperature of 10 °C was favorable to wastewater treatment and energy recovery by Scenedesmus sp. Z-4. When molasses wastewater with optimal COD concentration of 8000 mg L-1, initial inoculation ratio of 15%, and C/N ratio of 15 was used to cultivate microalgae, the maximum removal rate of COD, TN, and TP at 10 °C reached 87.2, 90.5, and 88.6%, respectively. In addition, lipid content of 28.9% and lipid productivity of 94.4 mg L-1 day-1 were obtained. Conclusions: Scenedesmus sp. Z-4 had good adaptability to low temperature conditions, and showed great potential to realize simultaneous wastewater treatment and lipid production at low temperatures. The proposed approach in the study was simple compared to other wastewater treatment methods, and this potential novel process was still efficient to remove COD, N, and P at low temperatures. Thus, it had a vital significance for the wastewater treatment in low temperature regions. [ABSTRACT FROM AUTHOR]

Background: Nutrient limitation, such as nitrogen depletion, is the most widely used method for improving microalgae fatty acid production; however, these harsh conditions also inhibit algal growth significantly and even kill cells at all. To avoid these problems, we used artificial microRNA (amiRNA) technology as a useful tool to manipulate metabolic pathways to increase fatty acid contents effectively in the green microalga Chlamydomonas reinhardtii. We down-regulated the expression of phosphoenolpyruvate carboxylase (PEPC), which catalyzes the formation of oxaloacetate from phosphoenolpyruvate and regulates carbon flux. Results: amiRNAs against two CrPEPC genes were designed and transformed into Chlamydomonas cells and amiRNAs were induced by heat shock treatment. The transcription levels of amiRNAs increased 16-28 times, resulting in the remarkable decreases of the expression of CrPEPCs. In the end, inhibiting the expression of the CrPEPC genes dramatically increased the total fatty acid content in the transgenic algae by 28.7-48.6%, which mostly increased the content of C16-C22 fatty acids. Furthermore, the highest content was that of C18:3n3 with an average increase of 35.75%, while C20-C22 fatty acid content significantly increased by 85-160%. Conclusions: Overall our results suggest that heat shock treatment induced the expression of amiRNAs, which can effectively down-regulate the expression of CrPEPCs in C. reinhardtii, resulting in an increase of fatty acid synthesis with the most significant increase occurring for C16 to C22 fatty acids. [ABSTRACT FROM AUTHOR]

Background: Ashbya gossypii is a filamentous fungus that is currently exploited for the industrial production of riboflavin. The utilization of A. gossypii as a microbial biocatalyst is further supported by its ability to grow in low-cost feedstocks, inexpensive downstream processing and the availability of an ease to use molecular toolbox for genetic and genomic modifications. Consequently, A. gossypii has been also introduced as an ideal biotechnological chassis for the production of inosine, folic acid, and microbial oils. However, A. gossypii cannot use xylose, the most common pentose in hydrolysates of plant biomass. Results: In this work, we aimed at designing A. gossypii strains able to utilize xylose as the carbon source for the production of biolipids. An endogenous xylose utilization pathway was identified and overexpressed, resulting in an A. gossypii xylose-metabolizing strain showing prominent conversion rates of xylose to xylitol (up to 97% after 48 h). In addition, metabolic flux channeling from xylulose-5-phosphate to acetyl-CoA, using aheterologous phosphoketolase pathway, increased the lipid content in the xylose-metabolizing strain a 54% over the parental strain growing in glucose-based media. This increase raised to 69% when lipid accumulation was further boosted by blocking the beta-oxidation pathway. Conclusions: Ashbya gossypii has been engineered for the utilization of xylose. We present here a proof-of-concept study for the production of microbial oils from xylose in A. gossypii, thus introducing a novel biocatalyst with very promising properties in developing consolidated bioprocessing to produce fine chemicals and biofuels from xyloserich hydrolysates of plant biomass. [ABSTRACT FROM AUTHOR]

Background: Yarrowia lipolytica is an ascomycete yeast used in biotechnological research for its abilities to secrete high concentrations of proteins and accumulate lipids. Genetic tools have been made in a variety of backgrounds with varying similarity to a comprehensively sequenced strain. Results: We have developed a set of genetic and molecular tools in order to expand capabilities of Y. lipolytica for both biological research and industrial bioengineering applications. In this work, we generated a set of isogenic auxotrophic strains with decreased non-homologous end joining for targeted DNA incorporation. Genome sequencing, assembly, and annotation of this genetic background uncovers previously unidentified genes in Y. lipolytica. To complement these strains, we constructed plasmids with Y. lipolytica-optimized superfolder GFP for targeted overexpression and fluorescent tagging. We used these tools to build the "Yarrowia lipolytica Cell Atlas," a collection of strains with endogenous fluorescently tagged organelles in the same genetic background, in order to define organelle morphology in live cells. Conclusions: These molecular and isogenetic tools are useful for live assessment of organelle-specific protein expression, and for localization of lipid biosynthetic enzymes or other proteins in Y. lipolytica. This work provides the Yarrowia community with tools for cell biology and metabolism research in Y. lipolytica for further development of biofuels and natural products. [ABSTRACT FROM AUTHOR]

Rising oil prices and concerns over climate change have resulted in more emphasis on research into renewable biofuels from microalgae. Unlike plants, green microalgae have higher biomass productivity, will not compete with food and agriculture, and do not require fertile land for cultivation. However, microalgae biofuels currently suffer from high capital and operating costs due to low yields and costly extraction methods. Microalgae grown under optimal conditions produce large amounts of biomass but with low neutral lipid content, while microalgae grown in nutrient starvation accumulate high levels of neutral lipids but are slow growing. Producing lipids while maintaining high growth rates is vital for biofuel production because high biomass productivity increases yield per harvest volume while high lipid content decreases the cost of extraction per unit product. Therefore, there is a need for metabolic engineering of microalgae to constitutively produce high amounts of lipids without sacrificing growth. Substrate availability is a rate-limiting step in balancing growth and fatty acid (FA) production because both biomass and FA synthesis pathways compete for the same substrates, namely acetyl-CoA and NADPH. In this review, we discuss the efforts made for improving biofuel production in plants and microorganisms, the challenges faced in achieving lipid productivity, and the important role of precursor supply for FA synthesis. The main focus is placed on the enzymes which catalyzed the reactions supplying acetyl-CoA and NADPH. [ABSTRACT FROM AUTHOR]

Background: Current biological pathways to produce biofuel intermediates amenable to separations and catalytic upgrading to hydrocarbon fuels are not cost effective. Previously, oleaginous yeasts have been investigated primarily for lipid production. However, yeasts store neutral lipids intracellularly making recovery difficult and expensive. In addition, once recovered from the cells, lipids are difficult to blend directly with the existing fuels without upgrading. We have, therefore, begun to investigate secreted fatty acid-derived products which can be easily recovered and upgraded to fuels. Results: In this study, we successfully demonstrate the production of fatty alcohols by the oleaginous yeasts, Yarrowia lipolytica and Lipomyces starkeyi, through expression of the fatty acyl-CoA reductase gene from Marinobactor aquaeolei VT8. This strategy resulted in the production of 167 and 770 mg/L of fatty alcohols in shake flask from Y. lipolytica and L starkeyi, respectively. When using a dodecane overlay during fermentation, 92 and 99% of total fatty alcohols produced by Y. lipolytica and L. starkeyi, respectively, were extracted into the dodecane phase, which compares favorably to the 3 and 50% recovered, respectively, without the dodecane layer. In both oleaginous yeasts, long chain length, saturated fatty alcohols, i.e., hexadecanol (C16:0) and octadecanol (C18:0), were predominant and accounted for more than 85% of the total fatty alcohols produced. To the best of our knowledge, this is the first report of fatty alcohol production in L. starkeyi. Conclusion: This work demonstrates that the oleaginous yeasts, Y. lipolytica and L. starkeyi, can serve as platform organisms for the production of fatty acid-derived biofuels and bioproducts. [ABSTRACT FROM AUTHOR]

Background: The oleaginous yeast, Yarrowia lipolytica, has been utilized as an industrial host for about 60 years for various applications. Recently, the metabolic engineering of this host has become increasingly popular due to its ability to accumulate lipids as well as improvements made toward developing new genetic tools. Y. lipolytica can robustly metabolize glucose, glycerol, and even different lipid classes. However, little is known about its xylose metabolizing capability. Given the desirability of having a robust xylose utilizing strain of Y. lipolytica, we performed a comprehensive investigation and elucidation of the existing components of its xylose metabolic pathway. Results: A quick and efficient means of determining functionality of the candidate xylose pathway genes (XYR, XDH, and XKS) from Y. lipolytica was desirable. We challenged Escherichia coli mutants lacking either the xylose isomerase (xylA) gene or the xylulose kinase (xylB) gene to grow on xylose minimal media by expressing the candidate genes from Y. lipolytica. We showed that the XKS of Y. lipolytica is able to rescue xylose growth of E. coli ?xylB, and the XDH enabled growth on xylitol, but not on xylose, of E. coli ?xylA. Overexpression of XKS and XDH in Y. lipolytica improved growth on xylitol, indicating that expression of the native enzymes was limiting. Overexpression of XKS and XDH in Y. lipolytica also enables robust growth on xylose under high nitrogen conditions without the need for adaptation. These results prove that a complete xylose pathway exists in Y. lipolytica, but the pathway is poorly expressed. To elucidate the XYR gene, we applied the E. coli ?xylA xylose growth challenge with 14 candidate XYR genes and XDH. The XYR2 candidate was able to rescue growth of E. coli ?xylA xylose on minimal media. Conclusions: While a native xylose pathway exists in Y. lipolytica, the microorganism's inability to grow robustly on xylose is an effect of cryptic genetic circuits that control expression of key enzymes in the metabolic pathway. We have characterized the key enzymes associated with xylose metabolism and demonstrated that gene regulatory issues can be overcome using strong hybrid promoters to attain robust growth on xylose without adaptation. [ABSTRACT FROM AUTHOR]

Background: Industrial biotechnology that is able to provide environmentally friendly bio-based products has attracted more attention in replacing petroleum-based industries. Currently, most of the carbon sources used for fermentation-based bioprocesses are obtained from agricultural commodities that are used as foodstuff for human beings. Lignocellulose-derived sugars as the non-food, green, and sustainable alternative carbon sources have great potential to avoid this dilemma for producing the renewable, bio-based hydrocarbon fuel precursors, such as microbial lipid. Efficient bioconversion of lignocellulose-based sugars into lipids is one of the critical parameters for industrial application. Therefore, the fed-batch cultivation, which is a common method used in industrial applications, was investigated to achieve a high cell density culture along with high lipid yield and productivity. Results: In this study, several fed-batch strategies were explored to improve lipid production using lignocellulosic hydrolysates derived from corn stover. Compared to the batch culture giving a lipid yield of 0.19 g/g, the dissolvedoxygen- stat feeding mode increased the lipid yield to 0.23 g/g and the lipid productivity to 0.33 g/L/h. The pulse feeding mode further improved lipid productivity to 0.35 g/L/h and the yield to 0.24 g/g. However, the highest lipid yield (0.29 g/g) and productivity (0.4 g/L/h) were achieved using an automated online sugar control feeding mode, which gave a dry cell weight of 54 g/L and lipid content of 59 % (w/w). The major fatty acids of the lipid derived from lignocellulosic hydrolysates were predominately palmitic acid and oleic acid, which are similar to those of conventional oilseed plants. Conclusions: Our results suggest that the fed-batch feeding strategy can strongly influence the lipid production. The online sugar control feeding mode was the most appealing strategy for high cell density, lipid yield, and lipid productivity using lignocellulosic hydrolysates as the sole carbon source. [ABSTRACT FROM AUTHOR]

Background: Commercial production of microalgal biodiesel is not yet economically viable, largely because of low storage lipid yield in microalgae mass cultivation. Selection of lipid-rich microalgae, thus, becomes one of the key research topics for microalgal biodiesel production. However, the laboratory screening protocols alone cannot predict the ability of the strains to dominate and perform in outdoor ponds. Comprehensive assessment of microalgae species should be performed not only under the laboratory conditions, but also in the fields. Results: Laboratory investigations using a bubbled column photobioreactor indicated the microalga Graesiella sp. WBG-1 to be the most productive species among the 63 Chlorophyta strains. In a 10 L reactor, mimicking the industrial circular pond, Graesiella sp. WBG-1 produced 12.03 g biomass m-2 day-1 and 5.44 g lipids (45.23 % DW) m-2 day-1 under 15 mol m-2 day-1 artificial light irradiations. The lipid content decreased to ∼34 % DW when the microalga was cultured in 30 L tank PBR under natural solar irradiations, but the decline of lipid content with scaling up was the minimum among the tested strains. Based on these results, the microalga was further tested for its lipid production and culture competitiveness using a pilot-scale raceway pond (200 m2 illuminated area, culture volume 40,000 L). Consequently, Graesiella sp. WBG-1 maintained a high lipid content (33.4 % DW), of which ∼90 % was storage TAGs. Results from the outdoor experiments indicated the nice adaptability of the Graesiella sp. WBG-1 to strong and fluctuating natural solar irradiance and temperature, and also demonstrated several other features, such as large cell size (easy for harvest and resistant to swallow by protozoa) and tolerance to high culture pH (helpful to CO2 fixation). Conclusions: Graesiella sp. WBG-1 was a promising strain capable of accumulating large amount of storage lipid under nature solar irradiance and temperature. The high lipid content of 33.4 % DW was achieved for the first time in pilot-scale raceway pond. The results also provide evidence for the feasibility of using low-cost raceway pond for autotrophic cultivation of microalgae for biodiesel production. [ABSTRACT FROM AUTHOR]

Background: The marine diatom, Phaeodactylum tricornutum, has become a model for studying lipid metabolism and its triacylglycerol (TAG) synthesis pathway makes it an ideal target for metabolic engineering to improve lipid productivity. However, the genetic background and metabolic networks of fatty acid biosynthesis in diatoms are not well understood. Glycerol-3-phosphate acyltransferase (GPAT) is the critical enzyme that catalyzes the first step of TAG formation. So far, characterization of GPAT in marine microalgae has not been reported, especially at the level of comprehensive sequence-structure and functional analysis. Results: A GPAT was cloned from P. tricornutum and overexpressed in P. tricornutum. Volumes of oil bodies were produced and the neutral lipid content was increased by twofold determined by Nile red fluorescence staining. Fatty acid composition was analyzed by GC-MS, which showed significantly higher proportion of unsaturated fatty acids compared to wild type. Conclusion: These results suggested that the identified GPAT could upregulate TAG biosynthesis in P. tricornutum. Moreover, this study offers insight into the lipid metabolism of diatoms and supports the role of microalgal strains for biofuels production. [ABSTRACT FROM AUTHOR]

Background: Algae have attracted attention as sustainable producers of lipid-containing biomass for food, animal feed, and for biofuels. Parachlorella kessleri, a unicellular green alga belonging to the class Trebouxiophyceae, achieves very high biomass, lipid, and starch productivity levels. However, further biotechnological exploitation has been hampered by a lack of genomic information. Results: Here, we sequenced the whole genome and transcriptome, and analyzed the behavior of P. kessleri NIES- 2152 under lipid production-inducing conditions. The assembly includes 13,057 protein-coding genes in a 62.5-Mbp nuclear genome. Under conditions of sulfur deprivation, lipid accumulation was correlated with the transcriptomic induction of enzymes involved in sulfur metabolism, triacylglycerol (TAG) synthesis, autophagy, and remodeling of light-harvesting complexes. Conclusions: Three-dimensional transmission electron microscopy (3D-TEM) revealed extensive alterations in cellular anatomy accompanying lipid hyperaccumulation. The present 3D-TEM results, together with transcriptomic data support the finding that upregulation of TAG synthesis and autophagy are potential key mediators of the hyperaccumulation of lipids under conditions of nutrient stress. [ABSTRACT FROM AUTHOR]

Background: Fatty acid composition is an important physiological parameter of microalgae, which is taken as the third generation alternative resource of biodiesel. To boost microalgal research and applications, a convenient, rapid, and acid-catalyzed transesterification procedure that satisfies the demand for the analysis of the fatty acid composition of lipids with micro-scale samples in the high-throughput screening of microalgal strains is needed, along with the evaluation of the physiological status of microalgae in response to nutrient stress. Results: The reaction conditions of transesterification via a micro-mixer reactor were optimized as follows: 90 °C reaction temperature, 20 min reaction time, 6:1 volume ratio of H2SO4-methanol to lipid-in-hexane, and a Y-type micromixer with a 20-m-long extended loop that has a 0.3 mm diameter. The minimum amount of sample was decreased to 30 µg lipids. The new approach was successfully applied to the fatty acid composition analysis of soybean oil and microalgal lipids. Definitely, it could be applied to acyl related oils from different sources. Conclusion: Here, we have developed a simple and rapid method for the analysis of the fatty acid composition of lipids. The new method requires less than 20 min for transesterification and a minimum of only 30 µg lipid sample. Furthermore, a high-throughput process can be easily realized by numbering up the micro-mixer reactors. The micromixer reactor has great potential for applications not only in large-scale biodiesel production but also for the microscale analysis of microalgae fatty acid compositions. [ABSTRACT FROM AUTHOR]

Background: Microalgae are considered promising alternative energy sources because they consume CO2 and accumulate large amounts of lipids that can be used as biofuel. Nannochloropsis is a particularly promising microalga due to its high growth rate and lipid content, and the availability of genomic information. Transcription factors (TFs) are global regulators of biological pathways by up- or down-regulation of related genes. Among these, basic helix-loophelix (bHLH) TFs regulate growth, development, and stress responses in plants and animals, and have been identified in microalgae. We identified two bHLH TFs in the genome of N. salina CCMP1776, NsbHLH1, and NsbHLH2, and characterized functions of NsbHLH2 that may be involved in growth and nutrient uptake. Results: We obtained NsbHLH2 overexpressing transformants of N. salina CCMP1776 by particle bombardment and confirmed that these were stable transformants. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting using antibodies against the FLAG tag that was attached at the end of the coding sequence confirmed the expression of the NsbHLH2 protein under various culture conditions. The qRT-PCR results also indicated that the endogenous and transgenic expression of NsbHLH2 was reduced under stressed conditions. Overexpression of NsbHLH2 led to increased growth rate in the early growth period, and concomitantly higher nutrient uptake, than wild type (WT). These enhanced growth and nutrient uptake resulted in increased productivities of biomass and FAME. For example, one of the transformants, NsbHLH2 3-6, showed increased biomass productivity by 36 % under the normal condition, and FAME productivity by 33 % under nitrogen limitation condition. Conclusively, the improved growth in the transformants can be associated with the enhanced nutrient uptake. We are currently assessing their potential for scale-up cultivation with positive outcomes. Conclusion: Overexpression of NsbHLH2 led to enhanced growth rate and nutrient uptake during the early growth phase, and increased biomass and FAME productivity, especially in the later period under normal and stressed conditions. Based on these results, we postulate that NsbHLH2 can be employed for the industrial production of biodiesel from N. salina. [ABSTRACT FROM AUTHOR]

Background: The colonial microalga Botryococcus braunii has been brought to people's attention for its conspicuous ability to accumulate a variety of lipids including hydrocarbons. B. braunii strains are classified into 3 races based on the types of hydrocarbons. A and B races are known to accumulate high level of lipids. However, their extreme slow growth rate has impeded its application for renewable biofuel production. Results: In this study, we report the transcriptomic response of a moderately growing subisolate from the culture of Botryococcus sp. CCALA-779 upon nitrogen deprivation (ND). We show that the subisolate has an average growth rate of 0.52 g l-1 day-1 under photoautotrophic growth conditions and lipid content is enhanced to 75 % of CDW upon ND. Both rDNA sequence and hydrocarbon composition analyses indicate that the subisolate belongs to A race B. braunii. Hence, it is designated as B. braunii 779. We show that B. braunii 779 transcriptome shares homology to majority of the A race but not B race B. braunii ESTs, suggesting that transcriptomes of A race differ from that of B race. We found that many homologous ESTs between A races 779 and Bot-88 are unknown sequences, implying that A race contains many unknown genes. Pathway-based transcriptomic analysis indicates that energy metabolisms are among the top expressed functions in log-phase cells, indicating that the slow growth rate is a result that energy flow is directed to lipid biosynthesis but not population growth. Upon ND, reconfiguration of metabolisms for reducing power is apparent, suggesting that B. braunii 779 is rapidly adapting under ND condition by transcriptomic reprogramming. Conclusions: Taken together, our result shows that the subisolate B. braunii 779, similar to the Gottingen strain, is useful for biofuel production. Difference between transcriptomes of A and B races implies that different races of B. braunii strains belong to different sub-species. Furthermore, there are many novel genes that are unique to A race, suggesting that sequences of many enzymes involved in hydrocarbon biosynthesis are not currently known. We propose that B. braunii transcriptomes provide a rich source for discovery of novel genes involved in hydrocarbon biosynthesis. [ABSTRACT FROM AUTHOR]

Background: Lignocellulose is known to be an abundant source of glucose and xylose for biofuels. Yeasts can convert glucose into bioethanol. However, bioconversion of xylose by yeasts is not very efficient, to say nothing of the presence of both glucose and xylose. Efficient utilization of xylose is one of the critical factors for reducing the cost of biofuel from lignocelluloses. However, few natural microorganisms preferentially convert xylose to ethanol. The simultaneous utilization of both glucose and xylose is the pivotal goal in the production of biofuels. Results: In this paper, we found that 97.3 % of the glucose and 93.8 % of the xylose in our experiments was consumed by black soldier fly (BSF) simultaneously. The content of lipid reached its highest level (34.60 %) when 6 % xylose was added into the standard feed. 200 g of rice straw was pretreated with 1 % KOH, followed by enzymatic hydrolysis for fermentation of ethanol, the residue from this fermentation was then fed to BSF for lipid accumulation. In total, 10.9 g of bioethanol and 4.3 g of biodiesel were obtained. Conclusions: The results of this study suggest that BSF is a very promising organism for use in converting lignocellulose into lipid for biodiesel production. [ABSTRACT FROM AUTHOR]

Background: Lignocellulosic biomass has long been recognized as a potential sustainable source of sugars for biofuels. However, many physicochemical structural and compositional factors inhibit the enzymatic digestibility of the lignocellulosic biomass. In this study, efficient pretreatment method of rice straw (RS) was developed and the RS hydrolysate was applied in the cultivation of microalgae for lipid production. Results: Gamma ray irradiation (GRI) and alkali solution were used for the pretreatment, and saccharification was carried out with lignocellulolytic enzymes. When RS was pretreated by combined GRI and alkali method, the glucose and xylose saccharification yield after enzymatic hydrolysis increased up to 91.65 and 98.84 %, respectively. The enzymatic hydrolysate from the RS pretreated with the combined method was used to cultivate Chlorella protothecoides for lipid production. The maximum concentrations of biomass and fatty acid methyl ester of cells were 6.51 and 2.95 g/L, respectively. The lipid content of C. protothecoides from RS hydrolysate was comparable to that from glucose, and the lipid composition was similar between different carbon sources. Conclusion: These results demonstrate that the combined pretreatment with gamma irradiation was highly effective in preparing hydrolysate, and the rice straw hydrolysate could be used as an alternative carbon source for microalgal lipid production for biofuel. [ABSTRACT FROM AUTHOR]

Background: Yarrowia lipolytica, one of the most widely studied "nonconventional" oleaginous yeast species, is unable to grow on cellobiose. Engineering cellobiose-degrading ability into this yeast is a vital step towards the development of cellulolytic biocatalysts suitable for consolidated bioprocessing. Results: In the present work, we identified six genes encoding putative ß-glucosidases in the Y. lipolytica genome. To study these, homologous expression was attempted in Y. lipolytica JMY1212 Zeta. Two strains overexpressing BGL1 (YALI0F16027g) and BGL2 (YALI0B14289g) produced ß-glucosidase activity and were able to degrade cellobiose, while the other four did not display any detectable activity. The two active ß-glucosidases, one of which was mainly cell-associated while the other was present in the extracellular medium, were purified and characterized. The two Bgls were most active at 40-45°C and pH 4.0-4.5, and exhibited hydrolytic activity on various ß-glycoside substrates. Specifically, Bgl1 displayed 12.5-fold higher catalytic efficiency on cellobiose than Bgl2. Significantly, in experiments where cellobiose or cellulose (performed in the presence of a ß-glucosidase-deficient commercial cellulase cocktail produced by Trichoderma reseei) was used as carbon source for aerobic cultivation, Y. lipolytica ?pox co-expressing BGL1 and BGL2 grew better than the Y. lipolytica strains expressing single BGLs. The specific growth rate and biomass yield of Y. lipolytica JMY1212 co-expressing BGL1 and BGL2 were 0.15 h-1 and 0.50 g-DCW/g-cellobiose, respectively, similar to that of the control grown on glucose. Conclusions: We conclude that the bi-functional Y. lipolytica developed in the current study represents a vital step towards the creation of a cellulolytic yeast strain that can be used for lipid production from lignocellulosic biomass. When used in combination with commercial cellulolytic cocktails, this strain will no doubt reduce enzyme requirements and thus costs. [ABSTRACT FROM AUTHOR]

Background: Microalgae have received considerable interest as a source of biofuel production. The unicellular green alga Pseudochoricystis ellipsoidea (non-validated scientific name) strain Obi appears to be suitable for large-scale cultivation in outdoor open ponds for biodiesel production because it accumulates lipids to more than 30 % of dry cell weight under nitrogen-depleted conditions. It also grows rapidly under acidic conditions at which most protozoan grazers of microalgae may not be tolerant. The lipid productivity of this alga could be improved using genetic engineering techniques; however, genetically modified organisms are the subject of regulation by specific laws. Therefore, the aim of this study was to develop a self-cloning-based positive selection system for the breeding of P. ellipsoidea. Results: In this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system. The cDNA of the uridine monophosphate synthase gene (PeUMPS) of P. ellipsoidea was cloned downstream of the promoter of either a beta-tubulin gene (PeTUBULIN1) or the gene for the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (PeRBCS) to construct the pUT1 or pUT2 plasmid, respectively. These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates. Use of Noble agar as the solidifying agent was essential to avoid the development of false-positive colonies. It took more than 6 weeks for the formation of colonies of pUT1 transformants, whereas pUT2 transformants formed colonies in 2 weeks. Real-time PCR revealed that there were more PeUMPS transcripts in pUT2 transformants than in pUT1 transformants. Uracil synthesis (Ura+) transformants were also obtained using a gene cassette consisting solely of PeUMPS flanked by the PeRBCS promoter and terminator. Conclusions: A self-cloning-based positive selection system for the genetic transformation of P. ellipsoidea was developed. Self-cloned P. ellipsoidea strains will require less-stringent containment measures for large-scale outdoor cultivation. [ABSTRACT FROM AUTHOR]

Background: Rising CO2 concentration was reported to increase phytoplankton growth rate as well as lipid productivity. This has raised questions regarding the NADPH supply for high lipid synthesis as well as rapid growth of algal cells. Results: In this study, growth, lipid content, photosynthetic performance, the activity, and expression of key enzymes in Calvin cycle and oxidative pentose phosphate pathway (OPPP) were analyzed in the marine diatom Phaeodactylum tricornutum under three different CO2 concentrations (low CO2 (0.015 %), mid CO2 (atmospheric, 0.035 %) and high CO2 (0.15 %)). Both the growth rate and lipid content of P. tricornutum increased significantly under the high CO2 concentration. Enzyme activity and mRNA expression of three Calvin cycle-related enzymes (Rubisco, 3-phosphoglyceric phosphokinase (PGK), phosphoribulokinase (PRK)) were also increased under high CO2 cultivation, which suggested the enhancement of Calvin cycle activity. This may account for the observed rapid growth rate. In addition, high activity and mRNA expression of G6PDH and 6PGDH, which produce NADPH through OPPP, were observed in high CO2 cultured cells. These results indicate OPPP was enhanced and might play an important role in lipid synthesis under high CO2 concentration. Conclusions: The oxidative pentose phosphate pathway may participate in the lipid accumulation in rapid-growth P. tricornutum cells in high CO2 concentration. [ABSTRACT FROM AUTHOR]

Background: Large-scale algal biofuel production has been limited, among other factors, by the availability of inorganic carbon in the culture medium at concentrations higher than achievable with atmospheric CO2. Life cycle analyses have concluded that costs associated with supplying CO2 to algal cultures are significant contributors to the overall energy consumption. Results: A two-phase optimal growth and lipid accumulation scenario is presented, which (1) enhances the growth rate and (2) the triacylglyceride (TAG) accumulation rate in the oleaginous Chlorophyte Chlorella vulgaris strain UTEX 395, by growing the organism in the presence of low concentrations of NaHCO3 (5 mM) and controlling the pH of the system with a periodic gas sparge of 5 % CO2 (v/v). Once cultures reached the desired cell densities, which can be "fine-tuned" based on initial nutrient concentrations, cultures were switched to a lipid accumulation metabolism through the addition of 50 mM NaHCO3. This two-phase approach increased the specific growth rate of C. vulgaris by 69 % compared to cultures sparged continuously with 5 % CO2 (v/v); further, biomass productivity (gL-1 day-1) was increased by 27 %. Total biodiesel potential [assessed as total fatty acid methyl ester (FAME) produced] was increased from 53.3 to 61 % (FAME biomass-1) under the optimized conditions; biodiesel productivity (g FAMEL-1 day-1) was increased by 7.7 %. A bicarbonate salt screen revealed that American Chemical Society (ACS) and industrial grade NaHCO3 induced the highest TAG accumulation (% w/w), whereas Na2CO3 did not induce significant TAG accumulation. NH4HCO3 had a negative effect on cell health presumably due to ammonia toxicity. The raw, unrefined form of trona, NaHCO3·Na2CO3 (sodium sesquicarbonate) induced TAG accumulation, albeit to a slightly lower extent than the more refined forms of sodium bicarbonate. Conclusions: The strategic addition of sodium bicarbonate was found to enhance growth and lipid accumulation rates in cultures of C. vulgaris, when compared to traditional culturing strategies, which rely on continuously sparging algal cultures with elevated concentrations of CO2(g). This work presents a two-phased, improved photoautotrophic growth and lipid accumulation approach, which may result in an overall increase in algal biofuel productivity. [ABSTRACT FROM AUTHOR]

Background: Many waste streams have a relatively high vegetable oil content, which is a potential resource that should be recovered. Microbial storage compound production for the recovery of lipids from lipid-water emulsions with open (unsterilized) microbial cultures was investigated in a sequencing batch reactor using a diluted vegetable oil emulsion as model substrate. Results: After feeding, triacylglycerides (TAG) were accumulated intracellular by the microbial enrichment culture and subsequently used for growth in the remainder of the sequencing batch cycle. Roughly 50% of the added TAG could be recovered as intracellular lipids in this culture. The maximum lipid storage capacity of the enrichment culture was 54% on volatile suspended solids (VSS) mass basis in a separate fed-batch accumulation experiment. The microbial community was dominated by a lipolytic fungus, Trichosporon gracile, that was responsible for intracellular lipid accumulation but also a significant fraction of lipolytic and long chain fatty-acid-utilizing bacteria was present. Conclusion: Herewith, we demonstrate an effective strategy for enrichment of a microbial community that can accumulate significant amounts of lipids from wastewaters without the need for sterilization of substrates or equipment. Further optimization of this process will make recovery of lipids from wastewater possible. [ABSTRACT FROM AUTHOR]

Background: Marine microalgae are among the most promising lipid sources for biodiesel production because they can be grown on nonarable land without the use of potable water. Marine microalgae also harvest solar energy efficiently with a high growth rate, converting CO2 into lipids stored in the cells. Both light intensity and nitrogen availability strongly affect the growth, lipid accumulation, and fatty acid composition of oleaginous microalgae. However, very few studies have systematically examined how to optimize lipid productivity by adjusting irradiance intensity, and the metabolic dynamics that may lead to improved lipid accumulation in microalgae have not been elucidated. Little is known about the mechanism of lipid synthesis regulation in microalgae. Moreover, few studies have assessed the potential of using marine microalgae as oil producers. Results: In this work, a newly isolated marine microalga, Chlamydomonas sp. JSC4, was selected as a potential lipid producer, and the effect of photobioreactor operations on cell growth and lipid production was investigated. The combined effects of light intensity and nitrogen depletion stresses on growth and lipid accumulation were further explored in an effort to markedly improve lipid production and quality. The optimal lipid productivity and content attained were 312 mg L-1 d-1 and 43.1% per unit dry cell weight, respectively. This lipid productivity is the highest ever reported for marine microalgae. Metabolic intermediates were profiled over time to observe transient changes during lipid accumulation triggered by combined stresses. Finally, metabolite turnover was also assessed using an in vivo 13C-labeling technique to directly measure the flow of carbon during lipid biosynthesis under stress associated with light intensity and nitrogen deficiency. Conclusions: This work demonstrates the synergistic integration of cultivation and dynamic metabolic profiling technologies to develop a simple and effective strategy for enhancing oil production in a marine microalga. The knowledge obtained from this study could be useful in assessing the feasibility of marine microalgae biodiesel production and for understanding the links between dynamic metabolic profiles and lipid biosynthesis during the course of microalgal cultivation. [ABSTRACT FROM AUTHOR]

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

Background: Photoautotrophic microalgae are a promising avenue for sustained biodiesel production, but are compromised by low yields of biomass and lipids at present. We are developing a chemical approach to improve microalgal accumulation of feedstock lipids as well as high-value alpha-linolenic acid which in turn might provide a driving force for biodiesel production. Results: We demonstrate the effectiveness of the small bioactive molecule "acetylcholine" on accumulation of biomass, total lipids, and alpha-linolenic acid in Chlorella sorokiniana. The effectiveness exists in different species of Chlorella. Moreover, the precursor and analogs of acetylcholine display increased effectiveness at higher applied doses, with maximal increases by 126, 80, and 60% over controls for biomass, total lipids, and alpha-linolenic acid, respectively. Production of calculated biodiesel was also improved by the precursor and analogs of acetylcholine. The biodiesel quality affected by changes in microalgal fatty acid composition was addressed. Conclusion: The chemical approach described here could improve the lipid yield and biodiesel production of photoautotrophic microalgae if combined with current genetic approaches. [ABSTRACT FROM AUTHOR]

Background The concept of adaptive evolution implies underlying genetic mutations conferring a selective advantage to an organism under particular environmental conditions. Thus, a flow cytometry-based strategy was used to study the adaptive evolution in Chlamydomonas reinhardtii wild type CC124 and a starchless mutant sta6-1 cells, with respect to lipid metabolism under nitrogen (N) deplete and replete conditions. Results The successive sorting and regeneration of top 25000 high-lipid content cells of CC124 and sta6-1 combined with nitrogen starvation led to the generation of a new population with improved lipid content when compared to the original populations, ~175% and ~50% lipid increase in sta6-1 and CC124, respectively. During adaptive evolution period, the major fatty acid components observed in cells were C16:0, C16:1, C18:0, and C18:1-3, and elemental analysis revealed that cellular carbon to nitrogen ratio increased at the end of adaptive evolution period. In order to gain an insight into highly stimulated intracellular lipid accumulation in CC124 and sta6-1 resulting from the adaptive evolution, proteomics analyses of newly generated artificial high-lipid content populations were performed. Functional classifications showed the heightened regulation of the major chlorophyll enzymes and the enzymes involved in carbon fixation and uptake including chlorophyll-ab-binding proteins, and Rubisco activase. The key control protein (periplasmic L-amino acid oxidase (LAO1)) of carbon-nitrogen integration was specifically over-expressed. Glutathione-S-transferases and esterase, the enzymes involved in lipid-metabolism and lipid-body associated proteins, were also induced during adaptive evolution. Conclusions Adaptive evolution results demonstrate the potential role of photosynthesis in terms of carbon partitioning/flux/fixation and carbon/nitrogen metabolism during lipid accumulation in microalgae and this strategy can be a new tool to develop C. reinhardtii strains and other microalgal strains with desired phenotypes such as high lipid accumulation. [ABSTRACT FROM AUTHOR]

Background Biodiesel production from marine microalgae has received much attention as microalgae can be cultivated on non-arable land without the use of potable water, and with the additional benefits of mitigating CO2 emissions and yielding biomass. However, there is still a lack of effective operational strategies to promote lipid accumulation in marine microalgae, which are suitable for making biodiesel since they are mainly composed of saturated and monounsaturated fatty acids. Moreover, the regulatory mechanisms involved in lipid biosynthesis in microalgae under environmental stress are not well understood. Results In this work, the combined effects of salinity and nitrogen depletion stresses on lipid accumulation of a newly isolated marine microalga, Chlamydomonas sp. JSC4, were explored. Metabolic intermediates were profiled over time to observe transient changes during the lipid accumulation triggered by the combination of the two stresses. An innovative cultivation strategy (denoted salinity-gradient operation) was also employed to markedly improve the lipid accumulation and lipid quality of the microalga, which attained an optimal lipid productivity of 223.2 mg L-1 d-1 and a lipid content of 59.4% per dry cell weight. This performance is significantly higher than reported in most related studies. Conclusions This work demonstrated the synergistic integration of biological and engineering technologies to develop a simple and effective strategy for the enhancement of oil production in marine microalgae. [ABSTRACT FROM AUTHOR]

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]

Background Microalgae can accumulate considerable amounts of lipids under different nutrient-deficient conditions, making them as one of the most promising sustainable sources for biofuel production. These inducible processes provide a powerful experimental basis for fully understanding the mechanisms of physiological acclimation, lipid hyperaccumulation and gene expression in algae. In this study, three nutrient-deficiency strategies, viz nitrogen-, phosphorus- and iron-deficiency were applied to trigger the lipid hyperaccumulation in an oleaginous Chlorella pyrenoidosa. Regular patterns of growth characteristics, lipid accumulation, physiological parameters, as well as the expression patterns of lipid biosynthesis-related genes were fully analyzed and compared. Results Our results showed that all the nutrient stress conditions could enhance the lipid content considerably compared with the control. The total lipid and neutral lipid contents exhibit the most marked increment under nitrogen deficiency, achieving 50.32% and 34.29% of dry cell weight at the end of cultivation, respectively. Both photosynthesis indicators and reactive oxygen species parameters reveal that physiological stress turned up when exposed to nutrient depletions. Time-course transcript patterns of lipid biosynthesis-related genes showed that diverse expression dynamics probably contributes to the different lipidic phenotypes under stress conditions. By analyzing the correlation between lipid content and gene expression level, we pinpoint several genes viz. rbsL, me g6562, accA, accD, dgat g2354, dgat g3280 and dgat g7063, which encode corresponding enzymes or subunits of malic enzyme, ACCase and diacylglycerol acyltransferase in the de novo TAG biosynthesis pathway, are highly related to lipid accumulation and might be exploited as target genes for genetic modification. Conclusion This study provided us not only a comprehensive picture of adaptive mechanisms from physiological perspective, but also a number of targeted genes that can be used for a systematic metabolic engineering. Besides, our results also represented the feasibility of lipid production through trophic transition cultivation modes, throwing light on a two-stage microalgal lipid production strategy with which heterotrophy stage provides sufficient robust seed and nitrogen-starvation photoautotrophy stage enhances the overall lipid productivity. [ABSTRACT FROM AUTHOR]