Shake Flask Cultures Research Articles

Engineering Vibrio alginolyticus as a novel chassis for PHB production from starch.

Vibrio alginolyticus LHF01 was engineered to efficiently produce poly-3-hydroxybutyrate (PHB) from starch in this study. Firstly, the ability of Vibrio alginolyticus LHF01 to directly accumulate PHB using soluble starch as the carbon source was explored, and the highest PHB titer of 2.06g/L was obtained in 18h shake flask cultivation. Then, with the analysis of genomic information of V. alginolyticus LHF01, the PHB synthesis operon and amylase genes were identified. Subsequently, the effects of overexpressing PHB synthesis operon and amylase on PHB production were studied. Especially, with the co-expression of PHB synthesis operon and amylase, the starch consumption rate was improved and the PHB titer was more than doubled. The addition of 20g/L insoluble corn starch could be exhausted in 6-7h cultivation, and the PHB titer was 4.32g/L. To the best of our knowledge, V. alginolyticus was firstly engineered to produce PHB with the direct utilization of starch, and this stain can be considered as a novel host to produce PHB using starch as the raw material.

Use of agricultural wastes in rhamnolipid production by Pseudomonas aeruginosa USM-AR2

This study investigates agricultural wastes as potential fermentation substrates to provide a cheaper solution in rhamnolipid production by P. aeruginosa USM-AR2 while simultaneously attempting to recycle waste. Rhamnolipid production was estimated by orcinol assay, oil spreading test (OST), and emulsification index (E24). The wastes used were waste cooking oil and glycerine pitch. Rhamnolipid fermentation was carried out in shake flask culture using minimal salt medium (MSM) supplemented with 3% (v/v) agricultural wastes as the sole carbon source and NaNO3 as the nitrogen source. MSM supplemented with waste cooking oil exhibited higher biomass, while glycerine pitch resulted in higher rhamnolipid production. Rhamnolipid from MSM supplemented with waste cooking oil demonstrated higher diameter of clearing zone in the OST than glycerine pitch. It exhibited high emulsification activity on waste cooking oil at 63.3 ± 0.03%, with glycerine pitch recording higher E24 at 71.5 ± 0.03%. In conclusion, P. aeruginosa USM-AR2 was able to utilize agricultural wastes to produce appreciable amounts of rhamnolipid.

Escherichia coli aceE variants coding pyruvate dehydrogenase improve the generation of pyruvate-derived acetoin.

Several chromosomally expressed AceE variants were constructed in Escherichia coli ΔldhA ΔpoxB ΔppsA and compared using glucose as the sole carbon source. These variants were examined in shake flask cultures for growth rate, pyruvate accumulation, and acetoin production via heterologous expression of the budA and budB genes from Enterobacter cloacae ssp. dissolvens. The best acetoin-producing strains were subsequently studied in controlled batch culture at the one-liter scale. PDH variant strains attained up to four-fold greater acetoin than the strain expressing the wild-type PDH. In a repeated batch process, the H106V PDH variant strain attained over 43g/L of pyruvate-derived products, acetoin (38.5g/L) and 2R,3R-butanediol (5.0g/L), corresponding to an effective concentration of 59g/L considering the dilution. The acetoin yield from glucose was 0.29g/g with a volumetric productivity of 0.9g/L·h (0.34g/g and 1.0g/L·h total products). The results demonstrate a new tool in pathway engineering, the modification of a key metabolic enzyme to improve the formation of a product via a kinetically slow, introduced pathway. Direct modification of the pathway enzyme offers an alternative to promoter engineering in cases where the promoter is involved in a complex regulatory network.

Microbial Synthesis of l-Fucose with High Productivity by a Metabolically Engineered Escherichia coli

l-Fucose is a natural deoxy hexose found in a variety of organisms. It possesses many physiological effects and has potential applications in pharmaceutical, cosmetic, and food industries. Microbial synthesis via metabolic engineering attracts increasing attention for efficient production of important chemicals. Previously, we reported the construction of a metabolically engineered Escherichia coli strain with high 2'-fucosyllactose productivity. Herein, we further introduced Bifidobacterium bifidum α-l-fucosidase via both plasmid expression and genomic integration and blocked the l-fucose assimilation pathway by deleting fucI, fucK, and rhaA. The highest l-fucose titers reached 6.31 and 51.05 g/L in shake-flask and fed-batch cultivation, respectively. l-Fucose synthesis was little affected by lactose added, and there was almost no 2'-fucosyllactose residue throughout the cultivation processes. The l-fucose productivity reached 0.76 g/L/h, indicating significant potential for large-scale industrial applications.

Metabolic Engineering of De Novo Pathway for the Production of 2'-Fucosyllactose in Escherichia coli.

2'-Fucosyllactose (2'-FL), one of the most abundant oligosaccharides in human milk, has gained increased attention owing to its nutraceutical and pharmaceutical potential. However, limited availability and high-cost of preparation have limited its widespread application and in-depth investigation of its potential functions. Here, a modular pathway engineering was implemented to construct an Escherichia coli strain to improve the biosynthesis titer of 2'-FL. Before overexpression of manB, manC, gmd, wcaG, and heterologous expression of futC, genes wcaJ and lacZ encoding UDP-glucose lipid carrier transferase and β-galactosidase, respectively, were inactivated from E. coli BL21 (DE3) with the CRISPR-Cas9 system, which inhibited the production of 2'-FL. The results showed that final shake flask culture yielded a 3.8-fold increase in 2'-FL (0.98g/L) from the engineered strain ELC07. Fed-batch fermentation conditions were optimized in a 3-L bioreactor. The highest titer of 2'-FL (18.22g/L) was obtained, corresponding to a yield of 0.25g/g glycerol and a substrate conversion of 0.88g/g lactose.

Discovery and Heterologous Expression of Unspecific Peroxygenases

Since 2004, unspecific peroxygenases, in short UPOs (EC. 1.11.2.1), have been explored. UPOs are closing a gap between P450 monooxygenases and chloroperoxidases. These enzymes are highly active biocatalysts for the selective oxyfunctionalisation of C–H, C=C and C-C bonds. UPOs are secreted fungal proteins and Komagataella phaffii (Pichia pastoris) is an ideal host for high throughput screening approaches and UPO production. Heterologous overexpression of 26 new UPOs by K. phaffii was performed in deep well plate cultivation and shake flask cultivation up to 50 mL volume. Enzymes were screened using colorimetric assays with 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol (DMP), naphthalene and 5-nitro-1,3-benzodioxole (NBD) as reporter substrates. The PaDa-I (AaeUPO mutant) and HspUPO were used as benchmarks to find interesting new enzymes with complementary activity profiles as well as good producing strains. Herein we show that six UPOs from Psathyrella aberdarensis, Coprinopsis marcescibilis, Aspergillus novoparasiticus, Dendrothele bispora and Aspergillus brasiliensis are particularly active.

Novel CaLB-like Lipase Found Using ProspectBIO, a Software for Genome-Based Bioprospection

Enzymes have been highly demanded in diverse applications such as in the food, pharmaceutical, and industrial fuel sectors. Thus, in silico bioprospecting emerges as an efficient strategy for discovering new enzyme candidates. A new program called ProspectBIO was developed for this purpose as it can find non-annotated sequences by searching for homologs of a model enzyme directly in genomes. Here we describe the ProspectBIO software methodology and the experimental validation by prospecting for novel lipases by sequence homology to Candida antarctica lipase B (CaLB) and conserved motifs. As expected, we observed that the new bioprospecting software could find more sequences (1672) than a conventional similarity-based search in a protein database (733). Additionally, the absence of patent protection was introduced as a criterion resulting in the final selection of a putative lipase-encoding gene from Ustilago hordei (UhL). Expression of UhL in Pichia pastoris resulted in the production of an enzyme with activity towards a tributyrin substrate. The recombinant enzyme activity levels were 4-fold improved when lowering the temperature and increasing methanol concentrations during the induction phase in shake-flask cultures. Protein sequence alignment and structural modeling showed that the recombinant enzyme has high similarity and capability of adjustment to the structure of CaLB. However, amino acid substitutions identified in the active pocket entrance may be responsible for the differences in the substrate specificities of the two enzymes. Thus, the ProspectBIO software allowed the finding of a new promising lipase for biotechnological application without the need for laborious and expensive conventional bioprospecting experimental steps.

Chromosome evolution of Escherichia coli Nissle 1917 for high-level production of heparosan.

Heparosan is a crucial-polysaccharide precursor for the chemoenzymatic synthesis of heparin, a widely used anticoagulant drug. Presently, heparosan is mainly extracted with the potential risk of contamination from Escherichia coli strain K5, a pathogenic bacterium causing urinary tract infection. Here, a nonpathogenic probiotic, E. coli strain Nissle 1917 (EcN), was metabolically engineered to carry multiple copies of the 19-kb kps locus and produce heparosan to 9.1 g/L in fed-batch fermentation. Chromosome evolution driven by antibiotics was employed to amplify the kps locus, which governed the synthesis and export of heparosan from EcN at 21 mg L-1 OD-1 . The average copy number of kps locus increased from 1 to 24 copies per cell, which produced up to 104 mg L-1 OD-1 of heparosan in the shaking flask cultures of engineered strains. The following in-frame deletion of recA stabilized the recombinant duplicates of chromosomal kps locus and the productivity of heparosan in continuous culture for at least 56 generations. Fed-batch fermentation of the engineered strain EcN8 was carried out to bring the yield of heparosan up to 9.1 g/L. Heparosan from the fermentation culture was further purified at a 75% overall recovery. The structure of purified heparosan was characterized and further modified by N-sulfotransferase with 3'-phosphoadenosine-5'-phosphosulfate as the sulfo-donor. The analysis of element composition showed that heparosan was N-sulfated by over 80%. These results indicated that duplicating large DNA cassettes up to 19-kb, followed by high-cell-density fermentation, was promising in the large-scale preparation of chemicals and could be adapted to engineer other industrial-interest bacteria metabolically.

Heterologous expression and characterization of an M4 family extracellular metalloprotease for detergent application.

Proteolytic enzymes stand out as the most widely employed category utilized in manufacturing industry. A new protease was separated from Planococcus sp.11815 strain and named as nprS-15615 in this research. The gene of this protease has not been reported, and its enzymatic properties have been studied for the first time. To enhance enzyme production, the Planococcus sp. protease gene was expressed in Bacillus licheniformis 2709. The expression level of nprS-15615 was observed under the control of regulatory elements PaprE. nprS-15615 protease activity reached 1186.24±32.87 U/mL after 48 hours of cultivation in shake flasks which was nearly four times the output of the original bacteria (291.38±25.73U/mL). The optimum temperature and pH of the recombinant protease were 30 ℃ and 8.0, respectively.The enzyme exhibited the highest capacity for hydrolyzing casein and demonstrated resilience towards a NaCl concentration of 10.0% (wt/v). Furthermore, in the presence of 0.5% surfactants, the recombinant protease activity can maintain above 75%, and with the existence of 0.5% liquid detergents, there was basically no loss of enzyme activity which indicated that nprS-15615 had good compatibility with surfactants and liquid detergents. In addition, npS-15615 performed well in the washing experiment, and the washing effect at 20 ℃ can be significantly improved by adding crude enzyme solution in the washing process.

Development of high methanol-tolerance Pichia pastoris based on iterative adaptive laboratory evolution

High methanol-tolerant strains were obtained using iterative adaptive laboratory evolution (microbial microdroplet culture (MMC) and shake flask culture (SFC)).

High cell density cultivation of Bacillus subtilis NCIM 2063: Modeling, optimization and a scale-up procedure

Bacillus subtilis is a non-pathogenic, sporulating, Gram-positive bacteria with pronounced antimicrobial and metabolic activity and great potential for wide application in various fields. The aim of this paper was to determine the optimum B. subtilis NCIM 2063 growth conditions and to scale up biomass production from shake flasks to a bioreactor level. The critical growth para-meters and their interaction effects were studied using Box?Behnken experimental design and response surface methodology. Developed model equations were statistically significant with good prediction capability. It was found that during shake flask cultivation glucose should be added in concentration up to 5 g L-1 in DSM medium, OTR at 10 mol m-3 h-1 and temperature of 33?C, to achieve the maximum number of viable cells and spores. To scale up the process from shake flasks to the bioreactor level kLa was used as a main criterion. Scale up effect was evaluated by comparing the growth kinetics in the shake flasks and in a laboratory bioreactor. The total number of cells obtained in the bioreactor was 4.57x109 CFU mL-1 which is 1.41 times higher than the number of cells in the shake flasks (3.24x109 CFU mL-1), proving that the scale-up procedure was conducted successfully.

Evaluation of wool protein hydrolysate as peptone for production of microbial enzymes.

Peptones are one of the most expensive components of microbial culture media. The present study was conducted to test the usability of low-cost sheep wool peptone (SWP) as an organic nitrogen source in the production of six industrially important enzymes (lipase, amylase, tannase, pectinase, cellulase and invertase). SWP was prepared by alkaline hydrolysis and acid neutralization. Bacillus licheniformis and Aspergillus niger were selected as test microorganisms for enzyme production. To evaluate the efficacy of SWP in enzyme production, it was compared with commercial tryptone peptone (TP) in the shaking flask cultures of the test microorganisms. The optimum concentration of both SWP and TP was determined to be 8g/L for the production of B. licheniformis-derived enzymes, but 6g/L for the production of A. niger-derived enzymes. It was determined that SWP was superior to TP in the production of four enzymes (lipase, amylase, tannase and pectinase) of both B. licheniformis and A. niger. This is the first study about the usage of sheep wool protein hydrolysate (SWP) as an organic nitrogen source or a peptone in fermentative production of microbial enzymes.

Metabolic engineering of Escherichia coli for the production of (R)-α-lipoic acid.

To increase the production of (R)-α-lipoic acid directly from octanoic acid using engineered Escherichia coli with the regeneration of S-adenosylmethionine. The biosynthesis of (R)-α-lipoic acid (LA) in E. coli BL21(DE3) is improved by co-expression of lipoate-protein ligase A (LplA) from E. coli MG1655 and lipoate synthase (LipA) from Vibrio vulnificus. The engineered strain produces 20.99µgl-1 of LA in shake flask cultures. The titers of LA are increased to 169.28µgl-1 after the optimization of the medium components and fermentation conditions. We find that the [4Fe-4S] cluster is important for the activity of LipA and co-expression of iscSUA promotes the regeneration of the [4Fe-4S] cluster and leads to the highest LA titer of 589.30µgl-1. The method described here can be widely applied for the biosynthesis of (R)-α-lipoic acid and other metabolites.

A Predictive Batch Culture Growth and Biosynthesis for Bacillus cereus (ATCC 14579) using Response Surface Methodology

Optimization studies of Bacillus cereus (ATCC 14579) tolerance to stress phenomenon could serve as a basic prerequisite for its utilization in future biotechnological research. Successful cell culture at optimum parametric conditions was found to prepare the cells to withstand upper and lower range values depending on the need and purpose. This study was performed to develop a predictive optimum model for the growth of this mesophilic bacterium, B. cereus in batch culture shake flasks. The linear and mutual interactions effects of nutrient availability (4-16 g/L) and composition, temperature (30-40 oC) and agitation (140 - 200 rpm) collectively termed as growth regulators were sought using central composite face-centred design (CCFD) response surface methodology (RSM). The effectiveness of the independent variables on the dependent variable was weighted and validated using statistical and graphical indices which spelt a suitable predictive model for B. cereus growth and biosynthesis. This model provided an efficient and reliable approach for predicting the growth of B. cereus as a function of the growth-influencing markers. The results showed that the model term is highly significant at P >0.0001 and a well-correlated adjusted R2 and predicted R2 of less than 1.0 (0.9984). Moreover, the coefficient of determination value of only 1.45 % variability as well as agreed predictive (3.01) versus experimental (3.0) values depicted that the hidden (noise) effect was very minimal. Therefore, the model further confirmed the versatility of the isolate to simple growth nutrient within defined optimal physical operational parameters of simple shake flask culture.

Inoculation with phosphate‐solubilizing bacteria alters microbial community and activates soil phosphorus supply to promote maize growth

AbstractPhosphorus (P) is one of the essential macronutrients for plants, but agricultural soil generally lacks available P. This study aims to ascertain the impact of phosphate‐solubilizing bacterial inoculation on the development of maize, soil characteristics, P fractions, and the microbial population in the soil. In this experiment, we explored the P solubilizing ability of P‐solubilizing bacteria through liquid culture. In a maize pot experiment, soil P fractions and maize growth were examined, and a metagenomic sequence was constructed to analyze the soil microbial community and gene abundances and to link their relationship. In the shaking flask culture experiment, it was found that the Burkholderia (XQP35) and Raoultella (SQP80) have a strong capability for calcium phosphate and calcium phytate. Compared with the blank control, the XQP35 and SQP80 treatments could improve maize biomass and the soil nutrient status. Moreover, SQP80 treatments increased the soil acid phosphatase activity. SQP80 inoculation dramatically decreased the variety of the soil microbial community and the abundance of Acidobacteria. We found that the different treatments had no significant effect on the number of genes but that the SQP80 treatments significantly increased the P‐cycling pathway gene family phoA and phoD compared with the CK treatment. Our results demonstrated that inoculation with XQP35 and SQP80 could dissolve varieties of insoluble P and, together with functional pathways of P‐cycling, promote the transformation of P fractions and ultimately increase soil available P content to promote the development of maize.

Optimization of l-malic acid production from acetate with Aspergillus oryzae DSM 1863 using a pH-coupled feeding strategy

BackgroundMalic acid, a dicarboxylic acid mainly used in the food industry, is currently produced from fossil resources. The utilization of low-cost substrates derived from biomass could render microbial processes economic. Such feedstocks, like lignocellulosic hydrolysates or condensates of fast pyrolysis, can contain high concentrations of acetic acid. Acetate is a suitable substrate for l-malic acid production with the filamentous fungus Aspergillus oryzae DSM 1863, but concentrations obtained so far are low. An advantage of this carbon source is that it can be used for pH control and simultaneous substrate supply in the form of acetic acid. In this study, we therefore aimed to enhance l-malate production from acetate with A. oryzae by applying a pH-coupled feeding strategy.ResultsIn 2.5-L bioreactor fermentations, several feeding strategies were evaluated. Using a pH-coupled feed consisting of 10 M acetic acid, the malic acid concentration was increased about 5.3-fold compared to the batch process without pH control, resulting in a maximum titer of 29.53 ± 1.82 g/L after 264 h. However, it was not possible to keep both the pH and the substrate concentration constant during this fermentation. By using 10 M acetic acid set to a pH of 4.5, or with the repeated addition of NaOH, the substrate concentration could be maintained within a constant range, but these strategies did not prove beneficial as lower maximum titers and yields were obtained. Since cessation of malic acid production was observed in later fermentation stages despite carbon availability, a possible product inhibition was evaluated in shake flask cultivations. In these experiments, malate and succinate, which is a major by-product during malic acid production, were added at concentrations of up to 50 g/L, and it was found that A. oryzae is capable of organic acid production even at high product concentrations.ConclusionsThis study demonstrates that a suitable feeding strategy is necessary for efficient malic acid production from acetate. It illustrates the potential of acetate as carbon source for microbial production of the organic acid and provides useful insights which can serve as basis for further optimization.

Hierarchical dynamic regulation of Saccharomyces cerevisiaefor enhanced lutein biosynthesis.

Lutein, as a carotenoid with strong antioxidant capacity and an important component of macular pigment in the retina, has wide applications in pharmaceutical, food, feed, and cosmetics industries. Besides extraction from plant and algae, microbial fermentation using engineered cell factories to produce lutein has emerged as a promising route. However, intra-pathway competition between the lycopene cyclases and the conflict between cell growth and production are two major challenges. In our previous study, de novo synthesis of lutein had been achieved in Saccharomyces cerevisiae by dividing the pathway into two stages (δ-carotene formation and conversion) using temperature as the input signal to realize sequential cyclation of lycopene. However, lutein production was limited to microgram level, which is still too low to meet industrial demand. In this study, a dual-signal hierarchical dynamic regulation system was developed and applied to divide lutein biosynthesis into three stages in response to glucose concentration and culture temperature. By placing the genes involved in δ-carotene formation under the glucose-responsive ADH2 promoter and genes involved in the conversion of δ-carotene to lutein under temperature-responsive GAL promoters, the growth-production conflict and intra-pathway competition were simultaneously resolved. Meanwhile, the rate-limiting lycopene ε-cyclation and carotene hydroxylation reactions were improved by screening for lycopene ε-cyclase with higher activity and fine tuning of the P450 enzymes and their redox partners. Finally, a lutein titer of 19.92 mg/L (4.53 mg/g DCW) was obtained in shake-flask cultures using the engineered yeast strain YLutein-3S-6, which is the highest lutein titer ever reported in heterologous production systems.

Optimization of Culture Conditions and Wheat Bran Class Selection in the Production of Bacillus thuringiensis-Based Biopesticides

Bacillus thuringiensis is the leading microbial-based biopesticide, thanks to its parasporal crystal proteins or δ-endotoxins, which are toxic to insect larvae upon ingestion. Once in the insect larvae midgut, the crystal is solubilized by the alkaline pH and the δ-endotoxins activated by proteolytic cleavage. Thanks to its high efficiency as a biopesticide, several efforts have been made to enhance its growth and δ-endotoxins production, in various types of culture media. In this study, a culture medium based on wheat bran (WB), the by-product of cereal grain milling, was used to grow Bacillus thuringiensis and produce δ-endotoxins. Using the response surface methodology (RSM), the effects of three variables were evaluated: WB particles granulometry, their concentration, and their agitation in a 48-h shake-flask culture at 30 °C. Three response parameters were targeted: δ-endotoxins production, final culture pH, and dry-matter consumption. According to the RSM results, the optimum would be at 3.7 g WB/50 mL, with a granulometry above 680 μm and agitation between 170 and 270 rpm. This study is key to developing natural and cheap culture media that can be used at an industrial level for Bacillus thuringiensis-based biopesticides.

Biosynthesis of cannabinoid precursor olivetolic acid in genetically engineered Yarrowia lipolytica

Engineering microbes to produce plant-derived natural products provides an alternate solution to obtain bioactive products. Here we report a systematic approach to sequentially identify the rate-limiting steps and improve the biosynthesis of the cannabinoid precursor olivetolic acid (OLA) in Yarrowia lipolytica. We find that Pseudomonas sp LvaE encoding a short-chain acyl-CoA synthetase can efficiently convert hexanoic acid to hexanoyl-CoA. The co-expression of the acetyl-CoA carboxylase, the pyruvate dehydrogenase bypass, the NADPH-generating malic enzyme, as well as the activation of peroxisomal β-oxidation pathway and ATP export pathway are effective strategies to redirect carbon flux toward OLA synthesis. Implementation of these strategies led to an 83-fold increase in OLA titer, reaching 9.18 mg/L of OLA in shake flask culture. This work may serve as a baseline for engineering cannabinoids biosynthesis in oleaginous yeast species.

Biotechnological Conversions of Mizithra Second Cheese Whey by Wild-Type Non-Conventional Yeast Strains: Production of Yeast Cell Biomass, Single-Cell Oil and Polysaccharides

The cultivation of oleaginous yeasts on various agro-industrial residues and the subsequent production of microbial lipids (single-cell oils), which can be used as starting materials for the synthesis of both “second generation” biodiesel and various types of oleochemicals, is considered as one of the most important approaches of the Industrial Biotechnology, in terms of circular economy and green and sustainable development. In this study, seven wild-type non-conventional yeast strains were evaluated for their growth on a semi-defined medium with cheese whey lactose as a sole carbon source. Five of these strains were further batch-cultivated into the complex substrate that contained second cheese whey, which is the main by-product of Mizithra whey cheese manufacture, after centrifugation and filtration without any extra mineral salts, carbon, or nitrogen source addition. All these five strains grown in second cheese whey produced mainly yeast biomass and to lesser extent microbial lipids and other interesting metabolites, such as polysaccharides. The strain Cryptococcus curvatus ATCC 20509 produced the highest total dry weight (TDW) amount (22.4 g/L), that contained 3.4 g/L of intra-cellular lipids, followed by C. curvatus NRRL Y-1511, which produced 20.6 g/L of TDW and 3.2 g/L lipids. A novel, non-previously systematically studied strain, namely Papiliotrema laurentii NRRL Y-2536, produced significant quantities of TDW (22.0 g/L) and, interestingly, secreted quantities of exopolysaccharides. Fed-batch shake-flask cultivation of C. curvatus ATCC 20509 in pretreated second cheese whey, pulse-supplemented with condensed cheese whey-derived lactose, led to the significant TDW quantity of 38.1 g/L that contained c. 57% w/w of total lipids (lipids at a concentration 21.7 g/L were produced). Cellular lipids of all microorganisms, mainly stored as triacylglycerols, contained in variable quantities the fatty acids Δ9C18:1, C16:0, Δ9,12C18:2 and C18:0, constituting perfect candidates for the synthesis of “second generation” biodiesel.