Improvement of lipid production from an oil-producing filamentous fungus, <Emphasis Type="Italic">Penicillium brevicompactum</Emphasis> NRC 829, through central composite statistical design

In the present study, 13 filamentous fungi were screened for their lipid production and an oleaginous fungus, Penicillium brevicompactum NRC 829, was found to be the highest lipid producer. Screening of various agro-industrial residues was performed and sunflower oil cake proved to be the best substrate for lipid production. A central composite design was employed to investigate the optimum concentrations of the most significant medium components required to improve the lipid production by P. brevicompactum. The results clearly revealed that the maximal lipid production of 8.014 ± 0.06 gL^?1 (representing 57.6% lipid/dry biomass) was achieved by the fungus when grown for 6 days at 30 °C under static condition in a medium containing sunflower oil cake, NaNO₃ and KCl at final concentrations of 8, 0.75 and 0.25 gL^?1, respectively. Gas chromatography-mass spectrometry analysis of P. brevicompactum lipid indicated that linoleic acid (LA) (C18:2–6, 9) was the most abundant fatty acid, accounting for up to 62% of the total fatty acid profile, followed by palmitoleic acid (C16:1, 16%) and linolenic acid (C18:3, 8%). These results suggest that P. brevicompactum NRC 829 may have potential for commercial development for the production of LA by fermentation using cheap raw material.     

Renewable microbial lipid production from <Emphasis Type="Italic">Oleaginous Yeast</Emphasis>: some surfactants greatly improved lipid production of <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis>

Microbial oil is drawing increasing interest worldwide as an alternative non-food oil feedstock for biodiesel industry. Nowadays researchers have been increasingly focused on the improvement of microbial oil production process. Oleaginous yeast Rhodosporidium toruloides (R. toruloides) is considered an important candidate due to its excellent capabilities of lipid accumulation, broad adaptabilities to various carbon substrates, and the potential of co-production of some pigments. In present work, the individual effects of non-ionic, cationic, and anionic surfactant on cell growth and lipid accumulation of R. toruloides were investigated for the first time. Interesting results were noticed when some anionic surfactants were supplemented. The most significant effect was observed with addition of 0.2 % (w/v) sodium lignosulfonate, that biomass concentration, lipid concentration, and lipid yield was increased by 25.1, 44.9, and 15.7 %, respectively. The fatty acid compositions of R. toruloides lipids remained unchanged, which is similar to that of vegetable oils, and is considered potential feedstock for biodiesel preparation.     

Characterization of cell structural change,growth, lipid accumulation,and pigment profile of a novel oleaginous microalga, <Emphasis Type="Italic">Vischeria stellata</Emphasis> (Eustigmatophyceae), cultured with different initial nitrate supplies

The appropriate microalgal species and the optimal nitrogen supply in culture medium are vital factors in maximizing biomass and metabolite productivities in microalgae. Vischeria stellata is an edaphic unicellular eustigmatophycean microalga. Cytological and ultrastructural characteristics and the effects of different initial nitrate-nitrogen concentrations on growth, lipid accumulation, fatty acid profile, and pigment composition were investigated in the present study. The cell structures of V. stellata changed with the degree of nutrient utilization and growth phase. The initial nitrate concentration for the optimal growth of V. stellata ranged from 6.0 to 9.0 mM. The maximum total lipid (TLs), neutral lipid (NLs), and total fatty acid (TFAs) contents were 55.9, 51.9, and 44.7 % of dry biomass, respectively. The highest volumetric productivity of TLs, NLs, and TFAs reached 0.28, 0.25, and 0.21 g L^?1 day^?1, respectively. V. stellata had a suitable fatty acid profile for biodiesel production, as well as containing eicosapentaenoic acid (EPA) for nutraceutical applications. In addition, the content β-carotene, increased gradually as culture time was prolonged, resulting in its exclusive production at the end of cultivation. V. stellata is a promising microalgal strain for the production of biofuels and bioproducts.     

Optimization of aeration for biodiesel production by <Emphasis Type="Italic">Scenedesmus obliquus</Emphasis> grown in municipal wastewater

Despite the significant breakthroughs in research on microalgae as a feedstock for biodiesel, its production cost is still much higher than that of fossil diesel. One possible solution to overcome this problem is to optimize algal growth and lipid production in wastewater. The present study examines the optimization of pretreatment of municipal wastewater and aeration conditions in order to enhance the lipid productivity of Scenedesmus obliquus. Results showed that no significant differences were recorded in lipid productivity of S. obliquus grown in primary settled or sterilized municipal wastewater; however, ultrasound pretreatment of wastewater significantly decreased the lipid production. Whereas, aeration rates of 0.2 vvm significantly increased lipid content by 51 %, with respect to the non-aerated culture, which resulted in maximum lipid productivity (32.5 mg L^?1 day^?1). Furthermore, aeration enrichment by 2 % CO₂ resulted in increase of lipid productivity by 46 % over the CO₂ non-enriched aerated culture. Fatty acid profile showed that optimized aeration significantly enhanced monounsaturated fatty acid production, composed mainly of C18:1, by 1.8 times over the non-aerated S. obliquus culture with insignificant changes in polyunsaturated fatty acid proportion; suggesting better biodiesel characteristics for the optimized culture.     

Incubation of <Emphasis Type="Italic">Aquilaria subintegra</Emphasis> with Microbial Culture Supernatants Enhances Production of Volatile Compounds and Improves Quality of Agarwood Oil

Incubation with microbial culture supernatants improved essential oil yield from Aquilaria subintegra woodchips. The harvested woodchips were incubated with de man, rogosa and sharpe (MRS) agar, yeast mold (YM) agar medium and six different microbial culture supernatants obtained from Lactobacillus bulgaricus, L. acidophilus, Streptococcus thermophilus, Lactococcus lactis, Saccharomyces carlsbergensis and S. cerevisiae prior to hydrodistillation. Incubation with lactic acid bacteria supernatants provided higher yield of agarwood oil (0.45% w/w) than that obtained from yeast (0.25% w/w), agar media (0.23% w/w) and water (0.22% w/w). The composition of agarwood oil from all media and microbial supernatant incubations was investigated by using gas chromatography-mass spectrometry. Overall, three major volatile profiles were obtained, which corresponded to water soaking (control), as well as, both YM and MRS media, lactic acid bacteria, and yeast supernatant incubations. Sesquiterpenes and their oxygenated derivatives were key components of agarwood oil. Fifty-two volatile components were tentatively identified in all samples. Beta-agarofuran, α-eudesmol, karanone, α-agarofuran and agarospirol were major components present in most of the incubated samples, while S. cerevisiae-incubated A. subintegra provided higher amount of phenyl acetaldehyde. Microbial culture supernatant incubation numerically provided the highest yield of agarwood oil compared to water soaking traditional method, possibly resulting from activity of extracellular enzymes produced by the microbes. Incubation of agarwood with lactic acid bacteria supernatant significantly enhanced oil yields without changing volatile profile/composition of agarwood essential oil, thus this is a promising method for future use.     

Boosting fatty acid synthesis in <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> PD630 by overexpression of autologous thioesterases

Objectives

To explore the role of thioesterases in Rhodococcus opacus PD630 by endogenously overexpression in this bacteria for increased lipid production.

Results

Overexpression of four thioesterases from R. opacus PD630 in E. coli led to a 2- to 8-fold increase in C16:1 and C18:1 fatty acids while, when overexpressed in R. opacus PD630, only two recombinants had significant effect on the quantities and compositions of total fatty acid. The contents of total fatty acids (FAs) in two recombinants, pJTE2 (OPAG_00508 thioesterase) and pJTE4 (WP_012687673.1 thioesterase), were 400–460 mg/g (CDW) which is 1.5 times of wild-type strain PD630 (300-350 mg/g CDW), and 20–30 % (w/w) more than that of the control strain PDpJAM2 (330-370 mg/g CDW). The contents of 17:1 and 18:1 fatty acids increased by about 27 and 35 %, respectively, in pJTE2 and by 35 and 20 %, respectively, in pJTE4 compared with the control strain.

Conclusions

The engineered strains showed improved production of lipid (as total fatty acids), and could also tailor the composition of the fatty acid profile when cultured in mineral salts medium using glucose as sole carbon source.

     

Microalgal Biomass and Lipid Production on Dairy Effluent Using a Novel Microalga, <Emphasis Type="Italic">Chlorella</Emphasis> sp. Isolated from Dairy Wastewater

The present study focused on cost-effective production of microalgal biomass and lipid production on dairy effluent. The novel microalga, Chlorella sp. isolated from the dairy effluent showed high growth and lipid production on the undiluted and two-fold diluted dairy effluent which were four to five times higher than those of Chlorella vulgaris (control). The high growth of Chlorella sp. was thought to be possibly due to its heterotrophic growth capacity, high turbidity, COD, nutrients and trace elements. In contrast, C. vulgaris showed poor heterotrophic and photoautotrophic growth under the highly turbid conditions of dairy effluent. Both Chlorella sp. and C. vulgaris showed similar total FAME (mg FAME/g algal cells). The fatty acid composition analysis revealed that both Chlorella sp. and C. vulgaris possessed major C18 and C20 fatty acids which will be used for biodiesel production. Overall, the novel microalga, Chlorella sp. isolated from the dairy effluent showed high potential for cost-effective algal cultivation and lipid production on dairy effluent without any modification of process.     

Microalgae from the Selenastraceae as emerging candidates for biodiesel production: a mini review

Over the years, microalgae have been identified to be a potential source of commercially important products such as pigments, polysaccharides, polyunsaturated fatty acids and in particular, biofuels. Current demands for sustainable fuel sources and bioproducts has led to an extensive search for promising strains of microalgae for large scale cultivation. Prospective strains identified for these purposes were among others, mainly from the genera Hematococcus, Dunaliella, Botryococcus, Chlorella, Scenedesmus and Nannochloropsis. Recently, microalgae from the Selenastraceae emerged as potential candidates for biodiesel production. Strains from the Selenastraceae such as Monoraphidium sp. FXY-10, M. contortum SAG 47.80, Ankistrodesmus sp. SP2-15 and M. minutum were high biomass and lipid producers when cultivated under optimal conditions. A number of Selenastraceae strains were also reported to be suitable for cultivation in wastewater. This review highlights recent reports on potential strains from the Selenastraceae for biodiesel production and contrasts their biomass productivity, lipid productivity as well as fatty acid profile. Cultivation strategies employed to enhance their biomass and lipid productivity as well as to reduce feedstock cost are also discussed in this paper.     

Production of <Emphasis Type="Italic">cis</Emphasis>-Vaccenic Acid-oriented Unsaturated Fatty Acid in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Biodiesel is produced worldwide as an alternative energy fuel and substitute for petroleum. Biodiesel is often obtained from vegetable oil, but production of biodiesel from plants requires additional land for growing crops and can affect the global food supply. Consequently, it is necessary to develop appropriate microorganisms for the development of an alternative biodiesel feedstock. Escherichia coli is suitable for the production of biodiesel feedstocks since it can synthesize fatty acids for lipid production, grows well, and is amenable to genetic engineering. Recombinant E. coli was designed and constructed for the production of biodiesel with improved unsaturated fatty acid contents via regulation of the FAS pathway consisting of initiation, elongation, and termination steps. Here, we investigated the effects of fabA, fabB, and fabF gene expression on the production of unsaturated fatty acids and observed that the concentration of cis-vaccenic acid, a major component of unsaturated fatty acids, increased 1.77-fold compared to that of the control strain. We also introduced the genes which synthesize malonyl-ACP used during initiation step of fatty acid synthesis and the genes which produce free fatty acids during termination step to study the effect of combination of genes in elongation step and other steps. The total fatty acid content of this strain increased by 35.7% compared to that of the control strain. The amounts of unsaturated fatty acids and cis-vaccenic acid increased by 3.27 and 3.37-fold, respectively.     

<Emphasis Type="Italic">Rhodococcus</Emphasis> bacteria as a promising source of oils from olive mill wastes

The accumulation of triacylglycerols (TAG) is a common feature among actinobacteria belonging to Rhodococcus genus. Some rhodococcal species are able to produce significant amounts of those lipids from different single substrates, such as glucose, gluconate or hexadecane. In this study we analyzed the ability of different species to produce lipids from olive oil mill wastes (OMW), and the possibility to enhance lipid production by genetic engineering. OMW base medium prepared from alperujo, which exhibited high values of chemical oxygen demand (127,000 mg/l) and C/N ratio (508), supported good growth and TAG production by some rhodococci. R. opacus, R. wratislaviensis and R. jostii were more efficient at producing cell biomass (2.2–2.7 g/l) and lipids (77–83% of CDW, 1.8–2.2 g/l) from OMW than R. fascians, R. erythropolis and R. equi (1.1–1.6 g/l of cell biomass and 7.1–14.0% of CDW, 0.1–0.2 g/l of lipids). Overexpression of a gene coding for a fatty acid importer in R. jostii RHA1 promoted an increase of 2.2 fold of cellular biomass value with a concomitant increase in lipids production during cultivation of cells in OMW. This study demonstrates that the bioconversion of OMW to microbial lipids is feasible using more robust rhodococal strains. The efficiency of this bioconversion can be significantly enhanced by engineering strategies.     

Diversity and characterization of cultivable oleaginous yeasts isolated from mangrove forests

A total of 198 yeasts were isolated from 140 samples collected from 7 mangrove forests in 4 provinces of Thailand, and were found to belong to 30 genera, 45 described species and at least 12 undescribed species based on their 26S rRNA (D1/D2 domain) gene sequence. The most prevalent species was Candida tropicalis, followed by Candida pseudolambica and Rhodosporidium paludigena. Lipid accumulation, as determined by Nile red staining, of the isolated yeasts revealed that 69 and 18 strains were positive and strongly positive, respectively, while quantitative analysis of the intracellular lipid accumulated in the latter indicated that 10 of these strains, Pseudozyma tsukubaensis (YWT7-2 and YWT7-3), Rhodotorula sphaerocarpa (YWW6-1 and SFL14-1SF), Saitozyma podzolica (YWT1-1, NS3-3 and NS10-2), Prototheca zopfii var. hydrocarbonea OMS6-1 and Prototheca sp. (YMTW3-1 and YMTS5-2), were oleaginous. In this study we found that under nitrogen depletion condition (155 C/N ratio) Pseudozyma tsukubaensis YWT7-2 accumulated the highest level of intracellular lipid at 32.4% (w/w, dry cell weight), with a broadly similar fatty acid composition to that in palm oil.     

Culture medium influence on growth,fatty acid,and pigment composition of <Emphasis Type="Italic">Choricystis minor</Emphasis> var. <Emphasis Type="Italic">minor</Emphasis>: a suitable microalga for biodiesel production

The purposes of this study were to assess the influence of culture medium on biomass production, fatty acid, and pigment composition of Choricystis minor var. minor and to evaluate the use of this microalga as a source of fatty raw material for biodiesel production. Cultures of C. minor var. minor were grown using WC (Wright’s cryptophyte) and BBM (Bold’s Basal medium) media. BBM medium produced more biomass (984.3 mg L^?1) compared to the WC medium (525.7 mg L^?1). Despite this result, WC medium produced a higher methyl ester yield for biodiesel production than the BBM medium (170.0 and 90.2 mg g^?1 of biomass, respectively). The average percentage of fatty acids obtained using the WC medium (17.0 %) was similar to soybean (18.0 %) and with similar biomass fatty acid profile. However, for pigment production, carotenoids and chlorophyll concentrations were twice as high when using the BBM medium.     

Lipid production by microalgae <Emphasis Type="Italic">Chlorella protothecoides</Emphasis> with volatile fatty acids (VFAs) as carbon sources in heterotrophic cultivation and its economic assessment

Volatile fatty acids (VFAs) that can be derived from food wastes were used for microbial lipid production by Chlorella protothecoides in heterotrophic cultures. The usage of VFAs as carbon sources for lipid accumulation was investigated in batch cultures. Culture medium, culture temperature, and nitrogen sources were explored for lipid production in the heterotrophic cultivation. The concentration and the ratio of VFAs exhibited significant influence on cell growth and lipid accumulation. The highest lipid yield coefficient and lipid content of C. protothecoides grown on VFAs were 0.187 g/g and 48.7 %, respectively. The lipid content and fatty acids produced using VFAs as carbon sources were similar to those seen on growth and production using glucose. The techno-economic analysis indicates that the biodiesel derived from the lipids produced by heterotrophic C. protothecoides with VFAs as carbon sources is very promising and competitive with other biofuels and fossil fuels.     

Association study between copy number variation and beef fatty acid profile of Nellore cattle

The aim of this study was to analyze the association between the copy number variation regions (CNVRs) and fatty acid profile phenotypes for saturated (SFA), monosaturated (MUFA), polyunsaturated (PUFA), ω6 and ω3 fatty acids, PUFA/SFA and ω6/ω3 ratios, as well as for their sums, in Nellore cattle (Bos primigenius indicus). A total of 963 males were finished in feedlot and slaughtered with approximately 2 years of age. Animals were genotyped with the BovineHD BeadChip (Illumina Inc., San Diego, CA, USA). The copy number variation (CNV) detection was performed using the PennCNV algorithm. Log R ratio (LRR) and allele B frequency (BAF) were used to estimate the CNVs. The association analyses were done using the CNVRuler software and applying a logistic regression model. The phenotype was adjusted using a linear model considering the fixed effects of contemporary group and the animal age at slaughter. The fatty acid profile was analyzed on samples of longissimus thoracis muscle using gas chromatography with a 100-m capillary column. For the association analysis, the adjusted phenotypic values were considered for the traits, while the data was adjusted for the effects of the farm and year of birth, management groups at birth, weaning, and superannuation. A total of 186 CNVRs were significant for SFA (43), MUFA (42), PUFA (66), and omega fatty acid (35) groups, totaling 278 known genes. On the basis of the results, several genes were associated with several fatty acids of different saturations. Olfactory receptor genes were associated with C12:0, C14:0, and C18:0 fatty acids. The SAMD8 and BSCL2 genes, both related to lipid metabolic process, were associated with C12:0. The RAPGEF6 gene was found to be associated with C18:2 cis-9 cis-12 n-6, and its function is related to regulation of GTPase activity. Among the results, we highlighted the olfactory receptor activity (GO:0004984), G-protein-coupled receptor activity (GO:0004930), potassium:proton antiporter activity (GO:0015386), sodium:proton antiporter activity (GO:0015385), and odorant-binding (GO:0005549) molecular functions. A large number of genes associated with fatty acid profile within the CNVRs were identified in this study. These findings must contribute to better elucidate the genetic mechanism underlying the fatty acid profile of intramuscular fat in Nellore cattle.     

Engineering <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> for the production of cyclopropanated fatty acids

Traditional synthesis of biodiesel competes with food sources and has limitations with storage, particularly due to limited oxidative stability. Microbial synthesis of lipids provides a platform to produce renewable fuel with improved properties from various renewable carbon sources. Specifically, biodiesel properties can be improved through the introduction of a cyclopropane ring in place of a double bond. In this study, we demonstrate the production of C19 cyclopropanated fatty acids in the oleaginous yeast Yarrowia lipolytica through the heterologous expression of the Escherichia coli cyclopropane fatty acid synthase. Ultimately, we establish a strain capable of 3.03?±?0.26 g/L C19 cyclopropanated fatty acid production in bioreactor fermentation where this functionalized lipid comprises over 32% of the total lipid pool. This study provides a demonstration of the flexibility of lipid metabolism in Y. lipolytica to produce specialized fatty acids.     

Improving of lipid productivity of the biodiesel promising green microalga <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis> via low-energy ion implantation

High lipid content in microalgae is an essential parameter for adopting of microalgal biomass as a feedstock for biodiesel. Mutation is one approach to obtain desired algal strain with high lipid production. In this study, a mutant strain of Chlorella pyrenoidosa was isolated using 1.5?×?10¹⁵ ions cm^?2 s^?1 of N⁺ ion beam implantation technique, which has been widely used in mutagenesis of agricultural crops. N⁺ implantation slightly improved the growth of the mutant over the corresponding wild strain with significant increase in lipid content (32.4 % higher than the wild strain), which resulted in significant increase in lipid productivity by 35 %. In addition, ion implantation mutagenesis of C. pyrenoidosa resulted in 21.4 % decrease in total saturated fatty acids (SFAs) compared to the wild type, with a noticeable increase in polyunsaturated fatty acids (PUFAs). The increase in PUFAs was due mainly to stimulation of hexadecadienoic acid (C16:2) and octadecadienoic acid (C18:2) production. However, the SFA content of wild and mutant strains was 31.7 and 24.9 % of total fatty acids, respectively, highlighting the oxidative stability of biodiesel produced by both strains according to the European standards. Cultivation of C. pyrenoidosa mutant in selenite enrichment medium for five successive cultivation experiments showed insignificant changes in biomass productivity, lipid content, and lipid productivity alongside the study period, which confirms the genetic stability of the produced mutant. The present study confirmed the feasibility of generation of microalgae mutants with significant high lipid production using ion beam implantation.     

High-level productivity of <Emphasis Type="Italic">α,ω</Emphasis>-dodecanedioic acid with a newly isolated <Emphasis Type="Italic">Candida viswanathii</Emphasis> strain

α,ω-Dicarboxylic acids (DC) are versatile chemical intermediates with different chain lengths, which are well-known as polymer building block. In this work, a new strain with high productivity of DC was isolated from oil-contaminated soil. Based on the morphology and phylogenetic analyses of the internal transcribed spacer sequences, it was characterized as Candida viswanathii. It was found that the contribution of carbon flux to the cell growth and DC production from n-dodecane could be regulated by the sucrose and yeast extract concentrations in the medium, and besides the broth pH, a suitable proportioning of sucrose and yeast extract was the key to achieve the optimal transition from cell growth phase to DC production phase. By optimizing culture conditions in a 7.5-L bioreactor, a higher DC productivity of 1.59 g·L^?1 h^?1 with a corresponding concentration of 181.6 g/L was obtained. After the purification of DC from the culture, the results from gas chromatography–mass spectrometry, infrared spectroscopy and ¹H-NMR showed that α,ω-dodecanedioic acid (DC₁₂) was the major product of C. viswanathii ipe-1 using pure n-dodecane as substrate. For the first time, we reported that a high productivity of DC₁₂ could be produced by C. viswanathii.     

<Emphasis Type="Italic">Paradevosia shaoguanensis</Emphasis> gen. nov., sp. nov., Isolated from a Coking Wastewater

A Gram staining negative, rod-shaped, aerobic bacterial strain J5-3^T with a single polar flagellum was isolated from coking wastewater collected from Shaoguan, Guangdong, China. It was motile and capable of optimal growth at pH 6–8, 30 °C, and 0–2 % (w/v) NaCl. Its predominant fatty acids were 11-methyl C_18:1 ω7c (29.2 %), C_16:0 (20.6 %), C_19:0 cyclo ω8c (18.2 %), C_18:0 (11.0 %), and C_18:1 ω7c/C_18:1 ω6c (10.9 %) when grown on trypticase soy agar. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, two unknown glycolipids (GL1, GL2), and two unknown phospholipid (PL1, PL2). The predominant ubiquinone was Q-10, and the genome DNA G+C content was 61.7 mol %. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain J5-3^T belonged to the family Hyphomicrobiaceae in Alphaproteobacteria. It shared the 16S rRNA gene sequence similarities of 93.8–96.1 % with the genus Devosia, 94.5–94.8 % with the genus Pelagibacterium, and <92.0 % with all the other type strains in family Hyphomicrobiaceae. It can be distinguished from the closest phylogenetic neighbors based on several phenotypic and genotypic features, including α-galactosidase activity, tetracycline susceptibility, major fatty acid composition, polar lipid profile, DNA gyrase B subunit (gyrB) gene sequence, and random-amplified polymorphic DNA profile. Therefore, we consider strain J5-3^T to represent a novel species of a novel genus within the family Hyphomicrobiaceae, for which the name Paradevosia shaoguanensis gen. nov., sp. nov. is proposed. The type strain of Paradevosia shaoguanensis is J5-3^T (=CGMCC 1.12430^T =LMG 27409^T).     

Ammonium,nitrate, and urea play different roles for lipid accumulation in the nervonic acid—producing microalgae <Emphasis Type="Italic">Mychonastes afer</Emphasis> HSO-3-1

Producing valuable coproducts from oleaginous microalgae is an option to reduce the total cost of biofuel production. Here, the influence of nitrogen sources on biomass yield and lipid accumulation of a newly identified oleaginous green microalgal strain, Mychonastes afer HSO-3-1, was evaluated. Carbon assimilation and the following lipid biosynthesis of M. afer were inhibited to some extent under weak acidic conditions (6 < pH < 7) and any of the tested nitrogen source. The highest lipid productivity of 50.7 mg L^?1 day^?1 was achieved with a 17.6 mM nitrogen supplement in the form of urea. The cell polar lipid content was significantly higher than triacylglycerol (TAG), and saturated palmitic acid (C16:0) occupied a dominant position in the fatty acid profiles while culturing M. afer in acidic medium with NH₄ ⁺ as the nitrogen source. Under neutral conditions, the lipid productivities of M. afer cultivated in media containing 17.6 mM of NaNO₃, NH₄Cl, and NH₄NO₃ were 76.2, 77.5, and 79.0 mg L^?1 day^?1, respectively. The greatest TAG content (58.56%) of total lipids was obtained when NaNO₃ was used as the nitrogen source. There was no significant difference in the fatty acid composition of M. afer cells when they were cultivated in neutral media supplemented with NaNO₃, urea, NH₄Cl, and NH₄NO₃. Therefore, NH₄ ⁺ was not a suitable nitrogen source for M. afer cultivation due to the additional labor, working procedures, and alkali required to adjust the medium pH. Considering that using urea as nitrogen source could reduce the cost of nutrient salts substantially and urea can be taken up and utilized by most microalgae, it is a preferred nitrogen source. The major properties of biodiesel derived from M. afer HSO-3-1 met biodiesel quality, and nervonic acid concentrations remained at approximately 3.0% of total fatty acids.     

Omega-3 fatty acids concentrate production by enzyme-catalyzed ethanolysis of supercritical CO<Subscript>2</Subscript> extracted oyster oil

The separation of oil by a suitable technique from the Pacific oyster muscle is important for the utilization of the oil as a ω-3 polyunsaturated fatty acids (ω-3 PUFAs) source and production of bio-functional peptides/ oligosaccharides from oil-free residue. This study was conducted to prepare ω-3 PUFAs concentrate from supercritical carbon dioxide (SC-CO₂) extracted Pacific oyster oil by enzyme-catalyzed ethanolysis reactions. SC-CO₂ extractions were done at different temperatures and pressures to optimize suitable extraction conditions and extracted oils were compared with Soxhlet (n-hexane) extracted oil to evaluate the yield and quality. Oil extracted by SC-CO₂ at optimized conditions was used for ethanolysis reaction catalyzed by immobilized sn-1,3 specific lipases, namely Novozymes-435, Lipozyme TLIM, and Lipozyme RMIM to produce 2-monoacylglycerols (2-MAG) rich in ω-3 PUFAs. The optimum temperature and pressure for SC-CO₂ extractions of oyster oil was 50°C and 30 MPa. In this condition, the yield of oil was 5.96% and the acid, peroxide, free fatty acid, and p-anisidine values were 4.49 mg KOH/g, 4.72 meq/kg, 3.42%, and 10.03, respectively. The ω-3 PUFAs content significantly increased in 2-MAG obtained from Novozymes 435, Lipozyme TLIM, and Lipozyme RMIM to 43.03 ± 0.36, 45.95 ± 0.29, and 40.50 ± 0.77%, respectively (p < 0.05). A thin layer chromatography (TLC) analysis confirmed the production and separation of 2-MAG in the ethanolysis process. The ratio of total ω-3 to ω-6 fatty acids was almost twice in 2-MAG of SC-CO₂ extracted oyster oil. SC-CO₂ extracted Pacific oyster oil can be used for sn-1,3 specific lipases catalyzed ethanolysis to produce ω-3 PUFAs rich in 2-MAG.