Total lipid and fatty acid composition of seaweeds for the selection of species for oil‐based biofuel and bioproducts

We investigated the potential of seaweeds as feedstock for oil‐based products, and our results support macroalgae (seaweeds) as a biomass source for oil‐based bioproducts including biodiesel. Not only do several seaweeds have high total lipid content above 10% dry weight, but in the brown alga Spatoglossum macrodontum 50% of these lipids are in the form of extractable fatty acids. S. macrodontum had the highest fatty acid content (57.40 mg g^?1 dw) and a fatty acid profile rich in saturated fatty acids with a high content of C18:1, which is suitable as a biofuel feedstock. Similarly, the green seaweed Derbesia tenuissima has high levels of fatty acids (39.58 mg g^?1 dw), however, with a high proportion of PUFA (n‐3) (31% of total lipid) which are suitable as nutraceuticals or fish oil replacements. Across all species of algae the critical parameter of fatty acid content (measured as fatty acid methyl esters, FAME) was positively correlated (R² = 0.67) with total lipid content. However, the proportion of fatty acids to total lipid decreased markedly with total lipid content, generally between 30% and 50%, making it an inaccurate measure of the potential to identify seaweeds suitable for oil‐based bioproducts. Finally, we quantified within species variation of fatty acids across locations and sampling periods supporting either environmental effects on quantitative fatty acid profiles, or genotypes with specific quantitative fatty acid profiles, thereby opening the possibility to optimize the fatty acid content and quality for oil production through specific culture conditions and selective breeding.     

Feasibility of oleaginous fungi isolated from soil samples of Saudi Arabia for mycodiesel production

As the demand for biofuels for transportation is increasing, it is necessary to acquire technologies that will allow affordable production of biodiesel. Conventional biodiesel is mainly produced from vegetable oil by chemical transesterification, but this product has relatively low yield and is competing with agricultural land that can be used for food production. In the present study, 14 filamentous fungi were isolated from different types of soil from Al-Hassa, Saudi Arabia. Nile red staining revealed that lipid bodies were present in 6 of the 14 fungal isolates. Lipid extraction showed that 3 fungi were able to accumulate lipid >20% (wt/wt) of their dry cell mass (0.59–2.40 g/L). The profile of fatty acids revealed a high content of oleic (C18:1, n9), palmitic (C16:0) and linoleic (C18:2) acids similar to conventional vegetable oils used for biodiesel production. Isolate RY4, with the highest lipid yield was identified as Mucor circinelloides based on morphological characteristics confirmed with 18S rRNA gene sequencing. Sequence data of nucleotides were obtained from DNA sequencing submitted to GenBank [GenBank: AB916546.1]. Certain oil compounds were determined by FTIR spectroscopy.     

Lipid productivity and fatty acid composition-guided selection of Chlorella strains isolated from Malaysia for biodiesel production

The need to develop biomass-based domestic production of high-energy liquid fuels (biodiesel) for transportation can potentially be addressed by exploring microalgae with high lipid content. Selecting the strains with adequate oil yield and quality is of fundamental importance for a cost-efficient biofuel feedstock production based on microalgae. This work evaluated 29 strains of Chlorella isolated from Malaysia as feedstock for biodiesel based on volumetric lipid productivity and fatty acid profiles. Phylogenetic studies based on 18S rRNA gene revealed that majority of the strains belong to true Chlorella followed by Parachlorella. The strains were similarly separated into two groups based on fatty acid composition. Of the 18 true Chlorella strains, Chlorella UMACC187 had the highest palmitic acid (C16:0) content (71.3?±?4.2 % total fatty acids, TFA) followed by UMACC84 (70.1?±?0.7 %TFA), UMACC283 (63.8?±?0.7 %TFA), and UMACC001 (60.3?±?4.0 %TFA). Lipid productivity of the strains at exponential phase ranged from 34.53 to 230.38 mg L^?1 day^?1, with Chlorella UMACC050 attaining the highest lipid productivity. This study demonstrated that Chlorella UMACC050 is a promising candidate for biodiesel feedstock production.     

Lipid quality of the diatoms Skeletonema costatum and Navicula gregaria from the South Atlantic Coast (Argentina): evaluation of its suitability as biodiesel feedstock

Since the fatty acid ester profile of a given biofuel is relatively consistent with the source’s fatty acid profile, the properties of the biodiesel produced from a particular feedstock exhibit predictable quality. Thus, lipid fractions and the fatty acid composition of stationary growth-phase cultures of the local strains of the diatoms Skeletonema costatum and Navicula gregaria were analysed to evaluate their suitability as biodiesel feedstock. Total lipid content was 20.83 pg cell⁻¹ in S. costatum and 19.17 pg cell⁻¹ in N. gregaria. Neutral lipids were the main fraction of total lipids in both species, accounting for ca. 65% and 76%, respectively. S. costatum was predominant in saturated fatty acids (SFAs; 43.48 %) and monounsaturated fatty acids (MUFAs; 40.11%), while N. gregaria was predominant in MUFAs (54.85%), followed by SFAs (33.42%). In S. costatum, the main fatty acids in neutral lipid fraction were myristic, palmitic, palmitoleic and oleic acids, while the main ones in N. gregaria were palmitic and palmitoleic acids. The oils extracted from these species presented linolenic acid contents within biodiesel’s quality specifications. However, in neutral lipid fraction both species showed eicosapentaenoic acid levels higher than the required limit. The lipid quality analysed in both species suggests that a biodiesel derived from these oils may present an acceptable cetane number, but likely poor cold-flow properties. This baseline information is useful for future research tending to find more suitable conditions in order to improve oil yield. In addition, both estuarine species neither compete with agriculture for food nor require farmland nor fresh water.     

Oligounsaturated fatty acid production by selected strains of micromycetes

Fifteen strains of filamentous fungi from theCulture Collection of Fungi (Charles University, Prague) were tested for their lipid production, fatty acid composition with emphasis on accumulation of oligounsaturated fatty acids. All cultures contained palmitic (16:0), palmitoleic (16:1), stearic (18:0), oleic (18:1), linoleic (18:2) and γ-linolenic (18:3) acid (GLA). The mycelium ofCunninghamella elegans, Rhizopus arrhizus, Mortierella parvispora, M. elongata andM. alpina contained arachidonic acid (ARA) in the range of 2.3–33.5% of the total fatty acids. The strains used in our experiment were capable to accumulate a relatively high amount of intracellular lipid (9.6–20.1% in dry biomass). The highest content of GLA (22.3 mg/g) was found inMucor circinelloides. The strain ofM. alpina containing 47.1 mg/g of ARA could be considered as the best producer of ARA.     

Mucor circinelloides whole-cells as a biocatalyst for the production of ethyl esters based on babassu oil

The intracellular lipase production by Mucor circinelloides URM 4182 was investigated through a step-by-step strategy to attain immobilized whole-cells with high lipase activity. Physicochemical parameters, such as carbon and nitrogen sources, inoculum size and aeration, were studied to determine the optimum conditions for both lipase production and immobilization in polyurethane support. Olive oil and soybean peptone were found to be the best carbon and nitrogen sources, respectively, to enhance the intracellular lipase activity. Low inoculum level and poor aeration rate also provided suitable conditions to attain high lipase activity (64.8 ± 0.8 U g^?1). The transesterification activity of the immobilized whole- cells was assayed and optimal reaction conditions for the ethanolysis of babassu oil were determined by experimental design. Statistical analysis showed that M. circinelloides whole-cells were able to produce ethyl esters at all tested conditions, with the highest yield attained (98.1 %) at 35 °C using an 1:6 oil-to-ethanol molar ratio. The biocatalyst operational stability was also assayed in a continuous packed bed reactor (PBR) charged with glutaraldehyde (GA) and Aliquat-treated cells revealing half-life of 43.0 ± 0.5 and 20.0 ± 0.8 days, respectively. These results indicate the potential of immobilized M. circinelloides URM 4182 whole-cells as a low-cost alternative to conventional biocatalysts in the production of ethyl esters from babassu oil.     

Biodiesel production from Jatropha oil by catalytic and non-catalytic approaches: an overview

Biodiesel (fatty acids alkyl esters) is a promising alternative fuel to replace petroleum-based diesel that is obtained from renewable sources such as vegetable oil, animal fat and waste cooking oil. Vegetable oils are more suitable source for biodiesel production compared to animal fats and waste cooking since they are renewable in nature. However, there is a concern that biodiesel production from vegetable oil would disturb the food market. Oil from Jatropha curcas is an acceptable choice for biodiesel production because it is non-edible and can be easily grown in a harsh environment. Moreover, alkyl esters of jatropha oil meet the standard of biodiesel in many countries. Thus, the present paper provides a review on the transesterification methods for biodiesel production using jatropha oil as feedstock.     

EVALUATION OF BIOTECHNOLOGICAL POTENTIALS OF SOME INDUSTRIAL FUNGI IN ECONOMICAL LIPID ACCUMULATION AND BIOFUEL PRODUCTION AS A FIELD OF USE

Considering the vast number of scientific reports on various potential uses of fungi, there was an attempt to select the best lipid producer of some fungi at optimized conditions (Aspergillus versicolor, Rhizopus oryzae, Rhizopus arrhizus, Tramates versicolor). The aim was to offer new fields of use to the industries already culturing and using such materials. Aspergillus versicolor mycelia were found to be accumulating the highest amount of lipids. Experiments to improve lipid accumulation and transesterification properties were performed in molasses medium; the first steps were testing the effects of different pH values and different nitrogen sources on lipid accumulation. Various concentrations of KNO₃ (0.5, 1.0, 1.5 gL^?1) and molasses (6%, 8%, 10%) were tried in order to find the optimum carbon and nitrogen requirements. Maximum lipid content was 22.8% in the samples containing 6% molasses solution and 1.0 gL^?1 KNO₃ at pH 4 after 10 days of incubation. The highest fatty acid ethyl ester yield of these samples was 77% (5.0 ethanol:oil, 0.4 sulfuric acid:oil at 30°C for 6 hr). Since the crude lipids were rich in C16 and C18 fatty acids, this was considered as suitable feedstock for biodiesel production.     

Oil content,fatty acid composition and biodiesel properties among natural provenances of Siberian apricot (Prunus sibirica L.) from China

Siberian apricot (Prunus sibirica L.), an excellent woody oil plant unique to Asia, is well known for its ability to produce high‐oil seeds for use as a promising feedstock of biodiesel. Based on the investigation of natural Siberian apricot resources in China in the early stage, seeds of Siberian apricot from 74 geographic provenances which can fully reflect the overall information were collected. In this research, seeds oil content, fatty acid composition and biodiesel properties were evaluated, and the key environmental factors that caused the variation of these in different geographic provenance were analyzed. The oil content of Siberian apricot seeds is 45.48%–61.07%, and the average was 50.95% for all provenances. The characteristics of oil can identify and quantify eight fatty acids. The most abundant fatty acids were oleic acid (C18:1; 54.02%–76.54%), followed by linoleic acid (C18:2; 16.78%–38.49%) and erucic acid (C16:0; from 3.27% to 6.12%). Monounsaturated fatty acids are the most abundant in 54.75%–77.03% compared with saturated fatty acids and polyunsaturated fatty acids. The biodiesel properties of most provenance seeds meet the standards of the ASTM D6751 and GB/T 20828, and a few meet the standards of the EN14214. Through the clustering of oil content and fatty acid composition and the analysis of biodiesel properties indexes, it is concluded that KSK provenance is the most suitable for biodiesel production. The XBZ, HHE, AES, ZLQ and LD provenances may be preserved as potential biodiesel. RDA and VPA showed that the effects of environmental factors on the oil properties of Siberian apricot were ranked as terrain factor > climate factor > soil factor, among which longitude, latitude and altitude are the main terrain indicators. These evaluations can provide reference for the effective utilization and further development of Siberian apricot as a bioenergy feedstock.     

Supercritical fluid extraction of lipids from the heterotrophic microalga Crypthecodinium cohnii

Microalgae biomass can be a feasible source of ω‐3 fatty acids due to its stable and reliable composition. In the present study, the Crypthecodinium cohnii growth and docosahexaenoic acid (DHA, 22:6ω3) production in a 100 L glucose‐fed batch fermentation was evaluated. The lipid compounds were extracted by supercritical carbon dioxide (SC‐CO₂) from C. cohnii CCMP 316 biomas, was and their fatty acid composition was analysed. Supercritical fluid extraction runs were performed at temperatures of 313 and 323 K and pressures of 20.0, 25.0 and 30.0 MPa. The optimum extraction conditions were found to be 30.0 MPa and 323 K. Under those conditions, almost 50% of the total oil contained in the raw material was extracted after 3 h and the DHA composition attained 72% w/w of total fatty acids. The high DHA percentage of total fatty acids obtained by SC‐CO₂ suggested that this extraction method may be suitable for the production of C. cohnii value added products directed towards pharmaceutical purposes. Furthermore, the fatty acid composition of the remaining lipid fraction from the residual biomass with lower content in polyunsaturated fatty acids could be adequate for further uses as feedstock for biodiesel, contributing to the economy of the overall process suggesting an integrated biorefinery approach.     

Simultaneous conversion of free fatty acids and triglycerides to biodiesel by immobilized Aspergillus oryzae expressing Fusarium heterosporum lipase

The presence of high levels of free fatty acids (FFA) in oil is a barrier to one‐step biodiesel production. Undesirable soaps are formed during conventional chemical methods, and enzyme deactivation occurs when enzymatic methods are used. This work investigates an efficient technique to simultaneously convert a mixture of free fatty acids and triglycerides (TAG). A partial soybean hydrolysate containing 73.04% free fatty acids and 24.81% triglycerides was used as a substrate for the enzymatic production of fatty acid methyl ester (FAME). Whole‐cell Candida antarctica lipase B‐expressing Aspergillus oryzae, and Novozym 435 produced only 75.2 and 73.5% FAME, respectively. Fusarium heterosporum lipase‐expressing A. oryzae produced more than 93% FAME in 72 h using three molar equivalents of methanol. FFA and TAG were converted simultaneously in the presence of increasing water content that resulted from esterification. Therefore, F. heterosporum lipase with a noted high level of tolerance of water could be useful in the industrial production of biodiesel from feedstock that has high proportion of free fatty acids.     

Production of gamma-linolenic acid by Mucor circinelloides and Mucor rouxii with acetic acid as carbon substrate

Summary The production of gamma-linolenic acid (GLA) by Mucor circinelloides CBS 203.28 and M. rouxii CBS 416.77 in fed-batch cultures operated in pH-stat mode with acetic acid as carbon substrate and titrant compared favourably with the performance of M. circinelloides in batch culture on glucose. On acetic acid M. circinelloides accumulated up to 39.8 mg GLA/g biomass, with a crude oil content of 28% containing 91% neutral lipids. The GLA content of the neutral lipid fraction was 15.6%.     

Optimizing a continuous flow lipid extraction system (CFLES) used for extracting microalgal lipids

A laboratory‐made continuous flow lipid extraction system (CFLES) was devised to extract lipids from microalgae Nannochloropsis sp., a potential feedstock for biodiesel fuel, with a focus to assess the workable temperatures and pressures for future industrial applications. Using conventional solvents, the CFLES recovered 100% of the lipids extracted with conventional Soxhlet extraction. The optimum temperature and pressure were found to be 100 °C and 50 psi, respectively; conditions significantly lower than those normally used in pressurized liquid extractions requiring specialized equipment. Approximately 87% of the extracted oil was successfully transesterified into biodiesel fuel (fatty acid methyl esters). Preliminary calculations based on the tested lab‐scale system indicated savings in energy, solvent consumption, and extraction time as 96%, 80%, and more than 90%, respectively, as compared to Soxhlet extraction. However, the true cost savings can only be assessed at scaled up level. Energy efficiency of CFLES was calculated as 48.9%. Residual water (~70%) in the biomass had no effect on the extraction performance of CFLES, which is expected to help the process economics at scaled up application. The effect of temperature and pressure on the fatty acids profile of Nannochloropsis sp. is also discussed. Based on the existing literature, the authors believe that a pressurized liquid extraction system with continuous solvent flow has not been reported for lipid extraction from Nannochloropsis sp.     

Molecular Identification and Comparative Evaluation of Tropical Marine Microalgae for Biodiesel Production

Marine microalgae have emerged as important feedstock for liquid biofuel production. The identification of lipid-rich native microalgal species with high growth rate and optimal fatty acid profile and biodiesel properties is the most challenging step in microalgae-based biodiesel production. In this study, attempts have been made to bio-prospect the biodiesel production potential of marine and brackish water microalgal isolates from the west coast of India. A total of 14 microalgal species were isolated, identified using specific molecular markers and based on the lipid content; seven species with total lipid content above 20% of dry cell weight were selected for assessing biodiesel production potential in terms of lipid and biomass productivities, nile red fluorescence, fatty acid profile and biodiesel properties. On comparative analysis, the diatoms were proven to be promising based on the overall desirable properties for biodiesel production. The most potential strain Navicula phyllepta MACC8 with a total lipid content of 26.54 % of dry weight of biomass, the highest growth rate (0.58 day^?1) and lipid and biomass productivities of 114 and 431 mgL^?1 day^?1, respectively, was rich in fatty acids mainly of C16:0, C16:1 and C18:0 in the neutral lipid fraction, the most favoured fatty acids for ideal biodiesel properties. The biodiesel properties met the requirements of fuel quality standards based on empirical estimation. The marine diatoms hold a great promise as feedstock for large-scale biodiesel production along with valuable by-products in a biorefinery perspective, after augmenting lipid and biomass production through biochemical and genetic engineering approaches.     

Triacylglycerol content,productivity and fatty acid profile in Scenedesmus acutus PVUW12

A detailed lipid characterization of Scenedesmus acutus PVUW12, with emphasis on the evaluation of triacylglycerols (TAGs) as a biodiesel feedstock, is presented. When algal cells were grown in nitrogen-free medium (N stress), a lipid increase was detected that was mainly due to TAG accumulation. In situ fluorescence measurements allowed the kinetics and extent of neutral lipid accumulation to be followed. Under N stress, the productivity of total lipids and TAGs increased significantly (80.99 and 63.74 mg L^?1 day^?1, respectively) compared with controls (29.51 and 16.23 mg L^?1 day^?1, respectively). Monounsaturated fatty acids were the major fraction and increased further (49.74 %) in stressed cells, with oleic acid as the most abundant compound (46.97 %). The polyunsaturated fatty acid composition of this algal oil appears to meet the European Standard EN 14214. These results indicate that S. acutus oil meets the requirements for its use as a biodiesel feedstock. Since this strain was also proposed for wastewater bioremediation, this opens up the possibility of its use in an integrated system combined with biofuel production.     

Expression of <Emphasis Type="Italic">Mucor circinelloides</Emphasis> gene for Δ6 desaturase results in the accumulation of high levels of γ-linolenic acid in transgenic tobacco

γ-Linolenic acid (GLA; C18:3 Δ6,9,12) is a nutritionally important fatty acid (FA) playing a vital role in biological structures and cellular functions, which is not produced in oil seed crops. Many oil seed plants, however, produce significant quantities of linoleic acid, a FA that could be converted into GLA by the enzyme Δ6 desaturase, if it is present. As a first step to produce GLA in oil seed crops, we isolated a cDNA encoding the Δ6-FA desaturase from filamentous fungus Mucor circinelloides M29. Expression of this gene in transgenic tobacco resulted in the accumulation of GLA to the levels of 23.1% of the total FA. The results suggested that it is feasible to introduce the M. circinelloides Δ6 desaturase gene into conventional oil crop to produce a large amount of GLA for functional foods and pharmaceutical products. This text was submitted by the authors in English. Y.L. Hao, X.H. Mei, and Y.B. Luo contributed equally to this work.     

Malic enzyme activity is not the only bottleneck for lipid accumulation in the oleaginous fungus Mucor circinelloides

Commercial interest in microbial lipids is increasing due to their potential use as feedstock for biodiesel production. The supply of NADPH generated by malic enzyme (ME; NADP⁺-dependent; EC 1.1.1.40) has been postulated as being the rate-limiting step for fatty acid biosynthesis in oleaginous fungi, based mainly on data from the zygomycete Mucor circinelloides studies. This fungus contains five genes that code for six different ME isoforms. One of these genes, malA, codes for the isoforms III and IV, which have previously been associated with lipid accumulation. Following a strategy of targeted integration of an engineered malA gene, a stable strain overexpressing malA and showing high ME activity has been obtained, demonstrating the feasibility of this strategy to overexpress genes of biotechnological interest in M. circinelloides. This is the first report showing the integration and overexpression of a gene in Zygomycetes. Unexpectedly, the genetically modified strain showed a lipid content similar to that of a prototrophic non-overexpressing control strain, suggesting that another limiting step in the fatty acid synthesis pathway may have been revealed as a consequence of the elimination of malic enzyme-based bottleneck. Otherwise, the fact that prototrophic strains showed at least a 2.5-fold increase in lipid accumulation in comparison with leucine auxotrophic strains suggests that a wild-type leucine biosynthetic pathway is required for lipid accumulation. Moreover, increasing concentrations of leucine in culture medium increased growth of auxotrophs but failed to increase lipid content, suggesting that the leucine synthesized by the fungus is the only leucine available for lipid biosynthesis. These results support previous data postulating leucine metabolism as one of the pathways involved in the generation of the acetyl-CoA required for fatty acid biosynthesis.     

Moringa oleifera oil: a possible source of biodiesel

Biodiesel is an alternative to petroleum-based conventional diesel fuel and is defined as the mono-alkyl esters of vegetable oils and animal fats. Biodiesel has been prepared from numerous vegetable oils, such as canola (rapeseed), cottonseed, palm, peanut, soybean and sunflower oils as well as a variety of less common oils. In this work, Moringa oleifera oil is evaluated for the first time as potential feedstock for biodiesel. After acid pre-treatment to reduce the acid value of the M. oleifera oil, biodiesel was obtained by a standard transesterification procedure with methanol and an alkali catalyst at 60 degrees C and alcohol/oil ratio of 6:1. M. oleifera oil has a high content of oleic acid (>70%) with saturated fatty acids comprising most of the remaining fatty acid profile. As a result, the methyl esters (biodiesel) obtained from this oil exhibit a high cetane number of approximately 67, one of the highest found for a biodiesel fuel. Other fuel properties of biodiesel derived from M. oleifera such as cloud point, kinematic viscosity and oxidative stability were also determined and are discussed in light of biodiesel standards such as ASTM D6751 and EN 14214. The (1)H NMR spectrum of M. oleifera methyl esters is reported. Overall, M. oleifera oil appears to be an acceptable feedstock for biodiesel.     

A reconfigured Kennedy pathway which promotes efficient accumulation of medium‐chain fatty acids in leaf oils

Medium‐chain fatty acids (MCFA, C6‐14 fatty acids) are an ideal feedstock for biodiesel and broader oleochemicals. In recent decades, several studies have used transgenic engineering to produce MCFA in seeds oils, although these modifications result in unbalance membrane lipid profiles that impair oil yields and agronomic performance. Given the ability to engineer nonseed organs to produce oils, we have previously demonstrated that MCFA profiles can be produced in leaves, but this also results in unbalanced membrane lipid profiles and undesirable chlorosis and cell death. Here we demonstrate that the introduction of a diacylglycerol acyltransferase from oil palm, EgDGAT1, was necessary to channel nascent MCFA directly into leaf oils and therefore bypassing MCFA residing in membrane lipids. This pathway resulted in increased flux towards MCFA rich leaf oils, reduced MCFA in leaf membrane lipids and, crucially, the alleviation of chlorosis. Deep sequencing of African oil palm (Elaeis guineensis) and coconut palm (Cocos nucifera) generated candidate genes of interest, which were then tested for their ability to improve oil accumulation. Thioesterases were explored for the production of lauric acid (C12:0) and myristic (C14:0). The thioesterases from Umbellularia californica and Cinnamomum camphora produced a total of 52% C12:0 and 40% C14:0, respectively, in transient leaf assays. This study demonstrated that the introduction of a complete acyl‐CoA‐dependent pathway for the synthesis of MFCA‐rich oils avoided disturbing membrane homoeostasis and cell death phenotypes. This study outlines a transgenic strategy for the engineering of biomass crops with high levels of MCFA rich leaf oils.