Microbial lipids from cellulolytic oleaginous fungus Penicillium citrinum PKB20 as a potential feedstock for biodiesel production

Microbial lipids derived from oleaginous fungi are considered as an alternative feedstock for biodiesel production. We attempt to isolate a cellulolytic oleaginous fungi as a potential feedstock for biodiesel production. The fungus was identified by 5.8 S-ITS rRNA gene sequencing. The extracellular enzyme activities were recorded after every 24 h for 7 days. Nile red staining and fluorescence microscopy was used to visualise the lipid bodies within the fungal hyphae. A renewable heterogeneous base catalyst derived from Musa balbisiana cola peels was used for the transesterification of Penicillium citrinum PKB20 derived oil into biodiesel. GC-MS analysis was used to analyse the fatty acid methyl esters (FAME) profile of the transesterified lipids. Penicillium citrinum PKB20 was isolated from detritus rich soil of Assam, India. The endoglucanase, xylanase and β-glucosidase enzyme activities were found to be 292.83 ± 0.29, 111.72 ± 0.45 and 6.54 ± 0.13 U/mg respectively. The specific enzyme activity for extracellular lipase was found to be 3.12 ± 0.16 U/mg. It could accumulate up to 60.61% of lipids in nitrogen-limited medium (7.34 ± 0.45 g/L biomass production). The extracted lipids were converted to biodiesel with 89.3% conversion efficiency. The predominant fatty acids were oleic acid (30.09%), palmitic acid (20.25%) and linoleic acid (33.14%) suggesting a balance between oxidative stability and cold flow properties for suitable biodiesel quality. Penicillium citrinum PKB20 was found to be a potential feedstock for biodiesel production with desirable fuel properties. The cellulolytic nature could be utilised for simultaneous lipid production directly on cellulosic substrates.     

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.     

Using lipidomics to reveal details of lipid accumulation in developing Siberian apricot (Prunus sibirica L.) seed kernels

Siberian apricot seed kernel (SASK) contains a high of 50% oil with suitable fuel properties conformed to biodiesel standard. To date, Prunus sibirica is a novel non‐crop feedstock for biodiesel production in China. Here, oil contents and fatty acid (FA) compositions were identified in developing SASK from AS‐80 and AS‐84, at intervals of 1 week from 3 weeks after anthesis (WAA) to 9 weeks. The major differences in oil content between C18:1 and C18:2 levels were greater among the AS‐80 (32.69/15.48 g/100 g) than among the AS‐84 (25.78/13.15 g/100 g). Subsequently, the SASKs from 4, 6, and 8 WAA, respectively, representing early, middle, and late phases of oil accumulation, were selected as optimal samples for lipidomics analysis. It was notable that 18:1/18:1/18:2, 18:1/18:1/18:3, and 18:2/18:2/18:2 were the prominent compositions in triacylglycerol (TAG), and their higher content found among the AS‐80 was consistent with FA results. Although phosphatidic acid (PA) is directly connected with diacylglycerol (DAG) in Kennedy pathway, we found significant difference between PA and DAG compositions. The resulting molecular species differ in acyl composition depending on whether they were generated via phosphatidylcholine (PC) or Kennedy pathway. By qRT‐PCR analysis, the expression levels of FAD3, PDCT, and DAG‐CPT related to the biosynthesis of polyunsaturated fatty acids (PUFAs) showed a gradual decrease with SASK mature, explaining the drastic change of DAG‐18:3/18:3 content. Additionally, the lipidomics data coupled with qRT‐PCR analysis suggested that phospholipid:DAG acyltransferase may play a critical role in incorporation of PUFAs into sn‐3 of TAG. Our data contribute significantly to understand the underlying mechanisms of lipid accumulation in P. sibirica, and may also present strategies for engineering oil accumulation in oilseed plants.     

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.     

Assessing the potential of fatty acids produced by filamentous fungi as feedstock for biodiesel production

Increased costs and limited availability of traditional lipid sources for biodiesel production encourage researchers to find more sustainable feedstock at low prices. Microbial lipid stands out as feedstock replacement for vegetable oil to convert fatty acid esters. In this study, the potential of three isolates of filamentous fungi (Mucor circinelloides URM 4140, M. hiemalis URM 4144, and Penicillium citrinum URM 4126) has been assessed as single-cell oil (SCO) producers. M. circinelloides 4140 had the highest biomass concentration with lipid accumulation of up to 28?wt% at 120?hr of cultivation. The profile of fatty acids revealed a high content of saturated (SFA) and monounsaturated fatty acids (MUFA), including palmitic (C16:0, 33.2–44.1?wt%) and oleic (C18:1, 20.7–31.2?wt%) acids, with the absence of polyunsaturated fatty acids (PUFA) having more than four double bonds. Furthermore, the predicted properties of biodiesel generated from synthesized SCOs have been estimated by using empirical models which were in accordance with the limits imposed by the USA (ASTM D6715), European Union (EN 14214), and Brazilian (ANP 45/2014) standards. These results suggest that the assessed filamentous fungus strains can be considered as alternative feedstock sources for high-quality biofuel production.     

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.     

The potential of restaurant waste lipids as biodiesel feedstocks

Biodiesel is usually produced from food-grade vegetable oils that are more expensive than diesel fuel. Therefore, biodiesel produced from food-grade vegetable oil is currently not economically feasible. Waste cooking oils, restaurant grease and animal fats are potential feedstocks for biodiesel. These inexpensive feedstocks represent one-third of the US total fats and oil production, but are currently devoted mostly to industrial uses and animal feed. The characteristics of feedstock are very important during the initial research and production stage. Free fatty acids and moisture reduce the efficiency of transesterification in converting these feedstocks into biodiesel. Hence, this study was conducted to determine the level of these contaminants in feedstock samples from a rendering plant. Levels of free fatty acids varied from 0.7% to 41.8%, and moisture from 0.01% to 55.38%. These wide ranges indicate that an efficient process for converting waste grease and animal fats must tolerate a wide range of feedstock properties.     

Bottlenecks in erucic acid accumulation in genetically engineered ultrahigh erucic acid Crambe abyssinica

Erucic acid is a valuable industrial fatty acid with many applications. The main producers of this acid are today high erucic rapeseed (Brassica napus) and mustard (Brassica juncea), which have 45%–50% of erucic acid in their seed oils. Crambe abyssinica is an alternative promising producer of this acid as it has 55%–60% of erucic acid in its oil. Through genetic modification (GM) of three genes, we have previously increased the level of erucic acid to 71% (68 mol%) in Crambe seed oil. In this study, we further investigated different aspects of oil biosynthesis in the developing GM Crambe seeds in comparison with wild‐type (Wt) Crambe, rapeseed and safflower (Carthamus tinctorius). We show that Crambe seeds have very low phosphatidylcholine‐diacylglycerol interconversion, suggesting it to be the main reason why erucic acid is limited in the membrane lipids during oil biosynthesis. We further show that GM Crambe seeds have slower seed development than Wt, accompanied by slower oil accumulation during the first 20 days after flowering (DAF). Despite low accumulation of erucic acid during early stages of GM seed development, nearly 86 mol% of all fatty acids accumulated between 27 and 50 DAF was erucic acid, when 40% of the total oil is laid down. Likely bottlenecks in the accumulation of erucic acid during early stages of GM Crambe seed development are discussed.     

Phosphate solubilizing bacteria enhanced growth,oil yield,antioxidant properties and biodiesel quality of Kasumbha

Biodiesel is considered as a potential alternative energy source, but problem exists with the quantity and quality of feedstock used for it. To improve the feedstock quality of biodiesel, a field experiment was conducted under natural conditions. Cultivar Thori of kasumbha was used in the experiment. Commercialized biofertilizers were applied at the rate of 20 kg per acre and chemical fertilizer (diammonium phosphate) was applied as half dose (15 kg/ha). Results indicated that number of leaf plant^?1, leaf area, number of seeds capitulum^?1 was significantly increased by biofertilizer treatment alone (BF) and combine treatment of biofertilizer and chemical fertilizer (BFCF). Agronomic traits such as plant height, no. of branches of a plant, no. of capitulum/plant was improved significantly by BF treatment over the control. Maximum 1000 seed weight (41%) and seed yield (23%) were recorded in half dose of chemical fertilizers treatment (CFH). Seed oil content and seed phenolics were significantly improved by BF and CF treatments while maximum biodiesel yield was recorded by BF treatment. Maximum oleic acid was recorded by BF treatment while other fatty acids being maximum in control except linoleic acid in BFCF treatment. Results for specific gravity were non-significant while acid value and free fatty acid contents were substantially reduced by BF treatment as compared to other treatments. Maximum value of iodine number was recorded in BFCF treatment while tocopherol contents were improved by BF treatment. It is inferred that biofertilizer treatment alone perform better as compared to other treatments and 50% chemical fertilizer can be replaced using biofertilizer which is a good approach for sustainable environmental-friendly agriculture.     

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.     

Homogeneous,heterogeneous and enzymatic catalysis for transesterification of high free fatty acid oil (waste cooking oil) to biodiesel: A review

In the last few years, biodiesel has emerged as one of the most potential renewable energy to replace current petrol-derived diesel. It is a renewable, biodegradable and non-toxic fuel which can be easily produced through transesterification reaction. However, current commercial usage of refined vegetable oils for biodiesel production is impractical and uneconomical due to high feedstock cost and priority as food resources. Low-grade oil, typically waste cooking oil can be a better alternative; however, the high free fatty acids (FFA) content in waste cooking oil has become the main drawback for this potential feedstock. Therefore, this review paper is aimed to give an overview on the current status of biodiesel production and the potential of waste cooking oil as an alternative feedstock. Advantages and limitations of using homogeneous, heterogeneous and enzymatic transesterification on oil with high FFA (mostly waste cooking oil) are discussed in detail. It was found that using heterogeneous acid catalyst and enzyme are the best option to produce biodiesel from oil with high FFA as compared to the current commercial homogeneous base-catalyzed process. However, these heterogeneous acid and enzyme catalyze system still suffers from serious mass transfer limitation problems and therefore are not favorable for industrial application. Nevertheless, towards the end of this review paper, a few latest technological developments that have the potential to overcome the mass transfer limitation problem such as oscillatory flow reactor (OFR), ultrasonication, microwave reactor and co-solvent are reviewed. With proper research focus and development, waste cooking oil can indeed become the next ideal feedstock for biodiesel.     

Single cell oil production from rice hulls hydrolysate

Rice hull hydrolysate was used as feedstock for microbial lipids production using the oleaginous fungus Mortierella isabellina. Kinetic experiments were conducted in C/N ratios 35, 44 and 57 and the oil accumulation into fungal biomass was 36%, 51.2% and 64.3%, respectively. A detailed mathematical model was used in order to describe the lipid accumulation process. This model was able to predict reducing sugar and nitrogen consumption, fat-free biomass synthesis and lipid accumulation. Neutral lipids constitute the predominant lipid fraction, while the major fatty acids were oleic, palmitic and linoleic acid. Fatty acids of long aliphatic chain were not detected, thus the microbial oil produced is a promising feedstock for biodiesel production.     

Metabolic engineering of Escherichia coli for efficient free fatty acid production from glycerol

Crude glycerol, generated as waste by-product in biodiesel production process, has been considered as an important carbon source for converting to value-added bioproducts recently. Free fatty acids (FFAs) can be used as precursors for the production of biofuels or biochemicals. Microbial biosynthesis of FFAs can be achieved by introducing an acyl–acyl carrier protein thioesterase into Escherichia coli. In this study, the effect of metabolic manipulation of FFAs synthesis cycle, host genetic background and cofactor engineering on FFAs production using glycerol as feed stocks was investigated. The highest concentration of FFAs produced by the engineered stain reached 4.82 g/L with the yield of 29.55% (g FFAs/g glycerol), about 83% of the maximum theoretical pathway value by the type II fatty acid synthesis pathway. In addition, crude glycerol from biodiesel plant was also used as feedstock in this study. The FFA production was 3.53 g/L with a yield of 24.13%. The yield dropped slightly when crude glycerol was used as a carbon source instead of pure glycerol, while it still can reach about 68% of the maximum theoretical pathway yield.     

Modification of Apricot Kernel Oil by Lipase Catalyzed Ester Interchange

In this study, apricot kernel oil was extracted from die kernel of apricot fruits and its physico chemical properties were studied. In addition apricot kernel oil was reacted with stearic and palmitic acid in hexane in the presence immobilized porcine pancreatic lipase to give products enriched with these fatty acids.     

Synthesis of structured lipid with balanced omega-3: Omega-6 ratio by lipase-catalyzed acidolysis reaction: Optimization of reaction using response surface methodology

The present study deals with the production of structured lipid containing omega-3 and omega-6 fatty acids in the ratio of 1:1 by incorporating omega-3 fatty acids (α-linolenic acid) from linseed oil into groundnut oil using lipase (Lipozyme IM from Rhizomucor miehei) catalyzed acidolysis reaction in hexane. The reaction conditions were optimized by response surface methodology with a four-variable five-level central composite rotatable experimental design. The influence of four independent parameters, namely ratio of fatty acid concentrate from linseed to groundnut oil (0.66–1.98, w/w), reaction temperature (30–60 °C), enzyme concentration (1–5%) and reaction time (2–54 h) on omega-3 fatty acids incorporation into groundnut oil were optimized. Optimal conditions for the structured lipid containing omega-3 to omega-6 fatty acids in the ratio of 1:1 were determined to be; enzyme concentration 3.75% (w/w), temperature 37.5 °C, incubation time 30.81 h and ratio of free fatty acid concentrate from linseed oil to groundnut oil 1.16 (w/w).     

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.     

Biodiesel Production from Waste Cooking Oil Using Anion-Exchange Resin as Both Catalyst and Adsorbent

A novel production process of biodiesel fuel was developed using an expanded-bed reactor packed with an anion-exchange resin having catalytic and adsorption abilities. Waste cooking oil was used as a cheaper feedstock, and methanol was added at the stoichiometric molar ratio of 3:1. The main constituent of the feedstock, triglyceride was completely converted to the biodiesel by the resin??s catalytic ability. The impurities of the feedstock, free fatty acid, water, and dark brown pigment were removed from the product by the adsorption on the resin. In addition, the by-product glycerin was also removed from the product by the adsorption on the resin. The product eluted from the reactor was directly used for the standard tests of the biodiesel properties. The eluted product almost met the biodiesel quality standards (EN14214 in Europe and ASTM D6751 in North America). Thus, the proposed system permitted the simple production of biodiesel from the waste cooking oil without the upstream processing to remove the impurities (free fatty acid and water) and the downstream processing to remove the catalyst and by-products (glycerin and soap).     

Variation in the Fatty‐Acid Content in Seeds of Various Black,Red, and White Currant Varieties

Currant seeds, a by‐product of juice production, are recognized as a valuable source of oil rich in polyunsaturated fatty acids. We have evaluated 28 currant varieties for their oil content and fatty‐acid composition. The oil content in the seeds ranged from 18.2–27.7%, and no statistical difference between varieties of different fruit color were recorded. Furthermore, the estimated oil yields in the field production ranged from 26.4–212.4 kg/ha. The GC and GC/MS chemical profiles of the seed oils extracted from all examined varieties were common for currants. Linoleic acid (LA) was the major component, with contents ranging from 32.7–46.9% of total fatty acids, followed by α‐linolenic acid (ALA; 2.9–32.0 %), oleic acid (OA; 9.8–19.9%), γ‐linolenic acid (GLA; 3.3–18.5%), palmitic acid (PA; 4.4–8.1%), stearidonic acid (SDA; 2.2–4.7%), and stearic acid (SA; 1.2–2.4%). Quantitative differences in the fatty‐acid profiles between varieties of different fruit color were observed. Blackcurrant varieties showed significantly higher contents of LA, GLA, and PA than red and white currant varieties, whereas significantly higher amounts of ALA and OL were detected in the red and white varieties. Cluster analysis based on the chemical oil profiles joined the blackcurrants in one group, while most of the red and white cultivars joined in a second group at the same linkage distance.     

超临界CO_2萃取韭菜籽油成分的GC-MS分析

以索氏提取法为对照,采用超临界二氧化碳(SC-CO_2)萃取韭菜籽油,气相色谱-质谱联用技术(GC-MS)对韭菜籽油成分进行分析,NIST 02质谱数据库对其进行分析和鉴定.结果表明,SC-CO_2萃取压力为22.25 MPa、温度为40.40℃条件下萃取86.7 min时,萃取得率为17.52%,共分离鉴定出17种物质,其中,饱和脂肪酸以棕榈酸(6.25%)为主,占脂肪酸总量的 9.05%;不饱和脂肪酸主要是亚油酸(69.71%)和油酸(19.53%),占脂肪酸总量的90.50%.采用索氏提取得率为16.50%,共鉴定出10种物质,饱和脂肪酸以棕榈酸(7.22%)为主,占总脂肪酸量的9.84%;不饱和脂肪酸主要是亚油酸(69.34%)和油酸(20.12%),不饱和脂肪酸占脂肪酸总量的90.16%.另外SC-CO_2萃取韭菜籽油还检出单不饱和脂肪酸7-棕榈烯酸、角鲨烯和β-谷甾醇.