An effective acid catalyst for biodiesel production from impure raw feedstocks

Biodiesel consists of fatty acids short chain alkyl esters produced through transesterification and esterification of fats and oils. Production of biodiesel is strongly affected by the purity of raw lipids, and catalysts play important role in these processes. Although direct utilization of impure feedstocks is more economical, their use necessitates development of effective catalysts to overcome hindering influences of impurities. In this study, sulfuryl chloride, thionyl chloride, acetyl chloride, p-toluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, dimethylsulfate and sulfuric acid were investigated as catalysts for the production of biodiesel because acids have higher tolerance to water and free fatty acids in oils and can simultaneously catalyze both the esterification and transesterification reactions. Sulfuryl chloride was found to be an effective catalyst for production of biodiesel from soybean oil, its waste oil and microalgal lipids.     

From agro-industrial wastes to single cell oils: a step towards prospective biorefinery

The reserves of fossil-based fuels, which currently seem sufficient to meet the global demands, is inevitably on the verge of exhaustion. Contemporary raw material for alternate fuel like biodiesel is usually edible plant commodity oils, whose increasing public consumption rate raises the need of finding a non-edible and fungible alternate oil source. In this quest, single cell oils (SCO) from oleaginous yeasts and fungi can provide a sustainable alternate of not only functional but also valuable (polyunsaturated fatty acids (PUFA)-rich) lipids. Researches are been increasingly driven towards increasing the SCO yield in order to realize its commercial importance. However, bulk requirement of expensive synthetic carbon substrate, which inflates the overall SCO production cost, is the major limitation towards complete acceptance of this technology. Even though substrate cost minimization could make the SCO production profitable is uncertain, it is still essential to identify suitable cheap and abundant substrates in an attempt to potentially reduce the overall process economy. One of the most sought-after in-expensive carbon reservoirs, agro-industrial wastes, can be an attractive replacement to expensive synthetic carbon substrates in this regard. The present review assess these possibilities referring to the current experimental investigations on oleaginous yeasts, and fungi reported for conversion of agro-industrial feedstocks into triacylglycerols (TAGs) and PUFA-rich lipids. Multiple associated factors regulating lipid accumulation utilizing such substrates and impeding challenges has been analyzed. The review infers that production of bulk oil in combination to high-value fatty acids, co-production strategies for SCO and different microbial metabolites, and reutilization and value addition to spent wastes could possibly leverage the high operating costs and help in commencing a successful biorefinery. Rigorous research is nevertheless required whether it is PUFA-rich oil production (for competing with algal omega oils) or neutral bulk oil production (for overcoming yield limitations and managing process economy) to establish this potential source as future resource.     

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.     

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.     

Production of algae-based biodiesel using the continuous catalytic Mcgyan® process

This study demonstrates the production of algal biodiesel from Dunaliella tertiolecta, Nannochloropsis oculata, wild freshwater microalgae, and macroalgae lipids using a highly efficient continuous catalytic process. The heterogeneous catalytic process uses supercritical methanol and porous titania microspheres in a fixed bed reactor to catalyze the simultaneous transesterification and esterification of triacylglycerides and free fatty acids, respectively, to fatty acid methyl esters (biodiesel). Triacylglycerides and free fatty acids were converted to alkyl esters with up to 85% efficiency as measured by 300 MHz ¹H NMR spectroscopy. The lipid composition of the different algae was studied gravimetrically and by gas chromatography. The analysis showed that even though total lipids comprised upwards of 19% of algal dry weight the saponifiable lipids, and resulting biodiesel, comprised only 1% of dry weight. Thus highlighting the need to determine the triacylglyceride and free fatty acid content when considering microalgae for biodiesel production.     

Regulation and remodeling of intermediate metabolite and membrane lipid during NaCl-induced stress in freshwater microalga Micractinium sp. XJ-2 for biofuel production

Microalgae can accumulate a large fraction of reduced carbon as lipids under NaCl stress. This study investigated the mechanism of carbon allocation and reduction and triacylglycerol (TAG) accumulation in microalgae under NaCl-induced stress. Micractinium sp. XJ-2 was exposed to NaCl stress and cells were subjected to physiological, biochemical, and metabolic analyses to elucidate the stress-responsive mechanism. Lipid increased with NaCl concentration after 0-12 hr, then stabilized after 12–48 hr, and finally decreased after 48–72 hr, whereas TAG increased (0–48 hr) and then decreased (48–72 hr). Under NaCl-induced stress at lower concentrations, TAG accumulation, at first, mainly shown to rely on the carbon fixation through photosynthetic fixation, carbohydrate degradation, and membrane lipids remodeling. Moreover, carbon compounds generated by the degradation of some amino acids were reallocated and enhanced fatty acid synthesis. The remodeling of the membrane lipids of NaCl-induced microalgae relied on the following processes: (a) Increase in the amount of phospholipids and reduction in the amount of glycolipids and (b) extension of long-chain fatty acids. This study enhances our understanding of TAG production under NaCl stress and thus will provide a theoretical basis for the industrial application of NaCl-induced in the microalgal biodiesel industry.     

Altered lipid composition and enhanced lipid production in green microalga by introduction of brassica diacylglycerol acyltransferase 2

Higher lipid biosynthesis and accumulation are important to achieve economic viability of biofuel production via microalgae. To enhance lipid content, Chlamydomonas reinhardtii was genetically engineered with a key enzyme diacylglycerol acyltransferase (BnDGAT2) from Brassica napus, responsible for neutral lipid biosynthesis. The transformed colonies harbouring aph7 gene, screened on hygromycin‐supplemented medium, achieved transformation frequency of ~120 ± 10 colonies/1 × 10⁶ cells. Transgene integration and expression were confirmed by PCR, Southern blots, staining lipid droplets, proteins and spectro‐fluorometric analysis of Nile red‐stained cells. The neutral lipid is a major class (over 80% of total lipids) and most significant requirement for biodiesel production; this was remarkably higher in the transformed alga than the untransformed control. The levels of saturated fatty acids in the transformed alga decreased to about 7% while unsaturated fatty acids increased proportionately when compared to wild type cells. Polyunsaturated fatty acids, especially α‐linolenic acid, an essential omega‐3 fatty acid, were enhanced up to 12% in the transformed line. Nile red staining confirmed formation of a large number of lipid globules in the transformed alga. Evaluation of long‐term stability and vitality of the transgenic alga revealed that cryopreservation produced significantly higher quantity of lipid than those maintained continuously over 128 generations on solid medium. The overexpression of BnDGAT2 significantly altered the fatty acids profile in the transformed alga. Results of this study offer a valuable strategy of genetic manipulation for enhancing polyunsaturated fatty acids and neutral lipids for biofuel production in algae.     

Overproduction of free fatty acids in E. coli: implications for biodiesel production

Whereas microbial fermentation processes for producing ethanol and related alcohol biofuels are well established, biodiesel (methyl esters of fatty acids) is exclusively derived from plant oils. Slow cycle times for engineering oilseed metabolism and the excessive accumulation of glycerol as a byproduct are two major drawbacks of deriving biodiesel from plants. Although most bacteria produce fatty acids as cell envelope precursors, the biosynthesis of fatty acids is tightly regulated at multiple levels. By introducing four distinct genetic changes into the E. coli genome, we have engineered an efficient producer of fatty acids. Under fed-batch, defined media fermentation conditions, 2.5 g/L fatty acids were produced by this metabolically engineered E. coli strain, with a specific productivity of 0.024 g/h/g dry cell mass and a peak conversion efficiency of 4.8% of the carbon source into fatty acid products. At least 50% of the fatty acids produced were present in the free acid form.     

Advancing oleaginous microorganisms to produce lipid via metabolic engineering technology

With the depletion of global petroleum and its increasing price, biodiesel has been becoming one of the most promising biofuels for global fuels market. Researchers exploit oleaginous microorganisms for biodiesel production due to their short life cycle, less labor required, less affection by venue, and easier to scale up. Many oleaginous microorganisms can accumulate lipids, especially triacylglycerols (TAGs), which are the main materials for biodiesel production. This review is covering the related researches on different oleaginous microorganisms, such as yeast, mold, bacteria and microalgae, which might become the potential oil feedstocks for biodiesel production in the future, showing that biodiesel from oleaginous microorganisms has a great prospect in the development of biomass energy. Microbial oils biosynthesis process includes fatty acid synthesis approach and TAG synthesis approach. In addition, the strategies to increase lipids accumulation via metabolic engineering technology, involving the enhancement of fatty acid synthesis approach, the enhancement of TAG synthesis approach, the regulation of related TAG biosynthesis bypass approaches, the blocking of competing pathways and the multi-gene approach, are discussed in detail. It is suggested that DGAT and ME are the most promising targets for gene transformation, and reducing PEPC activity is observed to be beneficial for lipid production.     

FATTY ACIDS OF CUPHEA (LYTHRACEAE) SEED LIPIDS AND THEIR SYSTEMATIC SIGNIFICANCE

Fatty acid analyses of seed lipids in 46 species of Cuphea are presented, representing the first major survey of a molecular nature for the family. A remarkable diversity in composition is found, with seeds containing high amounts of several medium chain fatty acids. Lauric acid (12:0) predominates in 43% of the species studied, constituting 50–74% of the total fatty acid content. Capric acid (10:0) is the dominant fatty acid in 32% of the species, comprising as much as 87% of the total acid content. Caprylic acid (8:0) predominates in one section of the genus. The emphasis on production of fatty acids with carbon chain lengths of 12, ten, and eight carbon atoms is unique among plant genera studied to date. Among seven of the nine sections studied, one pattern of fatty acid composition predominates. Two sections have no characteristic pattern, supporting other evidence of their polyphyletic origin. The most significant systematic contribution is made by comparison of the predominate fatty acid components in the seed lipids. When used in conjunction with floral morphology, pollen studies, and chromosome number, it provides an important new basis on which to draw inferences of evolution and clarify present relationships within the genus. Additionally, a trend from the longer-chained, unsaturated linoleic acid (18:2) as a major lipid component to shorter-chained saturated capric and caprylic acids is correlated with increasing floral specialization. It is suggested that mutations in regulatory genes have occurred which cause fatty acid production in seeds to cease at progressively earlier stages, resulting in accumulation of large amounts of single fatty acids of progressively shorter carbon chain lengths.     

Current Prospects on Production of Microbial Lipid and Other Value-Added Products Using Crude Glycerol Obtained from Biodiesel Industries

Production of microbial lipids using crude glycerol from the biodiesel industry is reviewed in this paper. Approximately 10 wt.% of crude glycerol is obtained for every batch of biodiesel. The crude glycerol accumulated contains various impurities and hence cannot be used for any commercial applications without further purification. Its conversion via biological and chemical routes into valuable products has been studied by different researchers. Varieties of fungal, yeasts, and algal species have been used to produce microbial lipids from crude glycerol. However, research focus on screening a robust industrial oleaginous strain capable of doing this is still on-going. Due to its chemical similarity to vegetable oils, microbial lipids are considered a potential renewable feedstock for biodiesel production and for applications in food and pharmaceutical industries. Its conversion to polyols and subsequently to biobased polymers is also being explored. The rising price of vegetable oils, increasing energy demands, growing environmental concerns, and availability of crude glycerol as a cheap carbon substrate result in considerable potential for the application of these processes in the future.     

Effect of pH on neutral lipid and biomass accumulation in microalgal strains native to the Canadian prairies and the Athabasca oil sands

Algal biodiesel has been a subject of growing importance in the realm of renewable energy due to carbon capture properties and its potential for photosynthetic efficiency with high lipid output. This study identified five isolates of freshwater green algae, belonging to the Chlorellaceae, and measured the lipid classes and fatty acid profiles of these species to determine suitability for biodiesel production. To induce the greater accumulation of lipids, especially in the form of triacylglycerols (TAGs) desired for biodiesel, we examined the lipid accumulation in cells stressed by nitrogen limitation, sulfur deficiency, or pH stress. Increases in biomass were monitored in order to determine if adjusting pH incrementally over the course of the experiment had any effect on growth and lipid accumulation of several isolates. TAG accumulation was visually screened by Nile Red fluorescence and further assessed by gas chromatography. Lipid amounts were comparably equal or better for pH stress treatments than for standard nutrient-deprivation treatments. Incrementally adjusted pH over the course of growth triggered lipid accumulation comparable to constant pH stress treatments, yet biomass accumulation was equivalent to unstressed growth. One isolate obtained from the Athabasca oil-sands region of Alberta, OS4-2, is a good candidate for biodiesel production, having accumulated over 45 % of its dry weight as lipid, with over 80 % of the lipid as triacylglycerols, and contains an abundance of 18:1 fatty acids. This class of fatty acids improves the cold flow and oxidative stability of biodiesel and is ideal for biofuel used in a Canadian climate.     

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.     

Cryptococcus curvatus O3酵母菌培养及产油脂特性

生物柴油的发展, 导致全球油脂供求紧张。微生物油脂的甘三酯组成与植物油类似, 发展微生物油脂可部分缓解植物油脂供应压力。本文研究了Cryptococcus curvatus O3酵母利用葡萄糖为碳源生长和积累油脂的特性。Cryptococcus curvatus O3酵母在培养过程中能适应间歇式碳源流加方式达到高密度培养的目的, 但在相同培养条件下, 不同氮源能影响其代谢过程中糖到油脂转化的脂肪系数。Cryptococcus curvatus O3酵母利用葡萄糖作为碳源在30°C下摇瓶发酵, 菌体生物量为51.8 g/L, 油脂含量达65.1%。脂肪酸组成分析结果表明, 菌油富含饱和及低度不饱和长链脂肪酸, 其中饱和脂肪酸之和占总脂肪酸组成的64%左右, 其脂肪酸组成类似于可可脂, 这些结果对于利用产油微生物转化生物质获取如类可可脂等具有高附加值油脂的研究具有重要意义。     

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.     

加快微生物油脂研究为生物柴油产业提供廉价原料

当前国内外致力于发展生物柴油,因其性能优良,成为化石柴油的替代品。由于以植物油脂生产生物柴油原料成本占总成本的70％-85％,所以亟待开发廉价油脂资源。微生物油脂主要是微生物利用碳水化合物合成的甘油脂,其脂肪酸组成和植物油相近。产油微生物具有资源丰富、油脂含量高、碳源利用谱广等特点,开发潜力大。然而,目前微生物油脂生产成本偏高,研究工作仍以富含多不饱和脂肪酸的高附加值菌油为目标。随着现代分子生物学和生物化工技术的发展,对产油微生物菌种筛选、改良、代谢调控和发酵工程的研究日趋深入,将降低微生物油脂生产成本,为未来生物柴油产业提供廉价原料。     

Calculation of Biodiesel Fuel Characteristics Based on the Fatty Acid Composition of the Lipids of Some Biotechnologically Important Microorganisms

The interdependences between the structure of fatty acid and biofuel characteristics obtained from these fatty acids were briefly reviewed. The fatty acid compositions of the lipids of yeasts and phototrophic microorganisms were analyzed. The main parameters of the biodiesel (iodine value, cetane number, and kinematic viscosity) that can be made from the lipids of these microorganisms were calculated based on the data and compared to the current standards. The lipids of the yeast Rhodosporidium toruloides VKPM Y-3349 were shown to be the most suitable for biofuel production due to the composition and content of fatty acid. The possibilities of a decrease in the prime cost of microbial lipids (along with plant oils) that would make them competitive raw material for biofuel production were considered.     

Scenedesmus sp. NJ-1 isolated from Antarctica: a suitable renewable lipid source for biodiesel production

Microalgal lipids are promising alternative feedstocks for biodiesel production. Scenedesmus sp. NJ-1, an oil-rich freshwater microalga isolated from Antarctica, was identified to be a suitable candidate to produce biodiesel in this study. This strain could grow at temperatures ranging from 4 to 35?°C. With regular decrease in nitrate concentration in the medium, large quantities of triacylglycerols accumulated under batch culture conditions detected by thin layer chromatography and BODIPY 505/515 fluorescent staining. Scenedesmus sp. NJ-1 achieved the average biomass productivity of 0.105?g?l^?1?d^?1 (dry weight) and nearly the highest lipid content (35?% of dry cell weight) was reached at day 28 in the batch culture. Neutral lipids accounted for 78?% of total lipids, and C18:1 (n-9), C16:0 were the major fatty acids in total lipids, composing 37 and 20?% of total fatty acids of Scenedesmus sp. NJ-1 grown for 36?days, respectively. These results suggested that Scenedesmus sp. NJ-1 was a good source of microalgal oils for biodiesel production.