Production of lipid from N‐acetylglucosamine by Cryptococcus curvatus

N‐Acetylglucosamine (GlcNAc), the monomeric constituent of chitin, is rarely used as a carbon source for fermentation technology. In this study, we demonstrate that the oleaginous yeast Cryptococcus curvatus ATCC 20509 can produce intracellular lipid during the cultivation process and total lipid content can reach 54% on a GlcNAc‐based medium. Culture of C. curvatus under various conditions indicated that lipid accumulation also occurred at a relatively broad range of temperatures as well as relatively high initial GlcNAc concentrations. Fatty acid analysis indicated that the product was rich in palmitic acid, stearic acid, and oleic acid, closely resembling the composition of palm oil. More importantly, the lipid sample produced at 22 °C had a total saturated fatty acid content of 54.2 wt%, suggesting that it may be explored as cocoa‐butter equivalent. Our data suggested that GlcNAc could be used as a feedstock for industrial biotechnology and that C. curvatus ATCC 20509 is a strain capable of accumulating high intracellular lipid using this nitrogen‐rich renewable material. Practical applications: Microbial lipid is a versatile material, especially for biodiesel production. Stable and abundant renewable raw substrates remain to be explored for large‐scale production of microbial lipid. The present work reports lipid production using N‐acetylglucosamine (GlcNAc) by the oleaginous yeast Cryptococcus curvatus ATCC 20509 to yield up to 54% intracellular lipid content. More significantly, the lipid sample produced at 22 °C had a total saturated fatty acid content of 54.2 wt%, suggesting that it may be explored as cocoa‐butter equivalent. Our technology provides the opportunity to effectively convert GlcNAc, available from one of the most abundant renewable materials chitin, into lipid. This procedure should prove valuable in terms of renewable energy production as well as environmental pollution control.     

Lipids of oleaginous yeasts. Part II: Technology and potential applications

The process of lipid accumulation in the oleaginous yeasts cultivated in various fermentation configurations when either sugars and related compounds or hydrophobic substances are used as substrates is presented and kinetic models describing both de novo and ex novo lipid accumulation are analyzed. Technological aspects related with single cell oil (SCO) produced by oleaginous yeasts are depicted. The influence of culture parameters upon lipid production process is presented. Lipid production has been studied in batch, fed‐batch, and continuous cultivation systems using yeasts belonging to the species Lipomyces starkeyi, Rhodosporidium toruloides, Apiotrichum curvatum, Candida curvata, Cryptococcus curvatus, Trichosporon fermentans, and Yarrowia lipolytica. The potentiality of yeasts to produce SCO as starting material of 2nd generation biodiesel is indicated and discussed. Of significant importance is also the utilization of yeast lipids as substitutes of high added value exotic fats (e.g., cocoa butter). Lipid produced by the various yeasts presents, in general, similar composition with that of common vegetable oils being composed of unsaturated fatty acids, whereas cocoa butter is principally composed of saturated fatty acids, consequently the various strategies that are followed in order to increase the cellular saturated fatty acid content of the yeast lipid are presented and comprehensively discussed.     

Enhanced microbial oil production by activated sludge microorganisms via co‐fermentation of glucose and xylose

The co‐fermentation of glucose and xylose by activated sludge microorganisms for the production of microbial oils for use as biodiesel feedstock was investigated. Various carbon sources at initial concentration of 60 g/L and C:N ratio 70:1 were investigated: xylose, glucose, and 2:1 and 1:2 (by mass) glucose/xylose mixtures. Oil accumulation ranged between 12 to 22% CDW, the highest of which was obtained when xylose was the sole substrate used. Kinetic modeling of the fermentation data showed that specific growth and oil accumulation rates were similar in all substrate types and the lipid coefficient ranged from 0.02 to 0.06 g/g of sugar consumed. The fatty acid methyl ester yield and composition of the lipids showed their suitability for conversion to biodiesel. Based on the results, lignocellulose sugars could be used as fermentation substrates by activated sludge microorganisms for enhancing the oil content of sewage sludge for its use as a sustainable biofuel feedstock source. © 2013 American Institute of Chemical Engineers AIChE J, 59: 4036–4044, 2013     

New Biofuel Alternatives: Integrating Waste Management and Single Cell Oil Production

Concerns about greenhouse gas emissions have increased research efforts into alternatives in bio-based processes. With regard to transport fuel, bioethanol and biodiesel are still the main biofuels used. It is expected that future production of these biofuels will be based on processes using either non-food competing biomasses, or characterised by low CO₂ emissions. Many microorganisms, such as microalgae, yeast, bacteria and fungi, have the ability to accumulate oils under special culture conditions. Microbial oils might become one of the potential feed-stocks for biodiesel production in the near future. The use of these oils is currently under extensive research in order to reduce production costs associated with the fermentation process, which is a crucial factor to increase economic feasibility. An important way to reduce processing costs is the use of wastes as carbon sources. The aim of the present review is to describe the main aspects related to the use of different oleaginous microorganisms for lipid production and their performance when using bio-wastes. The possibilities for combining hydrogen (H₂) and lipid production are also explored in an attempt for improving the economic feasibility of the process.     

Use of Essential Oils From Various Plants to Change the Fatty Acids Profiles of Lipids Obtained From Oleaginous Yeasts

We studied the ability of seven essential oils to alter the fatty acid composition of lipids produced by an oleaginous yeast Rhodosporidium toruloides. All of the essential oils, except thyme, significantly increased the stearic acid content of the lipids. The amount of essential oils in the media determined the fatty acid composition obtained. Subsequently, we studied the effect of the major monoterpenes present in these essential oils. When R. toruloides was grown on limonene, a major monoterpene in orange essential oil, the composition of lipid obtained was found to be quite similar to natural orange essential oil. This proved that limonene has a major role in the changes in fatty acid profiles of the lipids. The effect of orange essential oil on another oleaginous yeast, Cryptococcus curvatus, was also carried out. Although the effect of the essential oil on the fatty acid composition and biomass (cell mass) was similar for both these two yeasts, the reduction of the activity of some enzymes involved in the metabolic pathways was quite different. From these results, it can be concluded that the effect of essential oils differs with species and it is possible to produce lipids with alternate fatty acid profiles suitable for different applications and with good market value.     

Lipid and fatty acid biosynthesis by Rhodotorula minuta

Demand for fatty acids is increasing at an annual rate of 17%, due to their increased use in the oleochemical and transport industries. Presently, vegetable oils are the major source of fatty acids, whereas lipids with fatty acids similar to those of some vegetable oils have been reported to be synthesized by oleaginous microorganisms. In the present study, the culturing conditions for the oleaginous yeast Rhodotorula minuta IIP-33 have been optimized. In contrast to the lipid accumulation characteristics of most oleaginous yeasts, a carbon-to-nitrogen ratio of 30 was favorable for maximal accumulation of lipids (48%) with 22.5% conversion of glucose as carbon substrate. The lipids contained fatty acids in the C₇–C₁₈ range, the relative composition of which varied with culture temperature.     

基于消耗碳氮比进行底物分配和油脂得率预测

微生物油脂作为胞内代谢产物,其脂肪酸组成和植物油类似,是生物柴油和油脂工业理想替代原料。为了进一步了解底物中碳氮比对油脂合成的影响,通过恒化培养的方法,研究了圆红冬孢酵母在不同稀释率条件下,消耗碳氮比和底物在油脂和非油生物量之间的分配以及和油脂得率之间的关系。通过碳、氢、氧和氮的化学反应计量学分析,并根据不同稀释率稳态时所消耗C/N比,构建了底物碳在油脂和非油生物量之间的分配模型。利用实验数据确定了模型参数:最大油脂碳得率Y_L^max为0.51 mol C·(mol C)^-1,最大菌体碳得率Y_L^max为0.52 mol C·(mol C)^-1,促使油脂合成的临界C/N比为12.1 mol C·(mol C)^-1。利用该分配模型预测不同消耗C/N比的油脂得率,预测值为实验值的 95.2%～116.7%,表明模型可靠性较好。     

Anthracene Biodegradation by Oleaginous Rhodococcus opacus for Biodiesel Production and Its Characterization

This study examined biodegradation of anthracene, a model low molecular weight polycyclic aromatic hydrocarbon (PAH) by oleaginous Rhodococcus opacus for biodiesel production. Specific biomass growth rate (µ) in the range of 0.0075–0.0185 h^?1 could be attained over the initial anthracene concentration (50–500 mg L^?1), along with 68–70.6% (w/w) lipid accumulation. 10% (v/v) inoculum size showed more positive effect than 5% (v/v) inoculum size on both anthracene biodegradation efficiency and lipid accumulation by R. opacus. ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy of the bacterial lipids revealed 82.25% saturated fatty acids content. Furthermore, the transesterified bacterial lipids predominantly consisted of methyl palmitate (32.4%) and methyl stearate (25.9%) as the major fatty acid methyl esters (FAMEs). Overall, this study revealed a very good potential of the bacterium for the production of biodiesel from PAH-containing wastewater.     

产油酵母利用廉价原料合成油脂的研究进展

利用微生物油脂生产的生物柴油是替代传统化石燃料的绿色可再生能源,产油酵母是极具潜力的产油微生物。然而利用葡萄糖作为碳源成本较高,限制了微生物油脂合成技术的规模化应用,因而寻找廉价原料至关重要。本文基于产油酵母合成油脂的代谢机理,重点介绍了产油酵母利用4种廉价原料,即木质纤维素、粗甘油、有机废水和挥发性脂肪酸合成微生物油脂的研究进展。针对这些来自工农业副产物的廉价原料,分别讨论了其复杂成分对产油酵母生长和油脂合成产生的促进或抑制作用,分析总结了高效预处理、基因工程改造菌体以及微生物共培养等解决方案。通过以上分析,阐明了产油酵母利用廉价原料合成油脂的优点,提出了针对复杂成分和高有机质浓度的抑制性问题的解决方法,理清了未来研究的方向,有助于进一步推动廉价原料用于产油酵母生产油脂及可再生能源技术的发展。     

Techno-Economic Evaluation of Biodiesel Production from Waste Cooking Oil—A Case Study of Hong Kong

Fossil fuel shortage is a major challenge worldwide. Therefore, research is currently underway to investigate potential renewable energy sources. Biodiesel is one of the major renewable energy sources that can be obtained from oils and fats by transesterification. However, biodiesel obtained from vegetable oils as feedstock is expensive. Thus, an alternative and inexpensive feedstock such as waste cooking oil (WCO) can be used as feedstock for biodiesel production. In this project, techno-economic analyses were performed on the biodiesel production in Hong Kong using WCO as a feedstock. Three different catalysts such as acid, base, and lipase were evaluated for the biodiesel production from WCO. These economic analyses were then compared to determine the most cost-effective method for the biodiesel production. The internal rate of return (IRR) sensitivity analyses on the WCO price and biodiesel price variation are performed. Acid was found to be the most cost-effective catalyst for the biodiesel production; whereas, lipase was the most expensive catalyst for biodiesel production. In the IRR sensitivity analyses, the acid catalyst can also acquire acceptable IRR despite the variation of the WCO and biodiesel prices.     

Green microalga Chlorella vulgaris as a potential feedstock for biodiesel

BACKGROUND: A major bottleneck in microalgal biodiesel production is lipid content, which is often low in microalgal species. The present study examines Chlorella vulgaris as a potential feedstock for biodiesel by identifying and evaluating the relationships between the critical variables that enhance the lipid yield, and characterizes the biodiesel produced for various properties. RESULTS: Factors affecting lipid accumulation in a green microalga, Chlorella vulgaris were examined. Multifactor optimization raised the lipid pool to 55% dry cell weight against 9% control. When C. vulgaris cells pre‐grown in glucose (0.7%)‐supplemented medium were transferred to the optimized condition at the second stage, the lipid yield was boosted to 1974 mg L^?1, a value almost 20‐fold higher than for the control. The transesterified C. vulgaris oil showed the presence of ～82% saturated fatty acids, with palmitate and stearate as major components, thus highlighting the oxidative stability of C. vulgaris biodiesel. The fuel properties (density, viscosity, acid value, iodine value, calorific value, cetane index, ash and water contents) are comparable with the international (ASTM and EN) and Indian (IS) biodiesel standards. CONCLUSION: C. vulgaris biomass with 55% lipid content and adequate fuel properties is potentially a renewable feedstock for biodiesel. Copyright © 2011 Society of Chemical Industry     

Lipase-Catalyzed Production of Biodiesel by Hydrolysis of Waste Cooking Oil Followed by Esterification of Free Fatty Acids

Biodiesel is conventionally produced by alkaline‐catalyzed transesterification, which requires high‐purity oils. However, low‐quality oils can be used as feedstocks for the production of biodiesel by enzyme‐catalyzed reactions. The use of enzymes has several advantages, such as the absence of saponification side reactions, production of high‐purity glycerol co‐product, and low‐cost downstream processing. In this work, biodiesel was produced from lipase‐catalyzed hydrolysis of waste cooking oil (WCO) followed by esterification of the hydrolyzed WCO (HWCO). The hydrolysis of acylglycerols was carried out at 30 °C in salt‐free water (WCO/water ratio of 1:4, v/v) and the esterification of HWCO was carried out at 40 °C with ethanol in a solvent‐free medium (HWCO/ethanol molar ratio of 1:7). The hydrolysis and esterification steps were carried out using immobilized Thermomyces lanuginosus lipase (TLL/WCO ratio of 1:5.6, w/w) and immobilized Candida antarctica lipase B (10 wt%, CALB/HWCO) as biocatalysts, respectively. The hydrolysis of acylglycerols was almost complete after 12 h (ca. 94 %), and in the esterification step, the conversion was around 90 % after 6 h. The purified biodiesel had 91.8 wt% of fatty acid ethyl esters, 0.53 wt% of acylglycerols, 0.003 wt% of free glycerol, viscosity of 4.59 cP, and acid value of 10.88 mg KOH/g. Reuse hydrolysis and esterification assays showed that the immobilized enzymes could be recycled five times in 10‐h batches, under the conditions described above. TLL was greatly inactivated under the assay conditions, whereas CALB remained fully active. The results showed that WCO is a promising feedstock for use in the production of biodiesel.     

Effects of Acetic Acid on Growth and Lipid Production by Cryptococcus albidus

The cell growth and lipid accumulation process of Cryptococcus albidus were investigated using acetic acid as the sole carbon source at different concentrations. C. albidus showed high tolerance to acetic acid at a high concentration of 30 g L^?1. The highest lipid content (32.69 ± 0.50 %) and lipid yield (0.96 ± 0.05 g L^?1) were both obtained in the medium with an initial acetic acid concentration of 30 g L^?1 on day five. Interestingly, the maximum lipid content and lipid yield was obtained on a different day in a medium with different acetic acid concentration. The fatty acid composition of the lipids accumulated by C. albidus was 16–23 % palmitic acid (C16:0), 3–5 % linolenic acid (C18:3), 42–51 % linoleic acid (C18:2) and 23–27 % oleic acid (C18:1), which was similar to that of soybean oil; thus, this microbial oil has great potential value as a renewable biodiesel feedstock. This work also provides valuable information for further research to use cheap substrates containing a high concentration of acetic acid (such as lignocellulosic hydrolysates), which is an economical and environmentally friendly form of microbial oil production.     

Non-Edible Plant Oils as New Sources for Biodiesel Production

Due to the concern on the availability of recoverable fossil fuel reserves and the environmental problems caused by the use those fossil fuels, considerable attention has been given to biodiesel production as an alternative to petrodiesel. However, as the biodiesel is produced from vegetable oils and animal fats, there are concerns that biodiesel feedstock may compete with food supply in the long-term. Hence, the recent focus is to find oil bearing plants that produce non-edible oils as the feedstock for biodiesel production. In this paper, two plant species, soapnut (Sapindus mukorossi) and jatropha (jatropha curcas, L.) are discussed as newer sources of oil for biodiesel production. Experimental analysis showed that both oils have great potential to be used as feedstock for biodiesel production. Fatty acid methyl ester (FAME) from cold pressed soapnut seed oil was envisaged as biodiesel source for the first time. Soapnut oil was found to have average of 9.1% free FA, 84.43% triglycerides, 4.88% sterol and 1.59% others. Jatropha oil contains approximately 14% free FA, approximately 5% higher than soapnut oil. Soapnut oil biodiesel contains approximately 85% unsaturated FA while jatropha oil biodiesel was found to have approximately 80% unsaturated FA. Oleic acid was found to be the dominant FA in both soapnut and jatropha biodiesel. Over 97% conversion to FAME was achieved for both soapnut and jatropha oil.     

产油微生物利用废弃物积累油脂的研究进展

微生物油脂是一种应用前景广阔的新型油脂资源, 具有制备功能性油脂和生物柴油的优点, 因此越来越受到人们的重视。但微生物油脂生产时所用原料成本高, 导致微生物油脂的产业化发展受到制约。因此寻找廉价易得的发酵基质将促进微生物油脂生产的工业化进程, 同时解决了日益严峻的能源与环境安全问题。本文简述了各种工业废水、剩余活性污泥及餐厨废弃物等废弃物的特点, 总结了产油微生物利用该类废弃物生产油脂的研究现状及可能存在的发酵工艺及经济成本问题, 指出了未来的发展方向是开发附加值产品及廉价高效的絮凝剂以降低油脂成本、探究利用微生物前期处理降解剩余污泥中的毒性物质、探讨酶与酸碱联合水解餐厨废弃物的工艺等。     

Extraction of Lipids from Municipal Wastewater Plant Microorganisms for Production of Biodiesel

Municipal wastewater treatment plants in the USA produce over 6.2 × 10⁶ t of dried sewage sludge every year. This microorganism-rich sludge is often landfilled or used as fertilizer. Recent restrictions on the use of sewage sludge, however, have resulted in increased disposal problems. Extraction of lipids from sludge yields an untapped source of cheap feedstock for biodiesel production. Solvents used for extraction in this study include n-hexane, methanol, acetone, and supercritical CO₂. The gravimetric yield of oil was low for nonpolar solvents, but use of polar solvents gave a considerably increased yield; however, the percentage of saponifiable material was less. Extraction of lipids with a mixture of n-hexane, methanol, and acetone gave the largest conversion to biodiesel compared with other solvent systems, 4.41% based on total dry weight of sludge. In situ transesterification of dried sludge resulted in a yield of 6.23%. If a 10% dry weight yield of fatty acid methyl esters is assumed, the amount of biodiesel available for production in the USA is 1.4 × 10⁶ m³/year. Outfitting 50% of municipal wastewater plants for lipid extraction and transesterification could result in enough biodiesel production to replace 0.5% of the national petroleum diesel demand (0.7 × 10⁶ m³).     

Comparison between conventional and ultrasonic preparation of beef tallow biodiesel

Tallow is biodiesel feedstock that, due to its highly centralized generation in slaughter/processing facilities and historically low prices, may have energetic, environmental, and economic advantages that could be exploited. Transesterification of fatty acids by means of ultrasonic energy has been used for biodiesel production from different vegetable oils. However, application of ultrasonic irradiation for biodiesel production from beef tallow has received little attention. In this work, the transesterification of beef tallow with methanol was performed in the presence of potassium hydroxide as a catalyst using ultrasound irradiation (400 W, 24 kHz). The reaction time, conversion and biodiesel quality were compared with that seen in conventional transesterification. The results indicated that the reaction conversion and biodiesel quality were similar; however, the use of ultrasonic irradiation decreased the reaction time, showing that this method may be a promising alternative to the conventional method.     

Prospects of non-catalytic supercritical methyl acetate process in biodiesel production

Conventional biodiesel production methods utilize alcohol as acyl acceptor and produces glycerol as side product. Hence, with escalating production of biodiesel throughout the world, it leads to oversupply of glycerol and subsequently causes devaluation in the market. In this study, methyl acetate was employed as acyl acceptor in non-catalytic supercritical methyl acetate (SCMA) process to produce fatty acid methyl esters (FAME) and side product of triacetin, a valuable fuel additive instead of glycerol. Consequently, the properties of biodiesel produced (FAME and triacetin) are superior compared to conventional biodiesel method (FAME only). In this research, the effects of reaction temperature, reaction time and molar ratio of methyl acetate to oil on the yield of biodiesel were investigated. Apart from that, the influence of impurities commonly found in waste oils/fats such as free fatty acids and water were studied as well and compared with methanol-based reactions of supercritical and heterogeneous catalysis. Results show that biodiesel yields in SCMA process could achieve 99 wt.% when the operating conditions were fixed at 400 °C/220 bar for reaction temperature, methyl acetate/oil molar ratio of 30:1 and 60 min of reaction time. Furthermore, SCMA did not suffer from adverse effect with the presence of impurities, proving that SCMA has a high tolerance towards contamination which is crucial to allow the utilization of inexpensive waste oils/fats as biodiesel feedstock.     

Biotechnology and oleochemicals: Changing patterns

Biotechnology will have a broad impact on the oleochemicals industry. Only a narrow range of this interface is discussed: (a) the use of organic solvents in the enzymatic synthesis of lipid derivatives, (b) the effect of the chemical nature of the feedstock on the production of microbial monoesters, and (c) temperature as a determinant of the level of unsaturation in biosynthetic lipids. Interest in running enzymatic reactions in high concentrations of organic solvents is increasing. The implications of such processes for the oleochemical industry is illustrated by examples of ester synthesis and interesterification of oils and fats. The dramatic effect of feedstock chemistry on the final monoester product mix produced byAcinetobacter sp. HO1-N is also discussed. Products resulting from usingn-alkanes (C₁₆−C₂₀), acetic and propionic acids and, most recently, ethanol and propanol, are illustrated. They range from a monoester mix resembling sperm oil to one similar to jojoba oil. In general, temperature inversely affects biolipid unsaturation: the low the temperature, the greater the unsaturation. The major function of this response is to preserve fluidity and function in biological membranes. The effect is universal in nature, occurring in animals, plants and microorganisms. Controlled laboratory studies have supported these observations made in nature. We have investigated the effect of temperature on the unsaturation of monoesters produced by the bacterium,Acimetobacter sp. HO1-N. The inverse relationship between temperature and unsaturation is clearly shown. The enzymatic basis for these results and the possibility of chemical or genetic modification of plants and microorganisms to produce more or less unsaturated lipids is briefly discussed. Organic solvents, feedstock chemistry and temperature stress in biocatalysis are but three of the variables at the interface of biotechnology and the oleochemicals industry that will cause changing patterns.     

Current scenario and potential of biodiesel production from waste cooking oil in Pakistan: An overview

Biodiesel utilization has been rapidly growing worldwide as the prime alternative to petrodiesel due to a global rise in diesel fuel demand along with hazardous emissions during its thermochemical conversion. Although, several debatable issues including feedstock availability and price, fuel and food competition, changes in land use and greenhouse gas emission have been raised by using edible as well as inedible feedstocks for the production of biodiesel. However, non-crop feedstocks could be a promising alternative. In this article, waste cooking oils have been recommended as a suitable option for biodiesel production bearing in mind the current national situation. The important factors such as the quantity of waste cooking oil produced, crude oil and vegetable oil import expenses, high-speed diesel imports, waste management issues and environmental hazards are considered. Moreover, process simulation and operating cost evaluation of an acid catalyzed biodiesel production unit are also conducted. The simulation results show that the production cost of waste cooking oil-based biodiesel is about 0.66USD·L^-1. We believe that the present overview would open new pathways and ideas for the development of biofuels from waste to energy approach in Pakistan.