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.     

Biotechnological processes for biodiesel production using alternative oils

As biodiesel (fatty acid methyl ester (FAME)) is mainly produced from edible vegetable oils, crop soils are used for its production, increasing deforestation and producing a fuel more expensive than diesel. The use of waste lipids such as waste frying oils, waste fats, and soapstock has been proposed as low-cost alternative feedstocks. Non-edible oils such as jatropha, pongamia, and rubber seed oil are also economically attractive. In addition, microalgae, bacteria, yeast, and fungi with 20% or higher lipid content are oleaginous microorganisms known as single cell oil and have been proposed as feedstocks for FAME production. Alternative feedstocks are characterized by their elevated acid value due to the high level of free fatty acid (FFA) content, causing undesirable saponification reactions when an alkaline catalyst is used in the transesterification reaction. The production of soap consumes the conventional catalyst, diminishing FAME production yield and simultaneously preventing the effective separation of the produced FAME from the glycerin phase. These problems could be solved using biological catalysts, such as lipases or whole-cell catalysts, avoiding soap production as the FFAs are esterified to FAME. In addition, by-product glycerol can be easily recovered, and the purification of FAME is simplified using biological catalysts.     

Ultra-sonication application in biodiesel production from heterotrophic oleaginous microorganisms

Utilization of microbial oil for biodiesel production has gained growing interest due to the increase in prices and the shortage of the oils and fats traditionally used in biodiesel production. However, it is still in the laboratory study stage due to the high cost of production. Employing organic wastes as raw materials to grow heterotrophic oleaginous microorganisms for further lipid production to produce biodiesel has been predicted to be a promising method for reducing costs. However, there are many obstacles including the low biodegradability of organic wastes, low lipid accumulation capacity of heterotrophic oleaginous microorganisms while using organic wastes, a great dependence on a high-energy consumption approach for biomass harvesting, utilization of toxic organic solvents for lipid extraction, and large amount of methanol required in trans-esterification and in-situ trans-esterifications. Ultra-sonication as a green technology has been extensively utilized to enhance bio-product production from organic wastes. In this article, ultra-sonication applications in biodiesel production steps with heterotrophic oleaginous microorganisms have been reviewed, and its impact, potential, and limitations on the process have been discussed.     

Analysis of technological schemes for the efficient production of added-value products from Colombian oleochemical feedstocks

In the world, the evolution of biodiesel demand influences the global market of edible feedstocks, affecting their commercial prices and availability and consequently food security. Colombia as a tropical country has the potential of producing multiple oleochemical feedstocks including both edible and non-edible vegetable oils and animal fats. However, the oleochemical industry in the country is based on a small range of feedstocks. In this paper, five oleochemical feedstocks were selected according to comparative criteria of national availability, potential oleochemical derivatives, projected expansion, impact on food security and land requirements. This analysis allowed obtaining oil palm, maize, jatropha, castorbean, and microalgae as promissory feedstocks. Then, multiproduct biorefineries were developed and economically and environmentally assessed, according to their production features. Thus, it was stated that the proposed configurations were able to support the development of the oleochemical industry in Colombia providing sustainable alternatives, which supply a growing demand of biodiesel and other oleochemical derivatives.     

Biotechnological production of biodiesel fuel using biocatalysed transesterification: A review

Biotechnological production of biodiesel has attracted considerable attention during the past decade compared to chemical-catalysed production since biocatalysis-mediated transesterification has many advantages. Currently, there are extensive reports on enzyme-catalysed transesterification for biodiesel production; the related research can be classified into immobilised-extracellular and immobilised-intracellular biocatalysis and this review focusses on these forms of biocatalyst for biodiesel production. The optimisation of the most important operating conditions affecting lipase-catalysed transesterification and the yield of alkyl esters, such as the type and form of lipase, the type of alcohol, the presence of organic solvents, the content of water in the oil, temperature and the presence of glycerol, are discussed. However, there is still a need to optimise lipase-catalysed transesterification and reduce the cost of lipase production before it is applied commercially. Optimisation research of lipase-catalysed transesterification could include development of new reactor systems with immobilised biocatalysts, the use of lipases tolerant to organic solvents, intracellular lipases (whole microbial cells) and genetically modified microorganisms (intelligent yeasts). Biodiesel fuel is expensive in comparison with petroleum-based fuel and 60–70% of the cost is associated with feedstock oil and enzyme. Therefore ways of reducing the cost of biodiesel with respect to enzyme and substrate oils reported in literature are also presented.     

Technical difficulties and solutions of direct transesterification process of microbial oil for biodiesel synthesis

Microbial oils are considered as alternative to vegetable oils or animal fats as biodiesel feedstock. Microalgae and oleaginous yeast are the main candidates of microbial oil producers’ community. However, biodiesel synthesis from these sources is associated with high cost and process complexity. The traditional transesterification method includes several steps such as biomass drying, cell disruption, oil extraction and solvent recovery. Therefore, direct transesterification or in situ transesterification, which combines all the steps in a single reactor, has been suggested to make the process cost effective. Nevertheless, the process is not applicable for large-scale biodiesel production having some difficulties such as high water content of biomass that makes the reaction rate slower and hurdles of cell disruption makes the efficiency of oil extraction lower. Additionally, it requires high heating energy in the solvent extraction and recovery stage. To resolve these difficulties, this review suggests the application of antimicrobial peptides and high electric fields to foster the microbial cell wall disruption.     

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.     

Plant triacylglycerols as feedstocks for the production of biofuels

Triacylglycerols produced by plants are one of the most energy-rich and abundant forms of reduced carbon available from nature. Given their chemical similarities, plant oils represent a logical substitute for conventional diesel, a non-renewable energy source. However, as plant oils are too viscous for use in modern diesel engines, they are converted to fatty acid esters. The resulting fuel is commonly referred to as biodiesel, and offers many advantages over conventional diesel. Chief among these is that biodiesel is derived from renewable sources. In addition, the production and subsequent consumption of biodiesel results in less greenhouse gas emission compared to conventional diesel. However, the widespread adoption of biodiesel faces a number of challenges. The biggest of these is a limited supply of biodiesel feedstocks. Thus, plant oil production needs to be greatly increased for biodiesel to replace a major proportion of the current and future fuel needs of the world. An increased understanding of how plants synthesize fatty acids and triacylglycerols will ultimately allow the development of novel energy crops. For example, knowledge of the regulation of oil synthesis has suggested ways to produce triacylglycerols in abundant non-seed tissues. Additionally, biodiesel has poor cold-temperature performance and low oxidative stability. Improving the fuel characteristics of biodiesel can be achieved by altering the fatty acid composition. In this regard, the generation of transgenic soybean lines with high oleic acid content represents one way in which plant biotechnology has already contributed to the improvement of biodiesel.     

Biodiesel production from seed oil of Cleome viscosa L

Edible oil seed crops, such as rapeseed, sunflower, soyabean and safflower and non-edible seed oil plantation crops Jatropha and Pongamia have proved to be internationally viable commercial sources of vegetable oils for biodiesel production. Considering the paucity of edible oils and unsustainability of arable land under perennial plantation of Jatropha and Pongamia in countries such as India, the prospects of seed oil producing Cleome viscosa, an annual wild short duration plant species of the Indogangetic plains, were evaluated for it to serve as a resource for biodiesel. The seeds of C. viscosa resourced from its natural populations growing in Rajasthan, Haryana and Delhi areas of Aravali range were solvent extracted to obtain the seed oil. The oil was observed to be similar in fatty acid composition to the non-edible oils of rubber, Jatropha and Pongamia plantation crops and soybean, sunflower, safflower, linseed and rapeseed edible oil plants in richness of unsaturated fatty acids. The Cleome oil shared the properties of viscosity, density, saponification and calorific values with the Jatropha and Pongamia oils, except that it was comparatively acidic. The C. viscosa biodiesel had the properties of standard biodiesel specified by ASTM and Indian Standard Bureau, except that it had low oxidation stability. It proved to be similar to Jatropha biodiesel except in cloud point, pour point, cold filter plugging point and oxidation stability. In view of the annual habit of species and biodiesel quality, it can be concluded that C. viscosa has prospects to be developed into a short-duration biodiesel crop.     

微生物在生物能源生产中的应用(英文)

生物可再生能源是最有前景的石油替代品之一.生物能源的生产原料包括:植物、有机废弃物和微生物.微生物在生物能源生产上有着广泛的应用,利用微生物制备的主要生物能源包括:生物柴油、生物乙醇、生物甲烷等.某些微生物如微藻和真菌可以生产大量油脂,这些油脂可以转化为生物柴油;有些微生物如酵母可以将糖类、淀粉以及纤维素转化为燃料乙醇,添加乙醇的汽油或柴油燃烧排放明显降低;还有些厌氧微生物可以将有机废弃物转化为甲烷,可用做家用燃气、车用燃气或发电.除此之外微生物还具有在生产能源的同时治理环境污染的优势.总之研究开发微生物在生物能源生产中的应用有利于世界可持续发展.     

Oleaginous yeasts for biodiesel: Current and future trends in biology and production

Production of biodiesel from edible plant oils is quickly expanding worldwide to fill a need for renewable, environmentally-friendly liquid transportation fuels. Due to concerns over use of edible commodities for fuels, production of biodiesel from non-edible oils including microbial oils is being developed. Microalgae biodiesel is approaching commercial viability, but has some inherent limitations such as requirements for sunlight. While yeast oils have been studied for decades, recent years have seen significant developments including discovery of new oleaginous yeast species and strains, greater understanding of the metabolic pathways that determine oleaginicity, optimization of cultivation processes for conversion of various types of waste plant biomass to oil using oleaginous yeasts, and development of strains with enhanced oil production. This review examines aspects of oleaginous yeasts not covered in depth in other recent reviews. Topics include the history of oleaginous yeast research, especially advances in the early 20th century; the phylogenetic diversity of oleaginous species, beyond the few species commonly studied; and physiological characteristics that should be considered when choosing yeast species and strains to be utilized for conversion of a given type of plant biomass to oleochemicals. Standardized terms are proposed for units that describe yeast cell mass and lipid production.     

Biodiesel production from various feedstocks and their effects on the fuel properties

Biodiesel, which is a new, renewable and biological origin alternative diesel fuel, has been receiving more attention all over the world due to the energy needs and environmental consciousness. Biodiesel is usually produced from food-grade vegetable oils using transesterification process. Using food-grade vegetable oils is not economically feasible since they are more expensive than diesel fuel. Therefore, it is said that the main obstacle for commercialization of biodiesel is its high cost. Waste cooking oils, restaurant greases, soapstocks and animal fats are potential feedstocks for biodiesel production to lower the cost of biodiesel. However, to produce fuel-grade biodiesel, the characteristics of feedstock are very important during the initial research and production stage since the fuel properties mainly depend on the feedstock properties. This review paper presents both biodiesel productions from various feedstocks and their effects on the fuel properties. JIMB 2008: BioEnergy - Special issue.     

An overview of lipid metabolism in yeasts and its impact on biotechnological processes

High energy prices, depletion of crude oil supplies, and price imbalance created by the increasing demand of plant oils or animal fat for biodiesel and specific lipid derivatives such as lubricants, adhesives, and plastics have given rise to heated debates on land-use practices and to environmental concerns about oil production strategies. However, commercialization of microbial oils with similar composition and energy value to plant and animal oils could have many advantages, such as being non-competitive with food, having shorter process cycle and being independent of season and climate factors. This review focuses on the ongoing research on different oleaginous yeasts producing high added value lipids and on the prospects of such microbial oils to be used in different biotechnological processes and applications. It covers the basic biochemical mechanisms of lipid synthesis and accumulation in these organisms, along with the latest insights on the metabolic processes involved. The key elements of lipid accumulation, the mechanisms suspected to confer the oleaginous character of the cell, and the potential metabolic routes enhancing lipid production are also extensively discussed.     

Rhizopus oryzae IFO 4697 whole cell catalyzed methanolysis of crude and acidified rapeseed oils for biodiesel production in tert-butanol system

Whole cell Rhizopus oryzae (R. oryzae) IFO4697 immobilized within biomass support particles (BSPs) was used as catalyst for biodiesel production in tert-butanol, in which the stability of the catalyst could be enhanced significantly. Different feedstocks (refined, crude and acidified rapeseed oils) were adopted further for biodiesel production in tert-butanol system and it was found that when acidified rapeseed oil was used as feedstocks, the reaction rate and final methyl ester (ME) yield were significantly higher than that of refined and crude rapeseed oil. Major differences among the aforementioned oils were found to be the contents of free fatty acid (FFA), water and phospholipids, which showed varied influences on whole cell mediated methanolysis for biodiesel production. The reaction rate increased with the increase of free fatty acid content in oils; water content had varied influence on reaction rate and biodiesel yield; using adsorbent to remove excessive water could increase biodiesel yield significantly (from 73 to 84%); it was also found interestingly that phospholipids contained in oils could increase the reaction rate to a certain extent.     

组学技术在产油微生物中的应用

微生物油脂是未来燃料和食品用油的重要潜在资源。近年来,随着系统生物学技术的快速发展,从全局角度理解产油微生物生理代谢及脂质积累的特征成为研究热点。组学技术作为系统生物学研究的重要工具被广泛用于揭示产油微生物脂质高效生产的机制研究中,这为产油微生物理性遗传改造和发酵过程控制提供了基础。文中对组学技术在产油微生物中的应用概况进行了综述,介绍了产油微生物组学分析常用的样品前处理及数据分析方法,综述了包括基因组、转录组、蛋白(修饰)组及代谢(脂质)组等在内的多种组学技术,以及组学数据基础上的数学模型在揭示产油微生物脂质高效生产机制中的研究,并对未来发展和应用进行了展望。     

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.     

Laurie oil resources

Lauric oils and their derivatives have many applications both in the food and chemical industries. The major sources and some alternative raw materials for this multi-billon dollar business are discussed in the light of their ability to supply future market needs. There should be ample supply of lauric oils—except when drought and possibly disease affect large areas of coconut plantations—because of the rapid increase in palm kernel oil production expected from the African oil palm during the next decade. Most other sources are unlikely to be important in the short term because of the generally adequate supply of lauric oils and the considerable amount of research still needed to convert the best options into viable crops. However, a dramatic effect on supply can be expected if it becomes possible to manipulate the appropriate genes from lauric oil producing species of Cuphea into a conventional oil crop like rape. Future demand for lauric oils will be affected by the relative price of other vegetable oils and petroleum feedstocks that can be used to replace them in the manufacture of an increasing number of end products in both the food and chemical industries.     

Oleaginous yeasts: Biodiversity and cultivation

Microbial lipids produced by oleaginous microorganisms, also called microbial oils and single cell oils (SCOs), are very promising sources for several oil industries. The exploration of efficient oleaginous yeast strains, meant to produce both high-quantity and high-quality lipids for the production of biodiesel, oleochemicals, and the other high value lipid products, have gained much attention. At present, the number of oleaginous yeast species that have been discovered is 8.2% of the total number of known yeast species, most of which have been isolated from their natural habitats. To explore high lipid producing yeasts, different methods, including high-throughput screening methods using colorimetric or fluorometric measures, have been developed. Understanding of the fatty acid composition profiles of lipids produced by oleaginous yeasts would help to define target lipid-related products. For lipid production, the employment of low-cost substrates suitable for yeast growth and lipid accumulation, and efficient cultivation processes are key factors for successfully increasing the amount of the accumulated lipid yield while decreasing the cost of production.     

厌氧产表面活性剂微生物提高原油采收率的研究进展

微生物强化采油(microbial enhanced oil recovery,MEOR)是近年来在国内外发展迅速的一项提高原油采收率技术。微生物在油藏中高效生产表面活性剂等驱油物质是微生物采油技术成功实施的关键之一。然而,油藏的缺/厌氧环境严重影响好氧表面活性剂产生菌在油藏原位的生存与代谢活性;油藏注空气会增加开采成本,且注入空气的作用时效和范围难以确定。因此,开发厌氧产表面活性剂菌种资源并强化其驱油效率对于提高原油采收率具有重要意义。本文综述了国内外近年来利用厌氧产表面活性剂微生物提高原油采收率的研究进展,简述了微生物厌氧产表面活性剂的相关驱油机理、菌种资源开发现状以及油藏原位驱油应用进展,并对当前的研究提出了一些思考。     

Study on the effect of cultivation parameters and pretreatment on Rhizopus oryzae cell-catalyzed transesterification of vegetable oils for biodiesel production

Enzymatic methanolysis of vegetable oils for biodiesel production has become a hot point recently, in which study on whole cell as catalyst is an important field. In this paper, whole cell (Rhizopus oryzae IFO 4697) was adopted directly as biocatalyst for biodiesel production. Effects of carbon source on cell growth and whole cell-catalyzed methanolysis of vegetable oils for biodiesel production were studied. The results showed that different oils contained in the cultivation medium had varied effects on the whole cell-catalyzed methanolysis of oils; with some specified oil as the carbon source for cell cultivation, those cells expressed higher catalytic activity in catalyzing the transesterification of the same oil for biodiesel production. The initial reaction rate was increased notably (204%) with oil pretreatment on the cells before catalyzing the reaction, which was possibly due to the improved mass transferring of substrates. Under the optimized conditions, the maximum methyl ester yield could reach 86%.