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.     

The effect of physicochemical conditions and nutrient sources on maximizing the growth and lipid productivity of green microalgae

Biodiesel is a renewable fuel produced mostly from edible and non‐edible vegetables, by transesterification of neutral lipids (triacylglycerols). However, vegetable oil‐based biodiesel production competes with food crops for arable land, increasing food prices and leading to biodiversity loss. The production of biodiesel from oleaginous microorganisms – particularly microalgae – has attracted attention due to the higher lipid productivity of these organisms, when compared with vegetables. Several environmental factors – including light, temperature, pH and the presence of nutrients (particularly nitrogen, phosphorus and iron) – influence directly the ability of microalgae to produce and store triacylglycerols and other lipids, and also modulate microalgal growth. Although some environmental factors affect several species in a similar manner, differential responses between species are frequent, highlighting the importance of identifying optimal cultivation conditions for each species, to balance growth and lipid productivity for biodiesel production. Here, we reviewed the particular influence of the physicochemical and nutritional factors on the growth and lipid productivity of different green oleaginous microalgae species.     

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: 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.     

产油微生物利用餐厨垃圾生产油脂的研究进展

餐厨垃圾中含有丰富的营养物质,经生物转化过程可以合成对人类有用的化学品。某些产油微生物可以处理餐厨垃圾生产油脂,同时合成高附加值代谢产物如多不饱和脂肪酸、角鲨烯和类胡萝卜素等。这不仅能够降低生产成本,而且提高了产物的经济价值,具有极大的工业化应用潜力。文中主要概括了目前餐厨垃圾的处理研究现状,综述了产油微生物发酵餐厨垃圾生产油脂的研究进展,并结合笔者的工作对未来该领域的发展进行了总结与展望,以期为今后的相关研究提供有益借鉴。     

A symbiotic gas exchange between bioreactors enhances microalgal biomass and lipid productivities: taking advantage of complementary nutritional modes

This paper describes the association of two bioreactors: one photoautotrophic and the other heterotrophic, connected by the gas phase and allowing an exchange of O₂ and CO₂ gases between them, benefiting from a symbiotic effect. The association of two bioreactors was proposed with the aim of improving the microalgae oil productivity for biodiesel production. The outlet gas flow from the autotrophic (O₂ enriched) bioreactor was used as the inlet gas flow for the heterotrophic bioreactor. In parallel, the outlet gas flow from another heterotrophic (CO₂ enriched) bioreactor was used as the inlet gas flow for the autotrophic bioreactor. Aside from using the air supplied from the auto- and hetero-trophic bioreactors as controls, one mixotrophic bioreactor was also studied and used as a model, for its claimed advantage of CO₂ and organic carbon being simultaneously assimilated. The microalga Chlorella protothecoides was chosen as a model due to its ability to grow under different nutritional modes (auto, hetero, and mixotrophic), and its ability to attain a high biomass productivity and lipid content, suitable for biodiesel production. The comparison between heterotrophic, autotrophic, and mixotrophic Chlorella protothecoides growth for lipid production revealed that heterotrophic growth achieved the highest biomass productivity and lipid content (>22%), and furthermore showed that these lipids had the most suitable fatty acid profile in order to produce high quality biodiesel. Both associations showed a higher biomass productivity (10–20%), when comparing the two separately operated bioreactors (controls) which occurred on the fourth day. A more remarkable result would have been seen if in actuality the two bioreactors had been inter-connected in a closed loop. The biomass productivity gain would have been 30% and the lipid productivity gain would have been 100%, as seen by comparing the productivities of the symbiotic assemblage with the sum of the two bioreactors operating separately (controls). These results show an advantage of the symbiotic bioreactors association towards a cost-effective microalgal biodiesel production.     

Production of oils and fats by oleaginous microorganisms with an emphasis given to the potential of the nonconventional yeast Yarrowia lipolytica

Recently, there has been a great upsurge of interest in studies related to several aspects of microbial lipid production, which is one of the top topics in relevant research fields due to the high demand of these fatty materials in food, medical, oleochemical and biofuel industries. Lipid accumulation by the so-called “oleaginous microorganisms” can generate more than 20% w/w of oil in dry biomass and is governed by a plethora of parameters, such as medium pH, incubation temperature, nutrient limitation and C/N (carbon/nitrogen) ratio, which drastically affect the lipid production bioprocess. Until now, considerable work has been undertaken to find the cheapest substrate to enable lipid fermentation by oleaginous microorganisms. This review principally details information regarding microbial lipids, suitable production conditions and focuses attention on using the yeast Yarrowia lipolytica to achieve these objectives. Lipid production by this yeast is discussed and the necessary conditions and suitable substrates are reviewed.     

Modeling lipid accumulation in oleaginous fungi in chemostat cultures: I. Development and validation of a chemostat model for Umbelopsis isabellina

Lipid-accumulating fungi may be able to produce biodiesel precursors from agricultural wastes. As a first step in understanding and evaluating their potential, a mathematical model was developed to describe growth, lipid accumulation and substrate consumption of the oleaginous fungus Umbelopsis isabellina (also known as Mortierella isabellina) in submerged chemostat cultures. Key points of the model are: (1) if the C-source supply rate is limited, maintenance has a higher priority than growth, which has a higher priority than lipid production; (2) the maximum specific lipid production rate of the fungus is independent of the actual specific growth rate. Model parameters were obtained from chemostat cultures of U. isabellina grown on mineral media with glucose and NH₄ ⁺. The model describes the results of chemostat cultures well for D > 0.04 h⁻¹, but it has not been validated for lower dilution rates because of practical problems with the filamentous fungus. Further validation using literature data for oleaginous yeasts is described in part II of this paper. Our model shows that not only the C/N-ratio of the feed, but also the dilution rate highly influences the lipid yield in chemostat cultures.     

Perspectives of microbial oils for biodiesel production

Biodiesel has become more attractive recently because of its environmental benefits, and the fact that it is made from renewable resources. Generally speaking, biodiesel is prepared through transesterification of vegetable oils or animal fats with short chain alcohols. However, the lack of oil feedstocks limits the large-scale development of biodiesel to some extent. Recently, much attention has been paid to the development of microbial, oils and it has been found that many microorganisms, such as algae, yeast, bacteria, and fungi, have the ability to accumulate oils under some special cultivation conditions. Compared to other plant oils, microbial oils have many advantages, such as short life cycle, less labor required, less affection by venue, season and climate, and easier to scale up. With the rapid expansion of biodiesel, microbial oils might become one of potential oil feedstocks for biodiesel production in the future, though there are many works associated with microorganisms producing oils need to be carried out further. This review is covering the related research about different oleaginous microorganisms producing oils, and the prospects of such microbial oils used for biodiesel production are also discussed.     

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.     

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。     

High‐yield lipid production from lignocellulosic biomass using engineered xylose‐utilizing Yarrowia lipolytica

Lignocellulosic biomass shows high potential as a renewable feedstock for use in biodiesel production via microbial fermentation. Yarrowia lipolytica, an emerging oleaginous yeast, has been engineered to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into lipids for lignocellulosic biodiesel production. Yet, the lipid yield from xylose or lignocellulosic biomass remains far lower than that from glucose. Here we developed an efficient xylose‐utilizing Y. lipolytica strain, expressing an isomerase‐based pathway, to achieve high‐yield lipid production from lignocellulosic biomass. The newly developed xylose‐utilizing Y. lipolytica, YSXID, produced 12.01 g/L lipids with a maximum yield of 0.16 g/g, the highest ever reported, from lignocellulosic hydrolysates. Consequently, this study shows the potential of isomerase‐based xylose‐utilizing Y. lipolytica for economical and sustainable production of biodiesel and oleochemicals from lignocellulosic biomass.     

Volatile fatty acids as novel building blocks for oil-based chemistry via oleaginous yeast fermentation

Microbial oils are proposed as a suitable alternative to petroleum-based chemistry in terms of environmental preservation. These oils have traditionally been studied using sugar-based feedstock, which implies high costs, substrate limitation, and high contamination risks. In this sense, low-cost carbon sources such as volatile fatty acids (VFAs) are envisaged as promising building blocks for lipid biosynthesis to produce oil-based bioproducts. VFAs can be generated from a wide variety of organic wastes through anaerobic digestion and further converted into lipids by oleaginous yeasts (OYs) in a fermentation process. These microorganisms can accumulate in the form of lipid bodies, lipids of up to 60% wt/wt of their biomass. In this context, OY is a promising biotechnological tool for biofuel and bioproduct generation using low-cost VFA media as substrates. This review covers recent advances in microbial oil production from VFAs. Production of VFAs via anaerobic digestion processes and the involved metabolic pathways are reviewed. The main challenges as well as recent approaches for lipid overproduction are also discussed.     

Lipid production from Jerusalem artichoke by <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> Y4

Jerusalem artichoke (JA) is a perennial herbaceous plant widely available as non-grain raw material. Microbial lipid has been suggested as a potential feedstock for large scale biodiesel production. This paper describes lipid production using JA tuber processed by oleaginous yeast Rhodosporidium toruloides Y4. Batch and fed-batch modes were tested with feeding of concentrated JA extracts or JA hydrolysates. Cultivation of R. toruloides Y4 with JA extracts gave a moderate cellular lipid content of 40% (w/w), whereas lipid titer and cellular lipid content reached 39.6 g l⁻¹ and 56.5% (w/w), respectively, when JA hydrolysates were fed. Our results suggested that JA tubers may be further explored as raw material for large scale microbial lipid production.     

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。     

Effects of cassava starch hydrolysate on cell growth and lipid accumulation of the heterotrophic microalgae <Emphasis Type="Italic">Chlorella protothecoides</Emphasis>

Heterotrophic fermentation of microalgae has been shown to accumulate high amounts of microalgal lipids, which are regarded as one of the most promising feedstocks for sustainable biodiesel production. To increase the biomass and reduce the cost of microalgal culture, the purpose of this study was to evaluate the possibility of using cassava starch hydrolysate (CSH) instead of glucose as carbon source for heterotrophic culture of Chlorella protothecoides in flasks. First, the two-step enzymatic process of hydrolysis of cassava starch by α-amylase and glucoamylase was optimized; the conversion efficiency for cassava starch was up to 97.7%, and over 80% of CSH was glucose. Subsequently, we compared heterotrophic cultures of C. protothecoiedes using glucose or CSH as carbon source. The results demonstrated that when using CSH as the organic carbon source, the highest biomass and the maximum total lipid yield obtained were 15.8 and 4.19 g/L, representing increases of 42.3 and 27.7%, respectively, compared to using glucose as the organic carbon source. This suggests that CSH is a better carbon source than glucose for heterotrophic Chlorella protothecoides.     

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.     

Eighteen new oleaginous yeast species

Of 1600 known species of yeasts, about 70 are known to be oleaginous, defined as being able to accumulate over 20 % intracellular lipids. These yeasts have value for fundamental and applied research. A survey of yeasts from the Phaff Yeast Culture Collection, University of California Davis was performed to identify additional oleaginous species within the Basidiomycota phylum. Fifty-nine strains belonging to 34 species were grown in lipid inducing media, and total cell mass, lipid yield and triacylglycerol profiles were determined. Thirty-two species accumulated at least 20 % lipid and 25 species accumulated over 40 % lipid by dry weight. Eighteen of these species were not previously reported to be oleaginous. Triacylglycerol profiles were suitable for biodiesel production. These results greatly expand the number of known oleaginous yeast species, and reveal the wealth of natural diversity of triacylglycerol profiles within wild-type oleaginous Basidiomycetes.