Microbial lipids from renewable resources: production and characterization

A number of microorganisms belonging to the genera of algae, yeast, bacteria, and fungi have ability to accumulate neutral lipids under specific cultivation conditions. The microbial lipids contain high fractions of polyunsaturated fatty acids and have the potential to serve as a source of significant quantities of transportation fuels. This paper reviews the current state of the art of this field. It summarizes the various microorganism used, feed stocks available, environmental factors that influence growth of cells and accumulation of lipids, major fatty acid composition of lipids, and the technology.     

Oily yeasts as oleaginous cell factories

Oily yeasts have been described to be able to accumulate lipids up to 20% of their cellular dry weight. These yeasts represent a minor proportion of the total yeast population, and only 5% of them have been reported as able to accumulate more than 25% of lipids. The oily yeast genera include Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces. More specifically, examples of oleaginous yeasts include the species: Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, and Yarrowia lipolytica. Yeast do exhibit advantages for lipid production over other microbial sources, namely, their duplication times are usually lower than 1 h, are much less affected than plants by season or climate conditions, and their cultures are more easily scaled up than those of microalgae. Additionally, some oily yeasts have been reported to accumulate oil up to 80% of their dry weight and can indeed generate different lipids from different carbon sources or from lipids present in the culture media. Thus, they can vary their lipid composition by replacing the fatty acids present in their triglycerides. Due to the diversity of microorganisms and growth conditions, oily yeasts can be useful for the production of triglycerides, surfactants, or polyunsaturated fatty acids.     

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.     

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.     

Strain Design of Ashbya gossypii for Single-Cell Oil Production

Single-cell oil (SCO) represents a sustainable alternative for the oil industry. Accordingly, the identification of microorganisms with either higher lipidogenic ability or novel capacities for the transformation of raw materials constitutes a major challenge for the field of oil biotechnology. With this in mind, here, we were prompted to address the lipidogenic profile of the filamentous hemiascomycete Ashbya gossypii, which is currently used for the microbial production of vitamins. We found that A. gossypii mostly accumulates unsaturated fatty acids (FAs), with more than 50% of the total FA content corresponding to oleic acid. In addition, we engineered A. gossypii strains both lacking the beta-oxidation pathway and also providing ATP-citrate lyase (ACL) activity to block the degradation of FA and to increase the cytosolic acetyl-coenzyme A (CoA) content, respectively. The lipidogenic profile of the newly developed strains demonstrates that the mere elimination of the beta-oxidation pathway in A. gossypii triggers a significant increase in lipid accumulation that can reach 70% of cell dry weight. The use of A. gossypii as a novel and robust tool for the production of added-value oils is further discussed.     

产油微生物油脂生物合成与代谢调控研究进展

自然界中少量微生物在适宜条件下产生并贮存质量超过其细胞干重 2 0 %的油脂 ,具有这种表型的菌种称为产油微生物。产油微生物利用可再生资源 ,得到的微生物油脂与植物油脂具有相似的脂肪酸组成 ,有的还含有丰富的多不饱和脂肪酸 ,具有广阔开发应用前景。简要介绍了产油微生物的种类和代谢特点 ,较详细地阐述了微生物产油机制和代谢调控途径的最新研究进展 ,并对微生物油脂研究的未来发展方向提出了初步见解     

The effect of lipid content on the elemental composition and energy capacity of yeast biomass

Oleaginous yeasts (18 strains) were grown in ethanol media under various cultivation conditions (33 biomass samples). It was found that lipid and lipid-free fractions of dry biomass have elemental composition and biomass reductivity very close to values which can be considered as biological constants. The energy content of dry biomass strongly depended on the total lipid content. When the lipid content was 64%, the energy value of dry biomass reached 73% of diesel oil; therefore, oleaginous microorganisms can be a promising source of biodiesel fuel. The approach used in this work makes it possible to determine the energy value of biomass by its elemental composition without application of laborious and expensive calorimetric measurements of combustion heats.     

广谱碳源产油酵母菌的筛选

对10株酵母菌利用不同单糖为碳源条件下菌体内积累油脂的能力进行了初步考察,并对菌油进行了分离和脂肪酸组成分析。实验发现,以葡萄糖为唯一碳源时有9株菌油脂含量超过自身细胞干重的20%,可以界定为产油微生物。其中6#菌(T.cutaneumAS2.571)利用葡萄糖发酵菌体油脂含量达到65%(W/W)。所有实验菌株都能同化多种单糖,其中1#菌(L.starkeyiAS2.1390)、4#菌(R.toruloidesAS2.1389)和11#菌(L.starkeyiAS2.1608)表现出对碳源利用的广谱性,能转化五碳糖木糖和阿拉伯糖并在菌体内积累油脂,油脂含量最高达到26%。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,其中棕榈酸、油酸和亚油酸三者之和占总脂肪酸组成的90%以上,脂肪酸组成分布类似于常见的植物油。这些结果对利用产油微生物转化木质纤维素水解混合糖获取油脂资源的研究具有重要意义。     

未培养环境微生物培养方法的研究进展

自然界中微生物种类繁多、功能多样、分布广泛,对人类健康安全、生态稳定和物种进化等发挥不可替代的作用.尽管微生物培养技术至今已有一百多年,然而由于各种限制因素的制约,目前成功分离培养的微生物仅占0.1％-1.0％,自然界中仍有十分丰富的微生物资源有待挖掘和开发利用.如何理解难培养微生物的制约因素并探索作用机制,同时借此开...     

Detection of microbial proteolytic activity by a cultivation plate assay in which different proteins adsorbed to a hydrophobic surface are used as substrates.

A screening technique for microbial proteases, the thin-layer enzyme assay cultivation technique, was developed. The inner surface of a polystyrene petri dish was coated with protein and then covered with a culture agar medium. The enzymes, produced during growth of the microorganisms, reach the protein-coated surface by diffusion in the agar. Degradation of the protein was visualized by condensation of water vapor on the surface after removal of the agar medium. The wettability of the enzyme-affected protein-coated polystyrene surface was decreased compared with the unaffected protein surface. Enzyme substrates used were fibrinogen, immunoglobulin G, egg albumin, human serum albumin, bovine serum albumin, hemoglobin, mucin, and gelatin. It was possible to use a variety of culture agar media, nonselective as well as selective, in the assay. The technique provides a sensitive, convenient, and inexpensive method for screening various microbial proteases. In addition, the technique can be used for screening proteolytic enzyme activity of specific microbial species in a mixed microbial sample as well as for studies of factors that influence the cultivation conditions for protease production and activity.     

Detection of microbial proteolytic activity by a cultivation plate assay in which different proteins adsorbed to a hydrophobic surface are used as substrates

A screening technique for microbial proteases, the thin-layer enzyme assay cultivation technique, was developed. The inner surface of a polystyrene petri dish was coated with protein and then covered with a culture agar medium. The enzymes, produced during growth of the microorganisms, reach the protein-coated surface by diffusion in the agar. Degradation of the protein was visualized by condensation of water vapor on the surface after removal of the agar medium. The wettability of the enzyme-affected protein-coated polystyrene surface was decreased compared with the unaffected protein surface. Enzyme substrates used were fibrinogen, immunoglobulin G, egg albumin, human serum albumin, bovine serum albumin, hemoglobin, mucin, and gelatin. It was possible to use a variety of culture agar media, nonselective as well as selective, in the assay. The technique provides a sensitive, convenient, and inexpensive method for screening various microbial proteases. In addition, the technique can be used for screening proteolytic enzyme activity of specific microbial species in a mixed microbial sample as well as for studies of factors that influence the cultivation conditions for protease production and activity.     

Biosorption and recycling of gold using various microorganisms

In order to obtain basic information on the biosorption and recycling of gold from aqueous systems using microbial cells, the biosorption of gold by various microorganisms was investigated. Of 75 strains of microorganisms tested (25 bacteria, 19 actinomycetes, 17 fungi and 14 yeasts), high abilities of gold biosorption from a solution containing hydrogen tetrachloroaurate (III) were found in some gram-negative bacterial strains, such as Acinetobacter calcoaceticus, Erwinia herbicola, Pseudomonas aeruginosa, and P. maltophilia. Most of the gram-positive bacteria, actinomycetes, fungi and yeasts had a lower ability for gold biosorption than gram-negative bacteria. On the other hand, all of the microorganisms tested adsorbed far smaller amounts of gold from a solution containing gold dicyanoaurate (I). The biosorption of gold from a solution containing hydrogen tetrachloroaurate (III) using P. maltophilia having a high adsorbing ability for gold was very rapid and was affected by the pH of the solution, external gold concentration, and cell amounts. P. maltophilia cells immobilized with polyacrylamide gel also have a high ability for gold biosorption. The gold adsorbed on the immobilized cells is easily desorbed with 0.1 M thiourea solution. The immobilized P. maltophilia cells can be used repeatedly in biosorption-desorption cycles.     

微生物培养技术研究进展

微生物在自然界中分布广、种类多、资源丰富,绝大多数在现有条件下还不能被培养。了解微生物新的培养技术,提高微生物可培养性具有重要意义。文章就微生物不易培养的主要原因及改进微生物培养条件、设计开发新型微生物培养技术等方面进行了综述,旨在为微生物资源的开发利用提供借鉴。     

花蜜微生物及其生态功能研究进展

花蜜是虫媒植物提供给传粉者最有效的报酬,对花蜜特征介导的植物-传粉者相互关系的研究已成为当今传粉生物学研究中最活跃的领域之一。开花植物分泌的原始花蜜是无菌的,不过一些微生物可经由空气传播至花蜜或(和)通过与传粉者的喙接触而聚集于花蜜中,并利用花蜜中的营养物质进行快速繁殖。花蜜的高渗透压环境导致花蜜中微生物(酵母菌,细菌)的物种多样性相对较低。此外,某些生物(传粉者组成,微生物间的竞争)与非生物因素(渗透压,糖组成,次生代谢物质,抗菌化合物,可利用氮源,温度,pH)也可影响花蜜中微生物群落的形成。花蜜中微生物的代谢活动能够改变花蜜物理(温度,粘度)与化学(pH,H_2O_2含量,糖组成和浓度,氨基酸组分和浓度,以及气味)特性,进而影响传粉者的访花行为与植物的繁殖适合度。因而,对花蜜中微生物及其生态功能的研究近年来颇受传粉生物学家的关注。在总结已发表研究成果的基础上,提出今后的研究有必要结合分子生物学与化学分析技术,以进一步揭示影响花蜜中微生物群落的潜在因素的作用机制,同时对花蜜微生物改变花蜜的物理、化学特性及植物-传粉者之间相互作用的可能原因进行更详尽的阐释,特别是对花蜜微生物在生态系统中所发挥的生态功能进行进一步的研究与认识。     

Selective accumulation of heavy metals by microorganisms

Summary An investigation of the removal and recovery of urnnium from aqueous systems using microbial biomass has been described previously (Nakajima et al. 1982). To establish which microorganisms accumulate the most uranium, we extended our investigation of uranium uptake to 83 species of microorganisms, 32 bacteria, 15 yeasts, 16 fungi and 20 actinomycetes. Of these 83 species of microorganisms tested, extremely high uranium-absorbing ability was found in Pseudomonas stutzeri, Neurospora sitophila, Streptomyces albus and Streptomyces viridochromogenes.The selective accumulation of heavy metal ions by various microorganisms has also been examined. Uranyl, mercury and lead ions were readily accumulated by almost all the species of microorganisms tested. Actinomycetes and fungi differ from many bacteria and most yeasts in their selective accumulation of uranium and mercury.In addition to this fundamental research, uranium recovery was investigated in immobilized Streptomyces albus, a microorganism with high uranium-uptake ability. These immobilized cells adsorbed uranium readily and selectively. The immobilized cells recovered uranium almost quantitatively and almost all uranium absorbed was desorbed with 0.1 M Na₂CO₃. The dry weight of the free cells decreased by 50% during 5 adsorption-desorption cycles. However, the dry weight of the immobilized cells decreased by only 2% during 5 cycles. These results showed that microbial cells are more stable after immobilization and can be used repeatedly for the process of uranium adsorption-desorption.     

活性污泥微生物菌群研究方法进展

活性污泥是活性污泥法处理污水系统的功能主体。人类对活性污泥微生物菌群的认识随着其研究方法的发展而逐步深入。传统培养方法只能检测到活性污泥中1％～15％的微生物。随着一系列基于免培养的分子生物学技术的出现,活性污泥中菌群的复杂性和多样性以惊人的速度被人们认识,大量依靠传统检测方法未能发现却在活性污泥中起关键作用的微生物逐渐被发现。许多模拟活性污泥菌群生存环境条件的现代培养技术开始发展,且已成功培养了一部分传统培养方法不能培养的细菌类群,这为研究基于免培养方法发现的大量新的微生物菌群的生理特性和作用机制提供了可能,也无疑将把人们对活性污泥菌群的认识推向一个新的层次．主要介绍活性污泥微生物菌群研究的一系列方法,从传统培养方法到基于免培养的现代分子生物学技术,再到现代培养技术,着重论述了现代分子生物学技术及其在活性污泥微生物菌群研究中的进展。     

从环境中分离培养微生物:培养基营养水平至关重要

根据目前的微生物学知识体系,利用现有的微生物培养技术仅仅能够分离和培养部分微生物,自然环境中绝大多数微生物暂不能培养。本文总结归纳了部分微生物培养技术和培养策略,包括采取生境隔离、延长培养时间以及模拟自然环境条件等方法,尤其是培养基的营养水平对可培养微生物数量及种类产生重要影响。简要总结了寡营养微生物及其生态意义,以及营养物浓度影响微生物生长的机理。提出可根据微生物的生态环境条件及细胞生理特性,设计合理的培养条件和培养方法,以及采用多种分离培养方法联合,以期最终提高环境微生物的可培养性。     

Molecular insight into activated sludge producing polyhydroxyalkanoates under aerobic–anaerobic conditions

One of the options enabling more economic production of polyhydroxyalkanoates compared to pure cultures is the application of mixed cultures. The use of a microbial community in a sequencing batch reactor has a few advantages: a simple process control, no necessity for sterile processing, and possibilities of using cheap substrates as a source of carbon. Nevertheless, while cultivation methods to achieve high PHAs biomass concentration and high productivity in wild and recombinant strains are defined, knowledge about the cultivation strategy for PHAs production by mixed culture and species composition of bacterial communities is still very limited. The main object of this study was to characterize on the molecular level the composition and activity of PHAs producing microorganism in activated sludge cultivated under oxygen limitation conditions. PHAs producers were detected using a PCR technique and the created PHA synthase gene library was analyzed by DNA sequencing. The obtained results indicate that PHAs-producers belonged to Pseudomonas sp., and possessed genes coding for mcl-PHA synthase. The kinetics of mcl-PHA synthase expression was relatively estimated using real-time PCR technology at several timepoints. Performed quantitative and qualitative analysis of total bacterial activity showed that there were differences in total activity during the process but differential expression of various groups of microorganisms examined by using DGGE was not observed.     

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.     

Yeast Lipids: Extraction,Quality Analysis,and Acceptability

Abstract

While a class of yeasts excrete lipid-containing surfactants, oleaginous yeasts produce and store lipids similar to vegetable oils and fats. Recovery of the oleaginous yeast lipids is problematic because of their intracellular nature and protection by well-knit biopolymers of the cell wall and other membrane systems. There is no suitable method of choice that ensures 100% recovery of intracellular lipids without affecting the native state during different unit operations. Several laboratory methods are available, but none can be adopted directly for commercial extractions due to technological limitations. However, as a result of the emergence of new downstream processing techniques, there is a positive indication for commercialization of yeast-lipid production in the future. Although most of the oleaginous yeasts are nonpathogenic, it is mandatory to analyze and report quality as well as toxicity of their lipids prior to market introduction as a component of human diet. This warrants specially formulated codes for edibility of yeast lipids and, in general, for similar products from other microbial sources.