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。     

原生质体紫外诱变选育可利用廉价碳源的高产油新菌株

【目的】研究并建立利用原生质体紫外诱变技术选育可利用廉价碳源发酵的高产油新菌株的方法。【方法】采用1.5%蜗牛酶和1.0%纤维素酶混合液水解去除细胞壁得到2A00015(近平滑假丝酵母,Candida parapsilosis)的原生质体,将其放于紫外灯下诱变及再生壁培养,筛选获得可利用廉价碳源发酵的高产油酵母,并采用气相色谱质谱联用法(GC-MS)测定其脂肪酸组成。【结果】突变效果最好的突变菌株2A00015/25用葡萄糖发酵培养7 d后,其生物量、油脂产率和产油量分别为17.77 g/L、58.12%和10.32 g/L,较原始菌株分别提高了12.45%、23.32%和38.68%;利用废糖蜜发酵培养,其生物量、油脂产率和产油量分别为18.54 g/L、49.44%和9.17 g/L,较原始菌株分别提高了9.09%、21.16%和32.18%。利用废糖蜜培养其产油效率虽低于利用葡萄糖培养,但从环境保护及原材料成本的角度考虑,用废糖蜜作为碳源发酵培养产生油脂更具优势。诱变菌株利用废糖蜜发酵后产生油脂经检测含有8种脂肪酸,其脂肪酸组成与植物油近似,其中不饱和脂肪酸含量占脂肪酸总量的82.4%。【结论】通过利用原生质体紫外诱变技术,成功选育出一株新的可利用廉价碳源的高产油海洋菌株,产油率达到49.4%,提高了21.2%。     

高效利用木糖产油的氮离子注入Mortierella alpina诱变选育研究

木糖的有效利用是木质纤维素全利用的基础。为了获得高效利用木糖产油的高山被孢霉菌株,通过多轮氮离子束诱变,筛选出一株有效利用木糖的产油高山被孢霉1502．8（MortiereUaalpina1502—8,MAIS02．8）,并研究了以葡萄糖／木糖（W／W,5／3）组成的混合糖为碳源,突变株生长和油脂积累的特性。采用单因子和正交实验对培养基组成和发酵条件进行了优化,结果表明,诱变菌在温度28oC,pH8．0,接种量8％,装液量30％,蛋白胨0．76％和豆粕1％,分批补糖的最优发酵条件下发酵9d,生物量和菌体油脂积累量分别达29．8g／L和11．7g／L,较出发菌提高了2．59倍和2．05倍,同时原料糖利用率达到99．4％。     

斯达氏油脂酵母利用混合糖发酵产油脂

研究了斯达氏油脂酵母Lipomyces starkeyi2#利用葡萄糖-木糖混合糖为碳源生长和油脂积累特性。L.star-keyi2#利用70 g/L葡萄糖和70 g/L木糖作为碳源在30℃下摇瓶发酵96 h,糖利用率均达90%以上,菌体生物量分别为14.1 g/L和13.1 g/L,油脂质量分数分别为55.7%和52.6%。相同条件下该菌株利用混合糖(葡萄糖46 g/L,木糖24 g/L)为碳源时总糖利用率、生物量和油脂质量分数分别为75.1%,15.0 g/L和40.0%。借助于P lackett-Burm an设计法和单因子实验法对培养条件进行了优化,结果表明发酵96 h混合糖利用率可达到97.3%,发酵120 h后混合糖利用率、生物量和菌体油脂质量分数分别达99.5%、19.0 g/L和52.6%。生物量得率和油脂得率分别达到27%和14%。     

转化N-乙酰-D-葡糖胺产油真菌的筛选

对21株真菌利用甲壳素解聚产物N-乙酰-D-葡糖胺（NAG）为碳源积累油脂的能力进行了筛选。碳源同化实验得到可同化NAG的真菌7株,进一步筛选出能利用NAG积累油脂的酵母3株。摇瓶实验表明,C. albidus ATCC 56298和T. fermentans CICC 1368利用NAG发酵菌体油脂含量可分别达到67%和48%。气相色谱分析表明菌油富含棕榈酸、硬脂酸和油酸,与常规植物油脂的脂肪酸组成相似。研究结果拓宽了微生物油脂发酵的原料。     

碳源对卷枝毛霉EIM-10γ-亚麻酸产量的影响

目的:研究碳源对卷枝毛霉脂肪酸产量的影响,为代谢调控卷枝毛霉生产Y-亚麻酸奠定基础.方法:测定卷枝毛霉在各种碳源、碳源浓度及碳氮比条件下生物量、油质产量及油脂中GLA含量.结果:卷枝毛霉EIM-10在以葡萄糖为碳源发酵时,油脂得率为2%,油脂中γ-亚麻酸含量为18%;以大豆油为碳源时,其生物量(干重)达到33g/L,油脂占菌丝体干重的35%,GLA的含量为3%.卷枝毛霉EIM-10不能利用醋酸和柠檬酸,可以利用醋酸钠和柠檬酸钠生长但不积累油脂.结论:卷枝毛霉EIM-10脂肪酸从头合成能力不强,能利用外界脂肪酸合成细胞内油脂.     

产油脂霉菌的筛选及其产油条件优化

从土壤样品中筛选到一株产油脂较高的霉菌,命名为菌株T-5。通过正交实验,确定最佳油脂产油条件为葡萄糖100 g/L,无机氮源1 g/L,p H 6.0,温度28℃。对最佳产油条件进行验证,油脂含量为30.56%,从而为大规模工业化发酵生产微生物油脂提供了可靠的试验数据。气相色谱分析显示油脂脂肪酸组成为棕榈酸25.10%,油酸25.17%,亚油酸20.99%,硬脂酸4.78%,这与植物脂肪酸组成很类似,因此可成为制备生物柴油的油源。     

微拟球藻富油藻株筛选及柱状光生物反应器培养评价研究

研究以亲脂性荧光染料BODIPY505/515和流式细胞仪为基础, 从多株诱变海洋微拟球藻(Nannochloropsis oceanica)中筛选到4株候选富油藻株(MT-1,2,3,4), 并利用柱状光生物反应器对诱变株的产油能力进行了综合评价。结果表明, 藻株筛选时最佳BODIPY505/515使用浓度为0.87 μg/mL, 染色时间为10min; 4株诱变株产油性能较野生株有较大提高, 其中MT-4油脂积累达到了干重的66%, 油脂产率比野生型藻株提高了45%, 达到了27.32 mg/(L·d)。4株诱变株的脂肪酸组成合适, 其中C16和C18之和占78%以上, 且主要以饱和脂肪酸和单不饱和脂肪酸为主; 多不饱和脂肪酸只占总脂肪酸的6%—8%, 非常适合生物柴油生产。研究提供了一种针对海洋微拟球藻富油藻株快速、有效的筛选方法, 并以此为基础筛选得到4株极具生物柴油生产潜力的候选藻株, 有望用于规模化生产。     

碳源、氮源对异养单针藻Monoraphidium sp. FXY-10油脂积累和脂肪酸组成的影响

研究了碳源与氮源对单针藻Monoraphidium sp. FXY-10异养培养的影响。以BG-11为基础培养基,通过添加不同类型、浓度梯度碳源和氮源,比较分析微藻生物量、油脂积累以及脂肪酸组成。结果表明,以葡萄糖作碳源,硝酸钠为氮源,微藻细胞积累的油脂是理想的生物柴油制备原料。硝酸钠浓度分别为1.00、3.00和5.00 g/L时,对油脂产量影响不显著(P>0.05)。葡萄糖浓度为10.00 g/L,硝酸钠为氮源油脂产量达到实验最高值0.84 g/L,其油脂脂肪酸组成主要由C16:0和C18:1等短链饱和脂肪酸和单不饱和脂肪酸组成,不饱和度值(DU)为61.98,相对偏低。     

蛋白核小球藻发酵产油脂的研究

从5种不同来源的小球藻中筛选到1株油脂产量较高的蛋白核小球藻Chlorella pyrenoi-dosa No.2.研究了培养基组成及培养条件对其细胞生长和油脂积累的影响.结果表明,最适培养基组成为(g/L):葡萄糖20,甘氨酸0.08,MgSO4·7H2O 0.4,K2HPO4 1.0,FeSO4·7H2O 0.004;适宜的培养温度,初始pH、摇床转速和光照强度分别为28℃、6.0、130 r/min和650 Lux.在上述优化条件下培养7 d,Chlorella pyrenoidosa No.2的生物量和油脂含量分别由优化前的3.73 g/L和40.15%提高到6.56 g/L和59.90%,油脂产量提高了162%.Chlorella pyrenoidosa No.2能以木糖为碳源产油脂,可望用于以木质纤维素等可再生生物质资源为原料生产油脂.气相色谱分析表明该油脂的脂肪酸组成与植物油相似,不饱和脂肪酸含量达71%左右,可作为生产生物柴油的原料.     

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.     

The proteome analysis of oleaginous yeast Lipomyces starkeyi

Oleaginous yeast Lipomyces starkeyi, a species in the Saccharomycetales order, has the capability to accumulate over 70% of its cell biomass as lipid under defined culture conditions. In this study, analysis of L. starkeyi AS 2.1560 proteome samples from different culture stages during a typical lipid production process was performed using an online multidimensional μRPLC/MS/MS method. Data searching against the proteome database of the yeast Saccharomyces cerevisiae led to the identification of 289 protein hits. Further comparative and semi-quantitative analysis under more stringent criteria revealed 81 proteins with significant expression-level changes. Among them, 52 proteins were upregulated and 29 proteins were downregulated. Gene ontology annotation indicated that global responses occurred when cells were exposed to the nitrogen deficiency environment for lipid production. Protein hits were annotated and largely concerned metabolic processes for alternative nitrogen sources usage or lipid accumulation. Many of the downregulated proteins were related to glycolysis, whereas the majority of the upregulated proteins were involved in proteolysis and peptidolysis, carbohydrate metabolism and lipid metabolism. Insights were provided in terms of cellular responses to nutrient availability as well as the basic biochemistry of lipid accumulation. This work presented potentially valuable information for understanding the biochemical events related to microbial oleaginity and rational engineering of oleaginous yeasts.     

Efficient oleaginous yeasts for lipid production from lignocellulosic sugars and effects of lignocellulose degradation compounds on growth and lipid production

Microbial lipid production using lignocellulosic biomass is considered an alternative for biodiesel production. In this study, 418 yeast strains were screened to find efficient oleaginous yeasts which accumulated large quantities of lipid when cultivated in lignocellulosic sugars. Preliminary screening by Nile red staining revealed that 142 strains contained many or large lipid bodies. These strains were selected for quantitative analysis of lipid accumulation by shaking flask cultivation in nitrogen-limited medium II containing 70 g/L glucose or xylose or mixture of glucose and xylose in a ratio of 2:1. Rhodosporidium fluviale DMKU-SP314 produced the highest lipid concentration of 7.9 g/L when cultivated in the mixture of glucose and xylose after 9 days of cultivation, which was 55.0% of dry biomass (14.3 g/L). The main composition of fatty acids were oleic acid (40.2%), palmitic acid (25.2%), linoleic acid (17.9%) and stearic acid (11.1%). Moreover, the strain DMKU-SP314 could grow and produce lipid in a medium containing predominantly lignocellulose degradation products, namely, acetic acid, formic acid, furfural, 5-hydroxymethylfurfural (5-HMF) and vanillin, with however, some inhibitory effects. This strain showed high tolerance to acetic acid, 5-HMF and vanillin. Therefore, R. fluviale DMKU-SP314 is a promising strain for lipid production from lignocellulosic hydrolysate.     

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.     

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。     

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.     

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.     

Carbon source utilization and inhibitor tolerance of 45 oleaginous yeast species

Conversion of lignocellulosic hydrolysates to lipids using oleaginous (high lipid) yeasts requires alignment of the hydrolysate composition with the characteristics of the yeast strain, including ability to utilize certain nutrients, ability to grow independently of costly nutrients such as vitamins, and ability to tolerate inhibitors. Some combination of these characteristics may be present in wild strains. In this study, 48 oleaginous yeast strains belonging to 45 species were tested for ability to utilize carbon sources associated with lignocellulosic hydrolysates, tolerate inhibitors, and grow in medium without supplemented vitamins. Some well-studied oleaginous yeast species, as well as some that have not been frequently utilized in research or industrial production, emerged as promising candidates for industrial use due to ability to utilize many carbon sources, including Cryptococcus aureus, Cryptococcus laurentii, Hannaella aff. zeae, Tremella encephala, and Trichosporon coremiiforme. Other species excelled in inhibitor tolerance, including Candida aff. tropicalis, Cyberlindnera jadinii, Metschnikowia pulcherrima, Schwanniomyces occidentalis and Wickerhamomyces ciferrii. No yeast tested could utilize all carbon sources and tolerate all inhibitors tested. These results indicate that yeast strains should be selected based on characteristics compatible with the composition of the targeted hydrolysate. Other factors to consider include the production of valuable co-products such as carotenoids, availability of genetic tools, biosafety level, and flocculation of the yeast strain. The data generated in this study will aid in aligning yeasts with compatible hydrolysates for conversion of carbohydrates to lipids to be used for biofuels and other oleochemicals.     

Endophytic Fungal Strains of Soybean for Lipid Production

Lipids created via microbial biosynthesis are a potential raw material to replace plant-based oil for biodiesel production. Oleaginous microbial species currently available are capable of accumulating high amount of lipids in their cell biomass, but rarely can directly utilize lignocellulosic biomass as substrates. Thus this research focused on the screening and selection of new fungal strains that generate both lipids and hydrolytic enzymes. To search for oleaginous fungal strains in the soybean plant, endophytic fungi and fungi close to the plant roots were studied as a microbial source. Among 33 endophytic fungal isolates screened from the soybean plant, 13 have high lipid content (>20 % dry biomass weight); among 38 fungal isolates screened from the soil surrounding the soybean roots, 14 have high lipid content. Also, five fungal isolates with both high lipid content and promising biomass production were selected for further studies on their cell growth, oil accumulation, lipid content and profile, utilization of various carbon sources, and cellulase production. The results indicate that most strains could utilize different types of carbon sources and some strains accumulated >40 % of the lipids based on the dry cell biomass weight. Among these promising strains, some Fusarium strains specifically showed considerable production of cellulase, which offers great potential for biodiesel production by directly utilizing inexpensive lignocellulosic material as feedstock.