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.     

High-productivity lipid production using mixed trophic state cultivation of <Emphasis Type="Italic">Auxenochlorella</Emphasis> (<Emphasis Type="Italic">Chlorella</Emphasis>) <Emphasis Type="Italic">protothecoides</Emphasis>

A mixed trophic state production process for algal lipids for use as feedstock for renewable biofuel production was developed and deployed at subpilot scale using a green microalga, Auxenochlorella (Chlorella) protothecoides. The process is composed of two separate stages: (1) the photoautotrophic stage, focused on biomass production in open ponds, and (2) the heterotrophic stage focused on lipid production and accumulation in aerobic bioreactors using fixed carbon substrates (e.g., sugar). The process achieved biomass and lipid productivities of 0.5 and 0.27 g/L/h that were, respectively, over 250 and 670 times higher than those obtained from the photoautotrophic cultivation stage. The biomass oil content (over 60 % w/DCW) following the two-stage process was predominantly monounsaturated fatty acids (~82 %) and largely free of contaminating pigments that is more suitable for biodiesel production than photosynthetically generated lipid. Similar process performances were obtained using cassava hydrolysate as an alternative feedstock to glucose.     

Evaluating the usability of 19 effluents for heterotrophic cultivation of microalgal consortia as biodiesel feedstock

A key reason inhibiting commercialization of algal oil as biodiesel feedstock, is cultivation cost. For this reason, the usability of 19 readily available industrial effluents (autoclaved and non-autoclaved) to support heterotrophic growth and lipid accumulation was evaluated using six mixed algal cultures. Autoclaved whey effluent was the best with 14.32 g biomass L^?1, 13.23% lipids, resulting in a lipid production of 1.91 g lipids L^?1. Biomass production and lipid accumulation were in many cases inverse, e.g. mixed algal culture termed TUT4 accumulating 84.25% lipids on autoclaved acid mine drainage, with very little biomass produced. Biomass production was dependent on the effluent type, whereas the lipid accumulation was influenced mostly by the specific mixed algal cultures. The fatty acid composition of the algal oil (fish cannery and whey effluents) showed high saturation, leading to acceptable cetane numbers, kinematic viscosity, good oxidative stability, but poor cold flow properties.     

Renewable microbial lipid production from <Emphasis Type="Italic">Oleaginous Yeast</Emphasis>: some surfactants greatly improved lipid production of <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis>

Microbial oil is drawing increasing interest worldwide as an alternative non-food oil feedstock for biodiesel industry. Nowadays researchers have been increasingly focused on the improvement of microbial oil production process. Oleaginous yeast Rhodosporidium toruloides (R. toruloides) is considered an important candidate due to its excellent capabilities of lipid accumulation, broad adaptabilities to various carbon substrates, and the potential of co-production of some pigments. In present work, the individual effects of non-ionic, cationic, and anionic surfactant on cell growth and lipid accumulation of R. toruloides were investigated for the first time. Interesting results were noticed when some anionic surfactants were supplemented. The most significant effect was observed with addition of 0.2 % (w/v) sodium lignosulfonate, that biomass concentration, lipid concentration, and lipid yield was increased by 25.1, 44.9, and 15.7 %, respectively. The fatty acid compositions of R. toruloides lipids remained unchanged, which is similar to that of vegetable oils, and is considered potential feedstock for biodiesel preparation.     

培养条件对产油微生物生长的影响

为了筛选出高产油菌株, 首先采用细胞形态学方法与细胞化学方法(苏丹III染色法)对4株高产油脂菌株进行初筛, 并通过索氏提取法对初筛菌株油脂含量进行分析, 确定M2菌株为实验菌株, 其油脂含量达53.09%。为了增加产油微生物油脂产量, 本试验考察了不同发酵条件对其细胞生长和油脂积累的影响。优化工艺参数为: 10° Bx玉米皮渣水解液为培养基质, 0.2% NaNO3为氮源, pH 6.0、28oC下发酵培养6 d, 微生物油脂含量75.21%, 菌体生物量30.40 g/L, 油脂产量22.86 g/L。气相色谱分析表明该油脂的脂肪酸组成与植物油相似, 主要含有16碳和18碳系脂肪酸, 可作为生物柴油的原料, 不饱和脂肪酸含量达68%, 可应用于医药化工领域。     

不同营养元素限制对圆红冬胞酵母油脂生产的影响

以产油酵母圆红冬胞酵母（Rhodosporidium toruloides）作为研究对象,系统地研究了氮、磷、硫限制对其油脂积累的影响,并在3L生物反应器上考察了R.toruloides在C/P摩尔比为1 133.3时初始葡萄糖浓度对油脂生产的影响。结果表明：氮、磷、硫中任意一种营养元素受限,均能促使R.toruloides在胞内积累高于自身干重60%的油脂;通过改变培养基的组成,可以调节油脂中脂肪酸的构成,使油脂中饱和脂肪酸比例高于70%或不饱和脂肪酸比例高于60%。就油脂生产强度及转化效率而言,磷限制优于氮限制或硫限制。当C/P摩尔比相同时,初始葡萄糖浓度越低越有利于油脂生产。对采用不同原料生产微生物油脂的技术有一定指导意义。     

The effect of volatile fatty acids as a sole carbon source on lipid accumulation by Cryptococcus albidus for biodiesel production

The use of volatile fatty acids (VFAs) for microbial lipid accumulation was investigated in flask cultures of Cryptococcus albidus. The optimum culture temperature and pH were 25 °C and pH 6.0, respectively, and the highest lipid content (27.8%) was obtained with ammonia chloride as a nitrogen source. The lipid yield coefficient on VFAs was 0.167 g/g of C. albidus with a VFAs (acetic, propionic, butyric acids) ratio of 8:1:1, which was in good agreement with a theoretically predicted lipid yield coefficient of the VFAs as a carbon source. The major fatty acids of the lipids accumulated by C. albidus were similar to those of soybean oil and jatropha oil. A preliminary cost analysis shows that VFAs-based biodiesel production is competitive with current palm and soybean based biodiesels. Further process development for lower aeration cost and higher lipid yield will make this process more economical.     

Microbial lipids from cellulolytic oleaginous fungus Penicillium citrinum PKB20 as a potential feedstock for biodiesel production

Microbial lipids derived from oleaginous fungi are considered as an alternative feedstock for biodiesel production. We attempt to isolate a cellulolytic oleaginous fungi as a potential feedstock for biodiesel production. The fungus was identified by 5.8 S-ITS rRNA gene sequencing. The extracellular enzyme activities were recorded after every 24 h for 7 days. Nile red staining and fluorescence microscopy was used to visualise the lipid bodies within the fungal hyphae. A renewable heterogeneous base catalyst derived from Musa balbisiana cola peels was used for the transesterification of Penicillium citrinum PKB20 derived oil into biodiesel. GC-MS analysis was used to analyse the fatty acid methyl esters (FAME) profile of the transesterified lipids. Penicillium citrinum PKB20 was isolated from detritus rich soil of Assam, India. The endoglucanase, xylanase and β-glucosidase enzyme activities were found to be 292.83 ± 0.29, 111.72 ± 0.45 and 6.54 ± 0.13 U/mg respectively. The specific enzyme activity for extracellular lipase was found to be 3.12 ± 0.16 U/mg. It could accumulate up to 60.61% of lipids in nitrogen-limited medium (7.34 ± 0.45 g/L biomass production). The extracted lipids were converted to biodiesel with 89.3% conversion efficiency. The predominant fatty acids were oleic acid (30.09%), palmitic acid (20.25%) and linoleic acid (33.14%) suggesting a balance between oxidative stability and cold flow properties for suitable biodiesel quality. Penicillium citrinum PKB20 was found to be a potential feedstock for biodiesel production with desirable fuel properties. The cellulolytic nature could be utilised for simultaneous lipid production directly on cellulosic substrates.     

Prediction of lipid accumulation-degradation in oleaginous micro-organisms growing on vegetable oils

Oleaginous micro-organisms (yeasts, moulds), in culture media having the carbon source as limited factor, degrade reserve lipids and produce new biomass, after the onset of carbon exhaustion from the medium. In this paper the process of lipid accumulation-degradation in oleaginous micro-organisms, growing on a vegatable oil was simulated. The model was integrated with 4 different methods and the parameters were optimised with the least squares method. It was found that the degradation of endocellular carbon pool is a very slow process characterised, however, by a good yield in fat-free biomass. Low values of the specific growth rates of the fat-free microbial mass, both from consumption of extra cellular and endocellular carbon pools, favourite the production of microbial lipid. The maximum of the specific rate of lipid accumulation is positively affected by the low values of the specific growth rate of the fat-free microbial mass from consumption of extra cellular carbon pool, but remained unaffected by the specific growth rate of the fat-free microbial mass from consumption of endocellular carbon pool. On the other hand, lipid production and specific rate of lipid accumulation are positively influenced by the high values of the specific rate of storage lipid formation. In conclusion, this numerical model can be used in the laboratory as pilot for planing further experimental work.     

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。     

Organic nitrogen of tomato waste hydrolysate enhances glucose uptake and lipid accumulation in Cunninghamella echinulata

Aims: To investigate the effect of organic nitrogen on lipogenesis during growth of Cunninghamella echinulata on tomato waste hydrolysate (TWH) media. Methods and Results: Cunninghamella echinulata grown on a TWH medium rapidly took up glucose and produced large amounts of lipids. However, when some quantities of the organic nitrogen were removed from TWH (by acid followed by alkaline precipitation of proteins) the uptake of glucose was dramatically reduced and large quantities of fungal biomass having low lipid content were produced. Nevertheless, when glycerol was used as carbon source instead of glucose, the uptake rate as well as the biomass production and the lipid accumulation processes were unaffected by the TWH organic nitrogen removal. Finally, when the fungus was grown on a glucose supplemented TWH medium that contained no assimilable organic nitrogen (after further precipitation of proteins with methanol), the produced biomass contained non-negligible quantities of lipids, although glucose uptake remained low. Lipid analysis showed that the produced lipids comprised mainly of neutral lipids, which were preferentially consumed during lipid turnover. Lipid production on the original TWH medium having glucose as carbon source was 0·48 g of lipid per gram of dry biomass, corresponding to 8·7 g of lipid per litre of growth medium. The produced lipids contained 11·7%γ-linolenic acid (GLA), hence the GLA yield was more than 1 g l⁻¹. Conclusions: Organic nitrogen compounds found in TWH favour glucose (but not glycerol) uptake and lipid accumulation in C. echinulata. Significance and Impact of the Study: Agro-industrial wastes containing organic nitrogen, such as tomato waste, are produced in vast amounts causing severe environmental problems. These wastes could be used as fermentation feedstock to produce microbial lipids.     

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。     

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。     

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.     

Single-cell oil production by Mortierella isabellina DSM 1414 using different sugars as carbon source

Pure carbon sources, especially carbohydrates which are raw materials deriving from agro-industrial processes, are often used for small-scale single-cell oil production by fermentation. The aim of this study was to investigate the effects of different pure carbon sources on cell growth, lipid accumulation, and γ-linolenic acid (GLA) production by the filamentous fungus Mortierella isabellina DSM 1414 (Deutsche Sammlung von Mikroorganismen). The sugars utilized in this study are found extensively and abundantly in nature, especially in food raw materials and, in consequence, in agro-food industry wastes or surpluses. Thus, the potential of many waste materials containing these sugars to be used in the production of single-cell oil by fermentation could also be evaluated. The effects of the sugars utilized on cell growth, biomass production, and lipid production were investigated. Fatty acids were also analysed in the lipids produced at the end of the fermentations. Results showed that the maximum biomass production was 10.80 g/L in lactose-based media, while the maximum oil production was 5.44 g/L in maltose-based media. Oleic (20.42%–42.94%), palmitic (14.96%–22.19%), and stearic (9.00%–26.92%) acids were the major fatty acids along with linoleic acid (11.35%–18.67%) and GLA (3.56%–8.04%). The production of GLA as the target fatty acid was remarkable. This study indicates that agro-industrial waste including most of the sugars utilized (except for arabinose and sucrose with lipid production of 0.81 and 0.28 g/L, respectively) can be employed for production of single-cell oil by M. isabellina DSM 1414 which contains a high amount of GLA.     

Direct bioconversion of rice residue from canteen waste into lipids by new amylolytic oleaginous yeast Sporidiobolus pararoseus KX709872

The new amylolytic oleaginous red yeast, Sporidiobolus pararoseus KX709872, produced both α-amylase (540?±?0.09?mU/mL) and amyloglucosidase (23?±?0.00?mU/mL) and showed good ability to directly convert rice residue from canteen waste to biomass and lipids. Effects of medium composition and cultivation conditions on growth and lipid accumulation for strain KX709872 were investigated under shaking flask and upscaling levels. At C?:?N ratio of 25?:?1, pH 5.45, 22.36°C, and 199.40?rpm for 7 days, volumetric production of biomass and lipids, lipid content, and lipid productivity reached 17.69?±?0.44, 8.35?±?0.19?g/L, 49.48?±?0.41% (w/w), and 1.67?±?0.11?g/L/day, respectively. Production of lipids was also implemented in 5.0-L stirred tank bioreactor with 2.5?L of optimized medium at 300?rpm and 3.0 vvm for 5 days. Volumetric production of biomass and lipids, lipid content, and lipid productivity were 16.33?±?0.49, 8.75?±?0.13?g/L, 56.61?±?0.04% (w/w), and 2.19?±?0.03?g/L/day, respectively. Meanwhile, the fatty acids of lipids from strain KX709872 had high oleic acid content (60?62%) which was similar to those of vegetable oils, indicating that these lipids are promising as an alternative biodiesel feedstock. Moreover, the biodiesel derived from lipids of strain KX709872 had properties satisfying the criteria of ASTM D6751 and EN 14214 standards.     

Examination of Thermophilic Methane-Producing Digesters by Analysis of Bacterial Lipids

Thermophilic methane-producing digesters were examined by the analysis of lipids to determine the microbial biomass, community structure, and nutritional status of the microbes within the digesters. The digesters received a daily feedstock of cattle feed and Bermuda grass, with some digesters receiving additional supplements of propionate, butyrate, or nitrate. Microbial biomass, measured as total extractable lipid phosphate, was decreased in slurries from digesters receiving continuous addition of the fermentation intermediates propionate or butyrate as compared with slurries from control digesters receiving the feedstock alone. In slurries from digesters that received continuous addition of nitrate, the microbial biomass was higher than in the slurries from control digesters. The control digesters had ca. 2.5 × 10¹¹ bacteria per g (dry weight) as determined from total extractable lipid phosphate. Shifts in microbial community structure were observed by analysis of ester-linked phospholipid fatty acids. Statistical analysis of the patterns of phospholipid fatty acids indicated that the digesters receiving different supplements could be distinguished from the control digester and from each other. Poly-β-hydroxybutyric acid, an indicator of metabolic stress, was detected in slurries from all the digesters. Slurries from the nitrate-amended digester had the highest concentration of poly-β-hydroxybutyric acid, whereas slurries from the propionate-amended digester had the lowest concentration. These chemical analyses offer a quantitative means to correlate shifts in microbial biomass, community structure, and nutritional status in complex fermentation systems to the production of a specific end product.     

A reconfigured Kennedy pathway which promotes efficient accumulation of medium‐chain fatty acids in leaf oils

Medium‐chain fatty acids (MCFA, C6‐14 fatty acids) are an ideal feedstock for biodiesel and broader oleochemicals. In recent decades, several studies have used transgenic engineering to produce MCFA in seeds oils, although these modifications result in unbalance membrane lipid profiles that impair oil yields and agronomic performance. Given the ability to engineer nonseed organs to produce oils, we have previously demonstrated that MCFA profiles can be produced in leaves, but this also results in unbalanced membrane lipid profiles and undesirable chlorosis and cell death. Here we demonstrate that the introduction of a diacylglycerol acyltransferase from oil palm, EgDGAT1, was necessary to channel nascent MCFA directly into leaf oils and therefore bypassing MCFA residing in membrane lipids. This pathway resulted in increased flux towards MCFA rich leaf oils, reduced MCFA in leaf membrane lipids and, crucially, the alleviation of chlorosis. Deep sequencing of African oil palm (Elaeis guineensis) and coconut palm (Cocos nucifera) generated candidate genes of interest, which were then tested for their ability to improve oil accumulation. Thioesterases were explored for the production of lauric acid (C12:0) and myristic (C14:0). The thioesterases from Umbellularia californica and Cinnamomum camphora produced a total of 52% C12:0 and 40% C14:0, respectively, in transient leaf assays. This study demonstrated that the introduction of a complete acyl‐CoA‐dependent pathway for the synthesis of MFCA‐rich oils avoided disturbing membrane homoeostasis and cell death phenotypes. This study outlines a transgenic strategy for the engineering of biomass crops with high levels of MCFA rich leaf oils.     

圆红冬孢酵母两阶段培养法生产微生物油脂

为缩短发酵时间,减少原料消耗,采用细胞增殖和油脂积累分离的两阶段模式,培养圆红冬孢酵母Rhodosporidium toruloides AS 2.1389生产微生物油脂。结果表明,细胞增殖阶段获得的R.toruloides AS 2.1389细胞,重悬接种在葡萄糖溶液中,可快速积累油脂,菌体油脂含量超过自身干重的55%。增殖阶段细胞的菌龄越高,产油能力越强。油脂积累阶段使用高浓度葡萄糖溶液或未灭菌的葡萄糖溶液,油脂合成可以有效进行。油脂中脂肪酸成分以含16和18个碳原子的长链脂肪酸为主,可作为制备生物柴油的新型原料。     

The boosted lipid accumulation in microalga <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> by a heterotrophy and nutrition-limitation transition cultivation regime

A model of heterotrophy and nutrition-limitation transition cultivation for efficient algal biomass and lipid production was proposed in this study, wherein sufficient robust heterotrophic-seed cells submitted into nitrogen-starvation induction for boosted lipid accumulation. The results demonstrated that heterotrophic-seed (HS) achieved specific growth rate of 1.35 day^?1 and biomass productivity of 1.93 mg/L/d, representing 6.42- and 32.16-fold, 2.01- and 2.75-fold more than that of photoautotrophic-seed (PS) and mixtrophic-seed (MS). Even though subsequent nutrition-limitation cultivation repressed the growth of HS, the overall lipid productivity caused by nitrogen-starvation was not offset by biomass loss. The most favorable lipid productivity (465.61 mg/L/d) of HS was 3.25 and 52.31 times higher than that of MS and PS. The high content of monounsaturated fatty acids (50.13%) over saturated and polyunsaturated fatty acids (totally 47.39%) in HS cells could provide superior oxidation stability and lower viscosity for biofuels generated from algal biomass feedstock. These findings suggested the feasibility of using heterotrophy and nutrition-limitation transition cultivation for enhancing the overall lipid productivity. Further, several critical enzymes (i.e. G3PDH, ME, and ACAD) were highly related to lipid accumulation and showed especially pronounced up-regulation or down-regulation expression in HS, which provide indications for shedding light on the molecular mechanisms of lipid accumulation and a prospective metabolic engineering for lipid production.