Effects of Different Oil Sources and Residues on Biomass and Metabolite Production by Yarrowia lipolytica YB 423-12

Yarrowia lipolytica is known to have the ability to assimilate hydrophobic substrates like triglycerides, fats, and oils, and to produce single-cell oils, lipases, and organic acids. The aim of the present study was to investigate the effects of different oil sources (borage, canola, sesame, Echium, and trout oils) and oil industry residues (olive pomace oil, hazelnut oil press cake, and sunflower seed oil cake) on the growth, lipid accumulation, and lipase and citric acid production by Y. lipolytica YB 423-12. The maximum biomass and lipid accumulation were observed with linseed oil. Among the tested oil sources and oil industry residues, hazelnut oil press cake was the best medium for lipase production. The Y. lipolytica YB 423-12 strain produced 12.32 ± 1.54 U/mL (lipase activity) of lipase on hazelnut oil press cake medium supplemented with glucose. The best substrate for citric acid production was found to be borage oil, with an output of 5.34 ± 0.94 g/L. The biotechnological production of valuable metabolites such as single-cell oil, lipase, and citric acid could be achieved by using these wastes and low-cost substrates with this strain. Furthermore, the cost of the bio-process could also be significantly reduced by the utilization of various low-cost raw materials, residues, wastes, and renewable resources as substrates for this yeast.     

食品检验用解脂耶氏酵母菌标准物质制备研究

目的 为满足实验室酵母菌日常检验质量控制需求及实验室间比对,制备解脂耶氏酵母菌检验用标准物质。方法 通过生化、基质辅助激光解吸电离飞行时间质谱及ITS位点测序对研究用菌株进行菌种鉴定确认后,刮取适宜菌苔于保护剂中混匀后冻干,制备10_⁴ CFU/样品浓度的标准物质。参照CNAS-GL003等标准要求进行均匀性检验后,采用单因素方差分析对结果进行评价。将样品放于25 ℃和37 ℃及-20 ℃、4 ℃条件下,分别进行运输稳定性检验及储存稳定性检验。依据国家标准GB 4789.15,将研制的本标准物质加入7类食品基质共20件样品中进行检验,验证真实食品样品中适用性。组织3家实验室,对本标准物质进行协作标定。结果CMCC98025经生化鉴定为解脂耶氏酵母菌,准确度为93%;MALDI-TOF MS鉴定结果为解脂耶氏酵母,分数为2.012;ITS位点测序结果与NCBI Genbank中已有序列比对,匹配最优结果为解脂耶氏酵母Yarrowia lipolytica(Accession number ：CP061015.1;Query Cover：95%;Ident：100%)。均匀性测试结果符合正态分布,通过单因子方差分析计算F_0.05(19,20)=2.137,F值为1.697,F＜F_0.05,符合均匀性要求。25 ℃及37 ℃培养箱中放置7天,标准物质中菌含量仍保持在10_⁴ CFU/样品,可保持稳定。标准物质于4 ℃储存28天及-20 ℃储存90天,菌含量为10_⁴ CFU/样品,复苏率均在91%以上,说明4 ℃放置较短时间及-20 ℃放置较长时间样品稳定。7类食品样品基质中加入本标准物质后进行检验,均可检出,回收率为81.3%。经三家实验室协作标定,本标准物质平均浓度为2.0～3.0×10_⁴ CFU/样品。结论 本研究制备的解脂耶氏酵母菌标准物质均匀性、稳定性、真实食品样品中应用验证及协作标定结果均符合要求,可应用于食品中解脂耶氏酵母菌定性检验,今后可作为实验室日常检验工作的阳性对照质控样品,也可作为实验室间比对样品发放,以保障日常检验工作结果可靠性,进一步提升检验机构人员的检验水平。     

Utilization of Whey and Grape Must for Citric Acid Production by Two Yarrowia lipolytica Strains

In this study, utilization of whey and grape must were investigated for citric acid production using Yarrowia lipolytica NBRC 1658 and a domestic strain Y. lipolytica 57. In addition to its use as a sole nutrient source, whey was also fortified with glucose or fructose as well as other nutrients. The best results for citric acid production were obtained in the medium containing whey supplemented with fructose. Maximum citric acid concentrations in this medium were 49.23 and 32.65 g/L for the domestic and NBRC 1658 strains, respectively. In grape must, maximum citric acid concentrations obtained using domestic and NBRC 1658 strains were 32.09 and 10.39 g/L, respectively. Both of the natural nutrient sources were found to be promising for utilization in citric acid production process. A domestic Turkish yeast strain was confirmed to be superior for citric acid production for the first time. This can be targeted for enhancing citric acid production efficiencies from locally available substrates such as whey or grape must.     

泡椒凤爪中解脂耶氏酵母的分离鉴定及生长特性分析

本研究对涨袋泡椒凤爪产品中的主要腐败菌及其生长特性进行了分析。采用传统的分离培养方法,筛选出主要的污染菌群,通过美兰染色镜检对细胞形态进行检测,利用26S rDNA D1/D2区序列测定及系统发育分析对分离菌株进行鉴定,采取ERIC-RAPD技术对分离菌株的分子遗传多态性进行分析,最后对优势菌株的生长特性进行研究。结果表明:从涨袋产品中共分离出40株酵母菌,分别编号为PJ1~PJ40,镜检发现所有菌株的细胞形态相同,均为椭圆形。经分子鉴定发现所分离菌株都为解脂耶氏酵母,且为两个基因分型,说明解脂耶氏酵母是该产品中的一种优势腐败菌,且污染源不同。同时,研究发现解脂耶氏酵母PJ1的最适生长pH值为4.5、最适生长温度为25℃、并且NaCl浓度的升高对该菌的生长起到一定的抑制作用。     

Distribution of Mixing Efficiency in a Split-Cylinder Gas-Lift Bioreactor with Immobilized Yarrowia Lipolytica Cells Used for Olive Oil Mill Wastewater Treatment

The distribution of mixing in a split-cylinder gas-lift bioreactor has been investigated for suspensions of immobilized Yarrowia lipolytica cells with particles diameter varying between 3 and 4.2 mm. The results indicated important variation of mixing time on the height of riser or downcomer, as well as different behavior of suspensions flows in these two regions. Therefore, for the riser, the mixing efficiency increases from its bottom to the top, allowing the biocatalyst particles with intermediate size (3.6-mm diameter) reaching the most intense circulation of suspension. The analysis of the suspension flow in the downcomer region revealed that the intermediate positions are associated with the highest mixing mainly for the largest immobilized yeast particles (4.2-mm diameter). In both cases, the influence of aeration on turbulence extent is positive only for air superficial velocity up to 1.05–1.35 × 10^?3 m/s, the magnitude of this effect being correlated with the biocatalyst size and position on the riser or downcomer. By means of the experimental data, mathematical correlations for mixing time have been proposed for each circulation region, taking into consideration both the operational parameters and the distance from the bioreactor bottom. These equations offer a good concordance with the experiment, the average deviation being of 5.82% for the riser and 6.06% for the downcomer zone.     

Interaction of an odorant lactone with model phospholipid bilayers and its strong fluidizing action in yeast membrane

Some odorant lactones are naturally present in fruits or in fermented products; they can also be used as food additives and can be produced by microorganisms at the industrial scale by biotechnological processes. Gamma-decalactone was previously shown to have antimicrobial properties. We determined by infrared spectroscopy measurements that this compound rapidly diffused into model phospholipid bilayers (within 2 min), modifying the general physical state of a dimyristoyl-L-alpha-phosphatidylcholine (DMPC) film. In vivo, the lactone strongly increased membrane fluidity in the model yeast Yarrowia lipolytica, as evaluated by fluorescence anisotropy measurements. This effect was more important than that of benzyl alcohol, which is known as a fluidizing agent in living cells, and may explain the toxic action of gamma-decalactone in microorganisms.     

Extraction of lipids from Yarrowia Lipolytica

BACKGROUND: Microorganisms have often been considered for the production of oils and fats as an alternative to agricultural and animal resources. Extraction experiments were performed using a strain of the yeast Yarrowia lipolytica (Y. lipolytica), a high‐lipid‐content yeast. Three different methods were tested: Soxhlet extraction, accelerated solvent extraction (ASE) and supercritical carbon dioxide (SCCO₂) extraction using ethanol as a co‐solvent. Also, high pressure solubility measurements in the systems ‘CO₂ + yeast oil’ and ‘CO₂ + ethanol + yeast oil’ were carried out. RESULTS: The solubility experiments determined that, at the conditions of the supercritical extractor (40 °C and 20 MPa), a maximum concentration of 10 mg of yeast oil per g of solvent can be expected in pure CO₂. 10% w/w of ethanol in the solvent mixture increased this value to almost 15 mg of yeast oil per g of solvent. Different pretreatments were necessary to obtain satisfactory yields in the extraction experiments. The Soxhlet and the ASE method were not able to complete the lipid extraction. The ‘SCCO₂ + ethanol’ extraction curves revealed the influence of the different pretreatments on the extraction mechanism. CONCLUSION: Evaluating the effectiveness of a given pretreatment, ASE reduced the amount of material and solvent used compared with Soxhlet. In all three cases, the best total extraction performance was obtained for the ethanol‐macerated yeast (EtM). Addition of ethanol to the solvent mixture enhanced the oil solubility. Oil can be extracted from Y. lipolytica in two different steps: a non‐selective ethanol extraction followed by TAG‐selective SCCO₂ purification. © 2012 Society of Chemical Industry