高产油脂酵母菌选育及摇瓶发酵条件的研究

经紫外线和ＥＭＳ复合诱变选育出一株高产油脂的优良酵母菌株,命名为Ｌｉｐｏｍｙｃｅｓ．Ｓｔａｒｋｅｙｉ　ＨＬ。通过摇瓶培养,对各项与菌体产油脂相关的因素作了单因子实验,确定了摇瓶发酵培养的最佳产油脂条件：碳源,废糖液１６５．７ｍｌ／Ｌ;氮源,硫酸铵１．０８ｇ／Ｌ;Ｃ／Ｎ：６１：１;培养温度为２８℃;接种量１０％;发酵时间９６ｈ; ｐＨ５．０。最后可得油脂产量 ５．９ｇ／Ｌ;菌体生物量 １１．０ｇ／Ｌ;油脂含量 ５３．６％。对菌体内油脂组成进行了气相色谱与质谱分析,结果如下：软脂酸３３．２％,棕     

Mathematical modelling of lipid production by oleaginous yeasts in continuous cultures

A mathematical model was constructed to describe the influence of the carbon to nitrogen ratio (C/N-ratio) of the growth medium on lipid production by oleaginous yeasts. To test this model and to determine some relevant model parameters, the oleaginous yeast Apiotrichum curvatum ATCC 20509 was grown in continuous cultures at various C/N-ratios and dilution rates. It appeared that when nitrogen is limiting for the formation of biomass, the remaining glucose can be converted to storage carbohydrate and storage lipid. No clear dependence of carbohydrate yield on the C/N-ratio could be demonstrated, but lipid yield increased gradually with increasing C/N-ratios.The maximal dilution rate for lipid producing yeast cells appeared to be optimal at relatively low C/N-ratios. It can be concluded that the experimental results fitted well with the mathematical model. By using this model, lipid yield and lipid production rate can be calculated at any C/N-ratio of the growth medium and optimum operation conditions can be predicted for the production of microbial lipids.     

Isolation and characterization of a bacterium that produces hydrocarbons extracellularly which are equivalent to light oil

A halotorelant bacterial strain that produces a significant amount of lipids from short-chain fatty acids was isolated from the sludge of a sewage disposal plant. This strain displayed a significant extracellular accumulation of lipids. The yield of lipids including hydrocarbons was highest (120% of cell dry weight) at the end of the linear growth phase. Fractionation of the lipids using thin-layer chromatography and subsequent gas chromatography showed that hydrocarbons were also obtained following an increase in total lipids. Their yield was the highest (50% of cell dry weight) in the linear growth phase. Additional analysis using infrared absorption spectrum and gas chromatography-mass spectrometry showed that the hydrocarbon fraction was composed of alkanes, such as C15H32, C18H38, C21H44, C22H46 and C24H50. Homology analysis of the 16s rDNA sequence as well as studies of the morphological and physiological characteristics indicated that the bacterium is a strain of Vibrio furnissii.     

Growth and biocatalytic activities of aerobic thermophilic populations in sewage sludge

Summary Enzymatic activities of aerobic thermophilic microorganisms are described and investigated for the development and control of sewage sludge treatment processes in batch and fed-batch cultures. Proteolytic activity is the main enzymatic activity in an aerobic thermophilic sewage sludge treatment process. It has an optimum at 80°C and can be found also during growth on synthetic media. The activity is correlated with the increase in ammonium in the particle-free fraction and the values of the respiratory quotients during cultivation either in sewage sludge or in a syntheticc medium. No other extracellular activities (lipase, amylase, pectinase and cellulase) were detected in the investigated sludge samples. Carbohydrates, lipids and other polymers were either not present in significant amounts or passed with only minor modifications through the treatment. Cultivations in sewage sludge were either oxygen or carbon limited. One strain able to excrete lysozyme was isolated. It might have a synergistic effect on the heat inactivation of pathogenic microorganisms (cryptic growth) although lytic activity remained very low. Two-thirds of the entire metabolic activity is due to degradation of insoluble matter. The utilization of particulate matter also has a positive influence on the efficiency of the process by reduction in dry matter and increase in water-removal properties. Even at extremely low aeration rates, the acidification effect was small. Only small amounts of isobutyrate, isovaleriate and 2-methylbutyrate were formed at extremely low aeration rates and caused an increase in the total volatile fatty acid content after 12 and 36 h cultivation time.     

不同氮源及氮浓度对真眼点藻纲微藻生长及油脂积累的影响

以真眼点藻纲8株微藻(类波氏真眼点藻(Eustigmatos cf. polyphem)、大真眼点藻(Eustigmatos magnus)、波氏真眼点藻(Eustigmatos polyphem)、魏氏真眼点藻(Eustigmatos vischeri)、斧形魏氏藻(Vischeria helvetica)、点状魏氏藻(Vischeria punctata)、星形魏氏藻(Vischeria stellata)和眼点拟微绿球藻(Nan-nochloropsis oculata))为研究材料, 用3种氮源(硝酸钠、碳酸氢铵或尿素)和4种氮浓度(18、9、6和3 mmol) 在改良的BG-11培养基中对藻细胞进行培养。比较分析这8株微藻在不同培养条件下的藻液pH、生物量、油脂含量、脂肪酸组成的差异, 从而筛选出适合该类微藻生长和油脂积累的最适氮源与最佳氮浓度。结果表明, 这8株微藻均能在3种氮源中生长, 但是随着培养时间延长, 以碳酸氢铵和尿素为氮源时藻液pH逐渐降低, 其变化范围为5.0—6.0, 而以硝酸钠为氮源时藻液pH保持在7.0—8.0, 变化不大。当以尿素为氮源培养时, 能获得较高的生物量, 但是不同藻株在不同尿素浓度时达到最高生物量。最高生物量是波氏真眼点藻(E. polyphem)在9 mmol时达到, 为10.96 g/L。总脂含量分析发现, 在低氮浓度下均能促进8株微藻油脂的积累, 真眼点藻属中的魏氏真眼点藻(E. vischeri)在8株藻中获得最高油脂含量, 达到59.24%。进一步对脂肪酸分析发现, 8株微藻总脂肪酸含量为细胞干重的50%—58%, 主要脂肪酸组成为豆蔻酸(C14鲶0)、棕榈酸(C16鲶0)、棕榈油酸(C16鲶1)、油酸(C18鲶1)和二十碳五烯酸(C20鲶5), 其中拟微绿球藻(N. oculata)细胞中棕榈酸的含量最高占总脂肪酸50%左右; 其他7株微藻细胞中棕榈油酸的含量较高, 其占总脂肪酸含量范围在40%—60%。8株微藻均表现出较高的生物量与油脂积累能力, 以尿素为氮源, 氮浓度为6 mmol时更有利于该类微藻生物量和油脂的积累。总体来说, 真眼点藻纲的微藻是一类极具潜力适合于微藻生物燃料生产的微藻, 而真眼点藻属藻株表现更为明显的优势。     

不同氮源对蛋白核小球藻生长、色素和中性脂肪积累的影响

单细胞微藻在生长发育过程中,所积累的中性脂肪具有潜在的生物燃料价值。不同氮源对藻类的生长具有显著影响。研究了氨态氮、尿素氮和硝酸态氮对蛋白核小球藻生长、色素和中性脂肪积累的影响。结果显示,不同氮源对培养液pH有显著影响,以氨态氮为氮源导致培养液pH降低,而硝酸态氮导致培养液pH升高,培养液pH的波动可被添加的Hepes所稳定,并促进以氨态氮为氮源的蛋白核小球藻的生长。尿素氮对蛋白核小球藻生长、色素积累的效果优于氨态氮和硝酸态氮,硝酸态氮在中性脂肪积累上优于尿素氮和氨态氮,添加Hepes对氮饥饿后蛋白核小球藻的中性脂肪积累无显著影响。     

Single cell oil production by Yarrowia lipolytica growing on an industrial derivative of animal fat in batch cultures

The growth of an oleaginous strain of Yarrowia lipolytica on an industrial fat composed of saturated free fatty acids (stearin) was studied. Lipid accumulation during primary anabolic growth was critically influenced by the medium pH and the incubation temperature. This process was independent of the nitrogen concentration in the culture medium, but was favored at a high carbon substrate level and at a low aeration rate. At pH 6 and a temperature of 28-33 degrees C, 9-12 g/l of dry biomass was produced, whereas significant quantities of lipids were accumulated inside the yeast cells (0.44-0.54 g of lipid per gram of biomass). The strain showed the tendency to degrade its storage lipids, although significant amounts of substrate fat, rich in stearic acid, remained unconsumed in the culture medium. Y. lipolytica presented a strong fatty acid specificity. The fatty acids C12:0, C14:0, and C16:0 were rapidly incorporated and mainly used for growth needs, while C18:0 was incorporated with reduced rates and was mainly accumulated as storage material. Reserve lipids, principally composed of triacylglycerols (55% w/w of total lipids) and free fatty acids (35% w/w), were rich in stearic acid (80% w/w), while negligible amounts of unsaturated fatty acids were detected. When industrial glycerol was used as co-substrate, together with stearin, unsaturated fatty acid concentration in the reserve lipid increased.     

一株对硝基苯胺降解菌Microbacterium sp. PNA8的分离鉴定及其降解条件研究

从污泥中分离得到一株能以对硝基苯胺为唯一碳源、氮源和能源生长的细菌菌株PNA8。经过对其形态特征、生理生化特性、以及16S rRNA序列分析, 该菌株初步鉴定为Microbacterium sp.。进一步研究表明, 菌株PNA8利用对硝基苯胺生长和降解的最适温度和pH分别是30°C和7.0。培养基中添加定量酵母膏有利于菌株的生长及其对对硝基苯胺的降解。最适条件下, 在培养液中添加0.4 g/L酵母膏, 4 d内0.3 mmol/L对硝基苯胺降解率可达100%。     

Enhanced phosphate uptake and polyphosphate accumulation in Burkholderia cepacia grown under low pH conditions

Of bacterial cells in a sample of activated sludge, 34% contained detectable intracellular polyphosphate inclusions following Neisser staining, when grown on glucose/mineral salts medium at pH 5.5; at pH 7.5 only 7% of cells visibly accumulated polyphosphate. In a sludge isolate of Burkholderia cepacia chosen for further study, maximal removal of phosphate and accumulation of polyphosphate occurred at pH 5.5; levels were up to 220% and 330% higher, respectively, than in cells grown at pH 7.5. During the early stationary phase of growth at pH 5.5 a maximum level of intracellular polyphosphate that comprised 13.6% of cellular dry weight was reached. Polyphosphate kinase activity was detected in actively growing cells only when cultured at pH 5.5. The phenomenon of acid-stimulated phosphate uptake and polyphosphate accumulation in this environmental bacterial population parallels observations previously made by us in the yeast Candida humicola and may thus represent a widespread microbial response to low external pH values.     

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.     

利用污泥提取液制备微生物絮凝剂

以污水厂剩余污泥作为培养基原料,经过一系列处理,探索微生物絮凝剂产生菌的最适发酵培养基配方,结果表明,污泥预处理条件以pH 12碱解条件最优,碳氮源产出量最大,补加8 g/L葡萄糖后灭菌,微生物絮凝剂产生菌LLin6可正常产絮,絮凝率达91.55%。该结果为降低微生物絮凝剂的制备成本,并实现污泥的减量化和污泥资源化利用提供了基础。     

Valine production from hydrocarbon byMicrococcus varians

A bacterium isolated from Assam (India) soil was found to accumulateL-valine in the growth medium and was identified asMicrococcus varians. The strain grew and accumulated valine in a purely synthetic medium, but supplementation with either casamino acids or yeast extract or with both, improved the yield. The entire fermentation period could be divided into a growth phase and a production (phase which could be prolonged by adjustment of pH to neutral range. Among the different hydrocarbon and nitrogen sources tested straight run gas-oil and ammonium sulphate, respectively, were found most suitable. Antibiotics inhibited growth but stimulated extracellular valine accumulation. Vitamins stimulated growth and valine yield and an inoculum level of 10 % was found to be optimal. The yield ofL-valine under optimal conditions was 2.95 g/L.     

城市污泥土地利用研究

通过培养试验、盆栽试验和田间试验系统地研究了污泥的组分特征、性质及其农地和城市园林绿化地利用对作物或绿化灌木、土壤肥力及其环境的影响,结果表明,污泥富含有机质和氮磷养分,且养分当季有效性介于化肥农家肥之间。施用污泥将明显提高土壤肥力,表现改善土壤物理性质;增加土壤肥机质和氮磷水平,并增加土壤生物活性,因此施用污泥的处理作物产量较高,并有利于后茬作物的稳健生长。但污泥施用也存在重金属、病原物等污染     

Thermotropic behavior of lipids and the morphology of Yersinia pseudotuberculosis cells with a high content of lysophosphatidylethanolamine

The content of lysophosphatidylethanolamine (LPE) in Y. pseudotuberculosis cells was found to increase during their growth at 8 degrees C under stationary conditions (without stirring the medium) and at 37 degrees C when the medium contained glucose. The maximum level of LPE (up to 45% of the total phospholipids) was observed in cells grown at 8 degrees C under stationary conditions. Such cells showed an enhanced growth rate, a reduced yield of biomass, an altered cell morphology, and an increased cell area. The cells contained unsaturated fatty acids, phosphatidylethanolamine (PE), and total phospholipids in small amounts, whereas neutral lipids and diphosphatidylglycerol were abundant. In addition, the cells contained an amount of methylated PE and phospholipids of unknown structure. Irrespective of whether the temperature for growth was low or high, the LPE-rich cells showed a high value (32-36 degrees C) of the maximum temperature of thermal transition of lipids (Tmax). This finding is indicative of a densification of the membrane lipid matrix of the LPE-rich cells. The suggestion is made that LPE is accumulated in glucose-fermenting bacterial cells in response to stress caused by oxygen deficiency and low pH values of the growth medium. The possible relationship between LPE accumulation and the virulence of Y. pseudotuberculosis cells grown at low temperatures is discussed.     

一株降解煤油菌株的筛选与鉴定

本文通过从污水处理厂活性污泥中分离得到一株高效降解轻油(煤油)细菌5092-2,通过形态学、16S rRNA鉴定该菌为Mangroveibacter sp.。进一步对其生长条件进行了研究,发现该菌在23℃到38℃、pH为5.0到9.0范围内生长无明显差异。在温度为35℃,pH 7.2~7.4,160 r/min培养7 d,菌株降解煤油的能力达到52.3%,具有进一步研究的价值。     

Accumulation of osmoprotectants and lipid pattern modulation in response to growth conditions by Halomonas pantelleriense

The effects of salinity, growth temperature, pH and composition of the medium on the accumulation of intracellular organic solutes, by nuclear magnetic resonance spectroscopy (NMR) in Halomonas pantelleriense were examined. The modulation of lipid pattern in different growth conditions was also reported. H. pantelleriense accumulated glycine betaine, ectoine, hydroxyectoine and glutamate. The type of osmoprotectant and the relative proportion depended on growth conditions. The main lipids identified by NMR studies were 1,2 diacylglycero-3-phosphorylethanolamine (PEA), 1,2 diacylglycero-3-phosphoryl-glycerol (PG) and cardiolipin, (DPG). The predominant fatty acids were C16:0 and C18:1, minor fatty acids were C16:1 and C18:0. The relative percentage of polar lipids and fatty acids were affected by growth conditions.     

Factors affecting the survival of Salmonella and Escherichia coli in anaerobically fermented pig waste

The survival of Salmonella typhimurium was investigated in acidogenic, anaerobically fermented pig wastes and in synthetic media, each containing volatile fatty acids (VFA). Salm. typhimurium survived at pH 6.8, but not at pH 4.0, when incubated at 37 degrees C for 24 h in either fermented or synthetic medium containing VFA. The minimum inhibiting concentration of VFA for Salm. typhimurium after 48 h incubation at 30 degrees C at pH 4.0 was 0.03 mol/l and for Escherichia coli it was 0.09 mol/l. Fermented pig wastes in a digester, maintained at pH 5.9, were inoculated with Salm. typhimurium and then incubated at 37 degrees C for 24 h. The pH was adjusted to either 4.0 or 5.0 and after a further 48 h at 30 degrees C, Salm. typhimurium survived at pH 5.0 but not at pH 4.0. It was concluded that pH is critical in determining the survival of this organism in acidogenic anaerobically fermented pig waste.     

The characteristics of a new non-spore-forming cellulolytic mesophilic anaerobe strain CM126 isolated from municipal sewage sludge

A new mesophilic anaerobic cellulolytic bacterium, CM126, was isolated from an anaerobic sewage sludge digester. The organism was non-spore-forming, rod-shaped, Gram-negative and motile with peritrichous flagella. It fermented microcrystalline Avicel cellulose, xylan, Solka floc cellulose, filter paper, L-arabinose, D-xylose, beta-methyl xyloside, D-glucose, cellobiose and xylitol and produced indole. The % G + C content was 36. Acetic acid, ethanol, lactic acid, pyruvic acid, carbon dioxide and hydrogen were produced as metabolic products. This strain could grow at 20-44.5 degrees C and at pH values 5.2-7.4 with optimal growth at 37-41.5 degrees C and pH 7. Both endoglucanase and xylanase were detected in the supernatant fluid of a culture grown on medium containing Avicel cellulose and cellobiose. Exoglucanase could not be found in either supernatant fluid or the cell lysate. When cellulose and cellobiose fermentation were compared, the enzyme production rate in cellobiose fermentation was higher than in cellulose fermentation. The optimum pH for both enzyme activities was 5.0, the optimum temperature was 40 degrees C for the endoglucanase and 50 degrees C for the xylanase. Both enzyme activities were inhibited at 70 degrees C Co-culture of this organism with a Methanosarcina sp. (A145) had no effect on cellulose degradation and both endoglucanase and xylanase were stable in the co-culture.     

Expression, purification and characterization of a recombinant Lipomyces starkey dextranase in Pichia pastoris

The DEX gene encoding an extracellular dextranase from Lipomyces starkeyi was cloned into vector pPIC9k-His6 and was expressed in Pichia pastoris GS115 strain under the control of AOX1 promoter. After 107 h of the 5L-scaled fermentation, wet cells weight of the recombinant P. pastoris Mut(+) strain reached to 588.6g/L, and the concentration of dextranase and enzyme activity in the supernatant were 0.46 g/L and 83900 U/L, respectively. The activity of dextranase was improved 17.56-fold by cation-exchange chromatography only with a final yield of 71.61% and the specific activity of the purified enzyme was 181.96 U/mg. The purified dextranase, analyzed by SDS-PAGE and Western blotting, showed only one homogeneous band. Then the factors affecting the dextranase activity were evaluated. The optimal temperature and pH was 30 degrees C and pH 4.5, respectively. Metal ions Al(3+), Cu(2+), Fe(3+), and SDS could completely inhibit the enzyme activity, whereas Mg(2+) enhanced 145% of the enzyme activity. These characters are much different from what was previously reported for the L. starkeyi dextranase that was either expressed in S. cerevisiae or purified from natural L. starkeyi.     

Effect of pH,Temperature, and Lead Concentration on the Bioremoval of Lead from Water Using Lemna Minor

This study examined the ability of the aquatic plant Lemna minor (duckweed) to remove soluble lead under various laboratory conditions. In a batch process L. minor was exposed to different pH values (4.5–8.0) and temperature (15–35°C) in presence of different lead concentrations (0.1–10.0 mg L^?1) for 168 h. The amount of biomass obtained in the study period on a dry weight basis, the concentrations of lead in tissue and in medium and net uptake of lead by Lemna all have been determined in each condition. The percentages of lead uptake ratios (PMU) and bioconcentration factors (BCF) were also calculated for these conditions. Bioaccumulated lead concentrations and the PMU were obtained at lowest pH of 4.5, and at 30°C. The highest accumulated lead concentration was found at pH 4.5 as 3.599 mg Pb g^?1 in 10.0 mg L^?1. It decreased to pH 6.0, but it did not change at pH 6.0–8.0 range. The maximum lead accumulation was obtained at 30°C as 8.622 mg Pb g^?1 in 10 mg L^?1 at pH 5.0, and the minimum was at 15°C as 0.291 mg g^?1 in 0.1 mg L^?1. Lead accumulation gradually increased with increasing lead in medium, but the opposite trend was observed for PMU. Lead accumulation increased up to 50 mg L^?1, but did not change significantly in the 50.0–100.0 mg L^?1 range. The lead uptake from water was modeled and the equation fit the experimental data very well.