Breeding high-yield oil-producing strain to use cheap carbon source by UV induced protoplast mutagenesis

【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%。[Objective] We used UV induced protoplast mutagenesis to study breed a new high-yielding lipid-producing strain which could use cheap carbon source.[Methods] Get the 1.5% glusulase and 1.0% cellulose solution.Hydrolyze to remove the cell wall and obtain the protoplast of 2A00015(Candida parapsilosis).Put it under the ultraviolet lamp for mutagenesis and cultivate regenrated wall.Then screen to get the high oil generated yeast which could be fermented by low-cost carbon source.Determine its fat acid components by gas chromatography with mass spectrometric(GC-MS).[Results] Cultivated the mutant strain with best mutation 2A00015/25 in the glucose.We found that the biomass, oil yield rate and oil production are separately 17.77 g/L, 58.12% and 10.32 g/L, which are separately 12.45%, 23.32% and 38.68% higher than the original strain.We have also cultivated the mutant strain in the waste molasses and found the biomass, oil yield rate and oil production are separately 18.54 g/L, 49.44% and 9.17 g/L, which are separately 9.09%, 21.16% and 32.18% higher than the original strain.The oil yield rate is lower in the waste molasses cultivation than that in glucose cultivation.However, in consideration of environment protection and cost of raw materials, the waste molasses is of much more advantages.It is tested that the fat generated from the waste molasses fermentation consists of eight kinds of fat acid.Its fat acid components are similar to the vegetable oil, in which the content of unsaturated fatty acid comprised 82.4% of the total fatty acid.[Conclusion] Basing on UV induced protoplast mutagenesis, we have successfully bred a new high-yielding oil strain which can make use of the low-cost carbon source, with its oil production rate 49.4% which has increased by 21.2%.深海(微)生物资源勘探与资源潜力评价项目(No.DY125-15-R-01

Determination of D-ribose by ion chromatography with integrated pulsed amperometric detection

采用离子色谱-积分脉冲安培检测法(IC-IPAd)测定d-核糖,采用CArbO PACTM PA10色谱柱进行分离,60 MMOl/l nA OH溶液为淋洗液,流速0.6 M l/MIn,柱温30℃,Ed3000安培检测器,Au工作电极,TI对电极,Ag/Ag Cl复合参比电极。以外标法定量,d-核糖质量浓度为3.60~43.2Mg/l范围内峰面积呈良好的线性关系(r2=0.9999),检出限及定量限分别为0.200、0.620 ng,精密度实验的相对标准偏差(rSd)为0.524%,平均回收率为101%。因此,IC-IPAd可用于d-核糖含量的测定,且操作简便、无需衍生、分离效果好、灵敏度高。An analytical method was proposed and established for determination of D-ribose by ion chromatography with integrated pulsed amperometric detection(IC-IPAD).The separation was performed on a 250 mm×4 mm Carbo PacTM PA10 column at 30 ℃ by HPLC equipped with an ED3000 detector.The mobile phase was 60 mmol/L sodium hydroxide with a flow rate of 0.6 m L/min.The quantification was performed by an external standard approach.The concentration linear range for D-ribose was 3.60~43.2 mg/L(R2=0.9999).The relative standard deviation of precision was 0.524%.The detection and quantification limits for D-ribose were 0.200 ng and 0.620 ng,respectively.The average recovery ration for D-ribose was 101%.IC-IPAD was proved to be suitable for the determination of D-ribose with its convenience,rapidness,and sensitivity.海洋公益性专项(201105029); 国家重大科学仪器专项(2013YQ170525); 厦门市海洋经济发展专项资金项目(13CZP003HJ05

Application of Gas-Contrasted and Window Technic in Diagnosing Gastro-Colon Lesion with CT

目的探讨空气造影低剂量CT扫描及低窗位图像诊断肠道病变的应用价值。方法126例疑有胃或结肠病患者随机分为2组,一组(69例)采用传统的水造影或无造影常规剂量CT扫描及常规窗位诊断,另一组(57例)采用空气造影低剂量CT扫描及低窗位图像诊断,以内窥镜检查为诊断标准,由资深医生采用单盲法评价诊断,比较2组图像的诊断自信度及正确率。结果医生对空气造影低剂量CT扫描及低窗位图像的诊断自信度和正确率明显优于传统的水造影或无造影常规剂量CT扫描常规窗位图像。结论充气造影低剂量CT及窗位技术诊断胃结肠病变明显优越于传统检查方法,值得提倡。 【英文摘要】 Objective To study the Value of low dose CT scanning with gas-contrasted and low window level in diagnosing gastro-colon lesions.Methods 126 cases suspected with gastro-colon lesions were randomly devided into two groups,group A(69 cases) were scanned with conventional water-contrast or non-contrast and conventional expose dose,and with conventional window level,group B(57 cases) were scanned with gas-contrasted and low expose dose,and using low window level.The images were evaluated by 2 experienced radiol..

Study on screening of oleaginous yeast and determination of intracellular lipid content by nile red dyeing

通讯作者：Tel: 86-592-2195679; :mail: [email protected][中文文摘]【目的】建立产油酵母筛选以及胞内油脂含量测定的简便方法。【方法】利用尼罗红与胞内油脂成分结合后在紫外光照射下发出荧光且荧光强弱与油脂含量相关的原理。通过在添加尼罗红的培养基中培养酵母,并观察菌落荧光的方法对385株深海酵母进行产油脂菌株筛选,利用26S rDNA D1/D2区序列分析方法对筛选获得的产油酵母菌株进行鉴定,并以其中的一株高产油脂酵母(2A00015)为试验菌株,建立了一套尼罗红染色快速测定油脂含量的方法。【结果】获得22株产油酵母,其中油脂含量最高可达62.9%,经分子鉴定后显示这22株酵母分别属于(Candida viswanathii)、近平滑假丝酵母(Candidaparapsilosis)、粘质红酵母(Rhodotorula mucilaginosa)、汉逊德巴利酵母(Debaryomyceshansenii)、季也蒙毕赤酵母(Pichia guilliermondii)以及Rhodosporidium paludigenum酵母。尼罗红染色快速测定油脂含量方法的最佳检测条件为:菌悬液OD600小于1.2,尼罗红浓度0.5 mg/L,染色时间5 min,激发波长488 nm,发射波长570 nm。该测定方法得到相对荧光强度与称重法得到油脂含量呈正相关性,R2=0.9637。[英文文摘][Objective] To establish a simple method to screen oleaginous yeast and determine the intracellular lipid content.[Methods] The study is based on the theory that the combina-tion of nile red and intracellular oil will emit fluorescence when induced by UV light and the fluoresence indensity is associated with the lipid content.We cultivate yeast in the culture medium added with nile red,and screen the oleaginous yeast strains from the 385 deep-sea yeasts by measns of obeserving the fluorescence of the yeast colony.We have identified the screened oleaginous yeast strains by the 26S rDNA D1/D2 series analysis method.Designating one of the oleaginous yeasts(2A00015) as the test strain,the lipid content rapid determination method by nile red dyeing was established.[Results] 22 oleaginous yeasts were obtained with the lipid content reaching up to 62.9%.Based on the molecular identification,it showed that the 22 yeasts are separately belong to Candida viswanathii,Candida parapailosis,Rhodotorla mucilaginosa,Debaryomyces hansenii,Pichia guilliermondii and Rhodosporidium paludi-genum.The optimum condition for lipid content determination by nile red dyeing is: bacterium suspension OD600 lower than 1.2,nile red concentration 0.5 mg/L,dyeing time 5 min,excit-ing wavelength 488 nm,emission wavelength 570 nm.The relative fluorescence intensity ob-tained by this method exhibits a positive association with the lipid content obtained by weigh-ing method,which can be explained as R2=0.9637.国家海洋局第三海洋研究所基本科研业务费专项资金项目(No.海三科2010009

Determination of Glucosamine Compounds by HPLC-nano-quantity Analyte Detector

通信作者:[email protected][中文文摘]核粒子计数检测器是基于水凝聚激光计数专利技术(WCPC),对低紫外吸收的化合物有较强的优势.建立了高效液相色谱-核粒子计数检测器(HPLC-NQAD)检测D-氨基葡萄糖盐酸盐和N-乙酰-D-氨基葡萄糖的方法.采用AsahipakNH2(250mm×4.6mm,5μm)色谱柱,V(乙腈)∶V(0.5%三乙胺水溶液)=75∶25为流动相,流速为0.8mL/min,进样量为10μL,10min内完成检测.在NQAD蒸发管温度55℃、气体压力200kPa条件下,D-氨基葡萄糖盐酸盐与N-乙酰-D-氨基葡萄糖之间的分离度2.32,前者的检出限为16ng,定量限为50ng,后者的检出限为8ng,定量限为24ng.D-氨基葡萄糖盐酸盐在10.03～2 005.20μg/mL范围内线性回归相关系数为0.999 5,N-乙酰-D-氨基葡萄糖在10.18～2 036.80μg/mL范围内线性回归相关系数为0.999 9.检测D-氨基葡萄糖盐酸盐和N-乙酰-D-氨基葡萄糖的精密度实验的相对标准偏差(RSD)分别为1.53%和1.38%,平均回收率分别为100.18%和101.02%.该方法用于D-氨基葡萄糖盐酸盐和N-乙酰-D-氨基葡萄糖的检测,可靠、快速、简便.[英文文摘].Nano-quantity analyte detector(NQAD),which was based on water condensation particle counter(WCPC) technology,was advantageous for detection of low UV-absorbing compounds.An effective HPLC method with NQAD used for the detection of D-glucosamine hydrochloride(GAH) and N-acetyl-D-glucosamine(GN) was developed.The separation was performed on a 250 mm×4.6 mm NH2 column with a diameter of 5 μm(Asahipak).Isocratic elution was conducted using acetonitrile/(0.5% triethylamine)(75∶25,by vol.) with a flow rate of 0.8mL/min,and the injection volume of 10μL.This HPLC process was completed within 10 min.The resolution of GAH and GN was 2.32when the evaporation temperature of NQAD was 55℃and the pressure was 200kPa.The detection limit was 16ng for GAH and 8ng for GN,and the quantitation limit was 50ng for GAH and 24ng for GN.GAH showed a good linearity in the range of 10.03~2005.20μg/mL (r=0.999 5,)and the linearity range for GN was 10.18~2036.80μg/mL(r=0.9999,).The relative standard deviation of precision for GAH and GN were 1.53% and 1.38%,respectively. The average recovery rates of GAH and GN were 100.18% and 101.02%,respectively.These results indicated that the HPLCNQAD method was a reliable,quick and simple method for the detection of GAH and GN.海洋公益性专项(201005022