重金属污染黑土中固氮菌及反硝化菌作用强度的测定

本文采用气相色谱法同时测定了重金属Cd、As、Pb和Cu污染黑土中固氮菌及反硝化菌的作用强度。为准确、快速地提出土壤中Cd、As、Pb和Cu等重金属毒害土壤微生物临界指标开辟了新的途径。     

麦田根际土壤反硝化作用对O_3熏气和太阳辐射减弱的响应

为研究臭氧浓度升高和太阳辐射减弱复合背景下,麦田土壤反硝化作用及N2O排放的变化,采用开顶箱(OTC)法和遮光网技术,设置3个臭氧浓度梯度及3个辐射减弱梯度,连续4a对小麦生长季麦田土壤进行臭氧浓度增加太阳辐射减弱以及它们的复合作用的试验。采用MPN(最大或然数)法测定反硝细菌的数量,用气相色谱法测定反硝化强度。结果显示反硝化细菌数量和反硝化强度受小麦生长发育的影响,在小麦成熟期收割后土壤反硝化细菌数量和反硝化强度增加得特别明显。O3连续作用3个生长季后,以及太阳辐射减弱处理,土壤反硝化菌和反硝化强度显著升高,N2O排放量显著增加。减弱的太阳辐射与增加的O3复合作用,在小麦的每个生育期均显著促进了反硝化菌数量增加和反硝化强度增强,促进率显著高于O3和遮荫的单独作用。结果说明,O3浓度增加以及太阳辐射减弱对土壤反硝化菌和反硝化强度均有一定的促进作用,减弱的太阳辐射与高浓度的O3两因素之间存在协作关系,太阳辐射减弱有利O3的吸收,增加O3伤害,促进反硝化过程。     

分离于河口区芦苇湿地1株好氧反硝化菌的鉴定及其反硝化特性

【目的】筛选高效脱氮且N_2O释放量少的好氧反硝化细菌,并对菌株的反硝化特性进行研究,可为河口湿地富营养化水体的生物修复提供技术支撑。【方法】经BTB培养基初筛和反硝化能力测定,从辽河河口区芦苇湿地土壤中分离得到1株具有较高反硝化能力的好氧反硝化菌C3。经形态观察、生理生化鉴定和16S rRNA序列分析,对菌株进行鉴定。研究温度、碳源、pH及C/N对其生长量、反硝化能力及N2O释放的影响。【结果】筛选得到的高效好氧反硝化细菌C3,经鉴定属于假单胞菌属(Pseudomonas sp.)。反硝化特性研究结果表明,该菌最适碳源为柠檬酸三钠,在温度为30°C、pH为7.0、C/N为10时生长速率和脱氮效率最高且N_2O释放量较少。在此条件下,该菌在36 h内使NO_3~–由179.55 mg/L降至5.08 mg/L,脱氮率高达97.17%。该菌株在整个反硝化过程中中间产物N_2O的最大累积量较低,为0.22 mg/L。【结论】从湿地土壤中分离所得好氧反硝化菌C3为假单胞菌属的1个种(Pseudomonas sp.),该菌株在高效除氮和低N_2O累积方面均具有明显优势,对后续河口湿地富营养化水体治理具有重要意义。     

硫化物抑制潮土反硝化过程中氧化亚氮还原的菌群机制

【背景】土壤中的反硝化作用形成气态产物N_2O和N_2,会导致氮素的气态损失,并造成温室效应。硫化物对土壤的N_2O还原具有抑制作用,但其对菌群和功能基因的影响机制还不清楚。【目的】研究有无外加碳源情况下,硫化物对反硝化作用中间产物(NO、N_2O)的积累、反硝化功能基因(narG、nirS、nirK和nosZ)表达量以及菌群结构的影响。【方法】分别设置不同量葡萄糖(0和1000mg-C/kg干重土壤)和硫化钠(0和150mg-S/kg干重土壤)添加的交叉处理,进行室内微宇宙培养实验,利用自动化培养与实时气体检测系统检测培养过程中NO、N_2O和N_2的积累量,通过反转录定量PCR测定反硝化功能基因表达量,利用MiSeq技术平台基于16S rRNA基因序列的高通量测序分析样品的菌群结构。【结果】硫化钠的添加显著抑制N_2O还原,但是其对于N_2O积累量没有显著影响,却显著降低了NO的积累量。硫化钠的添加短时间内在转录水平上显著抑制N_2O还原酶的活性,并且抑制固氮弧菌属(Azoarcus)、微枝形杆菌属(Microvirga)、剑菌属(Ensifer)、氮氢单胞菌属(Azohydromonas)、芽孢杆菌属(Bacillus)、斯科曼氏球菌属(Skermanella)、申氏杆菌属(Shinella)和西索恩氏菌属(Chthoniobacter)的基因转录,降低它们的转录本丰度,结合Kyoto Encyclopedia of Genes andGenomes(KEGG)数据库的查询结果,发现硫化钠的添加抑制了不产生N_2O的N_2O还原反硝化细菌的生长。【结论】堆肥或其他原因引起的土壤硫化物增加,导致反硝化过程N_2O还原被抑制的原因是由于其对氧化亚氮基因转录的抑制和对不同反硝化菌的选择作用,研究结果有助于认识硫化物对氮代谢影响的微生物机制。     

秸秆还田对土壤硝化特性和氨化特性及其相关菌群数量的影响

研究秸秆还田后土壤硝化特性和氨化特性的改变及其与相关菌群数量变化的关系,为进一步研究秸秆还田对土壤氮素循环的影响提供参考。测定秸秆还田后植株生长过程中不同时间土壤硝化特性和氨化特性,以及相应的硝化细菌、氨化细菌、固氮菌的数量,将结果与无秸秆的空白对照区进行比较、分析。整个植株生长过程中,秸秆还田土壤中相关菌群数量以及氨化作用强度和硝化作用强度均明显高于空白对照,其中氨化菌、硝化菌和固氮菌为空白对照的1.3~1.6倍;氨化作用强度和硝化作用强度分别为空白对照的1.1和1.8倍。结果表明,秸秆还田可以有效提高土壤氨化活性和硝化活性以及相关菌群的数量和活性,相关菌群数量的变化与土壤氨化活性和硝化活性呈正相关。     

不同底物与固氮酶及其钼铁蛋白相互作用的荧光光谱分析

 本文研究了不同底物(N_2,H_2,N_2O,NaN_3,C_2H_2)对棕色固氮菌固氮酶及其钼铁蛋白荧光光谱的影响。结果表明,上述底物均能络合在钼铁蛋白及固氮酶上,但络合程度不同,从而为固氮酶系统有多个不同的底物络合中心,底物络合中心在钼铁蛋白分子上,铁蛋白对钼铁蛋白有变构作用,提供了光谱学证据。     

土壤释放的 N_2O 的原位测定

N_2O 是大气成分之一,它由微生物的硝化-反硝化作用、燃烧和大气闪电等过程产生,其中土壤微生物反硝化作用是最主要的来源。土壤微生物反硝化作用产生N_2O,不仅导致土壤中肥料氮素的损失,而且由于其“温室效应”和对臭氧层的破坏,受到国内外研究者     

真菌反硝化过程及其驱动的N2O产生机制研究进展

真菌反硝化过程的发现打破了反硝化过程只发生在原核生物中的传统认识,是对全球微生物氮循环过程的重要补充。真菌参与的反硝化过程由于缺乏N_2O还原酶,其终产物为具有强辐射效应的温室气体N_2O。真菌在环境中分布广泛,生物量巨大,故真菌反硝化作用对全球N_2O释放通量的贡献是不容忽视的。近年来许多研究表明,真菌反硝化过程是自然环境中N_2O产生的重要途径。本文对反硝化真菌的发现、多样性及分布、产生N_2O的机制和活性测定方法等几个方面进行综述,并对未来的研究提出展望。     

过量施肥对设施菜田土壤菌群结构及N2O产生的影响

【背景】N_2O是一种很强的温室气体,其温室效应强度大约是CO_2的265倍。土壤氮肥施加量是影响N_2O排放的重要因素,而厌氧条件下微生物反硝化则是N_2O产生的重要途径。【目的】研究过量施肥条件下蔬菜大棚土壤菌群结构变化及其对N_2O气体排放的影响。【方法】利用自动化培养与实时气体检测系统(Robot)监测土壤厌氧培养过程中N_2O和N_2排放通量,比较过量施肥和减氮施肥模式下土壤N_2O排放模式的差异。通过Illumina二代测序平台对这2种不同施肥处理的土壤微生物群落进行高通量测序,研究不同施肥量对土壤菌群组成的影响。【结果】过量施肥土壤中硝酸盐的含量大约是减氮施肥土壤的2倍,通过添加硝酸盐使2种土壤的硝酸盐含量均为60 mg/kg或为200 mg/kg时,过量施肥土壤在厌氧培养前期N_2O气体的产生量及产生速度都明显高于减氮施肥土壤。另外,过量施肥导致土壤菌群结构发生显著改变,并且降低了土壤微生物的多样性。相对于减氮施肥,过量施肥方式富集了Rhodanobacter属的微生物。PICRUSt预测结果显示,传统施肥没有显著改变反硝化功能基因相对丰度。【结论】长期过量氮肥施用显著增加了土壤N_2O的排放,可能原因是施肥改变了包括氮转化相关微生物在内的土壤菌群组成,从而影响了土壤N_2O气体的形成与还原过程。     

碳氮添加对不同湿度条件下温带森林土壤融化过程中氧化亚氮排放的影响

采用室内土柱培养方法,研究不同土壤湿度(55%和80%土壤充水孔隙度,WFPS)条件下外源碳(葡萄糖,6.4 g C·m~(-2))和2种形态氮(NH_4Cl和KNO_3,4.5 g N·m~(-2))的添加对温带成熟阔叶红松混交林和次生白桦林土壤冻结后融化过程中氧化亚氮(N_2O)排放量的影响。结果表明:冻结过程会激发2种林分土壤融化初期N_2O的排放。随着土壤湿度的增加,2种林分土壤大量消耗硝态氮,反硝化作用强烈,导致融化初期N_2O激发效应的强度大,持续时间长,尤其是白桦林土壤。单施葡萄糖后,2种林分土壤大量消耗铵态氮和硝态氮,进而显著促进2种林分土壤融化初期N_2O的激发排放;随着土壤湿度的增加,葡萄糖对2种林分土壤N_2O累积排放量的促进作用减弱,这可能与高湿度条件下,冻结后融化过程中土壤释放大量溶解性有机碳(DOC)有关。低湿度条件下,2种林分土壤融化过程N_2O排放是铵态氮限制性的,即硝化潜势占主要优势,尤其是白桦林土壤;高湿度条件下,白桦林土壤具有很强的反硝化潜势,并且随着葡萄糖的施加,这种反硝化潜势加强。逐步回归分析显示:2种林分土壤冻结后融化过程N_2O累积排放量受到土壤pH、WFPS及水浸提DOC含量的影响,共同解释其66%的变化,并且与土壤水浸提溶解性有机氮含量呈显著的正相关;阔叶红松混交林土壤冻结后融化过程N_2O累积排放量与微生物生物氮呈显著负相关。综上可推测,温带森林土壤融化过程中N_2O的排放主要依赖于冻结处理后的土壤pH、WFPS以及溶解性有机质释放量的变化。     

巴柑檬果皮精油化学成分的研究

用 GC-MS-DS 联用技术、气相色谱双柱保留指数和气相色谱双柱标准品叠加实验,鉴定了我国首次引种成功的巴柑檬果皮精油的化学成分,测定了用不同方法提取的巴柑檬果皮精油各化学成分的相对含量。结果表明:引种的巴柑檬果皮精油的化学成分与国外文献值相符合。     

Studies on the Natural Flavoux Components of Psidium gunajava L. Fruit

In the study Amberlite XAD-4 resin was employed as an adsorbent, which adsorbed the natural flavour sent forth from Psidium guajava L. fruit. The eluant of the adsorbate was concentrated by using a K-D concentrator. The concentrate was analyzed by dual capillary gas chromatography-mass spectrometry and temperature programed retention index on dual columns. A total of 25 substances were identified, which amounted to 98% of the total area of .the peaks. Four acetals were reported for the first time as guava components, i.e., l,l-dietho- xymethane, l,l-diethoxyethane, 1,1-diethoxyhexane and acetaldehyde ethyl cis-3-hexenyl acetal. Evaluation at an odour port during GC showed that the sharp characteristic aroma sent forth from guava fruitis attributed to l,l-diethoxymethane and l,l-diethoxyethane principally. It was discussed that the method of combining the dual column GC-MS with the dual column retention index could increase the reliability of the identification results.     

蕨菜叶、茎中γ-氨基丁酸的提取分离及含量测定

用渗漉法对蕨菜叶、茎中的γ-氨基丁酸进行了提取,并用柱层析法对提取物进行了分离。采用双波长薄层扫描 法测定其含量,测得蕨菜叶、茎中γ-氨基丁酸的质量分数分别为0.319%、0.141%。采用该法测定γ-氨基丁酸操作简便、 快速,结果稳定。     

气相色谱法测定抗毒素中的间甲酚含量

采用气相色谱法测定抗毒素中的间甲酚含量。样品用苯甲醇作内标,经高氯酸沉淀蛋白、氯仿抽提后,通过HP-INNO-WAX柱,以氢火焰检测器测定间甲酚含量。内标峰与样品峰的分离度大于1.5,平均回收率及变异系数分别为100.067%和1.93%,测定线性范围0.0202~1.7588mg/ml,最小检测限为0.01272mg/ml,该方法准确快速,比化学方法好。     

Naturally occurring monoterpenoids in needles of Picea abies (L.) Karst

Summary An analysis was made of the effects of different sampling and extraction techniques on the amounts and pattern of monoterpenoids isolated from needles of Norway spruce. The following isolation and analysis procedure was finally adopted: liquid nitrogen-cooled needles were pulverized by a microdismembrator, extracted with pentane overnight at 2°–3°C and concentrated to a volume not less than 3 ml/g fresh weight on a Vigreux column. The crude extract was injected splitless (with solvent split) onto a cold programmed temperature vaporized (PTV) precolumn of a gas chromatograph and the vaporizable compounds heated to a capillary column. This method was tested for production of artefacts and quantitative extraction and applied to needles of eleven 80-year-old spruce trees.     

A new on-line dual enzymatic method for simultaneous quantification of cholesterol and triglycerides in lipoproteins by HPLC

We describe an on-line dual detection method using HPLC for lipoprotein analysis that allows simultaneous determination of cholesterol and triglyceride profiles from a single injection of sample. Two different gel permeation columns, TSKgel LipopropakXL and Superose 6HR, were applied to the dual detection system, evaluating analytical performance of the proposed method and the columns by analyzing serum samples from human and nonhuman subjects. Both TSK and Superose columns produced good within-day imprecision values less than 4.7% for cholesterol and 4.2% for triglyceride determination. Linear regression analysis showed the results from the Superose column (y) correlated well with those from the TSK column (x): y = 0.969x + 5.44 (r = 0.990) for total cholesterol (mg/dl), y = 1.08x - 11.14 (r = 0.985) for total triglycerides (mg/dl), and y = 1.093x - 0.06 (r = 0.978) for the ratios of triglycerides to cholesterol (mg/mg). Furthermore, the cholesterol and triglyceride profiles elucidated the differences in the resolution ability of the columns, which have not been apparent from a single lipid profile. We conclude that the dual detection concept with proper choice of column and enzymic reagents specific to the objectives of the particular study can facilitate studies of lipoprotein metabolism.     

Preparation and analysis of a volatile derivative of cysteic acid

Summary We have reported preparations and gas chromatographic analyses of volatile derivatives of sulfuric acid and taurine (Masuoka et al., 1988; 1989). By extending these studies, we have developed a method for the gas chromatographic determination of cysteic acid. Cysteic acid was converted to the N-isobutoxycarbonyl derivative by the reaction with isobutyl chloroformate in the presence of sodium hydroxide. After desalting with a cation-exchange column, the derivative was converted to the silver salt by reacting with silver oxide. The resulting silver salt was quantitatively esterified with methyl iodide in the presence of dimethyl sulfate and silver oxide. Dimethyl N-isobutoxy-carbonylcysteate [methyl 2-(N-isobutoxycarbonylamino)-3-(methoxysulfonyl) propanoate] formed was analyzed by gas chromatography. The calibration curve was linear up to 5.0µmol per ml of cysteic acid and the recovery was more than 95%.     

气相色谱法对轮状病毒疫苗中β-丙内酯测定的研究

用气相色谱法对轮状病毒疫苗生产过程中采用的β?丙内酯灭活剂进行残留量的检测,检测结果表明:采用HP-INNOWAX毛细管色谱柱(15m×0.53mm×1.0μm),氢火焰检测器检测轮状病毒疫苗中β?丙内酯是极其灵敏的,该法最低检测限为:1.43×10-4μl/μl。实验还证明:轮状病毒疫苗中已不存在β?丙内酯。     

Purification and quantitation of tumor necrosis factor receptor immunoadhesin using a combination of immunoaffinity and reversed-phase chromatography

The development of an automated, dual column assay to quantitate and recover the glycoprotein, tumor necrosis factor receptor immunoadhesin (TNFr-IgG) from monkey plasma, human serum, cell culture fluid and buffer samples is described. A combination of immunoaffinity and reversed-phase chromatographies are used. The targeted protein was captured using an anti-TNFr-1 monoclonal antibody immobilized on POROS resin. After non-specific adsorption had been reduced, the affinity column was placed in-line with a reversed-phase column and eluted with dilute acid. The reversed-phase column was subsequently eluted with an acetonitrile gradient and the TNFr-IgG collected and quantitated by comparison with peak areas of similarly treated standards. Detection was performed by measurement of absorbance at 214 nm. The dynamic range is from 0.5–15 μg total sample. Samples were quantitated and recovered from monkey and human pharmacokinetics samples, as well as from cell culture fluid and buffers. The lowest concentrations assayed were 100 ng ml⁻¹. Quantitation is reproducible, with a coefficient of variation of 2%. The procedure was used to develop a pharmacokinetic profile for the clearance of TNFr-IgG in humans and cynomolgus monkeys. Sufficient material was recovered such that the glycoforms could be identified. Additionally it has been used for process monitoring. The results compared favorably with data generated by ELISA. Optimization of the method and results are presented.     

重楼属植物甾体皂甙的高效液相色谱分析

应用高效液相层析技术,对重楼属十八个种植物的甾体皂甙进行了定性、定量分析。植物用甲醇提取,抽出物经DIAION柱,以90％甲醇洗出总甙。在HPLC上,用ODS柱先将总甙以用醇:水(9:1)洗脱分为三馏段,每馏再在ODS柱或Rp-8柱上以甲醇:水(8:2;7:3.5)洗脱,使各个皂甙成分完全分离。被分离的每个色谱峰与已知重楼皂甙的保留时间进行比较并配合HPLC的加入法,TLC分析及用HPLC制备少量样品做MS测定来加以定性鉴定。定量采用内标及校正曲线法。