A rapid method to quantify nitrifiers in activated sludge

Quantification of bacteria using Fluorescence In Situ Hybridization (FISH), confocal laser scanning microscopy (CLSM) and image analysis is very time consuming and requires the availability of an expensive microscope. Therefore, a rapid method to quantify nitrifying bacteria in activated sludge using FISH and epifluorescence microscopy was developed. The quantification of the biovolume is based on manual counting of the aggregates formed by nitrifying bacteria and determination of their size. The overall uncertainty of the method was evaluated as a function of the number of analyzed microscopic fields. It was found that 10-15 microscopic fields for ammonia-oxidizing bacteria and 6-8 microscopic fields for nitrite-oxidizing bacteria per sample were optimal regarding effort and accuracy. Accordingly, the time needed for one sample was only 5-15 min, compared to about 1h for the quantification with CLSM and image analysis. As a consequence, this method also allows for the measurement of extended time series with a reasonable effort. The comparison of the determined biovolume and the measured activity showed an explicit correlation.     

Rapid quantification of bacteria in activated sludge using fluorescence in situ hybridization and epifluorescence microscopy

A rapid quantification method for bacteria in activated sludge has been developed, based on fluorescence in situ hybridization (FISH) and epifluorescence microscopy. Samples are hybridized on slides and analyzed by direct microscopic observation. Abundance categories were designed based on digital images of the target organisms. These rating systems were developed for the filamentous bacteria Microthrix parvicella and for different morphotypes of nocardioform actinomycetes, but can easily be adapted to other types of microorganisms. Due to the quantification by direct microscopic observation, this method is suitable for samples that are difficult to be processed by semi-automated image analysis techniques, such as samples containing fluorescent debris, cells of different fluorescence intensities and target organisms that need partial enzymatic digestion prior to FISH. In contrast to commonly used rating systems consisting of photographs, the newly developed categories allow to quantitatively compare results of different categories and different organisms. The uncertainties of the results were calculated by a non-parametric bootstrap procedure; a thorough uncertainty analysis was performed including sample variability and operator subjectivity.     

Estimating the quantity of egg white and whey protein concentrate in prepared crabstick using ELISA

Indirect enzyme-linked immunosorbent assays (ELISA) technique was used to identify and quantify the use of dried egg white (DEW) and whey protein concentrate (WPC) in crabsticks. The use of SDS–PAGE for the quantification of protein additives has had limited success due to the high shear and high temperature processes of surimi crabstick. Monoclonal (anti-heat-denatured ovalbumin) and polyclonal (anti-β-lactoglobulin) antibodies were used. Antibodies showed no significant cross-reactivity with non-target crabstick proteins. An optimised extraction solution of 10% SDS and 2.5% 2-ME yielded high extractability with improved consistency. Quantification of DEW and WPC was achieved using the optimised extraction solution and indirect ELISA. Estimated DEW values were within 7% of actual values, WPC samples were within 17%. Inter-assay coefficients of variance for DEW ranged from 0.9% to 3.1% and those of the WPC were 1.0–8.0%.     

Quantitative GC-ECD Analysis of Halogenated Metabolites: Determination of Surface and Within-Thallus Elatol of Laurencia obtusa

Information on natural concentrations or variability of secondary metabolites in marine organisms may be important both to ecological/evolutionary and applied approaches. A gas chromatographic procedure with an electron capture detector (GC-ECD) was developed to quantify the sesquiterpenoid elatol at the surface and within-thalli of 70 specimens of the red seaweed Laurencia obtusa. The concentration of elatol was highest within-thalli [9.89 mg g⁻¹ of L. obtusa, dry weight (d.w.)], compared to lower values found at the surface [0.006 mg g⁻¹ of L.obtusa (d.w.), or 0.5–10.0 ng cm⁻²]. This method provides a rapid and inexpensive quantification of small quantities of elatol, and probably may also be used to quantify other halogenated compounds usually found in red seaweeds.     

Exploration of transmission efficiency and quantitative measurement of the Axis Ultra electron spectrometer

The estimation of transmission efficiency was done on an Axis Ultra electron spectrometer according to the parameters presented in the literatures. At the same time, Ag, Au, and Cu were measured as standard sample to check the real transmission efficiency. Compared the measured intensities with the estimated ones, it was found that the X-ray flux of dual anode Mg Kα on the samples should be much more than 6.0 × 10¹¹ photons mm⁻² s⁻¹, which was different from the data given in the literature. Transmission function correction was also done and the quantification results were tested with stoichiometric samples of NaCl and KI after the transmission function correction, and the results agree well with the essentially stoichiometric relation. The analytical approach and procedure can provide valuable reference for the quantification analysis and correction of conventional electron spectrometer.     

Electrochemical study of rat brain acetylcholinesterase inhibition by chlorofos: Kinetic aspects and analytical applications

An electrochemical sensor was applied for investigating the immobilized rat brain acetylcholinesterase inhibition by chlorofos. Two alternative routes were explored as response-generating reactions: (i) direct electrochemical oxidation of thiocholine produced upon acetylthiocholine enzymatic hydrolysis and (ii) reduction of the produced thiocholine with hexacyanoferrate (III), followed by hexacyanoferrate (II) electrochemical detection. The advantages of the direct way are simplicity and higher sensitivity compared to the indirect one, which however avoids the interferences because of the lower potential applied.Enzyme inhibition was identified as competitive, the increasing from 1.31 to 1.43 mmol L⁻¹ with chlorofos concentration in the range 0.2-1.0 mmol L⁻¹ and the maximal rate of the enzyme reaction remaining constant (I_max = 579.30 ± 5.71 μA) in the presence of chlorofos. The inhibition constant was calculated using the Dixon method (K_I = 10.07 mmol L⁻¹).The suppression of the acetylcholinesterase activity by the inhibitor, expressed as current decrease at a constant substrate concentration, was exploited for chlorofos quantification optimized by the design of experiments methodology. Optimal response was obtained for an acetylthiocholine concentration of 0.2 mmol L⁻¹, at 26 °C and pH 7.     

A model freed from endogenous reference gene for quantification of genetically modified DNA by real-time PCR. 1. Quantification of DNA from genetically modified organisms in haplo-species materials

This paper is the first part of a serial study investigating a quantification model freed from endogenous reference gene for genetically modified (GM) content by real-time polymerase chain reaction (PCR). The serial study involves two parts: (1) quantitative determination of GM DNA in haplo-species plant samples; (2) quantitative determination of GM DNA in multi-species plant samples. The paper describes a methodology to quantify the GM content in a DNA extract using on one hand real-time PCR to determine the amount of GM targets present and on the other hand absorbance reading at 260 nm to measure the total DNA present in sample. The ratio of both values is expressed as GM percentage. The most prominent dominance of the novel model is that the direct quantitative relation between the initial amount of target template in real-time PCR reaction (X ₀) and the content of GM DNA in tested material (X _SD) is established. Theoretical analysis indicates that the developed quantitative model relieved from the dependence on endogenous reference genes is suitable to quantify the GM content in haplo-species plant sample, in addition, it has the applicability in the quantitative detection of GM content in multi-species GM plant sample. A trail, in which 75 haplo-species GM plant samples were involved, was conducted to validate the suitability of the novel quantification model. The bias varied from 0.00 to 24.00% except a tested sample with lower level of GM content, and the precision expressed as coefficient of variation (CV) was from 2.81 to 25.00%. The limit of quantitation (LOQ) of the quantitative assay was as low as 0.1%. Compared with the previous papers and the performance requirements raised by European Network of GMO Laboratories (ENGL) for analytical methods of GMO testing, the results demonstrated that the established quantification model is a suitable alternative to the more traditional endogenous reference assay in the quantification of GM content in haplo-species plant sample.     

鄂尔多斯盆地靖边气田前石炭纪古地貌解释新模式

近年来,鄂尔多斯盆地靖边气田在原有古地貌解释模式下部署的多口勘探开发井钻探均告失利,二维地震储层预测准确率下滑至70%以下,制约了该区天然气勘探的进程。为此,在综合分析已有古地貌恢复方法优缺点的基础上,根据印模法、残余厚度法的基本原理,结合钻井、测井资料,创建了一种考虑古构造影响的定量古地貌恢复方法,并利用该方法对该气田进行古地貌恢复与侵蚀沟槽重新解释。研究结果表明:(1)该区下古生界储层沟槽解释模式由原来的"东西向大型侵蚀沟槽、南北向毛细沟槽模式"修正为"东西侵蚀主沟槽与局部侵蚀潜坑并存"新模式;(2)较之于原预测结果,下古生界侵蚀主沟槽东西向长度缩短近1/2,毛细沟槽发育范围较小,局部被半径介于1~15 km的潜坑替代;(3)据重新解释的成果,该区下古生界气藏可扩大含气面积305 km~2,预计可建天然气产能4.5×10~8 m~3/a。2016—2017年,利用该研究成果在原解释的沟槽内部署井位30口,目前已完钻8口井,7口井下古生界奥陶系马家沟组马五1+2亚段储层保存完整,其中5口井试气的平均无阻流量为14.3×104 m~3/d,证实新方法能准确地对该区古地貌进行定量化表征、提高储层预测的准确率。     

Sand-ejecting fire extinguisher parameter sensitivity analysis based on DOE and CFD-DEM coupling simulations

To get more accurate quantitative impact effects of selective parameters of the sand-ejecting fire extinguisher on the scattering results by the CFD-DEM coupling method, orthogonal experimental design, analysis of range and variance, full factorial design and the OFAT design were used in this paper. The single impact effects and mixed impact effects of blade number, blade incidence and sand mass flow on scattering vertical distance and inclination were analysed and concluded, as well as the details of the sand distribution. The results show that only the sand mass flow has the dominating influence on the vertical distance, while all three factors have no significant influence on the scattering inclination. The larger the sand mass flow is, the more obvious influence of air resistance and airflow from the outlet of the scattering unit can be shown, and the scattering bifurcation phenomenon can be displayed more obviously.