Methods biological

More elegant analytical methods exploit substances biological or biochemical properties. This is simple for acetylcholinesterase-inhibiting pesticides. Acetylcholinesterase is easy to measure, and the enzyme may be bought from suppliers or extracted from flies, earthworms, or vertebrate nervous tissue. The enzyme may be measured with and without addition of the extract containing the insecticide. Some plant materials may contain natural cholinesterase inhibitors (e.g., solanine in potato) that will interfere with this analysis if not removed. 

In order to improve the credibility of the analysis, the laboratories follow certain standards and are certified as GLP laboratories. Ring tests, whereby many laboratories analyze identical samples, are carried out and the results compared. 

A book on waste minimisation written twenty years ago would have had little, if any, mention of the use of biological methods in industrial effluent treatment. Recent years have seen a massive expansion in this field partly because of new microbiological research results and partly in a response to the demand for new methods and effluent clean-up. 

It has been known for a long time that microbes can accumulate heavy metals. For all microbes this is an essential part of their biochemistry (much in the same way metals are needed by humans, see section 14.5) and they have developed means of capturing metals from their environment. Some of these methods are extremely sophisticated, whereas others are simple but effective. Four mechanisms are broadly accepted as being the main pathways of metal accumulation  

Volatilisation is the microbial biochemical process that methylates heavy metals. Several methyl-metal complexes have significant vapour pressures at room temperature. In this way metals methylated by the microbes just boil ofF. The best known metals to be treated in this fashion are mercury, selenium, tellurium, arsenic and tin. Much of the biomethylation pathway is unknown it appears to be very complex [26]. As a result of the complexity of the volatilisation process it has not been used in effluent treatment and is not discussed further herein. 

Extracellular precipitation can also be described as biomineralisation. The mechanism can range from being very simple to extremely sophisticated. The most complex systems involve cells which can control the crystallisation of common minerals (for example calcium carbonate) to such an extent that exoskeleton structure and shells can be constructed [27]. However, these complex mechanisms have little use in effluent treatment to date. 

The simplest mechanism, and the one which is of importance in effluent clean-up, is extracellular precipitation of metal sulfides (see section 14.7) by sulfate reducing bacteria. These bacteria reduce sulfate to sulfide as part of a biochemical energy pathway (scheme 14.6). 

Directed Evolution to Increase the Substrate Range of Benzoylformate Decarboxylase from Pseudomonas putida 

Marion Wendotff, Thorsten Eggert, Martina Pohl, Caroia Dresen, Michael Muller, and Karl-Erich Jaeger 

Benzoylformate decarboxylase (BFD EC 4.1.1.7) belongs to the class of thiamine diphosphate (ThDP)-dependent enzymes. ThDP is the cofactor for a large number of enzymes, including pyruvate decarboxylase (PDC), benzaldehyde lyase (BAL), cyclohexane-1,2-dione hydrolase (CDH), acetohydroxyacid synthase (AHAS), and (lR,6] )-2-succinyl-6-hydroxy-2,4-cyclohexadiene-l-carboxylate synthase (SHCHC), which all catalyze the cleavage and formation of C-C bonds [1]. The underlying catalytic mechanism is summarized elsewhere [2] (see also Chapter 2.2.3). 

BFD from Pseudomonas putida has been characterized in detail with respect to its biochemical properties [4, 5] and 3D structure [6, 7]. Like other enzymes of this class, BFD is a homotetramer with a subunit size of about 56 kDa. The four active sites are formed at the interfaces of two subunits. The structure was published in 2003 [7] and contains the competitive inhibitor (R)-mandelate bound to the active sites, allowing model-based predictions about the interactions between active site residues and the substrate. 

The substrate range of BFD toward 2-substituted benzaldehydes could be increased by directed evolution, yielding a variant with a single amino acid 

Several variants of the two-hybrid strategy have been developed. Some result solely from technical differences (e.g., use of different reporters, DNA-binding domains, fusion of the bait to the activation domain rather than to the DNA-binding domain), while others allow, for example, for 

TABLE 3 J Ionic Liquids (ILs) used for Pretreatment of Lignocelluloses, Showing the General Formula, With the R and X for Each IL (Mora-Pale et al., 2011) 

1 -Butyl-3-methylimidazolium tetrafluoroborate [BMIM] BF4 Butyl Tetrafluoroborate 

1 -Butyl-3 -methylimidazolium hexafluorophosphate [BMIM] PFg Butyl Hexafluorophosphate 

FIGURE 3.8 Proposed mechanisms of lignoceUulose transformation by enzymes in white- 

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Both nicotinic acid and nicotinamide have been assayed by chemical and biological methods. Owing to the fact that niacin is found in many different forms in nature, it is important to indicate the specific analyte in question. For example, if biological assay procedures are used, it is necessary to indicate whether the analysis is to determine the quantity of nicotinic acid or if niacin activity is the desired result of the analysis. If nicotinic acid is desired, then a method specific for nicotinic acid should be used. If quantitation of niacin activity is the desired outcome, then all compounds (bound and unbound) which behave like niacin will assay biologically for this substance (1). 

Biological, spectroscopic, and chromatographic methods have been used to assay vitamin A and the carotenoids. Biological methods have traditionally been based on the growth response of vitamin A—deficient rats. The utiUty and shortcomings of this test have been reviewed (52,53). This test has found apphcabiUty for analogues of retinol (54,55). Carotenoids that function as provitamin A precursors can also be assayed by this test (56). 

The standard chemical and biological methods of analysis are those accepted by the JnitedStates Pharmacopeia XXIII as well as the ones accepted by the AO AC in 1995 (81—84). The USP method involves saponification of the sample (dry concentrate, premix, powder, capsule, tablet, or aqueous suspension) with aqueous alcohoHc KOH solvent extraction solvent removal chromatographic separation of vitamin D from extraneous ingredients and colormetric deterrnination with antimony trichloride and comparison with a solution of USP cholecalciferol reference standard. 

In wet-air oxidation, the aqueous mixture is heated under pressure ia the presence of air, which oxidi2es the organic material. The efficiency of the oxidation process is a function of reaction time and temperature. The oxidation products are generally less complex and can be treated by conventional biological methods (31). The reactor usually operates between 177 and 321°C with pressures of 2.52—20.8 MPa (350—3000 psig). 

E. Sherman and P. Wakem in C. Guthde and G. Pink, eds.. Guide to Yeast Genetics and Molecular Biology, Methods in Engymology, Vol. 194, Academic Press, New York, 1991, p. 38. 

It is obvious from the provisional risk assessment values for microcystins, and, being of the same order of magnitude of mammalian toxicity, similar values may be calculated for the cyanobacterial neurotoxins, that sensitive detection methods are required to detect these low concentrations of toxins. Of the biological methods of detection discussed earlier, the mouse and invertebrate bioassays are not sensitive enough without concentration of water samples, in that they are only able to detect mg of microcystins per litre. Only the immunoassays (ng-/rg 1 and the protein phosphatase inhibition assays (ng O... 

For the estimation of ephedrine in its salts or simple solutions, titration methods and a Kjeldahl estimation have been described by various authors. The formation of iodoform from ephedrine has been proposed as a method of estimation by Sanchez, and biological methods have been used by several authors. ... 

Physicochemical and Biological Methods for Study of Organotin(IV) Compounds... 

II. PHYSICOCHEMICAL AND BIOLOGICAL METHODS FOR STUDY OF ORGANOTIN(IV) COMPOUNDS... 

The use of molecular biology methods, described in Section 5.3 seems to be especially worthwhile as it offers novel possibilities of optimization on process adjustment. Directed evolution leads to the formation of new biocatalysts with improved characteristics (selectivity, activity, stability, etc.). Incorporation ofnon-proteinogenic amino acids makes it possible to reach beyond the repertoire of building blocks used by nature. The prospect of bioconjugate preparation offers the possibility to form functional clusters of enzymes and to perform multiple synthetic steps in one pot. 

Various physical, chemical, and biological methods have been used for the treatment of dye-containing wastewater. However, these conventional technologies have disadvantages like poor removal efficiency and high running cost. Therefore, low-cost sorbents which can bind dye molecules and be easily regenerated have been extensively searched and tested [3-7]. 

The reduction of ketones, aldehydes, and olefins has been extensively explored using chemical and biological methods. As the latter method, reduction by heterotrophic microbes has been widely used for the synthesis of chiral alcohols. On the contrary, the use of autotrophic photosynthetic organisms such as plant cell and algae is relatively rare and has not been explored because the method for cultivation is different from that of heterotrophic microbes. Therefore, the investigation using photosynthetic organisms may lead to novel biotransformations. 

Established in 1894, AOAC International is an independent association of scientists and organizations in the public and private sectors devoted to promoting methods validation and quality measurements in the analytical sciences. AOAC has a mission to ensure the development, testing, validation, and publication of reliable chemical and biological methods of analysis for foods, drugs, feed-stuffs, fertilizers, pesticides, water, forensic materials and other substances affecting public health and safety and the environment. 

Cleanup of major oil spills may be accomplished by physical, chemical, and biological methods. In this activity, you will focus on bioremediation, which is a method of using natural organisms to break down contaminants at the site. 

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