Application of comprehensive two-dimensional gas chromatography with mass spectrometric detection for the analysis of selected drug residues in wastewater and surface water

Pharmaceutical residues have become tightly controlled environmental contaminants in recent years, due to their increasing concentration in environmental components. This is mainly caused by their high level of production and everyday consumption. Therefore there is a need to apply new and sufficiently sensitive analytical methods, which can detect the presence of these contaminants even in very low concentrations. This study is focused on the application of a reliable analytical method for the analysis of 10 selected drug residues, mainly from the group of non-steroidal anti-inflammatory drugs (salicylic acid, acetylsalicylic acid, clofibric acid, ibuprofen, acetaminophen, caffeine, naproxen, mefenamic acid, ketoprofen, and dicofenac), in wastewaters and surface waters. This analytical method is based on solid phase extraction, derivatization by N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and finally analysis by comprehensive two-dimensional gas chromatography with Time-of-Flight mass spectrometric detection (GC×GC- TOF MS). Detection limits ranged from 0.18 to 5 ng/L depending on the compound and selected matrix. The method was successfully applied for detection of the presence of selected pharmaceuticals in the Svratka River and in wastewater from the wastewater treatment plant in Brno-Modrice, Czech Republic. The concentration of pharmaceuticals varied from one to several hundreds of ng/L in surface water and from one to several tens of μg/L in wastewater.     

Improved xenobiotic-degrading activity of Rhodococcus opacus strain 1cp after dormancy

The goals of the present work were as follows: to obtain the dormant forms of R. opacus 1cp; to study the phenotypic variability during their germination; to compare phenotypic variants during the growth on selective and elective media; and to reveal changes in the ability of the strain to destruct xenobiotics that had not been degradable before dormancy. It was shown that Rhodococcus opacus 1cp (the strain degrading chlorinated phenols) became able to utilize a broader spectrum of xenobiotics after storage in the dormant state. Germination of the dormant forms of R. opacus 1cp on an agarized medium was followed by emergence and development of phenotypic variants that could grow on 4-chlorophenol and 2,4,6-trichlorophenol without adaptation. The cells of R. opacus 1cp phenotypic variants also utilized all of the tested chlorinated phenols: 2,3-, 2,5-, and 2,6-dichloro-, 2,3,4- and 2,4,5-trichloro-, pentachlorophenol, and 1,2,4,5-tetrachlorobenzene in concentrations up to 60 mg/L, though at the lower rates than 4-CP and 2,4,6-TCP. The improved degradation of chlorinated phenols by R. opacus strain 1cp exposed to the growth arrest conditions demonstrates the significance of dormancy for further manifestation of the adaptive potential of populations. A new principle of selection of variants with improved biodegradative properties was proposed. It embraces introduction of the dormancy stage into the cell life cycle with subsequent direct inoculation of morphologically different colonies into the media with different toxicants, including those previously not degraded by the strain.     

Biodegradation potential of the genus Rhodococcus

A large number of aromatic compounds and organic nitriles, the two groups of compounds covered in this review, are intermediates, products, by-products or waste products of the chemical and pharmaceutical industries, agriculture and the processing of fossil fuels. The majority of these synthetic substances (xenobiotics) are toxic and their release and accumulation in the environment pose a serious threat to living organisms. Bioremediation using various bacterial strains of the genus Rhodococcus has proved to be a promising option for the clean-up of polluted sites. The large genomes of rhodococci, their redundant and versatile catabolic pathways, their ability to uptake and metabolize hydrophobic compounds, to form biofilms, to persist in adverse conditions and the availability of recently developed tools for genetic engineering in rhodococci make them suitable industrial microorganisms for biotransformations and the biodegradation of many organic compounds. The peripheral and central catabolic pathways in rhodococci are characterized for each type of aromatics (hydrocarbons, phenols, halogenated, nitroaromatic, and heterocyclic compounds) in this review. Pathways involved in the hydrolysis of nitrile pollutants (aliphatic nitriles, benzonitrile analogues) and the corresponding enzymes (nitrilase, nitrile hydratase) are described in detail. Examples of regulatory mechanisms for the expression of the catabolic genes are given. The strains that efficiently degrade the compounds in question are highlighted and examples of their use in biodegradation processes are presented.     

Biological consequences of increased natural radiation background for Microtus oeconomus Pall. populations

The results of long-term investigations (1981-1999) on the state of Microtus oeconomus Pall. (tundra vole) population, living under the increased natural radiation background for a long time (for more than 100 generations), are presented. Population density dynamics, morphophysiological parameters, state of the lipid peroxidation regulatory system in different tissues and the cytogenetic effects in bone marrow cells of animals have been analyzed. It is shown that tundra voles from the studied radioactively contaminated areas differ from those on natural radiation background area for the parameters measured. The results of this long-term investigation show that qualitatively new sub-populations of tundra vole on these areas have evolved, which are able to survive in radioactively contaminated environment.     

Effect of biosurfactants on crude oil desorption and mobilization in a soil system

Microbially produced biosurfactants were studied to enhance crude oil desorption and mobilization in model soil column systems. The ability of biosurfactants from Rhodococcus ruber to remove the oil from the soil core was 1.4-2.3 times greater than that of a synthetic surfactant of suitable properties, Tween 60. Biosurfactant-enhanced oil mobilization was temperature-related, and it was slower at 15 degrees C than at 22-28 degrees C. Mathematical modelling using a one-dimensional filtration model was applied to simulate the process of oil penetration through a soil column in the presence of (bio)surfactants. A strong positive correlation (R(2)=0.99) was found between surfactant penetration through oil-contaminated soil and oil removal activity. Biosurfactant was less adsorbed to soil components than synthetic surfactant, thus rapidly penetrating through the soil column and effectively removing 65-82% of crude oil. Chemical analysis showed that crude oil removed by biosurfactant contained a lower proportion of high-molecular-weight paraffins and asphaltenes, the most nonbiodegradable compounds, compared to initial oil composition. This result suggests that oil mobilized by biosurfactants could be easily biodegraded by soil bacteria. Rhodococcus biosurfactants can be used for in situ remediation of oil-contaminated soils.     

Decolorization of RBBR by plant cells and correlation with the transformation of PCBs

An extracellular H2O2-requiring Remazol Brilliant Blue R (RBBR) decolorizing enzyme activity was detected after cultivation of cells of various plant species both in liquid medium and when growing on agar plates containing RBBR. Level of the enzyme activity was compared with the ability to metabolize polychlorinated biphenyls (PCBs). The ability to decolorize RBBR was tested in the presence and absence of PCBs. The cultures with high PCB-transforming activity proved to exhibit RBBR oxidase much more resistant towards the influence of PCBs. In addition low activities of lignin peroxidase (LiP) and manganese dependent peroxidase (MnP) were detected in medium and in plant cells. No correlation of MnP and LiP activities with PCB degradation could be found. The RBBR decolorization could be used as a rough screening method for plant cultures able to metabolize PCBs.     

Effect of alkyl hydroxybenzenes on the properties of dioxygenases

The aim of the present work was to investigate the influence of alkylhydroxybenzenes (AHBs) and tyrosol, which belong to cell differentiation factors d(1) group of autoregulators on properties of biodegradation enzymes, catechol 1,2-dioxygenase (Cat 1,2-DO) and methylcatechol 1,2-dioxygenase (MCat 1,2-DO) of Rhodococcus opacus 6a. AHBs were found to have a greater effect on MCat 1,2-DO than on Cat 1,2-DO. It was expressed by more pronounced changes in the activity of MCat 1,2-DO with unsubstituted catechol at different AHB concentrations and by increasing thermostability of MCat 1,2-DO compared to Cat 1,2-DO under the protective action of AHBs. The compound C(7)-AHB shifted the maximum of dioxygenase activities towards higher temperatures and increased their operation optimum. AHBs changed the specificity constant of dioxygenases by decreasing/increasing the K(m)/V(max) value. For example, the increase in the V(max) value of 3,6-dichlorocatechol oxidation by Cat 1,2-DO in the presence of C(7)-AHB was 300-fold higher compared to the same reaction without AHB. The influence of cell differentiation factors on the properties of dimeric enzymes has been shown for the first time. It gives an idea of how the specificity of enzymes can be changed in vivo when strains contact new substrates. The work has shown the possibility of modification of the properties of dimeric enzymes towards the extension of enzyme activity with difficulty converted substrates or in more extreme conditions, which may be important for biotechnological processes.     

Variability of enzyme system of Nocardioform bacteria as a basis of their metabolic activity

The present review describes some aspects of organization of biodegradative pathways of Nocardioform microorganisms, first of all, with respect to their ability to degrade aromatic compounds, mostly methylbenzoate, chlorosubstituted phenols, and chlorinated biphenyls and the intermediates of their transformation: 4-chlorobenzoate and para-hydroxybenzoate. Various enzyme systems induced during degradation processes are defined. The ability of microorganisms to induce a few key enzymes under the influence of xenobiotics is described. This ability may increase the biodegradative potential of strains allowing them to survive in the changing environment or demonstrate to some extent the unspecific response of microorganisms to the effect of toxicants. Nocardioform microorganisms responsible for degradation of such persistent compounds as polychlorinated biphenyls, polyaromatic hydrocarbons, chlorinated benzoates and phenols and other xenobiotics are characterized. The possibility of using Nocardioform microorganisms in some aspects of biotechnology due to their ability to produce some compounds important for industry is also estimated.     

Orange G and Remazol Brilliant Blue R decolorization by white rot fungi Dichomitus squalens, Ischnoderma resinosum and Pleurotus calyptratus

Thirty different white rot strains were screened for Orange G and Remazol Brilliant Blue R (RBBR) decolorization on agar plates. Three promising strains, Dichomitus squalens, Ischnoderma resinosum and Pleurotus calyptratus, selected on the basis of this screening, were used for decolorization study in liquid media. All three strains efficiently decolorized both Orange G and RBBR, but they differed in decolorization capacity depending on cultivation conditions and ligninolytic enzyme production. Two different decolorization patterns were found in these strains: Orange G decolorization in I. resinosum and P. calyptratus was caused mainly by laccase, while RBBR decolorization was effected by manganese peroxidase (MnP); in D. squalens laccase and MnP cooperated in the decolorization processes.     

Monitoring of krypton-85 activity in the atmosphere around Prague

The results of measurements of Krypton-85 (⁸⁵Kr) concentrations in the ground-level air of Prague between 1983 and 1992 are presented and time-related changes analysed. The long-term trend in activity level of ⁸⁵Kr has been steadily increasing with a growth rate of 0.04 Bq.m^–3 (STP) per year. Some peaks of ⁸⁵Kr activity were observed due to the influence of undispersed radioactive plumes coming from distant sources. Short-term variations within a typical range of concentrations from 0.61 to 1.25 Bq.m^–3 (STP) were found to be seasonally dependent, with the maximum occurring in spring.