Methane emission from stems of European beech (Fagus sylvatica) offsets as much as half of methane oxidation in soil.

Trees are known to be atmospheric methane (CH4 ) emitters. Little is known about seasonal dynamics of tree CH4 fluxes and relationships to environmental conditions. That prevents the correct estimation of net annual tree and forest CH4 exchange. We aimed to explore the contribution of stem emissions to forest CH4 exchange. We determined seasonal CH4 fluxes of mature European beech (Fagus sylvatica) stems and adjacent soil in a typical temperate beech forest of the White Carpathians with high spatial heterogeneity in soil moisture. The beech stems were net annual CH4 sources, whereas the soil was a net CH4 sink. High CH4 emitters showed clear seasonality in their stem CH4 emissions that followed stem CO2 efflux. Elevated CH4 fluxes were detected during the vegetation season. Observed high spatial variability in stem CH4 emissions was neither explicably by soil CH4 exchange nor by CH4 concentrations, water content, or temperature studied in soil profiles near each measured tree. The stem CH4 emissions offset the soil CH4 uptake by up to 46.5% and on average by 13% on stand level. In Central Europe, widely grown beech contributes markedly to seasonal dynamics of ecosystem CH4 exchange. Its contribution should be included into forest greenhouse gas flux inventories.

Bioreactors based on immobilized fungi: bioremediation under non-sterile conditions.

White-rot fungi are renowned for their remarkable potential to degrade a wide range of organic pollutants. They are applicable in standard bioreactors offering both the use of the continuous mode of action and easy upscaling of the biodegradation process. The recent advance in this field consisted in the use of various fungi and different types of reactors in the treatment of real wastewaters. Most degradation studies involving white-rot fungi carried out so far used controlled, aseptic conditions. However, during bioremediation of real wastewaters, the degradation capacity of the fungi would be significantly affected by autochthonous microorganisms. Consequently, for the development of sustainable bioremediation technologies, it is important to understand the mechanisms involved in the intermicrobial interactions occurring during the bioremediation process. This review summarizes recent applications of white-rot fungi to biodegradation of recalcitrant organopollutants under non-sterile conditions describing the invading microorganism(s) and the way how they affect the stability and degradation efficiency of the fungal bioreactor cultures. In addition, studies where fungal cultures were exposed to defined microbial stress are also reported documenting the effect and mechanisms of microbial interactions. Advanced OMICs techniques, specifically the genomics and metabolomics analyses, are suggested to help in identification of the invading microorganisms and in discovery of mechanisms taking part in the interspecific interactions.

Ascorbic Acid and Glucosinolate Levels in New Czech Cabbage Cultivars: Effect of Production System and Fungal Infection.

Nutritional value and disease-preventive effects of cabbage are well-known. Levels of the antioxidant compounds ascorbic acid (AA) and glucosinolates (GSL) in new Czech cabbage cultivars were determined in the context of different production systems. The contents of AA and GSLs in cabbage biomass were determined by HPLC. Individual GSLs were identified according to their exact masses with sinigrin used as the external standard. Artificial infection with A. brassicicola generally raised the AA levels. The major GSLs (≥10 mg kg-1) were glucobrassicin, sinigrin, and glucoiberin. Indole and aliphatic GSLs were present, but no aromatic ones were detected. Ecological growth conditions and the artificial fungal infection increased the total content of GSLs and, also, of the methoxylated indole GSLs. Sulforaphane, iberin, indole-3-carbinol, and ascorbigen resulting from the hydrolysis of GSLs were found in both cultivars. The amounts and profiles of GSLs present in the two Czech cultivars demonstrated their good nutritional value. The decomposition products sulforaphane, iberin, indole-3-carbinol, and ascorbigen detected improve its health-promoting qualities and represent a suitable component of the human diet.

Mutual interactions of Pleurotus ostreatus with bacteria of activated sludge in solid-bed bioreactors.

White rot fungi are well known for their ability to degrade xenobiotics in pure cultures but few studies focus on their performance under bacterial stress in real wastewaters. This study investigated mutual interactions in co-cultures of Pleurotus ostreatus and activated sludge microbes in batch reactors and different culture media. Under the bacterial stress an increase in the dye decolorization efficiency (95 vs. 77.1 %) and a 2-fold elevated laccase activity (156.7 vs. 78.4 Ul(-1)) were observed in fungal-bacterial cultures compared to pure P. ostreatus despite a limited growth of bacteria in mixed cultures. According to 16S-rDNA analyses, P. ostreatus was able to alter the structure of bacterial communities. In malt extract-glucose medium the fungus inhibited growth of planktonic bacteria and prevented shifts in bacterial utilization of potential C-sources. A model bacterium, Rhodococcus erythropolis responded to fungal metabolites by down regulation of uridylate kinase and acetyl-CoA synthetase.

Porcine EEF1A1 and EEF1A2 genes: genomic structure, polymorphism, mapping and expression.

Eukaryotic translation elongation factor 1 alpha (EEF1A) plays a key role in protein synthesis. In higher vertebrates EEF1A occurs in two isoforms, EEF1A1 and EEF1A2, encoded by distinct genes. The purpose of this study was to compare the two porcine genes as for the genomic sequence, gene organization and mRNA expression in different tissues, as well as to search for polymorphism and chromosomal assignment. Standard methods of DNA and mRNA analysis were used. We determined the complete genomic sequence of the porcine EEF1A1 and EEF1A2 genes. The two genes differ in the lengths of transcription units (3102 and 8588 bp, respectively), but have similar genomic organization and their coding sequences are highly similar (78% identity of coding sequences and 92.4% identity of amino acid sequences). Several polymorphisms in the two genes were detected. EEF1A1 and EEF1A2 were mapped to SSC1p11.1 and SSC17q23.3, respectively. mRNA of EEF1A1 was expressed in all studied tissues (the highest expression was in 44-day fetal muscle and low expression in adult liver and brain), while EEF1A2 was expressed only in skeletal-muscle, tongue, heart, diaphragm and brain tissues. EEF1A2 was not expressed in fetal muscle tissue (44 days). In this paper results are provided on genomic sequences, genomic organization, polymorphism, chromosomal assignment and spatial and temporal expressions of the porcine EEF1A1 and EEF1A2 genes. Novel polymorphisms were described in both genes. Porcine EEF1A2 was studied for the first time.

A Comprehensive Study for Determination of Free Fatty Acids in Selected Biological Materials: A Review.

The quality of oil is highly dependent on its free fatty acid (FFA) content, especially due to increased restrictions on renewable fuels. As a result, there has been a growing interest in free fatty acid determination methods over the last few decades. While various standard methods are currently available, such as the American Oil Chemists Society (AOCS), International Union of Pure and Applied Chemistry (IUPAC), and Japan Oil Chemists' Society (JOCS), to obtain accurate results, there is a pressing need to investigate a fast, accurate, feasible, and eco-friendly methodology for determining FFA in biological materials. This is owing to inadequate characteristics of the methods, such as solvent consumption and reproducibility, among others. This study aims to investigate FFA determination methods to identify suitable approaches and introduce a fresh perspective.

Fungal microsomes in a biotransformation perspective: protein nature of membrane-associated reactions.

Microsomal fraction of fungal cells grabs the attention of many researchers for it contains enzymes that play a role in biotechnologically relevant processes. Microsomal enzymes, namely, CYP450s, were shown to metabolize a wide range of xenobiotic compounds, including PAHs, PCBs, dioxins, and endocrine disruptors, and take part in other fungal biotransformation reactions. However, little is known about the nature and regulation of these membrane-associated reactions. Advanced proteomic and post-genomic techniques make it possible to identify larger numbers of microsomal proteins and thus add to a deeper study of fungal intracellular processes. In this work, proteins that were identified through a shotgun proteomic approach in fungal microsomes under various culture conditions are reviewed. However, further research is still needed to fully understand the role of microsomes in fungal biodegradation and biotransformation reactions.

The Effect of Microwave Radiation on the Solidification of C-S-H Gels: Its Influence on the Solidified Cement Mixtures.

The present paper deals with the properties of hardened cement mixtures that have been exposed to microwave radiation. Microwaves fall under electromagnetic waves (EMW), and the main reason for using EMW radiation is to accelerate the drying of concrete as well as to reduce the time required to obtain the handling strength after it is removed from the mould. This paper is divided into two main parts. In the first part, three sets of cement samples were made. One set of samples solidified naturally in air and the second and third sets of samples were exposed to EMW radiation, with different exposure times for each. The solidification was then stopped, and the representation of the major minerals was experimentally determined. The second part of the experiment focuses on the properties of the hardened cement mixtures, both in terms of strength and physical properties. The experiment was carried out on two sets of samples. Each mixture was exposed to EMW radiation, the main differences being the exposure time and the position of the samples relative to the EMW generator. The aim of the experiments is to determine the resulting mechanical properties of the samples in comparison with those that were subjected to normal solidification in air. The data from these experiments suggest that microwave radiation can be used to accelerate the curing of concrete specimens, obtaining the handling strength in a relatively short time, but a reduction in the resulting strength can be expected compared to the reference specimens.

Mechanistic study of 17α-ethinylestradiol biodegradation by Pleurotus ostreatus: tracking of extracelullar and intracelullar degradation mechanisms.

The white rot fungus Pleurotus ostreatus is able to completely remove the synthetic hormone 17α-ethinylestradiol (EE2, 200 µg in 20 mL) from a liquid complex or mineral medium in 3 or 14 days, respectively. Its efficiency has also been documented in the removal of estrogenic activity that correlated with the EE2 degradation. A set of in vitro experiments using various cellular and enzyme fractions has been performed and the results showed that EE2 was degraded by isolated laccase (about 90% within 24 h). The degradation was also tested with concentrated extracellular liquid where degradation reached 50% mainly due to the laccase activity; however, after a supplementation with H2O2 and Mn²âº, residual manganese-dependent peroxidase activities (40 times lower than Lac) raised the degradation to 100%. Moreover, the intracellular fraction and also laccase-like activity associated with fungal mycelium were found to be efficient in the degradation too. Isolated microsomal proteins appeared to also be involved in the process. The degradation was completely suppressed in the presence of cytochrome P-450 inhibitors, piperonylbutoxide and carbon monoxide, indicating a role of this monooxygenase in the degradation process. Attention was also paid to monitoring of changes in the estrogenic activity during these particular in vitro experiments when mainly degradations related to ligninolytic enzymes were found to decrease the estrogenic activity with EE2 removal proportionally. Several novel metabolites of EE2 were detected using different chromatographic method with mass spectrometric techniques (LC-MS, GC-MS) including also [¹³C]-labeled substrates. The results document the involvement of various different simultaneous mechanisms in the EE2 degradation by P. ostreatus by both the ligninolytic system and the eukaryotic machinery of cytochromes P-450.

A Microsatellite Genotyping-Based Genetic Study of Interspecific Hybridization between the Red and Sika Deer in the Western Czech Republic.

Although inter-species hybrids between the red and sika deer can be phenotypically determined only exceptionally, there is the eventuality of identification via molecular genetic analysis. We used bi-parentally inherited microsatellite markers and a Bayesian statistical framework to re-examine the proportion of hybrids in the Czech red and sika deer populations. In total, 123 samples were collected, and the nuclear dataset consisted of 2668 allelic values. The number of alleles per locus ranged from 10 (BM1818) to 22 (BM888 and T193), yielding the mean of 16 alleles per locus across the deer. The mean allelic diversity of the red deer markedly exceeded that of the Japanese sika deer. Interspecific hybrids were detected, enabling us to confirm the genetic introgression of the sika deer into the red deer populations and vice versa in western Bohemia. The mean hybrid score equaled 10.6%, with 14.3% of the hybrids being among red deer-like individuals and 6.7% among sika-like ones. At two western Bohemian locations, namely, Doupovské hory and Slavkovský les, the total percentages of hybrid animals equaled 18.8 and 8.9, respectively. No red deer alleles were detected in the sika populations of the subregions of Kladská, Zlutice, and Lány. The NeighborNet network clearly separated the seven red and sika deer sampling populations according to the geography. The knowledge gained from the evaluated data is applicable in hunting management to reduce hybridization with the European deer.

Beta-lactam resistance development during the treatment processes of municipal wastewater treatment plants.

This work monitored the effect of a municipal and a village wastewater treatment plant (WWTP) technology on the fate of beta-lactam resistance genes in bacterial populations in different phases of the wastewater treatment process. In case of the municipal WWTP1, the bacteria possessing a high ampicillin resistance (minimal inhibitory concentration (MIC) values of 20 mg/mL) accumulated in the sedimentation tank, which was accompanied with a higher concentration of ampicillin in the wastewater samples (28.09 ng/L) and an increase in the relative abundance of the blaTEM gene in the bacterial population. However, an opposite trend was revealed with the blaNDM-1 gene, making the sedimentation processes of WWTP1 crucial only for the accumulation of the blaTEM gene. Similarly, the comparison with the WWTP2 showed that the accumulation of the ampicillin resistance in bacterial population probably depended on the WWTP technology and wastewater composition. Out of the four tested resistance genes (blaTEM, blaKPC, blaNDM-1, and blaOXA-48), blaTEM and blaNDM-1 genes were the only two detected in this study. According to NGS analysis of bacterial 16 S rRNA gene, Gammaproteobacteria dominated the ampicillin-resistant bacteria of the WWTP sedimentation tanks. Their relative abundance in the bacterial population also increased during the sedimentation processes in WWTP1. It could indicate the role of the bacterial taxon in ampicillin resistance accumulation in this WWTP and show that only 9.29% of the original bacterial population from the nitrification tank is involved in the documented shifts in beta-lactam resistance of the bacterial population.

New in vitro reporter gene bioassays for screening of hormonal active compounds in the environment.

Identification of chemicals with endocrine-disrupting activities in the past two decades has led to the need for sensitive assays for detection and monitoring of these activities in the environment. In vitro reporter gene assays represent a relatively fast and easy-to-perform method for detection of compounds that are able to bind to hormonal receptors and stimulate or silence their transactivation activity, thus interfering with the hormone signaling pathways. This paper reviews upgrades on reporter gene assays performed during the last decade. The utilization of new reporter genes (luciferase and green fluorescent protein coding genes) significantly improved the sensitivity of the tests and made them faster. Reporter gene assays now represent a high-throughput system for screening chemicals for hormonal activity. Finally, modification of test set-ups for testing anti-hormonal activities also enabled measurements of endocrine-disrupting activities in complex environmental samples such as sediments and wastewater treatment plant effluents.

Cultivation of Medicinal Mushrooms on Spruce Sawdust Fermented with a Liquid Digestate from Biogas Stations.

The aim of this work was to prepare a softwood substrate on which to grow edible and medicinal mushrooms. Liquid digestate from a biogas station was successfully used in spruce sawdust fermentation. Pleurotus ostreatus, P. eryngii, and Ganoderma lucidum were grown on the obtained substrates and their mycelia grew at rates similar to rates of growth on control beech sawdust; values ranged from 4.1 to 5.54 mm/day. A 6-week fermentation period was determined to be sufficient for removing volatile extractives from sawdust (76% removal efficiency), which elevated content was shown to be most critical for fungal growth. Removal of 47% of resinous compounds and a decrease in the carbon-to-nitrogen ratio in the growth substrate were found during sawdust fermentation in the presence of the liquid digestate. Among ligninolytic enzymes, the growth substrates produced here favored laccase produced by tested fungi. It follows that utilizing wastes from biogas production to reuse softwood wastes could make an environmentally friendly and economically viable biotechnology for producing mushrooms.

Metabolic profiling of Fusarium oxysporum f. sp. conglutinans race 2 in dual cultures with biocontrol agents Bacillus amyloliquefaciens, Pseudomonas aeruginosa, and Trichoderma harzianum.

There are increasing efforts to identify biocontrol-active microbial metabolites in order to improve strategies for biocontrol of phytopathogens. In this work, Fusarium oxysporum f. sp. conglutinans was confronted with three different biocontrol agents: Trichoderma harzianum, Bacillus amyloliquefaciens, and Pseudomonas aeruginosa in dual culture bioassays. Metabolites produced during the microbial interactions were screened by a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). T. harzianum exhibited the strongest inhibition of growth of F. oxysporum resulting in overlay of the pathogen colony with its mycelium. Recorded metabolite profiles suggested a direct attack of F. oxysporum mycelium by T. harzianum and B. amyloliquefaciens by means of membrane-attacking peptaibols and a set of antimicrobial lipopeptides and siderophores, respectively. The direct mode of the biocontrol activity of T. harzianum and B. amyloliquefaciens corresponded to their ability to suppress F. oxysporum production of mycotoxin beauvericin suggesting that this ability is not specific only for Trichoderma species. In the case of P. aeruginosa, siderophores pyoverdine E/D and two rhamnolipids were produced as major bacterial metabolites; the rhamnolipid production was blocked by F. oxysporum. The results showed that this type of biocontrol activity was the least effective against F. oxysporum. The effective application of MALDI-MS profiling to the screening of nonvolatile microbial metabolites produced during the interaction of the phytopathogen and the biocontrol microorganisms was demonstrated.

Verification of reference genes for relative quantification of gene expression by real-time reverse transcription PCR in the pig.

The aim of this study was to develop a set of reliable reference genes for quantification of mRNA expression in the pig. The mRNA expression stability in pig tissues was studied for 4 genes: EEF1A1, GAPDH, HPRT1 and TOP2B. The level of expression was characterized by Ct values for each gene and each tissue. By using the geNorm algorithm, the stability of the reference genes was determined in the diaphragm, heart, kidney, liver, lungs, longissimus muscle, and spleen. On the basis of this information, suitable reference genes can be selected for mRNA expression studies in relevant pig tissues.

Antibiotic Resistance in Czech Urban Wastewater Treatment Plants: Microbial and Molecular Genetic Characterization.

Quantitative changes in antibiotic resistance genes (ARGs) were investigated in six urban wastewater treatment plants (WWTPs) treating municipal and industrial wastewaters. In a selected WWTP, the fate of ARGs was studied in a 1-year time interval and in two phases of wastewater treatment process. Nine ARGs (tetW, tetO, tetA, tetB, tetM, blaTEM, ermB, sul1, and intl1) were quantified in total and their relative abundance assessed by ARG copies/16SrRNA copies. From the tetracycline resistance genes, tetW was the only one detected in all sampled WWTPs. Its relative abundance in the nitrification tank of WWTP5 was found stable during the 1-year period, but was lowered by secondary sedimentation processes in the wastewater treatment down to 24% compared to the nitrification tank. Bacterial isolates showing high tetracycline resistance (minimal inhibition concentrations >100 µg/mL) were identified as members of Acinetobacter, Klebsiella, Citrobacter, Bacillus, and Enterobacter genera. Dynamic shifts in the relative abundance of ermB and sul1 were also demonstrated in wastewater samples from WWTP5.

The implication of Dichomitus squalens laccase isoenzymes in dye decolorization by immobilized fungal cultures.

The study focuses on the production of ligninolytic enzymes and dye degradation capacity of Dichomitus squalens immobilized on polyurethane foam (PUF) or pine wood (PW) in a fixed bed reactor at a laboratory scale (working volume of 0.6l). Immobilization of fungal cultures on pine wood improved eminently laccase production in comparison to the liquid cultures. Immobilized D. squalens was able to decolorize an anthraquinone dye Remazol Brilliant Blue R and an azo dye Reactive Orange 16, however, only a limited decolorization of Copper(II)phthalocyanine dye was observed in both types of reactor cultures. The involvement of a laccase activity in dye decolorization was suggested. Further, two different chromatographical forms of laccases, Lc1 and Lc2, were isolated from PW cultures of D. squalens using a fast, two step FPLC method. Enzymes revealed identical molecular masses of 68 kDa (estimated by SDS-PAGE) and similar pI's, however, they differed in their catalytic properties such as pH dependence of the activity and ABTS oxidation rates. In this study, we demonstrated different dye decolorization capacities of Lc1 and Lc2 as well.

Cryptogamic stem covers may contribute to nitrous oxide consumption by mature beech trees.

Naturally produced by microbial processes in soil, nitrous oxide (N2O) is an important greenhouse gas contributing to climate change. Accordingly, there is a need to accurately quantify the capability of forest ecosystems to exchange N2O with the atmosphere. While N2O emissions from soils have been well studied, trees have so far been overlooked in N2O inventories. Here, we show that stems of mature beech trees (Fagus sylvatica) may act as a substantial sink of N2O from the atmosphere under conditions of soils consuming N2O. Consistent consumption of N2O by all stems investigated (ranging between -2.4 and -3.8 µg m-2 h-1) is a novel finding in contrast to current studies presenting trees as N2O emitters. To understand these fluxes, N2O exchange of photoautotrophic organisms associated with beech bark (lichens, mosses and algae) was quantified under laboratory conditions. All these organisms were net N2O sinks at full rehydration and temperature of 25 °C. The consumption rates were comparable to stem consumption rates measured under field conditions. Cryptogamic stem covers could be a relevant sink of N2O in European beech forests.

Reduction in the mutagenicity of synthetic dyes by successive treatment with activated sludge and the ligninolytic fungus, Irpex lacteus.

Synthetic dyes are released in wastewater from textile manufacturing plants, and many of these dyes are genotoxic. In the present study, the mutagenicity of azo, anthraquinone, and triphenyl methane dyes was investigated before and after successive biodegradation with activated sludge and the ligninolytic fungus, Irpex lacteus. Two biodegradation systems were used to reduce the genotoxicity of dyes that were not efficiently inactivated by activated sludge alone. Mutagenicity was monitored with the Salmonella reversion assay conducted with the base-pair substitution detector strains, TA100 and YG1042, and the frame-shift detector strains, TA98 and YG1041, with and without rat liver S9. All dyes except for Congo Red (CR) were mutagenic with S9 activation. Assays conducted with the dyes indicated that only the azo dye Reactive Orange 16 (RO16) was mutagenic in both TA98 and TA100. Methyl Red and Disperse Blue 3 (DB3) were mutagenic in TA98, YG1041 and YG1042, while Reactive Black 5 was mutagenic in YG1041 and YG1042. Remazol Brilliant Blue R (RBBR), Crystal violet (CV) and Bromophenol Blue (BPB) were mutagenic only in TA98, but the toxicity of the latter two dyes complicated the evaluation of their mutagenicity. CR was not mutagenic in any of the tester strains. Biodegradation studies conducted with RO16 and DB3 indicated that the two-step biodegradation process reduced the mutagenic potential of RO16 and DB3 to a greater extent than activated sludge alone; the mutagenicity of the two dyes was reduced by 95.2% and 77.8%, respectively, by the two-step process. These data indicate that the combined biodegradation process may be useful for reducing the mutagenicity associated with wastewater from textile factories that contain recalcitrant dyes.

A two-dimensional protein map of Pleurotus ostreatus microsomes-proteome dynamics.

Recent studies documented that several processes in filamentous fungi are connected with microsomal enzyme activities. In this work, microsomal subproteomes of Pleurotus ostreatus were analyzed by two-dimensional (2-D) polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. To assess proteome dynamics, microsomal proteins were isolated from fungal cultures after 7 and 12 days of cultivation. Additionally, 10 mg/L of 17α-ethinylestradiol (EE2) was treated with the cultures during 2 days. Despite the EE2 degradation by the fungus reached 97 and 76.3 % in 7- and 12-day-old cultures, respectively, only a minor effect on the composition of microsomal proteins was observed. The changes in protein maps related to ageing prevailed over those induced by EE2. Epoxide hydrolase, known to metabolize EE2, was detected in 12-day-old cultures only which suggests differences in EE2 degradation pathways utilized by fungal cultures of different age. The majority (32 %) of identified microsomal proteins were parts of mitochondrial energy metabolism.