Changes in the content of emerging pollutants and potentially hazardous substances during vermi/composting of a mixture of sewage sludge and moulded pulp.

Processing sewage sludge can be problematic due to its potential environmental toxicity. It may contain high concentrations of pharmaceuticals, polycyclic aromatic hydrocarbons, and heavy metals, as well as pathogenic microorganisms. However, it is a good source of organic matter and rich in microbial communities and enzymatic activity. This study deals with composting and vermicomposting of pre-composted mixtures of two different kinds of sewage sludge blended with moulded pulp in an operating composting plant. Of the total number and concentration of pollutants detected in individual piles, a large percentage of them were reduced by the composting process. The composting 2 process resulted in the greatest reduction in contaminating substances--a total of 19 substances by 4.39-90.4%. Some pharmaceuticals accumulated in earthworm bodies during vermicomposting; a total of 11 substances were detected. Atorvastatin showed the highest percentage reduction in compost 2 (90.4%), vermicompost 1 (65.2%) and vermicompost 2 (97.3%). Both composting and vermicomposting appeared to be effective for removal of heavy metals. A higher content of microbial phospholipid fatty acids (PLFAs) was found in composts than vermicomposts. There was a significant reduction in the content of pathogenic microorganisms in both processes, but the reduction in enterococci was not significant.

Changes in soil microbial community functionality and structure in a metal-polluted site: The effect of digestate and fly ash applications.

Soil from Trhové Dusníky (Príbram, Czech Republic) is characterized by its high polymetallic accumulations in Pb-Ag-Zn due to mining and smelting activities. In previous studies performed in our research group, we have evaluated the potential use of amendments that would reduce the mobility and availability of metals such as Hg. We have observed that the application of digestate and fly ash in metal-polluted soil has an impact in immobilizing these metals. However, until now we have lacked information about the effect of these amendments on soil microbial functionality and communities. The multi-contaminated soil was used to grow wheat in a pot experiment to evaluate the impact of digestate and fly ash application in soil microbial communities. Soil samples were collected after 30 and 60 days of treatment. The digestate application improved chemical attributes such as the content in total organic carbon (TOC), water soluble carbon (WSOC), total soluble carbon (C), total soluble nitrogen (N), and inorganic N forms (NO3(-)) as consequence of high content in C and N which is contained in digestate. Likewise, microbial activity was greatly enhanced by digestate application, as was physiological diversity. Bacterial and fungal communities were increased, and the microbial biomass was highly enhanced. These effects were evident after 30 and 60 days of treatment. In contrast, fly ash did not have a remarkable effect when compared to digestate, but soil microbial biomass was positively affected as a consequence of macro- and micro-nutrient sources applied by the addition of fly ash. This study indicates that digestate can be used successfully in the remediation of metal-contaminated soil.

Chemical and microbiological characterization of an aged PCB-contaminated soil.

This study was aimed at complex characterization of three soil samples (bulk soil, topsoil and rhizosphere soil) from a site historically contaminated with polychlorinated biphenyls (PCB). The bulk soil was the most highly contaminated, with a PCB concentration of 705.95 mg kg(-1), while the rhizosphere soil was the least contaminated (169.36 mg kg(-1)). PCB degradation intermediates, namely chlorobenzoic acids (CBAs), were detected in all the soil samples, suggesting the occurrence of microbial transformation processes over time. The higher content of organic carbon in the topsoil and rhizosphere soil than in the bulk soil could be linked to the reduced bioaccessibility (bioavailability) of these chlorinated pollutants. However, different proportions of the PCB congener contents and different bioaccessibility of the PCB homologues indicate microbial biotransformation of the compounds. The higher content of organic carbon probably also promoted the growth of microorganisms, as revealed by phospholipid fatty acid (PFLA) quantification. Tag-encoded pyrosequencing analysis showed that the bacterial community structure was significantly similar among the three soils and was predominated by Proteobacteria (44-48%) in all cases. Moreover, analysis at lower taxonomic levels pointed to the presence of genera (Sphingomonas, Bulkholderia, Arthrobacter, Bacillus) including members with reported PCB removal abilities. The fungal community was mostly represented by Basidiomycota and Ascomycota, which accounted for >80% of all the sequences detected in the three soils. Fungal taxa with biodegradation potential (Paxillus, Cryptococcus, Phoma, Mortierella) were also found. These results highlight the potential of the indigenous consortia present at the site as a starting point for PCB bioremediation processes.

Ecotoxicity and biodegradability of new brominated flame retardants: a review.

Brominated flame retardants (BFRs) have been routinely used as additives in a number of consumer products for several decades in order to reduce the risk of fire accidents. Concerns about the massive use of these substances have increased due to their possible toxicity, endocrine disrupting properties and occurrence in almost all the environmental compartments, including humans and wildlife organisms. Several conventional BFRs (e.g. polybrominated diphenylethers (PBDE)) have been included in the list of Persistent Organic Pollutants and their use has been restricted because of their established toxicity and environmental persistence. Over the past few years, these compounds have been replaced with "new" BFRs (NBFRs). Despite the fact that NBFRs are different chemical molecules than traditional BFRs, most of physical-chemical properties (e.g. aromatic moiety, halogen substitution, lipophilic character) are common to both groups; therefore, their fate in the environment is potentially similar to the banned BFRs. Therefore, this article has been compiled to summarize the published scientific data regarding the biodegradability of the most widely used NBFRs, a key factor in their potential persistency in the environment, and their ecotoxicological effects on humans and test organisms. The data reviewed here document that the mechanisms through NBFRs exibit their ecotoxicity and the processes leading to their biotransformation in the environment are still poorly understood. Thus emphasis is placed on the need for further research in these areas is therefore emphasized, in order to avoid the massive use of further potentially harmful and recalcitrant substances of anthropogenic origin.

Chlorobenzoic acid degradation by Lentinus (Panus) tigrinus: in vivo and in vitro mechanistic study-evidence for P-450 involvement in the transformation.

Aim of this work was to investigate the ability of Lentinus (Panus) tigrinus to degrade and detoxify a chlorobenzoate (CBA) mixture composed of mono-, di- and tri-chlorinated isomers. The degradation process was investigated as a function of both the growing medium (i.e. low N Kirk's and malt extract-glucose medium) and cultivation conditions (i.e. stationary and shaken cultures). The majority of CBAs were quantitatively degraded within the early 15 d from spiking with the notable exception of the double ortho-chlorinated compounds, 2,6-di-, 2,3,6-tri- and 2,4,6-tri-CBA. Analysis of the degradation intermediates indicated the occurrence of side chain reduction, hydroxylation and methylation reactions. Although CBAs stimulated laccase production, in vitro experiments with a purified L. tigrinus laccase isoenzyme demonstrated its inability to participate in the initial attack on CBAs even in the presence of redox mediators; similar results were found with a Mn-peroxidase isoenzyme. Conversely, prompt degradation was observed upon 1h incubation of CBAs with a purified microsomal fraction containing cytochrome P-450 monooxygenase. The nature of some reaction products (i.e. hydroxylated derivatives), the dependency of the reaction on NADPH and its susceptibility to either CO or piperonyl butoxide inhibition confirmed the involvement of L. tigrinus cytochrome P-450 in the early steps of CBA degradation.

Transcriptional response of lignin-degrading enzymes to 17α-ethinyloestradiol in two white rots.

Fungal, ligninolytic enzymes have attracted a great attention for their bioremediation capabilities. A deficient knowledge of regulation of enzyme production, however, hinders the use of ligninolytic fungi in bioremediation applications. In this work, a transcriptional analyses of laccase and manganese peroxidase (MnP) production by two white rots was combined with determination of pI of the enzymes and the evaluation of 17α-ethinyloestradiol (EE2) degradation to study regulation mechanisms used by fungi during EE2 degradation. In the cultures of Trametes versicolor the addition of EE2 caused an increase in laccase activity with a maximum of 34.2 ± 6.7 U g⁻¹ of dry mycelia that was observed after 2 days of cultivation. It corresponded to a 4.9 times higher transcription levels of a laccase-encoding gene (lacB) that were detected in the cultures at the same time. Simultaneously, pI values of the fungal laccases were altered in response to the EE2 treatment. Like T. versicolor, Irpex lacteus was also able to remove 10 mg l⁻¹ EE2 within 3 days of cultivation. While an increase to I. lacteus MnP activity and MnP gene transcription levels was observed at the later phase of the cultivation. It suggests another metabolic role of MnP but EE2 degradation.

Hormonal activities of new brominated flame retardants.

After the phase-out of two commercial mixtures of brominated flame retardants, an increasing number of alternative flame retardants have been introduced in commercial applications. None of them, however, has been thoroughly tested for its hormonal activity. We used two yeast reporter-gene assays to determine the potential of eleven compounds to interfere with estrogenic and androgenic pathways. Our data demonstrate the ability of 2,4,6-tribromophenol to lower the transcriptional activity of human estrogen and androgen receptors. A nominal IC(50) value of 14.1 µM for anti-estrogenic and 3.9 µM for anti-androgenic activity was obtained using the luciferase reporter. An IC(50) value of 9.2 µM was calculated for the anti-estrogenic activity measured by the ß-galactosidase assay. Of the tested chemicals, this study highlights the endocrine disrupting effects of 2,4,6-tribromophenol whose occurrence in the environment should be monitored.

Lentinus (Panus) tigrinus augmentation of a historically contaminated soil: matrix decontamination and structure and function of the resident bacterial community.

The ability of Lentinus tigrinus to grow and to degrade persistent aromatic hydrocarbons in aged contaminated soil was assessed in this study. L. tigrinus extensively colonized the soil; its degradation activity after 60 d incubation at 28°C, however, was mostly limited to dichloroaniline isomers, polychlorinated benzenes and diphenyl ether while the fungus was unable to deplete 9,10-anthracenedione and 7-H-benz[DE]anthracene-7-one which were the major soil contaminants. Although clean-up levels were limited, both density of cultivable heterotrophic bacteria and richness of the resident bacterial community in L. tigrinus microcosms (LtM) increased over time to a significantly larger extent than the respective amended incubation controls (1.9×10(9) CFU g(-1) vs. 1.0×10(9) CFU g(-1) and 37 vs. 16, respectively). Naphthalene- and catechol 2,3-dioxygenase gene copy numbers, however, decreased over time at a higher rate in LtM than in incubation controls likely due to a higher stimulation on heterotrophs than xenobiotics-degrading community members.

Trachydiscus minutus, a new biotechnological source of eicosapentaenoic acid.

The yellow-green alga Trachydiscus minutus (class Xanthophyta) was cultivated in a standard medium and in media without sulfur and nitrogen. Its yield after a 16-d cultivation reached 13 g dry mass per 1 L medium. The content of oligoenoic ('polyenoic') fatty acid (PUFA), i.e. eicosapentaenoic (EPA), was in excess of 35 % of total fatty acids; the productivity was thus 88 mg/L per d. This result makes the alga a very prospective organism that may serve as a new biotechnological source of single cell oil.

Irpex lacteus, a white-rot fungus with biotechnological potential--review.

White-rot fungi that are efficient lignin degraders responsible for its turnover in nature have appeared twice in the center of biotechnological research - first, when the lignin degradation process started being systematically investigated and major enzyme activities and mechanisms involved were described, and second, when the huge remediation potential of these organisms was established. Originally, Phanerochaete chrysosporium became a model organism, characterized by a secondary metabolism regulatory pattern triggered by nutrient (mostly nitrogen) limitation. Last decade brought evidence of more varied regulatory patterns in white-rot fungi when ligninolytic enzymes were also abundantly synthesized under conditions of nitrogen sufficiency. Gradually, research was focused on other species, among them Irpex lacteus showing a remarkable pollutant toxicity resistance and biodegradation efficiency. Systematic research has built up knowledge of biochemistry and biotechnological applicability of this fungus, stressing the need to critically summarize and estimate these scattered data. The review attempts to evaluate the information on I. lacteus focusing on various enzyme activities and bioremediation of organopollutants in water and soil environments, with the aim of mediating this knowledge to a broader microbiological audience.

Estrogenic and androgenic activity of PCBs, their chlorinated metabolites and other endocrine disruptors estimated with two in vitro yeast assays.

Investigations of environmental pollution by endocrine-disrupting chemicals are now in progress. Up to now, several in vitro bioassays have been developed for evaluation of the endocrine disruptive activity; however, there is still a lack of comparative studies of their sensitivity. In this work comparison of the estrogen screening assay based on beta-galactosidase expression and a bioluminescent estrogen screen revealed differences in the sensitivity and specificity of the two tests. With the beta-galactosidase screen a slight estrogen-like activity of Delor 103, a commercial mixture of PCB congeners, and a fungicide triclosan was measured whereas no activity was detected using the bioluminescent assay. A bioluminescent androgen test negated previously suggested androgenic potential of triclosan. Further, this work demonstrates the androgenic activity of Delor 103, with an EC(50) value of 2.29 x 10(-2)mg/L. On the other hand, chlorobenzoic acids (CBAs), representing potential PCB degradation metabolites, exhibited no androgenic activity but were slightly estrogenic. Their estrogenicity varied with their chemical structure, with 2,3-CBA, 2,3,6-CBA, 2,4,6-CBA and monochlorinated compounds exhibiting the highest activity. Thus the results indicated possible transitions of the hormonal activity of PCBs during bacterial degradation.

Biodegradation of methyl tert-butyl ether using bacterial strains.

Prospective methyl tert-butyl ether (MTBE) degrading bacterial strains and/or consortia were identified. The potential for aerobic degradation of MTBE was examined using bacterial isolates from contaminated soils and groundwater. Using the 16S rDNA protocol, two isolates capable of degrading MTBE (Rhodococcus pyridinivorans 4A and Achromobacter xylosoxidans 6A) were identified. The most efficient consortium of microorganisms was acquired from contaminated groundwater. The growth of both strains and the consortium on MTBE was supported by various organic substrates, and monitored using Bioscreen. The biochemical oxygen demand of the cultures was measured using OxiTop, and their MTBE concentrations were estimated by gas chromatography. After 3 weeks of aerobic cultivation using n-alkanes as cosubstrate, the concentration of MTBE in R. pyridinivorans 4A was reduced to 62.4 % of its initial amount (50 ppm).

Degradation of PAHs by ligninolytic enzymes of Irpex lacteus.

The ligninolytic fungus Irpex lacteus was shown as an efficient degrader of oligocyclic aromatic hydrocarbons (PAHs; 'polycyclic aromatic hydrocarbons') possessing 3-6 aromatic rings in complex liquid media. The strain produced mainly Mn-dependent peroxidase in media without pollutants. Activity of ligninolytic enzymes was higher in a N-limited medium. However, after contamination with PAHs (especially pyrene) the values increased and significant activity of Mn-independent peroxidase appeared in the complex medium. Other factors (such as the increase in nitrogen concentration or the presence of solvent(s) for dissolution of PAHs) had no effect. Cytochrome P-450 was detected in the microsomal fraction of biomass grown in the complex medium. The rate of PAH degradation was also affected by the presence of various combinations of PAHs. However, independently of the enzyme activities, anthracene was shown to have a positive influence on degradation of pyrene and fluoranthene.

Compost-mediated removal of polycyclic aromatic hydrocarbons from contaminated soil.

Compost-assisted remediation of a manufactured-gas plant soil contaminated with polycyclic aromatic hydrocarbons (PAHs) was performed in thermally insulated composting chamber using mushroom compost consisting wheat straw, chicken manure, and gypsum. The degradation of individual PAHs was in range of 20-60% at the end of 54 days of composting followed by further increase of PAH removal (37-80%) after another 100 days of maturation. Both chemical analysis of the contaminated soil for PAHs and ecotoxicity tests on bioluminescent bacteria, earthworms, and plant seeds were performed before and after the composting. After the composting, inhibition of bioluminescence decreased, whereas no significant change in toxicity was observed for earthworm survival and seed germination. Using bacterial culture of Escherichia coli K12 genotoxicity tests were performed on samples taken from different parts of the composting pile; after the composting the decrease in genotoxicity was observed only in the sample taken from upper part of the composted pile.

Mycoremediation of PAH-contaminated soil.

Out of a number of white-rot fungal cultures, strains of Irpex lacteus and Pleurotus ostreatus were selected for degradation of 7 three- and four-ring unsubstituted aromatic hydrocarbons (PAH) in two contaminated industrial soils. Respective data for removal of PAH in the two industrial soils by I. lacteus were: fluorene (41 and 67%), phenanthrene (20 and 56%), anthracene (29 and 49%), fluoranthene (29 and 57%), pyrene (24 and 42%), chrysene (16 and 32%) and benzo[a]anthracene (13 and 20%). In the same two industrial soils P. ostreatus degraded the PAH with respective removal figures of fluorene (26 and 35%), phenanthrene (0 and 20%), anthracene (19 and 53%), fluoranthene (29 and 31%), pyrene (22 and 42%), chrysene (0 and 42%) and benzo[a]anthracene (0 and 13%). The degradation of PAH was determined against concentration of PAH in non-treated contaminated soils after 14 weeks of incubation. The fungal degradation of PAH in soil was studied simultaneously with ecotoxicity evaluation of fungal treated and non-treated contaminated soils. Compared to non-treated contaminated soil, fungus-treated soil samples indicated decrease in inhibition of bioluminescence in luminescent bacteria (Vibrio fischerii) and increase in germinated mustard (Brassica alba) seeds.

Bioremediation of PAH-contaminated soil by composting: a case study.

Composting technique was used for bioremediation of industrial soil originating from a former tar-contaminated site. The composting process was regulated by aeration to keep optimal temperature gradient and concentrations of O2 and CO2 inside the composting pile. The efficiency of bioremediation was evaluated by performing analysis of 11 individual three- to six-ring unsubstituted aromatic hydrocarbons (PAH) and estimating of changes in ecotoxicity of the contaminated soil. After 42 d of composting, PAH with 3-4 rings were removed from 42 to 68%, other higher-molar mass PAH from 35 to 57%. Additional 100 d of compost maturation in open-air field did not result in a further decrease of PAH. Ecotoxicity tests performed with bioluminescent bacteria Vibrio fischerii showed a decrease in toxicity both after composting and maturation phases. However, toxicity tests on mustard-seed germination did not reveal any significant changes during composting and maturation phases.

Hydrocarbon deposition and soil microflora as affected by highway traffic.

The proximity of a busy highway (90,000 vehicles/day) increased the amount of polycyclic aromatic hydrocarbons (PAHs) in soil at the depth of 5-15 cm from 106 ng/g as a grassland background to 3095 ng/g dry soil at the highway verge (a sum of 10 PAH species). The PAH concentration was related to the distance from the source and exhibited a biphasic character, which is interpreted in terms of bimodal distribution of the exhaust microparticles with different rates of deposition. Similarly, the tendency of benz(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, and indeno(1,2,3-cd)pyrene to decrease their proportion with distance from the highway, in contrast to phenanthrene, fluoranthene, pyrene, benzo(a)pyrene, and benzo(g,h,i)perylene, was attributed to their prevalent localisation on the heavier particle fraction. The abundance of bacteria (8.33 x background) and fungi (3.17 x background) close to the highway is thought to be a consequence of hydrocarbon deposition from the traffic that serves as a significant energetic input into the soil. The elevated concentrations of hydrocarbon substrates, as indicated by PAHs, increased both the absolute and relative numbers of the microbial degraders of diesel fuel, biphenyl, naphthalene, and pyrene. Their maximum numbers at 0.5-1.5 m from the pavement reached 1.3 x 10(4), 1.2 x 10(5), 1.1 x 10(4), and 6.6 x 10(3) colony-forming units (CFU) or infection units per gramme dry soil, respectively. On the other hand, the number of anthracene degraders (1.1 x 10(3) CFU per g dry soil) remained close to the detection limit of the enumeration technique used (0.1-0.2 x 10(3) per g dry soil), consistently with the absence of anthracene and higher linear PAHs in the investigated soil samples. The amounts of persisting PAHs justify artificial inoculation with effective degrader strains in the vicinity of motorways.

Irpex lacteus, a white rot fungus applicable to water and soil bioremediation.

Growth parameters, ligninolytic enzyme activities and ability to degrade polycyclic aromatic hydrocarbons by the fungus Irpex lacteus were characterized and compared with those of other white rot fungi capable of rapid decolorization of poly R-478 and Remazol Brilliant Blue R dyes. I. lacteus was able to grow on mineral and complex media and efficiently colonized sterile and non-sterile soil by exploratory mycelium growing from a wheat straw inoculum. In shallow stationary cultures growing on high nitrogen mineral medium containing 45 mM ammonium as nitrogen source, the fungus produced lignin peroxidase (LIP), Mn-dependent peroxidase (MnP) and laccase simultaneously, the respective maximal activities of 70, 970 and 36 U/l being attained around day 18. Growing in nitrogen-limited medium (2.4 mM ammonium), no LIP was formed and levels of MnP and laccase decreased significantly. During growth in sterile soil, the fungus synthesized LIP and laccase but not MnP. I. lacteus efficiently removed three- and four-ringed PAHs from liquid media and artificially spiked soil. The variety of ligninolytic enzymes, robust growth, capability of soil colonization and resistance to inhibitory action of soil bacteria make I. lacteus a suitable fungal organism for use in bioremediation.

Extracellular oxidative enzyme production and PAH removal in soil by exploratory mycelium of white rot fungi.

Selected strains of three species of white rot fungi, Pleurotus ostreatus, Phanerochaete chrysosporium and Trametes versicolor, were grown in sterilized soil from straw inocula. The respective colonization rates and mycelium density values decreased in the above mentioned order. Three- and four-ringed PAHs at 50 ppm inhibited growth of fungi in soil to some extent. The activities of fungal MnP and laccase (units per g dry weight of straw or soil), extracted with 50 mM succinate-lactate buffer (pH 4.5), were 5 to 20-fold higher in straw compared to soil. The enzyme activities per g dry soil in P. ostreatus and T. versicolor were similar, in contrast to P. chrysosporium, where they were extremely low. Compared to the aerated controls, P. ostreatus strains reduced the levels of anthracene, pyrene and phenanthrene by 81-87%, 84-93% and 41-64% within 2 months, respectively. During degradation of anthracene, all P. ostreatus strains accumulated anthraquinone. PAH removal rates in P. chrysosporium and T. versicolor soil cultures were much lower.