Transformation of hydroxylated polychlorinated biphenyls by bacterial 2-hydroxybiphenyl 3-monooxygenase.

Monohydroxylated PCBs (OH-PCBs) are an (eco)toxicologically significant group of compounds, as they arise from the oxidation of polychlorinated biphenyls (PCBs) and, at the same time, may exert even more severe toxic effects than their parent PCB molecules. Despite having been widely detected in environmental samples, plants, and animals, information on the fate of OH-PCBs in the environment is scarce, including on the enzymatic machinery behind their degradation. To date, only a few bacterial taxa capable of OH-PCB transformation have been reported. In this study, we aimed to obtain a deeper insight into the transformation of OH-PCBs in soil bacteria and isolated a Pseudomonas sp. strain P1B16 based on its ability to use o-phenylphenol (2-PP) which, when exposed to the Delor 103-derived OH-PCB mixture, depleted a wide spectrum of mono-, di, and trichlorinated OH-PCBs. In the P1B16 genome, a region designated as hbp was identified, which bears a set of putative genes involved in the transformation of OH-PCBs, namely hbpA encoding for a putative flavin-dependent 2-hydroxybiphenyl monooxygenase, hbpC (2,3-dihydroxybiphenyl-1,2-dioxygenase), hbpD (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase), and the transcriptional activator-encoding gene hbpR. The hbpA coding sequence was heterologously expressed, purified, and its substrate specificity was investigated towards the Delor 103-derived OH-PCB mixture, individual OH-PCBs, and multiple (chlorinated) phenolics. Apart from 2-PP and 2-chlorophenol, HbpA was also demonstrated to transform a range of OH-PCBs, including a 3-hydroxy-2,2',4',5,5'-pentachlorobiphenyl. Importantly, this is the first direct evidence of HbpA homologs being involved in the degradation of OH-PCBs. Moreover, using a P1B16-based biosensor strain, the specific induction of hbp genes by 2-PP, 3-phenylphenol, 4-phenylphenol, and the OH-PCB mixture was demonstrated. This study provides direct evidence on the specific enzymatic machinery responsible for the transformation of OH-PCBs in bacteria, with many implications in ecotoxicology, environmental restoration, and microbial ecology in habitats burdened with PCB contamination.

Recent advances in PCB removal from historically contaminated environmental matrices.

Despite being drastically restricted in the 1970s, polychlorinated biphenyls (PCBs) still belong among the most hazardous contaminants. The chemical stability and dielectric properties of PCBs made them suitable for a number of applications, which then lead to their ubiquitous presence in the environment. PCBs are highly bioaccumulative and persistent, and their teratogenic, carcinogenic, and endocrine-disrupting features have been widely reported in the literature. This review discusses recent advances in different techniques and approaches to remediate historically contaminated matrices, which are one of the most problematic in regard to decontamination feasibility and efficiency. The current knowledge published in the literature shows that PCBs are not sufficiently removed from the environment by natural processes, and thus, the suitability of some approaches (e.g., natural attenuation) is limited. Physicochemical processes are still the most effective; however, their extensive use is constrained by their high cost and often their destructiveness toward the matrices. Despite their limited reliability, biological methods and their application in combinations with other techniques could be promising. The literature reviewed in this paper documents that a combination of techniques differing in their principles should be a future research direction. Other aspects discussed in this work include the incompleteness of some studies. More attention should be given to the evaluation of toxicity during these processes, particularly in terms of monitoring different modes of toxic action. In addition, decomposition mechanisms and products need to be sufficiently clarified before combined, tailor-made approaches can be employed.

Predominant Biphenyl Dioxygenase From Legacy Polychlorinated Biphenyl (PCB)-Contaminated Soil Is a Part of Unusual Gene Cluster and Transforms Flavone and Flavanone.

In this study, the diversity of bphA genes was assessed in a 13C-enriched metagenome upon stable isotope probing (SIP) of microbial populations in legacy PCB-contaminated soil with 13C-biphenyl (BP). In total, 13 bphA sequence variants (SVs) were identified in the final amplicon dataset. Of these, one SV comprised 59% of all sequences, and when it was translated into a protein sequence, it exhibited 87, 77.4, and 76.7% identity to its homologs from Pseudomonas furukawaii KF707, Cupriavidus sp. WS, and Pseudomonas alcaliphila B-367, respectively. This same BphA sequence also contained unusual amino acid residues, Alanine, Valine, and Serine in region III, which had been reported to be crucial for the substrate specificity of the corresponding biphenyl dioxygenase (BPDO), and was accordingly designated BphA_AVS. The DNA locus of 18 kbp containing the BphA_AVS-coding sequence retrieved from the metagenome was comprised of 16 ORFs and was most likely borne by Paraburkholderia sp. The BPDO corresponding to bphAE_AVS was cloned and heterologously expressed in E. coli, and its substrate specificity toward PCBs and a spectrum of flavonoids was assessed. Although depleting a rather narrow spectrum of PCB congeners, the efficient transformation of flavone and flavanone was demonstrated through dihydroxylation of the B-ring of the molecules. The homology-based functional assignment of the putative proteins encoded by the rest of ORFs in the AVS region suggests their potential involvement in the transformation of aromatic compounds, such as flavonoids. In conclusion, this study contributes to the body of information on the involvement of soil-borne BPDOs in the metabolism of flavonoid compounds, and our paper provides a more advanced context for understanding the interactions between plants, microbes and anthropogenic compounds in the soil.

Degradation Products of Polychlorinated Biphenyls and Their In Vitro Transformation by Ligninolytic Fungi.

Metabolites of polychlorinated biphenyls (PCBs)-hydroxylated PCBs (OH-PCBs), chlorobenzyl alcohols (CB-OHs), and chlorobenzaldehydes (CB-CHOs)-were incubated in vitro with the extracellular liquid of Pleurotus ostreatus, which contains mainly laccase and low manganese-dependent peroxidase (MnP) activity. The enzymes were able to decrease the amount of most of the tested OH-PCBs by > 80% within 1 h; the removal of more recalcitrant OH-PCBs was greatly enhanced by the addition of the laccase mediator syringaldehyde. Conversely, glutathione substantially hindered the reaction, suggesting that it acted as a laccase inhibitor. Hydroxylated dibenzofuran and chlorobenzoic acid were identified as transformation products of OH-PCBs. The extracellular enzymes also oxidized the CB-OHs to the corresponding CB-CHOs on the order of hours to days; however, the mediated and nonmediated setups exhibited only slight differences, and the participating enzymes could not be determined. When CB-CHOs were used as the substrates, only partial transformation was observed. In an additional experiment, the extracellular liquid of Irpex lacteus, which contains predominantly MnP, was able to efficiently transform CB-CHOs with the aid of glutathione; mono- and di-chloroacetophenones were detected as transformation products. These results demonstrate that extracellular enzymes of ligninolytic fungi can act on a wide range of PCB metabolites, emphasizing their potential for bioremediation.

The sensitivity of multiple ecotoxicological assays for evaluating Microcystis aeruginosa cellular algal organic matter and contribution of cyanotoxins to the toxicity.

Secondary metabolites of cyanobacteria and algae released during algal blooms often exhibit toxic effects, but only a small number of the metabolites are the subject of routine analytical screenings. Alternatively, ecotoxicological assays offer a better representation of the overall negative effects. The aim of this work was to compare multiple assays in their sensitivity towards cellular algal organic matter (COM) of the toxin-producing cyanobacterium Microcystis aeruginosa. Multiple endpoints were investigated: mortality, growth inhibition, bioluminescence inhibition, genotoxicity, endocrine-disrupting effects, oxidative stress, and the induction of ethoxyresorufin-O-deethylase (EROD). Three rainbow trout (Oncorhynchus mykiss) cell lines as well as representatives of bacteria, yeasts, algae, vascular plants, and crustaceans were employed, and the results were expressed per mg of dissolved organic carbon (DOC) in the COM. M. aeruginosa COM was toxic to the RTgill-W1, RTG-2, and RTL-W1 cell lines (EC50 values ranging from 0.48 ± 0.02 to 1.9 ± 0.1 mgDOC/L), to the crustacean Thamnocephalus platyurus (LC50 = 20 ± 1 mgDOC/L), and to Lepidium sativum (IC50 = 241 ± 13 mgDOC/L). In contrast, no effect was observed for bacteria and yeasts, and the growth of the alga Desmodesmus subspicatus was even stimulated. No genotoxicity, endocrine-disrupting effects or increase in oxidative stress or EROD activity was detected. The content of six microcystins (MC-LR, MC-RR, MC-YR, MC-LY, MC-LW, and MC-LF), anatoxin-a, cylindrospermopsin, and nodularin in the M. aeruginosa COM was determined by liquid chromatography-tandem mass spectrometry. An artificially prepared mixture of the detected cyanotoxins in the corresponding concentrations did not induce response in the O. mykiss cell lines and T. platyurus, suggesting that other cyanobacterial metabolites are responsible for the toxicity of M. aeruginosa.

Biodegradability of Dental Care Antimicrobial Agents Chlorhexidine and Octenidine by Ligninolytic Fungi.

Chlorhexidine (CHX) and octenidine (OCT), antimicrobial compounds used in oral care products (toothpastes and mouthwashes), were recently revealed to interfere with human sex hormone receptor pathways. Experiments employing model organisms-white-rot fungi Irpex lacteus and Pleurotus ostreatus-were carried out in order to investigate the biodegradability of these endocrine-disrupting compounds and the capability of the fungi and their extracellular enzyme apparatuses to biodegrade CHX and OCT. Up to 70% ± 6% of CHX was eliminated in comparison with a heat-killed control after 21 days of in vivo incubation. An additional in vitro experiment confirmed manganese-dependent peroxidase and laccase are partially responsible for the removal of CHX. Up to 48% ± 7% of OCT was removed in the same in vivo experiment, but the strong sorption of OCT on fungal biomass prevented a clear evaluation of the involvement of the fungi or extracellular enzymes. On the other hand, metabolites indicating the enzymatic transformation of both CHX and OCT were detected and their chemical structures were proposed by means of liquid chromatography-mass spectrometry. Complete biodegradation by the ligninolytic fungi was not achieved for any of the studied analytes, which emphasizes their recalcitrant character with low possibility to be removed from the environment.

Biodegradation of PCBs in contaminated water using spent oyster mushroom substrate and a trickle-bed bioreactor.

Due to their persistence, polychlorinated biphenyls (PCBs) represent a group of important environmental pollutants, but conventional physicochemical decontamination techniques for their removal are usually expensive. The main aim of this work was to develop a cost-effective method for PCB bioremediation, focusing on contaminated water and utilizing the well-known degradation capability of Pleurotus ostreatus (the oyster mushroom). For this purpose, the conditions of several laboratory-scale reactors (working volume 1 L) were optimized. Spent oyster mushroom substrate obtained from a commercial farm was used as a fungal inoculum and growth substrate. The highest degradation efficiency (87%) was recorded with a continuous low-flow setup, which was subsequently scaled up (working volume 500 L) and used for the treatment of 4000 L of real contaminated groundwater containing 0.1-1 µg/L of PCBs. This trickle-bed pilot-scale bioreactor was able to remove 82, 80, 65, and 30-50% of di-, tri-, tetra- and pentachlorinated PCB congeners, respectively. No degradation was observed for hexa- or heptachlorinated congeners. Multiple mono- and dichlorobenzoic acids (CBAs) were identified as transformation products by mass spectrometry, confirming the role of biodegradation in PCB removal. A Vibrio fischeri bioluminescence inhibition test revealed slight ecotoxicity of the primary reactor effluent (sampling after 24 h), which was quickly suppressed once the effluent passed through the reactor for the second time. Moreover, no other effluent exhibited toxicity for the rest of the experiment (71 days in total). Microbial analyses (phospholipid fatty acid analysis and next-generation sequencing) showed that P. ostreatus was able to degrade PCBs in the presence of an abundance of other fungal species as well as aerobic and anaerobic bacteria. Overall, this study proved the suitability of the use of spent oyster mushroom substrate in a bioremediation practice, even for pollutants as recalcitrant as PCBs.

Method for analysis of psychopharmaceuticals in real industrial wastewater and groundwater with suspended organic particulate matter using solid phase extraction disks extraction and ultra-high performance liquid chromatography/time-of-flight mass spectrometry.

A rapid and reliable analytical method was developed for the quantitative determination of psychopharmaceuticals, their precursors and by-products in real contaminated samples from a pharmaceutical company in Olomouc (Czech Republic), based on SPE disk extraction and detection by ultra performance liquid chromatography, combined with time-of-flight mass spectrometry. The target compounds were quantified in the real whole-water samples (water including suspended particles), both in the presence of suspended particulate matter (SPM) and high concentrations of other organic pollutants. A total of nine compounds were analyzed which consisted of three commonly used antidepressants (tricyclic antidepressants and antipsychotics), one antitussive agent and five by-products or precursors. At first, the SPE disk method was developed for the extraction of water samples (dissolved analytes, recovery 84-104%) and pressurised liquid extraction technique was verified for solid matrices (sludge samples, recovery 81-95%). In order to evaluate the SPE disk technique for whole water samples containing SPM, non contaminated groundwater samples were also loaded with different amounts (100 and 300mgL(-1)) of real contaminated sludge originating from the same locality. The recoveries from the whole-water samples obtained by SPE disk method ranged between 67 and 119% after the addition of the most contaminated sludge. The final method was applied to several real groundwater (whole-water) samples from the industrial area and high concentrations (up to 10(3)µgL(-1)) of the target compounds were detected. The results of this study document and indicate the feasibility of the SPE disk method for analysis of groundwater.