Blanka Vrchotova

Phytoremediation of Polychlorinated Biphenyls

MARTINA MACKOVA, DIANE BARRIAULT, KATERINA FRANCOVA, MICHEL SYLVESTRE, MONIKA MODER, BLANKA VRCHOTOVA, PETRA LOVECKA, JITKA NAJMANOVA, KATERINA DEMNEROVA, MARTINA NOVAKOVA, JAN REZEK AND TOMAS MACEK 1 Dept. Biochemistry and Microbiology., Faculty of Food and Biochemical Technology, ICT Prague, Technicka 3, Prague, 166 28 Czech Republic, 2 Dept. of Natural Products, Institute of Organic Chemistry and Biochemistry, Academy of Science of the Czech Republic, Flemingovo n. 2, 166 10 Prague, Czech Republic, E-mail: tom.macek@uochb.cas.cz Institut National de la Recherche Scientifique, INRS-IAF, 245 Boul. Hymus, Pointe-Claire, Quebec, Canada, H9R 1G6, 4 Laboratory of Analytical Chemistry, UFZ Leipzig-Halle, Permoserstrasse 17, Leipzig, Germany, 4

Pseudomonads Rule Degradation of Polyaromatic Hydrocarbons in Aerated Sediment.

Given that the degradation of aromatic pollutants in anaerobic environments such as sediment is generally very slow, aeration could be an efficient bioremediation option. Using stable isotope probing (SIP) coupled with pyrosequencing analysis of 16S rRNA genes, we identified naphthalene-utilizing populations in aerated polyaromatic hydrocarbon (PAH)-polluted sediment. The results showed that naphthalene was metabolized at both 10 and 20°C following oxygen delivery, with increased degradation at 20°C as compared to 10°C—a temperature more similar to that found in situ. Naphthalene-derived 13C was primarily assimilated by pseudomonads. Additionally, Stenotrophomonas, Acidovorax, Comamonas, and other minor taxa were determined to incorporate 13C throughout the measured time course. The majority of SIP-detected bacteria were also isolated in pure cultures, which facilitated more reliable identification of naphthalene-utilizing populations as well as proper differentiation between primary consumers and cross-feeders. The pseudomonads acquiring the majority of carbon were identified as Pseudomonas veronii and Pseudomonas gessardii. Stenotrophomonads and Acidovorax defluvii, however, were identified as cross-feeders unable to directly utilize naphthalene as a growth substrate. PAH degradation assays with the isolated bacteria revealed that all pseudomonads as well as Comamonas testosteroni degraded acenaphthene, fluorene, and phenanthrene in addition to naphthalene. Furthermore, P. veronii and C. testosteroni were capable of transforming anthracene, fluoranthene, and pyrene. Screening of isolates for naphthalene dioxygenase genes using a set of in-house designed primers for Gram-negative bacteria revealed the presence of such genes in pseudomonads and C. testosteroni. Overall, our results indicated an apparent dominance of pseudomonads in the sequestration of carbon from naphthalene and potential degradation of other PAHs upon aeration of the sediment at both 20 and 10°C.

Characterization of Transgenic Tobacco Plants Containing Bacterial bphc Gene and Study of Their Phytoremediation Ability

Genetically modified plants can serve as an efficient tool for remediation of diverse dangerous pollutants of the environment such as pesticides, heavy metals, explosives and persistent organic compounds. Transgenic lines of Nicotiana tabacum containing bacterial bphC gene from the degradation pathway of polychlorinated biphenyls (PCBs) were tested. The product of the bphC gene – enzyme 2,3-dihydroxybiphenyl-1,2-dioxygenase is responsible for cleaving of the biphenyl ring. The presence of bphC gene in transgenic plants was detected on DNA, RNA and protein level. The expression of the bphC/His gene was verified after purification of the enzyme from plants by affinity chromatography followed by a Western blot and immunochemical assay. The enzyme activity of isolated protein was detected.Efficient transformation of 2,3-DHB by transgenic plants was achieved and the lines also exhibited high production of biomass. The transgenic plants were more tolerant to the commercial PCBs mixture Delor 103 than non-transgenic tobacco. And finally, the higher decrease of total PCB content and especially congener 28 in real contaminated soil from a dumpsite was determined after cultivation of transgenic plant in comparison with non-transgenic tobacco. The substrate specificity of transgenic plants was the same as substrate specificity of BphC enzyme.

Organochlorinated pesticide degrading microorganisms isolated from contaminated soil.

Degradation of selected organochlorinated pesticides (γ-hexachlorocyclohexane - γ-HCH, dichlorodiphenyltrichloroethane - DDT, hexachlorobenzene - HCB) by soil microorganisms was studied. Bacterial strains isolated from contaminated soil from Klatovy-Luby, Hajek and Neratovice, Czech Republic, capable of growth on the selected pesticides were isolated and characterised. These isolates were subjected to characterisation and identification by MS MALDI-TOF of whole cells and sequence analysis of 16S rRNA genes. The isolates were screened by gas chromatography for their ability to degrade the selected pesticides. Some isolates were able to degrade pesticides, and the formation of degradation products (γ-pentachlorocyclohexane (γ-PCCH), dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD)) observed in liquid culture confirmed their degradation capability. The isolates and DNA samples isolated from the contaminated soil were also screened for the bphA1 gene (encoding biphenyl-2,3-dioxygenase, the first enzyme in the PCB degradation pathway) and its occurrence was demonstrated. The isolates were also screened for the presence of linA, encoding dehydrochlorinase, the first enzyme of the HCH degradation pathway. The linA gene could not be found in any of the tested isolates, possibly due to the high specificity of the primers used. The isolates with the most effective degradation abilities could be used for further in situ bioremediation experiments with contaminated soil.

Genomic damage induced in tobacco plants by chlorobenzoic acids—Metabolic products of polychlorinated biphenyls

Tobacco seedlings (Nicotiana tabacum var. xanthi) were treated for 24 h with mono-(2- and 3-CBA), di-(2,5- and 3,4-CBA), and tri-(2,4,6- and 2,3,5-CBA)-chlorobenzoic acids (CBAs) and with the mixture of polychlorinated biphenyls--Delor 103, or cultivated for 1 or 2 weeks in soil polluted with the CBAs. DNA damage in nuclei of leaves and roots was evaluated by the comet assay. A significant increase in DNA damage was observed only at concentrations of CBAs that caused withering of leaves or had lethal effects within 2-4 weeks after the treatments. As the application of CBAs did not induce somatic mutations, the induced DNA migration is probably caused by necrotic DNA fragmentation and not by DNA damage resulting in genetic alteration. In contrast, the application of the monofunctional alkylating agent ethyl methanesulphonate as a positive control resulted in a dose-response increase of DNA damage and an increase of somatic mutations. Thus, the EMS-produced DNA migration is probably associated with genotoxin-induced DNA fragmentation. The data demonstrate that the comet assay in plants should be conducted together with toxicity studies to distinguish between necrotic and genotoxin-induced DNA fragmentation. The content of 2,5-CBA in tobacco seedlings was measured by reverse-phase high pressure liquid chromatography.

Influence of Root Exudates on the Bacterial Degradation of Chlorobenzoic Acids

Degradation of chlorobenzoic acids (e.g., products of microbial degradation of PCB) by strains of microorganisms isolated from PCB contaminated soils was assessed. From seven bulk-soil isolates two strains unique in ability to degrade a wider range of chlorobenzoic acids than others were selected, individually and even in a complex mixture of 11 different chlorobenzoic acids. Such a feature is lacking in most tested degraders. To investigate the influence of vegetation on chlorobenzoic acids degraders, root exudates of two plant species known for supporting PCB degradation in soil were tested. While with individual chlorobenzoic acids the presence of plant exudates leads to a decrease of degradation yield, in case of a mixture of chlorobenzoic acids either a change in bacterial degradation specificity, associated with 3- and 4-chlorobenzoic acid, or an extension of the spectrum of degraded chlorobenzoic acids was observed.

Characterizing Biochar as Alternative Sorbent for Oil Spill Remediation

Biochar (BC) was characterized as a new carbonaceous material for the adsorption of toluene from water. The tested BC was produced from pine wood gasification, and its sorption ability was compared with that of more common carbonaceous materials such as activated carbon (AC). Both materials were characterized in terms of textural features and sorption abilities by kinetic and equilibrium tests. AC and BC showed high toluene removal from water. Kinetic tests demonstrated that BC is characterized by faster toluene removal than AC is. Textural features demonstrated that the porosity of AC is double that of BC. Nevertheless, equilibrium tests demonstrated that the sorption ability of BC is comparable with that of AC, so the materials’ porosity is not the only parameter that drives toluene adsorption. The specific adsorption ability (mg sorbed m−2 of surface) of the BC is higher than that of AC: toluene is more highly sorbed onto the biochar surface. Biochar is furthermore obtained from biomaterial thermally treated for making energy; this also makes the use of BC economically and environmentally convenient compared with AC, which, as a manufactured material, must be obtained in selected conditions for this type of application.

Native Phytoremediation Potential of Urtica dioica for Removal of PCBs and Heavy Metals Can Be Improved by Genetic Manipulations Using Constitutive CaMV 35S Promoter.

Although stinging nettle (Urtica dioica) has been shown to reduce HM (heavy metal) content in soil, its wider phytoremediation potential has been neglected. Urtica dioica was cultivated in soils contaminated with HMs or polychlorinated biphenyls (PCBs). After four months, up to 33% of the less chlorinated biphenyls and 8% of HMs (Zn, Pb, Cd) had been removed. Bacteria were isolated from the plant tissue, with the endophytic bacteria Bacillus shackletonii and Streptomyces badius shown to have the most significant effect. These bacteria demonstrated not only benefits for plant growth, but also extreme tolerance to As, Zn and Pb. Despite these results, the native phytoremediation potential of nettles could be improved by biotechnologies. Transient expression was used to investigate the functionality of the most common constitutive promoter, CaMV 35S in Urtica dioica. This showed the expression of the CUP and bphC transgenes. Collectively, our findings suggest that remediation by stinging nettle could have a much wider range of applications than previously thought.

Bioremediation of Chlorobenzoic Acids

Chlorobenzoic acids (CBAs) can be released into the environment from many different sources. One possible source of CBAs is usage as herbicides or insecticides in agriculture. As a herbicide was used 2,3,6-CBA [1]. CBAs may also be formed as intermediates during the degradation of same herbicides. Namely 2,6-CBA is formed as an intermediate during microbial degradation of dichlobenil [2], 2,5-CBA in the chlorambene degradation [3] or 4-CBA is a final degradation product of the insecticide DDT [4].