Microbial biodegradation and biotransformation reactions are essential to most bioremediation processes, yet the specific organisms, genes, and mechanisms involved are often not well understood. Stable isotope probing (SIP) enables researchers to directly link microbial metabolic capability to phylogenetic and metagenomic information within a community context by tracking isotopically labeled substances into phylogenetically and functionally informative biomarkers. SIP is thus applicable as a tool for the identification of active members of the microbial community and associated genes integral to the community functional potential, such as biodegradative processes. The rapid evolution of SIP over the last decade and integration with metagenomics provide researchers with a much deeper insight into potential biodegradative genes, processes, and applications, thereby enabling an improved mechanistic understanding that can facilitate advances in the field of bioremediation.

• MeSH
• Biodegradation, Environmental * MeSH
• Phylogeny MeSH
• Isotope Labeling methods   MeSH
• Environmental Pollutants metabolism   MeSH
• Metagenomics methods   trends   MeSH
• Carbon metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Environmental Pollutants MeSH
• Carbon MeSH

Bacteria were identified associated with biodegradation of aromatic pollutants biphenyl, benzoate, and naphthalene in a long-term polychlorinated biphenyl- and polyaromatic hydrocarbon-contaminated soil. In order to avoid biases of culture-based approaches, stable isotope probing was applied in combination with sequence analysis of 16 S rRNA gene pyrotags amplified from (13)C-enriched DNA fractions. Special attention was paid to pyrosequencing data analysis in order to eliminate the errors caused by either generation of amplicons (random errors caused by DNA polymerase, formation of chimeric sequences) or sequencing itself. Therefore, sample DNA was amplified, sequenced, and analyzed along with the DNA of a mock community constructed out of 8 bacterial strains. This warranted that appropriate tools and parameters were chosen for sequence data processing. (13)C-labeled metagenomes isolated after the incubation of soil samples with all three studied aromatics were largely dominated by Proteobacteria, namely sequences clustering with the genera Rhodanobacter Burkholderia, Pandoraea, Dyella as well as some Rudaea- and Skermanella-related ones. Pseudomonads were mostly labeled by (13)C from naphthalene and benzoate. The results of this study show that many biphenyl/benzoate-assimilating bacteria derive carbon also from naphthalene, pointing out broader biodegradation abilities of some soil microbiota. The results also demonstrate that, in addition to traditionally isolated genera of degradative bacteria, yet-to-be cultured bacteria are important players in bioremediation. Overall, the study contributes to our understanding of biodegradation processes in contaminated soil. At the same time our results show the importance of sequencing and analyzing a mock community in order to more correctly process and analyze sequence data.

• MeSH
• Bacteria genetics   isolation & purification   metabolism   MeSH
• Benzoates metabolism   MeSH
• Biphenyl Compounds metabolism   MeSH
• Biodegradation, Environmental MeSH
• DNA, Bacterial metabolism   MeSH
• Phylogeny MeSH
• Isotope Labeling MeSH
• Carbon Isotopes MeSH
• Soil Pollutants analysis   MeSH
• Naphthalenes metabolism   MeSH
• Soil Microbiology * MeSH
• Base Sequence MeSH
• Sequence Analysis, DNA MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Benzoates MeSH
• Biphenyl Compounds MeSH
• biphenyl MeSH Browser
• DNA, Bacterial MeSH
• Carbon Isotopes MeSH
• Soil Pollutants MeSH
• Naphthalenes MeSH
• naphthalene MeSH Browser

Bacteria that are able to utilize biphenyl as a sole source of carbon were extracted and isolated from polychlorinated biphenyl (PCB)-contaminated soil vegetated by horseradish. Isolates were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The usage of MALDI Biotyper for the classification of isolates was evaluated and compared to 16S rRNA gene sequence analysis. A wide spectrum of bacteria was isolated, with Arthrobacter, Serratia, Rhodococcus, and Rhizobium being predominant. Arthrobacter isolates also represented the most diverse group. The use of MALDI Biotyper in many cases permitted the identification at the level of species, which was not achieved by 16S rRNA gene sequence analyses. However, some isolates had to be identified by 16S rRNA gene analyses if MALDI Biotyper-based identification was at the level of probable or not reliable identification, usually due to a lack of reference spectra included in the database. Overall, this study shows the possibility of using MALDI-TOF MS and MALDI Biotyper for the fast and relatively nonlaborious identification/classification of soil isolates. At the same time, it demonstrates the dominant role of employing 16S rRNA gene analyses for the identification of recently isolated strains that can later fill the gaps in the protein-based identification databases.

• MeSH
• Armoracia MeSH
• Bacteria chemistry   classification   genetics   isolation & purification   MeSH
• Biphenyl Compounds metabolism   MeSH
• Biodiversity MeSH
• DNA, Bacterial chemistry   genetics   MeSH
• Soil Pollutants metabolism   MeSH
• Molecular Sequence Data MeSH
• Soil Microbiology * MeSH
• Rhizosphere MeSH
• DNA, Ribosomal chemistry   genetics   MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Sequence Analysis, DNA MeSH
• Cluster Analysis MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods   MeSH
• Bacterial Typing Techniques methods   MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Biphenyl Compounds MeSH
• biphenyl MeSH Browser
• DNA, Bacterial MeSH
• Soil Pollutants MeSH
• DNA, Ribosomal MeSH
• RNA, Ribosomal, 16S MeSH

The aim of this work was to construct transgenic plants with increased capabilities to degrade organic pollutants, such as polychlorinated biphenyls. The environmentally important gene of bacterial dioxygenase, the bphC gene, was chosen to clone into a plant of Nicotiana tabacum. The chosen bphC gene encodes 2,3-dihydroxybiphenyl-1,2-dioxygenase, which cleaves the aromatic ring of dihydroxybiphenyl, and we cloned it in fusion with the gene for β-glucuronidase (GUS), luciferase (LUC) or with a histidine tail. Several genetic constructs were designed and prepared and the possible expression of desired proteins in tobacco plants was studied by transient expression. We used genetic constructs successfully expressing dioxygenase's genes we used for preparation of transgenic tobacco plants by agrobacterial infection. The presence of transgenic DNA , mRNA and protein was determined in parental and the first filial generation of transgenic plants with the bphC gene. Properties of prepared transgenic plants will be further studied.

• Keywords
• 2,3-dihydroxybiphenyl-1,2-dioxygenase, Nicotiana tabacum, bphC, phytoremediation, transgenic plant,
• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Biodegradation, Environmental MeSH
• Burkholderiaceae enzymology   genetics   MeSH
• Dioxygenases genetics   metabolism   MeSH
• Plants, Genetically Modified enzymology   genetics   metabolism   MeSH
• Glucuronidase genetics   metabolism   MeSH
• Cloning, Molecular MeSH
• Luciferases genetics   metabolism   MeSH
• Polychlorinated Biphenyls metabolism   MeSH
• Recombinant Fusion Proteins genetics   metabolism   MeSH
• Nicotiana enzymology   genetics   metabolism   MeSH
• Green Fluorescent Proteins genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 2,3-dihydroxybiphenyl oxygenase MeSH Browser
• Bacterial Proteins MeSH
• Dioxygenases MeSH
• Glucuronidase MeSH
• Luciferases MeSH
• Polychlorinated Biphenyls MeSH
• Recombinant Fusion Proteins MeSH
• Green Fluorescent Proteins MeSH

PURPOSE: Polychlorinated biphenyls (PCBs) represent a large group of recalcitrant environmental pollutants, differing in the number of chlorine atoms bound to biphenyl ring. Due to their excellent technological properties, PCBs were used as heat-transfer media, for filling transformers and condensers, as paint additives, etc. With increasing knowledge of their toxicity, transfer to food chains and accumulation in living organisms, their production ended in most countries in the 1970s and in 1984 in the former Czechoslovakia. But even a quarter of century after the PCB production ceased, from contaminated areas, the volatile PCBs evaporate and contaminate much larger areas even at very distant parts of the world. For this reason, PCBs still represent a global problem. The main method of PCB removal from contaminated environment is at present the expensive incineration at high temperatures. With the aim of finding effective alternative approaches, we are studying biological methods for PCB removal from the environment. In this paper, we summarise 10 years of studies using long-term PCB-contaminated soil from a dumpsite in South Bohemia, targeted for the use of plants (phytoremediation) and their cooperation with microorganisms in the root zone (rhizoremediation). MATERIALS AND METHODS: Long-term contaminated soil from Lhenice dumpsite, more than hundred kilograms of homogenised material, was used in microcosms (pots and buckets), and field plots were established at the site. Tested plants include among others tobacco, black nightshade, horseradish, alfalfa and willow. Aseptic plant cell and tissue cultures were from the collection of the IOCB. Microorganisms were our own isolates. The paper summarises experiments done between 1998 and 2008 with real contaminated soil, both vegetated and non-vegetated. PCB analysis was performed by GC-ECD, metabolic products identified mostly using 2D-GC/MS-MS and synthetic standards, whereas molecular methods included quantitative PCR and sequencing. RESULTS: The soil was used both for preparation of field plots at the site and for greenhouse and laboratory tests in microcosms. The results include analyses of changes in PCB content in untreated and vegetated soil, PCB uptake and distribution in different parts of various plant species, analysis of products formed, identification and characterisation of cultivable and non-cultivable bacteria both in rhizosphere and in bulk soil. Different treatments and amendments were also tested. Experiments in real contaminated soil were accompanied by in vitro experiments using aseptic cultures of plant biomass, genetically modified (GM) plants and bacteria, to allow identification of players responsible for PCB metabolisation in soil. The time-span of the experiments allows extrapolating some of the results and drawing conclusions concerning the effectivity of exploitation of various plant species and treatments to remove PCBs from soils. DISCUSSION: The approach using plants proved to represent a viable alternative to costly incineration of PCB-contaminated soils. The recent studies using molecular methods show that plants are responsible for the composition of consortia of microorganisms present in their root zone, including those with ability to degrade the chlorinated aromatic compounds. CONCLUSIONS: In addition to uptake, accumulation and partial metabolisation of PCBs by plants, compounds produced by plants allow survival of microorganisms even in poor soils, serve as carbon and energy source, and can even induce the degradation pathways of different xenobiotics. Thus, the choice of proper plant species is crucial for effective cleaning of different polluted sites. Our study shows how the efficiency of PCB removal is dependent on the plant used. RECOMMENDATIONS AND PERSPECTIVES: The use of plants in biological remediation of different organic xenobiotics proved to be a useful approach. Further improvement can be expected by application of specifically tailored GM plants and use of selective conditions ensuring high remediation potential based on optimal composition of the soil microbial consortia designed for the needs of given site.

• MeSH
• Biodegradation, Environmental * MeSH
• Time Factors MeSH
• Plant Roots MeSH
• Soil Pollutants metabolism   MeSH
• Polychlorinated Biphenyls chemistry   metabolism   MeSH
• Soil analysis   MeSH
• Soil Microbiology MeSH
• Plants metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Soil Pollutants MeSH
• Polychlorinated Biphenyls MeSH
• Soil MeSH

DNA-based stable isotope probing in combination with terminal restriction fragment length polymorphism was used in order to identify members of the microbial community that metabolize biphenyl in the rhizosphere of horseradish (Armoracia rusticana) cultivated in soil contaminated with polychlorinated biphenyls (PCBs) compared to members of the microbial community in initial, uncultivated bulk soil. On the basis of early and recurrent detection of their 16S rRNA genes in clone libraries constructed from [(13)C]DNA, Hydrogenophaga spp. appeared to dominate biphenyl catabolism in the horseradish rhizosphere soil, whereas Paenibacillus spp. were the predominant biphenyl-utilizing bacteria in the initial bulk soil. Other bacteria found to derive carbon from biphenyl in this nutrient-amended microcosm-based study belonged mostly to the class Betaproteobacteria and were identified as Achromobacter spp., Variovorax spp., Methylovorus spp., or Methylophilus spp. Some bacteria that were unclassified at the genus level were also detected, and these bacteria may be members of undescribed genera. The deduced amino acid sequences of the biphenyl dioxygenase alpha subunits (BphA) from bacteria that incorporated [(13)C]into DNA in 3-day incubations of the soils with [(13)C]biphenyl are almost identical to that of Pseudomonas alcaligenes B-357. This suggests that the spectrum of the PCB congeners that can be degraded by these enzymes may be similar to that of strain B-357. These results demonstrate that altering the soil environment can result in the participation of different bacteria in the metabolism of biphenyl.

• MeSH
• Armoracia microbiology   MeSH
• Bacteria classification   genetics   isolation & purification   metabolism   MeSH
• Genes, Bacterial MeSH
• RNA, Bacterial genetics   MeSH
• Betaproteobacteria classification   genetics   isolation & purification   metabolism   MeSH
• Biphenyl Compounds metabolism   MeSH
• DNA, Bacterial genetics   MeSH
• DNA Primers genetics   MeSH
• Phylogeny MeSH
• Carbon Isotopes MeSH
• Soil Pollutants metabolism   MeSH
• Molecular Sequence Data MeSH
• Polychlorinated Biphenyls metabolism   MeSH
• Soil Microbiology * MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Base Sequence MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Bacterial MeSH
• Biphenyl Compounds MeSH
• biphenyl MeSH Browser
• DNA, Bacterial MeSH
• DNA Primers MeSH
• Carbon Isotopes MeSH
• Soil Pollutants MeSH
• Polychlorinated Biphenyls MeSH
• RNA, Ribosomal, 16S MeSH