A global survey reveals a divergent extradiol dioxygenase clade as a widespread complementary contributor to the biodegradation of mono- and polycyclic aromatic hydrocarbons.

Extradiol dioxygenation is a key reaction in the microbial aerobic degradation of mono- and polycyclic aromatic hydrocarbon catecholic derivatives. It has been reported that many bacterial enzymes exhibiting such converging functions act on a wide range of catecholic substrates. The present study reports a new subfamily of extradiol dioxygenases (EXDOs) with broad substrate specificity, the HrbC EXDOs. The new clade belongs to the XII cluster within family 2 of the vicinal oxygen chelate superfamily (EXDO-VC2), which is typically characterized by a preference for bicyclic substrates. Coding hrbC orthologs were isolated by activity-based screening of fosmid metagenomic libraries from large DNA fragments derived from heavily PAH-contaminated soils. They occurred as solitary genes within conserved sequences encoding enzymes for amino acid metabolism and were stably maintained in the chromosomes of the Betaproteobacteria lineages harboring them. Analysis of contaminated aquifers revealed coexpression of hrbC as a polycistronic mRNA component. The predicted open reading frames were verified by cloning and heterologous expression, confirming the expected molecular mass and meta-cleavage activity of the recombinant enzymes. Evolutionary analysis of the HrbC protein sequences grouped them into a discrete cluster of 1,2-dihydroxynaphthalene dioxygenases represented by a cultured PAH degrader, Rugosibacter aromaticivorans strain Ca6. The ecological importance and relevance of the new EXDO genes were confirmed by PCR-based mapping in different biogeographical localities contaminated with a variety of mono- and polycyclic aromatic compounds. The cosmopolitan distribution of hrbC in PAH-contaminated aquifers supports our hypothesis about its auxiliary role in the degradation of toxic catecholic intermediates, contributing to the composite EXDO catabolic capacity of the world's microbiomes.

Indirect Evidence Link PCB Dehalogenation with Geobacteraceae in Anaerobic Sediment-Free Microcosms.

Although polychlorinated biphenyls (PCBs) production was brought to a halt 30 years ago, recalcitrance to degradation makes them a major environmental pollutant at a global scale. Previous studies confirmed that organohalide-respiring bacteria (OHRB) were capable of utilizing chlorinated congeners as electron acceptor. OHRB belonging to the Phyla Chloroflexi and Firmicutes are nowadays considered as the main PCB-dechlorinating organisms. In this study, we aimed at exploring the involvement of other taxa in PCB dechlorination using sediment-free microcosms (SFMs) and the Delor PCB mixture. High rates of congener dehalogenation (up to 96%) were attained in long-term incubations of up to 692 days. Bacterial communities were dominated by Chloroflexi, Proteobacteria, and Firmicutes, among strictly simplified community structures composed of 12 major phyla only. In a first batch of SFMs, Dehalococcoides mccartyi closely affiliated with strains CG4 and CBDB1 was considered as the main actor associated with congener dehalogenation. Addition of 2-bromoethanesulfonate (BES), a known inhibitor of methanogenic activity in a second batch of SFMs had an adverse effect on the abundance of Dehalococcoides sp. Only two sequences affiliated to this Genus could be detected in two (out of six) BES-treated SFMs, contributing to a mere 0.04% of the communities. BES-treated SFMs showed very different community structures, especially in the contributions of organisms involved in fermentation and syntrophic activities. Indirect evidence provided by both statistical and phylogenetic analysis validated the implication of a new cluster of actors, distantly affiliated with the Family Geobacteraceae (Phylum δ-Proteobacteria), in the dehalogenation of low chlorinated PCB congeners. Members of this Family are known already for their dehalogenation capacity of chlorinated solvents. As a result, the present study widens the knowledge for the phylogenetic reservoir of indigenous PCB dechlorinating taxa.

Divergent PCB organohalide-respiring consortia enriched from the efflux channel of a former Delor manufacturer in Eastern Europe.

Polychlorinated biphenyl (PCB) organohalide-respiring communities from the efflux channel of a former Delor manufacturer in Eastern Slovakia were assessed using metagenomic, statistical and cultivation-adapted approaches. Multivariate analysis of environmental factors together with terminal restriction fragment length polymorphisms of the bacterial communities in the primary sediments revealed both temporal and spatial heterogeneity in the distribution of microbial populations, which reflects the dynamic pattern of contamination and altered conditions for biodegradation activity along the channel. Anaerobic microcosms were developed from eight sediments sampled along the channel, where high concentrations of PCBs - from 6.6 to 136mg/kg dry weight, were measured. PCB dehalorespiring activity, congruent with changes in the microbial composition in all microcosms, was detected. After 10 months of cultivation, the divergently evolved consortia achieved up to 35.9 percent reduction of the total PCB concentration. Phylogenetic-analysis of the active Chloroflexi-related organohalide-respiring bacteria by partial sequencing of 16S rRNA genes in cDNA from microcosms with the highest PCB dechlorination activity revealed diverse and unique complexity of the populations. The predominant organohalide respirers were either affiliated with Dehalococcoides sp. and Dehalococcoides-like group (DLG) organisms or were composed of currently unknown distant clades of DLG bacteria. The present study should encourage researchers to explore the full potential of the indigenous PCB dechlorinating populations to develop effective bioremediation approaches that can perform the complete mineralization of PCBs in polluted environments.

Functional adaptation of microbial communities from jet fuel-contaminated soil under bioremediation treatment: simulation of pollutant rebound.

To investigate the link between the functionality and the diversity of microbial communities under strong selective pressure from pollutants, two types of mesocosms that simulate natural attenuation and phytoremediation were generated using soil from a site highly contaminated with jet fuel and under air-sparging treatment. An increase in the petroleum hydrocarbon concentration from 4900 to 18,500 mg kg(-1) dw soil simulated a pollutant rebound (postremediation pollutant reversal due to residual contamination). Analysis of soil bacterial communities by denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments showed stronger changes and selection for a phylogenetically diverse microbial population in the mesocosms with pollutant-tolerant willow trees. Enumeration of the main subfamilies of catabolic genes characteristic to the site detected a rapid increase in the degradation potential of both systems. A marked increase in the abundance of genes encoding extradiol dioxygenases with a high affinity towards various catecholic substrates was found in the planted mesocosms. The observed adaptive response to the simulated pollutant rebound, characterized by increased catabolic gene abundance, but with different changes in the microbial structure, can be explained by functional redundancy in biodegrading microbial communities.

Metagenomics reveals diversity and abundance of meta-cleavage pathways in microbial communities from soil highly contaminated with jet fuel under air-sparging bioremediation.

The extradiol dioxygenase diversity of a site highly contaminated with aliphatic and aromatic hydrocarbons under air-sparging treatment was assessed by functional screening of a fosmid library in Escherichia coli with catechol as substrate. The 235 positive clones from inserts of DNA extracted from contaminated soil were equivalent to one extradiol dioxygenase-encoding gene per 3.6 Mb of DNA screened, indicating a strong selection for genes encoding this function. Three subfamilies were identified as being predominant, with 72, 55 and 43 fosmid inserts carrying genes, related to those encoding TbuE of Ralstonia pickettii PK01 (EXDO-D), IpbC of Pseudomonas sp. JR1 (EXDO-K2) or DbtC of Burkholderia sp. DBT1 (EXDO-Dbt), respectively, whereas genes encoding enzymes related to XylE of Pseudomonas putida mt-2 were not observed. Genes encoding oxygenases related to isopropylbenzene dioxygenases were usually colocalized with genes encoding EXDO-K2 dioxygenases. Functional analysis of representative proteins indicated a subcluster of EXDO-D proteins to show exceptional high affinity towards different catecholic substrates. Based on V(max)/K(m) specificity constants, a task-sharing between different extradiol dioxygenases in the community of the contaminated site can be supposed, attaining a complementary and community-balanced catalytic power against diverse catecholic derivatives, as necessary for effective degradation of mixtures of aromatics.

Enhancement of the microbial community biomass and diversity during air sparging bioremediation of a soil highly contaminated with kerosene and BTEX.

In order to obtain insights in complexity shifts taking place in natural microbial communities under strong selective pressure, soils from a former air force base in the Czech Republic, highly contaminated with jet fuel and at different stages of a bioremediation air sparging treatment, were analyzed. By tracking phospholipid fatty acids and 16S rRNA genes, a detailed monitoring of the changes in quantities and composition of the microbial communities developed at different stages of the bioventing treatment progress was performed. Depending on the length of the air sparging treatment that led to a significant reduction in the contamination level, we observed a clear shift in the soil microbial community being dominated by Pseudomonads under the harsh conditions of high aromatic contamination to a status of low aromatic concentrations, increased biomass content, and a complex composition with diverse bacterial taxonomical branches.

Alternative primer sets for PCR detection of genotypes involved in bacterial aerobic BTEX degradation: distribution of the genes in BTEX degrading isolates and in subsurface soils of a BTEX contaminated industrial site.

Eight new primer sets were designed for PCR detection of (i) mono-oxygenase and dioxygenase gene sequences involved in initial attack of bacterial aerobic BTEX degradation and of (ii) catechol 2,3-dioxygenase gene sequences responsible for meta-cleavage of the aromatic ring. The new primer sets allowed detection of the corresponding genotypes in soil with a detection limit of 10(3)-10(4) or 10(5)-10(6) gene copies g(-1) soil, assuming one copy of the gene per cell. The primer sets were used in PCR to assess the distribution of the catabolic genes in BTEX degrading bacterial strains and DNA extracts isolated from soils sampled from different locations and depths (vadose, capillary fringe and saturated zone) within a BTEX contaminated site. In both soil DNA and the isolates, tmoA-, xylM- and xylE1-like genes were the most frequently recovered BTEX catabolic genes. xylM and xylE1 were only recovered from material from the contaminated samples while tmoA was detected in material from both the contaminated and non-contaminated samples. The isolates, mainly obtained from the contaminated locations, belonged to the Actinobacteria or Proteobacteria (mainly Pseudomonas). The ability to degrade benzene was the most common BTEX degradation phenotype among them and its distribution was largely congruent with the distribution of the tmoA-like genotype. The presence of tmoA and xylM genes in phylogenetically distant strains indicated the occurrence of horizontal transfer of BTEX catabolic genes in the aquifer. Overall, these results show spatial variation in the composition of the BTEX degradation genes and hence in the type of BTEX degradation activity and pathway, at the examined site. They indicate that bacteria carrying specific pathways and primarily carrying tmoA/xylM/xylE1 genotypes, are being selected upon BTEX contamination.

Dynamics of an oligotrophic bacterial aquifer community during contact with a groundwater plume contaminated with benzene, toluene, ethylbenzene, and xylenes: an in situ mesocosm study.

An in situ mesocosm system was designed to monitor the in situ dynamics of the microbial community in polluted aquifers. The mesocosm system consists of a permeable membrane pocket filled with aquifer material and placed within a polypropylene holder, which is inserted below groundwater level in a monitoring well. After a specific time period, the microcosm is recovered from the well and its bacterial community is analyzed. Using this system, we examined the effect of benzene, toluene, ethylbenzene, and xylene (BTEX) contamination on the response of an aquifer bacterial community by denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA genes and PCR detection of BTEX degradation genes. Mesocosms were filled with nonsterile or sterile aquifer material derived from an uncontaminated area and positioned in a well located in either the uncontaminated area or a nearby contaminated area. In the contaminated area, the bacterial community in the microcosms rapidly evolved into a stable community identical to that in the adjacent aquifer but different from that in the uncontaminated area. At the contaminated location, bacteria with tmoA- and xylM/xylE1-like BTEX catabolic genotypes colonized the aquifer, while at the uncontaminated location only tmoA-like genotypes were detected. The communities in the mesocosms and in the aquifer adjacent to the wells in the contaminated area consisted mainly of Proteobacteria. At the uncontaminated location, Actinobacteria and Proteobacteria were found. Our results indicate that communities with long-term stability in their structures follow the contamination plume and rapidly colonize downstream areas upon contamination.