Dechlorination of Four Commercial Polychlorinated Biphenyl Mixtures (Aroclors) by Anaerobic Microorganisms from Sediments

The rate, extent, and pattern of dechlorination of four Aroclors by inocula prepared from two polychlorinated biphenyl (PCB)-contaminated sediments were compared. The four mixtures used, Aroclors 1242, 1248, 1254, and 1260, average approximately three, four, five, and six chlorines, respectively, per biphenyl molecule. All four Aroclors were dechlorinated with the loss of meta plus para chlorines ranging from 15 to 85%. Microorganisms from an Aroclor 1242-contaminated site in the upper Hudson River dechlorinated Aroclor 1242 to a greater extent than did microorganisms from Aroclor 1260-contaminated sediments from Silver Lake, Mass. The Silver Lake inoculum dechlorinated Aroclor 1260 more rapidly than the Hudson River inoculum did and showed a preferential removal of meta chlorines. For each inoculum the rate and extent of dechlorination tended to decrease as the degree of chlorination of the Aroclor increased, especially for Aroclor 1260. The maximal observed dechlorination rates were 0.3, 0.3, and 0.2 μg-atoms of Cl removed per g of sediment per week for Aroclors 1242, 1248, and 1254, respectively. The maximal observed dechlorination rates for Hudson River and Silver Lake organisms for Aroclor 1260 were 0.04 and 0.21 μg-atoms of Cl removed per g of sediment per week, respectively. The dechlorination patterns obtained suggested that the Hudson River microorganisms were more capable than the Silver Lake organisms of removing the last para chlorine. These results suggest that there are different PCB-dechlorinating microorganisms at different sites, with characteristic specificities for PCB dechlorination

Functional genes to assess nitrogen cycling and aromatic hydrocarbon degradation: primers and processing matter

Targeting sequencing to genes involved in key environmental processes, i.e. ecofunctional genes, provides an opportunity to sample nature’s gene guilds to greater depth and help link community structure to process-level outcomes. Vastly different approaches have been implemented for sequence processing and, ultimately, for taxonomic placement of these gene reads. The overall quality of next generation sequence analysis of functional genes is dependent on multiple steps and assumptions of unknown diversity. To illustrate current issues surrounding amplicon read processing we provide examples for three ecofunctional gene groups. A combination of in-silico, environmental and cultured strain sequences was used to test new primers targeting the dioxin and dibenzofuran degrading genes dxnA1, dbfA1, and carAa. The majority of obtained environmental sequences were classified into novel sequence clusters, illustrating the discovery value of the approach. For the nitrite reductase step in denitrification, the well-known nirK primers exhibited deficiencies in reference database coverage, illustrating the need to refine primer-binding sites and/or to design multiple primers, while nirS primers exhibited bias against five phyla. Amino acid-based OTU clustering of these two N-cycle genes from soil samples yielded only 114 unique nirK and 45 unique nirS genus-level groupings, likely a reflection of constricted primer coverage. Finally, supervised and non-supervised OTU analysis methods were compared using the nifH gene of nitrogen fixation, with generally similar outcomes, but the clustering (non-supervised) method yielded higher diversity estimates and stronger site-based differences. High throughput amplicon sequencing can provide inexpensive and rapid access to nature’s related sequences by circumventing the culturing barrier, but each unique gene requires individual considerations in terms of primer design and sequence processing and classification

Review, Evaluation, and Directions for Gene-Targeted Assembly for Ecological Analyses of Metagenomes

Shotgun metagenomics has greatly advanced our understanding of microbial communities over the last decade. Metagenomic analyses often include assembly and genome binning, computationally daunting tasks especially for big data from complex environments such as soil and sediments. In many studies, however, only a subset of genes and pathways involved in specific functions are of interest; thus, it is not necessary to attempt global assembly. In addition, methods that target genes can be computationally more efficient and produce more accurate assembly by leveraging rich databases, especially for those genes that are of broad interest such as those involved in biogeochemical cycles, biodegradation, and antibiotic resistance or used as phylogenetic markers. Here, we review six gene-targeted assemblers with unique algorithms for extracting and/or assembling targeted genes: Xander, MegaGTA, SAT-Assembler, HMM-GRASPx, GenSeed-HMM, and MEGAN. We tested these tools using two datasets with known genomes, a synthetic community of artificial reads derived from the genomes of 17 bacteria, shotgun sequence data from a mock community with 48 bacteria and 16 archaea genomes, and a large soil shotgun metagenomic dataset. We compared assemblies of a universal single copy gene (rplB) and two N cycle genes (nifH and nirK). We measured their computational efficiency, sensitivity, specificity, and chimera rate and found Xander and MegaGTA, which both use a probabilistic graph structure to model the genes, have the best overall performance with all three datasets, although MEGAN, a reference matching assembler, had better sensitivity with synthetic and mock community members chosen from its reference collection. Also, Xander and MegaGTA are the only tools that include post-assembly scripts tuned for common molecular ecology and diversity analyses. Additionally, we provide a mathematical model for estimating the probability of assembling targeted genes in a metagenome for estimating required sequencing depth

Degradation of Aroclor 1242 Dechlorination Products in Sediments by Burkholderia xenovorans LB400(ohb) and Rhodococcus sp. Strain RHA1(fcb)

Burkholderia xenovorans strain LB400, which possesses the biphenyl pathway, was engineered to contain the oxygenolytic ortho dehalogenation (ohb) operon, allowing it to grow on 2-chlorobenzoate and to completely mineralize 2-chlorobiphenyl. A two-stage anaerobic/aerobic biotreatment process for Aroclor 1242-contaminated sediment was simulated, and the degradation activities and genetic stabilities of LB400(ohb) and the previously constructed strain RHA1(fcb), capable of growth on 4-chlorobenzoate, were monitored during the aerobic phase. The population dynamics of both strains were also followed by selective plating and real-time PCR, with comparable results; populations of both recombinants increased in the contaminated sediment. Inoculation at different cell densities (10(4) or 10(6) cells g(−1) sediment) did not affect the extent of polychlorinated biphenyl (PCB) biodegradation. After 30 days, PCB removal rates for high and low inoculation densities were 57% and 54%, respectively, during the aerobic phase