The origin of aerobic methanotrophy within the Proteobacteria.

Aerobic methanotrophs play critical roles in the global carbon cycle, but despite their environmental ubiquity, they are phylogenetically restricted. Via bioinformatic analyses, it is shown that methanotrophy likely arose from methylotrophy from the lateral gene transfer of either of the two known forms of methane monooxygenase (particulate and soluble methane monooxygenases). Moreover, it appears that both known forms of pyrroloquinoline quinone-dependent methanol dehydrogenase (MeDH) found in methanotrophs-the calcium-containing Mxa-MeDH and the rare earth element-containing Xox-MeDH-were likely encoded in the genomes before the acquisition of the methane monooxygenases (MMOs), but that some methanotrophs subsequently received an additional copy of Xox-MeDH-encoding genes via lateral gene transfer. Further, data are presented that indicate the evolution of methanotrophy from methylotrophy not only required lateral transfer of genes encoding for methane monooxygenases, but also likely the pre-existence of a means of collecting copper. Given the emerging interest in valorizing methane via biological platforms, it is recommended that future strategies for heterologous expression of methane monooxygenase for conversion of methane to methanol also include cloning of genes encoding mechanism(s) of copper uptake, especially for expression of particulate methane monooxygenase.

Production of electrically-conductive nanoscale filaments by sulfate-reducing bacteria in the microbial fuel cell.

This study reports that the obligate anaerobic microorganism, Desulfovibrio desulfuricans, a predominant sulfate-reducing bacterium (SRB) in soils and sediments, can produce nanoscale bacterial appendages for extracellular electron transfer. These nanofilaments were electrically-conductive (5.81S·m(-1)) and allowed SRBs to directly colonize the surface of insoluble or solid electron acceptors. Thus, the direct extracellular electron transfer to the insoluble electrode in the microbial fuel cell (MFC) was possible without inorganic electron-shuttling mediators. The production of nanofilaments was stimulated when only insoluble electron acceptors were available for cellular respiration. These results suggest that when availability of a soluble electron acceptor for SRBs (SO4(2-)) is limited, D. desulfuricans initiates the production of conductive nanofilaments as an alternative strategy to transfer electrons to insoluble electron acceptors. The findings of this study contribute to understanding of the role of SRBs in the biotransformation of various substances in soils and sediments and in the MFC.

Enzymatic degradation of aromatic hydrocarbon intermediates using a recombinant dioxygenase immobilized onto surfactant-activated carbon nanotube.

This study examined the enzymatic decomposition of aromatic hydrocarbon intermediates (catechol, 4-chlorocatechol, and 3-methylcatechol) using a dioxygenase immobilized onto single-walled carbon nanotube (SWCNT). The surfaces of SWCNTs were activated with surfactants. The dioxygenase was obtained by recombinant technique: the corresponding gene was cloned from Arthrobacter chlorophenolicus A6, and the enzyme was overexpressed and purified subsequently. The enzyme immobilization yield was 62%, and the high level of enzyme activity was preserved (60-79%) after enzyme immobilization. Kinetic analyses showed that the substrate utilization rates and the catalytic efficiencies of the immobilized enzyme for all substrates (target aromatic hydrocarbon intermediates) tested were similar to those of the free enzyme, indicating that the loss of enzyme activity was minimal during enzyme immobilization. The immobilized enzyme was more stable than the free enzyme against abrupt changes in pH, temperature, and ionic strength. Moreover, it retained high enzyme activity even after repetitive use.

Noncovalent and covalent immobilization of oxygenase on single-walled carbon nanotube for enzymatic decomposition of aromatic hydrocarbon intermediates.

The decomposition of various aromatic hydrocarbon intermediates was examined using a recombinant oxidative enzyme immobilized on single-walled carbon nanotubes (SWCNTs). Hydroxyquinol 1,2-dioxygenase (CphA-I), which catalyzes ring cleavage of catechol and its analogues, was obtained from Arthrobacter chlorophenolicus A6 via cloning, overexpression, and subsequent purification. This recombinant enzyme was immobilized on SWCNTs by physical adsorption and covalent coupling in the absence and presence of N-hydroxysuccinimide. The immobilization yield was as high as 52.1%, and a high level of enzyme activity of up to 64.7% was preserved after immobilization. Kinetic analysis showed that the substrate utilization rates (vmax) and catalytic efficiencies (kcat/KM) of the immobilized enzyme for all substrates evaluated were similar to those of the free enzyme, indicating minimal loss of enzyme activity during immobilization. The immobilized enzyme was more stable toward extreme pH, temperature, and ionic strength conditions than the free enzyme. Thus, the oxidative enzyme immobilized on SWCNTs can be used as an effective and stable biocatalyst for the biochemical remediation process if further investigations would be carried out under field conditions.

Evaluation of soil flushing of complex contaminated soil: an experimental and modeling simulation study.

The removal of heavy metals (Zn and Pb) and heavy petroleum oils (HPOs) from a soil with complex contamination was examined by soil flushing. Desorption and transport behaviors of the complex contaminants were assessed by batch and continuous flow reactor experiments and through modeling simulations. Flushing a one-dimensional flow column packed with complex contaminated soil sequentially with citric acid then a surfactant resulted in the removal of 85.6% of Zn, 62% of Pb, and 31.6% of HPO. The desorption distribution coefficients, KUbatch and KLbatch, converged to constant values as Ce increased. An equilibrium model (ADR) and nonequilibrium models (TSNE and TRNE) were used to predict the desorption and transport of complex contaminants. The nonequilibrium models demonstrated better fits with the experimental values obtained from the column test than the equilibrium model. The ranges of KUbatch and KLbatch were very close to those of KUfit and KLfit determined from model simulations. The parameters (R, ß, ω, α, and f) determined from model simulations were useful for characterizing the transport of contaminants within the soil matrix. The results of this study provide useful information for the operational parameters of the flushing process for soils with complex contamination.

Enhanced current production by Desulfovibrio desulfuricans biofilm in a mediator-less microbial fuel cell.

In this study, a mediator-less microbial fuel cell (MFC) inoculated with a sulfate-reducing bacterium (SBR), Desulfovibrio desulfuricans, was equipped with bare and surface-treated graphite felt electrodes. Electrochemical treatment of the anode surface facilitated biofilm formation on the electrode, resulting in rapid and enhanced current production. The maximum current density of the treated anode was 233±24.2mA/m(2), which was 41% higher than that of the untreated anode. The electron transfer rate also increased from 2.45±0.04 to 3.0±0.02µmol of electrons/mg of protein·min. Biofilm formation on the treated anode was mainly due to the strong hydrogen or peptide bonds between the amide groups of bacterial materials (including cytochrome c) and carboxyl groups formed on the electrodes. These results provide useful information on direct electron transfer by SRB in a mediator-less MFC through cytochrome c and the effects of the electrochemical treatment of electrodes on MFC performance.

Effective biochemical decomposition of chlorinated aromatic hydrocarbons with a biocatalyst immobilized on a natural enzyme support.

The enzymatic decomposition of 4-chlorophenol metabolites using an immobilized biocatalyst was investigated in this study. Catechol 1,2-dioxygenase for ortho ring cleavage obtained via cloning of the corresponding gene cphA-I from Arthrobacter chlorophenolicus A6 was overexpressed and purified. It was found that the cphA-I enzyme could catalyze the degradation of catechol, 4-chlorocatechol, and 3-methylcatechol. The expressed enzyme was immobilized onto a natural enzyme support, fulvic acid-activated montmorillonite. The immobilization yield was as high as 63%, and the immobilized enzyme maintained high substrate utilization activity, with only a 15-24% reduction in the specific activity. Kinetic analysis demonstrated marginal differences in νmax and KM values for the free and immobilized enzymes, indicating that inactivation of the immobilized enzyme was minimal. The immobilized enzyme exhibited notably increased stability against changes in the surrounding environment (temperature, pH, and ionic strength). Our results provide useful information for the effective enzymatic biochemical treatment of hazardous organic compounds.

Gene delivery properties of end-modified poly(beta-amino ester)s.

Here, we present the synthesis of a library of end-modified poly(beta-amino ester)s and assess their utility as gene delivery vehicles. Polymers were synthesized using a rapid, two-step approach that involves initial preparation of an acrylate-terminated polymer followed by a postpolymerization amine-capping step to generate end-functionalized polymers. Using a highly efficient poly(beta-amino ester), C32, we show that the terminal amine can greatly affect and improve polymer properties relevant to gene delivery. Specifically, the in vitro transfection levels can be increased by 30% and the optimal polymer:DNA ratio lowered 5-fold by conjugation of the appropriate end group. The most effective modifications were made by grafting primary diamine molecules to the chain termini. The added charge and hydrophobicity of some derivatives enhanced DNA binding and resulted in the formation of polymer-DNA complexes less than 100 nm in diameter. In addition, cellular uptake was improved 5-fold over unmodified C32. The end-modified poly(beta-amino ester)s presented here are some of the most effective gene-delivery polycations, superior to polyethylenimine and previously reported poly(beta-amino ester)s. These results show that the end-modification of poly(beta-amino ester)s is a general strategy to alter functionality and improve the delivery performance of these materials.