Flow field dynamics and high ethanol content in gasohol blends enhance BTEX migration and biodegradation in groundwater.

Gasohol spills may easily descend through the soil column down and impact sensitive receptors as contaminants dissolve into the groundwater. Gasoline formulations are commonly blended with ethanol to alleviate environmental and economic issues associated with fossil fuels. However, the amount of ethanol added to gasoline and the groundwater hydraulic regime can significantly affect BTEX plume dynamics and lifespan. In this study, two long-term (5 and 10 years) field-scale gasohol releases with ethanol contents of 85% (E85) and 24% (E24), respectively, were assessed to discern the different dynamics undergone by gasohol blends. Statistical, geochemical, microbiological and trend approaches were employed to estimate the influence of groundwater flow variations on ethanol and dissolved BTEX transport, and the associated biodegradation rates of different gasohol blend spills. Ethanol and BTEX groundwater flow were quantified in terms of breakthrough curve characteristics, plume centroid positions and spreading, source depletion and mass degradation rates. In addition, bromide migration was evaluated to address the contribution of flow-driven dissolution. Results revealed that the high amount of ethanol along with a fast and dynamic flow exerted a flushing behavior that enhanced BTEX dissolution, migration (vertical and horizontal) and concentrations in groundwater. The higher amount of ethanol in E85 enhanced BTEX dissolution (and bioavailability) relative to E24 site and led to faster biodegradation rates, which can be explained by the cosolvency effect and metabolic flux dilution. Therefore, flow field dynamics and high ethanol content in gasohol blends enhance BTEX migration and biodegradation in gasohol-contaminated sites. The balance of these factors is crucial to determine fate and transport of contaminants in field sites. These findings suggest that hydraulic regime should be spatially and temporally characterized to support decisions on appropriate monitoring plan and remedial strategies for gasohol spills.

Ethanol content in different gasohol blend spills influences the decision-making on remediation technologies.

Gasohol blend spills with variable ethanol content exert different electron acceptor demands in groundwater and the distinct dynamics undergone by these blends underscores the need for field-based information to aid decision-making on suitable remediation technologies for each gasohol blend spill. In this study, a comparison of two gasohol releases (E10 (10:90 ethanol and gasoline, v/v) and E25 (25:75 ethanol and gasoline, v/v) under monitored natural attenuation (MNA) and nitrate biostimulation, respectively) was conducted to assess the most effective remediation strategy for each gasohol release. Microbial communities were assessed to support geochemical data as well as to enable the characterization of important population shifts that evolve during biodegradation processes in E25 and E10 field experiments. Results revealed that natural attenuation processes sufficiently supported ethanol and BTEX compounds biodegradation in E10 release, due to the lower biochemical oxygen demand they exert relative to E25 blend. In E25 release, nitrate reduction was largely responsible for BTEX and ethanol biodegradation, as intended. First-order decay constants demonstrated that ethanol degradation rates were similar (p < 0.05) for both remediation technologies (2.05 ±â€¯0.15 and 2.22 ±â€¯0.23, for E25 and E10, respectively) whilst BTEX compounds exhibited different degradation rates (p > 0.05) that were higher for the experiment under MNA (0.33 ±â€¯0.06 and 0.43 ±â€¯0.03, for E25 and E10, respectively). Therefore, ethanol content in different gasohol blends can influence the decision-making on the most suitable remediation technology, as MNA processes can be applied for the remediation of gasohol blends with lower ethanol content (i.e., 10% v/v), once the aquifer geochemical conditions provide a sufficient electron acceptor pool. To the best of our knowledge, this is the first field study to monitor two long-term gasohol releases over various time scales in order to assess feasible remediation technologies for each scenario.

Functional reconstitution of cell-free synthesized purified Kv channels.

The study of ion channel activity and the screening of possible inhibitor molecules require reliable methods for production of active channel proteins, their insertion into artificial membranes and for the measurement of their activity. Here we report on cell-free expression of soluble and active Kv1.1 and Kv1.3 channels and their efficient insertion into liposomes. Two complementary methods for the determination of the electrical activity of the proteoliposome-embedded channels were compared using Kv1.1 as a model system: (1) single channel recordings in droplet interface bilayers (DIB) and (2) measurement of the membrane voltage potential generated by a potassium ion diffusion potential using the voltage-sensitive fluorescent dye oxonol VI. Single channel recordings in DIBs proved unreliable because of the non-reproducible fusion of proteoliposomes with an artificial membrane. Therefore, the use of the optical indicator oxonol VI was adapted for 96 well microtiter plates using the ionophore valinomycin as a positive control. The activity of Kv1.1 and Kv1.3 channels was then monitored in the absence and presence of different venom toxins, demonstrating that fluorescent dyes can be used very efficiently when screening small molecules for their channel blocking activity.

A ring-shaped conduit connects the mother cell and forespore during sporulation in Bacillus subtilis.

During spore formation in Bacillus subtilis a transenvelope complex is assembled across the double membrane that separates the mother cell and forespore. This complex (called the "A-Q complex") is required to maintain forespore development and is composed of proteins with remote homology to components of type II, III, and IV secretion systems found in Gram-negative bacteria. Here, we show that one of these proteins, SpoIIIAG, which has remote homology to ring-forming proteins found in type III secretion systems, assembles into an oligomeric ring in the periplasmic-like space between the two membranes. Three-dimensional reconstruction of images generated by cryo-electron microscopy indicates that the SpoIIIAG ring has a cup-and-saucer architecture with a 6-nm central pore. Structural modeling of SpoIIIAG generated a 24-member ring with dimensions similar to those of the EM-derived saucer. Point mutations in the predicted oligomeric interface disrupted ring formation in vitro and impaired forespore gene expression and efficient spore formation in vivo. Taken together, our data provide strong support for the model in which the A-Q transenvelope complex contains a conduit that connects the mother cell and forespore. We propose that a set of stacked rings spans the intermembrane space, as has been found for type III secretion systems.

Biodiesel presence in the source zone hinders aromatic hydrocarbons attenuation in a B20-contaminated groundwater.

The behavior of biodiesel blend spills have received limited attention in spite of the increasing and widespread introduction of biodiesel to the transportation fuel matrix. In this work, a controlled field release of biodiesel B20 (100L of 20:80 v/v soybean biodiesel and diesel) was monitored over 6.2years to assess the behavior and natural attenuation of constituents of major concern (e.g., BTEX (benzene, toluene, ethyl-benzene and xylenes) and PAHs (polycyclic aromatic hydrocarbons)) in a sandy aquifer material. Biodiesel was preferentially biodegraded compared to diesel aromatic compounds with a concomitant increase in acetate, methane (near saturation limit (≈22mgL-1)) and dissolved BTEX and PAH concentrations in the source zone during the first 1.5 to 2.0years after the release. Benzene and benzo(a)pyrene concentrations remained above regulatory limits in the source zone until the end of the experiment (6.2years after the release). Compared to a previous adjacent 100-L release of ethanol-amended gasoline, biodiesel/diesel blend release resulted in a shorter BTEX plume, but with higher residual dissolved hydrocarbon concentrations near the source zone. This was attributed to greater persistence of viscous (and less mobile) biodiesel than the highly-soluble and mobile ethanol in the source zone. This persistence of biodiesel/diesel NAPL at the source zone slowed BTEX and PAH biodegradation (by the establishment of an anaerobic zone) but reduced the plume length by reducing mobility. This is the first field study to assess biodiesel/diesel blend (B20) behavior in groundwater and its effects on the biodegradation and plume length of priority groundwater pollutants.

Influência do biodiesel de soja na biodegradação anaeróbia do benzeno e tolueno / Soybean biodiesel effects on anaerobic biodegradation of benzene and toluene

A influência do biodiesel de soja na biodegradação dos hidrocarbonetos monoaromáticos benzeno e tolueno foi estudada sob condições anaeróbias em dois microcosmos montados com água subterrânea sintética, inóculo metanogênico, benzeno, tolueno e biodiesel. Na presença de biodiesel não foi observada biodegradação do benzeno e do tolueno. Com a biodegradação do biodiesel ocorreu a formação de acetato e metano, uso do sulfato e um aumento de 45 vezes no número de bactérias sulfato-redutoras. Esses resultados mostraram que, na mistura com benzeno e tolueno, o biodiesel foi biodegradado tanto sob condições de sulfato-redução quanto metanogênicas e que sua presença estimulou o crescimento da biomassa.

The effects of biodiesel on the biodegradation of benzene and toluene under anaerobic conditions were assessed using two microcosms constructed using synthetic groundwater, methanogenic inoculum and amended with benzene, toluene and biodiesel. In the presence of biodiesel, benzene and toluene degradation was substantially inhibited. Biodiesel degradation was followed by a production of acetate and methane, consumption of sulfate and a 45-fold increase in sulfate-reducing concentration. These results demonstrated that, in the presence of benzene and toluene, biodiesel was biodegraded under sulfate-reduction and methanogenic conditions and also stimulated biomass growth.

Nitrate addition to groundwater impacted by ethanol-blended fuel accelerates ethanol removal and mitigates the associated metabolic flux dilution and inhibition of BTEX biodegradation.

A comparison of two controlled ethanol-blended fuel releases under monitored natural attenuation (MNA) versus nitrate biostimulation (NB) illustrates the potential benefits of augmenting the electron acceptor pool with nitrate to accelerate ethanol removal and thus mitigate its inhibitory effects on BTEX biodegradation. Groundwater concentrations of ethanol and BTEX were measured 2 m downgradient of the source zones. In both field experiments, initial source-zone BTEX concentrations represented less than 5% of the dissolved total organic carbon (TOC) associated with the release, and measurable BTEX degradation occurred only after the ethanol fraction in the multicomponent substrate mixture decreased sharply. However, ethanol removal was faster in the nitrate amended plot (1.4 years) than under natural attenuation conditions (3.0 years), which led to faster BTEX degradation. This reflects, in part, that an abundant substrate (ethanol) can dilute the metabolic flux of target pollutants (BTEX) whose biodegradation rate eventually increases with its relative abundance after ethanol is preferentially consumed. The fate and transport of ethanol and benzene were accurately simulated in both releases using RT3D with our general substrate interaction module (GSIM) that considers metabolic flux dilution. Since source zone benzene concentrations are relatively low compared to those of ethanol (or its degradation byproduct, acetate), our simulations imply that the initial focus of cleanup efforts (after free-product recovery) should be to stimulate the degradation of ethanol (e.g., by nitrate addition) to decrease its fraction in the mixture and speed up BTEX biodegradation.

Rational design of enhanced photoresistance in a photoswitchable fluorescent protein.

Fluorescent proteins are particularly susceptible to photobleaching, the permanent loss of fluorescence emission resulting from photodestruction of the chromophore. In the case of Reversibly Switchable Fluorescent Proteins (RSFPs), which can be switched back and forth between a non-fluorescent and a fluorescent state, the achievable number of switching cycles is limited by photobleaching, a process known as photofatigue. Photofatigue has become a crucial limitation in a number of advanced applications based on repeated photoswitching of RSFPs, notably in the field of super-resolution fluorescence microscopy. Here, based on our previous structural investigation of photobleaching mechanisms in IrisFP, an RSFP also capable of green-to-red photoconversion, we present the rational design of a single-mutant IrisFP-M159A that displays considerably enhanced photostability. The results suggest that, under moderate illumination intensities, photobleaching of IrisFP-like Anthozoan fluorescent proteins such as EosFP, Dendra or Dronpa derivatives is mainly driven by an oxygen-dependent mechanism resulting in the irreversible sulfoxidation of methionine 159. The photofatigue decay profiles of IrisFP and its photoresistant mutant IrisFP-M159A were investigated in different experimental conditions, in vitro and in cellulo. Although the performance of the mutant was found to be always superior, the results showed switching behaviors strongly dependent on the nanoenvironment. Thus, in general, assessment of photostability and switching properties of RSFPs should be carried out in real experimental conditions.

Effects of nitrate injection on microbial enhanced oil recovery and oilfield reservoir souring.

Column experiments were utilized to investigate the effects of nitrate injection on sulfate-reducing bacteria (SRB) inhibition and microbial enhanced oil recovery (MEOR). An indigenous microbial consortium collected from the produced water of a Brazilian offshore field was used as inoculum. The presence of 150 mg/L volatile fatty acids (VFA´s) in the injection water contributed to a high biological electron acceptors demand and the establishment of anaerobic sulfate-reducing conditions. Continuous injection of nitrate (up to 25 mg/L) for 90 days did not inhibit souring. Contrariwise, in nitrogen-limiting conditions, the addition of nitrate stimulated the proliferation of δ-Proteobacteria (including SRB) and the associated sulfide concentration. Denitrification-specific nirK or nirS genes were not detected. A sharp decrease in water interfacial tension (from 20.8 to 14.5 mN/m) observed concomitantly with nitrate consumption and increased oil recovery (4.3 % v/v) demonstrated the benefits of nitrate injection on MEOR. Overall, the results support the notion that the addition of nitrate, at this particular oil reservoir, can benefit MEOR by stimulating the proliferation of fortuitous biosurfactant-producing bacteria. Higher nitrate concentrations exceeding the stoichiometric volatile fatty acid (VFA) biodegradation demands and/or the use of alternative biogenic souring control strategies may be necessary to warrant effective SRB inhibition down gradient from the injection wells.

Assessment of microbial communities associated with fermentative-methanogenic biodegradation of aromatic hydrocarbons in groundwater contaminated with a biodiesel blend (B20).

A controlled field experiment was conducted to assess the potential for fermentative-methanogenic biostimulation (by ammonium-acetate injection) to enhance biodegradation of benzene, toluene, ethylbenzene and xylenes (BTEX) as well as polycyclic aromatic hydrocarbons (PAHs) in groundwater contaminated with biodiesel B20 (20:80 v/v soybean biodiesel and diesel). Changes in microbial community structure were assessed by pyrosequencing 16S rRNA analyses. BTEX and PAH removal began 0.7 year following the release, concomitantly with the increase in the relative abundance of Desulfitobacterium and Geobacter spp. (from 5 to 52.7 % and 15.8 to 37.3 % of total Bacteria 16S rRNA, respectively), which are known to anaerobically degrade hydrocarbons. The accumulation of anaerobic metabolites acetate and hydrogen that could hinder the thermodynamic feasibility of BTEX and PAH biotransformations under fermentative/methanogenic conditions was apparently alleviated by the growing predominance of Methanosarcina. This suggests the importance of microbial population shifts that enrich microorganisms capable of interacting syntrophically to enhance the feasibility of fermentative-methanogenic bioremediation of biodiesel blend releases.

Activity of ceftaroline against Enterococcus faecium PBP5.

The opportunistic human pathogen Enterococcus faecium overproduces the low-affinity PBP5. In clinical strains, mutations in PBP5 further reduce its acylation rate by ß-lactams. Previous studies have reported that ceftaroline had poor inhibitory activity against ß-lactam-resistant E. faecium strains. In this study, we show that ceftaroline exhibits killing activity against our laboratory-derived ampicillin-resistant E. faecium mutant that overproduces a wild-type PBP5 and that ceftaroline inactivates PBP5 much faster than benzylpenicillin and faster than ceftobiprole.

Biostimulation of anaerobic BTEX biodegradation under fermentative methanogenic conditions at source-zone groundwater contaminated with a biodiesel blend (B20).

Field experiments were conducted to assess the potential for anaerobic biostimulation to enhance BTEX biodegradation under fermentative methanogenic conditions in groundwater impacted by a biodiesel blend (B20, consisting of 20 % v/v biodiesel and 80 % v/v diesel). B20 (100 L) was released at each of two plots through an area of 1 m(2) that was excavated down to the water table, 1.6 m below ground surface. One release was biostimulated with ammonium acetate, which was added weekly through injection wells near the source zone over 15 months. The other release was not biostimulated and served as a baseline control simulating natural attenuation. Ammonium acetate addition stimulated the development of strongly anaerobic conditions, as indicated by near-saturation methane concentrations. BTEX removal began within 8 months in the biostimulated source zone, but not in the natural attenuation control, where BTEX concentrations were still increasing (due to source dissolution) 2 years after the release. Phylogenetic analysis using quantitative PCR indicated an increase in concentration and relative abundance of Archaea (Crenarchaeota and Euryarchaeota), Geobacteraceae (Geobacter and Pelobacter spp.) and sulfate-reducing bacteria (Desulfovibrio, Desulfomicrobium, Desulfuromusa, and Desulfuromonas) in the biostimulated plot relative to the control. Apparently, biostimulation fortuitously enhanced the growth of putative anaerobic BTEX degraders and associated commensal microorganisms that consume acetate and H2, and enhance the thermodynamic feasibility of BTEX fermentation. This is the first field study to suggest that anaerobic-methanogenic biostimulation could enhance source zone bioremediation of groundwater aquifers impacted by biodiesel blends.

Interaction of ceftobiprole with the low-affinity PBP 5 of Enterococcus faecium.

Ceftobiprole is a new cephalosporin that exhibits a high level of affinity for methicillin-resistant Staphylococcus aureus PBP 2a. It was reported that ceftobiprole did not interact with a mutated form of the low-affinity protein Enterococcus faecium PBP 5 (PBP 5fm) that, when overexpressed, confers a beta-lactam resistance phenotype to the bacterium. Our results show that ceftobiprole binds to unmutated PBP 5fm to form a stable acyl-enzyme and that ceftobiprole is able to efficiently kill a penicillin-resistant Enterococcus faecium strain that produces this protein.

Biorremediação de águas subterrâneas impactadas por gasolina e etanol com o uso de nitrato / Nitrate bioremediation of groundwater impacted with gasoline and ethanol

Neste estudo, avaliou-se, durante 32 meses e por meio de um experimento de campo, a utilização da biorremediação com injeção de nitrato na recuperação de águas subterrâneas impactadas por gasolina com 25 por cento de etanol. Por meio da análise da massa e da distribuição espacial dos compostos dissolvidos, verificou-se que a bioestimulação influenciou positivamente na biodegradação do etanol e dos BTEX, evitou a formação de zonas altamente redutoras (90 por cento dos valores foram superiores a +100 mV) e impediu o avanço das plumas de BTEX e etanol na área monitorada. Os resultados indicam que a bioestimulação com nitrato é uma alternativa altamente eficiente para se remediarem águas subterrâneas impactadas por gasolina contendo etanol.

In this study, nitrate bioremediation in groundwater impacted with gasoline containing 25 percent ethanol was evaluated during 32 months in a field experiment. By means of mass and spatial distribution analysis of the dissolved compounds, biostimulation was found to have a positive influence on ethanol and BTEX biodegradation, and prevented the formation of highly reducing zones (90 percent of values were higher than + 100 mV) and BTEX and ethanol plume migration in the monitoring area. Results indicate that nitrate biostimulation is a highly efficient alternative in remediating groundwater impacted by gasohol.

Active site mapping of MraY, a member of the polyprenyl-phosphate N-acetylhexosamine 1-phosphate transferase superfamily, catalyzing the first membrane step of peptidoglycan biosynthesis.

The MraY transferase is an integral membrane protein that catalyzes an essential step of peptidoglycan biosynthesis, namely the transfer of the phospho-N-acetylmuramoyl-pentapeptide motif onto the undecaprenyl phosphate carrier lipid. It belongs to a large superfamily of eukaryotic and prokaryotic prenyl sugar transferases. No 3D structure has been reported for any member of this superfamily, and to date MraY is the only protein that has been successfully purified to homogeneity. Nineteen polar residues located in the five cytoplasmic segments of MraY appeared as invariants in the sequences of MraY orthologues. A certain number of these invariant residues were found to be conserved in the whole superfamily. To assess the importance of these residues in the catalytic process, site-directed mutagenesis was performed using the Bacillus subtilis MraY as a model. Fourteen residues were shown to be essential for MraY activity by an in vivo functional complementation assay using a constructed conditional mraY mutant strain. The corresponding mutant proteins were purified and biochemically characterized. None of these mutations did significantly affect the binding of the nucleotidic and lipidic substrates, but the k cat was dramatically reduced in almost all cases. The important residues for activity therefore appeared to be distributed in all the cytoplasmic segments, indicating that these five regions contribute to the structure of the catalytic site. Our data show that the D98 residue that is invariant in the whole superfamily should be involved in the deprotonation of the lipid substrate during the catalytic process.

Importância do etanol na atenuação natural de águas subterrâneas impactadas por gasolina / Importance of ethanol on natural attenuation of groundwater impacted by gasohol

Nesse estudo, foram avaliados os resultados de um experimento de derramamento controlado de gasolina brasileira em água subterrânea durante 6,5 anos de monitoramento. A exaustão do etanol, aos 32 meses de monitoramento, e a significativa redução de mais de 90 por cento da massa máxima dos compostos BTEX dissolvidos no meio, aos 79 meses, associadas ao uso dos receptores de elétrons e acúmulo de seus subprodutos metabólicos, demonstraram a eficácia da atenuação natural monitorada para contaminações de águas subterrâneas sem riscos imediatos a receptores críticos. Constatou-se ainda que a biodegradação do etanol permitiu a formação de uma biobarreira natural que, após a sua completa degradação, acelerou a taxa de biodegradação dos BTEX e impediu o avanço da pluma destes contaminantes.

In this study, results of 6.5 years of a controlled release experiment with Brazilian gasoline in groundwater were evaluated. Ethanol exhaustion after 32 months and the significant dissolved BTEX mass reduction of more than 90 percent after 79 months, associated with the electron acceptors use and their metabolic byproducts accumulation, demonstrated the efficiency of monitored natural attenuation for groundwater contamination without immediate risk to receptors. Moreover, ethanol degradation provided a natural biobarrier formation that increased BTEX biodegradation rate and prevented the BTEX plume expansion.

Fitorremediacion de acuiferos contaminados por benceno y etanol utiliando "sauce lloron" (Salix babylonica)

El objetivo del trabajo fue investigar el potencial del sauce lloron (salix babylonica-Familia Salicaceae) en el proceso de adsorcion directa por las raices de etanol y benceno,como una etapa inicial en el estudio de la fitorremediacion.Los resultados obtenidos indicaron reducciones de hasta un 99 por ciento para ambos contaminantes,tanto a nivel balance de masa como a nivel de concentracion

Fitorremediacion de acuiferos contaminados por benceno y etanol utiliando "sauce lloron" (Salix babylonica)

El objetivo del trabajo fue investigar el potencial del sauce lloron (salix babylonica-Familia Salicaceae) en el proceso de adsorcion directa por las raices de etanol y benceno,como una etapa inicial en el estudio de la fitorremediacion.Los resultados obtenidos indicaron reducciones de hasta un 99 por ciento para ambos contaminantes,tanto a nivel balance de masa como a nivel de concentracion