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Widespread pesticide use in agriculture is a major source of soil pollution, driving biodiversity loss and posing serious threads to human health. The recalcitrant nature of most of these pesticides demands for effective remediation strategies. In this study, we assess the ability of soil microbial fuel cell (SMFC) technology to bioremediate soil polluted by the model pesticide atrazine. To elucidate the degradation mechanism and consequently define effective implementation strategies, we provide the first comprehensive investigation of the SMFC performance, in which the monitoring of the electrochemical performance of the system is combined with Quadrupole Time-of-Flight (QTOF) mass spectrometry and microbial analyses. Our results show that, while both SMFC and natural attenuation lead to a reduction on atrazine levels, the SMFC modulates the activity of different microbial pathways. As a result, atrazine degradation by natural attenuation leads to high levels of deisoproylatrazine (DIPA), a very toxic degradation metabolite, while DIPA levels in soil treated by SMFC remain comparatively low. The beta diversity and differential abundance analyses revealed how the microbial community evolves over time in the SMFCs degrading atrazine, demonstrating the enrichment of electroactive taxa on the anode, and the enrichment of a mixture of electroactive and atrazine-degrading taxa at the cathode. The detection and taxonomic classification of peripheral atrazine degrading genes, atzA, atzB and atzC, was carried out in combination with the differential abundance analysis. Results revealed that these genes are likely harboured by members of the order Rhizobiales enriched at the cathode, thus promoting atrazine degradation via the conversion of hydroxyatrazine (HA) into N-isopropylammelide (NIPA), as confirmed by mass spectrometry data. Overall, the comprehensive approach adopted in this work, provides fundamental insights into the degradation pathways of atrazine in soil by SMFC technology, which is critical for practical applications, thus suggesting an effective approach to advance research in the field.

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A 30-month pilot study was conducted to evaluate the potential of in-situ metal(loid) removal through biostimulation of sulfate-reducing processes. The study took place at an industrial site in Flanders, Belgium, known for metal(loid) contamination in soil and groundwater. Biostimulation involved two incorporations of an organic substrate (emulsified vegetable oil) as electron donor and potassium bicarbonate to raise the pH of the groundwater by 1-1.5 units. The study focused on the most impacted permeable fine sand aquifer (8-9 m below groundwater level) confined by layers of non-permeable clay. The fine sands exhibited initially oxic conditions (50-200 mV), an acidic pH of 4.5 and sulfate concentrations ranging from 600 to 800 mg/L. At the central monitoring well, anoxic conditions (-200 to -400 mV) and a pH of 5.9 established shortly after the second substrate and reagent injection. Over the course of 12 months, there was a significant decrease in the concentration of arsenic (from 2500 to 12 µg/L), nickel (from 360 to <2 µg/L), zinc (from 78,000 to <2 µg/L), and sulfate (from 930 to 450 mg/L). Low levels of metal(loid)s were still present after 34 months (end of study). Mineralogical analysis indicated that the precipitates formed were amorphous in nature. Evidence for biologically driven metal(loid) precipitation was provided by compound specific stable isotope analysis of sulfate. In addition, changes in microbial populations were assessed using next-generation sequencing, revealing stimulation of native sulfate-reducing bacteria. These results highlight the potential of biostimulation for long-term in situ metal(loid) plume treatment/containment.

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We present in this work a kinetic model of the acetone-butanol-ethanol (ABE) fermentation based on enzyme kinetics expressions. The model includes the effect of the co-substrate NADH as a modulating factor of cellular metabolism. The simulations obtained with the model showed an adequate fit to the experimental data reported by several authors, matching or improving the results observed with previous models. In addition, this model does not require artificial mathematical strategies such as on-off functions to achieve a satisfactory fit of the ABE fermentation dynamics. The parametric sensitivity allowed to identify the direct glucose → acetyl-CoA → butyryl-CoA pathway as being more significant for butanol production than the acid re-assimilation pathway. Likewise, model simulations showed that the increase in NADH, due to glucose concentration, favors butanol production and selectivity, finding a maximum selectivity of 3.6, at NADH concentrations above 55 mM and glucose concentration of 126 mM. The introduction of NADH in the model would allow its use for the analysis of electrofermentation processes with Clostridium, since the model establishes a basis for representing changes in the intracellular redox potential from extracellular variables.

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Increasing energy demands and environmental pollution concerns press for sustainable and environmentally friendly technologies. Soil microbial fuel cell (SMFC) technology has great potential for carbon-neutral bioenergy generation and self-powered electrochemical bioremediation. In this study, an in-depth assessment on the effect of several carbon-based cathode materials on the electrochemical performance of SMFCs is provided for the first time. An innovative carbon nanofibers electrode doped with Fe (CNFFe) is used as cathode material in membrane-less SMFCs, and the performance of the resulting device is compared with SMFCs implementing either Pt-doped carbon cloth (PtC), carbon cloth, or graphite felt (GF) as the cathode. Electrochemical analyses are integrated with microbial analyses to assess the impact on both electrogenesis and microbial composition of the anodic and cathodic biofilm. The results show that CNFFe and PtC generate very stable performances, with a peak power density (with respect to the cathode geometric area) of 25.5 and 30.4 mW m-2, respectively. The best electrochemical performance was obtained with GF, with a peak power density of 87.3 mW m-2. Taxonomic profiling of the microbial communities revealed differences between anodic and cathodic communities. The anodes were predominantly enriched with Geobacter and Pseudomonas species, while cathodic communities were dominated by hydrogen-producing and hydrogenotrophic bacteria, indicating H2 cycling as a possible electron transfer mechanism. The presence of nitrate-reducing bacteria, combined with the results of cyclic voltammograms, suggests microbial nitrate reduction occurred on GF cathodes. The results of this study can contribute to the development of effective SMFC design strategies for field implementation.

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The rapid emergence of antibiotic resistant bacterial pathogens constitutes a critical problem in healthcare and requires the development of novel treatments. Potential strategies include the exploitation of microbial social interactions based on public goods, which are produced at a fitness cost by cooperative microorganisms, but can be exploited by cheaters that do not produce these goods. Cheater invasion has been proposed as a 'Trojan horse' approach to infiltrate pathogen populations with strains deploying built-in weaknesses (e.g., sensitiveness to antibiotics). However, previous attempts have been often unsuccessful because population invasion by cheaters was prevented by various mechanisms including the presence of spatial structure (e.g., growth in biofilms), which limits the diffusion and exploitation of public goods. Here we followed an alternative approach and examined whether the manipulation of public good uptake and not its production could result in potential 'Trojan horses' suitable for population invasion. We focused on the siderophore pyoverdine produced by the human pathogen Pseudomonas aeruginosa MPAO1 and manipulated its uptake by deleting and/or overexpressing the pyoverdine primary (FpvA) and secondary (FpvB) receptors. We found that receptor synthesis feeds back on pyoverdine production and uptake rates, which led to strains with altered pyoverdine-associated costs and benefits. Moreover, we found that the receptor FpvB was advantageous under iron-limited conditions but revealed hidden costs in the presence of an antibiotic stressor (gentamicin). As a consequence, FpvB mutants became the fittest strain under gentamicin exposure, displacing the wildtype in liquid cultures, and in biofilms and during infections of the wax moth larvae Galleria mellonella, which both represent structured environments. Our findings reveal that an evolutionary trade-off associated with the costs and benefits of a versatile pyoverdine uptake strategy can be harnessed for devising a Trojan-horse candidate for medical interventions.

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This work reports the influence of ultrasound alone and combined with ozone for the treatment of real abattoir wastewater. Three different frequencies were studied (44, 300 and 1000 kHz) at an applied power of 40 W. The injected ozone dose was fixed at 71 mg/L and the treatment time varied from 1 to 60 min. Using ultrasound alone, 300 kHz was the only frequency showing a reduction in chemical oxygen demand (COD, 18% reduction) and biological oxygen demand (BOD, 50% reduction), while no diminution in microbial content was measured for any of the frequencies studied. Combining ultrasound with ozone, on the contrary, led to a significant decrease in COD (44%) and BOD (78%) removal for the three frequencies under study. A complete inactivation of total coliforms (TC) was obtained, as well as a final value of 99 CFU/mL in total viable counts (TVC, 5 log reduction). That is, the ozonation-sonication combined system was the only treatment method (compared to sonication and ozonation alone) reaching direct discharge limits, as well as meeting drinking water standards for microbial disinfection (TC and TVC).

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Elucidating the effect of harsh environments on the activities of microorganisms is important in revealing how microbes withstand unfavorable conditions or evolve mechanisms to counteract those effects, many of which involve electron transfer phenomena. Here we show that the non-acidophilic and non-thermophilic Bacillus subtilis is able to maintain activity after being subjected to extreme temperatures (100°C for up to 8 h) and acidic environments (pH = 1.50 for over 2 years). In the process, our results suggest that B. subtilis utilizes an extracellular electron transfer as an electron communication pathway between B. subtilis and the environment that involves the cofactor nicotinamide adenine dinucleotide as an essential participant to maintain viability. Elucidation of the capability of the non-acidophilic and non-thermophilic strain to maintain viability under these extreme conditions could aid in understanding the cell responses to different environments from the perspective of energy conservation pathways.

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In this work, we expanded and updated a genome-scale metabolic model of Streptomyces clavuligerus. The model includes 1021 genes and 1494 biochemical reactions; genome-reaction information was curated and new features related to clavam metabolism and to the biomass synthesis equation were incorporated. The model was validated using experimental data from the literature and simulations were performed to predict cellular growth and clavulanic acid biosynthesis. Flux balance analysis (FBA) showed that limiting concentrations of phosphate and an excess of ammonia accumulation are unfavorable for growth and clavulanic acid biosynthesis. The evaluation of different objective functions for FBA showed that maximization of ATP yields the best predictions for cellular behavior in continuous cultures, while the maximization of growth rate provides better predictions for batch cultures. Through gene essentiality analysis, 130 essential genes were found using a limited in silico media, while 100 essential genes were identified in amino acid-supplemented media. Finally, a strain design was carried out to identify candidate genes to be overexpressed or knocked out so as to maximize antibiotic biosynthesis. Interestingly, potential metabolic engineering targets, identified in this study, have not been tested experimentally.

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Oleaginous microorganisms represent possible platforms for the sustainable production of oleochemicals and biofuels due to their metabolic robustness and the possibility to be engineered. Streptomyces coelicolor is among the narrow group of prokaryotes capable of accumulating triacylglycerol (TAG) as carbon and energy reserve. Although the pathways for TAG biosynthesis in this organism have been widely addressed, the set of genes required for their breakdown have remained elusive so far. Here, we identified and characterized three gene clusters involved in the ß-oxidation of fatty acids (FA). The role of each of the three different S. coelicolor FadAB proteins in FA catabolism was confirmed by complementation of an Escherichia coliΔfadBA mutant strain deficient in ß-oxidation. In S. coelicolor, the expression profile of the three gene clusters showed variation related with the stage of growth and the presence of FA in media. Flux balance analyses using a corrected version of the current S. coelicolor metabolic model containing detailed TAG biosynthesis reactions suggested the relevance of the identified fadAB genes in the accumulation of TAG. Thus, through the construction and analysis of fadAB knockout mutant strains, we obtained an S. coelicolor mutant that showed a 4.3-fold increase in the TAG content compared to the wild type strain grown under the same culture conditions.

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The chassis is the cellular host used as a recipient of engineered biological systems in synthetic biology. They are required to propagate the genetic information and to express the genes encoded in it. Despite being an essential element for the appropriate function of genetic circuits, the chassis is rarely considered in their design phase. Consequently, the circuits are transferred to model organisms commonly used in the laboratory, such as Escherichia coli, that may be suboptimal for a required function. In this review, we discuss some of the properties desirable in a versatile chassis and summarize some examples of alternative hosts for synthetic biology amenable for engineering. These properties include a suitable life style, a robust cell wall, good knowledge of its regulatory network as well as of the interplay of the host components with the exogenous circuits, and the possibility of developing whole-cell models and tuneable metabolic fluxes that could allow a better distribution of cellular resources (metabolites, ATP, nucleotides, amino acids, transcriptional and translational machinery). We highlight Pseudomonas putida, widely used in many different biotechnological applications as a prominent organism for synthetic biology due to its metabolic diversity, robustness and ease of manipulation.

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This study investigates the microbial community composition in the biofilms grown on two different support media in fixed biofilm reactors for aerobic wastewater treatment, using next generation sequencing (NGS) technology. The chemical composition of the new type of support medium (TDR) was found to be quite different from the conventionally used support medium (stone). The analysis of 16S rRNA gene fragments recovered from the laboratory scale biofilm system show that biofilm support media and temperature conditions influence bacterial community structure and composition. Greater bacterial diversity was observed under each condition, primarily due to the large number of sequences available and sustenance of rare species. There were 6 phyla found, with the highest relative abundance shown by the phylum Proteobacteria (52.71%) followed by Bacteroidetes (33.33%), Actinobacteria (4.65%), Firmicutes, Verrucomicrobia (3.1%) and Chloroflex (>1%). The dataset showed 17 genera of bacterial populations to be commonly shared under all conditions, suggesting the presence of a core microbial community in the biofilms for wastewater treatment. However, some genera in the biofilms on TDR were observed in high proportions, which may be attributed to its chemical composition, explaining the improved level of wastewater treatment. The findings show that the structure of microbial communities in biofilm systems for wastewater treatment is affected by the properties of support matrix.

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In this paper, we analyse how electric power production in microbial fuel cells (MFCs) depends on the composition of the anodic biofilm in terms of metabolic capabilities of identified sets of species. MFCs are a promising technology for organic waste treatment and sustainable bioelectricity production. Inoculated with natural communities, they present a complex microbial ecosystem with syntrophic interactions between microbes with different metabolic capabilities. Our results demonstrate that low-potential anaerobic respirators--that is those that are able to use terminal electron acceptors with a low redox potential--are important for good power production. Our results also confirm that community metabolism in MFCs with natural inoculum and fermentable feedstock is a two-stage system with fermentation followed by anode respiration.

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The relationship between the diversity of mixed-species microbial consortia and their electrogenic potential in the anodes of microbial fuel cells was examined using different diversity measures as predictors. Identical microbial fuel cells were sampled at multiple time-points. Biofilm and suspension communities were analysed by denaturing gradient gel electrophoresis to calculate the number and relative abundance of species. Shannon and Simpson indices and richness were examined for association with power using bivariate and multiple linear regression, with biofilm DNA as an additional variable. In simple bivariate regressions, the correlation of Shannon diversity of the biofilm and power is stronger (r=0.65, p=0.001) than between power and richness (r=0.39, p=0.076), or between power and the Simpson index (r=0.5, p=0.018). Using Shannon diversity and biofilm DNA as predictors of power, a regression model can be constructed (r=0.73, p<0.001). Ecological parameters such as the Shannon index are predictive of the electrogenic potential of microbial communities.

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The performance and dynamics of the bacterial communities in the biofilm and suspended culture in the anode chamber of sucrose-fed microbial fuel cells (MFCs) were studied by using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified partial 16S rRNA genes followed by species identification by sequencing. The power density of MFCs was correlated to the relative proportions of species obtained from DGGE analysis in order to detect bacterial species or taxonomic classes with important functional role in electricity production. Although replicate MFCs showed similarity in performance, cluster analysis of DGGE profiles revealed differences in the evolution of bacterial communities between replicate MFCs. No correlation was found between the proportion trends of specific species and the enhancement of power output. However, in all MFCs, putative exoelectrogenic denitrifiers and sulphate-reducers accounted for approximately 24% of the bacterial biofilm community at the end of the study. Pareto-Lorenz evenness distribution curves extracted from the DGGE patterns obtained from time course samples indicated community structures where shifts between functionally similar species occur, as observed within the predominant fermentative bacteria. These results suggest the presence of functional redundancy within the anodic communities, a probable indication that stable MFC performance can be maintained in changing environmental conditions. The capability of bacteria to adapt to electricity generation might be present among a wide range of bacteria.

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BACKGROUND: Constraint-based approaches facilitate the prediction of cellular metabolic capabilities, based, in turn on predictions of the repertoire of enzymes encoded in the genome. Recently, genome annotations have been used to reconstruct genome scale metabolic reaction networks for numerous species, including Homo sapiens, which allow simulations that provide valuable insights into topics, including predictions of gene essentiality of pathogens, interpretation of genetic polymorphism in metabolic disease syndromes and suggestions for novel approaches to microbial metabolic engineering. These constraint-based simulations are being integrated with the functional genomics portals, an activity that requires efficient implementation of the constraint-based simulations in the web-based environment. RESULTS: Here, we present Acorn, an open source (GNU GPL) grid computing system for constraint-based simulations of genome scale metabolic reaction networks within an interactive web environment. The grid-based architecture allows efficient execution of computationally intensive, iterative protocols such as Flux Variability Analysis, which can be readily scaled up as the numbers of models (and users) increase. The web interface uses AJAX, which facilitates efficient model browsing and other search functions, and intuitive implementation of appropriate simulation conditions. Research groups can install Acorn locally and create user accounts. Users can also import models in the familiar SBML format and link reaction formulas to major functional genomics portals of choice. Selected models and simulation results can be shared between different users and made publically available. Users can construct pathway map layouts and import them into the server using a desktop editor integrated within the system. Pathway maps are then used to visualise numerical results within the web environment. To illustrate these features we have deployed Acorn and created a web server allowing constraint based simulations of the genome scale metabolic reaction networks of E. coli, S. cerevisiae and M. tuberculosis. CONCLUSIONS: Acorn is a free software package, which can be installed by research groups to create a web based environment for computer simulations of genome scale metabolic reaction networks. It facilitates shared access to models and creation of publicly available constraint based modelling resources.

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The spatiotemporal development of a bacterial community in an exoelectrogenic biofilm was investigated in sucrose-fed longitudinal tubular microbial fuel cell reactors, consisting of two serially connected modules. The proportional changes in the microbial community composition were assessed by polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) and DNA sequencing in order to relate them to the performance and stability of the bioelectrochemical system. The reproducibility of duplicated reactors, evaluated by cluster analysis and Jaccard's coefficient, shows 80-90% similarity in species composition. Biofilm development through fed-batch start-up and subsequent stable continuous operation results in a population shift from γ-Proteobacteria- and Bacteroidetes- to Firmicutes-dominated communities, with other diverse species present at much lower relative proportions. DGGE patterns were analysed by range-weighted richness (Rr) and Pareto-Lorenz evenness distribution curves to investigate the evolution of the bacterial community. The first modules shifted from dominance by species closely related to Bacteroides graminisolvens, Raoultella ornithinolytica and Klebsiella sp. BM21 at the start of continuous-mode operation to a community dominated by Paludibacter propionicigenes-, Lactococcus sp.-, Pantoea agglomerans- and Klebsiella oxytoca-related species with stable power generation (6.0 W/m(3)) at day 97. Operational strategies that consider the dynamics of the population will provide useful parameters for evaluating system performance in the practical application of microbial fuel cells.

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UNLABELLED: Constraint-based modeling of genome-scale metabolic networks has been successfully used in numerous applications such as prediction of gene essentiality and metabolic engineering. We present SurreyFBA, which provides constraint-based simulations and network map visualization in a free, stand-alone software. In addition to basic simulation protocols, the tool also implements the analysis of minimal substrate and product sets, which is useful for metabolic engineering and prediction of nutritional requirements in complex in vivo environments, but not available in other commonly used programs. The SurreyFBA is based on a command line interface to the GLPK solver distributed as binary and source code for the three major operating systems. The command line tool, implemented in C++, is easily executed within scripting languages used in the bioinformatics community and provides efficient implementation of tasks requiring iterative calls to the linear programming solver. SurreyFBA includes JyMet, a graphics user interface allowing spreadsheet-based model presentation, visualization of numerical results on metabolic networks represented in the Petri net convention, as well as in charts and plots. AVAILABILITY: SurreyFBA is distributed under GNU GPL license and available from http://sysbio3.fhms.surrey.ac.uk/SurreyFBA.zip.

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The construction and characterization of a one-compartment fructose/air biological fuel cell (BFC) based on direct electron transfer is reported. The BFC employs bilirubin oxidase and d-fructose dehydrogenase adsorbed on a cellulose-multiwall carbon nanotube (MWCNT) matrix, reconstituted with an ionic liquid, as the biocathode and the bioanode for oxygen reduction and fructose oxidation reactions, respectively. The performance of the bioelectrode was investigated by chronoamperometric and cyclic voltammetric techniques in a standard three-electrode cell, and the polarization and long-term stability of the BFC was tested by potentiostatic discharge. An open circuit voltage of 663 mV and a maximum power density of 126 microWcm(-2) were obtained in buffer at pH 5.0. Using this regenerated cellulose-MWCNT matrix as the immobilization platform, this BFC has shown a relatively high performance and long-term stability compared with previous studies.

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Conductive cellulose-multiwalled carbon nanotube (MWCNT) matrix with a porous structure and good biocompatibility has been prepared using a room temperature ionic liquid (1-ethyl-3-methylimidazolium acetate) as solvent. Glucose oxidase (GOx) was encapsulated in this matrix and thereby immobilized on a glassy carbon surface. The direct electron transfer and electrocatalysis of the encapsulated GOx has been investigated using cyclic voltammetry and chronoamperometry. The GOx exhibited a pair of stable, well defined and nearly symmetric reversible redox peaks. The experimental results also demonstrate that the immobilized GOx retains its biocatalytic activity toward the oxidation of glucose and therefore can be employed in a glucose biosensor. The results show that the bioelectrode modified by the cellulose-MWCNT matrix has potential for use in biosensors and other bioelectronics devices.

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