Electrochemical biosensor for aerobic acetate detection.

There is an increasing demand on alternatives methods to animal testing. Numerous health parameters have been already studied using in vitro devices able to mimic the essential functions of the organs, being the real-time monitoring and response to stimuli their main limitations. Regarding the health of the gut, the short chain fatty acids, and particularly acetate, have emerged as key biomarkers to evaluate gut healthiness and disease development, although the number of acetate biosensors is still very low. This article presents a microbial biosensor based on fully biocompatible materials which is able to detect acetate in aerobic conditions in the range between 11 and 50 mM, and without compromising the viability and function of either bacteria (>90% viability) or mammalian cells (>80% viability). The detection mechanism is based on the metabolism of acetate by Escherichia coli bacteria immobilized on the transducer surface. Ferricyanide is used as a redox mediator to transfer electrons from the acetate metabolism in the bacterial cells to the transducer. High bacterial concentrations are immobilized in the transducer surface (109 cfu mL-1) by electrodeposition of conductive alginate hydrogels doped with reduced graphene oxide. The results show successful outcomes to exploit bacteria as a biosensing tool, based on the use of inkjet printed transducers, biocompatible materials and cell entrapment technologies.

Fast fabrication of reusable polyethersulfone microbial biosensors through biocompatible phase separation.

In biosensors fabrication, entrapment in polymeric matrices allows efficient immobilization of the biorecognition elements without compromising their structure and activity. When considering living cells, the biocompatibility of both the matrix and the polymerization procedure are additional critical factors. Bio-polymeric gels (e.g. alginate) are biocompatible and polymerize under mild conditions, but they have poor stability. Most synthetic polymers (e.g. PVA), on the other hand, present improved stability at the expense of complex protocols involving chemical/physical treatments that decrease their biological compatibility. In an attempt to explore new solutions to this problem we have developed a procedure for the immobilization of bacterial cells in polyethersulfone (PES) using phase separation. The technology has been tested successfully in the construction of a bacterial biosensor for toxicity assessment. Biosensors were coated with a 300  µm bacteria-containing PES membrane, using non-solvent induced phase separation (membrane thickness ≈ 300 µm). With this method, up to 2.3 × 106 cells were immobilized in the electrode surface with an entrapment efficiency of 8.2%, without compromising cell integrity or viability. Biosensing was performed electrochemically through ferricyanide respirometry, with metabolically-active entrapped bacteria reducing ferricyanide in the presence of glucose. PES biosensors showed good stability and reusability during dry frozen storage for up to 1 month. The analytical performance of the sensors was assessed carrying out a toxicity assay in which 3,5-dichlorophenol (DCP) was used as a model toxic compound. The biosensor provided a concentration-dependent response to DCP with half-maximal effective concentration (EC50) of 9.2 ppm, well in agreement with reported values. This entrapment methodology is susceptible of mass production and allows easy and repetitive production of robust and sensitive bacterial biosensors.

Electro-addressable conductive alginate hydrogel for bacterial trapping and general toxicity determination.

In biosensors development, alginate hydrogels are a first choice for enabling stable biomolecules entrapment in biocompatible membranes obtained under soft physiological conditions. Although widely exploited, most alginate membranes are isolating and poorly repetitive, which limit their application in biosensing. Significant steps forward on improving repeatability and conductivity have been performed, but to date there is no single protocol for controlled deposition of live cells in replicable conductive alginate layers. Here, cell electrotrapping in conductive alginate hydrogels is examined in order to overcome these limitations. Conductive alginate-coated electrodes are obtained after potentiostatic electrodeposition of graphite-doped alginate samples (up to 4% graphite). The presence of graphite reduces electrode passivation and improves the electrochemical response of the sensor, although still significantly lower than that recorded with the naked electrode. Bacterial electrotrapping in the conductive matrix is highly efficient (4.4 × 107 cells per gel) and repetitive (CV < 0.5%), and does not compromise bacterial integrity or activity (cell viability = 56%). Biosensing based on ferricyanide respirometry yielded a four times increase in biosensor response with respect to non-conductive alginate membrane, providing toxicity values completely comparable to those reported. Cell electrotrapping in conductive hydrogels represents a step forward towards in high-sensitive cell-based biosensors development with important influence in environmental analysis, food and beverage industry as well as clinical diagnosis.

Paper-based chromatic toxicity bioassay by analysis of bacterial ferricyanide reduction.

Water quality assessment requires a continuous and strict analysis of samples to guarantee compliance with established standards. Nowadays, the increasing number of pollutants and their synergistic effects lead to the development general toxicity bioassays capable to analyse water pollution as a whole. Current general toxicity methods, e.g. Microtox(®), rely on long operation protocols, the use of complex and expensive instrumentation and sample pre-treatment, which should be transported to the laboratory for analysis. These requirements delay sample analysis and hence, the response to avoid an environmental catastrophe. In an attempt to solve it, a fast (15 min) and low-cost toxicity bioassay based on the chromatic changes associated to bacterial ferricyanide reduction is here presented. E. coli cells (used as model bacteria) were stably trapped on low-cost paper matrices (cellulose-based paper discs, PDs) and remained viable for long times (1 month at -20 °C). Apart from bacterial carrier, paper matrices also acted as a fluidic element, allowing fluid management without the need of external pumps. Bioassay evaluation was performed using copper as model toxic agent. Chromatic changes associated to bacterial ferricyanide reduction were determined by three different transduction methods, i.e. (i) optical reflectometry (as reference method), (ii) image analysis and (iii) visual inspection. In all cases, bioassay results (in terms of half maximal effective concentrations, EC50) were in agreement with already reported data, confirming the good performance of the bioassay. The validation of the bioassay was performed by analysis of real samples from natural sources, which were analysed and compared with a reference method (i.e. Microtox). Obtained results showed agreement for about 70% of toxic samples and 80% of non-toxic samples, which may validate the use of this simple and quick protocol in the determination of general toxicity. The minimum instrumentation requirements and the simplicity of the bioassay open the possibility of in-situ water toxicity assessment with a fast and low-cost protocol.

Fast and sensitive optical toxicity bioassay based on dual wavelength analysis of bacterial ferricyanide reduction kinetics.

Global urban and industrial growth, with the associated environmental contamination, is promoting the development of rapid and inexpensive general toxicity methods. Current microbial methodologies for general toxicity determination rely on either bioluminescent bacteria and specific medium solution (i.e. Microtox(®)) or low sensitivity and diffusion limited protocols (i.e. amperometric microbial respirometry). In this work, fast and sensitive optical toxicity bioassay based on dual wavelength analysis of bacterial ferricyanide reduction kinetics is presented, using Escherichia coli as a bacterial model. Ferricyanide reduction kinetic analysis (variation of ferricyanide absorption with time), much more sensitive than single absorbance measurements, allowed for direct and fast toxicity determination without pre-incubation steps (assay time=10 min) and minimizing biomass interference. Dual wavelength analysis at 405 (ferricyanide and biomass) and 550 nm (biomass), allowed for ferricyanide monitoring without interference of biomass scattering. On the other hand, refractive index (RI) matching with saccharose reduced bacterial light scattering around 50%, expanding the analytical linear range in the determination of absorbent molecules. With this method, different toxicants such as metals and organic compounds were analyzed with good sensitivities. Half maximal effective concentrations (EC50) obtained after 10 min bioassay, 2.9, 1.0, 0.7 and 18.3 mg L(-1) for copper, zinc, acetic acid and 2-phenylethanol respectively, were in agreement with previously reported values for longer bioassays (around 60 min). This method represents a promising alternative for fast and sensitive water toxicity monitoring, opening the possibility of quick in situ analysis.

Resolution of binary mixtures of microorganisms using electrochemical impedance spectroscopy and artificial neural networks.

This work describes the resolution of binary mixtures of microorganisms using electrochemical impedance spectroscopy (EIS) and artificial neural networks (ANNs) for the processing of data. Pseudomonas aeruginosa, Staphylococcus aureus and Saccharomyces cerevisiae were chosen as models for Gram-negative bacteria, Gram-positive bacteria and yeasts, respectively. In this study, best results were obtained when entering the imaginary component of the impedance at each frequency (strongly related to the capacitive elements of the electrical equivalent circuit) into backpropagation neural networks made up by two hidden layers. The optimal configuration of these layers respectively used the radbas and the logsig transfer functions with 4 or 6 neurons in the first hidden layer and 10 neurons in the second one. In all cases, good prediction ability was obtained with correlation coefficients better than 0.989 when comparing the predicted and the expected values for a set of six external test samples not used in the training process.

Impedimetric approach for quantifying low bacteria concentrations based on the changes produced in the electrode-solution interface during the pre-attachment stage.

This paper describes an approach for quantifying low concentrations of bacteria, particularly Escherichia coli, based on the measurement of the initial attachment of bacteria to platinum surfaces, using impedance spectroscopy. The value of the interface capacitance in the pre-attachment stage (before 1min of attachment) showed correlation with suspended concentration of bacteria from 10(1) to 10(7)CFUmL(-1) (colony forming units per mL). This method was found to be sensitive to the attachment time, to the applied potential and to the size of the counter electrode. The sensor lifetime was also evaluated.

Presence of opportunistic oil-degrading microorganisms operating at the initial steps of oil extraction and handling / Presencia de microorganismos oportunistas degradadores de petróleo en la extracción y manipulación iniciales de petróleo

Hydrocarbon-degrading microorganisms from natural environments have been isolated and identified using culture-dependent or molecular techniques. However, there has been little research into the occurrence of microorganisms incorporated into crude oil in the initial steps of extraction and handling, which can reduce the quality of stored petroleum. In the present study, a packed-column reactor filled with autoclaved perlite soaked with crude oil was subjected to a continuous flow of sterile medium in order to determine the presence of potential hydrocarbon degraders. Microorganisms developed on the surface of the perlite within a period of 73 days. DNA was extracted from the biofilm and then PCR-amplified using 16S rRNA bacterial and archaeal primers and 18S rRNA eukaryotic primers. No amplification was obtained using archaeal primers. However, denaturing gradient gel electrophoresis (DGGE) revealed the presence of unique bands indicating bacterial and eukaryotic amplification. Excision of these bands, sequencing, and subsequent BLAST search showed that they corresponded to Bacillus sp. and Aspergillus versicolor. The fungus was later isolated from intact perlite in agar plates. A bacterial clone library was used to confirm the presence in the biofilm of a unique hydrocarbon-degrading bacterium closely related to Bacillus sp. Analysis of the petroleum components by gas chromatography showed that there n-alkanes, aromatic hydrocarbons, and carbazoles were degraded (AU)

Algunos autores han aislado e identificado microorganismos degradadores de petróleo utilizando técnicas moleculares o dependientes de cultivo. Sin embargo, se ha investigado poco la presencia de microorganismos que entran en contacto con el petróleo en las fases iniciales de su extracción y manipulación, circunstancia que puede reducir la calidad del petróleo almacenado. Mediante un reactor con una columna cargada con perlita esterilizada en autoclave y empapada de petróleo, y sujeto a un flujo continuo de medio estéril, determinamos la presencia de posibles degradadores de hidrocarburos. En la superficie de la perlita se desarrollaron microorganismos en un período de 73 días. Se extrajo el DNA del biofilm, y se amplificó por PCR con cebadores para el 16S rRNA de bacterias y arqueas y con cebadores para el 18S rRNA de eucariotas. No se obtuvo ampliación del DNA de arqueas, pero el análisis por electroforesis en gel con gradiente desnaturalizante (DGGE) reveló la presencia de bandas únicas en la amplificación de bacterias y eucariotas. La escisión de estas bandas, la secuenciación y la subsiguiente búsqueda en BLAST demostraron que correspondían a Bacillus sp. y a Aspergillus versicolor. El hongo fue aislado posteriormente en placas de agar a partir de perlita intacta. También se llevó a cabo una biblioteca genética bacteriana, que confirmó la presencia de una única bacteria degradadora de petróleo en el biofilm, muy próxima a Bacillus sp. El análisis de los componentes del petróleo por cromatografía de gases mostró que había habido degradación de n-alcanos, hidrocarburos aromáticos y carbazoles (AU)