Development and Validation of an On-Line Water Toxicity Sensor with Immobilized Luminescent Bacteria for On-Line Surface Water Monitoring.

Surface water used for drinking water production is frequently monitored in The Netherlands using whole organism biomonitors, with for exampleDaphnia magnaorDreissenamussels, which respond to changes in the water quality. However, not all human-relevant toxic compounds can be detected by these biomonitors. Therefore, a new on-line biosensor has been developed, containing immobilized genetically modified bacteria, which respond to genotoxicity in the water by emitting luminescence. The performance of this sensor was tested under laboratory conditions, as well as under field conditions at a monitoring station along the river Meuse in The Netherlands. The sensor was robust and easy to clean, with inert materials, temperature control and nutrient feed for the reporter organisms. The bacteria were immobilized in sol-gel on either an optical fiber or a glass slide and then continuously exposed to water. Since the glass slide was more sensitive and robust, only this setup was used in the field. The sensor responded to spikes of genotoxic compounds in the water with a minimal detectable concentration of 0.01 mg/L mitomycin C in the laboratory and 0.1 mg/L mitomycin C in the field. With further optimization, which should include a reduction in daily maintenance, the sensor has the potential to become a useful addition to the currently available biomonitors

Exploiting bioluminescence to enhance the analytical performance of whole-cell and cell-free biosensors for environmental and point-of-care applications

The routine health monitoring of living organisms and environment has become one of the major concerns of public interest. Therefore, there has been an increasing demand for fast and easy to perform monitoring technologies. The current available analytical techniques generally offer accurate and precise results; however, they often require clean samples and sophisticated equipment. Thus, they are not suitable for on site, real-time, cost-effective routine monitoring. To this end, biosensors represent suitable analytical alternative tools. Biosensors are analytical devices integrating a biological recognition element (i.e. antibody, receptor, cell) and a transducer able to convert the biological response into an easily measurable analytical signal. These tools can easily quantify an analyte or a class of analytes of interest even in a complex matrix, like clinical or environmental samples, thanks to the specificity of the biological components. Whole-cell biosensors among others offer unique features such as low cost of production and provide comprehensive functional information (i.e. detection of unclassified compounds and synergistic effects, information about the bioavailable concentration). During this PhD, several bioengineered whole-cell biosensors have been developed and optimized for environmental and point-of-care applications. Analytical performance of biosensors have been improved (i.e. low limit of detection, faster response time and wider dynamic range) thanks to synthetic biology and genetic engineering tools. Bacterial, yeast and 3D cell cultures of mammalian cell lines have been tailored at the molecular level to improve robustness and predictivity. Several reporter genes, i.e. colorimetric, fluorescent and bioluminescent proteins, have been also profiled for finding the best candidate for each point-of-need application. Furthermore, spectral resolution of different optical reporter proteins has been exploited and multiplex detection has been achieved. The inclusion of viability control strains provided a suitable tool for assessing non-specific effects on cell viability, correcting the analytical signal and increasing the analytical performance of ready-to-use cartridges

Biosensors for Environmental Monitoring

Real-time and reliable detection of molecular compounds and bacteria is essential in modern environmental monitoring. For rapid analyses, biosensing devices combining high selectivity of biomolecular recognition and sensitivity of modern signal-detection technologies offer a promising platform. Biosensors allow rapid on-site detection of pollutants and provide potential for better understanding of the environmental processes, including the fate and transport of contaminants.This book, including 12 chapters from 37 authors, introduces different biosensor-based technologies applied for environmental analyses

Development of a whole-cell based biosensor technique for assessment of bioavailability and toxicity of heavy metals in soil

A thesis submitted to the University of Bedfordshire in partial fulfilment of the requirements for the degree of Doctor of PhilosophyThe aim of this study was to develop a suitable monitoring protocol for mediated amperometric whole-cell biosensors for in situ assessment of heavy metals in soil. E. coli 8277, Pseudomonas 9773, Pseudomonas 9046 and Pseudomonas 8917 were screened as biosensor catalysts to select the sensitive biosensor configurations to heavy metals. A new protocol was developed for monitoring heavy metals in defined solution, soil pore water, and in situ in soil. This study also demonstrated the applications of mediated amperometric bacterial biosensors for in situ assessing the bioavailability and toxicity of heavy metals in freshly spiked soils or historically contaminated soils, and mixture toxicities of heavy metals. It was found that the biosensors incorporating selected bacterial strains were appropriately sensitive to copper, but less sensitive to Zn, Pb, and Hg, compared to Microtox assay. The advantage of the mediated amperometric bacterial biosensor system is its in situ application in soils. The present study demonstrated that soil pore water does not accurately reflect conditions of soil ecosystem, and that in situ bioassays are more reliable for determining the bioavailability and toxicity of heavy metals. This is the first reported use of disposable whole cell biosensors for in situ heavy metal bioavailability and toxicity assessment. The biosensor protocol developed here can be adapted to allow the incorporation of dfferent bacterial biocatalysts for applications in soil quality assessment, screening of sites for contamination ‘hot spots’, and the evaluation of soil degradation or rehabilitation from metal pollution. Mediated amperometric bacterial biosensors are not analyte specific, their response reflecting the metabolic impact of the combined chemical and physical properties of the environment to which they are exposed. In assessing the toxicity of soil samples from fields using these biosensors, it is vital to get appropriate control soil samples. The conditions of soil samples also need to be well defined. The sensitivity of the mediated amperometric whole-cell biosensors to heavy metals need to be further improved. Investigations are also required to determine how the natural conditions affect the application of the biosensor system in the field

Development and characterization of a self-bioluminescent heavy metal cyanobacterial bioreporter strain

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 07-09-2017Esta tesis tiene embargado el acceso al texto completo hasta el 07-03-2019The thesis was supported by the Spanish government (proyects MICIN CGL2010-15675 and MINECO CTM2013-45775-C2-2-R) and the Dirección General de Universidades e Investigación de la Comunidad de Madrid, Research Network (Comunidad de Madrid S-2009/AMB/1511

Light dazzles from the black box: Whole-cell biosensors are ready to inform on fundamental soil biological processes

Whole-cell biosensors are natural or engineered microorganisms producing signals in response to specific stimuli. This review introduces the use of whole-cell biosensors for the study of the soil system, discuss the recent developments and some current limitations and draws future prospects of the whole-cell biosensors for application to the study of the agro-ecosystems. The review focuses mainly on the lux- and gfp-inserted whole-cell biosensors producing bioluminescence and multicoloured fluorescent proteins, which allow an easy and reproducible detection of the signals from a large number of prokaryotic and eukaryotic soil-borne microorganisms. This review also points out how the whole-cell biosensors indicate the bioavailability of selected analyte, an information that cannot be straight forwardly extrapolated using the chemical methods of soil analysis. However, regardless of the immense progress in biotechnology and genetics that allows to construct whole-cell biosensors for virtually detecting any chemical at ultra low concentrations, the soil still remains the most extreme natural system to be studied with these biotechnological analytical tools. Although a lack of standardization for most of the constructed whole-cell biosensors along with the scarce knowledge of their performance concur to prevent their use in the official methods of soil and environmental analysis, owing to their stability and selectivity we restate that the whole-cell biosensors are ready to provide information on the main processes occurring in soil, and represent unprecedented sensitive tools for improving agriculture and for soil monitorin

Les biocapteurs appliqués au contrôle des eaux: Revue - État de l'art

Cet article présente l'ensemble des biocapteurs en cours d'étude et proposés pour le contrôle en continu, automatisé et in situ de la qualité des eaux. Le principe des systèmes, étudiés jusqu'ici majoritairement en laboratoire et sur pilote, sera donné avec leurs performances au plan sensibilité et spécificité de détection des polluants hydriques. Ces performances conditionnent leur domaine d'application : les systèmes très sensibles étant affectés au contrôle des eaux d'alimentation et des eaux souterraines, les moins sensibles au contrôle des effluents très contaminés.Les biocapteurs peuvent se caractériser par deux de leurs composantes principales :- le réactif biologique ou biocatalyseur, sensible au(x) polluant(s); - le détecteur appelé transducteur, qui traduit la réponse biologique du biocatalyseur en un signal électrique. Le transducteur peut être de type optique, électrochimique, ampérométrique principalement, ou piézoélectrique. Trois grands types de biocapteurs peuvent être distingués selon la nature du biocatalyseur :- les bioréacteurs, basés sur l'étude des réponses comportementales des vertébrés (poissons) et d'autres organismes aquatiques (microcrustacés, bivalves): - les biosondes cellulaires reposant sur l'étude des fonctions métaboliques telles que la respiration, la bioluminescence, la photosynthèse de microorganismes immobilisés (bactéries, microalgues, levures) ou libres (boues activées) dans le milieu analysé: - les biocapteurs "d'affinité" basés sur l'utilisation d'enzymes ou d'anticorps, chargés de détecter respectivement les substrats et inhibiteurs enzymatiques spécifiques, ou les substances antigéniques vis à vis desquelles les anticorps ont été développés. Ces systèmes sont, par principe, les plus spécifiques mais aussi les plus sensibles. Ils ne couvrent, cependant, qu'une gamme encore très limitée de micropolluants hydriques. Le degré d'autonomie d'un biocapteur, sa facilité d'utilisation et de maintenance et sa fiabilité, sont des éléments qui rentrent en ligne de compte dans les performances. Ces qualités devront être évaluées lors de la phase de validation in situ, essentielle et déterminante pour juger de l'intérêt du système en conditions de fonctionnement réel.ContextThis paper reviews the use of biosensors for environmental biomonitoring and especially for the detection of water pollutants. These systems are developed in view of on-line applications, continuous and real time analysis. The principle and the design of the different systems proposed for this purpose are described with their performances deduced from pilot or in situ studies carried out up to now. Automation and autonomy, sensitivity and specificity are critical points that will determine the success of their applications in biomonitoring and the kind of application that can be envisaged. It is necessary they require minimal human intervention for maintenance and working . The more sensitive systems can be used for the monitoring of drinking and ground waters, the less sensitive ones for the monitoring of complex effluents, more heavily contaminated.Biosensors can be distinguished on the basis of the type of biocatalyst associated with thetransducer: the biological signal delivered by the biocatalyst is transmitted to a detector, also called transducer. The transducer, which may be an optical, electrochemical or piezoelectrical detector, transforms the biological response into an electric signal. This signal can be easily amplified and interpreted in terms of the toxicity and level of pollution of the analyzed sample.Three categories of biosensors can be defined:- biosensors using aquatic vertebrates and invertebrates: fish, microcrustacea, bivalves. Their behavior in the tested medium is studied as the criterion for toxicity; - cellular sensors, measuring physiological and biochemical functions such as respiration, bioluminescence, and photosynthesis, in microorganisms immobilized on the transducer (bacteria, yeast, microalgae,..) or suspended in the tested medium (activated sludge); - biosensors measuring an "affinity" response and a specific binding between enzyme/substrate or antibody/antigen. These systems use enzymes or antibodies immobilized in close contact with the transducer; they may detect the (analogs of) enzymatic substrates and inhibitors, or the (analogs of) antigenic substances binding to the antibody. These systems appear promising on the basis of their sensitivity. At present they can be applied for the detection of triazines and phenols. Such systems need to be developed and extended to other pollutants in order to cover the wide range of aquatic contaminants. User-friendliness, attendance and maintenance requirements, and service life are other critical aspects affecting the performances of a biosensor. These qualities need to be evaluated during the validation step of the equipment. In situ validation is essential for evaluating the relevance of the system in environmental biomonitoring and its applications. It is probable that among the numerous systems proposed as biosensors, only a few will be considered as suitable tools for on-line monitoring of waters

Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production

Many high-value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimise the productivity of these cultures it is important to monitor cellular metabolism, for example the utilisation of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness. Therefore, we developed a whole-cell Escherichia coli lactate biosensor based on the lldPRD operon and successfully used it to monitor lactate concentration in mammalian cell cultures. Using real samples and analytical validation we demonstrate that our biosensor can be used for absolute quantification of metabolites in complex samples with high accuracy, sensitivity and robustness. Importantly, our whole-cell biosensor was able to detect lactate at concentrations more than two orders of magnitude lower than the industry standard method, making it useful for monitoring lactate concentrations in early phase culture. Given the importance of lactate in a variety of both industrial and clinical contexts we anticipate that our whole-cell biosensor can be used to address a range of interesting biological questions. It also serves as a blueprint for how to capitalise on the wealth of genetic operons for metabolite sensing available in Nature for the development of other whole-cell biosensors

Microbial biosensors for wastewater monitoring: mini-review

Research on the use of microbial biosensors for monitoring wastewater contaminants is a topic that covers few publications compared to their applicability in other fields, such as biomedical research. For this reason, a systematic analysis of the topic was carried out, for which research-type articles were reviewed during the period 2012 to September 2022. For this, different search platforms were used, including PubMed, ScienceDirect, Springer Link, and Scopus, and through the use of search equations a relevant bibliography was located. After that, the research articles were selected based on exclusion criteria. As a result, it was found that, of the 126 articles, only 16 articles were strictly related to the topic, since there was a duplication of articles among the different databases. It was possible to demonstrate the usefulness of microorganisms as components of biosensors to monitor BOD, heavy metals, and inorganic contaminants in wastewater that also had a high sensitivity. Additionally, recombinant DNA techniques were shown to improve the performance of this type of biosensor and can finally be coupled to other emerging technologies, such as microbial fuel cells (MFCs). In conclusion, it was established that microbial biosensors have high acceptability and monitoring characteristics that make them a useful tool to detect low concentrations of pollutants in wastewater that can also provide results in real-time, thus generating forms of ecological safety and social responsibility in companies where wastewater is generated.Campus Trujill