Screening of metallic pollution in complex environmental samples through a transcriptomic fingerprint method.

Characterizing waste ecotoxicity is laborious because of both the undefined nature of environmental samples and the diversity of contaminants that can be present. With regard to these limitations, traditional approaches do not provide information about the nature of the pollution encountered. To improve such assessments, a fluorescent library of 1870 transcriptomic reporters from Escherichia coli K12 MG1655 was used to report the ecotoxic status of environmental samples. The reliability of the approach was evaluated with 6 metallic pollutants (As, Cu, Cd, Hg, Pb, Zn) used alone and in mixture in pure and complex matrices. A total of 18 synthetic samples were used to characterize the specificity of the resulting metallic contamination fingerprints. Metallic contamination impacted 4.5 to 10.2% of the whole transcriptomic fingerprint of E. coli. The analysis revealed that a subset of 175 transcriptomic reporters is sufficient to characterize metallic contamination, regardless of the nature of the sample. A statistical model distinguished patterns due to metallic contamination and provided information about the level of toxicity with 93 to 98% confidence. The use of the transcriptomic assessment was validated for 17 complex matrices with various toxicities and metal contaminants, such as activated sludge, wastewater effluent, soil, wood and river water. The presence of metals and their associated toxicity, which seems linked to their bioavailabilities, were thereby determined. This method constitutes a possible tool to screen unknown complex samples for their metallic status and identify those for which a deeper characterization must be achieved by the use of traditional biosensors and analytical methods.

Rapid BOD assessment with a microbial array coupled to a neural machine learning system.

The domestic usage of water generates approximately 310 km3 of wastewater worldwide (2015, AQUASTAT, Food and Agriculture Organization of United Nations). This sewage contains an important organic load due to the use of this water; this organic load is characterized using a standard method, namely, the biological oxygen demand measurement (BOD5). The BOD5 provides information about the biodegradable organic load (standard ISO 5815). However, this measurement protocol is very time-consuming (5 days) and may produce variability in approximately 20% of results mainly due to variation in the environmental inocula. To remedy these limitations, this work proposes an innovative concept relying on the implementation of a set of rigorously selected bacterial strains. This publication depicts the different steps used in this study, from bio-indicator selection to validation with real wastewater samples. The results obtained in the final step show a strong correlation between the developed approach and the reference method (ISO 5815) with a correlation rate of approximately 0.9. In addition, the optimization of the experimental conditions and the use of controlled strains (8 selected strains) allow significant reduction in the duration of the BOD5 analysis, with only 3 h required for the proposed method versus 5 days for the reference method. This technological breakthrough should simplify the monitoring of wastewater treatment plants and provide quicker, easier and more coherent control in terms of the treatment time.

Numerical modeling of the dynamic response of a bioluminescent bacterial biosensor.

Water quality and water management are worldwide issues. The analysis of pollutants and in particular, heavy metals, is generally conducted by sensitive but expensive physicochemical methods. Other alternative methods of analysis, such as microbial biosensors, have been developed for their potential simplicity and expected moderate cost. Using a biosensor for a long time generates many changes in the growth of the immobilized bacteria and consequently alters the robustness of the detection. This work simulated the operation of a biosensor for the long-term detection of cadmium and improved our understanding of the bioluminescence reaction dynamics of bioreporter bacteria inside an agarose matrix. The choice of the numerical tools is justified by the difficulty to measure experimentally in every condition the biosensor functioning during a long time (several days). The numerical simulation of a biomass profile is made by coupling the diffusion equation and the consumption/reaction of the nutrients by the bacteria. The numerical results show very good agreement with the experimental profiles. The growth model verified that the bacterial growth is conditioned by both the diffusion and the consumption of the nutrients. Thus, there is a high bacterial density in the first millimeter of the immobilization matrix. The growth model has been very useful for the development of the bioluminescence model inside the gel and shows that a concentration of oxygen greater than or equal to 22 % of saturation is required to maintain a significant level of bioluminescence. A continuous feeding of nutrients during the process of detection of cadmium leads to a biofilm which reduces the diffusion of nutrients and restricts the presence of oxygen from the first layer of the agarose (1 mm) and affects the intensity of the bioluminescent reaction. The main advantage of this work is to link experimental works with numerical models of growth and bioluminescence in order to provide a general purpose model to understand, anticipate, or predict the dysfunction of a biosensor using immobilized bioluminescent bioreporter in a matrix.

Design of a toxicity biosensor based on Aliivibrio fischeri entrapped in a disposable card.

The degradation of the marine environment is a subject of concern for the European authorities primarily because of its contamination by hydrocarbons. The traditional methods (ISO 11348 standard) of general toxicity assessment are unsuitable in a context of in situ monitoring, such as seaports or bathing zones. Consequently, to address this issue, bacterial biosensors appear to be pertinent tools. This article presents the design of an innovative bioluminescent biosensor dedicated to in situ toxicity monitoring. This biosensor is based on the entrapment of the wild marine bioluminescent bacterial strain Aliivibrio fischeri ATCC® 49387™ in an agarose matrix within a disposable card. A pre-study was needed to select the most biological parameters. In particular, the regenerating medium's composition and the hydrogel concentration needed for the bacterial entrapment (mechanical resistance) were optimized. Based on these data, the ability of the bacterial reporter to assess the sample toxicity was demonstrated using naphthalene as a chemical model. The biosensor's results show a lower sensitivity to naphthalene (EC50 = 95 mg/L) compared with the results obtained using the reference method (EC50 = 43 mg/L). With this architecture, the biosensor is an interesting compromise among low maintenance, ease of use, appropriate sensitivity, relatively low cost and the ability to control online toxicity.

High throughput and miniaturised systems for biodegradability assessments.

The society demands safer products with a better ecological profile. Regulatory criteria have been developed to prevent risks for human health and the environment, for example, within the framework of the European regulation REACH (Regulation (EC) No 1907, 2006). This has driven industry to consider the development of high throughput screening methodologies for assessing chemical biodegradability. These new screening methodologies must be scalable for miniaturisation, reproducible and as reliable as existing procedures for enhanced biodegradability assessment. Here, we evaluate two alternative systems that can be scaled for high throughput screening and conveniently miniaturised to limit costs in comparison with traditional testing. These systems are based on two dyes as follows: an invasive fluorescent dyes that serves as a cellular activity marker (a resazurin-like dye reagent) and a noninvasive fluorescent oxygen optosensor dye (an optical sensor). The advantages and limitations of these platforms for biodegradability assessment are presented. Our results confirm the feasibility of these systems for evaluating and screening chemicals for ready biodegradability. The optosensor is a miniaturised version of a component already used in traditional ready biodegradability testing, whereas the resazurin dye offers an interesting new screening mechanism for chemical concentrations greater than 10 mg/l that are not amenable to traditional closed bottle tests. The use of these approaches allows generalisation of high throughput screening methodologies to meet the need of developing new compounds with a favourable ecological profile and also assessment for regulatory purpose.

Online detection of metals in environmental samples: comparing two concepts of bioluminescent bacterial biosensors.

In this study, we compared two bacterial biosensors designed for the environmental monitoring of metals: Lumisens III and Lumisens IV. These two biosensors are based on the same bacterial sensors (inducible or constitutive bacterial strains) but with a different conservation mode. The results showed that the biosensor Lumisens III using immobilized cells in agarose hydrogel, allowed to detect artificial mercury contaminations on the limited period of 7 days in laboratory conditions with a reproducibility of 40%. With environmental samples, bioluminescence of the immobilized bacteria inside the biosensor was strongly limited by the environmental microflora because of the lack of oxygen, limiting the use of the biosensor to 2 days. The biosensor of the last generation, Lumisens IV, using freeze-dried bacteria in a disposable card allowed a stable detection during 10 days with 3% of reproducibility of the bioluminescence signal both in laboratory conditions and environmental samples. One analysis was performed in only 90 min against 360 min for Lumisens III. Nevertheless, the lack of specificity of the promoter, which regulates the bioluminescent reporter genes, limits the metal detection. We addressed the problem by using Lumisens IV and a data analysis software namely Metalsoft, developed in previous works. Thanks to this analytical software, Lumisens IV was a reliable online biosensor for the multidetection of Cd, As, Hg, and Cu.

Improvement of the identification of four heavy metals in environmental samples by using predictive decision tree models coupled with a set of five bioluminescent bacteria.

A primary statistical model based on the crossings between the different detection ranges of a set of five bioluminescent bacterial strains was developed to identify and quantify four metals which were at several concentrations in different mixtures: cadmium, arsenic III, mercury, and copper. Four specific decision trees based on the CHAID algorithm (CHi-squared Automatic Interaction Detector type) which compose this model were designed from a database of 576 experiments (192 different mixture conditions). A specific software, 'Metalsoft', helped us choose the best decision tree and a user-friendly way to identify the metal. To validate this innovative approach, 18 environmental samples containing a mixture of these metals were submitted to a bioassay and to standardized chemical methods. The results show on average a high correlation of 98.6% for the qualitative metal identification and 94.2% for the quantification. The results are particularly encouraging, and our model is able to provide semiquantitative information after only 60 min without pretreatments of samples.

A multi-channel bioluminescent bacterial biosensor for the on-line detection of metals and toxicity. Part I: design and optimization of bioluminescent bacterial strains.

This study describes the construction of inducible bioluminescent strains via genetic engineering along with their characterization and optimization in the detection of heavy metals. Firstly, a preliminary comparative study enabled us to select a suitable carbon substrate from pyruvate, glucose, citrate, diluted Luria-Bertani, and acetate. The latter carbon source provided the best induction ratios for comparison. Results showed that the three constructed inducible strains, Escherichia coli DH1 pBzntlux, pBarslux, and pBcoplux, were usable when conducting a bioassay after a 14-h overnight culture at 30 °C. Utilizing these sensors gave a range of 12 detected heavy metals including several cross-detections. Detection limits for each metal were often close to and sometimes lower than the European standards for water pollution. Finally, in order to maintain sensitive bacteria within the future biosensor-measuring cell, the agarose immobilization matrix was compared to polyvinyl alcohol (PVA). Agarose was selected because the detection limits of the bioluminescent strains were not affected, in contrast to PVA. Specific detection and cross-detection ranges determined in this study will form the basis of a multiple metals detection system by the new multi-channel Lumisens3 biosensor.