Toward the future of OECD/ISO biodegradability testing-new approaches and developments.

In the past decades, industrial and scientific communities have developed a complex standardized system (e.g., OECD, ISO, CEN) to evaluate the biodegradability of chemical substances. This system includes for OECD three levels of testing (ready and inherent biodegradability tests, simulation tests). It was adopted by many countries and is completely integrated into European legislation (registration, evaluation, authorization, and restriction of chemicals, REACH). Nevertheless, the different tests have certain deficiencies, and the question arises of how accurately these tests display the situation in the real environment and how the results can be used for predictions. This review will focus on the technical advantages and weaknesses of current tests concerning the technical setup, the inoculum characterization, and its biodegradation potential as well as the use of adequate reference compounds. A special focus of the article will be on combined test systems offering enhanced possibilities to predict biodegradation. The properties of microbial inocula are critically discussed, and a new concept concerning the biodegradation adaptation potential (BAP) of inocula is proposed. Furthermore, a probability model and different in silico QSAR (quantitative structure-activity relationships) models to predict biodegradation from chemical structures are reviewed. Another focus lies on the biodegradation of difficult single compounds and mixtures of chemicals like UVCBs (unknown or variable composition, complex reaction products, or biological materials) which will be an important challenge for the forthcoming decades. KEY POINTS: • There are many technical points to be improved in OECD/ISO biodegradation tests • The proper characterization of inocula is a crucial point in biodegradation tests • Combined biodegradation test systems offer extended possibilities for biodegradation tests.

Development and Application of an Automated Raman Sensor for Bioprocess Monitoring: From the Laboratory to an Algae Production Platform.

Microalgae provide valuable bio-components with economic and environmental benefits. The monitoring of microalgal production is mostly performed using different sensors and analytical methods that, although very powerful, are limited to qualified users. This study proposes an automated Raman spectroscopy-based sensor for the online monitoring of microalgal production. For this purpose, an in situ system with a sampling station was made of a light-tight optical chamber connected to a Raman probe. Microalgal cultures were routed to this chamber by pipes connected to pumps and valves controlled and programmed by a computer. The developed approach was evaluated on Parachlorella kessleri under different culture conditions at a laboratory and an industrial algal platform. As a result, more than 4000 Raman spectra were generated and analysed by statistical methods. These spectra reflected the physiological state of the cells and demonstrate the ability of the developed sensor to monitor the physiology of microalgal cells and their intracellular molecules of interest in a complex production environment.

Development and Automation of a Bacterial Biosensor to the Targeting of the Pollutants Toxic Effects by Portable Raman Spectrometer.

Water quality monitoring requires a rapid and sensitive method that can detect multiple hazardous pollutants at trace levels. This study aims to develop a new generation of biosensors using a low-cost fiber-optic Raman device. An automatic measurement system was thus conceived, built and successfully tested with toxic substances of three different types: antibiotics, heavy metals and herbicides. Raman spectroscopy provides a multiparametric view of metabolic responses of biological organisms to these toxic agents through their spectral fingerprints. Spectral analysis identified the most susceptible macromolecules in an E. coli model strain, providing a way to determine specific toxic effects in microorganisms. The automation of Raman analysis reduces the number of spectra required per sample and the measurement time: for four samples, time was cut from 3 h to 35 min by using a multi-well sample holder without intervention from an operator. The correct classifications were, respectively, 99%, 82% and 93% for the different concentrations of norfloxacin, while the results were 85%, 93% and 81% for copper and 92%, 90% and 96% for 3,5-dichlorophenol at the three tested concentrations. The work initiated here advances the technology needed to use Raman spectroscopy coupled with bioassays so that together, they can advance field toxicological testing.

Enzymatic electrochemical biosensor for glyphosate detection based on acid phosphatase inhibition.

A novel enzymatic electrochemical biosensor was fabricated for the indirect detection of glyphosate-based acid phosphatase inhibition. The biosensor was constructed on a screen-printed carbon electrode modified with silver nanoparticles, decorated with electrochemically reduced graphene oxide, and chemically immobilized with acid phosphatase via glutaraldehyde cross-linking. We measured the oxidation current by chronoamperometry. The current arose from the enzymatic reaction of acid phosphatase and the enzyme-substrate disodium phenyl phosphate. The biosensing response is a decrease in signal resulting from inhibition of acid phosphatase in the presence of glyphosate inhibitor. The inhibition of acid phosphatase by glyphosate was investigated as a reversible competitive-type reaction based on the Lineweaver-Burk equation. Computational docking confirmed that glyphosate was the inhibitor bound in the substrate-binding pocket of acid phosphatase and that it was able to inhibit the enzyme efficiently. Additionally, the established method was applied to the selective analysis of glyphosate in actual samples with satisfactory results following a standard method.

Characterization of an easy-to-use method for the routine analysis of the central metabolism using an affordable low-resolution GC-MS system: application to Arthrospira platensis.

We developed an easy-to-use method for the routine analysis of the central metabolism using an affordable low-resolution GC-MS system run in SIM mode. The profiling approach was optimized for the derivatization protocol of some 60 targeted metabolites. The performance of two silylation reagents (MSTFA and BSTFA) that allowed the comprehensive derivatization of 42 key intermediary metabolites of the 60 initially targeted (organic acids, phosphate derivatives, monosaccharides and amino acids) was measured. The experimental results unequivocally showed that the MSTFA reagent met mandatory criteria including ease of handling (a very simple one-step protocol was developed), comprehensiveness of derivatization (the 42 compounds covered the extended metabolic pathways of the central carbon metabolism, with a coverage percentage ranging from 17% for the worst to 90% for the best result), optimized response coefficient of the whole derivatives (median value greater than the others by one order of magnitude) and repeatability of the protocol (RSD value below 25% for the whole procedure). When tested in real conditions (cyanobacteria polar extract), the experimental results showed that the profiling methodology was adequately repeatable (RSD = 35%) to ensure quantification results comparable with much more sensitive analytical techniques (capillary electrophoresis/mass spectrometry and liquid chromatography/triple quadrupole mass spectrometry system), while needing only about twice the quantity of biomass. Graphical abstract Schematic overview of an easy-to-use profiling method for the routine analysis of the central metabolism using a low-resolution GC-MS system.

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.

The diversity and functions of choline sulphatases in microorganisms.

Choline sulphates have two putative roles in microorganisms: as a reservoir of C, N and S and as osmoprotectants. Although there is no established connection to date regarding the relative distribution of these two functions in microbial communities, this information is crucial in determining the role of choline sulphate in soils, particularly in cultivated soils where S is limiting. Therefore, in order to establish such a connection, the diversity of choline sulphatase (betC) genes was investigated in this study using numerous fully sequenced microbes available in GenBank. Our genomic analyses revealed unequivocally that the betICBA operon is restricted to Rhizobiaceae family members, which live under symbiotic conditions that prevent elemental depletion. Together with the uniform genetic organisation of the betICBA operon in Rhizobiaceae, BetC appears to be both utilised for osmoprotection or S replenishment. In contrast, betC in a wide variety of free-living microbes (including fungi, archaea and bacteria) was found in a cassette encoding only BetC and a choline sulphate transporter, a configuration that appears to be responsible for fulfilling elemental S requirements. Lastly, the relatively high number of BetC sequences available allowed the identification of a specific signature sequence that discriminates between these two functions and also globally defines some conserved motifs in microbial choline sulphatases. Due to the widespread presence of BetC in microbes and the wide repartition of the betC cassette system, the potential importance of choline sulphatase in global S recycling requires further clarification.

Unravelling the matrix effect of fresh sampled cells for in vivo unbiased FTIR determination of the absolute concentration of total lipid content of microalgae.

Over the past years, the substitution of the classical biochemical quantification techniques by Fourier transform infrared (FTIR) spectroscopy has been widely studied on microalgae because of its tremendous application potential for bioprocess monitoring. In the present work, mandatory aspects that have never been approached by FTIR end-users working onto fresh biomass were assessed. We demonstrated first that fresh cells' FTIR spectra main characteristics could be severely and unspecifically altered when the properties of the sampled biomass were not monitored. Microscopy indicated that important cell reorganization could occur when diminishing the cells density of the sample. Molecular probing approach suggested that such a modification could provoke an alteration of the hydrogen-bonding network of the sample. The sample heterogeneity was found to impact also the shape and intensity of the recorded FTIR bands, participating then to a matrix effect uncharacterized until now. In the second part of our study, we selected FTIR spectra not influenced by this matrix effect and the corresponding accurate calibration data obtained by the whole cell analytical procedure to elaborate an optimized total lipid quantification PLS-R model. Results demonstrated that our strategy could provide a small volume sampling (1 mL of fresh culture), rapid (within minutes), robust (physiological condition independent), and accurate (as accurate as the reference method could be) FTIR absolute quantification method to determine the fresh microalgae intracellular total lipid content. To validate our unbiased FTIR approach, a photobioprocess monitoring pipeline was developed and allowed assessing the effect of light attenuation on total lipid production by the marine microalga Nannochloropsis oculata.

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.

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.

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 II: technical development and proof of concept of the biosensor.

This research study deals with the on-line detection of heavy metals and toxicity within the context of environmental pollution monitoring. It describes the construction and the proof of concept of a multi-channel bioluminescent bacterial biosensor in immobilized phase: Lumisens3. This new versatile device, designed for the non-stop analysis of water pollution, enables the insertion of any bioluminescent strains (inducible or constitutive), immobilized in a multi-well removable card. The technical design of Lumisens3 has benefited from both a classical and a robust approach and includes four main parts: (1) a dedicated removable card contains 64 wells, 3 mm in depth, arranged in eight grooves within which bacteria are immobilized, (2) this card is incubated on a Pelletier block with a CCD cooled camera on top for bioluminescence monitoring, (3) a fluidic network feeds the card with the sample to be analyzed and finally (4) a dedicated computer interface, BIOLUX 1.0, controls all the elements of the biosensor, allowing it to operate autonomously. The proof of concept of this biosensor was performed using a set of four bioluminescent bacteria (Escherichia coli DH1 pBzntlux, pBarslux, pBcoplux, and E. coli XL1 pBfiluxCDABE) in the online detection of CdCl(2) 0.5 µM and As(2)O(3) 5 µM from an influent. When considering metals individually, the "fingerprints" from the biosensor were as expected. However, when metals were mixed together, cross reaction and synergistic effects were detected. This biosensor allowed us to demonstrate the simultaneous on-line cross detection of one or several heavy metals as well as the measurement of the overall toxicity of the sample.

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.

Study of structural changes of gluten proteins during bread dough mixing by Raman spectroscopy.

The aim of the present study was to evaluate Raman spectroscopy in determining changes that occur in the structure of gluten proteins induced during bread dough mixing. Raman spectra were measured directly within the dough. Three particular phases of mixing were studied: under-mixing, optimum mixing and over-mixing. A thiol blocking reagent, Tris(2-carboxyethyl)phosphine (TCEP) was then used to reduce disulphide bonds within proteins to confirm the important role of disulphide bridges in gluten network formation. For the control dough, the most important changes occurred during the optimum mixing phase when an increase in intermolecular disulphide bonds, anti-parallel ß-sheet and α-helix structures was observed, combined with the hydrophobic burial of tryptophan and tyrosine residues. The addition of TCEP appeared to effectively reduce the formation of intermolecular disulphide bonds, anti-parallel ß-sheet and α-helix structures and lead to a more disordered secondary protein structure.

An ultrasensitive immunosensor based on manganese dioxide-graphene nanoplatelets and core shell Fe3O4@Au nanoparticles for label-free detection of carcinoembryonic antigen.

A novel electrochemical immunosensor was developed for label-free detection of carcinoembryonic antigen (CEA) as a cancer biomarker. The designed immunosensor was based on CEA antibody (anti-CEA) anchored with core shell Fe3O4@Au nanoparticles which were immobilized on a screen-printed carbon electrode modified with manganese dioxide decorating on graphene nanoplatelets (SPCE/GNP-MnO2/Fe3O4@Au-antiCEA). The SPCE was placed onto a home-made electrode holder for easy handling. The approach was based on direct binding of CEA to a fixed amount of anti-CEA on the modified electrode for the specific detection using linear sweep voltammetry (LSV) and electrochemical impedance spectroscopy (EIS) monitored in a solution containing 5 mM [Fe(CN)63-/4-] prepared in 0.1 M phosphate buffer at pH 7.4. The difference in signal response owing to the redox reaction of [Fe(CN)6]3-/4- before and after interaction with CEA was regarded as the immunosensor response corresponding directly to the CEA concentration. Under optimized conditions, the linear range of 0.001-100 ng/mL, and the detection limits of 0.10 pg/mL (LSV) and 0.30 pg/mL (EIS) were evaluated. The applicability of the immunosensor was verified by well-corresponding determination of CEA in diluted human serum samples by electrochemiluminescence (ECL) immunoassay. Therefore, the proposed immunosensor could be suitable enough for a real sample analysis of CEA.

Degradation of a mixture of hydrocarbons, gasoline, and diesel oil additives by Rhodococcus aetherivorans and Rhodococcus wratislaviensis.

Two strains, identified as Rhodococcus wratislaviensis IFP 2016 and Rhodococcus aetherivorans IFP 2017, were isolated from a microbial consortium that degraded 15 petroleum compounds or additives when provided in a mixture containing 16 compounds (benzene, toluene, ethylbenzene, m-xylene, p-xylene, o-xylene, octane, hexadecane, 2,2,4-trimethylpentane [isooctane], cyclohexane, cyclohexanol, naphthalene, methyl tert-butyl ether [MTBE], ethyl tert-butyl ether [ETBE], tert-butyl alcohol [TBA], and 2-ethylhexyl nitrate [2-EHN]). The strains had broad degradation capacities toward the compounds, including the more recalcitrant ones, MTBE, ETBE, isooctane, cyclohexane, and 2-EHN. R. wratislaviensis IFP 2016 degraded and mineralized to different extents 11 of the compounds when provided individually, sometimes requiring 2,2,4,4,6,8,8-heptamethylnonane (HMN) as a cosolvent. R. aetherivorans IFP 2017 degraded a reduced spectrum of substrates. The coculture of the two strains degraded completely 13 compounds, isooctane and 2-EHN were partially degraded (30% and 73%, respectively), and only TBA was not degraded. Significant MTBE and ETBE degradation rates, 14.3 and 116.1 mumol of ether degraded h(-1) g(-1) (dry weight), respectively, were measured for R. aetherivorans IFP 2017. The presence of benzene, toluene, ethylbenzene, and xylenes (BTEXs) had a detrimental effect on ETBE and MTBE biodegradation, whereas octane had a positive effect on the MTBE biodegradation by R. wratislaviensis IFP 2016. BTEXs had either beneficial or detrimental effects on their own degradation by R. wratislaviensis IFP 2016. Potential genes involved in hydrocarbon degradation in the two strains were identified and partially sequenced.

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.

The ygaVP genes of Escherichia coli form a tributyltin-inducible operon.

A tributyltin (TBT) luxAB transcriptional fusion in Escherichia coli revealed that a TBT-activated promoter is located upstream of two cotranscribed orphan genes, ygaV and ygaP. We demonstrate that transcription from the promoter upstream of ygaVP is constitutive in a ygaVP mutant, suggesting that YgaV is an autoregulated, TBT-inducible repressor.

Microplate freeze-dried cyanobacterial bioassay for fresh-waters environmental monitoring.

Microorganisms have been very useful in environmental monitoring due to their constant sensing of the surrounding environment, their easy maintenance and low cost. Some freeze-dried toxicity kits based on naturally bioluminescent bacteria are commercially available and commonly used to assess the toxicity of environmental samples such as Microtox (Aliivibrio fischeri) or ToxScreen (Photobacterium leiognathi), however, due to the marine origin of these bacteria, they could not be the most appropriate for fresh-waters monitoring. Cyanobacteria are one of the most representative microorganisms of aquatic environments, and are well suited for detecting contaminants in aqueous samples. This study presents the development and application of the first freeze-dried cyanobacterial bioassay for fresh-water contaminants detection. The effects of different cell growth phases, cryoprotectant solutions, freezing protocols, rehydration solutions and incubation conditions methods were evaluated and the best combination of these parameters for freeze-drying was selected. The study includes detailed characterization of sensitivity towards reference pollutants, as well as, comparison with the standard assays. Moreover, long-term viability and sensitivity were evaluated after 3 years of storage. Freeze-dried cyanobacteria showed, in general, higher sensitivity than the standard assays and viability of the cells remained after 3 years of storage. Finally, the validation of the bioassay using a wastewater sample was also evaluated. Freeze-drying of cyanobacteria in 96-well plates presents a simple, fast and multi-assay method for environmental monitoring.