2024-03-28T17:06:06Zhttps://www.tdx.cat/oai/requestoai:www.tdx.cat:10803/4561782017-11-09T15:13:57Zcom_10803_120col_10803_130
TDX (Tesis Doctorals en Xarxa)
author
Pujol Vila, Ferran
authoremail
ferran.pujol@uab.cat
authoremailshow
true
director
Mas Gordi, Jordi
director
Muñoz Berbel, Xavier
authorsendemail
true
2017-11-09T14:35:41Z
2017-11-09T14:35:41Z
2017-09-28
9788449073632
http://hdl.handle.net/10803/456178
This thesis proposes the use of compounds that change their colour when reduced by living cells, i.e. electrochromic electron acceptors, in the development of fast and low-cost microbial toxicity bioassays suitable for in situ analysis. Electrochromism enables the monitoring of cell respiration by simple colorimetric measurement or even with the bare eye. Thus, allowing for toxicity determination without the need for complex instrumentation. Hexacyanoferrate compounds have been selected from the wide spectrum of reported electrochromic electron acceptors due to their suitable solubility and stability.
To this end, a microbial acute toxicity bioassay was developed based on metabolic reduction of ferricyanide and optical determination with standard laboratory equipment, using Escherichia coli (E. coli) as model bacterium. Interferences in the optical measurement due to biomass light scattering were minimized by dual wavelength detection at 405 nm (ferricyanide absorption and biomass scattering) and 550 nm (biomass scattering). On the other hand, modification of the refractive index (RI) of the medium until matching with refractive index of bacterial cells with (i.e. RI matching) was achieved by addition of 27% (w/v) sucrose, which reduced bacterial light scattering around 50%. The toxic impact of various compounds on E. coli was determined by analysis of ferricyanide reduction kinetics (variation of ferricyanide absorption with time) and single absorbance measurements. Kinetic analysis of bacterial ferricyanide reduction allowed for fast assays (assay time of 10 min) with half maximal effective concentrations (EC50) similar to standard methods (i.e. biolumiscence inhibition test) for organic and inorganic toxic compounds.
Technological implementation of the microbial toxicity bioassay was carried out by developing a low-cost miniaturized optofluidic analysis system. The optofluidic system was composed of a poly(methyl methacrylate) (PMMA) optofluidic structure incorporating discrete auxiliary optical elements (i.e. light emitting diodes, LEDs, and detectors) and an electronic circuit enabling for subtraction of ambient light interference. The optical performance of the analytical system for ferricyanide determination was tested. It was insensitive to environmental light changes and compared favourably to commercially available instrumentation. The simplicity, portability and robustness of the analysis system make it suitable for fast and low-cost determination of toxic pollutants in situ.
To reduce bioassay instrumentation requirements, we designed a low-cost solid system by trapping bacteria in hygroscopic paper matrices. E. coli cells were stably trapped on low-cost paper matrices (cellulose-based paper discs) and remained viable for long times (1 month when stored at -20 ºC). Apart of acting as bacterial carriers, paper matrices also acted as a fluidic element, allowing fluid management without the need of external pumps. Optical properties of individual paper matrices were analysed showing good comparability between them. Chromatic changes associated with bacterial ferricyanide reduction were determined by three different transduction methods, i.e. optical reflectometry, image analysis and visual inspection. Validation of the bioassay was performed by analysis of real samples from natural sources (i.e. wastewater influents/effluents and leachates from contaminated soils) using the mentioned transduction methods and the bioluminiscence inhibition test (Microtox, as standard method). Toxicity values obtained showed good agreement between them and with our reference method (70% of coincidence in toxic samples and 80% in non-toxic samples). The use of a light and inexpensive material and minimum instrumentation requirements of the bioassay make it a true low-cost method for in-situ assessment of toxic water pollution.
eng
Bioassajos microbians
Bioensayos microbianos
Microbial bioassays
Contamintants tòxics
Contaminantes tóxicos
Toxic pollutants
Electrónicos
Electrochromic
Electorn
Acceptor
Fast and low-cost microbial toxicity bioassays based on electrochromic electron acceptors
info:eu-repo/semantics/doctoralThesis info:eu-repo/semantics/publishedVersion
URL
https://www.tdx.cat/bitstream/10803/456178/1/fpv1de1.pdf
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fpv1de1.pdf