Luca Bolelli

Improved detection of toxic chemicals using bioluminescent bacteria

Abstract A sensitive, rapid and simple bioluminescent (BL) assay using bioluminescent bacteria to detect the toxic activity of several chemicals is described. This assay is based on the measurement of inhibition of light production of a bioluminescent bacterial strain, isolated from seawater, in the presence of different toxins like heavy metals, organic chemicals, such as benzene, toluene, ethylbenzene, xylene (BTEX) and a wide range of pesticides in environmental samples. The improvement with respect to other commercial and non-commercial bioluminescent assays consists of the possibility to work at room temperature without the need to thermostat, thus allowing the use of simpler and low cost instruments, or to improve the assay using a microplate format, which makes it possible to analyse several samples also continuously for several hours. Using lyophilised bacteria, the assay is performed in less than an hour, without any bacterial cultivation, which makes the test suitable for rapid and sensitive evaluation of chemical pollutants in environmental samples.

COMPARISON OF ANALYTICAL METHODS IN DETERMINING TOTAL ANTIOXIDANT CAPACITY IN RED WINE

ABSTRACT Total antioxidant capacity was measured in red wine samples by two analytical methods: chemiluminescent (CL) assay based on the luminol/peroxidase system and a spectrophotometric (SP) method based on the use of crocin. The imprecision, expressed as coefficient of variation (CV) for each method, was similar (1.2%), whereas the detection limit (LOD) was 35 and 0.3 µmol of Trolox for SP and CL methods, respectively. In addition, the chemiluminescent method has the advantage of being simple, rapid, sensitive and inexpensive. Different samples of red wine were analysed and both methods showed that the antioxidant capacity is related to the total polyphenol content and varies over time depending on storage conditions.

Optimal conditions for stability of photoemission and freeze drying of two luminescent bacteria for use in a biosensor

Bioluminescent bacteria have been used for many years for biotoxicological analysis. One of the main concerns with this microorganism is the low experimental repeatability when subjected to external factors. The aim of the present study was to obtain accurate, sensitive, and repeatable measurements with stable signals (during the detection and over days) for application in a water-analysis device for the detection of pollutants. Growth conditions were tested and optimized. An optimal freeze-drying procedure for the constitutive bioluminescent bacteria Vibrio fischeri and Photobacterium phosphoreum was developed. The luminescence stability after rehydration was also investigated. Freeze drying was found to be a critical process in survival and signal stability of luminescent bacteria; for this reason, different suspension fluids and various bacterial pellet/suspension fluid ratios (g/ml) were evaluated. The toxicity of heavy metals and organic compounds in water was determined to investigate the applicability of a test based on bacteria obtained in this way, comparing the data with legal limits. A scale-up process was developed with industrial technology: freeze-dried bacteria that emitted a stable luminous signal after rehydration were obtained. Moreover, the median effective concentration (EC50) was calculated with these bacteria.

Automated and manual luminescent assay of antioxidant capacity: analytical features by comparison

The analytical performances of a manual and a partially automated chemiluminescent (CL) assay, of total antioxidant capacity (TAC) were assessed. In both cases the light emitting reaction involved luminol, horseradish peroxidase and hydrogen peroxyde, but the emission kinetics and the parameters taken into account to calculate TAC values were completely different. The major characteristics expressing the quality of the two analytical methods, i.e. inaccuracy, repeteability and reproducibility, sensitivity, time required for the analysis and detection limit, were estimated by using standard solutions of Trolox. The reliability of the automated method, in comparison with the more validated manual one, was demonstrated testing food samples such as honey, wine and dietary supplements and performing a statistical analysis of the results. The comparison of the two series of data by t-test resulted in p values in the range 0.1-0.01. The time required for the analysis of each sample was reduced to one third using the automated method.

Trace analysis of pollutants by use of honeybees, immunoassays, and chemiluminescence detection

AbstractSpecific and sensitive analysis to reveal and monitor the wide variety of chemical contaminants polluting all environment compartments, feed, and food is urgently required because of the increasing attention devoted to the environment and health protection. Our research group has been involved in monitoring the presence and distribution of agrochemicals by monitoring beehives distributed throughout the area studied. Honeybees have been used both as biosensors, because the pesticides affect their viability, and as “contaminant collectors” for all environmental pollutants. We focused our research on the development of analytical procedures able to reveal and quantify pesticides in different samples but with a special attention to the complex honeybee matrix. Specific extraction and purification procedures have been developed and some are still under optimization. The analytes of interest were determined by gas or liquid chromatographic methods and by compound-specific or group-specific immunoassays in the ELISA format, the analytical performance of which was improved by introducing luminescence detection. The range of chemiluminescent immunoassays developed was extended to include the determination of completely different pollutants, for example explosives, volatile organic compounds (including benzene, toluene, ethylbenzene, xylenes), and components of plastics, for example bisphenol A. An easier and portable format, a lateral flow immunoassay (LFIA) was added to the ELISA format to increase application flexibility in these assays. Aspects of the novelty, the specific characteristics, the analytical performance, and possible future development of the different chromatographic and immunological methods are described and discussed. FigMap of a territory monitored by using honeybees, showing the areas covered by each beehive station (circles) and thedifferent agricultural products included in it (different colours).

Bioremediation of hydrocarbons contaminated waters and soils: monitoring by luminescent bacteria test

Bioremediation has proven successful in numerous applications to petroleum hydrocarbons or chlorinated aromatic hydrocarbons contaminated soils. There is increasing interest in application of biotoxicity tests for ecological assessment and for supporting management decisions for remediation. Luminescent assays, light-emitting bacteria in particular, can be a suitable tool for environmental analysis, and in vivo luminescence is a rapid and precise indicator of the toxic effects of xenobiotic on micro-organisms. In this study, three different strains of marine bioluminescent bacteria have been employed to follow the changes in biotoxicity occurring during the laboratory scale bioremediation of water and soil samples contaminated by hydrocarbons and collected at an industrial area. The degradation was made by hydrocarbons degrading bacteria, both of commercial sources and isolated from polluted water and soils. The samples were treated for 45 days. The toxicity of the samples, before and after the bioremediation, was determined directly on water samples or on the extracts of soil samples. The yield of extraction by different solvents (acetone, dioxane, ethanol and dichloromethane) was evaluated by the bioluminescent test. The measurements were carried out using a microplate format both for short time of contact (60 minutes, acute toxicity) and for longer time intervals (24 hours, chronic toxicity). The results have been expressed as percentage of inhibition with respect to the blank emission (100% emission). Original and treated samples have been analysed by gas chromatography to assess the hydrocarbons (C > 12 and Poly Chlorinated Biphenyls, PCB) content. The autochthonous bacteria isolated from polluted samples proved less effective, due to the short time for selection in remediation activity with respect to the commercial ones, but their capacity to degrade long chain hydrocarbons was satisfactory. The presented laboratory study can be applied also in case of on-field conditions.

Airborne particulate matter biotoxicity estimated by chemometric analysis on bacterial luminescence data

In this work, PM10 samples previously subjected to thorough chemical speciation and receptor modelling, have been investigated for their bio-toxicity using an inhibition test based on bacterial luminescence modulation when in contact with airborne particulate samples. The variation of light emission intensity from a luminescent bacteria strain, the Photobacterium phosphoreum, is proposed as an efficient proxy for the quantification of bio-toxic effects induced by airborne particulate matter. PM10 samples characterized by definite levels of pollutants from the pertaining air shed were found to induce a decrease in the bacterial bioluminescence intensity, expressed as percentage of Inhibition Ratio (IR%). This behaviour suggests the decay of this energy-consuming activity because of a toxic effect. Cluster analysis on chemical composition and IR% data provides evidence of a statistically significant association between the adverse effects on living cells and the range of specific chemical species in PM10.

Checking syrup adulteration of honey using bioluminescent bacteria and chemometrics

Accomplishing the Italian law to verify honey quality is onerous, because it requires measuring many chemical and physical parameters. On the contrary, bioluminescence-based analytical methods allow for rapid and inexpensive analysis. Bioluminescence has never been applied before to verify honey adulteration. The application of chemometrics to analytical methods based on bioluminescence has been here explored for this scope. Several honey samples were prepared, in which sugar syrup was added without exceeding legal limits: in this case, univariate analysis prescribed by the law cannot reveal the fraud. All samples were subjected to measurements of parameters prescribed by the law and also to bioluminescence analysis, executed using the Vibrio fischeri bacterium, one of the most common bioluminescent bacteria. Principal components analysis, linear discriminant analysis, and partial least square regression were applied to discriminate sugar-added honeys with respect to natural honeys, both by regulated physicochemical parameters and by bioluminescence ones. The feasibility of combining bioluminescence and multivariate analysis for a rapid screening of honey authenticity was demonstrated.

Control of microbial contamination in drinking water from microfiltering dispensers by dialysis ultrafilters

Abstract Tap water filtering devices are widely employed to improve odor and taste of tap water, or to obtain refrigerated or sparkling drinking water. The presence of disinfectants-resistant bacteria in tap water is responsible of the biofilm formation inside tubes and tanks. The consequent contamination of dispensed water is a well-known hygiene problem because of the quite constant presence of potentially pathogenic bacteria likes P. aeruginosa. In this study, we tested the technical feasibility and effectiveness of the addition to different commercial devices of a packaged polysulphone fibers filter. We aimed to find a simple solution to implement the quality of the delivered water. Water contamination levels were determined in a wide selection of microfiltered water dispensers and we selected among them a representative group of 10 devices, new or in use. The packaged ultrafilter was introduced in about half of them, to monitor, when possible, in parallel the contamination levels and flow rate of a couple of identical units, with and without the filter. The placement of the dialysis filters resulted feasible at different positions along the water circuits of the variously designed filtration units. Delivered water resulted completely free from bacteria when the filter was placed exactly at, or very close to, the outlet in spite of the inner surfaces contamination. This performance was not obtained in presence of a more or less long tract of water circuits downstream the ultrafilter: a significant but not complete reduction of the plate count numbers was observed. The filters worked in continue over the whole study period, ten months, showing exactly the same efficiency. Moreover, the flow rate in presence of the filter was quite unaffected. The addition of this kind of filter to already in use water dispensers was technically easy, and its use can be recommended in all cases a simple but reliable water sanitization is requested.