The technique of microsegmented flow was applied for the generation of two- and higher dimensional concentration spaces for the screening of toxic effects of selected substances on the bacterium Escherichia coli at the nanolitre scale. Up to about 5000 distinct experiments with different combinations of effector-concentrations could be realized in a single experimental run. This was done with the help of a computer program controlling the flow rates of effector-containing syringe pumps and resulted in the formation of multi-dimensional concentration spaces in segment sequences. Prior to the application of this technique for toxicological studies on E. coli the accuracy of this method was tested by simulation experiments with up to five dissolved dyes with different spectral properties. Photometric microflow-through measurement of dye distribution inside the concentration spaces allowed the monitoring of microfluid segment compositions. Finally, we used this technique for the investigation of interferences of the antibiotics ampicillin and chloramphenicol towards E. coli cultures and their modulation by silver nanoparticles by measuring bacterial autofluorescence. Each concentration point in this three-dimensional concentration space was represented by 4 or 5 single segments. Thus, a high reliability of the measured dose/response relations was achieved. As a result, a complex response pattern was discovered including synergistic and compensatory effects as well as the modulation of the range of stimulation of bacterial growth by a sublethal dose of chloramphenicol by silver nanoparticles.
International audienceThe cultivation of the monocellular green alga Chlorella vulgaris was implemented into microfluid segments to demonstrate the possibility of an automated screening of toxic effects of the common algaecide CuCl2. Therefore, the nutritional as well as light and carbon dioxide requirements of the algae had to be adapted to the microfluidic device. Generally, sequences of about 350 fluid segments with single volumes of about 500nL were applied for the doseresponse experiments. The growth of algae cultures inside microfluidic segments was non-invasively measured by microflow through techniques using two different optical channels. A multi-endpoint detection was realized by the photometric characterization of cell density by transmission measurements and the measurement of density of autofluorescent cells. The different methods revealed comparable half maximal effective concentrations (EC50) in the range between 34.6 and 39.9 mu g/mL for the toxicity of CuCl2 to the green algae C. vulgaris. By reference experiments in microtiter plates lower EC50 were achieved presumably caused by increased alkalinity of the growth medium due to higher photosynthesis. The results show that the microsegmented flow technique is well suited for the automated determination of dose/response functions for microorganisms like C. vulgaris and for the application of multi-endpoint procedures at the nanoliter scale
The cultivation and growth behavior of metal-tolerant strains of Streptomyce acidiscabies E13 and Streptomyces sp. F4 were studied under droplet-based microfluidics conditions. It was shown that the technique of micro segmented flow is well suited for the investigation of dependence of bacterial growth on different concentrations of either single metal ions or combinations of them. This study confirms higher tolerance to Zn than to Cu by our test organism. The highly resolved dose-response curves reflect two transitions between the different growth behaviors, separating initial responses to Cu concentration ranges into those with (a) intense growth, (b) moderate growth, and (c) growth inhibition. For Streptomyces sp. F4, an initial stimulation was shown in the sublethal range of zinc sulfate. Two-dimensional screenings using computer-controlled fluid actuation and in situ micro flow-through fluorimetry reflected a strong growth stimulation of strain F4 by zinc sulfate in the presence of sublethal Cu concentrations. This stimulatory effect on binary mixtures may be useful in providing optimal growth conditions in bioremediation procedures.
The combination of micro-segmented flow with miniaturized flow-through multisensor-technology has been utilized for metabolite profiling of soil bacteria. Series of sub-μl segments were generated containing soil sample slurry from historic copper mining sites and exposed to heavy metal salts of copper and nickel. Segments were examined for bacterial growth and spectral properties as well as for the effect of heavy metal-treatment after different incubation times. In order to evaluate microbial growth, extinction was recorded with 4 different spectral channels. Fluorescence was measured using a microflow-through fluorometer to detect both growth and production of fluorescent dyes or metabolites. The incidence of single segments with enhanced absorption in one of the spectral channels or enhanced fluorescence was scored to detect soil microorganisms with interesting properties for further screening. The study could show that the number of vegetated segments, the density of microorganisms in the segments after cultivation and the spectral response are different for separate soil samples and different metals. Thus, the highly parallelized and miniaturized segmented flow method is a promising tool for profiling of soil samples with regard to identifying micro-organisms with interesting profiles for secondary metabolite-production.
Summary1. One of the most important constituents in soil is the microflora, mainly containing bacteria and fungi with high metabolic versatility and very complex intra-and interspecific interactions. Co-occurrence of several microorganism species in soil regulates growth or suppression of single species, either by mutual tolerance or by induction of defence mechanisms, which may result in the release of secondary metabolites for growth suppression of coexisting species. Accumulations of heavy metals in soils can further affect the growth of soil microbial communities; this however is strongly dependent on the capability of micro-organisms to tolerate heavy metals. Until now, there is no fast and reliable method available to study the growth of microbial communities in highly resolved concentration spaces with environmentally relevant toxic substances such as heavy metals and to identify the tolerance thresholds of micro-organism communities of selected soils. 2. Here, we present a new methodological approach for the assessment of the growth-response behaviour of soil microbial communities in response to increasing heavy metal concentration (copper) using the droplet-based micro-segmented flow technique. Therefore, micro-organism-containing soil slurries from contact with metal artefacts from archaeological excavations and from the surface of early copper-mining areas were studied by separate cultivation in segments in the sub-lL range and growth, and fluorescence was characterized after cultivation by combined micro-flow-through photometry and fluorimetry. 3. Highly resolved dose-response data provided copper tolerance thresholds of the soil communities of the different soils. Concentration-dependent growth patterns of the micro-organisms in the segments could be observed and allowed to distinguish response groups with characteristic distribution of photometric and fluorimetric measurement values. 4. It is assumed that these response groups are caused by a sample characteristic growth of metal-tolerant microbial communities with characteristic critical metal concentrations for growth inhibition. The clear transitions between the groups in small concentration intervals are probably due to sharp transitions between growth and no growth of dominant micro-organism species at the critical metal concentration. The investigations demonstrate the potential of droplet-based microfluidic techniques for ultra-miniaturized ecological studies and its suitability for the assessment of tolerance thresholds of soil microbial communities from heavy metal-contaminated areas.
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