Microfluidically supported characterization of responses of Rhodococcus erythropolis strains isolated from different soils on Cu-, Ni-, and Co-stress

We present a new methodological approach for the assessment of the susceptibility of Rhodococcus erythropolis strains from specific sampling sites in response to increasing heavy metal concentration (Cu2+, Ni2+, and Co2+) using the droplet-based microfluid technique. All isolates belong to the species R. erythropolis identified by Sanger sequencing of the 16S rRNA. The tiny step-wise variation of metal concentrations from zero to the lower mM range in 500 nL droplets not only provided accurate data for critical metal ion concentrations but also resulted in a detailed visualization of the concentration-dependent response of bacterial growth and autofluorescence activity. As a result, some of the isolates showed similar characteristics in heavy metal tolerance against Cu2+, Ni2+, and Co2+. However, significantly different heavy metal tolerances were found for other strains. Surprisingly, samples from the surface soil of ancient copper mining areas supplied mostly strains with a moderate sensitivity to Cu2+, Ni2+, and Co2+, but in contrast, a soil sample from an excavation site of a medieval city that had been covered for about eight centuries showed an extremely high tolerance against cobalt ion (up to 36 mM). The differences among the strains not only may be regarded as results of adaptation to the different environmental conditions faced by the strains in nature but also seem to be related to ancient human activities and temporal partial decoupling of soil elements from the surface. This investigation confirmed that microfluidic screening offers empirical characterization of properties from same species which has been isolated from sites known to have different human activities in the past

Extremophiles in soil communities of former copper mining sites of the East Harz region (Germany) reflected by re-analyzed 16S rRNA data

The east and southeast rim of Harz mountains (Germany) are marked by a high density of former copper mining places dating back from the late 20th century to the middle age. A set of 18 soil samples from pre- and early industrial mining places and one sample from an industrial mine dump have been selected for investigation by 16S rRNA and compared with six samples from non-mining areas. Although most of the soil samples from the old mines show pH values around 7, RNA profiling reflects many operational taxonomical units (OTUs) belonging to acidophilic genera. For some of these OTUs, similarities were found with their abundances in the comparative samples, while others show significant differences. In addition to pH-dependent bacteria, thermophilic, psychrophilic, and halophilic types were observed. Among these OTUs, several DNA sequences are related to bacteria which are reported to show the ability to metabolize special substrates. Some OTUs absent in comparative samples from limestone substrates, among them Thaumarchaeota were present in the soil group from ancient mines with pH > 7. In contrast, acidophilic types have been found in a sample from a copper slag deposit, e.g., the polymer degrading bacterium Granulicella and Acidicaldus, which is thermophilic, too. Soil samples of the group of pre-industrial mines supplied some less abundant, interesting OTUs as the polymer-degrading Povalibacter and the halophilic Lewinella and Halobacteriovorax. A particularly high number of bacteria (OTUs) which had not been detected in other samples were found at an industrial copper mine dump, among them many halophilic and psychrophilic types. In summary, the results show that soil samples from the ancient copper mining places contain soil bacterial communities that could be a promising source in the search for microorganisms with valuable metabolic capabilities

Three soil bacterial communities from an archaeological excavation site of an ancient coal mine near Bennstedt (Germany) characterized by 16S r-RNA sequencing

This metagenomics investigation of three closely adjacent sampling sites from an archaeological excavation of a pre-industrial coal mining exploration shaft provides detailed information on the composition of the local soil bacterial communities. The observed significant differences between the samples, reflected in the 16S r-RNA analyses, were consistent with the archaeologically observed situation distinguishing the coal seam, the rapidly deposited bright sediment inside an exploration shaft, and the topsoil sediment. In general, the soils were characterized by a dominance of Proteobacteria , Actinobacteria , Acidobacteria , and Archaea , whereas the coal seam was characterized by the highest proportion of Proteobacteria ; the topsoil was characterized by very high proportions of Archaea —in particular, Nitrosotaleaceae —and Acidobacteria, mainly of Subgroup 2. Interestingly, the samples of the fast-deposited bright sediment showed a rank function of OTU abundances with disproportional values in the lower abundance range. This could be interpreted as a reflection of the rapid redeposition of soil material during the refilling of the exploration shaft in the composition of the soil bacterial community. This interpretation is supported by the observation of a comparatively high proportion of reads relating to bacteria known to be alkaliphilic in this soil material. In summary, these investigations confirm that metagenomic analyses of soil material from archaeological excavations can provide valuable information about the local soil bacterial communities and the historical human impacts on them

A droplet-based microfluidic platform enables high-throughput combinatorial optimization of cyanobacterial cultivation

Cyanobacteria are fast-growing, genetically accessible, photoautotrophs. Therefore, they have attracted interest as sustainable production platforms. However, the lack of techniques to systematically optimize cultivation parameters in a high-throughput manner is holding back progress towards industrialization. To overcome this bottleneck, here we introduce a droplet-based microfluidic platform capable of one- (1D) and two-dimension (2D) screening of key parameters in cyanobacterial cultivation. We successfully grew three different unicellular, biotechnologically relevant, cyanobacteria: Synechocystis sp. PCC 6803, Synechococcus elongatus UTEX 2973 and Synechococcus sp. UTEX 3154. This was followed by a highly-resolved 1D screening of nitrate, phosphate, carbonate, and salt concentrations. The 1D screening results suggested that nitrate and/or phosphate may be limiting nutrients in standard cultivation media. Finally, we use 2D screening to determine the optimal N:P ratio of BG-11. Application of the improved medium composition in a high-density cultivation setup led to an increase in biomass yield of up to 15.7%. This study demonstrates that droplet-based microfluidics can decrease the volume required for cyanobacterial cultivation and screening up to a thousand times while significantly increasing the multiplexing capacity. Going forward, microfluidics have the potential to play a significant role in the industrial exploitation of cyanobacteria

Investigation of mixture toxicity of widely used drugs caffeine and ampicillin in the presence of an ACE inhibitor on bacterial growth using droplet-based microfluidic technique

Droplet-based microfluidic technique is very suitable for a variety of screening processes. The cultivation within nanoliter segments and multidimensional microtoxicological screening of the Gram-negative bacterium Escherichia coli were studied under droplet-based bicrofluidic conditions. In order to evaluate the toxicity of the binary and ternary mixtures of antibiotic ampicillin and caffeine in the presence of the angiotensin-converting enzyme inhibitor captopril, a time-resolved optical double endpoint detection unit was applied. It included a microflow-through fluorimeter and photometer, which can simultaneously analyze changes in the endogenous autofluorescence signal and the cell density of E. coli cultivated inside 450-nl microfluid segments. As a result, strong nonlinear combination effects and a concentration-dependent antagonistic effect, as well as formation of activity summits on bolographic maps, were found. Our findings confirm the importance of multiparameter investigations for toxicological studies and could be taken into account in medical practice

Microfluidic chamber design for controlled droplet expansion and coalescence

The defined formation and expansion of droplets are essential operations for droplet-based screening assays. The volumetric expansion of droplets causes a dilution of the ingredients. Dilution is required for the generation of concentration graduation which is mandatory for many different assay protocols. Here, we describe the design of a microfluidic operation unit based on a bypassed chamber and its operation modes. The different operation modes enable the defined formation of sub-µL droplets on the one hand and the expansion of low nL to sub-µL droplets by controlled coalescence on the other. In this way the chamber acts as fluidic interface between two fluidic network parts dimensioned for different droplet volumes. Hence, channel confined droplets of about 30–40 nL from the first network part were expanded to cannel confined droplets of about 500 to about 2500 nL in the second network part. Four different operation modes were realized: (a) flow rate independent droplet formation in a self-controlled way caused by the bypassed chamber design, (b) single droplet expansion mode, (c) multiple droplet expansion mode, and (d) multiple droplet coalescence mode. The last mode was used for the automated coalescence of 12 droplets of about 40 nL volume to produce a highly ordered output sequence with individual droplet volumes of about 500 nL volume. The experimental investigation confirmed a high tolerance of the developed chamber against the variation of key parameters of the dispersed-phase like salt content, pH value and fluid viscosity. The presented fluidic chamber provides a solution for the problem of bridging different droplet volumes in a fluidic networ

Single use droplet Based Microfluidics – Screening Tools for Biotechnology and Life-Sciences

In the recent years droplet-based microfluidic techniques were developed and used for various screening purposes mainly in the field of life-sciences and biotechnology. Therefore, droplet volumes in a range from pL to μL were used and different technology platforms were necessary to produce the required fluidic devices which made of various materials. Traditionally, lithography combined with etching processes were used to produce precise glass or silicon devices. But, hot embossing and injection molding techniques are better suited for the production of single use devices. Recently, 3D-printing technologies are under development for the generation of individual costumes made microfluidic devices in low numbers for reasonable prices. Here, we present an example for a complex droplet based screening network designed for bacterial screening purposes received by CD-production technologies. General principles of droplet based processing, fluidic devise units and peripheral analytical techniques will be introduced as well as details for the screening of soil bacteria will be given. As an outlook the future microfluidic techniques for monitoring or continuous sampling form large volume bioprocesses will be given

From microtiter plates to droplets - there and back again

Droplet-based microfluidic screening techniques can benefit from interfacing established microtiter plate-based screening and sample management workflows. Interfacing tools are required both for loading preconfigured microtiter-plate (MTP)-based sample collections into droplets and for dispensing the used droplets samples back into MTPs for subsequent storage or further processing. Here, we present a collection of Digital Microfluidic Pipetting Tips (DMPTs) with integrated facilities for droplet generation and manipulation together with a robotic system for its operation. This combination serves as a bidirectional sampling interface for sample transfer from wells into droplets (w2d) and vice versa droplets into wells (d2w). The DMPT were designed to fit into 96-deep-well MTPs and prepared from glass by means of microsystems technology. The aspirated samples are converted into the channel-confined droplets’ sequences separated by an immiscible carrier medium. To comply with the demands of dose-response assays, up to three additional assay compound solutions can be added to the sample droplets. To enable different procedural assay protocols, four different DMPT variants were made. In this way, droplet series with gradually changing composition can be generated for, e.g., 2D screening purposes. The developed DMPT and their common fluidic connector are described here. To handle the opposite transfer d2w, a robotic transfer system was set up and is described briefly