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Köhler, Michael; Ehrhardt, Linda; Günther, Mike
Archaeal and extremophilic bacteria from different archaeological excavation sites. - In: International journal of molecular sciences, ISSN 1422-0067, Bd. 24 (2023), 6, 5519, S. 1-18

Beside natural factors, human activities are important for the development of microbiomes. Thus, local soil bacterial communities are affected by recent activities such as agriculture, mining and industry. In addition, ancient human impacts dating back centuries or millennia have changed soils and can emboss the recent bacterial communities up to now, representing a certain long-term "memory of soil". Soil samples from five different archaeological excavation places were investigated for the presence of Archaea with a Next Generation Sequencing (NGS) analysis of the DNA coding for 16S r-RNA sequences. It was found that the abundance of Archaea differs strongly between less than one and more than 40 percent of bacteria. A Principal Component Analysis (PCA) of all samples shows that the archaeological excavation places can be distinguished from each other by the archaeal component of soil bacterial communities, which presents a typical pattern for each place. Most samples are marked by the dominance of Crenarchaeota, which are presented mainly by ammonia-related types. High contents of Nanoarchaeaota have been observed in one ash deposit of a historical saline and all samples of a historical tannery area. These samples are also marked by a significant presence of Dadabacteria. The specific abundancies of special Archaea - among them ammonia-oxidizing and sulphur-related types - are due obviously to former human activities and support the concept of the "ecological memory of soil".

Xie, Ting; Köhler, Michael; Heyder, Stefan; Günther, Mike; Cao-Riehmer, Jialan
Microfluidically-assisted isolation and characterization of Achromobacter spanius from soils for microbial degradation of synthetic polymers and organic solvents. - In: Environments, ISSN 2076-3298, Bd. 9 (2022), 12, 147, S. 1-17

A micro segmented-flow approach was utilized for the isolation soil bacteria that can degrade synthetic polymers as polyethylene glycols (PEG) and polyacrylamide (PAM). We had been able to obtain many strains; among them, five Achromobacter spanius strains from soil samples of specific sampling sites that were connected with ancient human impacts. In addition to the characterization of community responses and isolating single strains, this microfluidic approach allowed for investigation of the susceptibility of Achromobacter spanius strains against three synthetic polymers, including PEG, PAM, and Polyvinylpyrrolidone (PVP) and two organic solvents known as 1,4-dioxane and diglyme. The small stepwise variation of effector concentrations in 500 nL droplets provides a detailed reflection of the concentration-dependent response of bacterial growth and endogenous autofluorescence activity. As a result, all five strains can use PEG600 as carbon source. Furthermore, all strains showed similar dose-response characteristics in 1,4-dioxane and diglyme. However, significantly different PAM- and PVP-tolerances were found for these strains. Samples from the surface soil of prehistorical rampart areas supplied a strain capable of degradation of PEG, PVP, and PAM. This study demonstrates on the one hand, the potential of microsegment flow for miniaturized dose-response screening studies and its ability to detect novel strains, and on the other hand, two of five isolated Achromobacter spanius strains may be useful in providing optimal growth conditions in bioremediation and biodegradation processes.

Khan, Nida Zaman; Martin, Daniel; Pliquett, Uwe; Zaikou, Yahor; Thomas, Nacke; Heinrich, Doris; Köhler, Michael; Nguyen, Thi-Huong
High-frequency contactless sensor for the detection of Heparin-induced thrombocytopenia antibodies via platelet aggregation. - In: International journal of molecular sciences, ISSN 1422-0067, Bd. 23 (2022), 22, 14395, insges. 13 S.

Heparin-induced thrombocytopenia (HIT), a severe autoimmune disorder, occurs in patients undergoing heparin therapy. The presence of platelet-activating antibodies against platelet factor 4/Heparin in the blood confirms patients suffering from HIT. The most widely used methods for HIT diagnosis are immunoassays but the results only suit to rule out HIT as the assays provide only around 50% specificity. To confirm HIT, samples with positive results in immunoassays are retested in functional assays (>98% specificity) that track platelet-activating antibodies via platelet aggregation. However, the protocols in functional assays are either time-consuming (due to the requirement of the detection of serotonin release) or require highly trained staff for the visualization of platelets. Here, we applied a cheap and easy-to-use contactless sensor, which employs high-frequency microwaves to detect the changes in the resonant frequency caused by platelet aggregation/activation. Analysis of change in conductivity and permittivity allowed us to distinguish between HIT-like (KKO) and non-HIT-like (RTO) antibodies. KKO caused a stronger reduction of conductivity of platelet samples than RTO. Our results imply that the high-frequency contactless sensor can be a promising approach for the development of a better and easier method for the detection of HIT.

Richter, Felix; Chen, Minqian; Schaub, Patrick; Wüst, Florian; Zhang, Di; Schneider, Steffen; Groß, Gregor Alexander; Mäder, Patrick; Dovzhenko, Oleksandr; Palme, Klaus; Köhler, Michael; Cao-Riehmer, Jialan
Induction of embryogenic development in haploid microspore stem cells in droplet-based microfluidics. - In: Lab on a chip, ISSN 1473-0189, Bd. 22 (2022), 22, S. 4292-4305

This work presents the application of droplet-based microfluidics for the cultivation of microspores from Brassica napus using the doubled haploid technology. Under stress conditions (e.g. heat shock) or by chemical induction a certain fraction of the microspores can be reprogrammed and androgenesis can be induced. This process is an important approach for plant breeding because desired plant properties can be anchored in the germline on a genetic level. However, the reprogramming rate of the microspores is generally very low, increasing it by specific stimulation is, therefore, both a necessary and challenging task. In order to accelerate the optimisation and development process, the application of droplet-based microfluidics can be a promising tool. Here, we used a tube-based microfluidic system for the generation and cultivation of microspores inside nL-droplets. Different factors like cell density, tube material and heat shock conditions were investigated to improve the yield of vital plant organoids. Evaluation and analysis of the stimuli response were done on an image base aided by an artificial intelligence cell detection algorithm. Droplet-based microfluidics allowed us to apply large concentration programs in small test volumes and to screen the best conditions for reprogramming cells by the histone deacetylase inhibitor trichostatin A and for enhancing the yield of vital microspores in droplets. An enhanced reprogramming rate was found under the heat shock conditions at 32 &ring;C for about 3 to 6 days. In addition, the comparative experiment with MTP showed that droplet cultivation with lower cell density (<10 cells per droplet) or adding media after 3 or 6 days significantly positively affects the microspore growth and embryo rate inside 120 nL droplets. Finally, the developed embryos could be removed from the droplets and further grown into mature plants. Overall, we demonstrated that the droplet-based tube system is suitable for implementation in an automated, miniaturized system to achieve the induction of embryogenic development in haploid microspore stem cells of Brassica napus.

Cao-Riehmer, Jialan; Russo, David A.; Xie, Ting; Groß, Gregor Alexander; Zedler, Julie
A droplet-based microfluidic platform enables high-throughput combinatorial optimization of cyanobacterial cultivation. - In: Scientific reports, ISSN 2045-2322, Bd. 12 (2022), 15536, insges. 12 S.

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.

Cao-Riehmer, Jialan; Pliquett, Uwe; Yang, Lin; Wiedemeier, Stefan; Cahill, Brian; Köhler, Michael
Contactless optical and impedimetric sensing for droplet-based dose-response investigations of microorganisms. - In: Sensors and actuators, ISSN 0925-4005, Bd. 372 (2022), 132688

The principle of droplet-based microfluidics was used for the characterization of dose/response functions of the soil bacteria Rhodococcus sp. and Chromobacterium vaccinii using a combination of optical and electrical sensors for the detection of bacterial growth and metabolic activity. For electrical characterization, a micro flow-through impedance module was developed which assessed the response of bacterial populations inside 500 nL fluid segments without direct galvanic contact between the electrodes and the electrolyte. It was found that the impedance sensor can detect an increase in cell density and is particularly suited for monitoring the metabolic response due to changes in the cultivation medium inside the separated fluid segments. Due to this sensitivity, the sensor is useful for investigating growing bacteria or cell cultures in small fluid compartments and obtaining highly resolved dose-response functions by microfluid segment sequences. The impedimetric data agree well with the optical data concerning the characteristic response of bacteria populations in the different concentration regions of heavy metal ions. However, the sensor supplies valuable complementary data on metabolic activity in case of low or negligible cell division rates.

Ehrhardt, Linda; Günther, Mike; Böhme, Manfred; Köhler, Michael; Cao-Riehmer, Jialan
Three soil bacterial communities from an archaeological excavation site of an ancient coal mine near Bennstedt (Germany) characterized by 16S r-RNA sequencing. - In: Environments, ISSN 2076-3298, Bd. 9 (2022), 9, 115, S. 1-19

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.

Mazétyté-Stasinskiené, Raminta; Freiberger, Emma; Täuscher, Eric; Köhler, Michael
Four-level structural hierarchy: microfluidically supported synthesis of polymer particle architectures incorporating fluorescence-labeled components and metal nanoparticles. - In: Langmuir, ISSN 1520-5827, Bd. 38 (2022), 29, S. 8794-8804

Hierarchical assemblies of functional polymer particles are promising due to their surface as well as physicochemical properties. However, hierarchical composites are complex and challenging to form due to the many steps necessary for integrating different components into one system. Highly structured four-level composite particles were formed in a four-step process. First of all, gold (Au) nanoparticles, poly(methyl methacrylate) (PMMA) nanoparticles, and poly(tripropylene glycol diacrylate) (poly-TPGDA) microparticles were individually synthesized. By applying microfluidic techniques, polymer nano- and microparticles were formed with tunable size and surface properties. Afterwards, the negatively charged gold nanoparticles and PMMA particles functionalized with a positively charged surface were mixed to form Au/PMMA assemblies. The Au/PMMA composites were mixed and incubated with poly-TPGDA microparticles to form ternary Au/PMMA/poly-TPGDA assemblies. For the formation of composite-containing microparticles, Au/PMMA/poly-TPGDA composites were dispersed in an aqueous acrylamide-methylenebisacrylamide solution. Monomer droplets were formed in a co-flow microfluidic device and photopolymerized by UV light. In this way, hierarchically structured four-level composites consisting of four different size ranges - 0.025/0.8/30/1000 μm - were obtained. By functionalizing polymer nano- and microparticles with different fluorescent dyes, it was possible to visualize the same composite particle under two different excitation modes (λex = 395-440 and λex = 510-560 nm). The Au/PMMA/poly-TPGDA composite-embedded polyacrylamide microparticles can be potentially used as a model for the creation of composite particles for sensing, catalysis, multilabeling, and biomedical applications.

Henkel, Thomas; Mayer, Günter; Hampl, Jörg; Cao-Riehmer, Jialan; Ehrhardt, Linda; Schober, Andreas; Groß, Gregor Alexander
From microtiter plates to droplets - there and back again. - In: Micromachines, ISSN 2072-666X, Bd. 13 (2022), 7, 1022, S. 1-13

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.

Cao-Riehmer, Jialan; Chande, Charmi; Köhler, Michael
Microtoxicology by microfluidic instrumentation: a review. - In: Lab on a chip, ISSN 1473-0189, Bd. 22 (2022), 14, S. 2600-2623

Microtoxicology is concerned with the toxic effects of small amounts of substances. This review paper discusses the application of small amounts of noxious substances for toxicological investigation in small volumes. The vigorous development of miniaturized methods in microfluidics over the last two decades involves chip-based devices, micro droplet-based procedures, and the use of micro-segmented flow for microtoxicological studies. The studies have shown that the microfluidic approach is particularly valuable for highly parallelized and combinatorial dose-response screenings. Accurate dosing and mixing of effector substances in large numbers of microcompartments supplies detailed data of dose-response functions by highly concentration-resolved assays and allows evaluation of stochastic responses in case of small separated cell ensembles and single cell experiments. The investigations demonstrate that very different biological targets can be studied using miniaturized approaches, among them bacteria, eukaryotic microorganisms, cell cultures from tissues of multicellular organisms, stem cells, and early embryonic states. Cultivation and effector exposure tests can be performed in small volumes over weeks and months, confirming that the microfluicial strategy is also applicable for slow-growing organisms. Here, the state of the art of miniaturized toxicology, particularly for studying antibiotic susceptibility, drug toxicity testing in the miniaturized system like organ-on-chip, environmental toxicology, and the characterization of combinatorial effects by two and multi-dimensional screenings, is discussed. Additionally, this review points out the practical limitations of the microtoxicology platform and discusses perspectives on future opportunities and challenges.