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Köhler, Michael; Ehrhardt, Linda; Cao-Riehmer, Jialan; Möller, Frances; Schüler, Tim; Günther, Mike
Beta-diversity enhancement by archaeological structures: bacterial communities of an historical tannery area of the city of Jena (Germany) reflect the ancient human impact. - In: Ecologies, ISSN 2673-4133, Bd. 4 (2023), 2, S. 325-343

Soil samples taken during archaeological investigations of a historical tannery area in the eastern suburb of the medieval city of Jena have been investigated by 16S r-RNA gene profiling. The analyses supplied a large spectrum of interesting bacteria, among them Patescibacteria, Methylomirabilota, Asgardarchaeota, Zixibacteria, Sideroxydans and Sulfurifustis. Samples taken from soil inside the residues of large vats show large differences in comparison to the environmental soil. The PCAs for different abundance classes clearly reflect the higher similarity between the bacterial communities of the outside-vat soils in comparison with three of the inside-vat soil communities. Two of the in-side vat soils are distinguishable from the other samples by separate use of each abundance class, but classes of lower abundance are better applicable than the highly abundant bacteria for distinguishing the sampling sites by PCA, in general. This effect could be interpreted by the assumption that less abundant types in the 16S r-RNA data tend to be more related to an earlier state of soil development than the more abundant and might be, therefore, better suited for conclusions on the state of the soils in an earlier local situation. In addition, the analyses allowed identification of specific features of each single sampling site. In one site specifically, DNA hints of animal residue-related bacteria were found. Obviously, the special situation in the in-site vat soils contributes to the diversity of the place, and enhances its Beta-diversity. Very high abundancies of several ammonia-metabolizing and of sulphur compound-oxidizing genera in the metagenomics data can be interpreted as an echo of the former tannery activities using urine and processing keratin-rich animal materials. In summary, it can be concluded that the 16S r-RNA analysis of such archaeological places can supply a lot of data related to ancient human impacts, representing a kind of “ecological memory of soil”.

Tsierkezos, Nikos; Freiberger, Emma; Ritter, Uwe; Krischok, Stefan; Ullmann, Fabian; Köhler, Michael
Application of nitrogen-doped multi-walled carbon nanotubes decorated with gold nanoparticles in biosensing. - In: Journal of solid state electrochemistry, ISSN 1433-0768, Bd. 27 (2023), 10, S. 2645-2658

Novel films consisting of nitrogen-doped multi-walled carbon nanotubes (N-MWCNTs) were fabricated by means of chemical vapor deposition technique and decorated with gold nanoparticles (AuNPs) possessing diameter of 14.0 nm. Electron optical microscopy analysis reveals that decoration of N-MWCNTs with AuNPs does not have any influence on their bamboo-shaped configuration. The electrochemical response of fabricated composite films, further denoted as N-MWCNTs/AuNPs, towards oxidation of dopamine (DA) to dopamine-o-quinone (DAQ) in the presence of ascorbic acid (AA) and uric acid (UA) was probed in real pig serum by means of cyclic voltammetry (CV) and square wave voltammetry (SWV). The findings demonstrate that N-MWCNTs/AuNPs exhibit slightly greater electrochemical response and sensitivity towards DA/DAQ compared to unmodified N-MWCNTs. It is, consequently, obvious that AuNPs improve significantly the electrochemical response and detection ability of N-MWCNTs. The electrochemical response of N-MWCNTs/AuNPs towards DA/DAQ seems to be significantly greater compared to that of conventional electrodes, such as platinum and glassy carbon. The findings reveal that N-MWCNTs/AuNPs could serve as powerful analytical sensor enabling analysis of DA in real serum samples.

Xie, Ting; Ehrhardt, Linda; Günther, Mike; Köhler, Michael; Cao-Riehmer, Jialan
Current to biomass: media optimization and strain selection from cathode-associated microbial communities in a two-chamber electro-cultivation reactor. - In: Environments, ISSN 2076-3298, Bd. 10 (2023), 6, 97, S. 1-19

Cathode-associated microbial communities (caMCs) are the functional key elements in the conversion of excess electrical energy into biomass. In this study, we investigated the development of electrochemical caMCs based on two-chamber microbial electrolytic cells (MECs) after optimization of media composition. Microbial communities obtained from a historical soil sample were inoculated into the cathode chamber of MECs. The inorganic medium with (A) carbon dioxide in air or (B) 100 mM sodium bicarbonate as carbon source was used in the absence of any organic carbon source. After 12 days of operation, the experimental results showed that (1) the bacterial community in group B exhibited lush growth and (2) a single strain TX168 Epilithonimonas bovis isolated from group A indicated electrochemical activity and synthesized large volumes of biomass using sodium bicarbonate. We also analyzed the caMCs of the MECs and reference samples without electro-cultivation using 16S rRNA gene sequencing. The results showed that the caMCs of MECs in groups A and B were dominated by the genera Acinetobacter and Pseudomonas. The caMCs were further inoculated and cultured on different agars to isolate specific electroactive bacterial strains. Overall, our study highlights the possibility of converting excess energy into biomass by electro-cultivation and the importance of selecting appropriate media to enrich specific microbial communities and single strains in MECs.

Mazétyté-Stasinskiené, Raminta; Kronfeld, Klaus-Peter; Köhler, Michael
Five-level structural hierarchy: microfluidically supported synthesis of core-shell microparticles containing nested set of dispersed metal and polymer micro and nanoparticles. - In: Particle & particle systems characterization, ISSN 1521-4117, Bd. 14 (2023), 10, 2300030, S. 1-13

This study presents the development of a hierarchical design concept for the synthesis of multi-scale polymer particles with up to five levels of organization. The synthesis of core-shell microparticles containing nested sets of dispersed metal and polymer micro- and nanoparticles is achieved through in situ photopolymerization using a double co-axial capillaries microfluidic device. The flow rates of the carrier, shell, and core phases are optimized to control particle size and result in stable core-shell particles with well-dispersed three-level composites in the shell matrix. The robustness and reversibility of these core-shell particles are demonstrated through five cycles of drying and re-swelling, showing that the size and structure of core-shell particles remain unchanged. Additionally, the permeability and mobility of dye molecules within the shell matrix are tested and showed that different molecular weight dyes have different penetration times. This study highlights the potential of microfluidics as a powerful tool for the controlled and precise synthesis of complex structured materials and demonstrates the versatility and potential of these core-shell particles for sensing applications as particle-based surface-enhanced Raman scattering (SERS).

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, S. 1-12

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.