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Many disciplines have become increasingly interested in cyanobacteria, due to their ability to fix CO2 while using water and sunlight as electron and energy sources. Further, several species of cyanobacteria are also capable of fixing molecular nitrogen, making them independent of the addition of nitrate or ammonia. Thereby they hold huge potential as sustainable biocatalysts. Here, we look into a dual-species biofilm consisting of filamentous diazotrophic cyanobacteria Tolypothrix sp. PCC 7712 and heterotrophic bacteria Pseudomonas taiwanensis VLB 120 growing in a capillary biofilm reactor. Such systems have been reported to enable high cell densities continuous process operation. By combining confocal laser scanning and helium-ion microscopy with a proteomics approach, we examined these organisms’ interactions under two nitrogen-feeding strategies: N2-fixing and nitrate assimilation. Not only did Pseudomonas facilitate the biofilm formation by forming a carpet layer on the surface area but also did N2-fixing biofilms show greater attachment to the surface. Pseudomonas proteins related to surface and cell attachments were observed in N2-fixing biofilms in particular. Furthermore, co-localized biofilm cells displayed a resilient response to extra shear forces induced by segmented media/air flows. This study highlights the role of Pseudomonas in the initial attachment process, as well as the effects of different nitrogen-feeding strategies and operation regimes on biofilm composition and growth.

Microbial interactions impact the functioning of both natural and engineered systems, yet our ability to directly monitor these highly dynamic and spatially resolved interactions in living cells is very limited. Here, we developed a synergistic approach coupling single-cell Raman microspectroscopy with 15N2 and 13CO2 stable isotope probing in a microfluidic culture system (RMCS-SIP) for live tracking of the occurrence, rate, and physiological shift of metabolic interactions in active microbial assemblages. Quantitative and robust Raman biomarkers specific for N2 and CO2 fixation in both model and bloom-forming diazotrophic cyanobacteria were established and cross-validated. By designing a prototype microfluidic chip allowing simultaneous microbial cultivation and single-cell Raman acquisition, we achieved temporal tracking of both intercellular (between heterocyst and vegetative cells of cyanobacteria) and interspecies N and C metabolite exchange (from diazotroph to heterotroph). Moreover, single-cell N and C fixation and bidirectional transfer rate in living cells were quantified via SIP-induced characteristic Raman shifts. Remarkably, RMCS captured physiological responses of metabolically active cells to nutrient stimuli through comprehensive metabolic profiling, providing multimodal information on the evolution of microbial interactions and functions under fluctuating conditions. This noninvasive RMCS-SIP is an advantageous approach for live-cell imaging and represents an important advancement in the single-cell microbiology field. This platform can be extended for real-time tracking of a wide range of microbial interactions with single-cell resolution and advances the understanding and manipulation of microbial interactions for societal benefit.

The spread of bacteria with antibiotic resistance genes (ARGs) in aquatic ecosystems is of growing concern as this can pose a risk of transmission to humans and animals. While the impact of wastewater treatment plant (WWTP) effluent on ARG abundance in surface waters has been studied extensively, less is known about the fate of ARGs in biofilms. The proximity and dense growth of microorganisms in combination with the accumulation of higher antibiotic concentrations in biofilms might render biofilms a reservoir for ARGs. Seasonal parameters such as water temperature, precipitation, and antibiotic concentrations should be considered as well, as they may further influence the fate of ARGs in aquatic ecosystems. Here we investigated the effect of WWTP effluent on the abundance of the sulfonamide resistance genes sul1 and sul2, and the integrase gene intI1 in biofilm and surface water compartments of a river in Germany with a gradient of anthropogenic impact using quantitative PCR. Furthermore, we analyzed the bacterial community structure in both compartments via 16S rRNA gene amplicon sequencing, following the river downstream. Additionally, conventional water parameters and sulfonamide concentrations were measured, and seasonal aspects were considered by comparing the fate of ARGs and bacterial community diversity in the surface water compartment between the summer and winter season. Our results show that biofilm compartments near the WWTP had a higher relative abundance of ARGs (up to 4.7%) than surface waters (<2.8%). Sulfonamide resistance genes were more persistent further downstream (>10 km) of the WWTP in the hot and dry summer season than in winter. This finding is likely a consequence of the higher proportion of wastewater and thus wastewater-derived microorganisms in the river during summer periods. We observed distinct bacterial communities and ARG abundance between the biofilm and surface water compartment, but even greater variations when con

Currently there are sparse regulations regarding the discharge of antibiotics from wastewater treatment plants (WWTP) into river systems, making surface waters a latent reservoir for antibiotics and antibiotic resistance genes (ARGs). To better understand factors that influence the fate of ARGs in the environment and to foster surveillance of antibiotic resistance spreading in such habitats, several indicator genes have been proposed, including the integrase gene intI1 and the sulfonamide resistance genes sul1 and sul2.Here we used quantitative PCR and long-read nanopore sequencing to monitor the abundance of these indicator genes and ARGs present as class 1 integron gene cassettes in a river system from pristine source to WWTP impacted water. ARG abundance was compared with the dynamics of the microbial communities determined via 16S rRNA gene amplicon sequencing, conventional water parameters and the concentration of sulfamethoxazole (SMX), sulfamethazine (SMZ) and sulfadiazine (SDZ).Our results show that WWTP effluent was the principal source of all three sulfonamides with highest concentrations for SMX (median 8.6 ng/L), and of the indicator genes sul1, sul2 and intI1 with median relative abundance to 16S rRNA gene of 0.55, 0.77 and 0.65 %, respectively. Downstream from the WWTP, water quality improved constantly, including lower sulfonamide concentrations, decreasing abundances of sul1 and sul2 and lower numbers and diversity of ARGs in the class 1 integron. The riverine microbial community partially recovered after receiving WWTP effluent, which was consolidated by a microbiome recovery model. Surprisingly, the relative abundance of intI1 increased 3 fold over 13 km of the river stretch, suggesting an internal gene multiplication.We found no evidence that low amounts of sulfonamides in the aquatic environment stimulate the maintenance or even spread of corresponding ARGs. Nevertheless, class 1 integrons carrying various ARGs were still present 13 km downstream

Despite the strongly accumulating evidence for microbial signatures in metabolic tissues, including the blood, suggesting a novel paradigm for metabolic disease development, the notion of a core blood bacterial signature in health and disease remains a contentious concept. Recent studies clearly demonstrate that under a strict contamination-free environment, methods such as 16 S rRNA gene sequencing, fluorescence in-situ hybridization, transmission electron microscopy, and several more, allied with advanced bioinformatics tools, allow unambiguous detection and quantification of bacteria and bacterial DNA in human tissues. Bacterial load and compositional changes in the blood have been reported for numerous disease states, suggesting that bacteria and their components may partially induce systemic inflammation in cardiometabolic disease. This concept has been so far primarily based on measurements of surrogate parameters. It is now highly desirable to translate the current knowledge into diagnostic, prognostic, and therapeutic approaches. This review addresses the potential clinical relevance of a blood bacterial signature pertinent to cardiometabolic diseases and outcomes and new avenues for translational approaches. It discusses pitfalls related to research in low bacterial biomass while proposing mitigation strategies for future research and application approaches.

The template synthesis with different concentrations of heterofunctional oligoperoxide NVP-co-VEP-co-GMA (2-tert-butylperoxy-5-methyl-1-hexene-3-yne) was performed to obtain inorganic nanoparticles of controlled size. An increase of polymer concentration provides the decrease of nanoparticle size and polydispersity index. As a result of template synthesis, the polymer molecules were adsorbed unto the nanoparticle surface forming irreversibly attached functional shell protecting the nanoparticles from aggregation. Such the polymer coated nanoparticles showed significantly lower aggregation even after high-temperature annealing, which is necessary to increase their luminescence efficiency. The luminescence spectral-kinetic study of YBO3–Ce, YBO3–Ce, Tb and YBO3–Ce,Tb, Eu nanoparticles revealed an efficient nonradiative transfer of excitation energy from Ce to Tb ions in YBO3–Ce, Tb and from Ce to Tb to Eu in YBO3–Ce,Tb, Eu nanoscale host. The potential use of YBO3–Ce, YBO3–Ce, Tb and YBO3–Ce,Tb, Eu nanoparticles for biological imaging was demonstrated by the luminescence microscopy.

Metabolic heterogeneity (MH) occurs when isogenic microbial populations display cell-to-cell differences in metabolic traits, albeit exposed to homogeneous conditions. Despite the increasing focus on MH, its triggering factors remain largely unknown. In the present thesis, I used stable isotope probing and chemical imaging with nanoscale Secondary Ion Mass Spectrometry (nanoSIMS) to study MH at single-cell level, in model organisms, synthetic and natural communities, to understand i) how abiotic factors, biotic interactions and antibiotics exposure influence MH and ii) its potential ecological role. Moreover, I optimized sample preparation for chemical and high-resolution imaging and suggested two different indices as ‘unit measure’ of MH. As results, I have shown for the first time that MH is displayed by microorganisms under favorable growth conditions, although none of the tested abiotic factors prevailed as the main trigger of MH. I brought insights on how biotic interactions play a role in the functional heterogeneity using bacteria pseudo-fungi co-cultures. I found that antibiotics reduce Carbon and Nitrogen assimilation rates of targeted phylogenetic groups in river-water communities, while increasing their MH, pointing to its ecological importance in natural environments. To conclude, I provided novel insights on the phenomenon of MH and its dynamics at different ecological scales.:Abbreviation list Summary Introduction Knowledge gaps Results and discussion - Optimization of sample preparation - Validation of quantitation methods - Abiotic factors shaping metabolic heterogeneity in bacterial populations - Influence of biotic factors in shaping heterogeneity - Metabolic Heterogeneity and ecophysiology of natural microbial populations influenced by emerging contaminants Conclusions Outlook Bibliography Appendix Acknowledgments Curriculum Vitae List of publications

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Microbial populations often display different degrees of heterogeneity in their substrate assimilation, that is, anabolic heterogeneity. It has been shown that nutrient limitations are a relevant trigger for this behavior. Here we explore the dynamics of anabolic heterogeneity under nutrient replete conditions. We applied time-resolved stable isotope probing and nanoscale Secondary Ion Mass Spectrometry (nanoSIMS) to quantify substrate assimilation by individual cells of Pseudomonas putida, P. stutzeri and Thauera aromatica. Acetate and benzoate at different concentrations were used as substrates. Anabolic heterogeneity was quantified by the cumulative differentiation tendency index. We observed two major, opposing trends of anabolic heterogeneity over time. Most often, microbial populations started as highly heterogeneous, with heterogeneity decreasing by various degrees over time. The second, less frequently observed trend, saw microbial populations starting at low or very low heterogeneity, and remaining largely stable over time. We explain these trends as an interplay of metabolic history (e.g. former growth substrate or other nutrient limitations) and metabolic fitness (i.e. the fine-tuning of metabolic pathways to process a defined growth substrate). Our results offer a new viewpoint on the intra-population functional diversification often encountered in the environment, and suggests that some microbial populations may be intrinsically heterogeneous.

The microbiome has emerged as an environmental factor contributing to obesity and type 2 diabetes (T2D). Increasing evidence suggests links between circulating bacterial components (i.e., bacterial DNA), cardiometabolic disease, and blunted response to metabolic interventions. In this aspect, thorough next-generation sequencing-based and contaminant-aware approaches are lacking. To address this, we tested whether bacterial DNA could be amplified in the blood of subjects with obesity and high metabolic risk under strict experimental and analytical control and whether a putative bacterial signature is related to metabolic improvement after bariatric surgery.Subjects undergoing bariatric surgery were recruited into sex- and BMI-matched subgroups with (n = 24) or without T2D (n = 24). Bacterial DNA in the blood was quantified and prokaryotic 16S rRNA gene amplicons were sequenced. A contaminant-aware approach was applied to derive a compositional microbial signature from bacterial sequences in all subjects at baseline and at 3 and 12 months after surgery. We modeled associations between bacterial load and composition with host metabolic and anthropometric markers. We further tested whether compositional shifts were related to weight loss response and T2D remission. Lastly, bacteria were visualized in blood samples using catalyzed reporter deposition (CARD)-fluorescence in situ hybridization (FISH). The contaminant-aware blood bacterial signature was associated with metabolic health. Based on bacterial phyla and genera detected in the blood samples, a metabolic syndrome classification index score was derived and shown to robustly classify subjects along their actual clinical group. T2D was characterized by decreased bacterial richness and loss of genera associated with improved metabolic health. Weight loss and metabolic improvement following bariatric surgery were associated with an early and stable increase of these genera in parallel with improvements in key cardiomet

Most microorganisms in the biosphere remain uncultured and poorly characterized. Although the surge in genome sequences has enabled insights into the genetic and metabolic properties of uncultured microorganisms, their physiology and ecological roles cannot be determined without direct probing of their activities in natural habitats. Here we employed an experimental framework coupling genome reconstruction and activity assays to characterize the largely uncultured microorganisms responsible for aerobic biodegradation of biphenyl as a proxy for a large class of environmental pollutants, polychlorinated biphenyls. We used 13C-labeled biphenyl in contaminated soils and traced the flow of pollutant-derived carbon into active cells using single-cell analyses and protein–stable isotope probing. The detection of 13C-enriched proteins linked biphenyl biodegradation to the uncultured Alphaproteobacteria clade UBA11222, which we found to host a distinctive biphenyl dioxygenase gene widely retrieved from contaminated environments. The same approach indicated the capacity of Azoarcus species to oxidize biphenyl and suggested similar metabolic abilities for species of Rugosibacter. Biphenyl oxidation would thus represent formerly unrecognized ecological functions of both genera. The quantitative role of these microorganisms in pollutant degradation was resolved using single-cell-based uptake measurements. Our strategy advances our understanding of microbially mediated biodegradation processes and has general application potential for elucidating the ecological roles of uncultured microorganisms in their natural habitats.