Distinct microbiota assembly and functional patterns in disease-resistant and susceptible varieties of tobacco.

The plant microbiota is believed to be an accessory genome that extends plant functions, forming holobionts together with the host plant. Plant disease resistance, therefore, is inextricably linked with plant microbiota, which play important roles in plant growth and health. To explore the relationship between plant microbiota and disease resistance, we investigated the tobacco microbiome of two varieties with contrasting disease-resistance levels to bacterial wilt and black shank diseases. Comparative microbiome analysis indicated that the resistant variety assembled a distinct microbiota with higher network complexity and diversity. While Pseudomonas and Ensifer, which contain biocontrol and beneficial members, were enriched in the rhizosphere of the resistant variety, Ralstonia, a genus including the known causative pathogen, was enriched in the susceptible variety. Metagenome sequencing revealed that biocontrol functions, such as hydrogen cyanide synthase, pyochelin biosynthesis, and arthrofactin-type cyclic lipopeptide synthetase, were more abundant in the resistant variety. Further analysis indicated that contigs encoding the corresponding genes were mostly assigned to Pseudomonas. Among all the metagenome-assembled genomes, positive selection was suggested in the genome assigned to Pseudomonas only in the rhizosphere of the resistant variety. The search of biosynthetic gene clusters in the Pseudomonas genome revealed a non-ribosomal peptide synthetase, the compound of which was brabantamide A, with known antimicrobial activity. Collectively, our study suggests that the plant microbiota might be involved in microbe-mediated disease resistance. Particularly, our results highlight Pseudomonas in the rhizosphere of the disease-resistant variety as a promising biocontrol candidate. Our study may facilitate further screening of bacterial isolates and the targeted design of microbial communities.

Taxonomic and functional assembly cues enrich the endophytic tobacco microbiota across epiphytic compartments.

The plant microbiome plays a critical role in plant growth, development, and health, with endophytes being recognized as essential members due to their close interactions with host plants. However, knowledge gaps remain in understanding the mechanisms driving the colonization and establishment of endophytic communities. To address this issue, we investigated the microbiota of tobacco roots and leaves, including both epiphytic and endophytic microorganisms. We found that Actinobacteria and Alphaproteobacteria were significantly enriched in the root endosphere. Additionally, we identified higher abundances of functional traits involved in antibiotic synthesis, plant cell wall degradation, iron metabolism, secretion systems, and nicotine degradation enzymes in the endosphere. We further studied metagenome-assembled genomes from the rhizosphere and root endosphere, revealing a greater diversity of secondary metabolites in bacteria within the root endosphere. Together, this study provides insights into the taxonomic and functional assembly cues that may contribute to shaping the endophytic plant microbiota.IMPORTANCEThe presence of diverse microorganisms within plant tissues under natural conditions is a well-established fact. However, due to the plant immune system's barrier and the unique microhabitat of the plant interior, it remains unclear what specific characteristics bacteria require to successfully colonize and thrive in the plant endosphere. Recognizing the significance of unraveling these functional features, our study focused on investigating the enriched traits in the endophytic microbiota compared to the epiphytes. Through our research, we have successfully identified the taxonomic and functional assembly cues that drive the enrichment of the endophytic microbiota across the epiphytic compartments. These findings shed new light on the intricate mechanisms of endophyte colonization, thereby deepening our understanding of plant-microbe interactions and paving the way for further advancements in microbiome manipulation.

Metagenomic analysis characterizes resistomes of an acidic, multimetal(loid)-enriched coal source mine drainage treatment system.

Heavy metal(loid) contaminations caused by mine activities are potential hot spots of antibiotic resistance genes (ARGs) because of heavy metal(loid)-induced co-selection of ARGs and heavy metal(loid) resistance genes (MRGs). This study used high-throughput metagenomic sequencing to analyze the resistome characteristics of a coal source acid mine drainage passive treatment system. The multidrug efflux mechanism dominated the antibiotic resistome, and a highly diverse heavy metal(loid) resistome was dominated by mercury-, iron-, and arsenic--associated resistance. Correlation analysis indicated that mobile gene elements had a greater influence on the dynamic of MRGs than ARGs. Among the metagenome-assembled genomes, six potential pathogens carrying multiple resistance genes resistant to several antibiotics and heavy metal(loid)s were recovered. Pseudomonas spp. contained the highest numbers of resistance genes, with resistance to two types of antibiotics and 12 types of heavy metal(loid)s. Thus, high contents of heavy metal(loid)s drove the co-selection of ARGs and MRGs. The occurrence of potential pathogens containing multiple resistance genes might increase the risk of ARG dissemination in the environment.

Coal-source acid mine drainage reduced the soil multidrug-dominated antibiotic resistome but increased the heavy metal(loid) resistome and energy production-related metabolism.

A recent global scale study found that mining-impacted environments have multi-antibiotic resistance gene (ARG)-dominated resistomes with an abundance similar to urban sewage but much higher than freshwater sediment. These findings raised concern that mining may increase the risk of ARG environmental proliferation. The current study assessed how typical multimetal(loid)-enriched coal-source acid mine drainage (AMD) contamination affects soil resistomes by comparing with background soils unaffected by AMD. Both contaminated and background soils have multidrug-dominated antibiotic resistomes attributed to the acidic environment. AMD-contaminated soils had a lower relative abundance of ARGs (47.45 ± 23.34 ×/Gb) than background soils (85.47 ± 19.71 ×/Gb) but held high-level heavy metal(loid) resistance genes (MRGs, 133.29 ± 29.36 ×/Gb) and transposase- and insertion sequence-dominated mobile genetic elements (MGEs, 188.51 ± 21.81 ×/Gb), which was 56.26 % and 412.12 % higher than background soils, respectively. Procrustes analysis showed that the microbial community and MGEs exerted more influence on driving heavy metal(loid) resistome variation than antibiotic resistome. The microbial community increased energy production-related metabolism to fulfill the increasing energy needs required by acid and heavy metal(loid) resistance. Horizontal gene transfer (HGT) events primarily exchanged energy- and information-related genes to adapt to the harsh AMD environment. These findings provide new insight into the risk of ARG proliferation in mining environments.

Effect and mechanism of simultaneous cadmium-tetracycline removal by a self-assembled microbial-photocatalytic coupling system.

Electrochemical bacteria Shewanella oneidensis MR-4 (MR-4) was used to biologically generate cadmium sulfide (bio-CdS) nanocrystals and construct a self-assembled intimately coupled photocatalysis-biodegradation system (SA-ICPB) to remove cadmium (Cd) and tetracycline hydrochloride (TCH) from wastewater. The characterization using EDS, TEM, XRD, XPS, and UV-vis confirmed the successful CdS bio-synthesis and its visible-light response capacity (520 nm). 98.4% of Cd2+ (2 mM) was removed during bio-CdS generation within 30 min. The electrochemical analysis confirmed the photoelectric response capability of the bio-CdS as well as its photocatalytic efficiency. Under visible light, SA-ICPB entirely eliminated TCH (30 mg/L). In 2 h, 87.2% and 43.0% of TCH were removed separately with and without oxygen. 55.7% more chemical oxygen demand (COD) was removed with oxygen participation, indicating the degradation intermediates elimination by SA-ICPB required oxygen participation. Biodegradation dominated the process under aerobic circumstances. Electron paramagnetic resonance analysis indicated that h+ and ·O2- played a decisive role in photocatalytic degradation. Mass spectrometry analysis proved that TCH was dehydrated, dealkylated, and ring-opened before mineralizing. In conclusion, MR-4 can spontaneously generate SA-ICPB and rapidly-deeply eliminate antibiotics by coupling photocatalytic and microbial degradation. Such an approach was efficient for the deep degradation of persistent organic pollutants with antimicrobial properties.

High-Efficiency Utilization of Waste Tobacco Stems to Synthesize Novel Biomass-Based Carbon Dots for Precise Detection of Tetracycline Antibiotic Residues.

Recycling waste biomass into valuable products (e.g., nanomaterials) is of considerable theoretical and practical significance to achieve future sustainable development. Here, we propose a one-pot hydrothermal synthesis route to convert waste tobacco stems into biomass-based N, S-codoped carbon dots (C-dots) with the assistance of carbon black. Unlike most of the previously reported luminescent C-dots, these biomass-based C-dots showed a satisfactory stability, as well as an excitation-independent fluorescence emission at ~520 nm. Furthermore, they demonstrated a pH-dependent fluorescence emission ability, offering a scaffold to design pH-responsive assays. Moreover, these as-synthesized biomass-based C-dots exhibited a fluorescence response ability toward tetracycline antibiotics (TCs, e.g., TC, CTC, and OTC) through the inner filter effect (IFE), thereby allowing for the establishment a smart analytical platform to sensitively and selectively monitor residual TCs in real environmental water samples. In this study, we explored the conversion of waste tobacco stems into sustainable biomass-based C-dots to develop simple, efficient, label-free, reliable, low-cost, and eco-friendly analytical platforms for environmental pollution traceability analysis, which might provide a novel insight to resolve the ecological and environmental issues derived from waste tobacco stems.

Rhizosphere Microbial Communities and Geochemical Constraining Mechanism of Antimony Mine Waste-Adapted Plants in Southwestern China.

Antimony (Sb) and arsenic (As) are two hazardous metalloid elements, and the biogeochemical cycle of Sb and As can be better understood by studying plant rhizosphere microorganisms associated with Sb mine waste. In the current study, samples of three types of mine waste-Sb mine tailing, waste rocks, and smelting slag-and associated rhizosphere microorganisms of adapted plants were collected from Qinglong Sb mine, southwest China. 16S rRNA was sequenced and used to study the composition of the mine waste microbial community. The most abundant phylum in all samples was Proteobacteria, followed by Bacteroidota, Acidobacteriota, and Actinobacteriota. The community composition varied among different mine waste types. Gammaproteobacteria was the most abundant microorganism in tailings, Actinobacteria was mainly distributed in waste rock, and Saccharimonadia, Acidobacteriae, and Ktedonobacteria were mainly present in slag. At the family level, the vast majority of Hydrogenophilaceae were found in tailings, Ktedonobacteraceae, Chthoniobacteraceae, and Acidobacteriaceae (Subgroup 1) were mostly found in slag, and Pseudomonadaceae and Micrococcaceae were mainly found in waste rock. Actinobacteriota and Arthrobacter are important taxa for reducing heavy metal(loid) mobility, vegetation restoration, and self-sustaining ecosystem construction on antimony mine waste. The high concentrations of Sb and As reduce microbial diversity.

Effect of Environmental pH on Mineralization of Anaerobic Iron-Oxidizing Bacteria.

Freshwater lakes are often polluted with various heavy metals in the Anthropocene. The iron-oxidizing microorganisms and their mineralized products can coprecipitate with many heavy metals, including Al, Zn, Cu, Cd, and Cr. As such, microbial iron oxidation can exert a profound impact on environmental remediation. The environmental pH is a key determinant regulating microbial growth and mineralization and then influences the structure of the final mineralized products of anaerobic iron-oxidizing bacteria. Freshwater lakes, in general, are neutral-pH environments. Understanding the effects of varying pH on the mineralization of iron-oxidizing bacteria under neutrophilic conditions could aid in finding out the optimal pH values that promote the coprecipitation of heavy metals. Here, two typical neutrophilic Fe(II)-oxidizing bacteria, the nitrate-reducing Acidovorax sp. strain BoFeN1 and the anoxygenic phototrophic Rhodobacter ferrooxidans strain SW2, were selected for studying how their growth and mineralization response to slight changes in circumneutral pH. By employing focused ion beam/scanning electron microscopy (FIB-SEM) and transmission electron microscopy (TEM), we examined the interplay between pH changes and anaerobic iron-oxidizing bacteria and observed that pH can significantly impact the microbial mineralization process and vice versa. Further, pH-dependent changes in the structure of mineralized products of bacterial iron oxidation were observed. Our study could provide mechanical insights into how to manipulate microbial iron oxidation for facilitating remediation of heavy metals in the environment.

Soft-Landing Dynamic Analysis of a Manned Lunar Lander Em-Ploying Energy Absorption Materials of Carbon Nanotube Buckypaper.

With the rapid development of the aerospace field, traditional energy absorption materials are becoming more and more inadequate and cannot meet the requirements of having a light weight, high energy absorption efficiency, and high energy absorption density. Since existing studies have shown that carbon nanotube (CNT) buckypaper is a promising candidate for energy absorption, owing to its extremely high energy absorption efficiency and remarkable mass density of energy absorption, this study explores the application of buckypaper as the landing buffer material in a manned lunar lander. Firstly, coarse-grained molecular dynamics simulations were implemented to investigate the compression stress-strain relationships of buckypapers with different densities and the effect of the compression rate within the range of the landing velocity. Then, based on a self-designed manned lunar lander, buckypapers of appropriate densities were selected to be the energy absorption materials within the landing mechanisms of the lander. For comparison, suitable aluminum honeycomb materials, the most common energy absorption materials in lunar landers, were determined for the same landing mechanisms. Afterwards, the two soft-landing multibody dynamic models are established, respectively, and their soft-landing performances under three severe landing cases are analyzed, respectively. The results depicted that the landers, respectively, adopting the two energy absorption materials well, satisfy the soft-landing performance requirements in all the cases. It is worth mentioning that the lander employing the buckypaper is proved to demonstrate a better soft-landing performance, mainly reflected in reducing the mass of the energy absorption element by 8.14 kg and lowing the maximum center-of-mass overload of the lander by 0.54 g.

Secondary Sulfate Minerals from Pyrite Oxidation in Lanmuchang Hg-Tl Deposit, Southwest Guizhou Province, China: Geochemistry and Environmental Significance.

Thallium (Tl) is a highly toxic trace metal posing a significant threat to human health. Tl pollution in soils and chronic Tl poisoning related to Tl-rich sulfides weathering in the Lanmuchang mine of southwest Guizhou province, China, have been intensively studied in recent years. And yet, there are few studies on the role of secondary sulfate minerals associated with Tl mobility in this area. The sulfate minerals were characterized by XRD and SEM-EDS. The concentrations of Tl and other elements were determined by ICP-MS. The results show that sulfate minerals are predominantly melanterite, halotrichite, and fibroferrite. The average contents of Tl in rock, sulfate minerals, and soil samples were 156.4, 0.11, and 72.1 µg g-1, respectively. This study suggests that Tl in the mineralized rocks entered soils by pyrite oxidation with less scavenged of the sulfate minerals. The dissolution of the ferric sulfate minerals accelerates pyrite oxidation and maintains soil acidity, and this likely enhances Tl mobility from soil to crops.

The substrate-dependent regulatory effects of the AfeI/R system in Acidithiobacillus ferrooxidans reveals the novel regulation strategy of quorum sensing in acidophiles.

A LuxI/R-like quorum sensing (QS) system (AfeI/R) has been reported in the acidophilic and chemoautotrophic Acidithiobacillus spp. However, the function of AfeI/R remains unclear because of the difficulties in the genetic manipulation of these bacteria. Here, we constructed different afeI mutants of the sulfur- and iron-oxidizer A. ferrooxidans, identified the N-acyl homoserine lactones (acyl-HSLs) synthesized by AfeI, and determined the regulatory effects of AfeI/R on genes expression, extracellular polymeric substance synthesis, energy metabolism, cell growth and population density of A. ferrooxidans in different energy substrates. Acyl-HSLs-mediated distinct regulation strategies were employed to influence bacterial metabolism and cell growth of A. ferrooxidans cultivated in either sulfur or ferrous iron. Based on these findings, an energy-substrate-dependent regulation mode of AfeI/R in A. ferrooxidans was illuminated that AfeI/R could produce different types of acyl-HSLs and employ specific acyl-HSLs to regulate specific genes in response to different energy substrates. The discovery of the AfeI/R-mediated substrate-dependent regulatory mode expands our knowledge on the function of QS system in the chemoautotrophic sulfur- and ferrous iron-oxidizing bacteria, and provides new insights in understanding energy metabolism modulation, population control, bacteria-driven bioleaching process, and the coevolution between the acidophiles and their acidic habitats.

Novel Fluorescein-Based Fluorescent Probe for Detecting H2S and Its Real Applications in Blood Plasma and Biological Imaging.

A broad-spectrum fluorescent probe, which can be applied to monitoring H2S in various biological systems, has been rationally designed and synthesized. This specific probe was applied to localize the endogenous H2S in living Raw264.7 macrophage cells, HepG2 cells, and H9C2 cells. At the same time, the probe has successfully visualized CBS- and CSE-induced endogenous H2S production and monitored CBS and CSE activity in H9C2 cells. This probe could serve as a powerful molecular imaging tool to further explore the physiological function and the molecular mechanisms of endogenous H2S in living animal systems.

A novel approach to study the structure-property relationships and applications in living systems of modular Cu(2+) fluorescent probes.

A series of Cu(2+) probe which contains 9 probes have been synthesized and established. All the probes were synthesized using Rhodamine B as the fluorophore, conjugated to various differently substituted cinnamyl aldehyde with C=N Schiff base structural motif as their core moiety. The structure-property relationships of these probes have been investigated. The change of optical properties, caused by different electronic effect and steric effect of the recognition group, has been analyzed systematically. DFT calculation simulation of the Ring-Close and Ring-Open form of all the probes have been employed to illuminate, summarize and confirm these correlations between optical properties and molecular structures. In addition, biological experiment demonstrated that all the probes have a high potential for both sensitive and selective detection, mapping of adsorbed Cu(2+) both in vivo and environmental microbial systems. This approach provides a significant strategy for studying structure-property relationships and guiding the synthesis of probes with various optical properties.

Submicron-Scale Heterogeneities in Nickel Sorption of Various Cell-Mineral Aggregates Formed by Fe(II)-Oxidizing Bacteria.

Fe(II)-oxidizing bacteria form biogenic cell-mineral aggregates (CMAs) composed of microbial cells, extracellular organic compounds, and ferric iron minerals. CMAs are capable of immobilizing large quantities of heavy metals, such as nickel, via sorption processes. CMAs play an important role for the fate of heavy metals in the environment, particularly in systems characterized by elevated concentrations of dissolved metals, such as mine drainage or contaminated sediments. We applied scanning transmission (soft) X-ray microscopy (STXM) spectrotomography for detailed 3D chemical mapping of nickel sorbed to CMAs on the submicron scale. We analyzed different CMAs produced by phototrophic or nitrate-reducing microbial Fe(II) oxidation and, in addition, a twisted stalk structure obtained from an environmental biofilm. Nickel showed a heterogeneous distribution and was found to be preferentially sorbed to biogenically precipitated iron minerals such as Fe(III)-(oxyhydr)oxides and, to a minor extent, associated with organic compounds. Some distinct nickel accumulations were identified on the surfaces of CMAs. Additional information obtained from scatter plots and angular distance maps, showing variations in the nickel-iron and nickel-organic carbon ratios, also revealed a general correlation between nickel and iron. Although a high correlation between nickel and iron was observed in 2D maps, 3D maps revealed this to be partly due to projection artifacts. In summary, by combining different approaches for data analysis, we unambiguously showed the heterogeneous sorption behavior of nickel to CMAs.

Solvent-dependent turn-on probe for dual monitoring of Ag(+) and Zn(2+) in living biological samples.

A novel, solvent-dependent "off-on" probe with benzoylthiourea moiety as the functional receptor and fluorescein as the fluorophore was designed for monitoring of Ag(+) in EtOH-H2O (2:8, v/v) solution and Zn(2+) in CH3CN-H2O (2:8, v/v) solution at physiological range with sufficient selectivity and sensitivity. The Ag(+) promoted desulfurization of thiosemicarbazide functionality in formation of the 1,3,4-oxadiazole and the coordination of Zn(2+) to the O atom and N atom of the spoirolactam moiety and the S atom of the benzoylthiourea moiety were investigated to be the power that promoted the fluorescent enhancement. This probe was tested highly suitable for mapping Ag(+) and Zn(2+) in living human osteosarcoma MG-63 cells and microbial cell-EPS-mineral aggregates, thus, providing a wonderful candidate for tracking Ag(+) and Zn(2+) in biological organisms and processes.

Visualizing tributyltin (TBT) in bacterial aggregates by specific rhodamine-based fluorescent probes.

Here we present the first examples of fluorescent and colorimetric probes for microscopic TBT imaging. The fluorescent probes are highly selective and sensitive to TBT and have successfully been applied for imaging of TBT in bacterial Rhodobacter ferrooxidans sp. strain SW2 cell-EPS-mineral aggregates and in cell suspensions of the marine cyanobacterium Synechococcus PCC 7002 by using confocal laser scanning microscopy.

Characterization of the physiology and cell-mineral interactions of the marine anoxygenic phototrophic Fe(II) oxidizer Rhodovulum iodosum--implications for Precambrian Fe(II) oxidation.

Anoxygenic phototrophic Fe(II)-oxidizing bacteria (photoferrotrophs) are suggested to have contributed to the deposition of banded iron formations (BIFs) from oxygen-poor seawater. However, most studies evaluating the contribution of photoferrotrophs to Precambrian Fe(II) oxidation have used freshwater and not marine strains. Therefore, we investigated the physiology and mineral products of Fe(II) oxidation by the marine photoferrotroph Rhodovulum iodosum. Poorly crystalline Fe(III) minerals formed initially and transformed to more crystalline goethite over time. During Fe(II) oxidation, cell surfaces were largely free of minerals. Instead, the minerals were co-localized with EPS suggesting that EPS plays a critical role in preventing cell encrustation, likely by binding Fe(III) and directing precipitation away from cell surfaces. Fe(II) oxidation rates increased with increasing initial Fe(II) concentration (0.43-4.07 mM) under a light intensity of 12 µmol quanta m(-2) s(-1). Rates also increased as light intensity increased (from 3 to 20 µmol quanta m(-2) s(-1)), while the addition of Si did not significantly change Fe(II) oxidation rates. These results elaborate on how the physical and chemical conditions present in the Precambrian ocean controlled the activity of marine photoferrotrophs and confirm the possibility that such microorganisms could have oxidized Fe(II), generating the primary Fe(III) minerals that were then deposited to some Precambrian BIFs.

Synchrotron-based chemical nano-tomography of microbial cell-mineral aggregates in their natural, hydrated state.

Chemical nano-tomography of microbial cells in their natural, hydrated state provides direct evidence of metabolic and chemical processes. Cells of the nitrate-reducing Acidovorax sp. strain BoFeN1 were cultured in the presence of ferrous iron. Bacterial reduction of nitrate causes precipitation of Fe(III)-(oxyhydr)oxides in the periplasm and in direct vicinity of the cells. Nanoliter aliquots of cell-suspension were injected into custom-designed sample holders wherein polyimide membranes collapse around the cells by capillary forces. The immobilized, hydrated cells were analyzed by synchrotron-based scanning transmission X-ray microscopy in combination with angle-scan tomography. This approach provides three-dimensional (3D) maps of the chemical species in the sample by employing their intrinsic near-edge X-ray absorption properties. The cells were scanned through the focus of a monochromatic soft X-ray beam at different, chemically specific X-ray energies to acquire projection images of their corresponding X-ray absorbance. Based on these images, chemical composition maps were then calculated. Acquiring projections at different tilt angles allowed for 3D reconstruction of the chemical composition. Our approach allows for 3D chemical mapping of hydrated samples and thus provides direct evidence for the localization of metabolic and chemical processes in situ.

Six-membered spirocycle triggered probe for visualizing Hg2+ in living cells and bacteria-EPS-mineral aggregates.

A novel rhodamine based probe with a unique six-membered spirocycle was rationally designed for detection of Hg(2+) with greatly improved selectivity, sensitivity, and photostability. The probe has been shown to be suitable for Hg(2+) imaging in living cells and mapping Hg(2+) distribution in living cell-EPS-mineral aggregates under anoxic conditions.

Mapping of heavy metal ion sorption to cell-extracellular polymeric substance-mineral aggregates by using metal-selective fluorescent probes and confocal laser scanning microscopy.

Biofilms, organic matter, iron/aluminum oxides, and clay minerals bind toxic heavy metal ions and control their fate and bioavailability in the environment. The spatial relationship of metal ions to biomacromolecules such as extracellular polymeric substances (EPS) in biofilms with microbial cells and biogenic minerals is complex and occurs at the micro- and submicrometer scale. Here, we review the application of highly selective and sensitive metal fluorescent probes for confocal laser scanning microscopy (CLSM) that were originally developed for use in life sciences and propose their suitability as a powerful tool for mapping heavy metals in environmental biofilms and cell-EPS-mineral aggregates (CEMAs). The benefit of using metal fluorescent dyes in combination with CLSM imaging over other techniques such as electron microscopy is that environmental samples can be analyzed in their natural hydrated state, avoiding artifacts such as aggregation from drying that is necessary for analytical electron microscopy. In this minireview, we present data for a group of sensitive fluorescent probes highly specific for Fe(3+), Cu(2+), Zn(2+), and Hg(2+), illustrating the potential of their application in environmental science. We evaluate their application in combination with other fluorescent probes that label constituents of CEMAs such as DNA or polysaccharides and provide selection guidelines for potential combinations of fluorescent probes. Correlation analysis of spatially resolved heavy metal distributions with EPS and biogenic minerals in their natural, hydrated state will further our understanding of the behavior of metals in environmental systems since it allows for identifying bonding sites in complex, heterogeneous systems.