Binding of heavy metal ions in aggregates of microbial cells, EPS and biogenic iron minerals measured in-situ using metal- and glycoconjugates-specific fluorophores

Likai Hao ^ab, Y. Guo ^ab, J. M. Byrne ^c, F. Zeitvogel ^b, G. Schmid ^b, P. Ingino ^bg, J. Li ^d, T. R. Neu ^e, E. D. Swanner ^f, A. Kappler ^c and M. Obst ^g

^a Institute of Geochemistry, Chinese Academy of Sciences, PR China

^bEnvironmental Analytical Microscopy, Eberhard Karls University Tuebingen, Germany

^c Geomicrobiology, Eberhard Karls University Tuebingen, Germany

^d College of Chemistry & Materials Science, Northwest University, PR China

^e Department of River Ecology, Helmholtz Centre for Environmental Research, Germany

^f Geobiology, Department of Geological and Atmospheric Sciences, Iowa State University, USA

^g Bayreuth Center of Ecology and Environmental Research, University of Bayreuth, Germany

haolikai@mail.gyig.ac.cn

Biofilms and microbial aggregate, which contains of organic matter, iron/aluminum oxides, and clay minerals, are common at bulk or microscale chemical interfaces, which all of these components bind toxic heavy metal ions and control their fate and bioavailability in the environment due to the high sorption capacity and binding capacity of cells, EPS, and minerals. 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, it also remains unclear to which of these component(s) the metals will bind in complex microbial aggregates and biofilm. To clarify this question, our present study focuses on 3D mapping of heavy metals sorbed to cells, glycoconjugates that comprise the majority of EPS constituents, and Fe(III) mineral aggregates formed by the phototrophic Fe(II)-oxidizing bacteria Rhodobacter ferrooxidans SW2 using confocal laser scanning microscopy in combination with metal-specific fluorophores. To evaluate the influence of glycoconjugates, microbial cell surfaces, and (biogenic) Fe(III) minerals, and the availability of ferrous and ferric iron on heavy metal sorption. To provide detailed knowledge on the spatial distribution of metal ions in the microbial aggregates at the sub-lm scale, which is essential to understand the underlying mechanisms of microbe–mineral–metal interactions. Statistical analysis revealed that all heavy metals tested showed relatively similar sorption behaviour that was affected by the presence of sorbed ferrous and ferric iron. Our results showed that in addition to the mineral surfaces, both bacterial cell surfaces and the glycoconjugates provided most of sorption sites for heavy metals. Simultaneously, ferrous and ferric iron ions competed with the heavy metals for sorption sites on the organic compounds. In summary, we have developed and applied highly selective and sensitive metal fluorescent probes for confocal laser scanning microscopy for mapping heavy metals in environmental biofilms and cell-EPS-mineral aggregates; the information obtained by the present approach using a microbial model system provides important information to better understand the interactions between heavy metals and biofilms, and microbial formed Fe(III) minerals and heavy metals in complex natural environments. 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 analysed in their natural hydrated state, avoiding artifacts such as aggregation from drying that is necessary for analytical electron microscopy. Correlation analysis of spatially resolved heavy metal distributions with EPS and biogenic minerals in their natural hydrated state will further our understanding of the behaviour of metals in environmental systems.