Using Flexible Gold-Titanium Reaction Cells to Simulate Pressure-Dependent Microbial Activity in the Context of Subsurface Biomining

Laboratory studies investigating subsurface microbial processes, such as metal leaching in deep ore deposits (biomining), share common and challenging ...

Federal Institute for Geosciences and Natural Resources

Bacterial adhesion is a key step to prevent environmental problems called acid mine drainage or to improve leaching efficiency in industry, since it initiates and enhances bioleaching. Thus, to analyze bacterial adhesion and to understand this process is crucial. In this study atomic force microscopy equipped with a pyrite or chalcopyrite tip was applied to study the adhesion of Sulfobacillus thermosulfidooxidans. The results illustrate that planktonic cells of both pyrite- and sulfur-grown cells of S. thermosulfidooxidans show more affinity to pyrite than to chalcopyrite (adhesion forces 2 nN versus 0.13 nN). However, the interactions between bacteria and chalcopyrite can be strengthened, if the bacteria are brought into contact with the chalcopyrite. The biofilm cells show low affinity to either pyrite or chalcopyrite. A high content of proteins in the extracellular polymeric substances collected from planktonic cells of S. thermosulfidooxidans and a low content of proteins collected from biofilm EPS indicates that proteins play an important role in initial adhesion. Analysis of adhesion force-distance curves reveal that adhesion by pyrite-grown cells is a complex interaction involving several bonding forces.

Investigation on adhesion of Sulfobacillus thermosulfidooxidans via atomic force microscopy equipped with mineral probes.

Bioleaching of metal sulfides represents an interfacial process where biofilm formation is important in the initial steps of this process. In technical applications of bioleaching, such as reactor leaching in the temperature range of 50 up to 90 °C and also in (self-heating) heaps, thermophilic archaea play an important role and often are the leaching organisms of choice. Nevertheless, to date there is little information available on the interactions between thermoacidophilic archaea and their natural mineral substrates such as pyrite. Especially for extracellular polymeric substances (EPS) of archaea and their biofilms in bioleaching environments information is rather limited. The present work focused on investigations of biofilm dynamics and EPS production of the thermoacidophilic archaeon Acidianus sp. DSM 29099 under bioleaching conditions. The results show that biofilms are dispersed non-homogeneously on pyrite. Large parts of the pyrite surfaces remain free of cells. Cell detachment from pyrite results in microbial "footprints" which, based on lectin binding assays, consist of mannose, glucose and fucose containing compounds. A monolayer biofilm develops on pyrite after 2-4 days of incubation. In addition, the pyrite surface is covered with a layer of organic compounds. EPS analysis indicates the presence of proteins, polysaccharides and uronic acids, the composition of which varies according to substrate and lifestyle (i.e. planktonic, biofilm cells). This report provides insight into EPS and biofilm characteristics of thermophilic archaea and improves understanding of the mineral-microbial-biofilm interfacial interactions in extreme environments. Moreover, the results on interaction dynamics of archaeal microbial consortia will facilitate the understanding of thermophilic bioleaching.

Biofilm dynamics and EPS production of a thermoacidophilic bioleaching archaeon.

Biooxidation of reduced inorganic sulfur compounds (RISCs) by thermoacidophiles is of particular interest for the biomining industry and for environmental issues, e.g., formation of acid mine drainage (AMD). Up to now, interfacial interactions of acidophiles with elemental sulfur as well as the mechanisms of sulfur oxidation by acidophiles, especially thermoacidophiles, are not yet fully clear. This work focused on how a crenarchaeal isolate  sp. DSM 29099 interacts with elemental sulfur. Analysis by Confocal laser scanning microscopy (CLSM) and Atomic force microscopy (AFM) in combination with Epifluorescence microscopy (EFM) shows that biofilms on elemental sulfur are characterized by single colonies and a monolayer in first stage and later on 3-D structures with a diameter of up to 100 μm. The analysis of extracellular polymeric substances (EPS) by a non-destructive lectin approach (fluorescence lectin-barcoding analysis) using several fluorochromes shows that intial attachment was featured by footprints rich in biofilm cells that were embedded in an EPS matrix consisting of various glycoconjugates. Wet chemistry data indicate that carbohydrates, proteins, lipids and uronic acids are the main components. Attenuated reflectance (ATR)-Fourier transformation infrared spectroscopy (FTIR) and high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) indicate glucose and mannose as the main monosaccharides in EPS polysaccharides. EPS composition as well as sugar types in EPS vary according to substrate (sulfur or tetrathionate) and lifestyle (biofilms and planktonic cells). This study provides information on the building blocks/make up as well as dynamics of biofilms of thermoacidophilic archaea in extremely acidic environments.

Insight Into Interactions of Thermoacidophilic Archaea With Elemental Sulfur: Biofilm Dynamics and EPS Analysis.

Euonymus japonicus Thunb. is a woody and ornamental plant popular in China, Europe and North America. Powdery mildew is one of the most serious diseases that affect E. japonicus growth. In this study, the diseased and apparently healthy leaves were collected from E. japonicus planted in a greenbelt in Beijing, and the effect of powdery mildew on the epiphytic microbial community was investigated by using Illumina sequencing. The results showed that the healthy leaves (HL) harbored greater bacterial and fungal diversity than diseased leaves (DL). Furthermore, both bacterial and fungal communities in DL exhibited significantly different structures from those in HL. The relative abundance of several bacterial phyla (Proteobacteria and Firmicutes) and fungal phyla (Ascomycota and Basidiomycota) were altered by powdery mildew. At the genus level, most genera decreased as powdery mildew pathogen Erysiphe increased, while the genera Kocuria and Exiguobacterium markedly increased. Leaf properties, especially protein content was found to significantly affect beta-diversity of the bacterial and fungal community. Network analysis revealed that positive bacterial interactions in DL were stronger than those in HL samples. Insights into the underlying the indigenous microbial phyllosphere populations of E. japonicus response to powdery mildew will help in the development of methods for controlling plant diseases.

Ruiyong Zhang

Federal Institute for Geosciences and Natural Resources

Using Flexible Gold-Titanium Reaction Cells to Simulate Pressure-Dependent Microbial Activity in the Context of Subsurface Biomining