Sören Bellenberg

AHL signaling molecules with a large acyl chain enhance biofilm formation on sulfur and metal sulfides by the bioleaching bacterium Acidithiobacillus ferrooxidans.

Biofilm formation plays a pivotal role in bioleaching activities of bacteria in both industrial and natural environments. Here, by visualizing attached bacterial cells on energetic substrates with different microscopy techniques, we obtained the first direct evidence that it is possible to positively modulate biofilm formation of the extremophilic bacterium Acidithiobacillus ferrooxidans on sulfur and pyrite surfaces by using Quorum Sensing molecules of the N-acylhomoserine lactone type (AHLs). Our results revealed that AHL-signaling molecules with a long acyl chain (12 or 14 carbons) increased the adhesion of A. ferrooxidans cells to these substrates. In addition, Card-Fish experiments demonstrated that C14-AHL improved the adhesion of indigenous A. ferrooxidans cells from a mixed bioleaching community to pyrite. Finally, we demonstrated that this improvement of cell adhesion is correlated with an increased production of extracellular polymeric substances. Our results open up a promising means to develop new strategies for the improvement of bioleaching efficiency and metal recovery, which could also be used to control environmental damage caused by acid mine/rock drainage.

Shotgun proteomics study of early biofilm formation process of Acidithiobacillus ferrooxidans ATCC 23270 on pyrite

Acidithiobacillus ferrooxidans is a chemolithoautotrophic, mesophilic Gram‐negative bacterium able to oxidize ferrous iron, sulfur, and metal sulfides. It forms monolayer biofilms where extracellular polymeric substances are essential for cell attachment and metal sulfide leaching. High‐throughput proteomics has been applied to study the early process of biofilm formation on pyrite by At. ferrooxidans ATCC 23270. After 24 h contact with the mineral, planktonic and sessile (biofilm) cell subpopulations were separated and proteins extracted. In total, 1319 proteins were detected in both samples. Sixty‐two of these were found to be increased in biofilms. Additionally, 25 proteins were found to be decreased in the biofilm cell subpopulation. Three transcriptional factors were found to be increased or decreased among both cell subpopulations, suggesting their potential involvement in the regulation of these processes. Although no significant differences were observed for the known proteins related to ferrous iron and sulfur oxidation pathways among both cell subpopulations, the results presented here show that the early steps of At. ferrooxidans biofilm formation consist of a set of metabolic adaptations following cell attachment to the mineral surface. Functions such as extracellular polymeric substances biosynthesis seem to be pivotal. This first high‐throughput proteomic study may also contribute to the annotation of several unknown At. ferrooxidans proteins found.

Biofilm formation, communication and interactions of leaching bacteria during colonization of pyrite and sulfur surfaces

Bioleaching of metal sulfides is an interfacial process where biofilm formation is considered to be important in the initial steps of this process. Among the factors regulating biofilm formation, molecular cell-to-cell communication such as quorum sensing is involved. A functional LuxIR-type I quorum sensing system is present in Acidithiobacillus ferrooxidans. However, cell-to-cell communication among different species of acidophilic mineral-oxidizing bacteria has not been studied in detail. These aspects were the scope of this study with emphasis on the effects exerted by the external addition of mixtures of synthetic N-acyl-homoserine-lactones on pure and binary cultures. Results revealed that some mixtures had inhibitory effects on pyrite leaching. Some of them correlated with changes in biofilm formation patterns on pyrite coupons. We also provide evidence that A. thiooxidans and Acidiferrobacter spp. produce N-acyl-homoserine-lactones. In addition, the observation that A. thiooxidans cells attached more readily to pyrite pre-colonized by living iron-oxidizing acidophiles than to heat-inactivated or biofilm-free pyrite grains suggests that other interactions also occur. Our experiments show that pre-cultivation conditions influence A. ferrooxidans attachment to pre-colonized pyrite surfaces. The understanding of cell-to-cell communication may consequently be used to develop attempts to influence biomining/bioremediation processes.

Erratum to: Visualization and analysis of EPS glycoconjugates of the thermoacidophilic archaeon Sulfolobus metallicus

Biofilms are surface-associated colonies of microorganisms embedded in a matrix of extracellular polymeric substances (EPS). As EPS mediate the contact between cells and surfaces, an understanding of their composition and production is of particular interest. In this study, the EPS components of Sulfolobus metallicus DSM 6482(T) forming biofilms on elemental sulfur (S(0)) were investigated by confocal laser scanning microscopy (CLSM). In order to visualize cell and EPS distributions, biofilm cells were stained with various dyes specific for glycoconjugates, proteins, nucleic acids and lipids. Biofilm cells on S(0) were heterogeneously distributed and characterized as individual cells, microcolonies, and large clusters up to a hundred micrometers in diameter. The glycoconjugates in biofilms were detected by fluorescence lectin-binding analysis (FLBA). Screening of 72 commercially available lectins resulted in the selection of 21 lectins useful for staining biofilms of S. metallicus (T). Capsular EPS from planktonic cells were mainly composed of carbohydrates and proteins. In contrast, colloidal EPS from planktonic cells were dominated by carbohydrates. Proteins were found to be major components in EPS from biofilms on S(0). Using specific probes combined with CLSM, we showed that extracellular proteins and nucleic acids were present in the EPS matrix. Finally, we showed that S. metallicus (T) cells were embedded in a flexible EPS matrix. This study provides new insights into archaeal biofilms and EPS composition and properties with respect to their interactions with S(0).

Change of Extracellular Polymeric Substances Composition of Thiobacillus thioparus in Presence of Sulfur and Steel

The process of microbially influenced corrosion (MIC) of metallic underground constructions is often related with biofilm formation and metabolic activity of sulfur cycle bacteria. The extracellular polymeric substances (EPS) produced by these bacteria in biofilms on metallic surfaces establish the interfacial reaction space in which relevant corrosion processes occur. The EPS composition of the sulfur oxidizing bacterium Thiobacillus thioparus DSM 505 varies according to growth conditions. Presence of elemental sulfur and mild steel are demonstrated here as stimulants for variation of the EPS composition. The distribution of sugar moieties in the EPS of planktonic and biofilm grown cells of Thiobacillus thioparus DSM 505 was observed by fluorescently labeled lectin binding assays. The strongest signal was found with the PWM lectin (Pokeweed, Phytolacca americana) specific for Poly- N-Acetylglucosamine (PNAG). Cell associated proteins, visualized by Thiazine Red staining, were observed in the planktonic growth mode. Low amounts of EPS proteins from planktonic were detected. The observed influence of elemental sulfur and mild steel on the EPS composition in biofilm forming cells can be suggested to have a pivotal role in interfacial processes such as (bio) corrosion. Understanding factors that mediate changes in the composition and surface associated structural features of EPS as a consequence of working materials may help to develop a new strategy for biocorrosion prevention.

Multi-omics reveal the lifestyle of the acidophilic, mineral-oxidizing model species Leptospirillum ferriphilumT.

ABSTRACT Leptospirillum ferriphilum plays a major role in acidic, metal-rich environments, where it represents one of the most prevalent iron oxidizers. These milieus include acid rock and mine drainage as well as biomining operations. Despite its perceived importance, no complete genome sequence of the type strain of this model species is available, limiting the possibilities to investigate the strategies and adaptations that Leptospirillum ferriphilum DSM 14647T (here referred to as Leptospirillum ferriphilumT) applies to survive and compete in its niche. This study presents a complete, circular genome of Leptospirillum ferriphilumT obtained by PacBio single-molecule real-time (SMRT) long-read sequencing for use as a high-quality reference. Analysis of the functionally annotated genome, mRNA transcripts, and protein concentrations revealed a previously undiscovered nitrogenase cluster for atmospheric nitrogen fixation and elucidated metabolic systems taking part in energy conservation, carbon fixation, pH homeostasis, heavy metal tolerance, the oxidative stress response, chemotaxis and motility, quorum sensing, and biofilm formation. Additionally, mRNA transcript counts and protein concentrations were compared between cells grown in continuous culture using ferrous iron as the substrate and those grown in bioleaching cultures containing chalcopyrite (CuFeS2). Adaptations of Leptospirillum ferriphilumT to growth on chalcopyrite included the possibly enhanced production of reducing power, reduced carbon dioxide fixation, as well as elevated levels of RNA transcripts and proteins involved in heavy metal resistance, with special emphasis on copper efflux systems. Finally, the expression and translation of genes responsible for chemotaxis and motility were enhanced. IMPORTANCE Leptospirillum ferriphilum is one of the most important iron oxidizers in the context of acidic and metal-rich environments during moderately thermophilic biomining. A high-quality circular genome of Leptospirillum ferriphilumT coupled with functional omics data provides new insights into its metabolic properties, such as the novel identification of genes for atmospheric nitrogen fixation, and represents an essential step for further accurate proteomic and transcriptomic investigation of this acidophile model species in the future. Additionally, light is shed on adaptation strategies of Leptospirillum ferriphilumT for growth on the copper mineral chalcopyrite. These data can be applied to deepen our understanding and optimization of bioleaching and biooxidation, techniques that present sustainable and environmentally friendly alternatives to many traditional methods for metal extraction.

Biofilm formation and interspecies interactions in mixed cultures of thermo-acidophilic archaea Acidianus spp. and Sulfolobus metallicus.

The understanding of biofilm formation by bioleaching microorganisms is of great importance for influencing mineral dissolution rates and to prevent acid mine drainage (AMD). Thermo-acidophilic archaea such as Acidianus, Sulfolobus and Metallosphaera are of special interest due to their ability to perform leaching at high temperatures, thereby enhancing leaching rates. In this work, leaching experiments and visualization by microscopy of cell attachment and biofilm formation patterns of the crenarchaeotes Sulfolobus metallicus DSM 6482(T) and the Acidianus isolates DSM 29038 and DSM 29099 in pure and mixed cultures on sulfur or pyrite were studied. Confocal laser scanning microscopy (CLSM) combined with fluorescent dyes as well as fluorescently labeled lectins were used to visualize different components (e.g. DNA, proteins or glycoconjugates) of the aforementioned species. The data indicate that cell attachment and the subsequently formed biofilms were species- and substrate-dependent. Pyrite leaching experiments coupled with pre-colonization and further inoculation with a second species suggest that both species may negatively influence each other during pyrite leaching with respect to initial attachment and pyrite dissolution rates. In addition, the investigation of binary biofilms on pyrite showed that both species were heterogeneously distributed on pyrite surfaces in the form of individual cells or microcolonies. Physical contact between the two species seems to occur, as revealed by specific lectins able to specifically bind single species within mixed cultures.

The Biofilm Lifestyle of Acidophilic Metal/Sulfur-Oxidizing Microorganisms

Microrganisms are well known for their unique ability to thrive in different lifestyles (e.g. planktonic or sessile) and environments, even within extreme ones. The most common and widespread lifestyle of microbes on earth is in form of biofilms, associated colonies of microorganisms embedded in a matrix of extracellular polymeric substances (EPS). Extremely acidophilic metal/sulfur-oxidizing microorganisms (AMOM) thrive in special ecological niches characterized by harsh conditions such as low pH (below 3) and high concentration of heavy metals across a broad range of temperatures. The molecular mechanisms controlling biofilm formation in acidophilic leaching bacteria are starting to be elucidated while these operating in archaea are far less explored. In this chapter we provide an overview about the biofilm lifestyle of AMOM. This includes surface sciences, microscopy, cell-cell communication, interspecies interactions as well as molecular and high-throughput studies. Current knowledge on the EPS composition and biofilm visualization of acidophiles is also included. Future perspectives in this field include the elucidation of EPS biosynthesis pathways and a comprehensive analysis of the chemical nature of the EPS polymers. Cell-cell communication and microbial interactions within multispecies biofilms of acidophiles are considered to be crucial determinants in controlling the metabolic activity of AMOM. Either for their biotechnological applications in biomining or in mitigation of acid mine drainage (AMD) generation, further studies in both fields may presumably reveal key perspectives to influence and control bioleaching of sulfide minerals.

Microbial Community Composition on Lignite before and after the Addition of Phosphate Mining Wastes

Empirical field tests in which phosphate mining wastes (NPR) were added to various sulphidic mining wastes resulted in neutral drainage. A biofilm was documented on waste rock covering the pyrite. This was likely the cause of the improved effluents. NPR was added to sterile columns containing pyritic lignite and spiked with granulated pyrite. The biological columns were inoculated with 108 cells/g of lignite-cultured microbes from Rio Agrio, Argentina. The microbes were maintained at 30°C using pyrite as sole energy source. The microbial populations were monitored with MPN before NPR addition and 2, 4 and 10 weeks later. After 45 days strong bioleaching was evident, as measured by electrical conductivity. Concurrently, a shift in the microbial populations took place. In the presence of NPR about 90% of populations consisted of heterotrophic neutrophiles covering the pyrite surfaces with a 10 μm thick biofilm, whereas without NPR, 99% of the populations were iron oxidizing acidophiles in mono-layered biofilms. These results are relevant to controlling processes in heap leaching and acid rock drainage.

Attachment to Minerals and Biofilm Development of Extremely Acidophilic Archaea

Biofilm development of F. acidiphilum BRGM4 on polycarbonate filters floating on liquid medium and pyrite surfaces were studied by confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM) combined with epifluorescence microscopy (EFM). Results show that F. acidiphilum biofilms were heterogeneously distributed, and varied among different growth conditions, such as inorganic phosphate (Pi) starvation and glucose supplementation. Biofilm and planktonic cells showed significant morphological differences. Capsular EPS were observed in both biofilm and planktonic cells. Cells showed preferential attachment to the cracks/defects of pyrite surfaces.