Effect of energy source, salt concentration and loading force on colloidal interactions between Acidithiobacillus ferrooxidans cells and mineral surfaces

https://doi.org/10.1016/j.colsurfb.2015.05.026Get rights and content

Highlights

  • Distinct retraction patterns of Acidithiobacillus ferrooxidans grown with different energy sources were observed.

  • Interaction forces between A. ferrooxidans and minerals with bacterial probes were quantified.

  • The conformation of surface biopolymers was affected by salt concentration.

Abstract

The surface appendages and extracellular polymeric substances of cells play an important role in the bacterial adhesion process. In this work, colloidal forces and nanomechanical properties of Acidithiobacillus ferrooxidans (A. f) interacted with silicon wafer and pyrite (FeS2) surfaces in solutions of varying salt concentrations were quantitatively examined using the bacterial probe technique with atomic force microscopy. A. f cells were cultured with either ferrous sulfate or elemental sulfur as key energy sources. Our results show that A. f cells grown with ferrous ion and elemental sulfur exhibit distinctive retraction force vs separation distance curves with stair-step and saw tooth shapes, respectively. During the approach of bacterial probes to the substrate surfaces, surface appendages and biopolymers of cells are sequentially compressed. The conformations of surface appendages and biopolymers are significantly influenced by the salt concentrations.

Introduction

Bacterial adhesion to mineral surfaces is of great importance to the growth of bacteria in natural habitats and in many industrial applications [1], [2]. In these processes, the bacterial surface largely determines the adhesion process by the surface appendages (e.g. pili and flagella) and extracellular polymeric substances. In the bioleaching process, Acidithiobacillus ferrooxidans (A. f) was the first described metal sulfide oxidizing microorganism, which is affiliated with the Gram-negative γ-Proteobacteria. It is one of the most important species in the bioleaching of sulfide ores operating at temperatures less than 40 °C [3]. A. f is endowed with a remarkably broad metabolic capacity, as it can live on the oxidation of ferrous salts, elemental sulfur and a variety of sulfide minerals [4], [5], [6].

Various growth substrates may induce physiological differences in the chemical composition of cell surfaces, which reflects the response of cells in optimizing nutrient uptake. Research associated with macroscopic assays of bacterial adhesion [7], [8], analysis of chemical compositions for cell surface biopolymers [8], [9] and characterization of cell surface structures [10], [11] has been well documented in the literature. However, the effect of different energy sources on bacterial adhesion behavior and the correlation between surface properties and fundamental interacting forces have not been fully resolved at the nanoscale.

In the past decade, remarkable developments in atomic force microscopy (AFM) have made it a versatile tool in determining the surface structures and specific interactions of biological samples under near-physiological conditions [12], [13]. AFM is capable of sensing picoNewton forces in aqueous solutions, and the obtained force–separation curves can provide information on the adhesive and nanomechanical properties of biological samples [14], [15], [16], [17], [18]. Tipped cantilevers have been extensively used as indenters to probe the elastic properties of different bacterial cells such as Escherichia coli [19], [20], Pseudomonas aeruginosa [21] and Shewanella putrefaciens [18]. Alternatively, a cell probe [20], [22] can be used to measure the overall mechanical properties of the cell. A colloidal probe (a microsphere glued onto the end of a cantilever) is often used to indent larger mammal cells [17], [23]. However, the use of AFM to investigate the nanomechanical properties of bioleaching bacteria has been rarely reported.

The goal of this study is to relate the adhesion behavior and nanomechanical interactions to the biophysical responses of bacterial cells to the change in environmental conditions (energy sources and salt concentrations). To this end, we performed AFM force measurements using bacterial probes constructed with A. f cells grown with different energy sources of Fe2+ and S0, and exposed the bacterial probes to solutions of various salt concentrations. The shape of the retraction curves, adhesion forces and Young's moduli of cell surface biopolymers were compared with A. f cells grown in different energy sources. Interesting features such as sequential “jump-in” events of approaching curves and distinct retraction curve patterns of A. f cells grown with the energy sources were obtained. Overall, our findings quantitatively describe the adhesion behaviors of A. f on mineral surfaces and the nanomechanical properties may help further the understanding of responses of cell surface appendages to environmental stimuli.

Section snippets

Microorganism and growth conditions

A. f was kindly provided by Professor Guohua Gu (School of Mineral Processing and Bioengineering, Central South University, China). Cells were cultured at 30 °C in 9 K medium (pH 2.0) [24]: (NH4)2SO4, 3 g/L; KCl, 0.1 g/L; K2HPO4·3H2O, 0.5 g/L; MgSO4·7H2O, 0.5 g/L; Ca(NO3)2 0.01 g/L. Bacteria were grown with 4.47% (w/v) FeSO4 and 3% (w/v) elemental sulfur as energy source, respectively. A. f cells were incubated on a rotary shaker at 170 rpm to their mid-exponential growth phase.

Substrate preparation

The silicon wafers (100

Bacterial probes

The SEM image in Fig. 2A shows a cell-coated colloidal probe which was used for a series of force measurements. The contact area of the microsphere was covered by bacterial cells, thus the measured force curves can reflect the true bacterial–mineral interactions. Fig. 2C displays the typical approach and retraction force curves recorded from a ferrous ion-grown cell probe. Far from the substrate surface, the bacterial probe senses no interaction forces between the surfaces. As the probe

Conclusions

In this study, the fundamental interaction forces between A. f cells cultured with different energy sources and the substrates were directly quantified with the bacterial probe technique. Our results show that the conformational changes in biopolymers due to the salt concentration are important factors in influencing the surface potentials, adhesion behavior and the softness of the bacterial cells. This research provides fundamental understanding and evidence that different energy sources and

Acknowledgements

The authors gratefully acknowledge The University of Queensland Postgraduate Scholarship (UQRS). We thank Mr. Guozhao Ji for advice on data processing.

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