Attachment of Leptospirillum sp. to chemically modified pyrite surfaces. Fast and simple electrochemical monitoring of bacterial-mineral interactions

Highlights

• Surface modified pyrite with different ratio of S/Fe compounds was exposed to bacteria.

• EIS spectra allow following the first steps of bacteria-electrode attachment.

• Phase angle changes (single frequency) were correlated to attachment and direct bacterial count.

• Best attachment was found when S and Fe compounds are in the oxidized pyrite surface.

Abstract

Bacterial cell attachment that results in biofilm formation is the first step of bacteria-mineral interaction, and it is known that they strongly depend on the chemical characteristics of the mineral surface. In some industrial processes, like those used in biohydrometallurgy, the minerals are present in different chemical oxidation states, especially when heterogeneous low-grade ore deposits and mining tailings are used as starting materials for microbial inoculation. This study describes a strategy for monitoring bacterial attachment to pyrite (FeS₂) and surface modified pyrite weathered in the culture growth medium used (pH 1.8), by means of a non-invasive electrochemical technique such as electrochemical impedance spectroscopy (EIS). The EIS evaluation of Leptospirillum sp. interaction with pyrite and surface modified pyrite weathered electrodes revealed significant changes at low frequencies, depending of the compounds presented over the four samples used, including unmodified pyrite and three surface modified samples. Once the frequency at which the adhesion process can become uncover was selected (0.1 and 0.05 Hz), the phase angle variation at such frequency was determined using different microbial concentrations. Different microbial attachment values were obtained for the different electrodes and related to the initial inoculum (2 × 10⁸ cells/mL), as follow: FeS₂/Fe(OH)_n,S⁰ (38 ± 3.2%); FeS₂/Fe(OH)_n (31 ± 5.5%); FeS₂ (27 ± 4.7%) and FeS₂/S⁰ (18 ± 3.8%). Microbial attachment to unmodified and modified surfaces, evaluated by EIS, was corroborated with the traditional method of bacterial attachment evaluation by cell count in a Neubauer chamber (r² = ~0.9). This strategy could be used in developing sensors for the fast and efficient bacterial attachment evaluation in minerals.

Introduction

The use of biohydrometallurgy processes in mining has increased over the last decades because of the need to process low-grade refractory ores, whose chemical or thermal processing is unprofitable (Gentina and Acevedo, 2016; Tao and Dongwei, 2014). Processes like mineral biooxidation require the presence of microorganisms that act as biocatalysts in mineral oxidation processes (Olson et al., 2003; Rohwerder et al., 2003). They are mostly acidophilic (pH 1.3 to 2.5), chemolithotrophic microorganisms, meaning that their source of energy is the oxidation of sulfur (Acidithiobacillus thiooxidans), iron (Leptospirillum ferrooxidans and L. ferriphilum), and iron‑sulfur (Acidithiobacillus ferrooxidans, among others). Due to their metabolic activity, these microorganisms are responsible for process efficiency (Rohwerder et al., 2003).

One of the most important stages in the development of biohydrometallurgy processes is the selection and evaluation of microorganisms or indigenous microbial communities that exhibit the greatest ability to solubilize metals from minerals (Vera et al., 2013). The realization of such studies may take weeks or months since they are based on a metabolic response of individual cells and their capacity for surface attachment and colonization, as well as on the structure of a biofilm formed by these cells on a mineral (Zhao et al., 2013). A fast way to determine the bacterial ability to form biofilms is by evaluating one of its initial phases, such as cell attachment to the mineral, a phenomenon that takes place within a few hours (Li et al., 2018, Li et al., 2016; Saavedra et al., 2018a).

A biofilm is a complex, heterogeneous, four-dimensional (spatiotemporal) microenvironment made up of interacting sessile microbial populations or communities (Garrett et al., 2008; Lara et al., 2010), embedded in an extracellular matrix composed of extracellular polymeric substances (EPS). The fourth dimension, the time, reflects changes in the microbial population that brings a dynamic risk to biofilms. In contrast, the biofilm heterogeneity includes the physical and chemical gradients that determine distribution and abundance of cells and macromolecules (Lara et al., 2010).

Biofilm formation occurs in different stages, described as (i) attachment to surface, (ii) formation of monolayer and production of matrix, (iii) microcolony formation and (iv) the formation of a mature biofilm (Vasudevan, 2014). These stages can take different times depending on the microorganism type, the metabolic state, and the interacting surface. In the case of microorganisms that participate in biomining processes (A. ferrooxidans, A. thiooxidans, L. ferrooxidans, among others), completing these four stages can take months due to their slow growth; the first stage is the fastest, being able to last only a few hours (Florian et al., 2011). Bacterial attachment is the first step in the establishment of a biofilm over a mineral, which occurs typically in the first hours of microbial/mineral encounter; several authors have shown that the future efficiency of metal recovery depends strongly on it (Castro and Donati, 2016; Florian et al., 2011; Vera et al., 2013; Zhu et al., 2015). This is a complex process in which electrostatic and hydrophobic/hydrophilic forces, biological mechanisms, and chemical reactions interact (Diao et al., 2014; Figueredo et al., 2018; Garrett et al., 2008). After the attachment to the surface occurs, the microorganisms produce EPS that serve as a glue and supporting shell on the mineral to form the biofilm and increasing bioleaching efficiency (Aguirre et al., 2018; Lara et al., 2010). Biofilm formation has two important and positive effects in biohydrometallurgical process; first, the EPS matrix provided a protective environment for the microorganism against metal ions presented in the solution, by both exclusion and chelating phenomena (Saavedra et al., 2020); second, the EPS create a microenvironment where iron is accumulated in high concentrations (e.g., more than 53 g/ L of iron) (Sand and Gehrke, 2006), therefore the ferric ions provokes a localized oxidation on the surface of the mineral, commonly known as pitting (Saavedra et al., 2018a). Moreover, the attachment of microorganisms to pyrite can be influenced by the different pyrite corrosion chemical species; the attachment to passivated materials (as S° and polysulfides) is impaired (Saavedra et al., 2018b).

Once the biofilm is formed, it exhibits special characteristics that allow the constituent bacteria to chemically attack the mineral more efficiently. The presence and distribution of the biofilm have been reported to be related to the process efficiency (Gehrke et al., 2001; Saavedra et al., 2013), and even pitting on the mineral surface has been reported as a result of corrosion caused by biofilm bacteria (Saavedra et al., 2018a).

In a previous study we reported the results of the interaction between an abiotic culture medium typically used in biohydrometallurgical processes, and pyrite and modified pyrite surfaces, by mimicking different oxidation states of the mineral (Saavedra et al., 2018b). The interaction between the culture medium and pyrite caused significant chemical changes to the mineral surface as well as the formation of chemical species with passive behavior, such as elemental sulfur, and jarosite. This passive behavior was shown in an electrochemical study as a significant decrease in charge values, associated with mineral oxidation under weathering conditions due to the culture medium; some authors have suggested that microbial activity (as well biofilm establishment) can slow down the passivation of the mineral surface (Tao and Dongwey, 2014).

Bacterial cell attachment and surface colonization that results in biofilm formation are the first steps of bacteria-mineral interaction, and it is known that they strongly depend on the chemical characteristics of the mineral surface. The process of cell attachment and biofilm formation on surfaces can be evaluated by optical methods (Florian et al., 2011; Ramsden et al., 1995), atomic force microscopy (Fang et al., 2000; Zhang et al., 2007) or certain electrochemical analyses (Kim et al., 2011; Wan et al., 2016), among others. Most of these methods are destructive, time-consuming, and/or require sample preparation. A new method that can replace or complement the aforementioned at the microbiological laboratory, which does not require expensive equipment and/or highly trained staff is desirable. Electrochemical instrumentation allowing EIS experiments are now available from multiple providers and relatively low competitive cost, with software allowing automatization and simultaneous (by multiplexing) measurement of an elevated number of samples with a single potentiostat. EIS is a non-destructive technique, capable of monitoring the time-lapse of the attachment process by using a single sample, simplifying the analytical set-up, and limiting workload, so the selection of better microbial strain/s or natural/artificial consortia (for a given mineral) in simulated lab conditions can be performed.

EIS has been previously used to characterize bacterial attachment, on conducting materials, e.g., adhesion of pathogenic bacteria onto indium tin oxide (ITO) or gold electrodes (Barreiros dos Santos et al., 2015; Koyama et al., 2013). There are just a few EIS studies on sulfide minerals (Ramírez-Aldaba et al., 2018), and they have focused on the characterization of initial phases of bioleaching and not on the monitoring of microbial adhesion (Arena et al., 2016).

Previously published work has shown the use of EIS to characterize bioleaching process, where different minerals relevant to the biohydrometallurgy industry were used. Changes in time constants at low frequencies (10–0.01 Hz), showing capacitance increase and slight resistance decrease have been observed in arsenopyrite oxidation in presence of A. thiooxidans (Ramírez-Aldaba et al., 2016), similar results were obtained using chalcopyrite (López-Cázares et al., 2017). Different results (capacitance decrease and slight resistance increase) have been obtained when chalcopyrite (Bevilaqua et al., 2004) or bornite oxidation was mediated by A. ferrooxidans (Bevilaqua et al., 2009). The obtained results are strongly dependent of several factors, including the mineral composition, crystallographic structure, time lapse of the experiment, surface oxidation grade, media composition, and metabolic characteristics of the microorganism/s used, among other experimental and biological factors.

The search for new monitoring systems opens an opportunity for increasing the efficiency and decreasing the times of characterization and follow-up of microbial adhesion processes. This study establishes the feasibility of using the EIS technique as a method for real-time monitoring of the attachment of chemolithotrophic (acidophilic, iron-oxidizing) bacteria Leptospirillum sp. to pyrite and surface modified pyrite minerals, emulating the conditions the mineral is exposed to in industrial processes. It aims to lay the foundations for the development of biosensors that would allow a rapid selection of biooxidizing bacteria based on their capability for attachment to mineral surfaces.