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Microbial transformations of Fe in the biogeosphere
Different groups of bacteria are responsible for direct Fe(II) oxidation in presence of oxygen (O2) or of Fe(III) reduction in anaerobic conditions, in a large range of pH (Figure I-3).
The biological catalysis of Fe oxidation/reduction is thought to be responsible for the formation of most naturally occurring insoluble Fe(II, III) oxides and consequently plays a key role in the biogeochemical cycling of Fe (Kappler and Straub, 2005; Li et al., 2015). Fe(II) can function as an electron source for iron-oxidizing bacteria under both oxic and anoxic conditions and Fe(III) can function as a terminal electron acceptor under anoxic conditions for iron-reducing bacteria (Weber et al., 2006). Variable pH values also select iron-oxidizing/reducing bacteria, and below pH 3, acidophilic groups of bacteria e.g., Acidithiobacillus and Sulfolobus (sulfur oxidation) thrive in acidic pH and help in dissolving the iron minerals e.g., pyrite (FeS2) from the solid phase into the aqueous phase (Brock et al., 1972; Kelly and Wood, 2000; Rodríguez et al., 2003). Moreover, iron-reducing bacteria Acidiphilium was isolated from acidic coal mine drainage and was able to reduce ferric iron under anoxic acidic environments even possibly in oxygen-containing condition (Johnson and Bridge, 2002; Wichlacz et al., 1986). Microbial ferrous Fe oxidation provides energy to acidophiles such as Leptospirillum which is well known to achieve Fe(II) oxidation at pH below 3 (Ojumu and Petersen, 2011; Van Scherpenzeel et al., 1998). In the pH range 3-6, Acidobacterium is an acidophilic, chemoorganotrophic bacterial genus isolated from acidic mineral environments and capable of dissimilatory iron reduction under anaerobic conditions (Coupland and Johnson, 2008; Kishimoto et al., 1991). The bacterial species of the genus Gallionella are typical iron-oxidizing bacteria that grow under neutral (pH 6.5-8) or moderately acidic (pH 4) conditions (De Vet et al., 2011; Fabisch et al., 2013). The genus Leptothrix contains “iron-oxidizing” or “model Mn(II)-oxidizing” species and is ubiquitously distributed in the aquatic environment (El Gheriany et al., 2009; Ghiorse, 1984). Leptothrix can be readily found in sites with a circumneutral pH, an oxygen gradient and a source of reduced Fe and manganese minerals (Emerson and Moyer, 1997; Sawayama et al., 2011). Finally, Geobacter and Shewanella are the two most studied dissimilatory Fe reducing genera under anaerobic and near-neutral pH conditions up to now (Engel et al., 2019; Han et al., 2018; Jiang et al., 2020; Li et al., 2012).
Influence of redox conditions and organic matter on iron reduction
Iron redox reactions have the potential to support substantial microbial populations in soil and sedimentary environments, as Fe is the fourth most abundant element in the Earth’s crust (Weber et al., 2006). Motomuraand and Yokoi (1969) have suggested that the different forms of ferrous Fe in flooded soils have a physical and chemical influence on the development and stabilization of soil structure, which in turn exerts an influence on soil productivity (Gotoh and Patrick Jr, 1974; Motomura and Yokoi, 1969). It is also well established now that flooded soils in anaerobic conditions are subjected to a succession of Fe transformations from the ferric to ferrous state under reducing conditions caused by a wide variety of facultative anaerobic soil bacteria. Fe reduction can be the result of bacterial metabolism, Fe functioning as an electron acceptor in dissimilatory iron reduction (DIR). Redox potential (oxidation-reduction) has a marked effect on microbial Fe behavior because a change in redox status in soils implies changing the availability of electron acceptors. As shown on the electron donors/acceptors tower in Figure I-4, Fe(III) may be used as an electron acceptor after depletion of oxygen, nitrate and manganese oxides. Pett-Ridge and Silver (2006) have indicated that some flexible microorganisms were able to respire/ferment or use multiple electron acceptors under fluctuating redox conditions, thus microbial populations can be periodically activated and inactivated, which in turn quickly alters the nature and rate of key biogeochemical transformations (DeAngelis et al., 2010; Husson, 2013; Pett-Ridge et al., 2006).
Ginn et al. (2017) studied the influence of oxygen variations on Fe speciation in soils from the Luquillo Critical Zone Observatory (Puerto Rico) through batch experiments in flasks inoculated with Shewanella. Shewanella cultures reduced Fe(III) much faster under redox fluctuations (cycles of oxic-anoxic conditions) than the oxic controls from soils (Ginn et al., 2017).
Moreover, several mechanisms are involved in the dynamics of metals and metalloids connected with the iron cycle: (1) dissolution of Fe(III) oxides can release the adsorbed/co-precipitated elements; (2) DIRB may directly change the speciation of the associated elements, because they can use both Fe(III) and other metals/metalloids as electron acceptors (Lovley, 2008), and (3) the natural microbial communities include both DIRB and other bacteria that use In soils, oxidation of FeII(aq) can induce the formation of colloidal complexes of Fe and organic matter (Peiffer et al., 1999; Pullin and Cabaniss, 2003). Dissolved organic matter appears to be a key factor in the control of the Fe(III)-oxyhydroxide dissolution rate. More specifically, organic matter, by strongly binding Fe(II), prevents Fe(II) readsorption and subsequent Fe secondary mineral formation, both of which are known to strongly decrease Fe(III)-oxyhydroxide dissolution rates (Davranche et al, 2013). In presence of humic substances, Fe particles of nanometric size (colloids) are formed in the humic matrix (Pédrot et al., 2011). Bioreduction experiments demonstrated that bacteria (Shewanella putrefaciens CIP 80.40 T) were able to reduce these Fe nanoparticles associated with humic organic matter about eight times faster than pure nano-lepidocrocite. These results suggest that in natural environments organic matter influence the type of iron phases formed in presence of oxygen, and the rate of Fe(II) production in anaerobic conditions. Alternance of redox conditions can lead to the formation of colloids in soils, composed of natural organic matter, complexed iron and other soil elements such as Al, Ti or Si (Thompson et al., 2006a). Moreover, under reducing conditions, the pH rise can be a key factor controlling organic matter solubilization during Mn-and Fe-oxyhydroxides reductive dissolution, as observed by Grybos et al., 2009 with a wetland soil. Pédrot et al. (2008) performed soil column experiments and showed that some trace elements, in particular Pb, Ti and U, were mobilized by humic acids containing iron nanoparticles. Humic substances directly or indirectly promoted the colloidal transport of insoluble trace elements either by binding trace elements or by stabilizing a ferric carrier phase (Pédrot et al., 2008).
Classification of Fe-minerals
Fe and Fe-minerals are common components in several compartments of the critical zone (e.g. soils, sediments and aquifers) and are present in many different mineralogical forms. Many different scientific disciplines are interested in Fe oxides (Figure I-6). There are sixteen known Fe oxides, oxy-hydroxides and hydroxides with different mineral structures which are listed in Table I-1 (Bonneville, 2005; Cornell and Schwertmann, 2003b; Fernández-Remolar, 2015).
Fe(III) (oxyhydr)oxides differ by their crystallinity, initially amorphous (the first formed after oxidation of Fe(II), maturation under different conditions then leads to other forms. Iron oxides polymorphs differ in the organization of the Fe(OH)n building blocks as shown in Figure I-7.
In flooded soils, the presence of Fe(III) will induce precipitation of Fe(II) from carbonate. Baas Becking et al. (1960) reported the formation of the Fe(II)/Fe(III) hydroxy-carbonates siderite and maghemite under natural aqueous environments and Halama et al. (2016) suggested that diagenetic magnetite in banded iron formations was possibly formed by microbial Fe(III) reduction during early diagenesis (Becking et al., 1960; Helama et al., 2016). Moreover, various concentrations of Fe(II) and forms/amounts of total Fe(III) might cause other phases such as magnetite/siderite/green rust to be formed in microbial (biotic) iron reduction systems (Mortimer et al., 2011; Ona-Nguema et al., 2002; Taylor, 1980). In addition, oxidation leads to the formation of either goethite, lepidocrocite, ferrihydrite or mixtures of these phases, depending on the mode of oxidation, and the presence of impurities also may result in the release of initially co-precipitated ions (Senn et al., 2017; Taylor, 1980).
Ferrihydrite is an amorphous or weakly crystalline Fe mineral (Bonneville, 2005). Structural studies carried out by (Feitknecht et al., 1973) indicated that 2-line ferrihydrite consists of local coherently scattered regions formed by four planar Fe(O,OH)6 octahedrons and Michel et al. indicated that ferrihydrite is nano-crystalline with a delta-Keggin local structure (Michel et al., 2007; Michel et al., 2011). Whereas, in goethite each Fe(III) ion is surrounded by three O2- and three OH- resulting in FeO3(OH3) octahedrons (Bonneville, 2005). Moreover, (Hiemstra, 2013) elucidated the surface structure of ferrihydrite particles whose faces have a much higher site density of singly-coordinated FeOH(H) groups in comparison to the main faces of goethite. Therefore, the related adsorption capacity per unit surface area of ferrihydrite is much higher than that of goethite. Lepidocrocite is metastable with respect to goethite which consists of double chains of Fe(O, OH)6 octahedrons running parallel to the c-axis. Hematite consists of layers of Fe(O)6 octahedrons and it has a similar thermodynamic stability to goethite, thus it is very often found in association with goethite (Tardy and Nahon, 1985).
Dissolution of Fe(III) (oxyhydr)oxides and iron cycling in surface environments
Fe (oxyhydr)oxides are common components in several compartments of the critical zone (e.g. soils, sediments and aquifers) and are present in many different mineralogical forms. Understanding biogeochemical behavior and iron cycling is fundamental for many scientific communities (Bonneville et al., 2004; Roden et al., 2004). Indeed, the mobility of trace elements (TE) is partly controlled by iron speciation, mineralogy and reactivity (Cornell and Schwertmann, 2003b).
Abiotic dissolution
Fe(III) (oxyhydr)oxides can be dissolved by surface protonation, a dissolution mechanism depending on pH conditions that can be enhanced by organic acids and anions (Cl-) (Zinder et al., 1986). Besides, two other dissolution mechanisms have also been reported, ligand promoted dissolution and bulk reductive dissolution (Afonso et al., 1990; Holmén and Casey, 1996; Kraemer, 2004). (Siffert and Sulzberger, 1991) indicated that reductive dissolution of hematite in the presence of oxalate occurs as a photocatalytic process, and Holmén & Casey studied the rate of goethite dissolution in the presence of acetohydroxamic acid in different pH conditions. The rate of reductive dissolution of several synthetic Fe(III) (oxyhydr)oxides in 10 mM ascorbate at pH 3 has been shown to occur according to the order ferrihydrite> lepidocrocite > goethite> hematite (Bonneville et al., 2004; Larsen and Postma, 2001).
In natural environments, reduction of Fe(III) (oxyhydr)oxides may also be linked to the presence of hydrogen sulfide (H2S/HS-) produced by sulfate-reducing bacteria (Dos Santos Afonso and Stumm, 1992; Neal et al., 2001).
Biotic dissolution
The natural solubility of crystalline Fe (oxyhydr)oxides is low. However, interactions with microbes and organic substances can improve the formation of soluble Fe(III) and increase the availability of Fe and associated TEs (Colombo et al., 2014). Biogeochemical aspects of Fe cycling in the major microbially mediated and abiotic reactions have been extensively covered (Melton et al., 2014), together with Fe redox transformations and availability of TEs (Zhang et al., 2012), as well as Fe redox cycling in bacteriogenic Fe oxide-rich sediments (Gault et al., 2011). In aerobic environments at circumneutral pH conditions, Fe is generally relatively stable and highly insoluble in the form of (oxyhydr)oxides (e.g., Fe(OH)3, FeOOH, Fe2O3). However, in anaerobic conditions these minerals can be reductively dissolved (Roden et al., 2004; Roden and Wetzel, 2002) by microbial and abiotic pathways (Bonneville et al., 2004; Hansel et al., 2004; Shi et al., 2016; Thompson et al., 2006b). Microbial Fe(III) reduction is an important mechanism for iron cycling: heterotrophic Fe(III)-reducing bacteria could convert solid-phase Fe(III) minerals into dissolved and solid Fe(II) phases during their metabolic processes (Lovley, 1997). In particular, dissolutive reduction of iron (oxyhydr)oxides can be driven by dissimilatory iron reducing bacteria (DIRB), significantly contributing to the biogeochemical cycle of Fe and subsequent TE cycling (Cooper et al., 2006; Ghorbanzadeh et al., 2017; Levar et al., 2017). Microbial dissimilatory iron reduction (DIR) is an ubiquitous biogeochemical process in suboxic environments (Crosby et al., 2005; Lovley, 2000; Schilling et al., 2019; Wilkins et al., 2006). DIRB use Fe (oxyhydr)oxides as electron acceptors instead of oxygen for oxidizing organic matter. In general, for ferrihydrite and other short-range ordered (SRO) poorly crystallized iron minerals, the microbial Fe(III) reduction rates are more rapid (typically within hours), than the microbial reduction of the well-ordered minerals hematite (α-Fe2O3), goethite (α-FeOOH) (e.g., several months), and lepidocrocite (γ-FeOOH) (Ginn et al., 2017; Roden, 2006a). The rate of Fe(III) reduction will influence mobility of TEs initially immobilized on or in Fe (oxyhydr)oxides through adsorption or co-precipitation. Crystallinity, specific surface area and size among other factors may influence the reactivity of Fe (oxyhydr)oxides in relation to the metabolic activity and diversity of DIRB (Aino et al., 2018; Cutting et al., 2009), as detailed in the next section.
Secondary minerals formed during the bio-reduction of Fe oxides
The first product of the bioreduction of iron oxides is soluble Fe(II). This chemical species can sorb to residual Fe(III)-oxides, or be involved in (bio)geochemical reactions generating secondary minerals (Table I-4) such as green rust (Génin et al., 1998; Ona-Nguema, et al., 2002), vivianite, siderite, magnetite (Fredrickson et al., 1998; Maitte et al., 2015; Urrutia et al., 1998; Zachara et al., 1998), chukanovite (O’Loughlin et al., 2010). The type of secondary product depends on the composition of the liquid phase, the presence of complexants, and on bacterial activity.
Table of contents :
Chapter 0: General introduction and objectives
Chapter I: Background knowledge
I-1 Biogeochemical cycle of Fe
I-1.1 Global Fe cycle
I-1.2 Microbial transformations of Fe in the biogeosphere
I-1.3 Influence of redox conditions and organic matter on iron reduction
I-2 Classification of Fe-minerals
I-3 Dissolution of Fe(III) (oxyhydr)oxides and iron cycling in surface environments
I-3.1 Abiotic dissolution
I-3.2 Biotic dissolution
I-3.3 Secondary minerals formed during the bio-reduction of Fe oxides
I-4 Iron-reducing bacteria (IRB)
I-4.1 Dissimilatory Iron-Reducing Bacteria (DIRB)
I-4.2 Iron reduction by fermentative bacteria
I-4.3 The genus Shewanella
I-4.3 The genus Geobacter
I-4.4 Comparison of the genera Shewanella and Geobacter
I-4.5 Primers for the detection and quantification of Shewanella and Geobacter
I-4.6 Biofilms of iron reducing bacteria
I-4.7 Studies involving complex iron reducing microbial communities
I-5 Mechanisms of microbial Fe(III) reduction
I-6 Cycling of Fe and mobility of associated As, Cr, Cd and other trace elements
I-6.1 Association of As, Cr and Cd with Fe-Oxides
I.6.2 Transformations of As, Cr and Cd by bacteria
I-6.3 Mobility of trace elements during Fe-oxides microbial dissolution
I-7 Positioning of the PhD thesis in regards to the state of the art
Chapter II: General materials and methods
II-1 Site information and soil / sediment sampling
II-2. Enrichment of iron-reducing bacteria (IRB) and subculture
II-3 Iron (oxyhydr)oxides and laboratory synthesis
II-3.1 Ferrihydrite
II-3.2 Lepidocrocite
II-3.3 Goethite and hematite
II-4 Physico-chemical analysis: pH, Eh, Fe(II)/FeT, As, Cr and Cd
II-5 BET surface areas
II-6 SEM-EDS and SEM observations
II-6.1 Observation of iron (oxyhydr)oxides
II-6.2 Bacteria Observations
II-7 57Fe Mössbauer spectrometry
II-8 Diversity and physiology of bacteria
II-8.1 Observation and counting of bacteria by Thoma cell
II-8.2 DNA extraction and PCR amplifications
II-8.3 CE-SSCP fingerprints
II-8.4 Bacterial 16S rRNA gene quantification
II-8.5 Detection of Shewanella and Geobacter
II-8.6 Quantification of Shewanella and Geobacter by qPCR
Chapter III: Experiments in slurry with four different iron oxides
III-1 Introduction
III-2 Specific Materials and Methods
III-2.1 Characterization of the environmental source of bacteria
Iron extraction in soils/sediments samples
III-2.2 Synthetic Fe(III) (oxyhydr)oxides and bacterial inocula
III-2.3 IRB incubation experiments
III-2.4 Fe analyses and pH/Eh monitoring
III-2.5 Determination of iron oxides solubilisation parameters
III-2.6 SEM-EDS observation and Mössbauer spectrometry
III-2.7 Biological analyses
III-2.8 Statistics
III-3 Results
III-3.1 Characterization of the environmental sources of bacteria
III-3.2 Dissolution of Fe (oxyhydr)oxides
III-3.3 Biological parameters
III-3.4 Mineral SEM-EDS observation
III-3.5 Mössbauer spectroscopy
III-4 Discussion
III-4.1 Influence of the type of iron oxide on bacterial iron solubilisation
III-4.2 Bacterial communities
III-4.3 Geobacter and Shewanella 16S genes abundances
III-4.4 Relation between iron solubilisation effectiveness and Geobacter and Shewanella 16S gene abundances
III-4.5 SEM observations of Fe (oxyhydr)oxides and Mössbauer spectroscopy
III-5 Conclusions and perspective
IV: Experiments with ferrihydrite fixed on slides
IV-1 Introduction
IV-2 Specific materials and methods
IV-2.1 Slide preparation with Fe(III) (oxyhydr)oxides
IV-2.2 Slides incubation experiments
IV-2.3 Monitoring
IV-2.4 DNA extraction and molecular analysis
IV-3 Experimental results
IV-3.1 Bacterial growth
IV-3.2 Physico-chemical monitoring
IV-3.3 Bacterial observations and molecular analysis
IV-3.4 Mineral SEM-EDS observations
IV-4 Discussion
IV-4.1 Fe dissolution
IV-4.2 Distribution of Shewanella and Geobacter 16S gene copies in the liquid medium and in the biofilm
IV-4.3 SEM observation of the solid particles
IV-5 Conclusion and perspective
Chapter V: Mobility of As, Cr and Cd adsorbed on Fe (oxyhydr)oxides submitted to IRB
V-1. Abstract
V-2. Introduction
V-3 Specific materials and methods
V-3.1 Adsorption of As, Cr and Cd on synthetic iron (oxyhydr)oxides
V-3.1.1 Preparation of TEs stock solution
V-3.1.2 Adsorption of TEs to iron oxyhydr(oxides)
V-3.2 Columns experimental setup
V-3.2.1 Preparation of Fe (oxyhydr)oxides
V-3.2.2 Preparation of silica gel and sand matrix
V-3.2.3 Column setup and experimental conditions
V-3.2.4 Monitoring
V-3.2.5 SEM-EDS observation and Mössbauer spectrometry
V-3.2.6 Biological analyses
V-4 Experimental results
V-4.1 Adsorption experimental results
V-4.2 Column experiments
V-4.2.1 Visual evolution of the columns
V-4.2.2 Spatial and temporal evolution of iron and absorbed elements in columns
V-4.2.3 Relationship between remaining Fe and TEs in columns
V-4.2.3 Biological Parameters
V-4.2.4 Mineral SEM-EDS observation
V-4.2.5 Mössbauer spectroscopy
IV-5 Discussion
IV-5.1 Spatial and temporal aspects of iron reduction of ferrihydrite and goethite
IV 5.2 Impact of iron reduction on behavior and mobilities of TEs
IV-5.3 Distribution of global bacterial biomass and two targeted IRB (Shewanella and Geobacter 16S genes) in the columns
IV-6 Conclusions and perspective
Chapter VI: Conclusions and perspectives
