Low-oxygen and chemical kinetic constraints on the geochemical niche of neutrophilic iron(II) oxidizing microorganisms

Abstract

Neutrophilic iron oxidizing bacteria (FeOB) must actively compete with rapid abiotic processes governing Fe(II) oxidation and as a result have adapted to primarily inhabit low-O₂ environments where they can more successfully compete with abiotic Fe(II) oxidation. The spatial distribution of these microorganisms can be observed through the chemical gradients they affect, as measured using in situ voltammetric analysis for dissolved Fe(II), Fe(III), O₂, and FeS_(aq). Field and laboratory determination of the chemical environments inhabited by the FeOB were coupled with detailed kinetic competition studies for abiotic and biotic oxidation processes using a pure culture of FeOB to quantify the geochemical niche these organisms inhabit. In gradient culture tubes, the maximum oxygen levels, which were associated with growth bands of Sideroxydans lithotrophicus (ES-1, a novel FeOB), were 15–50 μM. Kinetic measurements made on S. lithotrophicus compared biotic/abiotic (killed control) Fe oxidation rates. The biotic rate can be a significant and measurable fraction of the total Fe oxidation rate below O₂ concentrations of approximately 50 μM, but biotic Fe(II) oxidation (via the biotic/abiotic rate comparison) becomes difficult to detect at higher O₂ levels. These results are further supported by observations of conditions supporting FeOB communities in field settings. Variablity in cell densities and cellular activity as well as variations in hydrous ferrous oxide mineral quantities significantly affect the laboratory kinetic rates. The microbial habitat (or geochemical niche) where FeOB are active is thus largely controlled by the competition between abiotic and biotic kinetics, which are dependent on Fe(II) concentration, P_O2, temperature and pH in addition to the surface area of hydrous ferric oxide minerals and the cell density/activity of FeOB. Additional field and lab culture observations suggest a potentially important role for the iron–sulfide aqueous molecular cluster, FeS_(aq), in the overall cycling of iron associated with the environments these microorganisms inhabit.

Introduction

Central to addressing questions about the role microorganisms play in the cycling of elements on any scale are the specific geochemical settings in which organisms actively metabolize redox species. Iron cycling is a process of intense interest that has implications for deciphering large changes in ocean and atmospheric chemistry through deep time, the identification of microbial activity on other planets such as Mars, and the mobility of a host of contaminants in modern earth settings (Straub et al., 2001, Benison and LaClair, 2003, Edwards et al., 2004, Emerson and Weiss, 2004, Kappler and Newman, 2004, Roden et al., 2004, Ferris, 2005, Kump and Seyfried, 2005, Rouxel et al., 2005, Rouxel et al., 2006, Yamaguchi and Ohmoto, 2006, Stucki et al., 2007, Neubauer et al., 2008). In any of these environments geochemical control on microbial ecology can be manifested in diverse ways, but one key factor is to consider the rates at which organisms can utilize existing substrates including electron donors and acceptors to garner energy for growth. Conversely, the ecology and physiology of iron-utilizing microbes may significantly impact the geochemistry as microorganisms themselves are responsible for controlling the rates of different processes, which influence the gradients of elements coincident to their individual niche. Competition between microorganisms and/or competition between microbial metabolic reactions and abiotic reactions are thus an important part of deciphering iron cycling and can be investigated in terms of the relative kinetics of individual processes.

In any natural system, the cycling of elements is controlled not only by the microorganisms that can catalyze reactions, but also by the formation of specific aqueous complexes and minerals that can affect what form these elements are present in. Iron and sulfur are quite commonly associated with each other in different environments – and the settings neutrophlic iron oxidizers are found in are no different. Reduced iron and sulfur strongly interact, and the solubility product (log K) for the first Fe–S mineral to form in these environments (mackinawite) via:

$${\text{Fe}}^{2+}+{\text{HS}}^{-}\to {\text{FeS}}_{\text{mackinawite}}+{\text{H}}^{+}$$

is reported from various experiments as 2.13 ± 0.27 (at pH between 6.5 and 8; Wolthers et al., 2005), 3.00 ± 0.12 (Davison et al., 1999), and 3.88–3.98 (Benning et al., 2000, recomputed by Rickard, 2006) while a recent report by Chen and Liu (2005) tabulate 46 field measurements between 2.20 and 3.83. It is also well established that in a number of environments metastable concentrations of polynuclear clusters can exist in solutions associated with mineral dissolution and precipitation (Luther et al., 1999, Rozan et al., 2000, Luther et al., 2002, Furrer et al., 2002, Navrotsky, 2004). In the iron–sulfide system, iron sulfide clusters are often abbreviated FeS_(aq) (after Theberge and Luther, 1997), although it is important to realize that this notation likely represents a continuum of polynuclear species of differing stoichiometry and charge (i.e. some combination of different Fe_xS_y species). Iron sulfide clusters are known to exist in a number of marine and freshwater environments (Theberge and Luther, 1997, De Vitre et al., 1988, Davison et al., 1999, Luther et al., 2003, Druschel et al., 2004, Luther and Rickard, 2005, Roesler et al., 2007), and are thought to play a significant role in the precipitation of iron sulfide minerals (Rickard and Luther, 1997, Butler et al., 2004, Rickard, 2006, Roesler et al., 2007). Rickard (2006) recently showed that FeS_(aq) establishes an equilibrium with the Fe–S mineral mackinawite which is the first Fe–S mineral formed in low temperature reducing environments (Rickard, 2006).

Historically, microorganisms that utilize ferrous iron as a substrate are well known, in part as a few species have distinctive morphologies, for example, stalk forming Gallionella spp. and sheath forming Leptothrix spp. In freshwater, these organisms grow as dense communities in mat-like structures where anoxic water bearing Fe(II) comes into contact with air, such as often happens in wetlands or springs. The mat structure and density is dependent upon the flow conditions, when flow is rapid (>0.5 m/s) the mats will be quite dense; however, under slow flow, the mats may exist as loose aggregations of floculant iron oxyhydroxides (hydrous ferric oxides, HFO). It is in this context that neutrophilic Fe-oxidizing bacteria (FeOB) provide an interesting example of a group of microbes that are restricted in their ecological niche due to kinetic constraints on their energy source (Kirby et al., 1999, Burke and Banwart, 2002, Neubauer et al., 2002, Neubauer et al., 2008, Edwards et al., 2004, James and Ferris, 2004, Ferris, 2005). These organisms must outcompete abiotic reactions which consume Fe(II) in oxic and suboxic settings at circumneutral pH conditions (Emerson and Revsbech, 1994, Edwards et al., 2004, James and Ferris, 2004, Ferris, 2005). This competition is sensitive to pH as the kinetics of Fe(II) oxidation with O₂ are described by the rate law:

$$\frac{\mathrm{d}[{\text{Fe}}^{2+}]}{\mathrm{d}t}=-k[{\text{Fe}}^{2+}][{\text{O}}_2][{\text{OH}}^{-}{]}^2$$

where k = 8.0 × 10¹³L² mol⁻² atm⁻¹ at 25 °C (Singer and Stumm, 1970). It is these chemical realities, which restrict neutrophilic FeOB to inhabit suboxic microhabitats, or niches, where low [O₂] allow biotic oxidation rates to further outpace the abiotic rates of Fe(II) oxidation (James and Ferris, 2004, Roden et al., 2004, Ferris, 2005).

Several studies have investigated different aspects of the kinetic competition surrounding neutrophilic Fe oxidation both in the field and in the laboratory, and these have provided valuable insight to these processes (Emerson and Revsbech, 1994, Neubauer et al., 2002, James and Ferris, 2004, Roden et al., 2004, Rentz et al., 2007). However, previous studies have not attempted to understand geochemical conditions in natural microbial mat communities of FeOB and relate these back to kinetics of Fe oxidation in pure cultures of a lithotrophic Fe-oxidizing bacterium. To do this necessitates measuring both profiles of chemical species and their reaction kinetics. This study used voltammetry to make detailed field and laboratory measurements in an effort to decipher the specific environmental niche of neutrophilic FeOB, and the specific chemical conditions that an isolate, Sideroxydans lithotrophicus, grows best in when cultured in gradients that mimic natural conditions, and finally, to determine the kinetics of biotic vs. abiotic rates in the context of field and culture results.