Influence of organics and silica on Fe(II) oxidation rates and cell–mineral aggregate formation by the green-sulfur Fe(II)-oxidizing bacterium Chlorobium ferrooxidans KoFox – Implications for Fe(II) oxidation in ancient oceans

Highlights

• No encrustation of cells in Fe(II)-oxidizing co-culture by Fe minerals.

• Presence of cell–mineral aggregates.

• The presence of organics alters mineral morphology but not mineral identity.

• Silica does enhance Fe(II) oxidation rates possibly by lowering Fe(II) toxicity.

• Lack of cell encrustation makes preservation of cells as microfossils unlikely.

Abstract

Most studies on microbial phototrophic Fe(II) oxidation (photoferrotrophy) have focused on purple bacteria, but recent evidence points to the importance of green-sulfur bacteria (GSB). Their recovery from modern ferruginous environments suggests that these photoferrotrophs can offer insights into how their ancient counterparts grew in Archean oceans at the time of banded iron formation (BIF) deposition. It is unknown, however, how Fe(II) oxidation rates, cell–mineral aggregate formation, and Fe-mineralogy vary under environmental conditions reminiscent of the geological past. To address this, we studied the Fe(II)-oxidizer Chlorobium ferrooxidans KoFox, a GSB living in co-culture with the heterotrophic Geospirillum strain KoFum. We investigated the mineralogy of Fe(III) metabolic products at low/high light intensity, and in the presence of dissolved silica and/or fumarate. Silica and fumarate influenced the crystallinity and particle size of the produced Fe(III) minerals. The presence of silica also enhanced Fe(II) oxidation rates, especially at high light intensities, potentially by lowering Fe(II)-toxicity to the cells. Electron microscopic imaging showed no encrustation of either KoFox or KoFum cells with Fe(III)-minerals, though weak associations were observed suggesting co-sedimentation of Fe(III) with at least some biomass via these aggregates, which could support diagenetic Fe(III)-reduction. Given that GSB are presumably one of the most ancient photosynthetic organisms, and pre-date cyanobacteria, our findings, on the one hand, strengthen arguments for photoferrotrophic activity as a likely mechanism for BIF deposition on a predominantly anoxic early Earth, but, on the other hand, also suggest that preservation of remnants of Fe(II)-oxidizing GSB as microfossils in the rock record is unlikely.

Introduction

Geochemical conditions of the Precambrian oceans were different from today, with dissolved silica concentrations at least 1 mM (Jones et al., 2015), and potentially approaching the saturation state of amorphous silica (up to 2.2 mM) (Siever, 1962), while dissolved Fe(II) concentrations ranged from 50 μM (Holland, 1973) to ∼1 mM (Morris, 1993). Under these siliceous and ferruginous conditions the deposition of banded iron formations (BIF) took place (see Bekker et al., 2014 for review). Several mechanisms have been put forth to explain Fe(II) oxidation in Precambrian oceans: (1) abiotic or microbiological reactions with O₂ produced by cyanobacteria, (2) abiotic, UV-induced photo-oxidation, or (3) direct photosynthetic utilization of Fe(II) by phototrophic Fe(II)-oxidizers, the so called photoferrotrophs. Emerging evidence suggests that photoferrotrophs were the most probable drivers for the deposition of the precursor ferric oxyhydroxide layers that led to BIF before the Great Oxidation Event, some 2.45 billion years ago (Czaja et al., 2013, Kappler et al., 2005).

Photoferrotrophic organisms use light energy and Fe(II) as an electron donor for CO₂ reduction and the production of cell biomass (Widdel et al., 1993). This type of metabolism was shown to be widespread amongst freshwater and marine phototrophic bacteria, including purple sulfur bacteria (Croal et al., 2004), purple non-sulfur bacteria (Poulain and Newman, 2009, Widdel et al., 1993, Wu et al., 2014) and green sulfur bacteria (GSB) (Crowe et al., 2008, Heising et al., 1999). To date, the bulk of our understanding on how photoferrotrophs metabolize, and under what environmental conditions, has mainly come from the study of purple non-sulfur bacteria. However, it has been shown that in modern ferruginous freshwater lakes, GSB were present in the anoxic layers of the photic zone, where they can play an important role for the biogeochemical Fe and C cycles in these environments (Crowe et al., 2008, Llirós et al., 2015). Due to their adaptation to low light conditions (Llirós et al., 2015), GSB are perfectly suited for such conditions, much better than purple-sulfur and purple non-sulfur bacteria, underpinning their importance in ferruginous environments.

Only one strain of GSB capable of phototrophic Fe(II) oxidation has been studied in detail (Heising et al., 1999). This Chlorobium ferrooxidans strain KoFox was shown to oxidize Fe(II) at very low light intensities (>50 lux) (Hegler et al., 2008). It also grows in co-culture with Geospirillum sp. strain KoFum; the latter grows by fermenting fumarate to organic acids, which in turn, enhances Fe(II) oxidation by KoFox (Heising et al., 1999). It is known from abiotic Fe(II) oxidation experiments that the presence of such organics during Fe(II) oxidation influences the structure, particle size, and crystallinity of Fe(III) minerals (Mikutta et al., 2008).

Previous studies have additionally revealed that in the Fe(II)-oxidizing co-culture KoFox/KoFum, the cell surfaces of the fermenting strain KoFum become thinly encrusted in Fe(III) minerals after Fe(II) oxidation. In contrast, cells of the Fe(II)-oxidizing KoFox remained largely free of Fe(III) particles, with the exception of sparse, flat mineral particles (Schädler et al., 2009). This suggests that in this co-culture it is probably the non-Fe(II)-oxidizing partner and not the Fe(II)-oxidizer that will leave a trace in the rock record as a mineral-encrusted microfossil. However, these previous biomineralization studies have not taken into account the complex chemistry of ancient seawater, and how the presence of organic compounds consumed and produced by KoFum influences the mineralogy of the resulting ferric oxyhydroxides, or the influence that different activities of the Fe(II)-oxidizer might have on encrustation. For instance, dissolved silica has a high affinity for iron and it can influence the association of the cells with Fe(III) minerals (Eickhoff et al., 2014, Mayer and Jarrell, 1996). As the Precambrian oceans were Si-rich, its presence might have influenced cell–mineral interactions and thereby needs to be considered when conducting such experiments.

This study, therefore, aims to answer the following questions: (i) how do dissolved silica, fumarate (as an organic model compound that is fermented by KoFum), and variable light intensities impact the activity of the Fe(II)-oxidizer and thus influence Fe(II) oxidation rates, encrustation patterns and cell–mineral interactions in the KoFox/KoFum co-culture, and (ii) which minerals are formed during Fe(II) oxidation in the presence and absence of dissolved silica and organics.