Photoferrotrophs -phototrophic Fe(II)-oxidizing bacteria- are considered to have played an important role in creating the world’s largest iron deposits -banded iron formations or BIFs- during early Earth history. At that time no oxygen was in the atmosphere which meant the ocean was drastically different from today. For example, it was full of ferrous iron, which would have been oxidized to form iron oxides when exposed to oxygen. Shortly before the Great oxygen event -GOE- at 2.4 billion years ago when it is thought oxygen began to become produced by cyanobacteria on a large scale, some oxygen began to be present in local niches. This not only led to more oxygen being around, but led to more oxidized nitrogen-compounds as well like nitrate (NO₃^-). In modern environments, nitrate is used by different kinds of iron oxidizers, known collectively as the nitrate-reducing Fe(II)-oxidizing bacteria. On early Earth, as in modern habitats, these bacteria would be in direct competition for ferrous iron with photoferrotrophs.

However, we had no idea who would ‘win’ in this scenario. We wanted to test what happened if we would put both bacteria together: Would one out compete the other or would they share the iron?

In our first experiment we put both Fe(II)-oxidizers together and limited nitrate. We used Rhodobater ferrooxidans SW2, a photoferrotroph, and the KS culture, an autotrophic nitrate-reducing Fe(II)-oxidizing consortium. Surprisingly, we not only observed that the nitrate-reducer could use the iron faster, but no photoferrotrophs grew at all in their presence. Something must have been killing the photoferrotrophs! This observation started our detective work to search for the toxic substance. We equipped us with lab coats, gloves and magnifying glasses and began investigating.

Image 1: Schematic of bacteria inspecting a BIF and wondering how it was formed.

First, we ruled out that something we added during inoculation of the nitrate-reducing Fe(II)-oxidizing  bacteria was affecting the growth of the photoferrotrophs and established that the toxin had to be something that was produced during denitrification.

Image 2: Fe(II) oxidation in the KS culture alone, R. ferrooxidans SW2 alone and in a mixed culture containing both KS and R. ferrooxidans SW2. Aqueous- and gas-phase time-series measured (m) and simulated (s) concentration data for the phototrophic oxidation of Fe(II) by R. ferrooxidans SW2, the nitrate-dependent oxidation of Fe(II) by KS and in the incubation containing a mixture of KS and R. ferrooxidans SW2.

Numerous intermediate products produced during the nitrate reduction, NO₂^-, NO, N₂O or N₂ could be responsible. And indeed, in an additional setup where we exchanged the headspace frequently, photoferrotrophs grew together with Nred Fe(II)-oxidizer, showing it must be some kind of gas!

In the next steps we set up more experiments with the addition of NO, NO₂^- and N₂O,  to our photoferrotroph. With N₂O and NO₂^-no inhibition could be observed, but in the setups with NO inhibited growth could be observed. By methodically testing potential toxins we could reveal that NO (nitric oxide) was the culprit.

To understand whether this might be important in the environment, we also had to test whether other combinations of photoferrotrophs and nitrate-reducer were affected. We showed that all types of nitrate-reducing Fe(II) oxidation could drive this effect and that other photoferrotrophs were also affected to varying degrees. By looking at the genomes of all known phototrophs, we showed that our bacteria were not particularly weak when it came to detoxifying nitric oxide, they have very standard capabilities. Through this bioinformatics we could show that most phototrophs are likely susceptible to NO toxciity.

Consequences for the photoferrotrophs during early earth

As the ocean’s photic zone became more oxygenated before the Great Oxidation Event (GOE), nitrate levels increased due to high primary productivity. This led to the growth of nitrate-reducing bacteria that competed with photoferrotrophs for iron (Fe(II)) as an electron donor. These bacteria produced nitric oxide (NO), a toxin, which further marginalized photoferrotrophs.

In the context of banded iron formations (BIFs), photoferrotrophs were initially the main drivers of Fe(II) oxidation in the photic zone. As nitrate levels rose and denitrification intensified, photoferrotrophs were pushed away from areas with high primary productivity, where cyanobacteria thrived, and oxygen and oxidized nitrogen species accumulated. Although photoferrotrophs still had access to upwelling Fe(II), they were limited by other trace elements from continental weathering. It is hypothesized that nitrate-reducing Fe(II) oxidation could have compensated for BIF deposition after photoferrotrophs were inhibited, but the spread of cyanobacteria and deep ocean oxygenation eventually led to the end of BIF formation.