# Bacterial nitrite production oxidizes Fe(II) bioremediating acidic abandoned coal mine drainage

## TL;DR

Bacteria in acidic coal mine water produce nitrite, which chemically oxidizes iron, helping to clean up polluted water.

## Contribution

The study identifies nitrate-dependent iron oxidation as a widespread and previously underappreciated mechanism in acidic mine drainage remediation.

## Key findings

- Bacteria in acidic passive remediation systems produce nitrite, which oxidizes Fe(II) to Fe(III).
- NDFO-capable bacteria, including Paraburkholderia spp., are prevalent in acidic AMD systems.
- NRIO provides a simple and effective mechanism for iron removal in acidic conditions.

## Abstract

Passive remediation systems (PRSs) treating either acidic or neutral abandoned coal mine drainage (AMD) are colonized by bacteria that can bioremediate iron (Fe) through chemical cycling. Due to the low pH in acidic AMD, iron oxidation from soluble Fe(II) to precipitated Fe(III) is mainly directed by microbial oxidation. Less well described are biotic reactions that lead to iron remediation through abiotic secondary reactions. We describe here iron oxidation in acidic AMD that is mediated by the bacterial reduction of nitrate to nitrite followed by the geochemical oxidation of Fe(II). Within an acidic PRS, 4,560 bacteria cultured from the microbial community were screened for their ability to oxidize iron and to perform nitrate-dependent iron oxidation (NDFO). Iron oxidation in the culturable community was observed in every pond of the system, ranging from 2.1% to 11.4%, and NDFO was observed in every pond, ranging from 1.4% to 6.0% of the culturable bacteria. Five NDFO isolates were purified and identified as Paraburkholderia spp. One of our isolates, Paraburkholderia sp. AV18 was shown to drive NDFO through the bacterial production of nitrite that in turn chemically oxidizes Fe(II) (nitrate reduction-iron oxidation; NRIO). AV18 expressed nitrate reductase, napA, concurrent to nitrite production. Burkholderiales are found by 16S rRNA gene sequencing in every pond of the PRS. The frequency of NDFO metabolism in the culturable microbial community and abundance of Burkholderiales in the PRS suggest nitrite producers contribute to the bioremediation of iron in acidic AMD and may be an unharnessed opportunity to increase iron bioremediation in acidic conditions.

Our study sheds light on a poorly defined biogeochemical interaction, nitrate-dependent iron oxidation (NDFO), that has been described in several environments. We show that bacterial nitrate reduction produces nitrite, which can chemically oxidize ferrous iron, leading to insoluble ferric iron. We show that bacteria capable of the nitrate reduction-iron oxidation (NRIO) reactions are prevalent throughout multiple passive remediation systems that treat acidic coal mine drainage, indicating this may be a widespread mechanism for iron removal under acidic conditions. In acidic coal mine remediation, iron precipitation has been shown to be solely bacterially mediated, and NRIO provides a simple mechanism for aerobic oxidation of iron in these conditions.