# High-density biomass as a substrate for stimulating biosulfidogenesis in the deep layer of stratified acidic pit lakes

## TL;DR

This study explores using high-density biomass to stimulate sulfide production in acidic pit lakes, helping remove harmful metals and metalloids from water.

## Contribution

The study introduces a novel method of directly delivering high-density biomass to stimulate biosulfidogenesis in acidic pit lakes.

## Key findings

- High-density biomass led to faster sulfide production compared to biocomponents.
- Biosulfidogenesis effectively removed Zn and As contaminants from solution.
- Desulfosporosinus bacteria were enriched and likely responsible for sulfide production.

## Abstract

Accumulation of harmful metal(loid)s in acidic pit lakes (APLs) is a serious environmental issue in mining districts. This lab-based study evaluated a novel method to stimulate dissimilatory sulfate reduction to promote the formation of sparingly soluble metal(loid)-sulfide minerals in the permanently stratified deep layer of Cueva de la Mora (CM), an APL in the Iberian Pyrite Belt in Spain. Solid-phase biomass was selected because it can be pressed into high-density forms that are dense enough to settle into the deep layer of a lake. This “direct delivery” of electron donor overcomes the current “indirect method” to stimulate algae growth in the upper layer and wait for algae to die and settle into the deep layer. We added the microalgae Coccomyxa onubensis (predominant in CM), Euglena gracilis (another acid-tolerant microalgae), and Lemna obscura (duckweed), as well as model biocomponents (amino acids, monosaccharides, and lipids) as substrates to stimulate biological sulfide production (biosulfidogenesis). We found that compared with biocomponents, high-density biomass required a shorter lag time before it was utilized. Temporal patterns of the production of sulfide and volatile fatty acids with high-density biomass were similar to patterns with amino acids, suggesting that amino acids may be the preferred substrate among the biocomponent monomers for the microbial community. Biosulfidogenesis led to the complete removal of metal(loid)s (Zn and As) contaminants from solution, mimicking the chemical composition of the deep layer. Desulfosporosinus, the only acid-tolerant sulfate-reducing bacteria (SRB) identified in situ, was significantly enriched in the laboratory setup and presumably responsible for biosulfidogenesis.

Remediation of high concentrations of harmful metal(loid)s in acidic pit lakes is challenging. This research presents a novel strategy by supplying high-density biomass as a carbon source and electron donor to stimulate biological dissimilatory sulfate reduction in acidic pit lakes in the Iberian Pyrite Belt. The formation of biogenic sulfide precipitates dissolved metal(loid)s in the acidic pit lakes. This approach is feasible in meromictic acidic pit lakes, where precipitated metal(loid)s would remain sequestered in bottom sediments. However, the deep layer of acidic pit lakes is often oligotrophic with respect to organic carbon. Pelletized high-density biomass can be added to the top layer of the lake and transported to the deep layer. This strategy offers practical and adaptable guidance for the bioremediation of persistent metal(loid) contamination in acidic pit lakes.