The removal of iron and phosphate from culture medium by Rhodococcus ruber SiAl

Technical Summary: Selective Removal of Iron and Phosphate by Rhodococcus ruber SiAl

Problem Statement
The accumulation of heavy metals and phosphate by microorganisms is a critical mechanism for the bioremediation of polluted waters. While various bacteria are known to sorb metals or remove phosphates, there is a need to identify strains capable of selective accumulation, particularly for iron and phosphate, which are common contaminants in natural and industrial waters. Iron is abundant in the Earth's crust and prevalent in industrial waste, while excess phosphate leads to eutrophication. Although mechanisms such as biosorption, bioaccumulation, precipitation, and chelation (via siderophores or metalloproteins) are known, the interrelationship between metal and phosphate removal by specific bacterial strains remains under-explored. This study addresses the specific capability of the bacterium Rhodococcus ruber SiAl, isolated from corroded aluminum-silicon alloy, to selectively remove dissolved iron, magnesium, manganese, and phosphate from culture media.

Methodology
The study utilized the R. ruber SiAl strain, maintained on R2A agar and identified via whole-genome sequencing (GenBank accession GCA_053668425.1). The strain was cultivated in a defined liquid medium containing specific concentrations of iron (Fe³⁺), magnesium (Mg²⁺), manganese (Mn²⁺), and phosphate (Pi). Experiments were conducted under aerobic conditions at 28°C with shaking.

To assess accumulation, the researchers measured:

1. Residual concentrations of metals and phosphate in the culture supernatant using atomic absorption spectroscopy (for Fe, Mg, Mn) and colorimetric assays (for Pi).
2. Biomass content of these elements after harvesting and washing the cells.
3. Intracellular phosphorus fractions, distinguishing between inorganic phosphate (Pi), acid-soluble polyphosphate (polyP1), and acid-insoluble polyphosphate (polyP2).
4. Genomic analysis using KEGG GhostKOALA, RAST, and antiSMASH to identify gene clusters related to siderophore synthesis, iron transport (e.g., fecBCDE, efeUOB, sitABC), phosphate transport (Pst and Pit systems), and manganese homeostasis (mntH, mntR).

Key Results
The study demonstrated distinct selectivity in the accumulation capabilities of R. ruber SiAl:

• Iron Accumulation: The strain exhibited high efficiency in removing iron. When cultured in a medium with 2.0 mM Fe³⁺, up to 99% of the iron was removed from the solution and associated with the biomass. This removal was independent of the initial phosphate concentration (0.1–6 mM).
• Phosphate Accumulation: The strain removed significant amounts of phosphate, but the efficiency was modulated by the presence of specific metals. In the presence of 2 mM Fe³⁺, 75% of the initial phosphate was removed. In contrast, removal dropped to 20% with 2 mM Mg²⁺ and 10% with 2 mM Mn²⁺.
• Magnesium and Manganese: The strain showed minimal accumulation of magnesium (≤5% removal). Crucially, it did not remove manganese ions from the medium; manganese levels remained unchanged, and the presence of manganese salts (2 mM) actually inhibited bacterial growth and reduced biomass yield.
• Intracellular Composition: The accumulated phosphate in the biomass was primarily in the form of inorganic phosphate (Pi), not polyphosphates. The polyphosphate content (polyP1 and polyP2) was low, suggesting that inorganic polyphosphates are not the primary mechanism for phosphate removal in this strain.
• Genomic Insights: The genome analysis supported the experimental findings:
  • Iron: Two siderophore biosynthesis gene clusters (analogous to heterobactin and coelichelin) and multiple iron transport systems (including ABC transporters and Fe²⁺/Fe³⁺ specific systems) were identified, explaining the high iron uptake.
  • Phosphate: The genome contains the pst operon (high-affinity transport) and putative pit genes (low-affinity transport), consistent with the observed phosphate accumulation.
  • Manganese: The presence of the mntH transporter and mntR regulator suggests an active homeostasis system that represses manganese uptake under high concentrations, preventing toxicity.

Significance and Claims
The authors conclude that Rhodococcus ruber SiAl is a promising candidate for the bioremediation of waters contaminated with dissolved iron and phosphate. The strain's ability to selectively precipitate iron (likely as iron phosphate or iron oxides) while excluding manganese distinguishes it from other known iron-manganese bacteria, such as Sphaerotilus montanus, which remove manganese but not iron.

The study posits that the specific metabolic processes of R. ruber SiAl could contribute to the natural formation of phosphorites, including marine phosphatic mineral deposits. Furthermore, the strain represents a potential tool for water treatment technologies aimed at removing dissolved iron and phosphate simultaneously. The research highlights that while the strain accumulates phosphate, the process is not driven by intracellular polyphosphate storage but rather by the formation of metal-phosphate complexes or direct absorption into cell structures. The findings suggest that the interplay between iron uptake and phosphate removal is a specific trait of this strain, warranting further investigation into the underlying mechanisms for practical application in water purification.