Enriching Acidophilic Fe(II)-oxidizing Bacteria in No-flow, Fed-batch Systems.

Low-pH microbial Fe(II) oxidation occurs naturally in some Fe(II)-rich acid mine drainage (AMD) ecosystems across so-called terraced iron formations. Indigenous acidophilic Fe(II)-oxidizing bacterial communities can be incorporated into both passive and active treatments to remove Fe from the AMD solution. Here, we present a protocol of enriching acidophilic Fe(II)-oxidizing bacteria in no-flow, fed-batch systems. Mixed cultures of naturally occurring microbes are enriched from the fresh surface sediments at AMD sites using a chemo-static bioreactor (Eppendorf BioFlo®/Celligen® 115 Fermentor) with respect to constant stirring speed, temperature, pH and unlimited dissolved oxygen. Ferrous sulfate is discontinuously added to the reactor as the primary substrate to enrich for acidophilic Fe(II)-oxidizing bacteria. Successfully and efficiently enriching acidophilic Fe(II)-oxidizing bacteria helps to exploit this biogeochemical process into AMD treatment systems.

Geochemical and Temporal Influences on the Enrichment of Acidophilic Iron-Oxidizing Bacterial Communities.

UNLABELLED: Two acid mine drainage (AMD) sites in the Appalachian bituminous coal basin were selected to enrich for Fe(II)-oxidizing microbes and measure rates of low-pH Fe(II) oxidation in chemostatic bioreactors. Microbial communities were enriched for 74 to 128 days in fed-batch mode, then switched to flowthrough mode (additional 52 to 138 d) to measure rates of Fe(II) oxidation as a function of pH (2.1 to 4.2) and influent Fe(II) concentration (80 to 2,400 mg/liter). Biofilm samples were collected throughout these operations, and the microbial community structure was analyzed to evaluate impacts of geochemistry and incubation time. Alpha diversity decreased as the pH decreased and as the Fe(II) concentration increased, coincident with conditions that attained the highest rates of Fe(II) oxidation. The distribution of the seven most abundant bacterial genera could be explained by a combination of pH and Fe(II) concentration. Acidithiobacillus, Ferrovum, Gallionella, Leptospirillum, Ferrimicrobium, Acidiphilium, and Acidocella were all found to be restricted within specific bounds of pH and Fe(II) concentration. Temporal distance, defined as the cumulative number of pore volumes from the start of flowthrough mode, appeared to be as important as geochemical conditions in controlling microbial community structure. Both alpha and beta diversities of microbial communities were significantly correlated to temporal distance in the flowthrough experiments. Even after long-term operation under nearly identical geochemical conditions, microbial communities enriched from the different sites remained distinct. While these microbial communities were enriched from sites that displayed markedly different field rates of Fe(II) oxidation, rates of Fe(II) oxidation measured in laboratory bioreactors were essentially the same. These results suggest that the performance of suspended-growth bioreactors for AMD treatment may not be strongly dependent on the inoculum used for reactor startup. IMPORTANCE: This study showed that different microbial communities enriched from two sites maintained distinct microbial community traits inherited from their respective seed materials. Long-term operation (up to 128 days of fed-batch enrichment followed by up to 138 days of flowthrough experiments) of these two systems did not lead to the same, or even more similar, microbial communities. However, these bioreactors did oxidize Fe(II) and remove total iron [Fe(T)] at very similar rates. These results suggest that the performance of suspended-growth bioreactors for AMD treatment may not be strongly dependent on the inoculum used for reactor startup. This would be advantageous, because system performance should be well constrained and predictable for many different sites.

Thermodynamic controls on the kinetics of microbial low-pH Fe(II) oxidation.

Acid mine drainage (AMD) is a major worldwide environmental threat to surface and groundwater quality. Microbial low-pH Fe(II) oxidation could be exploited for cost-effective AMD treatment; however, its use is limited because of uncertainties associated with its rate and ability to remove Fe from solution. We developed a thermodynamic-based framework to evaluate the kinetics of low-pH Fe(II) oxidation. We measured the kinetics of low-pH Fe(II) oxidation at five sites in the Appalachian Coal Basin in the US and three sites in the Iberian Pyrite Belt in Spain and found that the fastest rates of Fe(II) oxidation occurred at the sites with the lowest pH values. Thermodynamic calculations showed that the Gibbs free energy of Fe(II) oxidation (ΔG(oxidation)) was also most negative at the sites with the lowest pH values. We then conducted two series of microbial Fe(II) oxidation experiments in laboratory-scale chemostatic bioreactors operated through a series of pH values (2.1-4.2) and found the same relationships between Fe(II) oxidation kinetics, ΔG(oxidation), and pH. Conditions that favored the fastest rates of Fe(II) oxidation coincided with higher Fe(III) solubility. The solubility of Fe(III) minerals, thus plays an important role on Fe(II) oxidation kinetics. Methods to incorporate microbial low-pH Fe(II) oxidation into active and passive AMD treatment systems are discussed in the context of these findings. This study presents a simplified model that describes the relationship between free energy and microbial kinetics and should be broadly applicable to many biogeochemical systems.