Abstract
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.
Keywords: Acid mine drainage, Enrichment, Bioreactor, Fe(II)-oxidizing bacteria, Bioremediation
Background
Low-pH microbial Fe(II) oxidation can be incorporated into AMD passive treatment systems by enhancing the development of terraced iron formations (DeSa et al., 2010; Brown et al., 2011; Larson et al., 2014a and 2014b). For extremely difficult-to-treat AMD (very low pH, very high concentrations of Fe(II) and associated metals), an active treatment bioreactor is required by enriching acidophilic Fe(II)-oxidizing bacterial communities. This process can effectively change a high acidity, high metals discharge into a moderate acidity (still low pH), low metals discharge (Sheng et al., 2016).Acidophilic aerobic Fe(II) oxidizers Acidithiobacillus spp., Leptospirillum spp., and Ferrovum myxofaciens have all been enriched in both suspended growth and fixed-film laboratory-scale bioreactors for AMD treatment (Hedrich and Johnson, 2012; Heinzel et al., 2009a and 2009b; Janneck et al., 2010; Tischler et al., 2013). For instance, Hedrich and Johnson (2012) designed an AMD remediation system that integrated low-pH Fe(II) oxidation and Fe removal in a multi-reactor system. A pure culture of the Fe(II)-oxidizer Ferrovum myxofaciens was enriched in first suspended-growth reactor. Heinzel et al. (2009a and 2009b), Janneck et al. (2010) and Tischler et al. (2013) all developed a natural mixed community of Fe(II)-oxidizers with porous fixed-film media in a pilot-scale reactor. A protocol of enriching mixed culture acidophilic Fe(II)-oxidizing bacteria in no-flow, fed-batch systems without fixed-film media is suggested here in a chemostatic bioreactor with controlled hydrogeochemical conditions (Sheng et al., 2016).
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Notes
Recipes
Acknowledgments
This protocol is adapted from Sheng et al. (2016). This work is partially supported by the US Office of Surface Mining Reclamation and Enforcement under Cooperative Agreement S11AC20005, by the China Scholarship Council (to Y.S.), and by the Appalachian Research Initiative for Environmental Science (ARIES). ARIES is an industrial affiliates program at Virginia Tech, supported by members that include companies in the energy sector. The opinions and recommendations expressed herein are solely those of the authors and do not imply any endorsement by ARIES.
References
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