Biogeochemical and kinetics characterization of sulfate reducing microbial communities enriched from mine drainages

[摘要] English: Acid mine drainage (AMD) or acid rock drainage (ARD) is a global challenge contaminating a lot of the fresh surface and groundwater. The drainage is characterized by low pH, high metal and sulfate concentrations. It is a consequence of most mining activities as lead source of AMD. The metals and sulfate in the drainage result from oxidation of metal sulfide containing rocks also referred to as host rock. Oxidation of the host rock occurs during mining by water and oxygen or naturally where the oxidation process occur through weathering and both processes can be accelerated by iron oxidising bacteria such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans. The A. ferrooxidans catalyses the oxidation of iron containing sulfide minerals such as pyrite and arsenopyrite releasing iron, arsenic and sulfur which gets oxidized into sulfate generating sulfuric acid that lowers the pH of the water. The acidic environment induces dissolution of other metals. In most coal mines, AMD contains some neutralizing minerals such as calcium oxide (lime), calcium carbonate (calcite) and sodium carbonate which raise the pH of the water but do not precipitate all of the metals and sulfate. The pH of water can be raised to between 5 and 8 hence the drainage is termed Non-acid mine drainage (NMD).Toxic metals in AMD and NMD contaminate streams and rivers where they affect aquatic and terrestrial life. Treatments of these drainages have been developed and characterized into biotic and abiotic systems where chemical and biological methods are used. In this study, attention was given to extending knowledge that can contribute to developing and extending biotic remediation systems where sulfate-reducing bacteria (SRB) are employed to interact with AMD or NMD. SRB are a diverse group of microorganisms used in bioremediation and have been studied widely. SRB play a major role in the reversal process of AMD and NMD formation by reducing the sulfate concentrations. SRB use sulfate as their terminal electron acceptor releasing sulfide in a gas form that dissolves in the solution when the pH is above 4. This process occurs optimally in anaerobic environments and the dissolved sulfide, reacts with dissolved metals in the drainage forming metal sulfide precipitates. The sulfate reduction and metal removal processes from the drainages can be affected by numerous factors and a few discussed were explored and the knowledge base extended in this study. Environmental conditions always have an effect on most activities performed by biological entities. For sulfate reduction, pH, temperature, carbon source type and availability, metal concentrations and redox conditions have direct effects on SRB activities.In this study, three mine drainage study sites with generic names: Site-Ex, Site-Ka and Site-Po were selected. Water samples from the three sites as well as a sludge sample from Site-Po were collected and characterized chemically. Drainage from Site-Ex had characteristics of NMD while Site-Ka and Site-Po water samples had AMD characteristics. High concentrations of sulfate and transition metals were detected in the AMD samples. Microscopic analysis revealed high microbial cell counts in NMD and lower cell counts in AMD samples that could most probably be directly related to the diversity and toxicity of metals and low pH of the drainages. Molecular analysis revealed the presence of various SRB species in the drainages and sludge samples including the well-studied Desulfovibrio sp. Two media compositions: Postgate medium B (PSGM) and Anaerobic sulfate reducing medium (ASRM) were used to enrich anaerobic bacterial communities. Acclimation process with three passaging intervals of 20 days was conducted in anaerobic serum vials. After the third passaging stage, molecular identification of the enriched cells was performed and results revealed successful enrichment of SRB and other anaerobic (some novel) bacteria within the consortia. Scanning electron microscopy (SEM) was used to morphologically characterize the biogenic precipitates from the tertiary cultures. The SEM results showed bacterial biofilm associated with precipitates similar to those identified as framboid pyrite precursors.The enriched consortia from the three study sites were co-cultured in PSGM and ASRM respectively. The best bacterial growth was achieved in PSGM which was then used for subsequent experiments as the medium of choice. The sulfate reducing capabilities of the enriched SRB were tested in PSGM with sulfate concentrations ranging between 2 000 mg/L to 4 000 mg/L. An average of 72% sulfate reduction was achieved in all experiments with a positive response of SRB to higher sulfate concentrations. Effects of pH and temperature on sulfate reduction were evaluated at pH of 3.5 and 6.2 and temperatures of 10ºC and 25ºC. Low pH conditions showed negative effects on sulfate reduction activity and bacterial growth even when temperature was raised to 25ºC. Optimum SRB activity was observed in the experiment where pH was 6.2 at 25ºC. The results confirmed higher sulfate reducing conditions at higher pH (6.2) and temperature (25ºC). The carbon source utilisation by the enriched SRB between glycerol and sodium lactate was evaluated in batch operated bioreactors. The best sulfate reduction activity by SRB was observed when glycerol was used as a sole carbon source yielding greater amounts of dissolved sulfide concentrations. Glycerol was then used further as the main carbon source in PSGM. Metal-microbe interactions were evaluated where higher concentrations of zinc (Zn2+) and iron (Fe2+) were introduced in the bioreactors. Results showed 100%, 85% and 40% sulfate reduction in experiments where no metals, 200 mg/L of iron and 200 mg/L of zinc were added respectively. Effects of high Zn2+ concentrations were similar to those exerted by low pH conditions. However, 90% zinc and 97% iron were removed from the medium through biogenic precipitation. Precipitates were characterized by SEM, Transmission Electron Microscopy (TEM), X-ray Diffraction (XRD) and Energy Dispersive X-ray Spectroscopy (EDX) which confirmed the presence of biologically induced precipitates.Results in this study can be used to model the activity of SRB in evaluated conditions that affect sulfate reduction. The enriched bacterial communities also showed great potential to be used in the up-scaled 'reactors to reduce sulfate concentration while indirectly precipitating dissolved metals in AMD.

[发布机构] University of the Free State