Article pubs.acs.org/est
pH and Organic Carbon Dose Rates Control Microbially Driven Bioremediation Efficacy in Alkaline Bauxite Residue Talitha C. Santini,*,†,‡ Laura I. Malcolm,†,‡ Gene W. Tyson,§ and Lesley A. Warren⊥ †
School of Geography, Planning, and Environmental Management, ‡Centre for Mined Land Rehabilitation, and §Australian Centre for Ecogenomics, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia ⊥ Lassonde Institute of Mining, The University of Toronto, 35 Saint George Street, Toronto, Ontario M5S 1A4, Canada S Supporting Information *
ABSTRACT: Bioremediation of alkaline tailings, based on fermentative microbial metabolisms, is a novel strategy for achieving rapid pH neutralization and thus improving environmental outcomes associated with mining and refining activities. Laboratory-scale bioreactors containing bauxite residue (an alkaline, saline tailings material generated as a byproduct of alumina refining), to which a diverse microbial inoculum was added, were used in this study to identify key factors (pH, salinity, organic carbon supply) controlling the rates and extent of microbially driven pH neutralization (bioremediation) in alkaline tailings. Initial tailings pH and organic carbon dose rates both significantly affected bioremediation extent and efficiency with lower minimum pHs and higher extents of pH neutralization occurring under low initial pH or high organic carbon conditions. Rates of pH neutralization (up to 0.13 mM H+ produced per day with pH decreasing from 9.5 to ≤6.5 in three days) were significantly higher in low initial pH treatments. Representatives of the Bacillaceae and Enterobacteriaceae, which contain many known facultative anaerobes and fermenters, were identified as key contributors to 2,3-butanediol and/or mixed acid fermentation as the major mechanism(s) of pH neutralization. Initial pH and salinity significantly influenced microbial community successional trajectories, and microbial community structure was significantly related to markers of fermentation activity. This study provides the first experimental demonstration of bioremediation in bauxite residue, identifying pH and organic carbon dose rates as key controls on bioremediation efficacy, and will enable future development of bioreactor technologies at full field scale.
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INTRODUCTION Developing low-cost, rapid, novel strategies for the remediation and subsequent reuse of tailings is crucial to the environmental sustainability of the minerals industry. An estimated 1.7 billion tonnes of alkaline tailings are generated annually through mining and refining activities, expanding the 1 million km2 of land surface area already occupied by mine wastes globally.1,2 Bauxite residue is an alkaline (average pH: 11.3) byproduct of the Bayer process and, like many other sources of alkaline tailings such as gold tailings, chromite ore processing residue, uranium mill tailings, and steel slag,2 is saline-sodic (average electrical conductivity: 7.4 mS cm−1), fine-grained (particle size: 2−100 μm; average specific surface area: 32.7 m2 g−1), and contains low concentrations of organic carbon (average organic carbon content: 0.3 wt %).2−5 Over 120 million tonnes of bauxite residue are produced worldwide each year, adding to the estimated 3 billion tonnes already in storage.6 Bauxite residue remediation typically focuses on decreasing pH and salinity and improving aggregation with a view to transforming it into a soillike material. Remediation is advantageous not only for direct revegetation of residue storage facilities but also in the event of unplanned releases associated with geotechnical failures,2 such as © XXXX American Chemical Society
in Ajka, Hungary, where the alkalinity and salinity of bauxite residue dispersed over agricultural areas had deleterious impacts on vegetation and soil biota.7−12 Microbially driven bioremediation has recently been identified as a novel strategy to neutralize pH in bauxite residue.3 Neutralization of pH, through fermentative microbial metabolisms producing organic acids and CO2(g) from an organic carbon substrate (Table 1),13−20 is a novel, promising target for bioremediation of alkaline tailings.3 In situ acid production through organic carbon fermentation offers several advantages over other potential acid-generating pathways (e.g., sulfur oxidation) in that organic carbon substrates are more readily available on site and in that fermentation of organic carbon is known to occur over a wider range of environmental conditions (e.g., aeration). Developing a rapid, efficient bioremediation process is contingent on increasing microbial community biomass and diversity and identifying the optimal environmental Received: April 21, 2016 Revised: August 31, 2016 Accepted: September 8, 2016
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DOI: 10.1021/acs.est.6b01973 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology Table 1. Common Microbial Fermentation Pathways for Glucose Substrates fermentation pathway 1: alcoholicacetic
acid yielda
chemical equations
C6H12O6 → 2CH3CH 2OH + 2CO2 (1)
4
2CH3CH 2OH + O2 → 2CH3CO2 H + 2H 2O (2)
characteristic microbial taxa Saccharomyces, Zymomonas Acetobacteraceae
2: acetic acid
C6H12O6 → 3CH3CO2 H (3)
3
Clostridium, Acetobacteraceae
3: homolactic
C6H12O6 → 3CH3CHOHCO2 H (4)
3
Lactobacillus
4: heterolactic
C6H12O6 → CH3CHOHCO2 H + CH3CH 2OH + CO2 (5)
2
Lactobacillus, Leuconostoc
5: mixed acid 6: 2,3butanediol
2C6H12O6 → CH3CHOHCO2 H + CH3CH 2OH + CH3CO2 H + CO2 HCH 2CH 2CO2 H + CO2 + H 2 (6) 3C6H12O6 → CH3CHOHCHOHCH3 + CO2 HCH 2CH 2CO2 H + CH3CH 2OH + CH3CHOHCO2 H + CH3CO2 H + 3CO2 + 2H 2 (7)
21/2
Enterobacteriaceae: Shigella, Citrobacter, Escherichia, Proteus, Salmonella, Yersinia
21/3
Enterobacteriaceae: Klebsiella, Enterobacter, Erwinia, Serratia
a
Acid yield: number of moles of H+ equivalents (calculated as sum of acetic and lactic acids, monoprotic above pH 7; succinic acid, diprotic above pH 7; and CO2(g)) produced per mole of glucose consumed.
conditions to support proliferation of fermentative microbial guilds in alkaline tailings. Given the low initial microbial community biomass and diversity in fresh tailings,21−25 bioaugmentation (through the addition of a diverse microbial inoculum) is likely to more rapidly initiate bioremediation than relying solely on the microbial communities present in fresh tailings.3,26−28 Most previous studies of microbial fermentation have been conducted at circumneutral pH (pH 4−8), focusing on the effects of either pH, salinity, or organic carbon supply as individual factors in isolation from each other on the efficiency of fermentation by single microbial strains rather than by mixed communities.29−34 In the context of bioremediation, bioaugmentation with a diverse inoculum generally yields more predictable and controllable results in bioreactors than augmentation with single strains,35−37 necessitating investigation of fermentation efficiency by a mixed community under the more extreme geochemical conditions present in tailings than have been used in previous studies. Decreasing pH and salinity or increasing organic carbon supply all impose financial costs to tailings remediation; therefore, understanding their combined and individual effects on microbial fermentation efficiency, particularly at alkaline pH (pH ≥9), is key to optimizing microbially driven pH neutralization in tailings. The paucity of information on organic carbon metabolism under combined high pH (pH >8.5) and high salt conditions has been previously identified as a limitation in the development of biotechnological applications.38 High pH and high salt conditions pose challenges for microbial survival and growth, which can be overcome via a number of energetically expensive adaptations.39−44 Lowering the initial pH and/or salinity of tailings through existing chemical and physical amendments is therefore expected to improve bioremediation efficiency; however, it is not clear whether pH, salinity, or both should be lowered to achieve optimal efficiency.3 Further, microbial communities are expected to follow distinct trajectories during bioremediation in response to variations in pH and salinity, which may influence the dominant microbial fermentation pathway (Table 1) being used by the community and thus the overall process efficiency. Organic carbon dose rates are expected to be a key factor controlling bioremediation efficacy in that organic carbon provides an energy source for fermentative guilds in the microbial community and in that carbon supply and conversion efficiency to organic acids and CO2(g) will determine the final pH in bioremediated tailings.3
Previous studies with individual strains have also demonstrated a concentration-dependent relationship between organic carbon and the fermentation pathway used.30 This study aimed to develop microbially driven pH neutralization strategies for alkaline tailings using bauxite residue (alumina refining tailings) as a model tailings system. Given the body of previous research indicating that introducing an inoculum is likely to be far more successful in driving bioremediation than relying upon the native microbial community, this study focused on the role of environmental factors in controlling bioremediation efficacy. The role of different inocula in driving pH neutralization will be presented in a future study. The objectives of this laboratory bioreactor study were to (a) identify environmental variables (pH, salinity, organic carbon dose rate) controlling the rates and extent of pH neutralization via microbially driven fermentation (bioremediation) in bauxite residue, (b) identify the major fermentative pathway by which pH neutralization is achieved in bauxite residue and key microbial phyla involved in fermentation, and (c) investigate microbial community dynamics during bioremediation of bauxite residue. Laboratory scale bioreactors were established to compare the effects of initial bauxite residue pH and salinity and organic carbon dose rate on rates and extent of pH neutralization. Fermentation products were analyzed to elucidate pathways by which pH neutralization was achieved and quantify carbon use efficiency. Microbial community structure was analyzed to identify key phyla involved in fermentation, as identified by increases in relative abundance during the bioremediation experiments, and to evaluate the effects of pH, salinity, and organic carbon dose rate on microbial community composition.
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MATERIALS AND METHODS Experimental Bioreactors. Bauxite residue and soil samples used in the preparation of experimental bioreactors were collected aseptically from an alumina refinery in Western Australia and shipped to laboratories at the University of Queensland under refrigerated (4 °C) conditions. Soil from an area nearby the alumina refinery from which the bauxite residue was collected, which has not been affected by human activities and supports native vegetation cover, was used as a diverse inoculum for bioaugmentation in this study. Three factors (each containing two levels: “high” and “low”) were modified in the experimental bioreactors, (1) initial bauxite residue pH, (2)
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DOI: 10.1021/acs.est.6b01973 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
Figure 1. Bauxite residue pH during bioremediation in (a) high and (b) low pH treatments. Values displayed are the means of three replicates; error bars indicate ±1 standard error of the mean.
rate was set at half this value (7.5 g/L). Nutrient media also contained peptone (2.5 g/L), yeast extract (2 g/L), KH2PO4 (0.02 g/L), and CaCO3 (0.01 g/L) and was filter sterilized (
