Microbially-driven strategies for bioremediation of ...

Accepted Manuscript

Title: Microbially-driven strategies for bioremediation ofbauxite residue

Author: Talitha C. Santini Janice L. Kerr Lesley A. Warren

PII: S0304-3894(15)00212-5DOI: http://dx.doi.org/doi:10.1016/j.jhazmat.2015.03.024Reference: HAZMAT 16672

To appear in: Journal of Hazardous Materials

Received date: 9-12-2014Revised date: 12-2-2015Accepted date: 12-3-2015

Please cite this article as: Talitha C.Santini, Janice L.Kerr, Lesley A.Warren,Microbially-driven strategies for bioremediation of bauxite residue, Journal ofHazardous Materials http://dx.doi.org/10.1016/j.jhazmat.2015.03.024

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http://dx.doi.org/doi:10.1016/j.jhazmat.2015.03.024

http://dx.doi.org/10.1016/j.jhazmat.2015.03.024

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Microbially-driven strategies for bioremediation of bauxite residue

Talitha C. Santini1,2,3, *, Janice L. Kerr1, Lesley A. Warren4

1 Centre for Mined Land Rehabilitation, Sir James Foots Building, The University of Queensland, St Lucia QLD 4072, Australia

2 School of Geography, Planning, and Environmental Management, Steele Building, The University of Queensland, St Lucia QLD 4072, Australia

3 School of Earth and Environment, The University of Western Australia, 35 Stirling Hwy, Crawley WA 6009, Australia

4 School of Geography and Earth Sciences, McMaster University, 1280 Main Street West, Hamilton ON L8S 4K1, Canada

* corresponding author: [email protected]; phone: +61 7 3346 1467; fax: +61 7 3365 6899

Abstract

Globally, 3 Gt of bauxite residue is currently in storage, with an additional 120 Mt generated every year. Bauxite residue is an alkaline, saline, sodic, massive, and fine grained material with little organic carbon or plant nutrients. To date, remediation of bauxite residue has focussed on the use of chemical and physical amendments to address high pH, high salinity, and poor drainage and aeration. No studies to date have evaluated the potential for microbial communities to contribute to remediation as part of a combined approach integrating chemical, physical, and biological amendments.

This review considers natural alkaline, saline environments that present similar challenges for microbial survival and evaluates candidate microorganisms that are both adapted for survival in these environments and have the capacity to carry out beneficial metabolisms in bauxite residue. Fermentation, sulfur oxidation, and extracellular polymeric substance production emerge as promising pathways for bioremediation whether employed individually or in combination. A combination of bioaugmentation (addition of inocula from other alkaline, saline environments) and biostimulation (addition of nutrients to promote microbial growth and activity) of the native community in bauxite residue is recommended as the approach most likely to be successful in promoting bioremediation of bauxite residue.

Keywords

Bioremediation; bauxite residue; bioaugmentation; biostimulation; geomicrobiology

Abbreviations

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ADP: adenosine diphosphate; ATP: adenosine triphosphate; EC: electrical conductivity; EPS: extracellular polymeric substance; ESP: exchangeable sodium percentage

1. Introduction

1.1 Production and management of bauxite residue

In 2013, 259 Mt of bauxite was mined globally, the largest production being from Australia [1] (Figure 1). For every metric tonne of aluminium metal produced from bauxite, two tonnes of bauxite residue (also referred to as red mud or alumina refining tailings) are generated [2]. After more than 110 years of commercial aluminium production, bauxite residue storage facilities worldwide currently hold an estimated 3 Gt of residue and are increasing by approximately 120 Mt per year [3]. The large and continually growing mass of stored bauxite residue highlights the need for effective remediation strategies to manage the environmental impacts of aluminium production and contribute to industry sustainability.

[Suggested location for Figure 1]

As a byproduct of the Bayer process used for alumina refining, bauxite residue is a highly alkaline (pH ≈ 11.3), saline (electrical conductivity ≈ 7.4 mS cm-1), sodic (exchangeable sodium percentage ≈ 69 %), massive (bulk density ≈ 2.5 g cm-3), and fine grained (specific surface area ≈ 32.7 m2 g-1) tailings material [4]. Bauxite residue pore water is dominated by the cations Na+ (major), K+, Ca2+ and Mg2+, and the anions Al(OH)4

-, SO42-, CO3

2-, and OH- [5]. Major minerals present in bauxite residue include a mixture of residual minerals from the parent bauxite (hematite, goethite, quartz, kaolinite, anatase, rutile, undigested gibbsite, boehmite, or diaspore) as well as precipitates formed during the Bayer process (perovskite, calcite, tricalcium aluminate, and zeolitic desilication product minerals such as sodalite and cancrinite) [4]. With the exception of perovskite, Bayer process precipitate minerals dissolve slowly during rainfall leaching and weathering of bauxite residue and release salts (Na+, Ca2+, various anions depending on mineral composition) and alkalinity (in the form of CO3

2- and OH-) to pore water solutions, maintaining the high pH and salinity of bauxite residue over time [4, 6]. The chemical and physical properties of bauxite residue pose significant challenges for remediation, the aim of which is to establish and maintain a vegetation cover after closure of tailings storage facilities, and convert the land area occupied by tailings to an alternative use. This requires transforming bauxite residue from tailings to a soil material with chemical, physical, and biological properties similar to those of productive forest and grassland soils (Figure 2). Specific remediation physico-chemical targets include [7]: pH between 5.5 and 9.0; electrical conductivity < 4 mS cm-1; exchangeable sodium percentage < 9.5 %; and bulk density of < 1.6 g cm-3. These are all values typical of ranges observed in well-functioning soils derived from bedrock parent materials. Without targeted strategies, natural weathering processes will drive remediation and soil processes as shown in the conceptual model of soil formation in Figure 2. Chemical, physical, and biological amendments as depicted in

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Figure 2 offer opportunities to accelerate soil formation rates and thus achieve the same remediation goals in a shorter timeframe.


Applications of various chemical and physical amendments to accelerate remediation and soil development in bauxite residue have been studied for nearly four decades [6, 8-16] (Figure 2). The focus of these studies was to lower salinity, sodicity, and alkalinity, and to encourage structure development. The application of gypsum, combined with tillage and irrigation, addresses several of these targets simultaneously, by providing a source of Ca2+ to displace Na+, as well as facilitating export of alkaline, saline-sodic pore water [7, 17, 18], and development of stable soil structure [18]. Plants grown in amended residues had increased seedling emergence rates and dry weight [15]. Organic amendments such as hay have also been successful in improving soil properties and encouraging the development of microbial and plant biomass [19]. Combinations of inorganic and organic amendments have also been evaluated. In combination with gypsum, mushroom compost and sewage sludge were also effective in improving soil chemistry and structure and in promoting plant growth [9, 11, 18-20]. In addition to providing a carbon source and improving structure and drainage, organic amendments such as compost and topsoil may act as microbial inoculants, providing a viable microbial community to perform vital soil ecosystem functions such as nutrient cycling [21], but this has not yet been investigated (Figure 2). The roles of microbial communities in contributing to remediation goals have been largely ignored in research to date, with research confined to an examination of the responses of any extant microbial community to remediation strategies [21, 22] rather than evaluating the extent to which these microbial communities play active roles in remediation. Furthermore, no studies have evaluated the survivability of microbial inoculants in bauxite residue, or factors which influence survival and the extent to which microbial inoculants may influence remediation of bauxite residue.

1.2 Microbial community responses and roles in remediation

Microorganisms are important components of all ecosystems, playing a vital role in soil development (pedogenesis) and in supporting plant nutrition (Figure 2). Interactions between microbial communities and the environments which they inhabit are often difficult to disentangle as they influence one another in complex ways. For example, environmental conditions influence microbial community structure and function by imposing selection pressures and regulating supply of substrates and removal of products, and microbial communities in turn influence environmental conditions through the reactions of their metabolic products with components of the surrounding environment. Investigation of the feedbacks between environmental conditions and microbial communities, especially in a geochemically dynamic and extreme environment

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such as tailings, therefore requires careful experimental design to understand the directions and relative magnitude of microbial and geochemical processes that contribute to remediation and how these might change over time. The initially extreme geochemical and physical conditions dominant in tailings creates strong selection pressures, such that initial colonisers in bauxite residue are likely to be haloalkaliphilic or haloalkalitolerant species that are well-adapted to survive in such extreme conditions. Although consisting of a narrow range of species, this specialised initial microbial community therefore has the potential to be highly active under extreme conditions and rapidly alter environmental conditions through excretion of metabolic products. Understanding threshold environmental conditions beyond which the survival and growth of this initial community are compromised is critical to incorporating microbially-driven remediation pathways into a holistic remediation strategy in concert with chemical and physical amendments. The identity and tolerances of initial colonisers are currently unknown, and is an area of active research. During remediation, microbial community diversity has previously been demonstrated to increase as environmental conditions become less extreme [21, 22], indicating the potential for feedbacks to exist between microbial communities and their environment which might usefully be exploited in the development of microbially-driven remediation strategies. Furthermore, a shift from autotroph dominated soil microbial communities to a dominantly heterotrophic community consuming organic carbon either aerobically or anaerobically has been associated with improved soil ecosystem stability [23], which is an important goal of remediation and thus a key part of successful rehabilitation strategies.

Increases in microbial biomass carbon, microbial respiration and microbial enzyme activity (dehydrogenase) are also indicative of successful remediation of contaminated soils [24], and higher microbial diversity is correlated with higher soil quality [25]. Plant communities rely on microbial communities for the ongoing supply, mobilisation and translocation of inorganic nutrients. Moreover, many plant species benefit from symbiotic associations with soil microorganisms such as mycorrhizal fungi and rhizobia [26]. Microorganisms have been successfully used as a seed inoculant to promote plant growth and reduce the requirement for compost amendment in tailings [27], identifying the promise of such biological strategies. Beyond benefits to the vegetation community, soil microorganisms are also capable of influencing soil chemistry to alter pH and sequester toxins. Microbial activity contributes to pH neutralization in acidic tailings [27] and evidence to date indicates that this is also likely to be the case for alkaline bauxite residues. Alkaliphiles using carbohydrate resources have been shown to reduce the pH of their culture medium by secreting organic acids [28, 29], and Hamdy and Williams [30, 31] successfully decreased pH in bauxite residue by supplying carbohydrates and nutrients to the native microbial community. The exact mechanisms by which pH was decreased in bauxite residue (hypothesised to be organic acid release), and the active members of the microbial community responsible for causing this pH decrease were not evaluated [30, 31]. Hamdy and Williams [30, 31] relied upon the metabolic capacity of the native microbial community to drive remediation, although direct application of microbial inoculants known to carry out metabolisms of interest in achieving remediation goals may improve remediation efficiency and provide a more consistent and controllable outcome. Optimal inoculant sources are currently unknown, but given that high salinity and alkalinity present major challenges for

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microbial growth and survival in bauxite residue, exploration of microbial communities in geochemically analogous environments, and evaluation of haloalkaliphilic or haloalkalitolerant pure cultures for their potential to contribute to geochemical and physical remediation targets is a logical first step.

The enhancement of microbial contributions to remediation presents a promising pathway by which overall remediation efficacy may be increased. To enable development of microbially assisted strategies for bioremediation of bauxite residue, the following is required:

1. an improved understanding of potential contributions of microorganisms (native or introduced) to remediation processes such as aggregation and pH decline;

2. a comparison of candidate microorganisms that could drive these remediation processes and their suitability to the challenging physicochemical conditions presented by bauxite residue; and,

3. an evaluation of inoculation (bioaugmentation) or biostimulation processes including potential for supporting succession in the microbial community as remediation progresses.

With all this in mind, the aim of this review is to identify pathways for the microbially-assisted remediation of bauxite residue, with each requirement above addressed in sections below. This review provides a synthesis of scientific data currently available from studies of individual organisms, microbial communities, and processes occurring in similar environments to evaluate the technical feasibility of a microbially-driven approach for bioremediation of bauxite residue. It is intended that identification of key knowledge gaps here will provide guidance for the focus future research efforts in this field, and ultimately enable implementation of a cost-effective strategy at field scale.

2. Bioremediation pathways in bauxite residue

In a diverse microbial community that includes bacteria, fungi, archaea and other organisms, hosting potentially wide ranging metabolic capabilities, a variety of biological processes may be targeted to reduce the pH, sodicity and salinity of bauxite residues, and increase aggregation, thus contributing to achievement of rehabilitation goals as outlined in Section 1.1. These processes include: (a) production of organic acids; (b) production of inorganic acids; (c) production of carbon dioxide; (d) production of extracellular polymeric substances (EPS) that bind soil particles into stable aggregates; and (e) altering the ionic balance though solubilisation of minerals and selective uptake of ions from solution (dashed box, Figure 2). Mechanisms by which these processes may contribute to in situ remediation of bauxite residue, and controls or limitations on their efficacy, are discussed below and linked back to the conceptual model of soil formation and remediation, followed by a summary of the likelihood of success of these processes and research needs.

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2.1 Production of organic acids

The production of organic acids by fermentative metabolisms is of interest to bioremediation of bauxite residues because they may assist in decreasing residue pH by reaction of H+ with various sources of alkalinity in both the bauxite residue pore water and solids [32] (Equations 1-6; Figure 3).

Pore water hydroxide: H+ + OH- H2O (1)

Pore water carbonate: H+ + CO32- HCO3

- (2)

Calcite: 2H+ + CaCO3(s) Ca2+ + CO2(g) + H2O (3)

Tricalcium aluminate: 6H+ + Ca3Al2OH12(s) 3Ca2+ + 2Al(OH)3(s) + 6H2O (4)

Desilication product minerals (represented here by hydroxysodalite):

2H+ + Na8(AlSiO4)6(OH)2(s) Na6(AlSiO4)6.2H2O(s) + 2Na+ (5)

6H+ + Na6(AlSiO4)6.2H2O(s) + 4H2O 6Na+ + 6Al(OH)3(s) + 6SiO2(s) (6)

Organic acids are a common product of the decomposition of organic matter such as plant residues. The specific acids produced (acetic, propionic, lactic (all monoprotic), and citric (triprotic) acids) will vary depending on the organism, the substrate provided and the enzymes used by the organism to degrade the substrate (Table 1). Laboratory experiments can be used to identify the acids and estimate the molar yield, thus enabling calculation of theoretical pH change due to microbial activity in a given medium.


Acetic, propionic, and lactic acids can all be produced via anaerobic pathways, making them well suited to the poorly aerated bauxite residue environment. Biological fermentation, under aerobic conditions, is a major source of acetic acid and therefore the biochemical mechanisms of production are well understood. However, acetic acid can also be produced via an anaerobic pathway yielding three moles of acetic acid per mole of glucose (Equation 7, with glucose as substrate) by Moorella thermoacetica and other species, potentially using (ligno-)cellulosic substrates such as hay or woodchips added to bauxite residue during remediation [32, 33, 34]), making the anaerobic pathway an efficient option for pH neutralisation in bauxite residue as part of a combined bioremediation strategy.

C6H12O6 3CH3COOH (7)

Lactic acid is most commonly produced by the lactic acid bacteria (Lactobacillaceae) including the genera Streptococcus, Pediococcus, Lactobacillus and Leuconostoc.. Most lactic acid bacteria favour anaerobic conditions for fermentation, yielding up to two moles of lactic acid per mole of

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glucose assuming strict homolactic fermentation (Equation 8) via the Embden-Meyerhof-Parnas pathway rather than heterolactic fermentation via the pentose phosphate pathway (Equation 9) [35]. The CO2 produced via pentose phosphate pathway may also contribute to pH neutralization as discussed in Section 2.3. Lactic acid can also be produced aerobically by fungi such as Aspergillus niger, which in fresh bauxite residues would require that neutralisation occurs in an aerated bioreactor, or tillage and addition of amendments to create and stabilise soil aggregates prior to addition of the microbial inoculant. Many lactic acid bacteria survive in alkaline, saline environments such as soda lakes, making them of particular interest for bioremediation of bauxite residues. A variety of different carbon substrates can be utilized for lactic acid production [35], making this pathway useful if applied in combination with organic amendments.

C6H12O6 2CH3CHOHCOOH (8)

C6H12O6 CH3CHOHCOOH + CH3CH2OH + CO2 (9)

Species from the genus Propionibacter are able to produce propionic acid from glucose or lactose via the anaerobic dicarboxylic acid pathway (Equation 10). This pathway also yields a range of other acidic products including acetic acid, carbon dioxide and succinic acid (where lactose is the substrate) which can also contribute to pH neutralization. Although propionic acid production yields a mixture of acidic products, overall acid generation is similar to that produced by anaerobic pathways for acetic and lactic acid production [32].

3C6H12O6 4CH3CH2COOH + 2CH3COO- + 2CO2 +2H2O (10)

Citric acid (C6H8O7) is produced aerobically by microorganisms such as Penicillia spp., Aspergillus spp., Candida spp. and Arthrobacter spp. [36]; however, many of these species have strict culture requirements [e.g. 37] such as trace metal concentrations, high aeration, and low pH, which would be difficult and expensive to meet in the bauxite residue environment. Stimulating the activity of citric acid-producing microorganisms is therefore not recommended for bioremediation of bauxite residues.

[Suggested location for Table 1]

Overall, organic acid production from the degradation of organic carbon sources via anaerobic (fermentative) metabolic pathways is a good candidate for accelerating pH neutralisation and remediation of bauxite residue as many of these pathways favour anaerobic conditions, and natural rates of acid production can easily be accelerated through addition of organic C substrates.

2.2 Production of inorganic acids

The direct addition of inorganic acids to bauxite residue achieves pH neutralisation via Equations 1-6 above and is a relatively expensive remediation strategy; however, some microorganisms can support the production of inorganic acids such as H2SO4 in situ, via chemoautotrophic or chemoheterotrophic metabolisms. In particular, the metabolic reactions of iron and sulfur

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oxidising bacteria (IOB and SOB) produce sulfuric acid [47], and may assist in microbially-driven remediation of bauxite residues by neutralising pH (Figure 4). Research examining the activities of SOB under alkaline and saline conditions has largely been confined to soda lake environments [48]. Sulfur oxidising bacteria that inhabit soda lakes (and are therefore tolerant of high pH, high salinity conditions) have been found to oxidise thiosulfate (S2O3

2-) [49-51], as well as other sulfur species including sulfite (SO3

2-) [51, 52] (Equations 11-13):

S2O32- + 5H2O → 2SO4

2- + 10H+ + 8e- (11)

2S2O32- + 3O2 + 2H2O → 2S2O3

2- + 2SO42- + 4+ + 4e- + 2S3O6

2- (12)

SO32- + H2O → SO4

2- + 2H+ + 2e- (13)

T. thioparus, a sulfur oxidising bacterium, oxidises elemental sulfur (S0) to sulfite and then sulphuric acid over a pH range of 6.0-9.0 (Equation 14 and 15) [53]. T. novellus has also been found to produce acid from S0 at pH ranging from 5-11 (optimum at pH 8) [54].

S⁰ + O2 + H2O → H2SO3 (14)

H2SO3 + ½O2→ H2SO4 (15)


A mixed culture, or a step-wise series of inoculations with various Thiobacillus spp. as the bauxite residue pH decreases, may be effective in reducing the pH of bauxite residues, following a similar strategy to that which was successfully employed in the heap leaching of copper [55]; however, optimisation of inoculation pH will be required as addition of the sulfur substrate (S0) only [12, 16], and substrate plus organic material [12] have yielded mixed results for reducing the pH of bauxite residue. This is likely to be related to the buffering capacity of the residue (dependent on the concentrations of various sources of alkalinity in bauxite residue as outlined in Equations 1-6) and will be variable between sites. Depending on the sulfur substrate, rates of application for neutralisation will range from 1.0 to 20.2 g/kg bauxite residue, based on a requirement for 0.315 moles of H+ per kilogram of residue [32, 54] (Table 2). As this is an aerobic process, tillage (for in situ bioremediation) or sparging (for bioreactor-based remediation) will be required to ensure that neutralisation via iron or sulfur-based oxidative metabolisms proceeds efficiently.


Iron oxidising bacteria (including Thiobacillus ferrooxidans) are well-studied due to their involvement in the oxidation of pyrite (FeS2; Equation 16) during the formation of acid mine drainage waters in sulfidic ores and tailings [56]. Iron oxidising bacteria play an integral role in

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the formation of acid mine drainage by oxidising Fe2+ to Fe3+, which is a more powerful oxidant for pyrite than oxygen (Equation 17), and is in fact the rate-limiting step in oxidation and acidification processes [56].

4FeS2 + 15O2 + 14H2O → 4Fe(OH)3 + 8SO42- + 16H+ (16)

4Fe2+ + O2 + 4H+ → 4Fe3+ + 2H2O (17)

As with sulfur oxidising bacteria, iron oxidising bacteria would require aerated conditions and addition of a substrate (FeS2 in this case). However, most ferrous iron-oxidising bacteria show greatest activity at low pH (≤ 3.5) due to inhibition of Fe2+ oxidation at higher pH by formation of ferric iron precipitates on cell surfaces [57]. Sulfur oxidation is therefore a more promising pathway for in situ acid generation in bauxite residues than iron oxidation.

Although chemoautotrophic pathways for acid production are attractive in the bioremediation of bauxite residue because they do not require organic carbon as a substrate (which is very low in fresh bauxite residue), they do require addition of an inorganic substrate (reduced Fe or S compounds) and also require aerated conditions. Production of inorganic acids is technically feasible, but therefore practically more challenging than other bioremediation options.

2.3 Production of carbon dioxide

Carbon dioxide is produced by microorganisms in soils during respiration (aerobic) (Equation 18) and fermentation (anaerobic) (Equation 19). Carbon dioxide dissolves in water to form carbonic acid (H2CO3) and subsequently dissociates to produce H+ and HCO3

- (Equation 20). Precipitation of metal carbonates may also occur (Equation 21). These reactions will make a positive contribution to remediation of bauxite residue by neutralising pH (Figure 5).

Aerobic respirationa: C6H12O6 + 6O2 → 6CO2 + 6H2O (18)

Anaerobic fermentationa: C6H12O6 → 2C2H5OH + 2CO2 (19)

CO2(g) + H2O(l) ↔ H2CO3(aq) ↔ H+

(aq) + HCO3-(aq) (20)

M2+(aq) + HCO3

-(aq) ↔ H+

(aq) + MCO3(s) (21)

a Glucose used here as representative organic matter substrate.

M2+ represents a divalent metal cation.


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Addition of CO2 to thickened residue slurry prior to deposition in storage facilities has already been implemented at refinery scale due to the benefits that it confers to bauxite residue, including decreased pH and increased shear strength [58, 59]. As is also the case for organic and inorganic acids as H+ sources (Equations 1-6), the mechanisms by which carbonation decreases bauxite residue pH are well known from studies conducted under abiotic conditions, including Equations 20 and 21 above, and Equation 22 for the neutralisation of tricalcium aluminate (Ca3Al2(OH)12), which is one of the major long-term sources of alkalinity in bauxite residues [60]. The kinetics of this reaction are relatively slow, dependent on the partial pressure of CO2 in reaction vessels amongst other variables [59-64].

Ca3Al2(OH)12 (s) + 3CO2 (g) ↔ 3CaCO3 (s) + 2Al(OH)3 (s) + 3H2O(l) (22)

In comparison, relatively little research has examined the potential for biogenic CO2 to decrease the pH of bauxite residue. Increased partial pressure of CO2 within residue pore spaces resulting from CO2 production either through microbially driven aerobic respiration (Equation 18) or anaerobic fermentation (Equation 19) of organic matter could potentially drive pH decrease. These biologically mediated reactions consume organic matter, which may include sugars, proteins, lipids and other complex carbohydrates. Equations 17 and 18 identify that, molecule for molecule, aerobic respiration produces three times as much carbon dioxide as fermentation, although organic products of fermentation reactions may be further utilised as an energy source by other organisms [65]. In many instances, fermentation products such as alcohols are oxidised to carboxylic acids and then to H2(g) and CO2(g). Thus, respiration generates CO2 quickly but may result in large net losses to the atmosphere whereas fermentation leads to indirect and prolonged CO2 generation. Given the slow kinetics of some reactions involved in the carbonation of bauxite residue [60], fermentation may be preferable to respiration for neutralising alkalinity stored in tricalcium aluminate. The anoxic and waterlogged conditions present in unremediated bauxite residues are also likely to favour fermentation over aerobic respiration.

Microbial communities in bauxite residue are carbon-limited and will require addition of a carbon source to drive CO2 production [66]. Bacterial CO2 production, and the potential for microbial CO2 to neutralise bauxite residues, is expected to increase in parallel with diversification of the initial microbial community away from a narrow, autotroph dominated community towards a diverse, heterotroph dominated community (Table 3). Aerobic respiration converts substrate carbon into carbon dioxide with an approximate efficiency of 30 % [67] and 44 g CO2 can be generated from 100 g of glucose (Equation 18). Assuming that 0.315 moles of CO2 are required to neutralise 1 kg of bauxite residue to pH 7 [32] (although this value would vary with the acid neutralisation capacity of the bauxite residue, initial pH, and solid:liquid ratio [4]), 20 g of glucose would need to be added to each kilogram of residue over time. This is reasonably practical and therefore bacterial respiration within oxygenated surface layers or sparged bioreactors has the potential to make a positive contribution to remediation of bauxite residues. As with organic acid production, microbial CO2 production has good potential to contribute to pH neutralisation in bauxite residue as it will proceed under aerobic or anaerobic conditions, and addition of an organic C source is likely to accelerate CO2 generation.

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2.4 Production of EPS

Extracellular polymeric substances (EPS) produced by bacteria and fungi exuded from cells and assist in survival by providing habitat and mechanical stability, improving adhesion to surfaces, and cohesion between cells, and providing protection from biocides released by other organisms and extreme environmental conditions (desiccation, heavy metals, high/low pH) (Figure 6). Extracellular polymeric substances are mainly polysaccharides, proteins, nucleic acids and lipids [73]; however, the exact composition depends on the identity of the organisms within the matrix as well as their environmental conditions [74-77]. Temperature, pH, and nutrient concentration influence EPS and organic acid production in bacteria [78], whereas studies of fungi have indicated nutrients, pH and water potential are important drivers of EPS production [79]. Soluble extracellular enzymes are also dissolved in the EPS matrix, and actively degrade molecules within the matrix into forms that can be metabolised by specific organisms. In the context of bauxite residue remediation, EPS generation is of interest for its utility in improving particle aggregation and encouraging development of a well aerated soil with stable structure. Increased aggregation, and aggregate stability, has previously been observed as a result of EPS production in extreme and disturbed environments such as saline-sodic soils, and polychlorinated biphenyl-contaminated soils [80, 81]. Despite good potential for EPS generation to contribute to successful bioremediation of bauxite residue, only one study has attempted to examine this, using scanning electron microscopy to compare aggregation in a bioremediated bauxite residue compared to a control sample [31]. Biological activity did appear to improve aggregation from visual inspection; however, no methodical investigation of EPS materials, or statistical comparison of aggregation was performed.


In addition to promoting development of stable aggregates, EPS confer insulation from and resistance to extreme physiochemical factors within the organism’s immediate environment, such as temperature, pH and salinity [75]. This may be explained by the secretion of thermoprotectors and osmoprotectors, such as ectoine, that accumulate and provide protection for all organisms within the biofilm matrix [75]. Acidic components of the polymer matrix may also limit proton permeability, allowing growth to continue at unfavourable pH. EPS from sulfate reducing bacteria buffer pH within three zones: ~pH 3.0 (carboxyl groups), ~pH 7.0 (thiols, sulfinic and sulfonic acid groups), and pH 8.5-9.2 (thiol and amino groups), suggesting that they are important in protecting sulfate reducers from fluctuations in pH [82]. EPS produced by cyanobacteria tend to be hygroscopic and thus confer resistance to desiccation by decreasing the rate of water loss and providing a water source [83]. Microbial EPS have been studied extensively for their ability to sequester toxic metals in contaminated environments [83]. Carboxyl groups present in EPS complex with metal ions and remove them from solution. Specifically, cations such as Na+ (but

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also Pb, Ni and Cd [84]) bind to acidic moieties within polysaccharide chains. In EPS extracted from activated sludge, carboxylic and phosphoric groups in the exopolysaccharides bound metals at pH 7 [84]. EPS produced by sulfate reducing bacteria bind to Ca2+ and may be critical in the precipitation of calcium carbonate within microbial mats [82].

Soils from arid environments and saline-sodic soils have similar properties to many bauxite residues. Selection of EPS-generating microorganisms known to survive in these environments, with some degree of halo-/alkali-tolerance (Table 4), is likely to result in successful remediation outcomes. Cyanobacteria, ubiquitous in soils, produce EPS under the harsh physicochemical conditions in saline-sodic soils [83] and can be applied as part of soil management strategies to minimise wind erosion and contribute to N accumulation [85]. In particular, members of the genus Halomonas, within the halotolerant order Oceanospirillales, may be good prospects for improving aggregation in bauxite residue given that other members of the Halomonas genus are known to survive and grow in bauxite residue environments and they produce EPS at yields of up to 1.6 g/L [86].


Production of EPS is likely to make a useful contribution to bioremediation of bauxite residue by improving aggregation and modifying local environmental conditions to support diversification of microbial communities and EPS production is likely to be accelerated by providing an organic carbon source. As a stress response, EPS production may be decreased in the presence of other amendments e.g. inorganic fertilisers. Interactions (positive or negative) with other remediation strategies have not yet been evaluated in the bauxite residue environment.

2.5 Altering the ionic balance

Bauxite residue is sodic by nature, with high concentrations of Na+ in pore water and occupying cation exchange sites. If the goal of remediation is to transform bauxite residues into a suitable medium for plant growth, then exchangeable sodium percentage (ESP) must be reduced to ≤ 9.5 % (see Section 1.1). High ESP discourages formation of stable aggregates as it promotes dispersion, and is also problematic for revegetation [5]. Increasing Ca2+:Na+ ratio (usually by adding gypsum) is a common strategy to correct the exchangeable sodium percentage and improve ionic balance, by providing Ca2+ to displace Na+ adsorbed to soil surfaces and in pore water. Solubilisation of Ca from Ca-bearing minerals already present in bauxite residue, such as calcite (CaCO3), tricalcium aluminate (Ca3Al2(OH)12), cancrinite (Na6Ca2Al6Si6O24(CO3)2.2H2O), or perovskite (CaTiO3), may present one suitable alternative to direct application of soluble Ca2+ sources (Figure 7). Excluding lime sintered bauxite residues, typical CaO concentrations in bauxite residue range from 2-15 % wt [4], and assuming that all Ca can be solubilised, this would be a similarly effective Ca source to gypsum which significantly decreases pH and exchangeable sodium percentage, and improves plant nutrition and yield when added at concentrations of 1-10 %wt [8, 10, 12, 14, 95].

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Bacteria, fungi and other microorganisms can increase the concentration of ions in the soil solution through solubilisation of the mineral or organic components of the soil [96]. Fungal species including Phoma glomerata, Epicoccum nigrum, Penicillum chrysogenum, Cladosporium herbarum, Altemria tenuis and Aspergillus ustus alter calcite and dolomite crystals [97-99] by surface etching. The solubility of calcium is governed by the availability of carbonate ions in solution, which is in turn driven by pH and alkalinity. Microbial processes that increase alkalinity encourage the precipitation of calcite, whereas processes that reduce pH (increased dissolved inorganic carbon or organic acid production) encourage calcite dissolution [100]. Processes encouraging dissolution include fermentation, glycogen degradation and denitrification by sulfide oxidisers (Equations 23-25). These processes, as described by Dupraz et al. [100] are summarised below:

Aerobic heterotrophs: CaCO3 (s) + CH2O + O2 (g) → 2HCO3-

(aq) + Ca2+ (aq) (23)

Sulfide oxidisers: CaCO3 (s) + HCO3- (aq) + 3HS-

(aq) + 4O2 (g) → 2CH2O + Ca2+ (aq) + 3SO4

2- (aq) (24)

Fermenters: CaCO3 (s) + 3CH2O + H2O (l) → CH3CH2OH (l) + Ca2+ (aq)

2HCO3- (aq) (25)

CH2O represents organic carbon compounds from organic matter.

In addition to solubilising calcium, fungi may improve plant tolerance of sodic conditions through arbuscular and ectomycorrhizal associations, which will be beneficial to remediation. Arbuscular mycorrhizal fungi (AMF) can alter the internal ionic balance in their host plants under conditions of salt stress [101, 102], by selectively taking up K+ and Ca2+ and excluding Na+ [103], thus rendering them more tolerant to sodic soils. This approach has been successful in the revegetation of saline, low fertility soils [104, 105]. Few studies have examined the utility of microorganisms in altering the ionic balance in bauxite residues by the mechanisms above. The best studied example is that of the phosphate-solubilising and organic acid producing mould Aspergillus turbingensis, in combination with Bermuda grass (Cynodon dactylon) for remediation of bauxite residues. Inoculation with A. turbingenis has beneficial effects on both vegetation (increased biomass and leaf nutrient concentrations, decreased leaf metal concentrations) and bauxite residue (increased organic C, available N and P, decreased pH) when combined with other inorganic and organic amendments such as sewage sludge and gypsum [106, 107].

The solubilisation of cations from minerals in bauxite residue is tied to acid (inorganic and organic) acid production, and therefore requires further investigation as part of a combined bioremediation strategy. It can be assumed that provision of substrates for acid and CO2 production will also promote mineral solubilisation and decrease ESP as well as increasing availability of some macro- and micronutrients. Timescales over which mineral dissolution will occur are unknown and this limits evaluation of its utility in meeting plant and microbial nutrient requirements. Although arbuscular and ectomycorrhizal fungal associations have already been

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demonstrated to improve remediation of bauxite residue for at least some fungal-plant species combinations, the diversity of mechanisms by which mycorrhizal associations may improve plant tolerance of saline-sodic conditions in bauxite residue, plus the variety of factors influencing the efficacy of these mechanisms necessitates evaluation of mycorrhizal associations on a species by species basis.

2.6 Summary and research needs

Given the waterlogged, anaerobic conditions prevalent in fresh bauxite residue, metabolic pathways that can proceed under both aerobic and anaerobic conditions are most likely to be effective in achieving remediation goals (Table 5). Neutralisation of alkaline pH via organic acid or CO2 production appears promising as these may proceed without addition of substrates, in the hostile environmental conditions presented by unremediated bauxite residue. Inorganic acid production is unlikely without addition of reduced Fe or S substrates. EPS and ionic balance are possible but involve complex processes and inter-species and inter-kingdom interactions that require substantial further research before implementation at field scale. Evaluation of activity rates (microbial kinetics) are a key research need that will in part be determined by the interaction of environmental (salinity, pH, temperature) and community composition factors (competition, mutualism), and require focussed effort in bauxite residue to identify the most efficient approach for bioremediation in this material.


3. Candidate microorganisms for bioremediation of bauxite residue

In selecting or designing a microbial community with the potential to contribute towards achievement of remediation goals, both the biochemical mechanisms by which species can contribute to these goals as well as any environmental constraints to their survival and activity must be considered. Williams and Hamdy [30] suggested the following as screening criteria when selecting microbial inoculants for bioremediation of bauxite residues:

1. Motility; 2. Anaerobic or facultative; 3. Ability to produce pleasant smelling by-products; 4. Capable of surviving in an alkaline environment; and 5. Non-pathogenic

To this list, we suggest adding:

6. Capable of surviving in a saline environment 7. Ability to fix nutrients (e.g. N2 to NH4

+; CO2 to organic C)

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8. Generate extracellular polysaccharides to assist in aggregation or acid production to reduce soil pH

9. Does not produce toxic substances

And modifying 3. to read:

3. Produces benign gaseous products

Of the above list, some features are more important than others, and many are desirable rather than essential characteristics. In Section 3.1 and 3.2, we examine microbial adaptations to the bauxite residue environment (including criteria 2, 4, 6, 7, and 8) and potential sources of microbial inoculants, both natural and engineered.

3.1 Biological challenges posed by bauxite residue

3.1.1 High pH and alkalinity

Elevated pH presents a number of challenges to bacteria, fungi and other microorganisms living in the soil. These include (1) maintaining a neutral cytoplasmic pH; (2) producing alkali resistant endo- and exo-enzymes; and (3) modification of ATP synthesis to cope with a reversed pH gradient [108]. Although studies of the physiological adaptations of the fungi to alkaline environments are few, mechanisms for pH homeostasis in a number of bacterial species have been studied systematically over the past two decades. Thus, the following discussion is related mainly to bacterial pH homeostasis.

For Bacillus spp., the upper bound of pH tolerance is linked to increases in cytoplasmic pH [109], indicating that breakdown of systems which maintain a circumneutral pH within the cytoplasm inhibits normal cellular functioning. Homeostatic regulation of internal pH is achieved through a number of passive and active mechanisms [110]. Cells may exhibit a decreased prevalence of amino acids with basic moieties in contact with the cytoplasm, or include cell wall components such as tiechuronic acid and tiechuronopeptides, which reduce membrane permeability [109]. Some cells employ a Na+ driven pump that actively imports protons into the cell in exchange for sodium [111], while providing energy for the uptake of solutes and motility [112]. Thus, a supply of Na+ is required to maintain pH homeostasis in these organisms [113]. Many alkaliphiles can maintain an internal pH two to three units below the external pH [114].

Despite adaptation to maintain circumneutral pH, cytoplasmic pH often still rises as external pH increases. This may cause decreased activity for many internal enzymes through deprotonation of proteins. Many alkalitolerant bacteria produce enzymes that remain active at alkaline pH [29, 115]. Alkaliphiles must also be able to degrade external energy resources in order to grow and reproduce. This involves using extracellular enzymes, which are secreted from the cell to break down the substrate, followed by absorption of the smaller molecules across the cell membrane. To be effective, these exo-enzymes must maintain functionality in alkaline environments. A variety of studies have shown that bacteria are indeed able to produce such enzymes. These include alkaline extracellular proteases, starch degrading enzymes, cellulases, lipases, xylanases,

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pectinases and chitinases [29]. These enzymes are primarily used to degrade energy resources but industrial applications have been found for many of them. A wide range of fungal groups are also able to produce cellulases that function at alkaline pH [116].

ATP synthesis by ATP synthases is energised by the electrochemical gradients of protons and sodium ions across the cell membrane. These synthases are transmembrane protein complexes with two domains, the F0 domain within the membrane, and the F1 domain within the cytoplasm. The protons are passed along an electrochemical gradient from F0 to F1. In order to maintain internal pH, alkaliphiles pump protons into the cell via antiporter proteins, which exchange a sodium ion for a proton, while sodium ions enter the cell via separate proteins known as symporters that transport sodium ions into the cell at the same time as solutes such as organic matter [114]. The production of ATP is a reversible reaction that requires a flux of protons to drive the reaction in the ATP generating direction. The processes of pH regulation pumping protons into the cytoplasm create a reverse pH gradient (acid inside) for ATP synthesis, with the “downhill” direction of movement for protons now being F1 to F0 and favouring ADP generation. Alkaliphiles need to overcome this problem, and it is likely that different species use differing mechanisms (Figure 8). Better characterization of biochemical adaptations and pH growth ranges for various consortia, based on species-level understanding of these mechanisms, would enhance opportunities for their strategic inclusion in effective microbially-driven remediation strategies.

Alkaliphilic bacteria may at least partially compensate for reversal of the pH gradient by (a) maintaining an internal pH well above that of neutrophiles (optimum growth pH from 5.5 to 8) [112, 117] (b) possessing mechanisms for capturing protons from an highly alkaline environment [111, 118]; (c) minimising outward leakage of protons (enzyme repression) [108] and maintaining a high membrane potential [114]; (d) coupling Na+ expulsion to electron transport for pH homeostasis and energy transduction [119, 120], a pathway that proceeds better at high pH [112]; or (e) using a fermentation pathway [111].


3.1.2 Salinity and sodicity

Salinity is the dominant environmental factor influencing bacterial community diversity in a wide variety of terrestrial and aquatic environments [121-123]. Biochemical adaptations for survival in saline environments are all energetically expensive. Higher salinity in soil has been correlated with a reduced abundance of viable fungi (as colony forming units; CFU) [121] and altered diversity in bacteria [122]. The effects of salinity on microbial diversity differ with taxonomic resolution, with high species level bacterial diversity being observed in hypersaline environments, but lower diversity at higher taxonomic levels. These differences may be due to high ionic strength, variation in ionic composition or other factors contributing to overall salinity [122]. These conditions create metabolic challenges for microorganisms in terms of water loss and ion toxicity.

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Cells living in saline environments must adjust their internal water potential to be lower than their exterior, by either a ‘salt in’ (where K+ and Cl- are accumulated in the cytoplasm to maintain equivalent osmolality with the external environment which is usually NaCl-dominated) or ‘salt out’ (where organic osmotic solutes are synthesised or accumulated in the cytoplasm to maintain low intracellular salt concentrations) strategy [123]. The salt in strategy requires adaptation of intracellular systems (especially protein conformation and activity) to high salt concentrations; whereas the salt out strategy requires no unusual intracellular adaptations [124]. Both strategies have been observed in both Bacteria and Archaea [124]. Halophilic fungal species are able to cope in hypersaline environments by synthesising polyols to prevent loss of internal hydraulic pressure [125], by the uptake of sodium and potassium ions [126] or the extrusion of sodium ions in exchange for potassium ions (which is pH dependent). Fungi may also produce protective mucilage to protect reproductive structures from higher salinities and to anchor them together or to a substrate.

Fungi need to maintain internal osmotic pressure in order to generate the turgor required for nutrient transport and growth [127] and high salt concentrations decrease water potentials, leading to reduced turgor. High concentrations of sodium chloride are also inhibitory to fungal enzyme activities, asexual spore production and sexual reproduction. Many fungal species respond to elevated salinity by synthesising or absorbing organic compounds such as polyols (e.g. glycerol; [128] (Eumycota)) or amino acids such as proline (lower fungi) to maintain internal osmotic pressure. At high concentrations (>1 M), these compounds have no effect on enzyme activity and do not affect protein conformation. Halophilic fungi maintain a low sterol to phospholipid ratio in their plasma membrane, making it more fluid and potentially better at retaining glycerol. Higher salinities also stimulate a genetic mechanism [129] whereby phospholipid fatty acids in the plasma membrane are modified to be less saturated [130, 131]. Fungi may be able to preferentially exclude sodium and chloride from crossing their cell wall, preferentially transporting potassium, calcium and organic acids instead, or to pump sodium out of the cell by exchanging it for potassium. Genetic responses to increased salinity have been associated with the high osmolality glycerol (HOG) signalling pathway. This pathway senses and responds to increasing concentrations of NaCl and signals changes in the solubility of histidine kinases [132], mitochondrial metabolism, biogenesis [133] and the thickness of the plasma membrane [134].

Production of internal organic osmotic solutes by microorganisms is energetically expensive, as is maintenance of a steep Na+ and K+ gradient across the cytoplasm. Regardless of the osmotic adaptation strategy used, this energy expenditure has to be offset through energy yielded via metabolism. This has been invoked as an explanation for the apparent loss of functional capacity within microbial communities at high salinities: the synthesis of osmotic solutes, or maintenance of high intracellular K+ and Cl- concentrations places bioenergetic restrictions on which metabolisms will yield energy overall, and where the upper limits for salinity lie that will still enable these metabolisms to yield energy overall [135]. The salt in strategy is energetically more favourable at high salinities, and is utilised by the Halobacteriales (Archaea) and Haloanaerobiales (Bacteria). Several metabolic capacities are not yet known to occur at high

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salinities: autotrophic nitrification, aceticlastic methanogenesis, and proton-reducing acetogenesis [135]. Despite the apparent loss of functional capacity at high salinity, the exceptional diversity found in some hypersaline environments, such as the Guerrero Negro microbial mat [136, 137], reflects the presence of numerous environmental niches to which a diverse array of adaptations are employed across different phyla and kingdoms in order to survive. Understanding analogous niches in bauxite residue and microbial adaptations for survival in such niches as well as characterization of endemic microorganisms may help to develop targeted bioremediation strategies for bauxite residue.

3.1.3 Metal toxicity

The speciation of trace metals in bauxite residues is largely unexplored; however, chromium, vanadium and arsenic have been known to accumulate in supernatant liquor in residue storage areas [5]. Decreasing pH will assist in the removal of hexavalent chromium (present as Cr(OH)4

- at high pH) from solution through precipitation as Cr(OH)3. Decreasing pH will also assist in the removal of arsenate (As(V) species, usually AsO4

3-) and arsenite (As (III) species, usually AsO33-)

species from solution through absorption on variable charge iron oxide surfaces. Vanadium toxicity is less clear, although vanadate ion (VO4

3-) can behave similarly to PO43- due to its

similar size and charge and may therefore disrupt biochemical processes by substituting for PO43-.

Vanadate speciation is also pH sensitive, with the majority of vanadium present in forms other than vanadate below pH 10. These trace metals (Cr, As, V) are therefore likely to pose challenges for initial microbial colonisers, who through their contributions to pH decrease, will alleviate toxicity effects for other microorganisms during successional change in community structure.

Some alkaliphiles are able to produce siderophores, small molecules with a high affinity for specific metals, enabling them to accumulate potentially toxic metals such as iron, gallium and aluminium [136, 138]. Other bacteria secrete anions [139] or EPS that can bind with metals (Sr, Fe, Mg, Ca) [82] and make them less bioavailable. Some species of freshwater fungi are able to biosequester, avoid and tolerate heavy metals dissolved in stream water via internal and external detoxification pathways, and although fungal diversity often decreases in response to metal pollution, fungal biomass is impacted to a lesser degree [140]. Fungi tend to accumulate more biomass than bacteria, and this enables them to sequester more metals [141]. The physiological mechanisms used by fungi to cope with elevated metal concentrations are discussed in detail by Krauss and co-authors [140].

3.1.4 Nutrient availability and uptake

Fresh bauxite residue is inherently low in most macro and micronutrients required for microbial and plant growth [10, 142-144], especially nitrate, potassium, magnesium, manganese, and boron (Table 6); however, the potential effects of the high salinity, pH, and low oxygen conditions on the capacity of microbial communities to mobilise and take up what few nutrients are available

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remains unclear [145]. Residual aluminosilicate clay content in bauxite residue imparts limited potential for the sorption of micronutrient cations, and precipitation of carbonates and hydroxides (e.g. CaCO3, Mg(OH)2) also becomes a factor controlling nutrient availability at high pH [66, 146]. Nitrogen is a key limiting macronutrient for biological activity, and inorganic nitrogenous fertilisers have limited efficacy in addressing this deficiency. Applied NH4

+ is rapidly volatilised at high pH, and the lack of anion exchange capacity on both variable and permanent charge surfaces within bauxite residue at high pH causes rapid loss of applied NO3

- via leaching [146, 147]. Microbial and plant communities must therefore be equipped to respond quickly to applications of nitrogenous fertilisers. In deglaciated soils, N is also limiting to microbial and plant community establishment and diversification [148], and diazotrophic bacterial species such as the Cyanobacteria play important roles in N accumulation in these settings [149]. N2 fixation and conversion by diazotrophic bacterial species into organic N may provide a more stable and persistent source of N for microbial and plant communities in bauxite residue compared to inorganic fertilisers, however this has yet to be evaluated. The formation of microbial mats (complex and highly diverse assemblages of archaea, bacteria, fungi, and algae) at the surface of bauxite residue is likely to play a key role in accumulation of N as well as organic C, and also improve aggregation [21]. Microbial community activity within sandy bauxite residues is inhibited by low availability of organic carbon, and microbial communities compete strongly with plants for organic nitrogen sources such as amino acids [145], indicating that both C and N are limiting nutrients for biological activity in bauxite residue.

However, the combined stresses of nutrient deficiency and high pH and salinity may also have some effects of benefit to remediation, by stimulating microorganisms to produce acids and EPS as a strategy to mobilise macro-, micro- and trace nutrients including manganese and boron. The mould, Aspergillus niger can be stimulated to produce citric acid under phosphate limited conditions, and the resulting decrease in local pH assists in solubilising phosphorus [150], which may be present in bauxite residues in the form of apatite (Ca10(PO4)6(OH)2) [4]. The initial lack of nutrients present in bauxite residue will limit microbial community diversity and function; however, early colonisers are likely to play a key role in nutrient accumulation and mobilisation and in fact be more effective in remediating the high pH and structural problems due to nutrient stress. Microbial inoculants should also be selected to assist in nutrient accumulation and a capacity to tolerate or increase production of organic acids and/or EPS in response to nutrient limited conditions.


3.1.5 Drainage, aeration, and structure

Soil structure and texture mainly influence the microbial community via their impacts on soil aeration, matric water potential, water holding capacity, and habitat sizes. Soil properties relating to drainage and aeration were responsible for 59% of the variance in bacterial community composition between unamended and amended bauxite residues, indicating that amendment of

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physical properties through tillage and incorporation of coarse grained material assists in developing a more diverse and broadly functional microbial community [21]. The high bulk density, clay dominated texture, and lack of soil structure in fresh bauxite residues limits aeration and drainage, which favours obligate and facultative anaerobic and microaerophilic organisms, and stimulates fermentation pathways in fungi. For fungi, the massive soil structure in fresh bauxite residue may also inhibit the penetration of hyphae through soil pores and the production of fruiting bodies, which must often be forced upward through the soil prior to sporulation [163]. Smaller pore sizes within the matrix may favour bacterial colonisation over fungi due to the protection that these habitats afford them from predatory organisms [164]. Given that one of the goals of remediation is shifting from an autotrophic bacteria-dominated microbial community towards a heterotrophic community comprising both bacteria and fungi, providing a variety of habitat sizes through development of stable aggregates that contain both macro- and micropores is a key part of this process.

3.2 Identifying organisms with bioremediation potential

Given the many challenges for microbial survival and growth posed by bauxite residue, understanding microbial community structure and adaptations in geochemically analogous environments and within target end land use/ecosystems may be useful in the design of bauxite residue bioremediation strategies as well as the selection of inoculant species or consortia to carry out bioremediation. Haloalkalitolerant species isolated in pure culture are also likely to be well-adapted to bauxite residue and are therefore also considered as suitable inoculants. These environments, the properties of their microbial communities, and their suitability as sources of inoculants for bioremediation of bauxite residue are discussed below.

3.2.1 Organisms from saline, sodic and alkaline environments

The high pH, alkalinity, and salinity of bauxite residue arguably present the greatest challenges for microbial growth and survival. A wide range of microorganisms are known to tolerate alkaline, sodic and saline conditions, many being isolated from natural habitats having these properties. Haloalkalitolerant and haloalkaliphilic Bacteria, Archaea, and Fungi have been isolated from saline-sodic and alkaline environments including soils [121, 165, 166, 167], lakes [49, 51, 168, 169], and tailings including bauxite residues [170, 171]. Many of these haloalkalitolerant species exhibit diverse functional capacities of benefit to bioremediation of bauxite residues including fermentation and growth on a variety of carbon sources, N2 fixation, sulfur oxidation and EPS production. In principle, therefore, these organisms could be expected to survive in the bauxite residue environment and make a positive contribution to the overall remediation process, regardless of their physiological tolerances or metabolic capacities. The characteristics of several natural and engineered haloalkaline environments and pertinent features of the microorganisms which inhabit them are presented below along with a discussion of their utility in the bioremediation of bauxite residue.

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3.2.1.1 Soda lakes

Soda lakes are salt lakes with high carbonate alkalinity, a pH between 9 and 11 and moderate to extremely high salinity [172]. Most of these lakes are found in inland semi-arid and arid regions in local depressions. The water chemistry of salt lakes is dominated by sodium, carbonate/bicarbonate, chloride and sulfate. The origin of salt lakes determines their chemistry: thalassohaline lakes (derived from seawater) are NaCl-dominated, with low concentrations of Ca2+ and Mg2+, and pH in the neutral to slightly alkaline range (pH 7-8.5); whereas athalassohaline lakes (representing the majority of soda lakes) are derived from sources other than seawater and have much higher concentrations of Ca2+ and Mg2+, as well as being generally more alkaline (pH 9-11) [173]. These lakes are well buffered, and pH is relatively stable [51]. Numerous novel species of fungi [165] and bacteria [17, 49, 51] with specific adaptations to high salt and high pH (haloalkalitolerance or haloalkaliphily) have been isolated from these environments [172]. Within the sulfate reducing bacterial community (SRB), haloalkalitolerant bacteria have been found within the Proteobacteria, including delta- and gamma-Proteobacteria [174] and a diverse range of gamma-Proteobacteria have been studied within sulfur oxidising communities in soda lakes [49, 51, 175]. RNA-derived community profiles indicate that Cyanobacteria, Actinobacteria, Clostridia and Beta-Proteobacteria are active in the surface sediments of soda lakes, whereas alpha-Proteobacteria were indicated in deeper sediments (10-20 cm) [174]. The populations of these lakes tend to have an optimal pH tolerance between 8 and 10, which corresponds well with gypsum treated bauxite residues that have similar chemical characteristics (high Ca2+, high SO4

2-) to athalassohaline soda lakes. The community response to salinity is variable but the diversity of higher taxonomic levels decreased with increased salinity (between 60 and 200 mg/L) whereas microdiversity (within genera) increased at higher salinities [174]. Sulfur oxidising bacteria from soda lakes, many of which are chemolithotrophic [49], may be of particular interest to bauxite remediation strategies due to the acid producing metabolic reactions used in this pathway [54] (Section 2.2).

Fungal communities in soda lakes are poorly characterised in comparison to bacterial communities, but haloalkaliphilic fungi have been isolated from soda lakes. Sodiomyces alkalinus is a common alkaliphilic member of fungal communities in soda lakes across Asia and Africa, capable of utilising a wide variety of carbon sources [165]. Growth rates at pH 10 were higher on more complex media such as alpha-cellulose, cotton-seed hulls, apple pectin and lignin. This is a particularly useful feature when considering the role of fungi in organic matter cycling in soda lakes and bauxite residue receiving organic amendments. Fungi are the main organisms responsible for the breakdown of lignocellulose [176] and saprotrophic fungi generally prefer moderately acidic soil conditions [177]. Thus, it is necessary to ensure that alkalitolerant fungal decomposers capable of lignocellulose breakdown are present in bauxite residue receiving complex organic matter amendments as part of remediation strategies. This will ensure that composts (e.g. corn stalks, wood chips) can be readily broken down, providing substrates for fermentation pathways.

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3.2.1.2 Hypersaline lakes, salt pans and salterns

Hypersaline environments occur where water loss is dominated by evaporation. This includes terminal lakes (which may have fresh or marine inflows), salt pans (which occur on poorly drained desert soils) and salterns (locations used for commercial or historical salt production). The high salt concentration in these environments creates osmotic stress for microbial cells as discussed in Section 3.1.2.

Fungal communities in hypersaline environments appear to have a consistent and stable species composition regardless of location [126], potentially consisting of a core microbiome plus additional species adapted to local conditions. Local community variability has been related to the availability of nitrogen, phosphorus, dissolved oxygen, water potential, pH and season [178]. Candidate fungal members of a core microbiome in hypersaline environments include meristematic melanised yeast-like fungi (cells form spherical clumps; see [179]), non-melanised yeasts and filamentous genera. Black (melanised) yeasts are tolerant of UV light, low water potentials and high salinity, colonising seemingly inhospitable surfaces such as bare rock [180]. This makes them valuable colonisers of new surfaces that contribute to habitat formation for other microorganisms. Yeasts such the genera Candida, Debaryomyces, Metschnikowia and Pichia [181] are capable of fermenting organic matter within anaerobic sediments which is associated with production of organic acids. Filamentous forms, including Wallemia, Scopulariopsis, Alternaria, Eurotium, Emericella and Petromyces [126], are important in the breakdown of organic matter which provides carbon for other organisms, and in binding soil particles.

Solar salterns are also habitats for halophilic Bacteria and Archaea, which tolerate salinities up to 30% NaCl or more [182]. Many species found at higher salinities (including Halobacteriaceae spp. and Salinibacter ruber) have a characteristic red pigmentation [183] which protects them from intense light conditions within the salterns [184]. Halobacteriaceae spp. are also tolerant of alkaline conditions and some species are known to produce acid [185-187]; whereas Salinibacter ruber does neither [188]. Non-pigmented halophilic genera with potential applications for bauxite residue bioremediation include Vibrio, Flavobacterium, Alcaligenes, Alteromonas, Halomonas, Bacillus, Salinicoccus, Pseudomonas, and Chromobacterium [183], many of which have a broad pH tolerance (pH 5-11), produce EPS (0.001-700 mg/L, with high concentrations of glucose and uronic acids) and produce NH4

+ [189-195]. The prevalence of haloalkalitolerance combined with capacity to produce acids and/or EPS and fix nutrients including C and N suggests that organisms from hypersaline environments may be valuable in the remediation of bauxite residues, with various species having potential for tolerating the extreme conditions, lowering the pH, forming soil aggregates, improving drainage, fixing nutrients and stabilising the surface.

3.2.1.3 Deep sea hypersaline basins

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Deep sea hyper-saline basins form in sub-marine depressions where Miocene subterranean salt deposits slowly dissolve to create dense, hypersaline lenses below the marine water [196]. The high salinity, anoxic, low nutrient and often sulfidic conditions found in these habitats are similar to those in bauxite residues, suggesting that organisms in these habitats may be suitable candidates for residue remediation. However, organisms inhabiting depths in excess of 3 km below sea level, under zero light, high pressure, and low temperature conditions require additional adaptations for survival beyond those required for survival in bauxite residue, making this a unique environment [197]. Bacterial communities in these environments show high species diversity [198, 199] and a range of metabolic capacities including sulfate reduction, methanogenesis, and heterotrophy. Putative methane-sulfur cycling consortia consisting of species similar to those present in deep-sea basins and hydrothermal vent environments were dominant in poorly remediated bauxite residue samples at 90-150 cm depth below surface [21, 200], supporting the utility of deep sea environments as analogues for understanding the geomicrobiology of bauxite residue environments and identifying potential inoculants to promote metabolisms of benefit in bioremediation. Fungal communities in deep sea brines also show considerable diversity [197] and are distinct from communities found in other hypersaline environments, probably due to the dominance of polyextremophiles, which suggests that some of these microorganisms may be worthy of consideration in the bioremediation of bauxite residue.

3.2.1.4 Saline-sodic and alkaline soils

Alkalitolerant fungi are readily isolated from saline, alkaline soils, capable of growth at pH 10 and ≤ 15 % NaCl [166, 201, 202]. Haloalkalitolerance appears to be a common feature within the Acremonium cluster of fungi, particularly the Emericellopsis-clade [167, 201, 202]. Many of these species tolerate low as well as high pH values [167], which is useful in the context of bauxite residue bioremediation as it will allow successful inoculation with these species at the initially high pH in unamended bauxite residue as well as the continued growth and activity of these fungi as pH declines towards circumneutral values. In addition, these fungi contribute to carbon cycling and soil particle aggregation, and can also be beneficial in reducing salt stress and improving plant biomass production under saline conditions through mycorrhizal associations [203-206]. In a bauxite remediation scenario, the use of AM fungi as plant symbionts could potentially shorten the period of remediation required before plants are introduced.

Research investigating bacterial communities in alkaline, saline-sodic soils (distinct from salt and soda lake sediments) has mostly been associated with rehabilitation activities. Although studies in alkaline, saline soils are limited, bacterial communities in saline soils are dominated by representatives of the genus Lysobacter (13 % relative abundance) followed by Sphingomonas (5 %), Halomonas (3 %) and Gemmatimonas (3 %) [207]. Pseudomonas, Vibrio and Actinopolyspora are also common. Gram negative bacteria (including Bacillus, Micrococcus and Salinicoccus) occur more frequently in bacterial communities from saline soils than gram positive bacteria [208]. In agricultural settings, microbial biomass, diversity and metabolic efficiency decreases with increasing electrical conductivity (EC) and sodium adsorption ratio [209].

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Biomarker and DNA-based community finger-printing methods have shown that rehabilitation strategies designed to tackle the chemical and physical properties of alkaline, saline-sodic soils (for example, with biosolids or irrigation) also increases microbial community diversity and efficiency [210, 211]. Novel haloalkaliphilic and haloalkalitolerant bacteria have been isolated from these environments with the ability to produce organic acids from carbohydrates, and to produce EPS [80, 212, 213], and these species may therefore be useful during remediation of bauxite residue.

Archaea are common members of microbial communities in saline environments, with salt tolerance a defining feature of the family Halobacteriaceae and also found in other Archaeal lineages. Most studies of archaeal salt tolerance focus on salt and soda lakes and their sediments; studies from soils are rare. The genera Halorubrum and Thermofilum dominate archaeal communities in saline soils [207]. Genus Halorubrum includes alkaliphilic (3.9–4.3 M, pH 9–10), halophilic (23-25% w/v NaCl), chemoorganotrophic, aerobic nitrate reducers that produce acid from carbohydrates [214], and the genus Thermofilium includes anaerobic, sulfur respiring, extremely thermophilic and mildly acidophilic Archaea from hot springs [215]. Metagenomic 16S rDNA analysis of alkaline saline-sodic soil from the drained Lake Texcoco in Mexico (pH 9.8 – 11.7 and EC 22-150 mS/cm) indicated that the archaeal community included Natronococcus spp., Natronolimnobius spp., Natronobacterium spp., Natrinema spp., Natronomonas spp., Halovivax spp., Halalkalicoccus jeotgali and novel members of the Halobacteriaceae family [216]. Species from these genera include obligate alkaliphilic (pH 7.8 – 10.0), halophilic (8–30 % NaCl), chemoorganotrophic and obligate aerobic organisms capable of aerobic denitrification. Although many of the microbial species present in saline-sodic soils have not been thoroughly described or isolated in culture, it is clear that this habitat may yield organisms of great value in the remediation of bauxite residues due to the observed capacity of cultured representatives to perform key roles in nutrient cycling and acid production.

3.2.2 Remediated bauxite residues

Despite being a harsh environment for microorganisms, a variety of haloalkalitolerant aerobic and anaerobic bacteria have been isolated from bauxite residues at various stages of remediation, some with the capacity to contribute to remediation goals including pH neutralisation and improved aggregation through biostimulation (addition of nutrients) [27, 31, 151, 170, 171]. Remediation causes significant shifts in microbial community composition, increased diversity and increased biomass [20, 21, 172]. Bauxite residue microbial communities initially resemble those found in other alkaline, saline tailings such as uranium mill tailings and oil sands tailings, as well as salt and soda lakes, dominated by Gammaproteobacteria, Firmicutes, and Ascomycota, and become more diverse during rehabilitation to resemble microbial communities found in forest and grassland soils [21]. The dominance of haloalkalitolerant species decreases during remediation of bauxite residues, but are not entirely lost. Given the demonstrated capacity of native microbial communities to contribute to pH neutralisation and increased aggregation, inoculation of unremediated bauxite residue with remediated bauxite residue communities has

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good potential for supporting successful bioremediation. Direct transfer of microbial communities from remediated to unremediated bauxite residues via inoculation presents an opportunity to ‘jumpstart’ the bioremediation process by accelerating the diversification of the microbial community, introducing a mixed community containing haloalkalitolerant species as well as neutrophilic species, and a variety of functional capacities beyond those found in unremediated bauxite residues.

3.2.3 Native communities from target ecosystems

The goal of remediation activities for bauxite residues is to develop a level of functionality similar to that observed in local natural ecosystems or other desired end land uses. The debate regarding the requirement for restoration to natural conditions versus restoration to a desired level of community functionality is ongoing [173]. However, local landscapes having similar geomorphological and/or chemical characteristics to remediated bauxite residues provide both a suitable reference for benchmarking ecosystem functionality and a potential source of soil microorganisms for bioremediation. For bauxite residue sand, coastal dune systems have been used as natural analogues. Although microbial biomass and diversity of bauxite residue communities increase rapidly (<6 months) to levels equivalent to those in the coastal dunes, community composition and structure remained significantly different [145]. This may reflect the relative importance of stochastic and deterministic microbial community assembly processes during primary succession in bauxite residues. Although the two sites shared similar geographical locations, dispersal may have been limited, preventing the bauxite residue community from recruiting species found in the coastal dunes. Direct inoculation with microbial communities from reference or target ecosystems may assist in achieving a more reproducible outcome during bauxite residue remediation.

3.2.4 Pure cultures

Pure cultures of fungi and bacteria are available as actively growing or freeze dried microbial cultures for a fee from scientific and government organisations around the world. Many of these organisations have websites with searchable databases that allow users to easily determine the availability various species and strains and provide a brief species description. Pure cultures provide the advantage of being assured of the identity of the species used in laboratory determinations of the efficacy of individual species in meeting the remediation goals of surviving in bauxite residues, reducing residue pH and improving the quality of the residues as a medium for plant growth. Larger scale production of organisms for use in remediation is also more efficient where limitations to growth and culture conditions have already been well defined. However, a tailored, mixed community of microorganisms including bacteria and fungi may be technically challenging to design given the complexity of positive (mutualism, commensalism) and negative (parasitism, amensalism, competition) interactions possible [217]. In comparison, mixed cultures from existing alkaline, saline environments may be more successful as the

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assemblages present in these communities have presumably already self-selected for optimal combinations of interactions between species [217].

Based on the criteria outlined in Section 3, candidate bacteria and fungi for bioaugmentation approaches to remediation of bauxite residues have been ranked according to weightings of these criteria as shown in Table 7 and Table 9, based on information available in the literature on their tolerances and functions. Where both greenhouse gases and toxic gases are produced, the organism is scored to zero. These rankings for bacteria and fungi are shown in Table 8 and Table 10. Many of the cells in these tables have been left blank, indicating that information on this criterion for the species or genus was not found in the literature. Where no information was available, the criterion defaults to a zero score in the interests of a precautionary approach. Therefore, these rankings may show some bias towards organisms that have received more thorough examination in the literature to date.





4. Microbial community assembly, succession and interactions at field scale

4.1 Microbial community assembly and succession in extreme, engineered environments

Although the organisms/consortia identified in Section 3.2 are likely to make a positive contribution to bioremediation of bauxite residue on the basis of their physiological tolerances and metabolic capacities, microbial community assembly and successional processes will influence the ultimate success of bioremediation approaches at field scale. The low initial microbial biomass and diversity present in mine wastes and tailings [218-221], including bauxite residue [21, 145], means that they are essentially primary successional environments. Although engineered environments have received little attention to date, principles developed from the study of microbial community assembly in naturally developed primary successional environments such as deglaciated soils (glacial till) [149, 222, 223], volcanic soils [224, 225], and post-fire soils [226] are applicable to understanding and interpreting similar processes in engineered analogues. Conversely, the study of engineered primary successional environments may offer advantages over natural systems in that they provide a well-defined time zero, are likely to present stronger selection pressures than natural environments due to their extreme chemical and physical properties (very low or very high pH, high salinity, very fine or very coarse particle size), and are more likely to be dispersal limited owing to their spatial isolation within industrial settings.

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The assembly of microbial communities is driven by four major processes: dispersal, selection, drift, and diversification [227-229]. These will act in combination with environmental factors to determine the successional trajectory (both phylogenetic and functional) of microbial communities in bioreactors or field-scale tailings storage facilities and therefore the overall success of imposed bioremediation strategies. The rest of this section will focus on bioremediation of field-scale tailings deposits, although many of the general principles are equally applicable to remediation in bioreactors. As mentioned above, selection is likely to exert a stronger pressure on primary succession in engineered primary successional environments compared with natural environments, and dispersal is more likely to be limited. Overlain on assembly processes and environmental factors are differences in physiological characteristics (body size, metabolic capacities and tolerances) between kingdoms, which will also influence primary successional trajectories. Bacteria generally have a smaller body size than Fungi, and are dispersed more readily and consistently at local to global scales, as well as exhibiting a wider array of metabolic capabilities [230]. In the colonisation of natural environments, Bacteria generally show more deterministic assembly patterns whereas fungal communities exhibit relatively stochastic assembly patterns due to the strong influence of dispersal and priority effects [230]. Similar patterns have also been observed previously in the establishment of microbial communities in bauxite residue [21]. Temporal variation in the relative influence of stochastic and deterministic processes has also been proposed in natural systems, with bacterial communities exhibiting a short initial phase dominated by neutral processes (dispersal), followed by a longer phase dominated by niche processes (selection, based on chemical and physical environmental factors), and finally a maturation phase in which niche processes wane in dominance and assembly processes reach a stable equilibrium ([226]; Figure 9). In engineered systems, bacterial communities can be expected to show an overall more deterministic assembly pattern compared with natural systems based on the strong influence of selection based on extreme environmental characteristics, and the relatively minor influence of spatial isolation on bacterial dispersal rates (Figure 9). The initial phase in which dispersal dominates community assembly can also be expected to be shorter given the strength of environmental selection pressures in engineered systems. Fungal communities are likely to have a longer initial phase dominated by stochastic processes compared to bacterial communities, and this is expected to be particularly pronounced in engineered environments where spatial isolation is likely to impact dispersal rates. Both bacterial and fungal communities are likely to take longer to stabilise in more extreme environments and have an equilibrium endpoint more strongly influenced by stochastic processes relative to natural environments (Figure 9) due to changes in environmental properties during initial weathering of mine wastes and tailings (neutralisation of pH, export of salts through leaching) which continuously shift the range of environmental niches available for colonisation, combined with patchy dispersal of microbial propagules from surrounding environments.


4.2 Implications of assembly processes for the design of bioremediation strategies

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Understanding community assembly processes and the influence of environmental, spatial, and temporal factors on community succession is essential in the design of field-scale bioremediation strategies. Given that the initial phase of establishment is likely to be dominated by dispersal, targeting application of inoculants here will overcome potential priority effects that may emerge if inoculants are applied later during succession. Priority effects are a consequence of the order in which microbial propagules arrive and colonise a medium, and can generate different community structures from the same type and amount of inoculants if these are applied in different orders [231]. The identity and functional capacities of initial colonisers in mine wastes and tailings are currently unclear; however, the lack of organic C and other macro- and micronutrients in metalliferous mine wastes and tailings dictates that autotrophic bacterial species (dominated by free-living nitrogen fixers and carbon fixers) will be the first colonisers, as is also observed in similar natural environments [149-232].

Preconditioning of inoculants to the local environmental conditions (high pH, high salinity, high sodicity, low aeration in the case of bauxite residue) prior to application is likely to improve control over the outcome of inoculation [233]. The benefits of preconditioning in predictability of community assembly lends support to a bioremediation strategy based around inoculants derived from remediated bauxite residue, in which species are presumably already adapted to the bauxite residue environment. The timing of inoculant addition also needs to be considered in relation to the physiological tolerances of the constituent species, which may be better suited to a later stage of succession and ecosystem development in which the chemical and physical properties of the tailings are less extreme. Again, selection of an inoculant from remediated bauxite residue or a geochemically analogous natural environment will be less likely to encounter challenges associated with extreme environmental characteristics. Alternatively, manipulation of environmental properties (enhanced leaching and/or tillage to remove alkaline, saline pore water; addition of inorganic and organic amendments as fertilisers and to improve chemical and physical properties [6, 8-18, 21, 22, 31]) may alleviate some of these challenges and accelerate succession and remediation. Fertiliser addition hastened succession in deglaciated soil but did not significantly alter the final community composition [148].

An alternative to applying a single pulse of microbial inoculants is to integrate a multi-staged approach into existing remediation strategies. Remediation of bauxite residues is generally initiated with treatments intended to ameliorate unfavourable physical properties. These include tillage, incorporation of coarse grained material, and irrigation coupled with improved drainage [21]. Haloalkaliphilic N- and C- fixers as well as some fermenters could be applied to the surface layers of fresh, unamended bauxite residue at this early stage to lower pH, improve structure and increase the concentrations of bioavailable organic carbon and nitrogen. In a second stage, haloalkalitolerant microorganisms selected to promote nutrient cycling and availability could be applied in combination with organic amendments such as sewage sludge and inorganic amendments such as gypsum to simultaneously address Na:Ca ratio, improve flocculation, and decrease pH. This would improve physical and chemical conditions for plant growth in bauxite residue as well as reducing fertiliser requirements as remediation continues. Depletion of accumulated nutrient reserves poses a threat to the success and sustainability of bauxite residue

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rehabilitation, and has been observed even in well rehabilitated sites [200]. Ensuring that microbial communities can efficiently mobilise and transform nutrients is essential to avoiding long-term imbalances and managing the risk of rehabilitation failure or requirements for ongoing management. A third consortia of microorganisms, including organisms such as mycorrhiza and rhizobia, can be then applied to the rhizosphere layers to aid establishment and growth of plants through symbiotic relationships that improve nutrient acquisition and tolerance of unfavourable soil conditions [234].

4.3 Comparison of bioaugmentation and biostimulation approaches

Thus far, we have only considered bioaugmentation (the addition of a pre-grown microbial consortium that has been designed to achieve specific management goals) as a strategy for bioremediation of bauxite residue. Biostimulation (providing nutrients and physicochemical environment required to support the growth and activity of the native microbial community (or key members thereof) to achieve specific management goals) is another option for bioremediation (‘Targeted inoculation with key microbial species’, Figure 2). Bioaugmentation is best suited to environments that (a) do not have sufficient competent cells to achieve the remediation bioreactions or (b) where the native population does possess the necessary metabolic capacity to carry out metabolisms useful to remediation such as acid generation, EPS production, or contaminant degradation [235], which is likely to be true for most mine wastes and tailings due to their low initial biomass and diversity. Biostimulation is best suited to instances where the physicochemical properties of the environment are not adequate to support the metabolic processes required to achieve remediation. The outcome of these approaches will be influenced by environmental characteristics and the composition and functions of the native microbial population [235]. Although the environmental characteristics of bauxite residue are well known, the composition and functions of the native initial microbial population are poorly characterised at present. No previous study has compared the merits of biostimulation and/or bioaugmentation for the remediation of bauxite residue; however, addition of a simple organic carbon source and other nutrients was sufficient to stimulate the activity of native microbial communities in both weathered and fresh (initial stages of microbial community succession) bauxite residue [30, 31]. The identity of the microorganisms responsible for driving pH decrease in these experiments was not determined, so it remains unclear whether this strategy would be effective universally for the bioremediation of bauxite residues.

In the management of other wastes and contaminated soils, bioaugmentation is generally more effective than biostimulation, due to higher activity rates and the lack of a lag time required for growth of species responsible for carrying out bioremediation [236, 237]. Combinations of bioaugmentation and biostimulation have also been successful [238-240] although not always demonstrating substantial advantages over bioaugmentation alone. Given the almost universal improvement shown with bioaugmentation approaches, it is likely (given careful selection of the organisms and appropriate trials) that bioaugmentation will further improve upon the results achieved so far with biostimulation for remediation of bauxite residue. Whether bioaugmentation

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is best achieved with a remediated residue, a community derived from an external system, or a pure culture is likely to be a practical decision based on cost and ease of preparation and application as well as reproducibility of outcomes. A combination of biostimulation and bioaugmentation with remediated residues is suggested as the most appropriate model for future research and field trials based on pilot study results and ease of sourcing and applying the inoculum.

5. Conclusions and research needs

Microbially driven strategies for bioremediation of bauxite residue hold great promise for improving the speed and decreasing the cost of remediation. This review considered biochemical pathways by which microbial communities might contribute to remediation; constraints to microbial growth and activity; and sources of inoculants capable of carrying out metabolisms of interest whilst subject to environmental constraints. Finally, the likely effects of microbial community successional processes were evaluated in the light of the characterisations of the environment to be remediation and the microbes which inhabit it. These are basic steps that can be applied to the design of any tailings or contaminant bioremediation strategy. Tailings materials, owing to the harsh chemical and physical industrial processes by which they are generated, are initially almost devoid of microbial life, and can therefore be considered as a primary successional environment. Engineered environments like tailings have been poorly studied in comparison to natural environments; however, we can draw upon settings such as deglaciated till to suggest likely community trajectories and understand the relative importance of dispersal, selection, drift, and diversification during bioremediation. Engineered environments also have much to offer the field of microbial community assembly by providing a well-defined ‘time zero’ and unique environmental (extremely saline, extremely low or extremely high pH) and geographical characteristics (comparatively isolated from natural sources of inoculants) that may facilitate disentanglement of various assembly processes that are more tightly coupled in natural systems.

Neutralisation of pH through organic acid production during fermentation and CO2 production are particularly promising pathways for bioremediation of bauxite residue, requiring potentially no or little addition of substrates, and being carried out by microorganisms known to tolerate physicochemical conditions similar to those present in unamended bauxite residue. Three major aspects of bioremediation must be addressed through experimental studies to inform the final design of a field-scale bioremediation strategy:

(1) identity and functions of initial (pioneer) microbial communities, and primary successional dynamics of these communities;

(2) comparison of potential inoculants (including kinetics of metabolic processes) to support bioaugmentation-based strategies based around pH neutralisation and EPS generation;

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(3) comparison of bioaugmentation, biostimulation, and combined bioaugmentation-biostimulation approaches for the remediation of bauxite residue.

Research needs and the current state of knowledge in these fields are summarised in Table 11 along with a summary of their relevance to design of bioremediation strategies. Although previous studies have demonstrated the potential for biostimulation to rapidly decrease pH in bauxite residue, the challenge remains now to understand the wider spectrum of possibilities for the bioremediation of bauxite residue and develop strategies that will deliver consistent, controllable outcomes to assist in meeting multiple remediation goals. By addressing the research priorities identified in this review, the economic feasibility of implementing bioremediation strategies at field scale may be assessed and then compared with other approaches to improve the financial efficiency of tailings remediation.


Acknowledgements

This research was supported by funding from the International Aluminium Institute and the Natural Sciences and Engineering Research Council of Canada.

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Figure captions

Figure 1. Global bauxite production by country in 2013 [1]. Data for bauxite mine production from the USA during 2013 was not available.

Figure 2. Conceptual model of soil formation and remediation within bauxite residue tailings. Arrows represent positive forcings between processes contributing to soil formation (e.g. rainfall percolation causes leaching of entrained liquor, and removal of entrained liquor favours plant establishment, etc.). Green boxes and arrows represent known links based on studies published to date in bauxite residue; black boxes and arrows represent likely links based on studies published to date in bauxite residue or analogous environments; dotted arrows represent possible links for which there no studies are currently published in bauxite residue. Italicised text represents amendments applied as part of remediation strategies. Dashed box encapsulates processes relevant to microbially-driven bioremediation of bauxite residue. Blue background represents atmosphere; orange background represents bauxite residue. The location of boxes does not necessarily reflect spatial distribution in the environment.

Figure 3. Mechanisms by which organic acid generation may contribute to pH neutralisation in bauxite residue.

Figure 4. Mechanisms by which inorganic acid generation may contribute to pH neutralisation in bauxite residue.

Figure 5. Mechanisms by which CO2 production may contribute to pH neutralisation in bauxite residue.

Figure 6. Mechanisms by which EPS production may contribute to soil formation and remediation goals in bauxite residue.

Figure 7. Mechanisms by which ionic balance may be altered in bauxite residue.

53

Figure 8. Proton and sodium ion influx and efflux from a thermoalkaliphilic aerobe, Bacillus sp. TA2.A1 based on diagrams published by Hicks and co-authors [114]. The diagram illustrates how protons are passed from the F0 domain to the F1 domain of the synthase protein complex and thus imported into the cell, reducing the pH of the cytoplasm and generating cellular energy as ATP. At the same time, symporter proteins import sodium ions with other solutes while antiporter proteins exchange protons for sodium ions. Protons are lost from the cell due to respiration.

Figure 9. Hypothesised relative influence of stochastic and deterministic processes on microbial community assembly during primary succession in natural and engineered environments. Natural environments (glacial till, post-fire soils, volcanic soils) are assumed to be adjacent to well-functioning ecosystems. Engineered environments (mine wastes and tailings) are assumed to be at extremes of pH (very high or very low) and spatially isolated within industrial areas. Adapted from Ferrenberg et al. [226].

Fig. 1

54

Fig. 2

55

Fig. 3

56

Fig. 4

57

Fig. 5

58

Fig. 6

59

Fig. 7

60

Fig. 8

Fig. 9

61

Table 1.Species of fungi and bacteria known to produce organic acids (reference in parentheses) that may promote pH neutralisation in bauxite residue.

Species Acid generated

Metabolism Yield (g acid/g substrate)

Rate (g/L/hr)

Comment

Bacteria Moorellathermoacetica[38] Acetic Anaerobic;

Acetyl Co-A pathway [39]

0.90 0.80 Thermophilic; conducted at pH 6.8

Corynebacteriumhumireducens[37]

Citric Facultatively anaerobic

ND ND Alkaliphilic and halotolerant

Bacillus sp. WL-S20 [40] Lactic Micro-aerobic

0.99 1.04 Alkaliphilic

Corynebacteriumglutamicum[40]

Lactic Facultatively anaerobic

0.87 4.00 Conducted at pH 7.0

Halolactibacillushalophilus[41]

Lactic Anaerobic glycolysis

0.85 1.10 Alkaliphilic and halotolerant

Lactobacillus lactis[40] Lactic Anaerobic glycolysis

0.99 1.90

Bacillus licheniformis[42] Lactic ND 0.86 7.80 Propionibacteriumacidipropionici[42]

Propionic Anaerobic dicarboxylic acid pathway

ND 3.60 Conducted at pH 6.6

Fungi Aspergillusniger[43] Citric Aerobic 0.09 ND Conducted at pH

2.1 Aspergillusniger[40,44] Lactic Aerobic 0.95 3.30 Plant waste

substrate, conducted at pH 5.4

Candida lipolytica[43] Citric Aerobic 1.97 0.03 Conducted at pH 5.5

Candida boidinii[45] Lactic Alcohol fermentation

1.01 1.79 Requires aeration

Rhizopusoryzae[40] Lactic Aerobic 0.81 1.60 Requires aeration Saccharomyces cerevisiae[46]

Lactic Anaerobic fermentation

0.64 3.90 Engineered strain

ND: not determined

Table 2. Selected sulfur oxidising bacteria producing sulfuric acid under alkaline conditions (references in parentheses) and their substrate requirements for neutralising bauxite residue to pH 7, based on a requirement of 0.315 mol H+ kg-1 [32].

Species Substrate Reaction equation

Theoretical yield (g H2SO4/g

Substrate required to neutralise

Active pH

62

substrate) bauxite residue (g/kg)

Thiobacillusthiooxidans [50]

Sulfide (S2-) as FeS2

1 0.41 20.2 < 7.5

Thiobacillusthioparus[53]

Elemental sulfur (S0)

2,3 3.06 5.05 6.0-9.0

Thiobacillusnovellus [52]

Elemental sulfur (S0)

2,3 1.53 5.05 5.0-11.0

Thioalkalimicrobiumspp. [49, 54]

Thiosulfate (S2O3

2-) 4 1.73 1.01 >10.0

Thioalkalivibriospp. [49, 52]

Thiosulfate (S2O3

2-) 5 0.29 3.56 >10.0

Table 3.Observed basal respiration rates from soils in natural and disturbed systems. CO2 production rates have been converted from published units where required.

Soil type

Treatment CO2 production rate (mg CO2-C .kg−1.d-1)

Bauxite residue [66] Control 4.0* Bauxite residue [66] Compost and manure 16.0* Pb/Zn contaminated soil [68] Bare ground 0.1 Pb/Zn contaminated soil [68] Revegetated 5 years 0.2 Pb/Zn contaminated soil [68] Native forest 0.2 Pb/Cu contaminated soil [69] Control 2.4 Pb/Cu contaminated soil [69] Alkaline fly ashes plus peat 4.6 Grassland [70] Unmodified 22.3 Forest soil [71] Natural 23.3 – 148.8 Tropical forest, Malaysia [72] Lightly logged 2781 *: indicates estimated from graph. Table 4.Halophilic bacteria and their reported EPS production yields.

Species EPS productionyield (g/L)

Bacillus licheniformis[87] 0.18 Halomonasventosaev[88] 0.29 Anabaena flos-aquae [80] 0.43 Halomonasanticariensis[88] 0.50 Aphanocapsahalophytia[89] 0.80 Nostoc sp. [90] 0.91 Alteromonashispanica [91] 1.00 Salipigermucosus [92] 1.35 Idiomarinafontislapidosi [91] 1.45 Idiomarinaramblicola [91] 1.50 Halomonaseuryhalina [93] 1.60 Haloferaxmediterranei [94] 3.00

63

Table 5. Summary of biochemical mechanisms by which microbial communities may contribute to remediation targets in bauxite residue, likelihood and conditions favouring occurrence of these mechanisms in bauxite residue, and key research priorities for implementation at field scale. In all cases, identification of key microbial species responsible for these mechanisms (whether in native communities or added in inoculants), their minimum nutrient requirements, and their environmental tolerances remain to be determined.

Biochemical mechanism

Major remediation target

Likelihood of occurrence in bauxite residue

Conditions favouring mechanism

Research priorities

Organic acid production

pH neutralisation

Very likely based on laboratory studies [30, 31]

- Nutrient addition (organic C, N, etc.) will accelerate acid production

- Aeration may improve efficiency of acetic acid production; overall acid production likely favoured by anaerobic conditions

- Quantification of acid production rates and dependence on type/availability of nutrients provided

Production of CO2

pH neutralisation

Very likely based on laboratory studies [30, 31]

- Nutrient addition (organic C, N, etc.) will accelerate acid production

- Aeration may improve CO2 production efficiency

- Quantification of CO2 production rates and dependence on type/availability of nutrients provided

- Evaluation of capacity for biogenic CO2 to neutralise solid phase alkalinity

Inorganic acid production

pH neutralisation

Possible with addition of substrates and modification of environmental conditions based on laboratory studies [12, 16]

- Inorganic substrates (reduced Fe, S compounds) must be added

- Aerated conditions must be provided

- May require adjustment of initial residue pH to more circumneutral values

- Evaluation of maximum pH at which sulfur and iron oxidising bacteria maintain activity and growth

Production of EPS

Aggregation Possible based on laboratory studies [30, 31]

- High pH and salinity, low nutrient conditions in unremediated bauxite residue are

- Understanding how other amendments e.g. addition of organic matter and inorganic fertilisers

64

likely to favour EPS generation as a stress response

interact with EPS generation

- Evaluation of requirements for aeration

Altering ionic balance

Decreased sodicity

Possible based on laboratory studies [106, 107

- Tied to production of (in-) organic acids and CO2, therefore addition of substrates will accelerate alteration of ionic balance

- High initial concentrations of Ca, P bearing minerals in bauxite residue

- Evaluation of timescales required for solubilisation of minerals, and capacity to meet biological nutrient demand

- Evaluation of mechanisms and efficacy of arbuscular/ ectomycorrhizal associations in improving plant tolerance of saline-sodic conditions on a species by species basis

65

Table 6.Typical concentrations of macro- and micronutrients to support microbial and plant growth in unamended bauxite residues. For the purposes of calculations, concentrations below detection limits assumed a zero value.

Nutrient (speciation) Optimum concentration (lower limit or range)

Minimum Average Maximum n References

Macronutrients Total N 0.15-0.5 % wta[158] 0 0.13 0.47 7 [10, 66, 151, 152]N (extractable NH4

+) < 50 mg kg-1 b[159] 0 9.28 16.80 4 [66, 153-155]N (extractable NO3

-) 15-30 mg NO3- kg-1 a[158] 0 0.75 1.00 4 [66, 154-156]

P (available PO43-) 10-25 mg P kg-1 a[158] 0 6.70 11.80 5 [66, 151, 153, 154, 157]

K (exchangeable K+) 0.3-2.0 cmolc kg-1 a[158] 0 0.15 1.00 8 [66, 152, 154, 155, 157]Micronutrients S (extractable SO4

2-) > 30 mg kg-1c[160] - 119 - 1 [157] Ca (exchangeable Ca2+) 5-20 cmolc kg-1 a[158] 0 15.8 83.0 8 [66, 152, 154, 155, 157]Mg (exchangeable Mg2+) 1-8 cmolc kg-1 a[158] 0 0.4 1.2 8 [66, 152, 154, 155, 157]Fe (DTPA extractable Fe3+) > 2.5 mg kg-1 d[161] 2.6 13.0 74.0 8 [66, 143, 153, 154, 156, 157]Mn (DTPA extractable Mn2+) > 1 mg kg-1 d[161] 0 0.3 2.0 9 [10, 66, 143, 154Zn (DTPA extractable Zn2+) > 0.2 mg kg-1 d[161] 0.1 0.3 1.1 8 [66, 143, 154, 156, 157]Cu (DTPA extractable Cu2+) > 0.1 mg kg-1 d[161] 0.2 0.3 0.8 8 [66, 143, 154B (hot water or CaCl2 extractable B)

> 0.5 mg kg-1 e[171] 0 0.1 0.2 4 [66, 143, 157

n: number of samples from which data was collated.

Table 7.Scoring criteria for the selection of candidate bacteria for use in bioaugmentation for the purposes of supporting remediation of bauxite residue.

Characteristic Metric Level Score Motility Non-motile 0

Motile 1 Aerotolerance Aerobic 1

Micro-aerobic 2 Facultatively anaerobic 3 Anaerobic 4

Alkalitolerance Optimum growth pH pH<6.0 0 6.0<pH<8.0 1 8.0<pH<10.0 2 pH>10.0 3

Halotolerance Optimum growth salinity (NaCl %)

<1% 0 <5% 1 <10% 2 >10% 3

Production of extracellular polymeric substances

Type of EPS produced None 0 Polysaccharides 1 Acids 1 Acids and polysaccharides 3

Gas production Type of gas produced Produces a toxic gas (H2S) 0

66

Produces a greenhouse gas (CH4, CO2, N2O, NO2)

1

Produces only benign gases (N2)

2

Toxic byproducts Toxic metal ions 0 Organic biocides 1 No toxic byproducts 2

Pathogenicity Causes disease in plants, animals or humans

Pathogenic 0 Non-pathogenic 1

Nutrient fixation and metal sequestration

None 0 Fixes metals 1 Fixes C,N,P or S 2

Table 8. Scoring for candidate bacterial species based on the selection criteria detailed in Table 7. Species are sorted by overall score (higher score, more promising).

Bacterial species Mot

ility

Aer

otol

eran

ce

Gas

pro

duct

ion

Toxi

c by

prod

uct

Alk

alito

lera

nce

Path

ogen

icity

Hal

otol

eran

ce

Nut

rient

s and

m

etal

s fix

atio

n

EPS

prod

uctio

n

Ove

rall

scor

e

Exiguobacteriummarinum

1 3 2 2 3 1 12

Moorellathermoacetica

0 4 2 1 1 1 2 1 12

Bacillus licheniformis

1 3 1 1 2 2 1 11

Clostridium alkalicellum

0 4 1 2 0 1 2 1 11

Corynebacteriumhumireducens

0 3 3 3 1 10

Pediococcushalophilus

0 3 2 1 3 1 10

Bacillus vedderi 1 3 3 0 2 9 Exiguobacteriumaestuarii

1 3 2 2 1 9

Kocuriarosea 0 3 2 3 1 9 Leuconostocmesenteroides ssp. mesenteroides

0 3 1 0 1 1 3 9

Nesterenkoniaaethiopica

0 1 2 1 2 3 9

Salinicoccusalkaliphilus

0 1 2 1 2 2 1 9

67

Thioalkalivibriodenitrificans

1 3 2 1 2 9

Leuconostoclactis 0 3 0 1 1 3 8 Aeromonasveronii 1 3 1 0 1 1 7 Arcobacterhalophilus

1 4 1 1 7

Halomonasmagadiensis

1 1 0 2 3 7

Kocuria RM1 strain 0 1 3 1 1 0 1 7 Pediococcusacidilactici

0 3 2 1 1 7

Pseudomonas putida 1 1 0 2 3 7 Salinibacteriranicus 0 1 1 1 3 1 7 Salinibacterluteus 0 1 1 1 3 1 7 Salinibacterruber 0 1 1 1 3 1 7 Streptococcus salivarius subsp. Thermophilus

0 3 2 0 1 1 7

Sulfurimonasdenitrificans

3 1 2 0 1 7

Thioalkalimicrobiumsibiricum

2 2 1 2 7

Thioalkalivibrionitratis

1 1 2 1 2 7

Thioalkalivibrioversutus

1 1 2 1 2 7

Acidithiobacillusthiooxidans

1 1 0 1 2 1 6

Lactobacillus brevis 0 3 0 1 0 1 1 6 Lactobacillus plantarum

0 3 1 1 1 6

Leuconostocfallax 0 3 0 1 1 1 6 Nocardiopsisalkaliphila

0 1 3 1 1 6

Nocardiopsishalophila

0 1 1 1 2 1 6

Pseudomonas jessenii

1 1 1 0 3 6

Thioalkalimicrobiumaerophilum

1 2 1 2 6

Weissellaparamesenteroides

0 3 0 2 1 6

Bacillus aurantiacus 0 1 2 2 5 Flavobacteriumanhuiense

1 1 2 1 5

Halomonas desiderata

3 2 0 5

Lactobacillus paracasei ssp.

0 3 1 1 5

68

Table 9.Scoring criteria for the selection of candidate fungal species for use in bioaugmentation for the purposes of supporting remediation of bauxite residue.

Characteristic Metric Level Score Form Yeast 0

Filamentous 1 Aerotolerance Aerobic 1

Micro-aerobic 2 Facultatively anaerobic 3

Alkalitolerance Optimum growth pH pH<6.0 0 6.0<pH<8.0 1 8.0<pH<10.0 2 pH>10.0 3

Halotolerance Optimum growth salinity (NaCl %)

<1% 0 <5% 1 <10% 2 >10% 3

Production of extracellular polymeric substances

Type of EPS produced None 0 Polysaccharides 1 Acids 1 Acids and polysaccharides 3

Gas production Type of gas produced Produces a toxic gas (H2S) 0 Produces a greenhouse gas (CH4, CO2, N2O, NO2)

1

Produces only benign gases (N2)

2

Toxic byproducts Toxic metal ions 0 Organic biocides 1 No toxic byproducts 2

Pathogenicity Causes disease in Pathogenic 0

paracasei Nocardiopsisvalliformis

0 1 3 1 5

Propionibacteriumacidipropionici

0 3 1 0 1 5

Pseudomonas gingeri

1 1 0 3 5

Arcobacter sp. 1 2 0 1 4 Dietzianatronolimnaios

0 1 2 1 4

Starkeyanovella 0 1 2 1 4 Arcobactermarinus 1 1 1 3 Arthrobacterparaffineus

0 1 1 0 1 3

69

plants, animals or humans

Non-pathogenic 1

Nutrient fixation and metal sequestration

None 0 Sequesters metals 1 Fixes C,N,P or S 2

Table 10. Scoring for candidate fungal species based on the selection criteria detailed in Table 9. Species are sorted by overall score (higher score, more promising).

Fungal species Form

Aer

otol

eran

ce

Gas

pro

duct

ion

Toxi

c by

prod

uct

Alk

alito

lera

nce

Path

ogen

icity

Hal

otol

eran

ce

Nut

rient

sand

m

etal

s

EPS

prod

uctio

n

Ove

rall

scor

e

Penicilliumvariabile 1 1 1 3 1 3 1 11 Aspergillusniger 1 1 1 2 1 1 1 2 1 11 Sodiomycesalkalinus 1 1 1 3 3 0 9 Hortaeawerneckii 0 3 1 1 0 3 1 9 Candida versatilis 0 3 1 0 1 3 1 9 Antrodiavaillantii 1 2 1 2 0 3 9 Wallemiasebi 1 1 1 1 0 3 1 8 Aspergillusnidulans 1 1 1 2 2 1 8 Aspergillusflavus 1 1 1 1 0 1 2 1 8 Aspergillusamstelodami 1 1 1 1 1 2 1 8 Antrodiaalbida 1 2 1 1 0 3 8 Wallemiaichthyophaga 1 1 1 3 1 7 Paecilomyceslilacinus 1 1 1 3 0 1 7 Aureobasidiumpullulans 0 1 1 1 3 1 7 Trimmatostromasalinum 0 1 1 1 3 6 Phaetothecatriangularis 0 1 1 1 3 6 Penicilliumoxalicum 1 1 1 1 1 1 6 Fusariumoxysporum 1 1 3 0 1 6 Acremoniumalcalophilum 1 1 1 2 0 1 6 Wallemiamuriae 1 1 1 1 1 5 Penicilliumcitrinum 1 1 1 1 1 0 5 Fusariumbullatum 1 1 3 5 Aspergillus unguis 1 1 1 1 1 5 Aspergillussydowii 1 1 1 0 0 2 5 Aspergillusochraceus 1 1 1 1 1 5 Aspergilluscandidus 1 1 1 2 5 Aspergilluscaespitosus 1 1 1 2 5 Antrodiacinnamomea 1 2 1 0 0 0 1 5 Penicilliumchrysogenum 1 1 1 1 0 4 Cladosporiumsphaerospe 1 1 1 0 1 0 4

70

Table 11. Priority research needs which should be addressed in order to advance bioremediation design for improved management of bauxite residue.

Research need Current status Implications for bioremediation design

Identification and characterisation of physiological tolerances (pH, salinity, trace metals) of pioneer microbial species colonising bauxite residue

Some knowledge based on physiological tolerances of cultured species identified within limited studies of pioneer microbial communities in bauxite residue

Identification of threshold conditions beyond which pioneer microbial community diversity and function are severely compromised

Quantification of amendment volumes required for successful, rapid microbial and plant community establishment and diversification

Identification of where bioremediation fits within overall remediation strategies

Evaluation of microbial community structure and functions in geochemically analogous environments (including remediated bauxite residues) as potential inoculants for bioremediation of bauxite residue

No previous studies in bauxite residue

Determination of appropriate microbial inoculants for use in bioremediation

Comparison of biostimulation and/or bioaugmentation approaches for remediation of bauxite residue

Biostimulation demonstrated as effective for remediation; bioaugmentation unknown



Quantification of rates of nutrient (C, N)

No previous studies in Quantification of amendment volumes (including inorganic fertiliser) required for

rmum Aspergilluspenicillioides 1 1 1 1 4 Aspergillusmelleus 1 1 1 1 4 Aspergillusflavipes 1 1 1 1 4

71

accumulation and solubilisation (Ca, P) in pioneer microbial communities, and residues inoculated with key microbial species responsible for these functions

bauxite residue successful, rapid microbial and plant community establishment and diversification



Evaluation of microbial community dynamics during primary succession in bauxite residue

No previous studies in bauxite residue



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