Acid mine drainage is of growing concern for both developing and developed economies. Thus there is increasing pressure to develop alternative remediation strategies. Biological sulphidogenic mechanisms have long since been studied but, very few have been implemented on a large scale. Limitations are due to the inability to acquire a suitable, low cost, environmentally friendly, renewable carbon source. The present study investigated the use of an algae biomass generated by the HRAOP of an IAPS as a carbon source for the EBRU 00AB/06 SRB consortium. The algae biomass and consortium were utilized together to remediate simulated AMD. Remediation involved decreasing the sulphate and metal concentrations in solution and decreasing the acidity of a simulated AMD. Experiments were carried out to investigate the capability of the EBRU 00AB/06 SRB consortium for sulphate reduction and sulphide generation. The consortium produced colonies when grown under anaerobic conditions in Petri dishes containing modified lactate SRB medium. The SRB consortium reduced the sulphate concentration of modified Postgates medium B and generated sulphide. Further analysis of the EBRU 00AB/06 SRB consortium revealed that the consortium was minimally impacted at pH 5 and by sulphate and iron at 3 g.L-1 and 0.5 g.L-1 respectively. The EBRU 00AB/06 SRB consortium was exposed to Actinomycin D and Ethidium Bromide to determine whether transcription and translation of proteins was required for sulphate reduction. Results indicated that sulphide generation and sulphate reduction were inducible. Analysis of the algae biomass used in this study revealed the empirical formula C1.0H1.91N0.084S0.003O0.36 indicating a carbon source rich in the nutrients required to sustain microbial development. Light microscopy revealed that algae cell walls and in particular those of Pediastrum were susceptible to acid hydrolysis. Dinitrosalicylic acid, Nile red, Bradford and Ninhydrin assays were used to determine the reducing sugar, lipid, protein and amino acid content respectively, of the mixed algae biomass. Results showed that upon exposure of the biomass to simulated AMD at pH 1 and pH 3, the concentration of reducing sugars and amino acids in solution increased. Whereas levels of lipids remained unchanged while the protein concentration decreased, indicating that, upon exposure of algae biomass to AMD, simulated or otherwise, cells ruptured, proteins were hydrolyzed and polysaccharides were broken down to sugars which are immediately available for SRB utilization. Exposure of biomass to simulated AMD revealed further that the presence of algae biomass increased the pH of simulated AMD (pH 3) to pH 7.67 after 4 d. Likewise, the pH of simulated AMD at 1 increased to 1.77 after 2 d while pH of the neutral control increased to 8.1 after 4 d. A direct comparison between lactate and algae biomass revealed 94 % sulphate removal after 23 d in the presence of algae biomass while 82 % sulphate removal was measured in the presence of lactate. Thus the EBRU 00AB/06 SRB consortium successfully utilized algae biomass for sulphate reduction and sulphide generation. In another experiment to establish if the consortium could remediate simulated AMD (pH 5) containing 0.5 g.L-1 iron and 3 g.L-1 sulphate while utilizing an algae biomass as the carbon source no residual iron was detected after 14 d and by day 23, an 89.07 % reduction in sulphate was measured. The results of this investigation are discussed in terms of utilizing a readily available and renewable biomass in the form of microalgae produced in HRAOPs as an effective carbon source in the SRB catalysed remediation of AMD.

published_or_final_version / Environmental Management / Master / Master of Science in Environmental Management

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. 2014. / The aim of this dissertation was to determine the likely impacts that planting woodlands would have on the storage, transport and discharge of mine water and contaminants, over a plume originating from the West Complex tailings storage facility within the Vaal River mine lease. The hydrological model MIKE SHE was run on a grid comprising of 120m square cells, for a pre-woodland period from 2001 to 2010. Sulphate was used as an indicator of the contaminant plume concentrations and transport across the study area. Six future woodland planting scenarios (2025 to 2034) were then simulated to determine the effects of mature Searsia lancea, Eucalyptus dunnii and Tamarix usneoides, and different planting scenarios on the contaminant plume. Results indicated that planting these deep-rooted species will be effective in decreasing the groundwater levels, groundwater flux and the quantity of contaminants reaching the river. Before tree water and contaminant uptake can be further modelled with improved accuracy within MIKE SHE, the limitations of the use of only one contaminant uptake value for the vegetation needs to be overcome, so that different uptake rates among different tree species can be shown.

Dissimilatory sulphate reduction, brought about by the action of sulphate reducing bacteria (SRB) was used in the treatment of acid mine drainage (AMD) in a fluidised bed bioreactor. Biologically produced hydrogen sulphide and bicarbonate ions, by SRB, facilitated the precipitation of heavy metals and the generation of alkalinity in the synthetic acid mine water, respectively. The SRB that had been selected were able to utilize acetate as the sole carbon source and were capable of growing in the bioreactors at low pHs, facilitating an increase in the influent pH from 2.75-7.0 to 5.4-7.8, after a 24-hour hydraulic retention time (HRT). The precipitation efficiencies for Fe, Mn, Zn, Cu, Cr and Al after a HRT of 24 h as metal sulphides ranged between 84- 99% for influent pH values of between 4 and 7, and above 54% for influent pH values between 2.75 and 4. Microbial metabolic activity decreased with decreasing influent pH. This was inferred from the decreasing differences in chemical oxygen demand (COD) depletion rate over a 24 h HRT, as influent acidity levels approached pH 2.75. Molecular studies, using PCR-DGGE analysis on the microbial consortium in the bioreactor, revealed the presence of at least 8 different bacterial species in the consortium. Attempts at sequencing these bands yielded inconclusive results, with the bands showing sequence homology to a large number of previously uncultured and undescribed bacteria. Scanning electron microscopy confirmed the presence of bacteria of different morphology, as well as the presence of biofilms, which account for the heavy metal and low pH tolerances that the bacteria sustained.

A research report submitted to the Faculty of Science, in partial fulfilment of the requirements for the degree of Master of Science, University of the Witwatersrand. Johannesburg, 2016. / Mining and associated anthropogenic activities have improved the livelihoods and economy of many countries but negatively impacted the environment and caused detrimental effects on fresh and ground water systems through the generation of acid mine drainage (AMD). The study evaluated three macrophyte species of P. communis, S.corymbosus and T. capensis for uptake of Mg, P, S, Cr, Mn, Fe, Co, Ni, Cu, Zn, Mo, and Pb in acid mine drainage polluted water. The concentration of elements was also determined in sediments and water so as to calculate the bioconcentration and translocation factors in various parts of the macrophtes. The homogenised plant samples were milled using a Fritsch Pulverisette 6 Mill into pulverized powder and element analysis was done using Spectroscout Geo+ XRF Analyzer Pro. Sediment samples were digested with microwave assisted extraction and analysed by inductively coupled plasma optical emission spectrometry (ICP-OES). Water samples were analysed directly using ICP-OES after filteration with ICP-OES. The concentration levels of these elements in water were compared in all the sites to determine which section of the site (inflow, midflow and outflow) have high amount of the selected elements. The results indicated that elements distribution varied in all the points where the water samples were collected. The concentration level of sediment was compared to the concentration levels of elements in the roots, rhizomes and leaves to determine the translocation and bio concentration factor (TC and BCF). Drinking water quality standards by international organisations were also used as a guideline to compare the concentration levels of elements found in water. Iron (Fe), Nickel (Ni), Manganese (Mn) and Copper (Cu) to determine whether their concentrations in the water were above or below the acceptable levels. the concentrations of Fe, Ni, Mn and Cu were found to be above the international water quality standards for drinking water and their average concentrations was 2230, 282, 5950 and 14080 μg/l respectively. The study found out that in autumn, Mg, S, P, and Mo were highly accumulated by leaves of T. capensis, S. Corymbosus leaves and rhizomes as well as the P. communis leaves and the highest concentrations were 6.61, 72900, 2.00 μg/g respectively. In autumn, Co was the only element highly accumulated by the roots of T. capensis with the highest concentration of 342.80 μg/g. On the other hand, Cr and Fe, was highly accumulated by S. corymbosus roots with the highest concentration of 279.20 and 10.03 μg/g in summer. In summer, Cr, Mn, Ni, Cu, Zn and Pb were highly accumulated by the roots of P. communis and the concentrations were 279.20, 39390, 204.10, 299.50, 813.80 and 47.5 μg/g respectively. The results show that although the plant species accumulated the elements in various concentrations, there was no plant species that accumulated all the selected elements in higher concentrations than the other plant species. They all accumulated a variety of elements in varying amounts and stored them in their different parts. Finally, in all the three plant species analysed, the leaves were the best accumulator of Mg, S, and Mo, whilst the roots were the best accumulators of Cr, Fe, Co, Ni, Cu, Zn and Pb. Since the translocation and bioconcentration factors showed that the macrophyte species accumulated higher concentrations of elements than water and sediments, they can be regarded as hyperaccumulators. Macrophytes species can uptake and accumulate in their different parts various elements and they have the potential to clean the heavy metal polluted sites due to their phytostabilisation and phytoextraction abilities. / LG2017

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Masters of Science, 2017 / The tailoring of zeolites surface properties using organic functionalising agents displaying higher binding affinity for metal ions is a widely explored approach for water treatment. In this study, amine functionalised zeolites and phosphate functionalised zeolites were separately synthesised from similar natural zeolite precursors using reflux methods. The surface composition and morphological elucidations were achieved by characterising the adsorbents using Fourier Transform Infra-red spectroscopy (FTIR), thermogravimetric analysis (TGA), Zeta potential, Point of zero charge (pHPZC), and the Brunauer, Emmett and Teller analysis (BET). In case study 5.1, the sorption mechanisms of the uranyl ion onto amine functionalised zeolites (AMZ), activated carbon (AC) and natural zeolite (NZ) were studied as function of various environmental batch parameters. There was effective adsorption when uranium existed as uranyl ions: UO22+ and UO2OH+. The data fitted numerous kinetic and isotherm models suggesting that the equilibrium mechanisms were characteristic of a combination of chemisorption and physisorption for these three adsorbents. The Dubinin-Radushkevich (DR) model did not fit the data and therefore the energy values derived from it were not used to predict the mechanisms involved. However, the thermodynamic evaluations of parameters ∆H, ΔG and ∆S° showed that equilibrium mechanisms were exothermically, randomly and spontaneously favoured for all adsorbents at temperatures ranging between 22 and 40oC. The adsorption capacity of 0.452 mg g-1 was achieved at pH 3 by 500 mg AC dosage using 20 mL volume of 10 mg L-1 uranyl ion solution after equilibrating for 6 h within the temperature ranges of 22 to 30oC. Under the same conditions of sorbent dosage of 500 mg, uranyl solution volume of 20 mL and 10 mg L-1 U(VI) solution concentration, the maximum adsorption capacity of 0.506 mg g-1 for NZ and 0.480 mg g-1 for AMZ were both achieved at pH 4 after equilibration time of 21 h and 6 h with the optimum temperature range of 22 to 30oC, respectively. The model results predict that intraparticle diffusion thorough pores decreased in the order AC ˃ NZ ˃ AMZ while estimating that chemisorption occurred in a reverse order. On the basis of the modelled data, it was deduced that amine functionalisation of natural zeolites improves their chemisorption capability for uranyl ion and can therefore be used as a cost efficient adsorbent for small-scale remediation of contaminated aquatic systems. In another case study 5.2, the surface properties of successfully prepared aminomethyl phosphonic acid functionalised natural zeolite (APZ) were compared to those of commercial silica polyamine composites (SPC) for uranium uptake in batch aqueous solutions. The FTIR spectrum revealed that (3-aminotrimethyl) phosphonic acid functional groups were successfully grafted onto natural zeolite. The TGA analysis showed that the APZ had higher thermal stability and fewer active sites compared to SPC. The optimum adsorption capacity (qe) of 49 mg g-1 and 44 mg g-1 uranium was achieved using 25 mg SPC and 100 mg APZ, respectively at pH 4, 25oC after 1 and 6 h equilibrating time. The data best fitted the pseudo second-order kinetic model and Freundlich isotherm model. The thermodynamic studies showed that adsorption occurred chemically and exothermically for both APZ and SPC. The overall selectivity order for APZ was; Na ˃ Mn ≥ U ˃ Ca ˃ Fe and for SPC was; Fe ˃ Mn ≥ Ca ˃ U˃ Na. The findings showed that phosphate- and amine-functionalised zeolite bind strongly to uranium compared to the unmodified natural zeolite and other conventional adsorbents such as activated carbon. Their selectivity for this element was commendable. With further improvements in the synthetic protocols e.g. by using microwave-based methods, it should be possible to obtain functionalised zeolite that has superior properties to SPC. / XL2017

Mine-impacted water bodies are considered to be one of the most serious threats to the environment. These can be highly acidic and often contain elevated concentrations of sulfate and soluble metals. The microbial generation of H2S by reduction of more oxidized sulfur species, and consequent precipitation of metal sulfides, known as biosulfidogenesis, is a promising technology for remediating acid mine drainage (AMD). The objective of this work was to develop an integrated system for remediating a target AMD at an operating mine in northern Brazil using a single low pH anaerobic sulfidogenic bioreactor (aSRBR) and an aerobic manganese-oxidizing bioreactor. A synthetic version of the mine water, which contained 7.5 mM copper and lower concentrations (< 0.25 mM) of other transition metals (Zn, Ni, Co and Mn) was used in the experimental work. In the first stage, H2S generated in the aSRBR was delivered to an off-line vessel containing synthetic AMD, which removed > 99% copper (as CuS) while no co-precipitation of other metals was apparent. The partly-processed AMD was then dosed with glycerol and fed into the aSRBR where zinc, nickel and cobalt were precipitated. The effect of varying the pH and temperature of the bioreactor was examined, and > 99% of Ni, Zn and Co were precipitated in the aSRBR when it was maintained at pH 5.0 and 35ºC. The bacterial communities, which were included 4 species of acidophilic sulfate-reducing bacteria, varied in composition depending on how the bioreactor was operated, but were both robust and adaptable, and changes in temperature or pH had only short-term impact on its performance. Manganese was subsequently removed from the partly-remediated synthetic AMD using upflow bioreactors packed with Mn(IV)-coated pebbles from a freshwater stream which contained Mn(II)-oxidizers, such as the bacterium Leptothrix discosphora and a fungal isolate belonging to the order Pleosporales. This caused soluble Mn (II) to be oxidised to Mn (IV) and the precipitation of solid-phase Mn (IV) oxides. Under optimised conditions, over 99% manganese in the processed AMD was removed. Metal sulfides (ZnS, CoS and NiS) that had accumulated in the aSRBR over 2 years of operation were solubilised by oxidative (bio)leaching at low pH. With this, ~ 99% Zn, ~ 98% Ni and ~ 92% Co were re-solubilised, generating a concentrated lixiviant from which metals could be selectively recovered in further downstream processes. The use of methanol and ethanol either alone or in combination with glycerol were evaluated as alternative electron donors for biosulfidogenesis. Methanol was not consumed in the bioreactor, though sulfate reduction was not inhibited in the presence of up to 12 mM methanol. In contrast, ethanol was readily metabolised by the bacterial community and sulfate reduction rates were relatively high compared to glycerol. Two acidophilic algae were characterised and their potential to act as providers of electron donors for biosulfidogenesis was also evaluated. Although algal biomass was able to fuel sulfate reduction in pure cultures of aSRB and in the aSRBR, rates were much lower than when either glycerol or ethanol were used.

Difficulties associated with the study of microbial communities, such as low proportions of cultivable species, have been addressed in recent years with the advent of a range of sequencing technologies and bioinformatic tools. This is enabling previously unexplored communities to be characterised and utilised in a range of biotechnology applications. In this thesis bioinformatic methods were applied to two datasets of biotechnological interest: microbial communities found living with the oil-producing alga Botryococcus braunii and microbial communities in acid mine drainage (AMD). B. braunii is of high interest to the biofuel industry due to its ability to produce high amounts of oils, in the form of hydrocarbons. However, a number of factors, including low growth rates, have prevented its cultivation on an industrial scale. Studies show B. braunii lives in a consortium with numerous bacteria which may influence its growth. This thesis reports both whole genome analysis and 16S rRNA gene sequence analysis to gain a greater understanding of the B. braunii bacterial consortium. Bacteria have been identified, some of which had not previously been documented as living with B. braunii, and evidence is presented for ways in which they may influence growth of the alga, including B-vitamin synthesis and secretion systems. AMD is a worldwide problem, polluting the environment and negatively impacting on human health. This by-product of the mining industry is a problem in the South West of England, where disused metalliferous mines are now a source of AMD. Bioremediation of AMD is an active area of research; sulphur-reducing bacteria and other bacteria which can remove toxic metals from AMD can be utilised for this purpose. Identifying bacteria and archaea that are able to thrive in AMD and which also have these bioremediation properties is therefore of great importance. Metagenomic sequencing has been carried out on the microbial community living in AMD sediment at the Wheal Maid tailings lagoon near Penryn in Cornwall. From these data have been identified a diverse range of bacteria and archaea present at both the sediment surface level and at depth, including microorganisms closely related to taxa reported from metalliferous mines on other continents. Evidence has been found of sulphur-reducing bacteria and of pathways for various other bioremediation-linked processes.

Thesis (M.S.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains xiii, 119 p. : ill. (some col.), col. maps. Includes abstract. Includes bibliographical references (p. 105-110).

<p>Natural and constructed clay liners are routinely used to contain waste and wastewater. The impact of acidic solutions on the geochemistry and mineralogy of clays has been widely investigated in relation to acid mine drainage systems at pH > 1.0. The impact of sulfuric acid leachate characterized by pH < 1.0, including potentially negative pH values on the geochemistry and mineralogy of clays is, however, not clear.</p> <p>To address this deficiency a series of batch and diffusion cell studies, investigating the geochemical and mineralogical impacts of H₂SO₄ solutions (pH 5.0 to -3.0), were conducted on three mineralogically distinct clays (Kc, Km, and BK). Batch testing was conducted at seven pH treatments (5.0, 3.0, 1.0, 0.0, -1.0, -2.0 and -3.0) using standardized sulfuric acid solutions for four exposure periods (14, 90, 180, and 365 d). Aqueous geochemical, XRD, and Si and Al XANES analyses showed: increased dissolution of aluminosilicates with decreasing pH and increasing exposure period; preferential dissolution of aluminosilicate Al-octahedral layers relative to Si-tetrahedral layers; formation of an amorphous silica-like phase that was confined to the surface layer of the altered clay samples at pH ⤠0.0 and t ⥠90 d; and precipitation of anhydrite and a Al-SO₄-rich phase (pH ⤠-1.0, t ⥠90 d).</p> <p>The diffusive transport of H₂SO₄ (pH =1.0, -1.0, and -3.0) through the Kc and Km clays for 216 d was examined using single reservoir, constant concentration, diffusion cells. The diffusive transport of H⁺ within the cells was modeled using 1-D transport models that assumed no absorption, linear absorption, and non-linear absorption of H⁺. The absorption isotherms were calculated from the pH 5.0, 3.0, and 1.0 batch experiment results, which were assumed representative of H⁺ absorption at pH < 1.0. However, model results indicated that the batch test results can not account for the observed H⁺ consumption in all cells and greatly underestimate the amount of H⁺ consumption in the pH -1.0 and -3.0. In the Kc and Km diffusion cells, above-background Ca, Al, Fe, and Si aqueous concentrations were associated with depth intervals characterized by decreased pH values. Respective peak concentrations of 325, 403, 176, 11.7, and 1.38 x 10³ μmol g^-1 (Kc) and 32.4, 426, 199, 7.2, and 1.22 x 10³ μmol g^-1 (Km) were measured in the pH -3.0 cells. XRD results showed that the elevated concentrations corresponded to the loss of carbonates and montmorillonite peaks and decreased peak intensities for illite and kaolinite in depth intervals with pH ⤠1.0, in the Kc and Km pH -1.0 and -3.0 cells.</p> <p>The combined results of these studies indicated that the long-term diffusion of H₂SO₄ through clays at pH < 1.0 will result in a large amount of primary phase dissolution; however, this will be accompanied by precipitation of soluble Ca and Al sulfate salts and amorphous silica, especially at pH ⤠0.0. Additionally, the presence of even a small amount of carbonate will serve to greatly buffer the diffusive transport of H₂SO₄ through clays, even at a source pH of -3.0.</p>