Recently Completed Research Projects
Recently completed research projects at BART:-
- Development and application of a novel biological system for the removal of manganese from contaminated waters
- Bioseparation and recovery of metals from acid mine drainage using acidophilic microorganisms
- Microbiology of mineral biomining heaps
- Biogeochemistry and biodiversity of natural and constructed wetlands receiving acidic, metalliferous mine drainage
- Microbial life in anoxic acidic environments
- Bioremediation of acidic mine waters by sulphate reduction in novel, compost-based, field-scale bioreactors
- Bio-processing of copper ores and concentrates
- Construction and application of acidophilic bacterial populations for targeting mineral processing
- Bioremediation of acid mine drainage in constructed wetland ecosystems
(Barrie Johnson, Kevin Hallberg, Kathryn Wakeman, Owen Rowe, Ellie Jameson)
The recovery of metals from sulfide ores by microbes (“biomining”) is a growing industry worldwide. The European Union Framework 6-supported BioMinE project (http://biomine.brgm.fr) aims to provide a further understanding of biomining, from fundamental processes to engineering of bioreactors to improve metal recovery. Emphasis is placed on ores from significant deposits in Europe that contain valuable metals such as copper, zinc, silver and gold.
As part of the BioMinE project, BART is studying fundamentals of microbial leaching of metals from ores and concentrates, including rational culture design for the targeted release of metals from complex ores. In addition, new acidophilic microbes that can play a role in metal leaching are being evaluated.
Another aspect of research carried out by BART is focused on the selective recovery of metals from leach solutions using acidophilic sulfate-reducing bacteria (SRB). A goal of this research is to gain a greater understanding of the kinetics of sulfate reduction by the SRB and metal removal rates, leading to the development of a pilot plant for selective metal recovery. In addition, BART is searching for new acidophilic SRB that will expand the possibilities of metal recovery from various biomining activities.
The BioMinE project began in 2004 and finished at the end of 2008.
Development and application of a novel biological system for the removal of manganese from contaminated waters
Soluble manganese, principally Mn(II), in surface and ground waters, and in domestic water supplies, can cause nuisance and more severe (toxicity) problems if present in high concentrations. Regulatory authorities are increasingly focusing on manganese as a measure of metal-contamination of natural water courses, and the European Union has imposed an Environmental Quality Standard (EQS) for Mn(II) of 30
The aim of this research project is to develop self-sustaining, low-cost bioreactors that can be used in situ for passive removal of manganese from contaminated water courses of pH >5. Using a variety of techniques, these systems will be studied for their biological component, mineralogy, chemistry and engineering in order to produce such systems for the remediation of Mn(II) from waters of varying chemistry.
(NERC CASE studentship, with support from Rio Tinto plc).
(Barrie Johnson, Kevin Hallberg, Kathryn Wakeman)
BIOSHALE (http://bioshale.brgm.fr/) is a Specific Targeted Research Project of the 6th European Union Framework Programme on Research and Development (Integrating and Strengthening the European Research Area). The project aims at evaluating biotechnology for safe, clean and viable beneficiation of black shale ores in Europe and to propose an innovative, environmentally and socially favourable model of mining activities and metals recovery. European deposits of black shale ores contain considerable reserves of base and highly valuable rare and precious metals (Cu, Ni, Zn, Ag, Co, Au, Pt, Pd, etc.), of which Europe is the main consumer in the world.
Two major difficulties restrict the exploitation of such abundant resources. The first is the low efficiency of the conventional technology for recovering valuable metals, from mining extraction to metallurgical processing. The second is the environmental impact of conventional metal recovery techniques. The success of biohydrometallurgy (microbial metal recovery) lies in the efficiency of the process with a reduced impact on the environment compared to conventional techniques.
As part of the BIOSHALE project, BART is assessing the ability of acidophilic microbes to release metals from the ore material as well as the role of microbiology on the environmental impact of black shale mine wastes. Work has included the characterisation of microbial populations at black shale mine sites (including isolation of indigenous microorganisms), evaluation of mixed populations of acidophiles on the leaching of base metals from black shales, and study of microbial populations of pilot scale operations (110 ton pilot tower and 17,000 ton demonstration heap).
The BIOSHALE project started in 2004 and finished at the end of 2007.
The overall aim of this project is to utilize acidophilic microorganisms (including novel species and strains, isolated as part of the project) in the development of low-cost systems for bioremediating mine waters and the selective recovery of metals from contaminated water courses. The project involves a variety of laboratory and field based work, focussed mainly on Mynydd Parys (Anglesey, North Wales, UK) and the monitoring of water chemistry of the acid mine drainage (AMD) impacted stream Afon Goch (the Red river) draining the lowest level of the currently abandoned Parys mine. This site, once the largest copper mine in the world,
Isolation of sulfate reducing bacteria (SRB), in addition to a series of experiments examining both in situ and ex situ bacterially-catalysed oxidation of iron, has been carried out based on this site. This work has led to the testing in situ of a small-scale pilot scheme. Other sites in the UK, Spain and Portugal have also been examined using a combination of microbiological and biomolecular techniques, and chemical analysis.
The information from this research project will facilitate the development of a two-component bioremediation/biorecovery system that would selectively separate some of the more valuable metals from mixed metal wastewaters, such as AMD, catalysed by the biological processes of sulfate reduction and iron oxidation. The selective recovery of base metals from polluted streams will facilitate their recycling, and will contribute to the cost-effectiveness of the remediation system.
(NERC CASE studentship, with support from Paques bv, The Netherlands).
Some bacteria and archaea are able to accelerate the oxidative dissolution of metal sulfide minerals. Their activities are of considerable importance in the areas of both environmental and applied microbiology. They are responsible for one of most serious forms of environmental pollution (“acid mine drainage”), but are also used to extract metals from low-grade or difficult to treat ores (“biomining”).
A combination of direct isolation and enumeration on selective overlaid solid media and biomolecular techniques (using methods developed at the University of Wales, Bangor and elsewhere) are being used in the project to assess microbial biodiversity and heterogeneity in mineral leaching heaps in the U.K. and overseas. The data obtained will make a significant contribution to our understanding of how commercial mineral heap biomining functions, and how rates of metal extraction may be controlled to prevent acid mine drainage.
(NERC CASE studentship, with support from Rio Tinto plc).
Biogeochemistry and biodiversity of natural and constructed wetlands receiving acidic, metalliferous mine drainage
Background: Acid mine drainage (AMD) results of the oxidative dissolution of sulfide minerals, most notably pyrite (FeS2). Waters draining mines and mine spoils are often highly acidic (pH <3) and enriched with metals (iron, aluminium and various heavy metals) and sulfate.
The oxidation of sulfide minerals may occur abiotically, though the rate of oxidation is greatly enhanced (by a factor of up to 106) in the presence of certain microorganisms. Some sulfide minerals (e.g. sphalerite, ZnS) oxidise in aerated acidic liquors, whilst others (such as pyrite) are relatively stable in such conditions, but are oxidised by ferric iron (Fe3+). The microorganisms which accelerate the oxidative dissolution of sulfide minerals are therefore those acidophiles (due to the necessity of an acidic milieu) which oxidise ferrous iron (thereby re-generating ferric iron) and/or reduced inorganic sulfur compounds (producing sulfuric acid).
These lithotrophic ("rock eating") bacteria form only a part of the microbial community within AMD streams. Other microorganisms include heterotrophic (organic carbon-utilising) bacteria, some of which are able to reduce either ferric iron or sulfate, i.e. essentially reversing the reactions of pyrite oxidation. These latter microbes have the potential for remediating AMD pollution as they (i) generate alkalinity; e.g. Fe(OH)3 + e- --> Fe2+ + 3OH- and (ii) cause a variety of heavy metals to be precipitated (and thereby removed from solution) as highly insoluble sulfides (e.g. Cu2+ + HS- --> CuS + H+). Acidophiles which carry out iron and sulfate reduction are obligate or facultative anaerobes, and would be predicted to inhabit the more anoxic zones in AMD-impacted ecosystems.
At the abandoned Parys copper mine, Amlwch, NE Anglesey, active mining ceased over 150 years ago and AMD has been discharging into the surrounding streams and from these to the sea since then. AMD flows from several mine adits, and in some areas transverses an area of natural wetland (of approximately 40m2). Preliminary investigations have indicated that AMD undergoes significant modification when it passes through one of these wetland sites, as indicated by an increase in pH (from about 2.5 to 2.7) and lowering (by about 50%) in soluble iron. These changes indicate that alkali-generating and metal-precipitating microorganisms occur within the wetland.
Project aims: This project is quantifying the biogeochemical transformations of Fe, S and C (and fluxes of heavy metals and arsenic) through the wetland, and to isolate and characterise the indigenous microflora, using a combination of cultivation and molecular techniques. There are three main areas of research being undertaken:
(i) construction of a working hypothesis of biogeochemical transformations and fluxes within and through the wetland, using analytical data collected over a two-year period. This involves studying different ionic species of the significant elements, as well as various environmental parameters (pH, Eh, Dissolved Oxygen), to make predictions on the mobilisation/immobilisation of heavy metals, arsenic etc.
(ii) analysis of indigenous microflora, with an aim to characterise those involved in Fe/S oxido-reduction. This dual approach involves acidophilic cultivation techniques alongside the use of biomolecular techniques (FISH, T-RFLP, gene libraries). Isolates are being characterised by comparison of 16S rRNA genes with those of sequenced microbes.
(iii) Studying intact core samples from the Parys wetland, to measure Fe/S/C fluxes under controlled conditions.
(NERC CASE studentship, with support from Rio Tinto plc).
This research project is focusing on the biodiversity and metabolisms of acidophilic microorganisms that live in acidic, metal-rich wastewaters, such as acid mine drainage (AMD). Very little is known about this area of microbiology, although work at Bangor and elsewhere has shown previously that some acidophiles can use ferric iron as a terminal electron acceptor to support their growth in anoxic liquors.
The project has two main areas of research. In the first, we are examining the biodiversity of macroscopic “acid streamer” growths found in an underground cave in Trefriw, north Wales. Using a combination of classical microbiology (cultivation techniques) and molecular biology (construction of clone libraries, terminal restriction fragment length polymorphism (T-RFLP) and fluorescent in situ hydribisation (FISH)) we are discovering that these slime-like growths are composed of a diverse community of bacteria, some of which have not previously been cultivated or observed in acidic waters. Later in the project, microbial diversity of acid streamers in the nearby Cae Coch pyrite mine (where microbial growths are estimated to exceed 100 m3) will be examined.
The second major area is following up on work began by a previous doctoral graduate from BART (Anna Sen; see past researchers) in which we are examining the phenomenon of bacterial sulfidogenesis (reduction of sulfate to sulphide) at low pH. A mixed community of acid-tolerant sulfate reducing bacteria (SRB) and other acidophiles (that appear to grow syntrophically with the SRB) is currently being grown at pH 4 in a bioreactor, where it is being used to selectively remove zinc from a metal-rich synthetic mine water. This is the first demonstration of a sulfidogenic system operating at acidic pH values, and has considerable potential for metal recovery and decontamination of metal-rich effluents.
Bioremediation of acidic mine waters by sulphate reduction in novel, compost-based, field-scale bioreactors: The ASURE Project
(Kevin Hallberg, Barrie Johnson)
Surface water pollution by acidic, metalliferous drainage (commonly known as acid mine drainage or simply AMD) from abandoned mine sites is a widespread problem in former mining areas of the UK and many other countries world-wide. Latest estimates suggest more than 600 km of river reach in the UK, and more than 3000 km in Europe as a whole, are degraded by abandoned mine drainage. Direct public expenditure on mine water remediation in the UK currently exceeds
The most promising low-cost treatment methods developed to date are the “passive treatment techniques” which principally involve the construction of wetland ecosystems with characteristics favouring water quality improvement. The use of aerobic reed-beds to treat alkaline mine waters contaminated only with Fe is now well-established and is reasonably well understood in scientific terms. For the more acidic mine waters, which typically contain a wider range of contaminant metals, compost-based passive treatment systems are prescribed. These are intended to promote alkalinity generation and metal sulphide precipitation by means of microbial sulphate- and / or iron-reduction processes. Recent monitoring of such “compost wetlands” both by our own group and others has shown that these microbial reduction-based passive systems do quite effectively lower the concentrations of iron and aluminium in mine waters, whilst increasing their alkalinity and pH.
However, very important questions remain about the nature and long-term efficacy of the biogeochemical processes occurring within such systems. Key questions include the following: (1) which process(es) control(s) the raising of pH?; (2) what are the key “sink” processes for pollutant metals?; and (3) what role does the depletion of organic carbon sources play in the dynamics and life-span of a compost wetland system? The uncertainties represented by questions such as these appear to be acting as a disincentive to the wider uptake of reduction-based passive treatment systems.
By means of collaborative research with geochemists and civil engineers at the University of Newcastle upon Tyne and ourselves along with both problem owners (Scottish Coal, Rio Tinto) and consultants / contractors in the field of abandoned mine site reclamation (IMC Ltd, Parkhill Estates Ltd), we undertook fundamental research into the biogeochemical processes that occur in reduction-based passive treatment systems receiving acidic, metal-rich waters. The findings of the process-level studies will be contextualised for engineering practice by means of characterisation and modelling of treatment system performance, drawing upon extensive water quality databases and pre-existing modelling codes.
BART focused on the characterisation of indigenous microorganisms in the study systems using a combination of isolation/identification and biomolecular approaches. Variations within stratified sediments and temporal changes in biomass size, composition and activity were also monitored, in conjunction with the biogeochemical studies to be carried out by our collaborators. It is our aim that these results will provide the crucial information on microbial community membership and dynamics, against which the biogeochemical data can be interpreted. This will provide a rational basis for estimating the nature and rates of organic carbon transformation and utilisation in the long-term, and the catalytic effects of microbes on key Fe and S redox reactions. This is essential information upon which quantitative process-based modelling of treatment performance will be based.
This work has been carried out under the LINK Biorem 4 project “ASURE”, supported through the BBSRC (grant # 5/BRM18412) and with support from the industrial partners Rio Tinto Technology, The Scottish Coal Company Ltd., White Young Green Ltd. and Parkhill Estates Ltd. We also acknowledge the contributions made by our colleagues at the University of Newcastle to the ASURE Project.
(Bethan Stallwood, Barrie Johnson)
(This is a classified research project and further details cannot be disclosed.)
‘‘Biomining’’ is a form of mining (mineral processing) that utilises microorganisms to degrade metal sulfides for the enhanced recovery of metals with economic value. Biomining has developed into one of the most successful and important areas of biotechnology; the estimated 1999 global value of the process was about $10 billion. There are many advantages to using bioleaching for the extraction of metals in terms of cost-efficiency, simplicity, robustness, high performance and environmentally friendly alternative to conventional mineral processing methods.
My project involved the construction of logically-designed bioleaching populations to specifically target ores and minerals of commercial importance and tested the efficiencies of these systems (relative to ‘conventional’ microflora) using a combination of molecular and non-molecular approaches.
1) Bioleaching of pyrite by defined mixed cultures of moderately thermophilic acidophiles
Leaching of pyrite (FeS2) concentrate and ground rock pyrite has been investigated using defined pure cultures and consortia of four moderately thermophilic bacteria: (i) a thermotolerant Leptospirillum ferriphilum isolate (strain MT6); (ii) Acidithiobacillus caldus (strain KU); (iii) a novel Gram-positive bacterium `Caldibacillus ferrivorus' (strain GSM); (iv) a Sulfobacillus acidophilus isolate (strain NC). Parameters measured included total iron released from pyrite, Fe2+ and Fe3+ concentrations, dissolved organic carbon, pH, Eh and numbers of different bacterial species. Pure cultures of both strain MT6 and strain KU did not accelerate the pyrite concentrate dissolution, while both strain GSM and strain NC were able to do so, albeit at relatively slow rates and at low redox potentials. The most effective dissolution of pyrite was observed in mixed cultures that included strain MT6, all of which maintained high redox potentials. The data indicate that strain MT6 was the most significant in the consortia and that At. caldus, although active in generating acidity and numerically the dominant acidophile present in mixed cultures, contributed nothing either directly or indirectly to pyrite oxidation.
2) Exploitation of important iron-metabolising microorganisms and development of RFLP method for their differentiation
Research on the biooxidation of sulfide minerals has tended to be heavily biased towards Gram-negative bacteria, such as Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans. However, the research team at the UWB has been finding significant biodiversity which has potential important role in biomining. We have isolated and characterised a number of phylogenetically distinct Gram-positive iron-metabolising bacteria, some of which are novel genus. Also, we have isolated gram-negative bacteria, such as L. ferrooxidans and At. ferrooxidans, which, in contrast to other recognised species, have unique characteristics. Development of a rapid, simple and convenient method to differentiate such microbes would have significant importance in the quick examination of biodiversity in industrial samples. To this end, a RFLP (Restriction Fragment Length Polymorphism) protocol was developed.
(Kevin Hallberg, Barrie Johnson)
The problem of acidic minewater discharges (also known as acid mine drainage or "AMD"), which arise from weathering of sulfidic minerals as abandoned mines fill with water, is becoming increasingly prominent in the U.K. and beyond. AMD is characterised by high acidity and by high concentrations of iron and other toxic metals. Following a pollution event in 1992, caused by AMD leaking from the former Wheal Jane tin mine in Cornwall, long term remediation measures were developed and included the construction of an experimental passive treatment system, using constructed wetlands, to clean-up AMD before discharge to surface water. A Department of Trade and Industry ("DTI") LINK sponsored research consortium investigated the processes involved in the remediation of AMD by the treatment system. The consortium included BART and groups from the University of Reading, Imperial College London, Centre for Ecology and Hydrology and Camborne School of Mines.
BART’s research programme at the Wheal Jane site included assessing microbial population sizes and biodiversity in the source AMD and in the aerobic reed beds. This includes estimating total numbers of indigenous microflora and identifying isolates, where possible, to the genus/species level; examining seasonal fluctuations of microbial populations; and investigating any correlation with measured physicochemical parameters (metal loadings etc.). We used well-established solid media (e.g. D. B. Johnson, Journal of Microbiological Methods, 1995, vol. 23, pp. 205-218) for enumeration of acidophiles. We also developed novel solid media to apply to this type of ecosystem that has not been subject to such a microbial study before. The data obtained on the cultivatable population will be compared with direct counts of microbes obtained using molecular techniques, such as in situ hybridisation studies using the 16S rRNA gene as a marker. Preliminary data indicate that the predominant cultivatable microbes in the AMD and in the aerobic reed beds are previously undescribed moderately acidophilic iron-oxidising bacteria.
Solid media showing some moderately acidophilic iron-oxidising microbes (the orange or "rusty" colonies) isolated from the Wheal Jane treatment plant. Similar iron-oxidisers have been isolated from AMD of mild acidity (pH 4-5) at several sites in the U.K.
These microbes grow at a pH of about 4 and outnumber the more familiar extremely acidophilic iron oxidisers, such as Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans, by at least an order of magnitude in both water and sediment samples. These and a phylogenetically distinct group of moderately acidophilic iron-oxidisers are also found in AMD from other such sites around the U.K, including a wetland constructed for the treatment of iron-rich effluent from a coal mine at Ynysarwed, south Wales.
A variety of heterotrophic acidophiles have also been isolated from the reed beds. Many exhibited the typical colony morphologies of the well known acidophilic heterotrophs that belong to the genera Acidocella and Acidiphilium, while others belong to quite different genera.
Diversity of heterotrophic acidophiles found at the Wheal Jane treatment plant is indicated by the various colonies shown here. Note the differences in colours, shapes and sizes.
Since quantitative removal of iron is a key objective of the aerobic cells and the success of this process has considerable impact on the potential environmental impact of mine discharge waters, laboratory experiments were carried out to investigate the iron oxidation kinetics with these new moderately acidophilic iron-oxidisers. Such data will be used to compare with the iron oxidation and removal rates observed at the pilot plant to assess which microbes are important in the process. This will provide key information on operation (and future design) of such treatment systems to promote efficient iron removal.
We also investigated the ferric iron reduction potential of the heterotrophic acidophiles found in the aerobic cells as this will also have an impact on the iron removal efficiency of the treatment system. Other work carried out at Bangor included an examination of microbiological and chemical compositions of the effluents that drain the pilot plant, with a view to predicting their environmental impact. Initial analyses indicate that significant quantities of ferrous iron are present in these waters, and that large numbers of iron-oxidising acidophiles survive the anaerobic cells.
This combination could be detrimental to the environment as subsequent oxidation and hydrolysis of iron would result in a net increase of acidity of water draining these cells. This, in turn, will have significant impact on the performance of the "algal ponds", where the main objective is to increase water pH to the level (> pH 8) at which manganese will be precipitated, and therefore on the overall performance of the passive treatment plant. Further laboratory experiments will examine this potential problem. BART personnel involved directly in this project included: Drs Barrie Johnson and Kevin Hallberg and Mr Stewart Rolfe.
This work was supported by the LINK directorate (Grants # BTL/70/21 and 5/BRM18412). The final report for this project is available for reading and a further details of the research carried out can be found in a special issue of Science and the Total Environment.