Plenary Lecture

15th International Biohydrometallurgy Symposium (IBS 2003) September 14-19, Athens, Hellas

"Biohydrometallurgy: a sustainable technology in evolution"

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Biohydrometallurgy: a sustainable technology in evolution

Giovanni Rossi

Dipartimento di Geoingegneria e Tecnologie Ambientali – Università Piazza d’Armi, 19 – 09123 Cagliari, Italia

Abstract In the mid to late 1990’s biohydrometallurgy used to be considered an innovative

technology, but one can hardly continue to describe it as "innovative" today. Since the advent of biohydrometallurgical processing many major breakthroughs have been achieved and this economically profitable and sustainable technology now finds wide application in a variety of spheres, ranging from metal extraction to environmental remediation. Consequently, now that the pioneering days are over, what is required is a concerted effort by researchers to rationalize and optimize biohydrometallurgical processes.

Three main areas of application can be identified: (i) environmental protection; (ii) metal extraction from minerals and rocks: (iii) pre-treatment of minerals to make them amenable to further processing.

The fundamentals of biohydrometallurgy draw on a variety of disciplines, ranging from minerals engineering and mineralogy to microbiology, physical chemistry (with strong emphasis on surface science, colloid chemistry and electrochemistry) and solid-state physics. Researchers in all these fields have provided an equally important contribution to the development of this technology and this presentation endeavours to review their achievements and to briefly discuss those issues that remain open. Some of these issues continue to arouse controversy, stimulating the interest of scientists, which can also benefit other fields of science and technology.

Based on his experience, the author would like to emphasize the need to establish a Biohydrometallurgical Society, to act as a point of reference to all those, from industry and academia, who are involved in implementing and further developing the technology. The Society should also provide a forum for information flow to decision makers in industry, about the potentials of biohydrometallurgy. Though it relies on the exploitation of the complex synergies between microoorganisms and minerals, this technology, when properly applied, is simple to implement, operationally stable and cost-effective.

Finally, the author would like to invite academia to develop suitable curricula to ensure that new generations gain a specific working knowledge of biohydrometallurgy and industry to increase their funding for higher education and for private and academic research.

1. INTRODUCTION In this review I will describe the evolution of a technology, biohydrometallurgy,

which in my opinion offers promising prospects and can be highly rewarding for all those

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involved in providing humankind with the mineral resources indispensable for its progress with the minimum harm to the environment.

Of course, I will focus most of my considerations on the engineering aspects of biohydrometallurgy and related physical-chemistry, solid state physics and earth sciences implications for two reasons with which I hope you will concur: first, in three recent excellent papers [1-3], all the most important aspects of microbiology applied to Biohydrometallurgy have been exhaustively reviewed in such a masterly way that there is really nothing significant to add at the present time; second, the degree of maturity attained by biohydrometallurgy as a new technology is such that its engineering developments and problems warrant attention.

I would also like to point out that I have restricted the references to those necessary to justify some of my statements and that I have omitted many excellent contributions simply because otherwise this talk would have been more of a reference list than a presentation. In effect, on bioleaching kinetics alone I keep more than fifty papers in my files all of which deserve attention and mention in a paper concerning that topic.

The Venn diagram shown in Figure 1 provides a visual representation of the interconnections among the various branches of science from which the fundamentals of biohydrometallurgy are derived.

The engineering aspects are of paramount importance for commercial applications. In this regard three main groups of processes need to be distinguished: those concerned with metal extraction from rocks, those for mineral upgrading and environmental protection processes.

For the sake of clarity these three groups will be examined separately.

Figure 1. Venn-Euler diagram showing biohydrometallurgy "parenthood"

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2. ENVIRONMENTAL PROTECTION PROCESSES As is well known, the origins of biohydrometallurgy can be traced to the

environmental problem created by the pollution of the Ohio River due to acid drainage of coalmines located in the river basin.

Environmental protection continues to be one of the main areas of application of biohydrometallurgy and concerns either the development of remediation techniques aimed at low cost inhibition of dangerous effluents from old stopes or dumps or of systems for trapping toxic ions from effluents or for decontaminating polluted soils. Investigations into the properties of cell envelopes as adsorbents, of the so-called microbial derivatives [4,5], the proposed ingenious mathematical adsorption models, analysis of the factors influencing the processes, and the encouraging commercial applications [5] have opened new avenues and some promising research results have also been published recently [6,7] The interactions between microbes and solid surfaces play a very important role and three papers [8-10] on the fundamentals of this subject provide a useful background for those intending to advance in this field.

The commercial applications rely on the knowledge of the mechanisms and the extent of adsorption of chemical elements or compounds by microorganisms. Providing the required information to the practitioners is the basic task of microbiology.

2.1 The biological fundamentals Organisms and microorganisms play the role of ion traps and, to some extent, can be

considered the biological equivalents of inorganic exchange resins. This branch of biohydrometallurgy involves not only microorganisms but, more generally, all living things especially plants and algae. This distinctive feature already emerged at the time of what can be considered the First Symposium on Biohydrometallurgy, held in Braunschweig: on that occasion, the properties of the alga Hormidium fluitans (Gay) were described [11].

This is a broad field of research, as the ample literature published to date demonstrates. As pointed out by two recent reviews [5,12,13], the technology is promising but does not yet meet, for a number of reasons, the prerequisites for becoming a widespread cost-effective commercial application. For the time being the prospects of its application as a process in its own right seem to be limited. However, because of its characteristic feature of rapid intrinsic kinetics it is envisaged that biohydrometallurgy will be successfully integrated into water purification flowsheets consisting of hybrid technologies. For this type of technology the distinction between intra-biotechnological (IBT) or inter technological (IT) [12] depending on the type of associated processes can be helpful. The term IBT refers to biosorption, bioreduction or bioprecipitation, IT to biotechnology-based processes integrated with non-biotechnology based ones such as chemical precipitation, electrochemical processes, etc.

3. BIOHYDROMETALLURGY AS A DEVELOPMENT OF EXTRACTIVE METALLURGY From its origins, in the 1940’s, up to the 1980’s, most biohydrometallurgical research

work focussed on the ambitious target of developing an environmentally and cost-effective process that could compete with pyrometallurgy-based processes for metal extraction from ores and concentrates.

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All of us old-timers certainly recall the enthusiasm with which we carried out our investigations and the encouraging results achieved by the first in-situ and dump leaching operations. Dare I say that almost all mining or metallurgical engineers involved in biohydrometallurgical research tested the process whenever an abandoned copper mine was available. The encouraging results obtained in several mining operations in the late 1970’s, that are still being confirmed today by the latest developments, like the Quebrada Blanca Mine [14] and the relatively low cost of the equipment required, certainly justify the impatience of several of us to start working on concentrate bioleaching. In effect, the results were quite frustrating. I still recall the disappointment after several months of very hard work in a well-equipped laboratory in Northern Italy. There, in 1977, in great secrecy, an Italian mining company entrusted an international team, of which I was part, with the task of developing a chalcopyrite flotation concentrate bioleaching flowsheet. We never succeeded in obtaining a 95% copper leaching in a single STR operated in batch. It was only two decades later that I began to understand the reasons for our failure, which depended simply on our ignorance of certain aspects of solid state physics typical of chalcopyrite.

Subsequent research carried out in the light of the contributions of solid-state physicists and of new investigation methods like XPS, demonstrated the great potential of biohydrometallurgy.

3.1 Pre-treatment of run-of-mine ores or flotation concentrates and recovery of valuable metals The processes belonging to this group share the same type of problems and for this

reason I will treat them together. The technology of this branch of biohydrometallurgy has already found successful application in cost-effective commercial operations.

As summed up in the block diagram of Figure 2, their profitability depends on a series of interrelated factors whose investigation covers a wide range of basic sciences and of technological applications that had yet to be fully explored in the 1970’s.

At this juncture I would like to stress the point that I purposely avoided the distinction between "pure" and "applied" sciences, as authoritatively stated more than a century ago by Pasteur [15]: ......No, a thousand times no, there does not exist a category of science to which one can give the name applied science. There are science and applications of science bound together as the fruit of the tree which bears it .....

The most encouraging commercial successes have been achieved in the pre-treatment of gold-bearing complex sulphide ores - notoriously refractory to conventional processing - for the subsequent cyanidation step. The excellent performance of numerous commercial plants is well documented [16,17]. Table 1 provides a summary of these achievements. It is no exaggeration to say that much of the recent research that has contributed to elucidating a great many problems posed by this technology has culminated in these successes.

Research efforts directed to coal desulphurisation have produced some interesting results. A semi-commercial pilot plant, the first of its kind, jointly designed, built and operated in partnership between four European research groups in the framework of a project funded by the Commission of European Communities, demonstrated the practical

Table 1. Operating parameters of some commercial bioleaching operations

Plant and location Ore minerals Reactor type

% Solids concentration

Total useful bioreactor volume, m3

Daily throughput per bioreactor unit useful volume, tonn/m3.day

Residence time, hours Reference

Fairview South Africa

P, A STR 20 90 0.444 96 [16]

Sao Bento Brazil

A, P, Pr STR 20 580 0.138 21 [16,17]

Olympia Greece

Complex Cu, Fe, As, Zn, Pb sulphides

STR 20 15,936 (3 moduli of 4 1,328 m3

each STR’s)

0.048 96 [16,18]

Amantaytau Uzbechistan

STR 20 23,376 (4 moduli of 6,974 m3

each STR’s)

0.047 96 [17]

Wiluna Australia

P, A, Stb STR 20 6x470 = 2,820 0.045 120 [17]

Ashanti Sansu Ghana

A, P, Pr, Mrc STR 20 16,200 (3 moduli of 6 900 m3

each STR’s )

0.0444 96 [17,19]

P = Pyrite; A = Arsenopyrite; Pr = Pyrrhotite; Mrc = Marcasite; Stb = Stibnite; C = complex Cu, Zn, Pb, As, Fe sulfides

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feasibility of coal biodepyritization. Removal of the so-called "organic sulphur" from coal, though investigated in depth by the same research groups, was not as successful, but did provide the guidelines for future research [20-22]. Solving this problem is of great environmental and economic significance, as billions of tons of fossil coal could be utilized were it possible to remove the organic sulphur therefrom [23].

Figure 2. Block diagram showing the factors affecting reactor bioleaching profitability

3.2 Base metal recovery from minerals As far as I am aware, no commercial bioleaching plant has ever been built for the

extraction of base metals from mineral sulphides concentrates. One of the likely reasons for this is that the state of the art technology cannot yet compete with conventional pyrometallurgical processes. The problem here is obviously one of profitability.

As Figure 2 shows, two main factors affect process profitability: bioleaching kinetics and reactor characteristics. Both factors are currently being researched, though bioleaching kinetics has received far more attention and will be dealt with first.

The development of analytical expressions that mathematically relate the characteristics of: (i) microbial strains, (ii) solid substrate, (iii) process environment and (iv) reactor features, and their interactions with process kinetics has been and continues to be the main objective of bioleaching research. The composition of microbial populations and their synergies have been the almost exclusive hunting ground of microbiologists and can reasonably expected to be so in the future. A better understanding of the role played by the solid substrate requires the involvement of several fields of specialized non-microbiological knowledge, ranging from mineralogy associated to solid state physics, chemistry, electrochemistry and mineral engineering. Bioleaching is essentially the product of the the microbial population interacting with the solid substrate but the process

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is strongly conditioned by the physico-chemical environment, specifically the liquid phase pH and Eh, and consequently the related electrochemical phenomena, chemical composition, solids concentration, particle size distribution and evolution, and temperature.

3.2.1 Bioleaching microbiology Microorganisms can be considered, latu sensu, as the biocatalysts of mineral

oxidation and solubilization processes. Up to now the following research lines have been pursued: (i) identification of the microorganisms involved in the process, (ii) how microorganisms interact among themselves and with the solid substrate, (iii) development of the most suitable microorganisms or microbial associations, (iv) enumeration of bioleaching microorganisms.

Identification of the microorganisms involved in the bioleaching process that began with Temple and Hinkle’s discovery in 1948 still continues today. Major breakthroughs include the discovery of Leptospirillum ferrooxidans [24] and of the ability of Sulfolobus to bioleach metal sulphides [25]. These discoveries opened up a whole new world that continues to be investigated today. Incidentally, the discovery of L. ferrooxidans, may well be cited as yet another example of serendipity, because later investigations showed the organism to be quite atypical compared to those species that turned out to be so important in commercial operations [26].

The pioneering work on the physiology of Thiobacilli carried out by Kelly [citations in 27] the application of the theory of chemiosmotic mechanism to A. ferrooxidans proposed by Ingledew, Cox and Helling and by Ingledew [citations in 27] and the work by Don Kelly and Norris [citations in 27] on the moderate thermophiles and on the inhibition by ferrous and ferric ions have provided the tools for interpreting and the approaches for optimizing some laboratory and commercial performances. The results of investigations aimed at gaining a better understanding of the evolution of how bioleaching systems evolve in commercial operations carried out in CFSTR’s (work that appears to be continuing today) have provided a sound understanding of the roles of Thiobacilli and of Leptospirillum ferrooxidans [28-31] in metal sulphide bioleaching and of the importance of properly calibrating the physico-chemical environment on their oxidation activity. Undeniably, this information is invaluable to plant engineers.

Identification methodologies involving nucleic acids represent the turning point for unequivocally distinguishing between strains of microorganisms as well as between species and genera and were exhaustively reviewed in a recent paper [32]. The confirmation of the wide diversity among Thiobacillus ferrooxidans strains [33], already observed by a number of researchers and reported two decades ago [34], as well as among the Leptospirillum ferrooxidans strains [35] are significant achievements of this methodology. Another group of methodologies are those based on immunological methods.

This diversity is also of great practical significance. I still recall that about forty years ago, I was rather puzzled by the statement of a distinguished colleague as to the ubiquity of T. ferrooxidans. As matter of fact, I myself collected from numerous mines throughout Italy several strains of a microorganism that complied with the characteristics of T. ferrooxidans reported in the literature then available. However, the various strains responded quite differently under identical experimental conditions, a situation that seriously intrigued me, as at the time the differences in performance were inexplicable, and naturally I attributed it to some mistake in the laboratory procedure. The problem

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became dramatic when, a few years later, I was testing different strains for developing an industrial bioleaching process.

As an engineer I would like to remark, however, that a major drawback of these methodologies is the need for fairly specialized equipment and skilled personnel [32]. I still recall the sophisticated van that housed a fine mobile laboratory where my Dutch colleagues very effectively carried out real time monitoring of the microorganisms used in the Porto Torres coal biodepyritization semi-commercial scale pilot plant [21]. It appears to me that this type of monitoring is very difficult to propose for a normal industrial operation. One potential area that warrants investigation is the development of kind of "diagnostic kit" that requires training accessible to technicians with a bachelor degree education background.

The same considerations can be made for enumeration methodologies. In recent years microbial associations have been progressively identified and their

importance recognized. Thus, it has been ascertained that several strains of heterotrophs contribute significantly to the activity of autotrophs f.i. acting as scavengers of their metabolites sometimes behaving as inhibitors when in sufficiently high concentrations [36-39]. Looking on the optimistic side, mutualism and synergism [37] should be considered important factors, for process optimisation also in continuous bioreactor systems, which so far have not received the attention they deserve. This is one way of remedying the other bad habit of many biohydrometallurgists – and I regret to say that I am one of them – of using enrichment cultures using substrates such as ferrous iron, sulphur and pyrite for isolating acidophiles. As vigorously pointed out recently [38], this unfortunately widespread practice may result in the selection of a relatively narrow range of acidophiles, depriving the cultures of the contribution of some important bacteria.

The interactions between components of the microflora present in leaching operations were brought to light many years ago by Groudev [citations in 27]. In more recent times the existence and importance of this interaction seem to have been confirmed, in relation for instance to the role played by chemoorganotrophic microorganisms, such as Acidiphilium sp., [35] as a possible stimulating agent of Extracellular Polymeric Substance (EPS) excretion [40] by Leptospirillum ferrooxidans and as a kind of mineral surface scavenger/cleaner of the residual EPS after detachment of microorganisms [41] as well as the predatory activity of some protozoa on Thiobacilli.

There is no doubt that the advances made, that started with some fundamental work by Costerton [42] and Characklis [43] on the microbial capsula and its importance in cell attachment to solid surfaces, fuelled the as yet unresolved controversy, that dates back more than thirty years [44,45] that has generated the fascinating and sometimes heated dispute between the supporters of direct [46] and those of indirect attack [47,40]. This issue has generated some very interesting and to a certain extent instructive critical reviews of the research work carried out on the topic. Probably the solution lies, as often happens, in a compromise and the papers mentioned above are the first premonitory signs.

The mechanism by which microorganisms enhance metal sulphides oxidation and leaching are still surrounded by controversy. Consensus appears to have been momentarily reached on the current lack of evidence as to the existence of an enzyme that justifies the theory of the direct attack. The discovery of the extracellular polymeric substance (EPS) has given rise to some discrepancy in the interpretation of its function between the supporters of the purely electrochemical mechanism [47] and those who uphold a (surface-) chemical process where EPS is the localized environment cell/mineral surface for the action of an energy carrier (e.g. cysteine-based sulphur carrier) produced by some

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chemically-induced mechanism in the microorganism or for the local artificial increase in the concentration of an electron extracting agent (Fe3+) [46].

The peculiar properties of cysteine [48] appear, in this regard, extremely interesting and further research on this subject may be very rewarding.

Consensus on the important role played by the EPS is of paramount importance to the process engineer because this provides vital information for the correct design of the bioreactor, as will be discussed later on.

However, there still seems to be some disagreement as to the agents that generate the phenomena evolving within the EPS layer: Fe3+ concentration, hydrogen ion concentration, occurrence of thiol groups (e.g. cysteine).

Experimental evidence based on redox potential measurements seems to favour the purely electrochemical mechanism [47]. However, further experiments are warranted in the light of recent contributions suggesting that redox potential at the surface of an electrode differs from the bulk of the solution in which it is immersed [49]. Similarly, further work is required to gain a better understanding of the ability of Thiobacillus ferrooxidans to excrete cysteine or other thiol group compounds.

Hence, at least until the enzyme mediating the direct attack of the mineral by the microorganism is discovered, it seems quite appropriate to replace the term “direct attack” with the more cautious "direct contact" coined several decades ago in the very early days of Biohydrometallurgy [50].

Assessment of the significance of EPS is in my opinion a very important issue: the latest results obtained in research work being conducted by my group substantiate its crucial role in bioleaching. During the bioleaching of a gold-bearing pyrite/arsenopyrite concentrate in the new continuous bioreactor designed and constructed by our team, for which a patent is pending, the Biorotor [51] we came across a situation that we had never encountered when using batch bioreactors. The final leach liquor was observed to contain as much as 50 gram.dm-3 total iron, much higher than is consistent with its pH (0.9), with similar amounts of Fe3+, Fe2+. The intriguing point is that conventional analytical methods have proven unsatisfactory, owing to the fact that the iron seems to be encapsulated in something very similar to the recently reported glucuronic acid/iron ion complex containing 53 gram dm-3 [52,53], hence possibly the remnants of the EPS after pyrite corrosion and detachment of the microbial cell. This seems to prevent the precipitation of iron compounds that significantly impair bioleaching performance.

Five of these substances’ properties appear to be quite remarkable, from the practical process engineering standpoint: (i) the fact that their chemical composition and surface activity are influenced by the substratum (ii) the fact that they form a particular, enlarged reaction space for the microbial cells, (iii) the ability of the cells to replenish their capsular material in a few hours when they loose it for any reason (for instance owing to mechanical action), (iv) EPS mediate the contact and (v) the microorganism looses its "catalyst" action if, for any reason, it is deprived of the EPS. This latter property appears to be significant [3] from the practical viewpoint: in the STR’s, at high suspension solids concentration, abrasion may seriously affect microbial action by tearing away the EPS from the cells. Two remedies are possible: either to modify the bioreactor design so as minimize abrasion and shear stresses or to design, employing genetic engineering methods, microorganisms resistant to abrasion and shear stresses. The Biorotor was designed in an attempt to pursue the first remedy.

The opportunities offered by genetic engineering over the past twenty years, can be likened to medieval alchemists search for the philosophers’ stone with the obvious difference that genetic engineering is a very rigorous and well developed science. And like

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their search that contributed enormously to the development of modern chemistry, so too genetic engineering has provided a better understanding of the mechanisms underlying microbe-minerals interactions and enabled a far more accurate identification of microbial strains. Genetic engineering aims to develop microorganisms tailor-made for leaching, with fast kinetics and highly resistant to metal ions, the individual mineral. However, it needs to be said that the potential of "wild" or "indigenous" microorganisms has likely not yet been fully exploited through the development f.i. of suitable bioreactors.

Genetic engineering may contribute, for instance, to suitably modifying the bacterial genomes of those microorganisms whose attachment to the mineral surface depends on the EPS, so that they are tailor engineered for the particular mineral.

The observation, that as commercial biohydrometallurgical operations are not sterile, the risk of modified organisms being released into the environment may discourage or even prohibit the resort to such a technique is quite realistic [54].

A broad range of microbiological investigations has been conducted in the context of metal oxides bioleaching and the recovery of important elements from silicates as exhaustively discussed in a recent review paper by Ehrlich [2].

Attempts to use microorganisms for enhancing the solubilization kinetics of rocks, in particular of silicates, date back to the beginning of the last century, hence well before the discovery of Acidithiobacillus ferrooxidans. The solubilization of leucite, a potassium and aluminium silicate, to extract potassium and aluminium at low cost, was attempted in 1906 by De Grazia and Camiola [55] using molds. While the interesting properties of Acidithiobacillus ferrooxidans, were being investigated, "silicate" solubilizing bacteria were claimed to have been isolated from agricultural soils [56]. It seems that the silicate bacteria claimed by Aleksandrof and his school are in effect strains of Bacillus circulans, [57,58] but research in this area probably still has a long way to go before commercial applications can be contemplated. Some controversy still surrounds this discovery and compared to the advances made with Thiobacilli little progress has been reported in this field.

Over the past fifty years no major advances have been achieved in research on the use of molds or, at least, they do not offer any promising commercial prospects. One of the major drawbacks of this technology is the dramatic volume of biomass involved and the practical problems posed by its handling.

Some interesting proposals for applications of silicate solubilizing bacteria concerned the beneficiation of bauxites [59], but again no industrial applications have been reported.

An interesting niche is represented by the investigations into the microbial recovery of manganese from manganese oxide [60-62]. Research carried out in this field is unravelling the mechanisms of microbial oxides reduction, and the results obtained so far seem quite encouraging. This area warrants further investigation as it offers interesting commercial prospects, for instance for recovering manganese from ocean nodules.

3.2.2 Microbe/solid matter interactions As far as solid matter is concerned, a distinction needs be made between rocks

(minerals) and solid matter other than rocks. The latter is concerned more with environmental applications and has been dealt with above.

Mineral bioleaching is a subject that embraces several topics. As sulphide minerals oxidation is a physico-chemical process, a better understanding of how the presence of microorganisms enhances electron transfer from the mineral to the end acceptor is crucial for optimising any bioleaching operation.

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The amenability of a given mineral to oxidation is related to the solid-state physics of both its bulk and its surface. A number of recent contributions have provided an insight into the importance of the semiconductor nature of the minerals involved and the modifications produced by the presence of foreign elements in the crystal lattices [22,63-66].

Molecular orbital theory, valence bond theory and mineralogy may provide a decisive contribution to elucidating the solubility of metal sulphides in bioleaching systems.

Thus, based on their solid state physics, Sand et al. [40] were able to classify metal sulphides into two main groups: the first, consisting of pyrite, molybdenite and tungstenite, and the second of sphalerite, galena, chalcopyrite, hauerite, orpiment and realgar, exhibiting strong differences in solubility in acid.

One typical case is sphalerite: mining engineers and metallurgists are well aware of the fact that quite often sphalerite is not a stoichiometric compound of zinc and sulphur but contains dissolved iron in proportions that usually differ from one orebody to another. These varieties are called "marmatites" and have been widely investigated [67]. The importance of the proportion of iron dissolved in the crystal lattice of sphalerite had already been recognized in the mid 1950’s by flotation engineers. Now, in the light of considerations that have emerged in recent years [64], the importance of iron has been fully elucidated. The plot reported by Crundwell [68] demonstrating that the rate of reaction for the oxidative dissolution of marmatites is a linear function of the iron concentration in the zinc sulphide lattice, shows dramatically how iron affects the electrochemical, and hence the leaching behaviour, of marmatites. Similar considerations were made for pyrite [22]. Since the elements dissolved in the lattice are kinds of fingerprints of the mineral, it is clear that its origin warrants special attention and may well explain the differences in leaching behaviour.

Mechanisms based on the valence bond theory have recently been proposed [46,64] for the oxidation of pyrite sulphur moiety by iron(III) ions and, though requiring further refinements, they are worthy of mention in that they are on the right track to solving the problem. Further research will likely provide the information necessary for process modulation.

The surface structure and composition of minerals and their modifications during the bioleaching process as well as a kind of "activation" by some metals, like silver, have been recognized in some instances as important factors for the evolution of the process. Chalcopyrite is, in this respect, quite typical since the acidic ferric sulphate leaching or bioleaching evolve according to a well-documented pattern [27,69]. Copper dissolution proceeds in two phases: the initial phase is characterized by a relatively high dissolution rate, which can be expressed by a parabolic law. Over time, the rate decreases pronouncedly, the dissolution rate being represented by a gently sloping straight line. The dissolution rate of the first phase can only be restored by regrinding the leached residue, which usually contains no less than 50% of the copper in the initial feed. Among the explanations advanced for this behaviour, the one based on electroanalytical observations [70] seems to have been confirmed by XPS investigation of surface layers of bioleached chalcopyrite [71]. According to this model, during the first phase, a diffusion layer of copper-depleted chalcopyrite is formed on the mineral surface and it is this layer that governs the subsequent dissolution rate.

The presence of silver in the reacting suspension has a catalytic effect [72] that strongly enhances copper solubilization and is explained by the formation of conductive

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compounds in the retarding barrier, which neutralize its passivating effect by acting as channels for electron flow.

This is just one of many significant, still debated examples, of the interactions of a multiplicity of physico-chemical, electrochemical and solid-state physics factors.

The intriguing micrographs showing the preferential adhesion of microbial cells to areas where emergence of dislocations is visible [40, 44, 73] seem to justify further and more sophisticated research on this subject. The resort to instruments that, like XPS, provide information on the chemical bonding between elements in the surface layers, may unveil the importance of increasing the surface area-to-bulk volume ratio and the extent to which ions in solution interact with the mineral surface and affect oxidation and solubilization.

The importance of chemical composition is also significant, as sometimes the elements passing into solution are toxic to the microorganism, thus requiring continuous adaptation. The inhibitory action of these elements may affect the growth kinetics of the microbial populations and this again relates to the physiology of microorganisms.

3.2.3 Bioleaching kinetics In the light of the above considerations it is now possible to briefly discuss what may

be considered one of the key issues in biohydrometallurgy: the bioleaching kinetics from a process engineering standpoint. As this audience is well aware, numerous researchers have explored this topic, most of whom are chemical engineers or hydrometallurgists, and at a rough estimate more than 50 papers have been published on the subject over the past sixty years. Further research is expected to provide useful data for modulation of the process.

The dramatic increase in mineral sulphides oxidation and solubilization kinetics produced by the pseudocatalytic mediation of Thiobacillus sp. organisms has roused great interest in practitioners and researchers in extractive metallurgy since the very beginnings of biohydrometallurgy. In effect, it was just around this time that a greater awareness of environmental issues was developing, which was soon to place increasing constraints on new industrial projects. Up until then extractive metallurgy had been dominated by pyrometallurgical processes, which had a negative environmental impact.

Clearly the development of mathematical models capable of predicting the economic performance of the new processes became a pressing need.

As shown in Figure 2, the performance of a bioleaching process depends on a multiplicity of factors and, as pointed out in recent years [27,74], its kinetics are a combination of microbial growth kinetics, microbe-minerals and chemical compounds interaction kinetics and particulate solids oxidation and dissolution kinetics (a process that involves several branches of science and technology).

The early models, which already endeavoured to develop a general theory of bioleaching, were relatively simple [69,75-77] but deserve mention in recognition of the originality and the initiative of those researchers. Developments in subsequent years are well documented in several excellent reviews [74,78,79] and numerous of proposed models (fifteen) are exhaustively summarized in a recent review paper [80].

The above-mentioned classification of metal sulphides proposed by Sand et al. [40] and the continuing speculation surrounding the importance of the glycocalyx in the biosolubilization process, would appear to definitively preclude the possibility of developing a general kinetic theory that embraces the entire field of bioleaching. At this point in time it seems expedient to revisit the existing models in the light of the advances in the fundamentals: a deeper insight into some of these may well lead to their unification.

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There seems to be general consensus that bioleaching kinetics is the result of the combination of several subprocesses, the most important being ferrous iron oxidation, bacterial growth (planktonic and sessile), and mineral sulphide dissolution reaction.

The kinetic models The four recently published reviews mentioned above, propose a classification of the

kinetic models. Three of them concern ferrous iron oxidation and mineral bioleaching, the fourth ferrous iron oxidation.

Crundwell [81], proposes a classification criterion based on the relative importance of what are unanimously defined as "contact leaching" and "indirect leaching" [46] mechanisms and refers to two categories: (i) models that postulate a well defined mode of evolution of the leaching process (shrinking-core, propagating-pore) and derive bacterial growth; and (ii) models that postulate a well defined bacterial growth law (rate of growth related to attachment to- and detachment from the solid substrate, Monod law, the logistic equation) and derive the evolution of the leaching process.

Haddadin et al. [80] based their classification (concerning both ferrous iron oxidation kinetics and mineral bioleaching) on the mass balance of each reactional system and on the underlying assumptions of each model and identified three categories: (i) well agitated reactors operating on a liquid phase; (ii) well agitated reactors operating on liquid and solid phases; (iii) the so-called "biofilm reactors".

Hansford [74] considers three classes: (i) empirical models, based on the logistic equation; (ii) models based on attachment of microorganisms; (iii) the two subprocess mechanism (bacterial ferrous iron oxidation and chemical ferric leaching of the sulphide mineral, assumed to be an electrochemical process for which the Volmer-Butler equation holds) for pyrite bioleaching. The implications of this mechanism, as reported by Hansford [74] are rather interesting, insofar as it seems to be able to explain the findings that L. ferroooxidans is the dominant bacterial species in the bioleaching of arsenopyrite and pyrite [28,29] and that arsenopyrite is preferentially leached ahead of pyrite [82].

However, none of these bioleaching kinetics models take into account the population balance approach for describing in quantitative terms, by means of suitable distribution functions, the influence of material properties distribution on the overall behaviour of biohydrometallurgical systems, exhaustively described by extractive metallurgists in the late 1970’s [83,84].

The significance of the material properties appear to have been first understood by researchers investigating coal biodepyritization in relation to the size distribution of pyritic coal [85] but the population balance approach was not adopted.

Crundwell [81] deserves recognition for his model based on the population balance for particulate leaching, for the bacterial cell number and material balance describing solution reactants and products. This model was found to provide an excellent fit to experimental data reported by other researchers for pyrite bioleaching, although he used the shrinking-core rather than the more realistic propagating-pore mechanism [74].

It should however be pointed out that the population balance approach can also have pitfalls, associated for instance with the practical difficulty, of which mineral processing practitioners are well aware, of developing a realistic function for the grain size distribution of a mineral powder. These difficulties can increase the complexity of such a model making its application impracticable.

The kinetics of ferrous iron oxidation has long been recognized as playing a key role in bioleaching and several papers have been published on this subject over the past sixty

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years. I will not review every single paper here but will limit myself to a few general remarks, spotlighting the main features of this history, as they provide a good description of the evolution of biohydrometallurgy.

Over the years the significance of the following factors in bioleaching processes - listed in chronological order - has been recognized: Total initial iron concentration Temperature Type of culture (continuous or batch) Wall growth Total iron concentration Inhibition by ferric iron Threshold concentration of ferrous iron Product inhibition pH Bacterial concentration (mass or numeric) Bacterial decay Maintenance Carbon dioxide transfer

Nemati et al. [86] reviewed the work carried out on the kinetics of ferrous iron oxidation by Thiobacillus ferrooxidans distinguishing two major classes: (i) freely suspended cells; (ii) immobilized cells. Whereas, for the immobilized cells processes, these authors compared the performances of different types of reactors (packed-bed, fluidised-bed, rotating biological contactors), in the paper by Pesic et al. [87] the experimental set-up consisted of a thermostated 300 cm3 plexiglass cell containing 250 cm3 of ferrous solution and Thiobacillus ferrooxidans inoculum and provided with a magnetic stirrer: no information was provided on the rpm of this stirrer. In another paper [88] ferrous conversion kinetics was experimentally determined using a set-up consisting of a small, thermostated, reaction cell (volume 20 cc) containing 10 cc of reaction liquid plus cell suspension, sparging air through the liquid contained in the cell at a flow rate of 8,3 cm3.s-1. This set-up can be regarded as an "ideal" reactor, where the bacterial cells are very likely only subjected to a minimum of mechanical stress. It is possible that experimental conditions in both cases ensured a fairly quiescent environment thus avoiding undue stress to the bacterial cells. These operating conditions differ significantly from those existing in the particulate mineral suspension of an STR fitted, for instance, with a Rushton-type turbine.

Only recently, however, a mechanistically-based model adopting the initial rate method and relying on Michaelis-Menten kinetics [79,88] and a model based on Ingledew’s chemiosmotic theory, on the electrochemical theory and on the Monod and Michaelis- Menten models, take into account all of the above influencing factors and appear to provide a satisfactory fit to the bacterial ferrous iron oxidation data reported in the literature [74]. This is undoubtedly an important achievement, though the data fit refers to experiments carried out in a variety of bioreactors where, in terms of reactor dynamics, optimum operating conditions may well not have been ideal for bacterial cells. So, all the models for ferrous iron bioleaching kinetics or for mineral bioleaching kinetics appear to neglect the influence of reactor dynamics, which, as far as I am aware, was only mentioned [80] in relation to a paper [89] dealing with the geometry and operating characteristics of an air–lift bioreactor (Pachuca tank) designed by researchers of Bergbauforschung and still being used in my laboratory.

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Practically all the authors who have provided an overview of bioleaching kinetics models agree as to the difficulty of generalizing the results of the individual models, on account of the numerous influencing factors that characterize each substrate, such as mineralogy, solid-state physics, chemical composition, particle size, specific gravity, electrochemistry, and the microbial population (temperature, pH, strains, associated microbial strains…).

Moreover, several authors have demonstrated that strong shear stresses are generated in conventional bioreactors (STR’s and ALR’s) during agitation of the suspension and that, beyond a certain threshold, abrasion produced by solid particles has to be taken into account.

Such situations may become a limiting and determining factor seriously affecting both bacterial growth and bacterial adhesion and ultimately process kinetics. Quantification of these effects is an open and very likely promising research field, although I still doubt that general theories can be formulated. Probably, the most practicable and profitable way is to design suitable bioreactors where these effects are minimized by suitably selecting the proper operating conditions.

There is general consensus that bioleaching process kinetics is directly dependent upon the number of active microbial cells present in the system. Already about twenty years ago this number had been estimated to be in the order of magnitude of 1012 cells.cm-3 [85,90,91] which, as far as I know, has not yet been achieved. It is therefore reasonable to attempt to maximize microbial growth. The question then arises as to what is the maximum number of microorganisms that can be achieved and if the bioreactors currently in use are the most suitable for attaining this population density in steady state regime and how such a reactor should be designed.

The problems are very similar to those confronted by mineral beneficiation researchers and flotation plant designers and operators for about 100 years, from the very early days of flotation technology. Research has played a very important role in elucidating the problems, but no general theory could be developed. Basically, the flotation process is the same for any ore, but each ore requires equipment, reagents, and physico-chemical environment to be properly adjusted. Process performance can only be predicted by a wise combination of fundamentals (that, except for the biological agent, are practically the same as in biohydrometallurgy) and of bench- and laboratory scale pilot testing using devices that simulate commercial operation, that provide the experimental numeric parameters for the mathematical expressions for describing the specific process kinetics - supplemented by an up-to-date knowledge of microbiology.

The future progress of Biohydrometallurgy much depends on the solutions of these problems: the fascinating aspect but also the intriguing feature of biohydrometallurgy is its multidisciplinarity. It is fascinating because it reveals how intimate the liaison between the various branches of science and technology can be; intriguing, because this technology requires a working knowledge of several disciplines, that are sometimes so different from one another and that cannot be reasonably mastered by a single specialist.

Real advances will only be made possible by the cooperation of people skilled in the various facets of biohydrometallurgy. The timely exchange of information and a very complete documentation are the prerequisites for gaining an identity that this new technology seems to have not yet achieved. The importance of up-to-date documentation will prevent researchers from expensive and time-wasting repetition of investigations successfully carried out elsewhere. What I am going to say now may sound trivial, but it

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should be remembered that a well-documented failure is as useful as a successful achievement.

4. A BIOHYDROMETALLURGY SOCIETY Based on his experience, the author would like to emphasize the need to establish a

Biohydrometallurgical Society, to act as a point of reference to all those, from industry and academia, who are involved in implementing and further developing the technology. The Society should also provide a forum for information flow to decision makers in industry, about the potentials of biohydrometallurgy. Though it relies on the exploitation of the complex synergies between microoorganisms and minerals, this technology, when properly applied, is simple to implement, operationally stable and cost-effective.

One task of the Society might be, for instance, the preparation and further updating of a Recommended Standard Terminology and Nomenclature for Biohydrometallurgy, like the one published several years ago by the Institution of Chemical Engineers Fluid Mixing Group [92]. This idea came to mind when I was reading a comprehensive review written by a distinguished microbiologist, who proposed an important terminology, justifying his intervention with the fact that the related topic had been studied by researchers from diverse cultural backgrounds other than microbiology.

Last, but not least, the Society would be able to benefit from the experience and knowledge of all those involved in industry and academia to develop new university curricula aimed at training a new generation of specialists.

REFERENCES 1. Ehrlich, Hydrometallurgy, 59 (2-3) (2001) 127. 2. Ehrlich, Minerals and Metallurgical Processing, 19 (4) (2002) 220. 3. Rawlings, D. E., Ann. Rev. Microbiol., 56 (2002) 65. 4. Brierley, J.A., Brierley, C.L., and Goyak, G.M., Fundamental and Applied Bio-

hydrometallurgy, Lawrence, R.W., Branion, R.M.R. and Ebner, H.G. (Eds.), Elsevier, Amsterdam (1986) 291.

5. Tsezos, M., Microbial Mineral Recovery, Ehrlich, H. L. and Brierley, C. L., (Eds.), McGraw-Hill Publishing Company, New York, (14) (1990) 325.

6. Pümpel, T., Ebner, C., Pernfuß, B., Schinner, F., Diels, L., Keszthelyi, Z., Stankovic, A., Finlay, J. A., Macaskie, L. E., Tsezos, M., and Wouters, H., Hydrometallurgy, 59 (2-3) (2001) 383.

7. Hatzikioseyian, A., Tsezos, M., and Mavituna, F., Hydrometallurgy, 59 (2-3) (2001) 395.

8. van Loosdrecht, M.C.M. and Zehnder, A.J.B., Experientia, 46 (1990) 817. 9. van Loosdrecht, M.C.M., Norde, W., Lyklema, J. and Zehnder, A.J.B., Aquatic

Sciences, 52 (1) (1990) 103. 10. van Loosdrecht, M.C.M., Lyklema, J., Norde, W., Gosse, S. and Zehnder, A.J.B.,

Appl. Environm. Microbiology, 53 (8) (1987) 1893. 11. Madgwick, J.C., and Ralph, B.J., Conference Bioleaching, Schwartz, W. (Ed.)

Weinheim (Germany) Verlag Chemie, (1977) 85. 12. Tsezos, M., Hydrometallurgy, 59 (2-3) (2001) 241. 13. Volesky, B., Hydrometallurgy, 59 (2-3) (2001) 203. 14. Schnell, H.A., Biomining: Theory, Microbes and Industrial Processe, Rawlings, D. E.

(Ed.), Springer, Berlin, (11) (1997) 21. 15. Pasteur, L., La révue scientifique, 2e Sér. 1 (4) (1871) 73.

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16. van Answegen, P.C. and Marais, H.J., MINERAL BIOTECHNOLOGY, Kawatra, S.K. and Natarajan, K.A. (Eds.), Littleton, Colorado, USA Society for Mining, Metallurgy, and Exploration, Inc., (2001) 121.

17. Dew, D.W., Lawson, E.N., and Broadhurst, J.L., Biomining, Rawlings, D.E. (Ed.), Berlin, Springer, (1997) 45.

18. Taxiarchou, M., Adam, K., and Kontopoulos, A. Hydrometallurgy, 36 (2) 1994 169. 19. Nicholson, H.M., Smith, G.R., Stewart, R.J., Kock, F.W., and Marais, H.J., Biomine

’94, Australian Mineral Foundation: Glenside, South Australia, 1994 2-1. 20. Loi, G., Mura, A., Trois, P., and Rossi, G., Memorie dell'Associazione Mineraria Sarda

– Iglesias, I (1) (1994a) 29. 21. Loi, G., Mura, A., Trois, P., and Rossi, G., Fuel Processing Technology, 40 (1994b)

26. 22. Rossi, G., Fuel, 72 (12) (1993) 1581. 23. International Scientific Committee of the Fourth Intrntl.Symp. on Biological

Processing of Fossil Fuels, Rossi, G. (Guest Ed.), Coal Processing Technology, 40 (1994), 379.

24. Markosyan, G.E., Biol. Zh. Armenii, (1972) 25. 25. Brierley, C.L., and Murr, L.E., Science, 179 (1973) 488. 26. Rawlings, D.E., Biomining: Theory, Microbes and Industrial Processe, Rawlings, D.

E. (Ed.), Springer Berlin, (1997) 229. 27. Rossi, G., Biohydrometallurgy, McGraw-Hill GmbH, Hamburg, (1990). 28. Rawlings, D. E., Tributsch, H., and Hansford, G. S., Microbiology, 145 (1999) 5. 29. Boon, M., Brasser, H. J., Hansford, G. S., and Heijnen, J. J., Hydrometallurgy, 53 (1)

(1999) 57. 30. Breed, A. W., Dempers, C. J. N., Searby, G. E., Gardner, M. N., Rawlings, D. E., and

Hansford, G. S., Biotechnology and Bioengineering, 65 (1) (1999) 44. 31. Battaglia-Brunet, F., d'Hugues, P., Cabral, T., Cezac, P., Garcia, J. L., and Morin, D.

1998, Minerals Engineering, 11 (2) (1998) 195. 32. Jerez, C. A. 1997, Biomining: Theory, Microbes and Industrial Processes. Rawlings,

D. E. (Ed.), Springer, Berlin (1997) 281. 33. Kelly, D. P. and Wood, A. P., International Journal of Systematic and Evolutionary

Microbiology 50 (2000) 511. 34. Harrison, A. P. J., Archives of Microbiology, 131 (1982) 68. 35. Hallmann, R., Friedrich, A., Koops, H., Pommering-Röser, A., Rohde, K., Zenneck,

C., and Sand, W., Geomicrobiology Journal, 10 (1992) 193. 36. Johnson, B. and Roberto, F. F., Biomining: Theory, Microbes and Industrial Processe,

Rawlings, D. E. (Ed.), Springer Berlin, (13) (1997) 260. 37. Johnson, D. B., FEMS Microbiol. Ecol., 35 (1998) 307. 38. Johnson, D.B., Hydrometallurgy, 59 (2-3) (2001) 147. 39. Johnson, B., Bacelar, Nicolau P., Okibe, N., Yahya, A., and Hallberg, K.B.,

Biohydrometallurgy - “Fundamentals, Technology and Sustainable Development”, Proceedings of the International Biohydrometallurgy Symposium IBS-2001, Part A, Ciminelli, V. S. T. and Garcia Jr., O. (Eds.), vol. A, Elsevier Amsterdam, (2001) 461.

40. Sand, W., Gehrke, T., Jozsa, P.-G. and Schippers, A., Hydrometallurgy, 59 (2-3) (2001) 159.

41. Sand W, Jozsa P-G, and Schippers A., Biohydrometallurgy and the Environment toward the Mining of the 21st Century, Amsterdam, Elsevier, Vol. A (1999) 27.

42. Costerton, J. W., Irvin, R. T., and Chen, K.-J., Ann Rev Microbiol, 35 (1981) 299. 43. Characklis, W. G., Biotechnology and Bioengineering, 23 (1981) 1923. 44. Berry, V. K. and Murr, L. E., Metallurgical Transactions, B, 6B, (1975) 488.

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20

45. Bennett, C. and Tributsch, H., J. Bacteriology, 134 (1) (1978) 310. 46. Tributsch, H., Hydrometallurgy, 59 (2-3) (2001) 177. 47. Crundwell, F.K., Biohydrometallurgy - “Fundamentals, Technology and Sustainable

Development”, Proceedings of the International Biohydrometallurgy Symposium IBS-2001, Part A, Ciminelli, V.S.T. and Garcia Jr., O. (Eds.), Elsevier, Amsterdam (2001) 149.

48. Rojas-Chapana, J. A., and Tributsch, H., 35 (2000) 815. 49. Nicol, M. J. and Lázaro, I., Hydrometallurgy, 63 (1) (2002) 15. 50. Silverman, M.P., and Ehrlich, H.L., Advan. Appl. Microbiol., 6 (1964) 153. 51. Loi, G., Trois, P., and Rossi, G., Biohydrometallurgical Processing, Vargas, T., Jedrez,

C.A., Wiertz, J.V. and Toledo, H. (Eds.), University of Santiago, Chile, Vol. 1, (1995) 263.

52. Gehrke, T., Hallmann, R., Kinzler, K., and Sand, W., Appl. Environ. Microbiol., 65 (1997) 159.

53. Gehrke, T., Telegdi, J., Thierry, D., and Sand, W., Appl. Environ. Microbiol., 64, 7 (1998) 2743.

54. Rawlings, D.E., Hydrometallurgy, (2-3) 59 (2001) 187. 55. De Grazia, S. and Camiola, G., Le stazioni sperimentali agrarie italiane, 39 (9) (1906)

829. 56. Aleksandrov, V.G., Kiev Izv. Akad. Nauk. S. S. S. R., (1958) 62. 57. Tesic, Z.P. and Todorovic, M.S., Zemljiste i biljka, 1 (1) (1952) 3. 58. Tesic, Z.P. and Todorovic, M.S., Zemljiste i biljka, 8 (1-3) (1958) 233. 59. Groudeva, V.I. and Groudev, S.N., 5th International Congress of ICSOBA, Zagreb,

Yugoslavia, Academie Yugoslave des Sciences et des Arts, 83 (18) 257. 60. Remezov, V. D., Agate, A. D., and Yurchenko, V. A., Biogeotechnology of Metals –

Manual, Karavaiko, G. I., Rossi, G., Agate, A. D., Groudev, S. N., and Avakyan, Z. A. (Eds.), Centre for International Projects – GKNT Moscow U.S.S.R., (1988) 304.

61. Ehrlich, H. L. (and Rossi, G.), Microbial Mineral Recovery, Ehrlich, H. L., and Brierley, C. L. (Eds.), (7), MacGraw-Hill Publishing Company, New York, (1990) 149.

62. Ehrlich HL., Biohydrometallurgical Technologies, Torma, A. E., Apel, M. L., and Brierley, C. L. (Eds.), v. 2, The Minerals, Metals & Materials Society, Warrendale, Penna, USA, (1993) 415.

63. Pridmore, D.F., and Shuey, R.T., American Mineralogist, 61 (1976) 248. 64. Crundwell, F.K., AIChE Journal, 34(7) (1988) 1128. 65. Nesterovich, L. G., Biogeotechnology of Metals - Manual. Karavaiko, G. I., Rossi, G.,

Agate, A. D., Groudev, S. N., and Avakyan, Z. A. (Eds.), Centre for International Projects GKNT, Moscow, U.S.S.R., (1988) 101.

66. Thomas, B., Ellmer, K., Bohne, W., Rohrich, J., Kunst, M., Tributsch, H., Solid State Communications, 111 (1999) 235-240.

67. Kullerud, G., Norsk Geologisk Tidsskrift, 32 (1953) 61. 68. Crundwell, F.K., Hydrometallurgy, 21(2) (1988) 155. 69. McElroy, R.O. and Bruynesteyn, A., Metallurgical Applications of Bacterial Leaching

and Related Microbiological Phenomena, Murr.L.A., Torma, A. E., and Brierley, J. A. (Eds.) Academic Press, New York (1978) 441.

70. Ammou-Chokroum, M., Cambazoglu, M. and Steinmetz, D., Bull. Soc. Fr. Miner. Cristallogr. 100 (1977) 161.

71. De Filippo, D., Rossi, A., Rossi, G., and Trois, P., International Biotechnology Symposium Proceedings, Durand, G., Bobichon, G., and Florent, J. (Eds.), J. Soc. Française de Microbiologie, Paris, France (1988) 1131.

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21

72. Miller, J.D., and Portillo, H.Q., Proceedings XIII Int. Min. Proc. Cong., Vol. 2 (A), Laskowski, J. (Ed.), Elsevier Scientific Publishing Comp., (1979) 851.

73. Rojas-Chapana, J.A., and Tributsch, H., Biohydrometallurgy and the Environment toward the Mining of the 21st Century, Amsterdam, Elsevier, Vol. 2 (1999) 597.

74. Hansford, G. S., Biomining. Rawlings, D. E., Springer: Berlin, (8) (1997) 153. 75. Torma, A. E., CANMET, Ottawa, Canada (1985) 5. 76. Torma, A. E. and Panneton, J. J., Ministère des Richesses Naturelles - Direction

Générale des Mines-Centre de Recherches Minérales, Québec, Canada BLS-1 (1973). 77. Legault, G., and Torma, A.E., 421ème Congrès Annuel, Association Canadienne

Française pour l'Avancement des Sciences, Province de Québec (1974) 1. 78. Barrett, J., Hughes, M. N., Karavaiko, G. I., and Spencer, P. A., Metal Extraction by

Bacterial Oxidation of Minerals. Ellis Horwood Limited, NewYork. (1993) 103. 79. Nemati, M, and Webb, C., Biotechnology Letters, 20 (9) (1998) 873. 80. Haddadin, J., Dagot, C., and Fick, M., Enzime and Microbial Technology, 17 (1995)

290. 81. Crundwell, F.K., The Chemical Engineering Journal, 54 (1994) 207. 82. Miller, D. M. and Hansford, G. S., Minerals Engineering 5 (7) (1992) 737. 83. Sepulveda, J. E. and Herbst, J. A., AIchE Symposium Series, 74 (173) (1978) 41. 84. Herbst, J. A., Rate Processes of Extractive Metallurgy. Sohn, H. Y. and Wadsworth,

M. E. (Eds.) Plenum Press New York, (2) (1979) 53. 85. Andrews G.F., Bioprocessing of Coal Workshop III, Idaho National Engineering

Laboratory, Idaho Falls, ID, USA (1988) 234. 86. Nemati, M., Harrison, S.T.L., Hansford, G.S. and Webb, C., Biochemical Engineering

Journal, 1 (1998) 171. 87. Pesic, B., Oliver, D.J., and Wichlacz, P., Biotechnology and Bioengineering, 33 (1989)

428. 88. Nemati, M. and Webb, C., Biotechnology and Bioengineering, 53, (5) (1997) 478. 89. Beyer, M., Ebner, H.G., and Klein, J., Appl. Microbiol. Biotechnol., 24 (1986) 342. 90. Andrews G.F. and Quintana J., First International Symposium on Biological

Processing of coal, E.P.R.I. Palo Alto, California, U.S.A., (1990) 5-69. 91. Stevens, C. J., Noah, K. S., and Andrews, G. F., Fuel, 72 (12) (1993) 1601. 92. Anonymous, The Chemical Engineer, (1980) 557.