BIOHYDROMETALLURGICAL PROCESSES: A PRACTICAL APPROACH

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2 BIOHYDROMETALLURGICAL PROCESSES: A PRACTICAL APPROACH Luis Gonzaga Santos Sobral Chemical Engineer, PhD. in Hydrometallurgy Senior Researcher at CETEM/MCTI Débora Monteiro de Oliveira Biologist, MSc. in Biotechnological Processes Researcher at CETEM/MCTI Carlos Eduardo Gomes de Souza Industrial Chemist Researcher at CETEM/MCTI CENTRE FOR MINERAL TECHNOLOGY - CETEM MINISTRY OF SCIENCE, TECHNOLOGY AND INNOVATION MCTI Rio de Janeiro 2011

3 Copyright 2011 CETEM/MCTI All right reserved The unauthorized reproduction of this publication, in whole or in part, constitutes a violation of copyright (Law 5.988). Valéria Cristina de Souza Formatting and Electronic Editing P2r - Marketing e Publicidade Cover and Graphic Design Information: CETEM Centro de Tecnologia Mineral Av. Pedro Calmon, 900 Cidade Universitária Rio de Janeiro RJ Homepage: Biohydrometallurgical processes: a practical approach/ed. Luis Gonzaga Santos Sobral, Débora Monteiro de Oliveira e Carlos Eduardo Gomes de Souza - Rio de Janeiro: CETEM/MCT, p.: il. 1. Bioleaching 2. Biooxidation 3. Biodegradation 4. Biodesulphurization 5. Microorganism 6. Heap leaching I. Centro de Tecnologia Mineral. II. Sobral, L.G. (Ed.). III. Oliveira, D.M. (Ed.) IV. Souza, C.E.G (E.d.). ISBN CDD

4 PREFACE With the efforts of several researchers and engineers from different countries, we are presenting to you in the present recollection, a basket of possibilities and process opportunities on what those microorganisms, as tiny workers, can do for you in your industrial mineral processing activities. They are literally billions of workers only claiming a nice physico-chemical environment and an energy source to perform their work. Their duties are to biooxidate minerals and concentrates releasing the base metals, as soluble species, from sulphide materials, or to convert precious metals bearing refractory ores or concentrates into cyanide soluble precious metals, allowing their recoveries by traditional cyanidation. The above processes can also be reversed with the work performed by another set of tiny workers, converting soluble metals into non soluble materials, stopping the migration of toxic soluble metals in effluents from soils, mines or tailings deposits, process known as bioremediation, and, in addition, the biodesulphurization of H 2S-bearing industrial gas streams. We are very pleased of opening for you a window with the possibilities in the field of microorganisms acting in industrial mining processes. The mineral industry has suffered in recent years two significant impacts: The raise on energy cost and more strict environmental regulations were in place for the metallurgical processing of sulphide ores and concentrates, mainly those containing arsenic. The above two impacts moved the mineral industry to develop alternative processes to roasting and smelting, and searching for alternative processes to avoid the high energy consumption grinding operation and flotation process of sulphide minerals. In parallel to the above restrictions, the depletion of high grade mineral resources, the mining industry is trying to develop processes to exploit low grade mineral deposits, most of them containing primary metals sulphides, task that appears to be solved by the action of another kingdom of microorganisms, the extreme thermophillic microorganisms, which are tiny workers that perform their jobs at a faster kinetic at high temperature processes. The industry led by Gencor from South Africa developed agitation bioleaching processes in stirred reactors (BIOX TM ) to convert the refractory precious metals concentrates into cyanide soluble feed for the existing cyanidation plants. The BIOX TM process developed by Gencor consisted in a series of agitation leaching tanks with concentrate pulps under high mechanical or aeration agitation, in the presence of mesophile bacteria and air, at a ph lower than 2. The above process was growing from modest 10 metric tons per day in the Fairview plant (RSA) in 1986, later expanded to 40 MT/d in 1991, with 100 cubic meters reactors, to the Ashanti Sansui plant (Obuasi in Ghana) treating 960 MT/d in 1000 cubic meters reactors. Other plants were developed such as Wiluna and Fosterville (Australia), Beaconsfield (Tasmania) for 70 MT/d gold concentrate, Kasese (Ghana) for 720

5 MT/y nickel cathodes using 1,380 cubic meters reactors, and plants in environment of below 0ºC such as Suzdal (Kazakhstan) and Talvivaara (Finland). The transfer of the agitation leaching technology to copper concentrates has not been developed at the same speed as that of gold concentrates. The Peñoles demo plant (Mexico) (2001) processes copper at the rate of 200 MT/y copper cathodes. A big effort was done in a joint venture Billiton/Codelco with the Alliance Copper Ltd. Demo Plant, for processing 100 MT/d of copper concentrate containing enargite and chalcopyrite mineral species, both refractory to mesophile bioleaching, in big reactors, 2,300 cubic meters built with ceramic materials using extreme thermophiles microorganisms such as archaea, and moderate thermophile microorganisms, working at temperatures range of 65 to 70ºC. The Alliance plant did not resulted in an incentive to decide to go to an industrial plant. The reason given by Alliance for not going ahead with a huge plant was the operational cost, as internally the bioleaching plant was competing negatively with the Codelco smelters cost for the treatment of same copper concentrates, in spite of the advantages of processing positively arsenic bearing concentrates. The above mentioned initiative has retarded the interest of new copper ventures to develop huge agitation bioleaching plants. Another reason is the presence of a new technology (GeoBiotics, LLC) that can do the same, but using a heap or a dump as bioreactor, with significant reduction in cost. Such technology uses controlled size inert materials where concentrate is coated on it, and those mineral agglomerates are used for raising an engineered heap with controlled microorganisms together with a system to capture inside the heap, being most of the heat generated during the sulphide minerals bio-oxidation reactions. In the case of ores bio-oxidation in dumps, there are reports mentioning the dissolution since the Roman Empire period, mainly in the current Rio Tinto area in west southern Spain. In the current times, the first operation of dump leaching with copper ores, with clear identification of the bioleaching process was in Bingham Young Cannon (Utah, USA) in 1967, where the copper bearing effluents were precipitated with iron scrap in the Kennecott Cones, being the SX/EW process for copper at the above time just developed. The low grade copper ores, were deposited in terraces, with vertical tubes to access air for the bio-reaction. A touristic tour for visitor was organized to observe the exiting steam leaving the tubes when the terraces were covered with snow. The copper industry acknowledges that the first heap bioleaching operation plant, for secondary copper ores, was raised in Los Aguirre 1983 (Chile). Since then, dozens of heaps and dumps were built and operated as bioleaching plants. There is certain lack of definitions of what a bioleaching process could be. Being the bioleaching a very flexible process, most heap leaching operations claim to have a number of microorganisms varying from 10 5 to 10 9 per gram of ore, being the later 10,000 bigger.

6 An important improvement in heap bioleaching was the introduction of forced air at the base of the heap, in 1993, most likely imported from the good results of air injection in stirred reactors. Many other improvements were introduced and universally adapted, such as plastic membranes covers, to capture heat and preventing water evaporation, drainage tubes to collect the solutions, network of drippers to irrigate the heap with solutions leaving behind the sprinklers, on/off irrigation system, and slowly adding more instrumentation to monitor the process. Another significant findings, was the ability of the microorganisms to adapt to new environments, or to improve their efficiency with time. The above ability allows our tiny workers to be flexible with changes in solution chemistry or ore mineralogy. In agitation leaching pilot plant tests it was observed that around 3% better copper extraction was obtained after one year. Using the above ability of those microorganisms, significant efforts are being done to adapt these to sodium chloride bearing solutions, even from using sea water or after recycling the solutions from the leaching chloride containing mineral species, such as atacamite (Cu 2Cl(OH) 3). Special mention must be placed in the future of bioleaching process applications using archaea microorganisms, also denominated extreme thermophile. Most people have the temptation to denominate the above microorganisms as bacteria, but they are a completely different species, some researchers claim that they belong to a different kingdom, indicating that archaea belongs to another branch of the life developing tree. Industrial experiences with dumps of low grade copper sulphide primary ores resulted in copper extractions of 30% after three years of leaching. Passivity affects chalcopyrite and the bio-reaction does not progress further. In controlled experiences at room temperature, same results were obtained, from 30 to 40% extraction in long leaching times, till the copper extraction curve is flatten, but if temperature is raised to 70 0 C, copper extraction raise up to 97% in 120 days. The above significant finding opens a new window for processing low grade primary copper ores. The pyrite oxidation reaction heap is noticeable, as an example it was reported that an ore bearing 2% of sulphur, considering the bioreaction running for 183 days, a constant heat is generated, which is equivalent to 54 W/cubic meter of ore. The aim is conducting the bio-reaction in the fastest possible way trapping most of the evolved heat, so as to reach temperatures of 60 0 C. There is a technology, known as HotHeap, that presents an algorithm to capture most of the evolved heat, manipulating simultaneously the irrigation and aeration mass aiming at raising the heap temperature up to 60 0 C, in a programmable ramped up model, depending on how much sulphide sulphur (fuel) is contained inside the heap.

7 The reward of the succeeding with the above technology will be the heap or dump leaching of primary copper ores that are below cut off to be processed by traditional grinding and flotation. There is a huge effort to be undertaken regarding the bioleaching of complex ore bodies, such as the poly-metallic ones, and at the same extent the separation of those metals being dissolved in a sort of PLS, such as arsenic, zinc, copper, nickel and cobalt. In the copper industry, the thermophile bioleaching process is a good alternative for arsenic containing ores or concentrates, bearing in mind that enargite (Cu 3AsS 4) is considered a refractory mineral species, which has low response to mesophile microorganisms. As it is always the case, the bioleaching tools are available for tailoring a solution for processing diverse base metals ores or concentrates, a job destined to process metallurgists. All the above has developed a parallel monitoring technology, comprising a device for measuring oxygen in exhaust air for evaluating the oxygen consumption in the dump or heap, for monitoring the health of our tiny workers, say, the Eh measurement or the viability or speciation of the microorganisms, the number of microorganisms per gram of ore or per cubic centimetre of solution, and the temperature profiles inside the heap. In the past all the monitoring equipment was connected by wires and the readings were also sent by wires, being the wiring a nightmare. Today all the information on the instrument readings are sent wireless to central points and from there via satellite technology to be computed in models in central offices, and the evaluation is instantaneously produced and reported to operators, sometimes with the most convenient process correction, say, to raise irrigation or reduce aeration. Once we opened the window to see the horizon to where the above technologies are conducting us, we realized that the there is no horizon but an endless continuous development that is stepwise converting our dreams into industrial realities. Enrique Carretero Civil Chemical Engineer PUCV M.S. Chemical Engineering UMR Manager Latin America G GeoBiotics, LLC

8 Posthumous homage Few years ago we had the unique opportunity to know Professor Oswaldo Garcia Junior during one of the IBS (International Biohydrometallurgy Symposium) meeting, and at that moment we realized the tremendous appreciation of the scientific community toward his knowledge on biohydrometallurgical processes. However, during our frequent literature survey on bioleaching it is quite clear his huge contribution for training new professionals at UNESP (São Paulo State University/Brazil) in the first place, and assisting the Brazilian Mining Sector in sorting out its production hassles. Our research group at CETEM (Centre for Mineral Technology, a research centre under the Brazilian Ministry of Science, Technology and Innovation) has been, for quite a while, taking advantage of his teaching showing us the way to reach the excellence on biohydrometallurgy. In fact, the idea of publishing this book arose from several discussions among our research groups bearing in mind his huge experience. However, all of sudden, our Lord took him from us, which for sure was a sort of an emergency, living us breathlessness. Nevertheless, we do believe, from heaven where he is, he will keep on enlightening and encouraging us to carry on contributing for the strengthening of the hydrometallurgy field the way he used to do. The Editors

9 VIII CONTENTS CHAPTER 1 CHALLENGES IN PRACTICING THE BIOLEACHING PROCESS Juan Rivadeneira 1. INTRODUCTION FACTORS AFFECTING THE BIOLEACHING INHIBITORS OF BIOLEACHING METABOLIC MECHANISMS OF BIOLEACHING IMPLEMENTATION OF THE GEOCOAT BIOLEACHING PROCESS REFERENCE CHAPTER 2 - MECHANISMS OF BIOLEACHING BASIC UNDERSTANDING AND POSSIBLE INDUSTRIAL IMPLICATIONS Maria A. Giaveno, Maria Sofia Urbieta and Edgardo Donati 1. THE TRADITIONAL MECHANISMS OF BIOLEACHING THE MECHANISMS CURRENTLY ACCEPTED MICROORGANISMS AND COMMERCIAL APPLICATION POSSIBLE INDUSTRIAL IMPLICATIONS REFERENCES CHAPTER 3 ADAPTABILITY OF BIOMINING ORGANISMS IN HYDROMETALLURGICAL PROCESSES Helen Watling 1. INTRODUCTION STIRRED TANKS AS MICROBIAL HABITATS HEAPS AS MICROBIAL HABITATS MICROBIAL ADAPTABILITY TO BIOPROCESSING ENVIRONMENTS SUMMARY ACKNOWLEDGEMENTS REFERENCES CHAPTER 4 MICROORGANISMS COUNTING TECHNIQUES IN HYDROMETALLURGY Pamela Chávez, Johanna Obreque, Jeannette Vera, Denisse Quiroga and Jorge Castro 1. INTRODUCTION COLLECTING SAMPLES KING OF SAMPLES EXTRACTION OF NUCLEIC ACIDS MICROBIOLOGICAL TECHNIQUES ENZYMATIC TECHNIQUE MOLECULAR TECHNIQUES REFERENCES... 80

10 IX CHAPTER 5 SCIENTIFIC MONITORING OF INDUSTRIAL BIOLEACHING PROCESS Cecilia Demergasso, Pamela Soto, Víctor Zepe and Pedro Galleguillos 1. INTRODUCTION ALTERNATIVES, DECISIONS AND SOME ACHIEVEMENTS COMPREHENSIVE DATA ANALYSIS/MICROBIAL DATA ANALYSIS THE MAIN CHALLENGES TO GO FURTHER AKNOWLEDGMENTS REFERENCES CHAPTER 6 BIOOXIDATION AMENABILITY TESTING Gregory J. Olson and Todd J. Harvey 1. INTRODUCTION OUTCOME OF AMENABILITY TEST WORK TESTING THE AMENABILITY OF GOLD ORES TOWARD BIOOXIDATION GENERAL CONSIDERATIONS TESTING THE AMENABILITY OF BASE METAL ORES TOWARD BIOLEACHING GENERAL CONSIDERATIONS PERFORMANCE OF LABORATORY AMENABILITY TESTING REFERENCES CHAPTER 7 BIOLEACHING OF METAL SULPHIDES ORES AND CONCENTRATES Enrique Carretero, Luis G. S. Sobral, Débora M. de Oliveira and Carlos E.G. de Souza 1. INTRODUCTION PREREQUISITE FOR A BIOLEACHING TEST WORK INDUSTRIAL PROCESSES CONCLUSIONS REFERENCES CHAPTER 8 - ELECTROCHEMICAL STUDIES OF SULPHIDE MINERALS IN THE PRESENCE AND ABSENCE OF A. FERROOXIDANS Denise Bevilaqua, Patricia Hatsue Suegama, Oswaldo Garcia Jr. and Assis Vicente Benedetti 1. INTRODUCTION ELECTROCHEMICAL TECHNIQUES EXPERIMENTAL RESULTS AND DISCUSSION CONCLUDING REMARKS ACKNOWLEDGMENT REFERENCES

11 X CHAPTER 9 BIOHYDROMETALLURGICAL APPLICATIONS OF IRON BIOREDUCTION Laura Castro, Felisa González, Antonio Ballester, Camino García-Balboa, Jesús Ángel Muñoz and María Luisa Blázquez 1. INTRODUCTION BIOREDUCTION OF IRON IN NATURE APPLICATION IRON BIOREDUCTION: SOME EXPERIMENTAL RESULTS ISOLATION OF Fe-REDUCER MICROORGANISMS BIOREDUCTION OF SOLUBLE IRON SOURCES BACTERIAL ADAPTATION TO ACIDIC PH BIOREDUCTION OF INSOLUBLE IRON SOURCES REFERENCES CHAPTER 10 BIOOXIDATION OF GOLD ORES Jaeheon Lee 1. INTRODUCTION MICROBIOLOGY FOR MINERAL PROCESSING STIRRED TANK REACTOR (STR) BIOOXIDATION BIOOXIDATION IN COLUMN MICROBIAL DEACTIVATION OF PREG-ROBBING CARBON NEWMONT S BIOHEAP NEVADA OPERATION REFERENCES CHAPTER 11 ARSENIC OXIDATION AND STABILIZATION OF REFRACTORY CONCENTRATES Paul I. Harvey, Quentin Graaff and Louis Pretorius 1. INTRODUCTION ARSENIC TREATMENT GEOCOAT ARSENIC TESTWORK REFERENCES CHAPTER 12 APPLICATION OF BIOTECHNOLOGY TO ENHANCE NUTRIENT BIOAVAILABILITY OF ROCK POWDER FOR CROP PRODUCTION SYSTEMS: A METHODOLOGICAL APPROACH Ricardo Melamed, Diego V. C. Cara, Luis G.S. Sobral, Francisco Lapido-Loureiro and José C. Gaspar 1. INTRODUCTION MASS BALANCE OF NUTRIENTS AS AFFECTED BY ROCK POWDER EFFICIENCY OF CARBONATITE POWDER TO SUPPLY K AND P APPLICATION OF BIOTECHNOLOGY TO ROCK POWDER THE SOIL AND THE MICROORGANISMS METHODOLOGY CONCLUSIONS REFERENCES

12 XI CHAPTER 13 BIODEGRADATION OF CRUDE OIL BEARING SOILS Willian Kohr 1. ORIGIN OF PETROLEUM GENERAL ASPECTS OF BIODEGRADATION CONTAMINATED SOILS AND UNDERGROUND BIOREMEDIATION FINAL REMARKS REFERENCES CHAPTER 14 BIO-DESULPHURISATION OF H 2 S BEARING INDUSTRIAL GAS STREAMS Martín Ramírez, Fernando Almenglo, Maikel Fernández, José Manuel Gómez, Domingo Cantero 1. INTRODUCTION MICROBIAL CICLING OF H 2 S BIOFILTRATION BIO-SR PROCESS OTHERS BIOPROCESSES CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

13 XII List of Contributors ANTONIO BALLESTER Material Science and Metallurgical Engineering Department, Chemical Science Faculty, Complutense de Madrid University, Madrid, Spain. ASSIS VICENTE BENEDETTI Chemical Institute, UNESP Univ. Estadual Paulista, São Paulo, Brazil. CAMINO GARCÍA-BALBOA Material Science and Metallurgical Engineering Department, Chemical Science Faculty, Complutense de Madrid University, Madrid, Spain. CARLOS EDUARDO G. DE SOUZA Centre for Mineral Technology/Brazilian Ministry of Science, Technology and Innovation, Brazil. CECILIA DEMERGASSO Biotechnology Centre, Católica del Norte University/Scientific and Technological Research Centre for the Mining Industry, Antofagasta, Chile. DÉBORA MONTEIRO DE OLIVEIRA Centre for Mineral Technology/Brazilian Ministry of Science, Technology and Innovation, Brazil. DENISE BEVILAQUA Chemical Institute, UNESP Univ. Estadual Paulista, São Paulo, Brazil. DENISSE QUIROGA Aguamarina S.A., Antofagasta, Chile. DIEGO V. CRESCENTE CARA Centre for Mineral Technology/Brazilian Ministry of Science, Technology and Innovation, Brazil. DOMINGO CANTER Department of Chemical Engineering and Food Technology, Natural Sciences Faculty. Universidad de Cádiz, Spain. EDGARDO DONATI Engineering Faculty, National University of Comahue, Buenos Aires, Argentina/CINDEFI (CCT LA PLATA-CONICET, UNLP)/Exact Sciences Faculty, National University of La Plata, La Plata, Argentina. ENRIQUE CARRETERO GeoBiotics, LLC, Colorado, United States.

14 XIII FELISA GONZÁLEZ Material Science and Metallurgical Engineering Department, Chemical Science Faculty, Complutense de Madrid University, Madrid, Spain. FERNANDO ALMENGLO Department of Chemical Engineering and Food Technology, Sciences Faculty. Cádiz University, Spain. FRANCISCO LAPIDO-LOUREIRO Centre for Mineral Technology/Brazilian Ministry of Science, Technology and Innovation, Brazil. GREGORY J. OLSON GeoBiotics, LLC, Colorado, United States. HELEN WATLING Parker Centre for Integrated Hydrometallurgy Solutions/CSIRO Minerals Down Under Flagship, Australia. JAEHEON LEE Newmont Mining Corporation, Colorado, United States. JEANNETTE VERA Aguamarina S.A., Antofagasta, Chile. JESÚS ÁNGEL MUÑOZ Material Science and Metallurgical Engineering Department, Chemical Science Faculty, Complutense de Madrid University, Madrid, Spain. JOHANNA OBREQUE Aguamarina S.A., Antofagasta, Chile. JORGE CASTRO Aguamarina S.A., Antofagasta, Chile. JOSÉ CARLOS GASPAR Geosciences Institute, Brasília University, Brazil. JOSÉ MANUEL GÓMEZ Department of Chemical Engineering and Food Technology, Natural Sciences Faculty. Universidad de Cádiz, Spain. JUAN RIVADENEIRA Catholic University of Chile, Santiago, Chile. LAURA CASTRO Material Science and Metallurgical Engineering Department, Chemical Science Faculty, Complutense de Madrid University, Madrid, Spain.

15 XIV LOUIS PRETORIUS GeoBiotics, LLC, Colorado, United States. LUIS G. S. SOBRAL Centre for Mineral Technology/Brazilian Ministry of Science, Technology and Innovation, Brazil. MAIKEL FERNÁNDEZ Department of Chemical Engineering and Food Technology, Sciences Faculty. Cádiz University, Spain/Study Centre of Industrial Biotechnology, Natural Sciences Faculty. Universidad de Oriente. Santiago de Cuba, Cuba. MARIA ALEJANDRA GIAVENO Engineering Faculty, National University of Comahue, Buenos Aires, Argentina/CINDEFI (CCT LA PLATA-CONICET, UNLP)/Exact Sciences Faculty, National University of La Plata, La Plata, Argentina. MARÍA LUISA BLÁZQUEZ Material Science and Metallurgical Engineering Department, Chemical Science Faculty, Complutense de Madrid University, Madrid, Spain. MARIA SOFIA URBIETA Engineering Faculty, National University of Comahue, Buenos Aires, Argentina/CINDEFI (CCT LA PLATA-CONICET, UNLP)/Exact Sciences Faculty, National University of La Plata, La Plata, Argentina. MARTÍN RAMÍREZ Department of Chemical Engineering and Food Technology, Sciences Faculty. Cádiz University, Spain. OSWALDO GARCIA Jr. (in memorian) Chemical Institute, UNESP Univ. Estadual Paulista, São Paulo, Brazil. PAMELA CHÁVEZ Aguamarina S.A., Antofagasta, Chile. PAMELA SOTO Biotechnology Centre, Católica del Norte University/Scientific and Technological Research Centre for the Mining Industry, Antofagasta, Chile. PATRICIA HATSUE SUEGAMA Exact Science and Technology Faculty, UFGD - Univ.Federal da Grande Dourado, Brazil. PAUL I. HARVEY GeoBiotics, LLC, Colorado, United States. PEDRO GALLEGUILLOS Biotechnology Centre, Católica del Norte University/Scientific and Technological Research Centre for the Mining Industry, Antofagasta, Chile.

16 XV QUENTIN GRAAFF GeoBiotics, LLC, Colorado, United States. RICARDO MELAMED Secretary of Politics and Programs of Research and Development, Ministry of Science and Technology of Brazil. TODD J. HARVEY GeoBiotics, LLC, Colorado, United States. VÍCTOR ZEPEDA Biotechnology Centre, Católica del Norte University/Scientific and Technological Research Centre for the Mining Industry, Antofagasta, Chile. WILLIAN KOHR Geo Fossil Fuels, LLC, United States.

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18 Biohydrometallurgical applications of iron bioreduction Introdution Anthropogenic activities account for the emission of four major types of greenhouse gases (GHG) with high contaminant potential due to their long permanence in the atmosphere: carbon dioxide (CO 2), methane (CH 4), nitrous oxide (N 2O) and halocarbons (fluorine, chlorine or bromine bearing gases). For the three former gases, the global concentration in the atmosphere has increased exponentially since In 2005, those concentrations exceeded, considerably, the values from natural sources recorded for the last 10,000 years (Figure 1). Spain, for instance, has doubled the CO 2 emissions in the last 13 years and significantly, but to a lesser extent, the emissions of methane and nitrogen oxide. Quantitatively speaking, the largest contaminant gas in the atmosphere is carbon dioxide (CO 2). According to the Synthesis Report 2007, the last of the four ones elaborated by IPCC (Intergovernmental Panel on Climate Change of United Nations), this gas represented in the last decade more than three fourths the global emissions of anthropogenic GHG (Ministry of Environment). The global emissions of CO 2 by sectors is given in Figure 2, and shows that both power supply derived from fossil fuels combustion and industry are the anthropogenic activities with a higher impact (45.3%) on the total emission. In turn, within the industrial sector, the available data corresponding to emissions of gases produced by the mining and metallurgical activities reach almost 8% of total. There is a generalized scientific consensus with respect to the idea that our way of producing and consuming energy is having an impact on global climate change. The potential effect of climate change is important as an environmental issue but also because of its economic and social consequences. As a global warming, it would also require a consensus worldwide response. Indeed, climate change cannot be precisely quantified since some uncertainties remain unsolved. Thus, the available information should be sufficient to adopt immediate resolutions, including the agreement known as "principle of precaution" referred in Article 3 of the Framework Convention on Climate Change (Naciones Unidas, 1992).

19 160 Laura Castro, Felisa González, Antonio Ballester, Camino García-Balboa, Jesús Ángel Muñoz and María Luisa Blázquez Figure 1. Concentrations of CO 2, CH 4 and N 2 O in the atmosphere over the last 10,000 years and since 1750.

20 Biohydrometallurgical applications of iron bioreduction 161 Figure 2. Contribution of different sectors to total emissions of anthropogenic GHG in 2004, in terms of CO 2 equivalents. In response to these challenges, the international community has reached two legal agreements: the mentioned United Nations Framework Convention on Climate Change, adopted in 1992 and operative since 1994, and the Kyoto Protocol, which establishes the practical basis to apply for the agreements reached in the former Convention. The Kyoto Protocol, adopted in 1997, establishes, for the first time, a reduction in net GHG emissions for the principal developing countries and economies in transition. The promulgation of new guidelines for enforcing an exhaustive control of anthropogenic activities is probably the most evident consequence of this changing movement. In this way, there have been regulations on the International Trading of Emission Rights, based on aspects, more or less complex, derived from the Kyoto Protocol. Furthermore, the UN Climate Change Summit celebrated in Copenhagen in November 2009, was an inflexion point and, in January 2010, the twenty seven EU ministries for the Environment reached the compromise of reducing CO 2 emissions by 80-95% in a medium term, with respect to levels of 1990, and a threshold value for the year 2050 with the aim of working for a low carbon economy. A consequence of the Emission Rights is that countries can obtain bonus through activities led to promote the development of new cleaner technologies. That is the case of research proposals focused on GHG emission reduction made by countries that have signed and ratified the Kyoto Protocol. In this way, the work shown in the present chapter is related with the search of cleaner environmental alternative routes for the beneficiation of iron ores. Iron is the fourth element most abundant in the earth s crust after Si, O and Al, averaging around 5%. This is probably the main reason for its importance for living processes (Castillo et al., 2005). Iron is found in nature in numerous minerals such as: hematite (Fe 2o 3), magnetite (Fe 3O 4), limonite (FeO(OH)), siderite (FeCO 3), pyrite (FeS 2) or ilmenite (FeTiO 3). Iron is mainly extracted to produce steel, an alloy where iron acts as the matrix element combined with other metallic and non metallic elements. Among the great variety of steels designed for specific requirements, most have been developed over the past ten years (International Iron and Steel Institute, 2008). This fact coincides with the sustainable increase in global steel production, around 3-5%, which, in turn, is a consequence of its larger consumption promoted mainly by emerging Asian countries

21 162 Laura Castro, Felisa González, Antonio Ballester, Camino García-Balboa, Jesús Ángel Muñoz and María Luisa Blázquez undergoing a rapid economic growth, China, in particular, the world leader in steel production (IISI Committee on Economics Studies, 2007). At present, steel is mainly produced by two routes: the classical iron and steel metallurgical plants, based on the sequence blast furnace-conversion-secondary metallurgy from iron-bearing minerals, and the electric steel mill that employs scrap and/or pre-reduced iron as raw materials fed into the electric furnace. On average, 1.7 tons of CO 2 are emitted per ton of steel produced and according to the International Energy Agency (IEA), this industry accounts, approximately, for 4-5% of the total emissions of CO 2 worldwide (Sustainability Report, 2008). In spite of the technological advances experimented by the iron and steel industries in the last 25 years, the emissions of CO 2 will have to be revised, sooner or later, and a more environmentally friendly process should be implemented. 2. Bioreduction of Iron in Nature There is a growing interest in developing biological technologies, also known as clean technologies, for dissolving and recovering valuable metals contained in ores, since they are non-polluting and economically feasible alternatives (Duménigo et. al., 2006). The ability of microorganisms to gain energy by changing the oxidation state of inorganic species is known since more than one century ago. In this sense, there is an emerging research field related to the natural iron cycle which could have a direct effect on the emission of greenhouse gases to the atmosphere. The importance of the iron cycle in natural processes has been recognized by several authors (Malki et al., 2005). The conversion between ferrous and ferric species takes place through redox reactions determined by environmental conditions: aeration, redox potential and ph (figure 3). Both type of reactions are complementary aspects in the biosphere and affect both metal mobility and metal distribution and accumulation in different sites. These transformations of iron-bearing compounds strongly depend on microbial activity. In acidic aerobic environments, microbial iron oxidation combined with the oxidation of sulphur reduced species is the predominant process. Ferrous ion oxidation can be catalyzed by different microorganisms depending on environmental conditions: Acithiobacillus and Leptospirillum ferrooxidans at moderate temperatures and in acid environments, Gallionella at neutral ph and Sulfolobus in acidic and thermophilic environments. These aerobic Fe- and S-oxidizing microorganisms have been known, studied and used since long time ago, especially in bioleaching processes of sulphide minerals.

22 Biohydrometallurgical applications of iron bioreduction 163 Figure 3. Biogeochemical cycle of iron. In anaerobic environments, the iron cycle is dominated by iron reduction coupled to the oxidation of some organic compound to carbon dioxide. In the absence of organic matter, ferric oxides usually are accumulated in sediments since the Fe-reducing activity decreases considerably. Most of the processes involved in iron reduction are performed by microorganisms able to respire iron. The ability of some organisms to use metals as electron acceptors in anaerobic conditions is a more recent finding (Lovley, 2002; Lloyd et al., 2003). Iron bioreduction was initially associated to the activity of diverse microbial consortia found in deep zones, as aquifers (Ghiorse, 1989) as subsoil (Ghiose and Wilson, 1988), which could be involved in the transport of environmental toxic contaminants and, thus, have a fundamental role in geochemical processes. The key role played by these microbial communities in the field of bioremediation or biotechnology has been recognized very recently (Fredichson et al., 1991; Fredichson, 1992). Iron is a key element in different natural processes as proven by the great abundance of its oxides in the earth crust. The microbial reduction of ferric iron and other metallic oxidized forms has a crucial participation in metal biogeochemical cycles but also in the final fate of organic matter and nutrients in a wide variety of environments. The use of metals as final electron acceptors is known as metal dissimilatory reduction which differs from the metal reduction associated to its retention inside cells known as assimilatory reduction (Lovley, 2002). Until recently, the reduction of Fe(III) in sedimentary environments and in the absence of oxygen was interpreted as due to abiotic processes controlled by changes of ph and redox potential. At present, microbial respiration, using Fe(III) oxides as final electron acceptor, is recognized as an important process in anoxic environments in which ferric iron reduction is mainly catalyzed by microorganisms (Villas-Boas and Sánchez, 2006; Luu and Ramsay, 2003). Thermodynamically speaking, the reduction of Fe(III) in this type of systems is more favourable than the reduction of other chemical species such as sulphate ion; however, this oxidizing agent produces less energy than the reduction of oxygen or nitrate. Geochemical and microbiological evidences suggest that reduction of Fe(III) might be a very early form of respiration on Earth and is a good candidate in the search for life on other planets. Nowadays, it is known that Fe(III) can be the dominant electron acceptor in the microbial respiration of many surface environments. Therefore, Fe(III)-reducing

23 164 Laura Castro, Felisa González, Antonio Ballester, Camino García-Balboa, Jesús Ángel Muñoz and María Luisa Blázquez microbial consortia can be responsible for most of the oxidized organic matter in those environments (Lloyd, 2003). Bacteria of genus Geobacter and Shewanella were the first organisms described that use the energy conservation mechanism for its growth (Venkateswaran, et al., 1999; Geobacter Project). Both genera include numerous species: G. metallireducens, G. bremensis, G. pelóphilus, G. sulfurreducens etc., in the former, and S. algae, S. amazonensis, S. frigidimarina, S. gelidimarina, S. oneidensis, S. putrefaciens, S. béntica, S. hanedai, S. pealeana, S. woodyi etc., in the latter case. Geobacter metallireducens, probably the most studied bacteria, is a strictly anaerobic bacteria belonging to δ-proteobacteria, Gram-negative, without motility and unable to form spores. It was the first microorganism described able to oxidize organic compounds containing multiple carbon atoms to carbon dioxide and using Fe(III) oxides as the sole electron acceptor. G. metallireducens and other Geobacter species isolated later provide a model to explain the geological transformations experimented by iron on the Earth s crust, such as the massive accumulation of magnetite. Since pioneering studies in 1980 s, a great number of microorganisms have been isolated with the ability to grow using Fe(III) as electron acceptor. All bacteria of genus Shewanella are also Gram-negative and do not form spores but are motile because of the presence of a flagellum. S. putrefaciens, an anaerobic facultative bacteria that can grow both in the presence or absence of oxygen, belong to γ- Proteobacteria using Fe(III), Mn(IV), or U(VI), as electron acceptor during its growth. Newly discovered Fe(III)-reducing bacteria are: Bacillus infernus, Deferribacter thermophilus (both thermophiles able to grow in profound depths) and Thermoterrabacterium ferrireducens. On the other hand, Geoglobus ahangari and Ferroglobus placidus were the first two hiperthermophilic bacteria able to oxidize acetate anaerobically using ferric oxide as the sole electron acceptor. Moreover, acidophilic bacteria such as Acidithiobacillus ferrooxidans can obtain energy indistinctly whether from the oxidation of Fe(II) to Fe(III) in aerobic environments, or from the reduction of ferric iron using sulphur as electron donor. Different electron donors can be used by bacteria: complex organic compounds, fermentable substances (sugars such as glucose, amino acids etc.), aromatic compounds (toluene, benzene), fatty acids of long or short chain, acetate, hydrogen, organic contaminants etc. Finally, however, the preferred electron donor will depend on the type of bacteria and environmental conditions, among other factors. The mechanism involved in the reduction of oxidized iron forms by Fe-reducing bacteria, which is determinant in order to control the rate of the process, is not completely understood yet (Lovley, 1997). The reduction process of Fe(III) to Fe(II) is affected by a great number of variables, among the most important are: the raw material (both minerals of Fe(III) and organic compounds acting as electron donors), environmental conditions (depth, temperature, ph, concentration of Fe(III) etc.) and type of microorganism involved in the reduction process. A key factor in the reduction process is the access of bacteria to the insoluble Fe(III) compound. On the other hand, most electron acceptors (oxygen, nitrate and sulphate) used by bacteria are soluble and can reach the inner cell. Nevertheless, for microorganisms that use insoluble solids (such as hematite, -Fe 2O 3, and goethite, -FeOOH) as electron acceptors, its dissolution and then transport of ferric ion to the bacterial cell cannot be

24 Biohydrometallurgical applications of iron bioreduction 165 the only explanation. Therefore, bacteria should use different strategies to transfer electrons to minerals during its respiration. Based on the great phylogenetic variety of Fe(III)-reducing microorganisms, there must be more than one strategy for ferric ion reduction (Luu and Ramsay, 2003). The mechanisms of Fe(III) reduction have been studied in detail for Shewanella oneidensis and Geobacter sulfurreducens (Lloyd et al., 2003; Lloyd, 2003). Despite that the reductase enzyme has not been yet identified, the participation of c-type cytochromes in the transport of Fe(III) has been demonstrated. In certain cases, the outer membrane or the surface of cells have shown to be actively participating in the direct electron transfer to the Fe(III) oxides which are very insoluble at ph near neutrality (Figure 4). Additionally to the direct transfer, bacteria can use soluble electron shuttles, organic compounds such as quinones, which can be transferred between metal-reducing microorganisms and mineral surface. Furthermore, bacteria can also dissolve minerals through chelating molecules produced by themselves. In this way, the addition of synthetic chelates can stimulate the electron transfer to iron minerals. According to this, iron bioreduction can take place without having direct contact (Figure 4) between the microorganism and the solid (Lloyd, 2003) Figure 4. Mechanism of actuation of Fe(III)-reducing microorganisms: A) Direct and B) Indirect (Lloyd, 2003). 3. Application The blast furnace process for making pig iron is being questioned since several years ago, although certainly not very deeply. In such process, the working temperature and the gas composition to reduce iron oxide ores are achieved by consuming large amounts of carbon as coke, which finally is emitted into the atmosphere as CO 2. This agrees with the fact that the primary iron industry is one of the most pollutants. Certainly the technological advances achieved in the iron and steel industry in the last 25 years were mainly focused on solving some drawbacks, especially those related to atmospheric pollution. In this way, some advances in the steelmaking process include: derivados

25 166 Laura Castro, Felisa González, Antonio Ballester, Camino García-Balboa, Jesús Ángel Muñoz and María Luisa Blázquez An increase in energy efficiency. An increase of the recycled material volume. Improvements in the environmental protection techniques used. A substantial reduction of CO 2 emissions, nearly 50% (Sustainability Report, 2008). However, the new processes for making steel and secondary products are approaching to the top limit of technology, especially with regard to iron and steel industries. Thus, considering the new environmental requirements that have to be fulfilled from 2012, according to Kyoto protocol, it is expected great research efforts by the companies related with that area. In the medium term, advances should come from technological transfer while, in the long term, new viable and sustainable technologies should be implemented. Having in mind that the solubilisation of insoluble Fe(III) oxides in aqueous solutions can take place through an electron transfer process, a biotechnological alternative to the GHG emissions by the iron industry could be the use Fe(III)-reducing microorganisms. However, the possibility of applying this process will require a deep knowledge of environmental conditions such as: microorganisms involved, microbial action mechanisms, chemistry of products formed, and those variables that allow the optimization of the bioreduction process of iron minerals. Furthermore, this is an unexplored and totally novel via for the metal recovery. There is a lack of work with respect to other alternatives to produce iron different from conventional pyrometallurgical methods. A research group of the Massachussets Institute of Technology (MIT) has proposed a new route to produce iron using molten salts electrolysis to decompose the iron oxides (Khetpal et al., 2004). Working at lab scale and at 1450ºC, Khetpal et al. (2004), were able to produce metallic iron in the cathode plus the evolution of oxygen in the anode in an electrolytic cell using platinum as cathode electrode and a mixture of oxides (FeO MgO CaO SiO 2) as electrolyte. According to these authors, the main advantage of this process was the absence of emissions of greenhouse gases to the atmosphere. Nevertheless, there was no mention to the source of electricity used in the hypothetic case the process reaches an industrial scale. This point is crucial since the energy provided by fossil fuels introduces as many problems as the own blast furnace for producing iron. The lack of bibliographic data with respect to new routes for iron making that lower significantly the emissions of CO 2 opens all kind of possibilities on this subject. For that reason, the new technologies should be effective by its own but also from an energetic and environmental point of view. That should be the case of the microbiological treatment of Fe(III) minerals as a way of dissolving iron as ferrous ion. Fe(II) is more soluble in aqueous solution than Fe(III) and, thus, the product obtained in the bioleaching of iron ores should be more easily leached with the standard hydrometallurgical acid reagents. Nevertheless, this route has not been used yet and could be a good starting point. The first studies on bioreduction of iron oxides date back to the end of the 20th century. A pioneer work published in 1998 attempted to deepen in the interaction between Pseudomonas and different iron minerals (Arnold et al., 1988). Nowadays, however, those microorganisms have not a key role in the bioreduction process. Later, new studies reported the mechanism by which microorganisms access insoluble Fe(III) as an energy source (Nevin and Lovley, 2002; Luu and Ramsay, 2003). In addition, some works have shown in detail the diverse prokaryotic microorganisms able to reduce both Fe(III) and Mn(IV) (Lovley, 2006). Recent studies have focused on the role play by iron microbial

26 Biohydrometallurgical applications of iron bioreduction 167 reduction in the dissolution of different insoluble Fe(III) sources: hydroxisulphates (Jones et al., 2006), oxyhydroxides (Bonneville et al., 2009), or even iron retained in bauxites (Papassiopi et al., 2010); others point to the formation of magnetite during dissimilatory dissolution of hematite (Behrends and Van Cappellen, 2007). In short, the latest studies on iron bioreduction have basically confirmed the interest of the scientific community in this subject and continue pushing towards the exploration of biotechnological routes for the metal recovery. Finally, other important areas where iron-reducing microorganisms could be applied are: bioremediation phenomena, both in soils and in aquifers, of organic contaminants, but especially of metals, and the so-called microbial fuel cells. Biodegradation of organic contaminants is a process relatively well documented that has been implemented successfully at real scale. Nevertheless, the technical basis of natural attenuation of heavy metals is far less known. Despite heavy metals are persistent in nature and are not destroyed by biological processes, many metals can be bio-reduced or bio-oxidized to less toxic and less mobile forms (Álvarez and Illman, 2006). Bioremediation of soils might be important in sites contaminated with mining-bearing sulphide residues, especially pyritic wastes (FeS 2). Thus, the iron and sulphur biogeochemical cycles become essential for a correct interpretation of acid drainages generation or the precipitation of dissolved metals (Ahmad et al., 2005). On the other hand, the basis of microbial fuel cells can be found in certain microorganisms that besides transfer electrons to natural metal oxides, e.g. iron, can transfer electrons to an electrode (Lloyd et al., 2003). Some members of the Geobacteraceae family can conserve energy for growth by coupling the oxidation of acetate or aromatic compounds with the electron transfer to an electrode (Bond et al., 2002; Bond and Lovley, 2003). Geobacter creates a biofilm on the electrode surface which is very stable and produces electricity. This new way of respiration has potential industrial application for electricity generation from organic wastes (Lovley, 2006). According to this new technology, bacteria able to oxidize organic matter colonize the anode supplying electrons to the electrode. Then, it works like a galvanic cell that produces electric energy from chemical energy and has the additional advantage of avoiding the contaminant effects produced by the combustion of fossil fuels. All these studies point out to the continuous progress achieved in this field and to the great possibilities offered by this type of systems to produce energy from the growth of microorganisms. In consequence, the application possibilities of bioreduction processes are enormous. In some cases, research studies are in an advanced stage, in others there is hardly experimental results. The following sections collect experimental results concerning iron bioreduction of both soluble and insoluble sources. 4. Iron Bioreduction: Some Experimental Results Currently, biotechnological solutions are a good choice to environmental and economic problems related with materials and human health. Biotechnologically speaking, the use of dissimilatory Fe(III)-reducers for iron extraction from recalcitrant ores represents a low-cost environmentally friendly technology. Therefore, bioleaching of iron minerals and bioremediation techniques could be applied to solve environmental issues related to mineral processing activities.

27 168 Laura Castro, Felisa González, Antonio Ballester, Camino García-Balboa, Jesús Ángel Muñoz and María Luisa Blázquez The studies on iron bioreduction performed by the Biohydrometallurgy Research Group of Complutense University of Madrid have been focused on the screening and adaptation of bacterial species in order to improve the solubilisation of iron compounds. Among all terminal electron acceptors, ferric iron is the most naturally abundant in many subsurface environments. Even in anaerobic environments where Fe(III) is not very common, ferric iron reduction may still be the dominant electron accepting process. Iron is a unique terminal electron acceptor as it can be readily re-oxidized and returned to sediments by settling, providing a mechanism for Fe(III) regeneration and for the movement of oxidizing potential into anaerobic zones. The iron cycle was reproduced in both weathering and attenuation processes, at laboratory scale, by using an indigenous microbiota isolated from an extremely acidic site (García-Balboa et al., 2009). In this study, the chemical and microbiological reactions that occur naturally, when a metal sulphide is discharged onto a natural soil, were investigated, with special emphasis on iron cycle. For that purpose, enrichment cultures able to complete the iron cycle at highly acidic environments were obtained. According to the model proposed (Figure 5), a synergistic effect occurs between Fe-oxidizing bacteria and Fe-reducing bacteria coupled to the weathering of a pyritic waste and to the attenuating effect of discharge, respectively. In addition, bacteria collected were able to reduce iron even at low ph values. Figure 5. Model proposed for the cycle of iron at an acidic environment. In the dissimilatory Fe(III) reduction process, microorganisms transfer electrons to an external source of ferric iron, which is reduced to ferrous iron but without iron assimilation. For instance, the use of lactate as electron donor and iron (in soluble or insoluble form, e.g., hematite) as electron acceptor can be represented by the following reactions: (1) (2)

28 Biohydrometallurgical applications of iron bioreduction 169 Dissimilatory iron-reducing bacteria grown on iron oxides or soluble ferric iron sources are associated to the formation of biogenic Fe(II) and/or Fe(III) as siderite (FeCO 3) or vivianite (Fe 3(PO 4) 2.8(H 2O). The composition of aqueous media where microbial iron reduction takes place has a strong impact on the rate and extent of iron reduction and the chemistry of the solids produced, which can provide a feedback control mechanism on microbial metabolism. 5. Isolation of Fe-reducer Microorganisms The aim of isolation was to obtain not strictly anaerobic species but facultative anaerobes with a greater flexibility and versatility to facilitate up-scaling from culture conditions to industrial bioreactors. Moreover, the recovery of metal of interest in soluble form is a key factor for the industrial implementation of anaerobic bioleaching. An interesting aspect for applying dissimilatory Fe(III) reduction would be the production of soluble products after mineral dissolution due to a considerable amount of precipitates generated during the process have detrimental blocking effects on valves, piping and other components of the reactor. Several strategies can be used to overcome these difficulties and the research group is mainly working in two directions: the adaptation of the bacterial cultures to lower ph values and the improvement of bioleaching species with the ability to hardly produce iron precipitates. The first activity developed was the sampling and the enrichment of cultures. The experiments were performed using a natural consortium and several pure strains isolated and identified as Aeromonas hydrophyla, Serratia fonticola, Clostridium celerecrescens and Clostridium amygdalinum. The samples for the microbial studies were collected from the edge of an open-pit lake surrounding an extinct mine site named Brunita (formerly a source of Pb-Zn ores) near La Unión (Murcia, Spain). The artificial lake, formed after the shutdown of the mineral activity, has a capacity of approximately 622,000 m 3 of water at ph and with a high MgSO 4 concentration (Robles-Arenas, 2007). Molecular analysis by Denaturing Gradient Gel Electrophoresis (DGGE) of the bacterial prokaryotic consortium revealed low species richness with a maximum of three different bands, both in bacterial and archaeal fingerprints. An explanation could be that the culture employed in the molecular analysis was successively adapted to grow under very restricted conditions and probably contained only some of the species originally present in the bacterial population. In contrast, the genus Clostridium was the most abundant and two of the isolated corresponded to C. celerecrescens and C. amygdalinum sp. Clostridia species can use different electron acceptors exhibiting frequently predominance in enrichment cultures (Wang et al., 2009).

29 170 Laura Castro, Felisa González, Antonio Ballester, Camino García-Balboa, Jesús Ángel Muñoz and María Luisa Blázquez Table 1. DGGE analysis corresponding to the bacterial consortia culture. Bacteria BLAST Band pb Close sequence Coverage (%) Maximum identity (%) Number of access to NCBI Database Relative abundance (%) Selenomona from anoxic bulk soil 16S rrna gene (strain SB90) Clostridium from anoxic bulk soil 16S rrna gene (strain XB90) AJ FJ Clostridium sulfatireducens 16S ribosomal RNA AY gene, partial sequence 4, 2, 5 - Not identified bands Archaea Band 3 pb 457 Ferroplasma acidiphilium strain DR1 16S rrna gene, partial sequence AY , 2 - Not identified bands Bioreduction of Soluble Iron Sources Soluble ferric iron forms are frequently used to grow ferric reducing bacteria. Despite soluble Fe(III) do not represent an environmentally significant form of iron, it allows culturing these bacteria easily. In our research, ferric citrate, a commonly ferric soluble form, was used (García-Balboa et al., 2010a). The prokaryotic consortium reduced Fe(III)-citrate quickly and effectively, and complete iron bio-reduction was achieved in 36 hours. However, ferrous iron did not remain in solution and a white precipitate, a mixture of vivianite and siderite, was formed. A. hydrophila showed the best reduction efficiency of the four isolates tested: 82% after 2 days and a maximum level of bioreduction of 93% after 3 days. Unlikely, S. fonticola achieved 48% of Fe(III) reduction in 15 days and a maximum reduction of 59% in 27 days. Cultures of C. celerecrescens reduced Fe(III) slowly and only about 30% of bioreduction was achieved in 21 days and no further reduction took place in the following 20 days. By contrast, C. amydalinum showed, like A. hydrophila, a great efficiency with 93% of reduction in 3 days. S. fonticola and C. celerecrescens behaved similarly: both microorganisms were able to reduce Fe(IIII) via a dissimilatory pathway but the fermentative pathway was predominant. In spite that the reaction yield using A. hydrophila and C. amygdalinum was similar and the kinetics of iron reduction was excellent, there was a very important difference between them: the formation of precipitates. While C. amydalinum formed a brownish precipitate, A. hydrophila presented the additional advantage that Fe(II) remained in solution. As previously mentioned, this is an interesting result as it shows that iron ores can be reduced without the need of extreme ph and extra energy inputs. Many respiratory microorganisms that grow anaerobically with sulphate serving as the electron acceptor also have the ability to enzymatically reduce ferric iron but they cannot grow with Fe(III) as sole electron acceptor.

30 Biohydrometallurgical applications of iron bioreduction 171 The effect of sulphate reduction on Fe(III) reduction was studied using dissimilatory ferric- and sulphate-reducing bacterial cultures collected from an open-pit lake near La Unión (Murcia, Spain) (García-Balboa et al., 2010b). The study was an attempt to improve the biological reduction rate of Fe(III) as a biotechnological alternative to the reduction step in the steel-making process. The results obtained show a successive reduction of ferric and sulphates. Ferric iron was firstly reduced whereas sulphate reduction started after stabilization of the ferrous iron concentration. Thus, none synergetic effect was detected. This result makes disadvantageous the employment of mixed cultures where ferric and sulphate activities are present in order to obtain higher ferric bioreduction efficiencies, due to the potential enhancing role of sulphate reducers is diminished as a consequence of a competition effect. 7. Bacterial Adaptation to Acidic ph Most of the natural environments present circumneutral values of ph and in these circumstances, ferric iron is highly insoluble and occurs in a variety of amorphous and crystalline mineral forms. The main exception to this are extremely acidic environments (ph<2.5) where appreciable amounts of soluble ferric iron may occur, such as in streams draining coal and metal mines, and some geothermal areas. The origin of isolated bacteria used in our work was a soil surrounding an extinct mine area in an acidic medium. However, these bacteria with the ability to reduce ferric iron to ferrous were neutrophilic. An important aspect to improve the efficiency of these processes is the adaptation of iron-reducing bacteria in growth culture mediums at low ph. The bacterial cultures were progressively adapted to moderately acidic ph values (García-Balboa et al., 2010a). The consortium was grown successfully at ph 4.5, and culture adaptation through successive transfers was reached at ph values as low as 3.8. The kinetics of iron reduction at this ph became slower (1 week versus 3 days at ph 7).The successive adaptation of A. hydrophila to grow at moderately acidic ph was also successful. A concentration of 2.5 g/l of Fe(II) was reached in 6 days in cultures grown at ph 5.5 and with 3 g/l of ferric citrate, which is a promising result for the application in ore dissolution. S. fonticola was adapted to ph 6.0 and the maximum bio-reduction level of 30% was reached in 12 days. C. celerecrescens adapted to ph 4.8 was able to reduce 30% of ferric iron in 7 days, and a maximum concentration of 1.30 g/l of Fe(II) was measured after 21 days. C. amygdalinum presented approximately the same iron reduction kinetics at ph 7.0 and 4.5, reaching 90% of ferrous iron formation after 2 days. In all cases, cultures grown at low ph values shifted to the medium ph up to reach the optimum ph of each species, normally around 6.8. Another interesting aspect that is being developed is the use of microorganisms found in extremely acidic environments, iron- and sulphur-oxidizing acidophilic bacteria, and their application in biological leaching. Microbiological studies in these conditions have focused predominantly on microbial dissimilatory oxidation of iron and sulphur, mainly due to the great importance of these reactions in the bio-processing of metal ores (e.g., copper, uranium and gold ores). Bacteria can also be used in the biological desulphurization of coal or the weathering of mineral metal sulphides. Nevertheless, it has also been reported the iron reduction by acidophilic microorganism. Under anaerobic conditions, ferric iron can replace oxygen as an electron acceptor for the oxidation of elemental sulphur. At ph 2, the following reaction takes place:

31 172 Laura Castro, Felisa González, Antonio Ballester, Camino García-Balboa, Jesús Ángel Muñoz and María Luisa Blázquez ( G = -314 kj/mol) (3) In spite that several facultative anaerobic acidophilic bacteria have been isolated, the mechanisms by which microorganisms access and reduce ferric iron are poorly understood and this is a promising line of research. 8. Bioreduction of Insoluble Iron Sources One of the main challenges for the industrial implementation of anaerobic bioleaching, as well as a decisive research stage, is the adaptation of microbial cultures to grow on iron minerals instead of soluble sources of ferric iron. Neutrophilic Fe-reducing bacteria have also been shown to cause the reductive dissolution of some ferric iron minerals. Iron(III) oxyhydroxides occur in soils and sediments in a variety of forms, ranging from amorphous phases, such as ferrihydrite (FeOOH. 4H 2O), to well-crystallized minerals, such as hematite (α-fe 2O 3) or goethite (FeOOH). Several authors have studied the effect of different mineral properties: crystallinity, particle size, specific surface area and solubility, exhibiting the dependence of Fe(III) reduction kinetics on oxyhydroxide variations (Larsen and Postma, 2001; Bonneville et al., 2004; Bonneville et al., 2009;). Amorphous ferric compounds are reduced preferentially over crystalline Fe(III) oxyhydroxides and, among the crystalline ferric phases, the reactivity towards microbial iron reduction decreases according to the sequence: lepidocrocite (γ-feo(oh)) > hematite > goethite. Firstly, a comparative study on the iron reducing ability of pure anaerobic strains of Geobacter metallireducens and Bacillus infernus, from Oregon Collection of Methanogens, was investigated using magnetite with or without the addition of ferric pyrophosphate (Fe 4(P 2O 7) 3) as electron acceptor (Crespo et al., 2007). It was observed that the bioreduction process was not affected by the type of bacterial strain used. Magnetite is not a final product in the dissimilatory reduction of ferric oxides; then, it is also susceptible to reduction and the presence of vivianite from the biogenic Fe(II) was detected. In later studies, the cultures collected from the abandoned mine site near La Unión (Murcia, Spain) were grown on different insoluble ferric sources. In this case, special efforts were made in the utilization of jarosite as ferric iron acceptor. The reduction of this mineral has hardly ever been reported and could be attractive since jarosite is an important sulphate mineral, which occurs in acidic mine drainage environments and in the hydrometallurgy of zinc. Moreover, jarosite could contain lead, silver and gold in its composition and these metals could be released by bacteria and recovered from solution. In experiments carried out with hematite, pure cultures were unable to reduce the mineral due to its high crystallinity; only the bacterial consortium showed the ability of developing the transformation (García-Balboa et al., 2010a). The mixed culture was adapted to grow on two different oxyhydroxides without any other source of electron acceptor: ferrihydrite, an amorphous mineral synthesized in the laboratory by neutralizing FeCl 3, and, ammonium jarosites produced by a pure culture of Sulfolobus metallicus. A 50% of bioreduction was achieved for ferrihydrite and 60% for jarosite (Figure 6a) (García-Balboa et al., 2009). Finally, the Fe(III) reducing ability of the mixed culture was studied using an iron ore from San Isidro (Venezuela), which was formed by a residual concentration of iron oxides and hydroxides from ferruginous quartzites containing mainly two phases, goethite and hematite (Laguna et al., 2011). However,

32 Biohydrometallurgical applications of iron bioreduction 173 ferrous iron in solution was undetectable in the test with all ferric iron in solid form (Figure 6b). These experiments confirmed that variations in mineral properties affect the microbial reduction rate. Figure 6. Dissimilatory reduction kinetics of: (a) ferrihydrite and jarosite and (b) a natural iron ore using the bacterial consortium. On the other hand, the culture of A. hydrophila was able to completely reduce ferrihydrite in 7 days using a 75:25 liquid/solid ratio, and also with a 50:50 L/S ratio. None precipitates was observed and Fe(II) and Fe(III) species remained in solution. In addition, this culture was able to reduce ammonium jarosite but not hematite. The results obtained would be an indication that microbial catalysis of anaerobic iron leaching has commercial potential. The ability of keeping ferrous iron in solution may offer an economically and environmentally friendly alternative to traditional leaching processes Mechanism of Ferric Iron Bioreduction: Bacteria-Mineral Interaction The mechanisms by which microorganisms can access and reduce insoluble iron sources are poorly understood. Initially, it was considered that microorganisms could reduce ferric oxides exclusively by direct contact. However, recent findings on the role played by extracellular electron shuttling and Fe(III)-chelating compounds, as intermediary between bacterial cells and mineral, have changed this opinion. These intermediary compounds include microbiological secreted compounds or exogenous electron shuttling agents. In addition, the production of microbial extracellular polymeric substances (EPS), with a high affinity for binding metal cations, is being studied. Humic substances are ubiquitous in soils and sediments and are formed during plants degradation, animals and microorganisms. They can be described as dark-colored, acidic, predominantly aromatic, hydrophilic and chemically complex. The presence of humic substances significantly improves ferric iron reduction when coupled to organic carbon oxidation due to chelation of Fe(III). These substances serve as electron shuttles between the cells and the ferric oxides. We used AQDS, an analogue humic acid exhibiting extracellular quinones that can serve as electron acceptor for microbial respiration. Cells transfer electron to AQDS, generating anthrahydroquinone-2,6-disulphonate (AHQDS). AHDS can, then, shuttle electrons to Fe(III), regenerating AQDS. In our studies, the ability of the isolated cultures to perform the reduction of ferric iron, from oxyhydroxides in the absence of direct contact, was investigated using dialysis membranes to separate the ferric solid from cells.

33 174 Laura Castro, Felisa González, Antonio Ballester, Camino García-Balboa, Jesús Ángel Muñoz and María Luisa Blázquez In addition, the isolated cultures can produce exopolymeric substances (EPS) that could catalyze the dissolution of jarosites. In this case, the use of dialysis membranes could stimulate the production of EPS when bacterial cells are in the presence of Fe(III). FEG images of A. hydrophila and S. putrefaciens (collection culture), showing biogenic material (Figure 7), could explain the special feature of these cultures to keep ferrous iron in solution. On the other hand, cells of C. amygdalinum free of biogenic material (Figure 7d) would be in agreement with the slow iron reduction rates observed. (A) (B) (C) (D) Figure 7. FEG images of different bacterial cultures grown on jarosite: (a) Shewanella putrefaciens, (b) bacterial consortium, (c) Aeromonas hydrophyla, (d) Clostridium amygdalinum. Our results point to the fact that bacteria might contact mineral surfaces in several ways and that the direct contact between cells and iron oxides seems not always necessary. Microorganisms can also use exogenous electron shuttles that, despite of facilitating the ferric iron reduction, may have important environmental implications.

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