ENVIRONMENTAL BIOTECHNOLOGY ASSIGNMENT ON

BIOMINING

BIOMINING - One of the marvels of Biotechnology of present day for environment-friendly mining and mineral processing industry.

Submitted to: Dr. Sumedha Mohan

Submitted by: Darpan Raghav

BSB/09/109

ABSTRACT
Biohydrometallurgy can be defined as the field of applications resulting from the control of natural (biochemical) processes of interactions between microbes and minerals to recover valuable metals. Many biotechnology-derived processes use microorganisms to help ease the usage of harmful chemicals in various industrial processes. The mining industry uses microorganisms and their natural ability to digest, absorb, and change the quality of different chemicals and metals, to refine ores. Microorganisms have been introduced to various areas of the mining industry with phenomenal success. Advances in biotechnology have permitted the extraction of metals from low-grade ores, improved recovery rates at operations, and reduced operating costs.

Biomining is the extraction of specific metals from their ores through biological means usually bacteria. Although it is a new technique used by the mining industry to extract minerals such as copper, uranium and gold from their ores but nowadays biomining occupies an increasingly important place among the available mining technologies. Today biomining is no longer a promising technology but an actual economical alternative for treating specific mineral ores. Traditional extractions involve many expensive steps such as roasting and smelting, which requires sufficient concentrations of elements in ores while low concentrations are not a problem for bacteria because they simply ignore the waste which surrounds the metals, attaining extraction yields of over 90% in some cases. In simple language we can say that biomining is a mixture of both Chemistry and Microbiology.

INTRODUCTION
Our ability to harness the natural capability of certain microbes to decompose a variety of mineral deposits is an old process that dates back to Roman times in the first century BC and probably the Phoenicians before that. Those early miners used microbial activity to leach copper from ore without being aware that microbes were involved but now with increasing research trend in mineral biotechnology our interest toward the phenomena has grown up and has given some potential results which bring the revolution in mining industry. As the world wide high grade ore reserves are falling day at appalling rate as most are worked out because of high metal demand, traditional techniques like pyrometellury and chemical processings are becoming more and more economically in viable. Micro organisms bear a clear advantage over it, as not only they offer a economically viable option but is also clean technology. Microbes such as bacteria and fungi convert metal compounds into their water-soluble forms and are biocatalysts of these leaching processes. Additionally, applying microbiological solubilisation processes, it is possible to recover metal values from industrial wastes which can serve as secondary raw materials. Now with the gaining importance this mechanism is being used to recover metals such as gold, copper, iron, uranium. Following diagram explains complete biomining process in brief:

Bio-mining is an application of biotechnology in recovery of various minerals from ore. Biomining is defined as extracting mineral ores or enhancing the mineral recovery from mines using microorganisms instead of traditional mining methods. Copper was the first metal extracted using microorganisms in the ancient past in the Mediterranean region. Bio-mining is becoming popular because it is cheap, reliable, efficient, safe, and environmentally friendly, unlike traditional mining methods. The efficiency of bio-mining can be increased either by finding suitable strains of microorganisms or by genetically modifying existing microorganisms, made possible due to rapid advances in the field of biotechnology and microbiology. Ores of high quality are rapidly being depleted and bio-mining allows environmentally friendly ways of extracting metals from low-grade ores (ores that have small amounts of valuable metals scattered throughout). Bio-mining includes two different chemical processes called bioleaching and bio oxidation. Thus, bio-mining is an application of biotechnology and is also known as microbial leaching or alternately, bio oxidation.

DEFINITION OF BIO-MINING

Bio-mining is defined as a general term used to describe the use of micro organisms to facilitate the extraction of metals from sulfide or iron containing ores.

It involves two processes : a. Bioleaching b. Bio-oxidation

HISTORICAL PERSPECTIVE
The first report where bio-mining have been involved is given by Roman writer Gaius Plinius Secundus in 13th century. During 15th century Georgius Agricola describes in his work de re metallica, the technique for recovery of copper. In 1878, in Portugal hydrometallurgical metal extraction were taken into consideration. In 1922, the oxidation of metal sulfides was described when mobilisation of zinc from zinc sulfide was investigated to be microbially mediated. In 1947, thiobacillus ferroxidans was identified as a part of microbial community found in acid mine drainage by Colmer and Hinkle. Zimmerley et.al in 1958 had granted first patent. In 1985, a major multidisciplinary and multi-institutional Chilean project on bacterial leaching of copper. In 1993, the first copper mine exploited solely by bacterial mining technology was developed in Minera Pudahuel in Chile. In 2002, a copper biomining project is carried out jointly by CODELCO and BHP Billiton. In Feb.2002, a series demonstration plant was commissioned in Hutti gold mines in Karnataka, India.

Woodcut from the book de re metallica written by Georgius Agricola (14941555) illustrating the manual recovery of copper-containing mine effluents which are collected in wooden basins and concentrated in the sun.

MICROORGANISMS IN BIOMINING
There are different types of bacteria present in nature that oxidize metal sulfides and solubilize minerals, thus, helping in their extraction from the ores. It is very important to select suitable microorganisms to ensure the success of biomining, a process which requires knowledge of properties of microorganisms, both physiological and biochemical. Bacteria are found to be the most suitable microorganisms that can be used for extraction of metals in bio-mining.

Characteristics of the bacteria used in bio-mining:

 Mineral extraction involves the production of high temperatures so the bacteria should be able to survive the heat, hence, they should be thermophilic.

 Bio-mining involves using strong acids and alkalis, hence, bacteria should be chemophilic.

 Bacteria should produce energy from inorganic compounds, hence, should also be autotrophic.

 The bacteria should be able to adhere to the solid surfaces or have the ability to form biofilms.

Identification of Bacteria Useful for Biomining Operations

There are wide varieties of bacteria with varying capabilities existing on earth; therefore, it is essential to identify precisely the types that can perform bio-oxidation/bioleaching effectively. Thiobacillus ferrooxidans is a chemophilic, moderately thermophilic bacteria which can produce energy from oxidation of inorganic compounds like sulfur and iron. It is the most

commonly used bacteria in biomining. Several other bacteria such as T.thioxidans, Thermothrix thiopara, Sulfolobus acidocaldarius and Sulfolobus. brierleyi are also widely used to extract various minerals.

Thermothrix thiopara is an extremely thermophilic bacteria that can survive very high temperatures between 60-75C and is used in extraction of sulfur. Techniques like genetic engineering and conjugation are used to produce bacteria with desired characteristics to increase the rate of bio-oxidation thus increasing the mineral yield through biomining. It is also important to identify biomining bacteria present in colonies of other bacteria. Techniques developed for this purpose include: (a) Immunoflourescence, (b) Dot immunoassay, and (c) Dot-blot hybridization.  Immunofluorescence - This technique is generally used to identify specific antibodies or antigens present in biological fluids. Fluorescent antibodies are used to identify biomining bacteria.

 Dot Immunoassay - This technique is used to identify ore-adhering bacteria like T.ferrooxidans and T.thiooxidans. The bacteria are applied in the form of dots on a nitrocellulose film. Antigen-antibody reaction is carried out on the film and then treated with a secondary antibody to make the reaction visible by producing a color. The sample can be approximated by comparison of the test sample with that of a known sample.

 Dot-blot Hybridization- This is a DNA based technique to identify biomining bacteria such as T.ferrooxidans. The bacteria are isolated from samples of ores and soil treated with sodium dodecyl sulfate (SDS). The cells are disrupted to extract DNA and the extracted DNA is then purified. The DNA obtained from ore sample is fixed on nitrocellulose membrane using southern blotting technique. Genetic probes are used to identify and distinguish various biomining bacteria used in this procedure. The DNA fragments on the membrane are treated with standard probes.

GENERAL PROPERTIES OF SOME MICROBES USED

Acidothiobacillus ferroxidans Chemolithotrophic microorganism, Rod shaped, non spore forming, gram negative, motile and single pore flagellated, CO2 is carbon source, Ferrous iron is oxidized, Ammonium as nitrogen source, pH varies between 2-8.

Acidithiobacillus caldus Cant oxidize Fe2+ and are highly acidophilic, Optimal growth temperature is 45C, Grow mixotrophically using yeast extract or glucose.

Acidothiobacillus thiooxidans Chemolithotrophic, Cant oxidise Fe2+, Gram negative,

Can only use reduced sulfur as electron donor, Maximum growth temperature is 35C, Very acid tolerant (pH 0.5).

Leptospirillum ferrooxidans Gram negative, Very acidophilic (optimum pH 1.5-1.8), Can only oxidise Fe2+ not S2-, They use O2/H2O couple (+820 mV) as electron acceptor. Thus they are obligate aerobes.

Sulfolobus ambivalens Grow autotrophically at pH 1.3 and temperature 50-90 C, Derive energy from oxidation of sulfur compounds and of ferrous iron.

Example of Metal Sulphide Minerals.

Iron sulfides: Pyrite FeS2 Pyrrhotite Fe7S8-FeS Mixed Iron sulfides : Calcopyrite CuFeS2 Bornite Cu5FeS4 Stannite Cu2FeSnS4

Arsenopyrite FeAsS Other metal Sulfides : Chalcolite Cu2S Covellite CuS Enargite Cu3AsS4 Galena PbS Millerite NiS Cinnabar HgS Molybdenite MoS2 Argentite Ag2S

BIO-MINING RECOVERY
Minerals are recovered from ores by the microorganisms mainly by two mechanisms: (a) Oxidation and (b) Reduction.

(a) Oxidation
The microorganisms like T.ferroxidans and T.thioxidans are used to release iron and sulfur respectively. T.ferroxidans oxidize ferrous ion to ferric ion.

4Fe++ + O2 + 4H+

Fe+++ + 2H2O

The bacteria attach to the surface of the ore and oxidize by a direct and indirect method.

Direct Method

In this method the ore is oxidized by the microorganisms due to the direct contact with the compound. 2FeS2 + 7O2 + 2H2O 2FeSO4 + 2H2SO4

Indirect Method

In this method the mineral is indirectly oxidized by an agent that is produced by direct oxidation. For example, the ferric ion produced by the above reaction is a powerful oxidizing agent and can release sulfur from the metal sulfides. Thus production of ferric ion indirectly causes oxidation of metal sulfide resulting in the breaking of the crystal lattice of the heavy metal sulfide and separating the heavy metal and sulfur. CuS + Fe+++ Cu+ + S + Fe++

This method is further divided into 2 different mechanisms:

Thiosulfate mechanism:

It is used for the oxidation of acid insoluble metal sulfides such as pyrite. In this mechanism solubilization is through ferric iron attack on acid insoluble metal sulfides, with thiosulfate being the main intermediate and sulfates the main end product. FeS2+6Fe3++3H2O S2O32- +8Fe3+ + 5H2O S2O32+ + 7Fe2+ + 6H+ 2SO42- + 8Fe2+ + 10H+

Polysulfide mechanism:

It is used for the oxidation of acid soluble metal sulfides such as calcopyrite or galena. In this mechanism solublization of acid soluble metal sulfide is through a combined attack by ferric iron and protons with elemental sulfur as the main intermediate.  2MS + 2Fe3+ + 2H+  H2Sn+ 2Fe3+  0.25S8+ 3O2 + 2H2O 2M2+ + H2Sn + 2Fe2+ 0.25S8 + 2Fe2+ + 2H+ 2SO42- + 4H+

(b) Reduction
Bacteria like Desulfovibro desulfuricans play an active role in reduction of sulfates which results in the formation of hydrogen sulfides. 4H2 + H2SO4 H2S + 4H2O

FACTORS AFFECTING BIOMINING

Success of biomining and efficiency in recovery of minerals depends on various factors some of which are discussed below.

(a) Choice of Bacteria This is the most important factor that determines the success of bioleaching. Suitable bacteria that can survive at high temperatures, acid concentrations, high concentrations of heavy metals, remaining active under such circumstances, are to be selected to ensure successful bioleaching.

(b) Crystal Lattice Energy This determines the mechanical stability and degree of solubility of the sulfides. The sulfide ores with lower crystal lattice energy have higher solubility, hence, are easily extracted into solution by the action of bacteria.

(c) Surface Area Rate of oxidation by the bacteria depends on the particle size of the ore. The rate increases with reduction in size of the ore and vice-versa.

(d) Ore Composition Composition of ore such as concentration of sulfides, amount of mineral present, and the extent of contamination, has direct effect on the rate of bio-oxidation.

(e) Acidity Bio-oxidation requires a pH of 2.5-3 for maximum results. The rate of bio-oxidation decreases significantly if the Ph is not in this range since the activity of acidophilic bacteria is reduced.

(f) Temperature The bacteria used in biomining are either mesophilic or thermophilic. Optimum temperature is required for bio-oxidation to proceed at a fast rate. Optimum temperature range for a given bacteria is between 25-35 C depending on the type of ore being selected. The rate of bio oxidation is reduced significantly if the temperature is above or below the optimum temperature.

(g) Aeration The bacteria used in biomining are aerobic thus require an abundant supply of oxygen for survival and growth. Oxygen can be provided by aerators and pipes. Mechanical agitation is also an effective method to provide continuous air supply uniformly and also to mix the contents.

(h) Solid-liquid Ratio The ratio of ore/sulfide to the leach solution (water + acid solution + bacteria inoculum) should be maintained at optimum level to ensure that bio-oxidation proceeds at maximum speed. The leach solution containing leached minerals should be removed periodically and replaced with new solution.

(i) Surfactant Adding small amounts of surfactants like Tween 20 to the leaching process increases the rate of bio-oxidation of minerals from sulfide ores. The surfactants decrease the surface tension of the leach solution, thus, wetting the ore and resulting in increased bacterial contact which ultimately increases the rate of bio-oxidation.

TECHNOLOGY ASSOCIATED WITH BIO-MINING

With the advancement in technology bio-mining uses different modes of processing, some of which are discussed below:

Stirred Tank Biomining

This method is used for leaching from substrates with high mineral concentration. Since the method is expensive and time consuming, substrates with lower concentration are not used for leaching. Copper and refractory gold ores are well suited for this type of method. Special types of stirred tank bioreactors lined with rubber or corrosion resistant steel and insulated with cooling pipes or cooling jackets are used for this purpose. Thiobacillus is the commonly used bacteria. Since it is aerobic the bioreactor is provided with an abundant supply of oxygen throughout the process provided by aerators, pumps and blowers. This is a multi-step process consisting of large numbers of bioreactors connected to each other. The substrate moves from one reactor to another and in the final stage it is washed with water and treated with a variety of chemicals to recover the mineral. The name is fairly self-explanatory, as the process requires constructing large aerated tanks that are generally arranged in a series, so that runoff from one tank serves as raw material for the next. In this way, the reactor can operate in continuous flow mode, with fresh ore being added to the first tank while the runoff from the final tank is removed and treated. The ore to be processed is generally crushed to a very small particle size, to ensure that the solids remain suspended in the liquid medium. Mineral nutrients in the form of (NH4)2SO4 and KH2PO4 are also added to the tanks to ensure maximal microbial density is maintained. Due to the extremely high cost of stirred tank reactors, they are only used for highly valuable materials. For gold extraction for example, this technique is usually used when the ore body contains high concentrations of arsenopyrite (AsFeS).

Bioheaps
Bioheaps are large amounts of low grade ore and effluents from extraction processes that contain trace amounts of minerals. Such effluents are usually stacked in large open space heaps and treated with microorganisms to extract the minerals. Bioheaps are also called biopiles, biomounds and biocells. They are also used for biodegradation of petroleum and chemical wastes. The low grade ores like refractory sulfide gold ore and chalocite ore (copper) are crushed first to reduce the size then treated with acid to promote growth and multiplication of chemophilic bacteria. The crushed and acid-treated ore is then agglomerated so that the finer particles get attached to the coarser ones, and then treated with water or other effluent liquid. This is done to optimize moisture content in the ore bacteria that is inoculated along with the liquid. The ore is then stacked in large heaps of 2-10 feet high with aerating tubes to provide air supply to the bacteria thus promoting bio-oxidation.

Photograph of copper heap leaching operation at Cerro Colorado, Chile

Advantages Heap reactors are cheaper to construct, to operate and are therefore more suited to treatment of lower grade ores. Disadvantages It is difficult to maintain the solution pH within the range 1.8-2.2. Heap reactors are more difficult to inoculate than are tank reactors. If the levels of acids used during agglomeration are too high, cell viability can be reduced. In the heap reactors, large particle sizes, less effective aeration and reduced process control make the process less efficient than stirred tank reactors.

In-situ Bio-Mining
In this method the mineral is extracted directly from the mine instead of collecting the ore and transferring to an extracting facility away from the site of the mine. In-situ bio-mining is usually done to extract trace amounts of minerals present in the ores after a conventional extraction process is completed. The mine is blasted to reduce the ore size and to increase permeability and is then treated with water and acid solution with bacterial inoculum. Air supply is provided using pipes or shafts. Bio-oxidation takes place in-situ due to growing bacteria and results in the extraction of mineral from the ore. Advantages The ore dont need to be removed from the ground. It is most economic when old mine workings can be used to access the low grade deposits. Disadvantages Requires very specific ore body characteristics. Recoveries are typically low. Time consuming.

METALS RECOVERED IN BIOMINING

Biomining of Copper
Copper was the first metal extracted by bioleaching. It is the metal most commonly extracted from oxide ores by this method. In the United States, alone, about 11% of copper is produced from low grade ores by bioleaching technique every year. Copper is available in mines across the world in more than 350 types of ores, but it is mainly present along with sulfur. Copper from low-grade ores like copper sulfide minerals is most commonly extracted by bio-oxidation since it is not economically viable to use conventional metallurgical techniques.

Bio-mining of copper

Biomining of Gold
Bio-oxidation of refractory gold ores to extract gold is carried out by a commercial procedure called BIOX developed by GENCOR S.A Ltd Johannesburg South Africa in an effort to replace existing procedures which posed severe pollution problems. The BIOX process had several advantages over existing procedures including lower cost.

Microbially Enhanced Oil Recovery (MEOR)

Recent technological developments have helped to make possible the recovery of oil. Using microorganisms is one such technique to improve the recovery process hence called microbial enhanced oil recovery (MEOR). It was discovered in 1926 that microorganisms can be used in the petroleum industry to enhance oil recovery, but the concept became popular only after the 1950s. Microbes can enhance the recovery of petroleum products directly or indirectly.

FUTURE PROSPECTS
Although mining is one of humankind's oldest activities, the techniques used to extract minerals haven't changed substantially for centuries. Ores are dug from the earth, crushed, and then minerals such as copper and gold are extracted by extreme heat or toxic chemicals. The environmental and health effects of traditional mining technologies have been deleterious. In the past few years, the mining industry has been turning to a more efficient and environmentally salubrious method for extracting minerals from ores: microorganisms that leach them out. Using a bacterium such as Thiobacillus ferooxidans to leach copper from mine tailings has improved recovery rates and reduced operating costs. Moreover, it permits extraction from low grade ores an important consideration in the face of the depletion of high grade ores. Thiobacillus ferooxidans, which is naturally present in certain sulfur-containing materials, gets energy by oxidizing inorganic materials, such as copper sulfide minerals. This process releases acid and an oxidizing solution of ferric ions, which can wash out metals from crude ore. Poor quality copper ore, which is bound up in a sulfide matrix, is dumped outside a mine and treated with sulfuric acid to encourage the growth of T. ferooxidans. As the bacteria chew up the ore, copper is released and collected in solution. The sulfuric acid is recycled. Currently 25% of all copper worldwide, worth more than $1 billion annually, is produced through bio-processing. This ranks it as one of the most important applications of biotechnology today. Bio-processing is also being used to economically extract gold from very low grade, sulfidic gold ores, once thought to be worthless. To increase the efficiency of biomining, the search is on for bacterial strains that are better suited to large-scale operations. Bio-processing releases a great deal of heat, and this can slow down or kill the bacteria currently being used. Researchers are turning to heat-loving thermophilic bacteria found in hot springs and around oceanic vents to solve this problem. These bacteria thrive in temperatures up to 100 degrees Celsius or higher and could function in a high temperature oxidative environment. Another effort is underway to find - or genetically engineer bacterial strains that can stand up to heavy metals such as mercury, cadmium, and arsenic, which poison microbes and slow the bio-processing.

CONCLUSION
Bio-mining is the sustainable, biotechnological process utilizing microorganisms to remove metals from sulfide mineral ores and concentrates. The development of bio-mining has progressed from poorly designed dumps to highly engineered heaps and stirred tank reactors in an industrially important biotechnological process. The release of metals from sulfide minerals is catalyzed by iron oxidizing acidophilic (optimum pH for growth) microorganisms that act in consortia with heterotrophic and sulfur oxidizing acidophiles in a mixed culture. The microorganisms catalyze metal release by regenerating ferric iron that oxidizes the mineral sulfide bond to produce metals and reduced inorganic sulfur compounds. The other microorganisms in the mixed culture may oxidize the reduced inorganic sulfur compounds to sulfuric acid that is the source of the acid in these environments. Now, tremendous improvements in biomining are expected with continued research in identifying bacterial strains better suited for individual applications and large-scale operations as well as in the genetic engineering of bacterial strains that can stand up to high temperature processes and heavy metals such as arsenic, mercury, or cadmium. Bio-mining has become one of the premier mining technologies, and the future appears bright. The potential applications of biotechnology to mining and processing are countless. Some examples of past projects in biotechnology include a biologically assisted in situ mining program, biodegradation methods, passive bioremediation of acid rock drainage, and bioleaching of ores and concentrates. Research often results in technology implementation for greater efficiency and productivity or novel solutions to complex problems. Additional capabilities include the bioleaching of metals from sulfide materials, phosphate ore bio-processing, and the bio-concentration of metals from solutions. One project recently under investigation is the use of biological methods for the reduction of sulfur in coalcleaning applications. From in situ-mining to mineral processing and treatment technology, biotechnology provides innovative and cost effective industry solutions.

BIBLIOGRAPHY
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Websites Used:
http://knol.google.com/k/partha-das-sharma/biomining/oml631csgjs www.ebookee.com.cn/Biomining_142912.html www.scribd.com www.docs-finder.com www.wikipedia.org