THESIS RESEARCH PROPOSAL

THESIS RESEARCH PROPOSAL

Topic: Isolation and Molecular Characterization of biooxidizing Bacteria, ‘Acidithiobacillus ferrooxidans’ from the AngloGold-Ashanti Gold Mines.

 

Introduction:

Acidithiobacilli are a common group of microorganisms in acidic mines and are active in the degradation of a range of sulfide minerals. The most important one is Acidithiobacillus ferrooxidans which is a Gram negative chemolithotrophic bacterium (Brierley, 1978). Acidithiobacillus ferrooxidans is a major participant in consortia of microorganisms used for the industrial recovery of copper and gold (bioleaching and/or biomining) (Rawlings, 2002). It is a γ-proteobacterium using energy from the oxidation of iron- and sulfur-containing minerals for growth (Rohwerder et al., 2003). It thrives optimally at 30°C and at extremely low pH (pH 1–2) (Rohwerder et al., 2003), and fixes both carbon and nitrogen from the atmosphere. It solubilizes copper and other metals from rocks and plays an important role in nutrient and metal biogeochemical cycling in acid environments. Its ability to oxidize Fe2+ to Fe3+ and elemental sulfur in acidic solution is well known. The bacteria obtain energy through the electrons released as a consequence of the oxidation process (Rohwerder et al., 2003). It is abundant in natural environments associated with pyritic ore bodies, coal deposits, and their acidified drainages (Gonzalez-Toril et al., 2003 and Davis et al., 2000)

 

The processing of metal-containing ores and concentrates of metal-containing ores using microbiological technology, biomining has been recognized as an alternative to the traditional physico-chemical methods of mineral processing. Commercial practices of biomining are usually partitioned into two separate processes;

 

• Biooxidation: - Uses microorganisms, not to extract metals, but to make the metals ready for extraction. Oxidation is the chemical reaction in which an element is changed by the addition of oxygen. Rust is an example of the oxidization of iron. Biooxidation is mainly used in gold mining. Gold is often found in ores with gold particles scattered throughout, called refractory ores, and the small particles of gold are covered by insoluble minerals. These minerals make the extraction difficult. Therefore, microorganisms that can "eat away" at the mineral coating are used to pre-treat the gold ores before they can be extracted.

 

• Bioleaching: - It is the extraction of specific metals from their ores through the use of bacteria. Bioleaching is a technique used by the mining industry to extract minerals from their ores. Traditional extractions involve many expensive steps such as roasting & smelting, which require sufficient concentrations of elements in ores. 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. These microorganisms actually gain energy by breaking down minerals into their constituent elements. The company simply collects the ions out of the solution after the bacteria have finished.

 

Both processes use naturally-occurring microorganisms to extract metals from sulfide-bearing minerals. Mineral biooxidation refers to the process when it is applied to enhance the extraction of gold and silver, whereas bioleaching usually refers to the extraction of base metals, such as zinc, copper, and nickel.

Bioleaching are commercially proven biohydrometallurgical or biomining processes that are economic alternatives to smelting, roasting, and pressure oxidation to treat base- and precious-metals associated with sulfide minerals.

 

 

 

Mechanism of Biooxidation and Bacterial Growth

The growth rate is proportional to the ferric ion formation. However, there is a corresponding decrease in soluble total iron with time due to the precipitation of ferric iron as jarosite (KFe3(SO4)2(OH)6). Initially, the pH increases due to the consumption of H+ by the bacteria and thereafter, it decreased owing to the abiotic hydrolysis of ferric sulfate.

The bacterial catalysed oxidation of ferrous to ferric ion can be expressed as follows:

2Fe2+ + ½ O2 + 2H+                                                                       2Fe3+ + H2O                                           1

The abiotic hydrolysis of ferric sulfate follows this reaction:

Fe3+ + 3H2O                                                                                     Fe(OH)3 + 3H+.                            2

The following reaction shows the formation of jarosite:

3F3+ + 2SO42- + K+ + 6H2O                                                         KFe3(SO4)2(OH)6 + 6H+.              3

The oxidation of elemental sulfur by the bacteria is an acid producing reaction, discharging sulfuric acid into the solution. The pH of the solution decreases sharply after a growth period of about 90 hrs. and a pH as low as 0.5 is observed after 10 days growth. Interestingly, there has been a report of a decrease in the number of cells in the liquid phase during the initial growth period, which is attributed to the adsorption of cells on the solid sulfur. The growth is accompanied by a decrease in pH. The oxidation of elemental sulphur can also be presented as:

2So + 3O2 + 2H2O                                                                       2H2SO4                                          4

When it comes to the cells grown in a pyrite medium, the cell number increases with time and the amount of soluble ferrous iron becomes almost negligible. The reactions for the oxidation of pyrite by direct and indirect mechanisms are as follows;

Direct:

FeS2 + 15/4O2 + ½H2O                                                         Fe3+ + 2SO42- + H+                            5 (biological)

Indirect:

FeS2 + 2F3+                                                                                     3Fe2+ + 2So                                          6 (chemical)

2Fe2+ + ½O2 + 2H+                                                                       2Fe3+ + H2O                                          7 (biological)

2So 3O2 + 2H2O                                                                       2H2SO4                                          8 (biological)

FeS2 + 14Fe3+ + 8H2O                                                        15Fe2+ + 2SO42- + 16H+              9 (chemical)

15Fe2+ + 15/4 O2 + 15H+                                                         15Fe3+ + 15/2H2)                             10 (biological)

 

 

 

A. ferrooxidans and its proposed role in bioleaching. The chemolithoautotrophic metabolism of A. ferrooxidans results in the oxidation/reduction of iron and sulfur compounds and the solubilization of copper and other commercially valuable metals in a process called bioleaching or biomining. It also results in the production of acidified solutions in pristine environments and acid mine drainage in bioleaching operations. A) Model of copper bioleaching by A. ferrooxidans. B) Oxidation/reduction reactions carried out by A. ferrooxidans. The scheme provided here presents the basic concepts of bioleaching and further details are provided in the review [4].

 

(Valdés et al., 2008)

 

 

Genome-based models for the oxidation of ferrous iron and reduced inorganic sulfur Compounds (RISCs). Schematic representation of enzymes and electron transfer proteins involved in the oxidation of (A) ferrous iron and (B) reduced inorganic sulfur compounds (RISCs). Proteins and protein complexes are described in the text.

 

(Valdés et al., 2008)

Uses of Biomining:

Currently, 25% of all copper worldwide is produced through biomining. The process is used on a variety of other metals such as gold & uranium. Biomining is not yet a proven or profitable technology to apply to other metals such as zinc, nickel and cobalt. Some advantages of biomining over traditional methods include reduced noxious gas production, and the elimination of toxic liquid waste produced as a result of chemical leaching. Biomining, however, is slower than traditional mining techniques and is not applicable to a wide variety of ores.

 

Amplified fragment length polymorphisms (AFLPs)

 

Amplified fragment length polymorphism (AFLP) analysis was developed by a team led by Marc Zabeau at Keygene N.V., Wageningen, The Netherlands (Vos et al., 1995; Zabeau and Vos, 1993). AFLP is a variation of Random Amplified Polymorphic DNA (RAPD) which is able to detect restriction site polymorphisms without prior sequence knowledge using PCR amplification for detection of restriction fragment (Blears et al., 1998; Mueller and Wolfenbarger, 1999; Vos et al., 1995; Zabeau and Vos, 1993). Here, the template for a PCR reaction is a restriction enzyme-digested genomic DNA. The primers (random primers) contain the restriction enzyme recognition site as well as additional ‘arbitrary’ nucleotides that extend beyond the restriction site. The fixed portion gives the primer stability and the random portion allows it to detect many loci. Amplified products are resolved by polyacrylamide gel electrophoresis. AFLP analysis is one of the robust multiple-locus fingerprinting techniques among genetic marker techniques that have been evaluated for genotypic characterization (Koeleman et al., 1997).

 

 

 

DNA Sequencing:

The term DNA sequencing refers to methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a molecule of DNA. The first DNA sequences were obtained by academic researchers, using laborious methods based on 2-dimensional chromatography (Sanger and Coulson, 1975 & Sanger et al., 1977). Following the development of dye-based sequencing methods with automated analysis, DNA sequencing has become easier and orders of magnitude faster. Knowledge of DNA sequences of genes and other parts of the genome of organisms has become indispensable for basic research studying biological processes, as well as in applied fields such as diagnostic or forensic research. The advent of DNA sequencing has significantly accelerated biological research and discovery. The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of the whole genomes (Hall, 2007 and Ju J et al., 1995).

Molecular charactrization of microorganisms involves the study of the genetic constitution of the respective microobes in order to put them into specific families. In the research involving At. ferrooxidans, the nucleotide sequence of the rDNA gene has been used to characterize a said bacteria as At. ferrooxidans. Research has shown that the base sequence of the rusticyanide gene (rus), ferrous iron oxidizing gene (iro) and the cytochrome oxygenase gene (Cox C) varry from one strain to the other hence these genes are made used of in characterizing the bacteria. Research has also shown that the variation in the genotypes of these genes directly correspons to their biooxidation potentials (Chen et al., 2007).

 

Rationale: Research has shown that the efficiency of biooxidation involving bacteria depends on the bacteria strain found in a particular environment where the raw material is also found. Yet in Ghana here the Anglo Gold Ashanti Gold mining company still relies on Acidithiobacillus ferrooxidans strain imported from South Africa for its operations.

 

Aim: To identify a local strain of the iron and sulpho-oxidizing bacteria Acidithiobacillus ferroxidans with a better biooxidation potential.

 

Objectives:

1. To isolate and identify the iron and sulpho-oxidizing bacteria Acidithiobacclilus ferooxidans. from the reactor tanks.
2. To isolate and identify local strains of this bacterial specie from the arsenopyrite and underground sulphide ores.
3. To determine possible similarities and differences in the genome between the local isolates and those present in the reactor tanks.
4. To determine any differences in biooxidizing activity / potential of the isolates.

 

Methods:

Sample Collection:

Gold ore samples (crushed ores) would be collected into polythene bags.

Slurry from reactor tank (50 ml) would be collected into sterile centrifuge tubes and transported within 5 hours to be stored at 4 ºC until use.

 

Isolation Purification and Maintenance of Ferrous Iron Thioxidans Bacteria.

Isolation: Slurry (5 ml) and /or 3 g (ore) would be put into 100 ml of 9K enrichment medium supplement with ferrous iron sulphate (9K -.Fe2+), pH ≤ 1.8 in a 50 ml Erlenmeyer flask.

The culture would then be incubated at room temperature (25-30 ºC) for 8 - 10 days with continuous agitation at a speed of 100 rpm on a rotary shaker.

 

Purification: Bacteria suspension (100 μl) at log phase would be inoculated by spreading on ferrous agar and / or ferrous agarose plates.

The inoculated plates would be incubated at 37 ºC for 4 to 6 days during which single cell colonies would be expected to have appeared.

Single colonies would be transferred with a sterile Pasteur pipette into sterile 9K – F2+ liquid medium in a 24 well tissue culture plate (Becton Dickinson & Company, USA) and incubated at room temperature (25 – 30 ºC) with continuous agitation on a rotary shaker for about 3 weeks for profuse bacteria growth.

The procedure above would be repeated for 3 more times to ensure purity of the isolates (Baron & Leuking, 1990). Light microscopy (magnification. X 1000, objective X 100) would be used to confirm purity of the isolates based on their morphology (Goebel & Stackebrant, 1994).

 

Maintenance: The isolates would be maintained using the 9K – Fe 2+ liquid medium.

Here cultures of the isolates would be prepared every two weeks by sub-culturing 10 ml of an ongoing culture into 100 ml of 9K liquid medium in a 500 ml Erlenmeyer flask.

The cultures would be continuously agitated on a rotary shaker at 100 rpm, room temperature (25 – 30 ºC) and opened 2 hours daily to allow fresh air to circulate.

 

Preservation of Isolates: Sample (100 ml) of each isolate at stationary phase of growth would be centrifuged at 10, 000 g for 60 minutes and the pellet re-suspended in 2 ml of 9 K liquid medium of pH 6.5. Sample (100 μl) of the re-suspended isolates would be pipetted into a 5 ml glass vials and frozen at -20 ºC then freeze-dried at -105 ºC and stored at 4 ºC until use.

 

Isolation and Purification of Genomic DNA of the Isolates: Modification of Sambrook et al., (1989) and Flook et al., (1992) protocol would be used as used by Asmah (1998) unpublished.

Bacteria cells (100 ml of culture) at stationary phase would be harvested by centrifugation at 2, 900g for 60 minutes at 4 ºC.

The pellet would be rinsed 3 times with 1 M Tris-HCl, pH 7.6 and re-suspended in 200 μl of Bender buffer at 4 ºC in Eppendorf tubes for lysis.

Proteinase K (10 μl) would be added to the lysed cells to denature every protein except itself. This would be done to protect the integrity of the DNA.

The homogenate would be incubated at 64 ºC for 60 minutes and spun in a micro-fuge for 10 minutes.

Pre-chilled 8 M potassium acetate (30 μl) would be added, tapped to mix well and left to stand on ice for 45 minutes before centrifuging at 12, 000 g for 10 minutes to pellet cell debris.

The supernatant would be pipetted out into a fresh Eppendorf tubes with the addition of 2 X volume of cold absolute ethanol and kept at -50 ºC for 30 minutes to precipitate the DNA.

The DNA would be collected by centrifugation at 12, 000 g for 10 minutes and air dried.

DNA pellet would be re-suspended in Tris-EDTA buffer (50 μl) pH 7.6 with 10 mg/ml DNAase free RNAase solution and further purified by phenol-chloroform extraction:

To the sample would be added an equal volume of phenol-chloroform mixture (1:1 v/v), and the contents mixed till an emulsion forms. The mixture (emulsified) would be centrifuged for 5 minutes in a micro-fuge to separate the organic phase from the aqueous phase containing the DNA. The aqueous phase would be transferred into a fresh tube and the whole process repeated for about 3 times then with only chloroform. Purified genomic DNA would be precipitated by the addition of 1 / 10 volume of sodium acetate, pH 5.2 and 2 X volume of cold absolute ethanol to the aqueous phase and keeping the mixture at -20 ºC overnight. DNA would be recovered by centrifugation at 12, 000 g for 10 minutes, washed with 70 % ethanol, air dried at room temperature then dissolved in TE buffer (30 μl) and stored at -20 ºC.

NB: purity of the DNA would be determined as described by Rodriquez and Tait (1983).

 

Molecular Manipulations of Isolated DNA

Amplified Fragment Length Plolymorphiss (AFLP)

• Polymerase Chain Reaction: The Primers;

 

Gene	Primer sequences	GenBank Accession number
16S rRNA	Forward: 5′-acgctggcggcatgcctaac-3′ Reverse: 5′- ttggtatggcaccattggcg-3′	AJ278724
iro	Forward: 5′-cctaaggagaaggctatg-3′ Reverse: 5′-cgctatctcatgctgactt-3′	AJ006456
rus	Forward: 5′-cggctggttctactcaggtt-3′ Reverse: 5′-ccagacaacaaccatccagg-3′	AJ006456
coxC	Forward: 5′-cggctggttctactcaggtt-3′ Reverse: 5′-ccagacaacaaccatccagg-3′	AJ006456

 

PCR reaction mixture shall contain 50 – 100 ng of genomic DNA in a 20 μl reaction mixture with 1.5 mM MgCl2, 20 mM dNTPs and 5 pmoles of each primer with 2.5 U, Taq polymerase (invitrogen).

Amplification conditions shall be: Initial denaturation (94 ºC for 5 minutes), initial annealing (55 ºC for 30 seconds) and initial extension (72 ºC for 45 seconds). The whole process would be repeated for 30 more cycles with the variation of the denaturation time from 5 minutes to 30 seconds throughout.

 

• Restriction Enzyme Digestion of PCR Products: The PCR products would be treated with random restruction enzymes as shown below.

The method used by Sambrook et al., (1989) would be used with slight modifications.

DNA solution (10 μl each) would be placed in sterile tubes, mixed with 8 μl sterile distilled water to give 18 μl DNA.

10 X restriction buffer (2 μl) would be added and mixed by tapping. The appropriate restriction endonuclease (1 μl) would be added directly into the reaction tubes and incubated at 37 ºC overnight.

The reaction would be stopped by heating the reaction mixture at 70 ºC for about 5 minutes and 4 μl of 6 X gel loading buffer added to each reaction mixture.

Digested DNA can be stored at -20 ºC until use.

 

• Gel Electrophoresis of Digested DNA:

PCR products would be analyzed by electrophoresis in 1 % agarose gel with the bands visualized by illuminating ethidium bromidestained gels on a UV transilluminator.

 

Sequencing of the Amplified Genes:

DNA fragments shown as bands from the electrophoresis would be extracted from agarose gels using a Wizard PCR Preps kit (Promega) and sequenced by using the respective primers with an automatic sequencer (at Noguchi Memorial Institute of Medical Research) according to the protocol recommended by the manufacturer base on the method of Sanger et al. (1977).

 

Determination of Biooxidation Potential of Isolates

For the first part, ferrous iron would be used as the sole energy source, the content of Fe3+ in the medium would then be determined as indicator for growth of At. ferrooxidans strain.

The second part shall have elemental sulphur (S0) as the sole energy source, and the pH of the medium would be measured as the indicator of biological activity.

The third and final part of the assay shall have pyrite as the energy source and here the total Fe (Fe2++Fe3+) content and pH of the medium would be determined as indicators of biological activity.

The total Fe (Fe2++Fe3+) content would be determined by using atomic absorption spectrometry according to the protocol recommended by the manufacturer (The Second Optic Apparatus Beijing, China). The Fe2+/Fe3+ ratio (the content of Fe3+) would be determined by complexometric titration using potassium dichromate (Zhang et al., 2005). The pH value would be determined with a pH/ISE testing instrument (828 model, Orion Research).

 

Expected Results:

I hope to isolate and characterize a local strain of the ferrous iron and sulphur oxidizing bacteria Acidithiobacillus ferrooxidans that has higher biooxidizing potential than the exotic strain used by the Anglo Gold Ashanti mining company.

 

Budget:

Item	Specifics	Individual Cost / US $		Total Cost / US $
Equipment	1. 2. 3.
Reagents	1. 2. 3.
Transportation
Miscellaneous

 

 

 

 

 

 

 

Reference

Brierley, C.L. (1978). Bacterial Leaching. Crit. Rev. Microbiol.; 6, 207 – 262.

 

Chen H., Yang B. & Chen X. (2007). Identification and Characterization of Four Strains of Acidithiobacillus ferrooxidans Isolated from Different Sites in China. Microbiol Res

 

Davis R.A. Jr., Welty A.T., Borrego J., Morales J.A., Pendon J.G.& Ryan J.G. (2000): Rio Tinto Estuary (Spain): 5000 Years of Pollution. Environ. Geol., 39(10):1107-1116.

 

Gonzalez-Toril E., Llobet-Brossa E., Casamayor E.O., Amann R. & Amils R. (2003): Microbial Ecology of an Extreme Acidic Environment, the Tinto River. Appl Environ Microbiol, 69(8):4853-4865.

 

Hall N. (2007). "Advanced Sequencing Technologies and Their Wider Impact in Microbiology". J. Exp. Biol. 210 (Pt 9): 1518–1525.

 

Valdés J., Pedroso1 I., Quatrini1 R., Dodson R.J., Tettelin H., Blake R., Jonathan A Eisen J.A. & David S Holmes D.S. (2008). Acidithiobacillus ferrooxidans Metabolism: from Genome Sequence to Industrial Applications. BMC Genomics, 9:597

 

 

 

 

Ju J., Ruan C., Fuller C.W., Glazer A.N., & Mathies R.A. (1995). "Fluorescence Energy Transfer Dye-Labeled Primers for DNA Sequencing and Analysis". Proc. Natl. Acad. Sci. 92 (10): 4347 – 4351

 

Rawlings D.E. (2002). Heavy Metal Mining Using Microbes. Annu Rev Microbiol , 56:65-91.

 

Rohwerder T., Gehrke T., Kinzler K. & Sand W. (2003). Bioleaching Review Part A: Progress in Bioleaching: Fundamentals and Mechanisms of Bacterial Metal Sulfide Oxidation. Appl Microbiol Biotechnol, 63(3):239-248.

 

Sanger F. & Coulson A.R. (1975). "A Rapid Method for Determining Sequences in DNA by Primed Synthesis with DNA Polymerase". J. Mol. Biol. 94 (3): 441 – 448.

 

Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA Sequencing With Chain-Terminating Inhibitors. Proc Natl Acad Sci ;74, 5463–5467.