Abstract:
The present invention discloses a method to identify and quantify environmental microorganisms useful in biomining processes. These microorganisms are basically 10, belonging to Bacteria:  Acidiphilium  sp.,  Leptospirillum  sp.,  Sulfobacillus  sp.,  Acidithiobacillus ferrooxidans  and  Acidithiobacillus thiooxidans;  and Archaea:  Acidianus  sp.,  Ferroplasma  sp.,  Metallosphaera  sp.,  Sulfolobus  sp. and  Thermoplasma  sp.  
     The method comprises performing a two-stage PCR known as nested PCR, where in the first stage, called primary PCR, 16S ribosomal DNA sequences (nucleotides 27 to 1492, with  E. coli  rDNA numbering) are amplified using universal primers for the Bacteria and Archaea kingdoms. In the second stage, these primary amplicons are used as template in qPCR reactions, called secondary PCR, in which internal universal primers for either Bacteria or Archaea kingdoms, as it corresponds, and specific primers designed in our laboratories for different taxons to be determined are used. The first PCR linearly multiplies 16S sequences from bacteria or archaea, thus increasing template abundance for the secondary PCR keeping the original microorganism proportion of the sample. This gives a higher sensitivity to the process when compared to the case of directly using taxon-specific primers on the sample. With qPCR results and other data obtained from the analyzed sample, the microorganism concentration of each analyzed taxon present in the sample is calculated using a mathematical formula.

Description:
FIELD OF THE INVENTION  
       [0001]     The present invention discloses a method to identify and quantify microorganisms useful in biomining processes that are present in a given sample. This method is presented as a useful tool in biomining, in every case where the present microbiological population needs to be evaluated, whether on the mineral, in solutions, in bioleaching heaps, in biomining laboratories or in any other circumstance that involves the use of such microorganisms.  
       BACKGROUND OF THE INVENTION  
       [0002]     Biomining is, in general terms, the use of microorganisms for metal recovery from mineral ores. Its most traditional expression is bioleaching, but not only this process is understood as biomining, but also the monitoring and intervention in such process, as these techniques are complex and are under constant development; and also laboratory research associated to process improvement or the development of new methodologies.  
         [0003]     Until now, bioleaching continues to be the most important process in biomining field, and is defined as a method to solubilize metals from complex matrixes in an acid medium, using direct or indirect microorganism action. The microorganisms that are useful in these processes belong to Bacteria or Archaea kingdoms, and fulfill two basic conditions: they are acidophilic and chemolithotrophic.  
       Microbiological Diversity in Communities Associated to Bioleaching Processes  
       [0004]     Various microorganisms have been described to be useful in bioleaching processes, and ten taxons could be identified among them: 3 genera and 2 species from the Bacteria kingdom, namely  Acidiphilium  sp.,  Leptospirillum  sp.,  Sulfobacillus  sp. genera and  Acidithiobacillus ferrooxidans  and  Acidithiobacillus thiooxidans  species, and five genera from the Archaea kingdom, namely  Acidianus  sp.,  Ferroplasma  sp.,  Metallosphaera  sp.,  Sulfolobus  sp. and  Thermoplasma  sp. (Rawlings D E. Heavy metal mining using microbes. Annu Rev Microbiol. 2002; 56:65-91; Rawlings D E. Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates. Microb Cell Fact. May 6, 2005; 4(1):13).  
       Factors Determining Diversity and Metabolic Activity of the Microbial Community Associated to a Bioleaching Process  
       [0005]     Each of the above mentioned genera or species catalyzes different reactions and require in its turn different conditions to perform such reaction, which could be, for instance, aerobic or anaerobic, or could require some specific nutrient. Therefore, the environmental conditions in which a bioleaching process is performed will modify the bacterial composition of the community.  
         [0006]     Additionally, the participation of microorganisms in a bioleaching process has been proposed to be direct and/or indirect (Rawlings D E. Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates. Microb Cell Fact. May 6, 2005; 4(1):13). When the action is direct, microorganisms directly oxidize the target metal or its counter-ion, in both cases liberating into the solution a target metal ion. On the other hand, when the action is indirect, the substrate of the microorganism is not the target metal neither its counter-ion, but instead chemical conditions are generated that allow the solubilization of said metal, either by acidification of the medium (e.g., by generating sulfuric acid) or by the generation of an oxidizing agent that ultimately interacts with the salt (metal and counter-ion) to be solubilized.  
         [0007]     Regarding this aspect, it is possible that the bacterial community changes its species composition as a function of the bioleaching type being performed in different mineral samples and/or the environmental conditions in which this process is carried out.  
         [0008]     For instance,  Acidithiobacillus  species are able to catalyze the oxidation of reduced sulfur compounds (e.g., sulfide, elemental sulfur, thionates, etc.) using oxygen as electronic acceptor and generating sulfuric acid as final product and reducing species like sulfite and thiosulfate as intermediate products, which allows the solubilization of metals associated to sulfides in the mineral.  Acidithiobacillus ferrooxidans  and  Leptospirillum ferrooxidans  are able to catalyze the oxidation of iron(II) to iron(III) using oxygen as electron acceptor, being the generated iron(III) a great oxidizing agent that can oxidize sulfides in the mineral or any other compound to be oxidized.  
         [0009]     The usual mining practice in bioleaching processes is to leave a mineral heap in an acid medium, generally sulfuric acid, and constantly remove the acid medium to recover the metal by electrolysis. Usually heaps in which the recovery yield of the metal is efficient are obtained, and also “inefficient” heaps that have a low yield under the same operation conditions and characteristics of the substrate to be leached. The explanation to this unequal result requires the elucidation of differences in abundance and types of species in the microbiological community between both heaps. In this way, the low yield problem could be explained by the microbial community composition, and could be solved in its turn by inoculation of microorganisms that catalyze the reaction to be maintained during the bioleaching process. However, a method that enables to quantify the population of archaea and bacteria useful in biomining processes is not available up to this date. In this patent, a method is described that solves the technical problem previously described, by designing a method to identify and quantify the presence of known microorganisms that are most relevant in biomining processes, namely the bacteria:  Acidiphilium  sp.,  Leptospirillum  sp.,  Sulfobacillus  sp.,  Acidithiobacillus ferrooxidans  and  Acidithiobacillus thiooxidans;  and the archaea:  Acidianus  sp.,  Ferroplasma  sp.,  Metallosphaera  sp.,  Sulfolobus  sp. and  Thermoplasma  sp.  
         [0010]     Nested polymerase chain reaction (PCR) was the technique selected to develop this method. In this technique, a conserved genome region of the microorganisms is firstly amplified in a first PCR reaction, either on bacteria or archaea. We have selected gene 16SrDNA as the conserved region. Then, taxon-specific primers (targeting genera or species) are used to identify the presence of target microorganisms in a second PCR reaction. This second PCR reaction is performed using an equipment that allows measuring the increase of amplified product in each amplification cycle, and this information allows the quantification, by interpolation, of the original abundance of the target genome in the sample being analyzed. PCR reaction under these conditions is called quantitative PCR or qPCR.  
         [0011]     A critical step in nested PCR technique is the design of primers for the second amplification reaction, which have to be specific for the taxon to be determined, and this aspect has a vital importance in this particular case, as the samples to which the process will be applied will usually be metagenomic samples. Therefore, it is necessary to reduce the possibility of primer unspecific hybridization to sequences present in the genome of microorganisms that have not yet been identified in the community. We have generated two fundamental tools for the design of these primers: firstly, a depurated 16SrDNA sequence database obtained from all disclosed 16SrDNA sequences; and a computational program for primer design that uses as input such database and allows designing thermodynamically stable taxon specific primers.  
         [0012]     In the state of the art there are many examples of the application of nested PCR or qPCR, but none of them is focused to bacteria or archaea useful in biomining processes. For instance, J. L. M. Rodrigues et al (Journal of Microbiological Methods 51 (2002) 181-189) describe a qPCR to detect and quantify PCB-degrading  Rhodococcus  present in soil, where the 16SrDNA gene belonging to the strain with the target activity is sequenced, specific primers for said sequence are designed and qPCR reactions are carried out using said primers. In this document, a direct qPCR approach is used, instead of a nested qPCR, and it is directed to other type of microorganisms, whose handling has been widely studied and many techniques for DNA extraction are available. Another document that uses a similar approach is Patent Application EP 1 484 416, which discloses a method for the detection and quantification of pathogen bacteria and fungi present in an environment sample using qPCR. The method comprises the extraction of DNA from bacteria and fungi present in an environment sample, obtaining specific sense and antisense primers for each of the taxons to be detected and quantified; and performing qPCR reactions using a pair of primers for each of the target pathogens.  
         [0013]     Although it is possible to enumerate documents in which microorganisms are identified and quantified using quantitative PCR techniques, as they are well known techniques in the art, the relevant point is the generation of a depurated database that allows to design specific primers and has not been implemented before for the identification of microorganisms useful in biomining processes, which is subject matter of this invention.  
       SUMMARY OF THE INVENTION  
       [0014]     The present invention discloses a method to identify and quantify environmental microorganisms useful in biomining processes. These microorganisms are basically 10, belonging to Bacteria:  Acidiphilium  sp.,  Leptospirillum  sp.,  Sulfobacillus  sp.,  Acidithiobacillus ferrooxidans  and  Acidithiobacillus thiooxidans;  and Archaea:  Acidianus  sp.,  Ferroplasma  sp.,  Metallosphaera  sp.,  Sulfolobus  sp. and  Thermoplasma  sp.  
         [0015]     The method comprises performing a two-stage PCR known as nested PCR, where in the first stage, called primary PCR, 16S ribosomal DNA sequences (nucleotides 27 to 1492, with  E. coli  rDNA numbering) are amplified using universal primers for the Bacteria and Archaea kingdoms. In the second stage, these primary amplicons are used as template in qPCR reactions, called secondary PCR, in which internal universal primers for either Bacteria or Archaea kingdoms, as it corresponds, and specific primers designed in our laboratories for different taxons to be determined are used. The first PCR linearly multiplies 16S sequences from bacteria or archaea, thus increasing template abundance for the secondary PCR keeping the original microorganism proportion of the sample. This gives a higher sensitivity to the process when compared to the case of directly using taxon-specific primers on the sample. However, the method also works and is applicable without the primary PCR, and therefore this stage may be optional.  
         [0016]     With qPCR results and other data obtained from the analyzed sample, the microorganism concentration of each analyzed taxon present in the sample is calculated using a mathematical formula. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0017]     FIGS.  1  to  7  show the results of Example 1, where the presence of  Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans, Leptospirillum  sp.,  Acidiphilium  sp. and total bacteria has been quantified in 7 different samples. In each Figure, results for each solid sample (MS), identified as MS-1, MS-2, MS-3, MS-4 and MS-5, and each liquid sample (ML), identified as ML-1 and ML-2, are plotted. Each plot shows quantified taxons in the abscissa and microorganism number per sample unit in logarithmic scale in the ordinate. Data giving origin to plots are shown in Tables 26 to 32.  
         [0018]     From these results it is possible to observe which one is the predominant species in each of the mineral samples from bioleaching heaps (MS-1 to MS-5) and from liquid samples recovered from bioleaching effluents (ML-1 and ML-2). This value can also be correlated to total bacteria found in said samples. Thus, in 6 over 7 samples  Leptospirillum  sp. predominance is observed, and even more, only this microorganism genus is present in one of the liquid samples. Only one of the solid samples (MS-2) shows  A. thiooxidans  predominance, which leads to the conclusion that  Leptospirillum  sp. is the most abundant microorganism in this type of mineral samples.  
         [0019]      FIGS. 8 and 9  show the results of Example 2, where the presence of  Sulfobacillus  sp,  Sulfolobus  sp,  Ferroplasma  sp., total bacteria and total archaea was quantified on 2 different samples obtained from bioleaching heap mineral. In each Figure, results for each solid sample (MS), identified as MS-6 and MS-7 are plotted. Each plot shows quantified taxons in the abscissa and microorganism number per sample unit in logarithmic scale in the ordinate. Data used for generating these plots are shown in Tables 45 and 46. From these results it can be concluded that the presence of microorganisms belonging to the Archaea kingdom is minority in both samples, compared to those belonging to the Bacteria kingdom. However, specific determinations of the genus  Sulfolobus  (archaea) in sample MS-6 are slightly higher than the number of bacteria belonging to the genus  Sulfobacillus,  which indicates the presence of a high number of bacteria from other genera in the sample. Likewise, a microorganism belonging to the genus  Ferroplasma  is detected in sample MS-7, and it is absent in the former sample. Again, these data could give an explanation to specific behaviors of the community that is present in the analyzed mineral.  
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0020]     As has been anticipated, the invention relates to a method that allows the identification and quantification of essential microorganisms in biomining processes. These essential microorganisms belong to 10 taxons, the genera  Acidiphilium  sp.,  Leptospirillum  sp.,  Sulfobacillus  sp. and the species  Acidithiobacillus ferrooxidans  and  Acidithiobacillus thiooxidans  belonging to the Bacteria kingdom; and the genera  Acidianus  sp.,  Ferroplasma  sp,  Metallosphaera  sp,  Sulfolobus  sp. and  Thermoplasma  sp. belonging to the Archaea kingdom.  
         [0021]     As previously indicated, a method to identify and quantify biomining microorganisms would have applications in different industrial tasks and areas. For instance, a good tool for suitable control of bioleaching process could be the identification of microorganisms that are present in a bioleaching heap and how abundant they are, as it could be established whether is necessary to inoculate some particular microorganism or simply determine which nutrients should be added to the mixture, thus maximizing the quantity of mineral recovered in the process. The idea is to correlate the recovery efficiency of different metals present in the heap with the composition of the microbiological community in the heap, referred to the number and type of present individuals.  
         [0022]     In general terms, samples to be analyzed in the method of the invention will be biomining samples, but this does not limit the scope of the invention, as the described method could be applied any time that one or more of the 10 taxons subject of this invention is to be identified and quantified.  
         [0023]     In the description of the invention, all oligonucleotide sequences are written in direction 5′ to 3′. Described oligonucleotides correspond to primers for PCR reactions, which can be sense or antisense primers, which could be indicated specifically (e.g., as table titles) or alternatively by including letter “F” for sense or forward primers and “R” for antisense or reverse primers in the name of the primer.  
         [0024]     The following is the description of each of the stages of the method in detail.  
         [0000]     DNA Preparation.  
         [0025]     In a first stage, it is necessary to extract DNA from the sample. Different methods to extract DNA from mineral or soil samples have been disclosed and any of them can be used, considering in each case the particular nature of the sample (Appl Environ Microbiol. July 2003; 69(7):4183-9; Biotechniques. April 2005; 38(4):579-86). In the case that total extracted DNA (from mineral samples, being e.g. grounded chalcopyrite type 1 or other) is turbid or has a yellow or orange color, it is recommended to repurify the sample using any existing purification technique; in our laboratories this step is performed using commercial DNA purification columns. The purified fraction is resuspended in sterile nuclease-free H 2 O.  
         [0026]     Once total DNA samples have been purified, total DNA present in the sample should be quantified; again, this quantification could be performed using any existing method to quantify DNA; in our laboratories it is done by spectrophotometry.  
         [0027]     After quantifying total DNA present in the sample, an aliquot is taken and diluted to a concentration suitable for the method, which finally ranges from 0.5 to 40 ng/μl, preferably from 1 to 30 ng/μl, and most preferably from 1 to 10 ng/μl. The dilution must be done using sterile nuclease-free water.  
         [0000]     Primary PCR.  
         [0028]     This stage is optional and could be skipped, however in our case it is advantageous to perform it as it restrict the analyzed subject universe and increases the sensitivity of the method. Using the DNA sample previously prepared at least one of the primary PCR is performed, one of them using primers to amplify 16S region from the Bacteria kingdom (“universal Bacteria primers”) and the other using primers to amplify 16S region from the Archaea kingdom (“universal Archaea primers”).  
         [0029]     Primary PCRs are intended to amplify the region coding for 16S ribosomal RNA, for which any primer pair could be used in primary PCR that fulfill the described requirements; in our laboratories universal primers shown in the list included in Table 1 are preferentially selected.  
                                                                       TABLE 1                       Primary PCR                                    Bacteria primers                Eub27-F 1     AGA GTT TGA TCC TGG CTC AG               Univ1492-R 1     GGT TAC CTT GTT ACG ACT T                            Archaea primers                Arch21-F 2     TTC CGG TTG ATC C(CT)G CCG GA               Univ1492-R 1     GGT TAC CTT GTT ACG ACT T                           1 Bond P., 2000, Appl Environ Microbiol. 66(9):3842-9.                  2 Delong, E.F., 1992, Proc. Nac. Acad. Sci. USA 89: 5685-9.             
 
         [0030]     It is important that primary PCR should be linear, i.e., amplification does not reach saturation, as the original proportion in the sample should be kept.  
         [0031]     This PCR is also applied on a negative control, containing sterile water instead of DNA, and a five-point calibration curve, formed by a master mix and four serial dilutions thereof. The master mix is specific for each kingdom, Bacteria or Archaea, and is formed by a standard DNA mix belonging to each of the taxons to be determined. This means that in the PCR using universal Bacteria primers, the standard DNA mix used in the master mix will contain pure DNA extracted from all bacteria to be identified and quantified, as  Acidiphilium  sp.,  Leptospirillum  sp.,  Sulfobacillus  sp.,  Acidithiobacillus ferrooxidans  and  Acidithiobacillus thiooxidans;  whereas in the primary PCR using universal Archaea primers the master mix will contain DNA from all Archaea to be identified and quantified, as  Acidianus  sp.,  Ferroplasma  sp,  Metallosphaera  sp,  Sulfolobus  sp. and  Thermoplasma  sp.  
         [0032]     The master mix optimally contains from 1 to 100 ng of DNA from each of the strains, and preferably contains 100 ng of total DNA, although it is possible to work with higher or lower amounts. The calibration curve will be used in the quantification that will be performed with secondary PCR and corresponds to the master mix in concentrations 1×, 0.1×, 0.01×, 0.001× and 0.0001×. Each one of these dilutions is subjected to the primary PCR.  
         [0033]     Preferably, each primary PCR is carried out using 1 μl of DNA, either for the sample or the calibration curve, or 1 μl of water for the negative control, plus 24 μl of the reaction mix whose composition is described in Table 2.  
                           TABLE 2                                       Sterile nuclease-free H 2 O   18.35 μl            PCR Buffer 10x   2.5 μl           MgCl 2  (50 mM)   1.5 μl           dNTPs (10 mM each)   0.5 μl           Primer Eubac27F (10 μM)   0.5 μl           Primer Univ1492R (10 μM)   0.5 μl           Hot Start Taq (5 U/μl)   0.15 μl                       
 
         [0034]     Primary PCR cycles are described in Table 3.  
                                                 TABLE 3                                       Temperature               Step   (° C.)   Time (s)                                        1. Initial denaturation   95   120           2. Denaturation   95   30           3. Alignment           3.1. for Bacteria   56   30           3.2. for Archaea   57   30           4. Extension   72   120                      
 
         [0035]     Wherein steps 2 to 4 are repeated from 15 to 20 times, avoiding saturation.  
         [0036]     Once this primary PCR has been performed, the sequence of region 16S of all bacteria and archaea present in the original sample has been amplified.  
         [0000]     Secondary PCR.  
         [0037]     Then, a plurality of PCR is carried out, specific for each taxon to be identified, using specific primers that amplify inside the 16S rDNA region amplified in the primary PCR.  
         [0038]     In this stage it is crucial to have specific and efficient primers to amplify the target fragment that have no cross-reaction with organisms from other taxons and are thermodynamically stable, i.e. do not form hairpins, homodimers or heterodimers. The primers used in this application have been designed using the method disclosed in Patent Application CL 2102-2005 filled by Biosigma; as said method guarantees the efficiency and specificity of the designed primers.  
         [0039]     In each primary PCR a reaction has been carried out to amplify each of the samples, 5 point of the calibration curve and one negative control. Each secondary PCR will be performed on all the reaction products of each corresponding primary PCR reaction. Advantageously, all reactions are carried out in duplicate, and a negative control is added.  
         [0040]     When we say that secondary PCR is performed on the corresponding primary PCR, we mean that if our target taxon to be amplified in the secondary PCR belongs to the Archaea kingdom, then we will use the primary PCR reaction products for archaea. Likewise, if the taxon to be quantified is a bacterium, we will use the primary PCR reaction products for bacteria in the secondary PCR.  
         [0041]     It is important to point out that the method of the invention can be carried out to identify and quantify either all the described taxons or only one of them, and also all the possible intermediate combinations, and as a consequence every one of these options will remain being comprised inside the scope of the present invention.  
         [0042]     The secondary PCR is a quantitative PCR (qPCR), therefore it should be performed in a suitable thermocycler and using fluorescent reagents for qPCR. There are different commercially available alternatives, either for equipment or reagents, and any of them can be selected to carry out the present method.  
         [0043]     For each secondary PCR reaction the following mix is prepared:  
                           TABLE 4                                       Sterile nuclease-free H 2 O   10.5 μl           Sense primer (10 μM)    0.5 μl           Antisense primer (10 μM)    0.5 μl           qPCR reagent   12.5 μl                      
 
         [0044]     To the mix described in Table 4, 1 μl of primary PCR reaction product or sterile water for the qPCR blank is added.  
       Primers for the Secondary PCR  
       [0045]     As previously indicated, the requirements to be fulfilled by each primer pair selected for the secondary PCR are: being specific for each taxon, having no cross-reactivity and being thermodynamically stable to assure primer availability in the PCR reaction. Our laboratory has developed a primer design program that gives a large amount of primers fulfilling these requirements. The method of the invention can be performed by combining any sense primer with any antisense primer designed by our program. In following tables, we give 20 sense primers and 20 antisense primers for each taxon, where any possible combination thereof could be selected for the qPCR. (Note: the sequences of the designed primers have been compared, by using Blast from NCBI, with previously existent sequence disclosures, thus guaranteeing its novelty as primers.)  
         [0046]     Bacteria Kingdom:  
                                       TABLE 5                             Acidiphilium  sp.            Sense primers   Antisense primers                    CAA CCA CGG TCG GGT CAG   TCT CTG ACC CGA CCG TGG           A   TT               GAC CTT AAG TTG ATG CGC   TCA ACT TAA GGT CAA ACC       T   AA               AGT CAA CCA CGG TCG GGT   GGA GCT TAT TCT GCG GGT       C   A               GGT TTG ACC TTA AGT TGA   GCA TCA ACT TAA GGT CAA       TG   AC               CTT AAG TTG ATG CGC TAA   AGC GCA TCA ACT TAA GGT       C   CA               GGC AGT CAA CCA CGG TCG   GTT AGC GCA TCA ACT TAA       G   GG               CGA TGC TGA GCT GAT CCT   CCG ACC GTG GTT GAC TGC       G   C               AAG TTG ATG CGC TAA CCG   GGA TCA GCT CAG CAT CGC       C   TG               AAA GTC GCC TAA GGA GGA   TCA GGA TCA GCT CAG CAT       G   CG               GTC GCC TAA GGA GGA GCC   CGG TTA GCG CAT CAA CTT       T   A               AAG GAG GAG CCT GCG TCT   GGC TCC TCC TTA GGC GAC       G   TT               AGG AGC CTG CGT CTG ATT   GTT GAC TGC CTC CTT GCG       A   GT               AGG AGG CAG TCA ACC ACG   TCC TCC TTA GGC GAC TTT       GT   CG               GCG AAA GTC GCC TAA GGA   GTG GTT GAC TGC CTC CTT       G   GC               GCC TAA GGA GGA GCC TGC   ACC GTG GTT GAC TGC CTC       GT   CT               GCA AGG AGG CAG TCA ACC   GCA GGC TCC TCC TTA GGC       A   GA               GCA AGT CGC TCG GGC AGT   GAC GCA GGC TCC TCC TTA       A   GG               ACC CGT AGG AAT CTA TCC   TCA GAC GCA GGC TCC TCC       T   TT               GCA CAG TCA GGC GTG AAA   TGC TAC TGC CCG AGC GAC       TA   TT               ACA CAT GCA AGT CGC TCG   TGA CCC GAC CGT GGT TGA       GG   C                  
 
         [0047]    
       
         
               
             
               
               
             
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                   
               
               
                   Leptospirillum  sp. 
               
             
          
           
               
                 Sense primers 
                 Antisense primers 
               
               
                   
               
             
          
           
               
                 TGA GGG GAC TGC CAG CGA 
                 CTA GAC GGG TAC CTT GTT 
                   
               
               
                 C 
                 AC 
               
               
                   
               
               
                 TAA ATA TCC CCG ATG ACG 
                 CCG TCA TCG GGG ATA TTT 
               
               
                 G 
                 A 
               
               
                   
               
               
                 TTG TCC GGA ACC GTG AAG 
                 TTC ACG GTT CCG GAC AAT 
               
               
                 GG 
                 AT 
               
               
                   
               
               
                 GGA ACC GTG AAG GGT TTC 
                 CGG TTC CGG ACA ATA TTC 
               
               
                 G 
                 G 
               
               
                   
               
               
                 CCG AAT ATT GTC CGG AAC 
                 CCC TTC ACG GTT CCG GAC 
               
               
                 C 
                 AA 
               
               
                   
               
               
                 CGA CAG AGT TTG ATC GTG 
                 CCA CGA TCA AAC TCT GTC 
               
               
                 G 
                 GA 
               
               
                   
               
               
                 AAT ATT GTC CGG AAC CGT 
                 AAA CCC TTC ACG GTT CCG 
               
               
                 G 
                 GA 
               
               
                   
               
               
                 TCC GGA ACC GTG AAG GGT 
                 TTC CGG ACA ATA TTC GGT 
               
               
                 T 
                 AT 
               
               
                   
               
               
                 AAA TCG GGC CAT CAC ACA 
                 CCG AAA CCC TTC ACG GTT 
               
               
                 G 
                 CC 
               
               
                   
               
               
                 CAA AGA GAC TGG CAG ACT 
                 TAG TCT GCC AGT CTC TTT 
               
               
                 AGA 
                 GGC 
               
               
                   
               
               
                 TCG GGC CAT CAC ACA GGT 
                 GCA CCT GTG TGA TGG CCC 
               
               
                 G 
                 GAT 
               
               
                   
               
               
                 AGA GAC TGG CAG ACT AGA 
                 CTC TAG TCT GCC AGT CTC 
               
               
                 G 
                 TTT 
               
               
                   
               
               
                 GGG GGG GCA ATA CCG AAT 
                 GCA GCA CCT GTG TGA TGG 
               
               
                 AGA 
                 CCC 
               
               
                   
               
               
                 ATA TCA AAT AAA TAT CCC 
                 CCT GTG TGA TGG CCC GAT 
               
               
                 CG 
                 TT 
               
               
                   
               
               
                 AAG GGA TAT CGA ATA AAT 
                 TCT ATT CGG TAT TGC CCC 
               
               
                 AT 
                 CCC 
               
               
                   
               
               
                 CTA GAG GCT GGG AGA GGG 
                 CCC CTT TCG GTT CCC TAC 
               
               
                 AAG 
                 TCG 
               
               
                   
               
               
                 GAC GCA GCA ACG CCA GCA 
                 TCC CTC TCC CAG CCT CTA 
               
               
                 GTG 
                 GTC 
               
               
                   
               
               
                 AAA TAA ATA TCC CCG ATG 
                 TCG GGG ATA TTT ATT TGA 
               
               
                 A 
                 T 
               
               
                   
               
               
                 CAG TGT GGG AAG AAG GCT 
                 CAT ACC TTG GGC GGC TCC 
               
               
                 TTC 
                 CT 
               
               
                   
               
               
                 AAC AAG GTA CCC GTC TAG 
                 CAG CCT CTA GTC TGC CAG 
               
               
                 A 
                 T 
               
               
                   
               
             
          
         
       
     
         [0048]    
       
         
               
             
               
               
             
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                   
               
               
                   Sulfobacillus  sp. 
               
             
          
           
               
                 Sense primers 
                 Antisense primers 
               
               
                   
               
             
          
           
               
                 CGA AGG CGG TGC ACT GGC 
                 CAG TGC ACC GCC TTC GCC 
                   
               
               
                 C 
                 A 
               
               
                   
               
               
                 GTG GCG AAG GCG GTG CAC 
                 GGC CAG TGC ACC GCC TTC 
               
               
                 T 
                 G 
               
               
                   
               
               
                 AGG TGT CGC GGG GGT CCA 
                 GGT GGA CCC CCG CGA CAC 
               
               
                 CC 
                 C 
               
               
                   
               
               
                 TGT CTG TCG GGA CGA GGA 
                 GGT CCT CGT CCC GAC AGA 
               
               
                 C 
                 C 
               
               
                   
               
               
                 GAG GGC AGG AGA GGT GCA 
                 CAT GCA CCT CTC CTG CCC 
               
               
                 T 
                 TC 
               
               
                   
               
               
                 GTC CAC CTC GCG GTG CCG 
                 TTA GCT CCG GCA CCG CGA 
               
               
                 G 
                 GG 
               
               
                   
               
               
                 CAC CTC GCG GTG CCG GAG 
                 GCG AGG TGG ACC CCC GCG 
               
               
                 C 
                 A 
               
               
                   
               
               
                 GGG GGT CCA CCT CGC GGT 
                 TGC ACC GCC TTC GCC ACC 
               
               
                 GC 
                 G 
               
               
                   
               
               
                 CTC GCG GTG CCG GAG CTA 
                 CGT ATC CAT CGT TTA CGG 
               
               
                 A 
                 CG 
               
               
                   
               
               
                 TGT CGC GGG GGT CCA CCT 
                 GAC CCC CGC GAC ACC TCG 
               
               
                 C 
                 TA 
               
               
                   
               
               
                 GGA TAC GAG GTG TCG CGG 
                 GAG TGC GTT AGC TCC GGC 
               
               
                 G 
                 AC 
               
               
                   
               
               
                 CGG AGC TAA CGC ACT CAG 
                 TCC ACC AGG AAT TCC ATG 
               
               
                 T 
                 C 
               
               
                   
               
               
                 GTA AAC GAT GGA TAC GAG 
                 GCC AGG CCA GTG CAC CGC 
               
               
                 GT 
                 C 
               
               
                   
               
               
                 TGA GTG GGG GAT ATC GGG 
                 CCA GGA ATT CCA TGC ACC 
               
               
                 C 
                 TC 
               
               
                   
               
               
                 TAC GAG GTG TCG CGG GGG 
                 CCT CGT ATC CAT CGT TTA 
               
               
                 T 
                 CG 
               
               
                   
               
               
                 AGC TAA CGC ACT CAG TAT 
                 ACT GAG TGC GTT AGC TCC 
               
               
                 C 
                 GG 
               
               
                   
               
               
                 ACG ATG GAT ACG AGG TGT 
                 GAT ACT GAG TGC GTT AGC 
               
               
                 CG 
                 TC 
               
               
                   
               
               
                 GTG CCG GAG CTA ACG CAC 
                 GCG ACA CCT CGT ATC CAT 
               
               
                 TC 
                 CG 
               
               
                   
               
               
                 AGG TGC ATG GAA TTC CTG 
                 CGG GAT ACT GAG TGC GTT 
               
               
                 GT 
                 AG 
               
               
                   
               
               
                 TGC ATG GAA TTC CTG GTG 
                 GCC CGA TAT CCC CCA CTC 
               
               
                 GA 
                 A 
               
               
                   
               
             
          
         
       
     
         [0049]    
       
         
               
             
               
               
             
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                   
               
               
                 
                   Acidithiobacillus ferrooxidans 
                 
               
             
          
           
               
                 Sense primers 
                 Antisense primers 
               
               
                   
               
             
          
           
               
                 CGG GTT CTA ATA CAA TCT 
                 AGA ACC CGC CTT TTC GTC 
                   
               
               
                 G 
                 CT 
               
               
                   
               
               
                 AGG ACG AAA AGG CGG GTT 
                 CCG CCT TTT CGT CCT CCA 
               
               
                 CT 
                 C 
               
               
                   
               
               
                 GTG GAG GAC GAA AAG GCG 
                 CAG ATT GTA TTA GAA CCC 
               
               
                 G 
                 G 
               
               
                   
               
               
                 ACG AAA AGG CGG GTT CTA 
                 ATT AGA ACC CGC CTT TTC 
               
               
                 AT 
                 GT 
               
               
                   
               
               
                 AAA AGG CGG GTT CTA ATA 
                 TGT ATT AGA ACC CGC CTT 
               
               
                 CA 
                 TT 
               
               
                   
               
               
                 AGG CGG GTT CTA ATA CAA 
                 CTC TGC AGA ATT CCG GAC 
               
               
                 T 
                 AT 
               
               
                   
               
               
                 TTC TAA TAC AAT CTG CTG 
                 AAC AGC AGA TTG TAT TAG 
               
               
                 TT 
                 AA 
               
               
                   
               
               
                 TAA TAC AAT CTG CTG TTG 
                 GTC AAC AGC AGA TTG TAT 
               
               
                 AC 
                 TA 
               
               
                   
               
               
                 TAC AAT CTG CTG TTG ACG 
                 CAC GTC AAC AGC AGA TTG 
               
               
                 TG 
                 TA 
               
               
                   
               
               
                 AAT CTG CTG TTG ACG TGA 
                 ATT CAC GTC AAC AGC AGA 
               
               
                 AT 
                 TT 
               
               
                   
               
               
                 CGC TAA GGG AGG AGC CTA 
                 GTA GGC TCC TCC CTT AGC 
               
               
                 CG 
                 GC 
               
               
                   
               
               
                 GCG GAC TAG AGT ATG GGA 
                 GCTC CTC CCT TAG CGC GAG 
               
               
                 G 
               
               
                   
               
               
                 CTA GAG TAT GGG AGA GGG 
                 CCA TAC TCT AGT CCG CCG 
               
               
                 TG 
                 GT 
               
               
                   
               
               
                 CCT CGC GCT AAG GGA GGA 
                 TCT AGT CCG CCG GTT TCC 
               
               
                 G 
                 A 
               
               
                   
               
               
                 GGC GGA CTA GAG TAT GGG 
                 GAC GTA GGC TCC TCC CTT 
               
               
                 AG 
                 AG 
               
               
                   
               
               
                 GGG AGG AGC CTA CGT CTG 
                 TAC TCT AGT CCG CCG GTT 
               
               
                 AT 
                 T 
               
               
                   
               
               
                 CGC GCT AAG GGA GGA GCC 
                 TCA GAC GTA GGC TCC TCC 
               
               
                 T 
                 CT 
               
               
                   
               
               
                 CGG ACC TCG CGC TAA GGG 
                 CCT CCC TTA GCG CGA GGT 
               
               
                 AG 
                 CC 
               
               
                   
               
               
                 GGC GGA CTA GAG TAT GGG 
                 TAG TGC GCC GGT TTC CAC 
               
               
                 A 
                 C 
               
               
                   
               
               
                 TAA GGG AGG AGC CTA CGT 
                 ATT GTA TTA GAA CCC GCC 
               
               
                 CT 
                 T 
               
               
                   
               
             
          
         
       
     
         [0050]    
       
         
               
             
               
               
             
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                   
               
               
                 
                   Acidithiobacillus thiooxidans 
                 
               
             
          
           
               
                 Sense primers 
                 Antisense primers 
               
               
                   
               
             
          
           
               
                 GGG AGA CGA AAA GGT AAT 
                 ATC CCC CGG TTT CTC CCT 
                   
               
               
                 CG 
                 C 
               
               
                   
               
               
                 AAA GTT CTT TCG GTG ACG 
                 ATA TTA GCG ATT ACC TTT 
               
               
                 GG 
                 T 
               
               
                   
               
               
                 CGG GGA AGG TTG ATA TGT 
                 CAA CCT TCC CCG TCA CCG 
               
               
                 TA 
                 AA 
               
               
                   
               
               
                 GAG GGA GAA ACC GGG GGA 
                 CCG AAG ATC CCC CGG TTT 
               
               
                 T 
                 CT 
               
               
                   
               
               
                 AAT CGC TAA TAT CGG TTA 
                 CTC CAA TAG CAC GAG GTC 
               
               
                 C 
                 CG 
               
               
                   
               
               
                 CCG GGG GAT CTT CGG ACC 
                 ACC GAT ATT AGC GAT TAC 
               
               
                 TC 
                 CT 
               
               
                   
               
               
                 TAA TAT CGCC TGC TGT TGA 
                 AAG ATC CCC CGG TTT CTC 
               
               
                 C 
                 C 
               
               
                   
               
               
                 TCG GTG ACG GGG AAG GTT 
                 TAT CAA CCT TCC CCG TCA 
               
               
                 G 
                 CC 
               
               
                   
               
               
                 GGA GAA ACC GGG GGA TCT 
                 GGT TTC TCC CTC AGG ACG 
               
               
                 T 
                 TA 
               
               
                   
               
               
                 ACG TCC TGA GGG AGA AAC 
                 GGT CCG AAG ATC CCC CGG 
               
               
                 CG 
                 TT 
               
               
                   
               
               
                 AGA CGA AAA GGT AAT CGC 
                 TTT CAC GAC AGA CCT AAT 
               
               
                 TA 
                 G 
               
               
                   
               
               
                 GTG ACG GGG AAG GTT GAT 
                 GTA ACC GAT ATT AGC GAT 
               
               
                 A 
                 TA 
               
               
                   
               
               
                 GAA ACC GGG GGA TCT TCG 
                 ACA TAT CAA CCT TCC CCG 
               
               
                 G 
                 TC 
               
               
                   
               
               
                 TCC TGA GGG AGA AAC CGG 
                 CCC GGT TTC TCC CTC AGG 
               
               
                 GG 
                 AC 
               
               
                   
               
               
                 CGA AAA GGT AAT CGC TAA 
                 GCG ATT ACC TTT TCG TCT 
               
               
                 TA 
                 CC 
               
               
                   
               
               
                 AAA GGT AAT CGC TAA TAT 
                 CCC CGT CAC CGA AAG AAC 
               
               
                 CG 
                 TT 
               
               
                   
               
               
                 TCG TGG GAG ACG AAA AGG 
                 TTA ACA TAT CAA CCT TCC 
               
               
                 TA 
                 CC 
               
               
                   
               
               
                 CGG ACC TCG TGC TAT TGG 
                 TTA GCG ATT ACC TTT TCG 
               
               
                 AG 
                 TC 
               
               
                   
               
               
                 GTT CTT TCG GTG ACG GGG 
                 CTT CCC CGT CAC CGA AAG 
               
               
                 A 
                 AA 
               
               
                   
               
               
                 CTT TCG GTG ACG GGG AAG 
                 ATT ACC TTT TCG TCT CCC 
               
               
                 G 
                 AC 
               
               
                   
               
             
          
         
       
     
         [0051]     Archaea Kingdom:  
                                       TABLE 10                             Acidianus  sp.            Sense primers   Antisense primers                    GGG AAA CCG TGA GGG CGC   CCG CAT TGG GGA CGT TTC           T   GCG               GCG AAA CGT CCC CAA TGC   GCG CCC TCA CGG TTT CCC       GG   GCA               CCG CAG GGA AAC CGG TAA   CCG CAT TGG GGA CGT TTC       GCC   GCG               CCC GGG AAA GGG CAG TGA   GCG CCC TCA CGG TTT CCC       TA   GCA               GGG AAA GGG CAG TGA TAC   TTC CCG CAT TGG GGA CGT       T   TTC               AAT CCG GGG CAG GCG AAG   TAG CGC CCT CAC GGT TTC       GG   CC               AGG GTA CTG GAA CGT CCC   GGC TTA CCG GTT TCC CTG       TT   CG               AAG CGT CCG GCC AGA ACG   CTG CCC TTT CCC GGG TTG       CGC   A               CGC CTA AAG GGG CAT GGG   TCA CTG CCC TTT CCC GGG       CT   T               GGC TAT TTC CCG CTC ATG   GTA TCA CTG CCC TTT CCC       CC   G               CGT ACG CCC TCG GGT AAG   GCC CGG GTC TTT AAG CAG       AGG   TG               AAC GGC CCG CCA AAC CGA   CTC CCG CCC CCT AGC CCT       TA   GCA               AGC CGG CCC TGC AAG TCA   CCC GGG ATC TGT GGA TTT       C   CGC               CAC TGC TTA AAG ACC CGG   TAC CCG AGG GCG TAC GAC       G   T               GGA GCT AAT CCG GGG CAG   CCT CTT ACC CGA GGG CGT       GCG   ACG               AAA CCG TGA GGG CGC TAC   TTC GCC TGC CCC GGA TTA       CC   G               AGG CGA AGG GTA CTG GAA   GGC GGC AGG CTT ACC GGT       CGT   TTC               ACC CCC AGT GCT CCC GAA   CGG ATT AGC TCC AGT TTC       AG   CCG               CCC TTC GCC TAA AGG GGC   GGA CGT TCC AGT ACC CTT       ATG   C               GCA TGG GCT ATT TCC CGC   CCC CGG ATT AGC TCC AGT       TCA   TT               GGG AAA CCG TGA GGG CGC   TAC CCT TCG CCT GCC CCG       T   GAT               GCG AAA CGT CCC CAA TGC   CCA TGC CCC TTT AGG CGA       GG   A                  
 
         [0052]    
       
         
               
             
               
               
             
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                   
               
               
                   Ferroplasma  sp. 
               
             
          
           
               
                 Sense primers 
                 Antisense primers 
               
               
                   
               
             
          
           
               
                 AGA GTC AAC CTG ACG AGC 
                 AAG CTC GTC AGG TTG ACT 
                   
               
               
                 TTA 
                 CT 
               
               
                   
               
               
                 GTC AAC CTG ACG AGC TTA 
                 GTA AGC TCG TCA GGT TGA 
               
               
                 CTC 
                 C 
               
               
                   
               
               
                 TGA GAG TCA ACC TGA CGA 
                 CGA GTA AGC TCG TCA GGT 
               
               
                 GC 
                 T 
               
               
                   
               
               
                 GAG CTT ACT CGA TAG CAG 
                 CTG CTA TCG AGT AAG CTC 
               
               
                 GAG 
                 G 
               
               
                   
               
               
                 TTT AAT TCG AGA GGG TTA 
                 TTT AAC CCT CTC GAA TTA 
               
               
                 A 
                 A 
               
               
                   
               
               
                 CTT ACT CGA TAG CAG GAG 
                 CTC CTG CTA TCG AGT AAG 
               
               
                 AGG 
                 C 
               
               
                   
               
               
                 AAT CAA ATC TGA TGT CGG 
                 TCA GAT TTG ATT TAA CCC 
               
               
                 TGA 
                 TC 
               
               
                   
               
               
                 GGT TAA ATC AAA TCT GAT 
                 ACC CTC CTC ACC GAC ATC 
               
               
                 G 
                 AG 
               
               
                   
               
               
                 TTC GAG AGG GTT AAA TCA 
                 ACA TCA GAT TTG ATT TAA 
               
               
                 AAT 
                 C 
               
               
                   
               
               
                 CAA ATC TGA TGT CGG TGA 
                 CCG ACA TCA GAT TTG ATT 
               
               
                 GGA 
                 T 
               
               
                   
               
               
                 TAA ATC AAA TCT GAT GTC 
                 TGA TTT AAC CCT CTC GAA 
               
               
                 G 
                 T 
               
               
                   
               
               
                 GAG AGG GTT AAA TCA AAT 
                 TCA CCG ACA TCA GAT TTG 
               
               
                 CTG 
                 A 
               
               
                   
               
               
                 ATC TGA TGT CGG TGA GGA 
                 ATT TGA TTT AAC CCT CTC 
               
               
                 GGG 
                 G 
               
               
                   
               
               
                 AAT TCG AGA GGG TTA AAT 
                 CTA CCT GAT AGG TTG CAG 
               
               
                 C 
                 ACT 
               
               
                   
               
               
                 GAT GTC GGT GAG GAG GGT 
                 GCA CCA CCT CTC TGC TAT 
               
               
                 T 
                 CG 
               
               
                   
               
               
                 GAG GGA TGG CAG TGT CGG 
                 ATC CCT CAA CGG AAA AGC 
               
               
                 A 
                 A 
               
               
                   
               
               
                 TGG CCA AGA CTT TTC TCA 
                 ACA CTT AAA GTG AAC GCC 
               
               
                 T 
                 CT 
               
               
                   
               
               
                 GAT GAG TCT GCA ACC TAT 
                 TCG CTC CGA CAC TGC CAT 
               
               
                 CA 
                 C 
               
               
                   
               
               
                 TAG CAG AGA GGT GGT GCA 
                 CCG ATC TCA TGT CTT GCA 
               
               
                 TGG 
                 GT 
               
               
                   
               
               
                 ACG GCC ACT GCT ATC AAG 
                 ATG AGA AAA GTC TTG GCC 
               
               
                 TTC 
                 A 
               
               
                   
               
             
          
         
       
     
         [0053]    
       
         
               
             
               
               
             
               
               
               
             
           
               
                 TABLE 12 
               
             
             
               
                   
               
               
                   
               
               
                   Metallosphaera  sp. 
               
             
          
           
               
                 Sense primers 
                 Antisense primers 
               
               
                   
               
             
          
           
               
                 AGG GCG TTA CCC CTA GTG 
                 GGC ACT AGG GGT AAC GCC 
                   
               
               
                 C 
                 C 
               
               
                   
               
               
                 TAC CCC TAG TGC CCT CGC 
                 AGA AGC TCG ACC TCC CAC 
               
               
                 A 
                 CC 
               
               
                   
               
               
                 GCG CCC GTA GCC GGC CTG 
                 TAC AGG CCG GCT ACG GGC 
               
               
                 TAA 
                 GC 
               
               
                   
               
               
                 GAG CTT CTC CTC CGC GAG 
                 AGC TCG ACC TCC CAC CCC 
               
               
                 GGG 
                 G 
               
               
                   
               
               
                 GCA CCA GGC GCG GAA CGT 
                 CCC CTC GCG GAG GAG AAG 
               
               
                 CCC 
                 C 
               
               
                   
               
               
                 GAG GTC GAG CTT CTC CTC 
                 TGC GAG GGC ACT AGG GGT 
               
               
                 CG 
                 A 
               
               
                   
               
               
                 CCC TAG TGC CCT CGC AAG 
                 TGA CTT TAC AGG CCG GCT 
               
               
                 A 
                 ACG 
               
               
                   
               
               
                 CCC GTA GCC GGC CTG TAA 
                 CAT GGC TTA GCC CTA CCC 
               
               
                 AGT 
                 CTA 
               
               
                   
               
               
                 CGG GGT GGG AGG TCG AGC 
                 AGG AGA AGC TCG ACC TCC 
               
               
                 TTC 
                 CA 
               
               
                   
               
               
                 GTC GAG CTT CTC CTC CGC 
                 GAC GTT CCG CGC CTG GTG 
               
               
                 GA 
                 C 
               
               
                   
               
               
                 GGT GGG AGG TCG AGC TTC 
                 CTT TAC AGG CCG GCT ACG 
               
               
                 TCC 
                 GG 
               
               
                   
               
               
                 TCG GGG TGG GAG GTC GAG 
                 TCT TGC GAG GGC ACT AGG 
               
               
                 C 
                 G 
               
               
                   
               
               
                 GCG TTA CCC CTA GTG CCC 
                 CGG AGG AGA AGC TCG ACC 
               
               
                 T 
                 TC 
               
               
                   
               
               
                 TAG GGG TAG GGC TAA GCC 
                 TCG CGG AGG AGA AGC TCG 
               
               
                 ATG 
                 AC 
               
               
                   
               
               
                 CGC ACC AGG CGC GGA ACG 
                 GAG GGC ACT AGG GGT AAC 
               
               
                 T 
                 G 
               
               
                   
               
               
                 GGG AGG TCG AGC TTC TCC 
                 ACC CCG AGG GGC AAG AGG 
               
               
                 T 
                 CC 
               
               
                   
               
               
                 AGG TGG AGG AAT AAG CGG 
                 GGG GTT ATC CAG ATC CCA 
               
               
                 GG 
                 AGG 
               
               
                   
               
               
                 GAA AGG TGG AGG AAT AAG 
                 GCC ACG CCC TCT TCC CGA 
               
               
                 C 
                 GA 
               
               
                   
               
               
                 GGG AGT CGT ACG CTC TCG 
                 GTT ATC CAG ATC CCA AGG 
               
               
                 GGA 
                 GC 
               
               
                   
               
               
                 CTA ACC TGC CCT TGG GAT 
                 CTT ATT CCT CCA CCT TTC 
               
               
                 CTG 
                 TGG 
               
               
                   
               
             
          
         
       
     
         [0054]    
       
         
               
             
               
               
             
               
               
               
             
           
               
                 TABLE 13 
               
             
             
               
                   
               
               
                   
               
               
                   Sulfolobus  sp. 
               
             
          
           
               
                 Sense primers 
                 Antisense primers 
               
               
                   
               
             
          
           
               
                 TAA ACC CTG CCG CAG TTG 
                 CCA ACT GCG GCA GGG TTT 
                   
               
               
                 G 
                 A 
               
               
                   
               
               
                 CCT TAA ACC CTG CCG CAG 
                 ACT GCG GCA GGG TTT AAG 
               
               
                 T 
                 G 
               
               
                   
               
               
                 GTC CTG GAA CGG TTC CTC 
                 CGA GGA ACC GTT CCA GGA 
               
               
                 G 
                 CTC 
               
               
                   
               
               
                 CTC TAC AAA GGC GGG GGA 
                 AAC CGT TCC AGG ACT CCT 
               
               
                 ATA 
                 CG 
               
               
                   
               
               
                 CTG GAA CGG TTC CTC GCT 
                 TCC AGG ACT CCT CGC CTA 
               
               
                 GA 
                 TGG 
               
               
                   
               
               
                 GGC GAG GAG TCC TGG AAC 
                 CCT TTG TAG AGC GGG GAA 
               
               
                 GGT 
                 A 
               
               
                   
               
               
                 TTT CCC CGC TCT ACA AAG 
                 AGC GAG GAA CCG TTC CAG 
               
               
                 G 
                 GA 
               
               
                   
               
               
                 TAC AAA GGC GGG GGA ATA 
                 CGT TCC AGG ACT CCT CGC 
               
               
                 AGC 
                 CTA 
               
               
                   
               
               
                 CGC TCT ACA AAG GCG GGG 
                 CCC CCG CCT TTG TAG AGC 
               
               
                 G 
                 G 
               
               
                   
               
               
                 ATA GGC GAG GAG TCC TGG 
                 TTC AGC GAG GAA CCG TTC 
               
               
                 AA 
                 CA 
               
               
                   
               
               
                 CCA TAG GCG AGG AGT CCT 
                 ATT CCC CCG CCT TTG TAG 
               
               
                 G 
                 A 
               
               
                   
               
               
                 GCT TTT CCC CGC TCT ACA 
                 TTG TAG AGC GGG GAA AAG 
               
               
                 A 
                 C 
               
               
                   
               
               
                 GCT AAC CTA CCC TGA GGA 
                 ATC TCC CTC CTC AGG GTA 
               
               
                 GG 
                 GGT 
               
               
                   
               
               
                 TCT CCC ATA GGC GAG GAG 
                 GGG TTA TCT CCC TCC TCA 
               
               
                 TC 
                 G 
               
               
                   
               
               
                 TGG CTA ACC TAC CCT GAG 
                 TCG CCT ATG GGA GAT TAT 
               
               
                 G 
                 C 
               
               
                   
               
               
                 ATA ATC TCC CAT AGG CGA 
                 TCA GGG TAG GTT AGC CAC 
               
               
                 G 
                 GT 
               
               
                   
               
               
                 TGA GGA GGG AGA TAA CCC 
                 CCT CAG GGT AGG TTA GCC 
               
               
                 CG 
                 A 
               
               
                   
               
               
                 ACA CGT GGC TAA CCT ACC 
                 CCG GGG TTA TCT CCC TCC 
               
               
                 CTG 
                 T 
               
               
                   
               
               
                 CCT GAG GAG GGA GAT AAC 
                 TCC TCG CCT ATG GGA GAT 
               
               
                 C 
                 T 
               
               
                   
               
               
                 AAA CTG GGG ATA ATC TCC 
                 CCT CCT CAG GGT AGG TTA 
               
               
                 C 
                 G 
               
               
                   
               
             
          
         
       
     
         [0055]    
       
         
               
             
               
               
             
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                   
               
               
                   Thermoplasma  sp. 
               
             
          
           
               
                 Sense primers 
                 Antisense primers 
               
               
                   
               
             
          
           
               
                 TCC TGA AAG GAC GAC CGG 
                 CAG GGG CAT ATT CAC CGT 
                   
               
               
                 TG 
                 AG 
               
               
                   
               
               
                 GGA CTG AGG GCT GTA ACT 
                 TCA GGA TTA CAG GAT TTT 
               
               
                 C 
                 A 
               
               
                   
               
               
                 GAG GTT GAA TGT ACT TTC 
                 ACC CTG AAA GTA CAT TCA 
               
               
                 AGG 
                 ACC 
               
               
                   
               
               
                 GGT GGC GAA AGC GTT CAA 
                 GCC ACC GGT CGT CCT TTC 
               
               
                 CT 
                 A 
               
               
                   
               
               
                 GCC CTC ACG AAT GTG GAT 
                 CTA GTT GAA CGC TTT CGC 
               
               
                 T 
                 C 
               
               
                   
               
               
                 ACC TCG AAA CCC GTT CGT 
                 TCG TCC TTT CAG GAT TAC 
               
               
                 AG 
                 AGG 
               
               
                   
               
               
                 TCC GTA GTA ATC GTA GGT 
                 ACG CTT TCG CCA CCG GTC 
               
               
                 C 
                 GTC 
               
               
                   
               
               
                 ATC CTG TAA TCC TGA AAG 
                 GGG TTT CGA GGT TAG CTT 
               
               
                 GAC 
                 C 
               
               
                   
               
               
                 GTA GTC AGG ACT GAG GGC 
                 CCC TCA GTC CTG ACT ACG 
               
               
                 TG 
                 A 
               
               
                   
               
               
                 AGG ACG ACC GGT GGC GAA 
                 CTG AAG ATT TAT AAG ACC 
               
               
                 AGC 
                 GG 
               
               
                   
               
               
                 TAA CTC GCC CTC ACG AAT 
                 TTA CAG CCC TCA GTC CTG 
               
               
                 GT 
                 ACT 
               
               
                   
               
               
                 GAA GGT GTT AAG TGG GTC 
                 AAT CCA CAT TCG TGA GGG 
               
               
                 A 
                 CGA 
               
               
                   
               
               
                 AAA CCC GTT CGT AGT CAG 
                 ATG GGG GTC TTG CTC GTT 
               
               
                 GAC 
                 AT 
               
               
                   
               
               
                 TAC GGT GAA TAT GCC CCT 
                 GCT GTT GAC CTA CGA TTA 
               
               
                 GC 
                 C 
               
               
                   
               
               
                 CAC TTG GTG TTG CTT CTC 
                 CCT ACG ATT ACT ACG GAA 
               
               
                 CGT 
                 TCC 
               
               
                   
               
               
                 GAT CAC TTT TAT TGA GTC 
                 ACC CAC TTA ACA CCT TCG 
               
               
                 T 
                 C 
               
               
                   
               
               
                 AGC ATC AGG AAT AAG GGC 
                 CCC AAG TCT TAC AGT CTC 
               
               
                 TG 
                 TT 
               
               
                   
               
               
                 AAG ACC CCC ATC TCT AAT 
                 CTA CCC TGA AAG TAC ATT 
               
               
                 T 
                 CA 
               
               
                   
               
               
                 CCG GTC TTA TAA ATC TTC 
                 CAG CCC TTA TTC CTG ATG 
               
               
                 A 
                 C 
               
               
                   
               
               
                 ATA ACG AGC AAG ACC CCC 
                 GGT CGT CCT TTC AGG ATT 
               
               
                 AT 
                 AC 
               
               
                   
               
             
          
         
       
     
         [0056]     In secondary PCRs a reaction is also included to quantify total bacteria or archaea present in the sample; in this case known universal primers are used for both kingdoms which are selected among the primers included in Table 15.  
                                           TABLE 15                       Secondary PCR                                    Bacteria primers                Eub27 1  F   AGA GTT TGA TCC TGG CTC AG                           Univ533-F 1     GTG CCA GCM GCC GCG GTA                       Bact358-F 2     CCT ACG GGA GGC AGC AG                       Univ907-R 3     CCG TCA ATT CCT TTG AGT T                       Bact338-R 4     GCT GCC TCC CGT AGG AGT                       Bact1387-R 5     GGG CGG WGT GTA CAA GGC                       Archaea primers           Arch344-F 6     ACG GGG CGC AGC AGG CGC GA                           Univ515-F 7     GTG CCA GCA GCC GCG GTA A                       Arch958-R 8     YCC GGC GTT GAM TCC AAT T                       Arch915-R 4     GTG CTC CCC CGC CAA TTC CT                       Univ534-R 5     ATT ACC GCG GCT GCT GG                           1 Bond P., 2000, Appl Environ Microbiol. 66(9):3842-9.                  2 Schauer M, 2003, Aquat Microb Ecol Vol. 31: 163-174.                  3 Nakagawa T, 2002, FEMS Microbiology Ecology 41:199-209.                  4 Schrenk MO, 1998, Science. 279:1519-22.                  5 Ellis R, 2003, Appl Environ Microbiol. 69(6):3223-30.                  6 Casamayor EO, 2002, Environ Microbiol. 4(6):338-48.                  7 Edwards K, 2003, Appl Environ Microbiol. 69(5):2906-13.                  8 Orphan VJ, 2001, Appl Environ Microbiol. 66(2):700-11.             
 
         [0057]     Each secondary PCR has a specific cycle, wherein the alignment temperature changes, said temperature being specific for each used primer pair. Table 16 summarizes general conditions for all qPCR cycles.  
                                                                         TABLE 16                                   Step   Temperature (° C.)   Time (s)                                    1   Initial denaturation   95   120        2   Denaturation   95   30       3   Alignment   (*)   30       4   Extension   72   40       5   Pre-reading   80   10       6   Reading   80   —                Repeat 40 times from step 2 to step 6 (qPCR cycle)            7   Denaturation curve   Between 70 and 100° C., reading each               0.2° C.                 (*) specific temperature for each used primer pair             
 
         [0058]     Duration curve carried out at the end of cycle 40, gives the Tm of the amplification product, and is also used to establish whether more than one amplification product is present in the amplified sample, as each would generate its own curve.  
         [0059]     The PCR thermocycler gives a result corresponding to DNA concentration present in each reaction, and this information is used to calculate the number of microorganisms present in the sample, which is called Q. This value is inferred by the computational program associated to the thermocycler based on: DNA concentration in calibration curve reactions and the cycle in which sample begins to amplify (or to exponentially increase its fluorescence value). The correlation between the logarithm of DNA concentration and the cycle in which amplification is observed generates a linear equation, from which DNA concentration in the analyzed samples is inferred.  
         [0000]     Calculation of the Number of Microorganisms Present in the Sample  
         [0060]     Taking into account the qPCR result and other data generated during the process, the inventors have developed a mathematical formula that allows calculating the exact number of microorganisms from a given taxon present in a given sample, specially a biomining sample. The formula is as follows:  
         Mo   /   Um     =       Q   ×   T         5   ·       10     -   6       ⁡     [     ng   ⁢     /     ⁢   mo     ]         ×   U   ×   Cm           
 
         [0061]     where: 
        Mo/Um is the number of microorganisms, either bacteria or archaea, per sample unit;     Q is the amount of initial DNA in nanograms that is present in each secondary PCR reaction, as determined by the program associated to the qPCR equipment;     T is the amount of total DNA extracted from the sample;     U is the amount of DNA used in the primary PCR reaction; and     C m  is the amount of biomining sample from which DNA was extracted, expressed in ml for liquid samples or in g for solid samples.     The number 5×10 −6  ng/mo is the average amount of DNA nanograms contained in the genome of a microorganism, according to Kuske et al. (1998).        
 
         [0068]     By applying the method of the invention, the number of microorganisms belonging to the taxons  Acidiphilium  sp.,  Leptospirillum  sp.,  Sulfobacillus  sp.  Acidithiobacillus ferrooxidans  and  Acidithiobacillus thiooxidans, Acidianus  sp.,  Ferroplasma  sp,  Sulfolobus  sp.,  Metallosphaera  sp, and/or  Thermoplasma  sp. present in a sample can be determined.  
       EXAMPLES  
     Example 1  
     Quantification of  Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans, Leptospirillum  sp. and  Acidiphilium  sp. Present in a Sample  
       [0069]     Five solid samples obtained from mineral bioleaching heaps (MS-1 to MS-5) and 2 liquid samples recovered from bioleaching effluents (ML-1 and ML-2) were analyzed and total DNA was extracted from each one.  
         [0070]     For all solid samples a further step was necessary, a re-purification of DNA, which consisted in a sample re-purification using any existing purification technique; in our laboratories this step is performed using commercial DNA purification columns to obtain a translucent appearance in the extraction solution.  
         [0071]     Then, total DNA was quantified in each sample using a NanoDrop 1.0 spectrophotometer. Total extracted DNA nanograms (T) are shown in Table 17 together with the initial sample volumes (C m ). Registered results were:  
                                                     TABLE 17                                   Sample   T   C m                                          MS-1   316.8   0.5   g           MS-2   370.4   0.5   g           MS-3   315.2   0.5   g           MS-4   526.4   0.5   g           MS-5   400   5.0   g           ML-1   2938   81.00   ml           ML-2   1114   76.55   ml                      
 
         [0072]     Each of these samples was diluted with sterile nuclease-free water in order to obtain a concentration between 0.5 and 30 ng/μl. Table 18 shows the final volume to which the DNA solution was brought and its final concentration.  
                                                 TABLE 18                                   Sample   Final volume (μl)   Concentration (ng/μl)                                        MS-1   80   3.96           MS-2   80   4.63           MS-3   80   3.94           MS-4   80   6.58           MS-5   80   5.00           ML-1   100   29.38           ML-2   100   11.14                      
 
         [0073]     A calibration curve was simultaneously prepared to allow the calculation of DNA concentration in experimental samples. Four serial dilutions were prepared from a standard DNA mix containing 25 ng of DNA from each of the following microorganisms:  Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans, Leptospirillum  sp. and  Acidiphilium  sp., in a final volume of 30 μl, to obtain 100 ng of total DNA in the standard sample, which in its turn is part of the calibration curve.  
         [0074]     More specifically, DNA was used from the following strains: 
         A. ferrooxidans  DSM 16786;      A. thiooxidans  DSM 504;      Leptospirillum  sp. DSM 1931 and      Acidiphilium acidophilus  DSMZ 700.        
 
         [0079]     The reaction mix for the primary PCR was prepared, wherein the amount of each constituent was multiplied by the total number of reactions to be carried out; a single reaction mix was prepared in order to homogenize reagent concentrations in the different PCR tubes. The reaction mix was aliquoted in 0.2 ml tubes, using a volume of 24 μl of reaction mix per tube.  
         [0080]     In the present Example, the following reactions were performed in duplicate: 
        a) seven reactions for the samples and     b) 5 reactions for the calibration curve, corresponding to standard DNA master mix concentrations of 1×, 0.1×, 0.01×, 0.001× and 0.0001×, and a blank, giving a total of 25 reactions.        
 
         [0083]     The prepared mix is shown in Table 19.  
                               TABLE 19                                   Reagent   1 reaction   25 reactions                           Sterile nuclease-free H 2 O   18.35 μl    458.75 μl            PCR Buffer 10x   2.5 μl   62.5 μl           MgCl 2  (50 mM)   1.5 μl   37.5 μl           dNTPs (10 mM each)   0.5 μl   12.5 μl           Primer Bacteria 27F (10 μM)   0.5 μl   12.5 μl           Primer Bacteria 1492R (10 μM)   0.5 μl   12.5 μl           Hot Start Taq (5 U/μl)   0.15 μl    3.75 μl                      
 
         [0084]     Used primers are described in Table 20.  
                           TABLE 20                       Microorganism   Alignment               to be   tempera-           determined   ture   Used primers                   Total bacteria   59° C.   Eubac27F:                   AGA GTT TGA TCC TGG CTC AG               Univ1492R:               GGT TAC CTT GTT ACG ACT T                  
 
         [0085]     This primary PCR reaction mix was homogenized and 25 aliquots were made with 24 μl each in 0.2 ml tubes appropriately labeled. To this mix 1 μl of sample DNA dilutions or 1 μl of calibration curve DNA was added as appropriate. To the primary PCR negative control 1 μl of sterile nuclease-free water was added instead of DNA.  
         [0086]     Reactions were incubated in a MJ Research PTC-100 thermocycler, with the following cycle program:  
                                                 TABLE 21                                       Temperature               Step   (° C.)   Time (s)                                        1. Initial denaturation   95   120           2. Denaturation   95   30           3. Alignment   56   30           4. Extension   72   120                      
 
         [0087]     Wherein steps 2 to 4 were repeated 18 times.  
         [0088]     Subsequently 5 secondary PCR were performed, one for each taxon:  Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans, Leptospirillum  sp.,  Acidiphilium  sp. and one for total bacteria, using specific primers for each of them, which hybridize inside the region amplified in the primary PCR. Sense and antisense primers were selected for the different taxons from those included in the description of the tables corresponding to each taxon, Tables 5, 6, 8 and 9 in this case. On the other hand, for total bacteria primers described in the literature were used, which were included in Table 15.  
         [0089]     Primers used for each taxon and their respective annealing temperatures are indicated in Table 22.  
                           TABLE 22                       Microorganism   Alignment               to be   tempera-           determined   ture   Used primers                   Total bacteria   56° C.   (P.1) 533-F:                   5′- GTG CCA GCA GCC GCG               GTA -3′               (P.2) 907-R:               5′- CCG TCA ATT CCT TTG               AGT T -3′                 A .  ferrooxidans     60° C.   (P.1) F:               5′- GTG GAG GAC GAA AAG               GCG G -3′               (P.2) R:               5′- ATT AGA ACC CGC CTT               TTC GT -3′                 A .  thiooxidans     56° C.   (P.1) F:               5′- AAA GGT AAT CGC TAA               TAT CG -3′               (P.2) R:               5′- ATT ACC TTT TCG TCT               CCC AC -3′                 Leptospirillum     58° C.   (P.1) F:       sp.       5′- AAC AAG GTA CCC GTC               TAG A -3′               (P.2) R:               5′- CTA GAC GGG TAC CTT               GTT AC -3′                 Acidiphilium     61° C.   (P.1) F:       sp.       5′- AGG AGG CAG TCA ACC               ACG GT -3′               (P.2) R:               5′- GTT AGC GCA TCA ACT               TAA GG -3′                  
 
         [0090]     One qPCR was carried out on each primary PCR reaction product for each taxon to be determined, these reactions being performed in duplicate. The qPCR was carried out using Mix SYBR Green qPCR. For each secondary PCR one duplicate per each one of the 25 primary PCR reactions is considered plus one control, which gives a total of 51 reactions. For each PCR the reaction mix shown in Table 23 is prepared, where primers are those that are corresponding according to Table 22.  
                                             TABLE 23                                   1 reaction   51 reactions                                        Sterile nuclease-free H 2 O   16.1 μl    821.1 μl            Primer 1 (10 μM)   0.5 μl   25.5 μl           Primer 2 (10 μM)   0.5 μl   25.5 μl           PCR Buffer 10x   2.5 μl   127.5 μl            MgCl 2  (50 mM)   1.5 μl   76.5 μl           dNTPs (10 mM each)   2.5 μl   127.5 μl            Hot Start Taq (5 U/μl)   0.15 μl    7.65 μl           SYBR Green qPCR 100x   0.25 μl    12.75 μl                       
 
         [0091]     This reaction mix was homogenized and aliquoted in 51 0.2 ml tubes, which were duly labeled. To each of the tubes 1 μl of primary PCR or 1 μl of sterile nuclease-free water for the blank was added.  
         [0092]     PCR tubes containing the reaction mix and sample were vortexed for 5 seconds and centrifuged for 1 minute at 2000 rpm, in order to homogenize and bring the reaction liquid to the bottom of the tube, respectively.  
         [0093]     Then, the tubes with secondary PCR reactions were subjected to temperature cycles for amplification. According to the microorganism to be determined, different primer pairs were used and therefore different amplification programs were used. In the following Table, amplification programs used in the different secondary PCR reactions are shown.  
                                                                         TABLE 24                                   Step   Temperature (° C.)   Time (s)                                    1   Initial denaturation   95   120        2   Denaturation   95   30       3   Alignment   (*)   30       4   Extension   72   40       5   Pre-reading   80   10       6   Reading   80   —                Repeat 40 times from step 2 to step 6 (qPCR cycle)            7   Denaturation curve   Between 70 and 100° C., reading each               0.2° C.                 (*) specific temperature for each used primer pair, as indicated in Table 22.             
 
         [0094]     When the qPCR is finished, all data generated by the qPCR thermocycler are stored; this data corresponds to Q and is shown in Table 25, wherein DNA amounts in nanograms used for each reaction are included (U).  
                                                         TABLE 25                                       Q                Sample   Total bacteria     A. ferrooxidans       A. thiooxidans       Leptospirillum  sp.     Acidiphilium  sp.   U               MS-1   187.68 × 10 −3     1.63 × 10 −3     0.13 × 10 −3     25.36 × 10 −3     0.03 × 10 −3     2       MS-2    33.91 × 10 −3     0.51 × 10 −3     2.33 × 10 −3      1.63 × 10 −3     0   2       MS-3   149.60 × 10 −3     0.59 × 10 −3     0.03 × 10 −3     10.46 × 10 −3     0.008 × 10 −3     2       MS-4    71.08 × 10 −3     0.01 × 10 −3     0.03 × 10 −3      6.23 × 10 −3     0.002 × 10 −3     2       MS-5   142.68 × 10 −3     0.29 × 10 −3     3.20 × 10 −3     15.10 × 10 −3     0   2       ML-1    9.20 × 10 −3     0   0    0.26 × 10 −3     0   2       ML-2    49.03 × 10 −3     0.05 × 10 −3     0    2.15 × 10 −3     0   2                  
 
       Calculation of the Number of Microorganisms Present in the Samples  
       [0095]     Taking into account the qPCR result and other data generated during the process, the following formula was applied, the meaning of which was defined above:  
         Mo   /   Um     =       Q   ×   T         5   ·       10     -   6       ⁡     [     ng   ⁢     /     ⁢   mo     ]         ×   U   ×   Cm           
 
         [0096]     According to this, the following microbiological populations were determined in the analyzed samples:  
                             TABLE 26                           MS-1                Bacteria   Mo./g of sample                       Total bacteria   1.19 × 10 7               A. ferrooxidans     1.03 × 10 5               A. thiooxidans     8.03 × 10 3               Leptospirillum  sp.   1.61 × 10 6               Acidiphilium  sp.   2.11 × 10 3                        
 
         [0097]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 27 
               
             
             
               
                   
               
               
                   
               
               
                 MS-2 
               
             
          
           
               
                   
                 Bacteria 
                 Mo./g of sample 
               
               
                   
                   
               
               
                   
                 Total bacteria 
                 2.51 × 10 6   
               
               
                   
                 
                   A. ferrooxidans 
                 
                 3.81 × 10 4   
               
               
                   
                 
                   A. thiooxidans 
                 
                 1.72 × 10 5   
               
               
                   
                   Leptospirillum  sp. 
                 1.21 × 10 5   
               
               
                   
                   Acidiphilium  sp. 
                 0 
               
               
                   
                   
               
             
          
         
       
     
         [0098]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 28 
               
             
             
               
                   
               
               
                   
               
               
                 MS-3 
               
             
          
           
               
                   
                 Bacteria 
                 Mo./g of sample 
               
               
                   
                   
               
               
                   
                 Total bacteria 
                 9.43 × 10 6   
               
               
                   
                 
                   A. ferrooxidans 
                 
                 3.70 × 10 4   
               
               
                   
                 
                   A. thiooxidans 
                 
                 2.11 × 10 3   
               
               
                   
                   Leptospirillum  sp. 
                 6.60 × 10 5   
               
               
                   
                   Acidiphilium  sp. 
                 5.07 × 10 2   
               
               
                   
                   
               
             
          
         
       
     
         [0099]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 29 
               
             
             
               
                   
               
               
                   
               
               
                 MS-4 
               
             
          
           
               
                   
                 Bacteria 
                 Mo./g of sample 
               
               
                   
                   
               
               
                   
                 Total bacteria 
                 7.48 × 10 6   
               
               
                   
                 
                   A. ferrooxidans 
                 
                 1.03 × 10 3   
               
               
                   
                 
                   A. thiooxidans 
                 
                 2.78 × 10 3   
               
               
                   
                   Leptospirillum  sp. 
                 6.56 × 10 5   
               
               
                   
                   Acidiphilium  sp. 
                 2.45 × 10 2   
               
               
                   
                   
               
             
          
         
       
     
         [0100]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 30 
               
             
             
               
                   
               
               
                   
               
               
                 MS-5 
               
             
          
           
               
                   
                 Bacteria 
                 Mo./g of sample 
               
               
                   
                   
               
               
                   
                 Total bacteria 
                 1.14 × 10 6   
               
               
                   
                 
                   A. ferrooxidans 
                 
                 3.31 × 10 3   
               
               
                   
                 
                   A. thiooxidans 
                 
                 2.56 × 10 4   
               
               
                   
                   Leptospirillum  sp. 
                 1.21 × 10 5   
               
               
                   
                   Acidiphilium  sp. 
                 0 
               
               
                   
                   
               
             
          
         
       
     
         [0101]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 31 
               
             
             
               
                   
               
               
                   
               
               
                 ML-1 
               
             
          
           
               
                   
                 Bacteria 
                 Mo./ml of sample 
               
               
                   
                   
               
               
                   
                 Total bacteria 
                 3.34 × 10 4   
               
               
                   
                 
                   A. ferrooxidans 
                 
                   0 × 10 0   
               
               
                   
                 
                   A. thiooxidans 
                 
                   0 × 10 0   
               
               
                   
                   Leptospirillum  sp. 
                 9.47 × 10 2   
               
               
                   
                   Acidiphilium  sp. 
                 0 
               
               
                   
                   
               
             
          
         
       
     
         [0102]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 32 
               
             
             
               
                   
               
               
                   
               
               
                 ML-2 
               
             
          
           
               
                   
                 Bacteria 
                 Mo./ml of sample 
               
               
                   
                   
               
               
                   
                 Total bacteria 
                 7.13 × 10 4   
               
               
                   
                 
                   A. ferrooxidans 
                 
                 6.69 × 10 1   
               
               
                   
                 
                   A. thiooxidans 
                 
                   0 × 10 0   
               
               
                   
                   Leptospirillum s p. 
                 3.12 × 10 3   
               
               
                   
                   Acidiphilium  sp. 
                 0 
               
               
                   
                   
               
             
          
         
       
     
         [0103]     FIGS.  1  to  7  are plots of the results described in Tables 26 to 32.  
       Example 2  
     Quantification of  Sulfobacillus  sp.,  Sulfolobus  sp., and  Ferroplasma  sp. in a Sample  
       [0104]     Two solid samples obtained from mineral bioleaching heaps (MS-6 and MS-7) were analyzed and total DNA was extracted from each one.  
         [0105]     A further DNA re-purification step was required to obtain a translucent appearance in the extraction solution.  
         [0106]     Then, total DNA was quantified in each sample using a NanoDrop 1.0 spectrophotometer. Total extracted DNA nanograms (T) are shown in Table 34 together with the initial sample volumes (C m ). Results are shown in Table 33.  
                       TABLE 33                       Sample   T   C m                     MS-6   426.8   0.5 g       MS-7   277.2   0.5 g                  
 
         [0107]     Each of these samples was diluted with sterile nuclease-free water in order to obtain a concentration between 0.5 and 30 ng/μl. Table 34 shows the final volume to which the DNA solution was brought and its final concentration.  
                       TABLE 34                       Sample   Final volume (μl)   Concentration (ng/μl)                   MS-1   80   5.34       MS-2   80   3.47                  
 
         [0108]     Two calibration curves were prepared simultaneously, one for the Bacteria kingdom and another for the Archaea kingdom, which allowed calculating DNA concentration in experimental samples. For the Bacteria kingdom 4 serial dilutions were carried out from a DNA standard, hereinafter called Bacteria standard, containing 100 ng of  Sulfobacillus  sp. DNA in a final volume of 30 μl, being the standard solution also part of the calibration curve.  
         [0109]     More specifically, DNA was used from the strain: 
         Sulfobacillus  sp. DSM 10 332        
 
         [0111]     For the Archaea kingdom, four serial dilutions were prepared from a standard DNA mix containing 50 ng of DNA from each of the following microorganisms:  Sulfolobus  sp. and  Ferroplasma  sp. in a final volume of 30 μl, obtaining 100 ng of total DNA in the standard mix, hereinafter called Archaea standard, which is also part of the calibration curve.  
         [0112]     More specifically, DNA was used from the following strains: 
         Sulfolobus  sp. DSM 6482 and      Ferroplasma  sp. DSM 12658.        
 
         [0115]     Then, reaction mixes for the primary PCR were prepared, wherein the amount of each constituent was multiplied by the total number of reactions to be carried out; a single reaction mix was prepared in order to homogenize reagent concentrations in the different PCR tubes. The reaction mix was aliquoted in 0.2 ml tubes, using a volume of 24 μl of reaction mix per tube.  
         [0116]     For the Bacteria kingdom and  Sulfobacillus  determination, the following reactions were set up in duplicate: 
        a) two reactions for the samples and     b) 5 reactions for the calibration curve, corresponding to the Bacteria standard solution in concentrations of 1×, 0.1×, 0.01×, 0.001× and 0.0001×, and a blank, giving a total of 15 reactions.        
 
         [0119]     The prepared mix is shown in Table 35.  
                                                     TABLE 35                                   Reagent   1 reaction   15 reactions                                        Sterile nuclease-free H 2 O   18.35 μl    275.25   μl           PCR Buffer 10×   2.5 μl   37.5   μl           MgCl 2  (50 mM)   1.5 μl   22.5   μl           dNTPs (10 mM each)   0.5 μl   7.5   μl           Primer Bacteria 27F (10 μM)   0.5 μl   7.5   μl           Primer Bacteria 1492R (10 μM)   0.5 μl   7.5   μl           Hot Start Taq (5 U/μl)   0.15 μl    2.25   μl                      
 
         [0120]     Primers were those described in Table 36:  
                           TABLE 36                       Microorganism   Alignment               to be   tempera-           determined   ture   Used primers                   Total bacteria   59° C.   Eubac27F:                   AGA GTT TGA TCC TGG CTC AG               Univ1492R:               GGT TAC CTT GTT ACG ACT T                  
 
         [0121]     This primary PCR reaction mix was homogenized and 15 aliquots were made with 24 μl each in 0.2 ml tubes appropriately labeled. To this mix 1 μl of sample DNA dilutions or 1 μl of calibration curve DNA was added as appropriate. To the negative control 1 μl of sterile nuclease-free water was added instead of DNA.  
         [0122]     Reactions were incubated in a MJ Research PTC-100 thermocycler, with the following cycle program:  
                                                 TABLE 37                                       Temperature               Step   (° C.)   Time (s)                                        1. Initial denaturation   95   120           2. Denaturation   95   30           3. Alignment   62   30           4. Extension   72   120                      
 
         [0123]     Wherein steps 2 to 4 were repeated 18 times.  
         [0124]     For the Archaea kingdom and  Sulfolobus  sp. and  Ferroplasma  sp. determination, the following reactions were set up in duplicate: 
        a) two reactions for the samples and     b) 5 reactions for the calibration curve, corresponding to the Archaea standard solution in concentrations of 1×, 0.1×, 0.01×, 0.001× and 0.0001 ×, and a blank, giving a total of 15 reactions.        
 
         [0127]     The prepared mix is shown in Table 38.  
                               TABLE 38                                   Reagent   1 reaction   15 reactions                           Sterile nuclease-free H 2 O   18.35 μl    275.25 μl             PCR Buffer 10x   2.5 μl   37.5 μl            MgCl 2  (50 mM)   1.5 μl   22.5 μl            dNTPs (10 mM each)   0.5 μl   7.5 μl           Primer Archaea 21F (10 μM)   0.5 μl   7.5 μl           Primer Archaea 1492R (10 μM)   0.5 μl   7.5 μl           Hot Start Taq (5 U/μl)   0.15 μl    2.25 μl                       
 
         [0128]     Primers were those described in Table 39:  
                           TABLE 39                       Microorganism   Alignment               to be   tempera-           determined   ture   Used primers                   Total archaea   57° C.   Arch21F:                   TTC CGG TTG ATC CTG CCG GA               Univ1492R:               GGT TAC CTT GTT ACG ACT T                  
 
         [0129]     This primary PCR reaction mix was homogenized and 15 aliquots were made with 24 μl each in 0.2 ml tubes appropriately labeled. To this mix 1 μl l of sample DNA dilutions or 1 μl of calibration curve DNA was added as appropriate. To the negative control 1 μl of sterile nuclease-free water was added instead of DNA.  
         [0130]     Reactions were incubated in a MJ Research PTC-100 thermocycler, with the following cycle program:  
                                                 TABLE 40                                       Temperature               Step   (° C.)   Time (s)                                        1. Initial denaturation   95   120           2. Denaturation   95   30           3. Alignment   57   30           4. Extension   72   120                      
 
         [0131]     Wherein steps 2 to 4 were repeated 18 times.  
         [0132]     Subsequently, 5 secondary PCR were performed, two on the primary PCR reaction product for the Bacteria kingdom, for  Sulfobacillus  sp. and for total bacteria; and three on the primary PCR reaction product for the Archaea kingdom, for  Sulfolobus  sp. and  Ferroplasma  sp. and for total archaea, using specific primers for each of them that hybridize inside the region amplified in the primary PCR. Sense and antisense primers were selected for the different genera from those included in the description of the tables corresponding to each taxon, Tables 7, 11 and 13 in this case. On the other hand, for total bacteria or archaea primers described in the literature were used, which were included in Table 15.  
         [0133]     Primers used for each taxon and their respective annealing temperatures are indicated in Table 41.  
                           TABLE 41                       Microorganism   Alignment               to be   tempera-           determined   ture   Used primers                   Total bacteria   59   (P.1) 27-F:                   5′- AGA GTT TGA TCC TGG               CTC AG -3′               (P.2) 338-R:               5′- GCT GCC TCC CGT AGG               AGT -3′                 Sulfobacillus     66   (P.1) F:       sp.       5′- AGG TGT CGC GGG GGT               CCA CC -3′               (P.2) R:               5′- CCA GGA ATT CCA TGC               ACC TC -3′               Total archaea   60   (P.1) 515-F:               5′- GTG CCA GCA GCC GCG               GTA A -3′               (P.2) 958-R:               5′- TCC GGC GTT GAA TCC               AAT T -3′                 Sulfolobus  sp.   60   (P.1) F:               5′- TAA ACC CTG CCG CAG               TTG G -3′               (P.2) R:               5′- CCA ACT GCG GCA GGG               TTT A -3′                 Ferroplasma  sp.   56   (P.1) F:               5′- GAT GTC GGT GAG GAG               GGT T -3′               (P.2) R:               5′- ATT TGA TTT AAC CCT               CTC G -3′                  
 
         [0134]     One qPCR was carried out on each primary PCR reaction product for each taxon to be determined, these reactions being performed in duplicate. The qPCR was carried out using Mix SYBR Green qPCR. For each secondary PCR one duplicate per each one of the 15 primary PCR reactions is considered plus one control, which gave a total of 31 reactions. For each PCR the reaction mix shown in Table 42 was prepared, where primers are those that are corresponding according to Table 41.  
                                             TABLE 42                                   1 reaction   31 reactions                                        Sterile nuclease-free H 2 O   16.1 μl    499.1 μl            Primer 1 (10 μM)   0.5 μl   15.5 μl           Primer 2 (10 μM)   0.5 μl   15.5 μl           PCR Buffer 10x   2.5 μl   77.5 μl           MgCl 2  (50 mM)   1.5 μl   46.5 μl           dNTPs (10 mM each)   2.5 μl   77.5 μl           Hot Start Taq (5 U/μl)   0.15 μl    4.65 μl           SYBR Green qPCR 100x   0.25 μl    7.75 μl                      
 
         [0135]     This reaction mix was homogenized and aliquoted in 31 0.2 ml tubes, which were duly labeled. To each of the tubes 1 μl of primary PCR or 1 μl of sterile nuclease-free water for the blank was added.  
         [0136]     PCR tubes containing the reaction mix and sample were vortexed for 5 seconds and centrifuged for 1 minute at 2000 rpm, in order to homogenize and bring the reaction liquid to the bottom of the tube, respectively.  
         [0137]     Then, the tubes with secondary PCR reactions were subjected to temperature cycles for amplification. According to the microorganism to be determined, different primer pairs were used and therefore different amplification programs were used. In the following Table, amplification programs used in the different secondary PCR reactions are shown.  
                                                                                 TABLE 43                                   Step   Temperature (° C.)   Time (s)                                    1   Initial denaturation   95   120       2   Denaturation   95   30       3   Alignment   (*)   30       4   Extension   72   40       5   Pre-reading   80   10       6   Reading   80   —                Repeat 40 times from step 2 to step 6 (qPCR cycle)                7   Denaturation curve   Between 70 and 100° C.,                   reading each 0.2° C.                 (*) specific temperature for each used primer pair, as indicated in Table 41.             
 
         [0138]     When the qPCR is finished, all data generated by the qPCR thermocycler are stored; this data corresponds to Q and is shown in Table 44, wherein DNA amounts in nanograms used for each reaction are included (U).  
                                                         TABLE 44                                       Q                Sample   Total bacteria     Sulfobacillus  sp.   Total archaea     Sulfolobus  sp.     Ferroplasma  sp   U               MS-6   20.26 × 10 −3     0.01 × 10 −3     0.71 × 10 −3     0.05 × 10 −3     0   2       MS-7   72.51 × 10 −3     0.11 × 10 −3     0.34 × 10 −3     0.03 × 10 −3     0.007 × 10 −3     2                  
 
       Calculation of the Number of Microorganisms Present in the Sample  
       [0139]     Taking into account the qPCR result and other data generated during the process, the following formula was applied, the meaning of which was defined above:  
         Mo   /   Um     =       Q   ×   T         5   ·       10     -   6       ⁡     [     ng   ⁢     /     ⁢   mo     ]         ×   U   ×   Cm           
 
         [0140]     According to this, the following microbiological populations were determined in the analyzed samples:  
                             TABLE 45                           MS-6                Microorganism   Mo./g of sample                       Total bacteria   1.73 × 10 6               Sulfobacillus  sp.   1.05 × 10 3             Total archaea   6.04 × 10 4               Sulfolobus  sp.   2.02 × 10 3               Ferroplasma  sp.   0                      
 
         [0141]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 46 
               
             
             
               
                   
               
               
                   
               
               
                 MS-7 
               
             
          
           
               
                   
                 Microorganism 
                 Mo./g of sample 
               
               
                   
                   
               
               
                   
                 Total bacteria 
                 4.02 × 10 6   
               
               
                   
                   Sulfobacillus  sp. 
                 6.00 × 10 3   
               
               
                   
                 Total archaea 
                 1.89 × 10 4   
               
               
                   
                   Sulfolobus  sp. 
                 1.76 × 10 3   
               
               
                   
                   Ferroplasma  sp. 
                 4.33 × 10 2   
               
               
                   
                   
               
             
          
         
       
     
         [0142]      FIGS. 8 and 9  are plots of the results described in Tables 45 and 46.