Method for the identification and quantification of microorganisms useful in biomining processes

FIELD OF THE INVENTION

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

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.

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

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

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.

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.

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.

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.

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.

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.

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.

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.

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

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. However, the method also works and is applicable without the primary PCR, and therefore this stage may be optional.

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

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.

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.

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

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.

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.

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.

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.

The following is the description of each of the stages of the method in detail.

DNA Preparation.

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 H₂O.

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.

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.

Primary PCR.

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”).

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 1Primary PCRBacteria primersEub27-F¹AGA GTT TGA TCC TGG CTC AGUniv1492-R¹GGT TAC CTT GTT ACG ACT TArchaea primersArch21-F²TTC CGG TTG ATC C(CT)G CCG GAUniv1492-R¹GGT TAC CTT GTT ACG ACT T
¹Bond P., 2000, Appl Environ Microbiol. 66(9):3842-9.

²Delong, E.F., 1992, Proc. Nac. Acad. Sci. USA 89: 5685-9.

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.

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.

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.

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 2Sterile nuclease-free H₂O18.35 μl PCR Buffer 10x2.5 μlMgCl₂(50 mM)1.5 μldNTPs (10 mM each)0.5 μlPrimer Eubac27F (10 μM)0.5 μlPrimer Univ1492R (10 μM)0.5 μlHot Start Taq (5 U/μl)0.15 μl

Primary PCR cycles are described in Table 3.

TABLE 3TemperatureStep(° C.)Time (s)1. Initial denaturation951202. Denaturation95303. Alignment3.1. for Bacteria56303.2. for Archaea57304. Extension72120

Wherein steps 2 to 4 are repeated from 15 to 20 times, avoiding saturation.

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.

Secondary PCR.

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.

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.

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.

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.

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.

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.

For each secondary PCR reaction the following mix is prepared:

TABLE 4Sterile nuclease-free H₂O10.5 μlSense primer (10 μM) 0.5 μlAntisense primer (10 μM) 0.5 μlqPCR reagent12.5 μl

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

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.)

Bacteria Kingdom:

TABLE 5Acidiphilium sp.Sense primersAntisense primersCAA CCA CGG TCG GGT CAGTCT CTG ACC CGA CCG TGGATTGAC CTT AAG TTG ATG CGCTCA ACT TAA GGT CAA ACCTAAAGT CAA CCA CGG TCG GGTGGA GCT TAT TCT GCG GGTCAGGT TTG ACC TTA AGT TGAGCA TCA ACT TAA GGT CAATGACCTT AAG TTG ATG CGC TAAAGC GCA TCA ACT TAA GGTCCAGGC AGT CAA CCA CGG TCGGTT AGC GCA TCA ACT TAAGGGCGA TGC TGA GCT GAT CCTCCG ACC GTG GTT GAC TGCGCAAG TTG ATG CGC TAA CCGGGA TCA GCT CAG CAT CGCCTGAAA GTC GCC TAA GGA GGATCA GGA TCA GCT CAG CATGCGGTC GCC TAA GGA GGA GCCCGG TTA GCG CAT CAA CTTTAAAG GAG GAG CCT GCG TCTGGC TCC TCC TTA GGC GACGTTAGG AGC CTG CGT CTG ATTGTT GAC TGC CTC CTT GCGAGTAGG AGG CAG TCA ACC ACGTCC TCC TTA GGC GAC TTTGTCGGCG AAA GTC GCC TAA GGAGTG GTT GAC TGC CTC CTTGGCGCC TAA GGA GGA GCC TGCACC GTG GTT GAC TGC CTCGTCTGCA AGG AGG CAG TCA ACCGCA GGC TCC TCC TTA GGCAGAGCA AGT CGC TCG GGC AGTGAC GCA GGC TCC TCC TTAAGGACC CGT AGG AAT CTA TCCTCA GAC GCA GGC TCC TCCTTTGCA CAG TCA GGC GTG AAATGC TAC TGC CCG AGC GACTATTACA CAT GCA AGT CGC TCGTGA CCC GAC CGT GGT TGAGGC

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

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

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

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

Archaea Kingdom:

TABLE 10Acidianus sp.Sense primersAntisense primersGGG AAA CCG TGA GGG CGCCCG CAT TGG GGA CGT TTCTGCGGCG AAA CGT CCC CAA TGCGCG CCC TCA CGG TTT CCCGGGCACCG CAG GGA AAC CGG TAACCG CAT TGG GGA CGT TTCGCCGCGCCC GGG AAA GGG CAG TGAGCG CCC TCA CGG TTT CCCTAGCAGGG AAA GGG CAG TGA TACTTC CCG CAT TGG GGA CGTTTTCAAT CCG GGG CAG GCG AAGTAG CGC CCT CAC GGT TTCGGCCAGG GTA CTG GAA CGT CCCGGC TTA CCG GTT TCC CTGTTCGAAG CGT CCG GCC AGA ACGCTG CCC TTT CCC GGG TTGCGCACGC CTA AAG GGG CAT GGGTCA CTG CCC TTT CCC GGGCTTGGC TAT TTC CCG CTC ATGGTA TCA CTG CCC TTT CCCCCGCGT ACG CCC TCG GGT AAGGCC CGG GTC TTT AAG CAGAGGTGAAC GGC CCG CCA AAC CGACTC CCG CCC CCT AGC CCTTAGCAAGC CGG CCC TGC AAG TCACCC GGG ATC TGT GGA TTTCCGCCAC TGC TTA AAG ACC CGGTAC CCG AGG GCG TAC GACGTGGA GCT AAT CCG GGG CAGCCT CTT ACC CGA GGG CGTGCGACGAAA CCG TGA GGG CGC TACTTC GCC TGC CCC GGA TTACCGAGG CGA AGG GTA CTG GAAGGC GGC AGG CTT ACC GGTCGTTTCACC CCC AGT GCT CCC GAACGG ATT AGC TCC AGT TTCAGCCGCCC TTC GCC TAA AGG GGCGGA CGT TCC AGT ACC CTTATGCGCA TGG GCT ATT TCC CGCCCC CGG ATT AGC TCC AGTTCATTGGG AAA CCG TGA GGG CGCTAC CCT TCG CCT GCC CCGTGATGCG AAA CGT CCC CAA TGCCCA TGC CCC TTT AGG CGAGGA

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

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

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

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

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 15Secondary PCRBacteria primersEub27¹FAGA GTT TGA TCC TGG CTC AGUniv533-F¹GTG CCA GCM GCC GCG GTABact358-F²CCT ACG GGA GGC AGC AGUniv907-R³CCG TCA ATT CCT TTG AGT TBact338-R⁴GCT GCC TCC CGT AGG AGTBact1387-R⁵GGG CGG WGT GTA CAA GGCArchaea primersArch344-F⁶ACG GGG CGC AGC AGG CGC GAUniv515-F⁷GTG CCA GCA GCC GCG GTA AArch958-R⁸YCC GGC GTT GAM TCC AAT TArch915-R⁴GTG CTC CCC CGC CAA TTC CTUniv534-R⁵ATT ACC GCG GCT GCT GG
¹Bond P., 2000, Appl Environ Microbiol. 66(9):3842-9.

²Schauer M, 2003, Aquat Microb Ecol Vol. 31: 163-174.

³Nakagawa T, 2002, FEMS Microbiology Ecology 41:199-209.

⁴Schrenk MO, 1998, Science. 279:1519-22.

⁵Ellis R, 2003, Appl Environ Microbiol. 69(6):3223-30.

⁶Casamayor EO, 2002, Environ Microbiol. 4(6):338-48.

⁷Edwards K, 2003, Appl Environ Microbiol. 69(5):2906-13.

⁸Orphan VJ, 2001, Appl Environ Microbiol. 66(2):700-11.

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 16StepTemperature (° C.)Time (s)1Initial denaturation95120 2Denaturation95303Alignment(*)304Extension72405Pre-reading80106Reading80—Repeat 40 times from step 2 to step 6 (qPCR cycle)7Denaturation curveBetween 70 and 100° C., reading each0.2° C.
(*) specific temperature for each used primer pair

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.

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.

Calculation of the Number of Microorganisms Present in the Sample

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:
$Mo / Um = \frac{Q \times T}{5 \cdot 10^{- 6} [ng / mo] \times U \times Cm}$

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_mis the amount of biomining sample from which DNA was extracted, expressed in ml for liquid samples or in g for solid samples.
- The number 5×10⁻⁶ng/mo is the average amount of DNA nanograms contained in the genome of a microorganism, according to Kuske et al. (1998).

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

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.

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.

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 17SampleTC_mMS-1316.80.5gMS-2370.40.5gMS-3315.20.5gMS-4526.40.5gMS-54005.0gML-1293881.00mlML-2111476.55ml

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 18SampleFinal volume (μl)Concentration (ng/μl)MS-1803.96MS-2804.63MS-3803.94MS-4806.58MS-5805.00ML-110029.38ML-210011.14

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.

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.

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.

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.

The prepared mix is shown in Table 19.

TABLE 19Reagent1 reaction25 reactionsSterile nuclease-free H₂O18.35 μl 458.75 μl PCR Buffer 10x2.5 μl62.5 μlMgCl₂(50 mM)1.5 μl37.5 μldNTPs (10 mM each)0.5 μl12.5 μlPrimer Bacteria 27F (10 μM)0.5 μl12.5 μlPrimer Bacteria 1492R (10 μM)0.5 μl12.5 μlHot Start Taq (5 U/μl)0.15 μl 3.75 μl

Used primers are described in Table 20.

TABLE 20MicroorganismAlignmentto betempera-determinedtureUsed primersTotal bacteria59° C.Eubac27F:AGA GTT TGA TCC TGG CTC AGUniv1492R:GGT TAC CTT GTT ACG ACT T

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.

Reactions were incubated in a MJ Research PTC-100 thermocycler, with the following cycle program:

TABLE 21TemperatureStep(° C.)Time (s)1. Initial denaturation951202. Denaturation95303. Alignment56304. Extension72120

Wherein steps 2 to 4 were repeated 18 times.

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.

Primers used for each taxon and their respective annealing temperatures are indicated in Table 22.

TABLE 22MicroorganismAlignmentto betempera-determinedtureUsed primersTotal bacteria56° C.(P.1) 533-F:5′- GTG CCA GCA GCC GCGGTA -3′(P.2) 907-R:5′- CCG TCA ATT CCT TTGAGT T -3′A. ferrooxidans60° C.(P.1) F:5′- GTG GAG GAC GAA AAGGCG G -3′(P.2) R:5′- ATT AGA ACC CGC CTTTTC GT -3′A. thiooxidans56° C.(P.1) F:5′- AAA GGT AAT CGC TAATAT CG -3′(P.2) R:5′- ATT ACC TTT TCG TCTCCC AC -3′Leptospirillum58° C.(P.1) F:sp.5′- AAC AAG GTA CCC GTCTAG A -3′(P.2) R:5′- CTA GAC GGG TAC CTTGTT AC -3′Acidiphilium61° C.(P.1) F:sp.5′- AGG AGG CAG TCA ACCACG GT -3′(P.2) R:5′- GTT AGC GCA TCA ACTTAA GG -3′

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 231 reaction51 reactionsSterile nuclease-free H₂O16.1 μl 821.1 μl Primer 1 (10 μM)0.5 μl25.5 μlPrimer 2 (10 μM)0.5 μl25.5 μlPCR Buffer 10x2.5 μl127.5 μl MgCl₂(50 mM)1.5 μl76.5 μldNTPs (10 mM each)2.5 μl127.5 μl Hot Start Taq (5 U/μl)0.15 μl 7.65 μlSYBR Green qPCR 100x0.25 μl 12.75 μl

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.

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.

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 24StepTemperature (° C.)Time (s)1Initial denaturation95120 2Denaturation95303Alignment(*)304Extension72405Pre-reading80106Reading80—Repeat 40 times from step 2 to step 6 (qPCR cycle)7Denaturation curveBetween 70 and 100° C., reading each0.2° C.
(*) specific temperature for each used primer pair, as indicated in Table 22.

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 25QSampleTotal bacteriaA. ferrooxidansA. thiooxidansLeptospirillum sp.Acidiphilium sp.UMS-1187.68 × 10⁻³1.63 × 10⁻³0.13 × 10⁻³25.36 × 10⁻³0.03 × 10⁻³2MS-2 33.91 × 10⁻³0.51 × 10⁻³2.33 × 10⁻³ 1.63 × 10⁻³02MS-3149.60 × 10⁻³0.59 × 10⁻³0.03 × 10⁻³10.46 × 10⁻³0.008 × 10⁻³2MS-4 71.08 × 10⁻³0.01 × 10⁻³0.03 × 10⁻³ 6.23 × 10⁻³0.002 × 10⁻³2MS-5142.68 × 10⁻³0.29 × 10⁻³3.20 × 10⁻³15.10 × 10⁻³02ML-1 9.20 × 10⁻³00 0.26 × 10⁻³02ML-2 49.03 × 10⁻³0.05 × 10⁻³0 2.15 × 10⁻³02

Calculation of the Number of Microorganisms Present in the Samples

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:
$Mo / Um = \frac{Q \times T}{5 \cdot 10^{- 6} [ng / mo] \times U \times Cm}$

According to this, the following microbiological populations were determined in the analyzed samples:

TABLE 26MS-1BacteriaMo./g of sampleTotal bacteria1.19 × 10⁷A. ferrooxidans1.03 × 10⁵A. thiooxidans8.03 × 10³Leptospirillum sp.1.61 × 10⁶Acidiphilium sp.2.11 × 10³

TABLE 27

MS-2

Bacteria
Mo./g of sample

Total bacteria
2.51 × 10⁶

A. ferrooxidans

3.81 × 10⁴

A. thiooxidans

1.72 × 10⁵

Leptospirillum sp.
1.21 × 10⁵

Acidiphilium sp.
0

TABLE 28

MS-3

Bacteria
Mo./g of sample

Total bacteria
9.43 × 10⁶

A. ferrooxidans

3.70 × 10⁴

A. thiooxidans

2.11 × 10³

Leptospirillum sp.
6.60 × 10⁵

Acidiphilium sp.
5.07 × 10²

TABLE 29

MS-4

Bacteria
Mo./g of sample

Total bacteria
7.48 × 10⁶

A. ferrooxidans

1.03 × 10³

A. thiooxidans

2.78 × 10³

Leptospirillum sp.
6.56 × 10⁵

Acidiphilium sp.
2.45 × 10²

TABLE 30

MS-5

Bacteria
Mo./g of sample

Total bacteria
1.14 × 10⁶

A. ferrooxidans

3.31 × 10³

A. thiooxidans

2.56 × 10⁴

Leptospirillum sp.
1.21 × 10⁵

Acidiphilium sp.
0

TABLE 31

ML-1

Bacteria
Mo./ml of sample

Total bacteria
3.34 × 10⁴

A. ferrooxidans

0 × 10⁰

A. thiooxidans

0 × 10⁰

Leptospirillum sp.
9.47 × 10²

Acidiphilium sp.
0

TABLE 32

ML-2

Bacteria
Mo./ml of sample

Total bacteria
7.13 × 10⁴

A. ferrooxidans

6.69 × 10¹

A. thiooxidans

0 × 10⁰

Leptospirillum sp.
3.12 × 10³

Acidiphilium sp.
0

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

Two solid samples obtained from mineral bioleaching heaps (MS-6 and MS-7) were analyzed and total DNA was extracted from each one.

A further DNA re-purification step was required to obtain a translucent appearance in the extraction solution.

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 33SampleTC_mMS-6426.80.5 gMS-7277.20.5 g

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 34SampleFinal volume (μl)Concentration (ng/μl)MS-1805.34MS-2803.47

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.

More specifically, DNA was used from the strain:

- Sulfobacillus sp. DSM 10 332

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.

More specifically, DNA was used from the following strains:

- Sulfolobus sp. DSM 6482 and
- Ferroplasma sp. DSM 12658.

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.

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.

The prepared mix is shown in Table 35.

TABLE 35Reagent1 reaction15 reactionsSterile nuclease-free H₂O18.35 μl 275.25μlPCR Buffer 10×2.5 μl37.5μlMgCl₂(50 mM)1.5 μl22.5μldNTPs (10 mM each)0.5 μl7.5μlPrimer Bacteria 27F (10 μM)0.5 μl7.5μlPrimer Bacteria 1492R (10 μM)0.5 μl7.5μlHot Start Taq (5 U/μl)0.15 μl 2.25μl

Primers were those described in Table 36:

TABLE 36MicroorganismAlignmentto betempera-determinedtureUsed primersTotal bacteria59° C.Eubac27F:AGA GTT TGA TCC TGG CTC AGUniv1492R:GGT TAC CTT GTT ACG ACT T

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.

Reactions were incubated in a MJ Research PTC-100 thermocycler, with the following cycle program:

TABLE 37TemperatureStep(° C.)Time (s)1. Initial denaturation951202. Denaturation95303. Alignment62304. Extension72120

Wherein steps 2 to 4 were repeated 18 times.

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.

The prepared mix is shown in Table 38.

TABLE 38Reagent1 reaction15 reactionsSterile nuclease-free H₂O18.35 μl 275.25 μl PCR Buffer 10x2.5 μl37.5 μl MgCl₂(50 mM)1.5 μl22.5 μl dNTPs (10 mM each)0.5 μl7.5 μlPrimer Archaea 21F (10 μM)0.5 μl7.5 μlPrimer Archaea 1492R (10 μM)0.5 μl7.5 μlHot Start Taq (5 U/μl)0.15 μl 2.25 μl

Primers were those described in Table 39:

TABLE 39MicroorganismAlignmentto betempera-determinedtureUsed primersTotal archaea57° C.Arch21F:TTC CGG TTG ATC CTG CCG GAUniv1492R:GGT TAC CTT GTT ACG ACT T

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.

Reactions were incubated in a MJ Research PTC-100 thermocycler, with the following cycle program:

TABLE 40TemperatureStep(° C.)Time (s)1. Initial denaturation951202. Denaturation95303. Alignment57304. Extension72120

Wherein steps 2 to 4 were repeated 18 times.

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.

Primers used for each taxon and their respective annealing temperatures are indicated in Table 41.

TABLE 41MicroorganismAlignmentto betempera-determinedtureUsed primersTotal bacteria59(P.1) 27-F:5′- AGA GTT TGA TCC TGGCTC AG -3′(P.2) 338-R:5′- GCT GCC TCC CGT AGGAGT -3′Sulfobacillus66(P.1) F:sp.5′- AGG TGT CGC GGG GGTCCA CC -3′(P.2) R:5′- CCA GGA ATT CCA TGCACC TC -3′Total archaea60(P.1) 515-F:5′- GTG CCA GCA GCC GCGGTA A -3′(P.2) 958-R:5′- TCC GGC GTT GAA TCCAAT T -3′Sulfolobus sp.60(P.1) F:5′- TAA ACC CTG CCG CAGTTG G -3′(P.2) R:5′- CCA ACT GCG GCA GGGTTT A -3′Ferroplasma sp.56(P.1) F:5′- GAT GTC GGT GAG GAGGGT T -3′(P.2) R:5′- ATT TGA TTT AAC CCTCTC G -3′

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 421 reaction31 reactionsSterile nuclease-free H₂O16.1 μl 499.1 μl Primer 1 (10 μM)0.5 μl15.5 μlPrimer 2 (10 μM)0.5 μl15.5 μlPCR Buffer 10x2.5 μl77.5 μlMgCl₂(50 mM)1.5 μl46.5 μldNTPs (10 mM each)2.5 μl77.5 μlHot Start Taq (5 U/μl)0.15 μl 4.65 μlSYBR Green qPCR 100x0.25 μl 7.75 μl

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.

TABLE 43StepTemperature (° C.)Time (s)1Initial denaturation951202Denaturation95303Alignment(*)304Extension72405Pre-reading80106Reading80—Repeat 40 times from step 2 to step 6 (qPCR cycle)7Denaturation curveBetween 70 and 100° C.,reading each 0.2° C.
(*) specific temperature for each used primer pair, as indicated in Table 41.

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 44QSampleTotal bacteriaSulfobacillus sp.Total archaeaSulfolobus sp.Ferroplasma spUMS-620.26 × 10⁻³0.01 × 10⁻³0.71 × 10⁻³0.05 × 10⁻³02MS-772.51 × 10⁻³0.11 × 10⁻³0.34 × 10⁻³0.03 × 10⁻³0.007 × 10⁻³2

Calculation of the Number of Microorganisms Present in the Sample

According to this, the following microbiological populations were determined in the analyzed samples:

TABLE 45MS-6MicroorganismMo./g of sampleTotal bacteria1.73 × 10⁶Sulfobacillus sp.1.05 × 10³Total archaea6.04 × 10⁴Sulfolobus sp.2.02 × 10³Ferroplasma sp.0

TABLE 46

MS-7

Microorganism
Mo./g of sample

Total bacteria
4.02 × 10⁶

Sulfobacillus sp.
6.00 × 10³

Total archaea
1.89 × 10⁴

Sulfolobus sp.
1.76 × 10³

Ferroplasma sp.
4.33 × 10²

FIGS. 8 and 9 are plots of the results described in Tables 45 and 46.

Method for the identification and quantification of microorganisms useful in biomining processes

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