(19) united states (12) patent application publication (10) · pdf file ·...

(19) United States US 201101361.25A1

(12) Patent Application Publication (10) Pub. No.: US 2011/0136125 A1 Parada ValdecantOS et al. (43) Pub. Date: Jun. 9, 2011

(54) METHOD FOR THE IDENTIFICATION AND (30) Foreign Application Priority Data QUANTIFICATION OF MICROORGANISMS USEFULIN BOMINING PROCESSES Aug. 26, 2005 (CL) ................................... 2179-2005

(75) Inventors: Pilar Angelica Parada Valdecantos, Nunoa (CL); Katia Nicole Ehrenfeld Stolzenbach, Las Condes (CL): Igor Alejandro Pacheco Cruz, Recoleta (CL): Alejandro Eduardo Maass Sepúlveda, Penalolen (CL); Andrés Octavio Aravena Duarte, Nunoa (CL); Mauricio Alejandro Gonzales Canales, Penalolen (CL): Servet Martinez Aguilera, La Cisterna (CL)

(73) Assignee: BIOSIGMA S.A, Colina (CL)

(21) Appl. No.: 12/945.407

(22) Filed: Nov. 12, 2010

Related U.S. Application Data (63) Continuation-in-part of application No. 1 1/509,870,

filed on Aug. 25, 2006, now abandoned.

Publication Classification

(51) Int. Cl. CI2O I/68 (2006.01)

(52) U.S. Cl. ....................................................... 435/6.12

(57) 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., Sulfobacil lus sp., Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, and Archaea: Acidianus sp., Ferroplasma sp., Metallosphaera sp., Sulfolobus sp. and Thermoplasma sp.

The method comprises performing a PCR with specific prim ers designed in our laboratories for different taxons SEQID No. 4 to SEQID No.: 407. With qPCR results and other data obtained from the analyzed sample, the microorganism con centration of each analyzed taxon present in the sample is calculated using a mathematical formula.

US 2011/O 1361.25 A1

METHOD FOR THE DENTIFICATION AND QUANTIFICATION OF MICROORGANISMS

USEFUL IN BOMINING PROCESSES

0001. This application is a Continuation-in-Part of U.S. Ser. No. 1 1/509,870, which claims benefit of Serial No. 2179 2005, filed 26 Aug. 2005 in Chile and which applications are incorporated herein by reference. To the extent appropriate, a claim of priority is made to each of the above disclosed applications.

FIELD OF THE INVENTION

0002 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

0003 Biomining is, in general terms, the use of microor ganisms for metal recovery from mineral ores. Its most tra ditional expression is bioleaching, but not only this process is understood as biomining, but also the monitoring and inter vention 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. 0004. 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 condi tions: they are acidophilic and chemolithotrophic. 0005 Microbiological Diversity in Communities Associ ated to Bioleaching Processes. 0006 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 Bac teria 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., Ferro plasma sp., Metallosphaera sp., Sulfolobus sp. and Thermo plasma 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-oxidiz ing microorganisms used for the recovery of metals from minerals and their concentrates. Microb Cell Fact. 2005 May 6;4(1):13). 0007 Factors Determining Diversity and Metabolic Activity of the Microbial Community Associated to a Bioleaching Process. 0008 Each of the above mentioned genera or species cata lyzes different reactions and require in its turn different con ditions to perform such reaction, which could be, for instance, aerobic or anaerobic, or could require Some specific nutrient.

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Therefore, the environmental conditions in which a bioleach ing process is performed will modify the bacterial composi tion of the community. 0009. 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. 2005 May 6;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 metalion. 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 acidifi cation 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. 0010 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. 0011 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 prod uct and reducing species like Sulfite and thiosulfate as inter mediate products, which allows the solubilization of metals associated to sulfides in the mineral. Acidithiobacillus fer rooxidans and Leptospirillum ferrooxidans are able to cata lyze the oxidation of iron(II) to iron(III) using oxygen as electron acceptor, being the generated iron(III) a great oxi dizing agent that can oxidize sulfides in the mineral or any other compound to be oxidized. 0012. 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 micro bial 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. 0013. 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 micro organisms 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. 0014 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

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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 infor mation 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 quan titative PCR or qPCR. 0015. 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 metage nomic samples. Therefore, it is necessary to reduce the pos sibility 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. 0016. 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 Microbio logical 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 avail able. Another document that uses a similar approach is Patent Application EP 1484 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. 0017 Although it is possible to enumerate documents in which microorganisms are identified and quantified using quantitative PCR techniques, as they are well known tech niques 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 Sub ject matter of this invention.

DETAILED DESCRIPTION OF THE INVENTION

0018. 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 gen

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era Acidianus sp., Ferroplasma sp. Metallosphaera sp., Sul folobus sp. and Thermoplasma sp. belonging to the Archaea kingdom. 0019. 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 individu als. 0020. 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 quan tified. 0021. In the description of the invention, all oligonucle otide 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. 0022. The following is the description of each of the stages of the method in detail. (0023 DNA Preparation. 0024. 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. 2003 July; 69(7):4183-9: Biotechniques. 2005 April: 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 re-purify the sample using any existing purification technique; in our labo ratories this step is performed using commercial DNA puri fication columns. The purified fraction is resuspended inster ile nuclease-free H.O. 0025. 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 spectro photometry. 0026. 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/ul, pref erably from 1 to 30 ng/ul, and most preferably from 1 to 10 ngful. The dilution must be done using sterile nuclease-free Water.

0027 All determinations of biomining microorganisms presented here are based in a specific recognition of a frag ment of 16S rDNA genes being unique for the taxon under analysis and not presenting any cross reaction with other microorganisms. The method claims the protection of several oligonucleotide primers that specifically amplify a fragment of 16S rDNA being used as identification tag. This method is

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even useful when analyzing complex samples with DNA coming from different microorganisms. The method com prises a standard curve construction, a qPCR reaction with the designed specific primers and the final data transformation. 0028. The standard curve is constructed using the specific PCR product fragment of the 16S rDNA gene produced by the designed primers in different dilutions of a mixed DNA stan dard sample. 0029 PCR Reaction. 0030 A plurality of PCR is carried out, specific for each taxon to be identified, using specific primers that amplify inside the 16S rDNA region. 0031. 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 thermo dynamically 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. 0032. PCR will be performed on all the reaction products. Advantageously, all reactions are carried out in duplicate, and a negative control is added. 0033. 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. 0034. The PCR is a quantitative PCR (qPCR), therefore it should be performed in a suitable thermocycler and using fluorescent reagents for qPCR. There are different commer

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cially available alternatives, either for equipment or reagents, and any of them can be selected to carry out the present method. 0035. For the PCR reaction the following mix is prepared:

TABLE 4

Sterile nuclease-free H2O 10.5 ul Sense primer (10M) 0.5 ul Antisense primer (10M) 0.5 ul qPCR reagent 12.5 ul

To the mix described in Table 4, 1 ul of DNA or sterile water for the qPCR blank is added.

Primers for the PCR

0036. As previously indicated, the requirements to be full filled by each primer pair selected for the 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 com pared, by using Blast from NCBI, with previously existent sequence disclosures, thus guaranteeing its novelty as prim ers.) 0037 Bacteria Kingdom:

TABLE 5

Acidiphilium sp.

Sense primers Seq ID Nos. Antisense primers Seq ID Nos.

CAA CCA CGG TCG GGT CAG A (SEQ

GAC CTT AAG TTG ATG CGC T (SEQ

AGT CAA CCA CGG TCG GGT C (SEQ

GGT TTG ACC TTA AGT TGA (SEQ TG

CTT. AAG TTG ATG CGC TAA C (SEQ

GGC AGT CAA CCA CGG TCG G (SEQ

CGA TGC TGA GCT GAT CCT G (SEQ

AAG TTG ATG CGC TAA CCG C (SEQ

AAA GTC GCC TAA GGA. GGA G (SEQ

GTC GCC TAA GGA GGA GCC T (SEQ

AAG GAG GAG CCT GCG TCT G (SEQ

AGG AGC CTG CGT CTG ATT A (SEQ

AGG AGG CAG TCA ACC ACG (SEQ GT

GCG AAA GTC GCC TAA GGA G (SEQ

D NO: 4) TCT CTG ACC CGA CCG TGG TT (SEO ID NO. 24)

D NO; 5) TCA ACT TAA GGT CAA ACC AA (SEO ID NO: 25)

D NO : 6) GGA GCT TAT TCT GCG GGT A (SEO ID NO: 26)

D NO: 7) GCA TCA ACT TAA GGT CAA AC (SEO ID NO: 27)

D NO: 8) AGC GCA TCA ACT TAA GGT CA (SEO ID NO: 28)

D NO: 9) GTT AGC GCA TCA ACT TAA GG (SEO ID NO : 29)

D NO : 1.O.) CCG ACC GTG GTT GAC TGC C (SEO ID NO : 3O)

D NO : 11) GGA TCA GCT CAG CAT CGC TG (SEO ID NO : 31)

D NO : 12) TCA GGA TCA GCT CAG CAT CG (SEO ID NO : 32)

D NO : 13) CGG TTA. GCG CAT CAA CTTA (SEO ID NO : 33)

D NO : 14) GGC. TCC TCC TTA. GGC GAC TT (SEO ID NO. 34)

D NO : 15) GTT GAC TGC CTC CTT GCG GT (SEO ID NO : 35)

D NO : 16) TCC TCC TTA. GGC GAC TTT CG (SEO ID NO : 36)

D NO: 17) GTG GTT GAC TGC CTC CTT GC (SEQ ID NO : 37)

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TABLE 14 - continued

Thermoplasma sp.

Sense primers Seq ID Nos. Anti sense primers Seq ID Nos.

TAA CTC GCC CTC. ACG AAT GT (SEO ID NO : 378) TTA. CAG CCC TCA GTC CTG ACT (SEO ID NO: 398)

GAA. GGT GTT AAG TGG GTC A (SEO ID NO : 379) AAT CCA CAT TCG TGA GGG CGA (SEO ID NO: 399)

AAA CCC GTT CGT AGT CAG GAC (SEO ID NO: 38O) ATG GGG GTC TTG CTC GTT AT (SEQ ID NO: 4 OO)

TAC GGT GAA. TAT GCC CCT GC (SEO ID NO: 381) GCT GTT GAC CTA CGA TTA. C (SEQ ID NO: 401)

CAC TTG GTG TTG CTT CTC CGT (SEO ID NO: 382) CCT ACG ATT ACT ACG GAA TCC (SEO ID NO. 4 O2)

GAT CAC TTT TAT TGA GTC T (SEO ID NO: 383) ACC CAC TTA ACA CCT TCG C (SEQ ID NO: 4 O3)

AGC ATC AGG AAT AAG GGC TG (SEO ID NO: 384) CCC AAG TCT TAC AGT CTC TT (SEQ ID NO: 4O4)

AAG ACC CCC ATC. TCT AAT T (SEO ID NO: 385) CTA CCC TGA AAG TAC ATT CA (SEQ ID NO: 405)

CCG GTC TTA TAA ATC TTC A (SEO ID NO: 386). CAG CCC TTA TTC CTG ATG C (SEQ ID NO: 406)

ATA ACG. AGC AAG ACC CCC AT (SEO ID NO: 387). GGT CGT CCT TTC AGG ATT AC (SEO ID NO: 4 O7)

In PCRs a reaction is also included to quantify total bacteria or archaea present in the sample; in this case known universal TABLE 1.5 - continued primers are used for both kingdoms which are selected among

PCR the primers included in Table 15.

Seq ID Nos. TABLE 1.5

Univ534-R ATT ACC GCG GCT (SEO ID NO: 417) PCR GCT GG

Seq ID Nos.

Bacteria primers Each PCR has a specific cycle, wherein the alignment ten perature changes, said temperature being specific for each

Eub27 F AGA GTT TGA TCC (SEO ID NO : 1) used primer pair. Table 16 Summarizes general conditions for TGG CTC AG all qPCR cycles.

Univ533-F" GTG CCA GCM GCC (SEO ID NO : 408) GCG GTA TABLE 16

Bact358-F CCT ACG GGA. GGC (SEQ ID NO: 409) Step Temperature (C.) Time (s) AGC AG 1 Initial denaturation 95 120

2 Denaturation 95 30 Univeo 7-R. CCG TCA ATT CCT (SEO ID NO : 41O) 3 Alignment (*) 30

TTG. AGT T 4 Extension 72 40 5 Pre-reading 8O 10

Bact338-R GCT GCC TCC CGT (SEO ID NO : 411) 6 Reading 8O AGG AGT Repeat 40 times from step 2 to step 6 (qPCR cycle)

7 Denaturation curve Between 70 and 100° C., reading each Bact1387-R. GGG CGG WGT GTA (SEQ ID NO: 412) O.2°C.

CAA. GGC () specific temperature for each used primer pair

Archaea primers Denaturation curve carried out at the end of cycle 40, gives the

Arch344-F ACG GGG CGC AGC (SEQ ID NO: 413) Tm of the amplification product, and is also used to establish AGG CGC GA whether more than one amplification product is present in the

amplified sample, as each would generate its own curve. 7 Univs1.5-F GTG CCA GCA GCC (SEQ ID NO: 414) 0039. The qPCR thermocycler gives a result correspond

GCG GTA A ing to DNA concentration present in each reaction, and this Arch958-R YCC GGC GTT GAM (SEO ID NO : 415) information 1s used to calculate the number of microorgan

TCC AAT T sms present in the sample, which is called Q. This value is inferred by the computational program associated to the ther

Arch915-R' GTG CTC CCC CGC (SEO ID NO : 416) mocycler based on: DNA concentration in calibration curve CAA TTC CT reactions and the cycle in which sample begins to amplify (or

to exponentially increase its fluorescence value). The corre

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lation 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. 0040 Calculation of the Number of Microorganisms Present in the Sample. 0041 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 num ber of microorganisms from a given taxon present in a given sample, specially a biomining sample. 0042. By applying the method of the invention, the num ber of microorganisms belonging to the taxons Acidiphilium

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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. 0043. The method comprises a standard curve construc tion, a qPCR reaction with the designed specific primers and the final data transformation. 0044) The standard curve is constructed using the specific PCR product fragment of the 16S rDNA gene produced by the designed primers in different dilutions of a mixed DNA stan dard sample. 0045. The standard curve obtained with the dilutions of the standard DNA mixture will have a profile as the follow

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Cd s

a. s

c s H ''

d

30

28

26 -

24

22 -

2O -

18

16

14 -

12

is s k k

12

y = 1.0017x - 0.0398

15

C(T) para Est. Antiguos

25 30

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Standard DNA mixture Diution 1:10 Dilution 1:100 Dilution 1:1 OOO Dilution 1:10000 Dilution 1:1 OOOOO

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Whereas C(T) is the value given by the equipment that is related to the DNA concentration of the samples used with known concentration (different dilutions) of the same taxon. One standard curve has to be made for each taxon to be analyzed. 0046. Using this information and the following equation it

is possible to correlate the concentration of DNA with the number of molecules of 16S rRNA and therefore microor ganisms of specific taxon. The standard curve can contain a mixture of DNAs coming from different species but even in this case one can be able to determine the number of mol ecules of specific specie knowing the length of the PCR product to be amplified by the specific primers and the con centration of initial DNA as it was added for the standard CUV.

6.023x105unlock Angul (1) B: 660ginot : 1 x 10, QN of molecules F

Whereas

0047 1 bp=660 g/mol I0048 6.023x10' molecules or copies (Avogadro num

ber) 0049 A-DNA concentration in ng/uL 0050 B-Length of the PCR product

0051. As only A and B are variables, formula can be expressed as:

(Anglu) (2) One of molecules = 9.126x10cing: inolecing

For problem samples the qPCR reaction is performed accord ing to the best conditions recommended for the PCR product to be amplify and the result obtained as a C(T) value is then interpolated in the standard curve to obtain the concentration of specific DNA fragment tag present in the sample. With the concentration and equation (2) one can obtain the number of molecules present in a sample as determined by the qPCR reaction. Finally to estimate microorganisms concentration the follow ing equation must be applied:

N QN molecules *Tng (3) Ceutni) = . . . . . Cmolecules/cel * Ung * Cn(mL)-(g)

0.052 N=Estimated Concentration of microorganisms. 0053 Q-Number of Molecules as calculated with the standard curve using the C(T) value given by the equip ment.

0054 T=Total DNA isolated from original sample. 0055 U=Amount of DNA used in the qPCR reaction. 0056 C=Number of molecules or copies by genome of the specific DNA fragment detected.

0057 Cm=Amount in “g” or “ml” of the processed sample.

0058. In all cases number of molecules present by genome's microorganisms (C) is known either by sequence available publicly or Southern blot performed in our lab. For

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example, in the case of A. ferrooxidans it is known that the specie DSM 16786 Wenelen has only one copy of 16S rDNA gene but in the case of Leptospirillum DSM 17947Yagan it is reported to have 2 copies of the 16S rDNA gene.

EXAMPLES

Example 1

Quantification of Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans, Leptospirillum sp. and Acidiphilium sp. Present in a Biomining Sample

0059. Two solid samples obtained from mineral bioleach ing heaps (SS-1 and SS-2) and 2 liquid samples recovered from bioleaching effluents (LS-1 and LS-2) were analyzed and total DNA was extracted from each sample. For all solid samples a further step was necessary, a re-puri fication 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.

Total DNA was quantified in each sample using a NanoDrop 1.0 spectrophotometer. Total extracted DNA nanograms (T) are shown in Table 1-1 together with the initial sample vol umes (C). Registered results were:

TABLE 1-1

Sample T Cn

SS-1 543.2 100 g

SS-2 1660.4 20 g

LS-1 11365.2 45 mL.

LS-2 16364.6 45 mL.

Each of these samples was diluted with sterile nuclease-free water in order to obtain a concentration in the range 0.5 to 30 ng/ul. Table 1-2 shows the final volume to which the DNA Solution was brought and its final concentration.

TABLE 1-2

Sample Final volume (l) Concentration (ngful)

SS-1 70 7.76

SS-2 70 23.72

LS-1 421 27

LS-2 545.5 30

A calibration curve was simultaneously prepared for each taxon to allow the calculation of molecule number of the target gene in experimental samples. The standard DNA was prepared for each taxon using PCR products obtained with specific primers shown in table 1-3.

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TABLE 1-3

Microorganism to be Alignment determined temperature Used primers

Total bacteria GCA GCC GCG GTA-3

ATT CCT TTG. AGT T-3'

A. ferrooxidans 6 Oc C. (P.13) F: GGG AGA. GGG TG-3

(P. 6) R: ATT CCG GAC AT-3'

A. thiooxidans 6 o C. (P. 7) F: TGC TGT TGA C-3'

(P.11) R: AGA CCT AAT G-3

Leptospirillum sp. 588 C. (P. 4) F: AAG GGT TTC G-3

(P. 2) R: GGG ATA TTT A-3

Acidiphilium sp. 618 C. (P.10) F: GGA. GGA, GCC T-3'

(P. 3) R: TCT GCG GGT A-3'

The PCR reaction to obtain the PCR products was carried out using as substrate the respective genomic DNA (200 ng) from each of the following microorganisms: Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans, Leptospirillum sp. and Acidiphilium sp. The composition of PCR mix is detailed in table 1-4. The PCR product was purified using commercial DNA purification columns and concentration of the respective PCR products was determinate using Nano Drop 1.0 spectrophotometer.

TABLE 1-4

Reagent 1 reaction

Sterile nuclease-free H2O 18.35 ul PCR Buffer 10x 2.5 ul MgCl2 (50 mM) 1.5 ul dNTPs (10 mM each) 0.5 ul Primer Forward* (10 M) 0.5 ul Primer Reverse (10M) 0.5 ul Hot Start Taq (5 U?ul) 0.15 ul

*Used primers are described in Table 1-3.

More specifically, the PCR products were obtained using genomic DNA from the following strains:

0060 A. ferrooxidans DSM 16786; 0061 A. thiooxidans DSM 504; 0062 Leptospirillum sp. DSM 1931 and 0063 Acidiphilium acidophilus DSMZ 700.

The number of molecules for each PCR product was calcu lated using the equation (2) described above. Each standard curves was prepared using five serial dilutions containing 1x10 molecules of specific PCR products in a final volume of 20 ul, which is included in the calibration CUV.

6 o C. (P.1) 533-F: 5' - GTG CCA

(P. 2) 907-R: 5' - CCG TCA

5 - CTA GAG TAT

s' - CTC TGC AGA

s' - TAA TAT CGCC

s" - TTT CAC GAC

5 - GGA ACC GTG

s' - CCG TCA TCG

s' - GTC GCC TAA

5 - GGA GCT TAT

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Amplify lengths (bp)

374

365

146

315

263

0064. A reaction “master mix' for the quantitative PCR was prepared wherein the amount of each constituent was multiplied by the total number of reactions to be carried out 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 Jul of reaction mix per tube. Subsequently 5 quantitative 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. Sense and antisense primers were selected for the different taxons from those designed specifically by our method. Primers used for each taxon and their respective annealing temperatures are indicated in Table 1-3.

In the present Example, the following reactions were per formed in duplicate:

0065 0.066 b) Five reactions for each point of calibration curve (n-6), corresponding to standard DNA mastermix concentrations of 1x10, 1x10", 1x10, 1x10 and 1x10" molecules, and a blank.

0067. One quantitative PCR reaction was carried out on each sample and on standard DNA, these reactions being performed in duplicate. The qPCR was carried out using Mix SYBR Green qPCR. Each quantitative PCR reaction was carried out by duplicate which gives a total of 40 reactions per each taxon.

a) Five reaction for each sample (n=4) and

The quantitative PCR reaction mix for each taxon is shown in Table 1-5, where primers used are those described on Table 1-3.

US 2011/O 1361.25 A1

TABLE 1-5

1 reaction 20 reactions

Sterile nuclease-free H2O 16.1 ul 322 Jul Primer 1 (10 M) 0.5 ul 10 ul Primer 2 (10 M) 0.5 ul 10 ul PCR Buffer 10x 2.5 ul 50 ul MgCl2 (50 mM) 1.5 ul 30 ul dNTPs (10 mM each) 2.5 ul 50 ul Hot Start Taq (5 U?ul) 0.15 ul 3 ul SYBR Green qPCR 100x 0.25 ul 5 ul

This reaction mix was homogenized and aliquoted in 200.2 ml tubes, which were duly labeled. To each of the tubes 1 ul of DNA sample dilution or 1 ul of sterile nuclease-free water for the blank was added. PCR tubes containing the reaction mix and sample were shaked at Vortex for 5 seconds and centrifuged for 1 minute at 2000xg, in order to homogenize and bring the reaction liquid to the bottom of the tube, respectively. Then, the tubes with quantitative PCR reactions were sub jected to temperature cycles for amplification (table 1-6). 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 PCR reactions are shown.

TABLE 1-6

Step Temperature (C.) Time (s)

1 Initial denaturation 95 120 2 Denaturation 95 30 3 Alignment (*) 30 4 Extension 72 40 5 Pre-reading 8O 10 6 Reading 8O

Repeat 40 times from step 2 to step 6 (qPCR cycle) 7 Denaturation curve Range between 70 and 100° C., reading

each 0.2°C.

() specific temperature for each used primer pair, as indicated in Table 1-3,

When the quantitative PCR is finished, all data generated by the qPCR thermocycler are stored, from standard curve is calculated the number of molecules for target gene into each sample; this data corresponds to Q and is shown in Table 1-7. wherein DNA amounts in nanograms used for each reaction are included (U).

Jun. 9, 2011 15

0068 Calculation of the Number of Microorganisms Present in the Samples. 0069. Taking into account the qPCR result and data gen erated during the process, the following formula described as equation (3) can be applied:

Noel (mL)-(g) = QN molecules *Tng unt - . Cmolecules/cel * Ung * Cn(mL)-(g)

0070 N=Estimated Concentration of microorganisms. (0071 Q-Number of Molecules as calculated with the

standard curve using the C(T) value given by the equip ment.

0.072 T-Total DNA isolated from original sample. 0.073 U=Amount of DNA used in the qPCR reaction. 0.074 C=Number of molecules or copies by genome of the specific DNA fragment detected. (see table 1-8)

0075 Cm=Amount in or “ml of the processed sample

9.

TABLE 1-8

Number of copies of 16S rDNA gene in the genome of different taxons as described in literature or determined by Southern blot.

Microorganisms C

A. ferrooxidans A. thiooxidans Acidiphillium Leptospirillaim Bacteria total

*The number of 16S rDNA copies by genome of total bacteria was estimated as an average according to our observations,

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

TABLE 1-9

SS-1

Bacteria Mo.g. of sample

Total bacteria S.27E--OS A. ferrooxidans 1.OOE--O3 A. thiooxidans 1.29E-04 Leptospirilium sp. S.2SE--O2 Acidiphilium sp. 4.94E--04

TABLE 1-7

Q

Total Leptospirillum Acidiphilium

Sample bacteria A. ferrooxidans A. thiooxidans sp. Sp. U

SS-1 4.85E-05 O.OOSE-OS O.06E--OS O.OOSE-OS O4SE--OS 2.5

SS-2 3.48E--OS O.O47E--OS O.O13E--OS O.71E--OS O.34E--OS 2.5

LS-1 3.12E--OS O.SSE--OS O O.OO6E--OS O 2.5

LS-2 2.88E--OS O.82E--OS O O.OO6E--OS O 2.5

US 2011/O 1361.25 A1

TABLE 1-10

SS-2


Total bacteria 5.78E--06

A. ferrooxidans 1.SSE--OS A. thiooxidans 4.15E-04

Leptospirilium sp. 1.17E--O6 Acidiphilium sp. S.71E--OS

TABLE 1-11

LS-1

Bacteria Mo./ml of sample

Total bacteria 1.57E--O7 A. ferrooxidans 5.61E--O6 A. thiooxidans O.OOE--OO Leptospirilium sp. 3.02E--04 Acidiphilium sp. O.OOE--OO

TABLE 1-12

LS-2


Total bacteria 2.09E-07 A. ferrooxidans 1.19E--O7 A. thiooxidans O.OOE--OO Leptospirilium sp. 4.77E--04 Acidiphilium sp. O.OOE--OO

Microorganism to be determined

Total bacteria

Sulfobacillus sp.

Total archaea

Sulfolobus sp.

16

temperature Used primers

Jun. 9, 2011

Example 2 Quantification of Sulfobacillus sp., Sulfolobus sp.,

and Ferroplasma sp. in a Sample 0076 One sample obtained from mineral bioleaching heap (SS-3) and one liquid sample recovered from bioleach ing effluents (LS-3) 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. Total DNA was quantified in each sample using a NanoDrop 1.0 spectrophotometer. Total extracted DNA nanograms (T) are shown in Table 2-1 together with the initial sample vol umes (C).

TABLE 2-1

Sample T Cn

SS-3 537.25 20 g LS-3 1621.13 50 mL.

Each of these samples was diluted with sterile nuclease-free water in order to obtain a concentration in the range of 0.5 to 30 ng/ul. Table 2-2 shows the final volume to which the DNA Solution was brought and its final concentration.

TABLE 2-2


SS-3 70 7.67 LS-3 540 30

Five calibration curves were prepared for quantitative PCR that includes total Bacteria, Sulfobacillus, total Archaea, Fer roplasma and Sulfolobus. These calibration curves were used for calculating the molecule number of target gene in experi mental samples. The standard DNA was prepared for each taxon using PCR products obtained with specific primers described in table 2-3.

TABLE 2-3

Alignment Amplify lengths (bp)

59 (P. 1) 27-F: 5'-AGA GTT TGA 374 TCC TGG CTC AG-3'

(P. 2) 338-R 5 " - GCT GCC TCC CGT AGG AGT-3'

66 (P.1) F : 5'- CGA AGG CGG TGC 122 ACT GGC C-3

(P. 3) R: 5'-GGT GGA CCC CCG CGA CAC C-3

6 O (P.1) 515-F: 5' - GTG CCA GCA 443 GCC GCG GTA A-3

(P. 2) 958-R: 5'-TCC GGC GTT GAA TCC AAT T-3'

6 O (P. 3) F : 5' - GTC CTG GAA CGG 282 TTC CTC G-3

(P.9) R. 5" - CCC CCG CCT TTG TAG AGC G-3

US 2011/O 1361.25 A1

TABLE 2-3 - continued

Jun. 9, 2011

Microorganism to be Alignment Amplify determined temperature Used primers lengths (bp)

Ferroplasma sp. 56 (P.17) F : 5'-TGG CCA AGA CTT 2O4. TTC TCA T-3

(P.19) R: CTT GCA GT-3'

The PCR reaction to obtain the PCR products was carried out using the respective primers and as Substrate the respective genomic DNA (200 ng) from each of the following microor ganisms: Sulfobacillus, Ferroplasma y Sulfolobus. Substrate for PCR products for standard representing Total Archaea was obtained from the mix of the genomic DNA containing 100ng of Ferroplasma and 100 ng of Sulfolobus. Substrate for PCR product representing Total Bacteria was obtained using DNA from Sulfobacillus (200 ng). The composition of PCR mix is detailed in table 2-4 and PCR amplification program in table 2-5. The respective PCR products were purified using commercial DNA purification columns and concentration of them was determinate using NanoDrop 1.0 spectrophotom eter.

TABLE 2-4

Reagent 1 reaction



TABLE 2-5

Temperature Step (° C.) Time (s)

1. Initial denaturation 95 120 2. Denaturation 95 30 3. Alignment 30 4. Extension 72 120

Wherein steps 2 to 4 are to be repeated 28 times, The alignment temperature used is according to pairs of primer detailed in table 2-3,

The number of molecules for each PCR product was calcu lated using the equation (2) described above. Each standard curves was prepared using five serial dilutions that containing 1x10 molecules of specific PCR products in a final volume of 20 ul, which in its turn is included in the calibration curve.

0077. A reaction “master mix” for the quantitative PCR was prepared wherein the amount of each constituent was multiplied by the total number of reactions to be carried out in order to homogenize reagent concentrations in the different

s' - CCG ATC. TCA TGT

PCR tubes. The reaction mix was aliquoted in 0.2 ml tubes, using a Volume of 24 Jul of reaction mix per tube. Subsequently 5 quantitative PCR were performed, one for each taxon: Sulfobacillus, Ferroplasma, Sulfolobus, Total Bacteria and one for Total Archaea, using specific primers for each of them. Sense and antisense primers were selected for the different genera from those included in the description of the tables corresponding to each taxon. Primers used for each taxon and their respective annealing temperatures are indi cated in Table 2-3.


0078 0079 b) Five reactions for each point of calibration curve (n-6), corresponding to standard DNA master mix concentrations of 1x10, 1x10", 1x10, 1x10 and 1x10" molecules, and a blank.

One quantitative PCR reaction was carried out on each sample and on standard DNA, these reactions being per formed in duplicate. The qPCR was carried out using Mix SYBR Green qPCR. Each quantitative PCR reaction was carried out by duplicate which gives a total of 16 reactions per each taxon.

a) Five reaction for each sample (n=2) and

The quantitative PCR reaction mix for each taxon is shown in Table 2-6, where primers are those that are corresponding according to Table 2-3.

TABLE 2-6


Sterile nuclease-free H2O 16.1 ul 257.6 ul Primer 1 (10 M) 0.5 ul 8 ul Primer 2 (10 M) 0.5 ul 8 ul PCR Buffer 10x 2.5 ul 40 ul MgCl2 (50 mM) 1.5 ul 24 ul dNTPs (10 mM each) 2.5 ul 40 ul Hot Start Taq (5 U?ul) 0.15 ul 2.4 ul SYBR Green qPCR 100x 0.25 ul 4 Jul

This reaction mix was homogenized and aliquoted in 200.2 ml tubes, which were duly labeled. To each of the tubes 1 ul of DNA sample dilution or 1 ul of sterile nuclease-free water for the blank was added.

PCR tubes containing the reaction mix and sample were shaked at Vortex for 5 seconds and centrifuged for 1 minute at 2000xg, in order to homogenize and bring the reaction liquid to the bottom of the tube, respectively. Then, the tubes with quantitative PCR reactions were sub jected to temperature cycles for amplification (table 2-7).

US 2011/O 1361.25 A1 18

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 PCR reactions are shown.

TABLE 2-7

Jun. 9, 2011

TABLE 2-7-continued


5 Pre-reading 8O 10 6 Reading 8O

Repeat 40 times from step 2 to step 6 (qPCR cycle) 7 Denaturation curve Between 70 and 100° C.,

reading each 0.2°C. Step Temperature (C.) Time (s) () specific temperature for each used primer pair, as indicated in Table 2-3.

1 Initial denaturation 95 120 When the quantitative PCR is finished, all data generated by 2 Denaturation 95 30 the qPCR thermocycler are stored, from standard curve is 3 Alignment (*) 30 calculated the number of molecules for target gene into each

sample; this data corresponds to Q and is shown in Table 2-8. 4 Extension 72 40 wherein DNA amounts in nanograms used for each reaction are included (U).

TABLE 2-8

-O-

Total Sulfolobus Ferroplasma Sample Total bacteria Sulfobacilius sp. archaea sp. sp U

SS-3 S.76E--OS O.14E--OS 1.61E--OS O O.O28E--OS 2.5 LS-3 16.4E--05 O.O18E--OS 16.8E--OS 0.27E--OS 4.7SE-OS 2

0080 Calculation of the Number of Microorganisms Present in the Sample. 0081. The standard curve obtained with the dilutions of the standard DNA mixture will have a profile as the follow ing:

US 2011/O 1361.25 A1 Jun. 9, 2011

| Standard DNA mixture y = 1.0017x - 0.0398 Diution 1:10

R2 = 1 Dilution 1:100 Dilution 1:1000 Dilution 1:1 OOOO Dilution 1:1 OOOOO

d > s 2

s c as

Whereas C(T) is the value given by the equipment that is related to the DNA ConCentration of the samples used with known

2O concentration (different dilutions) Of the Same taXOn. One Standard Curve has to be made for each taxon to be analyzed.

C(T) para Est. Antiguos

US 2011/O 1361.25 A1

0082. Using this information and the following equation it is possible to correlate the concentration of DNA with the number of molecules of 16S rRNA and therefore microor ganisms of specific taxon. The standard curve can contain a mixture of DNAs coming from different species but even in this case one can be able to determine the number of mol ecules of specific specie knowing the length of the PCR product to be amplified by the specific primers and the con centration of initial DNA as it was added for the standard CUV.

6.023x105unlock Angul (4) B: 660ginot : 1 x 10, QN of molecules F

Whereas

I0083. 1 bp=660 g/mol 10084) 6.023x10' molecules or copies (Avogadro num

ber) I0085 A-DNA concentration in ng/uL I0086 B-Length of the PCR product

0087 As only A and B are variables, formula can be expressed as:

(Anglu) (5) QNo of molecules = 9.126X 1Ol inolecing

For problem samples the qPCR reaction is performed accord ing to the best conditions recommended for the PCR product to be amplify and the result obtained as a C(T) value is then interpolated in the standard curve to obtain the concentration of specific DNA fragment tag present in the sample. With the concentration and equation (2) one can obtain the number of molecules present in a sample as determined by the qPCR reaction. Finally to estimate microorganisms concentration the follow ing equation must be applied:

N QN molecules *Tng (6) Ceit (mL), (g) Cmolecules/cel * Ung * Cn(mL)(g)

I0088 N=Estimated Concentration of microorganisms. I0089 Q-Number of Molecules as calculated with the

standard curve using the C(T) value given by the equip ment.

(0090 T=Total DNA isolated from original sample. (0091 U=Amount of DNA used in the qPCR reaction. 0092 C=Number of molecules or copies by genome of the specific DNA fragment detected.

(0093. Cm=Amount in “g” or “ml” of the processed sample.


Total bacteria

20 Jun. 9, 2011

0094. In all cases number of molecules present by genome's microorganisms (C) is known either by sequence available publicly or Southern blot performed in our lab. For example, in the case of A. ferrooxidans it is known that the specie DSM 16786 Wenelen has only one copy of 16S rDNA gene but in the case of Leptospirillum DSM 17947Yagan it is reported to have 2 copies of the 16S rDNA gene.

EXAMPLES

Example 1

Quantification of Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans, Leptospirillum sp. and Acidiphilium sp. Present in a Biomining Sample

0095. Two solid samples obtained from mineral bioleach ing heaps (SS-1 and SS-2) and 2 liquid samples recovered from bioleaching effluents (LS-1 and LS-2) were analyzed and total DNA was extracted from each sample. For all solid samples a further step was necessary, a re-puri fication 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. Total DNA was quantified in each sample using a NanoDrop 1.0 spectrophotometer. Total extracted DNA nanograms (T) are shown in Table 1-1 together with the initial sample vol umes (C). Registered results were:

TABLE 1-1

Sample T Cn

SS-1 543.2 100 g SS-2 1660.4 20 g LS-1 11365.2 45 mL. LS-2 16364.6 45 mL.

Each of these samples was diluted with sterile nuclease-free water in order to obtain a concentration in the range 0.5 to 30 ng/ul. Table 1-2 shows the final volume to which the DNA Solution was brought and its final concentration.

TABLE 1-2


SS-1 70 7.76 SS-2 70 23.72 LS-1 421 27 LS-2 545.5 30

A calibration curve was simultaneously prepared for each taxon to allow the calculation of molecule number of the target gene in experimental samples. The standard DNA was prepared for each taxon using PCR products obtained with specific primers shown in table 1-3.

TABLE 1-3

Alignment temperature Used primers


68 C. (P.1) 533-F: 5' - GTG CCA 374

US 2011/O 1361.25 A1


Microorganism to be Alignment determined temperature Used primers

(P. 2) 907-R: 5' - CCG TCA ATT CCT TTG. AGT T-3'

A. ferrooxidans 6 Oc C. (P.13) F : 5'- CTA GAG TAT GGG AGA. GGG TG-3

(P. 6) R: 5'- CTC TGC AGA ATT CCG GAC AT-3'

A. thiooxidans 6 o C. (P. 7) F: is "-TAA TAT CCCC TGC TGT TGA C-3'

(P.11) R: 5'-TTT CAC GAC AGA CCT AAT G-3

Leptospirillum sp. 588 C. (P. 4) F : 5'-GGA ACC GTG AAG GGT TTC G-3

(P. 2) R: 5'- CCG TCA TCG GGG ATA TTT A-3

Acidiphilium sp. 618 C. (P.10) F : 5' - GTC GCC TAA GGA. GGA, GCC T-3'

(P. 3) R: 5'-GGA GCT TAT

The PCR reaction to obtain the PCR products was carried out using as substrate the respective genomic DNA (200 ng) from each of the following microorganisms: Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans, Leptospirillum sp. and Acidiphilium sp. The composition of PCR mix is detailed in table 1-4. The PCR product was purified using commercial DNA purification columns and concentration of the respective PCR products was determinate using Nano Drop 1.0 spectrophotometer.

TABLE 1-4

Reagent 1 reaction



More specifically, the PCR products were obtained using genomic DNA from the following strains:

(0096 A. ferrooxidans DSM 16786; 0097 A. thiooxidans DSM 504; (0098. Leptospirillum sp. DSM 1931 and (0099. Acidiphilium acidophilus DSMZ 700.

The number of molecules for each PCR product was calcu lated using the equation (2) described above. Each standard curves was prepared using five serial dilutions containing 1x10 molecules of specific PCR products in a

Jun. 9, 2011


365

146

315

263

final volume of 20 ul, which is included in the calibration CUV.

0100. A reaction “master mix” for the quantitative PCR was prepared wherein the amount of each constituent was multiplied by the total number of reactions to be carried out 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 Jul of reaction mix per tube. Subsequently 5 quantitative 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. Sense and antisense primers were selected for the different taxons from those designed specifically by our method. Primers used for each taxon and their respective annealing temperatures are indicated in Table 1-3.


01.01 0102 d) Five reactions for each point of calibration curve (n-6), corresponding to standard DNA mastermix concentrations of 1x10, 1x10", 1x10, 1x10 and 1x10" molecules, and a blank.

0103) One quantitative PCR reaction was carried out on each sample and on standard DNA, these reactions being performed in duplicate. The qPCR was carried out using Mix SYBR Green qPCR. Each quantitative PCR reaction was carried out by duplicate which gives a total of 40 reactions per each taxon.

c) Five reaction for each sample (n=4) and

The quantitative PCR reaction mix for each taxon is shown in Table 1-5, where primers used are those described on Table 1-3.

US 2011/O 1361.25 A1

TABLE 1-5


Sterile nuclease-free H2O 16.1 ul 322 Jul Primer 1 (10 M) 0.5 ul 10 ul Primer 2 (10 M) 0.5 ul 10 ul PCR Buffer 10x 2.5 ul 50 ul MgCl2 (50 mM) 1.5 ul 30 ul dNTPs (10 mM each) 2.5 ul 50 ul Hot Start Taq (5 U?ul) 0.15 ul 3 ul SYBR Green qPCR 100x 0.25 ul 5 ul

This reaction mix was homogenized and aliquoted in 200.2 ml tubes, which were duly labeled. To each of the tubes 1 ul of DNA sample dilution or 1 ul of sterile nuclease-free water for the blank was added. PCR tubes containing the reaction mix and sample were shaked at Vortex for 5 seconds and centrifuged for 1 minute at 2000xg, in order to homogenize and bring the reaction liquid to the bottom of the tube, respectively. Then, the tubes with quantitative PCR reactions were sub jected to temperature cycles for amplification (table 1-6). 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 PCR reactions are shown.

TABLE 1-6



Repeat 40 times from step 2 to step 6 (qPCR cycle) 7 Denaturation curve Range between 70 and

100° C., reading each 0.2°C.

() specific temperature for each used primer pair, as indicated in Table 1-3,


22 Jun. 9, 2011

0104 Calculation of the Number of Microorganisms Present in the Samples. 0105 Taking into account the qPCR result and data gen erated during the process, the following formula described as equation (3) can be applied:

QN molecules *Tng Ncult = H. "' Coleculesce: Ungi. Cn(nl)-(g)

0106 N=Estimated Concentration of microorganisms. 0107 Q-Number of Molecules as calculated with the standard curve using the C(T) value given by the equip ment.

0.108 T-Total DNA isolated from original sample. 0109 U=Amount of DNA used in the qPCR reaction. 0110 C=Number of molecules or copies by genome of the specific DNA fragment detected. (see table 1-8)

0.111 Cm=Amount in ''g'' or “ml of the processed sample

TABLE 1-8

Number of copies of 16S rDNA gene in the genome of different taxons as described in literature or determined by Southern blot.

Microorganisms C

A. ferrooxidans A. thiooxidans Acidiphillium Leptospirillaim Bacteria total

*The number of 16S rDNA copies by genome of total bacteria was estimated as an average according to our observations,

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

TABLE 1-9

SS-1


Total bacteria S.27E--OS A. ferrooxidans 1.OOE--O3 A. thiooxidans 1.29E-04 Leptospirilium sp. S.2SE--O2 Acidiphilium sp. 4.94E--04

TABLE 1-7

Q

Total Leptospirillum Acidiphilium

Sample bacteria A. ferrooxidans A. thiooxidans sp. Sp. U

SS-1 4.85E-05 O.OOSE-OS O.06E--OS O.OOSE-OS O4SE--OS 2.5

SS-2 3.48E--OS O.O47E--OS O.O13E--OS O.71E--OS O.34E--OS 2.5

LS-1 3.12E--OS O.SSE--OS O O.OO6E--OS O 2.5

LS-2 2.88E--OS O.82E--OS O O.OO6E--OS O 2.5

US 2011/O 1361.25 A1

TABLE 1-10

SS-2



A. ferrooxidans 1.SSE--OS A. thiooxidans 4.15E-04

Leptospirilium sp. 1.17E--O6 Acidiphilium sp. S.71E--OS

TABLE 1-11

LS-1


Total bacteria 1.57E--O7 A. ferrooxidans 5.61E--O6 A. thiooxidans O.OOE--OO Leptospirilium sp. 3.02E--04 Acidiphilium sp. O.OOE--OO

TABLE 1-12

LS-2


Total bacteria 2.09E-07 A. ferrooxidans 1.19E--O7 A. thiooxidans O.OOE--OO Leptospirilium sp. 4.77E--04 Acidiphilium sp. O.OOE--OO


Total bacteria

Sulfobacillus sp.

Total archaea

Sulfolobus sp.

23

temperature Used primers

Jun. 9, 2011

Example 2 Quantification of Sulfobacillus sp., Sulfolobus sp.,

and Ferroplasma sp. in a Sample 0112 One sample obtained from mineral bioleaching heap (SS-3) and one liquid sample recovered from bioleach ing effluents (LS-3) 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. Total DNA was quantified in each sample using a NanoDrop 1.0 spectrophotometer. Total extracted DNA nanograms (T) are shown in Table 2-1 together with the initial sample vol umes (C).

TABLE 2-1

Sample T Cn

SS-3 537.25 20 g LS-3 1621.13 50 mL.

Each of these samples was diluted with sterile nuclease-free water in order to obtain a concentration in the range of 0.5 to 30 ng/ul. Table 2-2 shows the final volume to which the DNA Solution was brought and its final concentration.

TABLE 2-2


SS-3 70 7.67 LS-3 540 30

Five calibration curves were prepared for quantitative PCR that includes total Bacteria, Sulfobacillus, total Archaea, Fer roplasma and Sulfolobus. These calibration curves were used for calculating the molecule number of target gene in experi mental samples. The standard DNA was prepared for each taxon using PCR products obtained with specific primers described in table 2-3.

TABLE 2-3

Alignment Amplify lengths (bp)

59 (P. 1) 27-F: 5'-AGA GTT TGA 374 TCC TGG CTC AG-3'

(P. 2) 338-R 5 " - GCT GCC TCC CGT AGG AGT-3'

66 (P.1) F : 5'- CGA AGG CGG TGC 122 ACT GGC C-3

(P. 3) R: 5'-GGT GGA CCC CCG CGA CAC C-3

6 O (P.1) 515-F: 5' - GTG CCA GCA 443 GCC GCG GTA A-3

(P. 2) 958-R: 5'-TCC GGC GTT GAA TCC AAT T-3'

6 O (P. 3) F : 5' - GTC CTG GAA CGG 282 TTC CTC G-3

(P.9) R. 5" - CCC CCG CCT TTG TAG AGC G-3

US 2011/O 1361.25 A1 Jun. 9, 2011 24


Microorganism to be Alignment Amplify determined temperature Used primers lengths (bp)

Ferroplasma sp. 56 (P.17) F : 5'-TGG CCA AGA CTT 2O4. TTC TCA T-3

(P.19) R: CTT GCA GT-3'

The PCR reaction to obtain the PCR products was carried out using the respective primers and as Substrate the respective genomic DNA (200 ng) from each of the following microor ganisms: Sulfobacillus, Ferroplasma y Sulfolobus. Substrate for PCR products for standard representing Total Archaea was obtained from the mix of the genomic DNA containing 100 ng of Ferroplasma and 100 ng of Sulfolobus. Substrate for PCR product representing Total Bacteria was obtained using DNA from Sulfobacillus (200 ng). The composition of PCR mix is detailed in table 2-4 and PCR amplification pro gram in table 2-5. The respective PCR products were purified using commercial DNA purification columns and concentra tion of them was determinate using NanoDrop 1.0 spectro photometer.

TABLE 2-4

Reagent 1 reaction



TABLE 2-5

Temperature Step (° C.) Time (s)

1. Initial denaturation 95 120 2. Denaturation 95 30 3. Alignment 30 4. Extension 72 120

Wherein steps 2 to 4 are to be repeated 28 times, The alignment temperature used is according to pairs of primer detailed in table 2-3,

The number of molecules for each PCR product was calcu lated using the equation (2) described above. Each standard curves was prepared using five serial dilutions that containing 1x10 molecules of specific PCR products in a final volume of 20 ul, which in its turn is included in the calibration curve.

0113. A reaction “master mix' for the quantitative PCR was prepared wherein the amount of each constituent was multiplied by the total number of reactions to be carried out 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 Jul of reaction mix per tube.

s' - CCG ATC. TCA TGT

Subsequently 5 quantitative PCR were performed, one for each taxon: Sulfobacillus, Ferroplasma, Sulfolobus, Total Bacteria and one for Total Archaea, using specific primers for each of them. Sense and antisense primers were selected for the different genera from those included in the description of the tables corresponding to each taxon. Primers used for each taxon and their respective annealing temperatures are indi cated in Table 2-3.


0114 0115 d) Five reactions for each point of calibration curve (n-6), corresponding to standard DNA mastermix concentrations of 1x10, 1x107, 1x10, 1x10 and 1x10" molecules, and a blank.

One quantitative PCR reaction was carried out on each sample and on standard DNA, these reactions being per formed in duplicate. The qPCR was carried out using Mix SYBR Green qPCR. Each quantitative PCR reaction was carried out by duplicate which gives a total of 16 reactions per each taxon.

c) Five reaction for each sample (n=2) and

The quantitative PCR reaction mix for each taxon is shown in Table 2-6, where primers are those that are corresponding according to Table 2-3.

TABLE 2-6


Sterile nuclease-free H2O 16.1 ul 257.6 ul Primer 1 (10 M) 0.5 ul 8 Jul Primer 2 (10 M) 0.5 ul 8 Jul PCR Buffer 10x 2.5 ul 40 ul MgCl2 (50 mM) 1.5 ul 24 ul dNTPs (10 mM each) 2.5 ul 40 ul Hot Start Taq (5 U?ul) 0.15 ul 2.4 ul SYBR Green qPCR 100x 0.25 ul 4 Il

This reaction mix was homogenized and aliquoted in 200.2 ml tubes, which were duly labeled. To each of the tubes 1 ul of DNA sample dilution or 1 ul of sterile nuclease-free water for the blank was added.

PCR tubes containing the reaction mix and sample were shaked at Vortex for 5 seconds and centrifuged for 1 minute at 2000xg, in order to homogenize and bring the reaction liquid to the bottom of the tube, respectively. Then, the tubes with quantitative PCR reactions were sub jected to temperature cycles for amplification (table 2-7).

US 2011/O 1361.25 A1 25

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 PCR reactions are shown.

TABLE 2-7



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 2-3.


TABLE 2-8

Jun. 9, 2011

TABLE 2-9-continued

Number of copies of 16S rDNA gene in the genome of different taxas

Microorganisms C

Archaea total 2

Feroplasma Sulfolobus sp. 1

*The number of 16S rDNA of bacteria total and archaea total was estimated as average according our observations,

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

TABLE 2-10

SS-3

Microorganism Mo.g. of sample

Total bacteria 3.09E-06 Sulfobacillus sp. 7.72E--O4

-O-

Total Sulfolobus Ferroplasma Sample Total bacteria Sulfobacillus sp. archaea sp. sp U

SS-3 S.76E--OS O.14E--OS 1.61E--OS O O.O28E--OS 2.5 LS-3 16.4E--OS O.O18E--OS 16.8E--OS 0.27E--OS 4.7SE-OS 2

0116 Calculation of the Number of Microorganisms Present in the Sample. 0117 Taking into account the qPCR result and data gen erated during the process, the following formula described as equation (3) can be applied:

N QN molecules *Tng (3) Ceit (mL), (g) Cmolecules/cel * Ung * Cn(mL)(g)

0118 N=Estimated Concentration of microorganisms. 0119 Q-Number of Molecules as calculated with the standard curve using the C(T) value given by the equip ment.

I0120 T=Total DNA isolated from original sample. I0121 U=Amount of DNA used in the qPCR reaction. 0.122 C=Number of molecules or copies by genome of the specific DNA fragment detected. (see table 1-8)

I0123. Cm=Amount in ''g'' or “ml of the processed sample

TABLE 2-9

Number of copies of 16S rRNA gene in the genome of different taxas

Microorganisms C

Bacteria total 2 Sulfobacilius 1

TABLE 2-10-continued

SS-3

Microorganism Mo.g. of sample

Total archaea 8.6SE--OS

Stilfolobus sp. O Ferroplasma sp. 3.02E--04

TABLE 2-11

LS-3

Microorganism Mo./ml of sample


Sulfobacillus sp. 1.18E--OS Total archaea 109E-08

Stilfolobus sp. 3.53E--06 Ferroplasma sp. 6.16E--07

US 2011/O 1361.25 A1

SEQUENCE LISTING

<16O is NUMBER OF SEO ID NOS: 417

<210s, SEQ ID NO 1 &211s LENGTH: 2O &212s. TYPE: DNA

<213> ORGANISM: Artificial Sequence 22 Os. FEATURE:

<223> OTHER INFORMATION: PCR primer

<4 OOs, SEQUENCE: 1

agagtttgat cctggcticag

<210s, SEQ ID NO 2 &211s LENGTH: 19 &212s. TYPE: DNA



<4 OOs, SEQUENCE: 2

ggittaccttgttacgacitt



<223> OTHER INFORMATION: PCR primer 22 Os. FEATURE:

<221 > NAMEAKEY: misc feature <222s. LOCATION: (14) . . (14) 223s OTHER INFORMATION: n is c or t

<4 OOs, SEQUENCE: 3

titc.cggttga t c cngc.cgga



<223> OTHER INFORMATION: Sense primer

<4 OOs, SEQUENCE: 4

Calaccacggit cqggtcaga




<4 OOs, SEQUENCE: 5

gaccttalagt tatgcgct




26

19

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1

SEQUENCE: 6

agt calaccac ggtcgggt C

SEO ID NO 7 LENGTH: 2O TYPE: DNA

ORGANISM: Artificial Sequence FEATURE:

OTHER INFORMATION: Sense primer

SEQUENCE: 7

ggtttgacct taagttgatg

SEQ ID NO 8 LENGTH 19 TYPE: DNA



SEQUENCE: 8

cittaagttga tigcgctaac

SEO ID NO 9 LENGTH 19 TYPE: DNA



SEQUENCE: 9

ggcagt caac Cacggit cqg




SEQUENCE: 1.O

cgatgctgag CtgatcCtg




SEQUENCE: 11

aagttgatgc gct aaccgc




SEQUENCE: 12

aaagtc.gc.ct aaggaggag

27

- Continued

19

19

19

19

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1




SEQUENCE: 13

gtc.gc.ctaag gaggagcct




SEQUENCE: 14

aaggaggagc ctg.cgtctg




SEQUENCE: 15

aggagcctgc gtctgatta

SEQ ID NO 16 LENGTH: 2O TYPE: DNA



SEQUENCE: 16

aggaggcagt caaccacggit




SEQUENCE: 17

gcgaaagt cq cctaaggag




SEQUENCE: 18

gcctaaggag gag cctgcgt


28

- Continued

19

19

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1



<4 OOs, SEQUENCE: 19

gcaaggaggc agt caacca

<210s, SEQ ID NO 2 O &211s LENGTH: 19 &212s. TYPE: DNA



<4 OOs, SEQUENCE: 2O

gcaagttctgct cqggcagta





accc.gtagga at citat cot





gcacagticag gcgtgaaata





acacatgcaa gtc.gct cqgg



<223> OTHER INFORMATION: Antisense primer


t citctgaccc gaccgtggitt




29

- Continued

19

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1


t caacttaag gtcaaaccaa





ggagct tatt Ctgcgggta





gcatcaactt aaggtoaaac





agcgcatcaa cittaaggit ca





gttagcgcat Caacttalagg

<210s, SEQ ID NO 3 O &211s LENGTH: 19 &212s. TYPE: DNA




ccgaccgtgg ttgactgcc





ggat cagctic agcatcgctg


30

- Continued

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1




t caggat cag ct cagcatcg





cggittagcgc atcaactta





ggct cotcct tagg.cgacitt





gttgactgcc ticcittgcggit





t cct cottag gcgactitt cq




<4 OO > SEQUENCE: 37

gtggttgact gcct cottgc




31

- Continued

19

Jun. 9, 2011

US 2011/O 1361.25 A1


accotggttg act gcc tic ct





gCaggctic ct cct taggcga

<210s, SEQ ID NO 4 O &211s LENGTH: 2O &212s. TYPE: DNA



<4 OOs, SEQUENCE: 4 O

gacgcaggct cct cottagg





t cagacgcag gct cotcc tt





tgct actgcc cagogactt





tgaccc.gacc gtggttgac





tgaggggact gcc agcgac

32

- Continued

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1





taaat atc cc catgacgg





ttgtc.cggaa ccgtgaaggg




< 4 OO > SEQUENCE: 47

ggaaccgtga agggitttcg





ccgaat attgtc.cggalacc





cgacagagtt tatcgtgg





aatattgtcc ggalaccgtg


33

- Continued

19

19

19

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1




tccggalaccg talagggitt





aaatcgggcc at Cacacag





caaagagact ggcagactag a





tcgggc catc acacaggtg





agagactggc agacitagag





gggggggcaa taccgaatag a




34

- Continued

19

19

21

19

19

21

Jun. 9, 2011

US 2011/O 1361.25 A1

SEQUENCE: 57

at atcaaata aatat coccg




SEQUENCE: 58

aagggatat c gaataaat at

SEO ID NO 59 LENGTH: 21 TYPE: DNA



SEQUENCE: 59

Ctagaggctg. g.gagagggala g

SEQ ID NO 60 LENGTH: 21 TYPE: DNA



SEQUENCE: 6 O

gacgcagcaa cqc.ca.gcagt g




SEQUENCE: 61

aaataaatat coccgatga




SEQUENCE: 62

Cagtgtggga agaaggctitt C




SEQUENCE: 63

aacaagg tac cc.gtct aga

35

- Continued

21

21

19

21

19

Jun. 9, 2011

US 2011/O 1361.25 A1



OTHER INFORMATION: Antisense primer

SEQUENCE: 64

Ctagacgggt accttgttac




SEQUENCE: 65

cc.gt catcgg ggatattta




SEQUENCE: 66

ttcacggttc cqgacaat at




SEQUENCE: 67

cggttc.cgga caat attcg




SEQUENCE: 68

c cct tcacgg titc.cggacaa




SEQUENCE: 69

ccacgatcaa act ctdtcga

SEO ID NO 7 O LENGTH: 2O TYPE: DNA

36

- Continued

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1



<4 OO > SEQUENCE: 7 O

aaac cct tca cqgttc.cgga





titc.cggacaa tatt cqgitat





ccgaaaccct t cacggttcc





tagt ctdcca gtc.t.ctittgg c





gCacctgtgt gatggc.ccga t





ctictagt ctd c cagt ct citt t




37

- Continued

21

21

21

Jun. 9, 2011

US 2011/O 1361.25 A1


gcagcacctg ttgatggcc C





Cctgttgttgat ggc.ccgattt





tctatt cqgt attgcc cc cc c





cc cctitt.cgg titc cct actic g

<210s, SEQ ID NO 8O &211s LENGTH: 21 &212s. TYPE: DNA




t ccct citc cc agcctic tagt c





tcqgggat at ttatttgat





Cataccttgg gcggct Coct

38

- Continued

21

21

21

21

19

Jun. 9, 2011

US 2011/O 1361.25 A1




SEQUENCE: 83

cago ct ctag totgc.cagt




SEQUENCE: 84

cgaaggcggit gcactggcc




SEQUENCE: 85

gtgg.cgalagg C9gtgcact




SEQUENCE: 86

aggtgtc.gcg ggggtccacc




SEQUENCE: 87

tgtctgtcgg gacgaggac




SEQUENCE: 88

gagggcagga gaggtgcat


39

- Continued

19

19

19

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1








Caccitcgcgg to cqgagc





gggggtCCaC Ctcgcggtgc





Ctcgcggtgc cqgagctaa





tgtc.gcgggg gtccacct c





ggattacgagg titcgcggg




40

- Continued

19

19

19

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1

SEQUENCE: 95

cggagctaac gcacticagt




SEQUENCE: 96

gtaaacgatg gatacgaggit




SEQUENCE: 97

tgagtggggg at atcgggc




SEQUENCE: 98




SEQUENCE: 99

agctaacgca ct cagtatic




SEQUENCE: 1.OO

acgatggata caggtgtcg




SEQUENCE: 101

gtgc.cggagc taacgcactic

41

- Continued

19

19

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1




SEQUENCE: 1 O2

aggtgcatgg aatticctggit




SEQUENCE: 103

tgcatggaat tcc tigtgga




SEQUENCE: 104

cagtgcaccg cct tcgc.ca




SEQUENCE: 105

ggc.cagtgca ccgc.ct tcg




SEQUENCE: 106

ggtggaccCC cqcgacacc




SEQUENCE: 107

ggtc.ct citc cc.gacagac


42

- Continued

19

19

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1




catgcacctic ticctg.ccct c





ttagct Cogg Caccgc.gagg





gcgaggtgga CCC cc.gcga





tgcaccgcct tcgccaccg





cgitatic catc gtttacggcg





gaccc.ccg.cg acacct cqta




43

- Continued

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1

SEQUENCE: 114

gagtgcgitta gct Coggcac




SEQUENCE: 115

tccaccagga attic catgc




SEQUENCE: 116

gcc aggc.cag to accgc.c




SEQUENCE: 117

c caggaattic catgcacctic




SEQUENCE: 118

cct cqtat co atcgtttacg




SEQUENCE: 119

actgagtgcg ttagct Cogg




SEQUENCE: 12O

gatactgagt gcgittagctic

44

- Continued

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1





gccacacctic gitat coat cq





cgggat actg agtgcgittag





gcc.cgatat c ccc cactica

















45

- Continued

19

19

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1

































46

- Continued

19

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1

































47

- Continued

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1
































48

- Continued

19

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1

































49

- Continued

19

19

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1

































50

- Continued

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1






























gggagacgaa alaggtaatcg


51

- Continued

19

Jun. 9, 2011

US 2011/O 1361.25 A1




aaagttctitt C9gtgacggg





cggggalaggt tatatgtta





gagggagalaa CC9ggggat





aatcgctaat atcggittac





ccgggggatc titcggacctic





taatat cqcc togctgttgac




52

- Continued

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1


tCggtgacgg galaggttg





ggagaalaccg ggggat Ctt





acgt.cctgag ggagaalaccg





agacgaaaag gtaatcgcta





gtgacgggga aggttgata





gaaaccgggg gat ctitcgg





tCctgaggga gaalaccgggg

53

- Continued

19

19

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1




SEQUENCE: 178

cgaaaaggta atcgctaata




SEQUENCE: 179

aaaggtaatc gctaatat cq

SEQ ID NO 18O LENGTH: 2O TYPE: DNA



SEQUENCE: 180

tcgtgggaga caaaaggta




SEQUENCE: 181

cggacct cqt gct attggag




SEQUENCE: 182

gttctttcgg tacgggga




SEQUENCE: 183

Cttt C9gtga C9gggalagg


54

- Continued

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1




atc.ccc.cggit ttcticcict c





at attagciga ttacct titt





caacct tccc cqt caccgaa





ccgaagat.co. cccggtttct





CtcCaatagc acgaggtocg





accoat atta gcgattacct




55

- Continued

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1


aagat.ccc cc ggtttcticc





tat caacct t c ccc.gt cacc





ggtttctic cc ticaggacgta





ggtc.cgaaga t ccc.ccggitt





titt cacgaca gacctaatg





gtaaccata ttagcgatta





acatat caac ct tcc.ccgtc

56

- Continued

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1





cc.cggtttct c cct caggac





gcqattacct titt cqt ct co





cc.ccgt cacc gaaagaactt

<210s, SEQ ID NO 2 OO &211s LENGTH: 2O &212s. TYPE: DNA



<4 OOs, SEQUENCE: 2OO

ttalacatlatc. aac Ctt CCCC

<210s, SEQ ID NO 2 O1 &211s LENGTH: 2O &212s. TYPE: DNA



<4 OOs, SEQUENCE: 2O1

ttagcgatta ccttitt cqtc





citt.ccc.cgt.c accogaaagaa


57

- Continued

Jun. 9, 2011

US 2011/O 1361.25 A1




attaccttitt cqt citcc.cac





gggaaa.ccgt gagggcgct





gcgaaacgtc. cccaatgcgg





cc.gcagggaa accggtaagc C





cc.cgggaaag ggcagtgata





gggaaagggc agtgat act




58

- Continued

19

21

19

Jun. 9, 2011

US 2011/O 1361.25 A1


aatc.cggggg agg.cgaaggg





aggg tactgg aacgt.ccctt





aagcgt.ccgg C cagaacg.cg C





cgc.ctaaagg ggcatgggct





ggct attt co cqct catgcc





cgitacgcc ct cqggtaagag g





aacggc.ccgc caaaccgata

59

- Continued

21

21

Jun. 9, 2011

US 2011/O 1361.25 A1





agc.cggcc ct gcaagt cac





Cactgcttaa agaccc.ggg





ggagctaatc C9gggCaggc g





aalaccgtgag ggcgct accc





aggcgaaggg tactggaacg t





acccc.cagtg Ctc.ccgaaag


60

- Continued

19

19

21

21

Jun. 9, 2011

US 2011/O 1361.25 A1




CCCttcgc.ct aaaggggcat g





gcatgggcta titt cocqctic a





gggaaa.ccgt gagggcgct





gcgaaacgtc. cccaatgcgg





cc.gcattggg gacgtttcgc g





gcgc.cct cac ggttt ccc.gc a




61

- Continued

21

21

19

21

21

Jun. 9, 2011

US 2011/O 1361.25 A1

SEQUENCE: 228

cc.gcattggg gacgtttcgc g




SEQUENCE: 229

gcgc.cct cac ggttt ccc.gc a




SEQUENCE: 23 O

titc.ccgcatt ggggacgttt C




SEQUENCE: 231

tagcgc cct c acggtttccc




SEQUENCE: 232

ggct taccgg ttt Coctg.cg




SEQUENCE: 233

ctg.ccc.tttic ccgggttga




SEQUENCE: 234

t cactg.ccct titc.ccgggit

62

- Continued

21

21

21

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1




SEQUENCE: 235

gtat cactgc cctitt.ccc.g




SEQUENCE: 236

gcc.cgggit ct ttaa.gcagtg




SEQUENCE: 237

citcc.cgcc cc ctagocct go a




SEQUENCE: 238

cc.cgggat.ct gtggattt cq c




SEQUENCE: 239

taccc.gaggg C9tacgact




SEQUENCE: 24 O

Cctictt accc gagggcgtac g


63

- Continued

19

21

21

19

21

Jun. 9, 2011

US 2011/O 1361.25 A1




titcgc.ctgcc ccggattag





ggcggCaggc titaccggittt C





cggattagct c cagtttic cc g





ggacgttcca gtacccttic





cc.ccggatta gct coagttt





tacic ctitcgc ctdcc.ccgga t




64

- Continued

19

21

21

19

21

Jun. 9, 2011

US 2011/O 1361.25 A1


c catgc.ccct ttagg.cgaa





agagt caa.cc tacgagctt a





gtcaacct ga cdagct tact c





tgagagt caa cctgacgagc





gagott actic gataggagga g





tittaatticga gagggittaa





Cttact cat agcaggagag g

65

- Continued

19

21

21

21

19

21

Jun. 9, 2011

US 2011/O 1361.25 A1




SEQUENCE: 254

aatcaaatct gatgtcggtg a




SEQUENCE: 255

ggittaaatca aatctgatg




SEQUENCE: 256

titcgagaggg ttaaatcaaa t




SEQUENCE: 257

caaatctgat gtcggtgagg a




SEQUENCE: 258

taaatcaaat citgatgtcg




SEQUENCE: 259

gagagggitta aatcaaatct g


66

- Continued

21

19

21

21

19

21

Jun. 9, 2011

US 2011/O 1361.25 A1




atctgatgtc. g.gtgaggaggg





aatticgagag ggittaaatc





gatgtcggtg aggagggitt





gagggatggc agtgtcgga





tggccaagac ttittct cat





gatgagtctg caacctatica




67

- Continued

21

19

19

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1


tagcagagag gtggtgcatgg





acggcc actg citat caagtt c





aagctcgt.ca ggttgact ct





gtaa.gctcgt caggttgac





cgagtaagct cqt caggtt





Ctgctatcga gtaagctcg





titta accctic ticgaattaa

68

- Continued

21

21

19

19

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1





citcc togctat cqagtaagc





t cagatttga tittaac cct c




< 400 SEQUENCE: 275

accctic ct ca ccgacat cag





acat cagatt tdatttaac





ccga catcag atttgattit





tgatttaa.cc ct citcgaat


69

- Continued

19

19

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1




t caccgacat cagatttga





atttgattta accct citcg





Ctacctgata ggttgcagac t





gcaccacctic tictogctat cq





atcc ct caac ggaaaag.ca





acacttaaag togaacgcc ct




70

- Continued

19

19

21

19

Jun. 9, 2011

US 2011/O 1361.25 A1


tcqctic cqac act gccatc





ccgatctgat gtc.ttgcagt





atgagaaaag ticttggc.ca





agggcgttac C cctagtgc





tacic cc tagt gcc ct cqca





gcgc.ccgtag ccggcctgta a





gaggttct Co. tcc.gcgagggg

71

- Continued

19

19

19

19

21

21

Jun. 9, 2011

US 2011/O 1361.25 A1




SEQUENCE: 292

gcaccaggcg cggaacgt.cc C




SEQUENCE: 293

gaggtogagc titct cotc.cg




SEQUENCE: 294

c cct agtgcc ct cqcaaga




SEQUENCE: 295

cc.cgtagc.cg gcctgtaaag t




SEQUENCE: 296

cggggtggga gatcgagctt C




SEQUENCE: 297

gtcgagct tc. tcct cogcga


72

- Continued

21

19

21

21

Jun. 9, 2011

US 2011/O 1361.25 A1




ggtgggagglt c.gagct tctic C





tCggggtggg aggtoga.gc

<210s, SEQ ID NO 3 OO &211s LENGTH: 19 &212s. TYPE: DNA



<4 OOs, SEQUENCE: 3 OO

gcqttacc cc tagtgc cct

<210s, SEQ ID NO 3 O1 &211s LENGTH: 21 &212s. TYPE: DNA




tagggg tagg gctaagc.cat g





cgcaccaggc gcggaacgt.





gggaggtoga gct tct cot




73

- Continued

21

19

19

21

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1


aggtggagga ataagcgggg





gaaaggtgga ggaataagc





gggagt cqta cqct ct cqgg a




<4 OO > SEQUENCE: 3 O7

ctaacctgcc cittgggat.ct g





ggcact aggg gtaacgc.cc





agaa.gct cqa cct cocaccc





tacaggc.cgg ctacgggcgc

74

- Continued

19

21

21

19

Jun. 9, 2011

US 2011/O 1361.25 A1




SEQUENCE: 311

agct cq acct c ccaccc.cg




SEQUENCE: 312

CCCCtcgcgg aggaga agc




SEQUENCE: 313

tgcgagggca Ctaggggta




SEQUENCE: 314

tgactttaca ggc.cggctac g




SEQUENCE: 315

catggct tag ccctaccc ct a




SEQUENCE: 316

aggaga agct cacct coca


75

- Continued

19

19

19

21

21

Jun. 9, 2011

US 2011/O 1361.25 A1




gacgttcc.gc gcc tdgtgc





Ctttac aggc cqgctacggg





tcttgcgagg gCactaggg




<4 OOs, SEQUENCE: 32O

cggaggagaa gct cacctic





tcgcggagga gaagct cac





gagggc act a giggtaacg




76

- Continued

19

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1

SEQUENCE: 323

accc.cgaggg gcaa.gaggcc




SEQUENCE: 324

ggggittatcc agat.cccaag g




SEQUENCE: 325

gccacgcc ct citt cocq aga




SEQUENCE: 326

gttatccaga t cc caagggc




SEQUENCE: 327

cittatt cotc. cacctittctg g




SEQUENCE: 328

taalacc ctgc cqcagttgg




SEQUENCE: 329

ccittaaac cc togcc.gcagt

77

- Continued

21

21

19

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gtcCtggaac ggttcCtcg





Ctct acaaag gcgggggaat a





Ctggaacggit to Ctcgctga





ggcgaggagt CCtggaacgg t





titt.ccc.cgct c tacaaagg





tacaaaggcg ggggaataag C


78

- Continued

19

21

21

19

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Jun. 9, 2011

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cgct ct acaa aggcggggg





at aggcgagg agt cctggaa





c cat aggcga ggagtic ctg





gcttitt cocc gct ctacaa





gcta acctac Cctgaggagg





tctic ccatag gcgaggagtic




79

- Continued

19

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1


tggcta acct accCtgagg





ataatcto cc at aggcgag





tgaggaggga gata accc.cg





acacgtggct aacct accct g





Cctgaggagg gagatalacc





aaactgggga taatct coc





cCaactg.cgg Cagggittta

80

- Continued

19

19

21

19

19

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Jun. 9, 2011

US 2011/O 1361.25 A1





actg.cggcag ggtttalagg





cgagga accg titcCaggact c





aaccottcca ggactic ct cq





tccaggactic ct cqcctatgg





CCtttgtaga gcggggaaa

<210s, SEQ ID NO 3.54 &211s LENGTH: 2O &212s. TYPE: DNA




agcgaggaac C9ttcCagga


81

- Continued

19

21

21

19

Jun. 9, 2011

US 2011/O 1361.25 A1




cgttccagga citcct cqcct a





CCCCCCCtt ttagagcg





ttcagcgagg aaccgttc.ca





attic cc cc.gc citttgtaga





ttgtagagcg gggaaaagc





atct coct co toaggg tagg t

<210s, SEQ ID NO 3 61 &211s LENGTH: 19 &212s. TYPE: DNA



82

- Continued

21

19

19

19

21

Jun. 9, 2011

US 2011/O 1361.25 A1

SEQUENCE: 361

gggittatcto cotcct cag




SEQUENCE: 362

tcgc.ctatgg gagattatic




SEQUENCE: 363

t caggg tagg ttagcc acgt.




SEQUENCE: 364

Ccticaggg taggittagc.ca




SEQUENCE: 365

ccggggittat citc cct cot




SEQUENCE: 366

t cct cqccta tiggagatt




SEQUENCE: 367

Cctic ct cagg gtaggittag

83

- Continued

19

19

19

19

19

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Jun. 9, 2011

US 2011/O 1361.25 A1




SEQUENCE: 368

t cctgaaagg acgaccggtg




SEQUENCE: 369

ggactgaggg Ctgtaactic

SEO ID NO 37O LENGTH: 21 TYPE: DNA



SEQUENCE: 370

gaggttgaat gtactitt Cagg




SEQUENCE: 371

ggtggcgaaa gogttcaact




SEQUENCE: 372

gccCtcacga atgtggatt




SEQUENCE: 373

acct cqaaac ccgttcgtag


84

- Continued

19

21

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Jun. 9, 2011

US 2011/O 1361.25 A1




tcc.gtag taa ticgtaggit c





atcc td taat cotgaaagga c





gtag to agga citgagggctg





aggacga.ccg gtggcgaaag C





taactic gocc ticacgaatgt





gaaggtgtta agtgggtca

<210s, SEQ ID NO 38O &211s LENGTH: 21 &212s. TYPE: DNA



85

- Continued

19

21

21

19

Jun. 9, 2011

US 2011/O 1361.25 A1

SEQUENCE: 38O

aalaccc.gttc gtagt cagga C




SEQUENCE: 381

tacggtgaat atgc.ccctgc




SEQUENCE: 382

cacttggtgt togcttctic cq t




SEQUENCE: 383

gat cacttitt attgagtict




SEQUENCE: 384

agcatcagga ataagggctg




SEQUENCE: 385

aaga cc cc catctictaatt




SEQUENCE: 386

ccggtott at aaatcttca

86

- Continued

21

21

19

19

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Jun. 9, 2011

US 2011/O 1361.25 A1




SEQUENCE: 387

ataacgagca agaccc cc at




SEQUENCE: 388

Caggggcata t t caccgtag




SEQUENCE: 389

t caggattac aggattitta




SEQUENCE: 390

accctgaaag tacattcaac c




SEQUENCE: 391

gccaccggtc gtc.ctitt.ca




SEQUENCE: 392

c tagttgaac gctitt.cgc.c


87

- Continued

19

21

19

19

Jun. 9, 2011

US 2011/O 1361.25 A1




tcqtcc titt.c aggattacag g





acgctitt.cgc caccggtcgt c





gggttt cag gttagctt C





c cct cagt cc tdactacga





Ctgaagattt atalagaccgg





ttacagcc ct cagtcc togac t




88

- Continued

21

21

19

19

21

Jun. 9, 2011

US 2011/O 1361.25 A1


aatcCacatt C9tgagggcg a

<210s, SEQ ID NO 4 OO &211s LENGTH: 2O &212s. TYPE: DNA



<4 OOs, SEQUENCE: 4 OO

atgggggtct tctcgtt at

<210s, SEQ ID NO 4O1 &211s LENGTH: 19 &212s. TYPE: DNA



<4 OOs, SEQUENCE: 4 O1

gctgttgacc tacgattac





ccitacgatta citacggaatc c





acccacttaa caccitt cqc





cc caagttctt acagt ct citt





ctaccctgaaagtacattca

89

- Continued

21

19

21

19

Jun. 9, 2011

US 2011/O 1361.25 A1 Jun. 9, 2011 90

- Continued

<210s, SEQ ID NO 4O6 &211s LENGTH: 19 &212s. TYPE: DNA




cago cott at tcc tdatgc 19

<210s, SEQ ID NO 4O7 &211s LENGTH: 2O &212s. TYPE: DNA




ggtcgt.cct t t caggattac 2O





gtgc.ca.gcmg cc.gcggta 18





Cctacgggag gcagoag 17





cc.gt caattic ctittgagtt 19





gctgcctic cc gtaggagt 18


US 2011/O 1361.25 A1




gggcggWgtg tacaaggc

<210s, SEQ ID NO 413 &211s LENGTH: 2O

&212s. TYPE: DNA




acgggg.cgca gCaggcgcga

<210s, SEQ ID NO 414 &211s LENGTH: 19

&212s. TYPE: DNA




gtgc.ca.gcag cc.gcggtaa


&212s. TYPE: DNA




yccgg.cgttg amt coaatt

<210s, SEQ ID NO 416 &211s LENGTH: 2O

&212s. TYPE: DNA




gtgctic cc cc gccaattic ct


&212s. TYPE: DNA




attaccgcgg Ctgctgg

91

- Continued

18

19

19

17

Jun. 9, 2011

US 2011/O 1361.25 A1

What is claimed is: 1. Method to identify and quantify environmental micro

organisms useful in biomining processes, wherein said method comprises the steps of

(a) extracting DNA from a sample: (b) quantifying the extracted DNA; (c) performing a quantitative PCR (qPCR) technique,

using either said DNA sample, and specific primers of SEQ ID No. 4 to SEQID No. 407 for each taxon to be determined, where taxons are selected from: i. Bacteria: Total bacteria, Acidiphilium sp., Leptospir

illum sp., Sulfobacillus sp., Acidithiobacillus fer rooxidans and Acidithiobacillus thiooxidans, and

ii. Archaea: Total archaea, Acidianus sp., Ferroplasma sp, Metallosphaera sp., Sulfolobus sp. and Thermo plasma sp.,

(d) calculating the number of microorganisms in the sample that belong to each of the analyzed taxons according to formula (3)

92 Jun. 9, 2011

N QN molecules *Tng (3) Ceit (mL)-(g) Cmolecules/cel * Ung * Cn(mL)-(g)

wherein:

N=Estimated Concentration of microorganisms. Q=Number of Molecules as calculated with the standard

curve using the C(T) value given by the equipment. T=Total DNA isolated from original sample. U=Amount of DNA used in the qPCR reaction. C=Number of molecules or copies by genome of the spe

cific DNA fragment detected. Cm=Amount in ''g'' or “ml of the processed sample.

c c c c c

(19) united states (12) patent application publication (10) · pdf file ·...

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