Capsules of viable biomining microorganisms, with alginate and iron ions called biosigma bioleaching seeds (BBS) and their use for inoculation of these microorganisms in bioleaching processes

Abstract

The invention refers to viable biomining microorganisms encapsulated in alginate capsules, called BioSigma Bioleaching Seeds or BBS, wherein the alginate capsules have iron (II) and/or iron (III) ions as the cross-linking cations, and the usage of these capsules in the inoculation of these microorganisms in bioleaching processes.

Claims

1. A method of increasing bioleaching rate in a heap or dump under toxic conditions for microorganisms, wherein the method comprises the following steps: a) providing BioSigma Bioleaching Seeds (BBS) capsules that comprise a consortium of viable microorganisms in a matrix of alginate and iron ions, whether Fe(II) or Fe (III) or a mixture thereof and used as cross-linking cations, wherein the BBS capsules comprise a mixture of a solution of sodium alginate at a concentration of 0.2% to 3% with a suspension of microorganisms in a ratio of 4:1 to 1:4 by volume, and being the BBS capsules in a spherification solution that comprises Fe(II) or Fe(III) ions, or a mixture thereof; and wherein the consortium of microorganisms is present inside the BBS capsules in concentrations above 10.sup.3 microorganisms/mL of the solution of sodium alginate; and wherein the consortium of microorganisms is constituted by at least 1.0010.sup.3 microorganisms/mL of Acidithiobacillus ferrooxidans Wenelen DSM 16786, at least 9.7410.sup.6 microorganisms/mL of Acidithiobacillus thiooxidans, at least 1.0010.sup.3 microorganisms/mL of Acidiphilium spp., at least 1.0010.sup.3 microorganisms/mL of Leptospirillum spp., and at least 3.4210.sup.6 microorganisms/mL of Ferroplasma spp.; b) inoculating an ore in a bioleaching heap or dump with a proportion of BBS capsules between 0.01% and 99% by dry weight per gram of ore to form a homogeneous mixture of inoculum and ore, and then stockpiling the heap or dump after the inoculation is performed; and c) irrigating the stockpiled heap or dump with an irrigation solution comprising water and raffinate, wherein the raffinate is formed by Cu, Fe (III), Fe(II), SO.sub.4-2, Cl and heavy metals.

2. The method of claim 1, wherein the spherification solution comprises a total iron concentration of 0.1-30 g/L and a composition of 10-40% Fe (II) and 60-90% Fe(III).

3. The method of claim 1, wherein nutrients, additives or preservatives are added to the spherification solution prior to the generation of BBS capsules.

4. The method of claim 2, wherein nutrients, additives or preservatives are added to the spherification solution prior to the generation of BBS capsules.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1Acid resistance of BBS at different concentrations of sulfuric acid. BBS maintain their integrity at concentrations of up to 25% of sulfuric acid, which corresponds to of pH of 0.46. On the other hand, at concentrations of 50% of sulfuric acid, at pH of 1.16, the BBS are disintegrated.

(2) FIG. 2. Scanning electron microscopy of a complete BBS with a magnification of 45 (the lower bar corresponds to 1 mm).

(3) FIG. 3. Scanning electron microscopy of the surface of a BBS with a magnification of 3000 (the lower bar corresponds to 10 m). Microorganisms colonizing the surface of the BBS can be observed.

(4) FIG. 4. Light microscopy of a transversal cut of a stained BBS, with a magnification of 400. The microorganisms are encapsulated within the BBS and can colonize lumens that are part of their structure.

(5) FIG. 5. Fluorescence microscopy of a transversal cut of a BBS stained with LIVE/DEAD BacLight kit from Life technologies. The stain dyes live cells in green and dead cells in red. In this figure no red dyed cells were found, and all the marks correspond to live cells.

(6) FIG. 6. Chart representing in black diamonds the concentration of Acidithiobacillus thiooxidans (At.t) in the supernatants of cultures inoculated with 1.010.sup.8 microorganisms/mL of an inoculum of A. thiooxidans versus the open squares representing an equivalent concentration of encapsulated A. thiooxidans in BBS, and their growth behavior on time.

(7) FIG. 7. Incorporation of BBS in a column of mineral to be bioleached.

(8) FIG. 8. Comparison of the bioleaching activity in different inoculation conditions measured electrochemically by the redox potential (Eh, mV Ag/AgCl). Column 1 inoculated by irrigation with 1.010.sup.6 microorganisms/g of mineral, column 2 inoculated 1.010.sup.6 microorganisms/g of mineral in 5 ml of BBS (1%), Column 3 inoculated with 1.010.sup.6 microorganisms/g of mineral in 10 ml of BBS (2%), Column 4 inoculated with 1.010.sup.6 microorganisms/g of mineral in 20 ml of BBS (4%). Bioleaching activity is higher in the columns inoculated with BBS than in the column inoculated by irrigation. The inoculation with 1% of BBS in mineral shows the best performance.

(9) FIG. 9. Comparison of bioleaching activity in different inoculation conditions in the presence of raffinate: column C02, inoculated by irrigation with 1.010.sup.6 micro organisms/g of mineral, column C04 inoculated with 1.010.sup.6 microorganisms/g of mineral in 1% of BBS. Bioleaching activity starts 20 days earlier in the column inoculated with BBS when compared with to that the one inoculated by irrigation.

DESCRIPTION OF THE INVENTION

(10) The present invention describes viable biomining microorganisms encapsulated in alginate capsules, called BioSigma Bioleaching Seeds or BBS, where such capsules comprise a matrix of alginate and iron cations, either Fe(II) or Fe(III), which contain the biomining microorganisms. The pH of these capsules is between 1 and 2, according to the acidophilic conditions necessary for the biomining microorganisms. It has been found that the Fe(II), in spite of being in the cross-linking agent of alginate, is bioavailable and acts as substrate for encapsulated biomining iron oxidizing microorganisms. One advantage of these capsules is that they allow the microorganism inoculation to the mineral to be bioleached. The capsules facilitate the microorganisms handling and they preserve their integrity during transportation and inoculation, protecting the microorganisms from toxics present in the process, as well as extremely acid environments. Another advantage of the BBS is related to the storage of the microorganisms, since we have found that the microorganisms remain viable for years, in the same proportions they were encapsulated and without contamination. All the above represents a clear operational advantage on the existing methods of microorganisms inoculation, where the solutions that comprise the inoculum must be transported and incorporated.

(11) Within the bioleaching processes, a key step is the inoculation of the mineral to be bioleached with biomining microorganisms. Notwithstanding its importance, there are several problems associated to this step, for example, how to achieve the homogeneous inoculation in the entire mineral to be bioleached. Moreover, the toxins present in the process, either in the mineral or in the acidifying solutions (as raffinate or highly acid solutions), can affect the cellular viability, the generation of microorganisms consortia, which are made according to the mineral to be bioleached, and the handling of the microorganisms to be inoculated in the biomining operation.

(12) The traditional inoculation process of a bioleaching heap consists in irrigating the heap with sulfuric acid or other acid solution available in the operation and subsequently irrigate the heap with an inoculation solution which contains the biomining microorganisms. In this process the microorganisms are directly exposed to the extremely acid medium, with pH values under 1, and to the toxic chemicals which are either in the mineral to be bioleached or in the solutions used to irrigate the mineral, for example raffinate, which affects cellular viability and decimates the inoculated bacteria.

(13) Likewise, this method of inoculating microorganisms through irrigation has the disadvantage that it does not ensure homogeneous inoculation of the entire mineral. The permeability in the heap is not homogeneous because the minerals that form it have an irregular conformation. This makes the natural flow of the inoculation solution produce pseudo-channels in the inner part of the heap, creating sectors where the inoculation is successful and others within the same heap, where the inoculating solution cannot not reach.

(14) It is common that the microorganisms that are the most adequate to bioleach a mineral in particular, are actually a consortium of different specific microorganisms. These consortia can be grown in laboratory, but the desired ratio of each microorganisms in the inoculation solution is not always as expected, as the growth of some microorganism species often prevails over others.

(15) Finally the conventional inoculation has problems in the handling of the inoculation solution, which is liquid, and it must be generated in reactors within the same operation and transported by pipes or hoses to be able to inoculate the mineral.

(16) All the problems mentioned above are solved by this invention since the inventors have demonstrated that the invention capsules, BioSigma Bioleaching Seeds or BBS, protect the microorganisms from both the extreme acidity and the toxins.

(17) Moreover, as BBS are solid particles, they can be mixed with the mineral before stockpiling the heap, ensuring a homogeneous inoculation in the entire mineral, which increases the efficiency of the inoculation process and therefore the efficiency of bioleaching.

(18) The capsules of the invention, BioSigma Bioleaching Seeds may contain any microorganism or groups of microorganisms encapsulated at different ratios, as desired. They allow obtaining the consortia of specific encapsulated microorganisms to be able to homogeneously inoculate minerals that also have specific characteristics.

(19) Finally, as they are solid capsules they do not necessarily need to be generated in the same working area where the bioleaching is being done, and the problems associated to the transportation of the microorganisms to be inoculated are reduced.

(20) Additionally, the BBS can also contain, apart from the microorganism nutrients, additives or preservatives that permit an enhanced microbial activity.

(21) The BBS allow the encapsulation of any kind of biomining microorganism, either as a single cell cultures or in microorganism consortia, as the microorganism consortium being understood as both a consortium that is stable over time and also the simple operational combination of two or more single cell cultures obtained traditionally. Within the biomining microorganisms are: Acidiphilium spp., Leptospirillum spp., Sulfobacillus spp., Acidithiobacillus spp, specifically Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans; Acidianus spp., Ferroplasma spp., Metallosphaera spp., Sulfolobus spp. and Thermoplasma spp. The BBS allow obtaining concentrations of encapsulated biomining microorganisms of over 10.sup.3 microorganisms/g of capsules, specially over 10.sup.5 microorganisms/g of capsules, and preferentially over 10.sup.7 microorganisms/g of capsules.

(22) The method to generate BioSigma Bioleaching Seeds or BBS starts with obtaining a culture of biomining microorganisms, either a single cell culture or a consortium, in concentrations of over 10.sup.3 microorganisms/mL, especially over 10.sup.5 microorganisms/mL and preferentially over 10.sup.7 microorganisms/mL. The cultured cells are added by filtration or centrifugation to the solution of alginate in the desired concentration that can be over 10.sup.3 microorganisms/mL of solution to be encapsulated, especially over 10.sup.5 microorganisms/mL of solution to be encapsulated and preferentially over 10.sup.7 microorganisms/mL of solution to be encapsulated. The alginate concentration is between 0.2% and 3%, preferentially at 1.5% to form a solution of microorganisms and alginate, with a final alginate concentration between 0.2% and 3%, preferentially at 1%. The ratio between the alginate solution and the solution of biomining microorganisms to be encapsulated can vary between 4:1 and 1:4 in volume. Optionally nutrients, additives and preservatives can be added, such as metabolites, proteins, organic molecules and/or inorganic molecules and elements, etc.

(23) This mixture of microorganisms, alginate and optionally nutrients, additives or preservatives forms the solution that will be encapsulated. The solution to be encapsulated is added drop by drop and under smooth agitation to a solution of Fe(II) and/or Fe(III), which constitutes the spherification agent. The capsules are formed by contact of the solution to be encapsulated with the spherification agent, obtaining the BioSigma Bioleaching Seeds (BBS).

(24) The spherification solution comprises either Fe(II) and/or Fe(III) sulfate in concentrations from 0.1 g/L to 30 g/L. The combination where ferric sulphate represents 60 to 90% and ferrous sulphate represents between 40 and 10% relative in the solution is preferred.

(25) When the capsules comprise Fe (II) as the cross-linking cation, either alone or in combination with Fe (III), the Fe (II) can also act as substrate for the iron oxidizing microorganisms, such as Acidithiobacillus ferrooxidans, Leptospirillum spp., Sulfobacillus spp. and Ferroplasma spp. encapsulated in the BBS.

(26) The inventors have discovered that when encapsulating a complex consortium of microorganisms the ratio of the microorganism species within the BBS is similar to the ratio in the inoculum (see example 1) and therefore it is considered that the encapsulation is not selective for any type of microorganism in particular.

(27) As the inoculation of biomining microorganisms in the bioleaching process occurs in extremely acidic environments, the resistance of BBS was proven by exposing them directly to sulphuric acid. The results are shown in FIG. 1. It is observed that the BBS maintain their integrity when up to 25% of sulphuric acid is added, which corresponds to a pH of 0.46. On the other hand, the BBS are disintegrated when exposed to concentrations of 50% of sulphuric acid at a pH of 1.16. As the microorganisms without any protection can resist a pH near 1, the BBS can grant an additional protection for a pH of up to 0.46.

(28) The BioSigma Bioleaching Seeds (BBS) were studied by scanning electron, transmission and light microscopy techniques, and it was determined that the matrix of alginate and iron permit the proliferation, the traffic and adherence of the microorganisms. FIG. 2 shows an entire BBS with the microorganism Wenelen DSM 16786, an Acidithiobacillus ferrooxidans strain, property of BioSigma SA, where it is observed that the BBS presents ridges on its structure. FIG. 3 shows that there is colonization of the microorganisms over the surface of the BBS, which implies that the surface of the BBS allow adherence of the biomining microorganisms. Likewise, FIG. 4 shows a transversal cut of a BBS where the encapsulation of microorganisms within the BBS can be observed. The encapsulated microorganisms would be presented on both: the matrix of alginate and in interstices with microclimates isolated from the outdoor environmental conditions.

(29) When using the BBS to inoculate mineral, these can be used in a ratio between 0.01%-99% in weight per gram of mineral, preferentially in a ratio between 0.01 to 50% and in especial conditions in a ratio of between 0.01 to 10%.

(30) The BBS can be used to inoculate any media or system that requires the presence of biomining microorganisms, such as a bioleaching heap, a bioleaching reactor, a reactor of biomass generation, a flask, a bioleaching column, or other. An important additional advantage is that the BBS can be stored for a long time (years), without deteriorating the microorganisms inside it and without losing their activity or being contaminated by other species apart from the BBS.

(31) The medium or system inoculated with BBS operates in the usual way that is to say in the same way in which an inoculated system would operate in the traditional way with liquid cultures of biomining microorganisms.

EXAMPLES

Example 1. Encapsulation of a Consortium of Microorganisms

(32) A consortium of microorganisms constituted by Acidithiobacillus thiooxidans, Leptospirillum spp., Acidiphilium spp., Ferroplasma spp. and Wenelen DSM 16786 (strain of Acidithiobacillus ferrooxidans property of BioSigma SA.) was obtained. The concentrations of these microorganisms in the inoculum used were determined by qPCR as shown on Table 1.

(33) The cells were recollected by filtration and were washed twice in media KMD ((NH.sub.4).sub.2SO.sub.4, 247.5 mg/L; NaH.sub.2PO.sub.4.H.sub.2O, 36.5 mg/L; KH.sub.2PO.sub.4, 13.125 mg/L; MgSO.sub.4.7H.sub.2O, 25 mg/L; CaCl.sub.2, 5.25 mg/L) and were finally resuspend in 4 ml of KMD medium. The 4 ml containing the consortium were mixed with 6 mL of sodium alginate at 1.5% to form the solution to be encapsulated. The resulting solution was added drop by drop from a burette to a solution of 9 g/L of total iron with a proportion of 60% of ferric ions (Fe(III)) and 40% of ferrous ions (Fe(II)), applying slight agitation in a magnetic agitator and thus forming the BBS.

(34) To determine the concentration of microorganisms in the BBS an aliquot was taken from the BBS formed, which was dissolved in a solution of ethanol 70%. DNA was extracted and the concentration of each one of the microorganisms present was determined by qPCR. The results are shown on Table 1.

(35) TABLE-US-00001 TABLE 1 Inoculum BBS Microorganism (microorganisms/mL) (microorganisms/mL) Total Bacteria 3.54 10.sup.8 7.77 10.sup.7 A. ferrooxidans 1.00 10.sup.3> 1.00 10.sup.3> A. thiooxidans 5.99 10.sup.7 9.74 10.sup.6 Leptospirillum spp. 7.93 10.sup.5 1.00 10.sup.3> Acidiphilium spp. 4.91 10.sup.4 1.00 10.sup.3> Ferroplasma spp. 2.15 10.sup.7 3.42 10.sup.6 Wenelen DSM 16786 1.00 10.sup.3> 1.00 10.sup.3>

(36) The results show that the encapsulation is not selective for any specific type of microorganisms as the proportion of each specie is similar to that of the inoculum to be encapsulated. The results indicate that the BBS formed from a microbiological consortium are representative of such consortium. The minor differences are explained by the lower efficiency in DNA extraction from the BBS.

Example 2. Cell Activity in the BBS

(37) Some of the BBS formed in example 1 were stained with the LIVE/DEAD BacLight kit from Life Technologies, according to the instructions of the manufacturer. This kit dyes the live cells green and the dead cells red. In this case, no red cells were found. FIG. 5 shows the active cells inside the BBS, leading to the conclusion that the encapsulated microorganisms in the BBS continue to be active.

Example 3. Cell Viability

(38) To determine if the BBS are useful in the inoculation of microorganisms, the concentration of microorganisms was compared in the culture media of 1% S KMD ((NH.sub.4).sub.2SO.sub.4, 247.5 mg/L; NaH.sub.2PO.sub.4.H.sub.2O, 36.5 mg/L; KH.sub.2PO.sub.4, 13.125 mg/L; MgSO.sub.4.7H.sub.2O, 25 mg/L; CaCl.sub.2, 5.25 mg/L) inoculated directly with Acidithiobacillus thiooxidans or in the BBS encapsulated with Acidithiobacillus thiooxidans.

(39) In a flask containing 500 mL of 1% S KMD culture medium 110.sup.8 microorganisms/ml of Acidithiobacillus thiooxidans were inoculated. In a second flask containing 500 mL of 1% S KMD culture medium 5 mL of BBS with Acidithiobacillus thiooxidans encapsulated were inoculated at a concentration equivalent to the first flask. The microorganism concentration in the supernatant was measured daily during 18 days, the results are shown on FIG. 6.

(40) As expected, no microorganism was observed in the culture in the first days of inoculation with BBS. Notwithstanding in the 3.sup.rd day after inoculation concentrations higher than 10.sup.6 microorganisms/ml were observed for Acidithiobacillus thiooxidans. From day 12 on the concentration of microorganisms in the supernatant of both cultures were evened.

(41) This shows that the BBS allow the cellular viability of the encapsulated microorganisms. Also the it shows that the microorganisms are capable of leaving the BBS to colonize the surrounding medium. That is to say, it shows that the BBS are useful as inoculation agents.

Example 4. Inoculation of Bioleaching Columns

(42) To determine if the BBS are effective in the inoculation of microorganisms during bioleaching processes, 4 columns of mineral were prepared to be bioleached at laboratory scale. The columns were submitted to different inoculation conditions, firstly with the conventional inoculation method by irrigation of microorganisms culture and secondly with inoculation with the BBS formed in example 1. The conditions of each column are described below:

(43) 4 columns of 30 cm of high and 5.75 cm of diameter, were prepared with 500 g of crushed mineral with a granulometry of (1.27 cm). Once the columns were inoculated, the effluent solution was recirculated to the column (closed circuit).

(44) Column 1. Inoculation by Irrigation (Conventional)

(45) The mineral was agglomerated with acid water at pH1.4 which is equivalent to 5% humidity. An inoculum solution was prepared with 1 mL of the same consortium used to form the BBS of example 1, at a concentration of 110.sup.6 microorganisms/ml and it was diluted in 99 mL of a solution of 3 g/L Fe(II) and medium KMD ((NH.sub.4).sub.2SO.sub.4, 247.5 mg/L; NaH.sub.2PO.sub.4.H.sub.2O, 36.5 mg/L; KH.sub.2PO.sub.4, 13.125 mg/L; MgSO.sub.4.7H.sub.2O, 25 mg/L; CaCl.sub.2, 5.25 mg/L), at pH 1.4.

(46) The 100 ml solution previously prepared was used to inoculate the column in an open circuit. After the inoculation step, the circuit was closed. In the column, the recirculation solution was the accumulated effluent from the inoculation stage plus the required volume to reach 1000 mL of a solution of 3 g/L Fe(II) and medium KMD ((NH.sub.4).sub.2SO.sub.4, 247.5 mg/L; NaH.sub.2PO.sub.4.H.sub.2O, 36.5 mg/L; KH.sub.2PO.sub.4, 13.125 mg/L; MgSO.sub.4.7H.sub.2O, 25 mg/L; CaCl.sub.2, 5.25 mg/L), pH 1.4.

(47) Columns 2, 3 and 4: Inoculation by BBS.

(48) The mineral was agglomerated with acid water at pH 1.4, which is equivalent to 5% humidity. Firstly 50% of the acid water solution of was added (equivalent to 2.5% humidity) and it was mixed homogeneously by rolling the mineral over a plastic surface. Different volumes of BBS prepared in the same way as described on example 1 were added to each column: 5 mL in column 2 (BBS at 1%), 10 mL in column 3 (BBS at 2%) and 20 mL in column 4 (BBS at 4%) and mixed homogeneously. The concentration of microorganisms added to the mineral corresponds to 110.sup.6 microorganisms/g of mineral. The other 50% of the acid water solution was then added and mixed homogenously.

(49) The circuit was closed with 1000 mL of a solution of Fe(II) 3 g/L and medium KMD ((NH.sub.4).sub.2SO.sub.4, 247.5 mg/L; NaH.sub.2PO.sub.4.H.sub.2O, 36.5 mg/L; KH.sub.2PO.sub.4, 13.125 mg/L; MgSO.sub.4.7H.sub.2O, 25 mg/L; CaCl.sub.2, 5.25 mg/L), pH 1.4.

(50) FIG. 7 shows the BBS in the column.

(51) After the irrigation of the four columns has started, those remained in recirculation mode and the redox potential of the solution was measured daily (Eh, mV, Ag/AgCl), giving an indication of the bioleaching activity (oxidation of ferrous ion). The results are shown on FIG. 8. It is observed that the redox potential of the effluent solutions of the columns inoculated with BBS (in all different concentrations) increases faster (shorter operation time) than the column inoculated conventionally. This corresponds to a greater activity of the microorganisms inside the column, demonstrating the advantage of using this inoculation method.

Example 5. Inoculation of Bioleaching Columns in Presence of Toxins

(52) To determine whether the BBS are effective for the inoculation of microorganisms in bioleaching processes under toxic conditions, two mineral columns were prepared for bioleaching at laboratory scale. The columns were submitted to different inoculation conditions; firstly with the conventional inoculation by irrigation with the culture and secondly with inoculation with the BBS prepared as in example 1, using a method very similar to that described in example 4. 1% in weight of BBS per gram of mineral was added to the column inoculated with BBS. To evaluate the effect of the toxins on the BBS, 500 mL of the recirculating solution composed by Fe (II), 3 g/L and medium KMD ((NH.sub.4).sub.2SO.sub.4, 247.5 mg/L; NaH.sub.2PO.sub.4.H.sub.2O, 36.5 mg/L; KH.sub.2PO.sub.4, 13.125 mg/L; MgSO.sub.4.7H.sub.2O, 25 mg/L; CaCl.sub.2, 5.25 mg/L) at pH 1.4, was replaced by raffinate that is formed mostly by Cu, Fe (III), Fe(II), SO.sub.4-2, Cl and heavy metals.

(53) Once the two columns were set they were maintained in recirculation mode and Eh values were measured daily. The results are shown in FIG. 9. It is observed that the column inoculated with BBS starts its bioleaching activity around 20 days earlier than the column inoculated conventionally. This shows that the BBS acts protecting the microorganisms from the toxins present in the medium, permitting in this way their adaptation and subsequent colonization of the mineral with the corresponding bioleaching activity.