BIOLOGICAL PROCESS FOR PRODUCING MAGNETIC NANOPARTICLES

Abstract

A process for obtaining gold magnetic nanoparticles or of other metallic elements by fermenting a culture medium in the presence of producer microorganisms such as Brevibacterium halotolerans, Bacillus mojavensis, Bacillus subtilis subsp. Inaquosorum, Bacillus subtilis subsp. Spizizenii, Bacillus tequilensis, Bacillus amyloliquefaciens subsp. Amyloliquefaciens, Bacillus siamenensis, Bacillus amyloliquefaciens subsp. Plantarum, Bacillus subtilis, Bacillus subtilis subsp. Inaquosorum, Bacillus atrophaeus, or Bacillus vallismortis, inter alia. The process of the invention can be used to effectively control the size and shape of the nanostructure to be obtained, with production levels above those found at laboratory level.

Claims

1. A biotechnological process for obtaining magnetic nanoparticles which comprises obtaining magnetic nanoparticles from producer microorganisms, wherein the process comprises the steps of: selecting and preparing the producer microorganism; growing said microorganism's inoculum; sterilizing a culture medium; adding a salt of a metallic element of interest; fermenting the culture medium with the microorganism; centrifugation of the depleted broth; cell lysis to separate the obtained nanoparticles; sonication of the cellular remains; microfiltration and ultracentrifugation; and final presentation of the obtained magnetic nanoparticles.

2. The biotechnological process for obtaining magnetic nanoparticles according to claim 1, wherein the producer microorganism is selected from the group of bacteria comprising: Brevibacterium halotolerans, Bacillus mojavensis, Bacillus subtilis subsp. Inaquosorum, Bacillus subtilis subsp. Spizizenii, Bacillus tequilensis, Bacillus amyloliquefaciens subsp. Amyloliquefaciens, Bacillus siamenensis, Bacillus amyloliquefaciens subsp. Plantarum, Bacillus subtilis, Bacillus subtilis subsp. Inaquosorum, Bacillus atrophaeus, or Bacillus vallismortis, amongst others.

3. The biotechnological process for obtaining magnetic nanoparticles according to claim 2, wherein the preferred producer microorganism is a bacteria of the type Bacillus mojavensis having the indication CO01 before the Colección Española de Cultivos Tipo (CECT) under the deposit number CET 8698.

4. The biotechnological process for obtaining magnetic nanoparticles according to claim 1, wherein the growth of said microorganism's inoculum is carried out in a liquid culture medium, using carbon and nitrogen sources in a percentage ratio with respect to total volume of fermentation of between 1 and 60%.

5. The biotechnological process for obtaining magnetic nanoparticles according to claim 1, wherein the step of preparing and sterilizing the culture medium for the fermentation is carried out by dissolving in water the carbon sources, the nitrogen sources and salts of the metallic element of the magnetic nanoparticle of interest.

6. The biotechnological process for obtaining magnetic nanoparticles according to claim 1, wherein the process is for obtaining gold nanoparticles, for which, the salt of the metallic element (gold) of interest used is gold chloride, gold chloride hydrated, or mixtures thereof.

7. The biotechnological process for obtaining magnetic nanoparticles according to claim 1, wherein the step of sterilization of the culture medium is carried out at a preferred temperature of between 85° C. and 150° C., preferably in the order or 115° C. and 125° C. and a pressure of between 5 and 25 pounds/inch.sup.2 for a period of time of between 5 and 35 minutes.

8. The biotechnological process for obtaining magnetic nanoparticles according to claim 1, wherein the gold salt concentration in the culture medium is at a concentration of between 0.01 and 30 nM.

9. The biotechnological process for obtaining magnetic nanoparticles according to claim 1, wherein the reactor's operation conditions are of a stirring rate of between 50 and 160 rpm; a temperature of between 15 and 33° C. and a period of time of between 12 and 50 hours.

10. The biotechnological process for obtaining magnetic nanoparticles according to claim 1, wherein the reaction is carried out for a period of time until the final absorbance is in the order of 540 nm to obtain the difference of growth between the initial and final absorbance.

11. The biotechnological process for obtaining magnetic nanoparticles according to claim 1, wherein the fermentation is carried out: by batch culture or fed batch culture; in stirred tank-like bioreactors with one or more impellers, such as, but not limited to a marine propeller, flat pellets and/or Rushton and inclined type turbines; in controlled ranges of temperature of between 10 and 55° C.; at a pH of between 4 and 8; stirring rate of between 30 and 500 rpm; and dissolved oxygen (OD) of between 0.1-1.5 vvm; all in a period of time that can vary of between 5 and 200 hours.

12. The biotechnological process for obtaining magnetic nanoparticles according to claim 1, wherein the biomass or depleted fermentation broth is centrifuged to concentrate the biomass up to a rate of between 100 and 20,000 rpm, during a residence period of time of between 1 and 180 minutes, preferably in a discs centrifuge.

13. The biotechnological process for obtaining magnetic nanoparticles according to claim 1, wherein the intracellularly obtained nanoparticles are extracted by mechanic lysis, using devices such as glass, metal and plastic balls grinder, at rates of between 100 to 2,000 rpm and during a period of time of 1 to 120 minutes.

14. The biotechnological process for obtaining magnetic nanoparticles according to claim 13, wherein the cellular remains adhered to the nanoparticles are sonicated during a preferred period of time of between 5 and 240 minutes at a wavelength of between 1 and 90%.

15. The biotechnological process for obtaining magnetic nanoparticles according to claim 1, wherein the obtaining of the nanoparticles is carried out by filtering with a pore size of between 0.22 to 5 μm, followed by a tangential ultrafiltration system with various filters of pore diameter of between 0.1 to 0.6 μm, 0.05 to 0.26 μm and of 0.025 to 10 kDa.

16. The biotechnological process for obtaining magnetic nanoparticles according to claim 1, wherein the obtained nanoparticles in the first retainer are bigger than 100 nm, while those of the permeate are inferior to 100 nm.

17. The biotechnological process for obtaining magnetic nanoparticles according to claim 1, wherein stable and mono dispersed gold magnetic nanoparticles are obtained of between 1 and 100 nm, which can be presented in an aqueous suspension of sodium citrate, citric acid or PBS or in a powder.

Description

[0051] This and other objects of the present invention will be better understood and will be observed with more detail throughout the following chapter and claims.

[0052] FIG. 1 shows a simplified block diagram of the object process of the present invention, which was developed under the synthesis methodologies of mixed bioprocesses, such as heuristic, algorithmic, and evolutionary adapted in particular for obtaining nanoparticles by biological methods at the industrial level.

[0053] FIG. 2 shows a behaviour graph of the Bacillus mojavensis microorganism, which was part of the analysis of the kinetic parameters such as specific growth rate of the microorganism, biomass yield/substrate, biomass/product and substrate/product. Briefly, the obtained gold nanoparticles concentrations with respect to the biomass and substrate concentrations are observed according to the description for Example 2 of the present invention.

[0054] FIG. 3 shows images of some plasmon characteristic of nanoparticles solutions of an average size of 20 nm, useful for the characterization of the gold nanoparticles obtained according to the present invention.

[0055] FIGS. 4 to 9 show microphotographs obtained by TEM from the obtained nanoparticles according to the present invention. In said microphotographs the spherical nanoparticles can be appreciated, mono dispersed with a size distribution of 20 nm, corresponding to samples obtained from the solution to which the UV-vis from FIG. 3 was carried out.

DETAILED DESCRIPTION OF THE INVENTION

[0056] The present invention is related to a process for the production of nanostructures from biotechnological methods using selected or genetically modified producer microorganisms, in which there is particular reference to the production of gold magnetic nanoparticles, preferably crystals with a particle size of between 1 and 100 nm from microorganisms selected preferably from the group comprising the following types of bacteria: Brevibacterium halotolerans, Bacillus mojavensis, Bacillus subtilis subsp. Inaquosorum, Bacillus subtilis subsp. Spizizenii, Bacillus tequilensis, Bacillus amyloliquefaciens subsp. Amyloliquefaciens, Bacillus siamenensis, Bacillus amyloliquefaciens subsp. Plantarum, Bacillus subtilis, Bacillus subtilis subsp. Inaquosorum, Bacillus atrophaeus, or Bacillus vallismortis.

[0057] It is considered that the success of the process and the inventive merit of the present invention for obtaining gold nanoparticles with a yield up to scales superior to laboratory level not only because of the quantity but the quality of the obtained nanoparticles, must not be considered that they solely depend on scaling the bioprocess in a conventional manner, but that it involves attempting particular conditions which were complied with by using only a particular type of producer microorganism for obtaining gold nanoparticles.

[0058] Practically all the processes involving producer microorganisms of substances or active ingredients comprise steps, which derive from promoting the conditions of and the appropriate culture medium so that said microorganisms develop and generate within the biomass the product of interest. Generally, in accordance with the present invention it has been found that biotechnological processes for the production of nanomaterials, to be carried out at levels or scales that can be considered industrial, require very importantly the identification and control of diverse variables so as to obtain the intended products which have diverse particularities which are not proper of all kinds of biotechnological processes.

[0059] Traditionally, the biotechnological products must be produced in cells grown in culture, which in practice allows to dominate a series of aspects not only of the research and development of production methods of these compounds but rather put into practise diverse strategies for scaling up processes and obtain the desired products. An important aspect is to mention the obtaining of biotechnological products such as the nanostructures or nanoparticles (different to proteins or any other traditional biotechnological product), implying that the bioprocesses in question must have an extremely particular performance.

[0060] In the regular practise, during the research and development phase, usually the initial production methods are developed at small scale. With all the research and development data derived from these production steps, production methods are developed at a large scale with the purpose of obtaining a sufficient quantity of the product for the intended market. The process of increasing scale and manufacturing will follow the same guidelines of good manufacturing practice to guarantee the safety and purity of the product. Nevertheless how routinely it may be, in traditional practice the process of increasing a cell culture to scale can be very difficult and requires a lot of time, and so, a lot of time is needed before a product can be obtained. The full process of production of a biotechnological product is usually divided into two main parts: manufacturing (upstream) and transformation (downstream). The manufacturing processes suppose the production of the biotechnological product, with higher frequency by using cells (from microbes, insects or mammals), which grow in culture. The transformation processes comprise the recovery, purification, formulation and conditioning of the product of interest following either standard procedures but each case of particular product, as are the magnetic nanoparticles (different from proteins or any other biotechnological product) require novel and better proposals for the transformation and set up of the nanostructures in the shape, size, features, quality, amount such, for their later uses.

[0061] In a little more detailed sense, the production phase starts with the cells or microorganism, which are created or designed for obtaining the protein product. In the case of the present invention, strains of microorganisms have been identified, prepared and conditioned specifically targeted for obtaining metallic magnetic nanoparticles, particularly gold nanoparticles, following a biotechnological process, which allows obtaining the nanostructures of interest in the intended quantity and quality to be used in different applications, as will be described later.

[0062] Once the desired cell line or producer microorganism has been obtained, it is subjected to cryopreservation or any other conservation means of the producer elements, so as to create a cells or microorganism bank. In the case of the production of magnetic nanoparticle producer microorganisms of the present invention, the selection, characterization and handling of the producer microorganisms allows obtaining nanoparticles with an improved process such that it matches or surpasses known chemical processes, maintaining the controlled growth of the producer microorganisms to generate in the biomass the quantity and quality of intended nanoparticles. Usually, the physical environment where the cell cultures or microorganisms are grown is monitored and controlled, so that in the case of the employees in the present invention it has been developed for the automatic control levels so that they allow a liable performance of this process stage, so that the nanoparticles are obtained at levels of bioreactors.

[0063] In the transformation phase of the manufacturing process, the product is isolated from the cells or microorganisms which produced it. The proteins present inside the cell (intracellular proteins) require some special protocols with the purpose of extracting them for their purification. Normally, this includes to suddenly opening the cells so as to release the protein product, which then must be purified with the remaining components that exist within the cell. The proteins present in the exterior of the cell (extracellular proteins) are usually easier to isolate with procedures, considered at this level of the state of the art, as traditional. This definitely does not happen when talking about products from the bioreactor in the form of a biomass containing magnetic nanoparticles obtained by the biological action of the producer microorganisms. The object method of separation and purification of the present invention assumes that it is of more complex products than simple proteins, which require improvements and substantially specific elements, and of inventive skill to achieve the separation of said products from the biomass generated in the bioreactor.

[0064] The economy of the biotechnological processes depends in great measure of the bioseparation operations involved, so that the correct selection of these operations has a strong impact on the success of the processes. The bioseparation processes involve the recovery and purification of products from the bioreactor. The bioseparations comprise all the treatments that the culture broth requires to obtain a biotechnological product in the purity and activity conditions required. It can be said that the success of a biotechnological process depends in gran measure of the suitable selection of the bioseparation process.

[0065] Usually, in general three generations of biotechnological processes can be distinguished, with respect to the type of bioseparations that these involve. The first generation comprises the group of processes developed by cultures of non-recombinant organisms, whose products are obtained in active form even if they are intracellular or if they are secreted to the culture medium. In this generation the traditional biotechnology processes as well as the production of ethanol, enzymes, citric acid, and antibiotics, are found. The products of these processes are presented in high concentrations at the start of the separation stage and do not require an extreme purity for their sale.

[0066] The discoveries associated with Molecular Biology and Genetic Engineering achieved particularly in the last decades allow the placement of a second generation of Biotechnology products such as human insulin, growth hormone, and alpha interferon, among others. These are produced intracellularly using Escherichia coli recombinant cells. They are characterised for being found at low concentrations within the cell, have a high molecular weight, have properties similar to contaminants and require a high grade of purity. Moreover, when produced in the cell they don't have biological activity because they are peptide chains without the appropriate conformation or structure; which is translated in the need to apply further physicochemical treatments to achieve the product in its active state.

[0067] The third biotechnology generation can be characterized by processes whereby extracellular products are obtained from recombinant cells or producer microorganisms, most of which are eukaryotic. In these systems the capacity not only to exogenously produce the desired product, but to obtain it in active form, such is the case of magnetic nanoparticles, which form part of the objective of the present invention, have been observed. The biotechnological processes of both the second and third generation constantly demand the knowledge of the physicochemical properties of the products and their contaminants, with the objective of selecting the suitable separation operations because of the high grade of purity required by the products. In the case of the process of the present invention, the magnetic nanoparticles are preferably prepared by an improved centrifugation process and mechanic lysis under very particular conditions to obtain nanoparticles in the intended conditions.

[0068] The aspects of yield and purity of the products are basic to determine the viability of a bioprocess, since, to achieve the grade of purification required by this type of products, the purification must be carried out in various steps, generally.

[0069] Once isolated and purified, the products are subjected to analysis protocols confirming by characterization, the presence and properties of the obtained products. In the case of the object of the present invention, the magnetic nanoparticles are characterized and assessed in a way that allows demonstrating the obtaining of these nanostructures showing the viability and effectiveness of the process up to superior objective scales of the present invention. After this step, the obtained nanoparticles are susceptible of being functionalised and used in different uses and applications.

[0070] Therefore, in accordance with the present invention the start point is producer microorganisms of the type representing bacteria type Brevibacterium halotolerans, Bacillus mojavensis, Bacillus subtilis subsp. Inaquosorum, Bacillus subtilis subsp. Spizizenii, Bacillus tequilensis, Bacillus amyloliquefaciens subsp. Amyloliquefaciens, Bacillus siamenensis, Bacillus amyloliquefaciens subsp. Plantarum, Bacillus subtilis, Bacillus subtilis subsp. Inaquosorum, Bacillus atrophaeus, or Bacillus vallismortis, from which in an “upstream” process step in the presence of a suitable culture medium and with the precursor material of the metallic element of interest, a biomass is obtained containing metallic magnetic nanoparticles, preferably of gold.

[0071] According to what is shown in FIG. 1, a block diagram of the objective process of the present invention is shown, in which the block (1) comprises the feeding of the nanoparticle producer microorganisms. These microorganisms according to the present invention comprise bacteria preferably of the type Brevibacterium halotolerans, Bacillus mojavensis, Bacillus subtilis subsp. Inaquosorum, Bacillus subtilis subsp. Spizizenii, Bacillus tequilensis, Bacillus amyloliquefaciens subsp. Amyloliquefaciens, Bacillus siamenensis, Bacillus amyloliquefaciens subsp. Plantarum, Bacillus subtilis, Bacillus subtilis subsp. Inaquosorum, Bacillus atrophaeus, or Bacillus vallismortis. More particularly, it refers to a bacteria of the type Bacillus mojavensis which have been identified by request of the applicant and carried out with the method consisting of: direct amplification by PCR of the 16S ARNr gene, partial sequencing thereof (with readings on both directions) and analysis of the sequences (Arahal et al., 2008) and deposited under the CO01 indication before the International Autority for the Deposit of Microorganisms named Colección Española de Cultivos Tipo (CECT), under the number CET 8698.

[0072] In the initial “upstream” stage, the growth step of the microorganism's in question inoculum is carried out in a liquid culture medium, using carbon and nitrogen sources. The percentage ratio thereof with respect to the total volume of fermentation will be able to stay in a preferred range of between 1 and 60%.

[0073] The second step is the preparation and sterilization of the culture medium for the fermentation (2), which is carried out dissolving in water the carbon sources, the nitrogen sources and salts of the metallic element of the magnetic nanoparticle of interest, which in the case of obtaining gold nanoparticles the use of gold chloride, gold chloride hydrate is preferred, or mixtures thereof.

[0074] Afterwards, the sterilization of the culture medium at a preferred temperature of between 85° C. and 150° C., preferably in the order of 115° C. and 125° C. and a pressure of between 5 and 25 pounds/inch.sup.2 during a period of between 5 and 35 minutes, is carried out. Finally, the gold salts, such as, but not limited to gold chloride, gold chloride hydrate at a concentration of between 0.01 to 30 mM, are added to the cold culture medium, and it is put to stir at a speed such but not limited to 50, 80, 90, 100, 120, 140, 160 at a temperature such as but not determined as 15-20, 23, 27, 30, 33° C. during a period such but not limited to of 12, 24, 36, 48, 50 hours. Past the time, a final absorbance at 540 nm will be read to obtain the difference of growth between the initial and final absorbance.

[0075] According to the preferred embodiment of the present invention, the fermentation (3) is carried out by batch culture or fed batch culture, in stirred-tank like bioreactors with one or more impellers, such as, but not limited to a marine propel, flat pellets and/or Rushton type and tilted turbines, in controlled ranges (4) of temperature, pH, stirring and dissolved oxygen (OD) such as, but not limited to 10-55° C., 4-8, 30-500 rpm, and 0.1-1.5 vvm, respectively, during a period of time of from 5 to 200 hours.

[0076] In the stage called “downstream” after the fermentation, the first step is the centrifugation of the depleted broth (5) to concentrate the biomass. This is carried out at a speed of about 100 and 20 000 rpm, during a time of residence of between 1 and 180 minutes (6) in a discs centrifuge. Because the gold nanoparticles are produced intracellularly, the product must be extracted by mechanic lysis (7), which is carried out in devices such as, but not limited to, a ball grinder using pearls which can be of materials such as, but not limited to glass, metal and plastic under operation conditions (8) of turning speed and time of 100-2000 rpm and 1-120 minutes, respectively.

[0077] To separate the adhered cellular remains to the nanoparticles, these are sonicated (9) during a period, such as, but not limited to, of 5 to 240 minutes at a wavelength opening of between 1 and 90% (10). Afterwards, the culture medium is filtered (11) in at least three stages with the purpose of separating gold nanoparticles from the remaining components, as well as to generate define diameter ranges. For that, first, a microfiltration of the medium is carried out using a filtering medium with a pore diameter (12) such as, but not limited to, a range of between 0.22 and 5 μm; afterwards, a tangential ultrafiltration system is used, in which the medium is made to pass through a filter with a pore diameter such as, but not limited to of 0.1-0.06 μm. The obtained nanoparticles in the retainer (13) will be in the range of >100 nm, while those of the permeate are passed again through a filter of pore diameter such as, but not limited to, of 0.05 to 0.026 μm; the permeate can even be subjected to another filtration round through a filtering medium with a pore diameter such as, but not limited to, in a range of 0.025 μm to 10 kDa.

[0078] That is to say, once this stage is finished the gold nanoparticles will be obtained in the ranges such as, but not limited to, of 1 to 25 nm, 26 to 50 nm, 50 to 100 nm and >100 nm.

[0079] The final step is the formulation, which consists of the preparation of the intended gold nanoparticles for their final presentation, which can be an aqueous suspension with sodium citrate, citric acid or PBS or a powder presentation (14). If a suspension is desired, first the concentration is determined by UV-visible spectrophotometry and then it is taken to the required concentration obtaining the solution. If it is necessary to concentrate the nanoparticles, a centrifugation or lyophilisation can be carried out first. For the powder presentation, a lyophilisation at temperature and pressure ranges, such as but not limited to, from −80° C. to 20° C. and from 0.2 to 160 Pa, respectively, is carried out. Lastly, the gold nanoparticles are stored in closed containers for their final presentation.

[0080] To establish the above-described gold nanoparticles production process, it was important to first know the optimum conditions of preparation and growth of the microorganism, and the formulation of the production medium at laboratory level. Once these parameters were determined, the type of suitable culture medium for the production was established, as well as the purification method and finally, the formulation. According to this process, our invention is characterized for being suitable to operate processes for obtaining gold magnetic nanoparticles in volumes considered of scales superior to laboratory level, in reactors of the order of 7 to 10 litres.

[0081] To illustrate in more detail the above-described process and the results thereof put into practice, the following examples are provided as an explanation of the methods carried out to obtain the process for the biological production of the gold nanoparticles described in the present invention, as well as the methods for characterizing the obtained product.

Examples

Example 1: Method for Establishing the Optimum Production Conditions at Laboratory Level

[0082] The Bacillus mojavensis producer microorganism deposited under the indication CO01 before the International Authority for the Deposit of Microorganisms of name Colección Española de Cultivos Tipo (CECT), under the number CET 8698, was used, for which the optimum growth conditions and gold nanoparticles production rail, were established (upstream-fermentation-downstream) by an experimental design Box-Behnken (1960) for three factors (temperature, stirring speed and gold salts concentration) and three levels. That is, in total thirteen experiments in duplicate were carried out, of which 286 samples were obtained to which substrate, product and biomass concentrations were determined. Finally, thirteen more samples were further processed to determine the percentage of organic matter. Overall, a total of 1157 measurements were carried out.

[0083] The values for biomass and product were obtained with dry weight. The amount of substrate was determined by the 3,5-dinitrolsalicylic acid (DNS) technique described by Miller (1959), for which sucrose hydrolysis was previously carried out according to Godoy (2002) and ICONTEC (1994). The amount of organic matter was determined for each final sample of kinetics, by incineration (AOAC, 1990).

[0084] The separation of the nanoparticles was carried out in three stages: extraction, sonication and filtration. To carry out the wash, the samples were centrifuged at 3700 rpm for 20 minutes, the supernatant was decanted and the pellet was washed with distilled water, then it was centrifuged again under the same time and speed conditions. At the end, the liquid phase was removed.

[0085] For the extraction, two methods were tested: alkaline lysis and mechanical lysis. In the alkaline lysis the obtained cellular package was re-suspended in an equal proportion of volume of a NaOH solution at 20% and it was incubated at 37° C. for 4 hours. At the end of this time period, the cell suspension was subjected to sonication for 2 hours. The mechanic lysis was carried out adding an equal volume as glass pearls to the pellet and stirring in vortex for 5 minutes.

[0086] After carrying out the extraction, the samples were filtered, sonicated and serial-filtered with membranes of 0.22, 0.1, 0.05 and 0.025 μm. Afterwards, they were analysed with an electronic microscope (TEM and SEM).

[0087] The following table shows various samples that were handled according to the described process for obtaining gold nanoparticles.

TABLE-US-00001 TABLE 1 Box Behnken experimental design for the microorganism's kinetics Gold chloride Sucrose concentration concentration in Sample number Temperature (AuCl.sub.3) in medium medium 1 − 0 + 2 − + 0 3 − − 0 4 − 0 − 5 + − 0 6 + + 0 7 + 0 + 8 + 0 − 9 0 − + 10 0 + + 11 0 − − 12 0 + − 13 0 0 0 14 0 0 0 15 0 0 0

TABLE-US-00002 TABLE 2 Independent variables and their variation levels. Level Factor − 0 + Temperature 23° C. 28° C. 33° C. Gold chloride 0.5 mM 1.5 mM 3 mM concentration in the medium Sucrose 7.5 g/L 15 g/L 22.5 g/L concentration in the medium

[0088] Samples were taken every 1 or 2 hours according to the observed bacterial growth LP and the successive response variables were reported: growth specific rate (p); substrate consumption rate (qs); product generation rate (qp); and nanoparticle diameter.

[0089] The statistical analysis was carried out with support from MINITAB® 16 software; the obtained microorganism's kinetic parameters were: growth specific rate (p); gold nanoparticles performance with respect to an inductor (YPS); gold nanoparticles performance with respect to biomass (YPX); biomass performance with respect to a carbon source (YXS); Productivity (qP); and carbon source consumption-specific rate (qS).

[0090] The kinetic parameters were determined with the following methods: p.—dry weight before lysis; qp.—Dry weight-after lysis; qs.—3,5-dinitrosalicylic acid (DNS); Ypx.—Δp/Δx; Yxs.—consumed Δx/Δs; and Yps.—consumed Δp/Δs.

[0091] To calculate the growth specific rate the lineal form of the following formula was used:

[00001]$\frac{\mathrm{dx}}{\mathrm{dt}}=\mu .Math..Math.x$

wherein:

[0092] x=biomass concentration in g/L.

[0093] μ=growth specific rate in h−1.

[0094] To calculate the consumption specific rate of product generation, that is, of gold nanoparticles, (productivity) the following lineal formula was used:

[00002]$\frac{\mathrm{dp}}{\mathrm{dt}}=q_p.Math.x$

wherein:

[0095] x=biomass concentration in g/L.

[0096] qp=product generation specific rate in h−1.

[0097] To calculate the specific rate for substrate consumption the following lineal formula was used:

[00003]$\frac{\mathrm{ds}}{\mathrm{dt}}=-q_s.Math.x$

wherein:

[0098] x=biomass concentration in g/L.

[0099] qs=substrate consumption specific rate in h−1.

[0100] To determine the yields the following formulas were used:

[00004]$Y_{\mathrm{XS}}=\frac{\Delta .Math..Math.X}{\Delta .Math..Math.S}[=].Math..Math.g_{\mathrm{biomass}}.Math.\text{/}.Math.g_{\mathrm{sucrose}}$

[00005]$Y_{\mathrm{PC}}=\frac{\Delta .Math..Math.P}{\Delta .Math..Math.C}[=].Math.\frac{g_{\mathrm{AuNPts}}}{g_{{\mathrm{AuCl}}_3}}$$Y_{\mathrm{PX}}=\frac{\Delta .Math..Math.P}{\Delta .Math..Math.X}[=].Math.\frac{g_{\mathrm{AuNPts}}}{g_{\mathrm{biomasa}}}$

wherein:

[0101] S=amount of consumed carbon source, expressed in g.

[0102] P=amount of AuNPs generated, expressed in g.

[0103] X=amount of generated biomass, expressed in g.

[0104] C=amount of gold salt consumed, expressed in g.

Example 2. Method for Establishing the Optimum Production Conditions at Pilot Level

[0105] Once the optimum conditions for generating nanoparticles were determined, a batch culture in stirred tank-like bioreactors of 7 litres capacity, was established. As scaling up criteria, the geometric similarity and Reynolds Number were used, the latter because by maintaining the flow rate, the mixing, cut effort and air bubbles dispersion ensures that they are maintained constant, apart from favouring the micromixing.

[0106] In the case of the geometric similarity, the same ratios in the shape factors were maintained. Once the conditions at laboratory level, which gave the size and shape characteristics of the gold nanoparticles, were determined, fermentations were carried out in a stirring tank-like bioreactor, varying the conditions of stirring rate, pH and aeration, thereby obtaining the kinetic parameters (shown on Table 5) and the conditions with which nanoparticles with the desired sizes were obtained.

[0107] After specifying the fermentation process, the filtering process was established, and with that, the suspended organic matter from the product was eliminated and it was possible to separate the nanoparticles according to sizes.

[0108] Finally, various tests for the formulation were carried out, establishing the operating conditions of the drying equipment and the concentrations in the suspension with the purpose of obtaining a stable product.

TABLE-US-00003 TABLE 3 Box Behnken experimental design for the microorganism's kinetics Gold chloride Sucrose concentration concentration in Sample number Temperature (AuCl.sub.3) in medium medium 1 − 0 + 2 − + 0 3 − − 0 4 − 0 − 5 + − 0 6 + + 0 7 + 0 + 8 + 0 − 9 0 − + 10 0 + + 11 0 − − 12 0 + − 13 0 0 0 14 0 0 0 15 0 0 0

TABLE-US-00004 TABLE 4 Independent variables and their variation levels. Level Factor − 0 + Temperature 23° C. 28° C. 33° C. Gold chloride 0.5 mM 1.5 mM 3 mM concentration in the medium Sucrose 7.5 g/L 15 g/L 22.5 g/L concentration in the medium

[0109] Samples were taken every 1 or 2 hours according to the observed bacterial growth LP and the successive response variables were reported in the same manner of for the previous examples in terms of: growth specific rate (p); substrate consumption rate (qs); product generation rate (qp); and nanoparticle diameter.

[0110] Likewise, the statistical analysis was carried out with support from MINITAB® 16 software; the obtained microorganism's kinetic parameters were: growth specific rate (μ); gold nanoparticles performance with respect to an inductor (YPS); gold nanoparticles performance with respect to biomass (YPX); biomass performance with respect to a carbon source (YXS); Productivity (qP); and carbon source consumption-specific rate (qS).

[0111] Likewise, as in the previous Example 1, the kinetic parameters were determined; growth specific rate calculation, product generation consumption specific rate and substrate consumption specific rate.

[0112] In the graph appearing in FIG. 2 of the present invention, gold nanoparticles concentration and biomass and substrate concentrations with respect to time, all according to the product obtained as an embodiment described in this example, are shown.

[0113] The performance of determined parameters of the process for obtaining gold nanoparticles of the present invention with regards to Examples 1 and 2, at laboratory plant and pilot plant, respectively, were comparatively analysed.

TABLE-US-00005 TABLE 5 Kinetic parameters determined in a flask and a bioreactor. Parameter Pilot plant reactor Flask laboratory μ (1/h) 0.097 0.085 q.sub.p (1/h) 0.004 0.005 q.sub.s (1/h) 0.807 0.556 Y.sub.PS (g .sub.AuNPs/g .sub.AuCl3) 0.534 0.54 Y.sub.PX (g A.sub.uNPs/g .sub.biomass) 0.050 0.051 Y.sub.XS (g .sub.biomass/g .sub.sucrose) 0.293 0.29

Example 3. Gold Nanoparticles Characterization by UV-Visible Spectrophotometry

[0114] To characterize the gold nanoparticles by UV-Visible spectrophotometry, scanning at 400-800 nm wavelengths was carried out. The highest optical absorbance peak is shown in the ranges of 550-570 nm depending on the diameter. FIG. 3 shows spectra images to characterize the obtained gold nanoparticles, in which it is possible to acknowledge: (A) the established scan of a 400 to 600 nm wavelength; and (B) the established scan of a 400 to 800 nm wavelength.

Example 4. Gold Nanoparticles Characterization by Transmission Electron Microscopy

[0115] A small sample of the obtained product according to the nanoparticles obtained in Example 2 was taken, after the filtrations, it was placed on a copper rack, which was placed under the transmission electron microscope brand JEOL model J1100, and was taken to 220 kV. The nanoparticles were observed and appear in FIGS. 4 and 9 of the present application, in which their size, shape and mono dispersion can be shown in more detail, thereby demonstrating that not only gold nanoparticles are obtained in quantity but in quality, important according to the spirit of the present invention.

[0116] Having described the invention in detail, it is considered as novel and so it is claimed as property what is contained in the following claims.