MX2007011930A - Process for the joint culture of an association of microorganism, using pyrite (fes2) as and energy source . - Google Patents

Abstract

The invention publishes a process for the joint culture of an association of microorganisms using pyrite (FeS2) as an energy source. This invention particularly publishes the use of a pyrite ore as an energy source in the joint culture of an association of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans type isolated microorganisms known as Wenelen DSM 16786 and Licanantay DSM 17318 respectively.

Description

PROCESS FOR THE JOINT CROP OF A MICROORGANISM CONSORTIUM EMPIRING PIRATE (FES2) AS A SOURCE OF ENERGY.

FIELD OF THE INVENTION The invention discloses a process for the joint cultivation of a consortium of microorganisms using pyrite (FeS2) as an energy source. In particular, the invention discloses the use of a pyrite mineral as an energy source in the joint culture of a consortium of isolated microorganisms of the type Acidithiobacillus ferrooxidans and Acidi thiobacillus thiooxidans known as enelen DSM 16786 and Licanantay DSM 17318 respectively.

BACKGROUND OF THE INVENTION Typically, in the cultivation of microorganisms, artificial culture media, or expressly prepared media, are used frequently from high purity organic and / or inorganic chemicals. This usually aims to control to the maximum the variables related to the requirements of microorganisms, and to avoid all potential sources of contamination and inhibition of microbial growth.

For example, the growth of At. ferrooxidans and At. thiooxidans at laboratory scale has been respectively described by Silverman, M.P. & Lundgren D.G. 1959. "Studies on the chemoautotrophic iron bacterium ferrobacillus ferrooxidans I. An Improved Medium and a Harvesting Procedure for Securin g High Cell Yields". Journal of Bacteriology. 77: 642-647, and by Cook, T.M. 1964. "Growth of Thiobacillus thiooxidans in shaken culture". Journal of Bacteriology. 88: 620-623.

The previous approach is very suitable for the cultivation of microorganisms on a laboratory scale, and sometimes even on a pilot scale, but due to economic considerations it may become impractical, especially if it involves the production of biomass on a large scale. A common solution to this problem is to use technical reagents, which decreases the cost of the medium, but the potential sources of contamination increase, in addition to adding impurities that can inhibit the growth of microorganisms.

Thus, for the cultivation of microorganisms in industrial conditions, formulations based on ammonium sulfate and potassium phosphate of technical grade have been described (Hackl et al. 5. 089.412). In the same sense, Chilean patent applications CL2731-2004 and CL 2101-2005 respectively use culture media known as modified 9K (3.0 g / L of (NH4) 2S04, 0.5 g / L of K2HP04, 0.5 g / L of MgSO4 * 7H20, 0.1 g / L of KCl and 0.1 g / L of Ca (N03) 2, 30 g / L of FeS04-7H20) and 9KS (3, 0 g / L of (NH4) 2S04, 0.5 g / L of K2HP04, 0.5 g / L of MgSO4 * 7H20, 0.1 g / L of KCl, 0.1 g / L of Ca (N03) 2, 1% elemental sulfur or other reduced sulfur compound).

It is known that in cultures of microorganisms in media such as those indicated, the final concentration of biomass is limited by the concentration of the substrate used as an energy source and by the inhibition of growth exerted both by said substrate and by the products of the metabolism of that, generated during the microbial growth [LaCombe, J., Lueking, D. 1990. "Growth and aintenance of Thiobacillus ferrooxidans cells". Applied and Environmental Microbiology. 56: 2801-2806; Nagpal, S. 1997. "A structured model for Thiobacillus ferrooxidans growth on ferrous Iron". Biotechnology and Bioengineering. 53. 310-319].

On the other hand, the type of microorganisms obtained depends on the type of energy source used, iron in the form of Fe2 + compounds for iron oxidizing microorganisms, and sulfur compounds - in oxidation state -2, 0 and +4 - for sulfur oxidizing microorganisms.

This is a limitation for the design of a mixed biomass production process (iron and sulfur oxidant), since different strains impose different production conditions such as different substrates and pH.

Therefore, in the case that it is desired to cultivate two or more species of microorganisms, it is attractive to use the same culture medium, or even more, to cultivate the microorganisms together. In this way, the number of stages of the process is reduced, the complexity of the operation is simplified, and in some cases the characteristics of the underlying biochemistry can be taken advantage of.

Iron sulfides, such as pyrite (FeS2), which are reduced iron and sulfur sources, are therefore an interesting alternative for the production of mixed leaching biomass.

In the study of Chong, N., Karamanev, D.G., Margaritas, A. 2002. "Bffect of particle-particle shearing on the bioleaching of sulfide minerals". Biotechnology and Bioengineering 80: 349-357, the growth of microorganisms, such as At., Is demonstrated on a laboratory scale. ferrooxidans, on pyrite as an energy source, obtaining concentrations of microorganisms in the order of 108 cells / ml.

Schippers, A., Jozsa, P.G., Sand, W. 1996. "Sulfur chemistry in bacterial leaching of pyrite". Applied and Environmental Microbiology. 62: 3424-3431, propose the formation of thiosulfate (S2032 ~) during the pyrite degradation cycle. This compound can follow a series of abiotic reactions, or be used as an energy source by sulfur-oxidizing bacteria, which gives rise to the joint culture of ferrooxidant and thiooxidant microorganisms on pyrite.

For example, in the work of Bacelar-Nicolau, P. & Jonson, B. 1999. "Leaching of pyrite by acidophilic heterotrophic iron-oxidizing bacteria in puree and mixed cultures". Applied and Environmental Microbiology. 65: 585-590, the mixed culture of ferrooxidant and thiooxidant microorganisms on pyrite is presented.

From the chemical point of view, the decomposition of pyrite so that it can be used as an energy source by microorganisms of the Acidithiobacillus ferrooxidans type, the activity of said microorganisms is represented according to the following formula: FeS2 + 6Fe3 + + 3H20? 7Fe2 + + S2032"+ 6H + 7Fe2 + + 7/4 02 + 7H + -» 7Fe3 + + 7/2 H20 + At. Ferrooxidans FeS2 + 7/4 02 + H +? Fe3 + + S2032"+ 1/2 H20 + At. Ferrooxidans reaction (i) As can be seen in reaction (i), one of the products is thiosulfate, which contemplates sulfur in an intermediate oxidation state, and which, according to the following reaction, is useful as an energy source for microorganisms of the Acidithiobacillus type. thiooxidans: S2032 ~ + H20 + 202? 2S042 ~ + 2H + + At. thiooxidans reaction (ií) Finally, regarding the use of pyrite or materials that contain it, existing works raise different approaches, for example, in the patents WO0136693, WO0071763 and WO2004027100 its use as a source of sulfuric acid is proposed. In WO0136693 pyrite is associated to leaching systems in which sulfuric acid is not added; in WO0071763 its use is related to the replacement of the acid when the mineral presents a high demand for it; and in WO2004027100, its use is for the replacement of part of the necessary acid. In other documents such as US Pat. No. 6,110,253 and application US2005103162, pyrite is used as a mechanism for increasing the temperature of the cell, since it is biooxidated and generates heat, which according to said texts allows to practice bioleaching. with thermophilic microorganisms.

As far as we know, there is a lack of lower-cost culture media that make viable the large-scale production of microorganisms useful in bioleaching, and we are not aware of processes in which pyrite is effectively used as an energy source for the growth of mixed biomass.

BRIEF DESCRIPTION OF THE INVENTION For a greater understanding of the processes, it is understood as: a) ATCC: "American Type Culture Collection", American collection of cultures of microorganisms type. b) Bioleaching of minerals in rafts: process carried out in a pond with a false bottom where the mineral is loaded, flooding it with the leaching solution. which is circulated through the mineral particles, in the presence of acidophilic microorganisms, extracting the copper dissolved in an acid solution. c) Bioleaching of minerals in dumps: The minerals that are placed under the cut-off law, which are extracted from an "open pit" operation, are collected "run of mine" or with a primary crushing, in ravines that have appropriate characteristics to control the infiltration of solutions or surfaces where an impermeable folder has been previously installed and the Leaching solution is irrigated on the surface, in the presence of acidophilic microorganisms, extracting copper dissolved in an acid solution at the base. d) Biolixiviation of minerals in piles: In this process the crushed ore to a defined granulometry is collected on a waterproof surface, with a low slope and the leaching solution is irrigated on the surface, in the presence of acidophilic microorganisms, extracting the copper base dissolved in an acid solution. e) Bio-leaching of minerals "in si tu": Deposits of mineral in their natural state or that have been fractured, due to previous mining operations, are leached directly in their place, irrigating the leaching solution on the surface, in the presence of acidophilic microorganisms, extracting copper dissolved in an acid solution at the base. f) Biolixiviation of minerals in agitated tanks or reactors: the bioleaching process is carried out in a mechanically agitated pond where the finely divided mineral is mixed with the leaching solution, forming a pulp with a solids content of up to 20%, with the presence of Acidophilic microorganisms, extracting dissolved copper in an acidic solution. g) Biolixiviation of tailings dams: Tails coming from the flotation process and containing smaller amounts of the metal present in the ore are collected in dams, from where they are extracted to leach them either in piles or by agitation, in the presence of acidophilic microorganisms , extracting the copper dissolved in an acid solution. h) Biomass: mass of living organisms produced in a given area or volume. i) DSM: "Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH" German collection of cultures of microorganisms type. j) Inoculum: pure or mixed bacterial culture that will act as an active biological material during the bioleaching process. k) Passivation: Decrease in the leaching rate of a mineral as a consequence of the accumulation of layers of sulfur and poly-sulfides on its surface. 1) PLS: Aqueous solution generated in the bioleaching process that contains the metal ions leached from the ore. This solution constitutes the feeding of the solvent extraction plant. m) Refining: Aqueous solution depleted in copper resulting from the extraction process by solvent, n) Mixed energy source: substrate that allows the simultaneous growth of iron and sulfur oxidizing microorganisms, or) Mixed biomass: mass of microorganisms with the ability to oxidize reduced iron and sulfur compounds.

BRIEF DESCRIPTION OF THE FIGURES Figure 1: the growth curves of a consortium of microorganisms on a culture medium with various mixtures of ferrous sulfate and pyrite concentrate (I), according to that described in Example 1, are presented in this figure. .

Figure 2: The growth curve in batch mode of a consortium of microorganisms in culture medium modified with the incorporation of pyrite concentrate (II), according to what is described in Example 2, is presented in this figure.

Figure 3: The content of At is presented in this figure. ferrooxidans ENELEN DSM 16786 (black bars) and At. thiooxidans LICANANTAY DSM 17318 (white bars) in a continuously operated biomass propagation bioreactor, using modified culture medium with the incorporation of pyrite concentrate (II), as described in Example 3.

DETAILED DESCRIPTION OF THE INVENTION In order to produce on a large scale isolated microorganisms useful in the bioleaching of sulphided metal ores, a process has been developed, based on the use of bioreactors, in which it is possible to decrease the costs of the culture media for the growth of said microorganisms, using mixed energy sources.

This process consists in the use of a material containing pyrite, to replace a part of the standard culture medium, as a mixed energy source of two microorganisms of different types that grow together, namely, Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans.

This process also provides advantages related to the quantity of microorganisms, their adaptation to the solid phase, and also provides advantages related to the recovery of copper and obtaining the iron to the +3 oxidation state.

According to the present invention, Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans type microorganisms are cultured together with other microorganisms, using a culture medium modified with pyrite, which takes advantage of the presence and formation of suitable species as an energy source, respectively the iron (oxidation state +2) and the sulfur (oxidation state -2), and provides a series of advantages for the process of cultivation of microorganisms.

Considering that part of the conventional culture medium has been replaced by a material of low cost, it is obvious that this crop will be less expensive than the crop using conventional means. In addition, by cultivating two microorganisms simultaneously, there are also cost reductions related to facilities, reactors, control systems, etc., which otherwise would have to be duplicated.

Furthermore, the joint culture using pyrite allows to obtain a higher concentration of microorganisms than is normally obtained when the same microorganisms are grown separately. This is of economic importance, which can be evaluated by reducing the equipment needed to obtain a certain target concentration when new installations are projected, or by a greater production capacity in facilities that are in operation.

From the studies carried out, which are presented later in the examples, it is possible to affirm that the consortium of microorganisms that contemplates isolated microorganisms mixed with microorganisms native to the minerals grows normally in the modified medium with materials that contain it. The above constitutes an advance with respect to the state of the art, as it reduces Crop costs by reducing the costs of the culture medium.

On the other hand, according to the reactions set forth above, there will of course be a greater concentration of the species Acidithiobacillus thiooxidans, or equivalently, a greater relative growth of the species Acidithiobacillus thiooxidans. This may or may not be advantageous, depending on considerations about the subsequent processes in which the generated biomass is used. However, if desired or if necessary, the growth of the microorganisms can be balanced by the incorporation of Fe + 2 in the ferrous sulfate form (FeS04-7H20).

As noted, in practice the invention is verified by replacing a part of the standard culture medium of the microorganisms with a material containing pyrite. The fraction of the culture medium that is replaced is that corresponding to the iron and sulfur species, it being possible to replace it in a wide range, for example, in a modified culture medium according to the invention, it can be used between 1 and 20 g / L of pyrite (100% base).

On the other hand, and because the materials containing pyrite are mainly solids, an adaptation of the microorganisms to the oxidation of sulfides in solid phase is achieved. This adaptation is useful, and it is also a step forward in the technique, since the microorganisms are adapted to the solid phase, they will quickly populate the materials stored in piles, dumps, tailings dams or other operations "in si tu", in the which are used, decreasing the times associated with the leaching of them.

Finally, and in accordance with the reactions presented above, an enrichment of iron to the +3 oxidation state occurs in the culture medium. As is known in the art, the presence of Fe + 3 favors the leaching of secondary minerals, so this also represents an advantage over other processes.

The process of the present invention for the joint cultivation of a consortium of microorganisms of the Acidithiobacillus th.iooxida.ns type and of the Acidithiobacillus ferrooxidans type, using pyrite (FeS2) as an energy source is defined according to the following stages and operating conditions: a) preparing a culture medium for microorganisms of the Acidithiobacillus thiooxidans type and Acidithiobacillus ferrooxidans type by replacing a part of said culture with pyrite; b) the pH value of said medium is adjusted between 1.5 and 2.5; c) the culture medium is inoculated with a mixture of isolated microorganisms of the type Acidithiobacillus thiooxidans and the type Acidithiobacillus ferrooxidans, with or without other native microorganisms; d) the temperature is adjusted in a range between 25 and 35 ° C; e) an air stream enriched in C02 is passed between 0.20% and 0.80% C02.

The part of the culture medium, which is replaced in step (a), is that corresponding to reduced iron or sulfur compounds, such as ferrous sulfate and elemental sulfur.

In the process of the present invention the culture medium with pyrite considers an amount of pyrite between 1 and 20 grams per liter. The microorganisms of the type Acidithiobacillus thiooxidans and of the type Acidithiobacillus ferrooxidans that are cultivated are isolated microorganisms and more preferably the microorganism of the Acidithiobacillus thiooxidans type is Licanantay DSM 17318 and the microorganism of the Acidithiobacillus ferrooxidans type is Wenelen DSM 16786. The volume ratio of the inoculum of microorganisms to volume of culture medium is between 1:20 and 1: 5.

EXAMPLE 1 In order to determine the growth kinetics and the biomass yield of the Wenelen DSM 16786 and Licanantay DSM 17318 microorganism consortium using modified medium with the incorporation of the pyrite concentrate (I), an experiment is performed using the following protocol.

PROTOCOL To fulfill the proposed objective, a growth test was carried out, of the flask-agitated type. The growth of the mixture of strains was carried out in 100 ml flasks on 25 ml of a culture medium supplemented with mixtures of two energy sources: concentrate (I) of pyrite - whose characteristics are shown in Table 1 - and ferrous sulfate, FeS04. The mixtures of energy sources used are detailed in Table 2. The nutrient composition in the culture medium was as follows: 0.99g (NH4) 2S04 / L, 0.128g NaH2P04 · H20 / L, 0.0525g KH2P04 / L, 0, lg MgSO4 · 7H20 / L, 0.021g CaCl2 / L. The pH of the culture medium was adjusted to 1.8. The initial concentration of Wenelen DSM 16786 and Licanantay DSM 17318 strains in each flask was 2.5-107 cells / ml. The incubation of the flasks was carried out at 30 ° C in an orbital shaker operated at 200 rpm. Periodic monitoring of the biomass concentration in the flasks was carried out by means of microscopic counts in the Petroff-Hausser chamber, during a six-day interval.

Table 1: Mineralogical composition of pyrite concentrate (I) Minerals% Weight% Vol.% S% Cu% Fe% As% Mo | % Zn% Pb Chalcopyrite 1 1, 24 10.98 3.93 3,888 3.42 Calcosine 10.41 7.50 2.09 8.315 I Covelin 5.57 4.97 1, 87 3.705 I Bornite 7.74 6.23 1, 98 4.902 0.86 I Cu G Tenantite 0, 17 0, 15 0.04 0.088 0.034 Enargite 4.30 4.01 1, 40 2.075 0.816 I Pyrite 32.09 26.35 17, 13 14.95 I Molybdenite 2.34 2 , 04 0.94 1, 40 | Galena 0, 13 0.52 0.02 0, 1 1 Sphalerite 4, 13 4,23 1, 36 I 2,77 Hematite 0,09 0,07 0,07 i Limonite 0,27 0,30 0, 17 i Rutile 0, 15 0, 15 Ganga 21, 38 32,50 I Total 100.00 100.00 30.77 22.972 19.47 0.850 1, 40 | 2.77 0.11 Table 2: Mixtures of energy sources used in the growth assays of Example 1.

RESULTS OF EXAMPLE 1 As seen in Figure 1, the addition of pyrite concentrate (I) at concentrations of 2 and 5 g / L allows increasing the propagation speed of free biomass and the final titre of biomass obtained in a medium with an initial concentration of ferrous sulphate of 7.5 g / L. The addition of 10 g / L of concentrate (I) only makes it possible to increase the final titre, there being a delay in the propagation of free biomass, probably due to the adsorption of cells on the surface of the solid. In the case of the ferrous sulphate mixture of 7.5 g / L + concentrate (I) 5 g / L it is possible to obtain in six days more free biomass in comparison to a culture medium without concentrate (I) and with a concentration of ferrous sulfate of 15 g / L. That is to say, it is clearly established that it is possible to replace part of the ferrous sulfate of the medium with pyrite concentrate (I).

EXAMPLE 2 In order to determine the growth kinetics and the biomass yield of the Wenelen DSM 16786 and Licanantay DSM 17318 microorganism consortium using modified medium with the incorporation of the pyrite concentrate (II), an experiment is carried out using the following protocol.

PROTOCOL Bacterial growth was carried out in a 6 m3 reactor. The culture medium used in the propagation of the microorganisms was prepared by suspending pyrite concentrate (II) whose characteristics are shown in Table 3, at a pulp density of 1.25%, in a nutrient solution of the following composition: 75 g FeS04 / L, 0.99g (NH4) 2S04 / L, 0.128g NaH2P04 · H20 / L, 0.0525g KH2P04 / L, 0, lg MgSO4 · 7H20 / L, 0.021g CaCl2 / L. The pH of the culture medium was adjusted to 1.8.

Table 3: Mineralogical composition of pyrite concentrate (II) To start the culture, 5,400 L of culture medium were mixed with 600 L of bacterial inoculum, carrier of the microorganisms Wenelen DSM 16786 and Licanantay DSM 17318.

To allow the growth of microorganisms in the reactor, air enriched with 0.5% CO2 was supplied. The temperature of the reactor was controlled at 30 ° C. The pH in the reactor was controlled by the addition of H2SO4.

The reactor was operated in batch mode for 15 days. During the operation of the reactor, the growth of microorganisms was monitored by microscopic counting using a Petroff-Hausser chamber.

RESULTS OF EXAMPLE 2 As seen in Figure 2, there was a rapid increase in the concentration of microorganisms in the culture medium modified with pyrite concentrate, reaching a maximum concentration of microorganisms of l, 7xl09 cells / ml in 6 days. From the data obtained during the exponential growth phase it was possible to determine a specific growth rate of 0.069 h "1.

EXAMPLE 3 In order to demonstrate that the consortium of microorganisms in DSM 16786 and Licanantay DSM 17318 can effectively be propagated continuously using modified medium with the incorporation of a pyrite concentrate, an experiment is carried out using the following protocol.

PROTOCOL Bacterial growth was carried out in a 50 m3 reactor.

The culture medium used in the propagation of the microorganisms was prepared by suspending concentrate (II) of pyrite (at a pulp density of 0.125%) in a nutrient solution of the following composition: 8 g FeS04 / L, 0.99 g (NH4) 2S04 / L, 0.128 g NaH2P04 · H20 / L, 0.0525 g KH2P04 / L, 0, lg MgSO4 · 7H20 / L, 0.021g CaCl2 / L. The pH of the culture medium was adjusted to 1.8.

To start the culture, 44 m3 of culture medium was mixed with 6 m3 of bacterial inoculum, carrier of the microorganisms Wenelen DSM 16786 and Licanantay DSM 17318.

To allow the growth of the microorganisms in the reactor, air enriched with 0.5% C02 was supplied. The temperature of the reactor was controlled at 30 ° C. The pH in the reactor was controlled by the addition of H2SO4.

During the operation of the reactor, the growth of microorganisms was monitored by microscopic counting using a Petroff-Hausser chamber.

The characterization of the microorganisms present in the reactor was performed by the quantitative PCR technique (qPCR).

The reactor was operated in batch mode for 7 days, at the end of which the continuous operation of said reactor, by feeding the culture medium of the composition indicated at a rate of 360 L / h.

During the continuous operation phase of the reactor, samples were taken for characterization by qPCR.

RESULTS OF EXAMPLE 3 As can be seen in Figure 3, the continuous operation of a bioreactor, using a modified medium with the incorporation of pyrite concentrate, effectively allows the propagation of microorganisms of the At species. ferrooxidans and At. thiooxidans.

ADVANTAGES OF THE INVENTION In order to evaluate the decrease in costs of the culture medium resulting from the incorporation of a pyrite concentrate, a stack of 2000 tons is considered; irrigated with a flow of 480 L / h; with continuous inoculation at a concentration of 1-108 cells / mL.

The conditions indicated determine a production need of 360 L / h of a culture of microorganisms, at a concentration of 1.3-108 cells / ml. If a value of US $ 350 per ton of ferrous sulphate is considered, at a At the concentration of 8 g / 1 of ferrous sulphate, the total replacement of said reagent by concentrate (I) of pyrite would result in a saving of $ 8,830 per year. Typical tasks of copper mining involve the biolixivation of more than 2 million tons of ore per year (for example the Cerro Colorado site in Chile), so the savings associated with the use of pyrite instead of ferrous sulfate and a source of sulfur separately are more than US $ 8 million per year.

Claims (1)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as novelty, and therefore the content of the following is claimed as property: CLAIMS 1. Process for the joint cultivation of a consortium of microorganisms of the Acidithiobacillus thiooxidans type and of the Acidithiobacillus ferrooxidans type, CHARACTERIZED said process because it comprises the following stages: a) preparing a culture medium for microorganisms of the Acidithiobacillus thiooxidans type and of the type Acidithiobacillus ferrooxidans replacing a part of it with pyrite; b) adjust the pH value of said medium between 1.5 and 2.5; c) inoculating the culture medium with a mixture of microorganisms of the Acidithiobacillus thiooxidans type and the Acidithiobacillus ferrooxidans type, with or without other microorganisms; d) adjust the temperature to a temperature between 25 and 35 ° C; e) pass an air stream enriched in C02 with between 0.20% and 0.80% C02. Process according to claim 1, characterized in that the part of the culture medium that is replaced is that corresponding to reduced iron or sulfur compounds, such as ferrous sulfate and elemental sulfur. Process according to claim 1, CHARACTERIZED because the culture medium with pyrite considers an amount of pyrite between 1 and 20 grams per liter. Process according to claim 1, CHARACTERIZED because the microorganisms of the Acidithiobacillus thiooxidans type and of the type Acidithiobacillus ferrooxidans that are grown are isolated microorganisms. Process according to claim 4, characterized in that the microorganism of the Acidithiobacillus thiooxidans type is Licanantay DSM 17318 and the microorganism of the Acidithiobacillus ferrooxidans type is Wenelen DSM 16786. 6. Process according to claim 1, CHARACTERIZED because the culture pH is 1.8. Process according to the CHARACTERIZED claim because the temperature of the culture controls to be 30 ° C Process according to the CHARACTERIZED claim because the air is enriched with C02. Process according to the CHARACTERIZED claim because the ratio volume of inoculum microorganisms to volume of culture medium is between 1:20 and 1: 5.