BRPI0806002B1 - CONTINUOUS INDUSTRIAL PROCESS FOR PRODUCTION OF XANTHOMONAS CAMPESTRIS REACTIVATED CELLS - Google Patents

Abstract

continuous industrial process for production of reactivated cells of xanthomonas campestris. The present invention relates to a continuous industrial process for producing a constant and defined concentration dispersion of xanthomonas campestris cells, which dispersion is usable as inoculum in a fermentative process for producing xanthan gum under controlled conditions. The invention describes a controlled cell growth process in three interconnected reactors, one of which is periodically fed with the contents of a known volume ependorf of microorganism cells of interest and where the diluent of the entire process and any of the culture media. which permit cell growth and which may be used alone or in conjunction with the usual cell growth ym system for xanthomonas campestris or other xanthomonas species. This system is automatically controlled by measuring the content of the formed cells and predefining this cell content and leading the process to provide an adequate volume of a dispersion with a perfectly defined cell concentration for inoculation into the fermentation reactor.

Description

(54) Title: CONTINUOUS INDUSTRIAL PROCESS FOR PRODUCTION OF REACTIVATED XANTHOMONAS CAMPESTRIS CELLS (51) Int.CI .: C12M 1/34; 1/20 C12N; C12R 1/64 (73) Holder (s): QUANTAS BIOTECNOLOGIA S.A .. NATIONAL INDUSTRIAL LEARNING SERVICE, BAHIA REGIONAL DEPARTMENT - SENAI / DR / BA (72) Inventor (s): ALESSANDER ACACIO FERRO; MARINO TADEU FABI; JOSÉ FERNANDO BARRETO DA SILVA; LUCIO JOSÉ SOBRAL ROCHA; LUCA PESSOA BUZANELLI; EDSON QUIRINO BUZANELLI

1/37

CONTINUOUS INDUSTRIAL PROCESS FOR PRODUCTION OF REACTIVATED CELLS OF Xanthomonas campestris

Field of invention

The present invention relates to a continuous industrial system for the production of a dispersion of constant and defined concentration of Xanthomonas campestris cells, the dispersion being usable as an inoculum in a fermentation process for the production of Xanthan Gum under controlled conditions.

Xantana Gum is a biopolymer with uses in many branches of industry, such as food, cosmetics, pharmaceuticals, textiles, explosives and oil. In addition, such gum exhibits excellent characteristics as a viscosifier and has significant effect as a stabilizer in emulsions as well as excellent saline resistance and great resistance to pH changes, as well as stability to enzymatic action, which makes the use of xanthan gum as an additive increasing. important in a considerable plurality of processes and chemicals.

State of the art

Xanthan gum is produced by the action of bacteria of the species Xanthomonas campestris or other species of Xanthomonas in the process of aerobic fermentation from a medium where there is a source of carbon, a source of nitrogen and some mineral nutrients containing potassium, magnesium, phosphate and sulfur to its structure.

This biopolymer has a molecular mass in the range of 0.5 to 12 million daltons and consists molecularly of a chain of carbohydrates in which glucose, mannose and acid

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2/37 glycuronic in the ratio of 2.8: 2.8: 2 are arranged in a main chain of D-glucopyranose Glucan (glucopyranose link 1,4). There is a branch every two glucoses made up of an acetylated D-mannose that is linked to that glucose. To this acetylated D-mannose is connected a d-glucuronic acid and this is connected to a second pyruvatized D-mannose, as shown below:

D PIRUVATIZED MANNOSIS

STRUCTURE OF THE XANTAN GUM

The polymer is anionic and the sodium cation accompanies the organic molecule in most xanthan gum products. In addition, nitrogen contents of the order of 1% may also be present in xanthan gum, as a rule as a protein material associated with the main chain.

There are, in the state of the art, numerous publications

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3/37 referring to the production of water-based polysaccharides, of the xanthan gum type, US Patents 3,020,206, US 3,251,749, US 3,391,060, US 3,271,267, US 3,427,226, US

3,433,708, US 3,455,786, US 3,594,280, US 4,154,654 and US

4,282,321.

The usual procedure for the production of xanthan gum involves carrying out a fermentation in two different stages. First, the microorganism to be used is subjected to a suitable small-scale culture in order to obtain a relatively high concentration of bacteria. In this phase, the bacterium does not produce the exopolymer or produces it in a very low concentration. The population of microorganisms grows significantly at this stage. That is why it is called the germination or growth phase.

Thereafter, the material resulting from this first stage, having a relatively high concentration of bacteria, is inoculated into the larger reactor containing an appropriate culture medium and under controlled aeration and perfectly adjusted reaction conditions so that the fermentation stage itself develops. . This is the production or fermentation stage as it is also called.

Dozens of patents and articles of importance in the chemical literature related to this subject show that cell growth goes through two phases, the first being the log phase that lasts, from inoculation to 15 to 34 hours and the second, the stationary phase in which the growth is still there, but much less intense.

Papagianni et al., Process Biochem, 37,

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4/37

73-80 (2001), has a time of 42 to 44 hours to reach this lag phase of growth.

The article by Pons, Dussap and Gros, Biotec and Bioengineering, 33, 1989 clearly presents the relationship of bacterial growth, whether in terms of biomass content as a function of the parameters of the process itself. The authors highlight the importance and total dependence in relation to the nitrogen content consumed by the bacteria to define the biomass yield formed. A value for biomass yield expressed in grams of total microorganism cells formed / grams of nitrogen consumed, in the order of 8.48, is found by the authors and similar values are also highlighted by other research groups.

The authors also determine the maximum specific growth rate of cells as μ = 0.5 h ^-1 (first order velocity constant) which corresponds to a concentration of cells in the stationary phase after the log phase of the growth process. 1.8 g cells per liter of solution, in the growth medium used by them.

Several growth media are presented throughout the literature, with the YM or MY medium as initially named prevailing.

Piches and Pallent, Biotec and Bioeng., 28,1484, (1986), mentioning the work of Wiensroek and Klein. (US Patent No. 4,374,929) and using the growth medium consisting of KH ₂ PO ₄ 0.88, K ₂ SO ₄ 0.54,

MgSO ₄ .7H ₂ 0, CaCl ₂ .2H ₂ O 0.056, citric acid 0.167, FeCl ₃

0.01, glucose 22.5 and trace elements mg / kg (ZnSO ₄ 1

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5/37 as Zn, CuSO ₄ 0.6 as Cu, MnSO ₄ , H ₃ BO ₃ 0.1 as B, NaMoO ₄ 0.2 (as Mo) and KI 0.1 as I highlight a log to lag time 28-32 hours.

Weiss and Ollis, Biotec and Bioeng., 22,859, (1980), present the following equation:

X (t) = X0 e / Renda1.0 - (X0 /Xmax)(1.0 - e ^)]

Where X0 is the initial concentration of biomass in the inoculation;

X (t) is the concentration of cells at time t of cell growth;

Xmax is the maximum concentration of cells that

develop in a certain condition in middle in growth. In the work of Moraine and Rogovin, one graphic is 15 displayed showing that cell growth what part

from zero to increasing (log growth phase) reaches a maximum of 2.45 g / kg in approximately 40-45 hours of growth and remains at that level for up to 85 hours (growth lag phase) when it starts to fall and reaches 1, 6 in approximately 100 hours. For these authors, and considering the data from Moraine and Rogovin Biotec and Bioeng., 13, 381, (1971), the maximum concentration of cells,

Xmax is in the order of 2.45 g of cells per kilo of growth broth (0.25%).

Thompson and Ollis, Biotec and Bioeng., 22,875, (1980), use, as a growth medium, an aqueous solution of 2% dextrose, 0.8% of dry soluble diastyl extract, 0.5% of KaHPO ₄ and 0 , 01% MgSO ₄ ,

Cadmus et al., Biotec and Bioeng., 20, 103-1014, (1978), use the YM growth medium in two stages since

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6/37 a lyophilizate of Xanthomonas campestris cells and two initial stages of the growth phase and in the third stage of this phase, they use the YM-T medium which consists of 1.2% glucose, 0.15% yeast, 0, 15% malt extract,

0.25% peptone, 0.15% diamonic phosphate and 0.15% dipotassium phosphate and 0.005% anhydrous magnesium sulfate.

Moraine and Rogovin col., Biotec and Bioeng., 15, 225, (1973), present cell growth data and indicate that the maximum concentration of cells is obtained at a growth time of the order of 40-42 hours and state that the specific speed of cell formation is directly related to the nitrogen content of the organic nitrogen source used in the case the DDS with a maximum initial growth rate equal to 0.25 / hour and a maximum equivalent saturation of 0.008% of the nitrogen present in the DDS. The authors claim that almost half of the nitrogen available in the medium remains unchanged when cell growth ceases.

The Honnma, Higehiro and Kanji patent, US 5,580,763 is an excellent indication of a wide variety of growth and production media for xanthan gum. The authors recommend the use of ammoniacal nitrogen and an oily substance like soy milk.

The MY growth medium is used by the authors and consists of 0.3% malt, 0.3% yeast, 1% dextrose and 0.5% peptone.

Outline of the problem solution

Despite all this background of information on the cell growth process in the case of Xanthomonas campestris, it is verified that in all mentions of

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7/37 effective jobs and patents, the cell growth stage has no effective control during its realization. In addition, the actual concentration of cells of the microorganism at the end of the growth stage and which is effectively inoculated in the biopolymer production reactor is never mentioned or even unknown. Thus, it is based on empirical knowledge or the application of kinetic equations within complicated reaction models, as indicated above in the documents cited in the prior art and without taking into account the true content of cells that feed the fermentation reactor.

In the state of the art related to the synthesis of Xanthan Gum, empirical knowledge is used in the phases of cell growth itself that occur from the Petri dish to the inoculation itself and, thus, in the volume of inoculum added to the fermentation reactor that allows control of the manufacturing process.

This implies not having the value of the initial concentration of cells that is fed to the fermentation phase. It should be noted that the monitoring of the process takes place according to the time of growth, where the time that the cell mass must be left growing before being transferred to the fermenter is defined.

Therefore, one can have very variable cell concentrations when considering the cell growth stage.

The fact of having a concentration of cells that is not known at the end of the growth stage, which can vary significantly from batch to batch, makes the control of the entire process that directly involves the

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8/37 production step is always carried out empirically. Therefore, results are obtained with significant differences not only in the micro and macroscopic properties of the biopolymer obtained, but also reflecting in significant differences in quality and productivity of the xanthan gum obtained at the end of the process.

In order to solve this problem, said invention proposes a continuous system for the production of a dispersion of Xanthomonas campestris cells or other species of Xanthomonas and related bacterial microorganisms, in aqueous solution with a perfectly defined, adequate and constant cell concentration direct inoculation in the fermentation reactor for the production of Goma Xantana biopolymer.

In this way, it is always possible to introduce the same cell content (concentration) in the fermentation reactor leading to a significant gain in process control and product quality compared to the result of the productions according to the state of the art synthesis methodologies. xanthan gum. Thus, the invention establishes a control so that the cell concentration is quantified and known in the solution that is inoculated to the fermenter.

Brief description of the drawings

Figure 1 represents the process flow diagram for preparing a dispersion of Xanthomonas campestris cells of determined concentration of the present invention.

Detailed description of the invention

The continuous dispersion preparation system

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9/37 determined concentration of Xanthomonas campestris cells consists of three reactors (A), (B) and (C), a diluent storage tank (D), a continuous analyzer (R) of microorganism cells, a filter (F) and a level meter (U), in addition to the pipes, pumps and valves that allow its continuous operation.

The reactor (A) is the reactor for cell inoculation and feeding the continuous growth system of these cells.

Reactors (B) and (C) are the effective reactors that allow the continuous process of cell growth. The reactors (B) and (C) both have a V (liter) capacity. The volume V is on the order of 2 to 10 liters, better from 3 to 7 liters and even better from 4 to 6 liters.

The reactor volume (A) ranges from 1/20 to 1/10 of the reactor volume (B) and (C).

The volume V is generally equal to 1/10 of the volume of the corresponding fermentation reactor (E) when it has a capacity of 30 to 80 liters.

However, the invention is not restricted to these values only, and larger volumes can be used when larger amounts of inocula are used in the fermentation reactors of the xanthan gum industrial unit.

The aforementioned reactors (A, B and C) and also the diluent storage tank (D) are made of glass or stainless steel, usually 316, jacketed and with temperature control that allows working at defined and constant temperatures in the 26 to 30 ° C.

Such reactors do not have straight corners that cause

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10/37 scale or any similar process that may cause contamination of the medium contained therein.

Both the reactors and the diluent storage tank (D) are equipped with vents for the atmosphere, thus balancing the pressures, and the air entering the system is filtered and sterilized, protecting the system against natural contamination.

The reactors have magnetic stirrers perfectly defined in terms of shape and speed, thus providing an adequate and automatically controlled agitation speed to the medium.

The diluent storage tank (D) is able to feed each of the reactors (A, B and C) in order to maintain the dynamics of the process.

The supply to the reactor (A) that comes from the diluent storage tank (D) is controlled by the valve (1) and is performed by phase when the contents of the reactor (A) are transferred integrally to the reactor (B).

The reactor supply (B) that comes from the diluent storage tank (D) is controlled by the valve (2) and a liquid flow regulator system so that a defined flow is continuously fed to the reactor (B). This flow is established being in the order of 0.5 to 2.0 ml / min depending on the volume of the reactors (A) and (B) themselves.

A second piping also allows the diluent storage tank (D) to feed the reactor (B) by quickly introducing the diluent to the said reactor. The diluent flow is controlled by the valve (3).

The valve (3) when opened causes it to be inserted,

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11/37 in practically instantaneous time, a volume of diluent equivalent to the reactor capacity (B) which is regulated by the level meter (U). This valve (3) is opened whenever the contents of the reactor (C) are transferred to the transfer bottle (G) and from there to the fermentation reactor (E). Such transfers are carried out synchronously and with the transfer of the contents of the reactor (B) to the reactor (C).

The reactor supply (C) that comes from the diluent storage tank (D) is controlled by a liquid flow regulator system coupled to the analyzer (R) that continuously monitors the content of cells present in the reactor (C) .

The inline analyzer (R) is of the spectrophotometric type and is operable at a defined wavelength and fixed at 630 nm. This value is the wavelength value sensitive to small particle content in aqueous dispersion. He then points, continuously, the cell content in the reactor (C). The value indicated by the analyzer (R) acts on the diluent inlet valve (6) in that reactor (C). Every time the concentration of cells becomes greater than the defined value, which is a function of cell growth itself, the valve (6) that gives access to the reactor is opened and the diluent is introduced causing the concentration of cells to drop until the fixed value.

The filter (F) located between the reactors (B) and (C) operates in the direction from (C) to (B). Said filter is a 3 micron Millipore filter. Its function is to retain cell debris and debris that have been produced by the cell growth process so that the fluid

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12/37 goes to the reactor (B) to be clear and without these residues, being recycled to the reactor (C) sufficiently clean and free of such macroscopic by-products of the bacterial development process.

When the cell content in the reactor (B) is adequate, the entire content of the reactor (C) is transferred to the process sequence.

The cell content in the reactor (B) is considered adequate when it is equivalent to the pre-fixed cell content and corresponding to a residence time of the cells in the cell growth system (reactors B and C) from 28 to 42 hours, depending on the diluent and other conditions.

In general, the volume of the reactor (C) is fully transferred after the valve (4) that joins the reactors (B) and (C) is closed. This transfer is made quickly and ends the cell growth cycle initiated from the inoculation of the contents of the Ependorf to the reactor (A).

Once the transfer is made, the diluent is immediately introduced to the reactor (B). The valve (4) that joins the reactors (B) and (C) is opened again so that the growth cycle already developed in the reactor (A) and transferred to the reactor (B) continues uninterruptedly. In this way, the cell dispersion production system operates continuously.

Possible thinners to be used in the process

The diluent mentioned in the dispersion preparation process can, in principle, be any composition that fulfills the objective of allowing the proper growth of the microorganisms under consideration. Therefore, the diluent

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13/37 can be the classic growth medium called YM as well as other compositions as long as it allows the growth of cells in a controlled manner. Such dispersion obtained has a constant and predetermined concentration of the xanthomonas campestris cells.

The composition of the YM medium is as follows:

In the present invention, the YM medium is especially mentioned as a diluent. However, due to cost-benefit aspects, any aqueous medium, in principle, can be used as long as it contains a carbon source, a nitrogen source, some mineral nutrients that contain potassium, magnesium, phosphate and sulfur in its structure and a strain of bacteria of the genus and family xanthomonas. The carbon source must have a low carbohydrate content, being preferably a reducing sugar like glucose, but it can also be a disaccharide like sucrose, dextrose or any other. The source of mineral or organic nitrogen may at first come from a wide variety of possible nitrogenous substances, and may also contain the usual malt and yeast used for cell growth in numerous patents and works in the literature since the discovery of xanthan gum already known in the state of the technique. In the present invention the source of nitrogen used in the YM medium was peptone.

The classic composition of malt and yeast is shown below.

Mixed dilution media can also be used. As an example, we can mention the use of the YM dilution medium in the reactor (A) and the introduction of a second culture medium with a lower content of yeast and malt. Or else,

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14/37 this second culture medium can have zero levels of these two components, or these two components can be replaced by eventually another or other substances that can allow cell growth without blocking the subsequent production of xanthan gum to occur in the fermentation reactor (E) to which the complete reactor content (C) is transferred.

Operating examples presented below indicate some of the usable dispersants, not restricting this invention only to the dispersants indicated herein.

Operation details of the continuous process of preparation of dispersion of determined concentration of Xanthomonas campestris cells

More particularly, according to this invention, a dispersion containing a defined concentration of Xanthomonas campestris cells is continuously prepared in a controlled manner and available for inoculation in the xanthan gum biopolymer production reactor.

In this patent application, we refer to 20 experimental data corresponding to the production of Xantana Gum and, therefore, related to the microorganism Xanthomonas campestris and other microorganisms of related species of Xanthomonas. However, the invention also refers to all other systems of production of water-soluble biopolymers in terms of their growth stages of microorganisms that, with their fermentation, result in such exo-polymers of biological origin.

According to the procedure described in that invention, all operations are carried out in clean room conditions and all material is sterilized at 121-126 ° C for two hours

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15/37 before being used.

The system consists of three reactors (A), (B) and (C) and attachments made of suitable material, glass or 316 stainless steel and confined in a clean environment, preferably within a laminar flow system. The reactors are jacketed and the external fluid maintains the temperature accurately in the range of 27 to 28 ° C.

first glass reactor (A) of capacity V / 10 ml (where V is the volume of reactors B and C), contains a defined volume of the sterile solution of the YM medium and in it is added the content of 3 ml of an Ependorf kept in cryogenic vessel until the moment of this transfer.

The first reactor (A) is kept in agitation, with the magnetic stirrer specially defined for the desired rotation.

The second reactor (B), equipped with an analog magnetic stirring system, with a capacity of V liters, also jacketed and maintained at 27 ° C, is connected to the reactor (C) by suitable pipes, the flow being driven by pumps adapted to the desired flow rates and which can be of the piston mechanical type, peristaltic or other designs suitable for low flows of the order of 0.1 to 1 ml / min.

Preparation of the initial inoculum preserved in bottles

Ependorf

Performed in laminar flow in a fully sterile room. From a defined strain of the Xanthomonas campestrís bacterium, NRRL B1459, cell growth is carried out in a Petri dish, selecting colonies of appropriate size and after that, transferring an adequate amount of these cells to Erlenmeyer-sized cells.

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16/37 convenient, preferably 125 ml and kept in Shaker for 24 hours in YM growth medium.

Then, the contents of this Erlenmeyer are transferred to a 1000 ml Erlenmeyer and inoculated to 500 ml of solution

YM and placed in Shaker for another 24 hours.

Then, 3 ml volumes of this solution are transferred to Ependorf flasks, which are closed and kept in a cryogenic vessel for the necessary time and only removed at the moment of inoculation in the reaction system presented in the present application.

In this way, the stock of a few dozen, hundreds or even thousands of Ependorf flasks containing frozen solution of Xanthomonas campestris cells is kept in the cryogenic vessel and can be continuously sent, whenever necessary, to the respective process. After the continuous cell dispersion production system, with a known concentration, is in full operation, a volume of 1 to 2 liters of the product of that system is transferred to Ependorf flasks and these are kept in a cryogenic vessel.

Process start-up

In the example developed, the volume V chosen for reactors (B) and (C) was 5 liters. (V = 5 liters). The description is presented for any volume V in the range of 2 to 10 liters, preferably 3 to 5 liters.

The contents of an Ependorf are fed under absolutely clean conditions to the V / 10 liter reactor (A), and the flask is washed with the diluent. V / 15 ml of the diluent is added and the system is allowed to stir for 24 hours.

Then, the content of this reactor is entirely

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17/37 transferred to the reactor (B) by opening the valve (5). The reactor (B) contains V / 2 liters of the diluent at the start of the system and receives continuously, controlled by the valve (2), after transferring the contents from (A) to (B), a flow of 0.5 to 1, 5 ml / min of diluent and it is kept in agitation for another 24 hours keeping the valve (4) that joins the reactors (B) and (C) closed. The level meter (U) in the reactor (B) defines the opening moment of the valve (4) causing the liquid to enter the reactor (C) and start filling it. The fluid levels in the reactors (B) and (C) are equal and as the feeding in (B) continues, the level will rise until reaching the pipe (t) that joins (C) and (B) from the top and where a filter (F) is inserted. Before the filter (F) there is a pump that forces the liquid to pass through the filter (F) without losing travel speed.

The filtered fluid enters the reactor (B) closing the cycle.

Meanwhile, in the reactor (A) after transferring its contents to the reactor (B), a second load of diluent is fed through the tubing and with the opening of the respective valve (1) the cell growth is initiated by repeating the cycle transferring its volume to the reactor (B) after 24 hours as already described.

Continuous operation

The reactor (B) is fed continuously with the diluent solution at a flow rate of approximately 0.5 to 1.0 ml per minute, preferably 0.7 to 0.9 ml per minute for the volume of V = 5 liters controlled by an injection system of the type used in chromatographic apparatus of

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18/37 high performance liquid chromatography or flow analysis device (FIA).

Every 24-hour period, the contents of the reactor (A) are transferred to the reactor (B). The fluid cycles through the reactors (B) and (C) and is filtered through the filter (F).

The continuous analyzer (R) controls the content of cells in the reactor (C) and the valve (4) is kept open, making the fluid circulate in the appropriate direction and being filtered in the corresponding filter (F), eliminating the cellular residues from its constitution.

The pump and valve system is all controlled automatically although it allows manual operation.

If the cell content exceeds the appropriate value, a volume of diluent is added to the reactor (C) through the pipeline and the valve (6) to the point of diluting the solution until the concentration is adequate.

The fluid is circulated and cell growth is established and when the cell content indicated by the analyzer (R) in line is at the appropriate value, the volume V of the reactor (C) is transferred directly to the transfer flask (G). The transfer bottle (G) can be kept leaving the clean room and can be taken for direct inoculation in the fermentation reactor (E).

Always transfer to the transfer bottle (G) with the dispersion in the appropriate concentration.

For the transfer of this volume, the valves (6) and (4) are closed and the valve (7) is opened until the contents of the reactor (C) are exhausted. After that, the valve (4) is opened again and the liquid from the reactor (B) passes to the reactor (C) causing the cycle to start again with the addition

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19/37 of the supplementary volume of the diluent sufficiently so that the fluid begins to circulate through the filter (F) in the pipeline (t) again.

Thus, feeding the reactor (A) with the contents 5 of the Ependorf flask at appropriate times, on average every twenty-four hours, and adequately adjusting the volume of diluent and the flow rates of each flow, the system will always release a volume fixed V of aqueous dispersion of Xanthomonas campestris with a determined concentration and suitable for fermentation.

Operation and adjustment of the inline analyzer, process filter and Ependorfs content injection system, in the system

The reactor (C) is controlled by an automatic spectrophotometric analyzer (R) 15, operating in the visible range at 630 nm. By this analyzer (R), the cell concentration of this aqueous dispersion is continuously measured and regulated to a fixed value expressed in mg of cell per volume of aqueous dispersion in diluent, the content of which leads to the best performance of the production process of xanthan gum. This value is chosen experimentally and defined according to the diluent used.

An orientation value of 2.45 g of cells per kilo of growth broth (0.25%) already mentioned in the state of the art in the work of Moraine and Rogovin, serves as a basis for the standardization of the analyzer system and for the definition of the value to be achieved and fixed so that the system of this invention can produce. This value was adopted throughout the procedure, however, nothing prevents other values, in the range of 2-3 g cells per kilo of

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20/37 growth broth, be adopted and fixed for the operation of the system proposed in this invention.

This value of 2.45 g of cells per kilo of growth broth (0.25%) was experimentally determined by

Moraine and Rogovin and corresponds to the optimum content of cells present in YM fermentation broths after the growth stage and which provides the best fermentation performance when inoculated in the fermentation reactor loaded with the traditional SM medium known in the state of the art regarding the synthesis of xanthan gum. For this reason, it was pre-fixed for the desired concentration to be produced by the system of this invention and experimentally, it proved to be suitable for the respective operation. From then on, it was adopted in the entire sequence of operation of this process of production of dispersion of defined concentration of Xanthomonas campestris cells.

In the experimental sequence of standardization of the analyzer (R), an absorbance curve at 630 nm as a function of the cell mass is established. A turbidimetric analyzer can also be used for this reaction monitoring.

The valve (6) that joins the diluent storage tank (D) and the reactor (C) is thus activated depending on the concentration of cells measured in that reactor. If the value of this concentration, indicated by the analyzer (R) in line is higher than the desired value, the valve (6) is opened so that the diluent can be added to the reactor (C) sufficiently for the concentration to adjust to the value established. This procedure does not allow the viscosity of the dispersion to exceed the appropriate value for the

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21/37 smooth operation of the pump system that gives dynamics to the process.

Between the reactors, there is a Millipore filter (F) that eliminates particles with a diameter greater than

3 microns, containing the cells, which normally characterize the debris and waste produced by the microorganisms, so that the solution is recirculated. Filter media other than the Millipore filter can also be used in the filtration system proposed by this invention.

This filter has a self-cleaning system that allows frequent cleaning of the filter allowing its integrated operation throughout the process.

Thus, the concentration in the reactor (C) is always maintained at a defined value.

Every interval of approximately 36 to 46 hours, we have a dispersion volume of cells of the microorganism in the appropriate concentration and that can be transferred integrally to one of the fermentation reactors (E). This time interval can be longer or shorter depending on the cell development and the process parameters.

The excess cell dispersion is kept in a separate reservoir, at a temperature of 15 ° C, which is also periodically monitored by the spectroscopic (R) analyzer.

The contents of an Ependorf are transferred with the aid of a syringe to the reactor (A) equipped with a septum and injector with a liquid chromatography device, avoiding contamination and corresponding to a defined volume transfer and safely to the reactor (A) starting

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22/37 growth. After injection, the system including the syringe is carefully washed and sterilized and only installed at the time of inoculation of the contents of the next Ependorf.

Pasteurization of the diluent solution

The pasteurization of the diluent solution that feeds the continuous cell dispersion preparation system of determined concentration of Xanthomonas campestris is always carried out properly at 121 ° C, heating the carbohydrate, which is the glucose in case the diluent is the YM medium, separately from the rest of the composition.

Thus, in this case, glucose is pasteurized separately and added to the other constituents of the diluent solution at the time of preparation of the diluent solution and this solution being kept in a larger reservoir, also kept inside the environment and feeding the storage tank every 24 hours. diluent (D) so that the dispersant is always available for the operation of the system itself.

In all other diluent media, the carbohydrate is pasteurized in isolation and added to the aqueous medium of the other components.

The diluent media mentioned in the examples constitute only some of those used or possible to be used in the continuous dispersion preparation system of Xanthomonas campestris cells of determined concentration.

Each of these thinners may have slightly different procedures in the pasteurization process, which is perfectly understandable by a technician in the subject already

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23/37 accustomed to processes of this nature.

Preparation of Xanthomonas campestris cell dispersion of determined concentration in the continuous process of the present invention

With the process operating under suitable conditions, a dispersion of cells of perfectly adjusted concentration is produced. The concentration is given in mass of cells per volume of solution, being defined according to the cell growth kinetics itself and being perfectly controlled. This perfectly adjusted concentration is the optimal concentration to be inoculated directly into the fermentation reactor (E). With this, a greater quantity of Xanthan gum is obtained and the fermentation stage is better controlled, impacting the results of the entire process.

Thus, the greater reproducibility of the fermentation step is guaranteed since it always starts with the same initial concentration of cells, making the fermentation step much more controllable when compared to the syntheses of xanthan gum carried out in the prior art.

The system also allows you to set the control values to different reference points. For example, a cell dispersion with twice the cell concentration of the entire study and exactly defining the flow time conditions to obtain it.

Response curve: Cell concentration x

Absorbance

The results obtained are of cell concentration as a function of the measurements of the in-line analyzers at each growth time, as shown in table 1. This curve of

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24/37 response represents the way in which the absorbance of the solution varies with the concentration of the cells.

Table 1

Response curve g / kg of solution absorbance 0, 00 0.015 0.10 0.030 0.20 0.065 0.50 0.02 0.75 0. 175 1, 00 0.225 1.50 0.350 1.75 0.400 2.00 0.530 2.25 0.600 2.45 0.630 2.50 0.700 2.75 0.800 3.00 0.950 3.35 1.100 3.50 1. 115 3.75 1,200

This response curve, like all others, has a linearity range where there is a direct correlation between the data and a range in which the respective correlation loses linearity (usually at higher values). The linearity range of the curve is established from 0.50 to 3.00 g / kg of solution.

In the linearity range, the absorbance x concentration in grams / kg curve is reliable and provides the

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25/37 linear coefficient and the angular coefficient that were introduced in the device in line. The curve has a correlation coefficient of 0.9999. In this linearity range, it is noticed that the desired concentration point falls in the perfectly linear range and that lower concentrations than this also make the process work.

The value defined as the desired concentration is 2.50 g / kg of solution which corresponds to a measured absorbance of

0.700. This was also the value established for the automatic control point of the entire system. The value of 2.50 g / kg is the value that defines the end of the log phase of bacterial growth and the start of the lag phase of the same growth, according to data already known in the state of the art.

This concentration value is also the value of the optimal concentration of the cell dispersion in the system.

With this response curve and calibrated with a frequency of two days at most, cell growth and absorbance measurements of solutions are established in the analyzer (R) that accompanies the reactor (C).

The diluent compositions in the examples below are always for the diluent added to Reactors (B) and (C). The composition of the diluent in the Reactor (A) is always that of the YM culture medium, that is, 0.15% yeast extract,

0.15% malt extract, 0.25% peptone and 0.98% glucose. (3 g of yeast extract, 3 g of malt extract, 5 g of peptone, 20 g of anhydrous glucose, QSP for 1 liter with water).

Example 1

In a system according to figure 1, the reactor (A) has

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26/37 capacity of 100 ml, the reactor (B) and the reactor (C) are 4 liters and the storage tank (D) is 15 liters. The other accessories are compatible with these dimensions.

Initially, 15 liters of YM diluent solution 5 were prepared. However, each day, five liters of this solution are prepared, sterilized and placed in the storage tank (D).

O reactor (A) received 50 ml of the thinner YM. Beyond addition, the content of an Ependorf was transferred to the reactor 10 (A) with aid from a syringe and the agitation of referred

reactor was started.

After 24 hours, the contents of the reactor (A) were totally transferred to the reactor (B) which already contained 3 liters of the YM medium and was closed in its valve (4) in contact with the reactor (C).

The introduction of the diluent started at a flow rate of 1 ml / min and the reactor (B) was kept agitated for 24 hours when the valve (4) for the reactor (C) was opened. In the reactor (C) 2 liters of the diluent were added adding to the volume transferred from the reactor (B).

At that time, the content of a second Ependorf was also added to the reactor (A), starting cell growth in this reactor in the same way as with the previous Ependorf.

The liquid rose in the two reactors (B) and (C) and with 36 hours, reached the pipe (t) containing the filter (F) and the liquid was pumped through the filter (F) again to the reactor (B).

In the meantime, the contents of the reactor (A) were transferred to the reactor (B) and continued to grow in (B)

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The spectrophotometric (R) analyzer indicated absorbance values close to 0.66, with 0.70 being the value defined as the optimal cell concentration. Upon reaching

42 hours, the total volume of the system was equivalent to the two filled reactors and in this condition the absorbance indicated exactly 0.70.

At that moment, the valves were activated and the entire contents of the reactor (C), that is, 4 liters were transferred to the transfer bottle (G) and from there destined to the fermentation reactor (E).

liters of YM diluent were immediately introduced into the reactor (B) and the system continued to produce. At each moment when the absorbance reached 0.70 a cycle was repeated and an Ependorf was added to the system.

The diluent was initially prepared and was fed to the storage tank (D) keeping the tank at an adequate level.

The YM diluent has the known composition of 0.15% yeast extract, 0.15% malt extract, 0.25% peptone and 0.98% glucose.

Example 2

All conditions were kept the same as in Example 1, changing only one component of the diluent which in this case was the YM medium, where glucose was replaced by sucrose in the same content. With a practically equivalent time of growth, a control value was obtained with the analyzer at the same result as in Example 1.

Example 3

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All conditions were kept the same as in Example 1. In the reactor (A), the diluent used was YM. In the sequence of the process, that is, in reactors (B) and (C) and in the entire dynamic system, the diluent used was the medium recommended by Cadmus called YM-T. The YM-T diluent consists of 1.2% glucose, 0.15% yeast, 0.15% malt extract, 0.25% peptone, 0.15% diamonic phosphate and 0.15% dipotassium phosphate and 0.005% anhydrous magnesium sulfate.

With a growth time shorter than that of Example 1, (85% of the time of Example 1) the control value of the analyzer (R) was obtained, that is, the absorbance of 0.70.

Example 4

All conditions were kept the same as for

Example 1. In the reactor (A) the diluent used was YM. In the process sequence, that is, in reactors (B) and (C) and in the entire dynamic system, the diluent used was the YM-T medium. The YM-T diluent consists of 1.2% glucose, 0.15% yeast, 0.15% malt extract, 0.25% peptone,

0.15% of diamonic phosphate, 0.15% of dipotassium phosphate and

0.005% anhydrous magnesium sulfate.

With growth time less than that of Example 1 (80% of the time of Example 1), the analyzer control value (R) was obtained, that is, the absorbance of 0.70.

Example 5

All conditions were kept the same as in Example 1. In the reactor (A) the diluent used was YM. In the process sequence, that is, in reactors (B) and (C) and in the entire dynamic system, the diluent used was the medium

Modified YM-T. The modified YM-T thinner consists of

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1.2% sucrose, 0.15% yeast, 0.15% malt extract, 0.25% peptone, 0.15% diamonic phosphate, 0.15% dipotassium phosphate, 0.005% sulfate anhydrous magnesium and 0.05% citric acid.

With growth time less than that of Example 1 (80% of the time of Example 1), the analyzer control value (R) was obtained, that is, the absorbance of 0.70.

Example 6

All conditions were kept the same as in Example 1. The only difference is that, in this case, a turbidimetric analyzer was used to replace the spectrophotometric analyzer for the measurement and control of cell content. The turbidimetric analyzer was calibrated to the same values as the spectrophotometric analyzer used previously. This calibration also ensured that the system was also compatible and completely controlled by this alternative type of analyzer. However, it was found that the spectrophotometric analyzer provides data with faster response.

Results obtained

EXAMPLE 1 (CONDITION 1 - YM growth medium)

Absorbance curve and concentration Time (h) THE Conc. (g / kg) 10 0.020 0.05 20 0.280 0.09 25 0.275 1.20 30 0.350 1.50 35 0.400 1.75 40 0.530 2.00 45 0.600 2.25

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50 0.700 2.50 55 0.240 1.10 60 0.350 1.50 65 0.400 1.75 70 0.580 2.20 75 0.700 2.50 80 0.290 1.25 85 0.350 1.50 90 0.410 1.80 95 0.608 2.28 100 0.700 2.50 105 0.270 1.35 110 0.407 1.78 115 0.582 2.18 120 0.700 2.50

These data describe the continuous operation, although highlighting regular times every five hours and which illustrate the operation of the system under consideration, with the YM culture medium.

Both in this assay and in the others, in any culture medium, the analyzer setpoint was established at an absorbance value of 0.700.

The zero time value was measured after transferring the contents of the reactor (A) where the first one was inoculated

Ependorf, to the reactor (B). Thus, the absorbance in the analyzer (R) increased with cell growth itself, after feeding the system. In 50 hours, the absorbance value reached the control point. Thereafter, the diluent was added continuously in small volumes keeping the absorbance at the defined value.

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At this time of 50 hours, the cell dispersion was transferred / dispensed to the transfer flask (G) and a cell-free volume equivalent to that removed from the system, that is, 4 liters, was introduced from the storage tank.

5 storage (D) to the reactor (B) and then the circulation by all the system. Was observed that when accomplish this operation, the

The remaining concentration of cells falls, due to the dilution effect, to a value close to that of half and from then on it rises and after about 20 to 25 hours, it reaches the value of 2.50 g / kg again.

Between the first 10 to 50 hours which characterize the system start-up and correspond to the production of the first 4 liters, we have a productivity value of

80 ml / hour which is equivalent to 4000 ml every 50 hours.

From the second subsequent transfer and all the others (4 liters), the production is 160 ml / hour, that is, 4 liters every 25 hours on average.

The system is fed with new Ependorfs at regular intervals of 24 hours. As a result, there is a tendency in the system that the growth time will become shorter as the system works. This is as long as the filter is cleaned properly. In this case, cleaning was performed with 130 hours of system operation.

EXAMPLE 2 - CONDITION 2 (modified YM growth medium)

Absorbance and concentration curve Time (h) THE Conc. (g / kg) 10 0.020 0.06

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20 0.310 0.10 25 0.256 1.17 30 0.344 1.47 35 0.405 1.77 40 0.537 2.02 45 0.599 2.20 50 0.700 2.50 55 0.245 1.12 60 0.344 1.47 65 0.407 1.78 70 0.579 2.17 75 0.700 2.50 80 0.293 1.26 85 0.346 1.48 90 0.404 1.75 95 0.605 2.26 100 0.700 2.50 105 0.274 1.37 110 0.404 1.76 115 0.608 2.24 120 0.700 2.50

These data describe the operation in continuous, although highlighting regular times every five hours and which illustrate the functioning of the system under consideration, with the modified YM culture medium.

Between the first 10 to 50 hours which characterize the start of the system and correspond to the production of the first 4 liters, we have a productivity value of ml / hour which is equivalent to 4000 ml every 50 hours.

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From the second subsequent transfer and all the others (4 liters), the production is 160 ml / hour, that is, 4 liters every 25 hours on average.

EXAMPLE 3 - CONDITION 3 (YM-T growth medium)

Absorbance and concentration curve Time (h) THE Conc. (g / kg) 10 0.020 0.05 20 0.030 0.11 25 0.320 1.40 30 0.405 1.75 35 0.385 1.95 40 0.597 2.22 45 0.700 2.50 50 0.290 1.30 55 0.395 1.89 60 0.599 2.22 65 0.700 2.50 70 0.285 1.30 75 0.382 1.67 80 0.412 1.82 85 0.590 2.17 90 0.700 2.50 95 0.300 1.38 100 0.401 1.91 105 0.604 2.26 110 0.700 2.50 115 0.280 1.27 120 0.358 1.59

The same considerations as examples 1 and 2 apply to

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34/37 example 3.

Between the first 10 to 45 hours which characterize the system start-up and correspond to the production of the first 4 liters, we have a productivity value of

88.8 ml / hour which is equivalent to 4000 ml every 45 hours.

From the second subsequent transfer and all the others (4 liters), the production is 200 ml / hour, that is, 4 liters every 20 hours on average.

EXAMPLE 4 - CONDITION 4 (YM-T growth medium)

Absorbance and concentration curve Time (h) THE Conc. (g / kg) 10 0.022 0.06 20 0.034 0.13 25 0.315 1.36 30 0.380 1.79 35 0.410 1.99 40 0.590 2.19 45-47 0.700 2.50 50 0.315 1.36 55 0.401 1.91 60 0.600 2.24 65 0.700 2.50 70 0.310 1.34 75 0.376 1.78 80 0.405 1.96 85 0.593 2.22 90 0.700 2.50 95 0.308 1.32 100 0.408 1.97

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105 0.591 2.21 110 0.700 2.50 115 0.285 1.24 120 0.381 1.66

The same considerations for examples 1, 2 and 3 apply to example 4.

Between the first 10 to 45-47 hours which characterize the start of the system and correspond to the production of the first 4 liters, we have a productivity value of 88.8-85 ml / hour which is equivalent to 4000 ml every 45 hours.

From the second subsequent transfer and all other transfers (of 4 liters), the production is greater than 200 ml / hour, that is, 4 liters every 20 hours on average.

In these conditions, a productivity of at least 80% is obtained in relation to the productivity of example 1.

An insipient formation of xanthan gum is noted in this situation since the growth medium used in this example is the fermentation medium itself which will receive the cell concentrate in the fermentation reactor. Thus, the viscosity of the dispersion is greater than that observed in examples 1 to 3. There is no quantification of the content of xanthan gum present when the volume of 4 liters is dispensed into the transfer bottle (G). Also, no inconvenience is noted in this experimental condition in this regard. The increase in viscosity of the dispersion causes the need for more frequent cleaning of the filter (F) and a small loss of system pressure on the filter itself, which requires a higher pressure from the pump system. Even so, the whole set can operate at least 250 hours without a major maintenance stop.

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EXAMPLE 5 - CONDITION 5 (modified YM-T growth medium)

Absorbance and concentration curve Time (h) THE Conc. (g / kg) 10 0.024 0.07 20 0.037 0.15 25 0.320 1.43 30 0.376 1.78 35 0.410 1.98 40 0.595 2.23 45-47 0.700 2.50 50 0.305 1.27 55 0.400 1.92 60 0.597 2.26 65 0.700 2.50 70 0.312 1.35 75 0.358 1.62 80 0.390 1.86 85 0.580 2.17 90 0.700 2.50 95 0.310 1.33 100 0.400 1.97 105 0.599 2.28 110 0.700 2.50 115 0.290 1.27 120 0.350 1.66

The same considerations of examples 1, 2, 3 and 4 apply to example 5. The results obtained were also very similar to those of example 4.

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Between the first 10 to 45-47 hours which characterize the start of the system and correspond to the production of the first 4 liters, we have a productivity value of 88.8-85 ml / hour which is equivalent to 4000 ml every 45 hours.

From the second subsequent transfer and all other transfers (of 4 liters), the production is greater than 200 ml / hour, that is, 4 liters every 20 hours on average.

In these conditions, a productivity of at least 80% is obtained in relation to the productivity of example 1.

An insipient formation of xanthan gum is noted in this situation since the growth medium used in this example is the fermentation medium itself which will receive the cell concentrate in the fermentation reactor. Thus, the viscosity of the dispersion is greater than that observed in examples 1 to 3. There is no quantification of the content of xanthan gum present when the volume of 4 liters is dispensed into the transfer bottle (G). Also, no inconvenience is noted in this experimental condition in this regard. The increase in viscosity of the dispersion causes the need for more frequent cleaning of the filter (F) and a small loss of system pressure on the filter itself, which requires a higher pressure from the pump system. Even so, the whole set can operate at least 250 hours without a major maintenance stop.

It will be clear to those skilled in the art that variations in the culture medium can be made, without thereby escaping the inventive concept and scope of the invention. Thus, it is intended that the present invention covers such modifications and variations, as long as they are within the scope of the appended claims and their equivalents.

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Claims (7)

1. Industrial process for the continuous production of reactivated cells of Xanthomonas campestris characterized by comprising the following steps: (1) Reactivation of frozen Xanthomonas campestris cells in the reactor (A), which is the cell inoculation reactor and contains sterile culture medium, every 24 hours; (2) Transfer of the contents of the reactor (A) to the reactor (B) every 24 hours; (3) Adding diluent from the storage tank (D) to the reactor (B) until opening the valve on the reactor connection line (C) when the maximum permitted level indicated by the level meter (X) is reached; (4) Feeding the reactor (C) with the reactor contents (B) and with the diluent of the storage tank (D); (5) Circulation of the cell dispersion between the reactor (B) and (C) through the filter (F) to remove cell debris and circulation by the spectrophotometric analyzer to monitor the cell content; (6) Transfer of cell dispersion from the reactor (C) to the tank (G) when the concentration of cells reaches a value above 2 grams per kilogram of cell dispersion; (7) After step (6), the cell growth process in reactor B is restarted in step (2) with cells already reactivated in step (1) and all process steps are restarted so that the process proceeds continuously. 2. Process according to claim 1, characterized in that the reactors (B) and (C) are the reactors that allow the continuous process of cell growth. Petition 870170085532, of 11/7/2017, p. 10/50 2/4 3. Process according to claim 1 or 2, characterized by the fact that the volume of the reactor (A) is from 1/20 to 1/10 of the volume of the reactors (B) and (C). 4. Process, according to claim 1 or 3, 20 characterized by the fact that the volume of the reactors (B) and (C) are from 2 to 10 liters, preferably from 3 to 7 liters and more 5 preferably from 4 to 6 liters. 5. Process, according to claim 1, characterized by the fact that the volume of the fermentation reactor (E) is 30 to 80 liters. 6. Process, according to claim 1, characterized by the fact that the diluent storage tank (D) feeds each of the reactors (A), (B) and (C) in order to maintain 10 the dynamics of the process in steps (1), (2), (3) and (4). 7. Process, according to claim 1, characterized by the fact that the reactor supply (A) from the diluent storage tank (D) is controlled by the valve (1) being carried out in phase when the reactor content (A) is transferred entirely to the reactor (B). 15 8. Process according to claim 1, characterized by the fact that the reactor supply (B) from the diluent storage tank (D) is controlled by the valve (2) and by a liquid flow regulator system that a stream is continuously fed to the reactor (B). 9. Process according to claim 9, characterized in that the established flow 20 varies from 0.5 to 2.0 ml / min depending on the volume of the reactors (A) and (B). 10. Process according to claim 1, characterized by the fact that the diluent storage tank (D) also feeds the reactor (B) through a second pipe where the valve (3) controls the flow of diluent that enters the reactor (B). Petition 870170085532, of 11/7/2017, p. 11/50 3/4 11. Process according to claim 1, characterized by the fact that the reactor supply (C) from the diluent storage tank (D) is controlled by the valve (6) and by a liquid flow regulator system coupled to the analyzer (R) which continuously monitors the content of cells present in the reactor (C). 12. Process, according to claim 12, characterized by the fact that the in-line analyzer (R) is of the spectrophotometric type calibrated to operate at a wavelength of 630 nm. 13. Process, according to claim 12, characterized by the fact that the inline analyzer (R) can also be a turbidimetric analyzer. 14. Process, according to claim 1, characterized by the fact that the filter (F) is a 3 micrometer porosity filter. 15. Process, according to claim 14, characterized by the fact that the diluents used in the process are the culture medium consisting of 0.15% yeast extract, 0.15% malt extract, 0.25% peptone and 0.98% glucose or sucrose, or consisting of 1.2% glucose or sucrose, 0.15% yeast, 0.15% malt extract, 0.25% peptone, 0.15% diamonic phosphate, 0.15% dipotassium phosphate and 0.005% anhydrous magnesium sulfate. 16. Process, according to claim 1, characterized by the fact that the reactor (A), in step (1), contains a volume of the sterile solution of the medium consisting of 0.15% yeast extract, 0.15% malt extract, 0.25% peptone and 0.98% glucose and receive a dispersion of Xanthomonas campestris cells contained in a microtube preserved at -180 ^o C. 17. Process according to claim 16, characterized in that the microtube content is 0.5 to 3 ml. Petition 870170085532, of 11/7/2017, p. 12/50 4/4 18. Process according to any one of claims 11, 12 or 13, characterized by the fact that when the analyzer (R) indicates the cell content above 2 grams per kilogram of cell dispersion, the reactor content (C ) is transferred directly to the transfer tank (G) in step (6). 19. Process according to any one of claims 11, 12 or 13, characterized in that, when the analyzer (R) indicates a cell content above the value above 2 grams per kilogram, the valve (6) is open so that the diluent from the diluent tank (D) can be added to the reactor (C) so that the cell concentration decreases and adjusts to the value established in step (6). 20. Process according to claim 25, characterized in that the cell dispersion obtained has a concentration of 2.50 g of cells per kg of cell dispersion. 21. Process according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25 or 26, characterized by obtaining a cell dispersion production of at least 160 ml / hour in step (6). Petition 870170085532, of 11/7/2017, p. 13/50 1/1 ν- FIGURE 1