FR2611742A1 - Process for producing cultures enriched with microorganisms and/or with metabolites resulting from these cultures, apparatus for implementing this process and application of the latter, especially to the inoculation and re-inoculation of nitrifiers and water treatment tanks - Google Patents

Abstract

The present invention relates to a process for producing cultures enriched with microorganisms and/or with metabolites resulting from these cultures. According to this process, selected strains of microorganisms are cultured in a bioreactor in the presence of a substrate whose concentration is maintained at values which do not inhibit the growth of the bacteria, products capable of inhibiting the cells are removed from the culture medium and the microorganisms and/or the metabolites are recovered. The invention also relates to an apparatus for implementing this process. Application of this process especially to the inoculation and the re-inoculation of nitrifiers and water treatment tanks.

Description

 The present invention relates to a process for producing cultures enriched in microorganisms and / or metabolites resulting from these cultures, and in particular cultures enriched with nitrifying bacteria, as well as an apparatus for carrying out said method. The present invention further relates to the application of said method to the seeding of different biological media (and in particular to the seeding of nitrification basins) and for the production of metabolites in concentrated solution.

 For many years, the surface and deep waters have seen their nitrogen levels increase regularly and worryingly, and this phenomenon poses enormous pollution problems, both in the field of wastewater and drinking water. To remedy this problem, it is essential to ensure good purification of urban and industrial wastewater before their discharge into the receiving environment (rivers, lakes, oceans, etc ...). Similarly, prior to their introduction into drinking water systems, water intended for consumption often requires treatment for the elimination (or transformation) of nitrogen, to meet the standards for nitrogen compound levels. European drinking water standards, the ammonia level must be less than 0.5 mg / l, the nitrite content less than 0.1 mg / l and the nitrate content less than 50 mg / l.

 Various nitrogen removal (or transformation) processes are used, the most common and least expensive being biological processes. These biological processes involve certain microorganisms, the role of which is to transform the nitrogen present in water in the form of ammonia, into nitrates in a first stage called nitrification. If the nitrate concentration is too high, nitrification is followed by a second step, denitrification, which turns nitrates into nitrogen gas. Nitrification, which allows the oxidation of ammonia to nitrates, takes place in two stages. The first of these stages allows ammonia to be oxidized to nitrites if it is nitritation, whereas during the second Step n °, the nitrite is oxidized to nitrates.

Many microorganisms of the family of
Nitrobacteraceae are capable of promoting either of these two reactions, and the most commonly encountered microorganisms belong to the genera Nitrosomonas for nitritation and Nitrobacter for nitration. However, these microorganisms are very sensitive to variations in their environment, in particular to the quality of the effluent and the concentration of nitrogen compounds, until they cause their disappearance. In addition, the microorganisms have a high doubling time. The treatment of water containing ammoniacal nitrogen thus poses frequent problems of seeding or reseeding of the nitrification basins.

 It has therefore been proposed to seed or reseed water treatment basins with naturally occurring bacteria (but this is a long process). It has also been proposed to seed with activated sludge from nitrifiers or suspension of various bacteria to improve the functioning of these facilities, but none of these methods have led to results. satisfactory.

 It is therefore of the greatest interest to provide a process for the production of large quantities of nitrifying bacteria of the family Nitrobacteraceae, with a view to seeding or reseeding the nitrification basins, in order to alleviate the problems encountered in biological nitrification during the treatment of drinking water or waste water with high nitrogen content.

 The subject of the present invention is a process for producing cultures enriched in microorganisms and / or metabolites resulting from these cultures, characterized in that selected microorganism strains are cultured in a suitable bioreactor in the presence of a substrate. suitable for which the concentration is maintained at values which are not inhibitory of the growth of the bacteria, in that cells which can inhibit the cells, which accumulate in said medium during the fermentation, are removed from the culture medium, and in that the microorganisms and / or the metabolites are recovered in an appropriate manner.

 According to an advantageous embodiment of the process according to the present invention, the cultures of microorganisms are carried out with strains of nitrifying bacteria of the family Nitrobacteraceae.

According to an advantageous embodiment of this arrangement, the nitrifying microorganism cultures use nitritating bacteria of the Nitrosomonas genera,
Nitrosospira, Nitrosolobus, Nitrosovibrio and Nitrosococcus.

According to another advantageous form of this arrangement, the cultures of nitrifying microorganisms use nitrating bacteria of the Nitrobacter genera,
Nitrosococcus and Nitrospira.

 According to a preferred embodiment of the process according to the present invention, the optimum concentration of substrate in the culture medium is kept constant throughout the duration of the culture.

 According to another advantageous embodiment of this embodiment, the substrate concentration is kept optimal by adapting it to the amount of active biomass present.

 According to an advantageous arrangement of the process according to the invention, the control of the optimum concentration of the substrate medium is carried out by continuous dosing of the substrate, and addition of substrate in the medium when the concentration falls below a determined threshold. .

 According to an advantageous embodiment of the process according to the invention, the inhibitory products are removed from the culture medium by an appropriate separation technique, such as, in particular, filtration or centrifugation.

 According to an advantageous embodiment of this embodiment, the control of the presence of inhibiting products, and more particularly the toxic products of the metabolism of the culture, is achieved by continuous dosing and the separation is triggered when the concentration of said products in the culture medium reaches a predetermined rate.

 According to another advantageous embodiment of the process according to the invention, the culture comprises a plurality of successive fermentation stages which each consume a substrate charge introduced at the beginning of each of them with a new charge of medium of culture, in the biomass accumulated during the successive stages of fermentation, and the successive fermentation steps are separated by steps of elimination of the inhibiting products.

 According to yet another advantageous embodiment of the process according to the invention, the culture is carried out in the presence of a substrate whose concentration is kept constant in the culture medium by continuous dosing and addition (s) of substrate from the detecting a substrate concentration lower than a predetermined threshold, and the separation operations are triggered as soon as the concentration of inhibitor products, controlled by continuous dosing, reaches a predetermined threshold.

 According to an advantageous embodiment of the process according to the present invention, the culture is taken batchwise.

 According to another advantageous embodiment of the process according to the present invention, the culture is lifted continuously.

 According to an advantageous embodiment of the process according to the present invention, the cultures are carried out with strains of the genus Nitrobacter.

 According to another advantageous embodiment of the process according to the present invention, the cultures are carried out with strains of the genus Nitrosomonas.

 According to another advantageous embodiment of the process according to the present invention, co-cultures of Nitrosomonas and Nitrobacter are carried out.

 According to an advantageous embodiment of the process according to the present invention, the toxic products of the metabolism of nitrifying bacteria cultures are eliminated when their concentration in the culture medium reaches an inhibitory threshold of the metabolism of said culture.

 According to an advantageous embodiment of the process according to the present invention applied to the production of nitrifying bacteria, the substrate concentration of the culture medium of the nitrifying bacteria, expressed in nitrogen, is advantageously less than 2 g / liter.

 According to an advantageous embodiment of the process according to the present invention, nitrifying bacteria collected continuously or batchwise are either used immediately for seeding nitrification basins for the treatment of polluted or waste water, or preserved by freezing or lyophilization for delayed use.

 The present invention also relates to an apparatus for the production of cultures enriched in microorganisms and / or metabolites resulting from these cultures, characterized in that it comprises, in combination: - at least one reactor, provided with regulation means temperature and pH, controlled air supply means and stirring means, which is further provided with a feed in culture medium and substrate and an output of the inhibiting products and / or useful products of the metabolism of bacteria; at least one separating element of said products associated with said output; a system for continuously dosing the substrate and the aforesaid products respectively; - A control device, slaved to the aforesaid continuous metering system, the introduction of required amounts of substrate in the or a reactor and tripping the operation of the element, or a separating element.

 According to an advantageous embodiment of the apparatus according to the invention, the separating element is preferably an ultra- or microfiltration element.

 According to another advantageous embodiment of the apparatus according to the invention, the continuous metering system comprises a circuit for sampling small amounts of the medium contained in the reactor, associated with means for continuously quantifying the concentration of substrate and / or inhibitory products.

 According to an advantageous arrangement of this embodiment, said quantization means consist of calibrated probe batteries which emit electrical signals.

 According to another advantageous arrangement of this embodiment, said quantization means consist of at least one physicochemical colorimetric assay chain.

 According to yet another advantageous embodiment of the apparatus according to the invention, the aforesaid control device is a device taken from the group of programmable controllers and computers or microcomputers.

 In addition to the foregoing, the invention also comprises other provisions, which will emerge from the description which follows.

 The invention will be better understood with the aid of the additional description which follows, which refers to the appended drawing in which the single figure of the appended drawing represents a diagram of an advantageous embodiment of the apparatus according to the invention. present invention.

 it should be understood, however, that this drawing and the corresponding descriptive parts are given solely by way of illustration of the subject of the invention, of which they do not in any way constitute a limitation.

 The apparatus according to the present invention advantageously comprises at least one bioreactor 1 in which the cultivation of the desired microorganism or microorganisms takes place and the operation of which is controlled from a regulation cabinet 2 which controls and controls the air supply. , temperature, agitation and pH.

A volume of preculture equal to, for example, 10% of the volume of the bioreactor is introduced into the bioreactor 1, which preculture has been carried out batchwise on a medium containing an optimal initial concentration of substrate (for example 200 mg of nitrogen / l in the case of the production of nitrifying bacteria from a strain of
Nitrobacter Vinogradsky: agilis serotype or Nitrosomonas sp.) And the bioreactor is fed to culture medium from a reservoir 16 and substrate from a reservoir 7 '.

 The control of the concentrations of substrate and inhibitory products of the metabolism of the culture of microorganisms in the culture medium is carried out using the apparatus described in the following, as well as the control, determined by the dosage of said concentrations, the addition of new amounts of substrate, the operation of the separation system, including filtration, cultures of microorganisms produced and collection thereof.

 A sampling pump 3 takes a small fraction (about 0.1 to 0.5 ml / minute, for example) of the culture medium contained in the bioreactor 1 through a 3 'conduit. connected after the pump 3 to a continuous automatic dosing device 4, such as a "Technicon" or "Tecator" device for example, said fraction taken being possibly diluted before its introduction into the metering device, in a dilution circuit 4 '.

 The dosages of the substrate and the product of the metabolism of the microorganism contained in the fraction taken are made by colorimetry or spectrophotometry in the metering device 4 and an output is obtained at the output of said device in the form of signals expressed in voltage, controlled by a voltmeter. 5 '. The signals (between 0 and 5 volts, for example), which correspond to the dosing values, are converted by a digital analog converter 5 into digital values, which are themselves processed by a microcomputer 6 in order to express them in concentrations. .

 Said computer controls firstly a feed pump 7 which takes a determined amount of substrate contained in the reservoir 7 'with a pipe 7 "connected to the bioreactor 1, to feed the latter substrate.

 Said computer also controls the start of the separation operation, in particular by filtration, of the culture medium contained in the bioreactor 1 before starting the filtration operation, a level 9 probe associated with the bioreactor verifies that the latter is not empty, then the filtration pump 10, located between the bioreactor 1 and the filtration module 12 starts.

Said pump takes, via a conduit 10 ', the medium to be filtered contained in the bioreactor 1, after opening a valve 11 located between the bioreactor 1 and the pump 10, and conveys it into the filtration module 12 (Which advantageously consists for example of a multifibre filter cellulose diacetate). The filtrate circulates in a duct 13 ', after the opening of a valve 13, and its flow rate, as well as the filtered volume, are measured by a device advantageously comprising a collection container for the filtered volume 14, this container resting on a scale 8 connected by a transducer 8 'to the microcomputer 6. An additional valve 22 allows the collection container 14 to be emptied.

The unfiltered portion returns to bioreactor 1 via line 12 '. The measurement of the flow rate and the filtered volume can be carried out, according to an alternative embodiment, by a flowmeter at the outlet of the valve 13.

 According to an advantageous arrangement of the invention, the apparatus comprises a system which makes it possible, if necessary, to unclog the filtration module 12. From the culture medium contained in a reservoir 18 is sent under pressure, after opening a valve 19, at the low level of the filtration module 12; a portion of said medium passes through the filtration module 12 carrying with it the microorganisms adhering to the filter to return to the bioreactor 1 through the conduit 12 '. The filling of the unclogging system is carried out as follows: a filling pump 15 withdraws, through a conduit 15 ', a fraction of the culture medium contained in a storage enclosure 16, after the opening of a valve 17, and sends said medium into the tank 18 unclogging medium connected to a compressor 18'.Le reservoir 18 is connected, in an advantageous arrangement of the invention, by a conduit 20 '(after opening a valve 20) to an overflow container 21 , this system for suppressing the pressure in the tank 18.

 The bioreactor 1 is advantageously supplied to culture medium from the chamber 16, with the aid of the pump 10 which withdraws said medium via a conduit 23 'after opening a valve 23, conveying it through the module filtration 12, and the end in the bioreactor 1 through the conduit 12 '. Alternatively, the feed of the bioreactor 1 in culture medium could be done directly from the storage chamber 16 without passing through the filtration module 12.

 The apparatus comprises, according to an advantageous embodiment of the invention, a culture sampling system enriched in microorganisms or metabolites resulting from this culture, which comprises a pump 24 which sucks said culture by a conduit 24 'which ends in a storage container 25.

 An apparatus as described above by way of example with reference to the drawing, operates in the following manner: the bioreactor 1 is filled with culture medium, a determined amount of substrate and precultures of the microorganism or microorganisms intended for seed the crop. After setting the stirring, temperature, air supply and pH conditions using the control system 2, the cultivation begins.

 A small fraction, (of the order of 0.1 to 0.5 ml / min for example), of the culture medium in the bioreactor 1 is taken continuously by the sampling pump 3, and possibly diluted before its introduction into the continuous dosing device 4. The dosing values of the substrate and of the product, measured by colorimetry or spectrophotometry in said device 4, are transmitted in the form of voltage signals converted into digital values by the analog-digital converter 5, the latter being themselves processed by the microcomputer 6 and transformed into effective concentrations. If the substrate concentration in bioreactor 1 falls below a determined threshold, said microcomputer controls the feed pump 7 to supply bioreactor 1 with an appropriate amount of substrate. the bioreactor 1 exceeds a determined threshold, said microcomputer controls the triggering of the filtration operation.

 The filter medium is taken from the bioreactor 1 by a filtration pump 10, after a level 9 probe has verified that the bioreactor is not empty, then this medium is sent into a filtration module 12. The filtrate is collected in a collection container 14 which rests on a balance 8 connected to the microcomputer 6 by a transducer 8 'which successively transmits to it the values of the weighings obtained, the control of the weighing of the filtered liquid thus making it possible to follow the filtration rate snapshot and control the filtered volume. According to the digital indication given by the scale 8 and transmitted to the microcomputer by the transducer 8 ', the filtration is stopped or continued.

 When the filtration flow rate becomes too low at the end of the operation, the filtration module 12 is unclogged. The culture medium contained in a reservoir 18 is sent under pressure created by an associated compressor 18 '. at the reservoir 18, at high speed at the low level of the filtration module 12 and the unclogging of the filtration module 12 is thus carried out by reverse flow of the culture medium; a portion of the medium passes through the filtration module 12 to return to the bioreactor 1 as indicated above. A filling pump 15 then takes culture medium contained in a storage chamber 16, and sends it into the tank 18 declogging medium.

 Once the filtration operation has been carried out, the bioreactor 1 is filled with culture medium, using the pump 10 which withdraws said medium from the tank 16 by passing it through the filtration module 12, thereby performing an additional cleaning. Finally, a sampling system allows, according to an advantageous arrangement, by means of a pump 24, to collect the cultures enriched with microorganisms or metabolites resulting from these cultures.

 The invention will be better understood with the aid of the following description which refers to examples of the production process of cultures enriched in microorganisms and / or metabolites resulting from these cultures in accordance with the present invention. present invention.

 It should be understood, however, that these examples are given solely for the purpose of illustrating the object of the invention, of which they in no way constitute a limitation.

EXAMPLE 1: PRODUCTION OF MICROORGANISM NITROBACTER - The genus Nitrobacter groups bacteria from 0.8 to 2 μm
long, motionless in a general way. These bacteria,
aerobic, are chemolithotrophic and use for their
growth the inorganic carbon dissolved in the medium.

This bacterium is responsible for the transformation of
nitrites to nitrates, depending on the reaction
N02- + 1/2 2 -7N03 #, which is nitration, second
stage of biological nitrification. The strain used
been isolated from the drinking water
Aubergenville station.

The strain is likely to be inhibited by its substrate
(NO2-) and by its product (NO3-).

- Culture conditions
1. A culture medium adapted to the production of the microphone
Nitrobacter organism is a buffer mineral medium
laying down the following wording
Na2HPO4, 12H2O 12.6 g
KH2P04 0.5 g
Solution A 1 mi
(ZnSO4, 7H2O: 2 mg, CuSO4, 5H2O: 2 mg
Na 2 #NO 4, 2H 2 O: 2 mg; QSP 100 ml)
Solution B 10 mi
(MgSO4, 7H20: 2 g, QSP 100mi)
Solution C 5 mi
(FeSO4, 7H2O: 114 mg, EDTA: 206 mg;
QSP 1 liter) 2.Control conditions, provided by the device
constituting the control cabinet of the bioreactor whose
useful volume is 4 liters (the bioreactor used is
"BIOLAFFITTE" brand):
Temperature 3OeC
Air supply 0.5 vvm (liters of air / mn / liter of
reactor)
Stirring 300 rpm.

3. Seeding: Crops are sown at 10% with
pre-cultures carried out batchwise on the medium
defined above, added 203 mg of nitrogen per
liter.

Mise en oeuvre du procédé pour une culture en discontinu à
concentration de substrat constante avec filtrations
On utilise un bioréacteur "Biolafitte" de volume utile égal
à 4 litres qu'on remplit du milieu minéral tampon décrit
plus haut, additionné d'une quantité initiale de substrat
telle que la concentration en azote initiale So de substrat
dans le bioréacteur est fixée à 203 t 40 mg/l. Les nitrites
et les nitrates sont dosés en continu par une chaîne de do
sage colorimétrique physicochimique ("Technicon" par exem
ple).Les signaux (O à 5 volts) en sortie des colorimètres
sont convertis en valeurs numériques traitées par un micro
ordinateur ("Apple", par exemple) afin de les ramener à des
concentrations. Si la concentration en substrat, exprimée
en azote, est inférieure à la valeur So - 20% (soit une
concentration en azote d'environ 162 mg/l), valeur appelée
X, il y a déclenchement d'un ajout de substrat dont la
quantité est déterminée de la manière suivante : quantité
de l'ajout en substrat = 2 x (So - X), de façon à dépasser
d'autant la valeur moyenne nécessaire.La solution de subs
trat utilisée est une solution de NaNO2 concentrée (30,45 g
d'azote/1). Les filtrations sont déclenchées lorsque la
concentration en nitrates (produit) dépasse une concentra
tion exprimée en azote fixée & 812 mg/l (au-delà de 1,22 g
d'azote/l on observe une baisse conjointe de la croissance
et de l'activité).4. Source of nitrogen NaNO2
C02 carbon source dissolved
Oxygen source 02 dissolved
Energy source energy of the reaction

Implementation of the process for a batch culture at
constant substrate concentration with filtrations
A "Biolafitte" bioreactor of equal useful volume is used
to 4 liters filled with buffer mineral medium described
above, plus an initial amount of substrate
such as the initial nitrogen concentration So of substrate
in the bioreactor is set at 203 to 40 mg / l. Nitrites
and the nitrates are dosed continuously by a chain of do
wise physicochemical colorimetric ("Technicon" for example
The signals (0 to 5 volts) at the output of the colorimeters
are converted to numeric values processed by a microphone
computer ("Apple", for example) in order to bring them back to
concentrations. If the substrate concentration, expressed
nitrogen, is below the So - 20%
nitrogen concentration of about 162 mg / l), called
X, there is triggering of a substrate addition whose
quantity is determined as follows: quantity
of the addition in substrate = 2 x (So - X), so as to exceed
all the necessary average value. The solution of subs
used is a concentrated NaNO2 solution (30.45 g
nitrogen / 1). The filtrations are triggered when the
nitrate concentration (product) exceeds a concentration
in nitrogen at 812 mg / l (above 1.22 g
of nitrogen / l we observe a joint decline in growth
and activity).

A fully automated filtration sequence goes
rolls according to the following process:
- Verification of the volume of culture medium in organic
reactor (4.5 liters),
- Triggering the filtration,
- Estimate of the amount of filtrate passed (?, # Liters)
- Stop filtration,
- Reverse flow clogging of buffer mineral medium
(pressure: 2 to 3 bars, duration: 15 seconds),
- Filling the bioreactor with buffer mineral medium
through the filtration and filling module of the cir
cooked declogging,
- Addition of the substrate to obtain a concentration So,
then we proceed to restart the fermentation.

EXAMPLE 2 INCREASE IN BIOMASS CONCENTRATION IN
THE BIOREACTOR FOLLOWING DIFFERENT METHODS (FOR
MICROORGANISM NITROBACTER) Simple batch culture
The buffer mineral medium described in Example 1 is introduced
in the bioreactor in which 10% precultu
batchwise on the aforementioned medium, addi
of 203 mg nitrogen / liter. The culture is conducted jus
than at full consumption of the substrate by the micro
organization.

Results: after 100 hours, 13 mg / l of
biomass.

Batch culture fed
The buffer mineral medium described in Example 1 contained
in the bioreactor is inoculated at a rate of 10% with precookings as indicated above, supplemented with 203 mg of nitrogen / l and the culture consists of a succession of fermentations i as soon as the complete consumption of the substrate by the microorganism is detected, add substrate to the bioreactor. The inhibition by the nitrates produced blocks the reaction after 16 additions of substrate, which correspond to a final concentration of 4.7 g of nitrogen / l.

Results: after 460 hours, 85 mg / l of
biomass.

Batch culture with filtrations
The buffer mineral medium described in Example 1, supplemented with 203 mg of nitrogen / l, is introduced into the bioreactor which contains a strain of bacteria seeded with precultures, as indicated above. The culture consists of a succession of simple fermentations separated by filtrations: as soon as the complete consumption of the substrate by the microorganism is detected, the contents of the bioreactor are filtered. The biomass is stored inside the bioreactor and the filtrate is discharged to the sewer. The level of the bioreactor is supplemented by buffer mineral medium supplemented with nitrogen in a variable amount.

Results: after 2600 hours, 250 mg / l of
biomass.

Batch culture with constant substrate concentration
The bioreactor is fed with buffer mineral medium described in Example 1 and microorganisms seeded with precultures as described above. The substrate concentration in the bioreactor is set at 203 to 40 mg azo te / l. The nitrites and nitrates are continuously measured, as in Example 1, by a physico-chemical colorimetric assay chain ("Technicon" for example) and the addition of a determined quantity of substrate is automatically triggered when the concentration nitrite falls below a certain limit value.

 It can be seen that growth stops after 100 hours. for 220 mg / l of biomass obtained when the nitrogen concentration reaches 1.01 g / liter, and after 145 hours, for 260 mg / l of biomass obtained, when the nitrogen concentration reaches 1.78 g / l.

Batch culture - constant substrate concentration with filtrations
The same procedure is repeated as described in Example 1 and the filtrations are triggered when the nitrogen concentration exceeds 812 mg / l.

Results: After 500 hours, 3000 mg / l of
biomass
after 620 hours, 3900 mg / l of
biomass.

The results obtained with these different methods are shown in Table 1 below.

TABLE I

<tb><SEP> METHOD <SEP> TIME <SEP> BIOMASS <SEP> FACTOR <SEP> FROM <SEP> CONCENTRA
<tb><SEP> EMPLOYEE <SEP> (h) <SEP> (mg / l) <SEP> CONCENTRA- <SEP> TION / UNIT
<tb><SEP> TION <SEP> (t) <SEP><SEP> i <SEP><SEP> FROM <SEP> TIME (*)
<tb><SEP> Culture <SEP> en
<tb> 1 <SEP> batch <SEP> 100 <SEP> 13 <SEP> 1 <SEP> 1
<tb><SEP> Culture <SEP> en
<tb> 2 <SEP> batch <SEP> 460 <SEP> 85 <SEP> 6.5 <SEP> 1 <SEP><SEP> 1.4
<tb><SEP> powered
<tb><SEP> Culture <SEP> in <SEP> say
<tb> 3 <SEP> continuous <SEP> with <SEP> 2 <SEP> 600 <SEP> 250 <SEP> 19 <SEP> 0.7
<tb><SEP> filtrations
<tb><SEP> Culture <SEP> en
<tb> 4 <SEP> batch <SEP> to <SEP> 100 <SEP> 220 <SEP> 17 <SEP> 17.0
<tb><SEP> constant <SEP> constant <SEP> 145 <SEP> 260 <SEP> 20 <SEP> 13.8
<tb><SEP> Culture <SEP> en
<tb><SEP> batch <SEP> to <SEP> 500 <SEP> 3 <SEP> 000 <SEP> 230 <SEQ> 46.0
<tb> 5 <SEP> constant <SEP> constant <SEP> 620 <SEP> 3 <SEP> 900 <SEQ> 300 <SEQ> 48.4
<tb><SEP> with <SEP> filtrations
<tb> (*) the reference in biomass concentration and unit of
time corresponds to method 1
The table above shows the superiority of the con
form to the invention for obtaining significant quantities
microorganisms in a low volume reactor and
in a relatively short time.

EXAMPLE 3 PRODUCTION OF CO-CULTURES OF NITROSOMONAS AND
Nitrobacter
The culture medium used is the medium described in Example 1, supplemented with NH4 + ions in the form of (NH4) 2504, and inoculated with a coculture of Nitrobacter and Nitrosomonas. The Nitrosomonas bacteria present in the culture convert the ammonia into nitrite (N02-), which is the substrate of the bacterium Nitrobacter, and whose concentration, expressed in nitrogen, must be kept below about 200 mg / liter.

In this Example, the colorimetric assay chains assay NO2, which is both the product of Nitrosomonas and the Nitrobacter substrate, and the product (NO2- + NO3-, expressed as NOx ').

After 1 h, 1 g / l of biomass is obtained.

The nitrification-producing autotrophic bacteria (Nitrosomonas and Nitrobacter) obtained in large amounts in accordance with the present invention are used either immediately or at a later time (after freezing and lyophilization) to seed or re-seed the nitrification ponds for the treatment of wastewater. or polluted, with the following advantages - improvement of the investment / production ratio, - simplification of the transport phases, - reduction of the volumes to be treated for the conservation of cultures by freezing or lyopholisation, - reduction of the volumes to be stored, - reduction of volumes to implement during sowing
cements.

 The method according to the present invention can also be applied to other strains than those of nitrifying bacteria, for the purpose of sowing other systems (start of fermentation units on an industrial scale with a volume of preculture 300 less than that required in accordance with the prior art).

EXAMPLE 4 PRODUCTION OF METABOLITES
The process of the present invention can also be used for the production by microorganisms of concentrated solution metabolites in order to simplify the steps of purification of the product formed (sterilization by filtration, concentrated solution, accelerated production given the amount of microorganisms present in the bioreactor, examples: acetic acid, polysaccharides, vitamin B12, etc.).

- Case of an internal metabolite production with bacteria
Propionibacterium acidi-propioni bacteria are used on
a medium with a concentration of sugars (eg glu
cose) is 40 g / i, biomass production being limited
by the production of propionic or acetic acid.

This fermentation occurs with a succession of peri-o
aerobic-anaerobic. The triggering of filtration
occurs when the concentration of propionic acid de
pass 20 g / l. This bacterium accumulates vitamin B12 to
a concentration of about 100 pg / g of cell, which is
extracted after lysis of the cells and purified by chromato
graphy. The results obtained with the compliant apparatus
to the present invention are greater than 110 g / l of organic
mass (in conventional cultivated culture, 15 g / l of
biomass).

- Case of an external production of metabolites associated with the
bacteria culture
For example, it is possible to produce nitrites by the tank
Nitrosomonas serum, nitrates by the bacterium Nitro
bacter, or propionic acid by Propioni bacteria
bacterium acidi-propioni.

 As is apparent from the above, the invention is not limited to those of its modes of implementation, implementation and application which have just been described more explicitly; it encompasses all the variants that may come to the mind of the technician in the field, without departing from the scope or scope of the present invention.

Claims (2)

1o) Process for producing cultures enriched in microorganisms and / or metabolites resulting from these cultures, characterized in that selected microorganism strains are cultivated in a suitable bioreactor in the presence of a suitable substrate whose concentration is maintained at values which are not inhibitory of the growth of the bacteria, in that cells which can inhibit the cells which accumulate in said medium during the fermentation, are removed from the culture medium, and that microorganisms and / or metabolites are recovered in an appropriate manner. 22) Method according to claim 1, characterized in that the cultures of microorganisms are carried out with strains of nitrifying bacteria of the family Nitrobacteraceae.  30) Method according to claim 2, characterized in that the cultures of microorganisms use nitritating bacteria of the genera Nitrosomonas, Nitrosospira, Nitrosolobus, Nitrosovibrio and Nitrosococcus.  40) Method according to claim 2, characterized in that the cultures of microorganisms use nitrating bacteria of the genera Nitrobacter, Nitrosococcus and Nitrospira.  5N) Method according to claim 1, characterized in that the optimum concentration of substrate in the culture medium is kept constant throughout the duration of the culture.  6 ') Process according to claim 1, characterized in that the substrate concentration is maintained optimal by adapting it to the amount of active biomass present.  7 *) A method according to claim 1, characterized in that the control of the optimum concentration of the substrate medium is achieved by continuous dosing of the substrate, and addition of substrate in the medium when the concentration falls below a threshold determined.  8 ~) Method according to claim 1, characterized in that the inhibiting products are removed from the culture medium by an appropriate separation technique, such as, in particular, filtration or centrifugation.  9) Process according to claim 8, characterized in that the control of the presence of inhibitory products, and more particularly the toxic products of the metabolism of the culture, is carried out by continuous dosing and the separation is triggered when the concentration in said produced in the culture medium reaches a predetermined rate.  10 '> A method according to claim 1, characterized in that the culture comprises a plurality of successive fermentation steps which each consume a substrate charge introduced at the beginning of each of them with a new charge of culture medium, in the biomass accumulated during the successive stages of fermentation, and the successive fermentation steps are separated by removal steps of the inhibiting products.  11 ') Method according to claim 1, characterized in that the culture is carried out in the presence of substrate whose concentration is kept constant in the culture medium by continuous assay and addition (s) of the substrate as soon as the detection of a concentration of substrate below a predetermined threshold, and the separation operations are triggered as soon as the concentration of inhibitor products, controlled by continuous dosing, reaches a predetermined threshold.  12-) Method according to any one of claims 1 to 11, characterized in that the culture is taken batchwise.  13g) Method according to any one of claims 1 to 11, characterized in that the culture is taken continuously.  14 *) Method according to any one of claims 1 & 13, characterized in that the cultures are made with strains of the genus Nitrobacter.  Process according to any one of claims 1 to 13, characterized in that the cultures are carried out with strains of the genus Nitrosomonas.  16 *) Process according to any one of claims 1 to 13, characterized in that one carries out co-cultures of Nitrosomonas and Nitrobacter.  17 ') Process according to any one of claims 1 to 16, characterized in that the toxic products of the metabolism of cultures of nitrifying bacteria are removed when their concentration in the culture medium reaches a threshold inhibitory metabolism of said culture.  18 ') Process according to any one of claims 1 to 17, characterized in that the substrate concentration of the nitrifying bacteria culture medium, expressed as nitrogen, is advantageously less than 2 g / liter.  19g) Application of nitrifying bacteria produced by the process according to any one of claims 1 to 18, in the seeding or reseeding of nitrification basins.  20e) Application of the nitrifying bacteria produced by the method according to any one of claims 1 to 18, after preservation by freezing or lyophilization, the seeding or reseeding of nitrification basins.  21 ') Apparatus for the production of cultures enriched in microorganisms and / or metabolites resulting from these cultures, characterized in that it comprises, in combination: - at least one reactor, provided with means for regulating the temperature and the pH , air-controlled feed means and stirring means, which is further provided with a feed in culture medium and substrate and an output of the inhibiting products and / or useful products of the metabolism of bacteria ; at least one separating element of said products associated with said output; a system for continuously dosing the substrate and the aforesaid products respectively: a control device, slaved to the aforesaid continuous dosing system, of the introduction of the required quantities of substrate into the reactor or reactor, and operation of the element, or element, separation.  22 ~) Apparatus according to claim 21, characterized in that the separating element is preferably an element of ultra- or microfiltration.  23 |) Apparatus according to claim 21, characterized in that the continuous metering system comprises a circuit for sampling small amounts of the medium contained in the reactor, associated with means for continuously quantifying the concentration of the substrate and / or in inhibitory products.  24 ~) Apparatus according to claim 23, characterized in that the quantization means consist of calibrated probe batteries that emit electrical signals.  250) Apparatus according to claim 23, characterized in that the quantization means consist of at least one physicochemical colorimetric assay chain.  26 ') Apparatus according to any one of claims 21 to 25, characterized in that the control device is a device taken from the group of programmable controllers and computers or microcomputers.