EP3938488A1

EP3938488A1 - Method for producing microbial cellular biomass having flocculant properties

Info

Publication number: EP3938488A1
Application number: EP20725868.2A
Authority: EP
Inventors: Yolaine BESSIERE; Etienne Paul; Jean-Noël LOUVET; Elise BLANCHET; Evrard Mengelle; Simon DUBOS; Mansour BOUNOUBA; Carine SAUX; Ana MORGADO FERREIRA; Dores Cirne
Original assignee: Centre National de la Recherche Scientifique CNRS; Institut National des Sciences Appliquees de Toulouse; Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement
Current assignee: Centre National de la Recherche Scientifique CNRS; Institut National des Sciences Appliquees de Toulouse; Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement
Priority date: 2019-03-11
Filing date: 2020-03-10
Publication date: 2022-01-19
Also published as: US20220242759A1; FR3093732B1; WO2020183103A1; FR3093732A1

Abstract

The invention relates to microbial cellular biomass having flocculant properties, to a method for producing same, and to the use thereof in flocculation treatment.

Description

BIOMASS PRODUCTION PROCESS

MICROBIAL CELLULAR WITH PROPERTIES

FLOCCULANT

[0001] The object of the present invention relates to a microbial cellular biomass having flocculant properties, to its method of obtaining and to its use in the treatment by flocculation of an aqueous and / or oily solution or suspension.

[0002] The drinking water and wastewater treatment methods include

generally one or more steps to remove suspended matter and colloidal matter (so-called clarification step). Most of these clarification processes combine coagulation, flocculation and settling requiring both a primary coagulant, often inorganic salts, such as FcCL, and a flocculant builder, a polymer, the most widely used being anionic polyacrylamide. (P AM). It is estimated that from 1.2 to 7.2 kilotons of Fc CL are thus used each year in a water treatment plant treating an organic load corresponding to 1 million inhabitant equivalent, which represents a significant cost. .

[0003] In addition to the cost associated with these treatments, wastewater treatment plants (STEP) are

dependent on a supply of chemicals whose production and transport have an impact on the environment. The production and use of products manufactured within the wastewater treatment plant according to a circular economy principle with short circuit, consequently minimizing the environmental impacts and improving the environmental balance of the WWTP would be an important development. In general, any reduction in the consumption of reagents, in particular FcCL, would significantly improve the environmental performance of a WWTP. In addition, the synthesis of chemical products requires in the majority of cases, a lot of products and materials, and generates a lot of pollutants as well as wastewater which also requires treatment.

[0004] In addition, it is noted that certain synthetic polymer type flocculants can persist in the environment and some of their by-products can prove to be dangerous. The specific case of polyacrylamide (P AM) is representative because this polymer can degrade into acrylamide classified by the International Agency for Research on Cancer (IARC) as a probable group 2A carcinogen (Yuliani E., Imai T., Teeka J., Tomita S., Suprayogi Exopolysaccharide production from sweet potato-shochu distillery wastewater by Lactobacillus sakei CY 1, Biotechnology and Biotechnological Equipment, 2011, 25 (2), 2329-2333).

[0005] Thus, even if polyacrylamide and its derivatives produced from petroleum are the most widely used synthetic flocculants due to their high efficiency at low dosage and their easy handling, the search for alternative products is essential, both for the environmental protection aspect than that of health.

[0006] Thus, there is a need, in the clarification process, to develop

the use of coagulants / adjuvants / flocculants based on bioproducts, on the one hand, to reduce the dependence of the sewage treatment plant on added chemical reagents, and, on the other hand, to promote the use of biological polymers with lower impact on the environment and human health.

[0007] Bacterial exopolymers (EPS) are biopolymers which are secreted or

released by microbial cells and which partly remain more or less strongly associated with the surface of the cell (for example in capsule). These biopolymers are mainly composed of proteins, carbohydrates, lipids and / or nucleic acids.

[0008] Bacterial EPSs, and in particular the fraction comprising exopolysaccharides, may have excellent flocculant properties for certain and may thus be a satisfactory alternative to synthetic polymers such as PAM.

Most studies concerning the production of EPS illustrate the production of EPS through pure cultures, either by well-known organisms (Pseudomonas, Bacillus, lactic acid bacteria, fungi, etc.), or from isolates produced from various sources (activated sludge, soil, waste, etc.) and then used as pure cultures. Although microbial production of EPS is considered to be

respectful of the environment because based on the use of renewable carbon and in line with the concept of biorefinery, microbial EPS only constitute a minor fraction of the current market for flocculating polymers due to the high cost of their production and the difficulty of their recovery.

[0010] For this reason, many efforts have been devoted to the development of

cost-effective production processes using less expensive growing media. These substrates generally come from residues in liquid form such as syrups, molasses, juices, whey from cheese and wastewater in general or from solids such as lignocellulosic biomass and organic waste. These means of productions unfortunately have drawbacks. One of the major drawbacks lies in the use of so-called “pure” cultures, both in terms of the associated sterilization needs and in the inability of the pure bacterial strains to use the diversity of the substrates present.

[0011] There is thus a need to provide a product having flocculant properties.

sufficient to replace synthetic polymers that may have an impact on health, to overcome dependence on the supplier and to apply a principle of circular economy with short circuit by producing the product with flocculant properties where it is needed, to recover waste and by-products by producing products with flocculant properties from these residues, waste, by-products containing carbohydrates and possibly to be able to export them easily in order to increase the profitability of the wastewater treatment plant.

[0012] An object of the present invention thus relates to a process for producing a microbial cellular biomass having flocculant properties comprising the steps: a) introduction into a reactor:

- a substrate optionally comprising a microbial inoculum and comprising at least 30% by mass of oses, preferably chosen from the group comprising hexoses and pentoses; and

- optionally of nutrients for the microbial inoculum, the reaction medium obtained having a C / N molar ratio less than or equal to 10 and a dilution rate D less than or equal to 0.35h ^_1 ;

b) stirring the reaction medium obtained in a) and maintaining the C / N ratio to a value less than or equal to 10 and the dilution ratio D to a value less than or equal to 0.35H ^_1;

c) elimination of filamentous bacteria and possible filamentous fungi by sequential settling of the biomass obtained in b); and

obtaining microbial cellular biomass having flocculant properties.

By "substrate" is meant any hydrolyzate or mixture comprising a carbon source (oses), pasteurized or unpasteurized, purified or unpurified, filtered or unfiltered, comprising at least 30% by mass of sugars (oses) from any biomass. It can be for example a hydrolyzate of wood, straw and more generally any lignocellulosic biomass, of cereals, roots (eg beetroot), fruits but also residues or co-products and organic solid waste possibly biodegradable having a high fraction of sugars, in particular those indicated in the Report of the Agency for the environment and Energy Management (ADEME:

overview of co-products and biomass residues for use in chemistry and biobased materials sectors in France), September 2015. The substrate can, in certain cases, be a culture medium, if it includes all the nutrients for the microbial inoculum.

Advantageously, when the substrate is a hydrolyzate, it can be obtained by any method of chemical, thermal or biological hydrolysis known in the art such as for example an enzymatic hydrolysis in an acidic medium (Kumar, P., Barrett, DM,

Delwiche, M. J., & Stroeve, P., Methods for Pretreatment of Lignocellulosic Biomass for Efficient Hydrolysis and Biofuel Production. Industrial & Engineering Chemistry

Research, 2009, 48 (8), 3713-3729)

Advantageously, the substrate can comprise at least 40% by mass of oses. Preferably the substrate comprises at least 50% by mass of oses, and even more preferably from 70 to 100% by mass of oses, or even from 90 to 100%.

[0016] Advantageously, the substrate can comprise a hydrolyzate and / or oses.

By "ose" or "monosaccharide" is meant the monomer of carbohydrates. Osseous have at least 3 carbon atoms: they are poly hydroxyaldehydes (aldoses) or poly hydroxyketones (ketoses). They are not hydrolyzable. They are very soluble in water and generally have a sweetening power. They are distinguished by the length of their carbon chain. An n-carbon ose is made up of an unbranched carbon chain, n ranging from 3 to 7 carbons, with only single bonds. All the carbons carry an alcohol (OH) function except one which carries a carbonyl function. This therefore determines two categories of oses. The term “aldose” is used to refer to an ose whose carbonyl function is an aldehyde function; it is found on the first carbon (for example glucose). We call “ketose”, an ose whose carbonyl function is a ketone function, it is on the second carbon (for example fructose).

[0018] Advantageously, the oses can be chosen from the group comprising

hexoses and pentoses. For example, the substrate can comprise aldohexoses (example: glucose; galactose), ketohexoses (example: fructose; sorbose), aldopentoses (examples: xylose; arabinose; ribose) or even ketopentoses (example: ribulose; xylulose) and their mixtures.

[0019] Advantageously, the substrate can comprise a microbial inoculum, comprising in particular heterotrophic bacteria. The microorganisms of said substrate can be chosen from the group comprising gammaproteobacteria, flavobacteria, alphaproteobacteria and betaproteobacteria. For example, they can be bacteria belonging to taxonomic classes such as gammaproteobacteria, flavobacteria, alphaproteobacteria and betaproteobacteria. For example, the gammaproteobacteria can be Citrobacter, Klebsiella and Raoultella of the Enterobacteriaceae and Acinetobacter family of Moraxellaceae, the flavobacteria can be flavobacterium or flavobacteriaceae, the alphaproteobacteria can be Rhodobacter (Rhodobacteriaceae) etomonaceae Betaproteobacteria can be Comamonas (Comamonadaceae). Advantageously, the microorganisms selected during the implementation of the method according to the invention can for example be said previously mentioned heterotrophic bacteria, these microorganisms then being able to produce bacterial EPS.

By "biomass" is meant a mass comprising living microorganisms.

By "microbial cell biomass having flocculant properties" is meant biomass having a flocculating power containing microbial compounds comprising exopolysaccharides (EPS), pure or mixed with other substances (for example proteins) having flocculant properties and being able to replace synthetic polymers such as polyacrylamides for example.

By "bacterial exopolysaccharides" is meant EPS secreted or released by microbial cells and which remain in part more or less strongly associated with the surface of the cell. Bacterial exopolysaccharides can thus be produced by cells and accumulate around cells. These polysaccharides are homo- or heteropolysaccharides mainly composed of carbohydrates, glucose, galactose and mannose being the most common monomers. In the case of heteropolysaccharides, for example: D-glucose, D-galactose, L-rhamnose, sometimes with N-acetylglucosamine, N-acetylgalactosamine, or glucuronic acid, but also guluronic and manuronic acids are observed. Other neutral sugars (for example, rhamnose and fucose), certain uronic acids (mainly glucuronic acids and galacturonic) and osamines (N-acetylamino sugars) are also frequently encountered. In addition to carbohydrates, bacterial EPSs can contain several substituents (such as acyl and / or pyruvate groups). The presence of some of these acyl groups gives EPS an anionic character, increases its lipophilic character and affects its ability to interact with other polysaccharides and cations.

By "activated sludge" is meant a microbial biomass used for the treatment of wastewater which is organized in floc. Activated sludge is mainly composed of heterotrophic organisms, that is, organisms that use organic matter and acquire energy through oxidation or respiration, or fermentation. Some of these microorganisms produce EPS responsible for the observed flocculation of activated sludge.

By "undesirable microorganisms" is meant bacteria or fungi,

in particular filamentous bacteria and filamentous fungi, which do not exhibit any flocculant property, in particular not producing the EPS responsible for the observed flocculation of the activated sludge.

By "settling" is meant any process in which particles are separated by a difference in density, for example centrifugation, hydrocyclone or sedimentation.

Advantageously, step a) of the process according to the invention comprises an introduction into a reactor of a substrate and optionally of nutrients for the microbial inoculum. This mixture leads to the formation of a culture medium in the reactor (reaction medium). The substrate may in some cases meet the definition of culture medium.

[0027] Advantageously, the substrate can be supplemented by the introduction of elements

nutrients for the inoculum. Depending on the composition of the substrate, the introduction of nutrients makes it possible to obtain in the reactor a culture medium suitable for the proliferation of microorganisms. It can be a complementary source of carbon, potassium, phosphorus, nitrogen, sulfur, magnesium, calcium, iron, sodium, chloride and trace elements (salts of Cu , Zn, Co, Ni, B, Ti, Mo), water or a pH buffer. The nitrogen source can be mineral and is preferably in the form of an NH4 ⁺ ion (in equilibrium with NEE) but can also be in the form of nitrates or nitrites. The nitrogen source can also be organic (amino acids, peptides, proteins, nucleic acids). The nitrogen source can in particular be urea or a urea derivative (urine) or a nitrogen fertilizer. The substrate can also be a source of nitrogen. The nitrogen source can be a mixture of the aforementioned sources. The source of phosphorus can, for example, be phosphoric acid, phosphate or polyphosphate or a mixture thereof.

[0028] Advantageously, the C / N ratio of the mixture obtained in step a) is a ratio

molar. The values of C and N are expressed in moles. The C / N molar ratio may have a value less than or equal to 10, preferably in a range going from 1 to 10 and even more preferably in a range going from 7 to 10. The measurement of the reduced total nitrogen "N" (organic and inorganic) is done by the so-called Kjeldahl method. The measurement of reduced mineral nitrogen (NLL ⁺ ) is carried out by the Kjeldahl method or by ionic HPLC chromatography or even by the Nessler colorimetric method. Carbon "C" is measured by TOC (Total Organic Carbon). Only the biodegradable organic carbon, which is that which is degraded in the reactor, is taken into account in the value of “C”. The C / N ratio can be measured and adjusted on the basis of samples taken within the reactor or on the inputs and outputs of the reactor (for example on the basis of an input / output balance).

Advantageously, the dilution rate D in the reactor can be less than or equal to 0.35h ^_1 , preferably within a range ranging from 0.05 to 0.30h ^_1 , and even more preferably within a range ranging from 0.08 to 0.20h ^_1 or alternatively from 0.09 to 0.014h '. The dilution rate is the ratio between the liquid flow rate (m ³ .h ^_1 ) entering the reactor over the volume of the liquid of said reactor (m ³ ). In the case of a semi-continuous reactor, the dilution rate is calculated over the duration of the cycle. The liquid may optionally comprise suspended solids, including for example the substrate, the inoculum and other elements.

Advantageously, the pH in the reactor is within a range from 6 to 9, preferably from 6.5 to 8 or even from 6.8 to 7.8.

Advantageously, the mixture obtained in step a) has a C / P molar ratio of less than or equal to 600. The C / P ratio of the mixture obtained in step a) is a molar ratio. The values of C and P are expressed in moles. The C / P molar ratio may have a value less than or equal to 600, preferably less than or equal to 100 and even more preferably in a range of 40 to 100. The value of C is the same as in the C / N ratio. The measurement of phosphorus can be carried out by digestion in an acidic medium and in the presence of persulfate, the solubilized phosphorus is then determined by

colorimetry according to standard NF EN 14672 of December 2005. Generally, in the process according to the invention, when the dilution rate D increases, the required C / P ratio decreases. For example, when the dilution rate D is in a range going from 0.25 to 0.35 hrs ^_1 , the C / P ratio can be of the order of 40 to 70. When the dilution rate D is included in an interval ranging from 0.14 to 0.25 h ^_1 , the C / P ratio may be less than or equal to 600. When the dilution rate D is within an interval ranging from 0.09 to 0.14 h ^_1 , the ratio C / P can be of the order of 40 to 600. for example, when D is approximately 0,12h ^_1, C / P can be less than or equal to 100, preferably within a range of from 40 to 100 or in an interval going from 40 to 60.

[0032] Advantageously, step b) of the process according to the invention comprises stirring the reaction medium obtained at the end of step a). It can be mechanical or magnetic agitation.

Advantageously, in step b) according to the method of the invention, the stirring time can be in a range ranging from a few minutes to several hours, days or months. The duration of step b) may depend on how often step c) is performed.

Advantageously, throughout the duration of step b), the C / N ratio can be

maintained at a value less than or equal to 10, preferably within a range of 4 to 10 and even more preferably in a range of 7 to 10.

Advantageously, the C / N ratio of steps a) and b) is identical.

Advantageously, throughout the duration of step b), the dilution rate D can be maintained at a value less than or equal to 0.35h ^_1 , preferably within a range of 0.05 to 0, 30h ^_1 , and even more preferably in an interval ranging from 0.08 to 0.20h ^_1 or else from 0.09 to 0.14h ^_1 .

Advantageously, the dilution rate D of steps a) and b) is identical.

Advantageously, the imposition of a dilution rate D in the reactor fixes a residence time of the cells in the reactor and indirectly imposes a minimum specific growth rate m on the undecanted cells, thus allowing the elimination of the cells. unwanted. Advantageously, throughout the duration of step b), the C / P ratio can be maintained at a value less than or equal to 600, preferably less than or equal to 100 and even more preferably within a range of from 40 to 100.

Advantageously, the C / P ratio of steps a) and b) can be identical.

Advantageously, step c) of the method according to the invention can comprise a

elimination of filamentous bacteria and possible filamentous fungi, and possibly other undesirable micro-organisms, by sequential decantation of the biomass obtained in step b).

Advantageously, step c) of the method according to the invention can be carried out alternatively or simultaneously with step b). The method may comprise one or more iterations of steps a), b) and / or c), said steps a), b) and / or c) being consecutive or simultaneous. In particular, in the context of continuous operation, steps a) and b) can be simultaneous or consecutive.

Advantageously, step c) can be carried out directly in the reactor or else on the effluents from the reactor. When step c) is carried out in the reactor, the stirring is stopped and the biomass obtained in step b) is then allowed to settle in the reactor. The settling obtained, comprising filamentous bacteria and possible filamentous fungi, and possibly other undesirable microorganisms, is removed from the reactor, for example by partially purging the liquid volume of the reactor. When step c) is carried out on the effluents from the reactor, the decantate is then removed and the residue which contains in particular bacteria having flocculant properties is reinjected into the reactor. In this embodiment of the process, step c) can be carried out simultaneously with step a) and / or b) because the stirring does not need to be interrupted to allow the reaction medium to settle. . Step c) of the process of the invention makes it possible to prevent the accumulation of undesirable microorganisms, in particular filamentous microorganisms (bacteria or fungi) in the reactor. Step c) can also make it possible to eliminate other undesirable microorganisms whose settling rate is greater than that of the microorganisms produced during the process, in particular bacterial EPS.

[0044] Advantageously, in step c) according to the method of the invention, the duration of the

settling is sufficient to eliminate filamentous microorganisms having a sedimentation rate within an interval ranging from 0.1 meter per hour to 20 meters per hour and more generally between 0.5 m / h and 5 m / h.

By "sufficient settling time" is meant the time necessary for, in a given reactor, the undesirable microorganisms have time to reach the fraction of the reaction medium which will be purged. For example, the settling time of step c) for a reaction medium having a volume of 8 L and a height of 25 cm, of which 11% by volume is purged, is 4 to 5 minutes. The eliminated microorganisms are, for example, bacteria of the genus Sphaerotilus in particular, the species S. natans. Those skilled in the art will know how to adapt the duration of step c), depending on the type of filamentous microorganisms to be eliminated and the parameters of temperature, viscosity, volume and height of the reaction medium, in order to remove a sufficient quantity of microorganisms. filamentous, in particular also depending on the shape of the reactor and the fraction of volume of reaction medium purged.

Advantageously, step c) allows during the implementation of the method of

the invention, that the residence time of unwanted microorganisms is less than that of microorganisms having flocculant properties.

Advantageously, when the method comprises several steps c) according to the method of the invention, the frequency of implementation of step c) is preferably within an interval ranging from 1 to 60 times per day and from even more preferably in a range of 15 to 30 times per day.

Advantageously, the ratio t _c / t _b (duration of step c / duration of step b) has a value ranging from 0.01 to 0.40, preferably from 0.05 to 0.30 . The ratio t _c / t _b is defined as being the duration of a stage c) / the duration of a stage b), when stage c) is implemented in the reactor and as the sum of the durations of l step c) / the duration of step b), when step c) is carried out on the effluents from the reactor.

Advantageously, the production of the microbial cell biomass having flocculant properties occurs continuously during steps b) and / or c).

Advantageously, the method according to the invention can further comprise a step of inoculating the reactor with one or more microorganisms. This step

inoculation of the reactor can for example comprise the introduction of an activated sludge, of an inoculum resulting from another process for the biological treatment of wastewater, for example a biofilter, a bacterial bed, a lagooning process but also an extract of soil or waste, organic residues or else of an inoculum selected on simple sugar and ammonium ions, according to the process of the invention, preferably at a C / N molar ratio of less than or equal to 10. This inoculation step is a so-called priming step and can take place prior to step a), in particular when the substrate envisaged for the implementation of the method does not contain microorganisms or if they are not in sufficient number or sufficiently viable in the substrate for initiating the method according to the invention. During this step, the microorganism (s) of interest develop so as to be able to subsequently produce the biomass during the implementation of the method.

Advantageously, the concentration of microorganisms originating from the inoculum or from the substrate can generally represent at least 10 mg of viable cells in dry mass per liter of volume of liquid in the reactor.

Advantageously, the method according to the invention can further comprise a step of injecting into the reactor an additional dose or mixture of doses. This step can be implemented at any time in the implementation of the method to adjust the C / N ratio, or during the priming step or to promote the process of selection and production of microbial cell biomass. having flocculant properties.

Advantageously, the method according to the invention is implemented in a continuous or semi-continuous reactor. When operating in a continuous reactor, growth, selection of microorganisms and production of microbial cellular biomass having flocculant properties occurs during steps a), b) and c), within the same reactor. The method can also be implemented in several reactors, steps a), b) and c) being carried out in a first reactor, leading to the growth and selection of microorganisms. The step of producing microbial cell biomass having flocculant properties can then be carried out in a second reactor.

Advantageously, the method according to the invention can be implemented at a

temperature 4 to 55 ° C, preferably 10 to 30 ° C, more preferably at room temperature. The room temperature may vary depending on the location and the season, but is generally in the range of 15 to 25 ° C.

Advantageously, the method according to the invention can be implemented in a medium

open, ventilated. Aeration of the reaction medium can be carried out in the open air or under forced ventilation. The gas used for aeration can be air, or an oxygen enriched mixture. Preferably, the aeration is interrupted during step c) in order to promote settling.

Advantageously, the method of the invention is carried out in a sterile or non-sterile, preferably non-sterile, environment. An advantage of the method according to the invention is that it can be carried out in a non-sterile environment, for example an environment open to the outside and subject to contamination.

Advantageously, the method according to the invention can further comprise a step of completely emptying the reactor comprising cleaning the reactor and reintroducing the reaction medium. This step can in some cases prevent the formation of biofilms on the walls of the reactor, on the probes and on the blades of the mechanical stirrer or the magnetic stirrer.

[0058] Advantageously, the method according to the invention is implemented for a period of between 1 day and several months.

Advantageously, the method can have a minimum duration of implementation.

depending on the dilution rate D. Preferably, the process is carried out for a period of at least 4 * 1 / D. For example, when D = 0.12 h ¹ , the duration of the implementation of the method is at least 1.5 days. This duration can correspond to the duration of selection of the microorganisms which will then produce the EPS and will form the cellular microbial biomass having flocculant properties obtained according to the process of the invention.

[0060] Advantageously, the method according to the invention may further comprise a step of dehydration of the cellular microbial biomass having flocculant properties obtained according to the method of the invention. The dehydration step can be carried out by drying (heat supply by conduction, convection or radiation, or by the use of a gas) or by lyophilization.

Advantageously, the method according to the invention can further comprise any step allowing the conservation of the biomass according to the invention by lowering the activity of water, in particular as a solvent, in order to limit the biomass degradation reactions according to the invention. Advantageously, the method according to the invention can further comprise a step of treating wastewater. Said wastewater treatment step may comprise a step of contacting wastewater with a coagulant and the microbial cellular biomass having flocculant properties. Preferably, the step comprises contacting waste water with a coagulant and then with the microbial cellular biomass having flocculant properties.

[0063] The invention also consists of a microbial cellular biomass having flocculant properties obtained according to the process of the invention.

A flocculation effect is defined based on Stokes' law and showing an increase in the sedimentation rate of colloidal material and in suspension of standard elements (for example with a density of 1100 kg / m ³ ), such as as for example bacteria (diameter 1 mhi, settling speed = 1.5 10 ⁴ m / h), colloids (diameter 0.1 mhi, settling speed = 1.5 10 ⁶ m / h) or colloids ( diameter 0.01 mhi, settling speed = 1.5 10 ⁸ m / h). Advantageously, the microbial cellular biomass having flocculant properties obtained according to the process of the invention makes it possible to obtain particle diameters (flocs) of at least 100 mhi, generally between 100 mhi and 1 mm, leading to speeds of respective sedimentation of these same elements, under the same conditions, between 1.5 m / h and 150 m / h. These rates are compatible with sedimentation methods conventionally used in the art.

Advantageously, the microbial cell biomass having properties of

flocculant according to the invention comprises EPS, preferably bacterial exopolysaccharides. The microbial cellular biomass having flocculant properties according to the invention has a flocculant power equivalent to or even greater than that of the polymers produced by chemical synthesis conventionally used in purification stations, such as for example anionic polyacrylamide (for example Floerger- type AN 934 SH).

The term “polyacrylamide” is understood to mean a polymer corresponding to the formula [-CH2-CH (-

CONH ₂ ) -] _n , formed from acrylamide monomers. Depending on the intended use, the polyacrylamide can be functionalized. It can thus be ionic (cationic or anionic) or nonionic. The functionalized polyacrylamide can for example be a copolymer synthesized by copolymerization of acrylamide monomers and acrylic acid monomers or by hydrolysis of the amide group of the polyacrylamide. The WFP of type AN 934 SH is a functionalized, anionic polyacrylamide comprising approximately 30 mol% of fillers and having an average molecular mass Mw ranging from 1.3.10 ⁶ to 1.6.10 ^e Da. Its water content is less than 13%. It further has a viscosity ranging from 5.7 to 6.5 cPs and a density of 49.92 lbs / ft ³ (manufacturer data).

The term "flocculation" is understood to mean the physicochemical process during which materials in suspensions in a liquid agglomerate to form larger particles (flocs). The "flocculant power" consists of measuring the efficiency of the agglomeration effect of suspended matter. This flocculant power is for example often measured relative to references such as elimination of turbidity in the presence or absence of the flocculant in a sedimentation test. For example, PAM AN 934 SH (0.2ppm), associated with ferric chloride (20mg / L) allows a purification yield (1-turbidity / control turbidity without any addition)> 60% on ERU (Urban Residual Water) . PAM AN 934 SH (0.2ppm), associated with ferric chloride (2mg / L) allows a purification yield (1-turbidity / control turbidity without any addition)> 85% on kaolinite. Under the same conditions, cellular biomass

microbial having flocculant properties according to the invention gives equivalent results, or even greatly superior, from 60 to 99% on ERU and from 85 to 99% on kaolinite.

Advantageously, in the presence of a coagulant, the microbial cellular biomass having flocculant properties according to the invention has a flocculating power which leads to elimination of the turbidity of a waste water equal to at least 50% of the power. flocculant of anionic polyacrylamide (AN 934 SH) under the same conditions (ERU, temperature), preferably at least 100%, for example between 100% and 500%.

[0069] A further advantage of the microbial cell biomass having

Flocculant properties according to the invention is to lead to a sharp decrease in the chemical oxygen demand (COD) of the clarified ERUs, the COD being an indirect measure of the pollution of said ERUs.

Advantageously, the microbial cell biomass having properties of

flocculant according to the invention can be purified or unpurified before use.

Advantageously, the microbial cell biomass having properties of

flocculant can be in hydrated or dehydrated form. In dehydrated form, it offers the additional advantage of limiting the costs associated with its possible transport. The invention also relates to a use of the microbial cellular biomass according to the invention in the treatment of an aqueous and / or oily solution or suspension, in particular waste water (urban, industrial or agricultural), water. fluvial, storm water, runoff water, mining water, cleaning water, oils or gases. The microbial cell biomass according to the invention can be used in any type of aqueous solution or suspension requiring flocculation.

The invention also relates to a wastewater treatment process comprising a step of bringing an aqueous and / or oily solution or suspension into contact with a coagulant and a cellular microbial biomass according to the invention.

Advantageously, the coagulant can be any coagulant used in the known treatment methods. In particular, it can be chosen from the group comprising trivalent ions of iron and aluminum salts, preferably FcCh. The coagulant may also be chosen from the group comprising biopolymers or bio-based substances having the properties of coagulants (eg: tannins, chitin, modified starches).

Advantageously, the use or the treatment method according to the invention can be implemented to flocculate minerals and / or organic materials in contexts other than water treatment, in all environments where a flocculant is useful. In particular, the use or the method of treatment according to the invention can be implemented upstream of the filtration membranes to limit clogging or else to help eliminate water in the downstream stages of dehydration / drying of the sludge.

Advantageously, the use or the treatment process can comprise a step of rehydration or dilution of the cellular microbial biomass having flocculant properties according to the invention.

Figure 1 is a schematic representation of the implementation of the method of the invention comprising a continuous reactor, a supply system of minerals and trace elements, a substrate supply system, source of phosphorus and nitrogen (NPC), an air supply system, a mechanical agitator, a purge duct, a draw-off system, baffles and a device for controlling the dilution rate and the C / N and C / P ratios. FIG. 2 represents a photograph of the so-called "jar-test" apparatus used to perform the flocculation tests, the six beakers being filled with urban waste water.

[0079] FIG. 3 represents an analysis of the performance of biomasses obtained by the method according to the invention. On the x-axis is plotted the gain of the commercial polymer (P AM AN 934 SH) compared to the coagulant FcCh alone (in percentage), and on the y-axis is plotted the gain (%) of biomass according to l invention tested against FcCh alone.

FIG. 4 represents an enlarged view (Scale: 10 μm) of exopolysaccharides

bacteria included in a cellular microbial biomass according to the invention. Thanks to the Indian ink staining, the relatively compact layer which surrounds the bacterial wall and constitutes the capsule which is formed of viscous molecules produced by the bacteria can be observed (it is also possible to use other stains such as the Alcian blue which, at pH 2.5, adheres to

negatively charged macro molecules and then colors acidic polysaccharides blue).

FIG. 5 represents a comparison between A) a photograph of microscopic observations of a consortium dominated by Sphaerotilus (selection process carried out without selective purging, that is to say without step c) of the process) and B) a photography

microscopic observations of a biomass-dominated consortium with flocculant properties (selection process carried out with selective purging of filamentous bacteria); enlarged view (Scale: 10 pm). This comparison shows that in the absence of step c) of the process, filamentous bacteria and filamentous fungi are not eliminated.

The invention will be better understood on reading the non-limiting examples which follow.

Example 1: implementation of the process according to the invention

[0084] Inoculum and culture media

A stirred and aeration reactor is inoculated with an activated sludge from the Ginestous wastewater treatment plant, France) taken and stored according to Cavaillé et al (Cavaillé et al 2016. “Understanding of Polyhydroxybutyrate Production under Carbon and Phosphorus -Limited Growth Conditions in Non-Axenic Continuous Culture. ” Bioresource Technology 201 (February): 65-73.

https://doi.Org/10.1016/j.biortech.2015.l l.003).

The slurry is centrifuged at 4100 g for a period of 15 minutes and the supernatant is then removed. The pellet is collected and aliquots are frozen in liquid nitrogen and then stored at -20 ° C.

The inoculum thus prepared, before seeding the reactor, an aliquot is

thawed for 15h at a temperature of 4 ° C and then rinsed with a saline solution (0.9% w / v NaCl) thanks to three successive centrifugations (4100 g - 15 min). Before inoculation of the reactor, the sludge is fed-batch fed for 24 hours with additions of 1.8 g / L of an equimolar mixture of fructose and glucose.

The reactor is seeded with an initial activated sludge concentration of 1.5 g / L of volatile matter in suspension.

[0089] The culture medium is composed of a first solution containing the source of

carbon, nitrogen and phosphorus. This is a feed containing a carbon source (in the example an equimolar mixture of glucose and fructose), a phosphate buffer solution and ammonium added according to the degree of limitation by the nitrogen chosen, here from so as to obtain a consumed (C / N) ratio of 8.5 (molar).

A second solution is added containing the mineral salts and trace elements required for the microbial cultures (Table 1).

Table 1.

In order to assess the effect of constant microbial contamination on the efficiency of microbial selection, a continuous flow of dilute activated sludge is injected into the reactor. This flow rate is chosen to provide a flow of contaminating cells

corresponding to 5% of the reactor cell production (5% of r _x (g / (Lh) - 0.1g / L / h), ie approximately 0.005 g / L / h under stable conditions with a CODp around 0.8g / L in the reactor. Despite this continuous supply of contaminating cells, selection of cell biomass with flocculant properties occurs.

Production of cellular biomass having flocculant properties

The substrate used comprises the elements of Table 2 below:

Table 2.

The microorganisms selected by the method are essentially of the type

Comamonadaceae. These microorganisms produce bacterial EPS.

An 8L reactor of useful volume (10 L in total) (Figure 1) equipped with a jacket was used. Stirring set at 130 RPM is provided by Rushton turbines and two 5 cm wide counter-blades. To regulate the dissolved oxygen concentration to (2.5 ± 0.5) mg / L, flow meters (EL-FLOW®, Bronkhorst High-Tech B.V., France) were used. The temperature is regulated at 25 ° C ± 1 ° C, and the pH is maintained at 7.0 ± 0.3 with the addition of potassium hydroxide (KOH, IM). The operating conditions and the online data acquisition (dissolved oxygen concentration, pH and temperature) are controlled via acquisition software.

Twice a month, the reactors are emptied for cleaning in order to prevent the formation of biofilms on the walls, on the probes and on the blades. When deemed necessary, the supply hoses are replaced.

The reactors are controlled in a sequenced manner, with a feeding and stirring phase (55 min) followed by a settling phase (4 min 30 sec) then a purging phase (30 sec). The reactors are purged by means of 3 nozzles at the bottom of the tank, connected to the recovery tanks. At each cycle, the volume supplied is equivalent to purged volume of 960 mL (purge flow rate of 1.9 L / min). Since the useful volume is 8L, the hydraulic retention time is 8.33 h (dilution rate D = 0.12h ^_1 ).

The microbial cell biomass is obtained having flocculant A properties.

Example 2: Evaluation of the flocculant properties of cellular biomasses

microbial having flocculant properties according to the invention

[0100] The flocculant properties are evaluated on a representative effluent, here water.

urban wastewater (ERU).

[0101] The flocculation tests are carried out in jar-tests (Lavibond® - Amesbury, United Kingdom, Floc-Tester model, SN: 1013/61444) under conditions (stirring speeds, duration of the phases) identical for each combination of coagulant / flocculant. A

Photograph of the system called "jar-test" is shown in Figure 2.

[0102] The coagulant used is ferric chloride. For each test, 6 beakers filled with 900 mL of ERU are used. The first three beakers are used as a reference (n ° l: control (without addition), n ° 2: FcCh 20 mg / L alone, n ° 3 FcCh 20 mg / L + polyacrylamide AN934SH (named PAM) 0.2 mg / L) . The remaining three beakers are used for the assessment of biomass A at 3 different concentrations (# 4, # 5, # 6 FeC13 20 mg / L + biomass A). Biomass A is used directly without extraction or prior purification. The dosage is evaluated by measuring the total chemical oxygen demand (COD). Typically, concentrations of 1, 6 and 12 mg COD / L are used in the three test beakers.

[0103] After having agitated and measured the initial turbidity (Turb 0), the protocol comprises three main steps: (i) Addition of coagulant (FcCh - 20 mg / L) in all the beakers except the control and agitation at 150 rotations per minute (RPM) for 3 min; (ii) addition of the flocculant (PAM 0.2 mg / L - Biomass A 1-6-12 mgDCO / L in beakers 4 to 6) and stirring at 50 RPM for 15 minutes; (iii) 1 minute decantation and sample 2.2 cm from the surface for turbidity measurement (Turbx)

[0104] The results are analyzed in view of the purification yields (in%) calculated as follows:

[0105] Purification efficiency (% r) = (TurbO-Turbx) / TurbO (* 100)

[0106] The purification yields obtained are compared to the reference which contains

only ferric chloride (beaker 2). When the purification efficiency obtained with the biomass according to the invention is greater than that obtained with ferric chloride alone, a positive gain is calculated as follows:

[0107] GainBiomasseX (vs FcCh) = (% rx -% rF _e ci3) /% rF _e ci3

[0108] GainPAM (vs FeCL) = (% GRAM -% rFeci3) /% rF _e ci3

Finally, the performances obtained are systematically compared with those obtained with the PAM AN 934 SH: Performance compared to the PAM = GainBiomasseX / GainPAM. This result analysis makes it possible to overcome the very great variability of wastewater in terms of composition and their behavior with respect to the coagulants and flocculants used.

[0110] The representation method is given in Figure 3.

[0111] Four performance levels are defined: poor (less than 20% of the MAP gain), average (between 20% and 50% of the MAP), good (between 50% and 100% of the MAP) and excellent (greater than WFP gain).

[0112] Table 3 shows the results obtained with biomasses according to the invention (reference and variant 1 and 2), namely excellent flocculation performance (i.e. superior to that of PAM AN 934 SH). It also presents the operating conditions and results of two variants which also make it possible to obtain satisfactory performance.

Table 3. n / a = not determined.

In variant 2, thydrolyzate results from enzymatically hydrolyzed sieving residues (hemicellulases, cndo-b-1, 4-glucanascs, endo-P-D-xylanase).

[0114] Example 3: counter-examples

[0115] Table 4 shows some operating conditions which did not lead to the stable production of biomass exhibiting flocculant properties (cons-ex 1 and 2).

Table 4. n / a = not applicable.

Claims

[Claim 1] A method of producing a microbial cellular biomass having flocculant properties comprising the steps:

a) introduction into a reactor:

- optionally nutrients for the microbial inoculum,

the reaction medium obtained having a C / N molar ratio of less than or equal to 10 and a dilution rate D of less than or equal to 0.35h ^_1 ;

obtaining microbial cellular biomass having flocculant properties.

[Claim 2] The method of claim 1 further comprising aeration of the reaction medium during the process.

[Claim 3] A method according to any one of the preceding claims, implemented in an open environment.

[Claim 4] A method according to any one of the preceding claims, carried out in a non-sterile environment.

[Claim 5] A method according to any one of the preceding claims, in which the reaction medium of step a) comprises microorganisms selected from the group comprising gammaproteobacteria, flavobacteria,

alphaproteobacteria and betaproteobacteria.

[Claim 6] A method according to any one of the preceding claims,

further comprising a step of inoculating the reactor with one or more microorganisms, preferably chosen from the group comprising

gammaproteobacteria, flavobacteria, alphaproteobacteria and

betaproteobacteria, prior to step a).

[Claim 7] A method according to any one of the preceding claims, in which the substrate comprises a hydrolyzate and / or oses.

[Claim 8] A method according to any one of the preceding claims, further comprising a step of injecting into the reactor an ose or mixture of oses.

[Claim 9] A method according to any preceding claim, wherein the temperature in the reactor is 4 to 55 ° C, preferably 10 to 30 ° C, more preferably at room temperature.

[Claim 10] A method according to any of the claims

above, in which step c) is a settling inside the reactor, the settling being removed from the reactor or settling of the effluent leaving the reactor, the settling being removed and the residual then being reinjected into the reactor.

[Claim 11] A method according to any of the claims

above, further comprising a step of treating wastewater, said step of treating wastewater preferably comprising a step of contacting wastewater with a coagulant and microbial cell biomass having flocculant properties.

[Claim 12] Microbial cellular biomass having properties of

flocculant obtained by the process of any one of claims 1 to 10.

[Claim 13] Use of microbial cell biomass according to

claim 12 in the treatment of waste water.

[Claim 14] A method of treating wastewater comprising a step of contacting wastewater with a coagulant and a cellular microbial biomass according to claim 12.