FR2578552A1 - Processes and devices for the continuous production of biosurfactants - Google Patents

Abstract

The present invention relates to processes and devices for the continuous production of biosurfactants. A biotechnical device according to the invention comprises a reactor 1, positive displacement vacuum pumps P1, P2 which make it possible to prepare a mixture of hydrocarbons and water in defined proportions, a pre-emulsification loop 5, 6, a pump P3 for continuous removal from the reactor at a flow rate equal to the inlet flow rate, a prefilter 9 and two tangential ultrafiltration stages 10 and 11 mounted in series. The retentate R1 from the first stage passes into a decanter 12 and then through an ultrafiltration cell 13 and it is sent into a unit 15 for extraction of biosurfactants. The retentate R2 from the second stage, which contains living cells, is recycled in the reactor. The filtrate F2 from the second stage is sent into a reservoir 17 and part of it to the extraction unit 15 via a piping 20. One application is the production of biosurfactants as by-products of biological purification of waters polluted with hydrocarbons or their derivatives.

Description

 The present invention relates to methods and devices for continuously producing biosurfactants.

 The technical sector of the invention is that of biotechnologies.

 Biosurfactants are surfactants or emulsifiers that are secreted by microorganisms5.

 It is known that various micor-organxsmes growing on certain organic substrates, in particular on hydrocarbons or on carbohydrates or their derivatives, secrete biosurfactants.

 Demand for biosurfactants is important in the food, pharmaceutical and petroleum industries.

 For example, the use of biosurfactants in the sump recovery of oil contained in the deposits where the injection of biosurfactants makes it possible to reduce the viscosity of the oil, to emulsify it and to appreciably improve: the proportion of recovered oil . The biosurfactants can also be used to fight against water pollution by hydrocarbons and in this application, they have an efficiency much higher than that of.

surfactants chemical and, moreover, they are not toxic to the flora and fauna living in the water to be treated.

It is an object of the present invention to provide methods and devices that consistently produce large quantities of biosurfactants at an acceptable production cost,
Another object of the present invention is to produce biosurfactants as by-products of biological purification processes of effluents polluted by organic materials, in particular by hydrocarbons or carbohydrates.

The objectives of the invention are achieved by a prockdE which comprises the following steps:
in a reactor containing an organic substrate in an aqueous phase, a culture of microorganisms which degrade said substrate by exerting biosurfactants is carried out;
- Continuous introduction into said reactor and continuously withdraws a determined flow rate;
the continuous outflow is treated by ultrafiltration continuously from said reactor, so that a fraction containing the residual organic matter, a fraction containing the cells of live microorganisms and a fraction containing the biosurfactants in aqueous solution are obtained;
and the fraction containing the cells of live microorganisms is recycled to said reactor.

 In the case where the organic material of the substrate is composed of hydrocarbons, an emulsion of said hydrocarbons is formed in water containing between 1 and 18 g / l of hydrocarbons and, preferably, 12 g / l, which is introduced into said reactor and, after filtration, a portion of the biosurfactants is recycled upstream of the emulsifier.

 The devices according to the invention are of the known type comprising a reactor in which organic microorganisms are cultured on an organic substrate in the aqueous phase which degrade said substrate by excreting biosurfactants and means for introducing and withdrawing continuously into said reactor a flow determined from said substrate.

 A device according to the invention is characterized in that it comprises filtration means and tangential ultrafiltration means located at the outlet of said reactor which separate the liquid into several fractions, of which a first contains living microorganisms, a second of which contains biosurfactants in solution freed from cells of microorganisms and residual organic matter and said device further comprises means for recycling said first fraction into said reactor.

 In the case where the organic material of the substrate is composed of hydrocarbons, the filtration means comprise a pre-filter having a porosity between 8 and 15u which stops the heavy hydrocarbons and said ultrafiltration means comprise a first stage of tangential ultrafiltration having a porosity of between 0.7 and 8 and, preferably, of the order of 1.2 which separates the liquid into a first fraction which contains almost all the residual hydrocarbons and a filtrate freed from hydrocarbons and a second tangential ultrafiltration stage having a porosity of less than 0.5 which receives the filtrate and separates it into a fraction which contains almost all the cells of microorganisms present in the filtrate and a filtrate which contains biosurfactants in solution .

 In the latter case, a device according to the invention -con- port, in addition, qe pre-emulsification means located at the inlet of said reactor, which means comprise a high flow centrifugal pump and an emulsification chamber and it further comprises a volumetric pump which recycles, upstream of said pump, a determined bit rate of said filtrate containing the biosurfactants and the ratio between said flow rate and the flow rate of the new substrate entering said reactor is between 10 ut and 50 ut, preferably, the order of 16.

 The invention results in continuous biosurfactant production over a period of several months by means of biological reactors which admit a high degree of dilution and high organic matter contents, so that the yield is high and allows industrial production of biosurfactants.

 The methods and devices according to the invention make it possible to use, as substrate, waters polluted by hydrocarbons, by their derivatives (insecticides, pesticides, polychlorinated biphenyl) or by other organic materials based on carbohydrates or their derivatives. In this case, both water purification by biodegradation and, at the same time, biosurfactants and biomass which become by-products of purification are obtained.

 The methods and devices according to the invention are different from known methods and devices for the biological degradation of organic materials by microorganisms which excrete biosurfactants in that a large part of the biosurfactants, up to 60%, is isolated by ultrafiltration. residual organic matter and microorganism cells and makes it possible to obtain, by any known extraction process, biosurfactants that can be used in the food and pharmaceutical industries or in the petroleum industry, particularly for enhanced oil recovery. in the deposits.

Another difference lies in the fact that some of the living microorganism cells are recovered by ultrafiltration at the outlet of the reactor after they have been freed of residuals of non-biodegraded organic matter and are recycled to the reactor. which makes it possible to obtain a high rate of development of the micro-organisms in the reactor and to obtain a high processing capacity, for example an hourly processing capacity
3 equal to 100 liters per m of reactor volume, with water charged with 15 g / liter of hydrocarbons.

 In this case, by recovering 50% of the biosurfactants produced, an hourly production of biosurfactants of the order of 150 g.h-1 is obtained. m3 and one can also obtain a biomass production of the order of 350 g.h-1. m3 by recovering about 50% of the cells.

 The following description refers to the appended drawing which represents, without any limiting character, an exemplary embodiment of a device according to the invention for continuously producing bio-surfactants.

 The single figure is an overall diagram of a device according to the invention.

 This figure shows a device for continuously producing biosurfactants by fermentation of microorganisms in a reactor.

 Some strains of microorganisms are known to degrade certain organic substrates by excreting water-soluble biosurfactants.

 It is known, for example, that certain microorganisms taken from water polluted by hydrocarbons and cultured in a reactor in which a hydrocarbon emulsion is continuously introduced into the water degrade these hydrocarbons and excrete biosurfactants.

 It is also known that certain strains of microorganisms grown in a reactor on substrates containing carbohydrates or their derivatives excrete biosurfactants.

 it is specified that the process according to the invention can be applied to any organic substrate, in particular to substrates containing hydrocarbons and to microorganisms (bacteria, yeasts or fungi), capable of degrading these substrates by producing biosurfactants.

 The figure represents an example of a preferred device using a hydrocarbon-based substrate.

 It is specified that this choice is not limiting.

 The reference mark I represents a reactor or fermentor, in which a mixed bacterial population is cultivated in a non-sterile atmosphere, which has been obtained by sampling chronically polluted by discharges of hydrocarbons. it is specified that the nature of the microorganisms does not constitute a characteristic of the present invention and any strain or mixture of microorganism strains capable of degrading the hydrocarbons can be used.

 The reactor I comprises a stirrer 2Sexxample a mechanical stirrer, so that the liquid phase is stirred vigorously.

 The temperature in the reactor is maintained between 50C and 600C, preferably of the order of 300C.

 The figure represents an example in which microorganisms are cultivated on a substrate that is artificially produced by mixing hydrocarbons and water.

 Alternatively, hydrocarbon-polluted water which is degraded in the reactor can be used as the substrate and in this case the production of biosurfactants is a by-product of the purification.

 The mark 3 represents a tank or a tank of raw water which arrives through the pipe 4. A determined flow of water is taken by a positive displacement pump P1, the discharge of which is connected to a pre-emulsification device. ci comprises a high flow centrifugal pump 5 and a pre-emulsification tank 6.

Most of the outflow from the pump is recycled to the pre-emulsification tank and taken back to the suction of the pump thus forming a pre-emulsification loop. A flow equal to that of the P1 + P2 is sent into the reactor 1 through the pipe 7.

 The reference 8 represents a reservoir containing hydrocarbons or their derivatives, for example crude oil. A finished flow is pumped by a volumetric pump F2 and discharged into the pre-emulsification loop, the suction of the centrifugal pump 5.

 The respective speeds of the pumps Pi and P2 are adjusted so as to obtain an oil emulsion in water whose petroleum content is between 2 g 1 1 and 18 g l 1, and preferably of the order of 12 g. 1.

In addition, the flow rate of the pumps Pi and P2 is adjusted to obtain in the reactor a dilution ratio of between 0.1 and 0.5 h 1 and, preferably, of the order of 0.10 h 1 s c i.e., a time flow equal to 0.10 times the volume of the aqueous phase contained in the reaction
tor.

 A positive displacement pump P3 continuously draws into the reactor a flow rate equal to the inlet flow rate.

 The aqueous liquid phase pumped by the pump P3 contains residual hydrocarbons, living microorganism cells and biosurfactants in solution.

 This liquid phase passes through a prefilter 9, which is for example a filter cartridge having pores between 8p and 15p which retains a portion of residual hydrocarbons composed of heavy hydrocarbons, including asphaltenes.

This filter allows continuous operation of the order of several months before being unclogged or replaced without stopping the operation
The liquid exiting the prefilter 9 is sent into a tangential uxtrafiltration device which comprises two stages 10 and 11; The first stage 10 is composed for example of two ultrafiltration cartridges connected in series, having pores whose opening is between 0.7 and 8u and, preferably, of the order of 1.2.

 For example tangential ultrafiltration cartridges sold by MILLIPORE CORPORATION under the names "MILLIGARD" or "PELLICON" are used. The filter 10 separates the liquid into two fractions. A first fraction Ri which is the retentate of the filter contains all the residual hydrocarbons. This fraction also contains dissolved biosurfactants and microorganism cells.

 The second fraction F1 constituted by the filtrate is freed of hydrocarbons and contains only biosurfactants in solution and microorganisms.

 Fractions F1 and R1 normally correspond to about 50% of the flow rate entering the filter. By playing on valves, it is possible to slightly vary these proportions and obtain, for example, a filtrate flow F1 that can vary between 30% and 70% of the input flow rate and therefore contains between 30% and 70% of the cells and bio-surfactants. .

 The fraction R1 is collected in a settling tank 12 where the residual hydrocarbons are separated from the aqueous phase.

The aqueous phase is pumped after decantation by a volumetric pump PS and sent to a filtration unit comprising two stages 13 and 14. Each stage comprises several filter cartridges mounted in parallel. The first stage 13 is an ultrafiltration stage having a porosity of less than 0.5. It separates a retentate R3 which contains traces of residual hydrocarbons and cells. The filtrate of the filter 13 passes through a filter cartridge 14 having a porosity of less than 0.5. for example 0.22u. Retentate R4 contains the cells of microorganisms. The filtrate F4 contains biosurfactants a solution
The efficiency of the filtration unit 13, 14 is of the order of 60%.

The filtrate F4 containing the biosurfactants can be sent to an extraction unit 15, in which the biosurfactants are extracted by a known method, for example by means of solvents.
The filtrate F1 leaving the first ultrafiltration stage 10 is enybyed in a second tangential ultrafiltration stage 11, having a porosity such that it stops almost all. microorganisms, i.e., porosity less than 0'5n and preferably 0.22μ. The stage 11 consists for example of tangential ultrafiltration cartridges sold by MILLIPORE CORPO-
RATION, under the names "MILLIGARD" or "PELLICON".

 Stage II separates the liquid into two fractions in respective proportions which can range from 30% to 70% of the input rate.

 The first fraction R2, which is the retentate, contains in the living state the majority of the cells of microorganisms in the F1 fraction, that is to say about 50% of the cells taken from the reactor 1.

According to one characteristic of the invention, the retentate R2 is recycled into the reactor via a pipe
The second fraction F2, which consists of the filtrate, contains biosurfactants and 1 to 5% of microorganism cells.

 Fraction F2 is sent to a storage tank.17.

 A positive displacement pump P4 takes a determined flow rate in the reservoir 17. This flow rate is sent into a filter cartridge 18 which is for example a cartridge sold by MILLIPORE CORPORATION under the name "FLUOREX", having a porosity of 0.22. retains the small number of residual cells remaining in the filtrate F2. The filtrate F72 leaving the filter 18 is sent via a pipe 19, upstream of the emulsification unit, for example into the tank 3. The biosurfactants contained in the liquid F '2 contribute to the emulsification and the stability of the The ratio between the flow rate of the pump P1 and the flow rate of the pump P4 is between 10 and 50 and, preferably, of the order of 16.

 The figure represents a device operating with a hydrocarbon-based culture substrate that must be pre-emulsified before being introduced into the reactor. In this case, it is advantageous to recycle some of the biosurfactants upstream of the unit. pre-emulsification.

 In the case where the culture substrate is a soluble substrate such as for example sugars, it is not necessary to emulsify it. In this case, the device does not include by preemulsification unit or the loop 19 for recycling a portion of the bio-surfactants and the filtrate F-2 is sent entirely to the biosurfactant extraction unit 15 through the pipe 20.

 In total, a device according to the invention makes it possible to recover the biosurfactants included in the filtrate F4, which represent from Z to 35% of the total, and the greater part (95%) or all the biosurfactants included in the filtrate F 2 which represent 25 to 35% of the total.

 The device thus makes it possible to continuously extract 50% to 70% of the biosurfactants excreted by the microorganisms that have been freed from the residual substrate and to have recovered a portion of the cells in the living state for recycling them in the reactor. which makes it possible to maintain in the reactor a population of microorganisms in the exponential development phase.

 It will be noted that the retentate R1 contains a large proportion of cells that are found in the retentate R3. These cells can be recovered so that a device according to the invention makes it possible to produce biomass.

 Systematic tests have been carried out to study the influence of the hydrocarbon content of the substrate which is introduced into the reactor on the yield of biosurfactants and biomass.

 It is known from the literature that biosurfactants produced by hydrocarbon degradation are composed mainly of sugars and lipids. Biomass is composed mainly of proteins.

Table 1 attached corresponds to tests that were conducted with a dilution rate of 0.11 h
The first row of the table shows the substrate concentrations at the reactor inlet expressed in gl | in four trials with increasing concentration.

The second line shows the sugar contents. expressed in g 1-1. The third line shows the lipid contents in gl
The fourth line indicates the biomass in gl
The contents indicated in lines 2, 3 and 4 are the contents contained in the filtrates F2 and F4.

TABLE No.

<Tb>

<SEP> concentration of <SEP> Substrate <SEP> 7.5 <SEP> 12.2 <SEP> 14.5 <SEP> 18 <SEP>
<tb> Sugars <SEP> 1.84 <SEP> 2.7 <SEP> 2.1 <SEP> 1.6 <SEP>
<tb> Lipids <SEP> 0.228 <SEP> 0.320 <SEP> 0.258 <SEP> 0.192
<tb> Biomass <SEP> X <SEP> 5 <SEP> 7 <SEP> 5.7 <SEP> 4.2
<Tb>
These tests show that the best yields are obtained with a dilution ratio of the order of 0> 1 h -1 and for hydrocarbon concentrations of less than 18 g / l and, preferably, close to 12 g / l.

 An industrial plant according to the invention comprising 100 m3 of reactor volume that processes 10 m3 / hour of water containing 12 g / l of oil will produce 720 kg.h-1 of biosurfactants of which about 50% will be recoverable, a production of annual amount of about 125 tonnes of biosurfactants.

 In such an installation, it is also possible to recover about 50% of the biomass, ie about 1 T / day.

 The recycling of living cell microorganisms in the reactor makes it possible to increase the rate of growth of the microorganisms in the reactor and thus to use high substrate renewal rates of the order of 0.1 h. -1. hence, at constant reactor volume, a higher hourly degradation capacity.

 It is essential that the recycled fraction that contains the cells be removed before residual hydrocarbons are recycled, otherwise they will accumulate in the reactor and eventually inhibit the normal growth of the culture in the reactor.

Claims (9)

 A method for continuously producing biosurfactants, characterized in that  in a reactor containing an organic substrate in an aqueous phase, a culture of microorganisms which degrade said substrate by excreting biosurfactants is carried out;  - is introduced continuously into said reactor and is taken continuously a determined flow;  - The ultrafiltration process continuously outflow of said reactor, so that there is obtained a fraction containing the residual organic matter, a fraction containing the cells of live microorganisms and a fraction containing the biosurfactants in aqueous solution;  and the fraction containing the cells of live microorganisms is recycled to said reactor.  2. Process according to claim 1> characterized in that the hourly flow rate in said reactor corresponds to a dilution ratio between 0.1 -1 and 0.5 -1.  Process according to any one of claims 1 and 2, wherein the organic material of said substrate is composed of hydrocarbons, characterized in that an emulsion of said hydrocarbons in water containing between 1 and 18 g is formed It is preferred that hydrocrocides and, preferably, be added to said reactor and, after filtration, a portion of the biosurfactants be recycled upstream of the emulsifier.  4. Method according to any one of claims 1 to 3, characterized in that said substrate is water polluted by hydrocarbons which are degraded in said reactor resulting in a depollution effect.  5. Device for continuously producing biosurfactants of the type comprising a reactor in which micro-organisms which degrade said substrate by excreting biosurfactants and means for continuously introducing and withdrawing into said reactor are cultured on an organic substrate in the aqueous phase. a determined flow rate of said substrate, characterized in that it comprises filtration means and tangential ultrafiltration means located at the outlet of said reactor which separate the liquid into several fractions, a first containing living microorganism cells and a second of which contains biosurfactants in solution freed from the cells of microorganisms and residual organic materials and said device further comprises means for recycling said reactor said first fraction.  6. Device according to claim 5, for the preparation of biosurfactants from a hydrocarbon-based substrate, characterized in that said filtering means comprise a pre-filter (9) having a porosity of between 8 and 15 which stops the heavy hydrocarbons and said ultrafiltration means comprise a first tangential ultrafiltration stage (t0) having a porosity of between 0.7U and 8 and, preferably, of about 1.2y which separates the liquid into a first fraction (R1 ) which contains substantially all of the residual hydrocarbons and a hydrocarbon-freed filtrate (71) and a second tangential ultrafiltration stage t11) having a porosity of less than 0.5, which receives the filtrate (PI) and separates it in a fraction (R2) which contains almost all the cells of microorganisms present in the filtrate (F1) and a filtrate (F2) which contains biosurfactants in solution.  7. Device according to claim 6, characterized in that it further comprises pre-emulsification means located at the inlet of said reactor, which means comprise a centrifugal pump (5) with a high flow rate and a chamber (6). ) of emulsification and it further comprises a positive displacement pump (P4) which recycles upstream of said pump (5) a determined flow rate of said filtrate (F2) containing the biosurfactants and the ratio between said flow rate and the new substrate flow rate entering said reactor is between 10 and 50 and, preferably, of the order of 16.  8. Device according to claim 6, characterized in that it further comprises a decanter (12) which receives said first fraction (R1) leaving the first ultrafiltration stage (10), a centrifugal pump (P5 ) which sucks the decanted liquid, a tangential ultrafiltration cell (13) having a porosity of less than 0.5 which rids the liquid of traces of residual hydrocarbons and cells and the filtrate (F4) having passed through said stage (13) is sent to a biosurfactant extraction unit (15).  9. Device according to any one of claims 6 to 8, characterized in that it further comprises a biosurfactant extraction unit contained in said filtrates (Fl, F2 and F4).