WO1989010968A1

WO1989010968A1 - Process and installation for producing carbon dioxide and ethanol by multiple-stage continuous fermentation

Info

Publication number: WO1989010968A1
Application number: PCT/FR1989/000212
Authority: WO
Inventors: Charles Ghommidh; Catherine Fourtot; Jean-Marie Navarro; Jean-Marcel Cutayar; Jean Amen
Original assignee: L'air Liquide, Societe Anonyme Pour L'etude Et L'e; Oxigénio Do Brasil
Priority date: 1988-05-04
Filing date: 1989-05-02
Publication date: 1989-11-16
Also published as: ZA893238B; JPH02504107A; EP0365671A1; BR8906945A; FR2631040A1; AU3688289A; FR2631040B1

Abstract

In a process for multiple-stage continuous fermentation of a medium inoculated with microorganisms such as bacteria and yeast, each fermentation stage has a system for retaining microorganisms. The biomass is conveyed continuously or intermittently from one fermentation stage to the next, or the volume of each fermentation stage, in particular the first stage, is such that the critical concentration of the metabolite cannot be reached at the nominal operating output of the fermentation system. Application to the production of carbonic acid and ethanol.

Description

"PROCESS FOR PRODUCING CARBONIC ANHYDRIDE AND ETHANOL BY CONTINUOUS MULTI-STAGE FERMENTATION AND INSTALLATION"

The present invention relates to a process for producing carbon dioxide and ethanol by continuous multistage fermentation.

Although there is currently no doubt about the technical feasibility of producing ethanol by continuous fermentation, economically a number of constraints weigh on the development of this technique. Thus the substrate: sugar, starchy materials, must be converted, as completely as possible, into alcohol with a yield approaching the theoretical maximum production of 0.51 kg of ethanol per kg of glucose; similarly, the final ethanol content of fermentation juice must be greater than 60 g per liter, in order to minimize the energy cost of distillation and the cost of processing distilled vinasses.

In the fermentation field two types of microorganisms are of industrial interest. Yeasts, mainly of the genus Saccharomyces are well known and used for a very long time in brewing, winemaking ... And, bacteria belonging to the genus Zymomonas, arouse, because of their higher metabolic activity, an increasing interest.

At identical final ethanol concentrations, productivity and yield are the two main parameters that allow us to judge the efficiency of a fermentation process. Since the speed of production is closely linked to the quantity of active microorganisms present in the fermenters, continuous fermentation processes must increase the active microbial population in order to increase their productivity. To combat the dilution phenomena, which would tend to decrease the microbial concentration, generated by high dilution rates, these methods use techniques for retaining efficient microorganisms.

European patent application N ° EP. 0213005, offers a continuous fermentation process using flocculating microorganisms, the retention of which in the system is ensured by internal settling-recycling. This process achieves high concentrations of microorganisms. It is also insensitive to contaminants and avoids any mobile mechanical means essential in systems with external settling. In general, flocculation is a biological phenomenon by which microorganisms combine to form particles of biomass or cellular aggregates of microscopic size, called flocs, the size of these flocs generally varying from 0.2 to 6 mm, with a average value of 2 mm.

In continuous fermentation, in a single mixed tank, it is not possible to reach ethanol concentrations higher than 50-70 grams / liter; from this concentration called critical concentration, the mortality rate of microorganisms exceeds their growth rate; and this all the more that the ethanol concentration is high. The value of the critical concentration depends on the strain of microorganism, the raw material used and the fermentation conditions (temperature, oxygenation, etc.).

On the other hand, in a multi-stage fermenter, the alcoholic concentrations obtained largely exceed those obtained in a single-stage fermenter. Because, the first fermenters are used to generate the microorganisms which ensure, before their destruction, the complementary production in the following stages, indeed the production of ethanol continues either until exhaustion of the sugar, or until the death of the last microorganism.

The evolution of the concentration of biomass and ethanol as a function of time, for a culture of Zymomonas mobilis is represented in FIG. 1 of the appended drawing, the concentrations of biomass (X) in DO unit at 510 nm are plotted on the ordinate , as well as those of ethanol (C ₂ H ₅ OH) in grams / liter, and the time in hours is plotted on the abscissa (t / h). Curve 1 corresponds to the change in the concentration of total biomass, curve 2 to that of viable biomass and curve 3 to the concentration of ethanol.

The improvement in productivity generated by the direct recycling of the microorganisms from the effluent in the feed stream of a multi-stage system is only of marginal interest if the ethanol concentration of the effluent exceeds the critical concentration, the microorganisms having been, in fact, killed, at least partially, by ethanol.

To benefit both from the advantages brought by the confinement of microorganisms in the system and by the presence of multiple stages of fermentation, the recycling of biomass must take place at each stage, avoiding the transfer from a downstream stage to an upstream stage.

Among the many known methods, only those described in GB patent 2,125,064 and European application EP. 0213.005 have these technical characteristics.

The first of these two methods proposes a control strategy allowing rational optimization of the operation of a multi-stage system with confinement of microorganisms; while recognizing the need to transfer biomass from upstream to downstream, as is the case in all traditional multi-stage continuous processes. Indeed, in the absence of transfer, as could be obtained with a system for absolute retention of microorganisms (ultrafiltration membrane for example), the system would evolve spontaneously either towards clogging or blocking of any flow by excessive accumulation of biomass (for concentrations between 120 and 250 grams / liter of dry mass of microorganisms; this concentration being a function of the microbial strain and of the stirring system used), i.e. towards growth arrest caused by an increase excessive concentration of ethanol and / or associated metabolic inhibitor products.

This can be explained mathematically in a simple way by establishing the material balance on the mass of microorganisms, at the level of the first stage of fermentation for example:

- in the absence of transfer, the variation of the biomass concentration over time dX / dt, in the first fermentation stage is given by the relationship: dX / dt = μx where μ represents the growth rate of the microorganisms.

If we eliminate the trivial solution x = 0 gram / liter, an equilibrium state: dX / dt = 0 will be reached only for μ = 0 h. Under these conditions, the renewal of the microorganisms destroyed by the high concentrations of ethanol would no longer be ensured in the following stages.

The solution adopted in the British patent consists in slaving the operation of a microorganism transfer device (a pump for example), either to the ethanol concentration in the first stage of the system, or to the activity measured. by any means (the production speed of C0 for example). The control relates only to the first stage of fermentation.

A process for the production of carbon dioxide and ethanol has been found, by continuous multi-stage fermentation of a culture medium inoculated with microorganisms in the form of flocs, of the bacteria or yeast type with retention and recycling of microorganisms, the operation is independent of that of any measurement system.

According to an object of the invention, a controlled transfer (regular, continuous or timed) of the biomass is carried out from one fermentation stage to the next stage.

Under these conditions, the average transfer rate reduced to the volume unit of the fermentation stage is equal to the growth rate of the microorganisms when equilibrium is reached. The transfer law is defined on the basis of prior knowledge of the relationship between the concentration of ethanol and the growth rate of the microorganism. The variation of the biomass concentration over time dX / dt = isX - (q / v) .X, where it represents the growth rate of the microorganisms and q / v represents the average transfer rate reduced to the unit of volume of the fermenter. Balance is reached for q / v = LL

This type of transfer can be carried out either using a pump, or by opening a valve, or by any other suitable device.

In such a system, it is all the more necessary to transfer the active microorganisms from the upstream stages to the downstream stages that the final ethanol concentration is high, this in order to ensure the maintenance of the viability of the microorganisms in the last stages of the fermenter. Thus, in the first stage, the increase in the transfer rate q results in an increase in the growth rate / ι and a decrease in the alcoholic concentration.

This observation leads to maintaining in the first stage of the fermenter an alcohol concentration P all the lower the desired final concentration of ethanol is high.

For simplicity, it is assumed that there is a linear relationship between the growth rate | i and the ethanol concentration P: μ = μ _MAX (1-P / P _C ) in which μ _MAX is the maximum growth rate, without limitation and P is the critical ethanol concentration, from which microorganisms die.

Let P be the ethanol concentration to be reached at the outlet of the second stage. For economic reasons, P is greater than P. The growth rate in the second stage is therefore negative. In steady state, by neglecting biomass leaks, cell mortality is compensated by the supply of biomass from the first stage. A material balance on the second fermentation stage gives: qX ₁ + μ ₂ X ₂ V ₂ = 0 ⇒ q = - μ ₂ (X ₂ / X ₁ ) V ₂ where X and X are the biomass concentrations in the two stages , mainly determined by the characteristics of the retention system.

By replacing μ ₂ by its expression, we obtain:

It is clear from this equation that the concentration P will be lower the higher the concentration P.

According to another object of the invention, the volume of each fermentation stage, and of the first in particular, is calculated in such a way that, for the nominal operating flow rate of the fermentation system and for a determined final ethanol concentration, the transfer of biomass from the upstream stages to the downstream stages is effected by overflow as soon as the biomass concentration exceeds the retention capacities of the various stages. Such behavior implies a continuous growth of microorganisms in the first stage; the volume V ₁ will therefore be calculated from the following relation

:

from where :

V ₁ = volume of the first stage

Q = feed rate

P ₁ = ethanol concentration at equilibrium in the first stage v ₁ = specific fermentation activity of microorganisms

X _1max = limit concentration of microorganisms in the first stage.

For a flocculating yeast X _1max is around 90 to 120 g / liter.

For Zymomonas flocculant X _1max is around 70 to 80 g / liter.

P is defined in the same way as above. Furthermore, it is necessary that the microorganism retention system in each stage allows overflowing from one stage to another. This constraint therefore excludes the use of membrane type retention systems.

It is established that in systems where the microorganisms are present in the form of a solid phase (cells included, microbial films, flocs), concentration gradients of the substrates and products of metabolism appear, which can cause changes in the speed of reaction or even changes in metabolism.

In the case of alcoholic fermentation by flocculent yeasts, it has been shown that the gradients of concentration of glucose and ethanol were insufficient to appreciably modify the reaction kinetics.

On the other hand, for CO ₂ , the gradient is sufficient to cause the formation of bubbles at the center of the largest flocs. Traditionally, the fermenters used for the culture of flocculating microorganisms are of the fluidized bed reactors type: the flocs are kept in suspension in a tubular reactor of vertical axis by the upward flow of the fermentation medium. For microorganisms like Zymomonas, this operating principle is satisfactory: during the formation of the CO ₂ bubble, there is a local break in the floc. The cell-cell links being irreversibly broken, the floc bursts. There is therefore self-regulation of the particle size. With yeasts, the situation is different: if, during rupture, the floc is not subjected to sufficient shear stresses to separate the two fragments, the cells again adhere to each other after the bubble has left . The floc is therefore not necessarily divided. This can lead to formation of hollow spheroid flocs (0 = 0.5 - 1 cm, e = 2 mm), with fermentation activity, which occupy a large volume.

It is therefore important to control the size of the flocs, so as to avoid the appearance of inactive zones or the flotation of the aggregates, for the largest particles, while avoiding too intense disruption of the flocs which would lead to a significant loss of efficiency. It is generally unknown that the volume occupied by a given mass of flocculent yeasts is directly linked to the intensity of agitation. Figure 2 of the accompanying drawing illustrates this phenomenon through the volume occupied by the biomass, plotted on the ordinate V in liters, as a function of the sedimentation time in minutes t (min) plotted on the abscissa and the stirring speed ( rpm), curve 1 corresponds to a speed of 200 rpm, curve 2 to a speed of 300 rpm and curve 3 to a speed of 500 rpm. For the same population of flocculent yeasts, in a turbine reactor, the volume occupied by the sedimented flocs after stirring has stopped is a function of the sedimentation time, on the one hand, and of the prior rotation speed of the turbine. The sediment compacts slowly, the duration of this phenomenon is much longer than that of the mixture in a fermenter and does not allow a flock broken by agitation to regain its maximum compactness.

As a result, a. too intense agitation results in a loss of useful volume in the fermenter.

The agitation is optimal when it allows the breaking of the flocs weakened by the appearance of a CO ₂ bubble in their center.

This result is achieved:

- Either by pneumatic agitation by injecting a gas into the fermentation tank, with a flow rate between 0.2 and 2 volumes of gas per volume of medium and per minute. The gas can be air, or recycled fermentation CO ₂ or a mixture of the two;

- either by mechanical agitation using an agitator of the marine propeller type or of the anchor type, with a speed at the blade tip of the order of 0.1 to 0.2 m / s.

The stirring conditions are adjusted taking into account, on the one hand, the concentration of biomass to be kept in suspension, on the other hand the rate of fermentation which itself produces a gaseous release contributing to the stirring.

On the other hand, in the case of pneumatic agitation, it is also possible to control the size of the flocs by playing on the nature of the recirculation gas. When the gas used is recycled CO ₂ , the fermentation medium has a concentration of dissolved CO ₂ such that the transfer of CO ₂ from the floc to the liquid takes place slowly. The CO ₂ produced then accumulates within the flock until the flock bursts. The average size of the flocs will therefore be lower the higher the quantity of CO ₂ dissolved in the liquid. It is therefore possible by using at least partially the other gas than CO ₂ , for example air or a CO ₂ / air mixture, to obtain flocs of larger average size.

Still with a view to rationally increasing the yield of a multi-stage fermentation system with retention or confinement of microorganisms, it is advantageous to feed the substrate distributed over the different fermentation stages.

This in fact makes it possible to avoid any inhibition by excess of substrate at the level of the first stage. And by this means of the substrate supply, one controls the ethanol concentration (s) at equilibrium in the different stages.

Examples are given below which illustrate the invention without implied limitation. EXAMPLE 1.

Alcoholic fermentation with Zymomonas mobilis I 411 (Institut Pasteur) flocculated in a continuous two-stage fermenter, shown in Figure 3 of the accompanying drawing.

This installation includes the fermenters 1a, 1b, provided with internal decanters 2a, 2b and overflows 3a, 3b the overflow circuit 3, the feed 4a, 4b in nutritive medium constituted by the feed rings 4.1 at the base of the fermenters , the nutrient medium metering pumps 4.2, the water metering pump 4.3 and the preheater 4.4, and the gas flows 5a, 5b, on the circuit of which there are the rotameters 5.1 (used to measure the production of CO ₂ ) the condensers 5.2, the rotary masters 5.3 to measure the gas recirculation (CO ₂ recycling), in 5.4 a short circuit, the compressors 5.5, the filters 5.6 and the gas injection crowns 5.7 at the base of the fermenters. The installation also includes the foam control devices 6, the pH control devices 7, the temperature control devices 8, a biomass transfer pump 9, a timer 10, and orifices for sampling 11.1 of the nutrient media, 11.2 for the sampling of fermenters and 11.3 for the sampling of the overflow of the 1st stage and 3b for that of the overflow of the 2nd stage.

The fermentation was carried out in a first fermentation stage la, with a volume V ₁ of 1 liter, and in a second fermentation stage lb, with a volume V ₂ of 2 liters. The feed rate of the

1st stage of fermentation Q ₁ is 1.82 liters / hour and that of the second stage of fermentation Q2 is 0.32 liters / hour. The sugar concentration of the feed medium of the 1st fermentation stage S _o 1 is

150 g / 1, that of the feed medium of the second fermentation stage

S _o 2 is 540 g / l,

The ethanol concentration at the outlet of the 1st stage is 60 g / liter and that at the outlet of the second stage is 90 g / liter.

The transfer rate (q) of the biomass obtained by timed pumping from the first fermenter to the second is 0.02 liters / h; the second stage biomass is evacuated by overflow.

In the two fermenters the temperature is similar by 30 ° C. In the first stage of fermentation the pH is 4.9, and the pH of the second is 4.8.

The fermentation media are agitated through the fermentation gases, the recirculation rate being 1.6 vvm in the first stage and 0.8 wm in the second stage.

The overall ethanol productivity is equal to:

The residual sugars are at a concentration of 20 grams / liter.

The sugar use yield is 90.5% and the sugar conversion yield is 0.48 grams of ethanol per gram of sugar consumed. EXAMPLE 2

The experimental device is the same as above, but the feed rate of the first stage is brought to 3.3 1 / h and that of the second stage to 0.3 1 / h. The concentrations of the two sugar feeds are 150 and 650 g / l respectively.

The concentration of biomass in the first stage reaches 70 g / 1. The transfer of biomass is then made by overflow into the second stage. The ethanol concentration reaches 92 g / l in the second stage, with a residual sugar concentration of 10 g / l. The overall productivity is equal to 110 g / l / h.

The sugar use yield is 95% and the conversion yield is 0.505 grams of ethanol per gram of sugar consumed (ie 99% of the maximum theoretical yield). Example 3:

Alcoholic fermentation with a flocculated yeast Saccharomyces cerevisiae 38A (INRA MONTPELLIER IPV) in a continuous two-stage fermenter shown in FIG. 4 of the attached drawing.

The two-stage system comprises the fermenters 1a and 1b, the decanters 2a and 2b, the overflows 3a and 3b and the overflow circuit 3 between the fermenters la and lb, (flow rate Q1), the substrate supplies 4a and 4b (flow rates Q1 and Q2), the circuits for circulating the CO ₂ recycling gases 5a, 5b.

The installation further comprises in 6a and 6b the instantaneous means of measuring the CO ₂ produced in the first and the second fermenter, and by the circuit 7 where the flow rate at the outlet of the system is Q1 + Q2.

The fermentation is carried out under the same general conditions as above; the substrate is formed by the P2 sewers from the sugar refinery. The feed rate Q1 of the first fermentation stage is 1.45 liters / hour and that of the second stage Q2 is 1.30 liters / hour; the volume of the first stage being 2 liters and that of the second stage 5 liters.

Biomass transfers are ensured by overflow. The biomass concentration is 110 g / l in the first stage and 80 g / l in the second.

The sugar concentration of the first stage feed medium is 110 g / liter, and that of the second stage is 250 g / liter. The amount of residual sugar leaving the 2nd stage is 10 g / liter. The ethanol concentration at the outlet of the 1st stage P1 is 51 g / liter and that at the outlet of the 2nd stage is 80 g / liter.

Overall ethanol productivity:

The sugar utilization yield is 94.5% and the sugar conversion yield is 0.48 grams of ethanol per gram of sugar consumed (ie 90% of the maximum theoretical yield).

Claims

1. Process for the production of carbon dioxide and ethanol by continuous multi-stage fermentation of a culture medium inoculated with microorganisms in the form of flocs, of the bacteria or yeast type with retention and recycling of microorganisms, characterized in that one proceeds to a regular and continuous controlled transfer of the biomass from one fermentation stage to the next stage.

2. A method of producing carbon dioxide and ethanol by continuous multi-stage fermentation according to claim 1, characterized in that one proceeds to a timed controlled transfer of the biomass from one fermentation stage to the next stage.

3. A method for producing carbon dioxide and ethanol by continuous multi-stage fermentation according to claims 1 or 2, characterized in that the average rate of transfer of the biomass from one fermentation stage to the next stage is calculated from so as to compensate for the loss of viability of the biomass in the downstream stages.

4. Method for producing carbon dioxide and ethanol by continuous multi-stage fermentation of a culture medium inoculated with microorganisms of the bacteria or yeast type with retention and recycling of the microorganisms according to any one of claims 1 to 3, characterized in that the volume of each fermentation stage, and of the first in particular, is such that for the nominal operating flow rate of the fermentation system, and for a determined final ethanol concentration, the transfer of biomass from the upstream stages to the downstream stages 'performs by overflow.

5. A method of producing carbon dioxide and ethanol by continuous multi-stage fermentation according to any one of claims 1 to 4, characterized in that one proceeds to a substrate feed distributed over the different stages of fermentation.

6. A method of producing carbon dioxide and ethanol by continuous multi-stage fermentation according to claim 5, characterized in that by means of the supply of substrate, the equilibrium ethanol concentration is controlled in the different stages .

7. A method of producing carbon dioxide and ethanol by continuous multi-stage fermentation according to any one of claims 1 to 6 characterized in that the size and density of the flocs is controlled by injection of air, CO or a air mixture and CO ₂ with a flow rate between 0.2 and 2 vvm (gas volumes per culture volume and per minute) preferably 0.8 to 1.5 vvm

8. A method of producing carbon dioxide and ethanol by continuous multi-stage fermentation according to any one of claims 1 to 6, characterized in that the size and density of the flocs is controlled by mechanical stirring with a stirring mobile of the type marine propeller or anchor with a blade tip speed not exceeding 0.2 m / s.

9. Two-stage fermentation installation for the production of carbon dioxide and ethanol for the implementation of the process according to any one of claims 1 to 8, characterized in that it comprises the fermenters (1a) and 1b), provided with internal decanters (3a) and (3b) and the overflow circuit 3 between the two fermenters, nutrient medium supply circuits (4a) (4b) constituted by the supply rings (4.1) to the base of the fermenters, and fitted with nutrient medium metering pumps (4.2), water metering pumps (4.3) and a preheater (4.4), the circuits

(5a) and (5b) for recycling carbon dioxide provided with rotameters (5.1), condensers (5.2), filters (5.6) and rings for injecting recycling gas (5.7).

10. Two-stage fermentation installation according to claim 9, characterized in that it further comprises a biomass transfer pump (9) and a timer (10).