DE102006058465A1 - Process for the biotechnological production of ethanol - Google Patents

Abstract

The invention relates to a process for the biotechnological production of ethanol by a cyclic sequencing batch fermentation with the following successive phases in a cycle: a) settling phase (26) of the biomass (30), b) discharge phase (27) with volume reduction in the fermenter, c) filling phase (28) by substrate supply to the original volume and d) product formation phase (29) without inflow.

Description

The The invention relates to a method for biotechnological production of ethanol through a cyclic sequencing batch fermentation.

In fermentative alcohol synthesis, alcohol (ethanol) is biotechnologically produced by means of a carbohydrate microorganism serving as a substrate. Suitable microorganisms are, for example, the yeast cultures Saccharomyces cerevisiae, Pichia capsulata, Pichia angusta or the bacterium Zymomonas mobilis. In the DE 31 48 329 C2 is z. For example, a method for producing ethanol from fermentable substrates using Zymomonas mobilis bacterial strain ATCC 31,822 is described. In general, the alcohol synthesis is carried out anaerobically in batch mode. In this case, a final concentration of ethanol of about 10 to 12 vol .-% is achieved. The achievable productivity is determined both by the duration of the fermentation and by the time required for preceding and following processes. In the subsequent non-biological process stages is usually by thermal processes or hydrophilic polymer membranes concentration to pure alcohol.

The vast majority of ethanol production plants operate batchwise in so-called batch mode. In the batch ethanol fermentation high ethanol concentrations are achieved with a low productivity at the same time. A problem with fermentative alcohol syntheses is that the product ethanol during its formation process, both on the growth of the ethanol-forming microorganisms and on the product formation itself, inhibiting, ie inhibiting or retarding effects. Due to the onset of ethanol-induced growth inhibition of the cells in the fermentation process only low biomass concentrations of up to 15 g · l ^{-1 can be} achieved. Above all, the low productivity is due to the fact that the biomass has to be recaptured during each fermentation and thus is not adapted to the high glucose and ethanol concentrations. For the discontinuous fermentation, pre- and post-processing steps are required, which also has a negative effect on the productivity, since no product formation takes place during these process steps.

In addition to the discontinuous ethanol fermentation, there are increasingly other management strategies. From the DD 225 445 A1 discloses a method for obtaining fermentation ethanol by means of microorganisms from fermentable carbohydrates, which can be applied to fermentation processes, which are conducted discontinuously, semicontinuously or continuously. The DE 29 03 273 C2 includes a process for producing alcohol by continuous fermentation of a carbohydrate-containing substrate in a fermenter.

• 1. Teilweises Füllen des Fermenters mit Nährlösung und Ethanol bildenden Mikroorganismen,
• 2. Feed-Zugabe (d. h. Zusatz an Nährlösung) bis zum maximalen Füllvolumen des Fermenters,
• 3. Ablassen des Fermenters nach Abschluss der maximalen Alkoholbildung.

From a number of prior art documents, methods for fermentative ethanol production from sugars are known, which aim to increase productivity and alcohol yield. Ideally, these methods include strategies with addition of new substrate to a certain final value of the fermentation mass. By repeating the process, cyclic operations with respect to a fermenter arise. A classical cyclic fermentation cycle consists of the following phases:

• 1. Partial filling of the fermenter with nutrient solution and ethanol-forming microorganisms,
• 2. feed addition (ie addition of nutrient solution) up to the maximum filling volume of the fermenter,
• 3. Draining the fermenter after completion of maximum alcohol production.

In so-called fed-batch mode, the ethanol concentration is diluted by a continuous or semi-continuous feed addition and the resulting increase in the volume of liquid. The aim is to reduce the ethanol-induced inhibition of growth and product formation. The fed-batch processes permit ethanol fermentation in ethanol concentration ranges, in that the inhibition by ethanol due to a sequential feed and thus dilution with concentrated feed solution is not fully developed. When a certain volume is reached, the fermentation is continued up to the maximum ethanol concentration by adjusting the feed dosage. This is how it is added Alfenore et al. Appl. Microbiol. Biotechnol. (2002) 60: 67-72 described a fed-batch process in which the ethanol concentration is kept below a certain level to reduce the inhibition of growth and product formation. After an introductory batch fermentation with a glucose concentration of 100 g.l ^-1 , a highly concentrated feed solution containing 700 g.l ^{-1 of} glucose is fed sequentially when the substrate is limited. This so-called feeding strategy is used at the beginning of the fermentation of Nährmedienversorgung and in the further course of the dilution of the ethanol content. The process is ethanol-controlled, so that when reaching a defined ethanol concentration of 90 g · l ^-1, the feed solution is added until a sufficient dilution is reached. Thereby the ethanol inhibition can be reduced. However, this process strategy is limited by the reactor volume. Once the maximum volume has been reached, the fermentation is carried out to the final ethanol concentration, with the product being taken discontinuously at the end of the fermentation. A disadvantage of the productivity of the fed-batch process strategy is, in particular, the low starting concentrations of the biomass.

The Operation with cyclic loading of the fermenter within a predetermined filling volume range is referred to in biotechnology as a repeated fed-batch procedure. The Repeated fed-batch mode is characterized in that the Fermenter is not completely emptied when draining, but a part of the fermenter content remains in the fermenter. The concentrations Alcohol that typically occurs in the repeated fed-batch mode achieve similar Orders of magnitude like in the classical batch fermentation. Another downside to productivity exists for the fed-batch process and the repeated fed-batch method in that an increased control effort for Feed regulation and pre- and Post-processing processes are necessary for every fermentation.

In the field of ethanol fermentations with continuous operation, some methods are known with which the active biomass concentration and productivity should be increased. Essentially, this is done by immobilizing the biomass, the biomass being fixed to support materials or by intercalation. The immobilized biomass can remain in the reactor due to technical installations and be prevented from discharging. The biomass discharge through the continuous product removal and the resulting low biomass concentrations in the reactor are compensated by the biomass retention, whereby the productivity can be increased. The immobilization of the biomass generally takes place outside the reactor system. Achievable productivities in systems with immobilized biomass are due to Roca, C. Appl. Microbiol. Biotechnology (2003) 60; 560-563 known. However, the high productivity reported in this document belies a high overall productivity. Although a productivity of about 100 g · l ^-1 · h ^{-1 is} calculated, but the resulting alcohol concentration is at 10 to 15 g · l ^-1 . In order to achieve these high productivities and a complete metabolic rate, very low substrate concentrations and high dilution rates are generally used. As a result, only low ethanol concentrations result, which are below those of discontinuous processes. The low ethanol concentrations require a significant overhead in the work-up of the ethanol and allow foreign organisms to establish themselves in the system, severely compromising quality.

The The object underlying the invention is to provide a cost-effective and effective process for the biotechnological production of ethanol, That's a high productivity has, but without the disadvantages described above more continuous Have fermentations.

• a) Absetzphase der Mikroorganismen (Biomasse),
• b) Austragphase unter Volumenreduzierung im Fermenter,
• c) Auffüllphase durch Substratzufuhr bis zum ursprünglichen Volumen und
• d) Produktbildungsphase ohne Zufluss.

The object is achieved by a method for the biotechnological production of ethanol, which includes a cyclic sequencing batch fermentation. One cycle of the sequencing batch fermentation can be subdivided into the following successive phases:

• a) settling phase of the microorganisms (biomass),
• b) discharge phase with volume reduction in the fermenter,
• c) filling phase by substrate supply to the original volume and
• d) Product formation phase without inflow.

The Conception of the invention consists in the reduction of the ethanol caused inhibition of the growth of ethanol forming microorganisms and product formation. The inhibitory effect of the ethanol is reduced by the cyclic operational management according to the invention, in which a settling phase of the microorganisms is integrated. The is called, that the step b), the discharge phase, in which a separation a part of the fermenter content is carried out, a step a) in shape preceded by a settling phase in which the microorganisms previously settle.

• – gerichtete Strömungsverhältnisse im Fermenter mit hohen Zellkontakten,
• – Vergrößerung des hydrophoben Charakters und
• – Verringerung der negativen elektrischen Ladung in der Zellwand.

The microorganisms are caused by deliberate use of the change in cell surface properties during the course of fermentation and by targeted flow control for settling. Studies have shown that microorganisms - in particular the yeasts mentioned - are capable of forming cell aggregates in compact flake structures by agglutination. For this purpose, targeted flows are generated, which are triggered by the injection of air into a flow guide tube (riser). The microbiological processes that lead to agglutination are based on the same processes as cells can be fixed to carrier bodies. The flocculation of microorganisms is a natural process that z. B. is also used in beer brewing. By flocculation or agglutination densify the Cells so that they sediment faster in the absence of energy input (eg air) and facilitate product separation and biomass retention. The process of flocculation is based on the combination of molecules (flocculin and mannose residue) of two neighboring cells. The selected process control and media conditions lead to an adaptation of the surface properties by the microorganisms. The high physiological stress causes a strengthening of the cell wall structures (eg by incorporation of trehalose). High ethanol concentration also causes improved flocculation, while glucose has a negative effect on floc structures. These observations showed that flocculation is not a purely biochemical process, but that physical interactions also play a crucial role. So the cells must be able to contact each other to bind to each other. The following factors improve flocculation:

• Directed flow conditions in the fermenter with high cell contacts,
• - enlargement of the hydrophobic character and
• - Reduction of negative electric charge in the cell wall.

• – genetischer Hintergrund,
• – natürliche (biochemische) Faktoren und
• – physikalische Wechselwirkungen.

From this, three superordinate groups of influencing factors can be derived:

• - genetic background,
• - natural (biochemical) factors and
• - physical interactions.

Of the high selection pressure causes microorganisms (yeasts as well Foreign organisms) who were unable to get through the system high sedimentation rates (no good flocculation behavior) to be flushed out of the system.

• 1. Durch die zuflusslose Betriebsweise in der Produktbildungsphase in Schritt d) wird eine Ethanolkonzentration erreicht, die wesentlich höher als die Ethanolkonzentration bei kontinuierlicher Betriebsweise ist. Die hohe Ethanolkonzentration reduziert den notwendigen Aufwand für die weiteren nichtbiologischen Stufen des Verfahrens. Allerdings wird das Ethanolniveau unterhalb der Konzentration gehalten, die bei der diskontinuierlichen Betriebsweise erreicht wird, damit die Aktivität der Mikroorganismen für die quasikontinuierliche Betriebsweise erhalten bleibt. Ab einer Ethanolkonzentration von mehr als 6 Vol.-% kommt es zu einer wesentlichen Verlangsamung des Wachstums der Mikroorganismen durch die Inhibierung des Wachstums (Wachstumshemmung) aufgrund des Ethanols und in dessen Folge zu einer Inhibierung der Produktbildung. Der Einfluss der Ethanolkonzentration ist berechenbar, so dass eine Optimierung der Prozessführungsstrategie vorausschauend möglich ist.
• 2. Das Verfahren ist ein unsteriles Hochleistungsverfahren, bei dem kein fremder Mikroorganismus in der Lage ist, sich als Substratkonkurrent oder als schädigender Mikroorganismus durchzusetzen. Nahrungskonkurrenten oder störende Mikroorganismen werden, sofern sie sich nicht in der Absetzphase in Schritt a) mit der Produktionskultur absetzen, in der Austragsphase in Schritt b) mit dem flüssigen Überstand ausgetragen und somit in ihren möglichen störenden Einflüssen reduziert. Dadurch werden die Kosten für die Abwehr von Fremdkeimen reduziert.
• 3. Durch die ständige Wiederholung bei der zyklischen Betriebsweise wird die Produktionskultur in ihrer Leistungsfähigkeit trainiert. Der ständige Wechsel zwischen hoher Substratbelastung am Anfang der Produktbildungsphase in Schritt d) und hoher Produktkonzentration am Ende dieser Produktbildungsphase reduziert zudem die Gefahr der Veränderung der Produktionskultur durch Mutationen.

Surprisingly, it has been found that the driving method according to the invention contains some additional, unpredictable advantages. These are listed and explained below:

• 1. Due to the continuous operation in the product formation phase in step d), an ethanol concentration is achieved which is substantially higher than the ethanol concentration in continuous operation. The high ethanol concentration reduces the effort required for the further non-biological stages of the process. However, the ethanol level is kept below the concentration achieved in the batch mode to maintain the activity of the microorganisms for the quasi-continuous mode of operation. From an ethanol concentration of more than 6% by volume, there is a significant slowing down of the growth of the microorganisms by the inhibition of growth (growth inhibition) due to the ethanol and, as a result, to inhibition of product formation. The influence of the ethanol concentration is calculable, so that an optimization of the process management strategy is possible in a forward-looking manner.
• 2. The method is a non-sterile high performance process in which no foreign microorganism is able to prevail as a substrate competitor or as a damaging microorganism. Food competitors or interfering microorganisms, unless they settle in the settling phase in step a) with the production culture, discharged in the discharge phase in step b) with the liquid supernatant and thus reduced in their possible disturbing influences. This reduces the cost of defending against foreign germs.
• 3. By constantly repeating the cyclical mode of operation, the production culture is trained in its efficiency. The constant change between high substrate load at the beginning of the product formation phase in step d) and high product concentration at the end of this product formation phase also reduces the risk of altering the production culture by mutations.

For a high productivity in the ethanol formation is an optimally high concentration of active microbial biomass required. The discharge phase in step b) the process is used to increase the concentration of active Regulate biomass while maintaining a constant optimal level to keep. This is preferably done by having high biomass concentrations not only the supernatant in the fermenter with the product, but also partially discharged the excess biomass becomes. The regulation of the biomass concentration also ensures that that there is always a high level of active biomass in the system is. Dead or inactive cells are discharged.

Aerobic fermentation stimulates both biomass growth, which is necessary for active culture, and ethanol production. In anaerobic processes, the raw material used is reduced to ethanol. However, since most of the microorganisms usable here are so-called glucose-sensitive microorganisms, a limited aerobic mode of operation causes a faster product formation and thus contributes to a higher productivity. Glucose sensitive organisms can not control uptake of the substrate (eg, glucose). Therefore, they are easily inhibited by too high concentrations, which is especially an anaerobic Process adversely affects. A completely anaerobic metabolism has only a limited substrate metabolism, which works only with a 40 to 50% reduced enzyme activity. Due to the oversupply of substrate, however, these microorganisms are also aerobically brought to the so-called oxido-reductive formation of ethanol. This is vital for the microorganisms to reduce the high substrate concentration within the cells. Your respiratory capacity is limited; This is called a limitation of oxidation, since only part of the substrate can be "respired" without the formation of ethanol, and the substrate taken beyond the respiratory capacity must be converted to ethanol in a superflow or hyperflow reaction sufficient growth to compensate for biomass losses and to maintain an active culture that forms ethanol faster due to the superflow reaction The high substrate flow into the microorganisms causes repression despite the addition of oxygen, which leads to slower growth rates than at low substrate concentrations Thus, while biomass formation is higher than in anaerobic processes, it does not reach the velocities of aerobic processes at low substrate concentrations, and this effect is used to channel much energy into product formation despite biomass growth smoke, biomass and product formation is optimized for improved yield by adding oxygen.

Due to the aerobic ethanol formation in a loop reactor, in a particularly preferred embodiment of the method in semicontinuous operation, in addition to the product discharge in the discharge phase via the product stream from the fermenter medium, a second product discharge via a secondary product stream from the gas phase is possible. This continuous product discharge is achieved by gas stripping into a gas-air mixture. On the one hand there is an advantage in the cost-effective separation of the ethanol from the gas phase due to the fact that it can be concentrated higher immediately, on the other hand the deduction also reduces the inhibiting effect of the ethanol on the production culture. The gaseous ethanol is recovered by condensation along with other volatiles. The crude product obtained is free of particles, whereby subsequent treatment steps are minimized. The use of an air-CO ₂ mixture proved to be particularly advantageous as a gas stream for gas stripping. By increasing the CO ₂ content, the superflow reaction of the substrate to ethanol is further promoted, which in turn escapes more ethanol through the gas phase. The increase in the CO ₂ content is achieved by a preferred partial recycling of the exhaust air. The continuous gas discharge by the gas stripping leads to a condensation product containing at least 30 vol .-% ethanol. The cost and the costs for the subsequent concentration of the ethanol are thereby significantly reduced, especially since it must be considered that the primary energy costs - regardless of the substrate - form the main cost factor of any ethanol production.

to further increase productivity in conjunction with the reduction of waste costs will be beneficial the ingredients of the separated biomass (vinasse) used. The vinasse is the residue the concentration or distillation of a part of the fermenter content, the unused biomass, unused starting compounds and ethanol-containing water is formed. This is a preparation the resulting vinasse by hydrolysis and thickening before Return to the Reactor. In the vinasse are, among other important vitamins and Contain trace elements that increase growth, productivity and the Increase ethanol resistance of the production culture as biomass. By the repatriation will reduces the waste to be disposed of. The provision of the ingredients is preferably carried out by a thermal treatment, wherein determined was that by the presence of ethanol cell hydrolysis favored becomes. This results in a partial flow treatment of the fermenter content before distillation.

Further Details, features and advantages of the invention will become apparent the following description of embodiments with reference to the figures and the table 1.

It demonstrate:

table 1: the composition of the feed solution,

1 : a general flow pattern of a loop reactor,

2 : A flow chart of the process for the biotechnological production of ethanol and

3 : Sequence of Sequencing Batch Fermentation.

The ethanol fermentation is carried out with the yeast culture Saccaromyces cerevisiae at a pH in the range of pH2 to pH5 and a temperature of less than 45 ° C in sequencing batch reactor operation (SBR operation) under aerobic to quasi-anaerobic conditions carried out with CO ₂ circulation. The composition of the feed solution is shown in Table 1. The preferred selected ethanol concentration in the feed solution (substrate) is 200 g · l ^-1 . In a further embodiment of the process according to the invention, the ethanol fermentation is carried out analogously to the abovementioned process with a glucose concentration in the feed solution of 300 g.s ^-1 .

The reaction vessel is a loop reactor 1 , which generally has a flow pattern according to 1 and in an area with rising liquid - the riser 2 - As well as in an area with descending liquid - the so-called Downcomer 3 - is divided. The complete mixing of the fermenter medium in the loop reactor is ensured by a gas circulation according to the mammoth pump principle. The air intake takes place at the air inlet 4 at the bottom of the loop reactor 1 through a fine-pored nozzle in the riser 2 , The air outlet 5 is located at the top of the loop reactor 1 , The airflow 6 is adjusted so that a gas empty tube speed of 1 to 4 cm / s in the riser 2 established. Parallel to the air flow in the fermenter medium 10 the substrate current runs 11 ascending in the riser 2 and flowing down in the downcomer 3 , The loop reactor 1 is of a tempering jacket 7 with a temperature control input 8th in the lower area and a Temperiermittelausgang 9 in the upper part of the loop reactor 1 envelops.

A flow chart of the process for the biotechnological production of ethanol is in 2 shown. It is on the one hand supply air 12 over the gas line 13 into the loop reactor 1 initiated. For the management of the exhaust air 14 There are two different ways. On the one hand, the exhaust air 14 be guided so that they as exhaust air flow 14a is discharged from the system. About the setting of three-way valves 15 . 16 But also a circulation of the exhaust air 14 be achieved and thus a return of the exhaust air 14 over the exhaust air flow 14b in the gas line 13 and finally into the loop reactor 1 respectively.

According to 2 the substrate passes in the form of the feed solution via an inlet of the substrate current line 17 in the fermenter medium 10 , The product removal can in principle be done in two ways. On the one hand, ethanol passes through a product stream 18 from the fermenter medium 10 , On the other hand, ethanol can be extracted from the exhaust air 14 to be obtained. In the fermentation phase, this is in the exhaust air 14 contained mixture of volatile medium components in several phases condensed and as a secondary product stream 20 dissipated. This is the exhaust air 14 in countercurrent principle in coolers 19 or cooled in cold traps to 10 ° C to -20 ° C and recovered the condensate with an ethanol content of 25 to 70% by volume for further processing. The continuous ethanol discharge allows complete conversion of the glucose, with an ethanol concentration in the product removal from the reactor 1 is reached from 50 to 80 g · l ^-1 .

To further increase the productivity in connection with the reduction of waste costs are advantageously the ingredients of the separated biomass (vinasse 21 ) used. In this case, there is a treatment of the resulting vinasse 21 from the product stream 18 from the fermenter medium 10 is separated and in the Schlempeaufbereitungsanlage 22 passes through hydrolysis and thickening. Such a processed part 21a the vinasse 21 can in the loop reactor 1 over the substrate power line 17 to be led back. This allows the part to be disposed of 21b the vinasse 21 be significantly reduced.

About a correction media feed 23 in the upper part of the loop reactor 1 can correction media, such. As acids and bases for pH regulation and anti-foaming agents to control the formation of foam, in the fermenter medium 10 be initiated. As already in 1 shown, has the loop reactor 1 via a tempering jacket 7 for the temperature control of the loop reactor 1 , The temperature of the temperature control, the - in Temperiermittelkreislauf 24 guided - at the Temperiermitteleingang 8th in the tempering jacket 7 arrives and this at Temperiermittelausgang 9 leaves again is through the heat exchanger 25 regulated.

• a) Absetzphase 26 der Mikroorganismen (Biomasse),
• b) Austragphase 27 (Produktentnahme) unter Volumenreduzierung im Fermenter,
• c) Auffüllphase 28 durch Substratzufuhr bis zum ursprünglichen Volumen und
• d) Produktbildungsphase 29 (Fermentationsphase 29) ohne Zufluss.

One cycle of sequencing batch fermentation can be performed according to 3 subdivide into the following successive phases:

• a) settling phase 26 the microorganisms (biomass),
• b) Discharge phase 27 (Product removal) with volume reduction in the fermenter,
• c) refill phase 28 through substrate feed to the original volume and
• d) product development phase 29 (Fermentation phase 29 ) without inflow.

The fermentation phase 29 is through the product development phase 29 and the biomass growth ge features. The resulting exhaust air 14 is cooled and the condensate is either in the loop reactor 1 attributed or as already related to 2 as a secondary product stream 20 from the exhaust air 14 collected.

After complete consumption of the carbon source becomes the gas line 13 and thus the gas circulation is turned off and it takes place the settling phase 26 , The shutdown of the gas circulation causes due to the density difference between the biomass 30 and the rest of the fermentor medium 10 discontinuation of biomass 30 within a period of one to 60 minutes. The product removal 27 in the discharge phase 27 ends at 50% of the filling height of the reactor 1 , The deposited biomass 30 remains in the loop reactor 1 , In the following replenishment phase 28 the substrate is in the form of the feed solution or processed vinasse 21a from below over the substrate power line 17 into the loop reactor 1 pumped until the original level of the fermentor medium 10 is reached. The process is then followed by the fermentation phase 29 continued. The discontinuation of biomass 30 in the weaning phase 26 causes a biomass retention of 50 to 95%. After reaching a steady state (steady state) arises due to the growth of biomass 30 an active biomass concentration of about 30 to 50 g · l ^-1 .

1

loop reactor

2

riser

3

downcomer

4

air intake

5

air outlet

6

airflow

7

tempering

8th

Temperiermitteleingang

9

Temperiermittelausgang

10

fermenter medium

11

substrate current

12

supply air

13

gas pipe

14

exhaust

14a

exhaust air flow (discharged from the system)

14b

Exhaust air flow into the gas line 13

15

Three-way valve

16

Three-way valve

17

Substrate power line

18

Product stream from the fermenter medium 10

19

cooler

20

secondary product stream

21

mash

21a

processed part of the vinasse 21

21b

to be disposed of part of the vinasse 21

22

Schlempeaufbereitungsanlage

23

Correction media feed

24

temperature control medium

25

Heat exchanger

26

PCT

27

discharge phase, product withdrawal

28

filling phase, substrate feed

29

Product formation phase, fermentation phase

30

biomass

Table 1: ingredients concentration unit C ₆ H ₁₂ O ₆ 200 g · l ^-1 NH ₄ H ₂ PO ₄ 5000 mg · l ^-1 KH ₂ PO ₄ 2500 mg · l ^-1 MgSO ₄ .7H ₂ O 1000 mg · l ^-1 FeCl ₃ .6H ₂ O 2 mg · l ^-1 Ca (NO ₃ ) ₂ .4H ₂ O 20 mg · l ^-1 H ₃ BO ₃ 0.5 mg · l ^-1 CuSO ₄ .5H ₂ O 0.1 mg · l ^-1 KJ 0.1 mg · l ^-1 MnSO ₄ .4H ₂ O 0.4 mg · l ^-1 ZnSO ₄ 0.4 mg · l ^-1 Na ₂ MoO ₄ 0.2 mg · l ^-1 CoSO ₂ 0.1 mg · l ^-1

Claims (11)

Process for the biotechnological production of ethanol by a cyclic sequencing batch fermentation with the following successive phases in one cycle: a) settling phase ( 26 ) of biomass ( 30 ), b) Discharge phase ( 27 ) with volume reduction in the fermenter, c) filling phase ( 28 ) by substrate supply to the original volume and d) product formation phase ( 29 ) without inflow. Method according to claim 1, characterized in that in the discharge phase ( 27 ) the supernatant is discharged. Process according to claim 1 or 2, characterized in that the concentration of active biomass ( 30 ) in the discharge phase ( 27 ) at high biomass concentrations in the fermenter the settled biomass ( 30 ) is partially discharged. Method according to claim 3, characterized in that a part of the biomass ( 30 ) is excited by air intake to aerobic growth. Method according to one of claims 1 to 4, characterized in that in addition to the in the discharge phase ( 27 ) product discharge via the product stream ( 18 ) from the fermenter medium ( 10 ) additionally a second product discharge via a secondary product stream ( 20 ) takes place. Process according to claim 5, characterized in that the second product discharge via the secondary product stream ( 20 ) Is carried out by gas stripping with a gas-air mixture and that the ethanol contained in the gas-air mixture is obtained by condensation with other volatiles. A method according to claim 6, characterized in that as the gas stream for the gas stripping an air-carbon dioxide (CO ₂ ) mixture is used. Method according to one of claims 1 to 7, characterized in that for the purpose of CO ₂ enrichment in the fermenter, the exhaust air is at least partially recycled. Method according to one of claims 1 to 8, characterized in that the resulting vinasse ( 21 ) and thickened before entering the loop reactor ( 1 ) is returned. Method according to one of claims 1 to 9, characterized in that in the feed solution 200th g · l ^-1 glucose are included. Method according to one of claims 1 to 9, characterized in that in the feed solution 300 g · l ^-1 glucose are contained.