DE3824743C1 - Process for the production of biotechnological products by solid fermentation - Google Patents

Abstract

Process for the production of biotechnological products by solid fermentation of moist, granulated biomass formed from support composition and from substrate for the microorganisms employed for the fermentation, in which the metabolic products of the microorganisms are taken off by a gas while leaving the moist, granulated biomass at rest in a bed during a phase I, evacuating the tank with a vacuum pump and keeping the biomass under a preset vacuum during a phase II, feeding in the gas at a flow rate above the fluidisation point during a phase III, and repeating phases I to III when the gas has returned to the pressure chosen for phase I.

Description

The invention relates to a method for manufacturing biotechnological products through solid fermentation according to the preamble of claim 1 as it is from O. Moebus, M. Teuber: Der Gas / Fest Fluidized bed bioreactor. Microbiology Forum 9 (4) 187-197 (1986).

Solid fermentations are used in the present Registration viewed all biotechnological implementations, those in moist but still solid particles can be carried out. The scope of the given instructions is still on granular Restricted solid systems that are fluidizable or can be made fluidizable by adding flow agent can.  

For some forms of application in the biotechnological field the technology needs significant improvements. At the ethanolic fermentation z. B. is the removal of the formed ethanol restricted with the exhaust gas, that the gas has only a limited amount of ethanol (and Water) can transport. That means the maximum Yeast formation of ethanol in a fluidized bed fermenter depends on the gas flow. Another limitation concerns the selection of those for the gas / solid fluidized bed fermenter suitable microorganisms. The Application of the fluidized bed technique is generally due to this restricted that at higher gas speeds abrasion on the particles and on the container walls takes place. Of these, especially sensitive microorganisms affected. Hyphogenic mushrooms and in the case of Actinomycetes, abrasion was possible in our own experiments microscopically perfect in the peripheral area of the particles be determined. Practically unaffected only a few yeasts and yeast-like mushrooms remained (e.g. Oospora lactis, Hansenula anomala), of which known is that they contain chitin in their cell wall and may be resistant to abrasion for this reason. A third disadvantage arises from the fact that - especially with slow growing microorganisms in the lag phase - sufficient to operate the fluidized bed Air quantity must be present, but in relation on the supply of oxygen to the microorganisms represents an oversupply.

By installing a stirrer in the bed, the Defects not described above, not even then not if the stirrer is operated in a fixed bed.

The invention is therefore based on the object of a method specify with which - while maintaining the benefits fluidization - solid fermentation  can be carried out so that an abrasion of the particles avoided a discharge of volatile fermentation products not limited by the gas velocity and a better adjustment of the energy requirement for fermentation is made possible.

This object is achieved by the characterizing Part of claim 1 specified Features solved. The subclaims give advantageous Embodiments of the invention.

The invention is described below with reference to a drawing, reproduced in a plant for performing the method is explained in more detail.

The system consists of a fermenter 1 , a wind boiler 2 , a vacuum pump 3 , a quick-closing valve 4 for evacuating the fermenter, a quick-closing valve 5 for gassing the fermenter, a cellular wheel lock 6 for adding solids, and a cellular wheel lock 7 for Harvesting the biomass, a heat exchanger 8 arranged in the fermenter 1 , a distributor element 9 for the fluids, a three-way valve 10 for regulating the temperature in the fermenter with a bypass bridging the heat exchanger, a drain valve 11 , a relief valve 12 and one Inflow tray 13 for the fumigation of the fermenter.

The biomass is also used in a procedure according to the invention fluidized, in contrast to the known fluidized bed process however, the fluidization takes place as Single step of a periodic process in such a way that they are understood as re-gassing a tank can, the active particles before gassing as dormant Contains fill and was previously evacuated.  

During the fluidization, the quick-closing valve 4 at the fermenter outlet is closed. The second quick-closing valve supplies gas to the fermenter 1 via gas distributors at such a speed that the vortex point is exceeded and fluidization takes place. The gas penetrates the pores of the particles. This effect does not occur in the known fluidized bed. By spreading the pressure, the particles are driven apart until they fill the entire fermenter chamber before then collapsing again into a packed bed. In this way, the particle abrasion is also considerably reduced, in particular also because the period of fluidization is only a fraction of an entire period (period from one fluidization to the next).

The duration of the individual phases is different. Phases I and II are based on Type of fermentation (see examples in Table 2), the phase II is also of the constructive design of the Equipment or the size of the individual units dependent. The duration of phase III is exclusive due to construction.

Since the quick-closing valve 4 and the emptying valve 11 at the fermenter outlet are closed during the fluidization process, the devices, such as filters and separating organs, which are otherwise necessary in fluidized bed technology and catch the discharged particles and return them to the fermenter, are eliminated. In addition, the calming room of this size, which is often generously dimensioned above the fluidized bed, can be omitted. In the proposed method, the fermenter space must be large enough to accommodate the fluidized biomass.

An illustration of the mode of operation for the process shows Table 1. Should the proposed system be used for alcoholic fermentation, so is a Distilling off the ethanol in phase II of the process guaranteed. It works well for this design from the fact that the particles as a result of evaporation lower temperature than that sucked in by the vacuum pump Have vapors so that the environment of the particles is a may have a higher temperature than the microorganisms in aqueous systems can be expected. For vacuum operation a rough vacuum is sufficient with distillation.

Another effect also has a favorable effect on the Process out. During the ethanol / water vapors in a rough vacuum flow through the bed, there is an accumulation of the lower-boiling component ethanol in the Steam, while water prefers the particles remains.

The integration of the vacuum pump 3 enables a thermally favorable design of the process sequence, particularly in the case of alcoholic fermentation. In this case, the fermenter 1 can be designed in the form of a circulation evaporator. It contains elements for heating, as they are also used for the fluidized bed. The vapors from the fermenter 1 are sucked in with the vacuum pump and compressed against the atmospheric pressure present in the air boiler. The vapors are condensed in the pipeline system of the fermenter 1 by releasing heat to the fermenter content so that further liquid can evaporate. The energy required to operate the vacuum pump 3 is sufficient to carry out the entire process of fermentation and distillation of the alcohol / water mixture.

The system described here is particularly suitable be bio-alcohol in conjunction with a synthesis valuable, Natural products requiring adenosine triphosphate (ATP) to produce. ATP falls in the ethanolic Fermentation inevitably. This possibility of simultaneous Synthesis of biochemicals in the suggested sense could be interesting in the future. For synthesis There is a significant improvement in bio-alcohol of economy. In the case of simultaneous synthesis glutathione and ethanol are produced stoichiometrically two moles of ethanol and one mole of glutathione from one mole Glucose + precursor compounds.

S. cerevisiae breeds producing ethanol can be used for further syntheses are suitable microorganisms, since they are known is that cultures with these microorganisms in Fermentation approaches remain remarkably stable and many approaches can be carried out with them without the Cultures have to be newly grown. Because of the ethanol content the biomass is at risk of infection low. Except the use of these yeasts for already known ones Syntheses can be modified using the methods of genetic engineering Yeast, with synthetic possibilities for fabrics the animal and plant kingdom. Because it are cultural breeds that do not exist in nature occur, the modified yeasts pose no danger for the environment. As eukariotic microorganisms these yeasts are preferred for functional expression of eukaryotic genes from animal and Plant kingdom used.

Yeast factories in the Federal Republic of Germany, the Burning rights can be achieved by varying the oxygen content the ratio of baker's yeast in the supply air vary to ethanol. The alcohol can be used during the serving be separated. To do without the washing stage  it would be important, however, that the yeast itself is used as a fluidized granulate and accounted for Nutrient solutions are sprayed into the bed. A Using molasses would not be recommended.

Alcoholic fermentation can also take place when fully fumigated be carried out with air through defective yeasts because with these yeasts the Pasteur and Crabtree effects no longer work.

As a fluidizable carrier for yeasts and at the same time Ground and moistened grain products can be used as a substrate and a variety of other starch products, also pregelatinized starch can be used if suitable amylases and / or for digesting the starch Amylopeptidases can be added to the granules. The Use of cellulose-containing particles with an additive of cellulases as hydrolyzing enzymes is also feasible.

Open-celled formaldehyde resin foams, such as those for Soil improvement are used for growing nitrogen-binding bacteria - e.g. B. Azotobacter chroococcum - suitable. This bacterium grows in the loose structure of the resin.

Fluidizable immobilized microorganisms or parts of them can be used as a biologically active mass for biotechnological Implementation to be used. Can too the microorganisms themselves - e.g. B. granulated pressed yeast of the trade - without an additional carrier as an active particle form be used.  

For the reduction of the carbon dioxide content in the air in space ferries is the cultivation of green algae in one Fluid bed planned. The procedure described here enables fluidization in a plant with essential reduced space requirements and could therefore Fluidized bed are preferred.

Adapted to the needs of the microorganisms used can use water to regulate water activity and Nutrient solutions are sprayed into the particle mass.

Examples of the application of solid fermentation

Table 2 shows examples of the application of the Solid matter fermentation with its essential distinguishing features listed.

Processes mainly with oxygen in the air, those on breathing, oxidative fermentation or aerobic Implementation based on precursor compounds, run in phase I. In the predominantly aerobic processes Phases II and III are only switched on when the gas no longer flows evenly through the bed and "dead" areas have formed. This is particularly true on systems with mycelium-growing microorganisms to where it is easy to bridge between the particles (felting) can come. By the Interposing the fluidization is the bed renewed, the matting is stopped and thereby uniform gas flow guaranteed. Switching on and The course of the fluidization phase can be known Components of pneumatic conveyor technology are monitored.  

Processes with predominantly anaerobic metabolism are running in phase II. This includes fermentations with the Formation of volatile fermentation products are associated. In In the known fluidized bed processes, the speed is often sufficient of the propellant gas to the formed To remove fermentation product ethanol and thus the inhibition of To prevent fermentation intensity through your own fermentation product. In phase II, the fermentation product is continuously distilled off and thus avoided inhibition.

The Saccharomyces cerevisiae / sugar solution system by switching from phase I (with air) to phase II (with N₂ and CO₂) from cell mass to ethanol production switched. The periodic interposition of the Phases II and III in the case of biomass and phases III and I in the case of ethanol production is used for Implementation of fluidization. The same also applies if additionally precursor substances for the synthesis of Glutathione are entered.

Ground starch products are used instead of sugar solutions as a substrate and at the same time as a carrier for solid fermentation to produce ethanol. When using Saccharomyces cerevisiae is preferred pregelatinized starch with the addition of amylases and / or amylopeptidases used, however, can with Rhizopus javanicus native starch products without additional Enzymes are fermented. The Fermentation of the starch by Rhizopus spec. can, as well by Saccharomyces cerevisiae, carried out anoxidatively which is an exception with mushrooms. Therefore, the Process mainly take place in phase II.

For the green algae Chlorella vulgaris listed in Table 2 applies that they are at a sufficiently high light intensity except in phase I can also grow in phase II as they  under the conditions of photosynthesis itself oxygen produced and the partial pressure of CO₂ among the Conditions of the set rough vacuum are still sufficient is high to supply the algae. In contrast, in the dark Algae can only be supplied with an organic C source live, then they need enough Oxygen since they cannot ferment, so they are at Darkness predominantly must be kept in phase I.

Dissolved nutrients, precursor compounds and water to moisten the particles in each phase of the Processes listed in Table 2 via nozzles from above or sprayed into the bed below.

In addition, these liquids can also be sprinkled put into bed, but with the Limitation that the particles in contact with the free liquid phase is not softened and deformed because then the fluidizability of the particles get lost. This requirement is most likely with immobilization and, to a limited extent, also in the case of ground Starch products, but not for particles, which mainly consist of the microorganisms themselves. With the connection of a free aqueous phase Wastewater problems also arise in the past have already led to similar biotechnical Processes (e.g. yeast production) are no longer profitable were.

Aeration of the reactor with air during aerobic processes or phase I oxygen can go the same direction (from bottom to top) as in phase III but are below the vertebral point. Will the bed additionally sprinkled, so is the flowing down per unit of time Amount of liquid limited by the flood point. The liquid is above the flood point  discharged with the gas upwards.

There is a restriction due to vortex and / or flood points not if the system is in phase I as a trickle is operated, d. H. when liquid and gas Flows through the bed from top to bottom.

These variants are interesting for processes in which non-volatile fermentation products are formed, such as. B. in citric acid synthesis (Table 2). Become suitable Carrier for immobilizing the microorganisms and / or enzymes are used, so the sprinkling during fluidization will continue in phase III (Turbulence contact absorber).  

Table 1

Operating conditions for solid fermentation

Table 2

Examples of solid fermentation

Claims (8)

1. Process for the production of biotechnological products by solid fermentation of moist, granulated biomass formed from carrier mass and from substrate for the microorganisms used for the fermentation, in which the metabolic products of the microorganisms are removed by a gas, characterized by
Allowing the moist, granulated biomass to rest in a bed during phase I,
Evacuating the tank with a vacuum pump and maintaining the biomass at a given vacuum during a phase II,
Supplying the gas at a flow rate above the vortex point during a phase III, and
Repeat phases I to III when the gas has reached the pressure selected for phase I again. 2. The method according to claim 1, characterized in that that ground and moistened cereal products as a carrier mass and as a substrate for those used for fermentation Microorganisms are used, and that for Digestion of suitable amylases and / or amylopeptidases be added to the granules. 3. The method according to claim 2, characterized in that pregelatinized as a carrier mass and as a substrate Starch products are used. 4. The method according to claim 1, characterized in that cellulose as a carrier and as a substrate for the microorganisms used for fermentation and that suitable for the digestion of cellulose Cellulases are added to the granules. 5. The method according to claim 1, characterized in that as a carrier for the microorganisms porous plastics be used. 6. The method according to any one of the preceding claims, characterized in that the microorganisms themselves as a carrier and as a biotechnologically active component be used.   7. The method according to any one of the preceding claims characterized in that in the production of ethanol and yeast the alcohol distilled off during phase II becomes. 8. The method according to claim 7, characterized in that that breathing defective yeasts are used for ethanol production will.