DE10328552A1 - Producing defined enzyme mixture and/or metabolite mixture for fermentation of target substrates, by inoculating microorganisms in solid-phase bioreactor with inducer substrates and keeping mixed culture under appropriate conditions - Google Patents

Abstract

A culture method for producing a defined enzyme mixture and/or metabolite mixture optimized for the fermentation of one or more target substrates comprising contacting an inoculating mixed culture of microorganisms in a solid-phase bioreactor with target substrates, inducer substrates or any combination of target and inducer substrates, and by keeping the mixed culture under an appropriate conditions, is new. A culture method (M1) for producing a defined enzyme mixture and/or metabolite mixture optimized for the fermentation of one or more target substrates, by contacting an inoculating mixed culture of microorganisms in a solid-phase bioreactor with one or more target substrates, one or more inducer substrates or any combination of target and inducer substrates, and by keeping the mixed culture under an appropriate selection pressure by a suitable selection of the culturing parameters and by a specific induction by the target and/or inducer substrate and/or inhibition with appropriate inhibitors for a defined culturing time. Independent claims are also included for the following: (1) an enzyme mixture and a metabolite mixture obtainable by (M1); (2) a fermentation method for processing one or more target substrates comprising fermenting the target substrates with an enzyme mixture and/or metabolite mixture obtainable by (M1); (3) conserving (M2) enzyme-mixture produced in solid state fermentation comprising decreasing the water activity of substrates during the fermentation process, preferably by air flow through the substrate, and/or by a final drying step, preferably in a fluidized bed or belt dryer; (4) cultivating (M3) microorganisms at equal growth rates by adjusting the water activity; and (5) bioreactor (I), preferably for performing any one of (M1-M3), comprising a fermentation module for the fermentation of substrates under selection pressure, where the fermentation module comprises regulation units to adjust a fermentation environment, a feeding unit being connected to the fermentation module to feed the substrate, an induction module for adding reagents (agents conferring selection pressure) to the fermentation media, a harvesting module comprising outlet unit and a conveying unit to convey the media from the fermentation module through the induction module to the harvesting module.

Description

It There is a multitude of technological processes known in which enzymes previously only suboptimal or not at all used, although this of great economic like ecological Benefit would be. This includes For example, processes involving complex, biological, solid substrates changed or whose composition is very heterogeneous, the v. a. also contain degradable substances such as lignocellulose and / or the outer boundary layers (eg woody cell walls, Cuticles, etc.), which are barriers to enzymes and so on the bioavailability limit. For one effective treatment of these substrates would be specific to these substrates Optimized enzyme mixtures necessary. This is not yet as targeted and directed production by appropriate fermentations possible.

This Disadvantage is now remedied by a new cultivation procedure on the basis of a solid phase cultivation technique by means of special, likewise bioreactor according to the invention, a novel, modular and continuous screw reactor, to disposal is provided, characterized in that an adapted and stable, microbial culture, preferably a mixed culture of fungi, is prepared in such a way that these in the bioreactor according to the invention with one or more target substrates alone or in all possible combinations with other substrates or with them alone (taking under target substrates) those are understood that are used in a subsequent process are brought together) and under appropriate selection pressure by a suitable choice of the cultivation parameters as well as by specific Induction by the target substrate (s) mentioned above and / or Inhibition with special inhibitors after a given culture period a defined one, optimized for the target substrate (s)) Enzyme mixture and / or a Metabolitgemisch produced, which in the subsequent process for degradation of the target or the target continuously is used.

One important part of the present invention is further that through the continuous process control and modular design of the novel bioreactor, the temporal dependence of the production of Enzyme mixtures for their optimization with respect to the degradation of Target substrate, or the target substrates simultaneously or sequentially is used.

In recent decades, increased efforts have been made to treat biomass by enzymatic digestion (see for example DE19845207A1 ). Today, there are a number of approaches in which purified enzymes or enzyme mixtures or enzyme-producing microbial pure or mixed cultures for the treatment of biological substrates are used. Examples include the treatment of wood chips in the course of biopulping, the treatment of annual plants to improve the properties as feed or bagasse for the production of bioalcohol. However, all these processes are still suboptimal. The main reason for this is that the enzyme mixtures, microorganism cultures or microorganism mixed cultures used are not optimally adapted to the target substrates (eg the organisms and enzymes are not optimally adapted and selected to cold to very cold locations), which is e.g. B. in biopulping to very long treatment times of wood chips leads with correspondingly large footprint for the rental areas.

Lots biotechnological processes for the treatment of complex substrates (mostly plant material but also, for example, animal waste from animal slaughterhouses etc.) in addition to the mentioned enzyme mixtures (hydrolases, oxidoreductases) for preferably extensive degradation often in addition (Other than oxidoreductases) certain cofactors or mediators, the partly still unknown. That means that single enzymes or additions of mixtures to the process show only limited success, since only an interaction of previously adapted to the target substrates out and optimized enzyme mixtures along with these additional ones Factors can show optimal performance. But this is so far also not possible in an optimal way.

Out DE3539875A1 is z. Example, a method and an apparatus for the continuous production of an enzyme mixture for the treatment of biomass in biogas plants known. In this method, however, working with a pure culture of Aspergillus niger and without induction of this culture to the plant substrates used for the subsequent methanation. Therefore, it is not expected that the Enyzsmischung formed there is optimally adapted to the biomass utilizing process.

Optimal adapted to a complex, biological substrate and defined Enzyme mixtures and / or metabolite mixtures are only producible by Selection pressure on the producing microorganism populations (Mixed cultures) and by their induction by the corresponding Target substrates.

It is now well known that the application of selective pressure to mixed populations of microorganisms by setting defined environmental parameters in the course of culturing these organisms usually leads to certain population spectra. However, these population spectra are undirected and random.

As well is known that by the choice of cultural conditions certain Types of microorganisms enriched or contaminants are suppressed can.

Known it is also that during it the course of such cultures depending on the chosen selection conditions, but also in dependence from the substrate texture and the inductor availability (in non-constitutively formed proteins and metabolites) too time-varying metabolite spectra can occur. However, it is true Again, that these metabolite spectra are undirected and random.

method of the type mentioned are e.g. on defined solid nutrient media (agar plates → mutagenesis treatments, genetic transformations, etc.) as well as in the field of submerged culture used (in the form of chemostats or Turbidostaten also continuously). In the latter case, it is mainly cultivations of Pure cultures for the achievement of specific metabolic activities.

Bioreactors for the continuous conversion of solid substrates are known from various fields of application (food technology, composting technology, etc.). For example, the fermentation material is transported in composting about rollers. However, such methods do not allow sophisticated regulation of the cultivation parameters. The use of one or more screw conveyors in solid phase bioreactors is for example out DE10041977A1 . DE4308920A1 and DE 4208920A1 known. However, these bioreactors are not designed modular and also see no possibility of the subsequent addition of substrates and other materials for z. B. induction ago.

Detailed description the invention

These described disadvantages of the prior art are characterized by the in the following described inventive method and the bioreactor according to the invention for continuous implementation fixed this procedure.

It becomes a new cultivation method based on a solid-phase cultivation technique by means of special. also according to the invention bioreactor, a novel, modular and continuous screw reactor, provided. This is characterized by an adapted and stable, microbial culture, preferably a mixed culture of fungi, in the Is made that these in the bioreactor according to the invention with one or more target substrates alone or in all sorts Combinations with other substrates or alone with them alone (Under target substrates) are understood as those in a subsequent process) is brought together and under appropriate selection pressure by appropriate choice of cultivation parameters such as Moisture content (water activity), pH, temperature, oxygen availability, redox potential and nutrient composition etc. as well as by specific induction by the target substrate (s) mentioned above and / or inhibition with specific inhibitors after a given Culture duration to the target substrate (s) produced optimized enzyme mixture and / or a metabolite mixture, which in a subsequent process for degradation of the target or substrates is used continuously.

there special inhibitors are those as described e.g. in: R.Vogel; Natural enzyme inhibitors, Georg Thieme Verlag, 1984 and in: H. Zollner; Handbook of enzymes Inhibitors, VCH, 1989.

One important part of the invention is that the continuous generated enzyme-substrate-fungal mixtures either be used as such, or a separation of the Substrate-fungal mixture to obtain the liquid enzyme coctail takes place. Furthermore, it is possible the liquid obtained in such a way Enzyme mixtures to undergo further downstream processes (Purification by ultrafiltration, chromatography method Etc.).

Also is an important part of the invention that the natural contamination population of certain Target substrates / substrates is exploited to from desired germs with desired To select properties; still missing properties can through Addition of suitable inductors including the existing on this or selected germs are supplemented. This addition can at the beginning of the cultivation or during the cultivation. It can too Pure cultures naturally on these target substrates / substrates occurring, bred out of it or derived from collections or other strains of organisms (e.g. High-performance strains) which are suitable in their enzyme spectrum, are added.

An important part of the invention is further that the inventive method of ei ner can exist up to several stages of the process. These process steps can generally run simultaneously, ie it is worked with several target substrates / substrates used simultaneously, additional inducers and / or inhibitors can be added simultaneously or during the culture period. However, it is also possible to use substrates one behind the other, wherein not all have to be target substrates per se. This is made possible by the continuous process control and modular design of the novel bioreactor, which allows by its construction according to the invention additional substrate addition, addition of other inducers, inhibitors, addition of microorganism cultures, change of the selection pressure, etc. during the culture period. Associated with this is the important option according to the invention of preferably utilizing the temporal dependence of the quantitative or qualitative production of the enzyme mixtures as a manipulated variable for the regulation of the ongoing process itself and also of the downstream target processes.

at Growing with several process steps (eg 2-stage) depends on the Target process on suitable substrates by utilizing the natural Growing of these substrates and optionally by an additional, supportive Inoculation with pure cultures (also high performance strains and also from collections) and by setting selective conditions Choice of water activity, pH, redox potential, temperature, availability of oxygen, availability of inductors, etc. in a first phase, a corresponding mixed culture generated. The aim of this first phase of the procedure is to take one for the second Phase appropriate pool of microbial populations available put. The mixed culture is in the second stage of the process by adding another substrate, which also usually is the target substrate, and optionally adjusting others (as in the first stage) selective conditions by a suitable Choice of water activity, pH, redox potential, temperature, availability of oxygen, availability directed by inductors, etc. into a stable and adapted mixed culture (fine-tuning), after a given culture period, a defined spectrum Produces enzymes and / or metabolites that are used in the induction Target substrates is optimally adapted.

The inventive method is basically applicable to all technological processes involving biological Substrates changed or reacted and a treatment of these substrates by Enzyme is either the subject of the process itself, or as Pre-treatment or additional treatment can be used. In particular, that can inventive method for processes from the woodworking industry, pulp and paper industry, the textile industry, the leather industry, the animal-processing industry Industry, the detergent industry, the feed industry, the food industry, in wastewater, exhaust air and soil cleaning and in waste processing and processing of renewable raw materials.

Likewise is the bioreactor according to the invention across from the new state of the art in itself new and inventive. At the same time, the bioreactor for the continuous implementation of particular illustrated method of the invention suitable.

Other Reactor types for solid phase cultivation of microorganisms such as for example, Bioreactor for fermenting solids (PCT WO 01/19954), Bioreactor having at least two reaction chambers' (WO 02/100999 A2), 'cultivation procedure for microorganisms and bioreactor '(PCT / EP03 / 01663) as well as further known solid-state bioreactors (construction types are, for example, screw reactors, Drum reactors, tower reactors, falling film reactors, etc.), if appropriate modified or in the form of a cascade, also suitable in principle.

One Another important component of the present invention is that the inventive, continuous Bioreactor, optionally used as a cascade to the temporal dependence the production of the enzyme mixtures for their optimization with regard to on the degradation of the target substrate or target substrates simultaneously and sequentially, d. H. It will be possible to have one or more Enzyme formation process downstream processes simultaneously or sequentially with correspondingly optimized enzyme cocktails simultaneously or sequentially to supply.

The bioreactor according to the invention (according to 1 ) consists of any number of modules, with a total of 4 types of modules. All modules consist of a stainless steel tube o. Plastic tube ( 1 ), in which a screw with hollow shaft ( 2 ) is rotatably mounted. The snail is having harassment ( 3 ) equipped for vertical mixing of the fermentation. The individual modules and also the screw conveyor can be attached to each other via a suitable connection system. In the assembled state, the individual screw elements are driven together by a motor ( 4 ) emotional.

The fermentation module ( 5 ) has nozzle openings ( 6 ) on the hollow shaft of the screw through which the fermentation material can be ventilated. Further ventilation nozzles ( 7 ) are distributed in the lower half of the tube wall. In the upper half of the tube wall are spray nozzles ( 8th ) For the addition of liquid media arranged. The so-called induction module ( 9 ), which can be used in the method according to the invention for adding the substances according to the invention, has an opening ( 10 ) with closure ( 11 ) for the addition of solids on top of the tube wall. The drive module ( 12 ) has no nozzles. Here is also one of the two end faces ( 13 ) of the tube closed. There are the drive motor and the bearing ( 14 ) attached to the shaft. On top of the tube wall is a funnel ( 15 ) with closure ( 16 ) for the addition of solids and, if appropriate, for inoculum. The harvest module ( 17 ) also has no nozzles. Instead, there is an opening on the underside of the tube wall ( 18 ) with closure ( 19 ) attached to the removal of the fermentation. One of the two end faces ( 20 ) of the tube is closed. There is the counter bearing ( 21 ) attached to the hollow shaft. The bearing has a connection for ventilation ( 22 ).

The process according to the invention is carried out continuously in the bioreactor according to the invention as follows:
Either the target substrate itself or in admixture with other substrates or multiple target substrates as a mixture or substrates selected according to the target substrate are passed over the funnel (FIG. 15 ) in the drive module ( 12 ) introduced continuously. Optionally, a defined inoculum consisting of one or more pure microbial cultures is also added to the substrates. The fermentation material is adjusted before the start of the process with regard to the water activity, the pH and the redox potential. The fermentation mass is conveyed via a suitable number of fermentation modules by means of the screw elements ( 2 ) towards the harvest module ( 17 ). In the fermentation modules ( 5 ), the water activity, temperature, optionally the pH value and the oxygen concentration are continuously measured by means of suitable sensors. These parameters are set by controlling the aeration rate and the moisture content of the air vents ( 6 . 7 ) and the addition of water or buffer solutions via the spray nozzles ( 8th ). Via the correspondingly positioned induction module ( 9 ), further substrates (or target substrates) can be continuously introduced into the reactor for further inductive optimization of the resulting mixed culture. In the fermentation modules connected downstream of the induction module, other culture conditions may be adjusted such that a stable and defined microbial mixed culture is produced, which produces a defined enzyme and / or metabolite spectrum after a given culture period in such a way that it is present at the harvest module ( 17 ) is available for removal. The culture time is set by the rotational speed of the screw conveyor and the number of fermentation modules. Also, the bioreactor can be adapted to the Zielerfodernisse by a suitable number and composition of the modules. The delivery rate depends on the filling level of the reactor. The harvest module ( 17 ) removed fermentation material can be fed either directly or after appropriate treatment continuously to the target process.

in the Case of an arrangement of several inventive bioreactors in the form of a Cascade is the transition from one to the next Reactor provided as follows: The harvest module of the upstream Sub-reactor is connected to the feed module of the downstream Part of the reactor connected so that the harvesting module down to earth a movable chicane for flow division of the fermentation mass and the harvest module of the upstream sub-reactor two lockable outlets has. One of the two outlets is connected to the inlet of the downstream partial reactor and serves the continuation a portion of the substrate current within the process. The second Outlet serves to remove the other part of the substrate current for further use. The chicane is positioned the substrate stream is suitably portioned. Especially preferred will be the screw conveyors the partial reactors moved by a common drive.

Example of use:

Continuous production specific hydrolase cocktails for the optimization of biogas synthesis from renewable raw materials

The present invention consisting of the method according to the invention and the bioreactor according to the invention for continuous implementation of the method is described in detail by the following application example.

Renewable raw materials contain large quantities of persistent biopolymers such as lignocellulose, cellulose, hemicelluloses (xylan), pectin, cutin etc. These quantitatively important biopolymers, which form the main constituent or important fraction of rapeseed waste, straw or hay and are produced in large quantities in agriculture , so far can not be used in biogas plants or only suboptimal. In addition, their use as feed is partly limited by statutory regulations. The recovery of, for example, rapeseed residues in biogas plants would make it possible to cultivate rapeseed for biodiesel production on set-aside land on a larger scale, without existing agreements between the EU and the EU Affect the US.

The Conversion of, for example, rapeseed meal into biogas has so far been successful only moderately because anaerobically living bacteria are only conditionally able to their constituents to use efficiently as a carbon source. Also own others renewable raw materials such as straw etc. even less available carbon sources (Fiber structure) for the biogas production.

This Disadvantage should now future This will be remedied by the process of biogasassesis Rapeseed residues the inventive method - carried out in the bioreactor according to the invention - upstream becomes. this makes possible it to the operators of a biogas plant, as needed to those for biogas production used substrates optimally adapted enzyme mixtures themselves inexpensive manufacture.

According to the method is at a 2-phase growing preferred in the first stage of the process on crushed beet pulp (These themselves constitute residues from the processing of an agricultural product Products are available all year round to disposal) selectively prefers a mixed culture under suitable culture parameters consisting of such filamentous mushrooms produced, which produce hydrolytic enzymes. In a second Process stage is by adding the target substrate, here of rapeseed meal (which methanizes in the downstream process together with, for example, cattle manure is) selectively and selectively produces a conditioned mixed culture, the one adapted to the target substrate rapeseed meal spectrum hydrolytic Enzymes (cellulases, xylanases, chitinases, pectinases, proteases, Lipases, esterases) after a given culture period. The entire enzyme-containing fermentation material becomes the biogas process continuously added. The active enzymes close within the methanation stage the vegetable polymers of rapeseed meal on. By a suitable dosage of the enzyme mixture can also the Mirror free sugar in the methanation stage to an optimum Be set and maintained level. So can the production of Biogas based on rapeseed meal increases and sales times significantly shortened become.

By the new method can Furthermore many other untapped substrates like hay and straw Recycling in biogas plants are supplied.

The present invention consisting of the method according to the invention and the bioreactor according to the invention for continuous implementation of the method is not limited to the application described.

The described application is explained in more detail in the following example:

Example 1 (two-phase culture for the production of hydrolase mixtures for a subsequent biogas process)

20 g non-autoclaved pressed beet pulp with buffer solution brought to a total moisture content of 50% and pH 6.0 and at 30 ° C 11 days Incubate (1st phase). In this approach, a mixed culture develops from Penicillium chrysogenum, Eurotium amstelodami, Aspergillus niger and Aspergillus tubingesis. Various glucanases, xylanases, Proteases, pectinases, esterases and lipases are derived from this mixed culture secreted. This mixed culture is mixed with 10 g rapeseed meal buffer solution brought to 40% humidity and pH 5.5 induced (2nd phase). As a control, 10 g of pressed beet pulp added under otherwise identical conditions. After 6 days incubation at 30 ° C shows a dominance of Eurotium amstelodami and an increase of xylanase by a factor of three with constant cellulase activity in comparison for mixed culture (phase 1) and for control. Total hydrolytic activity measured as fluorescein diacetate hydrolysis, increases under the same conditions by a factor of 10. With this enzyme coctail could in the laboratory experiment Methane production based on rapeseed meal and cattle manure (im relationship 1: 1, total moisture content 65%) by 12% compared to the control (Enzymcoctail inactivated by heating). With the enzyme coctail by adding pressed beet pulp after all, instead of rapeseed meal, the result was a further increase in biogas yield of 4%.

Claims (15)

Novel culture method based on a solid-phase cultivation technique by means of a special, also inventive bioreactor, a novel, modular and continuous screw reactor, either individually or arranged in cascade, characterized in that an adapted and stable, microbial culture, preferably a mixed culture of fungi, in the Is made that this is brought together in the bioreactor according to the invention with one or more target substrates alone or in all possible combinations with other substrates or with these alone and under appropriate selection pressure by a suitable choice of cultivation parameters such as moisture content (water activity), pH, temperature , Oxygen availability, redox potential and nutrient composition, etc., as well as by specific induction by the target substrate (s) mentioned above and / or inhibition with specific inhibitors after a given Culture time, a defined enzyme mixture and / or a metabolite mixture which is optimized for the target substrate (s) is produced which is used continuously in a subsequent process for degrading the target substrate (s). Production of mixed cultures consisting of microorganisms for the preparation of enzyme mixtures and / or metabolite mixtures according to claim 1, characterized. that as substrates or Target substrates all possible Raw materials or waste natural (microbial, plant, animal or human) and not natural, industrial origin and mixtures thereof. Production of mixed cultures consisting of microorganisms for the preparation of enzyme mixtures and / or metabolite mixtures according to claim 1, characterized in that at least two microorganisms be used in mixed culture. Production of mixed cultures consisting of microorganisms for the preparation of enzyme mixtures and / or metabolite mixtures according to claim 3, characterized in that as microorganisms Fungi (Ascomycetes, Deuteromycetes) of the genera Penicillium spec., Aspergillus spec., Trichoderma spec. Fusarium spec., Eurotium spec. Absidia spec., Neurospora spec., Mucor sp., Chaetomium sp., Rhizopus sp., etc. are used Production of mixed cultures consisting of microorganisms for the preparation of enzyme mixtures and / or metabolite mixtures according to claim 4, characterized in that as microorganisms Fungi (Ascomycetes, Deuteromycetes) of the species Penicillium chrysogenum, Eurotium amstelodami, Aspergillus niger, Aspergillus tubingesis, Trichoderma harzianum, Trichoderma atroviride, Trichoderma reesei, Fusarium oxysporum be used. Production of mixed cultures consisting of microorganisms for the preparation of enzyme mixtures and / or metabolite mixtures according to claim 3, characterized in that as microorganisms Mushrooms (white rot fungi, brown rot fungi) of the genera Trametes spec., Pleurotus spec., Phanerochaete spec., Nematoloma spec. and Agaricus spec. etc. are used. Production of mixed cultures consisting of microorganisms for the preparation of enzyme mixtures and / or metabolite mixtures according to claim 3, characterized in that as microorganisms Bacteria (Actinomycetes) of the genera Streptomyces spec. Etc. be used. Production of mixed cultures consisting of microorganisms for the preparation of enzyme mixtures and / or metabolite mixtures according to claim 1, characterized in that the continuous enzyme-substrate-fungal mixtures used either as such or separation of the substrate-mushroom mixture for recovery of liquid Enzyme cctails done. Production of mixed cultures consisting of microorganisms for the preparation of enzyme mixtures and / or metabolite mixtures according to claim 1, characterized in that the method of one to several stages of the process can exist. Production of mixed cultures consisting of microorganisms for the preparation of enzyme mixtures and / or metabolite mixtures according to claim 1, characterized in that for the production of this Enzyme mixtures (hydrolytic enzyme cocktails) for supplementation a biogas plant a 2-phase-cultivation is performed. Production of mixed cultures consisting of microorganisms for the preparation of enzyme mixtures and / or metabolite mixtures according to claim 1, characterized in that the solid phase grows in a modular and continuous screw reactor after 1 , either individually or in cascade form, take place. Production of mixed cultures, consisting of microorganisms for the production of enzyme mixtures and / or metabolite mixtures according to claim 1 and 8, characterized in that the solid phase grows in a modular and continuous screw reactor after 1 either individually or arranged in a cascade done in the manner described. Production of mixed cultures, consisting of microorganisms for the production of enzyme mixtures and / or metabolite mixtures according to claim 1 and 4, characterized in that the solid phase grows in a modular and continuous screw reactor after 1 in the manner described for the continuous production of specific hydrolase-coctails for optimizing the following in a further process biogas synthesis from renewable raw materials. Production of mixed cultures consisting of microorganisms for the preparation of enzyme mixtures and / or metabolite mixtures according to claim 1, characterized in that the inventive, continuous Bioreactor is used as a cascade. Production of mixed cultures consisting of microorganisms for the production of enzyme Mixing and / or metabolite mixtures according to claim 1 and 9, characterized in that the solid phase grows in a modular and continuous screw reactor after 1 in the manner described for the continuous production of specific hydrolase cocktails and / or oxidoreductase cocktails for processes in the woodworking industry, the paper and pulp industry, the textile industry, the leather industry, the animal industry, the detergent industry, the feed industry, the food industry, in wastewater, exhaust air and soil cleaning and in the processing and processing of renewable raw materials.