WO2008019941A1 - Method for the separation and concentration of biomass - Google Patents

Abstract

The present invention relates to a method for the separation of enzymes and the concentration of biomass from a fermentation broth by means of dynamic cross flow filtration using rotating, ceramic overlapping membranes(2, 2').

Description

 Process for separation and concentration of biomass

The present invention relates to a process for the separation and concentration of biomass from a fermentation broth by means of dynamic cross-flow filtration with rotating ceramic membranes.

The purification of biotechnologically derived enzymes from fermentation broths usually takes place in several stages. Before the final purification and concentration stage, for example by ultrafiltration, unwanted biomass, in particular cells, cell components and other impurities must be separated.

Such methods for the recovery of recyclables from fermenter solutions are known. In a cross-flow membrane filtration of the dissolved valuable material is separated as permeate from the fermenter broth. Subsequently, a concentration of the valuable material in a further work-up plant, for example by precipitation or in an ultrafiltration or reverse osmosis. Both the microfiltration with pore sizes of 0.02-10 microns and the ultrafiltration with pore sizes of 0.001-0.02 microns are carried out with the aid of successively connected, so-called modules. In this case, the entering into the first module fermenter broth must be acted upon with a sufficiently high pressure, so that due to the pressure losses occurring in the modules even in the last module still a sufficiently large transmembrane pressure difference is ensured for separating the substance.

Applicant's EP 200032 A2 discloses a process for purifying fermenter solutions by means of cross-flow microfiltration through membranes. Accordingly, cross-flow microfiltration serves to separate the valuable material (permeate, in this case enzyme) passing through the membrane from the biomass (= retentate), which is retained by the membrane.

A particular embodiment of this method is described in EP 307737 A2 of the applicant. Accordingly, the cross-flow microfiltration is controlled by applying special pressure conditions. Also according to this embodiment, the enzyme is obtained as the recycled material from the impurities on the permeate side of the membrane. DE 10022258 A1 describes a method for purifying and / or concentrating protein inclusion bodies in a solution, which comprises passing the solution containing the protein inclusion body tangentially past one or more semipermeable membranes, so that the protein inclusion bodies are retained by the membranes and substances or pass through components having a smaller molecular weight or particle diameter, whereby a purified and / or concentrated protein inclusion body solution is obtained.

The methods known from the prior art, however, have high material losses and are therefore only of limited economic use.

A disadvantage of these known methods is also the problem of so-called fouling which always occurs in membrane processes. Due to the flow of material to the membrane surface, in addition to the recyclables, substances also reach the pores of the membrane, which due to their size can not pass through them. As a result, a complex composite covering layer is formed on the membrane surface, which more and more blocks the flow of material through the membrane, in particular of the valuable substance, during the concentration.

As a result, the retention (retention of valuable substances) is increased and the permeate flow is limited. In order to maintain the separation efficiency of the process, the concentration must be stopped at a relatively low degree of concentration and the membrane surfaces must be subjected to a purification.

In EP 200032 A2 of the applicant, a method is described which largely avoids this disadvantage. However, this method is based on the use of certain membrane materials and well-defined hydrodynamic conditions. Therefore, the method is only suitable for certain biotechnological products.

The object of the invention is therefore to provide a method for separating and concentrating biomass from a fermentation broth, which enables a low retention of the valuable material at high permeate flow and can be used universally. Furthermore, the method should be energy efficient and economically feasible. This object is achieved according to the invention by a process for the separation and concentration of biomass from a fermentation broth by means of dynamic cross-flow filtration (cross-flow filtration), which is characterized in that rotating ceramic filtration membranes are used.

The process according to the invention is particularly suitable for the concentration of proteins, preferably of enzymes, in particular of detergent enzymes.

If desired, a first concentration step can be carried out in an upstream centrifuge stage. According to the invention, the concentrate obtained from the centrifuging stage is preferably diafiltered with the addition of water and then further concentrated. Preferably, such an amount of water is added that the broth is diluted 1.5 to 4 times, more preferably 2 to 3 times. This addition of water to the retentate increases the yield and hence the efficiency of the filtration process. Quantities of the desired product in the retentate may still be recovered by this dilution step involving the addition of water to the retentate.

According to the invention, the membranes are designed such that the biomass is retained on the surface of the retentate and the solvent is obtained with the enzyme to be recovered as valuable material and optionally together with low molecular weight compounds on the permeate side. The permeate can then be further purified, for example subjected to ultrafiltration, directly for the final concentration of the enzyme in a manner known to the person skilled in the art.

Preferably, the rotating ceramic filtration membranes are used so that they partially overlap. Preferably, a ceramic membrane is used which has an average pore size in the range of 0.01 to 1 .mu.m, in particular from 0.1 to 0.2 microns.

During circulation of the broth along the membrane (crossflow filtration) it is concentrated as increased amounts of fluids present in the broth permeate through the membrane. An inventively suitable volumetric Einkonzentrierungsfaktor (VCF) ranges from 3 to 20, preferably from 3 to 5. This concentration may be at vorteilhaftesten at elevated temperatures, preferably at a temperature in the range of 1 ° C to 70 ⁰ C, more preferably from 35 ° C to 70 ⁰ C are performed. Particularly preferably, the process is started at a temperature of about ≦ 35 ° C and terminated at a temperature of about 70 ⁰ C.

The process is preferably carried out such that more than 60% of the enzyme permeates and the retentate has a solids content of more than 12%.

The Transmembrandruck is in carrying out the method according to the invention in the range of 0.2 to 10 bar, preferably in the range of 0.7 to 3.0 bar.

When carrying out the process according to the invention, shear rates in the range from 0.5 to 10 m / s, preferably in the range from 4 to 7 m / s, are achieved. The process according to the invention can be carried out continuously. Preferably, however, it is carried out as a batch process, that is, a large volume to be purified is introduced once and then worked up. A continuous system is less advantageous in enzyme recovery, because the fermenters are typically also prepared batchwise and harvested.

Membrane materials suitable according to the invention are in principle all from which ceramic membranes can be produced. However, the material of the membrane layer is preferably Ci-Al ₂ O ₃ , zirconium dioxide or titanium dioxide into consideration.

Rotary ceramic filtration membranes which can be used according to the invention are, for example, those which are also used in the devices described in DE 100 19 672 A1.

Devices of this type are used for the cross-flow permeation of flowable media. They comprise at least two shafts, on each of which many disk-shaped membrane bodies are arranged parallel to one another and at a mutual distance. The waves are hollow, and the membrane discs are made of ceramic material and are crossed by radial channels. There is a conductive connection between the radial channels and the interior of the hollow shaft. The liquid to be filtered passes through from the outside the porous material of the membrane body in the channels, and from there into the hollow shaft.

The said waves are parallel to each other, so that the membrane discs of two adjacent disk packages are arranged parallel to each other. In this case, the waves are arranged so close together that the discs of two disc packs mesh with each other like a toothing.

The discs need not have the stated construction of porous ceramic material. There are also applications in which some discs are constructed as so-called dummy discs. It is also conceivable to produce the discs from sieve bodies. Combinations of the types mentioned are conceivable, for example, the pairing Siebkörper-membrane body. The following is to be spoken only by "slices".

In order to achieve the highest possible output, as many disks as possible should be placed on a shaft. The discs must therefore be arranged as close as possible. On the other hand, however, make sure that the discs of one package do not touch the discs of the other package. Therefore, exact axial positioning of the discs on their shafts is required.

For the purpose of positioning spacers are arranged between two adjacent discs. Also in the sense of a precise axial positioning of the entire disc package is compressed, for example, by a nut which is screwed onto the respective hollow shaft on one end, and exerts a corresponding pressure on the plurality of discs with spacer elements located therebetween. Between mother and hollow shaft, a spring package can be switched.

Rotary ceramic filtration membranes which can preferably be used according to the invention are those which are also used in the devices described in WO 02005935 A2, to which reference is hereby made in their entirety.

By way of example, those preferably usable rotary ceramic filtration membranes are shown in FIGS. 1 to 3. FIG. 1 shows a device with filter plates in a schematic elevational view.

Figure 2 shows the article of Figure 1 in a plan view.

Figure 3 shows a modified embodiment of the article of Figure 1, again in plan view.

As can be seen from FIGS. 1 and 2, the device has two hollow shafts 1, 1 '. Each hollow shaft carries a package of disks 2, 2 '. The two shafts 1, 1 'and the disc packs are located in a container 10. The container has an inlet 10.1 and an outlet 10.2.

The two hollow shafts 1, 1 'are driven - see Figure 2, in a plan view of the ends of the shafts seen in a clockwise direction. You can also run both in a counter-clockwise direction. In any case, the circulation in one and the same sense of rotation is advantageous.

The discs are used for filtration. They are constructed of a porous ceramic material and have channels in their interior. The channels are connected to the interiors of the hollow shafts 1, 1 'in a conductive connection.

Through the inlet 10.1, the medium to be treated passes into the interior of the container 10. The filtrate / permeate then passes through the pores of the ceramic material in said channels and from there into the interior of the two hollow shafts 1, 1 '. It then exits at the upper ends of the hollow shafts - see the two arrows pointing upwards.

What can not penetrate through the pores of the ceramic material passes as a retentate to the outlet 10.2 of the container 10th

In the embodiment shown in FIG. 3, three shafts with three disk packs are shown. Again, the discs run in the same direction. But it could also be an opposite direction of rotation. The speeds of the packages consisting of shafts and disks can be variable, adapted to the needs of the product. They are preferably in the speed range between 10 and 1000 U / min.

All packages run in one and the same direction. Decisive here is the differential speed, which have two opposing surface elements of two discs in the overlapping region. This differential speed can be in the range between 1 and 20 m / s. It is best between 2.5 and 7 m / s.

The discs may be classic filter discs constructed of a porous ceramic material. But it may also be hollow screen body. Finally, at least some of the discs may be formed as so-called dummy discs.

The disks of the individual disk packs may be constructed of different filter media, which are different from each other in terms of their material or their deposition rates. This applies both within a chamber, and from one chamber to another.

For processing large volumes and for realizing a continuous mode of operation, several devices can be connected in parallel and / or in series.

Further inventively used rotary ceramic filtration membranes are those which are also used in the sold under the trade name RotoStream® by the company Canzler GmbH, Düren, or by the company. Andritz, Graz, devices

The use according to the invention of membrane disks mounted on rotating hollow shafts reduces membrane soiling as well as the consumption of energy in the flow channels and channel inlets typical in tube modules. Due to the self-cleaning effect, the blocking of the membrane is reduced without high energy input for generating high cross-flow velocities. In conventional cross-flow membrane systems used in industry, performance is often compromised due to membrane fouling or surface layering on the membrane surface. This leads to a reduction in the permeate rate. Conveniently, the thickness of the laminar overflow region at the membrane surface is reduced by increasing the flow rate of the food product along the membrane surface. However, this requires a high circulation flow and high electrical power consumption for the circulation pumps. Increasing concentrations usually also cause an increase in product viscosity. Thus, the energy expenditure is additionally increased, especially when large volumes of liquid are pumped at high speeds along the membrane.

The above-mentioned disadvantages are avoided by the dynamic cross-flow membrane filtration, for example by a filtration according to the RotoStream® principle.

Membrane discs are mounted on rotating hollow shafts. The discs on the adjacent shafts overlap. The food product is guided along the outer surface of the discs. Permeate enters through the membrane into the interior of the discs, the support structure, and is discharged through the hollow shaft. The support structure gives the windows the necessary mechanical stability. There is only a small flow resistance for the permeate when penetrating into the hollow shaft.

On the one hand, a high relative velocity between the disk surface and the liquid during rotation (cross flow velocity) is generated. On the other hand, extremely strong turbulences occur at the overlapping zones of the membranes. Since the product flow rate inside the module housing is small, the corresponding pressure loss also remains small, and the transmembrane pressure (TMP) is very uniform across all membrane discs.

Two distinct zones are active: the permeation zone, where the slices do not overlap, and the self-cleaning zone, where the topcoat is further broken apart beyond the high crossflow cycle cleaning effect. Thus, the cover layer thickness is controlled by the rotational speed.

The present invention thus has a number of advantages over the prior art: The cross flow cleaning effect and the TMP are independently adjustable.

Due to the small pressure loss in the system pressure housing and high-pressure pumps are usually not needed.

Likewise, a uniform TMP prevails over all membrane discs. High viscosity fluids can be treated.

Electrical energy savings of approx. 70 - 80% (compared to tubular membranes). Little need for space.

Production must be interrupted less frequently for cleaning. Long operating times with consistently high permeate rate.

Another object of the present invention is the use of a device according to the teaching of WO 02005935 A2 or DE 100 19 672 A1, or a device sold under the trade name RotoStream® by Canzler GmbH, Düren, or by the company. Andritz , Graz, for the separation and concentration of biomass from a fermentation broth by means of dynamic cross-flow filtration (cross-flow filtration).

The following example illustrates the invention without, however, limiting it to:

Example 1 :

Separation of Biomass and Simultaneous Protein Extraction from Concentrate of Centrifuge Preconcentration Stage.

1. The laboratory system comprises two shafts with the same direction of rotation and 0.136 m ²

2nd shear rate 6.5 m / s

3. Transmembrane pressure 0.5 bar

4. temperature 35 ° C

Diafiltration with water was carried out

Results:

Start: dry matter content 12.9%, sediment content 83.2%

End: dry matter content 14.9%, sediment content 96.8%

Protein yield 69.8%

Claims

claims 1. A process for the separation and concentration of biomass from a fermentation broth by means of dynamic cross-flow filtration, which is characterized in that one uses rotating ceramic filtration membranes. 2. The method according to claim 1, characterized in that one uses membranes, which are designed so that the biomass retained on the retentate side and the solvent is obtained with the recoverable as a valuable enzyme and optionally together with low molecular weight compounds on the permeate side. 3. The method according to claim 1 or 2, characterized in that one uses the rotating ceramic filtration membranes so that they partially overlap. 4. The method according to any one of the preceding claims, characterized in that one uses a ceramic membrane having an average pore size in the range of 0.01 to 1 .mu.m, in particular from 0.1 to 0.2 microns. 5. The method according to any one of the preceding claims, characterized in that one reaches a volumetric Einkonzentrierungsfaktor which is in the range of 3 to 20, preferably from 3 to 5. 6. The method according to any one of the preceding claims, characterized in that starting the process at a temperature about <35 ° C and terminated at a temperature of about 70 ⁰ C. 7. The method according to any one of the preceding claims, characterized in that one carries out the process so that more than 60% of the enzyme permeate and the retentate has a solids content of more than 12%. 8. The method according to any one of the preceding claims, characterized in that one carries out the method so that the transmembrane pressure in the range of 0.2 to 10 bar, preferably in the range of 0.7 to 3.0 bar. 9. The method according to any one of the preceding claims, characterized in that one carries out the method so that shear rates in the range of 0.5 to 10 m / s, preferably in the range of 4 to 7 m / s can be achieved. 10. The method according to any one of the preceding claims, characterized in that it is carried out as a batch process. 11. The method according to any one of the preceding claims, characterized in that one uses as membrane materials Ci-AI ₂ O ₃ , zirconium dioxide or titanium dioxide. 12. The method according to any one of the preceding claims, characterized in that one carries out a first concentration step in an upstream centrifuge stage and diafiltered from the centrifuge stage resulting concentrate preferably with the addition of water and then further concentrated.