WO2013079797A1

WO2013079797A1 - A material and method for immobilizing microbial cells

Info

Publication number: WO2013079797A1
Application number: PCT/FI2012/051181
Authority: WO
Inventors: Tom GRANSTRÖM; Tuomas UUKSULAINEN; Petri Haikola; Juhana Ahola
Original assignee: Kemira Oyj
Priority date: 2011-12-01
Filing date: 2012-11-29
Publication date: 2013-06-06

Abstract

The present invention provides an immobilizing matrix for immobilizing microbial cells, the matrix containing first supporting material comprising a layer of sheet-like porous material, and second cell-retaining material deposited onto the first supporting material, wherein the layers of the first supporting material and the second cell-retaining material are arranged into a rolled or folded structure allowing a sufficient flow of cell suspension medium through the immobilizing matrix. The present invention also provides a bioreactor for immobilizing microbial cells and methods for immobilizing cells of a microorganism on an immobilizing matrix in a bioreactor and for producing a fermentation product within a bioreactor.

Description

A material and method for immobilizing microbial cells

Field of the invention The present invention relates to an immobilizing matrix for immobilizing microbial cells. The present invention also relates to a bioreactor containing said immobilizing matrix and to methods for immobilizing microbial cells and for producing a fermentation product within a bioreactor. Background of the invention

The industrial biotechnology processes using microorganisms are generally based on the exploitation of the cells in the fermentation medium during the process. The classical fermentations suffer from various constraints such as low cell density, nutritional limitations, and batch-mode operations with high down time. It has been well recognized that the microbial cell density is of prime importance to attain higher volumetric productivities. The continuous fermentations with free-cells and cell recycle options aim to enhance the cell population inside the fermenter. However, the free-cell systems cannot operate under chemostatic mode that decou- pies specific growth rate and dilution rates (Ramakrishna and Prakasham, Microbial fermentations with immobilized cells, Current Science 77 (1), 1999 pp. 87- 100).

Over the last years the cell immobilizing technology has attracted attention of sev- eral research groups. This process eliminates most of the constraints faced with the free-cell systems. The remarkable advantage of the immobilizing technology is the freedom it has to determine the cell density prior to fermentation. It also facilitates operation of microbial fermentation on continuous mode without cell washout. The whole-cell immobilization process decouples microbial growth from cellu- lar synthesis of favored compounds.

The use of immobilized whole microbial cells and/or organelles eliminates the often tedious, time consuming, and expensive steps involved in isolation and purification of intracellular enzymes. In many cases the metabolic pathway for produc- ing the desired product requires several different enzymatic reactions and corresponding enzymes, such as ten in the case of the production of propionic acid from glycerol. The immobilization also tends to enhance the stability of the enzyme by retaining its natural catalytic surroundings during immobilization and subse- quent continuous operation. The ease of conversion of batch processes into a continuous mode and maintenance of high cell density without washout conditions even at very high dilution rates, are a few of the many advantages of immobilized cell systems. The metabolically active cell immobilization is particularly preferred where co-factors are necessary for the catalytic reactions. Since co-factor regeneration machinery is an integral function of the cell, its external supply is uneconomical. There is considerable evidence to indicate that the bound-cell systems are far more tolerant to perturbations in the reaction environment and similarly less susceptible to toxic substances present in the liquid medium.

The use of immobilized whole microbial cells and/or organelles also facilitates the use of extractive fermentation. Extractive fermentation is generally a process for producing a variety of chemical products by fermentation in which the product is removed from the fermentation medium as it is formed by liquid-liquid extraction using an extractant for the product which is immiscible with water.

The most extensively studied method in cell immobilization is the entrapment of microbial cells in polymer matrices. The matrices used are agar, alginate, carra- geenan, cellulose and its derivatives, collagen, gelatin, epoxy resin, photo cross- linkable resins, polyacrylamide, polyester, polystyrene and polyurethane. Among the above matrices, polyacrylamide has been widely used by several workers. This gel was first used for immobilization of enzymes. Later this technique was successfully applied to immobilization of lichen cells. As a rule, the entrapment methods are based on the inclusion of cells within a rigid network to prevent the cells from diffusing into surrounding medium while still allowing penetration of substrate (Ramakrishna and Prakasham, Microbial fermentations with immobilized cells, Current Science 77 (1), 1999 pp. 87-100).

In immobilized-cell systems, the substrate (starting material) and the end products produced by the microbial cells are in the mobile liquid phase, which flows through the bioreactor. The microbial cells are attached to a solid matrix in which a high population of cells can be maintained. Some materials known in the art include calcium alginate and carrageenan. US 5595893 discloses inert, solid supports that may be used for immobilizing cells of a microorganism to form a biofilm reactor. The supports are composed of a synthetic polymer, preferably a polyolefin such as polypropylene, in admixture with one or more plant-derived organic polymers such as corn fibers, oat hulls, cellu- lose, starch, and the like. The supports are substantially water-insoluble. The supports may be produced by combining a synthetic polymer and a plant-derived organic polymer in admixture to provide a composite, dough-like thermoplastic composition. The composition may be prepared, for example, in an extrusion mixer and co-extruded as an extrudate to form a shaped support, or the composition may be molded into a shaped article or support according to techniques known in the art, as for example, by compression molding, injection molding, and the like.

The biofilm reactor (i.e., support with attached microbial cells) may be used in a variety of systems, as for example, in a continuous fermentation process to produce a fermentation product such as lactic acid, acetic acid, citric acid, succinic acid, propionic acid, and other like organic acids, or ethanol or butanol-acetone, or other like organic alcohols; in a chemically contaminated stream for bioremedia- tion; in a waste treatment system to remove recalcitrant compounds and to reduce the biochemical oxygen demand; and the like. In such systems, the biofilm- covered support may be placed or positioned in a liquid medium that includes a substance, as for example, a carbohydrate or a contaminant substance such as petrochemicals, herbicides or pesticides, to be processed, i.e., metabolized, by the microorganism, and then the microorganism is allowed to substantially metabolize the substance to provide a microbial end-product.

One example of organic acid production is propionic acid production from glycerol. Zhang and Yang (Process Biochemistry 44 (2009) 1346-1351) describe such a process by using metabolically engineered Propionibacterium acidipropionici in free-cell fermentation and immobilized cell fermentation using fibrous-bed bioreac- tor. Propionic acid (also called propanoic acid) is a naturally occurring carboxylic acid with chemical formula CH₃CH₂COOH. It can be used as solvent, as food preservative or in the herbicide manufacture. Propionic acid is also useful as an intermediate in the production of other chemicals, especially polymers. Cellulose- acetate-propionate is a useful thermoplastic. Vinyl propionate is also used as monomer in (co)polymers with e.g. ethylene, vinyl chloride and (meth)acrylic esters. In more specialized applications it is also used to make pesticides and pharmaceuticals. The esters of propionic acid have fruit-like odors and are sometimes used as solvents or artificial flavorings. Brief description of the invention

The present invention provides an immobilizing matrix for immobilizing microbial cells, the immobilizing matrix containing first supporting material comprising a lay- er of sheet-like porous material, and second cell-retaining material deposited onto the first supporting material, wherein the layers of the first supporting material and the second cell-retaining material are arranged into a rolled or folded structure allowing a sufficient flow of cell suspension medium through the immobilizing matrix. The present invention also provides a bioreactor for immobilizing microbial cells, the bioreactor containing said immobilizing matrix.

The present invention also provides a method for immobilizing cells of a microorganism on said immobilizing matrix in a bioreactor.

The present invention also provides a method for producing a fermentation product within a bioreactor containing said immobilizing matrix.

It is an advantage of the invention that a high number of productive microbial cells can be packed into a very small area increasing the volumetric productivity of end product as well as reducing the costs of hardware investments.

It is another advantage of the invention that the inhibitory end product is continuously removed from the column reactor and replaced by fresh medium enhancing and prolonging the viability and production stage of the cells.

It is still another advantage of the invention that channeling problems generally occurring in such bioreactor matrices are avoided. Brief description of the drawings

Figure 1 shows a schematic diagram of a bioprocess for biobased propionic acid production. Propionibacterium acidipropionicii cells were grown in CSTR reactor (not shown) and cells were transferred to the immobilization column. Crude glyc- erol and nutrients were circulated through the high cell density column where pH was kept at 6.2 by using the base pump GA-4. Fermentation broth was collected to the product tank and formed acids were analyzed with HPLC. Detailed description of the invention

The present inventors have developed a method and an apparatus for biobased fermentation and production of useful products, such as organic acids, alcohols and the like. This apparatus is based on continuous immobilized cell column reactor, which allows increased productivity of organic acids. Cells are attached to optimized matrix, which allows sufficient flow through the matrix without channeling problems. Fermenting cells are in a continuous-flow stirred tank (CSTR) bioreactor until exponential growth phase is reached. The filling is inserted into the column reactor and cell suspension is circulated through the column filling material and returned to the CSTR reactor. The flow can be either 1 or 2-way, up or downwards or both ways. The cell loading can be continued until the CSTR bioreactor is empty of cells. In case of longer loading period there should always be an excess of substrate during the cell loading to prevent cell degradation. After the column filling is saturated by cells, the column is detached from the CSTR bioreactor and connected to substrate feeding and product tanks. The substrate feeding is initiated and product is collected from the products tank. The end product may be recovered with optimized extraction and distillation processes. The process is based on continuous feeding of substrate through the column filling and simultaneous conversion of substrate into product. The volumetric productivity is calculated by dividing the flow rate (m³/h) with the volume of the reactor (m³) and multiplying with the end concentration of the product. Using the apparatus of the invention the volumetric productivities up to 7 g/l have been obtained. These are significantly higher than volumetric productivities given in the relevant scientific literature.

The concept can be used e.g. for various organic acid productions from different sugar-rich biomasses, but a preferable process is propionic acid production from crude glycerol, which is derived from a FAME biodiesel production process. Crude glycerol contains typically 80 glycerol and by-products, such as organic matter and salts. In order to remove these by-products, RO membrane can be installed before the feeding tank directly to crude glycerol line and/or to the raffinate water line from the downstream section.

The present invention provides an immobilizing matrix for immobilizing microbial cells for production of useful fermentation products, such as organic acids, alcohols and the like, the matrix comprising first supporting material comprising a layer of sheet-like porous material, and second cell-retaining material deposited onto the first supporting material, wherein the layers of the first supporting material and the second cell-retaining material are arranged into a rolled or folded structure allowing a sufficient flow of cell suspension medium through the immobilizing matrix. The first supporting material and the second cell-retaining material are different and separate materials, not for example a copolymer of two materials.

The immobilizing matrix allows sufficient flow of the cultivation medium through the matrix without any channeling problems. Channeling occurs usually in packed col- umns when the flow through the matrix is not uniform but it forms channels at certain areas of the matrix. "Sufficient flow" therefore refers to substantially free flow having low resistance or drag in the matrix.

The first and second materials are arranged to a folded structure, such as a rolled or spun structure, which allows the flow through the structure. Such structure may also be called as spiral wound or convoluted matrix. Usually the flow direction is axial to the rolling or folding of the matrix.

The first supporting material supports the second cell retaining material and also maintains the substrate flow free thus avoiding channeling in the matrix. The first supporting material or framework may also be called an effluent grid or mesh, such as porous mesh framework. The supporting material may be any suitable sheet-like porous material, such as any plastic. In one embodiment the first supporting material comprises polyethylene, which may be in the form of a rigid plastic net with holes in it, such as small holes of about 0.1-30 mm, such as 1-10 mm, for example round or rectangular holes. If the second cell-retaining material is loose, the hole size of the net should be chosen accordingly to keep the material inside the net (the holes should be smaller than the diameter of the loose material pieces). The first supporting material may also be in the form of a perforated sheet, such as a polyethylene sheet, with perforations of about 0.1-10 mm, such as 0.2- 2 mm or 0.2-1 mm, for example about 0.3 mm or 0.6 mm (1/8 or ¼ inch). Such materials are generally commercially available. Other examples of the first supporting material include thermoplastics, such as acrylonitrile butadiene styrene, high density polyethylene, low density polyethylene, polyamide, polyamide-imide, polyaryletherketone, polycarbonate, polyether ether ketone, polyetherimide, polyethylene terephthalate, polyimide, polymethylmethacrylate, polypropylene, polysul- fone, polytetrafluoroethylene, polyvinyl chloride and self-reinforced polyphenylene, and polyester, phenolic plastics, epoxes and polyurethane. The cell-retaining material is the material to which the cells are mainly attached or immobilized in the matrix. The cell-retaining material may be for example selected from polypropylene, wood chip, clay or shale aggregate such as LECA (lighweight expanded clay aggregate), LESA, polyester, other thermoplastics, cotton etc. The cell-retaining material may be any suitable sheet-like material, or it may be loose material, such as said wood chips or clay or shale aggregates, which is supported by the first supporting material. The cell-retaining material may be porous or it has a large surface area. In one embodiment the cell-retaining material is polypropylene, which is soft and porous fabric material. Most commercial polypropylene is isotactic and has an intermediate level of crystallinity between that of low-density polyethylene (LDPE) and high-density polyethylene (HDPE). Polypropylene is normally tough and flexible, especially when copolymerized with ethylene. In another embodiment the cell- retaining material is polyester. The thickness and the amount of the fibers in the material can be varied by changing the production method.

In one embodiment the cell-retaining material is wood chip or similar lignocellulosic material. In one embodiment the cell-retaining material is fibrous material. A repre- senting example of wood chips is alder wood chips of about 2x10x13 mm in size. Such chips have high productivities and they gave steady flow through the column. Another example of wood chips includes bigger wood chips of about 5x20x30 mm in size. Generally the wood chips may have dimensions in the range of 1-30 mm, for example in the range of 1-20 mm. The wood chips may also act as the effluent grid and maintain the substrate flow free avoiding channeling in the matrix.

In one embodiment the cell-retaining material is clay or shale aggregate, preferably porous clay or shale aggregate, such as the ones sold by trade name LECA (Lightweight Expanded Clay Aggregate) or Lecastone. Lecastone is made by kiln firing small balls of clay which causes them to become extremely porous and durable. Generally the Leca beads are about 8-16 mm in size, but other sizes may be useful as well. Another example of such aggregates is the shale aggregate sold by trade name LESA (Lightweight Expanded Shale Aggregate). Generally the first supporting material and the second cell-retaining material are arranged as layer in layers before folding or rolling. The ratio of how much the first supporting material and the second cell-retaining material are used for constructing the matrix has a direct effect on how much cells can be retained in the matrix and subsequent final product concentration. In one embodiment the first supporting material is polyethylene and the second cell-retaining material is polypropylene. In such a case the preferred ratio between these two materials is one layer of polyethylene on every two layers of polypropylene, i.e. the ratio of first supporting material to second cell-retaining material is 1 :2. The ratio of the layers of the first supporting material to second cell-retaining material may be for example in the range of 3:1 to 1 :3, such as in the range of 2:1 to 1 :2. In one embodiment the ratio is 1 :1. In another embodiment the ratio is 1 :2. The folded or rolled matrix may form disc-like structures, which can be placed inside a vessel, container or column, such as a bioreactor. The vessel, container or column may contain one or more of the matrix structures and they may be positioned in line and may be separated by a dividing plate, for example at about every 50 cm. The dividing plate will support the structure but it will also contain perfora- tion or other openings to allow the flow through.

In one example the materials are rolled slightly larger in diameter than the diameter of the column or the like, such as about 1.5 cm larger. In one example the roll is packed in plastic which squeezes the matrix roll smaller than the diameter of the column. When the roll is placed into the column and the wrapping plastic is removed, the roll expands and fills the column tightly.

One embodiment of the invention provides a bioreactor, such as a bioreactor column, for immobilizing microbial cells for production, the bioreactor containing the immobilizing matrix placed inside the bioreactor. The bioreactor generally contains an inlet and an outlet for medium flow, such as the cell suspension medium flow. In one embodiment the inlet is in the first end of the bioreactor and the outlet in the second end of the bioreactor thus allowing the flow through the bioreactor and the immobilizing matrix inside. The instrumentation of the bioreactor may contain pH and temperature control. In one example the feeding of substrate and optionally a base is carried out at the upper part of the column. The removal of the product may also be carried out at the upper part of the column. Measurement of the pH may be carried out in a bypass tube of the column having a flow of up to 10-fold to the flow of the incoming substrate. In one embodiment the inlet and the outlet for cell suspension medium flow are both at the same end of the bioreactor, such as at the upper end. The bioreactor as used herein refers to any suitable container, (cultivation) vessel or column which may be used for cultivation of microbial cells, such as for fermentation. Generally the bioreactor may be called a bioreactor column or a biocolumn which terms may be used interchangeably.

The present invention further provides a method for immobilizing cells of a microorganism on an immobilizing matrix in a bioreactor, comprising providing said bioreactor containing the immobilizing matrix, and contacting the immobilizing matrix with an amount of a culture of said microorganism cells for a time period effective for cells to attach to the surface of the immobilizing matrix. The cells may be first cultivated in a separate container, such as in a continuous-flow stirred tank (CSTR) bioreactor, and then transferred to the bioreactor containing the immobilizing matrix. The present invention further provides a method for producing a fermentation product within a bioreactor, comprising providing said bioreactor containing the immobilizing matrix, providing microorganism cells in the bioreactor attached to the immobilizing matrix, providing a substrate to be fermented in the bioreactor, and allowing the microorganisms to ferment the substrate to produce the fermentation product. In one embodiment the production method is extractive fermentation wherein the fermentation product is continuously extracted.

In certain embodiments the system is adapted for batch, fed-batch or continuous cultures. The continuous culture may comprise continuous addition of fresh media and the continuous removal of the fermentation effluent from the culture. It may also further comprise recycling and reutilization of fermentation broth nutrients and minerals recovered.

Suitable microorganisms, microbes or microbial cells, as the terms may be used interchangeably, for use in fermentation processes and in the methods described herein include all fermenting microorganisms, such as bacteria and yeast. Non- limiting examples of such microorganisms (and fermentation products thereof) include Saccharomyces (ethanol and carbon dioxide), Streptococcus and Lactobacillus (lactic acid), Propionibacterium (propionic acid, acetic acid, and carbon diox- ide), Escherichia coli (acetic acid, lactic acid, succinic acid, ethanol, carbon dioxide, and hydrogen), Enterobacter (formic acid, ethanol, 2,3-butanediol, lactic acid, carbon dioxide, and hydrogen), and Clostridium (butyric acid, butyl alcohol, acetone, isopropanol, carbon dioxide, and hydrogen). In one embodiment the substrate is selected from glycerol, sugars, such as glucose, lactic acid, sucrose, and molasses, starch, maltodextrine, wheat flour and other carbohydrates, hydrocarbons and sources thereof. The substrate may be obtained by hydrolysis with enzymes or some other chemical method from larger polymers, such as carbohydrates.

In one embodiment the fermentation product is hydrogen. In one embodiment the fermentation product is an organic acid. In one embodiment the organic acid is selected from formic acid, acetic acid, citric acid, lactic acid, itaconic acid, malic acid, propionic acid, gluconic acid, fumaric acid, gibberelic acid, succinic acid, butyric acid and other dicarboxylic acids, adipic acid and amino acids. Generally organic acids that may be produced by fermentation include organic mono-, di- and tricarboxylic acids which optionally bear 1 or more, e.g. 1 , 2, 3 or 4, hydroxyl groups and have preferably 3 to 10 carbon atoms, for example tartaric acid, itaconic acid, succinic acid, fumaric acid, maleic acid, 2,5-furandicarboxylic acid, 3-hydroxypropionic acid, glutaric acid, laevulinic acid, lactic acid, propionic acid, gluconic acid, aconitic acid and diaminopimelic acid, citric acid; especially proteinogenic amino acids and non-proteinogenic amino ac- ids, preferably lysine, methionine, phenylalanine, tryptophan and threonine.

In one embodiment the organic acid is propionic acid. One exemplary microorganism especially suitable for propionic acid production is Propionibacterium aci- dipropionici.

In one embodiment the fermentation product is an alcohol, such as methanol, eth- anol, propanol, isopropanol, butanol, 1 ,2-propanediol 1 ,3-propanediol, 2,3- propanediol, 1 ,2-butanediol, 2,3-butanediol, 1 ,2,4-butanetriol, 1 ,2-pentanediol, 1 ,2- hexanediol, glycerol, n-pentanol and isomers thereof, n-hexanol and isomers thereof, n-heptanol and isomers thereof, n-octanol and isomers thereof.

In one embodiment the substrate is glycerol and the fermentation product is propionic acid. In some embodiments the fermentation product is aldehyde or ketone. The aldehyde may be selected from acetaldehyde, butyraldehyde, or propionaldehyde. The ketone may be selected from acetone or butanone. Examples Example 1. Biobased propionic acid was produced in an immobilization column bioreactor by using authentic crude glycerol from biodiesel manufacturing process. Propionibac- terium acidipropionicii were first cultivated [medium according to Quesada-Chanto et al. Microbial production of propionic acid and vitamin B12 using molasses or sugar (1994) Applied Microbiology and Biotechnology, Vol. 41 , Number 4, 378- 383, containing (per litre) saccharose 60 g, yeast extract 12 g, KH₂P0₄ 1 .0 g, K₂HPO₄ 1.0 g, MgS0₄ x 7H₂0 0.2 g, CoCI x 6H₂0 20 mg, FeS0₄ x 7H₂0 2.5 mg, 5,6-dimethylbenzimidazole 2.0 mg] in a continuous-flow stirred tank (CSTR) bioreactor, at +37°C, until exponential growth phase was reached. Cells were then transferred to a column reactor containing polypropylene-polyethylene matrix with ratio of 1 : 1 . This column filling allowed sufficient flow through the matrix without channeling problems. Cells attached to the column filling due to flow from the CSTR bioreactor through the column filling material and back to the CSTR reactor. The loading phase was continued until the CSTR bioreactor was empty of cells and the column filling was saturated with cells. At this point, the column was de- tached from the CSTR bioreactor and connected to the substrate feeding and product tanks (Figure 1 ). The substrate feeding media was kept similar to the cultivation media except saccharose was changed to crude (80%) glycerol. The feeding of the substrate and removing the product effluent took place in the headspace of the column. This feeding liquid was fed as 41.74 g/l, and the dilution rate through the column bioreactor was adjusted to 0.02 1/1, the process pH was maintained at 6.2 by using an on-line NaOH feeding device, installed into the head- space area of the column (Figure 1 ). The pH measuring electrode was installed into the circulation loop. The bioprocess temperature was maintained at +32°C. Table 1 shows the results from the fermentation. The yield is expressed as g(propionic acid) per g(glycerol) Table 1. Acids (HPLC results) in the fermentation broth. Samples were taken from the product tank.

Table 2 shows results from another test wherein raw glycerol was converted at 37°C. The characteristics for the bioprocess were: Productivity (Q) = -1 g/l/h, Propionic acid concentration = -28 g/l, Yield = -0.6. The formation of butyric acid was below the detection limit. The yield is expressed as g (propionic acid) per g (glycerol). D is the dilution rate (1/h) which indicates how long it takes to exchange the column volume (column work volume was 55.4 I).

Table 2. Acids (HPLC results) in the fermentation broth.

Succinic Acetic Propanol Butyric Feed D Glycerol Glycerol Propionic Yield Q acid acid (g/i) acid (l/h) 1/h (in) (out) acid (g/g) (vol)

(g/i) (g/i) (g/i) (g/i) (g/i) (g/i)

3.18 1.25 0.96 0.00 1.69 0.03 50.90 5.08 27.82 0.61 0.85

3,02 1.16 1.15 0.00 1.62 0.03 50.90 3.18 28.54 0.60 0.83

2.98 1.45 1.33 0.00 1.57 0.03 50.90 1.55 28.44 0.58 0.80

Claims

1. An immobilizing matrix for immobilizing microbial cells, the immobilizing matrix containing

- first supporting material comprising a layer of sheet-like porous material, and - second cell-retaining material deposited onto the first supporting material, wherein

the layers of the first supporting material and the second cell-retaining material are arranged into a rolled or folded structure allowing a sufficient flow of cell suspen- sion medium through the immobilizing matrix.

2. The immobilizing matrix of claim 1 , characterized in that the supporting material is selected from acrylonitrile butadiene styrene, high density polyethylene, low density polyethylene, polyamide, polyamide-imide, polyaryletherketone, poly- carbonate, polyether ether ketone, polyetherimide, polyethylene terephthalate, polyimide, polymethylmethacrylate, polypropylene, polysulfone, polytetrafluoro ethylene, polyvinyl chloride and self-reinforced polyphenylene, and polyester, phenolic plastics, epoxes and polyurethane.

3. The immobilizing matrix of claim 1 , characterized in that the supporting material is polyethylene.

4. The immobilizing matrix of any of the preceding claims, characterized in that the cell-retaining material is selected from polypropylene, wood chips, clay aggre- gate, shale aggregate, and polyester.

5. The immobilizing matrix of any of the preceding claims, characterized in that it contains cells of a microorganism attached to the surface of the immobilizing matrix.

6. A bioreactor for immobilizing microbial cells, characterized in that the biore- actor contains the immobilizing matrix of any of the preceding claims placed inside the bioreactor.

7. The bioreactor of claim 6, characterized in that it has an inlet and an outlet for medium flow.

8. A method for immobilizing cells of a microorganism on an immobilizing matrix in a bio reactor, characterized in that the method comprises

- providing the bioreactor of claim 6 or 7 containing an immobilizing matrix, and

- contacting the immobilizing matrix with an amount of a culture of said microor- ganism cells for a time period effective for cells to attach to the surface of the immobilizing matrix.

9. A method for producing a fermentation product within a bioreactor, characterized in that the method comprises

- providing the bioreactor of claim 6 or 7 containing an immobilizing matrix,

- providing microorganism cells in the bioreactor attached to the immobilizing matrix,

- providing a substrate to be fermented in the bioreactor, and

- allowing the microorganisms to ferment the substrate to produce the fermenta- tion product.

10. The method of claim 9, characterized in that the substrate is selected from glycerol, sugars, such as lactic acid, sucrose, and molasses, starch, wheat flour, and other carbohydrates and sources thereof.

11. The method of claim 9, characterized in that the fermentation product is an organic acid.

12. The method of claim 11 , characterized in that the organic acid is selected from formic acid, acetic acid, citric acid, lactic acid, itaconic acid, malic acid, propionic acid, gluconic acid, fumaric acid, gibberelic acid, succinic acid, butyric acid and other dicarboxylic acids, adipic acid and amino acids.

13. The method of claim 9, characterized in that the fermentation product is an alcohol.

14. The method of claim 13, characterized in that the alcohol is selected from methanol, ethanol, propanol, isopropanol, butanol, 1 ,2-propanediol 1 ,3- propanediol, 2,3-propanediol, 1 ,2-butanediol, 2,3-butanediol, 1 ,2,4-butanetriol, 1 ,2- pentanediol, 1 ,2-hexanediol, glycerol, n-pentanol and isomers thereof, n-hexanol and isomers thereof, n-heptanol and isomers thereof, n-octanol and isomers thereof.

15. The method of claim 9, characterized in that the substrate is glycerol and the fermentation product is propionic acid.