Method of removing cells from fermentation broth

Abstract: A method for improving the membrane permeation rate under milder pre-treatment conditions when membrane-separating cells from a fermentation broth obtained by culturing a microorganism belonging to the genus Escherichia, characterized in that polyethyleneimine is added to the fermentation broth before the membrane-separation of the cells. The method of the present invention can improve the permeation rate by from 1.5 to 4.0 times, avoiding the need for large-sized equipment, and increasing the efficiency of the membrane permeation equipment.

Description: Field of the Invention The present invention relates to the removal of cells from a fermentation broth, particularly of the genus Escherichia. Background In recent years, the technology of fermentative production using recombinant strains has advanced, and Escherichia coli (hereinafter abbreviated as "E coli"), which can easily be subjected to genetic manipulation, has been often used for this purpose. When the desired product has been excreted outside the cells at the end of the fermentation, the cells are usually removed by membrane separation or centrifugation after sterilization, and the resulting cell-free solution is subjected to the subsequent treatment steps. When the desired product is present inside of cells, the cells are lysed through freezing or using a homogenizer, a mill or the like, and then the cells and the cell debris are removed by membrane separation or centrifugation. In general, membrane separation can completely remove the cells to obtain a clear cell-free solution, as compared with centrifugation. However, where the permeation rate is low in membrane separation, a large-sized device is required. The membrane for removing cells is generally a microfiltration membrane (hereinafter abbreviated as "MF"), an ultrafiltration membrane (hereinafter abbreviated as "UF"), or the like. However, in the case of a fermentation broth obtained by culturing E coli strains, these membranes are problematic in that the permeation rate is low regardless of milling. It has been known hitherto that heating of the fermentation broth is effective as a pre-treatment for improving the permeation rate in removing cells from the fermentation broth through a membrane. For example, Japanese Laid-Open Patent Application (Kokai) No 91,196/1982 describes that when cells and high-molecular substances are separated through an ultrafiltration membrane, the membrane permeation rate is improved by heat-treating an inosine or guanosine fermentation broth with a pH of from 5.5 to 9.0 at from 90 to 110°C. Further, Japanese Laid-Open Patent Application (Kokai) No 78,588/1985 describes that the ultrafiltration membrane permeation rate is improved by heating the amino acid fermentation broth at from 50 to 100°C. However, as mentioned above, when removing cells from an E coli fermentation broth through a membrane, the permeation rate is extremely low, requiring a large-scale membrane treatment equipment. It is also possible to improve the permeation rate by an above-mentioned method, comprising pH adjustment and heat-treatment of the fermentation broth. However, this method is not very effective for E coli fermentation broth. Further, the heat-treatment is at times liable to cause coloration of the fermentation broth or the formation of small amounts of by-product impurities. Still further, when the desired product is thermally unstable, this method cannot be used. There has therefore been a need for a method in which, when cells, cell debris or dissolved high-molecular impurities are removed through a membrane in the treatment of a fermentation broth obtained by culturing a microorganism belonging to the genus Escherichia, the membrane permeation rate is improved through pre-treatment under mild conditions. Summary of the invention The present invention relates to a method in which membrane fouling substances are preliminarily treated, and the membrane permeation rate is improved when removing cells through the membrane, thereby shortening the treatment time and minimizing a size of an equipment. More particularly, the present invention provides a method for removing cells, cell debris or dissolved high-molecular impurities from a fermentation broth obtained by culturing a microorganism belonging to the genus Escherichia, by the use of a membrane to separate the cells etc, characterized in that polyethyleneimine(hereinafter abbreviated as "PEI") is added to the fermentation broth, and the mixture is then filtered through the membrane to separate the cells etc. Brief description of the drawings Fig 1 is a graph showing the relationship between the change in membrane permeation rate in UF filtration of a lysine fermentation broth over time and the amount of PEI added. Fig 2 is a graph showing the relationship between the change in membrane permeation rate in MF filtration of a leucine fermentation broth over time and the amount of PEI added. Detailed description of the invention The microorganism belonging to the genus Escherichia which is used in the present invention may be a wild strain or a mutant. A recombinant strain which is derived through cell fusion or genetic manipulation can also be used. An example of microorganisms belonging to the genus Escherichia is Escherichia coli. The desired product produced by this microorganism may be a product which permeates a UF membrane or an MF membrane used in the present invention. Examples include amino acids such as lysine, glutamic acid, isoleucine, valine and leucine; nucleic acids such as inosine and guanosine; organic acids such as lactic acid and malic acid; and vitamins. The fermentation broth which is used in the present invention can be prepared by culturing the above-mentioned microorganism in an appropriate medium. The medium is not particularly limited, and it may be an ordinary medium containing a carbon source, a nitrogen source, an inorganic ion and optionally an organic nutrient. The fermentation may be conducted by the method described in International Publication WO95/16042, EP-A-685,555, or Japanese Laid-Open Patent Application (Kokai) No 047,397/1996, under appropriate conditions for growth of the microorganism. Any carbon source can be used so long as it is applicable to the microorganism. Specific examples of the carbon source include saccharide such as glucose, fructose, sucrose and maltose; organic acids such as fumaric acid, citric acid, acetic acid and propionic acid; and salts thereof. Any nitrogen source can be used so long as it is applicable to the microorganism. Specific examples of the nitrogen source include inorganic acid ammonium salts such as ammonium sulfate and ammonium chloride; organic acid ammonium salts such as ammonium fumarate and ammonium citrate; nitric acid salts such as sodium nitrate and potassium nitrate; organic nitrogen compounds such as peptone, yeast extract, meat extract and corn steep liquor; and a mixture thereof. A nutrient source which is used in the ordinary fermentation may be used as required. Examples of the nutrient source include inorganic salts, trace metallic salts and vitamins. In general, PEI can be obtained in the form of an aqueous solution. However, when PEI is added to an E coli fermentation broth, it is advisable to add an aqueous solution containing from 1 to 50% of PEI, whereby an increase in the viscosity of the PEI aqueous solution is controlled and dilution of the fermentation broth is prevented. With respect to the addition of PEI, a predetermined amount of a 10% PEI aqueous solution can be added while the fermentation broth is stirred when the fermentation is completed. However, the method of the addition is not particularly limited so long as a uniform mixture can be obtained. The amount of PEI added is suitably between 0.0005 and 0.5% by weight, preferably between 0.001 and 0.05% by weight based on the E coli fermentation broth. PEI to be added may be water-soluble, and the average molecular weight thereof is not particularly limited. PEI having an average molecular weight of approximately from 10,000 to 100,000 is easy to obtain and to treat. When the membrane separation is conducted after the addition of PEI, the membrane permeation rate can be improved. Further, when the pH of the E coli fermentation broth after the addition of PEI is adjusted to from 3 to 9, preferably to from 4 to 7, the membrane permeation rate can be more improved. When the pH of the fermentation broth after the addition of PEI is near neutral, there is no need to readjust the pH. However, when the pH is readjusted to the above-mentioned range, a higher membrane permeation rate can be obtained at times. Thus, the readjustment of the pH can be conducted as required. An ordinary pH adjustor can be used to adjust the pH. Examples thereof include acids such as hydrochloric acid, sulfuric acid and phosphoric acid; and alkali compounds such as sodium hydroxide, potassium hydroxide and ammonia. The fermentation broth may be heat-treated before or after the addition of PEI. An appropriate heat-treatment temperature is between 50 and 130°C, and an appropriate heat-treatment time is between 10 and 20 minutes. This heat-treatment can improve the efficiency of mixing PEI and the permeation rate. The membrane to be used may be MF or UF. A flat membrane, a hollow fibre membrane, a tubular membrane or a spiral membrane can be used. The material of the membrane may be an organic material such as polysulfone, polyolefin, polyvinylidene difluoride or teflon, or an inorganic material such as ceramics. In the case of UF, a membrane having molecular weight cut off of from 1,000 to 500,000 is convenient. In the case of MF, a membrane having a pore diameter of from 0.05 to 1 µm is appropriate. The present invention is illustrated specifically by the following Examples. A test for membrane permeation in Example 1 and 2 was conducted by mounting two PTTK membranes (UF membranes made by Millipore, USA, molecular weight cut off 30,000) on a Minitan module made by Millipore. With respect to PEI, a 10% aqueous solution, obtained by diluting with pure water a 50% PEI solution having an average molecular weight of 50,000 (made by Sigma), was used. Example 1 L-lysine was produced by the method described in the Examples of International Publication No WO95/16042 using B-399/RSFD80 strain obtained by introducing plasmid/RSFD80 into E coli B-399 strain. The fermentation was conducted at a temperature of 37°C for 48 hours using a medium having the following formulation while stirring the mixture at from 114 to 116 rpm. Formulation of the medium for production of L-lysine: A: (NH 4 ) 2 SO 4 16 g/litre KH 2 PO 4 1 g/litre MgSO 4 ·7H 2 O 1 g/litre FeSO 4 ·7H 2 O 0.01 g/litre MnSO 4 ·5H 2 0 0.01 g/litre yeast extract (Difco) 2 g/litre L-methionine 0.5 g/litre The pH was adjusted to 7.0 with KOH, and the mixture was autoclaved at 115°C for 10 minutes (16/20 volume). B: 20% glucose (which was autoclaved at 115°C for 10 minutes) (4/20 volume) C: CaCO 3 by Japanese pharmacopeia (which was sterilized with dry heat at 180°C for 2 days) (30 g/litre). (A and B were mixed at a ratio of 4:1, and C was added thereto in an amount of 30 g/litre. Antibiotics (100 µg/ml of streptomycin and 5 µg/ml of kanamycin) were added to the mixture. The obtained mixture was used as the medium for production of L-lysine.) When the fermentation was completed, the concentration of L-lysine produced was 9.2 g/litre calculated as lysine hydrochloride. To each 300 ml of the thus-obtained lysine fermentation broth was added the above-mentioned 10% PEI solution in an amount of 0.32 g, 0.91 g or 1.6 g. The mixture was adjusted to a pH of 4.0 with 98% sulfuric acid, and was heated at 60°C for 20 minutes while being stirred. The concentrations of PEI in the fermentation broth after the addition of PEI in the above-mentioned amounts were approximately 100 ppm, 300 ppm and 500 ppm respectively. The thus-treated fermentation broths were used in the test for membrane permeation. The test for membrane permeation was conducted by feeding each of the above-treated fermentation broths to the above-mentioned UF module at a rate of 1,000 ml/min while maintaining the fermentation broth at 50°C and permeating it through the membrane at an average pressure of 0.9 kg-f/cm 2 . The change in the permeation rate was measured over the course of the permeation time of 30 minutes. The results are shown in Fig 1. As is apparent from Fig 1, in the case of the E coli fermentation broth, the membrane permeation rate abruptly decreased at the initial stage of the cell removal (within 5 minutes after the start-up), and later gradually became stable. With respect to the permeation rate after 30 minutes, the permeation rate with the addition of PEI was at most 4 times as high as that with no addition of PEI. The improvement of the permeation rate was remarkably observed with the addition of 100 ppm of PEI, and the permeation rate in this case was twice as high as that with no addition of PEI. Example 2 L-isoleucine was produced using E coli AJ12919 strain described in Published EP-A-685,555 or Japanese Laid-Open Patent Application (Kokai) No 047,397/1996. AJ12919 strain was spread on a culture medium containing 1% bactotrypton, 0.5% yeast extract, 0.5% NaCl, 1.5% agar, 100 µg/ml of streptomycin and 100 µg/ml of ampicillin, and was incubated at 37°C for from 18 to 24 hours. Then, a part of the broth was inoculated in 300 ml of a fermentation medium (containing 4% glucose, 1.6% ammonium sulfate, 0.1% potassium dihydrogen phosphate, 0.1% magnesium sulfate 7-hydrate, 0.001% ferrous sulfate 7-hydrate, 0.001% manganese sulfate 5-hydrate, 0.2% yeast extract and 3% calcium carbonate, pH 7.0) using a platinum loop, and was cultured at 37°C for 16 hours with air permeation of 300 ml/min while being stirred at 400 rpm to obtain a seed culture broth. The resulting seed culture broth was inoculated in a fermentation medium containing 6% glucose, 1.6% ammonium sulfate, 0.1% potassium dihydrogen phosphate, 0.1% magnesium sulfate 7-hydrate, 0.001% ferrous sulfate 7-hydrate, 0.001% manganese sulfate 5-hydrate and 0.2% yeast extract (pH 7.0). Glucose was fed in an appropriate amount at 37°C with air permeation of 300 ml/min while controlling the number of rotations such that the dissolved oxygen concentration in the medium reached 5% or more. The mixture was cultured for 23 hours while maintaining the pH at approximately 7.0 with ammonia gas. When the fermentation was completed, the concentration of L-isoleucine accumulated was 37 g/litre. The thus-obtained L-isoleucine fermentation broth was heated at 120°C for 10 minutes when the fermentation was completed, and the pH was adjusted to 4.0 with hydrochloric acid. To 300 ml of the thus-treated fermentation broth were added 0.3g of a 10% PEI solution, and the mixture was reheated at 60°C for 20 minutes. A fermentation broth which was heated at 60°C for 20 minutes with no addition of PEI was used as a control. The test for membrane permeation was conducted in the same manner as in Example 1. The results are shown in Table 1. Permeation time (min) Membrane permeation rate [liter/m 2 ·hour·(kg-f/cm 2 )] No addition of PEI Addition of PEI 1 75 96 5 60 76 10 55 69 20 50 62 30 48 58 As is clear from Table 1, the membrane permeation rate was increased with the addition of 100 ppm of PEI in the case of the isoleucine fermentation broth. Example 3 E coli FERM-P 5274 strain was resistant to β-2-thienylalanine and had an ability to produce L-leucine(disclosed in Japanese Laid-Open Patent Application (kokai) No 34,397/1987) was subjected to a mutation treatment with N-methyl-N'-nitro-N-nitrosoguanigine. Out of the mutated strains, a strain which was resistant to 4-azaleucine was isolated to obtain a strain having an improved ability to produce L-leucine. The thus-obtained strain was cultured at 31°C for 72 hours with stirring in a medium (containing 5g/dl glucose, (NH 4 ) 2 SO 4 2.5g/dl, KH 2 PO 4 0.2g/dl, MgSO 4 ·7H2O 0.1g/dl, yeast extract 0.05g/dl, thiamine hydrochloride 1mg/L, FeSO 4 ·7H 2 O 1mg/dl, MnSO 4 ·4H 2 O 1mg/dl, CaCO 3 2.5g/dl, pH 7.0). When the fermentation was completed, the concentration of L-leucine accumulated was 3.1g/dl. The above-mentioned 10% PEI solution was added to each 300 ml of the thus-obtained fermentation broth to prepare fermentation broth having a PEI concentration of 0, 200 and 1000 ppm respectively. The obtained broth was adjusted to a pH of 4.0 with 98% sulfuric acid and was heated at 60°C for 30 minutes while being stirred. The thus-treated broths were used in the test for membrane permeation. The test for membrane permeation was conducted by feeding each of the above-treated broths to a Pencil type PSP-003 module (pore diameter; 0.3 µm, membrane area; 150 cm 2 ) made by Asahi Chemical Industry Co Ltd, at a rate of 600 ml/min and an entrance pressure of 1.0 kg-f/cm 2 . The change of the permeation rate was measured over 60 minutes. The results are shown in Fig 2. As is apparent from Fig 2, the membrane permeation rate was increased with the addition of PEI in case of L-leucine fermentation broth. As shown above, by means of the present invention, the membrane permeation rate of the fermentation broth can be improved without using large-scale equipment. Further, it is also possible to increase the efficiency of the process for membrane removal of cells from a fermentation broth, and to minimize the size of the membrane separation equipment for removal of cells.

Claims: A method for removing cells from a fermentation broth obtained by culturing a microorganism belonging to the genus Escherichia, using a membrane to separate the cells, characterized in that polyethyleneimine is added to the fermentation broth, and the mixture is then filtered through the membrane to separate the cells. A method according to claim 1, wherein the pH of the fermentation broth is adjusted to from 3 to 7. The method of claim 1 or 2, wherein the membrane used is an ultrafiltration membrane or a microfiltration membrane. The method of any one of claims 1 to 3, wherein the amount of the added polyethyleneimine is between 0.0005 and 0.5% by weight based on the fermentation broth. The method of any one of claims 1 to 4, wherein the fermentation broth is heated at from 50 to 130°C before and/or after the addition of polyethyleneimine.

Inventors: NAKAMURA TOHRU, TANABE TOSHIYA, NAKAMURA,TOHRU, TANABE, TOSHIYA, Nakamura,Tohru c/o Technology &amp; Engeneering Lab., Tanabe, Toshiya c/o Technology&amp;Engeneering Lab.
Applicants: AJINOMOTO KK, AJINOMOTO CO., INC.
Application year: 1996
Main classification: C12N1
IPC codes: C12N1