JPH1042890A - Production of bacteria cellulose by aerobic culture under agitation under high oxygen-transfer coefficient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To promote a production rate of biocellulose in an aerobic culture under agitation. SOLUTION: Cellulosic materials are produced by culturing cellulose-producing bacteria at least for a predetermined period under a culturing condition containing 2wt.% of bacteria cellulose and having about 50-100/hr oxygen- transfer coefficient (KLa) measured by using a simulated solution having 15-20 poise plastic viscosity by using a horizontal type culturing device installed with a mixing blade on a horizontal shaft in a tank.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a microorganism capable of producing a cellulosic substance under culture conditions capable of achieving a high oxygen transfer capacity coefficient (KLa) (hereinafter referred to as "cellulose-producing bacteria"). A cellulosic substance (hereinafter referred to as "bacterial cellulose" or "B
C ". ).

[0002]

2. Description of the Related Art BC (bacterial cellulose) is edible, is used in the food field, and has excellent water-based dispersibility. It has industrial value in improving food stability, as a low calorie additive or as an emulsification stabilizing aid. BC is characterized in that the fibril fragment width is as small as about two digits compared to cellulose produced from wood pulp or the like. Therefore, the dissociated product of BC has various industrial uses as a reinforcing agent for polymers, particularly aqueous polymers, based on such structural and physical characteristics of microfibrils. A material obtained by solidifying such a cellulosic disagglomerated product into a paper or solid form exhibits a high tensile modulus, and therefore is expected to have excellent mechanical properties based on the structural characteristics of microfibrils, and is applicable to various industrial materials. is there.

A method for producing BC is disclosed in
No. 265990, JP-A-63-202394 and JP-B-6-43443 describe the method for producing BC. The nutrient medium suitable for culturing cellulose-producing bacteria includes a carbon source, peptone, yeast extract, Schramm / He containing sodium phosphate and citric acid.
strin medium (Schramm et al., J. General Biology, ll , p.
pp. 123-129, l954) are known. In addition, in such a nutrient medium, inositol, phytic acid and pyrroloquinoline quinone (PQQ), which are factors for promoting the production of cellulose by specific nutrients in the medium (Japanese Patent Publication No. 5-1718; , Vol.42, No.3, No.23
7-244), and further, carboxylic acid or a salt thereof (Japanese Patent Application No. 5-191467), invertase (Japanese Patent Application No. 5-331149), and methionine (Japanese Patent Application No. 5-335564). It has been found that the productivity of cellulosic substances is improved by adding. Conventionally, as a culture method for culturing microorganisms, stationary, shaking, aeration and stirring cultures and the like have been used. In addition, as a culture operation method, a so-called batch fermentation method,
Fed-batch fermentation, repeated batch fermentation, continuous fermentation, and the like have been used. In addition, as the stirring means, for example, an impeller (a stirring blade), an air lift fermenter, a pump-driven circulation of a fermentation broth, a combination of these means, and the like are used. Known types of impellers include portal blades, turbine blades, helical ribbon blades, screw blades, and the like.

[0004] In general, in an industrial fermentation process, the oxygen demand of the culture is satisfied by aeration and stirring. However, in many fermentation processes, the productivity is limited by the oxygen supply capacity of the fermenter, and therefore, it is considered important to consider factors that affect the oxygen supply during culturing of microorganisms. When oxygen in the air moves to the cells in the culture system, the oxygen transfer from the bubbles to the liquid phase is represented by the following equation.

(Equation 1) Here, CL is the dissolved oxygen concentration (mmol / l) in the culture solution,
t is time (hr),

(Equation 2) Is the change in dissolved oxygen concentration over a certain period of time, that is, the oxygen transfer rate (mmol / l · hr), KL is the oxygen transfer rate coefficient of the liquid film (cm / hr), and a is the gas-liquid interface area per unit volume (cm). ² / cm ³ ) and C ^* is the dissolved oxygen concentration (mmol / l) equilibrium with the oxygen partial pressure of the bubbles. KL is the reciprocal of the resistance of the transfer of oxygen from the gas phase to the liquid phase, and (C ^* -CL) can be regarded as the driving force for the oxygen to move against the resistance. Since it is very difficult to measure KL and a in a fermentation system, KLa obtained by multiplying these two terms is called an oxygen transfer capacity coefficient.
The KLa dimension is the reciprocal of time and is usually expressed in hr ^-1 . The oxygen transfer capacity coefficient is a measure of the oxygen transfer capacity of the fermenter. Under the same conditions, the oxygen transfer capacity of a fermenter with a larger KLa is higher.

[0005]

In conventional batch and fed-batch fermentation methods, particularly in aeration and agitation culture, the culture period is increased due to the accumulation of BC in the culture solution due to the cultivation of cellulose-producing bacteria. In the latter half of the period, the viscosity of the culture solution increases, and as a result, it becomes extremely difficult to uniformly and thoroughly agitate and mix the entire culture system. The production speed was decreasing. Therefore, the present inventors have developed a method for producing BC under a culture condition in which KLa, which is an index of oxygen supply ability, shows a high value in a culture solution containing BC, and completed the present invention. Was. That is, it has been reported that the presence of an antifoaming agent generally lowers KLa. However, by using a horizontal culture device, in a BC culture system, the presence of an antifoaming agent is reversed. Value increases and BC
It has been found that the production speed can be improved. This is because, in a normal culture solution of a cellulose-producing bacterium for producing BC, fine bubbles remain in the solution for a long time, so that the fluidity of the culture solution changes greatly and a new bubble solution generated by aeration is generated. Dispersion into the medium is inhibited and KLa decreases, but the concentration of the defoaming agent used in the conventional method (foam rises from the gas-liquid interface to the gas phase due to the foaming phenomenon, and finally the liquid flows out of the apparatus together with ventilation. It is considered that adding more than the concentration (a concentration that can suppress the phenomenon of partial release) promotes interface renewal of fine bubbles in the liquid phase, and as a result, KLa increases.

[0006]

That is, the present invention uses a horizontal culture apparatus having stirring blades on a horizontal axis in a tank,
At least constant under culturing conditions having an oxygen transfer capacity coefficient (KLa) of about 50-100 / hr as measured using a simulated liquid containing 2% by weight of bacterial cellulose and having a plastic viscosity of 15-20 poise. The present invention relates to a method for producing a cellulosic substance by culturing a cellulose-producing bacterium for a period. As one specific embodiment of the present invention, there is a method of culturing in the presence of an antifoaming agent using a horizontal culturing apparatus provided with a stirring blade on a horizontal axis in a tank. As the defoaming agent, conventionally known defoaming agents, for example, polypropylene glycol (P
PG), silicon, esters, alcohols, fatty acids and derivatives thereof, and the like. The concentration in the culture solution depends on the type of the antifoaming agent, but is preferably 0.001% by weight or more, more preferably. Is 0.005% by weight or more. In another specific embodiment, a horizontal culture device having stirring blades on a horizontal axis in a tank is used, and the air bubble ratio is 1 unit.
There is a method of culturing under a condition of 0% or less. The bubble ratio can be calculated by the following method. Calculation method of bubble ratio : Assuming that the apparent specific gravity of the simulation liquid containing bubbles is A, and the true specific gravity of the simulation liquid is B

(Equation 3)

[0007] As the portal type blade or the turbine blade provided in the horizontal culture apparatus, various types known in the art can be used. For example, as a turbine blade, besides a general Rushton turbine, a paddle without a disk, a disk with a disk directly above a paddle, a curved paddle, a scover, a marine impeller, or the like can be used. In the culturing apparatus of the present invention, the same type or different types of blades such as portal blades and turbine blades can be provided on the same shaft or separate shafts in the same tank. The horizontal axis can be used up to about 80 °. KL
“a” is an important index of general device performance in culture, and varies depending on properties of a control liquid, the shape of a stirring blade, and the number of rotations. The present inventors set up a simulated solution close to the desired liquidity of bacterial cellulose (a culture solution of a cellulose-producing bacterium), and evaluate it with KLa. be able to. If measured by this evaluation system, arbitrary conditions can be selected for the shape and rotation speed of the stirring blade.

Measurement method of KLa : 1/3 volume of glass jar fermenter (horizontal culture device) having a total volume of 2 L containing a simulation liquid containing 2% by weight of bacterial cellulose and having a plastic viscosity of 15 to 20 poise , And the measurement is carried out in a state where an antifoaming agent is appropriately added thereto. The simulated liquid in which the dissolved oxygen concentration was in a saturated state of 0 to 10% by passing nitrogen while rotating the stirring blade, and then the oxygen partial pressure of 20%
Air of ２１21% is ventilated, and the dissolved oxygen concentration that rises is measured using a dissolved oxygen electrode. KLa can be obtained from the above equation (1). For convenience, the dissolved oxygen concentration is measured every 5 to 30 seconds, and the dissolved oxygen concentration D at time t1 is calculated.
KLa is determined from O1 and the dissolved oxygen concentration DO2 at time t2 by the following equation. ((DO2-DO1) / (t2-t1)) / (C ^* −
(DO1 + DO2) / 2), unit (/ hr) (where C ^* is the dissolved oxygen concentration equilibrium with the oxygen partial pressure of the bubble)

In the present specification, “KLa is about 50-100 /
The term “culturing conditions that are hrs” is a numerical value obtained by the above-described measurement system as defined herein. It is possible for an expert to appropriately select at what time and for how long under the specific device conditions during the culture, depending on the type of the bacterial cell, the composition of the medium, the type of the culture device, and the like. Since the viscosity of the medium increases with the growth of the cells, it is usually constant at least when the cellulose concentration in the culture solution becomes 5 g / L or more, or when the production rate becomes 0.2 g / L / hr. It is preferable to achieve the above-specified range of KLa for a sufficient period to supply a sufficient oxygen to the culture system. It is also possible to set this particular device condition intermittently during the culture period. With the method of the present invention, extremely high production rates of biocellulose can be obtained.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION In carrying out the method of the present invention, in addition to the above-mentioned culturing method and culturing method, Japanese Patent Application No.
192287 describes a method for producing a cellulosic substance comprising circulating a culture solution containing bacterial cells between a culture device and a separation device such as a flotation device and an edge filter, wherein the separation device comprises: Characterized in that the cellulosic substance that is the product is separated from the cells and the culture solution,
A method for producing a cellulosic substance by culturing a cellulose-producing bacterium, which is described in Japanese Patent Application No. 6-192288. By continuously supplying a new culture solution having a volume substantially equal to the amount, the concentration of the cellulosic substance in the culture solution during the culture is reduced to, for example, 10 g /
L or a low oxygen consumption rate of 15 μmol / L / hr or more.

The cellulose-producing bacterium used in the present invention is, for example, Acetobacter xylinum subsp. Scrofermentans typified by BPR2001 strain.
entans ), Acetobacter xylinum ( Acetobacter)
xylinum) ATCC23768, Acetobacter xylinum ATCC23769, Acetobacter Pasutsurianusu (A. pasteurianus) ATCC10245, Acetobacter xylinum ATCC14851, acetic acid bacteria such as Acetobacter xylinum ATCC11142 and Acetobacter xylinum ATCC10821, other, Agrobacterium , Rhizobium, Sarsina, Pseudomonas, Achromobacter, Alcaligenes, Aerobacterium, Azotobacter and Zooglare, and their mutations by known methods using NTG (nitrosoguanidine) and the like. Various mutants. In addition, BPR2001
The strain was deposited on February 24, 1993 at the Patented Microorganisms Depositary Center, National Institute of Bioscience and Human-Technology, Ministry of International Trade and Industry (Accession No. FERM P-13466), and then 199
Deposits based on the Budapest Treaty on the International Recognition of Deposits on Patent Proceedings dated February 7, 2004 (accession number FERM B
P-4545).

The chemical mutation treatment method using a mutagen such as NTG includes, for example, Bio Factors, Vol. 1, p. 297-302.
(1988) and J. Gen. Microbiol, Vol. 135, p. 2917-2.
929 (1989). Therefore, those skilled in the art can obtain the mutant strain used in the present invention based on these known methods. The mutant strain used in the present invention can also be obtained by other mutation methods, for example, irradiation. In the composition of the medium used in the production method of the present invention, as a carbon source, sucrose, glucose, fructose, mannitol, sorbitol, galactose,
Maltose, erythrit, glycerin, ethylene glycol, ethanol and the like can be used alone or in combination. Furthermore, starch hydrolyzate containing these, citrus molasses, beet molasses, beet juice,
Sugar cane juice, fruit juices such as citrus fruits, and the like can be used in addition to sucrose. As the nitrogen source, an organic or inorganic nitrogen source such as ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, nitrate, and urea can be used.
t-Peptone, Bact-Soytone, Ye
Nitrogen-containing natural nutrients such as ast-Extract and soybean may be used. Amino acids, vitamins, fatty acids, nucleic acids, 2,7,9-tricarboxy-
1H pyrrolo [2,3,5] -quinoline-4,5-dione, sulphite pulp waste liquor, ligninsulfonic acid and the like may be added.

When an auxotrophic mutant that requires an amino acid or the like for growth is used, it is necessary to supplement the required nutrient. As the inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts, cobalt salts, molybdates, red blood salts, chelate metals and the like are used. Further, the above-mentioned cellulose production promoting factor can be appropriately added to the medium. For example, when acetic acid bacterium is used as a production bacterium, the pH of the culture is controlled at 3 to 7, preferably around 5. The culture temperature is 10 to 40 ° C,
Preferably, it is performed in the range of 25 to 35 ° C. The oxygen concentration supplied to the culture device is 1 to 100%, preferably 21 to 80%
Is fine. Those skilled in the art can appropriately select the composition ratio of each component in the medium, the inoculation of the cells into the medium, and the like, depending on the culture method.

The BC produced by the method of the present invention may be obtained by recovering the cells as they are, and may be subjected to a treatment for removing impurities other than the cellulosic substance containing the cells contained in the substance. To remove impurities,
Water washing, pressure dehydration, diluted acid washing, alkali washing, treatment with bleach such as sodium hypochlorite and hydrogen peroxide, treatment with cell lysing enzymes such as lysozyme, surface activity such as sodium lauryl sulfate and deoxycholic acid Impurities can be almost completely removed from the cellulosic material by performing treatment with an agent, washing with heat in the range of room temperature to 200 ° C. alone or in combination. The cellulosic substance thus obtained in the present invention includes those containing cellulose, a heteropolysaccharide having cellulose as a main chain, β-1,3, β
-1 and 2 glucans. In the case of the heteropolysaccharide, components other than cellulose include hexoses such as mannose, fructose, galactose, xylose, arabinose, rhamnose, and glucuronic acid, pentoses, and organic acids. In addition, these polysaccharides may be a single substance, or two or more polysaccharides may be mixed by hydrogen bonding or the like.

[0015]

The present invention will be described in more detail with reference to the following examples. Example 1 Using the horizontal culture device shown in FIG. 1 and the above-mentioned KLa measurement system, the required power was about 40 to 170 kg · cm / sec.
L was changed within the range, and how the value of KLa changed with or without the antifoaming agent was measured. The result is shown in FIG. It can be seen that when the required power is relatively high, the effect of the addition of the antifoaming agent becomes particularly remarkable. In a similar system, the effect of the concentration of the antifoaming agent on the value of KLa was measured. The result is shown in FIG. It can be seen that the effect of the addition of an antifoaming agent becomes particularly remarkable when the rotation speed is relatively high. Further, the effect of the concentration of the antifoaming agent on the bubble ratio was measured in the same system (rotation speed: 200 rpm). FIG. 4 shows the results.

[Brief description of the drawings]

FIG. 1 shows a horizontal culture apparatus used for a KLa measurement system.

FIG. 2 shows a change in the value of KLa with a change in required power.

FIG. 3 shows the effect of the concentration of an antifoaming agent on the value of KLa.

FIG. 4 shows the effect of the concentration of an antifoaming agent on the bubble ratio and the value of KLa.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Fumihiro Yoshinaga 3-2-1 Sakado, Takatsu-ku, Kawasaki City, Kanagawa Prefecture Biopolymer Research Inc.

Claims (4)

[Claims] 1. Using a horizontal culture apparatus having stirring blades on a horizontal axis in a tank, using a simulated liquid containing 2% by weight of bacterial cellulose and having a plastic viscosity of 15 to 20 poise. A method for producing a cellulosic substance by culturing a cellulose-producing bacterium for at least a certain period under a culture condition in which the measured oxygen transfer capacity coefficient (KLa) is about 50 to 100 / hr. 2. The method according to claim 1, wherein the cellulosic bacterium is cultured in the presence of an antifoaming agent for at least a certain period of time. 3. The method according to claim 2, wherein the concentration of the antifoaming agent is 0.001% by weight or more. 4. An air bubble ratio of 10% for at least a certain period.
The method according to claim 1, wherein the culture is performed under the following conditions.