JPH09173090A - Production of l (+)-lactic acid and manufacturing equipment therefor - Google Patents
Production of l (+)-lactic acid and manufacturing equipment thereforInfo
- Publication number
- JPH09173090A JPH09173090A JP35114095A JP35114095A JPH09173090A JP H09173090 A JPH09173090 A JP H09173090A JP 35114095 A JP35114095 A JP 35114095A JP 35114095 A JP35114095 A JP 35114095A JP H09173090 A JPH09173090 A JP H09173090A
- Authority
- JP
- Japan
- Prior art keywords
- lactic acid
- culture
- container
- air
- producing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J19/24—Stationary reactors without moving elements inside
- B01J19/2455—Stationary reactors without moving elements inside provoking a loop type movement of the reactants
- B01J19/246—Stationary reactors without moving elements inside provoking a loop type movement of the reactants internally, i.e. the mixture circulating inside the vessel such that the upward stream is separated physically from the downward stream(s)
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00049—Controlling or regulating processes
- B01J2219/00051—Controlling the temperature
- B01J2219/00074—Controlling the temperature by indirect heating or cooling employing heat exchange fluids
- B01J2219/00087—Controlling the temperature by indirect heating or cooling employing heat exchange fluids with heat exchange elements outside the reactor
- B01J2219/00094—Jackets
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/18—Details relating to the spatial orientation of the reactor
- B01J2219/185—Details relating to the spatial orientation of the reactor vertical
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/19—Details relating to the geometry of the reactor
- B01J2219/194—Details relating to the geometry of the reactor round
- B01J2219/1941—Details relating to the geometry of the reactor round circular or disk-shaped
- B01J2219/1943—Details relating to the geometry of the reactor round circular or disk-shaped cylindrical
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は好気的培養法による
乳酸の製造法及びそのために用いられる装置に関する。
より具体的に言えば、本発明は、気泡塔型バイオリアク
ターを用いてリゾプス（Rhizopus）属に属する真菌（カ
ビ）を回分培養、反復回分培養あるいは連続培養するこ
とにより、澱粉もしくはその糖化液またはグルコースか
らＬ（＋）−乳酸を効率よく生産する方法及びこの方法
に有用な気泡塔型バイオリアクターに関する。TECHNICAL FIELD The present invention relates to a method for producing lactic acid by an aerobic culture method and an apparatus used therefor.
More specifically, the present invention uses a bubble column bioreactor to perform batch culture, repeated batch culture, or continuous culture of a fungus (mold) belonging to the genus Rhizopus to obtain starch or its saccharified solution or The present invention relates to a method for efficiently producing L (+)-lactic acid from glucose and a bubble column bioreactor useful for this method.
【０００２】[0002]
【従来技術】最近、ポリ乳酸（ＰＬＡ）が生分解性ポリ
マーとして優れた性質を有していることが明らかとな
り、その原料としての乳酸が注目されている。ＰＬＡが
バイオポリマーとして商業生産される場合、その生産量
は現在のプラスチックスの２０％としても２４０万トン
と推定される。従って、原料としての乳酸もそれ以上の
数量が要求されることになるが、これは最も多く生産さ
れている発酵生産物であるグルタミン酸の約十倍であ
る。また、バイオプラスチックスの原料として用いる場
合、その価格はできるだけ低く抑えることが要求され
る。しかしながら、伝統的な乳酸製造法である乳酸菌
（ラクトバチルス（Lactobatilus）属、ストレプトコッ
カスStreptcoccus）属等に属する一群の乳酸産生細菌の
総称。）による嫌気発酵法では、量的にもコスト的に
も、このような要求に対応することができない。2. Description of the Related Art Recently, it has been clarified that polylactic acid (PLA) has excellent properties as a biodegradable polymer, and lactic acid as a raw material thereof has been receiving attention. If PLA is produced commercially as a biopolymer, its production is estimated to be 2.4 million tons, even 20% of current plastics. Therefore, a larger amount of lactic acid as a raw material is required, which is about ten times that of glutamic acid, which is the most produced fermentation product. In addition, when used as a raw material for bioplastics, it is required to keep the price as low as possible. However, lactic acid bacteria is a traditional lactic acid production process (Lactobacillus (Lactobatilus) genus Streptomyces cock <br/> scum Streptococcus) term for a group of lactic acid-producing bacteria belonging to the genus or the like. The anaerobic fermentation method according to (1) cannot meet such requirements both in terms of quantity and cost.
【０００３】クモノスカビとして知られるRhizopus属の
菌種も、グルコースから乳酸を生産することが知られて
いる。Rhizopus属による乳酸発酵は好気的であるため、
乳酸菌による嫌気発酵よりは効率的であることが期待で
きる。しかし、Rhizopus属のカビは菌糸塊を形成しやす
く、従来の形式の通気撹拌型バイオリアクターを用いて
大量培養しようとすると、槽内の構造物、たとえば邪魔
板や冷却コイルに巨大な菌糸塊が付着して基質や酸素な
どの物質移動を妨げたり、あるいは、槽内の菌体のほと
んどすべてが空気を含む菌糸塊として培養液の上部に浮
上してしまい、乳酸の生産効率が著しく低下するという
問題があった。It is also known that the Rhizopus sp. Strain known as Kumonosukubi produces lactic acid from glucose. Lactic acid fermentation by Rhizopus is aerobic, so
It can be expected to be more efficient than anaerobic fermentation with lactic acid bacteria. However, molds of the genus Rhizopus tend to form mycelium, and when large-scale culture is performed using a conventional type aeration-stirring bioreactor, huge mycelium is generated in the structures in the tank, such as baffles and cooling coils. It adheres to block the transfer of substances such as substrates and oxygen, or almost all of the bacterial cells in the tank float up to the top of the culture solution as a mycelial mass containing air, which significantly reduces the production efficiency of lactic acid. There was a problem.
【０００４】また、従来、安価な炭素源である澱粉を直
接資化できるRhizopus属の菌種は知られておらず、澱粉
等は発酵前にグルコース等に加水分解した上で用いる必
要があり、乳酸製造コストを低減する上での障害となっ
ていた。発酵槽に加水糖化酵素を添加しておく方法も知
られているが（特開平2-76592 号公報等）、この場合
も、添加する酵素が原料コストを押し上げる原因となる
上に、酵素を培地に添加するためにはその無菌濾過が必
要であり、そのために要する装置や工程等によりさらに
製造コストが増加するという問題がある。[0004] Further, conventionally, no species of Rhizopus genus capable of directly assimilating starch, which is an inexpensive carbon source, has been known, and starch or the like must be hydrolyzed to glucose or the like before fermentation before use. This has been an obstacle to reducing the production cost of lactic acid. There is also known a method in which a hydrosaccharifying enzyme is added to a fermenter (Japanese Patent Laid-Open No. 2-76592, etc.), but in this case as well, the enzyme to be added causes an increase in raw material cost and the enzyme is added to the medium. Aseptic filtration is required to add to the above, and there is a problem that the manufacturing cost is further increased due to the equipment and process required for that.
【０００５】[0005]
【解決しようとする課題】本発明は、発酵法による乳酸
製造方法において原料コストを低く抑えつつ生産効率を
高め、ＰＬＡ原料として要求される価格・生産量に見合
った、経済的でかつ大規模な実施が可能である乳酸製造
法を提供することを目的とする。[Problems to be Solved] The present invention is an economical and large-scale apparatus for producing lactic acid by a fermentation method, in which the raw material cost is kept low and the production efficiency is improved, and the price and production amount required as PLA raw materials are met. It is an object of the present invention to provide a lactic acid production method that can be carried out.
【０００６】[0006]
【課題を解決するための手段】本発明者らは、上記の問
題を解決するべく鋭意研究を重ねた結果、菌糸塊の形成
による問題点は気泡塔型バイオリアクターの利用により
軽減できること、特に特定構造の気泡塔型バイオリアク
ターが有効であること、また澱粉を原料として乳酸発酵
を行なうRhizopus属に属する菌株を見出し、さらに、気
泡塔型バイオリアクターによる乳酸発酵を実用化する上
での問題点と考えられたスケールアップに伴なう問題点
についても解決方法を見出し、本発明を完成するに至っ
た。As a result of intensive studies to solve the above problems, the present inventors have found that the problems due to the formation of mycelium can be reduced by using a bubble column type bioreactor. We found that the bubble column bioreactor with a structure is effective, and found a strain belonging to the genus Rhizopus that performs lactic acid fermentation using starch as a raw material. Furthermore, there were problems in practical application of lactic acid fermentation by the bubble column bioreactor. The inventors have found a solution to the problem associated with the considered scale-up and have completed the present invention.
【０００７】本発明は、以下の乳酸製造方法及びこの方
法において有用なバイオリアクターを提供する。 １） 底部から空気を吹き込む気泡塔型バイオリアクタ
ーを使用し、炭素源として澱粉もしくはその糖化液また
はグルコースを含有する水性培地でリゾプス（Rhizopu
s）属に属する菌を培養することを特徴とするＬ（＋）
−乳酸の製造法。 ２） 気泡塔型バイオリアクターとして、筒状の壁面部
と逆錐状の底面部と排気管を備えた蓋部とからなる容器
本体、容器本体内に容器壁面に概ね平行に固設されたド
ラフトチューブ、前記逆錐状の底面部とドラフトチュー
ブの下端との間に設けられたエアスパージャー、容器外
部からエアスパージャーに気体を送る通気管、容器外壁
面を覆う温度調節可能なジャケット、及び容器本体に連
通する少なくとも１本の通液管を有し、前記底面部の逆
錐の母線と鉛直線とのなす傾斜角θが２０〜４０°であ
り、容器本体内に対するドラフトチューブ内径の比が0.
5 ないし0.7 の範囲にあるリアクターを使用する前記１
に記載のＬ（＋）−乳酸の製造法。The present invention provides the following method for producing lactic acid and a bioreactor useful in this method. 1) Using a bubble column type bioreactor in which air is blown from the bottom, Rhizopu ( Rhizopu ) is used in an aqueous medium containing starch or its saccharified liquid or glucose as a carbon source.
s ) L (+) characterized by culturing a bacterium belonging to the genus
-Method for producing lactic acid. 2) As a bubble column type bioreactor, a container body composed of a cylindrical wall surface portion, an inverted cone-shaped bottom surface portion, and a lid portion provided with an exhaust pipe, and a draft fixed substantially parallel to the container wall surface in the container body. A tube, an air sparger provided between the bottom surface of the inverted pyramid and the lower end of the draft tube, a ventilation pipe for sending gas from the outside of the container to the air sparger, a temperature-adjustable jacket covering the outer wall surface of the container, and The container has at least one liquid passage communicating with the container body, the inclination angle θ formed by the vertical pyramid and the generatrix of the inverted cone of the bottom surface is 20 to 40 °, and the ratio of the inner diameter of the draft tube to the container body is Is 0.
1 using a reactor in the range of 5 to 0.7
The method for producing L (+)-lactic acid according to 1.
【０００８】３） 空気吹込み量が 0.125〜0.25ｖｖｍ
である前記１または２に記載のＬ（＋）−乳酸の製造
法。 ４） 培養する菌がリゾプス・オリザエ（Rhizopus ory
zae ）である前記１乃至３のいずれかの項に記載のＬ
（＋）−乳酸の製造法。 ５） 培養する菌株がリゾプス・オリザエＮＲＲＬ３９
５株（Rhizopus oryzaeNRRL395)である前記４に記載の
Ｌ（＋）−乳酸の製造法。 ６） 炭素源が澱粉である前記２乃至５のいずれかに記
載のＬ（＋）−乳酸の製造法。 ７） 所定期間培養を行なった後、空気の吹き込みを停
止して培養液中の菌体を沈降させて前記通液管から上澄
液のみを取り出し、取り出した液量に相当する量の培養
液を補充した後、空気吹き込みを再開して培養を繰り返
す前記１乃至６のいずれかの項に記載のＬ（＋）−乳酸
の製造法。3) The amount of air blown is 0.125 to 0.25 vvm
The method for producing L (+)-lactic acid according to 1 or 2 above. 4) The fungus to be cultivated is Rhizopus ory
L according to any one of 1 to 3 above, which is zae ).
A method for producing (+)-lactic acid. 5) The strain to be cultured is Rhizopus oryzae NRRL39
5. The method for producing L (+)-lactic acid according to 4 above, which is 5 strains ( Rhizopus oryzae NRRL395). 6) The method for producing L (+)-lactic acid according to any one of 2 to 5 above, wherein the carbon source is starch. 7) After culturing for a predetermined period of time, the blowing of air is stopped, the bacterial cells in the culture solution are allowed to settle, and only the supernatant liquid is taken out from the passage tube, and the amount of the culture solution corresponding to the taken-out liquid quantity 7. The method for producing L (+)-lactic acid according to any one of 1 to 6 above, wherein after replenishing the above, the air blowing is restarted and the culture is repeated.
【０００９】８） 培養液の引き抜きと補充とを連続的
に行なう前記１乃至６のいずれかの項に記載のＬ（＋）
−乳酸の製造法。 ９） リアクター内の菌体量が所定量に達するまでは前
記７に記載の方法によって菌の培養を行ない、菌体量が
所定量に達した後は前記８に記載の方法によって菌の培
養を行なう前記１乃至６のいずれかの項に記載のＬ
（＋）−乳酸の製造法。１０） 筒状の壁面部と逆錐状
の底面部と排気管を備えた蓋部からなる容器本体、容器
本体内に容器壁面に概ね平行に固設されたドラフトチュ
ーブ、前記逆錐状の底面部とドラフトチューブの下端と
の間に設けられたエアスパージャー、容器外部からエア
スパージャーに気体を送る通気管、容器外壁面を覆う温
度調節可能なジャケット、及び容器本体に連通する少な
くとも１本の通液管を有し、前記底面部の逆錐の母線と
鉛直線とのなす傾斜角θが２０〜４０°であり、容器本
体内径に対するドラフトチューブ内径の比が 0.5乃至
0.7の範囲にある気泡塔型バイオリアクター。8) L (+) described in any one of 1 to 6 above, in which the withdrawal and replenishment of the culture solution are carried out continuously.
-Method for producing lactic acid. 9) Cultivation of the bacterium by the method described in 7 above until the amount of the microbial cells in the reactor reaches a predetermined amount, and after reaching the predetermined amount, the culturing of the bacterium by the method described in 8 above. L according to any one of 1 to 6 above
A method for producing (+)-lactic acid. 10) A container body composed of a cylindrical wall surface portion, a reverse cone-shaped bottom surface portion, and a lid portion provided with an exhaust pipe, a draft tube fixed substantially parallel to the container wall surface in the container body, the reverse cone-shaped bottom surface. At least one communicating with the container body, an air sparger provided between the lower part of the draft tube and the lower part of the draft tube, a ventilation pipe for sending gas from the outside of the container to the air sparger, a jacket for adjusting the temperature of the outer wall of the container And an inclination angle θ formed by the vertical pyramid and the generatrix of the inverted cone on the bottom surface is 20 to 40 °, and the ratio of the inner diameter of the draft tube to the inner diameter of the container body is 0.5 to.
Bubble tower bioreactor in the range of 0.7.
【００１０】［気泡塔型バイオリアクター］本発明の方
法では、内部に冷却コイルや邪魔板あるいは撹拌翼など
の構造物を除去し可能な限り単純化した構造の、いわゆ
る気泡塔型反応槽をバイオリアクターとして用いる。気
泡塔型反応槽は、図２に示したように、反応槽１の底面
の一部にエアスパージャー（ガス分散器）２を設けたも
のであり、撹拌用の機械的駆動部分を有する通気撹拌型
反応槽とは異なる構造のものである。通常は、エアスパ
ージャーの上方に上下端を開口させたドラフトチューブ
３を設置する。使用に際しては、内部に液体を収容し、
エアスパージャーからガスを吹き込む。吹き込まれたガ
スは気泡となってドラフトチューブ内を浮上して上昇流
を生じる。表面に達した上昇流は、ドラフトチューブ上
端から周辺に向かって放射状に広がる流れとなり、反応
槽の壁面に達した後、下降流に転じる。上昇流と下降流
はドラフトチューブの管面により隔てられているため、
２つの流れは勢いをそがれることなく、槽内において効
率的な循環流を形成する。ガスは反応槽の蓋に設けられ
た排気管８から排出される。[Bubble tower type bioreactor] In the method of the present invention, a so-called bubble tower type reaction vessel having a structure as simple as possible by removing structures such as cooling coils, baffles, and stirring blades inside the bioreactor is used. Used as a reactor. As shown in FIG. 2, the bubble column type reaction tank is one in which an air sparger (gas disperser) 2 is provided on a part of the bottom surface of the reaction tank 1 and has a mechanical drive part for aeration. It has a different structure from the stirring reaction tank. Usually, a draft tube 3 having upper and lower ends opened is installed above the air sparger. When using, contain a liquid inside,
Blow gas from the air sparger. The blown gas becomes bubbles and floats in the draft tube to generate an upward flow. The upward flow reaching the surface becomes a flow that spreads radially from the upper end of the draft tube toward the periphery, reaches the wall surface of the reaction tank, and then changes to a downward flow. As the upflow and downflow are separated by the tube surface of the draft tube,
The two streams do not lose their momentum and form an efficient circulating flow in the vessel. The gas is discharged from the exhaust pipe 8 provided on the lid of the reaction tank.
【００１１】従来、このような気泡塔型反応槽は各種提
案されているが（例えば、特開平1-160476号公報）、乳
酸発酵用バイオリアクターに用いることを提案した例は
見られない。本発明の方法では、気泡塔型反応槽をRhiz
opus属の真菌種の培養に用いることにより、Rhizopus属
の培養において問題であった、菌糸塊の付着による生産
性低下の問題を解消することに成功した。本発明では既
存の気泡塔型反応槽も使用可能であるが、図１に示す構
造の反応槽が特に好ましい。Conventionally, various bubble column type reaction vessels have been proposed (for example, Japanese Patent Laid-Open No. 1-160476), but no example has been proposed for use in a bioreactor for lactic acid fermentation. In the method of the present invention, a bubble column reactor is used as a Rhiz reactor.
By using it for culturing a fungal species of the genus opus, we succeeded in solving the problem of productivity decrease due to attachment of mycelium, which was a problem in the culture of the genus Rhizopus . In the present invention, the existing bubble column type reaction tank can be used, but the reaction tank having the structure shown in FIG. 1 is particularly preferable.
【００１２】図１に示す本発明に好適な反応槽は、底部
を下部に向かって逆錐状に突出した形状を有する。底部
の逆錐の母線と鉛直線とのなす傾斜角θは２０〜４０°
の範囲が好ましい。２０°よりも小さいとリアクター底
部の円錐部の占める割合が大きくなり、リアクターの容
積効率が低下する。４０°を超えると菌体の沈降性が低
下する。また、反応槽の外壁の周囲にはこれと接して給
水口６及び排水口７を備えたジャケット５を設け、ジャ
ケット内に温水もしくは冷水を流して槽内の温度を制御
する。反応槽にはジャケット５を貫いてあるいはこれを
避けて容器本体に連通する少なくとも１本の通液管を４
を設け、必要により培養液の供給及び／または抜出しを
行なう。The reaction tank shown in FIG. 1 and suitable for the present invention has a shape in which the bottom portion projects downwardly in an inverted cone shape. The inclination angle θ between the generatrix of the inverted cone at the bottom and the vertical line is 20 to 40 °.
Is preferable. If the angle is smaller than 20 °, the conical portion of the bottom of the reactor occupies a large proportion, and the volumetric efficiency of the reactor decreases. If it exceeds 40 °, the sedimentation property of the bacterial cells will be reduced. Further, a jacket 5 having a water supply port 6 and a drainage port 7 is provided around the outer wall of the reaction tank so as to be in contact therewith, and hot water or cold water is caused to flow in the jacket to control the temperature in the tank. In the reaction tank, at least one liquid passage pipe that penetrates the jacket 5 or avoids it and communicates with the container body is provided.
Is provided, and the culture solution is supplied and / or withdrawn as necessary.
【００１３】反応槽の横断面形状は特に限定されず、円
形、楕円形、矩形のいずれでもよいが、液を均一に循環
させるためには断面を円形とし、反応槽全体としては円
筒状の形状とすることが好ましい。ドラフトチューブは
反応槽の壁面と平行に設けることが好ましく、反応槽の
内径に対するドラフトチューブ内径の比は 0.5乃至 0.7
程度であることが望ましい。内径比が0.5 未満では気液
が接触する時間が相対的に小さくなり酸素供給能力が低
下する。内径比が0.7 を超えると下降流の流速が低下
し、結果的に槽全体での酸素供給能力が低下する。反応
槽及び管系の材質は特に限定されず、例えば、ステンレ
ス材、鉄材あるいはゴムライニング鉄材等が用いられ
る。The cross-sectional shape of the reaction tank is not particularly limited, and may be circular, elliptical or rectangular. However, in order to circulate the liquid uniformly, the cross section is circular, and the reaction tank as a whole has a cylindrical shape. It is preferable that The draft tube is preferably installed parallel to the wall of the reaction tank, and the ratio of the inner diameter of the draft tube to the inner diameter of the reaction tank is 0.5 to 0.7.
Desirably. If the inner diameter ratio is less than 0.5, the gas-liquid contact time becomes relatively short, and the oxygen supply capacity decreases. When the inner diameter ratio exceeds 0.7, the flow velocity of the downflow is reduced, and as a result, the oxygen supply capacity of the entire tank is reduced. The materials of the reaction tank and the pipe system are not particularly limited, and, for example, a stainless material, an iron material, a rubber-lined iron material, or the like is used.
【００１４】また、後述の反復回分方式に用いる場合
は、特に、槽内径に対する容器本体部分の高さ（槽高）
の比を２以上とし、反応槽壁面の上下にずれた位置
ａ1 ，ａ2，……ａn に所定間隔でそれぞれ複数の通液
管を配置する。ｎの最適値は槽高にもよるが、通常は、
２〜１０程度が好ましい。図１ではｎ＝４の例を示し
た。このような構成を取ることにより、空気の吹込みを
停止した後、菌体の沈降に応じて上澄液を速やかに抜き
出すことが可能になる。When used in a repetitive batch method described later, the height of the container body relative to the inner diameter of the tank (tank height)
Ratio was 2 or more, shifted above and below the reaction vessel wall position a 1, a 2, respectively placing a plurality of liquid passage pipe at predetermined intervals in ...... a n. The optimum value of n depends on the tank height, but normally,
About 2 to 10 is preferable. FIG. 1 shows an example where n = 4. By adopting such a configuration, it is possible to promptly withdraw the supernatant liquid in accordance with the sedimentation of the bacterial cells after stopping the blowing of air.
【００１５】［乳酸生産菌種］培養菌種としてはリゾプ
ス（Rhizopus）属に属し、乳酸生産能を有する菌であれ
ば適宜使用しうる。リゾプス・オリザエ（Rhizopus ory
zae ）が好ましい。特に、ＮＲＲＬ３９５株（Rhizopus
oryzae NRRL395)が好ましい。本発明者らは、上記の菌
株が、澱粉を利用して乳酸を生産する能力を有すること
を見出した。[Lactic acid-producing bacterial species] As the cultured bacterial species, any bacteria belonging to the genus Rhizopus and capable of producing lactic acid can be appropriately used. Rhizopus ory
zae ) is preferred. In particular, the NRRL395 strain ( Rhizopus
oryzae NRRL 395) is preferred. The present inventors have found that the above strain has the ability to utilize starch to produce lactic acid.
【００１６】本菌株は、以下の特徴を有する。 (a) 培養的・形態的性質 培養時の形態的性質を表１に示す。培地としてはツァペ
ック寒天培地及び馬鈴薯ブドウ糖培地（バレイショ２０
ｇ、グルコース２０ｇ及び寒天１５ｇを蒸留水１Ｌ中に
含有するもの）をそれぞれオートクレーブ中１ｋｇ／ｃ
ｍ2 Ｇで１５分間滅菌処理したものを用いた。This strain has the following characteristics. (a) Culture and morphological properties Table 1 shows the morphological properties during culture. As the medium, Czapek agar medium and potato-glucose medium (potato 20
g, glucose 20 g, and agar 15 g in 1 L of distilled water) in an autoclave at 1 kg / c, respectively.
What was sterilized by m 2 G for 15 minutes was used.
【００１７】[0017]
【表１】 [Table 1]
【００１８】(b) 生理学的・化学分類的性質 (b-1) 増殖温度 培養温度が本菌株の生育に与える影響を表２に示す。な
お、培地としてはツァペック培地を用いた。(B) Physiological and chemical taxonomical properties (b-1) Growth temperature Table 2 shows the influence of the culture temperature on the growth of this strain. The culture medium used was Czapek medium.
【００１９】[0019]
【表２】 [Table 2]
【００２０】この結果より本菌株の生育限界温度は５０
℃と推定される。 (b-2) 増殖ｐＨ ツァペック培地を用い初発ｐＨを変えて２４℃で培養
し、ｐＨが本菌株の生育に与える影響を調べた。結果を
表３に示す。From these results, the growth limit temperature of this strain is 50
It is estimated to be ° C. (b-2) Proliferation pH Using a Czapek medium, the initial pH was changed and the cells were cultured at 24 ° C. to examine the effect of pH on the growth of this strain. Table 3 shows the results.
【００２１】[0021]
【表３】 [Table 3]
【００２２】(b-3) 各種炭水化物と増殖の適否 本菌株を２４℃で生育させることにより本菌株の各種糖
質に対する資化性を調べた。各種炭水化物を添加したポ
テト寒天培地における増殖の適否を表４に、各種炭水化
物を添加したツァペック培地における発酵によるガス発
生の有無を表５に示す。(B-3) Appropriateness of Propagation with Various Carbohydrates This strain was grown at 24 ° C. to examine the assimilation ability of this strain for various sugars. Appropriateness of growth in potato agar medium supplemented with various carbohydrates is shown in Table 4, and presence or absence of gas generation by fermentation in Czapek medium supplemented with various carbohydrates is shown in Table 5.
【００２３】[0023]
【表４】 [Table 4]
【００２４】[0024]
【表５】 [Table 5]
【００２５】［培養方法］乳酸の製造に際しては、ま
ず、前培養を行なって菌体量を増やし、しかる後、前記
の気泡塔型バイオリアクターに菌体を移して本培養を行
なう。 (1) 前培養Rhizopus 属の上記カビの胞子または菌糸は栄養源含有培
地に接種して好気的に増殖させることによって前培養す
る。栄養源としては、例えば、炭水化物、窒素源、無機
物等の通常使用される資化可能な栄養源を使用すること
ができる。炭素源としては、例えば澱粉、デキストリ
ン、グルコース、グリセリン等；窒素源としては、例え
ば、硫酸アンモニウム、硝酸ナトリウムなど；無機物と
しては、例えば、リン酸二水素カリウム、硫酸亜鉛、硫
酸マグネシウムなどの無機塩が使用できる。その他、Ｌ
（＋）−乳酸の生産を阻害しない有益な物質であれば、
公知のカビの培養において栄養源として使用されるいず
れの物質も使用できる。また、加熱滅菌時及び培養中に
おける発泡を抑えるため、シリコン、植物油等の消泡剤
を添加してもよい。[Culturing Method] In the production of lactic acid, first, pre-culture is carried out to increase the amount of bacterial cells, and then the bacterial cells are transferred to the bubble column type bioreactor to carry out main culture. (1) Preculture The above-mentioned fungi spores or mycelia of the genus Rhizopus are precultured by inoculating into a nutrient source-containing medium and aerobically growing. As the nutrient source, for example, a commonly used assimilable nutrient source such as a carbohydrate, a nitrogen source, and an inorganic substance can be used. Examples of the carbon source include starch, dextrin, glucose, glycerin, etc .; examples of the nitrogen source include ammonium sulfate, sodium nitrate and the like; and examples of the inorganic substance include inorganic salts such as potassium dihydrogen phosphate, zinc sulfate and magnesium sulfate. Can be used. Others, L
If it is a beneficial substance that does not inhibit the production of (+)-lactic acid,
Any substance used as a nutrient source in known mold cultures can be used. Further, in order to suppress foaming during heat sterilization and during culture, an antifoaming agent such as silicone or vegetable oil may be added.
【００２６】栄養源の配合割合は特に制約されるもので
はなく、広範囲にわたって変えることができる。使用す
る菌株にとって最適の栄養源の組成及び配合割合は簡単
な小規模実験により容易に決定することができる。栄養
培地は培養に先立ち滅菌後のｐＨが６〜７前後になるよ
うに苛性ソーダやアンモニアの水溶液を用いてｐＨを調
整するのが有利である。培養温度は菌株の増殖が実質的
に阻害されず乳酸を生産しうる範囲であれば特に制限さ
れるものでないが、一般に２０〜４０℃、好ましくは３
５〜４０℃の範囲内の温度が好適である。前培養では、
通常、本培養での培養液量に対して１０％程度の前培養
液を準備する。The mixing ratio of the nutrient source is not particularly limited and can be varied over a wide range. The optimum nutritional source composition and formulation for the strain used can be readily determined by simple small scale experiments. Prior to culturing, it is advantageous to adjust the pH of the nutrient medium by using an aqueous solution of caustic soda or ammonia so that the pH after sterilization will be around 6 to 7. The culture temperature is not particularly limited as long as it can produce lactic acid without substantially inhibiting the growth of the strain, but is generally 20 to 40 ° C., preferably 3
Temperatures within the range of 5-40 ° C are preferred. In preculture,
Usually, about 10% of the preculture liquid is prepared with respect to the amount of the culture liquid in the main culture.
【００２７】(2) 本培養 前培養で得られた菌体は、前記の気泡塔型リアクターに
て本培養され、乳酸を生産する。培養液中の乳酸は、例
えば真空蒸留法あるいはメチルエステル法等の常法によ
り単離し、精製される。本培養における培養液は、基本
的には前培養における栄養培地と同様の成分を含む水溶
液が用いられる。特に、前記 Rhizopus oryzae NRRL395
株を用いる場合は、炭素源として澱粉が使用可能であ
る。澱粉はジャガイモ、サツマイモ、トウモロコシ、コ
ムギ、タピオカ等の既知のいずれの原料から得られるも
のでもよい。澱粉あるいはその他の炭素源を５〜２０重
量％程度含む培養液が好ましい。５重量％未満では生産
効率が悪い。菌体量は、培養液中0.5 〜５重量％程度に
保つことが好ましい。0.5 重量％未満では乳酸生産速度
が低下し、５重量％を超えると対炭素源収率が著しく低
下する。(2) Main culture The microbial cells obtained in the pre-culture are main-cultured in the bubble column reactor to produce lactic acid. Lactic acid in the culture solution is isolated and purified by a conventional method such as a vacuum distillation method or a methyl ester method. As the culture medium in the main culture, an aqueous solution containing basically the same components as the nutrient medium in the preculture is used. In particular, said Rhizopus oryzae NRRL395
When using a strain, starch can be used as a carbon source. The starch may be obtained from any known raw material such as potato, sweet potato, corn, wheat and tapioca. A culture solution containing about 5 to 20% by weight of starch or another carbon source is preferable. If it is less than 5% by weight, the production efficiency is poor. It is preferable to keep the amount of cells in the culture solution at about 0.5 to 5% by weight. If it is less than 0.5% by weight, the production rate of lactic acid is reduced, and if it exceeds 5% by weight, the yield of carbon source is significantly reduced.
【００２８】本培養における空気の吹込み量は培養液量
に対する１分間当たりの容積量（ｖｖｍ）で0.125 〜0.
25の範囲が好ましい。0.125 ｖｖｍ未満では生産効率が
悪い。0.25ｖｖｍを超えると発泡が激しくなり、消泡の
ために大量の消泡剤の添加が必要となり、結果的に乳酸
の回収精製が困難になる。空気はエアスパージャに送る
前に例えば蒸気等を用いて加熱殺菌する。培養の温度条
件は前培養の場合と同様である。ｐＨは好ましくは５〜
８に維持されればよい。この目的のため、培地の滅菌前
に炭酸カルシウムを添加するかあるいは培養中にｐＨセ
ンサー及びｐＨコントローラーを用いて苛性ソーダの水
溶液あるいはアンモニア水などで自動的に適切なｐＨの
値に維持することが望ましい。The amount of air blown in the main culture is 0.125 to 0.10 in terms of volume (vvm) per minute with respect to the amount of culture solution.
A range of 25 is preferred. If it is less than 0.125 vvm, the production efficiency is poor. If it exceeds 0.25 vvm, foaming becomes severe and a large amount of antifoaming agent needs to be added for defoaming, resulting in difficulty in recovery and purification of lactic acid. The air is heat-sterilized using, for example, steam before being sent to the air sparger. The temperature conditions for the culture are the same as those for the preculture. The pH is preferably 5
It should be maintained at 8. For this purpose, it is desirable to add calcium carbonate before sterilization of the medium, or to automatically maintain an appropriate pH value with an aqueous solution of caustic soda or aqueous ammonia by using a pH sensor and a pH controller during the culture. .
【００２９】培養は回分方式、連続方式、反復回分方式
のいずれの態様によるものでもよいが、連続方式もしく
は反復回分方式またはこれらの方式を組み合わせた方法
が好ましい。ここで、回分（バッチ）方式とは、本培養
１回ごとに培養液全量の装入と抜出しを行なうものであ
る。連続方式は、本培養を行ないつつ、培養液の抜取り
と補充を行なうものである。培養条件にもよるが、培養
液の希釈率は、通常、0.1 〜1.0 （１／日）程度とする
のが好ましい。間欠的に抜取り及び／または補充を行な
ってもよい。連続方式では、菌体を取り出さずに乳酸の
生産を続けて行くため効率的であるが、培養液の抜き取
りの際に相当量の菌体が液に含まれて系外に出てしまう
ため、本培養開始当初から比較的高い濃度で菌体が培養
液中に存在している必要がある。前述の通り、本培養に
対する前培養率は１０％程度であり、フラスコ（１リッ
トル（以下Ｌと略記））内から立ち上げた場合、例えば
１００ＫＬの本培養に至るまでに、理論上１Ｌ、１０
Ｌ、１００Ｌ、１ΚＬ、１０ΚＬの前培養槽が必要とな
る。このため、労務費等の作業経費並びに装置コストが
膨大となり、製品のコスト増となる。The culture may be performed in any of a batch system, a continuous system and a repetitive batch system, but a continuous system, a repetitive batch system or a combination of these systems is preferable. Here, the batch system means that the whole amount of the culture solution is charged and withdrawn for each main culture. In the continuous method, the main culture is performed while the culture solution is withdrawn and supplemented. Although it depends on the culture conditions, the dilution ratio of the culture solution is usually preferably about 0.1 to 1.0 (1 / day). You may intermittently withdraw and / or replenish. The continuous method is efficient because it continues to produce lactic acid without removing the cells, but when extracting the culture solution, a considerable amount of cells are contained in the solution and go out of the system. From the beginning of the main culture, the bacterial cells must be present in the culture medium at a relatively high concentration. As described above, the pre-culture rate for the main culture is about 10%, and when it is started up from the inside of a flask (1 liter (hereinafter abbreviated as L)), for example, theoretically, 1 L, 10
L, 100L, 1K L, 10K L pre-incubator is required. Therefore, labor costs such as labor costs and device costs become enormous, resulting in an increase in product costs.
【００３０】本発明者らはこの問題を解決するため培養
液の一部を前培養液として再利用することにより前培養
操作を省略乃至軽減する方法（本明細書において「反復
回分方式」という。）を開発した。反復回分方式では、
培養液内の乳酸濃度や排ガス中のＣＯ2 濃度などを監視
しこれらの値が一定水準に達した後、空気の吹込みを停
止する。空気吹込みを停止すると上下の循環流が停止す
るため、培養液中の菌体が重力に従って沈降する。前記
の通り、反復回分方式用の反応槽には上下方向にずれた
位置に複数の抜出口ａ1 ，ａ2 ……ａn を設けるが、菌
体の沈降に応じ上方の抜出口から順次培養液を抜出して
いくことにより、実質的に菌体を含まない上澄液のみを
速やかに反応槽外に抜き出すことができる。培養液の全
量の１０〜９０％を抜き出し、抜出量に相当する量の培
養液を補充し、再び培養を繰り返すことにより培養を長
期間にわたって反復継続することができる。反復回分方
式では菌体が殆ど排出されないため、本培養当初の菌体
濃度が比較的低い水準であってもよく、前培養の培養量
を本培養の培養液量に対して１／１０以下に抑えること
が可能である。この結果、前培養における作業経費及び
装置コストを抑えてプロセス全体の製造コストを低減す
ることができる。In order to solve this problem, the present inventors have proposed a method of reusing a part of the culture solution as a preculture solution to omit or reduce the preculture operation (referred to as "repetitive batch method" in the present specification). ) Was developed. In the iterative batch method,
The lactic acid concentration in the culture solution and the CO 2 concentration in the exhaust gas are monitored, and after these values reach a certain level, the blowing of air is stopped. When the air blowing is stopped, the upper and lower circulation flows are stopped, so that the bacterial cells in the culture solution settle down due to gravity. As described above, a plurality of outlets a 1 , a 2 ... a n are provided at vertically displaced positions in the reaction tank for the repeated batch method, but the upper outlets are used for culturing sequentially according to the settling of bacterial cells. By withdrawing the liquid, only the supernatant liquid containing substantially no bacterial cells can be quickly withdrawn to the outside of the reaction tank. The culture can be repeated for a long period of time by extracting 10 to 90% of the total amount of the culture solution, supplementing the amount of the culture solution corresponding to the extracted amount, and repeating the culture again. Since the bacterial cells are hardly discharged in the repeated batch method, the bacterial cell concentration at the beginning of the main culture may be at a relatively low level, and the culture amount of the pre-culture should be 1/10 or less of the culture liquid volume of the main culture. It is possible to suppress. As a result, it is possible to reduce the work cost and the apparatus cost in the preculture and reduce the manufacturing cost of the entire process.
【００３１】[0031]
【実施例】以下実施例をもって本発明の実施の態様を説
明するが、これらは単なる例示であって本発明を何等制
限するものではない。実施例１ ：Ｌ（＋）−乳酸の回分発酵生産（気泡塔型バ
イオリアクター） 加熱滅菌した５００ｍｌ容三角フラスコ内に表６に示す
前培養培地１００ｍｌを入れ、乳酸生産能を有するRhiz
opus oryzae NRRL395 株のスラントをかきとって接種し
た（胞子濃度：１０7 個／ｍＬ）。これを３７℃でロー
タリーシェカー（回転数１７０ｒｐｍ，回転半径２ｃ
ｍ）で１５時間培養した。次いで、表６に示す生産培地
1.5 Ｌを入れた実容量 2.5Ｌ気泡塔型バイオリアクター
に、前記前培養液（菌体を含む。）を培地量の１０％
（ｖ／ｖ）になるように添加した。通気量は0.5 ｖｖ
ｍ、培養温度３５℃で通気のみの培養を行った。培養開
始後２４時間後にｐΗが急激に低下したので炭酸カルシ
ウムを添加し調整した。培養経過を図３に示す。４８時
間で８８ｇ／Ｌの乳酸を蓄積し、最終的には７２時間で
９２ｇ／Ｌ（１時間当たり1.27ｇ／Ｌ）の乳酸が得られ
た。仕込み澱粉（グルコースとして）あたりの収率は７
６％であった。The embodiments of the present invention will be described with reference to the following examples, but these are merely examples and do not limit the present invention in any way. Example 1 : Batch fermentation production of L (+)-lactic acid (bubble column bioreactor) In a heat-sterilized 500 ml Erlenmeyer flask, 100 ml of the preculture medium shown in Table 6 was put, and Rhiz capable of producing lactic acid was introduced.
A slant of opus oryzae NRRL395 strain was scraped off and inoculated (spore concentration: 10 7 cells / mL). Rotary shaker (rotation speed 170 rpm, rotation radius 2 c
M) was cultured for 15 hours. Then, the production medium shown in Table 6
10% of the amount of the culture medium containing the preculture liquid (including the bacterial cells) was placed in a 2.5 L bubble column type bioreactor containing 1.5 L.
(V / v) was added. Aeration rate is 0.5 vv
The culture was carried out only with aeration at a culture temperature of 35 ° C. Since 24 hours after the start of the culture, the pH decreased sharply, calcium carbonate was added to adjust. The progress of culture is shown in FIG. 88 g / L lactic acid was accumulated in 48 hours, and finally 92 g / L lactic acid was obtained in 72 hours (1.27 g / L per hour). Yield per charged starch (as glucose) is 7
It was 6%.
【００３２】[0032]
【表６】 [Table 6]
【００３３】比較例１：Ｌ（＋）−乳酸の回分発酵生産
（通気撹拌型バイオリアクター） 実施例１と同一条件下にRhizopus oryzae NRRL395 株の
前培養を行ない、表１に示した生産培地 1.5 Ｌを入れ
た 2.5 Ｌジャーファーメンター（丸菱バイオエンジ
（株）、東京）に前記前培養液（菌体を含む。）を培地
量の１０％（ｖ／ｖ）になるように添加した。回転数３
００ｒｐｍ、通気量 0.5ｖｖｍ、培養温度３５℃で通気
撹拌培養した。培養開始後２４時間目にｐΗを調整する
ために１００ｇの炭酸カルシウムを添加した。発酵経過
を図４に示す。前培養液を接種後２４時間目ぐらいから
糖の活発な消費が始まるのと平行して乳酸の生産が始ま
ったが、同時に培養液表面に大きな菌糸塊が形成されは
じめ、その後まもなく培養液中には菌体がほとんど見ら
れなくなった。この時点より澱粉の消費及び乳酸の生産
が緩慢となり、結果的に澱粉が完全に消失するまで１２
０時間かかった。最終的には８２ｇ／Ｌ（１時間当たり
0.68ｇ／Ｌ）の乳酸を生産した。仕込み澱粉（グルコー
スとして）あたりの乳酸の収率は６８％であった。 Comparative Example 1 : Batch fermentation production of L (+)-lactic acid (aeration-stirring bioreactor) Rhizopus oryzae NRRL395 strain was pre-cultured under the same conditions as in Example 1, and the production medium 1.5 shown in Table 1 was used. The pre-culture liquid (including the bacterial cells) was added to 2.5 L jar fermenter containing L (Maruhishi Bioengineering Co., Ltd., Tokyo) so as to be 10% (v / v) of the medium amount. Number of revolutions 3
The culture was performed with aeration and stirring at 00 rpm, an aeration amount of 0.5 vvm, and a culture temperature of 35 ° C. Twenty-four hours after the start of the culture, 100 g of calcium carbonate was added to adjust the pΗ. The fermentation process is shown in FIG. The production of lactic acid started in parallel with the active consumption of sugar about 24 hours after inoculation of the preculture liquid, but at the same time, a large mycelial mass began to form on the surface of the culture liquid, and shortly thereafter Almost no bacterial cells were seen. From this point, the consumption of starch and the production of lactic acid became sluggish, resulting in the complete disappearance of starch.
It took 0 hours. Eventually 82g / L (per hour
0.68 g / L) lactic acid was produced. The yield of lactic acid per charged starch (as glucose) was 68%.
【００３４】実施例２〜３ 通気量を0.125 及び0.25ｖｖｍと変えた他は実施例１と
同様の方法により乳酸発酵を行ない、当該気泡塔型バイ
オリアクターにおける最適通気量の検討を行なった。そ
の結果を窒素ガスを0.25ｖｖｍ吹き込んだ対照実験の結
果と共に表７に示す。 Examples 2 to 3 Lactic acid fermentation was carried out in the same manner as in Example 1 except that the aeration amount was changed to 0.125 and 0.25 vvm, and the optimum aeration amount in the bubble column type bioreactor was examined. The results are shown in Table 7 together with the result of the control experiment in which 0.25 vvm of nitrogen gas was blown.
【００３５】[0035]
【表７】 [Table 7]
【００３６】通気量が0.25ｖｖｍで最も高い乳酸の蓄積
量が得られ、消費した澱粉に対する収率も８５％を超
え、グルコースを原料とする発酵例を含め、これまで報
告されている中では最も高い収率で乳酸が得られた。−
般に、気泡塔型バイオリアクターを用いて微生物の培養
を行なう場合、スケールアップには通気量が最大の障害
となっている。つまり、通気量を大きくすると、送気の
ための空気圧縮機の建設コストやランニングコストが膨
大になると同時に発泡の制御が困難となる。この結果、
スケールアップが通常の通気撹拌槽に比較して容易であ
るという気泡塔型バイオリアクターのメリットが相殺さ
れる。本発明では、0.5 ｖｖｍ以下の少ない通気量で乳
酸の高収率生産が実現でき、気泡塔型バイオリアクター
を用いた乳酸製造の実用性が確認できた。The highest amount of lactic acid accumulated was obtained at an aeration rate of 0.25 vvm, and the yield based on the consumed starch exceeded 85%, and was the most reported so far including fermentation examples using glucose as a raw material. Lactic acid was obtained in high yield. −
Generally, when culturing microorganisms using a bubble column bioreactor, the aeration amount is the biggest obstacle to scale-up. That is, when the air flow rate is increased, the construction cost and running cost of the air compressor for air supply become enormous, and it becomes difficult to control foaming. As a result,
The merit of the bubble column type bioreactor that the scale-up is easier than that of an ordinary aeration and stirring tank is offset. In the present invention, high yield production of lactic acid can be realized with a small air flow rate of 0.5 vvm or less, and the practicality of lactic acid production using a bubble column bioreactor was confirmed.
【００３７】実施例４：乳酸の反復回分発酵生産（気泡
塔型バイオリアクター） 通気量を0.25ｖｖｍ、初発仕込量を2.3 Ｌとした以外は
すべて実施例１と同じ条件で気泡塔型バイオリアクター
（図１）による培養を開始した。発酵排ガス中の炭酸ガ
ス濃度をモニタリングし、ｌ％以下に下がった時点（培
養開始後４日目）を発酵終了とみなし、エアスパージャ
ーからの空気の吹込みを停止した。空気の吹込みを停止
すると菌体が沈降し始めるので、抜出口ａ1 ，ａ2 ，ａ
3，ａ4 から順次培養液を抜き出し（抜出量の合計：2.0
Ｌ）、抜出量と等量の培地を新たに補充し、再び通気
を行って培養を再開した。 Example 4 : Repeated batch fermentation production of lactic acid (bubble column type bioreactor) A bubble column type bioreactor under the same conditions as in Example 1 except that the aeration rate was 0.25 vvm and the initial charge was 2.3 L ( The cultivation according to FIG. 1) was started. The carbon dioxide concentration in the fermentation flue gas was monitored, and when it fell below 1% (4th day after the start of culture), it was considered that the fermentation had ended, and the blowing of air from the air sparger was stopped. When the blowing of air is stopped, the bacteria begin to settle, so the outlets a 1 , a 2 , a
The culture solution is sequentially withdrawn from 3 and a 4 (total withdrawal amount: 2.0
L), the medium was newly replenished with an amount equal to the amount taken out, and aeration was performed again to restart the culture.
【００３８】この反復回分培養を全部で１１回繰り返し
た。結果を図５に示す。ｌ回の回分培養に必要な日数は
回を経るにつれ短くなり、図示するように２〜４回目ま
ででは各２日間、５回目以降は各１日で培養が終了し
た。結局２３日間で１１回の反復回分培養が可能とな
り、全体として約５０ｇ／Ｌ／日（2.08ｇ／Ｌ／時間）
の乳酸を生産することができた。通常の回分培養では発
酵に3.5 日、リアクターの洗浄その他に 0.5日かかると
して２３日では約６回の回分生産が限界である。これに
対し、本発明による反復回分培養の生産性は既存の方法
の概ね２倍である。また、反復回分培養では前培養が必
要なのは初回だけであり、毎回前培養液を調製しなけれ
ばならない通常の回分培養に比較するとこの点でも有利
である。また、単なる連続方式では培養液抜取りの際に
菌体が逸出するが、反復回分培養ではそのような問題も
ない。This repeated batch culture was repeated 11 times in total. Results are shown in FIG. The number of days required for 1 batch culture became shorter as the number of times passed, and as shown in the figure, the culture was completed in 2 days for each of the 2nd to 4th cultures and 1 day in each of the 5th culture and thereafter. Eventually, it became possible to perform 11 batch cultures in 23 days, and about 50 g / L / day (2.08 g / L / hour) as a whole
Was able to produce lactic acid. Fermentation takes 3.5 days in normal batch culture, and 0.5 days for cleaning the reactor, etc., and batch production is limited to about 6 times in 23 days. In contrast, the productivity of the repeated batch culture according to the present invention is almost twice that of the existing method. Further, in the repeated batch culture, the pre-culture is required only for the first time, which is also advantageous in this respect as compared with the normal batch culture in which the pre-culture liquid has to be prepared each time. In addition, although the bacterial cells escape when the culture solution is drawn out in the simple continuous method, such a problem does not occur in the repeated batch culture.
【００３９】実施例５：乳酸の連続発酵生産（気泡塔型
バイオリアクター） 実施例４と同じ方法で気泡塔型バイオリアクターによる
回分培養を行なった。培養開始後３日目に排気ガス中の
炭酸ガス濃度が１％以下になっていることを確認して空
気吹込みを停止し、生成した菌体を塔底部に沈降させ
た。ついで培養液2.3Ｌを抜き出し、新たに生産培地 2.
3Ｌを入れて再び通気を始めて培養を再開した。この操
作を初めの回分発酵も含めて３回繰り返し、菌糸体濃度
を充分に高めた後、ｌ日当たり 2.5Ｌの割合で生産培地
を連続的に供給し、また塔上部より菌糸体を含まない液
を供給培地と等量引き抜いた。発酵の経過図を図６に示
す。最初の回分も含めてｌ箇月の連続運転が可能であっ
た。連続培養操作に切り替えてから４日後には定常状態
に到達し、排出液中乳酸濃度は８６〜８８ｇ／Ｌであっ
た。この時の乳酸の生産性は８６〜８７ｇ／Ｌ／日（3.
58〜3.62ｇ／Ｌ／時間）となり実施例１で示した回分培
養、実施例５に示した反復回分培養に比較してより高い
生産性を示した。 Example 5 : Continuous fermentation production of lactic acid (bubble column bioreactor) In the same manner as in Example 4, batch culture was carried out using a bubble column bioreactor. On the third day after the start of the culture, it was confirmed that the carbon dioxide concentration in the exhaust gas was 1% or less, air blowing was stopped, and the produced bacterial cells were allowed to settle to the bottom of the tower. Then, 2.3 L of the culture solution was drawn out and a new production medium was prepared 2.
After adding 3 L, aeration was started again to restart the culture. This operation is repeated 3 times including the first batch fermentation, the mycelium concentration is sufficiently increased, and then the production medium is continuously supplied at a rate of 2.5 L per 1 day, and the mycelium-free liquid is supplied from the upper part of the tower. Was withdrawn in an amount equal to the supply medium. FIG. 6 shows a fermentation process chart. It was possible to continuously operate for 1 month including the first batch. Four days after switching to the continuous culture operation, the steady state was reached, and the lactic acid concentration in the effluent was 86 to 88 g / L. The productivity of lactic acid at this time is 86 to 87 g / L / day (3.
58 to 3.62 g / L / hour), and higher productivity was exhibited as compared with the batch culture shown in Example 1 and the repeated batch culture shown in Example 5.
【００４０】[0040]
【発明の効果】本発明は、特定構造の気泡塔型バイオリ
アクターを用いてRhizopus属のカビを培養することによ
り、乳酸を効率的かつ大量に生産することを可能にした
ものである。また、特定の菌株を用いることにより、従
来の乳酸発酵では困難であった澱粉からの乳酸製造が可
能となり、原料コストを大きく低減することに成功し
た。さらに、本発明によれば反復回分培養方式あるいは
これと連続方式とを組み合わせた培養方式を採用するこ
とにより、前培養操作が省略乃至軽減され効率的な乳酸
製造ができる。INDUSTRIAL APPLICABILITY The present invention enables efficient and large-scale production of lactic acid by culturing Rhizopus mold using a bubble column bioreactor having a specific structure. In addition, by using a specific strain, it became possible to produce lactic acid from starch, which was difficult with conventional lactic acid fermentation, and succeeded in greatly reducing raw material costs. Furthermore, according to the present invention, by adopting a repeated batch culture method or a culture method combining this with a continuous method, the preculture operation can be omitted or reduced, and efficient lactic acid production can be performed.
【図１】 本発明の気泡塔型バイオリアクターの構造の
概略を示す模式的断面図である。FIG. 1 is a schematic cross-sectional view showing the outline of the structure of a bubble column bioreactor of the present invention.
【図２】 従来の気泡塔型バイオリアクターの構造を示
す模式的断面図である。FIG. 2 is a schematic cross-sectional view showing the structure of a conventional bubble column type bioreactor.
【図３】 気泡塔型バイオリアクターでの回分発酵経過
を示すグラフである。FIG. 3 is a graph showing the progress of batch fermentation in a bubble column bioreactor.
【図４】 ジャーファーメンターでの回分発酵経過を示
すグラフである。FIG. 4 is a graph showing the progress of batch fermentation in a jar fermenter.
【図５】 気泡塔型バイオリアクターでの反復回分発酵
経過を示すグラフである。FIG. 5 is a graph showing the process of repeated batch fermentation in a bubble column bioreactor.
【図６】 気泡塔型バイオリアクターでの連続発酵経過
を示すグラフである。FIG. 6 is a graph showing a continuous fermentation process in a bubble column bioreactor.
ｌ 気泡塔型バイオリアクター ２ スパージヤー ３ ドラフト管 ４ 培養液抜き口 ５ ジャケット ６ 温水（冷却水）入口 ７ 温水（冷却水）出口 ８ 排気管 ９ 圧縮空気（酸素／圧縮空気混合ガス）入口 l Bubble column bioreactor 2 Sparger 3 Draft tube 4 Culture solution outlet 5 Jacket 6 Hot water (cooling water) inlet 7 Hot water (cooling water) outlet 8 Exhaust pipe 9 Compressed air (oxygen / compressed air mixed gas) inlet
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｃ１２Ｒ 1:845） Ｃ０７Ｍ 7:00 ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 6 Identification code Internal reference number FI technical display area C12R 1: 845) C07M 7:00
Claims (10)
リアクターを使用し、炭素源として澱粉もしくはその糖
化液またはグルコースを含有する水性培地でリゾプス
（Rhizopus）属に属する菌を培養することを特徴とする
Ｌ（＋）−乳酸の製造法。1. A bacterium belonging to the genus Rhizopus is cultivated in an aqueous medium containing starch or its saccharified solution or glucose as a carbon source using a bubble column type bioreactor in which air is blown from the bottom. The method for producing L (+)-lactic acid.
の壁面部と逆錐状の底面部と排気管を備えた蓋部とから
なる容器本体、容器本体内に容器壁面に概ね平行に固設
されたドラフトチューブ、前記逆錐状の底面部とドラフ
トチューブの下端との間に設けられたエアスパージャ
ー、容器外部からエアスパージャーに気体を送る通気
管、容器外壁面を覆う温度調節可能なジャケット、及び
容器本体に連通する少なくとも１本の通液管を有し、前
記底面部の逆錐の母線と鉛直線とのなす傾斜角θが２０
〜４０°であり、容器本体内径に対するドラフトチュー
ブ内径の比が0.5 乃至0.7 の範囲にあるリアクターを使
用する請求項１に記載のＬ（＋）−乳酸の製造法。2. A bubble column type bioreactor, comprising a container main body comprising a cylindrical wall surface portion, an inverted cone-shaped bottom surface portion and a lid portion provided with an exhaust pipe, and fixed in the container main body substantially parallel to the wall surface of the container. Draft tube, an air sparger provided between the inverted pyramid-shaped bottom portion and the lower end of the draft tube, a ventilation pipe for sending gas from the outside of the container to the air sparger, and a temperature control for covering the outer wall surface of the container It has at least one liquid passage communicating with the jacket and the container body, and the inclination angle θ formed by the vertical pyramid and the generatrix of the inverted cone on the bottom surface is 20.
The process for producing L (+)-lactic acid according to claim 1, wherein a reactor having a ratio of the inner diameter of the draft tube to the inner diameter of the container body of -40 ° is in the range of 0.5 to 0.7.
る請求項１または２に記載のＬ（＋）−乳酸の製造法。3. The method for producing L (+)-lactic acid according to claim 1, wherein the amount of air blown is 0.125 to 0.25 vvm.
opus oryzae ）である請求項１乃至３のいずれかの項に
記載のＬ（＋）−乳酸の製造法。4. A bacterium to be cultivated is Rhizopus oryzae ( Rhiz
opus oryzae ), The method for producing L (+)-lactic acid according to any one of claims 1 to 3.
ＲＬ３９５株（Rhizopus oryzae NRRL395)である請求項
４に記載のＬ（＋）−乳酸の製造法。5. The strain to be cultured is Rhizopus oryzae NR.
The method for producing L (+)-lactic acid according to claim 4, which is RL395 strain ( Rhizopus oryzae NRRL395).
ずれかに記載のＬ（＋）−乳酸の製造法。6. The method for producing L (+)-lactic acid according to claim 2, wherein the carbon source is starch.
込みを停止して培養液中の菌体を沈降させて前記通液管
から上澄液のみを取り出し、取り出した液量に相当する
量の培養液を補充した後、空気吹き込みを再開して培養
を繰り返す請求項１乃至６のいずれかの項に記載のＬ
（＋）−乳酸の製造法。7. After culturing for a predetermined period of time, the blowing of air is stopped, the bacterial cells in the culture solution are allowed to settle, and only the supernatant liquid is taken out from the liquid passage, and the amount corresponding to the taken out liquid amount. 7. The L according to any one of claims 1 to 6, wherein after the culture solution is replenished, the air blowing is restarted and the culture is repeated.
A method for producing (+)-lactic acid.
なう請求項１乃至６のいずれかの項に記載のＬ（＋）−
乳酸の製造法。8. The L (+)-of claim 1, wherein the withdrawal and supplementation of the culture solution are performed continuously.
Method for producing lactic acid.
までは請求項７に記載の方法によって菌の培養を行な
い、菌体量が所定量に達した後は請求項８に記載の方法
によって菌の培養を行なう請求項１乃至６のいずれかの
項に記載のＬ（＋）−乳酸の製造法。9. The method according to claim 7, wherein the microorganism is cultured by the method according to claim 7 until the amount of bacterial cells in the reactor reaches a predetermined amount, and after the amount of bacterial cells reaches the predetermined amount, the method according to claim 8. The method for producing L (+)-lactic acid according to any one of claims 1 to 6, wherein the bacterium is cultured by the method.
管を備えた蓋部からなる容器本体、容器本体内に容器壁
面に概ね平行に固設されたドラフトチューブ、前記逆錐
状の底面部とドラフトチューブの下端との間に設けられ
たエアスパージャー、容器外部からエアスパージャーに
気体を送る通気管、容器外壁面を覆う温度調節可能なジ
ャケット、及び容器本体に連通する少なくとも１本の通
液管を有し、前記底面部の逆錐の母線と鉛直線とのなす
傾斜角θが２０〜４０°であり、容器本体内径に対する
ドラフトチューブ内径の比が0.5 乃至0.7 の範囲にある
気泡塔型バイオリアクター。10. A container body comprising a cylindrical wall surface portion, a reverse cone-shaped bottom surface portion, and a lid portion provided with an exhaust pipe, a draft tube fixed substantially parallel to the container wall surface in the container body, and the reverse cone. An air sparger provided between the bottom surface of the container and the lower end of the draft tube, a ventilation pipe for sending gas from the outside of the container to the air sparger, a temperature-adjustable jacket covering the outer wall surface of the container, and the container body. It has at least one liquid passage, the inclination angle θ between the generatrix of the inverted cone of the bottom surface and the vertical line is 20 to 40 °, and the ratio of the inner diameter of the draft tube to the inner diameter of the container body is 0.5 to 0.7. Bubble tower bioreactor in range.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP35114095A JPH09173090A (en) | 1995-12-27 | 1995-12-27 | Production of l (+)-lactic acid and manufacturing equipment therefor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP35114095A JPH09173090A (en) | 1995-12-27 | 1995-12-27 | Production of l (+)-lactic acid and manufacturing equipment therefor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH09173090A true JPH09173090A (en) | 1997-07-08 |
Family
ID=18415323
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP35114095A Pending JPH09173090A (en) | 1995-12-27 | 1995-12-27 | Production of l (+)-lactic acid and manufacturing equipment therefor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH09173090A (en) |
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| WO2017135316A1 (en) * | 2016-02-04 | 2017-08-10 | 花王株式会社 | Mutant of genus rhizopus |
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| JP5535619B2 (en) * | 2007-05-21 | 2014-07-02 | 株式会社明治 | Natural cheese manufacturing method |
| CN102198390A (en) * | 2010-03-26 | 2011-09-28 | 中国石油天然气股份有限公司 | Inner circulating type ethene polymerization reaction vessel for slurry process |
| WO2012008589A1 (en) * | 2010-07-16 | 2012-01-19 | 国立大学法人九州大学 | Method for producing l-lactose by means of lactobacillus under presence of pentose and cello-oligosaccharide |
| JP2013165719A (en) * | 2010-07-16 | 2013-08-29 | Sumitomo Corp | Method for producing l-lactic acid by lactic acid bacterium under presence of pentose cellooligosaccharides |
| JP5307295B2 (en) * | 2010-07-16 | 2013-10-02 | 住友商事株式会社 | Method for producing L-lactic acid by lactic acid bacteria in the presence of pentose and cellooligosaccharide |
| US9234219B2 (en) | 2010-07-16 | 2016-01-12 | Kyushu University, National University Corporation | Method for producing L-lactic acid by lactic acid bacterium under presence of pentose and cellooligosaccharides |
| JP2015136310A (en) * | 2014-01-21 | 2015-07-30 | 株式会社アウレオ | METHOD FOR PRODUCING β-GLUCAN |
| WO2017135316A1 (en) * | 2016-02-04 | 2017-08-10 | 花王株式会社 | Mutant of genus rhizopus |
| JPWO2017135316A1 (en) * | 2016-02-04 | 2018-11-29 | 花王株式会社 | Mutant Rhizopus sp |
| US10947522B2 (en) | 2016-02-04 | 2021-03-16 | Kao Corporation | Mutant of genus Rhizopus |
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