JPH11169671A - Separation of bacterial cell - Google Patents
Separation of bacterial cellInfo
- Publication number
- JPH11169671A JPH11169671A JP34141597A JP34141597A JPH11169671A JP H11169671 A JPH11169671 A JP H11169671A JP 34141597 A JP34141597 A JP 34141597A JP 34141597 A JP34141597 A JP 34141597A JP H11169671 A JPH11169671 A JP H11169671A
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- Prior art keywords
- water
- membrane
- added
- liq
- soluble polymer
- 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.)
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- Separation Using Semi-Permeable Membranes (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は菌体分離方法に係
り、特に、膜濾過により発酵液から菌体を分離すると共
に目的有価物を回収する方法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for separating cells, and more particularly to a method for separating cells from a fermentation solution by membrane filtration and recovering a valuable resource.
【０００２】[0002]
【従来の技術】発酵法によって生産した酵素などの有価
物、例えば、リパーゼ、セルラーゼ、キシラーゼ等の酵
素や生理活性ペプチド、蛋白質などを製品化する場合、
生産菌と発酵生産物とを分離して有価物を回収する必要
がある。従来、発酵液からの目的有価物の回収及び菌体
分離のための一般的な方法として、珪藻土濾過法があ
る。珪藻土濾過法では、多量の珪藻土を濾過助剤又はプ
リコート剤として使用するため、珪藻土の混入した菌体
が分離される。この珪藻土の混入した菌体は焼却処理す
ることができず、投棄処分するため、処分場の問題があ
る。2. Description of the Related Art When commercializing valuable materials such as enzymes produced by a fermentation method, for example, enzymes such as lipase, cellulase and xylase, physiologically active peptides and proteins,
It is necessary to separate production bacteria and fermentation products to recover valuable resources. BACKGROUND ART Conventionally, there is a diatomaceous earth filtration method as a general method for recovering a target valuable substance from a fermentation liquid and separating cells. In the diatomaceous earth filtration method, since a large amount of diatomaceous earth is used as a filter aid or a pre-coating agent, bacterial cells mixed with diatomaceous earth are separated. The diatomaceous earth-mixed cells cannot be incinerated and are discarded, so there is a problem with the disposal site.
【０００３】このため、珪藻土を使用しない分離方法と
して、ＭＦ（精密濾過）膜又はＵＦ（限外濾過）膜を用
いた膜分離法が検討されている。For this reason, as a separation method without using diatomaceous earth, a membrane separation method using an MF (microfiltration) membrane or a UF (ultrafiltration) membrane has been studied.
【０００４】膜分離法による発酵液からの菌体分離と目
的有価物の回収は、発酵液の膜濾過（有価物の透過と菌
体の濃縮）とダイアフィルトレーション（加水処理によ
る、濃縮液側に残った有価物の透過液側への回収）とに
よって行われる。即ち、まず、発酵液を膜分離処理して
有価物を透過液側に回収すると共に菌体を濃縮し、菌体
の濃縮がある程度進んだ後に、濃縮液側に水を添加（加
水処理）して濃縮液側に残留する有価物の透過液側への
回収を促進する。[0004] Separation of the cells from the fermented liquor and recovery of the target valuables by the membrane separation method include membrane filtration of the fermented liquor (permeation of valuables and concentration of the cells) and diafiltration (concentration of the concentrated liquid by a water treatment). (Recovering valuables remaining on the permeate side). That is, first, the fermented liquor is subjected to membrane separation treatment, valuable resources are collected on the permeate side, and the cells are concentrated. After the concentration of the cells has progressed to some extent, water is added to the concentrate side (water treatment). To promote the recovery of valuables remaining on the concentrate side to the permeate side.
【０００５】このようなダイアフィルトレーションに必
要な加水量（Ｖ1）は、初期の濃縮液量（Ｖ0）、初期の
濃縮液側の有価物濃度（Ｃ0）、目標とする濃縮液中の
有価物濃度（Ｃ1）及び膜による有価物の阻止率（Ｒ）
から、次のように算出される。[0005] The amount of water (V 1 ) necessary for such diafiltration includes an initial concentrated liquid amount (V 0 ), an initial concentrated liquid concentration (C 0 ), and a target concentrated liquid. Concentration of valuables in the medium (C 1 ) and rejection of valuables by membrane (R)
Is calculated as follows.
【０００６】 Ｖ1＝−ｌｎ（Ｃ1／Ｃ0）×Ｖ0／（１−Ｒ／１００） 従って、目標とする濃縮液中の有価物濃度（Ｃ1）と濃
縮液量（Ｖ0）が決められている場合、ダイアフィルト
レーションに必要な加水量（Ｖ1）は初期の有価物濃度
（Ｃ0）が低いほど、また、膜による有価物の阻止率
（Ｒ）が小さいほど少なくて足りる。V 1 = −ln (C 1 / C 0 ) × V 0 / (1−R / 100) Therefore, the concentration of the valuable substance (C 1 ) in the target concentrate and the amount of the concentrate (V 0 ) Is determined, the amount of water (V 1 ) necessary for diafiltration decreases as the initial value of the valuable material (C 0 ) decreases and as the rejection (R) of the valuable material by the membrane decreases. Is enough.
【０００７】[0007]
【発明が解決しようとする課題】発酵液の膜分離処理で
は、膜の孔が菌体や発酵生産物などによって閉塞し、目
的有価物が膜を透過せずに阻止されてしまうことがあ
る。目的有価物が膜で阻止されると、濃縮液側で目的有
価物が過度に濃縮されることにより、目的有価物が変性
したり、濃縮液中から目的有価物を回収するためのダイ
アフィルトレーションに必要な加水量が大幅に増加する
などの問題が生じる。この加水量の増加は設備の大型
化、処理時間の増大を招く。In the membrane separation treatment of the fermentation solution, the pores of the membrane may be blocked by cells or fermentation products, and the target valuables may be blocked without passing through the membrane. When the target valuables are blocked by the membrane, the target valuables are excessively concentrated on the concentrate side, thereby denaturing the target valuables or diafiltration for recovering the target valuables from the concentrate. There is a problem that the amount of water required for the filtration is greatly increased. This increase in the amount of water causes an increase in the size of the equipment and an increase in the processing time.
【０００８】本発明は上記従来の問題点を解決し、膜濾
過により発酵液から菌体を分離すると共に目的有価物を
回収する方法において、膜による目的有価物の阻止を抑
制し、ダイアフィルトレーションに必要な加水量を低減
して目的有価物の回収効率を高めることができる菌体分
離方法を提供することを目的とする。The present invention solves the above-mentioned conventional problems and provides a method for separating bacterial cells from a fermentation liquor by membrane filtration and recovering the target valuables. It is an object of the present invention to provide a method for separating cells which can reduce the amount of water required for the filtration and increase the efficiency of collecting the target valuables.
【０００９】[0009]
【課題を解決するための手段】本発明の菌体分離方法
は、発酵液を膜分離処理して菌体を濃縮液側に分離する
と共に、目的有価物を透過液側に回収する方法におい
て、該発酵液に水溶性ポリマーを添加して膜分離処理し
た後、濃縮液側に水を加えて膜分離処理することを特徴
とする。The method of separating cells according to the present invention is a method for separating a cell into a concentrated solution by subjecting a fermentation solution to membrane separation, and recovering a target valuable substance to a permeated solution. It is characterized in that after the water-soluble polymer is added to the fermentation solution and the membrane separation treatment is performed, water is added to the concentrated solution side and the membrane separation treatment is performed.
【００１０】発酵液に水溶性ポリマーを添加することに
より、発酵液中のコロイド状濁質等が除去されて、膜孔
の閉塞が防止され、その結果、膜による目的有価物の阻
止も抑制され、目的有価物の回収のためのダイアフィル
トレーションに必要な加水量が低減され、効率的な膜分
離処理を行える。[0010] By adding a water-soluble polymer to the fermentation liquor, the colloidal turbidity and the like in the fermentation liquor are removed, and the pores are prevented from being clogged. In addition, the amount of water required for diafiltration for recovering the target valuables is reduced, and efficient membrane separation can be performed.
【００１１】[0011]
【発明の実施の形態】以下に本発明の実施の形態を詳細
に説明する。Embodiments of the present invention will be described below in detail.
【００１２】本発明において、発酵液に添加する水溶性
ポリマーとしては、カチオン性、アニオン性等の各種の
水溶性ポリマーを用いることができる。例えば、カチオ
ン性水溶性ポリマーとしては、ポリエチレンイミン、ポ
リビニルアミン、ポリビニルアミジン、ポリ（メタ）ア
リルアミン、ハロゲン化ポリジアリルアンモニウム、ポ
リアミノアルキルメタクリレート、キトサンなどを用い
ることができる。また、アニオン性水溶性ポリマーとし
ては、ポリアクリル酸ナトリウム、カルボキシメチルセ
ルロースなどを用いることができる。In the present invention, as the water-soluble polymer to be added to the fermentation solution, various water-soluble polymers such as cationic and anionic can be used. For example, as the cationic water-soluble polymer, polyethyleneimine, polyvinylamine, polyvinylamidine, poly (meth) allylamine, polydiallylammonium halide, polyaminoalkyl methacrylate, chitosan and the like can be used. In addition, as the anionic water-soluble polymer, sodium polyacrylate, carboxymethyl cellulose, or the like can be used.
【００１３】通常の場合、水溶性ポリマーは所定濃度の
水溶液として発酵液に添加するが、この場合において、
ポリマー濃度に特に制限はないが、発酵液量の増加を抑
制するためには、なるべく高濃度の水溶液として添加す
るのが望ましい。ポリマー濃度は０．５重量％以上、と
りわけ５重量％以上とするのが望ましい。水溶液の取り
扱い性とくに粘性等を考慮した場合、ポリマー濃度は特
に５〜１０重量％であることが望ましい。Usually, the water-soluble polymer is added to the fermentation broth as an aqueous solution of a predetermined concentration.
Although there is no particular limitation on the polymer concentration, it is desirable to add as a highly concentrated aqueous solution in order to suppress an increase in the amount of fermentation liquor. The polymer concentration is preferably 0.5% by weight or more, particularly preferably 5% by weight or more. In consideration of the handleability of the aqueous solution, particularly the viscosity, the polymer concentration is particularly preferably 5 to 10% by weight.
【００１４】なお、水溶性ポリマーの分子量には特に制
限はないが、ポリマー水溶液の濃度を高くするために
は、分子量が小さいほうが取り扱い上有利であり、通常
は分子量３０万以下のもの、好ましくは１万〜２０万の
ものが用いられる。The molecular weight of the water-soluble polymer is not particularly limited. However, in order to increase the concentration of the aqueous polymer solution, a smaller molecular weight is more advantageous in handling, and usually has a molecular weight of 300,000 or less, preferably Those with 10,000 to 200,000 are used.
【００１５】発酵液に対する水溶性ポリマーの好適添加
量は、処理対象の発酵液により異なるため、発酵液毎に
予備実験等により適宜決定される。即ち、少量の水溶性
ポリマーを発酵液に添加した場合、水溶性ポリマーは菌
体と反応して液中には残留しないが、添加量がある量以
上になると菌体と反応しきれない水溶性ポリマーが液中
に残留するようになる。水溶性ポリマーの適正添加量
は、通常は液中にポリマーが残留しはじめる添加量付近
であるが、発酵液の性状によっては、ポリマーが残留し
ないほうが良いものや、若干残留する程度が良いものも
ある。The preferred amount of the water-soluble polymer to be added to the fermentation liquid varies depending on the fermentation liquid to be treated, and is appropriately determined by a preliminary experiment or the like for each fermentation liquid. That is, when a small amount of a water-soluble polymer is added to a fermentation solution, the water-soluble polymer reacts with the cells and does not remain in the liquid, but when the amount exceeds a certain amount, the water-soluble polymer does not react with the cells. The polymer will remain in the liquid. The appropriate addition amount of the water-soluble polymer is usually around the addition amount at which the polymer starts to remain in the solution, but depending on the properties of the fermentation solution, there are those in which it is better that the polymer does not remain, and those in which the polymer slightly remains. is there.
【００１６】本発明において、水溶性ポリマーを添加し
た後の発酵液の膜分離方法には特に制限はない。膜とし
てはＭＦ又はＵＦ膜が用いられる。膜素材にも制限はな
く、ポリオレフィン膜、ポリスルホン膜、テフロン膜、
セラミック膜などが用いられる。ＭＦ又はＵＦ膜の選
択、及び膜素材の選択は、目的有価物の分子量や発酵液
の性状などを加味して行われる。膜型式にも特に制限は
ないが、一般的には中空糸、チューブラー、スパイラ
ル、プレート＆フレーム型膜モジュールなどが用いられ
る。In the present invention, the method for membrane separation of the fermentation liquor after adding the water-soluble polymer is not particularly limited. An MF or UF film is used as the film. There is no limitation on the membrane material, and polyolefin membrane, polysulfone membrane, Teflon membrane,
A ceramic film or the like is used. The selection of the MF or UF membrane and the selection of the membrane material are performed in consideration of the molecular weight of the target valuable material, the properties of the fermentation solution, and the like. Although there is no particular limitation on the membrane type, hollow fiber, tubular, spiral, plate & frame type membrane modules and the like are generally used.
【００１７】水溶性ポリマーを添加した発酵液の膜分離
処理は、例えば次のような操作で行われる。[0017] The membrane separation treatment of the fermented liquor to which the water-soluble polymer has been added is performed, for example, by the following operation.
【００１８】まず、水溶性ポリマーを添加した発酵液を
クロスフローで膜モジュールに供給し、目的有価物を含
む透過液を回収する。これに伴い、濃縮液側に菌体が濃
縮されていく。所定の倍率まで濃縮が進んだ後、濃縮液
側に残存する有価物を更に回収するために水を加えてダ
イアフィルトレーションを行う。この場合、ダイアフィ
ルトレーションに必要な加水量や加水方法は、濃縮液中
の目的有価物の濃度、膜による目的有価物の阻止率、及
び目標とする目的有価物回収率等によって、適宜選択さ
れる。なお、加水方法は、連続加水であっても間欠加水
であっても良く、また、バッチ毎の加水であっても良
い。本発明方法によれば、低加水量で回収率９０％以上
を達成することが可能である。First, the fermentation liquor to which the water-soluble polymer has been added is supplied to the membrane module by cross flow, and the permeate containing the target valuables is collected. Along with this, the cells are concentrated on the concentrate side. After the concentration has progressed to a predetermined magnification, water is added to further collect valuable resources remaining on the concentrated liquid side, and diafiltration is performed. In this case, the amount and method of water required for diafiltration are appropriately selected depending on the concentration of the target valuable material in the concentrate, the rejection of the target valuable material by the membrane, and the target recovery ratio of the target valuable material. Is done. The water addition method may be continuous water addition or intermittent water addition, or may be water addition for each batch. According to the method of the present invention, it is possible to achieve a recovery rate of 90% or more with a low amount of water.
【００１９】透過液側に回収された目的有価物は、その
種類に応じて、ＵＦ膜又はＲＯ（逆浸透）膜、或いはイ
オン交換樹脂、晶析等により濃縮精製され、製品とされ
る。The target valuables collected on the permeate side are concentrated and purified by a UF membrane or a RO (reverse osmosis) membrane, an ion exchange resin, crystallization, or the like, depending on the kind thereof, to obtain a product.
【００２０】このような本発明の菌体分離方法は、発酵
液からの菌体の分離及び、蛋白質やリパーゼ、セルラー
ゼ、キシラーゼなどの酵素、生理活性ペプチド等の目的
有価物の回収に工業的に極めて有用である。The method for separating bacterial cells according to the present invention is industrially useful for separating bacterial cells from fermentation broth and recovering valuable resources such as proteins, enzymes such as lipase, cellulase and xylase, and physiologically active peptides. Extremely useful.
【００２１】[0021]
【実施例】以下に実施例及び比較例を挙げて本発明をよ
り具体的に説明する。The present invention will be described more specifically below with reference to examples and comparative examples.
【００２２】実施例１ 乾燥菌体濃度２重量％、ｐＨ６．２、電気伝導度４．３
ｍＳ／ｃｍの目的蛋白質含有発酵液１０Ｌに、５重量％
キトサン（分子量１０万）水溶液０．１Ｌを加えて１分
間撹拌した。キトサン水溶液添加後の発酵液中の目的蛋
白質濃度は９．５７ｇ／Ｌであった。Example 1 Dry cell concentration 2% by weight, pH 6.2, electric conductivity 4.3
5% by weight in 10 L of fermentation liquor containing the target protein of mS / cm
0.1 L of an aqueous solution of chitosan (molecular weight 100,000) was added thereto, followed by stirring for 1 minute. The concentration of the target protein in the fermentation broth after the addition of the chitosan aqueous solution was 9.57 g / L.
【００２３】膜面積０．０５ｍ2のポリオレフィン平膜
を装着したセルを用いて、キトサン水溶液を添加した発
酵液のクロスフロー濾過を行った。発酵液は膜面流束２
ｍ／秒で循環し、濾過圧力は１．０ｋｇ／ｃｍ2とし
た。また、発酵液の温度は２５℃で一定になるようにし
た。Using a cell equipped with a polyolefin flat membrane having a membrane area of 0.05 m 2 , cross-flow filtration of the fermentation liquor to which the chitosan aqueous solution was added was performed. Fermentation liquor is membrane flux 2
Circulation was performed at m / sec, and the filtration pressure was 1.0 kg / cm 2 . The temperature of the fermentation liquor was kept constant at 25 ° C.
【００２４】透過液を１．０Ｌずつ分取し、９．０Ｌの
透過液を回収したところで、濃縮液側に１．０Ｌのイオ
ン交換水を加えた。更に透過液１．０Ｌを回収したとこ
ろで、濃縮液側に１．０Ｌのイオン交換水を加えた。そ
の後、初期の発酵液中に含まれている目的蛋白質の９９
％が透過液側に回収されるまで、この加水を繰り返し
た。The permeate was separated by 1.0 L, and when 9.0 L of the permeate was collected, 1.0 L of ion-exchanged water was added to the concentrate side. Further, when 1.0 L of the permeate was recovered, 1.0 L of ion-exchanged water was added to the concentrate side. Then, 99% of the target protein contained in the initial fermentation broth
This water addition was repeated until% was recovered on the permeate side.
【００２５】透過液分取毎の各透過液中の目的蛋白質濃
度及び濃縮液中の蛋白質濃度と、これらの値から算出し
た膜による目的蛋白質の阻止率及び目的蛋白質の回収
量，回収率を表１に示した。なお、蛋白質回収率は透過
液をプロテインアッセイ（ＢＩＯ−ＲＡＤ）で分析して
求めた。The concentration of the target protein in each permeate and the concentration of the protein in the concentrate for each fractionation of the permeate, the inhibition rate of the target protein by the membrane, the amount of the target protein recovered and the recovery rate calculated from these values are shown in the table. 1 is shown. The protein recovery was determined by analyzing the permeate with a protein assay (BIO-RAD).
【００２６】[0026]
【表１】 [Table 1]
【００２７】比較例１ 発酵液にキトサン水溶液を添加しなかったこと以外は、
実施例１と同様にして膜分離処理を行い、処理結果を表
２に示した。Comparative Example 1 Except that no chitosan aqueous solution was added to the fermentation broth,
A membrane separation treatment was performed in the same manner as in Example 1, and the treatment results are shown in Table 2.
【００２８】[0028]
【表２】 [Table 2]
【００２９】表１，２の結果から明らかなようにキトサ
ン水溶液を添加した実施例１では、膜の目的蛋白質阻止
率は低く、９９％以上の目的蛋白質を回収するのに必要
な加水量は３．０Ｌで足りた。これに対し、キトサン水
溶液を添加していない比較例１では膜の目的蛋白質阻止
率が高く、９９％以上の目的蛋白質を回収するために必
要な加水量は８．０Ｌと実施例１に比べて多かった。As is clear from the results in Tables 1 and 2, in Example 1 in which the aqueous chitosan solution was added, the membrane had a low target protein rejection, and the amount of water required to recover 99% or more of the target protein was 3%. 0.0L was enough. On the other hand, in Comparative Example 1 in which the chitosan aqueous solution was not added, the target protein rejection of the membrane was high, and the amount of water required to recover 99% or more of the target protein was 8.0 L, which was lower than that in Example 1. There were many.
【００３０】[0030]
【発明の効果】以上詳述した通り、本発明の菌体分離方
法によれば、膜濾過及びダイアフィルトレーションによ
り発酵液から菌体を分離すると共に、目的有価物を回収
する方法において、膜による目的有価物の阻止を抑制
し、ダイアフィルトレーションに必要な加水量を大幅に
低減することができる。このため、膜分離処理に必要な
膜面積や循環ポンプ等の装置設備を縮小すると共に、処
理時間の短縮を図ることができ、目的有価物の回収効率
は格段に向上する。As described above in detail, according to the method for separating cells of the present invention, the method for separating cells from the fermentation solution by membrane filtration and diafiltration and recovering the target valuable substance is carried out by a method comprising the steps of: , The amount of water required for diafiltration can be significantly reduced. Therefore, the membrane area required for the membrane separation process and the equipment such as a circulating pump can be reduced, and the processing time can be shortened, so that the efficiency of recovering the target valuable material can be significantly improved.
Claims (1)
に分離すると共に、目的有価物を透過液側に回収する方
法において、 該発酵液に水溶性ポリマーを添加して膜分離処理した
後、濃縮液側に水を加えて膜分離処理することを特徴と
する菌体分離方法。1. A method for separating a bacterial cell into a concentrated liquid by subjecting a fermented liquid to membrane separation and recovering a valuable substance to a permeated liquid by adding a water-soluble polymer to the fermented liquid. A method for separating bacterial cells, comprising, after the treatment, adding water to the concentrated liquid side to perform a membrane separation treatment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP34141597A JP3371783B2 (en) | 1997-12-11 | 1997-12-11 | Cell isolation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP34141597A JP3371783B2 (en) | 1997-12-11 | 1997-12-11 | Cell isolation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH11169671A true JPH11169671A (en) | 1999-06-29 |
| JP3371783B2 JP3371783B2 (en) | 2003-01-27 |
Family
ID=18345902
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP34141597A Expired - Fee Related JP3371783B2 (en) | 1997-12-11 | 1997-12-11 | Cell isolation method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3371783B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015122981A (en) * | 2013-12-26 | 2015-07-06 | 花王株式会社 | Method of producing purified hydrophobic enzyme solution |
| JP2017200691A (en) * | 2016-02-04 | 2017-11-09 | ポール・コーポレーションＰａｌｌ Ｃｏｒｐｏｒａｔｉｏｎ | Inline diafiltration with multi-channel pump |
-
1997
- 1997-12-11 JP JP34141597A patent/JP3371783B2/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015122981A (en) * | 2013-12-26 | 2015-07-06 | 花王株式会社 | Method of producing purified hydrophobic enzyme solution |
| JP2017200691A (en) * | 2016-02-04 | 2017-11-09 | ポール・コーポレーションＰａｌｌ Ｃｏｒｐｏｒａｔｉｏｎ | Inline diafiltration with multi-channel pump |
| US10183108B2 (en) | 2016-02-04 | 2019-01-22 | Pall Corporation | Inline diafiltration with multi-channel pump |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3371783B2 (en) | 2003-01-27 |
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