WO1981000575A1

WO1981000575A1 - Magnetically immobilized microorganism and the use thereof

Info

Publication number: WO1981000575A1
Application number: PCT/SE1980/000215
Authority: WO
Inventors: P Larsson; K Mosbach
Original assignee: Berol Kemi Ab; P Larsson; K Mosbach
Priority date: 1979-08-24
Filing date: 1980-08-22
Publication date: 1981-03-05
Also published as: DK182781A; EP0041065A1; JPS56501191A; BR8009026A; FI802671A

Abstract

Process for the microbial fermentation of an organic substrate as well as to a microbial catalyst used in the process. The catalyst being of the immobilized microorganism type is magnetically influenced, a property which is used in order to separate the catalyst from the fermented organic substrate upon fermentation.

Description

MAGNETICALLY IMMOBILIZED MICROORGANISM AND THE USE THEREOF

The present invention relates to a process in the microbial fermentation of an organic medium and to a microbial catalyst suitable for use in said process.

The preparation of products such as ethanol, lactic acid, acetic acid, antibiotics, vitamins and steroids by means of microorganisms is an old well-known technique which recently has met with a renewed scientific interest, i.a. initiated by the world-wide problems of provision of certain raw materials. An example of a recently published report is "Rapid ethanol fermentation of cellulose hydrolysate. II. Product and substrate inhibition and optimization of fermentor design" by T.K. Ghose and R.D. Tyagi, Biotechnol. Bioeng. 21, 1401 (1979).

So-called immobilized microorganisms have been used earlier for the preparation of i.a. ethanol. Cf. "The immobilization of microbial cells, subcellular organelles and enzymes in calcium alginate gels" by M. Kierstan and C. Bucke, Bio- technol. Bioeng. 19, 387 (1977). Immobilization of a microorganism means that it is associated with a carrier material, e.g. polymeric materials, such as polyacrylamide or calcium alginate. By .using immobilized microorganisms several advantages are obtained; the microorganisms can e.g. be reused comparatively easy and the fermentation product obtained becomes more pure.

It has also been proposed to immobilize microorganisms by fixing them onto magnetic material. The amount of microorganism that can be fixed is, however, very low which is a hindrance to practical application. In the microbial fermentation of organic substrates containing considerable amounts of suspended solid particles, e.g. enzymatically or non-enzymatically hydrolyzed cellulose (parts of plants, recovery paper etc.) as well as starch that has been hydrolyzed correspondingly, many of the advantages normally attributed to immobilized microorganisms cannot be fully used since the solid particles for instance easily clogs reactor beds or make it difficult to separate the immobilized microorganisms upon completed fermentation. Likewise, in i.a. continuous fermentation processes it is difficult to keep the immobilized microorganism in the reactor without its passing along with the flow of fermented organic substrate out of the reactor.

In accordance with the present invention it has been shown to be possible to solve advantageously the problems associated with the use of immobilized microorganisms and to derive the full advantages of this technique. More particularly, the invention relates to a process in microbial fermentation of organic substrate, the fermentation being carried out with a microorganism that is immobilized in a carrier material together with a magnetically influenced substance so that the ratio between the weight of the micro organism and the weight of the magnetically influenced substance is within the range 5-500, preferably 10-200, an the microorganism and the fermented organic substrate being separated upon completion of the fermentation by means of a magnetic field.

The separation can e.g. be effected by keeping the microbial catalyst (being understood to comprise the micro- organism immobilized in the carrier material together with a magnetically influenced substance) in the reactor by magnetic forces while the reactor is emptied. Another way of effecting separation is to catch the microbial catalyst outside the reactor and then return it to the reactor for use in the fermentation of the next substrate batch. Still another variant is to keep the microbial catalyst in the reactor by means of a magnetic field while the organic substrate continuously is allowed to flow through the reactor at an adjustable rate.

In fermentation it is desirable, for economical reasons, to obtain as high proportions of the fermentation product as possible. Thus, when fermenting e.g. sugar (cellulose hydrolysate, starch hydrolysate) by using higher proportions of sugar in the organic substrate it is possible to obtain an ethanol concentration of 10-15% by volume. However, a great problem is that a high proportion of ethanol considerably reduces the fermentation rate. In the present invention it is easy to eliminate the problem of the inhibitory effect of the fermentation product by continuously removing it from the fermentation reactor and in that way keeping its concentration at a low level in the very reactor.

One way is to extract continuously the reactor contents during the course of the fermentation with an organic solvent removing the fermentation product formed. The solvent must of course be adapted to the fermentation product. Examples of frequently occuring solvents are alcohols such as ethanol, octanol, decanol and dodecanol; esters such as dibutyl phthalate and tributyl phosphate; aliphatic and/or aromatic hydrocarbons; chlorinated hydrocarbons such as bromoethane, 2-bromopropane and dichloroethane. The very combination of immobilized microorganisms and solvent extraction is essential since immobilization protects the microorganisms from being harmfully influenced by the solvent used. Owing to the fact that the microbial catalyst is magnetically influenced the catalyst and the organic substrate can be separated magnetically. Thus, a magnetic field can keep the microbial catalyst in the reactor while the organic substrate containing the fermentation product is pumped to an extraction unit and thereupon is returned to the reactor. The principle of magnetic separation allows a rapid and safe circulation of the organic substrate for a long. time. If instead a filter disc is used as separator, the pores of the filter disc are easily clogged by the suspended particles present leading to operating interruptions

Another way of keeping the concentration of the fermentation product low in the reactor is to boil off the same continuously from the organic medium in vacuum. The method of boiling off the fermentation product in vacuum from the organic substrate has been disclosed earlier for non-immobilized microorganisms (G.R. Cysewski and C.R. Wilke, Bio technol, Bioeng. 19 , 583 (1977)) but said technique has not been considered to be economically justifiable owing to the large amounts of energy required in order to pump away the carbon dioxide formed. When applying the present invention it is possible to make use of the advantages of vacuum boiling-off without the method being loaded with its disadvantages since one can let the fermentation reactor operate at atmospheric pressure and there let the greater part of the carbon dioxide escape with no energy consumption while the boiling-off of the fermentation product is carried out in a separate unit. The amount of carbon dioxide to be boiled off in vacuum can normally be restricted to 5-20% of the amount required in a conventional vacuum process. The fact that the fermentation step and the vacuum step are so easy to separate is based on the ease of keeping the microbial catalyst in the reactor while the fermented organic substrate is pumped on. When the fermented organic substrat is loaded with solid particles or viscosity-increasing substances the very magnetic separation is particularly suitable since it removes the problems easily occurred in filter separators owing to the pores being clogged.

The microbial catalyst according to the invention is charac- terized in that the microorganism is immobilized in a carrier material together with a magnetically influenced substance, the ratio between the weight of the microorganism and the weight of the magnetic substance being 5-500, pre- ferably 10-200. By incorporating the microorganism with the polymeric material it is possible to immobilize great amounts of microorganism. When preparing the catalyst the magnetic substance is suitably added in connection with the immobilization of the microorganism.

The carrier material is suitably a polymer, such as calcium alginate, polyacrylamide, polymethacrylate and carragenate whereas the magnetically influenced substance (being understood to be a material attracted by a magnetic field) can be iron filings, magnetite, ferrite, etc., preferably ferrite powder. The microorganisms to be used can be of widely different types. Thus, it has been shown that fungi and bacteria well maintain their catalytic activity when applying the present invention but use can also be made of other kinds of microorganisms, such as virus, algae and protozoa.

The present invention is further illustrated by the following examples.

Example 1

50 grams of Saccaromyces cerevisiae were suspended in 50 ml of water. Powdered ferrite coated with organic material (Ferrofluid) corresponding to an amount of 2 grams of iron oxide powder was mixed with the fungus suspension and the mixture was poured under stirring into 100 ml 3% sodium alginate solution. The viscous solution obtained was then added dropwise to 3 liters of 0.05 M calcium chloride solution, small round balls being formed. After 3 hours in

0.05 M calcium chloride the microbial catalyst was ready for use. Exempel 2

A microbial catalyst was prepared in accordance with Example 1, however with the exception that the ferrite powder used in Example 1 and coated with organic material was replaced by 2 grams of ferrite powder.

Example 3

A microbial catalyst prepared in accordance with Example 2 in an amount of 2 grams was mixed with 10 ml of 5% glucose solution containing 10 mM calcium chloride. The mixture was stirred in a vessel provided with a cover at 30º and the formation of ethanol was followed by means of gas chromatography. As reference the corresponding non-immobilized microorganism as well as microorganism immobilized according to Example 2 but in the absence of ferrite were used. As is evident from the table the magnetic catalyst is as effective as the non-magnetic one and almost as effective as free microorganism.

Example 4

Upon completion of the fermentation according to Example 3 the magnetic microbial catalyst was prepared by means of a strong permanent magnet, was washed with water while still attached to the magnet and then transferred to the next fermentation cycle. This separation could also be carried out in the presence of great amounts of solid particles. As solid particles use were made of sand, glas and wood chips, in the order mentioned, in amounts of 20% by volume.

Example 5

A 5% sugar solution in an amount of 10 ml was fermented with a microbial catalyst according to claim 1 (5 grams) in the presence of (test A) and in the absence of (test B) 50 ml of dodecanol. During the first stage the two fermen- tation processes proceeded equally fast but during the later stage of the fermentations the fermentation in test A proceeded about 20% fasterthan the fermentation in test B dependent on the beneficial effect of the removal of the ethanol from the aqueous phase.

Example 6

A fermentation process similar to that of Example 5 was carried out but using decanol instead of dodecanol while tests A and B were carried out using a starting concentration of 7% by weight of ethanol. During the continued fer- mentation of glucose test A proceeded more than twice as fast as test B. This illustrates the importance of extracting ethanol at high ethanol concentrations.

Example 7

A microbial catalyst according to Example 2 was kept magnetically in a reactor of the volume 100 ml through which there was continuously pumped a 10% glucose solution or a 10% glucose solution containing nutrients (0.25% yeast extract, 0.025% NH₄H₂PO₄, 0.0025% MgSO₄). The temperature was 30ºC. In the table below there is shown the ethanol- yielding capacity as a function of time. In the presence of nutrients the catalyst according to the invention has a good ethanol-yielding capacity during a long time.

Ethanol-yielding capacity (% of the initial capacity)

Example 8

Cells of the bacterium Zymomonas mobilis obtained from 24 hours cultivation at 30°C in a medium containing 5% glucose, 1% yeast extract and 0.1% (NH.J-SO. were immobilized in the same way as in Example 2.

Example 9

A microbial catalyst according to Example 8, immobilized Zymomonas mobilis but without magnetic substance and free Zymomonas mobilis cells were compared with respect to the ethanol-yielding capacity according to Example 3. The capacity of the immobilized bacterium was not influenced nega tively by the incorporation of magnetic material in the carrier material and was about 75% of the capacity of the free bacterium.

Claims

1. A process in the microbial fermentation of organic substrate in a reactor, characterized in that the fermentation is carried out in the presence of a microbial catalyst that is immobilized in a carrier material together with a magnetically Influenced substance, the ratio between the weight of the microorganism and the weight of the magnetic substance being within the ratio 5-500, preferably 10-200.

2. A process according to claim 1, characterized in that the magnetic substance being ferrite powder is added to the immobilized microorganism in connection with the immobilization.

3. A process according to claims 1-2, characterized in that the microbial catalyst is removed from the fermentation vessel.

4. A process according to claims 1-2, characterized in that the microbial catalyst is kept in the fermentation vessel while the fermentation medium is withdrawn from the reactor.

5. A process according to claims 1-4, characterized in that during the course of the fermentation the fermentation product obtained is removed from the fermented substrate by extraction with an organic solvent.

6. A process according to claims 1-4, characterized in that the fermented substrate continuously is pumped from the fermentation reactor to a vacuum distillation unit where the fermentation product is separated, and thereupon is returned to the fermentation reactor while the microbial catalyst is kept in the reactor.

7. Microbial catalyst, characterized in that the micro- organism is immobilized in a carrier material together with a magnetically influenced substance, the ratio between the weight of the microorganism and the weight of the magnetic material being within the ratio of 5-500, preferably 10-200.

8. A catalyst according to claim 7, characterized in that the magnetic substance is added to the immobilized microorganism in connection with the immobilization.

9. A catalyst according to claims 7-8, characterized in that the carrier material is a polymeric material, such as calcium alginate, polyacrylamide, polymethacrylate or carragenate.

10. A catalyst according to claims 7-9, characterized in that the magnetic material is ferrite powder.