SE438868B - SET TO MAKE ACETON AND BUTANOL FROM A CELLULOSAMENTAL - Google Patents

Description

It has now been found that greater concentration of an acetone-butanol mixture can be obtained in preparation from a cellulosic material by a one-step process, when the cellulosic material is simultaneously affected by cellulase and an acetone-butanol-forming microorganism.

The invention is characterized in that the cellulosic material and the acetone / butanol-producing microorganism are charged to a single reaction zone, and reacted simultaneously to form a product mixture containing acetone and butanol.

By "cellulosic material" for use as a starting material according to the present invention is meant cellulose or material consisting mainly of cellulose, such as wood, rice straw, rice husks, corn stalks, corn cobs and paper waste such as tongs, corrugated cardboard, magazines, short fibers from paper mills and fibrous waste and industries. It is convenient to pulverize or disintegrate the materials so that they can be used effectively as a substrate.

For saccharification of these cellulose substrates, any commercially available cellulase can be used, such as Cellulase Onozuka. A liquid enzyme preparation, such as a liquid filtrate or a solid extract from a culture of any cellulase-producing microorganism, such as T. viride, may be used. Likewise, a liquid or solid culture of T. viride itself can be used as a crude mixture without separation, extraction or purification.

As the acetone-butanol-producing microorganism for use with the cellulase, some well-known microorganism can be used, for example Clostridium acetobutylicum and Clostridium saccharoacetobutylicum.

In order for the cellulose substrate to be simultaneously affected by the cellulase and the acetone-butanol-producing microorganisms, an aqueous suspension containing 1-50% by weight of cellulose material, suitable amounts of any nitrogen source, inorganic salts and other nutrients is prepared and sterilized in a known manner to serve as a substrate.

A cellulase or a cellulase-containing solution is added to the substrate simultaneously with a pre-cultured, acetone-butanol-producing microorganism, so that the fermentation takes place.

Temperature and pH conditions depend on the microorganism and cellulase used, but usually the optimum conditions are between 30 and 55 ° C and pH 4-6. The fermentation times also vary depending on the type of cellulosic material, the acetone-butanol-producing microorganism and the activity of the cellulase, but usually the desired amount of acetone-butanol is formed in 48 hours.

As can be seen in the following examples, the present invention not only simplifies the production process but also reduces the inhibition of glucose by the cellulase reaction as well as by the acetone-butanol-producing microorganism as compared with the conventional two-step method, i.e. saccharification and fermentation. invention the sugar concentration in the medium is kept low during the fermentation process. As a result, the acetone-butanol yield of the cellulose increases markedly.

According to the invention, there is thus provided a method of producing acetone and butanol from a cellulosic material in an economical and advantageous process in one step.

The invention will be described in more detail below with reference to preferred embodiments.

Example Preparation of yeast culture Clostridium acetobutylicum IFO 3854, acetone-butanol-producing microorganism, was inoculated into 10 ml of sterile medium consisting of the following components and cultured anaerobically at 37 ° C for 3 days.

Medium composition Glycose 20 g Polypeptone 5 g KHZPO4 0.5 g K2HPO4 0.5 g Mgso4. 71x20 0.2 g MHSO 4. 4-5H2O 10 mg FeSO4. 7H 2 O 10 mg NaCl 10 mg Deionized water 1 liter pH 6.5 Of a medium consisting of the above components, 50 ml was charged to a 100 ml flask and 5 ml of the above culture solution was added. ....... ...-_._._-...._.._ ..._ »_ 7801262-2 and cultured anaerobically at 37 ° C for 3 days. This became the yeast culture. 1) Medium A. - I 1 A medium with the same composition as above but with 60 g of glucose instead of 20 g. 2) Medium B.

A medium with the same composition as above but with 100 g of cellulose powder, finer than 100 mesh, instead of glucose.

A commercially available cellulase was salted out with ammonium sulfate and desalted. It was then lyophilized to a pure cellulase.

One gram of this cellulase was dissolved in 100 ml of water at a pH adjusted to 4.5 and 10 g of cellulose powder <100 mesh was added. Thereafter, a saccharification was performed at 45 ° C for 48 hours. After completion of the pre-suction operation, the reaction solution was analyzed by liquid chromatography and 3.2 g of glucose were found to have formed. In other words, this cellulase had the above activity.

One gram of the cellulase described above and 5 ml of the yeast culture as above were added to 100 ml of the well stirred medium B.

The pH of the mixture was adjusted to 6.5 and the fermentation took place anaerobically at 37 ° C for 48 hours, checking that the pH did not fall below 4.5.

Comparative Example 1) The above-mentioned medium B was thoroughly stirred and 100 ml thereof was added with 3 g of the above-mentioned cellulase. The pH was adjusted to 4.5 and the mixture was saccharified at 45 ° C for 48 hours. When the resulting sugar solution was analyzed, a production of 6.3 g of glucose could be ascertained. The sugar solution was heat sterilized and 5 ml of the previously indicated yeast culture was added. Thereafter, .u. _ _ 0. an anaerobic acetone-nutanol fermentation at J7 C for 48 hours. 2) 5 ml of the yeast culture prepared according to the above were inoculated into 100 ml of the above-mentioned medium A which was heat sterilized. The pH was adjusted to 6.5 and the mixture was allowed to react anaerobically at 37 ° C for 48 hours checking that the pH did not fall below 4.5. The results of the experiments are summarized in the following table. The products were analyzed by gas chromatography. 78Û1262-2 5 Table Product Acetone B talol Substrate Cellulas mg / ml umgyml According to the invention cellulose 10 g 1 g 4.43 7.53 Example 1) cellulose 10 g 3 g 3.20 5.52 Example 2) glucose 6 g 3.24 5.50 As can be seen from the above table, according to the present invention, 1.4 times more acetone and butanol are produced than that obtained by the conventional method, where cellulose is first hydrolysed to glycos and then used as a substrate, Example 1 ), or the glucose itself was used as the substrate, Example 2). Accordingly, the present invention has the advantages that acetone and butanol are produced by a simple one-step process, that the yields of the products are considerably improved and that the cellulase consumption is less than in the conventional processes.

Claims (3)

7861262--2 Païæëïrer A method of producing acetone and butanol by saccharifying a cellulosic material in the presence of a cellulase, and fermenting the sugared cellulosic material in the presence of an acetone / butanol-producing microorganism, characterized in that the cellulosic material and the acetone / The butanol-producing microorganism is charged to a single reaction zone, and simultaneously roared to form a product mixture containing acetone and butanol. 2. A method according to claim 1, characterized in that the acetone and the butanol are recovered from the product mixture. 3. A method according to claim 1, characterized in that the amount of cellulosic material charged to the reaction mixture is of the order of 1 ~ 50% by weight, and that the reaction takes place at a pH of about 4-8 at a temperature of 30-55 ° C.