Method for Cultivation of Microorganism

The present invention relates to a method for cultivating microorganisms.

As is known, fibrous materials such as wood and non-wood plants, etc. are cooked under a high alkaline condition at an elevated temperature and pressure by the kraft pulping process, soda pulping process, oxygen-alkali pulping process, alkaline sulfite pulping process, or other alkaline pulping process, a large amount of the hemicellulose component and a portion of the cellulose component in addition to the lignin component contained in the fibrous materials are solubilized and decomposed, thereby dissolving in the extracted liquor or the spent liquor.

As is disclosed in "Tappi" Vol. 59, No. 9, 118-121 (1976), various organic acids including isosaccharinic acids and metasaccharinic acids mainly derived from carbohydrates are formed and dissolved in spent liquor in the course of alkaline pulping. The mechanism of decomposition and dissolution of carbohydrates in natural fibrous materials is known as the so-called peeling reaction. However, it has not been reported in the prior arts that these organic acids can be utilized under alkaline conditions by microorganisms.

The initial pH of an alkaline cooking liquor is approximately 14 due to the presence of caustic alkali. Although the caustic alkali contained in the cooking liquor is partially consumed by organic acids formed in the course of cooking, the liquor extracted from the cooking system at the initial and intermediate cooking stages and the spent liquor generally contain remaining alkali in a concentration of 2 through 30 g/l in terms of Na.sub.2 O and still are strongly alkaline having a pH of 10 through 14. Thus, the extracted liquor or the spent liquor derived from the alkaline pulping contain, as an organic substance, lignin and various organic acids and also contain a large amount of various salts.

The cellulose rich insoluble component obtained from the alkaline cooking is directly utilized as pulp in the production of paper. However, the remaining extracted liquor and spent liquor have heretofore been burned after concentration. Thus, only combustion energy has been recovered from the organic substances and only cooking chemicals have been recovered from the inorganic substances.

Accordingly, the object of the present invention is to effectively utilize the above-mentioned extracted liquor and spent liquor so as to cultivate microorganisms by utilizing the organic substances, especially various organic acids contained in the extracted liquor or spent liquor in an alkaline region i.e., without neutralizing the alkaline extracted liquor or spent liquor.

Other objects and advantages of the present invention will become clear from the following description.

In accordance with the present invention, there is provided a method for cultivating microorganisms comprising the steps of:

inoculating the microorganisms into a culture medium containing, as a carbon source, the extracted liquor or the spent liquor derived from alkaline pulping;

and cultivating the inoculated microorganisms in the culture medium, whereby organic acids contained in the extracted liquor or spent liquor are utilized.

The inventors have studied the microorganisms which may be commercially produced under alkaline conditions, desirably a pH of 8.0 through 12.5, using the alkaline extracted liquor or spent liquor containing salts in an high concentration. As a result of the classification and search of numerous strains of bacteria isolated from natural soils, the bacteria belonging to genera Bacillus, Arthrobacter, Corynebacterium or Brevibacterium, which are viable in an alkaline extracted liquor or spent liquor have been isolated from the natural soils at Shinonome, Koto-ku, Tokyo, Japan.

Typical examples of the bacteria usable in the present invention are the genus belonging to: Bacillus such as Bacillus sp. FERM-P No. 5861, which has been deposited since Jan. 30, 1981 in the Fermentation Research Institute (FRI) in Japan (all the numbers quoted as "FERM-P" hereinafter refer to the deposition numbers of said Institute) and Bacillus sp. FERM-P No. 5862 deposited on the same date: Arthrobacter such as Arthrobacter sp. FERM-P No. 5683 deposited on the same date, and Arthrobacter sp. FERM-BP No. 88, which has been deposited since Feb. 2, 1982 in the Fermentation Research Institute (FRI) (i.e. International Depository Authority under Budapest Treaty) as Tsukuba in Japan; Corynebacterium sp. FERM-BP NOs. 89 and 90 deposited on Feb. 2, 1982 in FRI under Budapest Treaty; and Brevibacterium FERM-BP Nos. 91 and 92 deposited on Feb. 2, 1982 in FRI under Budapest Treaty. All the depositions were made by Mr. Yukio Kita, one of the inventors.

The morphological characteristics of these bacteria are shown below. The test methods of these morphological characteristics and the classification of the bacteria were carried out according to the descriptions in N. R. Smith, R. E. Gordon & F. E. Clark "Aerobic Sporeforming Bacteria (United States Department of Agriculture, November, 1952)" and "Bergey's Manual of Determinative Bacteriology (8th edition, 1974)".

Both strains FERM-P Nos. 5861 and 5862 are aerobic, sporeforming gram positive, motile, rod shaped bacteria, with peritrichous flagella. It is clear that these two strains should belong to the genus Bacillus. The characteristic point of these bacteria was that they grew well in alkaline media rather than neutral media such as nutrient broth, the optimal pH for growth was about 10. The microbiological properties of these strains were similar to those of Bacillus subtilis. Both isolates, however, were distinguished from the typical strain B. subtilis in growth pH; Bacillus FEMP-P Nos. 5861 and 5862 grew better in alkaline media rather than in neutral media; and in Voges-Proskauer test; both isolates were negative but the typical strain B. subtilis is positive. The strain FERM-P No. 5861 was distinguished from the strain FERM-P No. 5862 in respect of cell size, production of H.sub.2 S and production of acid from carbohydrates. Therefore, it is clear that these two strains are not the same.

Strains FERM-P No. 5863 and FERM-BP No. 88 are aerobic, non sporeforming rods, given gram positive, non acid fast, pleomorphic and showing snapping division. Furthermore, these two bacteria do not attack cellulose and the cell walls do not contain meso-DAP. It is clear that these two strains should belong to the genus Arthrobacter. The strain FERM-P No. 5863 was distinguished from the strain FERM-BP No. 88 in respect of cultural characterization in gelatin stab and litmus milk. Therefore, it is clear that these two strains are not the same.

Strains FERM-BP Nos. 89 and 90 are aerobic, non sporeforming rods, gram positive, non acid fast, pleomorphic and showing snapping division. Furthermore these two bacteria do not attack cellulose and the cell walls contain meso-DAP. It is clear that these two strains should belong to the genus Corynebacterium. The strain FERM-BP No. 89 was distinguished from the strain FERM-BP No. 90 in respect of cultural characterization in nutrient broth and litmus milk. Therefore, it is clear that these two strains are not the same.

Strains FERM-BP Nos. 91 and 92 are aerobic, non sporeforming short rods, gram positive, non acid fast, non pleomorphic, no branching or fragmentation. It is clear that these two strains should belong to the genus Brevibacterium. The strain FERM-BP No. 91 was distinguished from the strain FERM-BP No. 92 in respect of cell size and reduction of nitrate to nitrite. Therefore, it is clear that these two strains are not the same.

Taxonomic characteristics of strains FERM-P Nos. 5861, 5862 and 5863 and FERM-BP Nos. 88, 89, 90, 91 and 92 were investigated according to the method described in "Bergey's Manual of Determinative Bacteriology". These isolates were partly different from the typical strains in some properties. The characteristic point of these isolates was that they grow well in alkaline media rather than neutral media such as nutrient both, the optimal pH for growth is about 10.

The important cultivation condition of these bacteria is the pH of the culture medium. That is to say, when these bacteria are cultivated, the growth of Bacillus sp. FERM-P No. 5861 and FERM-P No. 5862, Arthrobacter sp. FERM-P No. 5863 and FERM-BP No. 88, Corynebacterium FERM-BP Nos. 89 and 90 and Brevibacterium FERM-BP Nos. 91 and 92 are largely affected by the pH of the culture medium. The desirable pH of the culture medium is within the range from 8.0 through 12.5. This is very convenient in the case where the above-mentioned microorganisms grow well in the alkaline extracted liquor or spent liquor obtained from alkaline pulping. According to the present invention, although the alkaline extracted liquor or spent liquor is used as a main carbon source, it should be noted that other carbon source substances which can be utilized by the microorganisms can also be used together with the extracted liquor or spent liquor.

In actual cultivation according to the present invention, inorganic nitrogen compounds such as ammonium salts and nitrates and nitrogen-containing organic substances such as urea and casein can be used as a nitrogen source. Furthermore, inorganic salts such as calcium salts, magnesium salts, potassium salts, phosphates, manganese salts, zinc salts, iron salts, and copper salts and, optionally, substances necessary to grow microorganisms or growth promoting agents such as vitamines, amino acids, corn steep liquor and yeast extract can be desirably added to the culture medium.

The cultivation of the present invention can be carried out by, for example, inoculating bacteria capable of growing in the alkaline extracted liquor or spent liquor obtained from alkaline pulping into the culture medium mentioned above, then conducting aerobic cultivation at a temperature of, for example, about 40.degree. C. for 48 hours. Thus, the organic acids contained in the culture medium can be utilized. Thereafter, the cultivated product is subjected to a centrifugal operation at 5000 through 8000 rpm for 5 through 15 min, whereby the cultivated microbial bodies or cells are readily separated. The cells separated are collected and dried in a conventional manner to form the dried cells.

In order to further facilitate the cultivation operation and the separation and recovery operation of the cultivated bacteria according to the present invention, a pretreatment preventing the deposition or precipitation of the lignin component during the cultivation, such as the previous removal or oxidation of the lignin component in the alkaline extracted liquor or spent liquor can be advantageously carried out prior to the cultivation.

It has been determined that these cultivated cells obtained from the cultivation in the alkaline extracted liquor or spent liquor contain a large amount of crude proteins. Accordingly, the cultivated cells obtained from the cultivation of the present invention can be satisfactorily used as, for example, the protein-amino acid supply source, an animal feed, or a fish feed.

The present invention will be further illustrated by, but is by no means limited to, the following examples, in which all percentages are expressed on a weight basis unless otherwise specified.

Spent liquor derived from a soda pulping process and containing approximately 2.0% of organic acids, including isosaccharinic acid and metasaccharinic acid was adjusted in a pH of 10.0 by carbon dioxide. The precipitates formed by the addition of carbon dioxide were filtered through filter paper to obtain spent liquor having no substantial amount of suspended solids.

To the resultant spent liquor were added 0.2% of yeast extract, 0.15% of K.sub.2 HPO.sub.4, 0.05% of MgSO.sub.4 7H.sub.2 O, and 0.5% of KNO.sub.3. The pH thereof was then again adjusted to 10.0 by sodium carbonate.

Fifty ml portions of the culture medium prepared above were placed in 300 ml flasks and sterilized at a temperature of 120.degree. C. for 15 minutes. Bacillus sp. FERM-P Nos. 5861 and 5862 and Arthrobacter sp. FERM-P No. 5863 were independently inoculated into the culture mediums.

Bacterial strains were subjected to shake culture at a temperature of 40.degree. C. for 48 hours. The cells were centrifuged at 5000 rpm for 15 minutes and dried at a temperature of 100.degree. C. for 24 hours. The amounts of the dried cells were as follows.

The pH of each culture medium after the cultivation of the bacterium was 9.5 through 10.0. The amounts of the organic acids contained in the culture media after separating the cells of FERM-P Nos. 5861, 5862 and 5863 were 0.5%, 0.6%, and 0.2%, respectively.

As is clear from the above results, the organic acids contained in the spent liquor derived from the soda pulping process were utilized by the above-mentioned microorganisms under alkaline conditions, thereby being converted into the cells.

The crude protein contents in the dried cells of FERM-P Nos. 5861, 5862, and 5863 were 55%, 60%, and 55%, respectively.

Spent liquor derived from a kraft pulping process and containing approximately 3.0% of organic acids including isosaccharinic acid and metasaccharinic acid was adjusted in a pH of 9.5 by carbon dioxide. The precipitates formed by the addition of carbon dioxide were filtered through filter paper to obtain spent liquor having no substantial amount of suspended solids.

To the resultant spent liquor were added 0.2% of yeast extract, 0.15% of K.sub.2 HPO.sub.4, 0.05%, or MgSO.sub.4.7H.sub.2 O, and 0.2% of urea. The pH thereof was then again adjusted to 9.5 by sodium carbonate.

Fifty ml portions of the culture medium prepared above were placed in 300 ml flasks and sterilized at a temperature of 120.degree. C. for 15 minutes. Bacillus sp. FERM-P Nos. 5861 and 5862 and Arthrobacter sp. FERM-P No. 5863 were independently inoculated into the culture media.

After the inoculation, bacterial strains were subjected to shake culture at a temperature of 40.degree. C. for 48 hours. The cells were centrifuged at 5000 rpm for 15 minutes and dried at a temperature of 100.degree. C. for 24 hours. The amounts of and the crude protein contents in the dried cells were as follows.

The pH of each culture medium after the cultivation of the bacteria was 9.5 through 10.0.

As is clear from the above results, the organic acids contained in the spent liquor were effectively utilized by the above-mentioned microorganisms.

Example 1 was repeated, except that bacteria Arthrobacter FERM-BP No. 88, Corynebacterium FERM-BP Nos. 89 and 90 and Brevibacterium FERM-BP NOs. 91 and 92 were used and the content of the organic acids was approximately 5%. The results were as follows.

Example 2 was repeated except that bacteria Arthrobacter FERM-BP No. 88, Corynebacterium FERM-BP Nos. 89 and 90 and Brevibacterium FERM-BP Nos. 91 and 92 were used the content of the organic acids was approximately 6% and the pH was adjusted to 10.5. The results were as follows.

The Bacillus sp. FERM-P Nos. 5861 and 5862 and the 11 Arthrobacter sp. FERM-P No. 5863 were redeposited on Feb. 5, 1982, as an effective deposition date of May 1, 1981, in the Fermentation Research Institute (FRI) in Japan under the Budapest Treaty as FERM-BP Nos. 97, 98 and 99, respectively.