USE OF CELLULASE PREPARATIONS IN THE CULTIVATION AND USE OF CELLULOSE-PRODUCING MICRO-ORGANISMS

Abstract: Dense cultures of cellulose-producing microorganisms may be produced and used for various purposes by the processes of the present invention. Initially a nutrient medium for cellulose-producing microorganism is prepared which comprises a cellulase preparation. The nutrient medium is then inoculated with cellulose-producing micro­ organism. The inoculated medium is then incubated under aerobic conditions to facilitate microorganism growth and the production of a dense microorganism culture substantially free of cellulose pellicle. This dense culture may used as an inoculum for a large volume of suitable nutrient medium or may be harvested and the harvested microorganisms utilized, for example as a source of cellular components, for example, such as DNA or enzymes. When a larger volume of nutrient medium is inoculated, a subsequent aerobic incubation of the inoculated medium facilitates the large scale production of cellulose.

Description: The present invention involves the action of cellulolytic enzymes to degrade cellulose as it is being produced by cellulose-producing microorganisms such as Acetobacter xylinum , for example. The rationale relating to this invention is manifold. Organisms such as Acetobacter , which make large amounts of cellulose are rather difficult to transfer from one inoculum to the next because the cells frequently become entangled in the thick cellulosic pellicle. For large scale production of cellulose, a large inoculum is necessary for the final large scale cellulose production step. Cellulase enzyme preparations do not impede cell growth, yet with appropriate concentrations of cellulase preparation added to the growth medium, no resultant cellulose is observed. Using this system it is possible to obtain large numbers of cells which are physiologically intact and fully competent to synthesize cellulose once the cellulase preparation is either diluted or removed from the culture nutrient medium. The abstract "Cellulase and Cellulose-free Cell Production by Acetobacter xylinum " by Dillingham et al (1961) Bacteriological Proceedings: 67, describes effects of cellulases upon Acetobacter cellulose production; however, with further search of the literature no related subsequent publications giving any more details have been found. In this abstract, it is stated: The normal culture develops little or no turbidity below the white cellulose pellicle. In the presence of cellulase, no pellicle forms and a typical turbid cell suspension develops (viable count 10.0 x 10⁷ as compared to 112 x 10⁵ in 30 hours for normal cultures). This indicates about a nine-fold increase in cell density, ascribable to the presence of a partially purified cellulase from Myrothecium verrucaria . It has been suggested that Acetobacter xylinum be renamed as Acetobacter pasteurianus (see De Ley (1984) Bergey's Manual of Systematic Bacteriology, Williams and Wilkins, Baltimore/London, pp 268-274). These names are viewed as interchangeable for the purposes of this specification. According to the present invention, a nutrient medium in which cellulase-producing microorganisms are cultivated contains between 0.000375 and 0.015 units of cellulase per ml; the cellulase is at a level insufficient to hydrolyse substantially all of the cellulose, the rate of cellulose synthesis by microorganisms being cultivated increases and the cellulose produced may be of a more amorphous character. Cellulose-producing microorganisms usable in the practice of the present invention include prokaryotes such as those of the genus Acetobacter (preferred), Rhizobium , Agrobacterium , Pseudomonas or Alcaligenes . A sterile nutrient medium comprising a cellulase preparation is prepared. Cellulase preparations usable as a component of nutrient media should comprise the enzyme cellulase(beta-1,4-glucan glucanhydrolase, E.C.3.2.1.4) and, when desired, may also include cellobiohydrolase or beta-glucosidase (beta-D-glucoside glucohydrolase, E.C.3.2.1.21). Cellulase preparations may be obtained from organisms such as those of the genus Myrothecium , Trichoderma or Polyporus . More specifically, the cellulase preparations may be obtained from organisms of the species Myrothecium verrucaria , Trichoderma viride , Trichoderma reesei or Polyporus versicolor . The nutrient medium comprising the cellulase preparation (between 0.000375 and 0.015 units of cellulase per ml) is inoculated with cellulose-producing microorganism. This inoculated medium is then incubated under aerobic conditions and generally at between 20 and 40°C. The nutrient medium is preferably at a pH between 3 and 7. Nutrient media suitable for cultivation of cellulose-producing microorganisms include a broad variety of media well known in the art, such as those containing simple or complex carbohydrates, sources of nitrogen, minerals and vitamins. Many suitable sources of nitrogen, carbon, etc, for the Acetobacter are shown by De Ley in Bergey's Manual of Systematic Bacteriology, V 1, pp 268-274 (1984) Williams and Wilkins, Baltimore/London. The following Example illustrates the invention. Example Acetobacter xylinum was grown in media containing low levels of cellulase (1220B, Novo Industri A/S) at a concentration of 0.25 mg per 100 ml medium. It was observed that after 1 week at 28°C on rotary shake culture the amount of cellulose produced in the cellulase-culture was approximately 1.5 x greater than that of the control (minus cellulase) culture. The results are shown in the following Table. Cellulase-treated (Weight cellulose produced in grams) Control (No cellulase) (Weight cellulose produced in grams) .29340 .15182 .17114 .16593 .24530 .16729 .30743 .16978 .18312 .14490 .26404 Average .15994 Average Cellulase added to the culture medium in low concentrations promoted the yield of microbial cellulose. This was true when the starting inoculum of Acetobacter was very low (approximately 10,000 cells per flask). If the starting inoculum was greater or if the cultures were allowed to grow longer than one week, the increase in cellulose production over that of the control is much less and sometimes equal to or even less than the controls. This area needs further definition, but thus far illustrates that the amount of cellulose produced can be regulated by the addition of cellulolytic enzymes.

Claims: A process for producing microbial cellulose, which comprises cultivating cellulose-producing microorganisms in a nutrient medium including, per ml thereof, between 0.000375 and 0.015 units of cellulase, such that not all cellulose being produced is hydrolysed, but cellulose crystallisation is altered and the rate of cellulose production is thereby accelerated. The process of claim 1, wherein the nutrient medium has a pH between 3 and 7. The process of either preceding claim, wherein the cellulose-producing microorganisms is a prokaryote. The process of claim 3, wherein the prokaryote is of the genus Rhizobium , Agrobacterium , Pseudomonas , Alcaligenes or Acetobacter . The process of claim 4, wherein the genus is Acetobacter . The process of claim 3, wherein the prokaryote is of the species Acetobacter xylinum . The process of any preceding claim, wherein the cellulase is obtainable from an organism of the genus Myrothecium , Trichoderma or Polyporus . The process of claim 7, wherein the organism is of the species Myrothecium verrucaria , Trichoderma viride , Trichoderma reesi or Polyporus versicolor . The process of any preceding claim, wherein the cellulase-containing medium additionally comprises cellobiohydrolase and beta-glucosidase.

Inventors: BROWN, R. MALCOLM
Applicants: BOARD OF REGENTS THE UNIVERSITY OF TEXAS SYSTEM
Application year: 1987
Main classification: C12P19
IPC codes: C12P19, C12R1