Propagation of Bacillus popilliae in laboratory fermentors

Abstract

The insect pathogen Bacillus popilliae Dutky causes a fatal milky disease of Japanese beetle larvae. Spores of the bacterium offer a biological means of controlling this insect. While satisfactory sporulation in vitro has not yet been accomplished, conditions have been developed for the proliferation of vegetative cells in shaken flasks and aerated fermentors. Vegetative cultures are maintained by frequent transfer or by lyophilization. Media based on yeast extract are used routinely, but corn steep liquor and casein hydrolyzates afford comparable yields of 5 × 108 cells/ml. in 16–24 hr. Nutritional requirements have been established for growth in a synthetic medium. Oxygen availability affects the pathway of carbohydrate catabolism and is necessary for optimal growth. In rapidly growing cultures, a short period of maximum viability is characteristically followed by rapid death of the cells. When inoculum size and transfer time are suitably manipulated, viable cell yields reach 1–2 × 109/ml. Alternative methods of propagation, including the addition of particulate carbon, and procedures designed either to neutralize acids or to remove metabolic products by dialysis, do not markedly enhance the yield of cells per volume of medium, although viability may be prolonged.

Characteristics of the constituent substrains of Bacillus popilliae growing in batch and continuous cultures

“…Maximum vegetative populations of B. popilliae have been obtained in batch culture by using a rich medium containing 1.5% Difco yeast extract, 0.2% glucose, and 0.3% K2HPO4 (milky disease [MD] medium [9]). Since yeast extract alone will support significant growth, it has been impossible to formulate a medium in which glucose is truly limiting or in which yeast extract concentration can be varied without affecting growth.…”

“…Colonial studies used a rich medium containing 1.5% Difco yeast extract, 0.2% glucose (autoclaved sepa-Air out la] Exit tube baffle rately), 0.3% K2HPO4, and 0.2% Difco agar (MD agar medium [9]).…”

“…For example, B. popilliae did not form spores in shake-flask cultures of rich medium in which the average cell generation time or division time (td) was about 3 h or in a dialysis system when td was reduced to 2 h, the shortest time we have observed (9). Limited sporulation has occurred in flask cultures containing activated carbon (5) where td was about 3.5 h. In colonies where td was between 9 and 11 h, the incidence of sporulation was about 20% (10). Generation time of the sporulating cultures of Wyss (15) was similarly extended.…”

“…Large-scale fermentative production of infective spores of Bacillus popilliae Dutky would supply a proven biological insecticide (3,14) to control the Japanese beetle, Popillia japonica Newman. Suitable vegetative proliferation of this entomopathogen has been reported (9), and methods for obtaining limited sporulation in liquid and on solid medium have been described (5,10,15). Nevertheless, a means to induce extensive sporulation of B. popilliae growing in laboratory media has evaded discovery.…”

Preparation and Characterization of Intact and Free Spores of Bacillus Popilliae Dutky

“…Bacillz~s popilliae Dutky, the causative agent of milky disease in Japanese beetle larvae (Popillia japonica Newman), grows appreciably only under restricted conditions, has limited viability, and sporulates slightly, or not a t all, in laboratory cultures (11,12,15,17,18). Conversely, the organism grows well in larval hemolymph, and spore populations average about 2 X lo9 per larva (1) equivalent t o about 2 X 101° spores per milliliter of larval heinolymph.…”