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Growth characteristics of the insect pathogen, Bacillus popilliaeDutky, were studied by propagation in shaken flasks and in 2-liter fermentors. Maximal populations between 5 × 108and 2 × 109viable cells per milliliter of culture medium routinely were obtained in incubation periods of 18 to 24 hr at 30 C in a medium composed of 1.5% yeast extract, 0.6% K2HPO4, and 0.2% glucose or trehalose. The carbohydrate required for growth in liquid media was fermented with the formation of 2 meq of acid per mmole of carbohydrate utilized; acid products ordinarily were not subsequently metabolized. B. popilliaeis an aerobe, and the amount of growth obtained varied with aeration to an optimum at oxygen absorption rates of about 0.5. Maximal populations persist in a culture for periods of only 1 to 4 hr; cessation of growth was followed immediately by rapid death of cultures, so that less than 1% of the cells remained viable after 48 hr, and viability often was lost entirely by the end of 72 hr of incubation. No cytological evidence for spore formation was observed under any growth condition. Death was not associated with lysis of the cells, although extensive granulation ultimately occurred. Continuous neutralizaiton, augmented buffering, various techniques of dialysis, or slow feeding of the carbohydrate did not markedly alleviate the characteristic death of the cultures.

Spent mycelia of Blakeslea trisporarecovered from a previous fermentation enhanced carotene production by mated cultures of the same organism. Peak yields of 107 to 142 mg of carotene per 100 ml of medium were achieved in 6 days. β-Carotene constituted 92% of the total carotenoids produced. The enhancing substance was found present in an aqueous extract of the mycelium. Part of its activity was due to an organic acid fraction. Other microbial cells, molds, yeasts, and bacteria were also capable of enhancing carotene production.

Removal of aflatoxin B1from liquid cultures by resting and growing cells of Flavobacterium aurantiacumNRRL B-184 was studied. Spectrophotometic and thin-layer techniques served as aflatoxin assays. Cells grown in the presence of 5 ppm or higher levels of aflatoxin developed aberrant morphological forms. These toxin concentrations partially inhibited growth, and the nature of the inhibition suggested that aflatoxin interfered with cell wall synthesis. Incubation of 1.0 × 1011resting cells per milliliter with 7.0 μg/ml of aflatoxin B1during a 4-hr period facilitated complete toxin removal from a buffered aqueous medium. Autoclaved cells and cell wall preparations could remove a fraction of the aflatoxin of a test system. However, the toxin removed by autoclaved cells and cell walls could be extracted by washing with water but the aflatoxin B1removed by intact cells could not be extracted into the liquid phase. The uptake of aflatoxin B1by resting cells was sensitive to temperature and pH. Ruptured preparations of F. aurantiacumwere not able to remove or modify the aflatoxin in an aqueous solution.

The effects of antibiotics, sulfonamides, and other antimicrobial agents on vegetative cultures of five strains of milky disease bacteria were compared with those on Bacillus subtilisCohn emend. Prazmowski, Staphylococcus aureusRosenbach, Sarcina luteaSchroeter, Escherichia coli(Migula) Castellani and Chalmers, Saccharomyces pastorianusHansen, and Mucor ramannianusMoel. Similar numbers of viable cells of each organism were exposed to the test materials by use of an antibiotic-sensitivity disc method adapted from techniques recommended by the Food and Drug Administration in the Federal Register. The results suggest that vancomycin or ristocetin, as well as a few other materials, might be useful in controlling contamination either during culture of the fastidious milky disease bacteria or in large populations of vegetative cells undergoing treatment to induce sporulation. Inhibitory concentrations of vancomycin and ristocetin in shaken-tube tests were much lower than expected in comparison with results of sensitivity-disc tests on the milky disease bacteria. Sublethal concentrations of the two antibiotics elicited some morphological change in the bacteria.

A method has been developed for the production of aflatoxin by growing Aspergillus flavusNRRL 3145 on solid substrate wheat. Optimal yields of 900 μg of aflatoxin G1and 900 μg of aflatoxin B1per g of substrate were obtained in 4 to 5 days at 28 C. A study of aflatoxin production on hulls and groats of oats and on whole oats by A. flavusstrains NRRL 2999, NRRL 3000, and NRRL 3145 revealed that aflatoxin was produced on all three substrates, although production was very slight on hulls. Strain NRRL 3145 grown on solid substrate groats produced the largest amounts of aflatoxin: 580 μg of B1and 450 μg of G1per g of substrate. A densitometric method for reading thin-layer chromatographic plates is described; this is more objective and more accurate than the visual methods previously used for the determination of all four aflatoxins.

Yeasts, molds, bacteria, actinomycetes, algae, and fungal spores were screened for their ability to degrade aflatoxin. Some molds and mold spores partially transformed aflatoxin B1to new fluorescing compounds. Only one of the bacteria, Flavobacterium(aurantiacum?) NRRL B-184, removed aflatoxin from solution. Both growing and resting cells of B-184 took up toxin irreversibly. Toxin-contaminated milk, oil, peanut butter, peanuts, and corn were completely detoxified, and contaminated soybean was partially detoxified by addition of B-184. Duckling assays showed that detoxification of aflatoxin solutions by B-184 was complete, with no new toxic products being formed.

Yields of from 200 to 300 mg per liter of aflatoxins B1and G1were produced by two strains of Aspergillus flavusin 20-liter fermentors under proper conditions of inoculum (well-dispersed growth) and aeration (0.5 volume per volume per min of air, 300 rev/min, 30 psi back pressure, baffles). Peak yields were usually attained in 72 hr, after which the aflatoxin concentration declined rapidly. Degradation of aflatoxin depended primarily on mycelial lysis and high-aeration conditions. Cultures previously reported not to degrade aflatoxin could be induced to do so under these conditions. The percentage and rate of toxin degradation were independent of toxin concentration, and appeared to be nonenzymatic and nonspecific. Degradation simulating that occurring in the fermentor was achieved by reacting aflatoxin with peroxidized methyl esters of vegetable oil; initial degradation was rapid and appeared to involve a complex series of reactions.