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Vol. 38, No. 2
INDUSTRIAL AND ENGINEERING CHEMISTRY
optimum for growth and amylase production by the isolate; amylase activity identical with the highest reported in Table V (2.3 minutes) was obtained by 5-day incubation at 35’ C. The data of Table V show that with all but two of the supplementation treatments the activity attained was less than that found with the sodium-carbonate-neutralized stillage. Apparently the influential factor in these two instances was nitrogen (monosodium glutamate), I n most instances the addition of inorganic nutrients or of sucrose appeared to he detrimental at the levels tested. The data indicate that the particular thin stillage used r a s reasonably adequate for amylase production and required only neutralization. If supplementation were practiced with this material, it might be limited to additional nitrogen. COMPARISON OF AMYLASES
Some properties of the amylase produced on wheat bran by isolate 23 have been described ( g ) . To determine whether culturing in thin stillage effected any changes in these properties, amylases produced in thin stillage and on wheat bran were compared. The high saccharifying activity manifested by the amylase of isolate 23 grown on wheat bran was deemed one of its significant properties. That this property was retained after culturing in thin stillage is indicated by the production of fermentable sugars. Pressure meters were employed as previously (2) except that the enzyme solution and water brought the final volume of the reaction mixture to 25 ml. The enzyme solutions were aliquots (dextrinization times, 14 minutcs) from a corn thin stillage culture and from a water extract of a dried wheat bran culture. After the end of a 21-hour fermentation period, the pressures caused by the carbon dioxide evolved were closely comparablemanometer readings of 397 mm. of mercury for the thin stillage culture and 407 mm. for the wheat bran culture. The amylase inhibitor of Kneen and Sandstedt (6) derived from wheat does not inhibit the action of high-liquefying commercial bacterial amylases but does inhibit the action of the highsaccharifying amylase of isolate 23, Equal amounts of inhibitor reduced by 48% the dextrinization rate of t,he amylase of isolate 23 produced either in corn thin stillage or on wheat bran. The behavior of the amylases of isolate 23 derived from thin stillage and wheat bran were essentially identical with respect to saccharifying ability and response to inhibitor. For that reason it was considered that culturing in thin stillage had not affected the properties of the amylase. DISCUSSION
The studies on production of bacterial amylase using thin stillage as a medium have several significant’ features. The relations found between pellicle production and amylase secretion are Important. The formation of a pellicle appears to be a prime requisite for efficient production of amylase by the organism used. To utilize fully t,he capacity of this pellicle for amylase production, several techniques are practical. In addition to the customary shallow tray procedure, large volumes of nutrient liquid with relatively small surface area may be employed. To do this it is necessary to agitate the liquid in such a manner that (a) t’he amylase will be distributed through the body of the liquid, (b) fresh nutrients will be brought in contact with the pellicle, and ( c ) the pellicle itself will be relatively undisturbed and thus remain intact. Further modification of the liquid culture procedure might well be the use of a method similar to that known as the quick-vinegar process. The nutrient liquid can be dripped onto wood chips packed into a vertical tower. Provision is made for preforming the pellicle on the surface of the chips and likexise for aeration. A previous communication (2) demonstrated that amylase could be produced satisfactorily by culturing the organism on mois-
tened wheat bran. A number of procedures are possible for this type of work. The utility of any one of them depends upon the facilities available and the product desired. A semisolid wheat bran medium has certain advantages as to speed of amylase production: Alaximum activity was attained in 2 days as compared with 4 to 6 days for maximum amylase production in an undisturbed liquid medium. On the other hand, the rate of amylaae production in a liquid medium can be greatly accelerated by replacement of the medium with fresh nutrient. Also emphasized is the fact that various natural materials have potential value as bacterial nutrients. In addition to the extract of hydrolyzrd soybeans commonly used ( 2 1 ) , neutralized thin stillage is a satlafactory medium and is available as an industrial by-product in large quantities. It may be assumed that the type of amylase produced by a specific organism on all of these media will not vary appreciably. For example, when an extract of a dried wheat bran culture of isolate 23 was compared with a thin stillage culture of the same organism, the amylases produced had essentially the same properties. From an industrial standpoint, the prime requirements would appear to be rapid production of a desirable type of amylase IT ith a minimum of effort. The type of amylase is governed by the nature of the organism, and the quantity produced may be regulated by further selection to obtain better strains. Production features such as speed of turnover and the minimizing of contamination appear to be most satisfactorily achieved by the use of wheat bran. However, the production in liquid, either as CURtomarily done or nith one of the variants suggested, might meet industrial requirements with greater facility. Provided the importance of pellicle formation is kept in mind and the proper relation between the pellicle and source of nutrient is maintained, little difficulty should be involved in adapting any of thrse procedures in industry. LITERATURE CITED
(1) Bauernfeind, J. C., Smith, M e B., Garey, J. C., Baumgarten, W., Gustoff, I?. H., and Stone, L., Cereal Chem., 21, 421 (1944). (2) Beckord, L. D., Kneen, E., and Lewis, K . I
