In the search for bacteria possessing high amylolytic activity, some thousand or more isolates were screened on soluble starch agar and the extent of their activities determined qualitatively by the loss of medium response to Lugol\u27s iodine solution. The selected isolates were then grown on a liquid wheat bran medium and the resulting starch-dextrinizing activity measured quantitatively. No attempt was made to determine the properties of the various amylases. The primary purpose of this report is to point out the original sources from which the isolates were made and to correlate the nature of the substrates with the extent of amylase production by the bacteria

Beckord, L.D.

Peltier, George L.

English

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University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Papers in Microbiology Papers in the Biological Sciences August 1945 SOURCES OF AMYLASE-PRODUCING BACTERIA George L. Peltier University of Nebraska-Lincoln L.D. Beckord University of Nebraska-Lincoln Follow this and additional works at: https://digitalcommons.unl.edu/bioscimicro  Part of the Microbiology Commons Peltier, George L. and Beckord, L.D., "SOURCES OF AMYLASE-PRODUCING BACTERIA" (1945). Papers in Microbiology. 6. https://digitalcommons.unl.edu/bioscimicro/6 This Article is brought to you for free and open access by the Papers in the Biological Sciences at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Microbiology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. SOURCES OF AMYLASE-PRODUCING BACTERIA'GEORGE L. PELTIER AND L. D. BECKORDDepartments of Bacteriology and Agricultural Chemistry, University of Nebraska, LincolnReceived for publication August 29, 1945In the search for bacteria possessing high amylolytic activity, some thousandor more isolates were screened on soluble starch agar and the extent of theiractivities determined qualitatively by the loss of medium response to Lugol'siodine solution. The selected isolates were then grown on a liquid wheat branmedium and the resulting starch-dextrinizing activity measured quantitatively.No attempt was made to determine the properties of the various amylases. Theprimary purpose of this report is to point out the original sources from whichthe isolates were made and to correlate the nature of the substrates with theextent of amylase production by the bacteria.METHODSPreliminary testing on starch agar. A loopful of a 24-hour-old peptone beefextract broth suspension of each isolate was spotted at each of three points onthe surface of a soluble starch agar plate (2 g Lintner soluble starch per liter ofpeptone beef extract agar). After 48 hours' incubation at 30 C the plates wereflooded with Lugol's iodine solution (1 g iodine and 2 g potassium iodide madeup to a volume of 300 ml with water), the excess poured off after several minutes,and the diameter of the cleared zone around the bacterial colony noted. Allisolates showing a wide, cleared zone (approximately 10 to 15 mm) were retainedfor further culturing in a wheat bran medium.Preparation and inoculation of wheat bran medium. Experience with a varietyof media indicated that the evaluation of amylase activity was most comparableand consistent on a wheat bran medium prepared as follows: 50 grams of wheatbran per liter of distilled water was autoclaved (30 minutes at 121 C) and strainedthrough a fine cheesecloth. To one liter of the strained bran extract were added10 grams peptone (bacto) and 2 ml of phosphate buffer (15 g KH2PO4 and 35 gK2HO4 3H20 per 100 ml distilled water). This medium was dispensed in200-ml amounts into glass-covered preparation dishes (diameter 100 mm, depth50 mm) so that the liquid depth was 2.5 cm (Beckord, Peltier, and Kneen, 1945)and autoclaved for 20 minutes at 121 C. All stock cultures were carried onwheat bran agar slants made by adding 20 grams of agar per liter of wheat branmedium.Each dish was inoculated with a loopful from a 24-hour wheat bran agar slantand allowed to incubate (5 days at 35 C) quiescently in order to facilitate pellicleformation (Beckord, Peltier, and Kneen, 1945). The amylase-producing capac-ity of each isolate was desired in this investigation. Accordingly, dextrinizingactivity, which can be determined by a convenient method in the laboratory,1 Published with the approval of the Director as paper no. 387, Journal Series, NebraskaAgricultural Experiment Station.711 at UNIV OF NEBRASKA-LINCOLN on August 30, 2007 jb.asm.orgDownloaded from GEORGE L. PELTIER AND L. D. BECKORDwas selected as the criterion by which this particular property was to be as-certained.Evaluation of amylase activity. After incubation, a 10-ml aliquot of the mediumwas withdrawn from below the pellicle and tested directly for dextrinizing activ-ity by a method described previously (Beckord, Kneen, and Lewis, 1945).Amylase activity is expressed in terms of "dextrinization time," namely, thetime in minutes required by 10 ml of wheat bran medium to convert 20 ml ofTABLE 1Dextrinizing activity of liquid wheat bran culture8 of 265 bacterial isolates hydrolyzing-starchagarIncubation: 35 C, 5 daysISOLATES HYDRO- "DEXTRINIZATION TME" OF WEAT BRAN CULTURE MEDIUMSOURCE OE ISOLATION LYZING STARCCEHAGAR NO. 1-10 min 11-20 miin 21-30 min 31-60 mi 60+ miDManures*.................. 42 1 0 0 0 41Composts................... 26 0 0 0 0 26Sludges .................. 16 0 0 0 0 16Soils.................... 17 1 0 1 1 14Fish meal.................. 2 1 1 0 0 0Soybeans.................. 8 1 3 0 0 4Peanuts.................... 7 1 3 1 0 2Beet pulp.................. 2 0 0 0 0 2Flours.................... 7 3 0 1 0 3Starches................... 7 5 0 0 0 2Thinstillage............... 8 2 5 0 0 1Bread (ropy).17 17 0 0 0 0Laboratory contaminants.. 2 1 1 0 0 0Air (dust)t......... 60 4 11 2 3 40Insects$...... .... .. ........44 0 1 0 0 43Totals.265 37 25 5 4 194* Organisms isolated from the first 13 substrates by Lillian Wind, Mildred Penner, andIrma Tingelhoff, Dept. of Bacteriology, University of Nebraska.t From the collection of Floyd Schroeder, Preparator, Dept. of Bacteriology, Universityof Nebraska.t From the collection of Dr. Edward A. Steinhaus, Rocky Mountain Laboratory, Hamil-ton, Montana.1 per cent boiled starch (at 30 C and pH 6) to the point where the "red-brown"col9r is given with iodine. Occasionally certain cultures produced so muchamylase that it was necessary to use an aliquot smaller than 10 ml. Since thetime required for dextrinization is inversely proportional to the amount ofenzyme present, the dextrinization time for a 10-ml aliquot could be calculated.If the "end point" was not reached by one hour's action of a 10-ml aliquot, theactivity was recorded as 60+ minutes.RESULTSThe results are presented in the data of table 1. Of the 1,000 or more organ-isms isolated, 265 hydrolyzed starch agar. and were cultured further in' the wheat712 at UNIV OF NEBRASKA-LINCOLN on August 30, 2007 jb.asm.orgDownloaded from SOURCES OF AMYLIASE-PRODUCING BACTERIAbran medium. Thirty-seven of the 265 isolates had dextrinization times of 10minutes or less and were considered highly active. The majority of the cultureswere considered inactive since 194 gave a dextrinization time of more than60 minutes.Manures, composts, sludges, soils, and insects were poor natural sources. Of145 organisms isolated from these materials, 2 were active (1 to 10 minutes),1 was fairly active (11 to 20 minutes), and the remainder were inactive. Ropybread consistently yielded high amylase producers. From 4 different samples ofropy bread 17 isolations were made, all of which were very active. In fact, someof these isolates gave activities of less than 1 minute. Since one-fourth of the60 organisms from the air (isolated during July and August) were either activeor fairly active, air was one of the relatively good sources.In many instances, only a small number of isolations were made, so that thedata were not considered sufficient to draw any definite conclusions concerning aparticular source, but they do give an indication of the potentialities of the source.It would appear that fish meal, soybeans, peanuts, flours, starches, and thinstillage were good sources since a relatively high percentage of the isolates fromthese were amylase producers. Neither of the 2 isolates from beet pulp wereactive.From the data in table 1, the inference is gained that some sources yieldamylase-producing bacteria in greater numbers than others. The sources investi-gated may be rated in decreasing activity as follows: (1) ropy bread, (2) starchesand flours, (3) thin stillage, (4) soybeans and peanuts, and (5) air. Since therewas apparently a lack of amylase-producing organisms in manures, composts,sludges, and soils, the active bacteria from the air may have had their origin,principally, from plant materials rather than decomposed plant and animalresidues in the soil.Isolates showing superior amylase production gave the following preliminarycharacterization: gram-positive sporeforming bacilli, approximately one micronin length, with central spores usually smaller than the cell. Agar colonies wereeither small, smooth, glistening, and round, or intermediate in size, flat, rough,and irregular, or sometimes rather large, spreading, and mucoid. The greatmajority of isolates fermented glucose and sucrose with the production of acid.Lactose was not fermented. Most of them liquefied gelatin completely within72 hours. Provisionally, they all appear to fit into the Bacillus subtilis group.SUMMARYThe members of a recent collection of some 1,000 or more bacterial isolatesfrom a variety of different natural sources were grown on starch agar plates andthe zones of starch hydrolysis noted.Of this number, 265 hydrolyzed starch and were further cultured in a liquidwheat bran medium. The amount of amylase produced was determined fromthe dextrinizing activity of the culture medium.Of the 265 selected isolates grown in the wheat bran medium, 194 gave adextrinization time of 60+ minutes, whereas thirty-seven yielded the dextrini-713 at UNIV OF NEBRASKA-LINCOLN on August 30, 2007 jb.asm.orgDownloaded from 714 GEORGE L. PELTIER AND L. D. BECKORDzation time of 1 to 10 minutes; twenty-five, 11 to 20 minutes; five, 21 to 30 min_utes; and four, 31 to 60 minutes, respectively.Manures, composts, sludges, soils, and insects were extremely poor sources.Ropy bread, flour, starches, and other plant materials were excellent to fairsources. Exposure of plates to the air was fairly effective in picking up amylase-producing bacteria.A prelminary characterization of the high-amylase-producing isolates placedthem in the Bacilu8 mbtili8 group.REFERENCESBzCxORD, L. D., KNzEN, E., AND LEwis, K. H. 1945 Bacterial amylases. Production onwheat bran. Ind. Eng. Chem., Ind. Ed., 37, 692-696.BzCORD, L. D., PELTIER, G. L., AND KNSEN, E. 1945 Bacterial amylases. Productionin thin stillage. In press. at UNIV OF NEBRASKA-LINCOLN on August 30, 2007 jb.asm.orgDownloaded from 

SOURCES OF AMYLASE-PRODUCING BACTERIA

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SOURCES OF AMYLASE-PRODUCING BACTERIA

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