An Evaluation of - Propiolactone for the Sterilization of Fermentation Media 1

HIMMELFARB, PHILIP (University of Massachusetts, Amherst), R. B. READ, JR., AND WARREN LITSKY. An evaluation of 3-propiolactone for the sterilization of fermentation media. Appl. Microbiol. 9:534-537. 1961.-Twenty-five bacterial species were cultured in basal broth plus 1 of 19 different carbohydrates which were sterilized by Seitz filtration, autoclaving (112 C, 10 min), or exposure to 0.2% ,B-propiolactone (BPL). No significant differences were found either in the visual observations for acid and gas, pH, or titrable acidity determinations after 3 days of incubation with any of the three preparations tested. An effort was made to further determine the effect of BPL and heat on carbohydrates by assaying for glucose before and after treatment. Results indicated that glucose was not degraded by 0.2% BPL, however, it was shown that autoclave temperatures caused extensive degradation. Statistical treatment of the results from Warburg studies indicated that BPL-treated glucose showed no appreciable toxic effects, although the actual oxygen uptake was not as great as with Seitzor autoclavetreated glucose. The application of the BPL sterilization process was discussed. The effect of heat sterilization on reducing sugars employed in fermentation media has been the subject of many investigations. The decrease in concentration of these sugars due to reactions with other constituents of the heated media has been troublesome in precise measurements of fermentation processes. Attempts have been made to minimize carbohydrate degradation or reaction by the use of sterilization processes employing reduced heat exposures, filtration, or chemical agents. One of these chemical agents, 13-propiolactone (BPL) has been found in recent years to be an efficient sterilizing agent. Hartman, Piepes, and Wallbank (1951) reported that BPL possessed excellent virucidal and bactericidal properties, when used for the preparation of sterile whole blood and blood plasma. Curran and Evans (1956) indicated that BPL rapidly killed bacterial spores. Bernheim and Gale (1952) and LoGrippo and I Contribution no. 1299 of the University of Massachusetts, College of Agriculture Experiment Station. Hartman (1955) demonstrated the fungicidal activity of BPL against pathogenic and nonpathogenic fungi. BPL is currently being used to sterilize arterial homografts, according to the method suggested by Lo Grippo et al. (1955). Recently, BPL has been shown by Hoffman and Warshowsky (1958) to be an effective disinfectant when used in the vapor state. The purpose of this investigation was to determine the efficacy of BPL as a sterilizing agent of carbohydrate solutions with an evaluation of concomitant carbohydrate alteration. These alterations were evaluated by determining the ability of various bacteria to ferment the sterilized carbohydrate, the effect of BPL sterilization on glucose as measured by glucose oxidase activity, and the effect of BPL degradation products on bacterial respiration. MATERIALS AND METHODS Carbohydrates employed. Twenty per cent solutions of the carbohydrates listed below were prepared in distilled water and divided into three aliquots of 33.3 ml each. These were then sterilized by BPL, Seitz filtration (Pad Type ST, K6), and autoclaving at 112 C for 10 min, respectively. Included among the carbohydrates were adonitol, arabinose, cellobiose, dextrin, dulcitol, galactose, glucose, inositol, lactose, maltose, mannitol, melibiose, raffinose, rhamnose, salicin, sorbitol, sucrose, trehalose, and xylose. Procedure for sterilization with BPL. The carbohydrate solution was sterilized in the following manner: BPL (Good-rite)2 was diluted 1:10 with ice water and added to a 20 % carbohydrate solution to yield a final BPL concentration of 0.2 %1. This mixture was then placed in a 37 C water bath for 2 hr and shaken every 10 to 15 min. The sterilized carbohydrate solution was then incubated overnight at room temperature to insure complete BPL degradation. Fermentation of carbohydrates sterilized by BPL, Seitz, and autoclaving. The BPL-treated and Seitzfiltered carbohydrate solutions were added to tubes containing heat sterilized Purple Broth Base (Difco)3 to yield a final concentration of 0.5 % carbohydrate. 2 B. F. Goodrich Chemical Company, Cleveland, Ohio. BPL is a registered trade mark of the Celanese Corporation of America. 3 Difco Laboratories, Inc., Detrbit, Mich. 534 on O cber 5, 2017 by gest ht://aem .sm .rg/ D ow nladed fom ~BPL STERILIZATION OF FERMENTATION MEDIA53 The third carbohydrate aliquot was added to unsterile broth tubes and autoclaved (112 C, 10 min). To obtain a uniform pH of 7.0 for all three media it was necessary to pre-adjust the broth base to which BPL-treated carbohydrates were added because of the acidic properties of the BPL degradation products. To ascertain any change in the carbohydrates which would have been caused by the sterilization procedures, the following organisms were inoculated into each of the aforementioned sterilized substrates and the amount of acid production with or without gas, was determined: Aerobacter aerogenes, Bacillu-s cereus, TABLE 1. Fermentation patterns of some selected organisms: pH measurements after incubation for 3 days at 35 C