Radiation Resistance of Soil Azotobacter

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JouRNAL of BAcruzIoLoGY, May, 1965 Copyright @ 1985 American Society for Microbiology Vol. 89, No. 5 Printed in U.S.A. Radiation Resistance of Soil Azotobacter GERARD R.

1 JouRNAL of BAcruzIoLoGY, May, American Society for Microbiology Vol. 89, No. 5 Printed in U.S.A. Radiation Resistance of Soil Azotobacter GERARD R. VELA AND ORVILLE WYSS School of Aerospace Medicine, Brooks Air Force Base, Texas, and The University of Texas, Austin, Texas Received for publication 2 January 1965 ABSTRACT VELA, GERARD R. (School of Aerospace Medicine, Brooks Air Force Base, Tex.), AND ORVILLE Wyss. Radiation resistance of soilazotobacter. J. Bacteriol. 89: Quantitative recovery of Azotobacter from soils subjected to -y-radiation from a cobalt- 60 source showed the soil populations to be much more highly resistant than isolates from such cultures grown on laboratory media. Even in the encysted state, the laboratory populations were reduced 10,000-fold by exposure to 200 kr, whereas the soil populations were not measurably reduced by that dose. Almost all studies on radiation effects in microorganisms have been concerned with pure cultures of selected species growing under laboratory conditions. Studies on overall biological processes and mixed populations in native environments have been neglected. Goring and Clark (1952) incorporated p32 into soil and observed that very high doses of radiation were required to affect the metabolic activities of soil microorganisms; less active populations were more easily disturbed than rapidly proliferating ones. Stotzky and Mortensen (1959) found no gross changes in soil ph, CO2 evolution, or nitrogen mineralization in an organic soil subjected to doses of y rays ranging from 8 to 250 kr, but did observe a decreased rate of utilization of ammonium at the higher radiation doses which was correlated with an increase in the bacterial population and a decrease in the fungal population. Stanovick, Giddens, and McCreery (1961) concluded that the symbiotic nitrogenfixing bacteria are more susceptible to neutrons and y rays than are other soil organisms. Decreases in bacterial and fungal populations, nitrification, and sulfate production in soils exposed to 7-radiation were observed by Popenoe and Eno (1962). Bernard and Geller (1962) reported that doses of y rays exceeding 15 kr were injurious to the soil microflora and that doses of 100 kr were lethal to Azotobacter and the nitrifying bacteria in sandy soils. Other workers have irradiated microorganisms in their native habitat and have examined the survival of given segments of the microflora (Proctor and Goldblith, 1951; Davis, Sheldon, and Auerbach, 1956; Witkamp, 1961) but have not pursued the effects of radiation on individual species of the soil bacteria. From the literature cited, it appears that soil bacteria are very resistant to ionizing radiation. Some authors suggest that the soil protects the microflora therein, others state that resistance is a function of the rate of proliferation as determined by nutrient levels, whereas still others suggest that "natural" bacteria are more resistant to radiation than are "laboratory" bacteria. Vela and Wyss (1962) studied the kinetics of the destruction of microbes that carry on transformations of nitrogen in nature. The ability to fix nitrogen aerobically was destroyed by radiation doses that still permitted nitrification and ammonification. Nitrogen fixation was a discontinuous function of radiation dose; i.e., no nitrogen was fixed if all Azotobacter were killed, but any survival gave maximal fixation. On the other hand, nitrification was depressed as a continuous function of the radiation dose, indicating greater sensitivity of the more efficient nitrifiers. It appeared that such lessened efficiency was correlated with clumping, but unequivocal data on this point were not obtained. A third type of response was observed with the ammonification process in which the ability to release ammonia from urea was enhanced by doses up to 2000 kr. The radiation-resistant sporeformers proved to be efficient ammonifiers, and the destruction of the more sensitive, competing organisms yielded a soil that behaved as a more efficient biochemical entity for ammonification. These results support the findings of McLaren, Reshetko, and Huber (1957). This investigation has been continued (i) to determine the effect of graded doses of y rays on aerobic nonsymbiotic nitrogen-fixing bacteria in the soil, (ii) to compare the resistance of laboratory and soil Azotobacter, and (iii) to 1280

2 VOL. 89, 1965 RADIATION RESISTANCE OF SOIL AZOTOBACTER 1281 determine something of the nature of the differences. MATERIALS AND METHODS Soils. All soils studied were collected from areas that were not under cultivation. Each specimen consisted of 100 g of surface soil from each of 5 to 10 different sites in the area sampled. The soil was mixed, ground in a sterile mortar if necessary, and passed twice through a sterile 80-mesh sieve. For those experiments in which soil particles of known volume were required, that fraction of soil was used that passed through a 60-mesh sieve, but was retained by an 80-mesh sieve. Particles selected in this way measured about 250, on a side. Irradiation. A 600-c cobalt-60 source of -y-rays was employed in all experiments. The specimens, at ambient temperatures, were placed in a rotating chamber to insure uniform distribution of radiation dose and uniform exposure to air. Mixtures of water and soil or aqueous suspensions of bacteria were placed in rubber-stoppered tubes large enough to insure uniform oxygenation during the exposure. Dose-rate variations were accomplished by adjusting the source-to-sample distance, and total dose was determined by length of exposure. The dose rates varied from 0.8 to kr/min, and the total doses reported here are given as r6ntgens in air. Radiation dosimetry was checked with halogenated hydrocarbon chemical dosimeters (Sigaloff, 1957) which had in turn been calibrated against National Bureau of Standard radiation sources. The radiation delivered to these dosimeters at the rate of 1 kr/min has been shown to have a maximal variation of +5%. Microbiology. Unless otherwise stated, the medium of Augier (1956) was used to culture Azotobacter with the modification that 5 g of sucrose and 3 g of sodium benzoate were substituted for the mannitol (Darznick, 1959). For measuring nitrogen fixation, triplicate 1-g portions of soil were exposed to radiation and transferred to 2-liter Erlenmeyer flasks containing 100 ml of sterile 1% aqueous mannitol solution. The flasks were incubated at 28 C on a reciprocal shaker. The aerobic nitrogen-fixing ability retained by the soils was measured by micro-kjeldahl determinations. The amount of nitrogen fixed was corrected for evaporation of the culture liquid. Estimates of populations by the "most probable number" method were obtained by placing 1.0 g of soil in a small test tube. The tubes, after treatment, were placed in bottles containing 100 ml of sterile medium without carbon compounds. The bottles were shaken violently to break the tubes and then placed on a laboratory shaker for 20 min to disperse the soil. A decimal dilution series was prepared and 1 ml from each dilution of the series was transferred to each of 10 tubes containing 4 ml of the complete medium. The tubes were incubated in a slanted position in the 33 C incubator and checked for typical Azotobacter growth after 5 and 10 days. The most probable number of Azotobacter in the specimen under study was obtained from McCrady's table (Prescott, Winslow, and McCrady, 1946). The statistical significance of these numbers was derived by the method proposed by Eisenhart and Wilson (1943) based on the data of Halvorson and Ziegler (1933). Estimates of Azotobacter populations were also obtained by dilution plating or by distributing known weights of dispersed soil particles on the surfaces of multiple agar plates. In the latter procedure, the weight of soil inoculum and the number of plates employed for each measurement were such that each plate yielded no more than 10 colonies. The results obtained by these two plate methods were compatible and gave population estimates of the same magnitude as those obtained by the "most probable number" method. Counts of bacterial populations in soil-water mixtures or in wet soils were placed on a comparative basis by evaporating to dryness samples of the suspensions and weighing the residues so that bacterial counts could be expressed as organisms per gram of dry soil. Pure cultures of Azotobacter isolated from the soil were induced to form cysts by the method of Socolofsky and Wyss (1962). Cyst formation became evident on the 3rd or 4th day of incubation, TABLE 1. Azotobacter populations in irradiated soils and in mannitol solution inoculated with these soils Dose (kr) O* ,000 Azotobacter count after incubation for 0 days days (X 106) < days (X 107) < 100 * Control. A heat-sterilized control was also included and gave counts of zero.

1282 1282 VELA AND WYSS J. BACTERIOL. and the cysts matured by the 10th day of incubation as determined by the cyst stain of Vela and Wyss (1964).

3 VELA AND WYSS J. BACTERIOL. and the cysts matured by the 10th day of incubation as determined by the cyst stain of Vela and Wyss (1964). RESULTS Table 1 shows that doses of y rays ranging from 0 to 150 kr exerted only a slight effect on the initial soil Azotobacter, as shown by triplicate platings. When such soil samples were inoculated into a mannitol solution, vigorous growth of Azotobacter occurred. At 500 kr, surviving Azotobater cells were below the number required for our method of detection, and they failed to grow out in mannitol solution. This experiment was repeated with replicate samplings of the same soil and with samples of other Texas soils; the initial count always showed from 500 to 5,000 Azotobacter cells per g, and the radiation dose required for drastic reduction in this population always approached 500 kr. When a population survived radiation, the nitrogen-fixing ability was not impaired (Table 2). This resistance to -y rays was far beyond that reported for laboratory-grown Azotobacter by Socolofsky and Wyss (1962). Figure 1 shows survival curves obtained from laboratory-grown vegetative cells and cysts, as well as the survival curves of Azotobacter in soil. The laboratory-grown organisms were isolated from these same soils after exposure to 300 kr. The isolates were grown to yield the vegetative populations and the cysts of a density of 107 cells for use in these experiments. The initial populations of Azotobacter in these soils were about 5 X 103. The shape of the points on the lines shows the TABLE 2. Total nitrogen in 100 ml of mannitol solution inoculated with I-g samples of soil Treatment Days of culture after irradiation Heat-sterilized * No radiation kr kr * Results expressed as milligrams of nitrogen per gram of soil. KILOROENTGENS FIG. 1. Survival rates of irradiated Azotobacter. Lines 1 and 2 depict the survival of the natural populations in four different Texas soils. Line 8 deals with laboratory-grown cysts, and line 4 deals with laboratory-grown vegetative cells. The open and closed circles and the triangles in lines 8 and 4 represent cultures isolatedfrom the survivors at 800 krfrom the soils reported in line 1. source of the organisms used for the laboratory cultures. It is evident that a laboratory culture freshly isolated from a soil exposed to 300 kr (which killed only a small fraction of the Azotobacter therein) has a radiation resistance of a different order of magnitude than that it exhibited when in soil. Even when the laboratory cultures were induced to produce cysts, these resistant forms were much more radiationsensitive than were the soil populations. Whereas this figure shows results from three soils and isolates therefrom, the experiment has been repeated with several additional soils obtained from other sources. In each case the resistance of the mature soil populations and the laboratory cultures isolated from them showed the relationships indicated in Fig. 1. Results from the "most probable number" method agreed with the plate method. Laboratory populations are suspended as single cells prior to irradiation, whereas the soil populations may consist of colonies attached to soil particles; such colonies might not be dispersed by vigorous agitation in the dilution bottles and could yield death curves, as shown in Fig. 1, even if the organisms comprising them were as sensitive as the laboratory-grown populations. To resolve this, the soil samples were sieved to separate fractions of particles of uniform size, and these were dusted on the surface of plates of Azotobacter medium. From the number of particles planted and the fraction of particles from

$The results obtained with the 80- mesh fractions of two soils are tabulated in Table 3.$

4 VOL. 89, 1965 RADIATION RESISTANCE OF SOIL AZOTOBACTER 1283 which Azotobacter grew, the number of clones was determined. The results obtained with the 80- mesh fractions of two soils are tabulated in Table 3. About 40,000 particles of this fraction of these soils weigh 1 g; thus, the initial Azotobacter count is in the same range as that determined on the total soils by the other counting procedures. The particles used in this study had a maximal volume equal to 105 packed Azotobacter cysts. Based on the cyst death curve in Fig. 1, had the particles yielding colonies been entirely cysts with the resistance of the laboratory-grown organisms, survivors would not have been detected at radiation doses in excess of 200 kr. Pure cultures of Azotobacter spp. isolated from the survivors from irradiated soils were grown on encystment medium for 8 days; the cysts were scraped from the plates, washed three times with water, and added to samples of the same soils from which the culture was isolated. The inoculation was done by adding 0.1 ml of a cyst suspension containing about 1,000 cysts for each gram of soil. Autoclaved soils were included for controls. The soils were stored in paper bags for 30 days at room temperature, at which time they were subjected to 100 kr of y rays to determine whether the slow drying of cysts in the soil enhanced their radiation resistance. The results in Table 4 show that the soil Azotobacter were little affected by 100 kr but the cysts added to either autoclaved or nonautoclaved soil were decreased by at least 99%. Drying did not make the laboratory-grown cysts resistant nor did the soil protect them from the ionizing radiations. TABLE 3. Effect of radiation on Azotobacter on soil particles of about 2505 in diameter No. of No. of par- Fraction Radiationo tidles givin with Soil Dose particles mgs6a: ial planted growth Asotobacter fraction kr II , _ A ,007 0 _ TABIE 4. Radiation resistance of laboratorycultured Azotobacter cysts added to the soil and allowed to dry there* Specimen Soil A... Soil I... Soil J... Soil A... Soil I... Soil J... Sterile soil A... Sterile soil I. Sterile soil J... Cysts of isolate added None None None Aao0 I 0oo J200 Am0, I 00 Jsoo Most probable number of Asotobacter per g of soil Radiation dose of o kr ,120 1,928 2,632 2, ,126 1,226 Radiation dose of 100 kr <11 <11 <11 * A8,, is a strain of Azotobacter isolated from a sample of soil A that had been exposed to 300 kr of j'-radiation. TABLE 5. Change in radiation sensitivity of the soil Azotobacter during the 16 days following moistening of the soil to 60% of its water-holding capacity with 1% mannitol solution Days Azotobpg t1r 99% lethal dose kr > > >100 Conversely, soaking soils for 1 day at room temperature or 10 days in a refrigerator (4 C) did not render the Azotobacter strains sensitive to y rays, but, if soils were kept moist at room temperature, the radiation resistance disappeared. A soil was moistened to 50% of the water-holding capacity with a 1% mannitol solution and was kept at room temperature for 16 days. At intervals, samples of the soil were removed, subjected to y radiation at 10, 20, 40, 80, and 100 kr, and assayed for surviving Azotobacter. The results were plotted, and the doses for 90% destruction, as read from the curves, are presented in Table 5. Four days after the addition of aqueous mannitol, the radiation resistance declined to a value approaching that of laboratory vegetative cells, but by 16 days the resistance returned to that of the native soil populations. A number of native soils from Texas have been irradiated at a level of 200 kr and assayed for

5 1284 VELA AND WYSS J. BACTERIOL. TABLE 6. Distribution of radiation-resistant Azotobacter in Texas soils Soil Location Radiation dose* 0 kr 200 kr A NE Austin B N Austin C NW Austin 3 0 D Jollyville 0 0 E Manor H Austin (downtown) I San Marcos 10 9 J Brooks Air Force Base 10 6 K S Austin 10 8 O Jonestown 7 5 R Waco 9 4 S Corpus Christi 6 4 U Grandview * For each soil sample, 20 tubes were inoculated with 1 ml of a suspension of 1 g of soil in 99 ml of diluent. Ten tubes were irradiated and 10 were not. The numbers indicate the number of tubes giving Azotobacter growth. Five additional tubes were inoculated with 0.01 ml of a suspension of 1 g of soil in 99 ml of diluent. These tubes showed no growth. In the "most probable number" series, a result of five negative tubes at the 0.01-ml level indicates a population of fewer than 2,400 Azotobacter cells per g of soil. A population of this size will have survivors to 200 kr only if it is of the resistant type. Azotobacter by the planting of multiple broth tubes with 1 ml of a 1% suspension of the soil. As shown in Table 6, all soils but one contained Azotobacter at the detectable level, with the indicated number in each soil being fewer than 3,400 per gram. The survival of this population after 200 kr of radiation indicated that radiation resistance in soil Azotobacter is usual in Texas soils. DISCUSSION One of the interesting problems of microbiological survival is the relevance to practical problems of data collected with laboratory-grown strains. Ionizing radiations and soil Azotobacter serve as a convenient model for such studies. Survival can be assayed quantitatively without interference from the extensive soil flora, and the soil and its population do not interfere with the lethal action. The studies presented here support the view that "natural" organisms may differ greatly in resistance from laboratory-grown cultures. The difference observed with radiation resistance of Azotobacter was not restricted to one or a few soils; it is not due to artifacts in the counting, to protection offered by the soil colloids, or to the extent of drying. Because of the low numbers involved, it is not feasible to observe microscopically the soil Azotobacter. The possibility of a very small soil Azotobacter form cannot be dismissed. Suggestions of life cycles involving heat-resistant corpuscles have appeared in the literature, but documentation of such work has not been convincing. Our studies show clearly that they exist almost entirely in a state that is far more radiation-resistant than any laboratory forms thus far encountered. This resistance results when the organisms develop in the soil. Whether it will be possible to modify the cultural conditions of laboratory-grown cysts so that they attain the resistance of soil forms is now under study. ACKNOWLEDGMENT This investigation was supported by Public Health Service grant AI from the National Institute of Allergy and Infectious Diseases. LITERATURE ClTED AUGIER, J A propos de la numeration des azotobacter en milieu liquide. Ann. Inst. Pasteur 91: BERNARD, V. V., AND I. T. GELLER Diestvie gamma luchei na nekatorye gruppy pochvennoi mikroflory. (The effect of gamma rays on certain groups of soil microflora.) Agrobiologiya 4: DARZINEK, Y The carbon source in a medium for the quantitative assay of azotobacter in soil. Mikrobiologiya 28: DAVIS, R. J., V. L. SHELDON, AND S. I. AUERBACH Lethal effects of gamma radiation upon segments of a natural microbial population. J. Bacteriol. 72: EISENHART, C., AND P. W. WILSON Statistical methods and control in bacteriology. Bacteriol. Rev. 7: GORING, C. A. I., AND F. E. CLARK Radioactive phosphorus and the growth and metabolic activities of soil microorganisms. Soil Sci. Soc. Am. Proc. 16:7-9. HALVORSON, H. O., AND N. R. ZIEGLER Application of statistics to problems in bacteriology. III. A consideration of the accuracy of dilution data obtained by using several dilutions. J. Bacteriol. 26: McLAREN, A. D., L. RESHETKO, AND W. HUBER Sterilization of soil by irradiation with an electron beam, and some observations on soil enzyme activity. Soil Sci. 83: POPENOE, H., AND C. F. ENO The effect of gamma radiation on the microbial population of the soil. Soil Sci. Soc. Am. Proc. 26: PRESCOTT, W. C., C.-E. A. WINSLOW, AND M. H. MCCRADY Water bacteriology, 6th ed., p John Wiley & Sons, Inc., New York. PROCTOR, B. E., AND S. A. GOLDBLITH Food processing with ionizing radiations. Food Technol. 5:

VOL. 89, 1965 RADIATION RESISTANCE OF SOIL AZOTOBACTER 1285 SIGALOFF, S. C. 1957. Chemical systems for the measurement of penetrating radiations. School of Aviation Medicine, U.S. Air Force, Randolph Air Force Base, Tex.

6 VOL. 89, 1965 RADIATION RESISTANCE OF SOIL AZOTOBACTER 1285 SIGALOFF, S. C Chemical systems for the measurement of penetrating radiations. School of Aviation Medicine, U.S. Air Force, Randolph Air Force Base, Tex. Rept. No SOCOLOwFSKY, M. D., AND 0. WYSS Resistance of the Azotobacter cyst. J. Bacteriol. 84: STANOVICK, R., J. GIDDENS, AND R. A. MCCREERY Effect of ionizing radiation on soil microorganisms. Soil Sci. 91: STOTZKY, G., AND J. L. MORTENSEN Effect of gamma radiation on growth and metabolism of microorganisms in an organic soil. Soil Sci. Soc. Am. Proc. 23: VELA, G. R., AND 0. WYss The effect of gamma radiation on nitrogen transformation in soil. Bacteriol Proc., p. 24. VELA, G. R., AND 0. WYBS Improved stain for visualization of Azotobacter eneystment. J. Bacteriol. 87: WITKAMP, M Influence of radiation from an unshielded reactor on a natural microflora. Oak Ridge National Laboratory, Technical Manual No. 29.

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