INDUSTRIAL AND ENGINEERING CHEMISTRY.
lune, 1944
I n tobaccos fermented for snuff production, the original flora was rapidly supplanted by micrococci. As fermentation progressed, the latter declined in number and were supplanted by numerous yeastlike forms. The latter existed in practically pure culture a t the end of the processing of the snuff blend (sample 42-11) and comprised more than three fourths of the flora after 3 to 4 weeks (42-10 and 42-13). From the limited observations, it would appear t h a t t h e maximum increase in microorganisms occurs during the first week of fermentation and gradually dedines as the first floral types are replaced by t h e other forms. I n the fermentation of dark-fired tobacco (Type 22) for production of Italian-type cigars, yeastlike forms were found in only one sample (42-14). The original mixed flora was supplanted by micrococci, which persisted throughout. The presence of the B. sereus-B. megatherium group at the end of fermentation (42-16) probably indicates t h a t they pewisted as a minority and were dehected only as the count decreased. These results indicate t h a t microorganisms multiply on .fire-rured tobaccos under the proper conditions. The apparent contradiction in the bacterial count of the series comprising samples 42-14, 42-15, and 42-16, in which there is an anomalous increase in the fifth turning followed by a considerable decrease in the sixth turning, is explained by the fact that these are from different lots of tobacco. This fact is evidenced by the nicotine content, which is lower in 42-15 than i n 42-16; if the two turnings had originated from the same lot of cured tobacco, t h e relative nicotine contents would have been reversed. CONC L USlON S
The average content of volatile phenols in the unfermented fire-cured tobaccos was fifteen times as great as that of the aircured tobaccos. The volatile phenol content of fire-cured tobacco diminished during fermentation and aging. Latakia tobacco contained more volatile phenols and less nicotine than domestic samples. I n the unfermented samples the number of bacteria per gram was correlated with the content of volatile phenols. This rela-
559
tion did not hold without exception in actively fermenting tobacco, where t h e conditions were more complicated. There appears t o be no relation between the nicotine content and the bacterial count of t h e tobaccos studied. Micrococci were the predominating organisms on samples of fire-cured tobacco a t the height of fermentation. I n samples of tobacco fermented for snuff the micrococci increased initially but were supplanted by yeastlike forms as fermentation progressed. I n samples of tobacco fermented for use in Italian-type cigars, the micrococci gained ascendency and persisted t o t h e end. A few spore formers were detected a t completion of fermentation when the total count became relatively low. ACKNOWLEDGMENT
The authors express their thanks t o B. A. Brice and R. M. Chapman of this laboratory for helpful suggestions in measuring the absorption values of the phenolic solutions, and to W. W. Garner of this department for supplying t h e illustrations. LITERATURE CITED
Andreadis, T. B., Toole, E. J., Binopoulos, X., and Tsiropoulos, J., Z. Untersuch. Lebensm., 77, 262-72 (1939).
Avens, A. W., and Pearce, G. W., IND. ENQ.CHEM.,ANAL.ED., 11, 505-8 (1939). Dixon, L. F., Darkis, F. R., Wolf, F. A,, Hall, J. A., Jones, E. P., and Gross, P. M., IND.ENG.C H ~ M28, . , 180-9 (1936). Folin, O., and Ciocalteu, V., 3. Biol. Chern., 73,627-50 (1927). Harris, R. G., Haley, D. E., and Reid, J. J., Soil Sci. SOC.Am. Proc., 3, 183-6 (1938). Hawley, L. F., and Wise, L. E., “Chemistry of Wood”, New York, Chem. Catalog Co., 1926. Kissling, R., “Handbuch der Tabakkunde”, 5th ed., Berlin, P. Parey, 1925. Lehmann, K. B., Arch. Hyg., 68, 319-420 (1908-9) Molinari, E., Fach. Mitt. osterr. Tabakregie, 1936, No. 3, 14 (cited by Wenusch, 18). Reid, 3. J., McKinstry, D. W., and Haley, D. E., Pa. Agr. Expt. Sta., Bull. 356 (1938). Shmuk, A. A., and Semenova, V ., Krasnodar Vsesoiuzn Nauch. I
Issled. Inst. Tabach. Bull. 33 (1927).
Wenusch, A,, “Der Tabakrauch”, Bremen, A. Geist. 1939.
Influence of Nitrogen Oxides on
TOXICITY of OZONE R. D. WATSON1, Cornel1 University, Ithaca, N.Y
T
HE amount and importance of nitrogen oxides produced by ozonizers using a silent discharge have been a subject of controversy. The matter was brought t o the front by Thorp (6) who c,oncluded t h a t ozone plus nitrogen oxides may be more toxic than ozone alone. H e reported t h a t a mixture of ozone at a concentration of 3 parts per million, containing 47% oxides of nitrogen, had bactericidal properties, but pure ozone did not exhibit bactericidal properties below 50 p.p.m. This percentage of nitrogen oxides is a high yield as compared with other measurements reported in the literature. I n the interpretation of the tests of toxicity of ozone and nitrogen oxides t o Escherichia coli, Thorp apparently did not consider the possible effects of increased acidity which may have been large enough t o be toxic t o the bacteria or which may have increased their sensitivity t o ozone. I n contradiction, Ewell @) stated t h a t with dry air without sparks in the ozonizer little 1
Present address, Texas Agricultural Experiment Station, Substation
No. 2. Tyler, Texas.
nitrogen oxide is produced, no matter what dielectric is used. H e made a further statement t o the effect t h a t with a glass dielectric the germicidal action of a given quantity of ozone is the same, whether produced from dry air or from pure oxygen. H e never found more than a trace of nitrogen oxides and indicated: “There is no evidence t h a t ultraviolet light produces appreciable oxides of nitrogen in air, and for equal quantities of ozone the germicidal effect is the same, whether ozone is produced by a brush discharge in dry air or by wave length below 2000 A. in air.” Ewell (8) found t h a t ozone is highly toxic t o E. coli and that the toxicity depends on a concentration-time relation. Thorp (7), in reply t o Ewell’s criticism, interprets his data more fully. H e regards ozone a b a gas of low toxic limits. In his experiments ozone containing nitrogen oxides was much more toxic than ozone without nitrogen oxides. An ozonizer producing 4701, nitrogen oxides was used for the tests so that any variation in t h e toxicity t o the organisms caused by the oxides could be large enough to be detected easily. The general conclusion to be
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Experiments indicate that ozone is the toxic agent in ozonized air, and that there is no measurable difference in the toxicity to S.fructicola and E. coli of ozone produced from pure oxygen and from air when used at the same concentration. Nitrogen oxides have no direct effect on the toxicity of ozone. The additions of nitrous oxide and nitrogen pentoxide to ozone have no influence on the toxicity of ozone to S. fructicola spores. Of the possilde nitrogen oxides that might be produced by ozonizing air, only nitrous oxide and nitrogen pcntoxide could exist in the presence of ozone. Nitrogen pentoxide is produced by the ozonizers used and an absence of the nitrous-acidforming oxides, nitrogen dioxide and nitrogen trioxide, is demonstrated. Nitrogen oxides may affect the acidity of the test medium, in which case there could he an indirect influence on the toxicity of ozone to fungi. The acidity of the medium has a relatively unimportant effect on the toxicity of ozone over the pH range 4.3 to 7.95, but below 4,.3 there is an interaction between ozone and the acid in the medium which results in a large decrease in germination of the treated spores.
drawn from this article was that pure ozone was not so toxic t.o humans, mice, weevils, and E. coli as impure ozone. Thus there is a difference of opinion on two major issues: the toxicity of ozone itself, and the amount of nitrogen oxides produced in ozonizing a,ir and their influence on ozone toxicity. The purpose of these investiga,tions was to determine whether ozone is t80xicto fungi, t o what extent this toxicity is due to or altered by oxides of nitrogen, which oxides are present in ozonized air and the relative importance of each, and t o establish the basis of the effect of nitrogen oxides on the toxicity of ozone t o fungi. MATERIALS AND METHODS
I n the experiments reported, it was necessary to use a source of ozone that was free from all nitrogen oxides. The ozone, therefore, was made from electrolytically produced oxygen which gave no positive tests for nitrogen, and the ozone mas produced by a metal-glass-metal dielectrode commercial ozonizer (supplied by Electroaire Corporation) having a silent blue-corona discharge. The ozone mixtures were diluted greatly with air before they were passed through the test chambers in order to avoid the toxic action of high concentrations of oxygen and to make comparable the tests of the toxicity of ozone produced from pure oxygen and from air. The apparatus was made of glass. Gas flowmeters were used to regulate the concentrations of the gaseous mixtures. The gas mixtures were divided and equalized by orifices into the two test chambers, large glass desiccators. The gas mixtures entered the chambers at the bottom and were removed a t the top t o ensure circulation. A gas mixture could be equally divided and used in separate chambers for duplicate tests or another gas, such as a nitrogen oxide, could be added to one of these chambers and a comparison made between a n ozone-air mixture and a n ozoneair-nitrogen oxide mixture. The test organisms were Sclerotinia fructicola (Wint.) liehm and E. coli. The water was freshly redistilled in a Pyrex still and had a p H of 6.5 with more than a million-ohm resistance. The chemicals were all reagent grade. The method of spore germination was modified from Lin (4). Unless otherwise stated, the washed spores in 5 ml. of redistilled water or 5 ml. of the diluted bacterial suspension were exposed in Petri dishes to a continuous flow of a large volume of air and ozone in the test chambers. To measure the nitrogen oxides the ozonized air was passed through towers with sintered-glass plates containing a solution of sodium hydroxide which completely absorbed the nitro en oxides forming the relatively stable nitrate or nitrite salts. %itrous oxide is not absorbed by sodium hydroxide. The Griess test specific for the nitrite ion and sensitive t o 1 part salt in 100,000,000 parts of solution was used for the determination of the nitrite ion. The test for the presence of nitrate ion was tho blue color reaction with diphenylamine in concentrated
Vol. 36, No. 6
sulfuric acid. Since many oxidizing agents give a positive reartion, this test is not specific for nitrates. The interfering oxidizing agents were therefore removed by evaporation. The test is sensitive to 1 part of nitrate (or nitrite) in 1,000,000 parts of water. KO tests mere made to determine whether nitrous oxide was present in ozonized air. I n all cases comparable checks were made on the unozonizec! air to determine whether small concentrations of nitrogen oxides were present in the air supply. -411 tests on the air supply n w c negative. Two methods mere followed in comparing the toxicity of ozone produced from oxygen with that froin air, or a n ozone mixturr containing nitrogen oxides. First, ozone produced from oxygen was compared with the toxicity of a similar amount of ozone produced from air using the same ozonizer. Second, a flow of the same ozone stream was equally divided into two chambers, to one of which nitrogen oxides were added. The effect of the hydrogen-ion concentration of the .medium on the toxicity of ozone to S. fructicola spores was studied, using a 0.1 M potassium phosphate buffer. I t was desirable to keep the test medium simple to minimize any react,ion between ozon(i and large changes of hydrogen ion and substances in the mediim which might influence spore germination or the toxicity of ozone to the spores. The phosphate buffer was chosen because it is simple and the phosphate and potassium ions are known to h a w no important effect on germination. Each Petri plate contained 1 ml. of buffer and 4 ml. of spore suspension. The plates were exposed for 4 and 6 hours to a strea.m of pure ozone and air in the same chamber. One t8hirdof the plates served as checks on the effect of hydrogen-ion concentration alone on germination. T h e pH values of the media before and after treatment were defrimined by a Beckman glass electrode. KO change in pH occurred during treatment. A solution of potassium nitrate equivalent to the highesl concentration of pot,assium ion in the buffered solutions was included in the tests t o determine whether there was intcractiori between the potassium ion and ozone. More detailed methods and results were given in the author's thesis (8). Factors affecring the toxicity of o ~ o n elo fungi other than nitrogen oxid and the hydrogen-ion coirccrrt,rat,ionwere discussed in a r paper (9). RESULI'S O F
ozoxizmc
Kitrous oxide, nitrogen pentoxide, a.nd possibly the interrtietliate nitrogen peroxides are the only nitrogen oxides that can exkt in the presence of ozone. Tho others are oxidized t o higher forrw (5). If these oxidations were not rapid and complete, we wouid expect to find the salts of nitrous mid in a sodium hydroxide solution through which a mixture of ozone and nitrogen dioxide or nitrogen trioxide was passed. Negative tests for nitrites were obtained on such a solution. Similar results were obtained by GorodetskiX (S). Kitrous oxide is riot oxidized by ozone t o the acid oxides ( I ) . Nitrogen peroxides are said to be formed when nitrogen dioxide and oxygen pass through an ozonizer ( 1 ) . Because these nitmgen peroxides are apparent*ly short-lived or are merely intermediates, they are thought t o ha,ve little effect on the toxicity of ozone. Any effect of thew peroxides would have been includfi1 in the measurement of the toxicit,- of mixtures of nitrogen ppntoxide and ozone. TOXlClTY TO SPORES
The effect of nitrous oxide on toxicity t o spores of 8.fructwoict was determined by exposing the spores for 24 hours t o concentrations of 50 and 99.6% nitrous oxide. Nitrous oxide was found t o have no effect on germination of S.fructicola spores. When added t o the ozone stream it did not alter the toxicity of ozone to fungi or bacteria (Table I). Even when the concentration of nitrous oxide was 2 parts per 1000 parts of air, which was about onc hundred times that of the ozone concentration, there TYRS no noticeable difference in the toxicity of the two gas mixtures. Although no nitrous-acid-forming gases, such as nitrogen dioxide and trioxide, were found in ozonized air, the influence of these compounds on ozone toxicity was tested. Xitrogen dioxide was used and a t high concentrations was found to be toxic t o fungus spores. In water it forms both nitric and nitrous acid
