A NEW TYPE OF GASEOUS FERMENTATION OCCURRING DURING THE SALTING OF CUCUMBERS by JOHN LINCOLN ETCHELLS A THESIS Submit ted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Bacteriology ACKNCV/LEDGEMSNT The writer wishes to express his sincere appreciation to Dr. F. W. Fabian for his keen interest and helpful advise through­ out this investigation. Also, the writer is deeply indebted to those of the Agricultural Chemical Research Division of the Bureau of Agricultural Chemistry and Engineering of the United States De­ partment of Agriculture for making this study possible. Further­ more, grateful acknowledgement is made for the excellent coopera­ tion of the Department of Horticulture of the North Carolina, Agri­ cultural Experiment Station and especially to Dr. Ivan D. Jones, collaborator in the commercial phases of the work. Finally, thanks are due the Chas. F. Cates and Sons Company of Fa,ison, N. C. for the many facilities ma.de available throughout the salting studies. CONTENTS INTRODUCTION 1 HISTORICAL REVIEW 3 ISOLATION STUDIES 11 IDENTIFICATION STUDIES Procedure IS Morphological 22 Cultural 22 Physiological 23 Acid production 30 End-products 31 PRELIMINARY BIOCHEMICAL STUDIES Procedure 5I4 Results 61 PRINCIPAL BIOCHEMICAL STUDIES Procedure 71 Fermentations by different strains 7I4 Fermentations by the same strain 75 Effect of temperature 76 Effect of pH 78 Effect of salt 80 Effect of heating the culture medium 83 Effect of dextrose on themaltose fermentation 89 Fermentation of various carbon compounds 89 Effect of heat on the viability of cultures 93 CONTENTS (continued) FERMENTATIONS UNDER COMMERCIAL CONDITIONS 112 Procedure Fermentations in 60° Sal. "brines 118 Fermentations in ^40° Sal. brines 121 Fermentations in 20° Sal. brines 128 SUMMARY AND CONCLUSIONS lLO LITERATURE CITED lL 6 - INTRODUCTION Approximately 6,000,000 bushels* of pickling cucumbers are raised annually in the United States, representing a cash income of about $3,600,000 to the growers. Since only a small acreage, usually one to two acres, is planted per grower it is estimated that approxi­ mately 60,000 farmers are benefited by the pickling industry. The commercial salting procedure** in brief is as follows: Large wooden vats of 400 to 1000 bu. capacity are filled with green cucumbers, fitted with a false head and Ho per cent saturated salt brine added so as to come a few inches above the heads. The initial salt concentration is raised according to a set schedule so that the salt concentration reaches 50 to 70 per cent saturation by the end of the sixth week. An acid fermentation commences shortly after the cu­ cumbers are brined and continues for from two to six weeks. The initial inoculum of microorganisms for the fermentation comes from the cucumbers and from adhering particles of soil. The salt— tolerant organisms utilize as their nutritive material the solu­ ble constituents that diffuse into the brine as the result of the ac­ tion of salt on the cucumber tissue. The action of the microorganisms on the fermentable material in the brine brings about the production of various compounds (chiefly lactic acid, also acetic acid and alco­ hol) as well as the evolution of considerable quantities of gas. At the completion of the curing process (about 3 months), the cucumbers have changed from the green, opaque, air-filled, bouyant fruit to olive colored, translucent, air-free salt stock. * Agricultural Statistics, U.S.D.A., Washington, D. C. (1939). ** For M-0 per cent saturated brine treatment. -2It is clearly evident that the curing process is one of complex bacteriological, chemical and physical changes. Any one of these changes constitutes adequate basis for considerable fundamental inves­ tigation in an attempt to gain a better understanding of the principles involved during curing. Such studies would go a long way in placing the pickling industry in the group of scientifically controlled fermen­ tations . For the past 4o years research has dealt principally with the bacteriology of the lactic acid fermentation, with a very small propor­ tion of the work being carried out under plant conditions. Little or no attention has been given to the fact that probably fermentations, due to microorganisms other than the lactic acid bacteria, existed and could contribute to the general fermentation. That such is the case is evi­ denced by the fact that the true yeast fermentation, associated with the general cucumber fermentation, was not recognized by workers in this field until as late as 19^40 (12). Similarly, combined bacteriological and chemical investigations upon cucumber fermentations at certain salt concentrations point toward the inclusion of still another phase in the fermentation proper. The latter phase is brought about by a group of gas-producing salt-tolerant microorganisms and can be detected most easi­ ly by the evolution of ga.s which is composed of approximately equal parts of carbon dioxide and hydrogen. The isolation, identification and physiological studies of this group of organisms as well as the observations dealing with their typical fermentation under plant conditions constitute the basis for this study. -3- HISTORICAL REVIEW The literature on cucumber fermentations will be reviewed chiefly with respect to the bacteriological aspects of the fol­ lowing topics: Lactic acid fermentation; yeast fermentation and the hydrogen fer­ mentation. Until recently, bacteriological investigations on brine cucumber fermentations (for salt stock) have dealt principally with that phase of the fermentation resulting chiefly in the production of lactic acid. As early as 1S99 Aderhold. (l) studied the acid fermentation in dill brines and found that it was fostered by anaerobic conditions and that such conditions resulted in the production of more acid. In 1909 Kossowicz (2U) confirmed Aderhold's work and also studied the flora of the active fermentation in dill brines. In addition experiments on the use of pure cultures for starters for dill pickle fermentations were car­ ried out. The observations of these workers, while conducted on dill brines, are particularly interesting and can be considered of fundamental significance. Lactic A cid Fermentation.- Prior to 1913 there was little or no work published in this country on cucumber fermentations. In 1913* Rahn's (29) bacteriological study of cucumber fermentations revealed that actively fermenting brine contained as many as 200,000,000 acidforming bacteria per cc. and the acidity of the brine at the end of ac­ tive fermentation reached 0.6 to 1.2 per cent lactic acid. Brown (S), in 1916, in an abstract of an unpublished article, made a series of observations covering many aspects of cucumber fer­ mentations. In reference to the production of acid, he describes the -ir­ responsible microorganisms as short rods or cocci, arranged chiefly in chains and being facultative with respect to oxygen requirements. He further observed that the ratio of lactic to acetic acid was 2 to 1 and that other acids, propionic, butyric and benzoic occurred in traces. During the years 1919, 1920, 1922, Le Pevre (25, 26, 28) re­ ported his observations on a series of experiments. He pointed out that cucumbers, as they came in from the field carried numerous bac­ teria, the principal groups being; "lacto-bacilli", aerobic spore- formers, gas-formers, yeasts and molds. The "lacto-bacilli" were claimed to be the most tolerant of salt and were considered the most significant in bringing about acid production. Thirty degrees C. was found to be the optimum temperature for the "lacto-bacilli". The ad­ dition of fermentable sugar was reported to be advantageous in bring­ ing about a quicker onset of the fermentation and resulted in a higher degree of acidity. Le Fevre also pointed out that a higher acid con­ tent developed in the higher as compared to the lower salt content brines. Campbell (9 ) reported (a) that the microorganisms responsible for the souring of milk were concerned with the cucumber fermentation; (b) that the lactic group of organisms should be supplied with oxygen; (c) that yeasts played a desirable role in the fermentation. Le Fevre (27) took issue with Campbell (9) because of the letter's misconception of the bacteriological changes during cucumber fermentations. A careful inspection of Campbell's report (which included no data) shows that he was undoubtedly confusing true yeasts with Mycoderma (false yeasts) originating from the heavy surface scum. -5Tanner (36 ), 1926 , in a discussion of the curing process, claimed that the brine acidity resulted mainly from volatile acids rather than lactic acid. He was of the opinion that while lactic acid was generally mentioned as the predominating acid, this was usu­ ally done without adequate chemical examination. The curing process was said to be chiefly an acid-gas fermentation. Fabian (13), in 1930. presented a general discussion of the con­ siderations of pickle manufacture and referred principally to the ob­ servations of Le Fevre which have been discussed previously. Fabian also pointed out that the production of cucumber pickles was a scien­ tific process and a thorough knowledge of the fundamentals was essen­ tial for successful manufacture. The influence of salt was stressed both from the osmotic effect for withdrawing food material for bacteria and for its effect in encouraging lactic acid ba.cteria to the exclusion of putrefactive bacteria. During the same year Joslyn (23 ) presented a general article in relation to pickle manufacture. His discussion of the lactic fermentation was the same as that of earlier authors. It is well to point out that the work on the acid fermentation of brined cucumbers up to about 1930 consisted for the greater part of general discussions and opinions as viewed by the various authors and directed toward the industry with very little bacteriological or chemi­ cal data included as supporting evidence for the conclusions that were made. However, the work definitely presents many interesting and valu­ able contributions to the general understanding of the bacteriological processes of brine cucumber fermentations. Tanner and Eagle (37) in an excellent general review of the literature through 1926 more or less -6summed up the situation when they stated, "Despite the fact that this fermentation is an old one, much research may yet le done and must be done before it may be considered a controlled fermentation industry. There is a great need for combined microbiological and chemical inves­ tigations." They might also have added that there was a great need for investigations to be carried out under conditions which could be con­ sidered typical of the industry, a point which up until 1930 had not been given serious consideration. Although a routine differential plating medium suitable for de­ tecting acid-forming organisms (weak and strong and peptonizing bacteria) was reported and described in great detail by Ayers and Mudge (U) in 1920 , no adaption was made of this medium to cucumber fermentations for a full 12 years. Fabian and co-workers (l*+), in 1932, in perhaps the first systematic study of the microflora and chemical end-products of cucumber fermentations, used this medium to advantage in determining the populations of acid-forming organisms occurring in brines of in­ itial strength of 8 and 10.6 per cent salt concentration. In addition to the study of the effect of acids, bases and salts on the fermenta­ tion, chemical determinations were carried -out during the fermentations with respect to total acids, volatile acids, alcohol and reducing sug­ ars. The results of the investigations, in general, showed that the flora of the fermentation with respect to acid-forming organisms con­ sisted chiefly of weak acid-formers and that there was a greater number of acid-formers in the weaker brines, and they reached a maximum sooner than in the stronger brines. The chemical analyses showed that there v/ere greater amounts of non-volatile acids, volatile acids and alcohol in the weaker brines as compared, to the stronger brines. In 1935. Vahlteick, Haurand and Perry (38) attempted one of the first bacte­ riological studies conducted under commercial conditions. They stud­ ied the microflora during the curing of cucumbers salted at a brine concentration of 10 per cent. The data from their limited observa­ tions on two vats led them to believe that the following groups of microorganisms were present: type; High acid producers of the Lactobacillus low acid producers of the Leuconostoc type; round yeasts; ellip­ soidal yeasts (scum yeasts); spore-forming bacteria; and unclassified. The formation of acid during fermentation was attributed to acid-pro­ ducing organisms identified as Lactobacillus cucumeris and to two species of the Leuconostoc genus. Wustenfeld and Kreip (*+2), 19,33. divided the lactic acid bacteria into the following genera; Bacillus, Streptococcus, and Pedococcus. Furthermore, they stressed the desira­ bility of the addition of pure cultures as starters subsequent to thorough washing of the cucumbers at the time of salting. Jones (21), in 19*4-0, carried out extensive studies over a three year period at a commercial pickling plant on the salting of cucumbers in barrels. Fer­ mentations In initial brine concentrations of 20, 30. ^0, 60 an(i SO per cent saturation brought about increased acid production in the reverse order named. In addition, he proved that higher acidity resulted from the fermentations (at any given salt concentration) when cucumbers of the smaller sizes were employed. The reverse was true when the larger sizes of cucumbers were used. It is of interest to consider the effect of added sugar on the lactic acid fermentation. Numerous investigators (10, 15, l6 , 22, 26, -g- *4-2) have suggested that beneficial results may be obtained by the ad­ dition of sugar to cucumber fermentations and that these benefits may be reflected in an accelerated rate of acid production or in an in­ creased production of lactic acid. Also, it has been reported that both acceleration and increased acid production can be obtained. ever, How­ in some cases it would seem that not a sufficient amount of work was carried out under conditions- typical of the industry to justify the extensive conclusions presented. In contrast to those reporting bene­ ficial results, Vahlteick, Haurand and Perry (38) were unable to stimu­ late the acid fermentation by the addition of sugar to some of their commercial vats. Veldhuis and co-workers (*40) were unable to demon­ strate a significant increase in brine acidity in the resulting fermen­ tations to which sugar was added over that of the control lots. In their experiments sugar was added to different fermentations as follows: At the start, after 10 days, and in small amounts at short intervals during the fermentation. These investigators emphasized the importance of conducting well replicated experiments with adequate controls before conclusions might be drawn safely concerning the influence of addition of sugar to brines at the start or during fermentation. Yeast Fermentation.- Although true yeasts have been mentioned in the literature in connection with cucumber fermentations (Kossowicz (2*4); Hasbrouck (19); Riley (31); Brown (8 ); Le Fevre (27,28); Joslyn (23); Vahlteick et al. (38); Campbell (10) ), no systematic study as to their populations in brine was recorded until very recently. In 19*40, Etchells (12 ) showed that a part of the typical fermentation of cucumbers was brought about by yeasts. Yeast fermentations were found in brine treat- “ 9~ ments of 20 , J>0, 40 and 60 per cent saturation with respect to salt. It was shown that there was no direct correlation between hrine con­ centration and maximum number of yeasts present. In an earlier report, Veldhuis and Etchells (39) carried out probably the first systematic study of gases evolved during the fermentation of cucumbers salted un­ der what could be called commercial conditions at various salt concen­ trations (20 to SO per cent saturation). A definite correlation was reported between carbon dioxide production and the presence of typical yeast fermentations. Other interesting points of the above work, par­ ticularly that dealing with the presence of hydrogen in the evolved gases, will be discussed under the following topic heading. Hydrogen Fermentation.- A thorough review of the literature on cucumber fermentations up until 1939 reveals no study, either chemical or bacteriological, dealing with that phase of the fermentation during which a mixture of carbon dioxide and hydrogen is evolved. However, it may be noted that hydrogen was mentioned casually upon at least two oc­ casions when it was claimed to be a product of the undesirable micro­ organisms at the start of fermentations (3, 27)- Upon another occasion it was claimed present among numerous other by-products (2 3 ). The first conclusive evidence that showed hydrogen represented a portion of the evolved gas from certain cucumber fermentations was presented by Veldhuis and Etchells (39) i-n 1939- this work it was shown that hydrogen was produced in considerable quantities in all fermentations observed in 60 per cent saturation brines. Examination of gas from hollow cucumbers or "bloaters" from 60 per cent saturation brines showed it to have about the same composition with respect to carbon dioxide and hydrogen as the -10corresponding surface gas. The bacteriological investigations re­ ported were of a limited nature. It was pointed out however, that an organism could be isolated from this typical hydrogen fermentation which was capable of producing a relatively high percentage of hydrogen. -11- isoLATioH s t u d i e s From the historical review it is evident that, prior to 1939, no study had appeared dealing with either the chemical or "bacterio­ logical aspects of that phase of cucumber fermentations, during which hydrogen constituted a portion of the evolved gases. The original study (3 9 ). which dealt principally with analyses of the evolved gases from cucumber fermentations, showed that hydrogen represented a portion of the evolved gases, especially those carried out at 60° salometer brines*. The above report also mentioned that an organism could be iso­ lated which was associated with the production of hydrogen. It is of interest here to review somewhat completely the bacteriological prob­ lems involved during this work inasmuch as it was through continuation of the initial observations that the stock cultures were subsequently isolated and used to constitute a portion of the present work. The first observations (1937 season) on the gases evolved from cucumber fermentations were undertaken at the field laboratory located at the plant of the Chas. F. Cates Co., Faison, N. C. The various salt­ ing procedures were carried out in vats of approximately 85 b u . capacity Additional observations on commercial fermentations were made at the M t . Olive Pickle Company, located at M t . Olive, II. C. During the study of the composition of the evolved gases, it was found that, in addition to carbon dioxide, hydrogen was present in the gases from some of the fermentations. During this period, the bubbles forming on the surface of the vat brine would explode with a distinct report when lighted with a match. * Per cent saturation with respect to salt. -12Numerous attempts were made during the 1937 season to isolate the causative microorganisms from the fermentations evolving hydrogen hy the ordinary plating methods available, hut all proved futile. During the following season (1933), a vat salted at Uo° salometer and located at M t . Olive, a short distance from the field labora­ tory, was found to have a vigorous gaseous fermentation two days after having been put down and the gas analyses showed a relatively high per­ centage (3 7 .O per cent) of hydrogen present. Since this fermentation was thought to present an ideal opportunity for bacteriological study, plate counts were made at this time using nutritive caseinate agar (Difco) and tartaric acid agar. These media revealed the following groups of organisms present per cc. of brine: 22 million tiny acid- formers, 200 thousand peptonizing bacteria of the soil type, and 200 yeasts. Numerous isola.tions of the predominating organisms present v/ere made and tested in fermentation tubes, using cucumber juice broth as the liquid medium, but no visible gas was produced by any of the re­ sulting fermentations. Hence, it was assumed that the organisms pre­ dominating in the fermentation, as evidenced by the plate counts, were not responsible for the hydrogen evolution. Also, it was obvious that the routine aerobic plating methods ths.t had been devised for following the brine fermentations with respect to acid-formers, peptonizers and yeasts were not suitable for isolation of the hydrogen-producing organ­ isms. Whether the difficulty was due to growth requirements of the or­ ganisms in question or due to interference by other groups present in the brine, remained to be determined. Since the fermentation in t h e v a t described above w a s r e l a t i v e l y -13short, and also, since it was not on the premises where the laboratory was located, no further observations could be carried out. However, later on, in other vats, a more intensive effort was made to cultivate the microorganisms responsible for the hydrogen evolution. This in­ volved numerous platings and the employment of va.rious types of solid media. Finally, it was demonstrated that by use of strict anaerobic conditions*, a suitable differentiation of the brine microflora could be obtained and the organisms revealed in sufficient numbers so that typical colonies could be picked and studied. Once the colonial char­ acteristics were established, numerous strains were isolated by this method. Since the organisms proved'to be facultative anaerobes, it was obvious that other cultural difficulties prevented reasonably easy iso­ lations by ordinary aerobic cultural methods. Later studies showed that the plate counts indicated that the hydrogen-producing organisms were present in the brines in relatively low numbers as compared to the total numbers of microorganisms present. Also, it was found that the use of a culture medium suitable for the growth of the acid-forming bacteria, resulted in a low pH of the medium, which was inhibitive to the hydrogen group. Furthermore, cluded the acid-forming bacteria, the employment of a medium which ex­ encouraged the spreading, aerobic spore-forming types, making the plates valueless for numerical counts or isolation of the organisms producing hydrogen. The latter difficulty was particularly true under laboratory growth conditions prevailing at * Large desiccators used for anaerobic jars, freed of oxygen by the ad­ dition of excess of alkaline pyrogallol mixture followed by drawing a vacuum of 15 to 20 inches of mercury. (See Fig. 1.) -l4~ Tig. 1. Anaerobic culture jars used in the isolation studies. Desiccator, vacuum type °f Pyrex glass with tubulated cover, inside diameter of 2^0 mm., capacity about 2b Petri dishes. Opening in cover supplied with a two hole, No. 8 rubber stopper fitted with two pieces of ordinary glass tubing; a straight piece running to the bottom of the jar, used for the addition of tne alka­ li and a short, right angle piece for drawing and releas­ ing vacuum. Both tubes supplied with a short piece of vacuum rubber tubing and a screw clamp. Solutions in the order used; (a) U 00 cc. of pyrogallol solution, (lk2 g, per liter) placed in the bottom of the jar; (b) U 00 cc. of KOH solution (222 g. per liter) added through the straight tube. the field station during the hot, humid weather which is typical during the summer months of this section of North Carolina. Due to the type of spreading growth of the hydrogen-producing organisms (under the above climatic conditions) numerical counts of the colonies were further com­ plicated since a surface colony might spread in a film over the surface of the agar, the same being true for the bottom colonies. spite of the described cultural difficulties, However, in it was found possible upon a number of occasions to identify colonies of these organisms from the routine, aerobic brine platings. This was facilitated after having be ­ come thoroughly familiar with the various forms of colonial growth. During the 1938 season, the first attempt was made to reproduce the typical hydrogen fermentation by inoculation. A series of four 15 gal. kegs was divided into duplicate lots of two kegs each which were filled with discarded cucumber ends from the manufacture of fresh cucum­ ber pickle. Both lots were salted at 60° salometer brine concentration and were maintained at that salinity. an actively fermenting, Inoculation was with ^00 cc. of cucumber juice culture of one of.the hydrogen- producing strains, growing at 20° salometer brine concentration. The brine pH of one lot was kept at U.O to *4.5, while the other was left u n ­ adjusted. Typical hydrogen fermentations resulted in the unadjusted lot while vigorous yeast fermentations developed in those in which the pH was controlled. No claim is made with respect to complete reproduction of the hydrogen fermentations solely by inoculation since the organisms may have been present on the cucumbers in sufficient numbers to bring about such fermentation even though the ends received a thorough washing. This -16possibility was clearly pointed out in the case of a ^5 ends that was salted at the same time as the above lots. • cask of The brining xvas carried out at 50 ° salometer and a most vigorous hydrogen fermen­ tation was found on the second day after having been put down. How­ ever, these inoculated fermentations are worthy of mention since they gave considerable opportunity for close observation of the fermentation as well as providing the source of some of the strains of the isolated organisms. A total of 20 strains (Table 1, part A) were isolated by the anaerobic cultural methods from cucumber fermentations during the 1938 season. Of this number, eight were isolated from UoD salometer fermen­ tations, nine from 50 ° salometer fermentations and three from 60 ° salome­ ter fermentations. During the following season, nine additional strains were added to the collection of stock cultures (Table 1, part B). This group was divided as follows; two from 20 ° salometer fermentations and seven from 60° salometer fermentations. The strains isolated during 193S were studied in detail, while those isolated during 1939 were used chiefly for comparative studies, particularly in reference to gas evolu­ tion and composition. -17Table 1. Origin of Cultures Isolated from Cucumber Fermentations. 1 1 * A. 193S Season ' B . 1939 Season ! 1 1 1 1 1 1 Strain ' Brine con— ' Strain 1 Brine con1 centration ' 1 No. No. ' centration ------------ -1------------------Ul------------- 1—------------1 i ' 0 sal. 1 0 sa l. ' H-138 ' Ho 1 60 H-139 I I t ) 1 1 H-238 1 Ho ' 1 1 ' h- 3 3 8 1 I I ' H-H^S ' I Ho • H- 53S ' Ho H- 63 S H-738 H-S38 ' s H-1138 1 H- 123S H- 133S H- 1H38 H- 153S H -1638 1 > 1 ' H -1738 H -133S H -193S H -2038 1 1 1 • ' ' ■ 1 ' 1 1 H -2138 H-2238 1 1 •' H-339 60 ' H-H39 ' 60 H -639 I 1 ' t 1 1 1 1 ! ' 60 ' t t 1 1 I 20 H-739 t * t t 1 1 ' 1 60 H-539 1 ' 1 1 1 1 1 1 H~S39 60 1 t ' t ' 1 t t 1 1 1 1 1 1 ’ > <50 * 1 t ' 1 1 50 60 1 1 ' 50 t I 50 50 1 1 1 ' 50 t t 50 50 ' x 1 1 50 20 1 | t 60 I 1 t 1 « 1 60 1 t 1 1 t 1 • Ho f t • • I • ' « 1 ' 1 u ' ' | 1 | t t 1 1 Ho 1 t 1 ' ' t I * Ho I t ' Ho I 1 ' ' ' | t 1 ’ 1 H-239 | | 1 ■ J I ' ' ■ 60 H-939 ' 1 t * 1 1 ! 1 1 1 1 j 1 — 18- IDEFTIPICATIOF STUDIES Twenty strains (Ta,ble 1, part A.) were studied in detail with respect to morphological, cultural and physiological characteristics. The procedure followed, for the most part, was according to the recom­ mended bacteriological methods* (3*0 • When a particular method for a test or procedure different from this was used, the reference is indi­ cated by number. During the study dealing with the cultural characteristics ob­ served on the 10 solid and liquid media employed, complete observations with respect to the various characteristics were made for each of the 20 strains on each type of medium. This material was then analyzed and finally condensed into what might be called the typical characteristics for the type strain (H-1^3S) on the various media. presented (Tables 3 ~ H ) This material is accordance with the prescribed methods (S.A.B.). In the case of the biochemical or physiological tests, particu­ larly where an observation is recorded as either positive or negative, no attempt was made to list such reactions, for all 20 strains since for the most part they were either of one reaction or the other. In cases where clear cut differences were noted, the number of strains reacting in each direction is pointed out. It will be noted that strains H-13& and H-238 showed the most variance from the typical characteristics rep­ resentative of H-l^i-32 , particularly with reference to the following tests; methyl red, uric acid, and acid production. * Society of American Bacteriologists: Manual of Methods for Pure Cul­ ture Study of Bacteria. S.A.B., 7th ed., Geneva, N. Y-, 1932- -19The combined, morphological, cultural and physiological charac­ teristics of the 20 strains are presented in the summarized discussion of results. For the sake of completeness, additional data are included in the summary of results (such as fermentation reactions on the various carbon compounds) although this information is not fully discussed u n ­ til subsequent sections of this report. After establishing the purity of the cultures by successive platings and prior to inoculation onto the cultural media, the cultures were given two transfers; first on nutritive caseinate agar (with 8.0 cc. of 0.4 per cent brom cresol purple per liter) and then in cucumber juice broth. were made. From the latter, the inoculations onto the va.rious media A similar set of broth transfers was used at 24 hours of age for the morphological, staining and motility tests (Table 2). Growth on the initial transfer stabs was best at the top, being gray, glistening and entire, with alkaline reaction extending down into the stab. Growth in the stab was out from the line of puncture and was lobate to papillate. tube. An acid reaction prevailed in the deep part of the Gas was formed in some cases in the base of the tube and in other cases was also present as small bubbles in the surface growth. gas formed in the base of the stab, When the the amount was sufficient to split the agar and raise the stab to the top of the tube. Transfers made from these stabs into cucumber juice broth showed moderate growth in 12 hours and good to abundant growth in 24 hours. this time a delicate pellicle or film appeared. The turbidity and the amount of sediment markedly increased during the 12 hour period. Gas At -20formation caused the broth to be heavily charged and when shaken, a foam of two to three cm. in height would rise up the tube. remained turbid several days. The broth If the pellicle was disturbed, to be submerged, a new one formed in 24 hours. tening and sometimes flaky in appearance. so as The pellicle was glis­ Gas production was most vig­ orous during the first 2k to 48 hours. At ~{2 hours of age the cucumber juice transfers, described above, were inoculated into the following media and incubated at 35° C. Ob­ servations were recorded at three days, one week and two weeks. Culture medium PH ~ J"L“""1" ""' • 1 Nutritive caseinate agar slants (Difco) 6.6 1 Nutrient agar slants 6.2 3 i i 1 Dextrose-tryptone agar slants 1 (Difco) 6.8 ' 6.8 Cucumber juice agar slants* 1 'Table reference N o . 1 4 ' 5 i 1 Cucumber juice broth* i t Nutrient broth 1 Potato slants 1 Brom-cresol-purple milk 1 Plain gelatin stabs 6.6 6.8 • . ■ 6.6 1 1 1 6 1 ' 1 7 ' t 8 1 9 1 1 i 1 10 i 6.8 i ! 1 1 ' ' 1 1 » ! 11 ' i 1 t * Expressed juice from fresh cucumbers adjusted to pH 6 .8 , no addition­ al nutrients added. Por solid medium, 15 g. of agar added per liter of juice. Inasmuch as the descriptions (Tables 3 - H ) 01" the growth reac­ tions on the various media are self explanatory they will not be dis­ cussed in detail but will be summarized later. However, attention is -21called to a few of the more significant characteristics which are more or less typical of all strains. They are briefly as follows: (a) In nutritive caseinate ags.r stabs plus brom-cresol-purple, the surface growth appears to be about 0.5 cm. in area on the top of the stab; u n ­ derneath, the reaction is alkaline about one-third the way down the tube and acid in the lower part of the tube and may be accompanied by gas production. (b) In plain agar slants, there is filiform growth with typical cross-hatching or net-like structure which is irriaescent and translucent in character. Also, on this medium after about one week's time, there appears a spiked, feathery growth, down into the agar at irregular intervals along the slant. (c) In cucumber juice broth, there is rapid growth with pronounced turbidity and sediment. A deli­ cate film or pellicle follows ring formation; the broth is heavily charged with ga.s, rising up in a foam when shaken. Cultures remain tur­ bid for a number of days and have a large amount of sediment which is viscid in character. The odor of the cultures is of a sweetish charac­ ter and tends to be aromatic, resembling a yeast fermentation. After prolonged incubation on nutrient agar, the odor tends to become mildly putrefactive. (d) Growth in brom cresol purple milk shows slight acidi­ ty at the first reading (3 days) ■which increased after the one week pe­ riod and at two weeks there is coagulation with acid curd and evidence of trapped gas bubbles in the curd. (e) Gelatin stabs show slight to moderate growth at first, with a 0.5 cm. area of growth at the top of the stab. tube. Lenticular pockets are present one-third the wray down the Later, at two weeks, liquefaction starts from the top and the sur­ face of the gelatin drops down in a. cone shape. Marked liquefaction is found at three weeks. (g) Gas production in solid media containing dextrose is evidenced by the breaking up of the agar and the pushing up of the slants into the mouths of the tubes, also, by gas bubbles sometimes present directly in the surface growth on the slant. The summarized data with respect to morphological, cultural and physiological characteristics are as follows: Morphological Rods, 0.5 x 1.0; 0.75 x 1*5; 1-0 x 2.5 microns, occurring singly, in pairs, in chains of three elements or masses and groups. Rods appear rounded on the ends and show indications of bi-polar staining. In all cases (20 strains) the majority of the cells are of the smaller size. In the masses and clumps, a viscid material. it appears that the cells are held together by They are gram negative, non-encapsulated and motile. In old cultures (eight months) the cells appear slightly smaller although no other changes were noted and no spores were found. Cultural Nutritive caseinate agar colonies: Growth on the surface may spread in a flat veily film covering the whole agar surface in the cul­ ture dish, also colonies may range from 1 to 7 cm. in diameter in fleecy, arborescent, branched filaments of growth which are irridescent and trans­ lucent. Sub-surface colonies are small, O .5 x 1-0 x 2.0 mm. in size, lenticular in shape. However, growth on dextrose and plain agars (Rigs. 2 and 3 ) may vary somewhat as to the colonial characteristics described above, chiefly with respect to spreading of the colonies and the branch­ ing type of growth. In general, growth on the latter media is more reg­ ular, without pronounced branched filaments. Fig. 2. Surface, sub-surface and bottom colonies from a s t o c k c u l t u r e o f s t r a i n H-IU 3 S, h o u r s o l d , on n u t r i e n t agar . -25N u t r i t i v e c a s e i n a t e agar s l a n t s : s l i g h t l y spreading at base o f s l a n t , tening, fla t, Growth a bu nd an t, f i l i f o r m t o e d g e s f i n e l y l o b a t e t o wavy, smooth t o s l i g h t l y c o n t o u r e d from c e n t e r l i n e , v i s c i d to b u ty r o u s . Odor s w e e t t o a l c o h o l i c . g lis­ tran slu cen t, Acid r e a c t i o n i n b u tt o f s l a n t and a l k a l i n e b e n e a t h s u r f a c e growth; a p o r t i o n o f t h e a g a r b r o k e n or s p l i t i n b o t t o m due t o g a s p r o d u c t i o n . H utrient agar s l a n t s ; Growth mo der ate t o abundant ( n o t a s v i g o r ­ ous a s t h a t d e s c r i b e d f o r t h e a b o v e medium) f i l i f o r m , of slan t, glisten in g , fla t t o somewhat c o n t o u r e d , n e t - l i k e c r o s s - h a t c h i n g (some s t r a i n s s m o o th ) ; spreading at base f i n e l y r e t i c u l a t e d w ith irrid escen t, translucent w i t h s t r e a k b e i n g w h i t e t o g r a y i s h and b e i n g v i s c i d t o b u t y r o u s . or t o t h e medium, week, s w e e t t o a r o m a t i c odor; no gas d e t e c t e d . A f t e r one c r o s s - h a t c h i n g o r n e t - l i k e a p p e a r a n c e shows more c l e a r l y . w i t h numerous c o l o n i e s , Plates a f t e r s i x da ys i n c u b a t i o n a t room t e m p e r a t u r e have a m i l d p u t r e f a c t i v e odor ; a l s o , A f t e r two w e e k s , No c o l ­ t h e odor o f ammonia can be d e t e c t e d . th e m a j o r i t y o f t h e s t r a i n s gro w th a t i r r e g u l a r p o i n t s a l o n g t h e s t r e a k , show a s p i k e d t o f e a t h e r y e x t e n d i n g down i n t o t h e a - gar. D e x tr o s e -tr y p to n e agar s l a n t s : spreading, tening, Growth a b u n d a n t, f i l i f o r m smooth e d g e s u s u a l l y e n t i r e b u t may be wavy. l i q u i d in appearance, transparent. a c i d i n b a s e o f s l a n t and a l k a l i n e u n d e r n e a t h g r o w t h . ent i n b a s e o f tube r a i s i n g or b r e a k in g a g a r . base of the tube, orless. Growth g l i s ­ s u r f a c e smooth and t r a n s l u c e n t a s w e l l a s Streak g ra y ish w ith v i s c i d c o n s iste n c y , show an a l k a l i n e r e a c t i o n , to s w e e t i s h odo r, Gas may be p r e s ­ A f t e r one week a l l s t r a i n s i n c a s e s where t h e a g a r i s b r o k e n away i n t h e t h e low e r p o r t i o n shows a r e d u c t i o n o f i n d i c a t o r t o c o l ­ -26Cucumber j u i c e a g a r s l a n t s ; Growth a b u n d a n t , f i l i f o r m w i t h s l i g h t s p r e a d in g i n lower p o r t i o n o f s l a n t . Some s t r a i n s h a v e a wavy, tr a n s p a r e n t p e r ip h e r y a lo n g the edge o f th e s t r e a k , convex, b u t g e n e r a l l y smooth and f l a t ; contoured, tran slu cen t, irrid escen t, glisten in g slig h tly some s t r a i n s wavy t o m o d e r a t e l y gray to iv o r y c o lo r , v i s c i d to b u t y r o u s and no a p p a r e n t c o l o r g i v e n t o medium; s w e e t to a l c o h o l i c o d o r . Gas p r o d u c t i o n may s p l i t a g a r or p u s h s l a n t t o t o p o f t u b e , w i t h ' b u b b l e s b e i n g f o r m e d and t r a p p e d i n s u r f a c e g r o w t h . may d e v e l o p t h e n e t - l i k e , n u t r ie n t agar s l a n t s . in ch aracter, A f t e r one w e ek some s t r a i n s c r o s s -h a tc h e d appearance d e s c r ib e d f o r the A f t e r two w e e k s , a secondary, gr ow th a p p e a r s more s p r e a d i n g a r b o r e s c e n t gr ow th s h o w i n g from t h e e d g e o f th e primary s t r e a k . Cucumber j u i c e b r o t h : membrane o r p e l l i c l e , S t r o n g (3 p l u s ) w h ic h d r o p s or d i s i n t e g r a t e s a t e t o ab u nd an t s e d i m e n t , w h i t e , in a s w ir l. tu r b id it y w ith d e l i c a t e Gas p r e s e n t , in masses, i f distu rb ed . somewhat v i s c i d and r i s e s t h e b r o t h b e i n g h e a v i l y c h a r g e d and i f a foam r i s e s 3 to ^ CK1* i n h e i g h t . Moder­ A f t e r one week, p e l l i c l e shaken (re-grown s i n c e t u b e s were s h a k e n on t h e t h i r d d a y ) has a, g l i s t e n i n g l u s t r e . percep tib le still s w e e t i s h o do r o b s e r v e d . ex ists, A f t e r two w e ek s a m o d e r a t e c l o u d i n g s u r f a c e g r o w t h p r e s e n t but no t c o n t i n u o u s i n a l l s t r a i n s . N utrient broth: S l i g h t t o m o d e r a te t u r b i d i t y or d e l i c a t e p e l l i c l e p r e s e n t growth, i n a few s t r a i n s , (l p lu s), membrane a l s o some w i t h r i n g t h e l a t t e r h a v i n g v i l l o u s p r o j e c t i o n s down i n t o t h e b r o t h . r i n g may f a l l A in p a rt in to the b r o th . w ith ou t s u r fa c e growth. The The m a j o r i t y o f t h e s t r a i n s a r e No odor d e t e c t e d . S m a l l amount o f s e d i m e n t somewhat v i s c i d i n c h a r a c t e r when p r e s e n t i n s u f f i c i e n t amounts t o s w i r l . -27No g a s d e t e c t e d v i s u a l l y . ent c l o u d i n g . A f t e r one week, s l i g h t A f t e r two w e e k s, amount o f v i s c i d s e d i m e n t ; Potato s l a n t s : to mo der ate p e r s i s t ­ s l i g h t t o m o d e r a te c l o u d i n g , no s u r f a c e growth p r e s e n t . Growth m od er ate t o a b u n d a n t , f i l i f o r m , ir r e g u la r along the l i n e o f in o c u la tio n , glisten in g, r a i s e d , y e l l o w i s h to f a i n t o r an ge i n v a r i e d d e g r e e s strain s), some a p p e a r i v o r y t o cream i n c o l o r ; c i d t o b u t y r o u s d a r k e n i n g t h e medium. one w eek , Brom c r e s o l p u r p l e m i l k ( 1 1 ) : sligh tly smooth, s l i g h t l y (for d iffe r e n t consisten cy s l i g h t l y v i s ­ Odor s w e e t t o a l c o h o l i c . growth a p p e a r s t o b e t a n t o g o l d e n . curd a b s e n t , small A fter No change a f t e r two w eek s. S lig h tly acid in rea ctio n , acid r e n n e t curd a b s e n t , p e p t o n i z a t i o n a b s e n t , f i l m o f growth a t s u r f a c e w i t h gas b u b b l e s t r a p p e d u n d e r n e a t h . creased acid r e a c tio n , A f t e r one week an i n ­ s l i g h t c o a g u l a t i o n w i t h one s t r a i n . weeks s o l i d cur d w i t h a l l strain s, A f t e r two some e x t r u s i o n o f whey pr on ou nc ed acid r e a c tio n to in d ic a to r . Pla in g e la t in s t a b : Growth f i l i f o r m , f i n e l y p a p i l l a t e e d g e s , gas b u b b l e s i n up pe r o n e - h a l f t o o n e - t h i r d o f t h e s t a b a l o n g t h e l i n e of in o c u la tio n ; bubbles l e n t i c u l a r , tio n absent at four days. Liquefac­ A f t e r one week t h e s u r f a c e growth o f 0 . 2 to 0 . 5 cm. a r e a on t o p o f t h e s t a b i s bubbles s t i l l v i s i b l e . 0 . 2 t o 1 . 0 cm. i n s i z e . smooth, g r a y i s h and g l i s t e n i n g , gas A f t e r two weeks t h e g as b u b b l e s d i s a p p e a r and t h e s u r f a c e growth d r o p s , l e a v i n g a sunken a r e a . L iq u efa ctio n of napi- form c h a r a c t e r , p r o g r e s s i n g more i n some s t r a i n s t h a n i n o t h e r s a l t h o u g h not p r o n o u n c e d b e i n g o n l y 0 . 5 t o 1 . 0 cm. i n a r e a and e x t e n d i n g downward from 1 . 5 t o 3-0 cm. A f t e r t h r e e w e ek s t h e r e t i o n b e i n g c o m p l e t e i n two s t r a i n s , is considerable liq u e fa c ­ a l m o s t c o m p l e t e i n 12 s t r a i n s , and -28a b o u t one—h a l f c o m p l e t e i n t h e o t h e r s i x s t r a i n s "being i n f u n d i b u l i f o r m to s t r a t if o r m in c h a r a c t e r . P h ysiological Indol e ( 7 ) : N itrates Hot p r o d u c e d ("by a l l s t r a i n s ) . (^4-1): Reduced (b y a l l s t r a i n s ) . Hydrogen s u l p h i d e (U3 ) : C a t a l a s e ( 17) : P r odu ced ( a l l s t r a i n s ) . M ethy l r e d t e s t : fu l, Not p r o d u c e d (b y a l l s t r a i n s ) . N egative w ith m ajority of s t r a i n s , a fe w d o u b t ­ two (H- 138 and 23 S) p o s i t i v e . Voges-Proskauer t e s t : U ric a c id t e s t (2); (a ll strain s). U r i c a c i d can be u t i l i s e d a s t h e s o l e s o u r c e o f n i t r o g e n by most s t r a i n s . C it r ic ac id t e s t P ositive (2): H-138 and 233 d o u b t f u l . C i t r i c a c i d can be u t i l i z e d a s t h e s o l e s o u r c e o f c arb o n b y a l l s t r a i n s . E ffe c t of organic a c i d s : Growth u s u a l l y i n h i b i t e d b y a bo u t 0 . 0 5 p e r c e n t a c e t i c a c i d and 0 . 1 p e r c e n t l a c t i c a c i d . C l e a v a g e o f c ar bo n compounds: t i o n o f gas from: m altose, 1-ara'binose, d e x tr o s e , d- ma nn ose , d - m a n n i t o l , d -sorb itol, D e m o n s t r a t i o n o f c l e a v a g e by e v o l u ­ and 1 - x y l o s e ; raffin ose, d-galactose, rhamnose, lactose, saccharose, 1^; i n u l i n , starch, 11; lemon p e c t i n , no t a t t a c k e d by a n y o f t h e s t r a i n s . by t h e s t r a i n s t e s t e d . salacin , c l e a v a g e was d e m o n s t r a t e d f o r t h e f o l l o w i n g a d d i t i o n a l compounds by t h e i n d i c a t e d number o f s t r a i n s : glycerol, levulose, Likew ise, 2. dextrin, J>; M e l e z i t o s e was c e l l u l o s e was n o t a t t a c k e d The d e t a i l e d o b s e r v a t i o n s f o r th e ab ove compounds, w i t h t h e e x c e p t i o n o f p e c t i n and c e l l u l o s e , a r e shown i n Table 12. R e la tio n to oxygen: A erobic, f a c u l t a t i v e , under b o t h a e r o b i c and a n a e r o b i c c o n d i t i o n s . s t r a t e d by c u l t i v a t i o n w i l l grow abundantly- A n a e r o b i c growth demon­ i n s o l i d and l i q u i d me di a i n an a n a e r o b i c j a r ( l a r g e d e s i c c a t o r ) as d e s c r ib e d in F ig . 1. R elation to s a l t : ter (2 1 .1 per cent s a l t ) Growth i n c o n c e n t r a t i o n s upwards o f 80° s a l o m e i n normal cucumber f e r m e n t a t i o n s . un de r o r d i n a r y l a b o r a t o r y c o n d i t i o n s , u s i n g l i q u i d media, However, gro wth i s no t o b s e r v e d v i s u a l l y i n s a l t c o n c e n t r a t i o n s e x c e e d i n g a b o u t o n e - h a l f t h e a b o v e amount. Some s t r a i n s , may be made t o i n c r e a s e i n s a l t t o l e r ­ a n c e when s u b - c u l t u r e d s e r i a l l y i n l i q u i d media c o n t a i n i n g s a l t . Salt i s n o t n e c e s s a r y i n o r d i n a r y media f o r abundant g r o w th . Thermal d e a t h t i m e : 6 0° C . , I n s ta n ta n eo u s to s i x minutes exposure at d e p e n d i n g on t h e s t r a i n . C u l t u r e s a b l e t o w i t h s t a n d 50 ° C. f o r 10 m i n u t e s . Optimum g r o w th temp e r a t u r e : 30 t o 3 5 ° C. Ho gas p r o d u c e d a t U50 C. a l t h o u g h gr ow th may be n o t e d . Optimum pH c o n d i t i o n s : series, 5*1 t o 5*3* dextrose broth, Maximum g a s p r o d u c t i o n i n a b u f f e r e d E x c e l l e n t gas p r o d u ctio n occu rred in u n b u ffered e q u a l t o or e x c e e d i n g t h a t o f t h e b u f f e r e d l o t . The f o r e g o i n g d a t a v /it h r e s p e c t t o m o r p h o l o g i c a l , p h ysiological ch a ra cteristics c u l t u r a l and ( a s w e l l a s t h e b i o c h e m i c a l s t u d i e s on a c i d p r o d u c t i o n and e n d - p r o d u c t s o f t h e f e r m e n t a t i o n w hi c h a r e d i s c u s s e d s h o r t l y ) a s s u r e p o s i t i v e i d e n t i f i c a t i o n o f t h e 20 c u l t u r e s as b e l o n g i n g i n t h e ge nu s A e r o b a c t e r ( B e i j e r i n c k ) d e s c r i b e d i n B e r g e y ' s manual ( 6 ) . Only two t r u e s p e c i e s o f t h e genus a r e r e c o g n i z e d i n t h e above manual, t h e y a r e , A e r o b a c t e r a e r o g e n e s and A e r o b a c t e r c l o a c a e . B o t h s p e c i e s have -30a g r e a t number o f c h a r a c t e r i s t i c s i n common; h o w e v e r , t h e y d i f f e r c h i e f ­ l y w i t h r e g a r d t o f e r m e n t a t i o n o f g l y c e r o l and l i q u e f a c t i o n o f g e l a t i n . With A. a e r o g e n e s g l y c e r o l i s f e r m e n t e d and g e l a t i n i s n o t l i q u e f i e d v' n w h i l e w i t h A. c l o a c a e t h e r e v e r s e i s t r u e f o r t h e a c t i o n on b o t h com­ pounds . Taking in t o accou nt th e above r e a c t i o n s a s w e l l a s o th er charac­ teristics o f t h e group as a w h o l e , 18 o f t h e 20 c u l t u r e s ca n be c o n s i d ­ e r e d a l l i e d t o t h e s p e c i e s c l o a c a e a l t h o u g h t h e c h a r a c t e r i s t i c s a r e by no means i d e n t i c a l . W i t h i n t h e group a r e a number o f c u l t u r e s t h a t by v i r t u e o f t h e i r a c t i o n on g l y c e r o l and s t a r c h a s w e l l as o t h e r c h a r a c ­ t e r i s t i c s may be c o n s i d e r e d a s i n t e r m e d i a t e s and a l l i e d t o b o t h t h e s p e c i e s o f t h e genus A e r o b a c t e r , na m ely , A. a e r o g e n e s and A. c l o a c a e . The r e m a i n i n g two c u l t u r e s (H-133 and H-238) must b e c o n s i d e r e d a s f u r ­ t h e r v a r i e t i e s o f A. c l o a c a e . Acid p r o d u c t io n When v i e w e d i n th e l i g h t o f known b e h a v i o r o f t h e A e r o b a c t e r , th e r e s u l t s o f t h e f o l l o w i n g i n v e s t i g a t i o n upon a c i d p r o d u c t i o n from v a r i o u s compounds by a l l s t r a i n s becomes more u n d e r s t a n d a b l e . The t e s t s o l u t i o n s l i s t e d i n T a b l e 12 were t i t r a t e d a t t h e c o n ­ c l u s i o n o f t h e i n c u b a t i o n p e r i o d (two w e e k s ) and t h e v a l u e s w i t h r e ­ s p e c t to amounts o f N/lO a c i d or a l k a l i r e q u i r e d f o r n e u t r a l i z a t i o n a r e p r e s e n t e d i n T a b l e 13- In th e m a jo r ity o f th e c a s e s , the f i n a l r e a c tio n t o t h e i n d i c a t o r was a l k a l i n e , n e c e s s i t a t i n g t i t r a t i o n w i t h a c i d . two s t r a i n s , Only H-138 and H-238 ( v a r i e n t s ) showed any c o n s i s t e n c y i n f e r ­ m e n t i n g t h e compounds w i t h r e s u l t i n g a c i d p r o d u c t i o n whi ch was s t i l l p r e s e n t a t the time o f a n a l y s e s . In g e n e r a l , i t would a p p e a r t h a t f o r -31the m a j o r it y o f th e s t r a i n s t e s t e d , i n c u b a t i o n was n e g l i g i b l e or a b s e n t . vestigation , t h e f i n a l a c i d i t y a f t e r two weeks The r e s u l t s o f a s u b s e q u e n t i n ­ d e a l i n g w i t h t h e a c i d p r o d u c t i o n from d e x t r o s e a f t e r f o u r day s i n c u b a t i o n ( T a b l e 1*+), s e r v e t o c o n f i r m t h e p r e c e d i n g d a t a . In t h e c a s e o f t h e f e r m e n t a t i o n o f d e x t r o s e , out o f t h e 29 c u l t u r e s s t u d i e d , or Us p e r c e n t showed n e g a t i v e v a l u e s a f t e r c o r r e c t i n g f o r t h e co n­ tro l. The b a l a n c e o f t h e c u l t u r e s ( w i t h t h e e x c e p t i o n o f H-138 and H-238) were e i t h e r n e u t r a l or n e a r l y s o . I f a p p r e c i a b l e amounts o f a c i d w ere pr o d u c e d b y t h e A e r o b a c t e r cultu res, other than the e x c e p tio n s noted, i t was e i t h e r n e u t r a l i z e d by a l k a l i n e compounds fo r m e d or i t was d e s t r o y e d t o form o t h e r compounds-. P r e v i o u s work p o i n t s toward t h e l a t t e r c o n d i t i o n being: b r o u g h t a b o u t . The r e v e r s i o n o f a c i d i t y c a u s e d by t h e A e r o b a c t e r has b e e n shown by A ye rs and Rupp ( 5 ) * Also, t h e s e w or ke rs p o i n t e d out t h a t t h i s r e v e r s i o n c o u l d be a c c o m p l i s h e d under c o n d i t i o n s w h ic h were i n d e p e n d e n t o f a l k a l i production. F u rt h e rm o re , t h e y d e m o n s t r a t e d t h a t t h e a c e t i c acid, formed by t h e o r g a n is m s d u r i n g t h e d e x t r o s e f e r m e n t a t i o n was i n p a r t d e s t r o y e d . The f i n d i n g s o f R e y n o l d s and Werkman ( 3 0 ) c o n f i r m e d t h o s e o f A y e r s and Rupp and i n a d d i t i o n showed t h a t t h e r e v e r s i o n o f t h e a c c u m u l a t e d a c e t i c a c i d was ac co m p a n ie d by an i n c r e a s e i n a c e t y l m e t h y l c a r b i n o l and 2 , 3b u t y l e n e g l y c o l b r o u g h t a bo u t by c o n d e n s a t i o n and r e d u c t i o n . E n d - p r o d u c t s of_ t he fe r m e n t a t i o n I t has b e e n shown ( 1 8 , 32 ) t h a t t h e f e r m e n t s . t i o n o f d e x t r o s e by Aerobacter aerogenes y ie ld e d formic, as c a rb o n d i o x i d e , hy d r o g e n , acetylm ethylcarbinol - l a c t i c and s u c c i n i c a c i d s , as w e l l ethyl alcohol, Of t h e s e p r o d u c t s , 2, 3-butylene g ly c o l, and i t has b e e n s u g g e s t e d (3 2 ) - 32- t h a t s u c c i n i c a c i d p r o b a b l y r e s u l t e d from t h e p r o t e i n p r e s e n t i n t h e c u l t u r e medium. I n an i n v e s t i g a t i o n o f t h e d i s s i m i l a t i o n p r o d u c t s o f d e x t r o s e by A e r o b a c t e r i n d o l o g e n e s ( r e g a r d e d by t h e a u t h o r s o f B e r g e y ' s manual a s a v a r i e t y o f A. c l o a c a e ) , R e y n o l d s and Werkman ( 3 0 ) showed t h a t t h e f o l l o w i n g p r o d u c t s were formed; f o r m i c , e t h y l a l c o h o l , hydrogen, carbon d i o x i d e , butylene g ly c o l. a c e t i c and l a c t i c a c i d s , a c e t y l m e t h y l c a r b i n o l and 2, 3~ Of t h e s e p r o d u c t s , f o r m i c and a c e t i c a c i d s and a c e t y l ­ m e t h y l c a r b i n o l d e c r e a s e d i n amount s u b s e q u e n t t o p r i o r a c c u m u l a t i o n . The f o r m i c a c i d was dec ompose d t o c ar b on d i o x i d e and hydro ge n; the ace­ t i c a c i d t o a c e t y l m e t h j r l c a r b i n o l and t h e n t h e l a t t e r t o 2, 3 - b u t y l e n e gly co l. It i s e v i d e n t from t h e a b o v e d i s c u s s i o n t h a t t h e p r o d u c t s from t h e f e r m e n t a . t i o n o f d e x t r o s e by b o t h s p e c i e s o f t h e genus Ae roba c t e r ar e e s s e n t i a l l y t h e same. In the p r e se n t s t u d i e s , an i n v e s t i g a t i o n o f a. l i m i t e d n a t u r e upon t h e f e r m e n t a t i c n o f d e x t r o s e b y t y p e s t r a i n H-11*3 8 ( s e e T ab le 15) showed t h e f o l l o w i n g p r o d u c t s were formed; c a r b o n d i o x i d e , e t h y l a l c o h o l and e s t e r s strong t e s t (trace). A lso, the f e r m e n t e d l i q u o r gave a ( q u a l i t a t i v e ) for the presence of a c e ty lm e th y lc a r b in o l. The d e t e r m i n a t i o n f o r b u t y l a l c o n o l was n e g a t i v e . s t r a i n H—138 ( v a r i a n t ) , (probably, hy dro ge n, The f e r m e n t a t i o n by r e s u lt e d in the production of n o n - v o la t ile a c id s c h i e f l y l a c t i c ) and v o l a t i l e a c i d s i n a d d i t i o n t o t h e p r o d ­ u c t s m e n t i o n e d f o r s t r a i n H- ii*3 s . In view o f the e a r l i e r d i s c u s s i o n o f th e d e s t r u c t i o n of a c e t i c and f o r m i c a c i d s by t h e A e r o b a c t e r i t i s q u ite probable th a t t h i s behav­ i o r a c c o u n t s f o r t h e a b s e n c e o f a c i d s i n t h e c a s e o f t h e f o u r day f e r - -33m e n t a t i o n by s t r a i n H - l ^ S * at a l l u n lik e ly that l i t t l e I f s u c h i s t o b e pr esu me d, or no l a c t i c a c i d was p r o d u c e d , v e r s i o n o f t h e a c i d i t y was c o m p l e t e . by t h e v a r i a n t then i t i s no t since re­ However, w i t h t h e f e r m e n t a . t i o n ( H - 1 3 8 ) t h e ab ov e r e l a t i o n s h i p w i t h r e g a r d t o r e v e r s i o n o f a c i d i t y was n o t p r o n o u n c e d s i n c e a n a p p r e c i a b l e amount o f n o n - v o l a ­ t i l e a c i d a s w e l l a s a d e t e r m i n a b l e amount o f v o l a t i l e a c i d was fo u n d at th e co n clu sio n of the four-day ferm entation p erio d . Table 2. Culture No . Morphological Characteristics of the Cultures, Including Motility Determinations. Gram S tain M otility ('broth) S i z e r a n g e s i n m ic r o n s and arrangement H -138 + Rods: .75 x 1; 1 x 2 ; 1 x 3 ; o c c u r r i n g s i n g l y , some p a i r s , 3 ’ s &&& clumps and m a s s e s . H -2 3 8 + Rods: .75 x 1; 1 x 3 ; 1 x 4 ; o c c u r r i n g m o s t l y s i n g l y , fe w p a i r s , a l s o i n gr ou p s and c lu m p s. + Rods t o c o c c o - b a c i l l i : . 7 5 x *75 t o 1; few 1 x 3 ; almost o v a l, o c c u r r in g s i n g l y , p a ir s and c lu m p s. H-43S + Rods: .75 x 1; 1 x 1 . 2 5 ; 1 x 2 - 5 ; o c c u r r i n g s i n g l y , p a i r s and c lu m p s , ( e v i d e n c e o f b i p o l a r sta in in g .) H -53S + Rods: .75 x 1; 1 x 2 . 5 ; m o s t l y s i n g l y , a l s o clumps and m a s s e s , some i n 2 ' s and 3 ’ s and groups. + Same a s H-53S H-738 + Rods: .75 x 1; *75 x 2; . 7 5 x 2 . 5 ; o c c u r r i n g s i n g l y , 2 ' s , 3 ’ s and i n gr oup s and m a s se s and clum ps. H -838 + Rods: . 7 5 x 1 . 2 5 ; 1 x 2 . 0 ; m a s s e s and c lu m p s . H -113S + Rods: .50 x 1; . 7 5 x 1*5; 1 x 2 . 0 ; o c c u r r i n g s i n g l y , 2 ' s , g r o u p s , m a s s e s and clumps; some c e l l s c o c c i-to oval. + Rods: *75 x 1; 1 x 1 . 5 ; «75 x 2; 1 x 3 ; o c ­ c u r r i n g s i n g l y , 2 ' s , clumps and m a s s e s . Most c e l l s the sm aller s i z e . H -1 3 3 8 + Rods: .50 x 1; . 7 5 x 1 . 5 ; . 7 5 x 2 . 5 - 3 ; s m a l l e r s i z e i n t h e m a j o r i t y ; o c c u r r i n g s i n g l y , clumps gro ups and m a s s e s . H -11+38 + Rods: .50 X . 7 5 ; 1 x 1 . 2 5 ; 1 x 3 ; o c c u r r i n g s i n g l y , 2 ' s , clumps, m as se s and g r o u p s . H -1338 + Rods: .50 X . 7 5 ; *75 x 1 . 5 ; 1 x 2 ; few . 7 5 x 4 o c c u r r i n g s i n g l y , p a i r s , clumps and m a s s e s . 1 H -338 H - 63S H -1 2 3 8 - - - sin gly, 2's, in groups Table 2. C ulture No. Morphological Characteristics of the Cultures, Including Motility Determinations (continued). Gram S tain M otility (broth) S i z e r a n g e s i n m ic r o n s and ar rangement H - I 638 - + R od s: .50 x . 7 5 ; *75 x 1 . 5 ; *75 x 3; o c c u r r i n g s i n g l y , p a i r s , clum ps, gr o u p s and m a s s e s . H-173S - + Rods: .50 x . 7 5 ; .75 x 1 .2 5 ; p a i r s , gro ups and m a s s e s . ■ + R od s: .50 x . 7 5 ; s m a l l - t o o v a l a p p e a r a n c e t h e m a j o r i t y , a l s o some . 7 5 x 2 t o 3: o c c u r r i n g s i n g l y , p a i r s and m a s s e s . H - I 838 - . 7 5 x 2; s i n g l y , H-1938 - + R ods: .50 x . 7 5 : .75 x 2; . 7 5 x 3; o c c u r r i n g s i n g l y , p a i r s , 3 ' s > clumps and m a s s e s . H-2038 - + Rods: .50 x . 7 5 ; .75 X 1 . 5 ; *75 x 2 - 3 ; o c ­ c u r r i n g s i n g l y , p a i r s , m a s s e s , groups and clum ps. H-213S - + Rods: .75 x 1 - 1 . 5 ; i p a ir s , short chains, + Rods: . 7 5 x 1 - 1 - 5 ; 1 x 2 ; f e w 1 x 3 ; s i n g l y , p a i r s , g r o u p s , m a s s e s , and c lu m p s . Most c e l l s sm aller s i z e . H-223S N. B. - x 2.5 ; occurring s in g ly , gr ou p s and m a s s e s . The r o d s a p p e a r r o u n d e d on t h e e n d s and show i n d i c a t i o n s o f b i ­ polar sta in in g . In a l l c a s e s th e m a jo r ity o f the c e l l s are of the sm aller s i z e s l i s t e d . I n t h e m a s s e s or clumps i t a p p e a r s th a t the c e l l s are h e ld to g e th er by a v i s c i d m a t e r ia l. S p o re s absent. -36T a b l e 3* Growth o f C u l t u r e s , No s. H-13S Through 22 38 on NUTRITIVE CASEINATE AGAR SLANTS A f t e r t h r e e d a y s I n c u b a t i o n a t 3 5 ° C. GROWTH: Abundant t o m od er ate FORM: F ilif o r m to s l i g h t l y spreading a t the base of g r o w t h . Edge s f i n e l y l o b a t e t o wavy. LUSTRE: G listen in g ELEVATION: E lat TOPOGRAPHY: Smooth t o s l i g h t l y c o n t o u r e d from c e n t e r l i n e . OPTICAL CHARACTERISTICS: T ranslucent; CHROMOGENESIS: Grayish CONSISTENCY: V i s c i d to butyrous COLOR TO MEDIUM: Not d e t e r m i n e d , B . C . P . ODOR: Sweet t o a l c o h o l i c REACTION: A c i d i n t h e b u t t and a l k a l i n e b e n e a t h s u r f a c e g r ow th . i r r i d e s c e n c e n o t n o t e d on t h i s medium. in the agar. AFTER ONE WEEK INCUBATION Growth f i l i f o r m , s t a r t i n g t o s p r e a d from t h e b a s e upward, growth b e i n g smooth t o c o n t o u r e d a l o n g t h e e d g e s , b a t e and a p p e a r i n g wa vy. the l a t t e r b e in g f i n e l y l o ­ A l k a l i n e r e a c t i o n i n a l l t u b e s e x c e p t H-223 8, I 538, 33S and 138; o f t h e s e , a l l b u t 338 ha ve a c i d r e a c t i o n i n t h e p o r ­ t i o n o f t h e s l a n t b r o k e n away by g a s p r o d u c t i o n . T h e se a r e t h e s t r a i n s showing e v id e n c e o f g a s. AFTER TWO WEEKS INCUBATION Same a s f o r t h e one week o b s e r v a t i o n w i t h a l l t u b e s h a v i n g a l k a ­ lin e reaction . C u l t u r e s hav e s w e e t i s h o d o r , r e s e m b l i n g e s t e r s . -37Growth o f C u l t u r e s , N o s . H-138 Through 2238 on NUTRIENT AGAR SLANTS A f t e r t h r e e da ys I n c u b a t i o n a t 3 5 ° C. Table GROWTH: Moderate t o a b u n d a n t, ju ice FORM: F iliform , LUSTRE: G listen in g ELEVATION: Flat TOPOGRAPHY: C o nt ou re d, f i n e l y s t i p p l e d t o n e t - l i k e c r o s s h a t c h i n g , some s t r a i n s smooth* no t a s good a s on cucumber some s t r a i n s s p r e a d i n g i n b a s e o f s l a n t OPTICAL CHARACTERISTICS: Irridescent, CHROMOGENESIS: Streak b e in g w hite to g r a y ish CONSISTENCY: V is c id to butyrous COLOR TO MEDIUM: None ODOR: S w e e t i s h odor GAS: None d e t e c t e d v i s u a l l y translucent AFTER ONE WEEK INCUBATION Same a s f o r t h e t h r e e day d e s c r i p t i o n , ent. Most s t r a i n s s p r e a d i n g a t t h e b a s e , n e t - l i k e appearance. a little more growth p r e s ­ c l e a r l y s h o w i n g c r o s s - l i n e s or Odor m i l d l y p u t r e f a c t i v e i n c h a r a c t e r . AFTER TWO WEEKS INCUBATION S i m i l a r t o one week o b s e r v a t i o n . N ote d t h a t t h e m a j o r i t y o f t h e c u l t u r e s showed a s p i k e d t o f e a t h e r y g r o w th a t i r r e g u l a r p o i n t s a l o n g t h e gro wth t h a t e x t e n d down i n t o t h e a g a r . N o t e : - The same s p i k e d , feath­ e r y gr ow th n o t e d f o r cucumber j u i c e a g a r b u t l o n g e r and more f i l a m e n t o u s in n a tu r e . T h i s i s no t p r e s e n t i n t h e n u t r i t i v e c a s e i n a t e ag a r s l a n t s nor in the d e x tr o s e tryptone agar s l a n t s . -38T a b l e 5* Growth o f C u l t u r e s , N o s . H-138 Through 2 2 3 8 on DEXTROSE TRYPTONE AGAR SLANTS A f t e r t h r e e d a y s I n c u b a t i o n a t 3 5 ° C* GROWTH: Abundant FORM: E ilifo r m to spreading, may b e w ar y. LUSTRE: G listenin g, ELEVATION: Flat TOPOGRAPHY: Smooth OPTICAL CHARACTERISTICS: T r a n s l u c e n t and t r a n s p a r e n t CHROMOGENESIS: Grayish CONSISTENCY: V iscid COLOR TO MEDIUM: Not d e t e r m i n e d , B . C . P . i n medium ODOR: Sw eetish, REACTION: A cid i n b ase or b u t t , GAS: May b e p r e s e n t i n b a s e o f t u b e , may b r e a k a g a r o r c r a c k i t . sm ooth e d g e s , u s u a l l y e n t i r e , l i q u i d appearance resem bling e s t e r s a lk a lin e beneath a stroke. r a i s i n g t h e a g a r or AFTER ONE WEEK INCUBATION A l l t u b e s show a l k a l i n e r e a c t i o n , somewhere t h e a g a r i s b r o k e n away a t t h e b o t t o m and h a v i n g g a s p r e s e n t cator (B .C .P .) to c o l o r l e s s . Growth i s show a r e d u c t i o n o f t h e i n d i ­ s p r e a d a c r o s s t h e s l a n t up to t h e t o p o n e - t h i r d o f t h e t u b e and gr ow th a p p e a r s t o be g l i s t e n i n g and liq u id , growth i s o f v i s c i d c o n s i s t e n c y . AFTER TWO WEEKS INCUBATION Same a s f o r t h e one w e ek o b s e r v a t i o n s . -39Table 6 . Growth o f C u l t u r e s , N o s . H -1 3 8 T hrough 2 2 3 8 on CUCUMBER JUICE AGAR SLANTS A f t e r t h r e e d a y s I n c u b a t i o n a t 3 5 ° C- GROWTH: Most a b u n d a n t FORM: E i l i f o r m w i t h s l i g h t s p r e a d in g i n low er p a r t o f s t r e a k ; some s t r a i n s h a v e a wavy, t r a n s p a r e n t p e r ip h e r y a lo n g the s t r e a k . LUSTRE: G listen in g ELEVATION: S l i g h t l y convex TOPOGRAPHY: Smooth g e n e r a l l y , OPTICAL CHARACTERISTICS: T ranslucent, CHROMOGENESIS: Gray t o i v o r y i n c o l o r CONSISTENCY: V is c id to butyrous COLOR TO MEDIUM: None d e t e c t e d ODOR: Sweet to a l c o h o l i c GAS: May s p l i t the tube, some wavy, m o d e r a t e l y c o n t o u r e d . irrid escen t th e agar or push the s l a n t to the top o f g a s b u b b l e s may o c c u r i n t h e g r o w t h . AFTER ONE WEEK INCUBATION D e s c r i p t i o n f o r t h e t h i r d day s i m i l a r t o t h e o n e week r e s u l t s . S t r e a k s may b e f i l i f o r m or s p r e a d in g . Some s t r a i n s 1338, (338, ^38, F iliform growth s l i g h t l y co n v e x . and 1 9 3 8 ) h a v e a w r i n k l e d , cross-hatched, n e t - l i k e growth d e s c r ib e d o r i g i n a l l y f o r th e n u t r i e n t agar s l a n t s . s t r a i n s a r e g l i s t e n i n g an d i r r i d e s c e n t . A ll Odor a n d c o n s i s t e n c y t h e same as th ir d day. AFTER TWO WEEKS INCUBATION Same a s f o r t h e o n e w e e k , may b e a t e n d e n c y f o r more s p r e a d i n g g r o w th w h i c h i s c o m p l e t e i n some, b e in g a b o r e sc e n t in cha.racter. g ro w th f r o m t h e e d g e o f t h e gro wth -U o - T a b l e 7* Growth o f t h e C u l t u r e s , N o s . H -1 3 8 Through 2 2 3 8 i n CUCUMBER JUICE BROTH A f t e r t h r e e d a y s I n c u b a t i o n a t 350 C. SURFACE GROWTH: F i l m t o membrane o r p e l l i c l e , d e l i c a t e i n n a t u r e drops or d i s i n t e g r a t e s i f d is tu r b e d . CLOUDING: Strong, ODOR: R e s e m b l i n g cucumber J u i c e b r o t h SEDIMENT: W h i t e , m a s s e s , somewhat v i s c i d and r i s e s i n a s w ir l o f m asses. considerab le tu r b id ity . (+ ++ ) AMOUNT OF SEDIMENT: M o d e ra t e t o a b u n d a n t . TURBIDITY: 3+ GAS: Present REMARKS: The b r o t h o f t h e t y p e s t r a i n i s h e a v i l y c h a r g e d w i t h g a s ; i f s h a k e n , foam r i s e s 3 "to U cm. up the tube. AFTER ONE WEEK INCUBATION F ilm or p e l l i c l e p r e s e n t and o f g l i s t e n i n g l u s t r e , t h i s gr ow th r e ­ grown s i n c e t h e t u b e s w e r e s h a k e n on t h e t h i r d day when t h e t u b e s w ere shaken to d eterm in e th e c h a r a c te r o f th e sed im e n t. b eg in n in g to c l e a r . S t r a i n s H-238 and M o d e r a t e to a bu nda nt s e d i m e n t i n most t u b e s . s w e e tis h odor o b serv ed . Sligh t B a l a n c e o f d e s c r i p t i o n same. AFTER TWO WEEKS INCUBATION S i m i l a r t o t h e one w e ek i n c u b a t i o n d e s c r i p t i o n , s t i l l mo d er ate c l o u d i n g and s u r f a c e g r o w t h b u t n o t n e c e s s a r i l y c o n t i n u o u s . -41Table 8. Growth o f C u l t u r e s , N o s . H -1 3 8 Through 2 2 3 8 i n NUTRIENT BROTH A f t e r t h r e e d a y s I n c u b a t i o n a t 3 5 ° C• SURFACE GROWTH: Membrane o r d e l i c a t e p e l l i c l e i n a f e w s t r a i n s , a l s o a few s t r a i n s w i t h r i n g growth h a v in g v i l ­ l o u s p r o j e c t i o n s down i n t o t h e b r o t h , t h e r i n g may f a l l i n p a r t down i n t o t h e b r o t h . M ajority o f t h e c u l t u r e w i t h no s u r f a c e g r o w t h . CLOUDING: S l i g h t to moderate ODOR: None d e t e c t e d SEDIMENT: S m a l l amount, somewhat v i s c i d e no ug h t o s w i r l . AMOUNT OE SEDIMENT Small TURBIDITY: 1+ GAS: None d e t e c t e d v i s u a l l y . — i n c h a r a c t e r when Q U ­ ARTER ONE WEEK INCUBATION D e sc rip tio n sim ila r to those of the th ird day. have p e r s i s t e n t c lo u d i n g , ing i s slig h t, s l i g h t to moderate; E-138, Type s t r a i n s 238 and 33 S c l o u d ­ t h e named c u l t u r e s b e i n g t h e o n l y o n e s h a v i n g any t r a c e o f s u r f a c e g r o w t h i n t h e for m o f r i n g . no s u r f a c e g r o w t h . S e d im e n t v i s c i d , The b a l a n c e o f t h e c u l t u r e s h a v e s m a l l amount. AFTER TWO WEEKS INCUBATION Same a s f o r t h e one w eek o b s e r v a t i o n , ing, b e in g p e r s i s t e n t . s u r f a c e growth. slig h t t o m o d e r a te c l o u d ­ S m a l l amount o f s e d i m e n t w h i c h i s v i s c i d . No -42Table 9- Growth o f C u l t u r e s , N o s . H -1 3 8 Through 2 2 3 8 on POTATO SLANTS A f t e r t h r e e d a y s I n c u b a t i o n a t 3 5 ° C* GROWTH M o d e ra t e t o ab u nd an t FORM: F ilifo r m , s l i g h t l y ir r e g u la r alon g l i n e o f in­ ocu lation LUSTER: G listen in g TOPOGRAPHY: Smooth ELEVATION: S lig h tly raised CHROMOGENESIS: Y e llo w is h to f a i n t orange i n v a r i e d d e g r ee s i n d ifferen t strain s. Some a p p e a r i v o r y t o cream color. CONSISTENCY: S l i g h t l y v i s c i d to butyrous COLOR TO MEDIUM: Darkened, y e l l o w i s h to brow nish ODOR: S w e e t w i t h a l c o h o l i c aroma AFTER ONE WEEK INCUBATION S i m i l a r t o t h r e e day d e s c r i p t i o n , C h aracteristics sim ilar. no more g r o w t h n o t i c e a b l e . C o l o r s ee m s t o b e t a n t o l i g h t AFTER TWO WEEKS INCUBATION Same a s f o r t h e one w eek d e s c r i p t i o n . golden co lo r. -43Table 1 0 . Growth o f C u l t u r e s , N o s . H -13 8 Through 2 2 3 8 i n BROM-CRESOL PURPLE MILK A f t e r t h r e e d a y s I n ­ c u b a t i o n a t 3 5 ° C. REACTION: 3 days, s l i g h t l y acid; ACID CURD: 3 days, 0; iM- d a y s +. RENNET CURD: 3 days, 0; lU d a y s 0. PEPTONIZATION: 3 days, 0; 1^ d a y s 0. REMARKS: I n d i c a t o r changed to a c i d r e a c t i o n , t h a t o f a y e l ­ low ish c o lo r . There was a f i l m o r p e l l i c l e a t t h e su rfa ce w ith gas hubbies trapped underneath. lU d a y s , acid . AFTER ONE WEEK INCUBATION I n c r e a s e d a c i d r e a c t i o n over the th r e e days o b s e r v a t io n , S l i g h t c o a g u l a t i o n i n th e lo w e r p a r t o f H-1S38. + t o ++ +. O th e r s t r a i n s t h e same" a s a t t h e t h r e e d a y s o b s e r v a t i o n e x c e p t f o r more a c i d p r o d u c t i o n . No changes in c o n s is t e n c y . AFTER TWO WEEKS INCUBATION S o l i d c u r d i n a l l t u b e s w i t h or w i t h o u t e x t r u s i o n o f whey o r p r e s ­ ence o f gas b u b b l e s . A c id r e a c t i o n to th e i n d i c a t o r . AFTER THREE WEEKS INCUBATION A l l s t r a i n s h a v e f o r m e d a c i d c u r d and g a s b u b b l e s o r p o c k e t s v i s i ­ b le i n the curd, some e x t r u s i o n o f whe y. M - Table 1 1 . Growth o f C u l t u r e s , N o s . 13S Throug h 22 3S i n PLAIN GELATIN STABS A f t e r f o u r d a y s I n c u b a t i o n a t 3 5 ° C. GROWTH: Uniform, stab . LINE OF PUNCTURE: F ilifo rm , f i n e l y p a p illa t e edges. Gas b u b b l e s i n the upper o n e -h a lf to o n e -t h ir d p a r t o f the stab a lo n g the l i n e o f in o c u la tio n , bubbles l e n t i c u l a r , 0 . 2 t o 1 . 0 cm. i n d i a m e t e r . LIQUEFACTION: Absent a t fo u r days. DEGREE: None METHOD §USED: S t a b s k e p t a t 2 0 ° C- A f t e r i n o c u l a t i o n t h e y w e re kept a t th e above tem perature in water bath. COLOR TO MEDIUM: Unchanged. 0 . 2 5 t o O . 5O d i a m e t e r a r e a on s u r f a c e o f — 0— AFTER ONE WEEK INCUBATION Growth u n i f o r m , ish , g listen in g. 0 . 2 t o 0 . 5 cm. a r e a on t o p o f s l a n t , smooth g r a y ­ F i l i f o r m i n c h a r a c te r w i t h f i n e l y p a p i l l a t e e d g e s which a r e wavy o u t from t h e s t a b g r o w t h . ob servation s t i l l v i s i b l e ; Gas b u b b l e s p r e s e n t a t t h e t h r e e day no s i g n o f l i q u e f a c t i o n a t one w eek . AFTER TWO WEEKS INCUBATION At two w e e k s t h e g a s p o c k e t d i s a p p e a r e d an d t h e t o p growth on t h e g e l a t i n dropped l e a v i n g a sunken a r e a . L i q u e f a c t i o n i n napiform c h a ra c­ t e r a n d more p r o g r e s s e d i n some s t r a i n s t h a n i n o t h e r s . b e c o m i n g l i q u e f i e d fr o m t h e su n k en t o p o f g e l a t i n . Gas p o c k e t a r e a s -45Table 12. Gas Production from Various Carbon Compounds (Carbo­ hydrates, Alcohols and Glucosides). Methods; Compounds (1.0 per cent) made up in basal broth of 0.5 per cent tryptone and 0.2 per cent yeast extract dispensed in 5 cc. amounts into tubes containing insert tubes (4.5 x 0.8 cm.) and sterilized (10 lbs. pressure for 10 min.) Pigures shown below are expressed as per cent volume of the insert tubes and represent one-tenth of actual production. Each set of four figures (left to right) indicate production of gas at 1+8 hours, 8*+ hours, one week and two weeks. CARBON COMPOUNDS C ulture No . A rabinose Dextrose D extrin G lycerol G alactose H -138 4—2—2—2— 4 - 2 - 2 -1 1- 1- 1- + o-o-o-o 5-4-4-3 H- 238 3-2-2— 2 3-2-2-2 o-o-o-o o-o-o-o 4-3-3-3 H- 33S g_6 - 4 - 3 - 6 - 4 - 3 - 2- 0-0-0—0 0- + - 1-1 7—6—5—4— E—1+38 9-9-3-2 g - 7-3-2 o-o-o-o o-o-o-o 7-10-6-4 h- 5 3 8 7-4-4-2 7- 6- 2-1 o-o-o-o o-o-o-o 7-9-S-6 H- 63S 10-7-3-2 S - 5- 2-1 o-o-o-o 0—+—+ —+ 6-6-3-3 H-73S 7- 5- 2-1 IO-7-3-2 o-o-o-o 0- + - + - + 5- 9- g - 4 H-S3S 10—4—4—2 6- 7- 2-1 C -C - 0-0 o-o-o-o 10- 10- 7-5 H- 113S 10-4-4-3 6- 7- 1-1 o-o-o-o 0—+ — + — + 6- 5- 3-2 H - 123S 6 - 6 - 3 —1 10- 7- 4-2 0—0—0—0 o-o-o-o 6_ g - 9-6 H-1338 7- 4- 2-1 7- 4 - 2-2 0- 0- 0- + Q—0—+ — + g-io-6-4 H-ll+38 7-3-2-1 7 - 4 - 2-1 o-o-o-o ± - 1- 1-1 l O - g - 5-3 H-1538 7-4-3-+ 7- 5- 3- + 0- 0- 0- 0- l-l-l-l 5-l«_2-l E-1638 6- 3- 2- + S - 5- 3-1 0—0—0—0 + - 1- 1-1 10- 10- 7-3 K-1738 8—4—2 - 1 9- 6- 3-2 0-o-c-o 0- ± - + - + 10- 7- 4-3 H-1 83S 10-7-4-2 5 - 3 - 1 - 1- 0—0—0—0 0—+ - + - + 7-5-3-l H-193S 5- 6- S -2 10- 7- 4-3 0—0—0—+ 0—+—+—+ s-io-ic-6 H- 203 8 4- 3- 1-1 7- 4- 2-1 o-o-o-o 10- 10- 7-3 H- 2138 7—4- 2—2 6-3-2-1 0—C—+—+ 0—+—1— + +-1-1-1 H-223S 7-4-3-1 4 - 2 - 1-1 o-o-o-o 0- + - + - + 8- 6- 3-2 0 0 0 0 0 U ninoc. Controls (/> See footnotes at end of table. 7-3-2-1 -US- Table 12. Gas P r o d u c t i o n f r o m V a r i o u s Carbon Compounds ( C a r b o h y d r a t e s , A l c o h o l s and G l u c o s i d e s ) ( C o n t i n u e d ) . C ulture Ho. In u lin Lactose L evulose M altose Mannose H-138 +—+—+—0 1- 3- W 3-3-2-1 3—2—2—1 g - 6- 6-5 H-238 o-o-o-o 3- U - 3- U 7-6-4-3 3-2-2-3 6-5-4-3 H-33S o-o-o-o 4- 10- 10-7 4-3-1-1 8- 9- 10-8 8—6—4 —3 H-U38 o-o-o-o 7—8—3—6 10- 6- 3-2 10- 10- 7-5 g - 5- 2-2 H-53S o-o-o-o 1-3-5-4 10- 9- 6-3 5- g - 9 -7 2-5-3-1 H-638 0—0—0—0 3-7-2-6 io-s-5-3 10—10—7*-*6— 6 - 4 - 2 -1 H- 73 2 o-o-o-o 0- 1- 2-3 10-7-4-2 10—10—10—7 10- 10- 7-3 H-S38 o-o-o-o 2- 3—7-6 10- 6- 3-2 6- 8- 1C-7 H-113S O-O-O-O 2—3—3—5 10—8—4 —3 9- 10- 10-6 H -1 2 3 8 1- 1- + - + 0- 3- 5-6 I O - 7- 3-2 10- 10- 3-6 H-133S 0—0—0—0 3-S-9-6 4 - 2 - 1-1 7- g - 7-4 H - 1^38 o-o-o-o 5- 8- 10-5 10- 7- 4-2 10- 10- 9-4 10- 8- 6-3 H-15 3S o-o-o-o 5-7-9-6 7- 4- 2-1 10- 10- 3-6 8-6-4-2 H-163S o-o-o-o b - 5- 6-5 10- 6- 3-2 10- 10- 10-5 s - 5- 3-1 H-173S o-o-o-o 9-10-6-4 9-5-3-2 i o - s - 6-4 10- 6- 2-1 H - I 838 0-0-0-0 5-8-10-6 7-4-2-1 7—7—5—3 7 - 4 - 1-1 H-1S3S o-o-o-o 1- 2- 3-3 6- 3- 2-1 6-7-7-4 S - 5- 3-2 H-2038 0—0—0—0 5-7-9-9 10-10-5-2 10-10-6-4 10- 8- 5-2 H- 213S 1-1-+-1 5- 7- S -5 10-6-4-2 10- 10- 9-5 6 - 7 - 4 -2 E - 223S +-0-C -0 3—6- 6—3 7 - 4 - 2-1 7-7-6_3 7-4-2-l 0 0 0 0 0 Uninoc. C ontrols S e e ' f o o t n o t e s a t end o f t a b l e . 10-8-4-2 7- 4- 3-1 10- 7- 3-2 9- 6- 3-2 -47Table 12. Gas Production from Various Carbon Compounds (Carbo­ hydrates, Alcohols and Glucosides) (Continued). Culture No._________Mannitol Melezitose Raffinose Rhamnose Saccharose H-138 7_5.J4.-3 o-o-o-o 4-3-1-3 2-2-2-1 4- 4- 3- 2 E-23S 7_ 6- 5-3 0 —0— 0— 0 4- 3- 3-5 1-1-1-+ 2 - 2 — 2— 2 H-33S 7_ 7_Lj._3 o-o-o-o 3— 3— 3—3 2-2-2-1 s - 7- 3-2 E-438 S-5-2-2 o-o-o-o 8 - 8- 7-5 2-3-1-1 10-8-5-3 E-538 7_ 7_ 7_4 o-o-o-o 7-S-9-7 3-3-2-1 5-3-2-1 E-63S 6-7-4-3 O-o-o-o 10-10-7-6 3-3- 2-1 7- 4- 2-1 H - 73S 10-7-6-3 o-o-o-o 6-7-10-5 2-2-1— + S - 7- 6-4 E - S 38 10-7-5-4 o-o-o-o 3— 3— 5-3 2-2-1— 1 7-4- 2-1 E-1138 7- 6- 3-2 o-o-o-o 8-10-10-6 2-3-2— 1 10-7-3-2 E -1238 10-10-6-3 o-o-o-o 6 - 7- 4-5 2 - 2 — 7—1 7-4-1-1 E - 133S 6-5-3-2 o-o-o-o 5- S - 6-4 1-1-+— + 7- 6- 4- 2 H-1U38 1 0 - s - 5-3 O-o-o-o 7- 9- 10-7 2- 3- 3- s 7- 5- 3-2 E-1538 9— 7— 5—5 o-o-o-o 3- 4- 4-3 2- 3- 4-3 8-10-8-6 H-163S 10- 8- 5-4 o-o-o-o 6 - 6 - 6-4 +_+-+-+ 8 - 8- 7-3 E-1738 10-6-3-2 O-o-o-o 6_ 6- 7-4 l_l_+_+ 10-7-7-3 E - 1S38 10-7-5-4 O-O-O-O 6 - 6 - 8-4 “Kw»+— g _ 7_ 4-2 o-o-o-o 2- 3- 3-4 1-.+W4*— ’4* 4-3-1-1 E-193S 7- 9- 7-i; E-2038 10-8-5-3 o-o-o-o 6— 7— 7—7 1-1-1-+ g _ g _ 6-3 E-2138 10- 7- 6-4 O-o-o-o 4-4-5-5 2-2-3-2 S - 7- 6-4 E -2238 9-7-I1-3 O-O-O-O 4- 5— 7-5 1- 1-+-+ g _ 6_ 4-2 0 0 0 0 0 U ninoc. Controls See footnotes at end of table. -48Table 12. G-as Production from Various Carbon Compounds (Carbo­ hydrates, Alcohols and Glucosides) (Continued). C ulture No . S alacin S orb itol Starch X ylose Broth Control H-138 3-I1-3-3 5— 7— 7—7 1-1-1-1 5 - 5- 4-3 o-o-o-o H-238 3- 4~ 3-3 4- 7 - 7 - 6 1-1-1-2 4-4-3-3 o-o-o-o H - 33S 1-1— 2-2 7-g-io-s 1— 1 - 2 - 2 7- 7-7-7 o-o-o-o H - 43S 7-G-2-2 9- 10- 5-4 o-o-o-o o-o-o-o H - 53S 6 - 5- 4-3 10-10-S-7 o-o-o-o 7— 7— 7—7 7-7-7-g h ~ 63S 5- 4- 2- 2 s-9-7-6 o-o-o-o 7-S-7-7 0— 0— 0 — 0 h -738 s- 7 - 6 - 4 g-10-9-7 o-o-o-o G - 7- 7-7 o-o-o-o H - S 3S 7- 6- 4-3 g-7-7-5 o-o-o-o 6-s-s-s o-o-o-o H - 113S 7- 5- 3-2 7-10-10-6 o-o-o-o 7- g _ s -6 o-o-o-o H— 1238 7- 7- 7-4 s-io-s-6 1 - + - + — *- g-S-S-S o-o-o-o H-1338 G - 5- 4-3 8— 1 0 — 8— 6 8-S-9-9 o-o-o-o H-1438 2- 3- 3-3 10- 10- 8-4 1-1-1-1 8-10-10-10 o-o-o-o h -1538 2- 3- 3-3 io-s-7-6 0— 0— 0 — 1 s-9-10-10 o-o-o-o H-1638 7- 7- 5-3 9 - 9 - s-6 0-0-0-+ 8-8-8-6 o-o-o-o H-173S G - 6- 3-3 7-10-5-6 0-Q-0-+ 7- 7- g - g o-o-o-o H-1S 38 7- 5- 3-2 9-8-7-4 1-1-1-1 6-7-7-s o-o-o-o H - 193S 1 - 2-2-2 7-9-10-s 1-1-1-1 7— 6— 6 — 6 o-o-o-o H-2038 6- 4- 3-2 9-10-S-6 o-±-o-o S-S-S-S ----- H-2138 2- 3- 3-4 10-10-S-7 1-1-1-1 7-8-9-s o-o-o-o H -2238 2- 3- 3-3 9-10-7-6 o-o-o-o 8-8-8-6 o-o-o-o 0 0 0 U ninoc. C ontrols 0 0 lo st o-o-o-o + I n d i c a t i n g gas p r e s e n t , not s u f f i c i e n t to measure. 0 No g a s e v i d e n t . 1 10 p e r c e n t g a s ; 2, 20 p e r c e n t g a s , e t c . ± I n d ic a t in g d o u b tfu l gas p r o d u ctio n , very sm all bu bble. Note: I n m os t c a s e s , t h e f e r m e n t a t i o n i s r a p i d , t h e maximum volume o f g a s i s p r o d u c e d w i t h i n a b o u t 48 h r s . A f t e r t h i s p e r i o d t h e volume d e c r e a s e s due t o t h e g r e a t s o l u b i l i t y and r a p i d d i f f u s i o n o f c arb o n d ioxide. The r e m a i n i n g p o r t i o n o f g a s may b e a s su m ed t o be h y d ro ge n s i n c e l a t e r s t u d i e s showed o n l y two g a s e s p r o d u c e d . 4 Table 13* Acid Production, from Various Carbon Compounds (Carbohydrates Alcohols and Glucosides) After two weeks Incubation at 35° C M e t h o d s : Same medium a s o u t l i n e d i n T a b l e 12 i n c l u d i n g p r e p a r a t i o n o f c a r b o n c o m p o u n d s . At c o n c l u s i o n o f two w e e k s t h e c o n t e n t s (5 c c . ) o f e a c h t u b e w e r e t i t r a t e d w i t h N /l O NaOH, u s i n g p h en o lp h th a lein a s the in d ic a to r . Where t h e c u l t u r e s gave an a l k a l i n e r e a c t i o n to t h e i n d i ­ c a t o r an e s t i m a t i o n o f t h e amount o f a l k a l i was d e t e r m i n e d b y u s e o f N / 10 HC1. CARBON COMPOUNDS Dex­ Glyc­ trin erol Culture No. Arabin ose Dex­ trose H-138 0 . 8* 0 . 6* 0.5 H-23S 0 .4 0 . 8* H-333 0.7 H-U3S G alac­ tose Inulin Lac­ tose 0.7 1 . 0* 1.3 0.0 0.5 0.1 0.9* 0.5 0 . 1* 0.7 0.5 0 . 2* 0 . 6* 0.5 0.5 0.6 0.7 0.5 0 . 3* 0.6 0.5 0.6 H-53S 0.7 0.7 0 .5 0.1* 0.6 0.5 0.6 H-638 0.7 0.7 0 .4 0.3 0.7 0.5 o.7 h- 7 3 3 0.7 0.7 0 .2 0 .1 0 .6 0.5 0.2 H-S38 0.8 0 .8 0.5 0.2* 0.7 0.5 0 .5 B-1138 0.7 0.7 0.6 0 .1 0.7 0.5 0 .2 H-123S 0.7 0 .8 0 .2 0 .0 0 .5 0 .6 0 .2 H-133S 0.7 0 .8 0 .4 0 .2 0 .7 0 .6 0 .8 H-IU 38 0.8 0 .8 0.5 0.1* 0.3 0 . 3* 0-7 h- 1 5 3 8 0.7 0.7 0 .2 0 . 3* 0 .8 0 . 3* 0.5 h~ i 63S 0.7 0.7 0 .2 0 .1 0 .8 0.4* 0 .2 h- 1 7 3 8 0.7 0.7 0 .2 0 .1 0.7 0.5 0 .8 H-183S 0.7 0.7 0 .4 0 .1 0.7 0.4* 0 .6 h- 1 9 3 8 0.6 0 .8 0.6 0 .1* 0.6 0.5 0 .2 H- 203 8 0 .8 0 .7 0.5 0.1 0.7 0 . 3* 0.1 H- 213S 0.7 0.7 0.6 0 .0 0.7 0. 3* 0.6 H -22 38 0.6 0.7 0 .1 0 .0 0.7 0.6 o .6 U ninoc. C ontrols o .U * 0.4* 0.4* 0 . 3* 0.4* 0 . 3* 0.4* See footnote at end of table. -50Table 13♦ Acid Production, from Various Carbon Compounds (Carbohydrates, Alcohols and Glucosides) After two weeks Incubation at 35° C. (Continued). Levulose Malt­ ose Mannose Mann ito l H-138 0.6 0.4* 0.9* 0.9* H-238 0.6 0.1 1 . 0* 0.5 H-333 0.7 0.4 0.5 O H-438 0.7 0.7 0.6 H- 53S 0.6 0.5 H- 63S 0.6 H-738 M elezitose Rhamnose 0.5 0 .4 0.9* 0.6 0.4 0 .5 0.6 0.4 0.5 0.5 0.6 0.6 0 .5 0.5 0 .4 0.6 0 .4 0 .5 0.7 0.6 0.6 0.7 0.6 0.6 0.7 0.5 0.6 0.5 0.7 0.6 0.7 H-S 38 0 .8 0.2 0.6 0.6 0.5 0.6 0.7 H- 11 38 0.7 0.7 0.7 0.7 0.7 0.5 0.6 H—12 33 0.7 0.6 0.7 0.7 0.7 0.5 0.6 H-1338 0 .8 0.7 0.6 0.6 0.7 0.5 0.6 H—lU-38 0.7 0.7 0.7 0.7 0.7 0 .4 0.6 H-1538 0.7 0.6 0.6 0.7 0.7 0.2 0.3 H-1638 0.7 0.7 0.6 0.7 0.7 0.6 0.6 H- 173S 0 .8 0.8 0.6 0.6 0.7 0.5 0.6 H- 133S 0.7 O.S 0.6 0.6 0.6 0.6 0.6 H- 193S 0.7 0.5 0.7 0.6 0.6 0.0 0 . 5* H-2038 0.7 0.7 0.5 0.6 0.7 0.1 0.6 H- 213S 0.7 0.6 0.6 0.6 0.7 0.2 0.6 H-2238 0.7 0.6 0.6 0.6 0.6 0 .4 0.6 U ninoc. C ontrols 0.4* 0.4* 0 . 3* 0 . 3* 0 . 3* 0 . 3* 0 . 3* See footnote at end of table. VD R affinose * Culture No. -51Table 13- C ulture Ho . Acid Production from Various Carbon Compounds (Carbohydrates, Alcohols and Glucosides) After two weeks Incubation at 35° C* (Continued). Basal Saccha­ SalaSorbi­ Starch Broth X ylose cin rose tol Control H-13S 0.4* 0.5* 0.0 0.4* 0 . 8* 0.8 H-238 0.6 0.7* 0.4* 0 . 2* 0.7* 0.7 H-33S 0.8 0.5* 0.0 0.5* 0 . 2* 0.8 H-5+3S 0.0 0.3 0.5 0.3* 0.0 0.8 H-53S 0.2 0.3 0.5 0.4* 0 . 1* 0.8 h-638 0.7 0.3 0.1 0 . 5* 0 . 2* 0.8 H-73S 0 . 1* 0.0 0.0 0 . 5* 0 . 1* 0.8 H-S 38 0.7 0.0 0.1 0 . 8* 0.0 0.8 H-1138 0.8 0.1 0.2 0.5* 0.2 0.7 H- 123S 0.7 0 . 1* 0.1 0.7* 0 . 2* 0.8 H- 133S 0.0 0.1 0.1 0.2 0 . 1* 0.8 h-iM- 38' 0.7 0 . 5* 0.3 0.2 0.3* 1.0 H-1533 0.2 0 . 5* 0 . 2* 0 . 6* 0 . 2* 0 08 H- 163S 0 . 1* 0.2 0.1 0.4* 0.1 1.0 H-173S 0.0 0.1 0.1 0.4* 0 . 2* 0.8 H-1S3S 0.1 0.2 0.5 0 . 5* 0.0 1.0 H- 153 8 0.8 0.5* 0.1 0 . 6* 0 . 2* 0.8 H-2038 0.0 0.1 0.1 0.0 H-2138 0.0 0.3* 0.1 0.4* 0.0 0.8 H-2238 0.0 0 . 1* 0.4 0 . 5* 0.1 0.8 U ninoc. C ontrols 0.4* 0.3* 0 . 3* 0.4* 0.4* 0.4* * 0.8 Amount i n c c . o f N /l O NaOH r e q u i r e d t o n e u t r a l i z e a 10 c c . sa m p le . B a l a n c e o f t h e f i g u r e s show amount i n c c . o f H/lO KC1 r e q u i r e d to n e u t r a l i z e a 10 c c . s a m p l e . -5 2 - Table l 4 . A c i d P r o d u c t i o n fr om t h e F e r m e n t a t i o n o f D e x t r o s e B r o t h A f t e r f o u r d a y s I n c u b a t i o n a t 3 5 ° C. Medium: 0 . 5 p e r c e n t d e x t r o s e , 0 . 5 p e r c e n t KgHPOi)., t r y p t o n e and p e p t o n e 0 . 5 p e r c e n t e a c h . M e t h o d s : Two c c . a l i q u o t s d i l u t e d w i t h 35 c c . o f d i s t i l l e d w a ter, brought to a b o i l to expel COg. c o o l e d an d t i t r a t e d w i t h 0 . 1 1 1 N E&OH, u s ­ in g p h en o lp h th a lein as the in d ic a to r ; v a lu e s o b t a i n e d e x p r e s s e d a s grams l a c t i c p e r 1 0 0 c c . o f medium. pH d e t e r m i n a t i o n s b y t h e g l a s s e l e c ­ trode . C ulture ' Ho. F inal PH t 1 Broth 1 Control 1 Culture '• Ho. ' 1t 1 7.3S t Grams l a c t i c 1 p e r 100 c c . I ' 0.120 | H -2 3 8 5.92 1 .360 1 H -3 38 7.60 1 ! H-U38 0 0 6.38 K'l * t , H-138 c 1 t » K-1 73S * 0.180 , , E - 1 8 3 8 Final pH 1 Grams l a c t i c 1 p e r 100 c c . ' I C 1 1 ' 7.22 1 1 0.120 0.000 7.10 • . .130 .010 1 1 1 1 ' 1 .080 - . 0U0 ' 7.32 ' .100 -.020 ' 7.52 O O• -.050 , H -2 1 3 8 7-35 , .120 .000 , I H -53 8 7.52 .090 -.030 ; | H-2 238 * 7.02 ; .130 .010 ’ H- 63S 7 . 14-0 ' .080 - . 0^-0 ' ' H-139 7.02 ' .165 . 0U5 t' ' ! H-738 7.3S , .080 - . 0J+0 , ! H-239 j.kz ! .070 -.050 i I H-S3S 7.38 ' .080 - . 0U0 '11 H-339 6.85 ' .180 .060 ' H -1 1 3 8 6.92 * . 1U0 .020 ' > h- ^ 3 9 7.36 t .100 -.020 | H -1 23 8 6.96 ; .125 . 0 0 5 ; I H-539 7.02 ! .150 1 H-133S ! . H - l ^-38 7 . 5s ' .070 -.050 ' ' H-639 7.12 ' .130 .010 7 . 28 , .120 tt . 0 0 0 . . H-739 7-58 , .070 -.050 , ; H - 153S 7.^5 : .150 . 0 3 0 ; | H-839 7.08 ! .150 .030 I 7 .U 6 ■ .075 - . 0^5 1 1 -.020 ' 1 H-939 • .100 .11__ . * I, Values in c lu d e a c i d o f b r o t h c o n t r o l . C, C o r r e c t e d v a l u e s . *, C u l t u r e s t h r o u g h H -2 2 3 8 fr o m 1 9 3 8 s e r i e s , 1939 s e r i e s . ' H- 163S 1 J.k8 1 1 1 ' 1 ' O 1 * 1 ; 0 7.25 .070 . 2^40 't 1 H - 1 9 3 8 -.050 ' * H -2 0 3 8 ' 1 ..... _ ..L r e m a i n i n g c u l t u r e s from -53T a b le 15. F e r m e n t a t i o n E n d - P r o d u c t s b y S t r a i n s H- 1U3S ( t y p e s t r a i n ) and H-13S ( v a r i e p t ) fro m D e x t r o s e B r o t h A f t e r f o u r d a y s I n c u b a t i o n a t 3 5 ° C. Medium; F e r m e n t a t i o n s were c a r r i e d o u t i n one l i t e r f l a s k s c o n t a i n i n g ^00 c c . o f medium w i t h t h e f o l l o w i n g i n g r e d i e n t s p e r 100 c c ; 1 . 0 g . o f d e x t r o s e , 0 . 5 g . o f KgHPOi^, 0 . 2 g . o f y e a s t e x ­ t r a c t , 0.5 g . o f t r y p t o n e . M e t h o d s ; T i t r a t a b l e a c i d i t y by t h e method de ­ s c r i b e d i n Table l 4 . V o l a t i l e a c i d s and e s t e r s a c c o r d i n g t o t h e A .O . A .C . ( 3 ) ; E t h y l and B u t y l a l c o h o l s b y t h e method o f S t a h l y , Osborn and Werkman ( 3 5 ) 5 Gases e v o l v e d c o l l e c t e d o v e r s a t u ­ r a t e d a c i d i f i e d b r i n e s o l u t i o n and a n a l y z e d a c ­ c o r d i n g t o method d e s c r i b e d b y V e l d h u i s and E t c h e l l s (39)* pH d e t e r m i n a t i o n s b y t h e g l a s s electrod e. 1 1 P r o d u c t s i n grams p e r 100 c c . o f medium , Strain ru 0 0 1 Gases 1T i t r a t a b l e V o l a t i l e A lcohols 1 Esters t as e t h v l l a cid s as. 1 a cid ity ethyl 1 butyl 1 acetate 1 h2 acetic 'as l a c t i c ----------- 1. __ u.-------------- u ............... ....... - . 1 1 i 1 0.007 0.113 I 0 . 0 . 03** ! 0.388 a . H-lM-38 1 -O .O 65* 1 . 0.159 0 . 00U 0 . 0.02 0.027 1 0 . 26s* 0.073 ' . H-138 t 1 1 1 * V alues c o r r e c t e d f o r a c i d ( 0 . 1 0 2 g ./lO O ) p r e s e n t i n i t i a l l y in the medium. ** V a l u e s c o r r e c t e d f o r b l a n k ( 0 . 0 0 5 g . / l O O ) run on t h e medium. a Absent, d i s t i l l a t e a l k a l i n e . E . B . I n i t i a l pH o f t h e medium was 7 . IS a f t e r f o u r d a y s o f ferm entation b y s t r a i n s H - 1U38 and H-138 t h e v a l u e s were 7 . 6 s and 5* 33 r e s p e c ­ tiv ely . ' ' . 1 PRELIMINARY BIOCHEMICAL STUDIES I n t h e p r e s e n t i n v e s t i g a t i o n a. s t u d y o f t h e amount and c o m p o s i ­ t i o n o f g a s e s e v o l v e d fr om f e r m e n t a t i o n s ha s b e e n made by e m p l o y i n g q u a n t i t a t i v e m e th o ds o f a n a l y s e s . E e r m e n t a t i o n s r e s u l t i n g from m edi a c o n t a i n i n g v a r io u s carbon s o u r c e s , s u c h a s c a r b o h y d r a t e s , a l c o h o l s and g l u c o s i d e s h a v e b e e n e x a m in e d w i t h r e s p e c t t o t h e com pon ents o f t h e gases evolved . During th e fe r m e n t a t io n o f d e x t r o s e , s e v e r a l o f the f a c t o r s i n f l u e n c i n g th e fe r m e n ta tio n proper have been i n v e s t i g a t e d . T his c o n s t i t u t e s t h e p r i n c i p a l work i n v o l v e d a l t h o u g h o t h e r i n v e s t i g a ­ t i o n a l phases are in clu ded. P r e lim in a r y Experiments A c o n s i d e r a b l e amount o f p r e l i m i n a r y work wa,s c a r r i e d o u t r e l a ­ tive t o t h e p r e p a r a t i o n and m e th o d s o f h a n d l i n g o f a s u i t a b l e a p p a r a t u s f o r gas c o l l e c t i o n , d ition , s i n c e s p e c i f i c r e q u ir em en ts were n e c e s s a r y . I n ad­ a number o f p r e l i m i n a r y f e r m e n t a t i o n s t e s t i n g t h e a p p a r a t u s w ere conducted. Two s e t s o f e x p e r i m e n t a l f e r m e n t a t i o n s a r e a l l t h a t w i l l be reported. A d r a w i n g g i v i n g t h e s p e c i f i c a t i o n s o f t h e gas c o l l e c t i o n o u t f i t d e s i g n e d f o r t h e s t u d i e s i s shown i n F i g u r e 4 . P r io r to u s i n g , i t was s t e r i l i z e d a t 15 po u nd s p r e s s u r e f o r 10 m i n u t e s w i t h a n empty f l a s k a t ­ t a c h e d t o p r o t e c t t h e l o w e r p a r t o f U - t u b e F. A s m a l l c o t t o n p l u g was p l a c e d w e l l up i n t o t h e U - t u b e F t h a t s e p a r a t e s t h e c u l t u r e medium from the c o l l e c t i o n f l a s k A p r io r to a u to c la v in g . The s t e r i l e o u t f i t was handled as f o l l o w s : A sterile 50 c c . ro und b o t t o m e d f l a s k c o n t a i n i n g t h e c u l t u r e me- -55- (3 M C Q L L. E C T I C fV CUT F t T \£ — .• v - — « < - - • v P i g . H. The o u t f i t c o n s i s t s o f a one 50 c c . and two 250 c c . f l a s k s . F l a s k A f i t t e d w i t h a t h r e e h o l e , No. 5 rubber s to p p e r which i s s u p p lie d w ith a s h o r t r i g h t a n g le tube D f o r gas o u t l e t ; a s h o r t U-tube F f o r a t ­ t a c h i n g t h e 50 c c . ro un d b o t t o m c u l t u r e f l a s k C by means o f a No. 0 r u b b e r s t o p p e r ; a l o n g U - t u b e E f o r c o n n e c t i n g f l a s k s A and B . F l a s k B f i t t e d w i t h a two h o l e No. 5 r u b b e r s t o p p e r t h r o u g h w h i c h p a s s e s t h e Ut u b e f r o m f l a s k A and a n a i r o \ i t l e t t u b e G. A s h o r t p i e c e o f r u b b e r t u b i n g i s a t t a c h e d t o t u b e D and s u p ­ p l i e d w i t h a s c r e w clamp when f e r m e n t a t i o n i s s t a r t e d . - 56- diurn was i n o c u l a t e d and s u b s t i t u t e d f o r t h e empt y f l a s k . Saturated, a c i d i f i e d b r i n e s o l u t i o n was p l a c e d i n f l a s k B and was b r o u g h t up t o t h e b o t t o m o f t h e r u b b e r s t o p p e r i n f l a s k A t h r o u g h U - t u b e E an d up i n t o t h e g a s o u t l e t t u b e D b y s u c t i o n on a s h o r t p i e c e o f r u b b e r t u b i n g a t t a c h e d t o D. The g a s o u t l e t was t h e n s e a l e d w i t h a sc r e w c l a m p . c u b a t i o n was a t 3 5 ° C. In­ F i g u r e 5 shows an o u t f i t p r e p a r e d f o r i n c u b a t i o n . One o f t h e c h i e f a d v a n t a g e s o f t h i s t y p e o f a p p a r a t u s , s i m p l e c o n s t r u c t i o n and e a s e o f h a n d l i n g , is o th e r than i t s th at because o f i t s sm all s i z e and c o m p a c t n e s s a number o f i n d i v i d u a l f e r m e n t a t i o n s c a n b e c a r ­ r i e d o u t a t t h e same t i m e i n a l i m i t e d amount o f i n c u b a t i o n s p a c e . T r e a tm e n t o f C u l t u r e F l a s k s S e v e r a l gas c o l l e c t i o n o u t f i t s were c o n s t r u c t e d , f r e e fr o m l e a k a g e . t e s t e d an d f o u n d N e x t t o be c o n s i d e r e d was t h e d e v e l o p m e n t o f a s a t ­ i s f a c t o r y method o f . h a n d l i n g t h e c u l t u r e f l a . s k s i n o r d e r t o h a v e t h e maximum amount o f medium p r e s e n t and t h u s l e s s e n t h e amount o f a i r i n t r o ­ duced i n t o the produced g a s . A f t e r s e v e r a l m e th o d s w e re t r i e d , f o u n d p o s s i b l e t o s t e r i l i z e a s much a s 50 c c . i t was o f medium i n t h e 50 c c . c u l t u r e f l a s k w i t h o u t e x c e s s i v e b o i l i n g o r e v a p o r a t i o n u n d e r a n y one o f the f o l l o w i n g c o n d itio n s : (b) ( a ) when t h e f l a s k s w e r e p l u g g e d w i t h c o t t o n ; when t h e f l a s k s w e r e p l u g g e d w i t h r u b b e r s t o p p e r s s u p p l i e d w i t h s h o r t U -tubes plugged w ith co tto n ; ( c ) when t h e f l a s k s w e re a t t a c h e d t o t h e gas c o l l e c t i o n o u t f i t s . However, p r e l i m i n a r y e x p e r i m e n t s d e a l i n g w i t h t h e amount o f m e di ­ um t h a t c o u l d b e u s e d i n t h e f e r m e n t a t i o n s showed t h a t a p p r o x i m a t e l y 57- 58 c c . c o u l d b e u s e d i n t h e c u l t u r e f l a s k w i t h l i t t l e a r i s i n g fr om f r o t h i n g d u r i n g f e r m e n t a t i o n . or no d i f f i c u l t y T h i s volum e o f medium br ou gh t t h e l i q u i d l e v e l w e l l up i n t o t h e n e c k o f th e c u l t u r e f l a s k , le a v in g ap- Showing gas c o l l e c t i o n o u t f i t at the s t a r t c f fe r m e n t a t i o n -5 8 - p r o x i m a t e l y t h r e e t o fo u r c c . volume o f a i r s p a c e ( s e e T able l 6) w hich would have l i t t l e duced g a s . to e i g h t e f f e c t on t h e a n a l y s i s o f a 100 c c . vo lu m e o f p r o ­ I t was n e c e s s a r y t o a s e p t i c a l l y p i p e t t e t h e a d d i t i o n a l s e v e n c c . o f medium t o t h e o r i g i n a l l y s t e r i l i z e d ^0 c c . of cultu re medium i n t h e f e r m e n t a t i o n f l a s k . The a b o v e t r e a t m e n t o f c u l t u r e f l a s k s was u s e d i n t h e p r e l i m i n a r y ferm en tation s reported, the r o u t i n e s t u d i e s , one s t e p b e i n g e l i m i n a t e d . h e a te d to 8 0° C. i n a w a t e r b a t h , a t 10 p o u n d s p r e s s u r e f o r 10 m i n u t e s . were s u s c e p t i b l e to h e a t , tration . then s t e r i l i z e d by a u to cla v in g Where compounds w e r e u s e d t h a t s t e r i l i z a t i o n was c a r r i e d o u t b y S e i t z f i l ­ The s t e r i l i z e d s o l u t i o n s w e re t h e n p o u r e d a s e p t i c a l l y t o t h e g r a d u a t e d mark on t h e n e c k o f t h e s t e r i l e fla sk s. The f i n a l p r o c e d u r e was t h e s o l u t i o n s t o b e t e s t e d w ere t r a n s f e r r e d t o 125 c c . as f o ll o w s : fla sk s, ho w e v e r t h e p r o c e d u r e was s l i g h t l y m o d i f i e d f o r 50 c c . c a l i b r a t e d c u l t u r e A f t e r i n o c u l a t i o n t h e f l a s k s w e r e p l a c e d on t h e g a s c o l l e c t i o n o u tfits. ing f la s k s Saturated, a c i d i f i e d b r i n e s o l u t i o n was a d d e d t o t h e r e c e i v ­ and a f t e r t h e o u t f i t s w e re o b s e r v e d f o r on e ho ur f o r l e a k a g e th e y were p l a c e d i n th e i n c u b a t o r . Methods o f A n a l y s e s The m e th o d s o f a n a l y s e s f o r t h e g a s e s p r o d u c e d w e r e e s s e n t i a l l y t h e same f o r t h e p r e l i m i n a r y s t u d i e s a s i n t h e l a t e r more d e t a i l e d i n ­ vestigation s and w i l l b e o u t l i n e d i n d e t a i l a t t h i s t i m e . Any v a r i a ­ t i o n s fr o m t h e s e m e th o d s w i l l b e n o t e d i n c o n n e c t i o n w i t h t h e s t u d i e s w he r e t h e y o c c u r r e d . The g a s was m e a s u r e d and a n a l y z e d i n a m o d i f i e d W i l l i a m s g a s a n a l ­ ysis o u tfit, Model B- Carbon d i o x i d e was d e t e r m i n e d by a b s o r p t i o n i n a - 59- 40 p e r c e n t s o l u t i o n o f p o t a s s iu m h y d r o x id e . Oxyge n was d e t e r m i n e d "by a b s o r p t i o n i n a l k a l i n e p y r o g a l l o l s o l u t i o n . Hy drogen was d e t e r m i n e d by f i r i n g w it h added oxygen i n an e l e c t r i c sp ark e x p l o s i o n b u r e t t e . Methane o r o t h e r c o m b u s t i b l e h y d r o c a r b o n s w e r e d e t e r m i n e d b y p a s s i n g t h e p r o d u c t s fr om t h e h y d r o g e n d e t e r m i n a t i o n t h r o u g h p o t a s s i u m h y d r o x ­ id e and n o t i n g th e d e c r e a s e in volum e. T h i s d e t e r m i n a t i o n was a l w a y s n e g a t i v e and i s not rep o rted . was a n a l y z e d a s t h e a p p a r a t u s was d e s i g n e d f o r maximum e f f i c i e n c y a t t h i s volum e. When p o s s i b l e , A n a l y s e s o f s m a ll volum es o f gas a 100 c c . vo lu m e o f g a s ( 1 0 t o 20 c c . ) d u c e d a c o n s i d e r a b l e e r r o r , b r o u g h t a b o u t p r i n c i p a l l y by: in tro ­ (a) in su f­ f i c i e n t amount o f g a s a v a i l a b l e s o t h a t a p o r t i o n c a n b e u s e d f o r w a s h ­ in g o u t th e m a n ifo ld and (b) d i f f i c u l t y i n m a n ip u la tio n o f th e small amount o f g a s t h r o u g h t h e a b s o r p t i o n s o l u t i o n s contact. R esid ual (d is s o lv e d ) so a s to o b t a i n a d e q u ate g a s r e m a i n i n g i n t h e medium was d e t e r ­ m in e d b y b o i l i n g t h e c u l t u r e f l a s k an d t r a p p i n g t h e g a s d r i v e n o f f . some i n s t a n c e s in co rp o ra ted in th is In ga.s was a n a l y z e d s e p a r a t e l y w h i l e i n o t h e r s i t was t h e c o l l e c t e d g a s an d t h e t o t a l g a s a n a l y z e d . This method o f d e t e r m i n i n g t h e amount o f r e s i d u a l g a s was n o t w h o l l y a c c u r ­ a t e s i n c e a s m a l l amount o f g a s was d i r e c t l y a b o v e t h e l i q u i d , it so, gave a r e a s o n a b le however, i n d i c a t i o n o f t h e volu m e o f t h e d i s s o l v e d g a s . A l­ t h e a n a l y s e s o f t h e r e s i d u a l g a s as t o c o m p o n e n t s a r e n o t c o m p a r a b l e i n a c c u r a c y t o g a s a n a l y s e s r un on l a r g e r v o l u m e s . It s h o u l d be n o t e d a t th is tim e th a t in the gas a n a ly s e s , the oxy­ ge n v a l u e s w e re a l w a y s f o u n d t o b e b e l o w t h e amount known t o h a v e b e e n i n i t i a l l y p resen t at the s t a r t of ferm en ta tio n . That i s , if v a l u e was m u l t i p l i e d b y f o u r t o g i v e t h e e s t i m a t e d n i t r o g e n , t h e ox yg e n and t h e surn -6oo f t h e two v o l u m e s c o n s i d e r e d t o b e a i r , the f i g u r e d id not t o t a l to t h e amount o f a i r p r e s e n t a b o v e t h e c u l t u r e medium a t t h e s t a r t o f f e r ­ m entation. T h i s w o u ld i n d i c a t e t h a t some o f t h e o x y g e n was u t i l i z e d b y the fe r m e n ta tio n as w e l l as p o s s i b l y b e in g d i s s o l v e d in the l i q u i d s y s ­ te m . This c o n d it io n , tation s, e s p e c i a l l y i n a n a l y s e s o f g a s fr om a . c t i v e f e r m e n ­ f a v o r s a low er v a lu e o f gas a cco u n ted f o r in the a n a ly s e s s i n c e t h e r e m a i n i n g g a s was c a l c u l a t e d a s n i t r o g e n and t h i s f i g u r e was c a l c u ­ l a t e d fr o m t h e o x y g e n f o u n d p r e s e n t . U sually, the e f f i c i e n c y o f the gas a n a l y s e s r a n g e d from 9 5 - 9 9 P e r c e n t . A n a l y s e s o f Gas f r o m Cucumber J u i c e and D e x t r o s e B r o t h s The f i r s t p r e l i m i n a r y e x p e r i m e n t was c a r r i e d o u t t o d e t e r m i n e t h e amount an d c o m p o s i t i o n o f t h e g a s e s p r o d u c e d fr om f e r m e n t i n g cucumber ju ice broth. sterile tles The j u i c e had b e e n p r e v i o u s l y p r e p a r e d and was on hand i n 200 c c . am ou nts and was k e p t i n 12 o z . brown "s tubby" b e e r b o t ­ s u p p l i e d w i t h crown c l o s u r e s . ( s e e Table 17) h e a te d , The medium was a d j u s t e d t o pH 7*2* f i l t e r e d and f i n a l l y s t e r i l i z e d i n c u l t u r e f l a s k s a t 15 po u n d s p r e s s i i r e f o r 10 m i n u t e s . t h e v o l u m e s w e re b r o u g h t t o 57~ 5 3 c c . , s te r ile p ip ette. A fter coolin g, t h e a d d i t i o n s b e i n g made w i t h a The f l a s k s w ere i n o c u l a t e d w i t h one drop o f an em ul­ s i o n from a n o l d a g a r s l a n t o f s t r a i n H -1 !+3S. tig h tly fitte d i n 50 c c . amou nts The i n o c u l a t e d f l a s k s were to th e gas c o l l e c t i o n o u t f i t s a f t e r h avin g f i r s t the a u x i l i a r y f l a s k s . ( S e e P i g . H) rem oved A l l o u t f i t s were s u p p l i e d w i t h b r i n e s o l u t i o n and i n c u b a t e d a t 35° C. The s e c o n d e x p e r i m e n t was c o n d u c t e d t o d e t e r m i n e no t o n l y t h e a mount and c o m p o s i t i o n o f gas r e s u l t i n g from t h e f e r m e n t a t i o n o f d e x t r o s e * pH d e t e r m i n e d w i t h a g l a s s electrod e. « 6 i- but a l s o extract the r a te of gas e v o lu tio n - Tryptone ( 0 . 5 p e r c e n t ) and y e a s t ( 0.2 p e r c e n t ) w ere u s e d a s t h e b a s a l medium t o w h i c h 1.0 p e r c e n t d e x t r o s e was a d d e d . The b r o t h was p r e p a r e d i n 10 0 c c . am ou nt s and a d j u s t e d t o t h e d i f f e r e n t pH v a l u e s b y u s e o f s o d iu m h y d r o x i d e and h y d r o ­ c h l o r i c a c i d ( s e e Table 1 8 ) . D u p l i c a t e s e t s w e re a d j u s t e d t o t h e f o l ­ l o w i n g i n i t i a l pH v a l u e s 2 . 7 5 , was t h e n a d d e d t o s t e r i l e 5*^5, 6 . 8 0 and 9 * 0 5 - The a d j u s t e d b r o t h 50 c c . c u l t u r e f l a s k s i n 50 c c . am ou nt s and s t e r i l i z e d a t 15 p o u n d s f o r 10 m i n u t e s . A fter co o lin g , the f i n a l v o l­ umes w e r e b r o u g h t t o 57“ 5 S c c . b y t h e a d d i t i o n o f more b r o t h b y a p i ­ p ette. A l l s e t s w e r e i n o c u l a t e d w i t h o n e d r o p o f a 2U hour b r o t h c u l ­ t u r e o f s t r a i n H - l ^-38 an d t h e f l a s k s w e re h a n d l e d i n t h e manner p r e v i ­ o u s ly d e scr ib e d fo r the gas c o l l e c t i o n o u t f i t s . I n c u b a t i o n was a t 3 5 ° C. During th e f e r m e n t a t io n p e r io d th e b r in e l e v e l in th e gas r e c e i v ­ i n g f l a s k A ( s e e F i g u r e 3 ) was marked d a i l y a n d a t t h e c o n c l u s i o n o f t h e e x p e r i m e n t t h e r a t e o f gas p r o d u c t i o n was d e t e r m i n e d by b r i n e d i s p l a c e ­ ment . R esults The r e s u l t o f t h e f e r m e n t a t i o n s o f cucumber j u i c e b r o t h a s t o g a s e v o l u t i o n and c o m p o s i t i o n a r e shown i n T a b l e 19 . The mean v a l u e s o f f i v e d e t e r m i n a t i o n s show t h a t t h i s medium y i e l d e d g a s composed o f 8 1 . 0 p e r c e n t c a r b o n d i o x i d e and 16.H p e r c e n t h y d r o g e n . g e n t o c a r b o n d i o x i d e was 1 : 5 * The r a t i o of hydro­ There were v a r i a t i o n s b e tw e e n i n d i v i d u a l fe r m e n ta tio n s a lth o u g h not s u f f i c i e n t to be c o n s id e r e d s i g n i f i c a n t . The a n a l y s e s o f t h e r e s i d u a l g a s a r e shown i n T a b l e 20 and c o n s i s t e d p r i n c i ­ p a l l y o f carbon d io x i d e ( 8 3 - 0 P®r c e n t ) . -62T a b l e 21 g i v e s t h e r e s u l t s o f t h e f e r m e n t a t i o n s o f d e x t r o s e b r o t h w i t h r e s p e c t t o g a s p r o d u c t i o n and c o m p o s i t i o n . The p e r c e n t a g e s o f c a r ­ bon d i o x i d e and h y d r o g e n w e r e f o u n d t o b e s i m i l a r r e g a r d l e s s o f t h e i n i ­ t i a l pH a d j u s t m e n t . Ho s i g n i f i c a n t d i f f e r e n c e s w e re n o t e d t h a t w ere n o t exceeded by v a r i a t i o n s w it h in th e d u p l i c a t e s . c a r b o n d i o x i d e f o r t h e t h r e e l o t s a t pH 5 * 0 5 , 1 : 2 . 3 and 1 : 2 . 3 r e s p e c t i v e l y . the th re e r a t i o s . How ever, The r a t i o o f h y d r o g e n t o 6 . 8 0 and 9 - 0 5 w e r e : 1:2.1; T h e r e i s p r a c t i c a l l y no d i f f e r e n c e b e t w e e n t h e y do show a c o n s i d e r a b l e i n c r e a s e i n p r o ­ p o r t i o n o f h y d r o g e n when compared w i t h cucumber j u i c e b r o t h f e r m e n t a t i o n w h i c h was 1 : 5 The g a s p r o d u c t i o n f o r t h e a b o v e s e r i e s was r e c o r d e d a t up t o 1*+ d a y s and t h e r e s u l t s a r e shown i n T a b l e 2 2 . p r e se n ted g r a p h ic a lly i n Figure 6 . i n a l l l o t s was r a p i d , days' incu b ation . w ith l i t t l e in terv a ls This m a t e r i a l i s I t w i l l be seen th a t th e fe r m e n ta tio n gas b e in g produced a f t e r th r e e to fo u r The f e r m e n t a t i o n i n t h e u n a d j u s t e d l o t (pH 6 . 8 ) was t h e m os t r a p i d a n d showed some i n c r e a s e i n g a s p r o d u c t i o n compared w i t h t h e o t h e r l o t s a d j u s t e d t o pH 5 - 0 5 a n & 9 - 9 5 The most i m p o r t a n t r e s u l t s p e r im e n ts were: t o be n o t e d from t h e s e p r e l i m i n a r y e x ­ (a) a s a t i s f a c t o r y apparatus f o r stu d y in g in d iv id u a l f e r m e n t a t i o n s was d e v i s e d and t e s t e d ; ( b ) t h e g a s e s p r o d u c e d fr o m cucum­ b e r j u i c e a n d d e x t r o s e p r o v e d t o b e composed s o l e l y o f h y d r o g e n and c a r ­ b o n d i o x i d e from f e r m e n t a t i o n s v a r y , m e nt ed ; d e p e n d i n g up on t h e c a r b o n s o u r c e f e r ­ (d) th e f e r m e n t a t io n s tak e p la c e over a c o n s id e r a b le range w ith r e s p e c t t o i n i t i a l pH a d j u s t m e n t and ( e ) d u c t i o n was r a p i d , 72 h o u r s . d u r i n g f e r m e n t a t i o n t h e ga s p r o ­ t h e major p o r t i o n b e i n g p r o d u c e d w i t h i n a b o u t 1+8 to 6.6 / OO 80 4 * 10 /o / 3 E T/A A E // V 5 O AyS i 10. 1+ 9*5 t f t 1 0.2 i ' i ' i 1 1 | ' t * i ' 1 1 9.1 1 t 1 1 1+ . 1+ 0 . 1+ ' 8.3 0.0 * 1 1 ' | 7.0 1 1 1 3*8 Amount o f N 1 NaOH r ' t ' ' f 1 t 0.2 t 1 t I t t ' I • 1 t 2.9 3 .2 0 . 1+ 1 1 r 1 t I t ' t t i 1 ' t ' j ! 1 pH 2.0 5 . 1+ 1 t t 3 .0 ' t I 1 1 2 . 1+ O.S 7-0 I 1 I * ! < ) 1 ■ ! 1 1 ' 1 pH 1 1 i+.g f Amount o f H 1 / 1 0 NaOE | f ! ' i 1 ' ' 1 1 1 1 cc. ' t1 1 1 1 t t t 1 1 I 1 1 I 1 t 7*o 1 7*7 ' t « i ' t s.3 9.1 9*5 i •' ! ' 1 1 * Table 19. Analyses of Gas Produced from the Fermentation of Cucumber Juice Broth (pH 7*2) by Organism No. H-IU3S 1 1 ' ' 1 t 1 Flask No. 1 ' ' Yol. o f collected gas 1 cc. Carbon Dioxide * cc. Oxygen Hydrogen 6 jo 1 c c. Remainder gas* Ratio o f H2 :C02 ** 1 1 | cc. cc. 0.3 1 0.4 R 1:5.8 ' | 0.4 ' 0.9 1 4 1:4.0 ' * ' t ' 136 1 8 2 .2 112 14.1 ' 19 t ' 2 ' 3 1 1 1 2l4 78.0 167 19.6 ' 170 80.9 137 15.4 1 26 0.3 • 0.6 6 1:5-2 ' \ ' 42 1 4 ' 1 200 81.8 164 15.6 ' 31 0 . 4 1 0.8 X 4 1:5.2 5 ' 210 82.0 172 17.4 1 37 0.2 1 0.4 4 1:4.7 Mean ' 186 81.0 150 16.4 1 31 1 0 .3 ’ 1 ' I 0.6 5 * P r i n c i p a l l y n i t r o g e n from the a i r i n i t i a l l y p r e se n t above c u lt u r e medium. ** Calculated from the hydrogen and carbon d i o x i d e p e r c e n t a g e s . N.B. A na ly s is of the cucumber j u i c e showed 1 .9 2 grams o f red uc ing sugars per 100 c c . I ' 1 1:5-0 1 1 T able 20. ' ' F lask N o. ' 1 ' 2 , * A n a l y s e s o f H e s i d u a l Gas f r o m C u l t u r e Medium o f F e r m e n t a t i o n s Shown i n T a b l e 1 9 1 ' Y ol. of 1 R esid u al 1 gas* j -------------------------' cc. I ' | 1 Ca rbon d ioxid e $ ' 1 ■ I cc. ' Hydrogen 1 cc. 1 ’R e m a i n d e r ’ ' gas ' 1 f -J------------------- L 1 CC. 1 t I 21 86.1 » t 18 1 7-0 ' 1.5 lb so. 5 1 11 ' 6.2 1 0.9 1 t 2.1 1 t 1 t 1.5 t 3 ' 18 S 5.5 ' t 15 ' 8.7 * 1.6 > i.b r 1+ ' 2b gi+*7 ' t 20 ' 8.U ' 2 .0 • t 2.0 1 1 5 • ib 7S .5 • 11 ' 12.2 ' 1.7 • t 1.3 ; is S3.0 ; 15 1.5 ; 1.6 Mean 1 1 8.5 1 Gas d i s s o l v e d i n c u l t u r e medium a n d p r e s e n t a b o v e c u l t u r e medium. 1 ; Table 21. Analyses of Gas Produced from the Fermentation of One per cent Dextrose at Different Initial pH Values by Organism No. H-1438; Basal Broth of Tryptone and Yeast Extract. t 1 ' 1 Flask No. Amount of broth cc. ' 52.0 7 Mean PH Carbon dioxide * 'Remainder' R a t i o o f ' ' gas* ' H2 :C02 ** ' 1 I _ ._______ 4. J I t cc. cc. 1 1 Oxygen Hydrogen t cc. 30.5 38 0.4 75 34.8 43 0.4 ' cc. I 65.6 1 82 • 125 5.05 57.5 ' 124 5.05 57.2 . 125 5.05 63.O , 79 32.7 1+1 0.4 0.5 1 ! ♦ O K jD 1! i 10 ' Vol. of 'collected 1 gas ■ ■!■- ■ -- .1 1 CC. ! 1 0.5 0.5 4 1 t 5 2 56.5 . 132 6.80 66.8 1 92 29.1+ 39 0.3 0.4 , 1 58.5 ! 1^7 6.80 65.7 ! 97 29.5 43 0.3 0.4 1^3 6.80 66.3 1 95 29.5 1+1 0.3 0.4 57.O ! 1 1:2.1 , 1:2.3 , 1:2.2 , 1:2.3 ! 1:2.4 , 1: 2.2 , 1 7 I 4 57.0 , n4 9.05 65.7 . 75 29.4 34 0.3 0.3 3 58.0 ! 130 9.05 66.8 , 27 29.4 32 0.3 0.4 Mean 57.5 1 1 122 9.05 66. 3 ; 81 29.4 36 0.3 0.4 ; 5 1 1:2.3 5 58.0 1 1 0 2.75 0 1 1 0 0 0 0 0 1 1 0 * t 0 9 58.0 • 0 2.75 0 ' 0 0 0 0 0 0 ' 0 Mean 58.0 0 2.75 0 0 0 . 5 1 * ** ' 1 7 1 Mean 1:2.0 t 7 1 i ' t 5 1 , 1:2.2 t 0 0 1 5 0 P r i n c i p a l l y n i t r o g e n from t h e a i r i n i t i a l l y p r e s e n t above c u l t u r e medium. C a lc u la t e d from the hydrogen and carbon d i o x i d e p e r c e n t a g e s . 0 0 | ' Table 2 2 . 1 r i t t 1 t Flask No. 10 7 t■ i Mean 1 Amounts o f Gas Produced by Organism No. H~lk38 from D e x t r o s e B r o th ( l $ ) A d j u s t e d to D i f f e r e n t I n i t i a l pH V a l u e s . , Amount i of broth i in cc. ' 58.0 T o t a l v o l . o f gas c o l l e c t e d i n c c . by days pH ! 1 2 5.05 - 68 130 75 * 57-5 5.05 - 30 . 57.7 5. 0 5 . 1+9 t 103 (h2)t (89 ) ' 130 ! * 130 t 130 * 100 SI 1 130 1 111 1 122 1 1 130 1 1 . 121 , 126 115 ( 100) . ( 10 5) . (109) 1 ' 128 ' 13 V t 139 ! 139 ! 139 139 ; ' 13k ! ; 13s lk2 ‘ lk2 1 1 1 lk2 lk2 ' 1 • 136 1 (118) lkl ( 122) 1 lk l ’ ( 12 2) » lkl 1 (122) lk l (1 22 ) ’ 1 Ilk ’ Ilk ! ’ Ilk 1 Ilk ’ 126 ■ 126 t 126 126 t 6.80 ' 76 12k- ' i. 1 ; 58.5 6.80 ' 73 122 ' i Mean I 57.5 6.80 ' 75 ( 65) 123 ' 131 ( 106) ' ( 11k) k ' 57.0 9.05 ’ kk 91 3 > 58.0 9.05 ' 52 100 | 57.5 9.05 I kg (k2) * ** t 1 130 t lk * * . 108 , (9*0 56.5 (8 3) ..... ....... 8 1 ■ 106 » ( 92) ; 96 86 7 1 1 - - -f 126 , ( 109) . ' 2 ' Mean t 1 6 1 ' i t k 3 I 1 1 1 , 96 ' 96 1 1 106 • ’ 101 ’ ( 88 ) 1 107 1 (93) 1 1 118 1 120 ( 10k) 1 120 1 120 1 ( 10k) • (10k) —i— .... 1 122 120 ' (10k) ' -------------- L B a s a l medium o f t ry p t o n e ( 0 - 5 $ ) and y e a s t e x t r a c t ( 0 . 2 $ ) . T o t a l volume o f gas c o l l e c t e d , a f t e r lM- days f e r m e n t a t i o n . F i g u r e s i n p a r e n t h e s e s c a l c u l a t e d on b a s i s o f gas e v o l v e d from 50 c c . o f c u l t u r e medium. -71PRINCIPAL BIOCHEMICAL STUDIES The p r e l i m i n a r y i n v e s t i g a t i o n s w e re f o l l o w e d w i t h more e x t e n s i v e ferm en ta tio n s t u d ie s w ith r esp e c t to gas p r o d u c tio n , r a t e o f gas produc­ t i o n and c o m p o s i t i o n o f t h e e v o l v e d g a s e s . p e r i m e n t a l w ork , in r e l a t i o n to th e above g e n e r a l grouping, fo llo w in g phases: (a ) Ferm entations hy d i f f e r e n t t i o n s b y t h e same s t r a i n ; strain s; the e f f e c t o f (c ) tem perature, s a l t * upon th e f e r m e n t a t i o n o f d e x t r o s e ; c u l t u r e medium; More s p e c i f i c a l l y , (f) the ex­ covered the (b) fe r m e n ta ­ ( d ) pH and, (e) t h e e f f e c t o f h e a t up on t h e ( g ) t h e e f f e c t o f d e x t r o s e on t h e f e r m e n t a t i o n o f m a l t o s e ; ( h ) t h e f e r m e n t a t i o n o f v a r i o u s c a r b o n compounds. E x p e r i m e n t s somexirhat u n r e l a t e d to th e above fe r m en ta tio n s e r i e s d e a lin g w ith ( i ) h e a t o n t h e v i a b i l i t y o f two s t r a i n s a r e i n c l u d e d . the e f f e c t of The e x p e r i m e n t a l work w i l l b e p r e s e n t e d i n t h e o r d e r l i s t e d a b o v e i n T a b l e s 23 t o 3 3 . Procedure The g a s c o l l e c t i o n o u t f i t s , of r esid u a l gas, methods o f g a s a n a l y s e s , determ ination d e t e r m i n a t i o n o f t h e r a t e o f g a s e v o l u t i o n and t h e p r e ­ p a r a tio n o f the t e s t c a r b o n compounds ha ve a l l b e e n p r e v i o u s l y d e s c r i b e d . G e n e r a l p r o c e d u r e n o t p r e v i o u s l y d e s c r i b e d w i l l be p r e s e n t e d i n p art a t t h i s tim e; t h e b a l a n c e w i l l b e g i v e n when t h e i n d i v i d u a l e x p e r i ­ ments under c o n s i d e r a t i o n a r e ta k en up . The b a s a l b r o t h t o w h i c h t h e f e r m e n t a b l e c a r b o n compounds were ad d ed was m o d i f i e d s l i g h t l y yeast extract, (f ro m t h e o r i g i n a l d e s c r i p t i o n ) to e x c l u d e s i n c e t h i s m a t e r i a l had a t e n d e n c y t o pr o mo te f r o t h i n g i n the neck o f the c u lt u r e f l a s k s du rin g fe r m e n ta tio n . c o n s iste d s o le ly of tryptone The f i n a l b a s a l b r o t h ( 0-5 p e r c e n t ) , w h i c h was f o u n d s a t i s f a c t o r y * The e f f e c t o f s a l t up on t h e f e r m e n t a t i o n o f cucumber j u i c e i s a l s o cluded. . in­ -7 2 - f o r t h e gr ow th r e q u i r e m e n t s o f t h e o r g a n i s m s . For t h e f e r m e n t a t i o n s c a r r i e d o u t i n b u f f e r e d b r o t h , c e d u r e was a s f o l l o w s : A stock b u ffer s o lu tio n t a i n i n g 20 g . o f ammonium d i h y d r o g e n p h o s p h a t e , the pro­ ( 3 3 ) was p r e p a r e d c o n ­ U5 g . o f d i p o t a s s i u m h y d r o g e n p h o s p h a t e and Jl g . o f c i t r i c a c i d p e r l i t e r . One hu nd r e d c c . q u a n t i t i e s o f t h i s b u f f e r s o l u t i o n were a d j u s t e d t o t h e d e s i r e d pH v a l u e s w i t h 10 N s odi um h y d r o x i d e ( F i g u r e J). s t o c k s o l u t i o n s p e r 100 c c . o f b a s a l medium w ere u s u a l l y s u f f i c i e n t ob ta in the req u ired b u ffe r in g e f f e c t . of a l i t e r or more o f c u l t u r e medium, o n e -te n th o f the amounts o f t h e t h e b a s a l b r o t h an d t h e n a d j u s t t o t h e d e s i r e d pH. Ten c c . o f t h e a d j u s t e d to In ex p e rim en ts i n v o l v i n g th e u s e i t was f o u n d p r a c t i c a b l e t o u s e c h e m i c a l s l i s t e d a b o v e i n one l i t e r of t h i s amount w i t h 1 N sodium h y d r o x i d e T h i s p r o c e d u r e was u s e d t o o b t a i n t h e b u f f e r e d b r o t h (pH 5 * 1 5 ) u s e d i n a number o f t h e e x p e r i m e n t s . A l l gas. v o l u m e s r e p o r t e d w e r e c a l c u l a t e d on t h e b a s i s o f gas r e ­ s u l t i n g fr om 50 c c • o f c u l t u r e medium c o n t a i n i n g 1 . 0 p e r c e n t o f t h e c a r b o n compounds*, w h i c h w o u l d b e i n t e r m s 0 . 5 g* o f f e r m e n t a b l e c arb on source p resen t* * . 55.5 t o 5 8 . 0 c c . A ctually, t h e f e r m e n t a t i o n c u l t u r e f l a s k s h e l d from ( s e e T a b l e 16) , hence such a c a l c u l a t i o n i s w i t h t h e a c t u a l amount o f f e r m e n t a b l e c a r b o n s o u r c e t e s t e d . in keeping The c o n v e r ­ s i o n f a c t o r s u s e d i n t h e s e c a l c u l a t i o n s a r e shown i n T ab le 1 6 . d r o g e n an d c a r b o n d i o x i d e r a t i o s , * The h y ­ i n a l l c s . s e s , a r e b a s e d on t h e a c t u a l With t h e e x c e p t i o n o f l a c t o s e , r a f f i n o s e , rhamnose and s a l a c i n ( T a b l e 36 ) , h e r e t h e amount was i n c r e a s e d t o 3*0 p e r c e n t t o o b t a i n s u f f i ­ c i e n t gas f o r a n a l y s i s . ** Where cucumber j u i c e was e m p lo y e d a s t h e n u t r i e n t medium, t h e c a l c u l a ­ t i o n s were b a s e d on g a s p r o d u c t i o n from 50 c c . o f 2.0 p e r c e n t c a r b o ­ h y d r a t e ( e q u a l t o 1 . 0 g . o f CEO p r e s e n t ) . -73- n B u //b r so /7 }. A/Hi Hx POi - Z-0 070 & -Kx H P0H- 4-5 Citric Acid- 7-1 y ' " C C. A/a. OH F i g . 7 * PH c ha nge i n b u f f e r s o l u t i o n up on t h e a d d i t i o n o f NaOH. I , 10 c c . o f b u f f e r so'Ln. p l u s IN NaOH; I I , 10 0 c c . o f b u f f e r s o l n . p l u s ION NaOH. * - 7^ - volum es o f t h e s e g a s e s produced and in c l u d e d o t h e v o lv e d gas ( a l s o d e s ­ i g n a t e d a s c o l l e c t e d g a s ) and r e s i d u a l g a s . I n t h e maj or p o r t i o n o f t h e e x p e r i m e n t s , the gas analyzed repre­ s e n t e d an a l i q u o t fr om t h e r e s u l t i n g f e r m e n t a t i o n s and i n c l u d e d t h e r e s i d u a l gas in c o r p o r a te d a t the tim e o f a n a ly s e s . o f experim ents However, i n two s e t s (d and h ) r e s i d u a l g as was d e t e r m i n e d and a n a l y z e d s e p a ­ ra tely . A l l f e r m e n t a t io n s , w it h the e x c e p tio n o f th e tem perature s e r i e s (c), w e r e c o n d u c t e d a t 3 5 ° C. perature Gas v ol um es w e r e c o r r e c t e d t o room tem­ ( 2 3 ° C. f o r t h e s e e x p e r i m e n t s ) . C o r r e c t i o n s a.s t o v a r i a t i o n s i n b a r o m e t r i c p r e s s u r e were t o o s m a l l when a p p l i e d t o 100 c c . t o be c o n s i d e r e d s i g n i f i c a n t . growing, g a s v o lu m e s A l l i n o c u l a t i o n s w e re made from a c t i v e l y 2b t o *4-3 ho ur c u l t u r e s , i n o c u l a t i o n b e i n g made w i t h on e drop o f c u l t u r e ad d ed b y a s t e r i l e p i p e t t e . S t r a i n H-l^+38 was u s e d i n a l l e x ­ p e r im e n ts a s th e t e s t organism w i t h the e x c e p t io n o f the com parative study upon f e r m e n t a t i o n s b r o u g h t a b o u t b y s e v e r a l s t r a i n s i t y experim ents ( i ) , (a). In the v i a b i l ­ two s t r a i n s w e r e u s e d ; H - l ^ S and H - 1 3 8 . A l l pH d e t e r m i n a t i o n s w e re made w i t h a g l a s s e l e c t r o d e . R esults a . F e r m e n t a t i o n s by d i f f e r e n t s t r a i n s S e v e r a l s t r a i n s w ere i n v e s t i g a t e d w i t h r e s p e c t t o p r o d u c t i o n and com position o f gas. The f e r m e n t a t i o n s were c a r r i e d o u t i n b u f f e r e d (pH 5 . 15) d e x t r o s e ( 1.0 p e r c e n t ) b r o t h c o n t a i n i n g O.5 p e r c e n t t r y p t o n e . The f o l l o w i n g s t r a i n s w e r e u s e d : E - 7 3 9 , H-U39, H-&39, H- 63S, H - 1 3 3 8 , week a t 3 5 ° C. H-U38 and H-138 ( 1 9 3 8 s e r i e s ) . The g a s o u t f i t s w e r e marked a t (1939 s e r i e s ) , I n c u b a t i o n was f o r one 2^- hour i n t e r v a l s f o r r a t e -75of ga.s production. in the At the conclusion of the incubation period the gas d i f f e r e n t o u t f i t s was a n a l y z e d . The r e s u l t s a r e shown i n T a b l e s 23 24 a r e p r e s e n te d g r a p h ic a lly in Figure 3. an d 2 4 . The d a t a fr om T a b l e The c o m p o s i t i o n o f g a s was t h e same f o r a l l t h e f e r m e n t a t i o n s w i t h a l l t h e s t r a i n s e x c e p t s t r a i n H - I 38 . The g a s f r o m t h e s e s t r a i n s r a t i o s b e t w e e n 1 : 2 - 3 2 and 1 : 2 - 5 9 * showed h y d r o g e n t o c a r b o n d i o x i d e The maximum amount o f gas p r o d u c e d by f i v e o f t h e a b o v e group a l s o was q u i t e s i m i l a r , a b o u t 139 t o cc- However, i n b e h a v i o r was d e m o n s t r a t e d , t h e r a n g e b e i n g fr om i n t h e c a s e o f s t r a i n H-133 a d i f f e r e n c e n o t o n l y b y a d e c r e a s e i n t h e amount o f g a s p r o d u c e d b u t a l s o b y t h e p r o p o r t i o n o f h y d r o g e n i n t h e g a s w h i c h was greater (1 :1 .4 4 ). In g en e ra l, it i s noted (Figure 8) t h a t t h e f e r m e n t a t i o n w i t h a l l s t r a i n s i s r a p i d w i t h t h e major p o r t i o n o f t h e hours. days. In t h e s e fe r m en ta tio n s, A lso, little gas evolved w ith in 48 o r no g a s was p r o d u c e d a f t e r f o u r t h e g a s e v o l u t i o n c u r v e s show t h a t f i v e * o f t h e s e v e n s t r a i n s t e s t e d produ ced g a s volumes w e l l above b - F e r m e n t a t i o n s by t h e t h e 100 c c . r a n g e . same s t r a i n The r e s u l t s o f q u a d r u p l i c a t e f e r m e n t a t i o n s o f d e x t r o s e b r o t h by s t r a i n H - 1 4 3 3 a r e shown i n T a b l e 25; p a r t s A and B show g a s c o m p o s i t i o n and r a t e o f g a s e v o l u t i o n r e s p e c t i v e l y . The p e r c e n t a g e s o f c a r b o n d i o x i d e and h y d r o g e n a r e c om par abl e for a l l ferm en tation s, t h e g r e a t e s t v a r i a t i o n o b s e r v e d b e i n g a b o u t two per c e n t f o r each component. The r e l a t i o n s h i p o f t h e g a s e s i s p r e s e n t e d a g a in in th e hydrogen to carbon d i o x i d e r a t i o s . t w e e n f e r m e n t a t i o n s w e r e fr om 1 : 2 - 3 5 t o 1 : 2 . 4 9 , * Here t h e v a r i a t i o n s b e ­ such d i f f e r e n c e s b e in g Curve f o r H-4‘38 i n a d v e r t e n t l y o m i t t e d ; d a t a i n T ab le 24 i n d i c a t e s i t w o u l d be p r a c t i c a l l y i d e n t i c a l w i t h E - 1336- -76considered in s ig n if i c a n t . The d a t a fr om T a b le 2 5 , p a r t B a r e r e p r o d u c e d i n F i g u r e 9* f e r m e n t a t i o n s were r a p id ; hours. The a l l p r o d u c i n g w e l l o v e r 100 c c . o f g a s i n Three o f t h e f e r m e n t a t i o n s r e s u l t e d i n p r a c t i c a l l y t h e same amount o f e v o l v e d g a s ; t h e f o u r t h f e l l a l i t t l e s h o r t o f t h e amount a t ­ t a in e d by th e o t h e r s . c . The e f f e c t o f t e m p e r a tu r e on the fe r m en ta tio n of d e x t r o s e . The i n f l u e n c e o f t e m p e r a t u r e w i t h r e s p e c t t o g a s p r o d u c t i o n and com position i s shown i n T a b l e 2 6 . C. t o 1+5° C. w e r e e m p lo y e d . broth, E i g h t t e m p e r a t u r e s , r a n g i n g fr om 5° Experim ental procedure concernin g the i n o c u l a . t i o n and t h e s t r a i n u s e d ha ve b e e n p r e v i o u s l y d e s c r i b e d . The l o w e r and h i g h e r l i m i t a t i o n s f o r t h e f e r m e n t a t i o n y i e l d i n g , g a s were f o u n d t o b e 5° an(i ^ 5 ° C. Eo p e r c e p t i b l e g r o w t h was n o t e d a t t h e lo w e r l i m i t , w h ile a t the higher l i m i t , no g a s d e t e c t e d , p e r c e p t i b l e g r o w th was o b s e r v e d b u t e i t h e r e v o lv e d or r e s i d u a l . o f t h e p r o d u c e d g a s was r e s i d u a l , At 13° C. a l m o s t o n e - h a l f due t o t h e g r e a t e r s o l u b i l i t y o f c a r ­ bon d i o x i d e a t l o w e r t e m p e r a t u r e s . The t e m p e r a t u r e s 1 9 ° , C. ivere s i m i l a r w i t h r e s p e c t t o t h e t o t a l amount o f end o f f e r m e n t a t i o n . The c o m p o s i t i o n o f t h e gas ( 1 3 ° t o *+0c C .) was co mp ar abl e a s t o p e r c e n t a g e s o f h y d r o g e n and c a r b o n d i o x i d e f o u n d ; C02 P e r c e n t r e s p e c t i v e l y . 2 S . 1 t o 33 *5 E2 and 6E .6 t o 6 9 - 2 The h y d r o g e n and c a r b o n d i o x i d e r a t i o s l i k e ­ w i s e show s i m i l a r r e l a t i o n s h i p s f o r The e f f e c t o f t e m p e r a t u r e on tion is gas p r e s e n t a t the I t would a p p e a r t h a t t h e optimum t e m p e r a t u r e f o r g a s p r o d u c t i o n l i e s w i t h i n t h e 3 5 ° C. r a n g e . fr om a l l f e r m e n t a t i o n s 2U° and U0° th is series. t h e ga s e v o l v e d and r a t e o f e v o l u ­ shown i n T a b l e 27 and F i g u r e 1 0 . T h i r t y - f i v e d e g r e e s C. shows -7 7 - 140 /ZO CC. GAS' /OO 80 40 20 /o / 4 Z 3 T i * A E SM D A y s 6 F ig . 8. Gas e v o l u t i o n fr om t h e f e r m e n t a t i o n o f d e x t r o s e "by s e v e r a l s t r a i n s o f t h e s t o c k cultu re c o lle c t io n . CC. GAS' /OO H-14-38 60 40 20 to T/K\E "V O A V S Fig. 9Gas evolution from quaarun1icats fe: mentations of dextrose by strain II— 1-33. * -7 8 t h e most r a p i d e v o l u t i o n a s w e l l a s t h e maximum amount o f g a s e v o l v e d . F e r m e n t a t i o n s e i t h e r a b o v e ( 4 o ° C . ) or somewhat b e l o w (2U° C . ) t h e op­ timum ( 3 5 ° C . ) w e re c o n s i d e r a b l y r e t a r d e d and l e s s g a s was e v o l v e d . Gas e v o l u t i o n a t 1 9 ° C. was much s l o w e r t h a n a t 2U° C . , b u t a t t h e end o f t h e i n c u b a t i o n p e r i o d ( e i g h t d a y s ) t h e amount o f g a s e v o l v e d was a b o u t t h e same. At 13° C . , the gas produced d u rin g the f i r s t f o u r d a y s vias d i s s o l v e d i n t h e c u l t u r e medium. that t h is However, it is th ree to evident tem perature d e f i n i t e l y reta rd ed the fe r m en ta tio n . d . The e f f e c t o f pH on t h e f e r m e n t a t i o n of dextrose. For t h i s s e r i e s , 100 c c . amounts o f t h e s t o c k b u f f e r s o l u t i o n , prepared as p r e v io u s ly described, w e re a d j u s t e d w i t h 10 N sodium hy­ d r o x i d e t o c o v e r a r a n g e o f e i g h t pH v a l u e s . Ten c c . amounts a t e a c h d e s i r e d r a n g e w e r e s u p p l e m e n t e d w i t h 1.0 g . o f d e x t r o s e and 0.5 g . o f t r y p t o n e and made up t o 100 c c . v o l u m e . t o pH w ere made b e f o r e and a f t e r the d i f f e r e n t l o t s D eterm inations w ith r esp e c t sterilizin g . e x t e n d e d from 3*6 t o 8 . 85,• The f i n a l pH v a l u e s f o r An u n b u f f e r e d c o n t r o l (pH 6 . 8 ) was i n c l u d e d . The r e s u l t s show ( T a b l e 2 8 ) t h a t t h e f e r m e n t a . t i o n t a k e s p l a c e o v e r a c o n s i d e r a b l e pH r a n g e . No g r o w th r e s u l t e d a t pH 3*6 i n t h e a c i d r a n g e w h i l e pH 8 . 8 5 seemed t o approa.ch t h e l i m i t f o r a d e q u a t e gro wth i n the a l k a l i n e range; t h e l a t t e r b e i n g as su me d on t h e b a s i s o f marked d e ­ c r e a s e i n g a s e v o l u t i o n a t t h i s pH. Of t h e pH v a l u e s s t u d i e d , p e a r s t o b e t h e optimum i n t h e b u f f e r e d s e r i e s , 5-3 ap­ although the unbuffered c o n t r o l ( i n i t i a l pH 6 . 8 ) r e s u l t e d i n a s l i g h t l y g r e a t e r volume o f t o t a l gas p r o d u c e d . In g e n e r a l , carbon d i o x i d e r a t i o s it is shown from c o n s i d e r i n g t h e hyd rogen and t h a t no g r e a t d i f f e r e n c e i n gas c o m p o s i t i o n e x i s t e d , -79- IZO too 80 ZO to ~ r / as \ £ F ig . 10. E f f e c t o f t e m p e r a t u r e on g a s e v o l u t i o n f r o m t h e f e r m e n t a t i o n o f d e x t r o s e b y s t r a i n H-IU 38 . /OO 5,0 80 1 S 5 4.25 60 'TO . 8 . 05, <0 20 8 .8 5 to T 6 4 //V D A Y S F ig . 11. E f f e c t o f b u f f e r e d pH on g a s e v o l u t i o n fr o m t h e f e r m e n t a t i o n o f d e x t r o s e by s t r a i n H-IU 3S. O / 3 ~7~/ Art £T * -8 0 a l t hou gh t h e r e s ee ms t o be a s l i g h t in c r e a s e i n p r o p o r tio n o f hydrogen from t h e f e r m e n t a t i o n s w here t h e pH r a n g e i n c r e a s e s a b o v e 7*0; th is s ee ms p r o b a b l e f o r t h e l o w e r v a l u e , na m el y, U . 25 . The a n a l y s e s o f t h e r e s i d u a l gas from t h e f e r m e n t a t i o n s s e n t e d i n T a b l e 29 . lik ew ise, The amount o f r e s i d u a l gas in c r ea se s w ith are pre­ t h e more a l k a l i n e pH v a l u e s , due t o t h e g r e a t e r s o l u b i l i t y o f c a r b o n d i o x i d e . The p e r c e n t a g e o f c a r b o n d i o x i d e was f o u n d t o b e a b o u t t h e same i n a l l c a s e s and t h i s g a s c o n s t i t u t e d t h e major p o r t i o n o f t h e t o t a l r e s i d u a l gas . The r a t e o f g a s e v o l u t i o n f o r t h e b u f f e r e d s e r i e s i s g i v e n i n T a b l e 30* T h e s e d a t a ( w i t h t h e e x c e p t i o n o f pH 7 •55*) ?-r e p r e s e n t e d in Figure 11. A c o m p a r i s o n o f t h e g a s e v o l u t i o n c u r v e s shows t h a t t h e most r a p i d e v o l u t i o n o f g a s , a s w e l l a s t h e g r e a t e s t amount, r e s u l t e d from t h e f e r m e n t a t i o n a t pH 5*3- At pH 6.0 t h e r a t e o f g a s e v o l u t i o n was n o t p a r t i c u l a r l y i n f l u e n c e d a l t h o u g h t h e r e was a d e c r e a s e i n t h e amount o f g a s e v o l v e d . F e r m e n t a t i o n s a t pH 1+.25> 7*55 ( n o t shown) and 7 . 0 were comparable w i t h r e s p e c t to r a te of gas e v o lu tio n , t h e major p o r t i o n o f the gas b e i n g e v o lv ed w it h in th r e e to fo u r days. only a l i t t l e However, more t h a n o n e - h a l f t h e amount o f g a s was e v o l v e d from t h e s e f e r m e n t a t i o n s a s compared t o t h e optimum (pH 5-3)t h e more a l k a l i n e pH v a l u e s ( S . 0 5 and 8 . 8 5 ) The e f f e c t o f was shown most c l e a r l y by a marked d e c r e a s e i n g a s e v o l u t i o n . e * The e f f e c t o f s a l t (NaCl) on t h e f e r m e n ­ t a t i o n o f d e x t r o s e and cucumbe r j u i c e . C a l c u l a t e d amounts ( T a b l e 31 ) o f C.F. * s a l t were ad d ed t o 100 c c . Omitted for sake of clarity in presentation; followed 7-C very closely -81v o l u m e t r i c f l a s k s and made up t o vo lu m e w i t h 1 . 0 p e r c e n t d e x t r o s e b r o t h b u f f e r e d a t pH 5 *1 5; "the s a l t c o n c e n t r a t i o n s e m pl oy e d w e r e ; 20 a n d 25 p e r c e n t s a t u r a t i o n w i t h r e s p e c t t o s a l t . steriliza tio n , a t 3 5 ° C. 5. 10. This s e r i e s , 15. after was i n o c u l a t e d w i t h s t r a i n H-IU 3 S and i n c u b a t e d one week In a l l fe r m e n ta tio n s , t h e r e s i d u a l g a s was b o i l e d o u t and i n ­ c l u d e d i n t h e c o l l e c t e d gas a t t h e t i m e o f t h e a n a l y s e s . The r e s u l t s f o r t h e d e x t r o s e l o t s a r e shown i n T a b le 3 1 , p a r t A. T he re was a marked d e c r e a s e i n g a s p r o d u c t i o n a s t h e s a l t c o n c e n t r a t i o n i n c r e a s e d a b o v e 5 Pe r c e n t s a t u r a t i o n . S a lt co n cen tra tio n s as high as 20 and 25 p e r c e n t s a t u r a t e d r e s u l t e d i n no g r o w t h . in th is s e r ie s ( i n c l u d in g the c o n t r o l) In a l l fe r m e n ta tio n s t h e p e r c e n t a g e s o f h y d r o g e n and c a r b o n d i o x i d e were s i m i l a r w i t h t h e p o s s i b l e e x c e p t i o n o f t h o s e from t h e f e r m e n t a t i o n a t 15 p e r c e n t s a t u r a t i o n . p r o p o r t i o n o f hyd.rogen f o u n d was h i g h e r , In the l a t t e r c a s e , the howe ve r t h e volume o f g a s a - v a i l a b l e f o r a n a l y s i s was s m a l l ( 13 . I c c . ) w h i c h i n f l u e n c e d t h e a c ­ curacy o f the d ete r m in a tio n . I t i s o f p a r t i c u l a r i n t e r e s t t o n o t e t h e e f f e c t o f s a l t upon t h e b u f f e r e d pH ( F i g u r e 1 2 ) . In the d e x t r o s e l o t s , t h e pH v a l u e s d e c r e a s e d b e l o w t h e optimum ( 5-3 t o 5 . 15) w i t h t h e a d d i t i o n o f s a l t t o t h e e x t e n t o f 15 p e r c e n t s a t u r a t i o n and d e c r e a s e d f u r t h e r w i t h h i g h e r s a l t tration s. T h i s was n o t d e s i r a b l e s i n c e some i n h i b i t o r y e f f e c t o f t h e l o w e r pH v a l u e s was p r o b a b l y e x e r t e d u p on t h e f e r m e n t a t i o n s . ly , concen­ C ons eq ue nt ­ a s i m i l a r e x p e r i m e n t was s e t up u s i n g cucumber j u i c e b r o t h , p r e v i o u s ­ l y a d j u s t e d t o pH 5*9 s o t h a t t h e a d d i t i o n o f s a l t would n o t b r i n g t h e pH t o t h e i n h i b i t o r y r a n g e . In t h i s series it The r e s u l t s a r e shown i n T ab le 13, p a r t B . i s e v i d e n t t h a t t h e s a l t had t h e same g e n e r a l e f f e c t u p on g a s p r o d u c t i o n a s p r e v i o u s l y e x p e r i e n c e d w i t h t h e d e x t r o s e -82- / F ig, 12. Vac/- /S " % 2 0 SAT'f E f f e c t o f NaCl upon t h e pH o f d e x t r o s e b r o t h b u f f e r e d a t pH 5*15* -83lots ( p a r t A) A l t h o u g h a d e q u a t e g r o w t h and g a s p r o d u c t i o n r e s u l t e d i n t h e 20 p e r c e n t s a t u r a t e d l o t . S l i g h t g r o w th was n o t e d i n t h e 25 p e r c e n t s a t u r a t e d l o t hut e v i d e n t l y n o t s u f f i c i e n t f o r gas p r o d u c t io n . The s m a l l d i f f e r e n c e s i n g a s c o m p o s i t i o n a r e n o t c o n s i d e r e d p a r t i c u l a r l y sig n ifica n t. The e f f e c t o f s a l t on t h e pH o f t h e cucumber j u i c e b r o t h was s i m i l a r t o t h a t u p o n d e x t r o s e b r o t h . a d j u s t e d pH o f t h e f o r m e r was h i g h e r , However, s in c e the i n i t i a l t h e drop due t o s a l t d i d n o t b r i n g t h e f i n a l va-lues to t h e i n h i b i t o r y r a n g e f o r t h e f e r m e n t a t i o n s in th e high s a l t concentrations. T a b l e 3 2 p r e s e n t s t h e r e s u l t s w i t h r e s p e c t t o g a s e v o l v e d and t h e r a t e o f e v o l u t i o n f o r b o t h t h e d e x t r o s e and cucumber j u i c e l o t s . T h e s e date, a r e shown g r a p h i c a l l y i n F i g u r e 13 and a r e c a l c u l a t e d on t h e same b a s i s tate (50 c c . o f 1.0 p e r c e n t c a r b o h y d r a t e p r e s e n t ) t o f a c i l i ­ c o m p a r i s o n b e t w e e n t h e two l o t s o f media u s e d . It i s evident that s a l t a b o v e f i v e p e r c e n t s a t u r a t i o n e x e r t s a d e c i d e d e f f e c t up on t h e f e r m e n t a t i o n w h ich i s dem on strated by a r e d u c t i o n i n the gas e v o lv e d a s w e ll a s a r e t a r d a tio n in the ra te o f e v o lu tio n . of s a lt A part o f the e f f e c t i n t h e d e x t r o s e s e r i e s ca n be a t t r i b u t e d t o t h e i n f l u e n c e e x e r t e d u p on t h e pH o f t h e medium. f . The e f f e c t o f h e a t i n g t h e c u l t u r e medium. Upon s e v e r a l o c c a s i o n s , d e l a y e d f e r m e n t a t i o n s o f cucumber j u i c e have b een e x p e r i e n c e d . U su ally, a d e c r e a s e in gas e v o l u t i o n . s u c h f e r m e n t a t i o n s w ere a c c o m p a n ie d by A c h e c k o f t h e p r o c e d u r e t h a t was f o l l o w e d i n s u c h i n s t a n c e s r e v e a l e d t h a t t h e o n l y v a r i a b l e f a c t o r was t h e l e n g t h o f t i m e ’*' t h e j u i c e had b e e n s u b j e c t e d t o t h e u s u a l stea m p r e s s u r e (15 * Time i n c r e a s e s r e s u l t e d c h i e f l y l y opened, unused l o t s . through r e - s t e r i l i z a t i o n o f p r e v io u s ­ -84- DEXT/ZOSE' /OO /o 25 15 o JOO / 6 2 C L /C U M S E /l- J U I C E & & & TH v — -J- (C&3.CMO) j------- -6 - /o ~ -O' 25 zo 0 6 3 / 'T /M e /x / D A Y S P i g . 13. E f f e c t o f NaCl upon g a s e v o l u ­ t i o n fr om t h e f e r m e n t a t i o n o f d e x t r o s e ( u p p e r p a r t ) and cucumber J u i c e ( l o w e r part). Curves i d e n t i f i e d a c c o r d i n g t o per cent s a tu r a tio n o f s a l t . Ib .) e m p lo y e d f o r s t e r i l i z a t i o n . Consequently, a s t u d y was made t o d e t e r m i n e t h e e f f e c t o f h e a t up on cucumber J u i c e a s e v i d e n c e d b y s u b s e q u e n t f e r m e n t a t i o n w i t h and w ith out the p resen ce o f s a l t . In the s a l t s t u d i e s , t h e c h i e f aim l a y i n t h e p o s s i b i l i t y o f b r i n g i n g a b o u t a more v i g o r o u s f e r m e n t a t i o n i n t h e p r e s e n c e o f s a l t a t one o f t h e h i g h e r c o n c e n t r a t i o n s (15 p e r c e n t satu ration ), w i t h w h i c h o n l y m o d er ate s u c c e s s had b e e n p r e v i o u s l y (Table 3^) o b ta in e d . salt A lso, the p o s s i b i l i t y o f the in flu e n c e o f p la n t ( t h e g r a d e u s e d a t c o m m e r c i a l p l a n t s ) as compared t o C.P. s a l t was given a t t e n t i o n . Procedure F r e s h cu cumbers \\rere f r o z e n and thawed t o f a c i l i t a t e r e m o v i n g t h e J u i c e and t h e l a t t e r was c e n t r i f u g e d one hour a t 2 , 0 0 0 r . p . m . 250 c c . amounts and s t e r i l i z e d b y S e i t z f i l t r a t i o n . s e t up a s f o l l o w s : in to three parts; The f i r s t lot of S e itz f i l t e r e d t h e f i r s t wras u n h e a t e d , in F e r m e n t a t i o n s were J u i c e was d i v i d e d t h e s e c o n d was a u t o c l a v e d ^40 m i n u t e s a t 15 l b . p r e s s u r e and a t h i r d was a u t o c l a v e d 100 m i n u t e s a t t h e same p r e s s u r e . III r e sp e c tiv e ly . T h e s e t h r e e l o t s o f J u i c e were l a b e l e d I , I I and A s e c o n d l o t o f J u i c e was g i v e n t h e a b o v e t r e a t m e n t s and i n a d d i t i o n was s u p p l e m e n t e d w i t h s a l t t o t h e e x t e n t o f 15 p e r c e n t satu ration . T h e s e t h r e e l o t s w e re l a b e l e d IV, VI and V I I . A lso, one p o r t i o n o f u n h e a t e d J u i c e was i n c l u d e d i n w h ic h t h e s a l t ad d ed was ob­ t a i n e d from a c o m m e r c i a l p i c k l i n g p l a n t w h i c h was l i s t e d a s C gr ade mine s a l t . T h i s was l a b e l e d V. The i n o c u l a t i o n s f o r t h e s a l t series were made from a US t o 72 hour cucumber J u i c e b r o t h c u l t u r e o f s t r a i n H-IU 3 S g r o w i n g a t t h e a b ove s a l t t e r o n e week i n c u b a t i o n . con cen tration . A n a l y s e s were made a f ­ A l l a n a l y s e s i n c l u d e d r e s i d u a l g a s incor~por e t - -8 6 - ed i n t o t h e c o l l e c t e d g a s a t t h e t i m e o f a n a l y s e s . R esults The r e s u l t s ( T a b l e 3 3 ) show t h a t o f t h e f e r m e n t a t i o n s r e s u l t i n g from t h e S e i t z f i l t e r e d ju ice (I, I I and I I I ) , the unheated ( I ) ju ice gave a c o n s i d e r a b l e i n c r e a s e w i t h r e s p e c t t o t o t a l g a s p r o d u c t i o n . The d i f f e r e n c e b e t w e e n t h e *+0 and 100 m i n u t e h e a t e d l o t s was n o t o u t s t a n d ­ i n g , a l t h o u g h s l i g h t l y l e s s g a s ( a b o u t 20 c c . ) m in u t e t r e a t m e n t . was s i m i l a r ; r e s u l t e d fr om t h e 100 The c o m p o s i t i o n o f t h e g a s fr o m a l l t h r e e t r e a t m e n t s t h e h y d r o g e n and c a r b o n d i o x i d e r a t i o s f o r t h e l o t s j u i c e w e re a s f o l l o w s ; I, 1:3*50; l*'3-97 and I I I , 1:U .08. The d i f ­ f e r e n c e s w e re w e l l w i t h i n t h e l i m i t s or f e r m e n t a t i o n d i f f e r e n c e s . ferm entations in lo t s However, t h e 100 m in ut e h e a t i n g ( V I I ) m a r k e d l y d e c r e a s e d t h e amount o f g a s p r o d u c e d . found, The IV, V and VI show no p a r t i c u l a r d i f f e r e n c e w i t h r e s p e c t to t o t a l gas p ro d u ction . g a s fr o m t h i s s e r i e s , of The c o m p o s i t i o n o f t h e a s i n d i c a t e d by th e p e r c e n t a g e o f carbon d io x i d e shows no s i g n i f i c a n t d i f f e r e n c e s . u s u a l c o r r e l a t i o n between However, it i s e v id e n t th a t the t h e two g a s e s was a b s e n t s i n c e t h e i r sum l a c k s c o n s i d e r a b l e o f t o t a l i n g 100 p e r c e n t . T h i s p o i n t s to w ar d an e r r o r i n the d e t e r m in a t io n o f hydrogen, a lth o u g h the i d e n t i t y o f th e d i f f i c u l t y was n o t l e a r n e d . The r a t e o f g a s e v o l u t i o n f o r t h e f e r m e n t a t i o n s w i t h and w i t h o u t s a l t a r e p r e s e n t e d i n T a b l e 3h and F i g u r e l U . (p lant s a l t ) R e s u l t s from t r e a t m e n t V, i s o m i t t e d in as mu ch a s t h e y were s i m i l a r to IV. The r e ­ s u l t s show a v e r y s t r i k i n g r e l a t i o n s h i p w i t h r e g a r d t o t h e s p e e d o f f e r ­ m e n t a t i o n a s i n f l u e n c e d by h e a t i n t h e s a l t - f r e e t r e a t m e n t s . ho ur i n t e r v a l , the unheated j u ic e At t h e 4g ( I ) demonstrated a tw o -fo ld in crea se i n g a s e v o l u t i o n o v e r t h e Uo m in u te h e a t t r e a t m e n t ( I I ) and a t w e n t y - 300 WITHOUT SALT I 200 GAS ISO CC. Z 50 100 50 0 ( Z 3 DAYS 4 5 6 7 O / Z 3 4 5 6 7 D A Y S F i g . lh. E f f e c t o f a u t c s l a v i n g cucumber j u i c e w ith and w ith o u t s a l t - (15 p er cen t s a tu r a t i o n ) on gas e v o l u t i o n . I, unheated ; I I , ^0 min. h e a tin g ; I I I , 100 min. h e a t in g ; IV u n heated p lu s s a l t ; VI, 40 min. h e a t i n g p lu s s a l t ; V II, 100 min. p lu s s a l t . -S g - f i v e - f o l d i n c r e a s e o v e r t h e 100 m i n u t e h e a t t r e a t m e n t ( I I I ) . W h il e t h e f e r m e n t a t i o n o f t h e j u i c e r e c e i v i n g t h e 100 m in u t e heat treatm ent ( I I I ) e v o l v e d w i t h i n 30 c c . o f t h e amount o f g a s a s compared t o t h e M-0 m in u t e h e a t t r e a t m e n t ( I I ) , t h e l a t t e r was m ar ked ly r e t a r d e d i n gas e v o l u t i o n s i n c e o n l y a b o u t o n e - t e n t h t h e volume o f g a s was p r o d u c e d i n t h e t h r e e day i n t e r v a l . I n t h e s e r i e s t o w h i c h s a l t was ad d ed , shown, two o f t h e t r e a t m e n t s t h e u n h e a t e d (I V ) and t h e Uo m in u te h e a t e d ( V I ) , were co m pa r abl e a s t o t h e amount and r a t e o f g a s e v o l u t i o n . Here t h e major p o r t i o n o f t h e g a s was e v o l v e d d u r i n g t h e f i r s t t h r e e d a y s . I t i s noted that h e a t­ i n g t h e j u i c e f o r 100 m i n u t e s ( V I I ) r e s u l t e d i n v e r y l i t t l e g a s produc­ t i o n fr o m t h e s u b s e q u e n t f e r m e n t a t i o n . A summary o f t h e f i n d i n g s o f t h i s s t u d y r e v e a l t h e f o l l o w i n g : ( a ) C e r t a i n c h a n g e s o c c u r d u r i n g t h e h e a t i n g o f cucumber j u i c e w h ic h i n ­ f l u e n c e d t h e s p e e d o f f e r m e n t a t i o n a s w e l l a s t h e amount o f g a s e v o l v e d ; ( b ) t h e a b o v e i n f l u e n c e r e s u l t e d i n a much d e l a y e d f e r m e n t a t i o n i n j u i c e r e c e i v i n g 100 m i n . h e a t i n g a s compared to a 40 mi nu te h e a t i n g a l t h o u g h t h e f i n a l v o lu m e s o f g a s e v o l v e d a f t e r one w e ek w e r e c om pa ra bl e; under th e c o n d i t i o n s o f e x p e r im e n ta tio n , i t was no t p o s s i b l e t o b r i n g abo u t a more v i g o r o u s f e r m e n t a t i o n i n t h e p r e s e n c e o f s a l t sa t.) (c) (15 p e r c e n t i n u n h e a t e d j u i c e s i n c e t h e f e r m e n t a t i o n s r e s u l t i n g from u n h e a t e d and *+0 min. h e a t e d j u i c e were s i m i l a r w i t h r e s p e c t t o s p e e d o f f e r m e n t a ­ t i o n and t h e amount o f g a s e v o l v e d ; ( d ) no s i g n i f i c a n t i n f l u e n c e c o u l d be d e m o n s t r a t e d b y t h e t y p e o f s a l t employed ( p l a n t s a l t or C .P . s a l t ) . -9 0 - itia lly (after steriliza tio n ) an d a f t e r t h e g a s a n a l y s e s h a d "been com­ p leted . A tten tion tose, raffin ose, not y i e l d is ca lled t o t h e f a c t t h a t w i t h f o u r c o m po u nd s , rhamnose and s a l a c i n , su fficien t t h e one p e r c e n t s o l u t i o n s d i d g a s v o l u m e s f o r a n a l y s e s w h i c h w e re c o m p a r a b l e t o the volum es r e s u l t i n g from t h e o t h e r carb on compounds. cases, for t h e f e r m e n t a t i o n s were r e p e a t e d , c a l c u l a t e d on t h e b a s i s Hence, in th ese t r i p l i n g t h e o r i g i n a l amount e a c h compound s o t h a t i n t h e f i n a l r e s u l t s so lu tio n la c­ t h e f i g u r e s a r e shown o f t h e f e r m e n t a t i o n o f 50 c c . o f a 3*0 p e r c e n t ( 1 . 5 e* o f compound p r e s e n t ) . A lso, i n th e se four in stan ces, t h e r e s i d u a l g a s wa s i n c o r p o r a t e d i n t o t h e c o l l e c t e d g a s a t t h e t i m e o f a n a ly sis. However, the l a t t e r change o f procedure i s o f no p a r t i c u l a r conseq uence s i n c e th e f i n a l hydrogen to carbon d io x i d e r a t i o s f o r a l l compounds f e r m e n t e d w e r e c a l c u l a t e d fr om t h e t o t a l v o l u m e s o f t h e g a s q s produced, b o th e v o lv e d and r e s i d u a l . The and to r esu lts of the ferm en tation s w ith resp ect c o m p o s itio n a r e p r e s e n t e d i n Table 36. cap a b ility of the stances residual tested , rela tiv e t y p e s t r a i n H - 1U3 S t o f e r m e n t t h e d i f f e r e n t g a s w h i c h was s i m i l a r i n amount f o r a l l ily ferm ented, and lev u lo se; e v a lu a tio n * as sub­ a s e v i d e n c e d b y t h e amount o f g a s e v o l v e d ( e x c l u d i n g pounds w ere u t i l i z e d those A to gas e v o l u t i o n c a s e s w h e r e t h e com­ to any e x t e n t ) would be a s f o l l o w s : - dextrose, d-m ann itol, d -galactose, th o se l e s s r e a d i ly ferm ented, m oderately ferm ented, Those rea d ­ d-mannose, saccharose d - s o r b i t o l a n d 1- a r a b i n o s e ; m a l t o s e and x y l o s e ; th o se p o o r ly ferm ented, P o r t h e c o n d i t i o n s u n d e r wrh i c h t h i s s t u d y was c o n d u c t e d and b a s e d on tr.e e v o l v e d g a s f r o m t h e f e r m e n t a t i o n o f ^0 c c . o f t h e v a r i o u s s o l u t io n s . -91la cto se, raffin ose, rhamnose and. s a l a c i n . I n a. s t u d y o f t h e d a t a c o n c e r n i n g t h e c o m p o s i t i o n o f t h e gas e v o l v e d from t h e s e f e r m e n t a t i o n s , into i t w as f o u n d t h a t t h e compounds f e l l th re e g e n e ra l c l a s s i f i c a t i o n s w ith r e f e r e n c e to prod u ction o f hy­ drogen and carbon d i o x i d e . In th e f i r s t , the gas i s composed o f one volu m e o f h y d r o g e n a nd two v o l u m e s o f c a r b o n d i o x i d e compounds i n c l u d e d a r e : 1-ara.bin ose, d e x tr o se , d—mannose an d s a c c h a r o s e (1 p e n t o s e , In t h e sec o n d , the gas i s levu lose, composed o f o n e vo lu m e o f h y d r o g e n and (l;l) 1-xylose, raffin ose, s a l a c i n (2 p e n t o s e s , d-galactose, b h e x o s e s and 1 d i s a c c h a r i d e ) . one vo lu m e o f c a r b o n d i o x i d e r h a m n o se , m a l t o s e , ( 1 : 2 ) and t h e and t h e compounds i n c l u d e d a r e : 1 disaccharide, d-m annitol, d - s o r b i t o l and 1 trisacch arid e, 2 hexahydric a l ­ c o h o l s and 1 g l u c o s i d e ) . In the t h i r d , t h e g a s i s composed o f two v o l u m e s o f h y d r o g e n and one vo lu m e o f c a r b o n d i o x i d e la c to se ( 2 : 1 ) and o n l y one compound i s included; (d isacch arid e). The v o l u m e s o f r e s i d u a l g a s and t h e a n a l y s e s a s t o co m p on en ts from t h e a b o v e f e r m e n t a t i o n s su lts ( T a b l e 36 ) a r e shown i n T a b l e 37 . The r e ­ show t h a t c a r b o n d i o x i d e r e p r e s e n t s t h e m aj or p o r t i o n o f t h e g a s . The r a t e o f g a s e v o l u t i o n f o r t h e f e r m e n t a t i o n s p r e s e n t e d i n T a b l e 38 sh ow s, in general, w i t h i n 2k t o *18 h o u r s . t h a t t h e major p o r t i o n o f t h e g a s i s e v o l v e d I n most c a s e s , no ted a f t e r fo u r days o f i n c u b a t i o n . g r a p h i c a l l y i n F i g u r e 15 . little o r no g a s e v o l u t i o n was The d a t a fr om t h i s t a b l e a r e shown A ttention is c a l l e d to th e f a c t t h a t th e c u r v e s shown r e p r e s e n t t h e g a s e v o l v e d fr om t h e f e r m e n t a t i o n o f 50 c c . o f t h e t e s t c a r b o n compound s o l u t i o n s (l.O per c en t). _ M O /J O S A CCA/A E /O E S (PEA/TOSESj D/SA CC //A gjDBS JL JZ /OO s » c c - SA J D E X r& O S£ J J K f-M A M A /O S e ' JR d~ GALACTOSE - 72 4EVUL0SE 0 - TE./SACCEAR./OE - JZ5 CC. GAS - 100 o / 2 DAYS F i g . 15. i 5 0 / 2 3 DAYS 0 / Z 3 DAYS &a3 e v o l u t i o n from t h e f e r m e n t a t io n o f 50 c c . o f one p er c e n t s o l n s . o f v a r i o u s c a r to n compounds. -93i . E f f e c t o f h e a t on v i a b i l i t y o f broth c u l t u r e s . S e v e r a l e x p e r im e n ts were condu cted to determ ine th e therm al d e a th t i m e s f o r s t r a i n s H—1^+33 and H—13S . broth con ta in in g d extrose ( 0.5 p e r c e n t ) , KgHPO^ ( 0 . 5 p e r c e n t ) w e r e u s e d . cc. Twenty—f o u r hou r c u l t u r e s grown i n ( 0-5 p e r c e n t ) , tryptone In the p relim in a ry experim ents, and fiv e amounts o f t h e c u l t u r e s were p l a c e d i n a w a te r b a t h a t th e d e s i r e d tem perature. The w a t e r was c i r c u l a t e d b y a m ot o r d r i v e n s t i r r e r . t h e same t i m e t h e c u l t u r e s w e r e p l a c e d i n t h e b a t h , At a l i k e tube o f b ro th w i t h a n i n s e r t e d t h e r m o m e t e r wa s im m er s ed , w h i c h s e r v e d a s a t e m p e r a t u r e c o n tr o l fo r the c u ltu r e s . At t h e v a r i o u s t e m p e r a t u r e and t i m e i n t e r v a l s , s t a n d a r d l o o p s o f t h e b r o t h c u l t u r e s w e r e r em ov ed and t r a n s f e r r e d t o sterile t u b e s o f b r o t h and i n c u b a t e d Us .0 72 h o u r s a t 3 5 ° C- and ob­ served fo r growth. In th e f i r s t e x p e r i m e n t t h e w a t e r b a t h was k e p t a t 6 0 ° C. and l o o p s o f t h e b r o t h c u l t u r e s w ere r e m o v e d when t h e l a t t e r had r e a c h e d U0 °, 5 0 ° a n d 6 0 ° C. a s w e l l a s i n t e r v a l s o f tw o , f o u r , m i n u t e s a.t 6 0 ° 0 . six, e i g h t and 10 The t i m e s f o r t h e b r o t h t u b e s t o r e a c h Uo °, 6 0 ° C. w e r e o n e - h a l f m i n u t e , 50 ° and o n e m i n u t e and two and o n e - h a l f m i n u t e s r e ­ sp ectively. The r e s u l t s show ed t h a t s t r a i n H -1 3 3 s u r v i v e d 5 0 ° "but was k i l l e d b y e x p o s u r e t o 60 ° 0 - w h i l e s t r a i n H-1>+3S s u r v i v e d two m i n u t e s a t 60° C. b u t was k i l l e d b y an e x p o s u r e f o r f o u r m i n u t e s a t t h e same t e m p e r a t u r e . I n t h e n e x t e x p e r i m e n t , t h e w a t e r b a t h was m a i n t a i n e d a t 5®° C. and e x p o s u r e i n t e r v a l s u s e d w e re t w o , T h i s t e m p e r a t u r e was s e l e c t e d four, six , e i g h t and 10 m i n u t e s . In order to determ ine i t s l a r l y on s t r a i n H - 13S f o r l o n g e r e x p o s u r e i n t e r v a l s . eifect, p articu ­ -9 1 + - I n t h e a b o v e t e s t s H-1 3S s u r v i v e d a l l e x p o s u r e i n t e r v a l s . S t r a i n H - 1 ^ 3 3 s h ow ed t h e same r e s u l t s , it s u r v i v e d 60® C. f o r w h i c h was t o be e x p e c t e d s i n c e two m i n u t e s . In th e f i n a l experim en t, t h e t o t a l number o f o r g a n i s m s o f s t r a i n H-1433 s u r v i v i n g t e m p e r a t u r e s o f *i0°, 5 0 ° , f o u r an d s i x m i n u t e s wa s d e t e r m i n e d . h o u r b r o t h c u l t u r e w a s u s e d and a t 55°, 6 0 ° and 6 0 ° C. f o r t w o , In t h i s c a s e , e i g h t c c . o f a 2^ t h e a b o v e t e m p e r a t u r e s one c c . was removed and p l a t e d on o r d in a r y d e s t r o s e a g a r . The r e s u l t s o b t a i n e d a r e t a b u l a t e d below : 1 ! I i Temperature 1 1 0 c. t | In itia l* « (30°) j Survival Count i per cc. ' 1 , 920, 000,000 , 1 0 0 1 , 920, 000,000 1 1 ,0 0 8 ,000,000 50° 1 t 55° 6o° » , 60 ° ( f o r 2 m i n . ) , , 60° ( f o r 4 " ) , | 60° ( f o r 6 " ) , 793, 000,000 1 t , 1 1 ' 23,000 , 5,000 , 10 , 0 . i * | At s t a r t o f t h e e x p e r i m e n t . The a b o v e p l a t e c o u n t s p e r c c . , p l o t t e d l o g a r i t h m i c a l l y , shown i n F i g u r e l b . H e r e , t h e v e r y s h a r p downward t r e n d o f t h e s u r v i v a l curve in d ic a t e s th a t th e c r i t i c a l proached. are d e a t h t e m p e r a t u r e ( 60° ) i s b e i n g ap­ P o i n t s A and B r e p r e s e n t t h e o r g a n i s m s p r e s e n t a f t e r e x p o s u r e f o r two and f o u r m i n u t e s r e s p e c t i v e l y a t 6 0 ° C. -95- /OB /a <►A 30 40 jiS G E .E e s F i ' 3'. 1 6 . 60 SO C. E f f e c t o f t e m p e r a t u r e on t h e d e a t h r a t e o f s t r a i n B t f o u r min. e x p o su re A, two m i n . e x p o s u r e a t 60® C. a t 60° C. Table 23 • Fermentation of Dextrose Broth, Buffered at pH 5*^-5> C o llec tio n . , Several Strains of the Stock Culture 1 ■ 1 139.3 69.0 1 96.1 ,Ratio o f, Remainder Residual Oas , t gas , Hydrogen ,H2 :C02**, °2 gas accounted fo r , by a n a ly sis , I 1 * cc. i cc. cc. cc. t f 1 1 4.0 , 27.9 38.9 > 1:2.47 , .3 13.3 97.9 , H-U39 . 2 129.0 68.8 1 88.8 , 29.6 . - 3 145.7 70.0 ,102.0 , 28.0 , 4 102.5 70.3 , 72.1 ' K-1338 ' 5 M .f 1 E-^33 • 6 1 H-138 ’ 7 ,Outfit , Strain , Ho. Total v o l. of gas* cc. • h-739 h-639 , H-63S * Carbon dioxide P 1 cc. r 27.4 38.2 » 1 :2.32 , •3 I ! 40.8 , 1 :2.50 , .6 1 1 28.1 . 1 :2.56 , .3 71.0 >104.2 1 27. 3 l4o.O 70.8 - 99.1 33.3 55-7 > 18.5 1-7 12.3 99.4 2.3 ■ 15.6 100.0 2.0 12.3 99.2 40.1 < 1:2.59 ■ •3 2.1 17.0 99.3 ’ 27.3 38.2 > 1:2.59 1 •3 2.4 13.8 99.1 - 38.5 12.8 1 1:1.44 1 •3 1 ) 1.7 4.4 99.2 Values based on fermentation of 50 cc. of one per cent dextrose broth; gas volumes include residual gas. ** Calculated from volumes of H2 and COg. t P rin cip a lly nitrogen, also includes experimental error. Table 2H. 1 S tra in Hate o f Gas Production During Ferm entation o f D extrose Broth, B uffered at pH 15. by S everal S tra in s from Stock Culture C o lle c tio n . O u tfit No. i"\---------1t ' , 1 ia y T o t a l volume o f gas c o l l e c t e d i n c c . * 2 days 3 days 1 if days 5 days 6 d ays 7 days 1 1 H-739 1 , ! 49 107 113 . 116 116 116 120 , , h-^39 2 ,, 30 98 105 , 109 109 109 109 j , H-639 3 ,, 75 125 125 , 125 125 128 130 ! 1 H- 63S if '’ ! 39 75 78 ' SO 81 82 S3 | ' 121 121 121 128 ' 1 122 123 123 125 | ' 27 27 27 28 ' 1 ' H-1333 5 ;’ t 52 118 118 ' H-H38 6 11 ifO 115 119 1 h- 1 3 8 7 ;1 6 2if 26 * 1 1 1 1 Maximum amount o f gas c o l l e c t e d ; d oes not i n c l u d e r e s i d u a l g a s . Table 25 . Comparison o f Four Ferm entations o f D extrose Broth, B uffered a t pH 5*15, by S tra in H-i438 A. Gas composition 1 t , Total , volume , of gas* Carbon Dioxide t 1 cc. * cc. * cc. ■ 132.7 6s.6 92.4 29.3 37.6 1:2.45 . .5 1^9.5 70.0 104.7 28.5 42.6 , H-1^38 1 1 . 1 Hydrogen 2 °2 H2 5COg 1 1 , Ratio**, of , Remainder Residual Gas , gas^ gas accounted for, by an alysis , cc. 4 r 2.2 11.4 99-9 1:2.45 | .3 1-9 17.0 99.5 cc. cc. 3 ' l4s.6 70.2 104.3 28.1 4-1.8 1:2.49 ' .3 2.3 17.2 99.3 4 , 1I+3.0 69.0 9S.7 29-4 42.0 1:2.35 - .3 1.8 15.0 99.9 > ! B. Rate o f Gas Production 1 1 1 Total volume of gas c o lle c te d in c c . ^ ', h-1438 1 1 t 1 l day ( 2 days , 3 days 4 days 115 116 , 3^ ! 113 ' 1 2 ' ! 65 | 130 ' I 130 44 ' 120 * 122 124 | 126 128 * ** t tt | 5 days | 6 days 7 days 120 117 ’ 1 130 ' 1 130 124 ' t 126 ' t 128 | 120 131 ' 128 128 , 128 . 12S , Values based on fermentation of 50 cc. of one per cent dextrose broth; gas volumes include residual gas. Calculated from volumes of Hg and CC^P rincip ally Hg, also includes experimental error. Maximum amount of gas collected ; does not include residual gas. 52 , | 1 0 «—1 1 3 , t . 4 1 1 1 ' | T a b le 2 6 . E f f e c t o f Temperature Upon t h e P r o d u c t i o n and C o m p o sitio n o f t h e Gas from D e x t r o s e B r o th B u f f e r e d a t pH 5»15* 1 TeznVolume of 'perature c o lle c te d gas* 1 in C. cc. Carbon dioxide P Ratio of Hydrogen H2:C02 cc. cc. Gas ' 'Remainder Residual 1 gas** gas accounted for' °2 by an a ly sis ' » cc. cc. 1 cc. > 0 0 | 13° 60.0 67.6 4o.6 28.8 17*3 1:2-34 .2 ' 1*9 29.5 98.1 . ig° 106.9 65.0 69.5 33*5 35*8 lil.Sk .2 • 1.4 11.3 99*5 t 24 ° io 4.o 65.4 68.0 33*4 34.7 1:1-97 .2 ' 1.1 12.1 99*8 ' 30c 126.6 66.2 S3.8 31*9 4o.4 1 :2.07 *3 ' 1.1 18.4 99*1 ' 35° 127.O 69.2 87*3 28.1 35*7 1:2.46 *5 ' 2.9 13. C 99*3 64.6 61.1 32.4 30.7 1:1.99 .4 1 2.4 12.4 99*0 o O ^ 5 5° 94.6 1 h5° 0 * Values based on fermentation of 50 cc. of one per cent dextrose broth; gas volumes include residual gas. P rin cip ally also includes e x p 't. error. ** 0 ' Effect of Temperature on the Hate of Gas Production in Dextrose Broth, Buffered at pH 5*15. t t 1 t T o t a l volume o f gas c o lle c te d i n c c . t Temper ature 7“ , i n C. , 1 day 1 ' 1 , 13° igo 2 1|0 1 1 0* , 0* , 0* 3 7 25 1+1 63 ' 72 ' 1+1 ' 5U 96 101 1 * A l+QO , 1. 33 1+5 50 16 22 25 62 71 87 87 S7 92 92 102 105 105 1 ll4 111* 114 ' 74 7S 82 1 S3 1 1 98 1 1 114 ] ! No g as c o l l e c t e d , but r e s i d u a l gas p r e s e n t i n t h e medium. ' 1 65 57 t t I ' ' 8 days 1 1 1 108 7 days ' t | . 1 • 93 8 8 6 days 10 1 80 , 1 I t 35° 5 days 5 t 26 , 1 1 10 1 t 4 days I 1 30° , 1 t 1 ' 3 days 1 1 1 1 2 days ' 1 “OOI Table 27* Table 28. ! pH E ffe c t o f pH Upon the Production and Composition o f the Gas from B uffered D extrose B roth. , Yolume of , c o lle c te d t gas 1 cc. Carbon dioxide Hydrogen $ cc. P cc. Ratio of H giCOg °2 Remainder gas** cc. cc. Gas , accounted fo r , by analyses , f, ; 3.6 ' 0 . 4.25 . 54.0 54.0 36.8 38.5 21.5 1:1.71 .4 2.3 96.5 ' 5.3 107.0 65-4 78.4 3 3.O 36.3 1 :2.15 •3 1.4 99.9 87 .4 65*4 68.3 32.4 29.8 1:2.29 .4 1.4 99.9 < 6.0 t | ! 7*0 53.3 58.6 46.4 35-6 22.4 1 :2.07 .4 2.9 97.^ ! ; 7.55 59.8 56.4 44.6 39.7 25.2 1 :1.76 .2 2.2 98.1 ’ - s.C5 31-1 U8 - 2 26.8 42.4 16.2 1:1.65 .4 1.8 96.6 ; s .8 5 1 | 21.6 1+8.2 8.7 29.2 5*3 1:1.64 .4 4.2 88.4 | 1 6.8° ' 116.9 64.8 91.9 32.6 38.8 1 :2.36 .4 2.7 98.9 | * ** t i c Figures represent t o t a l volumes of COg and Hg produced by the fermentation, including c o l­ lected and residual gas. P rin cip a lly nitrogen from a ir present above culture medium at sta r t, also includes manipu­ la tio n and experimental error, Ratios calculated from to ta l volumes of COg and Hg produced from 50 cc. of one per cent dextrose broth. Residual gas included in orig in a l analyses of c o llected gas. Unbuffered control. -1 0 2 - Table 2 9 . pH ' 3-6 1 4 .25 ' 5.3 C o m p o s i t i o n of R e s i d u a l Gas f r o m the Ferment a t i o n s T a b u l a t e d in Table 28. I 1 1 V o lume o f ' 1 residual 1 1 gas* 1 1 cc. ' 1 1 0 ' ! \ ' f 9.8 ' 10.7 1 • 6.0 ' 7-0 ' • ' I 77.0 1 78.6 ' •Remain— ider gas 1 ** Hydrogen cc. 7.6 % 7. if cc. lif.O 15.2 ' | ' 1 CC * 8 .if 9*3 ' 11.1 9-7 t 1 . 5 1-0 1.3 l.lf ' 1 2 .2 1 0 .2 1 -6 1.5 • l.if f • 7.55 8.05 ' • 8 , 85* t 1 * 6 . 8C ' * ** i c 79-5 13-7 I | • t ’ 10 .9 1 1 .2 16.6 ' 1 79-0 | • 11.8 1 1 8 .7 1 1 ' ■ 1 5.5 2.3 1 t 1 ' 16.2 3-7 *7 t Gas d i s s o l v e d in culture m e d i u m a n d p r e s e n t above culture medium; all c a l c u l a t i o n s b a s e d on 5° cc- of one p e r cent d e x t r o s e b r o t h fermented. P r i n c i p a l l y air i n t r oduced d u r i n g a n a lysis manipulation, also i n c ludes e x p e r i m e n t a l error, R e s i d u a l gas a n a l y s e d w i t h the coll e c t e d gas. U n b u f f e r e d control. * 1 1 ' t O.S 1 1 1 1 8 6 .5 ’ t 1.3 1*5 ! 1 1 t I 80.0 1 t t 79-5 y t t ' ! 1 1 t •7 t t ’ t Carbon d io x i d e 1 1 .9 1 t -103- Table 30* ! Effect of pH Upon the Rate of Gas Production in the Fermentation of Dextrose in Broth. t 1 1 pH t t 1 t Total volume of gas collected i n cc .* i i i 1 day- ' 2 day s 1 3 days ' 4 days ' 5 days 1 6 days \ t ' 1 • 1 ' 6l ' 1 10 . ' | 53 ’ | 6.0 ' t 49 '7.0 ' 39 ' t 7*55 ' ! ' 1 S . 05 1 1 s .S5 ; ! 6 t 4.25 ' t 5-3 « 1 * c . 8 c 61 1+4 6l 101 ' 110. ' 1 110 ' | 110 76 ' I 85 ' 1 89 « 1 S9 ' 1 1+5 ' ) 49 ' t 53 39 * 1 46 1 1 53 56 ' t 16 • 23 1 1 26 ' I 1 1 ' | 2 ' I ’ | t 3* 1 ' 1 3 S3 1 1 ! 5 112 1 1 29 7 1 1 8 1 1 6l ' « 110 89 ' ' | 60 ' t 60 ' t 60 1 1 60 ' 1 32 ’ 1 32 ' I 1 1 1 * 1 ' 1 ' 9 9 I 1 1 8 ' r Figures based on 50 cc. of dextrose broth fermented, Unbuffered control. t 1 1 8 1 f Table 3 1 . E ffe c t o f S a lt (SfaCL) C oncentration Upon Gas Production and Composition During the Ferm entation o f D extrose Broth and Cucumber Ju ice Broth. S a lt c o n c e n t. j sat. g*jl00 cc. Carbon rii’ n ri rift V o l. o f ! c o lle c te d gas* 1 R s. gas 1 pH** 1 \ 1 Gas a c c ’ t .1 fo r by 1 a n a ly se s 1 C C . | - \ * 1 cc. f 1 ) cc. cc. cc. \ \ < 5.15 66.8 • SU.8 > 31.1 1 39.5 < 1 : 2 .Ik *3 2*3 1 3 .1 6 8 .0 8 5 .0 , 35.9 , 1 :2 .3 6 .5 3*6 13*0 r M ’ 23.9 ‘ 1 :1 * 9 7 ! t . 5.2 1 1 : 1.03 1 ! 1p■ t 1 ! _ t a..* 1 - 1- ____ *5 2*5 9 .6 ’ ^*75 99*0 — 2 .5 6.1 1 I+.65 9 3 .7 5 1 .3 3 125*0 1 10 2 .6 8 7 ^ .2 , 63.8 ' t ’ 02 R. gas 1 2 6 .9 , « TIvrirojs*fl'n 1 R a t io o f ' H2 :C0 2 D e x t r o s e b r o t h b u f f e r e d a t pH 5*15 cc. 0 1 \ t A. 0 1 15 1+.06 1 3 .1 20 5-1+7 0 25 6 .9 1 0 i+s.o . 1 —— 1 t 1 B. , 2 8 .7 ^7*3 '| 32*3 5. 1+ . U5.7 ! — 1 — 1 _ \ — — 0 ! — 0 1 ^*55 Cucumber j u i c e b r o t h u n b u f f e r e d ! 1 \ \ I 1 \ 1 99*0 98*7 x -■ 1 » 0 0 ' 5 1.33 2 0 8 .6 79.^ ' 165.6 ' 19*25 ' 1+0.3 ' 1:1+.10 .1+ 2 .3 16.7 • 5.7 9 9 .6 ! 10 2 .6 8 1 0 1 .5 8 0 .8 1 : 5*06 .1+ 2*9 ll+.O , 5.65 9 8 .2 ’ 15 1+.06 8.9 ‘ 1 : 5 - 5 3 J .1+ 5.0 8.5 ■ 5.6 97*8 . 20 b .2 ! •5 2.5 12.6 , 5.55 9 8 .1 ; 25 — — * (lo st) , 8 2 .0 , 16.0 , 16.2 , 63.6 79*2 ' 1+9.3 't 15.6 5. 1+7 29.O 7I+.6 , 2 1 .6 . 1U .5 ' 1 . 6.91 0 \ I 1 i I t — ! 1 1:5.11+ ! 5.9 0 ; 5.5 — Includes residual gas; calculations based on 5° cc. ofone per centdextrose broth, and 50 cc. of two per cent cucumber juice broth. ** pH values taken atstart of fermentation, drop in pH dueto influence of NaCl. R. Remainder gas. R s . Residual gas. , Table 3 2 . E ffe c t o f S a lt (HaCl) C oncentration Upon th e Hate o f Gas P rod u ction in the Ferm entation o f D extrose Broth and Cucumber J u ic e B roth. ' t S a lt co n cen tra tio n | $ sat . 1 1 T o t a l volum e o f g a s c o l l e c t e d i n c c . J g ./lO O c c . 1 day J 2 d ays J 3 days j 4 days J 5 days | 6 days 7 days { D e x t r o s e b r o th * \ t 1 0 t . 5 1 . t 0 72 103 1 .3 3 72 105 21 ’ ! 1 t 1 1 113 1 108 . 1 1 ll4 114 t ! 112 112 , I 1 0 2 I 64 64 64 , 4 5 6 7 0 ' 0 0 0 ' 0 ( 0 0 0 . ' 2 .6 8 46 15 1 4 .0 6 0d , 0d , 2 20 ' 5^7 0 ' 0 25 i 6 .9 1 0 ' t . 37 1 ll4 5S 10 0 t ' 1 « 0 0 1 I . Cucumber J u i c e b r o th * * 1 . 0 5 * 10 1 , 1 1 t 0 1 ! 1 1 1 (lo st) ! 50 133 ' 16 T4 , 2 29 ' 0d ! 180 I 1 189 ; 192 79 84 ' 88 33 4i 1 ' * 1 195 195 92 93 52 55 16 16 0 0 ; 1 . 15 1 1 ' 3 ' 11 0 1 0 . 0 ’ 20 * 25 * ** d F i g u r e s b a s e d on 50 c c . o f one p e r c e n t d e x t r o s e b r o t h . F i g u r e s b a s e d on 50 c c . o f two p e r c e n t cucumber j u i c e b r o t h , Ko c o l l e c t e d g a s , but g a s p r e s e n t i n t h e medium. j \ I 0 t 47 t 1 15 0 1 1 . Table 33* E ffe c t o f Heat and H ad Upon Gas Production and Composition in th e Fermentation o f Fresh Cucumber J u ic e . ' Treatm ent o f 'S e itz f i l t e r e d 1 ju ic e cc. , I Unheated 1 I I + ho min. 1 a u t o c la v i n g * * 1 I I I + 100 min. 1 a u to cla v in g . 2 7 5 .6 aP 11*0 cc. 212.2 1 1 Hydrogen * , 22.0 1 R a t io o f 1 Hg:COg cc. , 60.6 °2 R. R s. cc. cc. cc. In ­ itia l pH F in a l1 pH - , 1:3-50 .6 2.2 19.4 6 .3 5-95 , ; 1 : 3.97 .6 2 .9 17.7 6.1 5 .5 5 | 1 207.6 78.6 163.2 1 19.8 ’ 4 1 .1 \ ! 7 8 .6 1 ^ 5 .6 > 19.2 ' 1 :4 .0 8 .4 3 .6 i 4 .5 5.65 5 .5 » 1 IV Unheated + 1 1556 s a t . C.P. ' EaCl ■ 35.6 I 1 1 69.O 8 1 .7 5 6 .4 ■ 1 1 .6 • ' 1 :7 .0 5 .4 4 .2 13.5 6 .2 5 .4 - , V Same a s IV ,w ith p la n t s a l t 5 3 .7 8 3 .2 4 4 .7 1 ! 1: 9.33 .4 4 .1 9.9 6 .2 5.4 ! ’ 1 :7. 98 •3 3.6 5.85 5.3 ’ , 1 : 5-00 .2 •5 5-35 5 . 18 , i VII Same as VI 1 + 100 min. 1 a u to c lsv in g * ** R. R s. 8 .0 8 .3 '9 0 T - 1 8 5 .2 1 VI + 15# s a t . C * P. HaCl + 40 1 min. a u t o 1 c la v in g A Carbon d io x id e Volume o f c o lle c te d gas 1 I 1 6 4 .1 1U.5 83.5 7 9 .0 53.5 1 1 .5 1 10. 1+ ’ , 15.6 I i . 1 6.7 2.3 I n c lu d e s r e s i d u a l g a s; c a l c u l a t i o n s b ased upon 50 c c . o f j u i c e fe r m e n te d . A u to c la v e d a t 15 l b s . p r e s s u r e . Remainder g a s . R e s id u a l g a s . i4 .i 6.0 Table 3*1. i ' • E ffe c t o f Heat and NaCl Upon the Rate o f Gas Production in Fresh Cucumber J u ic e . Treatm ent o f S eitz f ilt e r e d ju ic e ■ ' ' T o t a l volume o f g a s c o l l e c t e d i n c c . * 1 day 1 2 days ' 3 days ! 1 , I Unheated I , ’ I I + 40 min. 1 a u to c la v in g ’ 1 \ \ 1 I I I + 100 min. i a u to c la v in g i1"" — --------------- . t 1 IV U nheated + 15$ ' s a t . C.P. UaCl ' , VI + 15$ s a t . G.P. NaCI + *+0 min. ( a u to c la v in g * i i ' ■ > - t , , VII Same a s VI + t t 100 m in. a u t o c l a v i n g , * d 88 | 202 1 1 1 ; ... ' ... 5 d ay s | 2h2 23s 1 169 1 187 i 6 days * | 2^9 ' ; 196 256 | ' 196 ' 168 , l 1 13 . 88 1 131 i 151 . t ? 1 1 1 1 1 21 ho ! 1 1 t * ' 1 1' 1 7 d a ys y 1 56 ' 56 . hg t 50 \ 56 * 5 F ig u r e s b a s e d on 50 c c . o f j u i c e fe r m e n te d , Ho c o l l e c t e d g a s , but r e s i d u a l gas p r e s e n t i n t h e medium. 8 I s * f ' 56 1 1 50 1 1 3 [ t 39 21 i 0d « . . 1 lh h 101 s 3 h days . ... 1 \ 20 23^ 1 1_ « 50 1 1 8 , S | Table 3 5 . E ffe c t o f D extrose ( 0 .1 g .) Upon M altose Ferm entation. A. T otal gas p rod u ction from d extrose and m altose m ixture. C a lc u la t e d amount o f gas r e s u l t i n g from 0 . 1 g . o f d e x t r o s e b a s e d on d e x t r o s e f e r m e n t a t i o n . . . 2g.O c c . C a lc u la t e d amount o f g as r e s u l t i n g from 0 . 4 g . o f m a lt o s e b a s e d on m a lt o s e f e r m e n t a t i o n 30*5 T o ta l* amount o f g a s p roduced from t h e m ix tu r e o f 0 . 1 g . o f d e x t r o s e and 0 . 4 g . o f m a lto se ' 5^*^ c c . 1 _________________ B. t 4 .1 c c . 1 D i f f e r e n c e b etw een f e r m e n t a t i o n . . . . R ate o f Gas P r o d u c t io n i n t h e M ixture o f D e x tr o s e and M a lto se Compared to M a lto se A lo n e . T o t a l volume o f c o l l e c t e d gas i n c c . ! 1 1 day 1 1 ' t 0.1 g . o f d e x t r o s e + 0 . 4 g . o f m a lt o s e ' 0.5 g . o f m a lt o s e a lo n e * I n c lu d e s r e s i d u a l g a s . 2 days 1 3 d ays . ' 13 19 1 12 24 \ t 4 days 5 days 1 6 days . ' ' "1 r ' 1 1 ! 24 30 28 31 32 ' 36 31 : 31 1 ! -S O T 58.5 c c . 1 T otal Table 3 6 . Production and Composition of Gas from Carbon Compounds in 0.5 Tryptone Broth by S tra in H—lM-3S. 1 Compound , (0* 5 g . ) V o l. o f c o lle c te d g as , , f> . CC.* 1 8 3 .1 5 9 .1 1 t 57 ^ D e x tr o s e 1 3 1 .3 6 6 .2 1 9 3 .3 ' d - G a la c t o s e 1 1 1 .5 60.8 | ' L actose^ 1+6.6 t L e v u lo s e cc. R a t io o f , Hydrogen . c c .* 3I+.I ' f Hg aCOg I °2 , Remainder , Gas a c , c o u n te d f o r , gas ,by a n a l y s i s 1 cc. 1 c c .* * , , , t 1 31.8 . 2 8 .7 1 :2 .0 0 ’ •3 ' 1 5* | 95.3 1+2.7 1 :2 .1 8 s •3 . 2 .2 . 99.0 7 3 .2 - 1 36.8 | 1+1.5 1 : 1.76 | .5 30.6 ' 1I+.2 ’ 61+.2 ' 30.1+ 2 .ll+ :l ' .3 ’| 1 .7 « 97.8 98.3 63.O , 69.0 , 3 1 .5 . 32.1 1 : 2 . ll+ , .1+ . M ! 96.5 ! M altose 30.5 36.6 j 1 5 .9 1 53*3 1 1 7 .* 1 .0 9 :1 | .2 ' t 2 .8 ; 92.9 1 1 d-Mannose 110.8 2 8 .1 ' ( 3 3 .7 1: 2.50 ' .1+ ’ 5 .5 • 96.7 50.0 , 67.1 1: 1.03 , •3 « 1.1+ . 99.6 ! | 1 1 d -M a n n ito l 131.8 | 6 6 .6 1 79^ | 1 1+8.6 1 6 9 .3 • » 99.6 2 *2 R a ffin o se ^ 5 8 .7 1+1.3 | 2I+.2 ' 5 3 -7 | 3 1 .5 1. 30:1 | •5 ' 1 2 .8 ; 97-2 1 Rhamnose*^ 52.8 1+1+.6 « 23.6 ' 1+9.5 ' 26.1 1 .1 0 :1 ' •3 • 2 .9 - 96.6 , S a cch a ro se 1 0 9 .7 b 3 .8 ! 75. 7 ! 29. 1+ ! 3 3 .5 1 :2 .2 5 ! •7 1 6.7 ; 96.2 1 S a la c in ^ 5 8 .6 1+2.7 ' 25.0 1 52. 1+ ' 30.7 1 .2 2 :1 ’ .2 1 1 2 .7 ; 97.1 r d -S o r b ito l 85.9 1+0.0 ' 1+1.3 ' 1 .2 2 :1 ' .1+ ’ 2 .3 • 99.3 1 1 - X y lo s e 3I+.6 32.3 . 1 8 .0 , 56.8 ' 5 0 .7 11 56.0 , 20.7 1 .1 5 :1 i .1+ . 3-6 ! 9^.9 * ** t tt ! ; p ' 1 ; - 1 l- A r a b i n o s e A Carbon d io x id e 60 I , 1 F ig u r e s r e p r e s e n t t o t a l volum es o f C02 and H2 p roduced from t h e f e r m e n t a t i o n s and in c l u d e c o l l e c t e d g as a s w e l l a s r e s i d u a l g a s . P r i n c i p a l l y n i t r o g e n from t h e a i r p r e s e n t above t h e c u l t u r e medium a t s t a r t o f f e r m e n t a t i o n , a l s o in c l u d e s e x p e r im e n ta l e r r o r , R a t i o s c a l c u l a t e d from th e t o t a l volum es o f Hg and C02 p roduced from 50 c c . o f one p er c e n t s o l u t i o n s , The o r i g i n a l gas volum es from 50 c c . o f one p e r c e n t s o l u t i o n s i n o rd er l i s t e d above were 9 »0 , 1 6 . 8 , 9*1 and I 3 . I c c . r e s p e c t i v e l y ; s i n c e t h e s e volum es were t o o s m a ll f o r a c c u r a t e a n a l y s e s th e fe r m e n ta ­ t i o n s were r e p e a t e d . F ig u r e s i n t h e above t a b l e a r e b a s e d on t o t a l g a s volume from f e r m e n t a t i o n o f 50 c c . Q u a n t it ie s o f t h r e e p e r c e n t s o l u t i o n s o f each c a r b o h y d r a te . Composition of Residual Gas From Fermentations Shown in Table 36. Compound 1 1 -A r a b in o s e Carbon d io x id e Volume o f r e sid u a l gas* cc. $ 1 ' cc. Hydrogen 1 I L e v u lo s e M a lto se d-Mannose d -M a n n ito l R a f f in o s e * * ! 72.0 ; 6.3; 8 .9 6 i.o ' 11 1 5 .4 — ' t1 1 9 .8 1 7 i.c ; 7-° 1 i , 7-6 1 6 l.4 1 4.7 » 14.3 ' 7 -9 : 71.0 i 1 J i 7-6 ■ 69.0 ' 5.2 ’ | t 1 1 1 1 1 *i i I .9 1 1.6 6 .9 5.05 .5 I' 3 .0 6.9 4.75 ■ ! I 7 .0 5 4.5 ** t 5 .8 ’ 1 n . 5 ; 5.6 , 7 .3 iM ’ 6 7 .^ ’ fI S a ia c in * * t — ! ' 9-7 ' f , 5 .7 ' 1 70.0 , 6 . 9 ’ 1 6 . 8 t I 6.95 ; 4.55 1 .1 ! 1 .8 MM * 4.55 .6 , 1*7 6.95 ! 4.7 1 .3 7.15 ; 4.9 7.15 . ■ 4.3 1 .1 ' 1 1 1 70.8 1 3 -9 ' 1 1 7.0 1.6 ! 19.1 1 1 .9 13.7 1 .3 . 4.6 1. 7 1 .2 ' ' 1I 1 1*1 '1 ' 8 .5 9-7 1 1 0 .5 | ! 1 iI cc. 6.65 t S a cch a ro se at f in is h o f f erm. 1 .4 ! ! at sta rt o f fe r m . .4 1 1 i ' 1 — I * — cc. pH o f s o l n . * 1 1 1 ( 1 - X y lo s e . S .8 1 0 .1 Rhamnose^ d -S o r b ito l 4 .2 8.3 . D extrose L a c to se * * - J 8 2 .0 , I 4 -C -a la cto se $ J i i Remainder gas 4.7 7.05 1 4.65 7.05 ! 4.35 ’ 1 0 .9 7.1 . 1.6 6.75 5.2 . 4.7 C-as d i s s o l v e d i n c u l t u r e media and p r e s e n t above t h e m ed ia. A l l c a l c u l a t i o n s b a s e d on 50 c c . o f b r o th c o n t a i n i n g one p e r cen t o f th e carbon compound. Hot s u f f i c i e n t g a s f o r a n a l y s i s . R e s id u a l gas in c lu d e d a t tim e o f a n a l y s e s o f c o l l e c t e d g a s . -on Table 37. -1 1 1 - Table 38- Rate of Gas Production Prom Different Carbon Compounds by Strain H-1U3 8 . T o t a l vo lu m e o f g a s c o l l e c t e d i n c c - 1 OUrrvmUmLiLnilU H. f il-A rabinose 1 da y 1 2 d a y s 1 1 35 t 62 t 1 39 D extrose 100 79 101 k 5 8 1 28 77 9s t 12 2*4 30 id—G a l a c t o s e 1 30 1 Lactose 1 1 L evulose , M altose . 3 da y s * 1+ d a y s \ , 8*4 79 1 1 131 131 . t 1 t . t 1 id—Mannose 1 39 . 109 111 t , id-M annitol 1 29 1 1 77 112 , 1 R affinose 1 1 12 lb 9 Rhamnose , 2 , Saccharose, 30 , S alacin , 0** 131 112 8 9 92 98 t 98 ! ! 1 . 31 1 111 1 132 ! 17 » 14 ! 31 31 111 111 132 132 15 16 9 I 1 3 b ! i . su 110 | 110 110 2 7 ! 10 12 13 1 72 1 32 1 !+ u , 12 ! 30 4s 1 57 63 ,1-X ylose , 10 1 19 26 ! 30 32 ** . 109 ,d -S orb itol * 131 1 1 1 113 1 . 1 . * , 5 days 1 6 days 1 X 8*4 • 8*4 1 , ! no , F i g u r e s b a s e d on 50 c c - o f b r o t h c o n t a i n i n g one p e r c e n t o f t h e c a r b o n compound. No c o l l e c t e d g a s , b u t r e s i d u a l g a s p r e s e n t i n t h e medium. -1 1 2 - EERMENTATIONS UNDER COMMERCIAL CONDITIONS The e x p e r i m e n t a l w o r k p r e s e n t e d up u n t i l w ith i s o l a t i o n , t h i s p o in t has d e a lt i d e n t i f i c a t i o n and b io c h e m ic a l s t u d i e s o f th e organism s r e s p o n s i b l e f o r t h e e v o l u t i o n o f h y d r o g e n d u r i n g cucumber f e r m e n t a t i o n s . The c o n c l u d i n g p o r t i o n o f t h e e x p e r i m e n t a l o b s e r v a t i o n s , d eals p r i n c i p a l l y w i t h t h e t y p i c a l f e r m e n t a t i o n s o f t h e s e o r g a n i s m s u n d e r com­ m ercial s a l t i n g c o n d itio n s. The f e r m e n t a t i o n s w e r e f o l l o w e d a s t o t h e i r p r o g r e s s iv e changes in gas e v o lu tio n , ga.s c o m p o s i t i o n an d y e a s t p o p u l a ­ tio n s . The w o r k r e p o r t e d i s b a s e d o n s t u d i e s made d u r i n g t h e 19I+O s e a ­ son. E xperim ental Procedure I n g e n e r a l t h e s a l t i n g p r o c e d u r e was a s f o l l o w s : V a t s o f S5 b u s h e l c a p a c i t y ( s e e F i g u r e 17 ) w e r e f i l l e d w i t h c u c u m b e r s , f i t t e d w i t h f a l s e h e a d s a n d s a l t b r i n e a d d e d s o a s t o come a f e w i n c h e s a b o v e t h e heads. I n i t i a l b r i n e c o n c e n t r a t i o n s o f 2 0 , 4 0 and 6 0 ° s a l o m e t e r ( p e r cen t s a t u r a t i o n w i t h r e s p e c t to s a l t ) were u s e d . sch ed ule, showing th e r a t e o f i n c r e a s e o f b r in e c o n c e n t r a t io n f o r each treatm ent i s shown i n T a b l e 3 9 - The s a l t i n g p r o c e d u r e s w i l l b e i n d i ­ c a t e d t h r o u g h o u t t h e work b y t h e i n i t i a l T a b l e 39* 1 t sa lt concen tration - S a l t i n g S c h e d u l e (19^+0). I n i t i a l brine concen tration 1 ' ' The a c t u a l s a l t i n g 0 salom eter j 1 1 20 ' ' , uo , « 60 1 Rate o f in c r e a s e of brin e concen tration 0 salom eter 1 2 , ' 1 1 1 1 ' up 1 0 ° p e r week t o 60° 5° p e r week t o 60° h e l d a t 60 ° Number o f vats follow ed ' 1 3 2 ' / i g . 17. E x p erim en ta l v a t s { 8p bu. c a p a c i t y ) o f t h e U. S. De^t. o f A g r i c . , l o c a t e d a t t h e Chas. F. C a tes Company, F a is o n , II. C. At the tim e t h e p h otog rap h ‘ as ta k e n , most o f t h e v a t s had h e e n p u t down and f e r m e n t a t io n was under way; two v a t s ( 5t h and 7th i n r i g h t row) a re b e i n g ore^sred p r i o r t o f i l l i n g w it h cucum bers. -lll+ In a d d it io n to th e v a ts l i s t e d above, s e v e n o t h e r s were f o l ­ lowed to t h e e x t e n t o f s i n g l e gas a n a l y s e s d u r in g a c t i v e f e r m e n t a t i o n . The l a t t e r v a t s w e r e l o c a t e d a t a n o t h e r p l a n t and w e r e s a l t e d s i m i l a . r t o t h e Uo° b r i n e t r e a t m e n t , e x c e p t t h a t s a l t and w a t e r w e r e a d d e d d u r - ■ i n g t h e f i l l i n g o f t h e v a t s w i t h c u c u m b e r s i n am ou nts s u f f i c i e n t to make a n i n i t i a l Uo° b r i n e . The p r e v a i l i n g b r i n e t e m p e r a t u r e d u r i n g a c t i v e f e r m e n t a t i o n ( t w o t o s i x w e e k s ) was w i t h i n t h e r a n g e o f jG t o 80 ° IP. A l l v a t s w ere u n sh eltered . The c o m p a r a t i v e g a s e v o l u t i o n s t u d i e s w e r e b a s e d on t h e amount o f gas c o l l e c t e d from a r e p r e s e n t a t i v e p o r t i o n o f th e s u r f a c e a r e a o f each v a t . salted , The p r o c e d u r e was a s f o l l o w s : an i n v e r t e d s t a i n l e s s At t h e t i m e e a c h v a t was s t e e l f u n n e l * 1^4- i n c h e s i n d i a m e t e r was p l a c e d j u s t b elow th e f a l s e head in th e v a t s . I t was h e l d i n p l a c e by r u n n i n g t h e d e l i v e r y t u b e end o f t h e f u n n e l t h r o u g h a h o l e b o r e d i n t h e head. The t r a p p e d g a s e s w e r e c o l l e c t e d o v e r b r i n e i n g l a s s b o t t l e s o f th ree to f i v e gallon cap acity. At r e g u l a r i n t e r v a l s the c o l l e c t e d g a s e s w e r e r e m o v e d b y d i s p l a c e m e n t i n t o g r a d u a t e d one g a l l o n c o n t a i n e r s and m e a s u r e d t o o n e - t e n t h o f one l i t e r . Gas e v o l u t i o n d e t e r m i n a t i o n s w e re c a r r i e d o u t from t h e t i m e t h e v a t s w e r e p u t down u n t i l s i g n i f i c a n t g a s e v o l u t i o n had c e a s e d ( a b o u t o ne m o n t h ) . Fo r t h e m aj or p o r t i o n o f t h e g a s a n a l y s e s ( Uo ° t o 60° t r e a t m e n t s ) , t h e s a m p l e s w e r e c o l l e c t e d fr om t h e s u r f a c e o f t h e b r i n e i n t h e f o l l o w ­ i n g man ner: * An i n v e r t e d g l a s s f u n n e l s u p p l i e d w i t h a s h o r t p i e c e o f r u b ­ The"~ratio""of t h e p o r t i o n o f t h e s u r f a c e a r e a o c c u p i e d b y t h e f u n n e l ( 153.9 s q . i n . ) t o t h e w h o l e s u r f a c e a r e a o f t h e v a t ( U , 0 7 1 - 5 s 9 • ^n -) was 1 : 26 . 4 . -115b e r t u b i n g and a p i n c h clamp was p l a c e d o v e r a c r a c k i n t h e f a l s e h e a d and t h e e v o l v e d g a s e s c o l l e c t e d b y d i s p l a c e m e n t . 200 c c . o f gas were c o l l e c t e d , ment i n t o When a p p r o x i m a t e l y t h e s am pl e was t r a , n s f e r r e d b y d i s p l a c e ­ th e r e c e i v i n g b o t t l e of the sam pling o u t f i t o f th e vat in Figure IS. shown on t h e t o p By h a v i n g a c o m p l e t e s e t o f f u n n e l s and g a s t r a n s f e r b o t t l e s r e s e r v e d f o r each v at under o b s e r v a t io n , samples cou ld b e t a k e n q u i c k l y an d a t r e g u l a r i n t e r v a l s and t h e g a s a n a l y z e d w i t h o u t unnecessary d elay. T h i s was o f c o n s i d e r a b l e h e l p i n t h e c a s e s where a n a l y s i s was made t h r e e a n d f o u r t i m e s a, da y d u r i n g a c t i v e gas e v o l u t i o n . The g a s a n a l y s e s f o r t h e two 2 0 ° f e r m e n t a t i o n s ( o f w h i c h o n l y one i s r e ­ p o r t e d ) w e r e o b t a i n e d from t h e t h r e e g a l l o n c a p a c i t y c o l l e c t i o n b o t t l e s , u s e d f o r t h e measurement o f g a s e v o l u t i o n . l y t h e same a s t h a t The p r o c e d u r e was e s s e n t i a l ­ ju st described. The me th od o f a n a l y s i s f o r t h e g a s s a m p l e s h a s b e e n d e s c r i b e d i n d e t a i l elsew here in t h i s r ep o r t. The d e t e r m i n a t i o n f o r o x y g e n was made on a l l s a m p l e s b u t t h e v a l u e s , b e i n g t o o s m a l l t o b e o f any s i g n i f i c a n c e , a r e o m i t t e d fr om t h e r e s u l t s . D uring a c t i v e e v o l u t i o n , w ay s f e l l b e t w e e n 0 . 2 and 0.U- p e r c e n t . th ese values a l ­ During slow e v o l u t i o n o f gas fr om t h e f e r m e n t a t i o n s , , e i t h e r a t t h e s t a r t o r d u r i n g t h e l a g b e tv 'e e n t h e hyd_rogen a n d y e a s t f e r m e n t a t i o n s or a t t h e c o n c l u s i o n o f t h e f e r m e n ­ t a t io n proper, the v a lu e s f o r oxygen in c r e a s e d s l i g h t l y . t o t h e more o r l e s s tissu e c o n s t a n t r a t e o f d i f f u s i o n o f a i r from t h e cucumber t h r o u g h o u t t h e c u r i n g p e r i o d and h e n c e o x y g e n w o u ld show up more c l e a r l y when t h e r e w a s s l i g h t y sis T h i s was due e v o l u t i o n due t o m i c r o b i a l a c t i v i t y . f o r m e th an e was made on a l l Anal­ g a s s a m p l e s and none was f o u n d . B r i n e s a m p l e s were t a k e n f o r b a c t e r i o l o g i c a l a n a l y s i s by i n s e r t i n g a p ie c e of s t a i n le s s s t e e l t u b i n g t h r o u g h an o p e n i n g i n t h e he ad o f t h e F i g . IS . T y p ic a l, v ig o r o u s hydrogen f e r m e n t a t io n i n a Uo® s a lo m e te r b r in e (Vat U ), Gas sa m p lin g t r a n s f e r o u t f i t (two 12 o z . b o t t l e s ta p ed t o g e t h e r ) i s shown a t t h e l e f t on th e c r o s s - b o a r d on th e top o f th e v a t . J u s t b elo w , submerged i n th e b r in e i s th e i n v e r t e d g l a s s f u n n e l u sed f o r c o l l e c t i n g s u r f a c e g a s s a m p le s . The f i v e g a l l o n b o t ­ t l e ( f u l l o f gas) s e t o v er th e s t a i n l e s s s t e e l f u n n e l i s shown in th e background. -117vat, down i n t o t h e b r i n e t o w a r d t h e c e n t e r o f t h e v a t and w i t h d r a w i n g th e b r i n e sample throu gh an a t t a c h e d p i e c e o f rubber t u b in g . Two 12 02 . b o t t l e s f u l l w e r e w i t h d r a w n b e f o r e t a k i n g t h e f i n a l s a m p l e . p lin gs Sam­ i n a l l c a s e s were s t a r t e d from about t h e time th e v a t s were put down and h e a d e d and w e r e c o n t i n u e d a t r e g u l a r i n t e r v a l s days) during the course o f fe r m en ta tio n . ( o n e t o two • A l l f e r m e n t a t i o n s w e re e x a m i n e d w i t h r e s p e c t t o y e a s t p o p u l a ­ tion s in the b r in e . T h i s was a c c o m p l i s h e d b y p l a t i n g d i l u t i o n s o f t h e b r i n e on t a r t a r i c a c i d a g a r * ( 2 0 ) . o r d i n a r y d e x t r o s e agar to w h ic h f i v e I n b r i e f t h i s medium c o n s i s t e d o f c c . of s t e r i l e f iv e per cent ta r ­ t a r i c a c i d w e r e ad d e d t o 10 0 c c . am ou nts o f t h e a g a r p r i o r t o p o u r i n g the p l a t e s . The a d d i t i o n o f t h e t a r t a r i c a c i d b r o u g h t t h e pH o f t h e medium t o a p p r o x i m a t e l y 3 - 7 , ganisms e x c e p t th e y e a s t s . ferm entations, it thus i n h i b i t i n g a l l th e u su a l b rin e or­ I n t h e c a s e o f b r i n e s a m p l e s from t h e 6 0 ° was f o u n d n e c e s s a r y t o d e c r e a s e t h e amount o f t a r ­ t a r i c a c i d t o t h r e e c c . p e r 100 c c . o f medium s i n c e t h e y e a s t s from t h e a b o v e f e r m e n t a t i o n s grew p o o r l y when t h e g r e a t e r amount o f a c i d was used. The y e a s t p l a t e s w e re u s u a l l y i n c u b a t e d t h r e e d a y s a t 3 5 ° C. and counted. I n c a s e s w h e r e g r o w t h was s p a r s e , t h e i n c u b a t i o n p e r i o d was extended to f i v e d a y s. Two d 0 ° f e r m e n t a t i o n s ( V a t s k and 6) w e r e f o l l o w e d w i t h r e s p e c t to th e p o p u l a t i o n s o f th e organisms r e s p o n s i b l e f o r th e e v o l u t i o n o f h y d r o g e n ( t h e A e r o b a c t e r )♦ An e s t i m a t i o n o f t h e i r numbers was b a s e d up­ on t h e c o l o n i a l c h a r a c t e r i s t i c s * Laboratory Manual (Methods I n t . A ssn . M ilk D e a le r s , p. on n u t r i t i v e of A n a l y s e s c a s e i n a t e agar ( D if e o ) , of M i l k a n d Its P r o d u c t s ) 81 ( 1 9 3 3 ) * m —118co n ta in in g eig h t cc- o f 0 . 4 p e r c e n t b r o m - c r e s o l - p u r p l e i n d i c a t o r add ed a t th e time o f p r e p a r a t i o n o f the a g a r . The f e r m e n t a t i o n s a t 2 0, 4 0 and 6 0 ° s a l o m e t e r b r i n e c o n c e n t r a t i o n w ill oe d i s c u s s e d i n t h e r e v e r s e o r d e r named. The r e s u l t s a r e shown i n g r a p h i c form w i t h r e s p e c t t o y e a s t p o p u l a t i o n s , the com position o f th e evo lved g a s . u n its, g a s e v o l u t i o n and T h i s m a t e r i a l i s p r e s e n t e d i n two t h e u p p e r one d e a l s w i t h y e a s t p o p u l a t i o n s and g a s e v o l u t i o n w h i l e t h e l o w e r o n e shows t h e c o m p o s i t i o n o f t h e e v o l v e d g a s w i t h r e ­ s p e c t t o c a r b o n d i o x i d e and h y d r o g e n . The t a b u l a r d a t a a r e i n c l u d e d f o r s p e c i f i c r e f e r e n c e ( T a b l e s 4 2 , 43 and 44-). The v a l u e s f o r y e a s t p o p u l a t i o n s a r e p l o t t e d l o g a r i t h m i c a l l y , p r i n c i p a l l y to f a c i l i t a t e s h o w i n g c o u n t s p r i o r and s u b s e q u e n t t o a c t i v e fe r m e n ta tio n as w e l l as counts th a t vary g r e a t ly during th e a c t i v e f e r ­ m entation. Coun ts l e s s t h a n 1 , 0 0 0 p e r c c . a r e shown b e l o w t h e d o u b l e l i n e drawn p a r a l l e l t o t h e a b s c i s s a , o p p o s i t e IT on t h e o r d i n a t e , val­ u e s l e s s t h a n 100 p e r c c . a r e n o t p l o t t e d . The ga s e v o l u t i o n v a l u e s f o r a l l f e r m e n t a t i o n s (20, 40 and 60 ° s a l o m e t e r t r e a t m e n t s ) r r e p l o t t e d a c c o r d i n g t o t h e same s c a l e l i t e r s d i v i d e d i n t o s e v e n 20 l i t e r o f the t y p i c a l , and 4 0 ° d ivision s). ( l4o This p e r m its comparison v i g o r o u s h y d r o g e n f e r m e n t a t i o n s a t 6 0 ° ( V a t s 3 and 9) ( V a t s 4 and 6 ) w i t h t h o s e a t 20 and 4 0 ° w h i c h r e s u l t e d i n l i t t l e h y d r o g e n e v o l u t i o n ( V a t s 8 and 1 0 ) . D iscu ssion of R esults 60° Salom eter B rin e s Over a p e r i o d o f s e v e r a l s e a s o n s , p r o b a b l y t h e 6 0° t r e a t m e n t shows t h e most c o n s i s t e n t b e h a v i o r w i t h r e s p e c t to what migh t be t e r m ed t h e -1 1 9 t y p i c a l h y d r o g e n f e r m e n t a t i o n and d u r i n g t h i s p h a s e o f t h e f e r m e n t a t i o n proper, th e e v o lv e d gas c o n s i s t s o f a p p r o x im a te ly eq u a l p o r t i o n s o f hy­ d rogen and carbon d i o x i d e . above s a l t The d a t a f o r d u p l i c a t e f e r m e n t a t i o n s a t t h e c o n c e n t r a t i o n a r e shown i n F i g u r e 19 . The m a t e r i a l i s p r e ­ s e n te d w ith r e s p e c t to p r o g r e s s iv e changes in y e a s t p o p u la tio n s , evolu tion gas ( u p p e r p a r t ) and c o m p o s i t i o n o f t h e g a s e v o l v e d ( l o w e r p a r t ) . E o t h f e r m e n t a t i o n s a r e s o s t r i k i n g l y s i m i l a r t h a t t h e y c a n be d i s c u s s e d jo in tly . The d a t a i n d i c a t e t h a t t h e g a s e o u s f e r m e n t a t i o n p r o p e r , i n g on a b o u t t h e 1 1 t h da y f o r b o t h v a t s , The f i r s t , start­ was d i v i d e d i n t o two p h a s e s . w h i c h c o v e r e d a p e r i o d o f a b o u t one w e ek , was b r o u g h t a b o u t by th e h y d r o g e n -p r o d u c in g b a c t e r i a o f th e genus A erob a.eter. T hr oug hou t t h i s phase o f a c t iv e gas e v o lu tio n , i t w i l l be noted (lower p a r t s of \ F i g u r e 1 9 ) t h a t t h e p e r c e n t a g e o f h y d r o g e n was r e l a t i v e l y h i g h (h o t o 50 p e r c e n t ) . During th e sh o r t in terval (two t o t h r e e d a y s ) o f v e r y slo w gas e v o l u t i o n t h a t f o ll o w e d th e above f e r m e n t a t io n , the gas th a t v'as c o l l e c t e d f o r a n a l y s e s came p r i n c i p a l l y b y d i f f u s i o n from t h e i n ­ t e r i o r s o f t h e " b l o a t e d " or h o l l o w cuc umbers w h i c h were fo r m e d d u r i n g th e a c t i v e hydrogen f e r m e n ta tio n . This a c c o u n ts f o r the p r e s e n c e of c o n s i d e r a b l e am ou nt s ( a b o u t 30 p e r c e n t ) of hydrogen during t h i s in­ terval . The a d v e n t o f t h e a c t i v e y e a s t f e r m e n t a t i o n , on a b o u t t h e l 6 t h to I S t h day, b r o u g h t about th e sec o n d p h a se o f a c t i v e gas e v o l u t i o n . This i s d e m o n s t r a t e d b y t h e upward t r e n d o f t h e g a s e v o l u t i o n c u r v e . The y e a s t f e r m e n t a t i o n c o v e r e d a p e r i o d o f a b o u t 10 t o 12 d a y s w hi c h compares f a v o r a b l y to th e p e r i o d d u r in g which p r o g r e s s i v e gas e v o l u t i o n SAL. *** LQ6 SAL. ^nc OF U>106 [u 1Z0 K Ci took i 8 0 . k/OOT o o P: 60 .. ^ / 0 7 v3 HCOt/AiT** -1 2 2 - Ays F ig . 21. Y east p o p u l a t i o n s , Aerobac t e r p o p u la t i o n s (cu rve la b e le d A ) , g a s e v o l u t i o n and a n a l y s e s o f t h e g a s fr o m a f e r m e n t a t i o n i n M-O* s a l o m e t e r b r i n e fr om Vat 6 . -124I t i s e v i d e n t from c o m p a r i s o n o f t h e two f e r m e n t a t i o n s i n V a t s f o u r an d s i x t h a t some v a r i a t i o n e x i s t e d w i t h r e g a r d t o t h e o n s e t o f the r e s p e c t iv e y e a st fe r m en ta tio n s. I n t h e c a s e o f t h e f o r m e r (Va t 4 ) t h e r e was an i n t e r v a l o f a b o u t n i n e da ys d u r i n g w h i c h g a s e v o l u t i o n was very s lig h t , p r i o r to the advent o f the y e a s t fe r m e n ta tio n . In the c a s e o f t h e l a t t e r f e r m e n t a t i o n ( V a t 6 ) t h i s i n t e r v a l was a b o u t f i v e days d u r a t io n . H ow ev er, in both c a se s, t h e amount o f g a s p r o d u c e d b y th e y e a s t f e r m e n t a t io n r e p r e s e n t e d o n ly a sm all p o r t i o n o f the t o t a l gas p r o d u ced by th e f e r m e n t a t io n p r o p e r . The a n a l y s e s o f t h e f e w s u r ­ f a c e g a s s a m p l e s t h a t c o u l d be c o l l e c t e d d u r i n g t h e a b o v e f e r m e n t a t i o n sho wed t h a t c a r b o n d i o x i d e was t h e p r i n c i p l e c o m p o n e n t. a m o u n ts o f h y d r o g e n w e r e a l s o f o u n d . However, sm all The p r e s e n c e o f t h e l a t t e r was due t o t h e sloxv d i f f u s i o n o f g a s fr o m t h e i n t e r i o r s o f t h e b l o a t e d cu­ cum ber s f o r m e d d u r i n g t h e p r e v i o u s h y d r o g e n f e r m e n t a t i o n . T h i s was t h e same r e l a t i o n s h i p p r e v i o u s l y shown f o r t h e 60° b r i n e s . Of t h e t o t a l amount o f g a s e v o l v e d from t h e 4 0 ° f e r m e n t a t i o n s , a p p r o x i m a t e l y f o u r - f i f t h s was p r o d u c e d b y t h e o r g a n i s m s o f t h e A e r o b a c t e r g r o u p w h i l e t h e r e m a i n d e r was p r o d u c e d by t h e y e a s t s . T h i s i s an i n ­ t e r e s t i n g o b s e r v a t i o n s i n c e i n t h e 60° b r i n e s a l m o s t t h e r e v e r s e was t r u e w h i l e i n b o t h c a s e s ( 4 0 ° and 60° b r i n e s ) the s i g n i f i c a n t yeast c o u n t s w e r e s i m i l a r i n numbers and t h e y o c c u r r e d o v e r c o m p a r a b le p e r i o d s o f tim e. In F ig u r e 22, gas e v o lu tio n curves fo r f e r m e n t a t i o n s a t 4 0 and 6 0 ° ( V a t s 6 , sin gle, a c t i v e hydrogen 9 , p r e v i o u s l y d i s c u s s e d ) a r e shown i n s u c h a manner a s t o e m p h a s i z e t h e amount o f ga s p r o d u c e d by e a c h p h a s e o f a c t i v e f e r m e n t a t i o n as w e l l a s th e d u r a t io n oi th e e v o l u t i o n . This 150 I5C 6D /5 D J2.5 /25 too —o -/—----- -klQC £>------- VAT-9, 60a6AL. / / 2 & C0Z £VOLV£D C0L £ VOL V£D Co 25 8V JO T) 25 D A Y S F i g . 2 2 . Comparison o f gas e v o l u t i o n from f e r m e n t a t io n s i n 40 and 60 ° s a lc m e t e r b r i n e s . L, l i t e r s o f g as c o l l e c t e d from th e 1*+ i n . diam. f u n n e ls ; D, tim e i n days f o r ea ch phase o f e v o l u ­ t i o n showing t h e H2 and C02 cu rve f o r t h e hydrogen f e r m e n t a t io n and t h e C02 curve f o r th e y e a s t f e r m e n t a t io n . g r a p h shows more c l e a r l y what h a s a l r e a d y "been d i s c u s s e d w i t h r e g a r d t o g a s e v o l u t i o n f o r t h e s e two t y p i c a l h y d r o g e n f e r m e n t a t i o n s . Based on t h e amount o f g a s t r a p p e d "by t h e lU i n c h f u n n e l ( a p p r o x i m a t e l y o n e t w e n t y —f i f t h o f t h e t o t a l f e r m e n t a t i o n ) , it i s n o t e d t h a t f o r t h e ^-0 ° f e r m e n t a t i o n , w h i c h s t a r t e d o n a h o u t t h e t h i r d d a y , a p p r o x i m a t e l y 100 liters o f g a s w e re c o l l e c t e d o f w h i c h t h e major p o r t i o n , a b o u t 85 l i t e r s , was p r o d u c e d b y t h e h y d r o g e n f e r m e n t a t i o n (H2 and C02 c u r v e ) . amount was p r o d u c e d i n a b o u t t h e f i r s t was r e c o r d e d . This s i x days a f t e r a c t i v e e v o l u t i o n The b a l a n c e o f t h e c o l l e c t e d g a s , a b o u t 18 l i t e r s , came from t h e s u b s e q u e n t y e a s t f e r m e n t a t i o n (C0 2 c u r v e ) o v e r a p e r i o d o f a b o u t two w e ek s t i m e ( 10t h t o 25t h d a y ) . The 6 0 ° f e r m e n t a t i o n , w h i c h s t a r t e d on a b o u t t h e t e n t h d a y , p r o ­ d u c e d g a s t o t h e e x t e n t t h a t a p p r o x i m a t e l y 130 l i t e r s w e re c o l l e c t e d fr o m t h e lU i n c h f u n n e l . Of t h i s amo un t, a b o u t 29 l i t e r s r e s u l t e d from t h e h y d r o g e n f e r m e n t a t i o n (H^ and C0 2 c u r v e ) d u r i n g a n i n t e r v a l o f ap­ p r o x i m a t e l y e i g h t d a y s ( 10t h t o 1 8 t h d ay ) w h i l e t h e major p o r t i o n , liter s, 93 was p r o d u c e d b y t h e y e a s t f e r m e n t a t i o n ( C02 c u r v e ) d u r i n g t h e s u b s e q u e n t 10 day p e r i o d ( 1 8 t h t o 2 3 t h d a y ) . Thus f a r , t h e m a t e r i a l p r e s e n t e d ha s d e a l t e n t i r e l y w i t h t h e t y ­ p i c a l , a c t i v e h y d r o g e n f e r m e n t a t i o n s i n 60 and ^0 ° b r i n e s . has been m entioned t h a t th e l a t t e r treatm ent su lt However, it ( U o ° ) may o r may n o t r e ­ i n t h e p r o d u c t i o n o f c o n s i d e r a b l e amounts o f h y d r o g e n . F i g u r e 23 s ho w s t h e b e h a v i o r o f a 4 0 ° f e r m e n t a t i o n w h i c h r e s u l t e d i n o n l y a s m a l l amount o f h y d r o g e n e v o l v e d . Here i t i s noted th a t the a c t i v e y e a st f e r ­ m e n t a t i o n s t a r t e d on a b o u t t h e e l e v e n t h day and c o n t i n u e d u n t i l a bo u t the tw e n ty -seco n d day. a cro g ressiv e in crease. D u r i n g t h i s p e r i o d t h e g a s e v o l u t i o n c u r v e shows The g a s a n a l y s e s show t h a t d u r i n g t h e a c t i v e ~ L/T E E S -127- -/O GAS £VOL U TION SAL. s# o_ . JOO D y < y s Fig. 2 3 . Yeast populations, gas evolution and analyses of the gas from a fermentation in *40° salometer hrine from Vat 10. m -1 2 8 f e r m e n ta tio n th e e v o lv e d gas c o n s i s t e d p r i n c i p a l l y o f carbon d io x id e , u s u a l l y w e l l above 90 p e r c e n t . On t h e e l e v e n t h day 6 . 2 p e r c e n t o f h y d r o g e n was n o t e d w h i l e t h r o u g h o u t t h e r e m a i n d e r o f t h e f e r m e n t a t i o n c o v e r e d b y t h e a n a l y s e s somewhat s m a l l e r amounts w e r e f o u n d . The t o t a l amount o f g a s c o l l e c t e d from t h e a b o v e f e r m e n t a t i o n was a p p r o x i m a t e l y 69 l i t e r s a s compared t o 99 l i t e r s from Vat f o u r and 104 l i t e r s f r o m Vat s i x fr om t h e two t y p i c a l h y d r o g e n f e r m e n t a t i o n s a t t h e same s a l t concen tration. 20° Salom eter B r in e s Some f e r m e n t a t i o n s r e s u l t i n g fr o m t h e 2 0 ° s a l t i n g t r e a t m e n t may h a v e s m a l l am ou nt s o f h y d r o g e n i n t h e e v o l v e d g a s w h i l e i n o t h e r s i t may b e w h o l l y a b s e n t . A t y p i c a l f e r m e n t a t i o n a t th e above s a l t concen­ t r a t i o n w h i c h d e m o n s t r a t e s t h e f i r s t b e h a v i o r c i t e d i s shown i n F i g u r e 214 . In t h i s c a se th e y e a s t fe r m e n ta tio n s t a r t e d a f t e r about th e f i f t h day an d c o n t i n u e d u n t i l t h e f i f t e e n t h d a y . The g a s e v o l u t i o n c u r v e r e ­ s p o n d s w i t h a n upward t r e n d d u r i n g t h e a c t i v e g r o w t h p h a s e o f t h e y e a s t s , a l t h o u g h compared t o o t h e r f e r m e n t a t i o n s p r e v i o u s l y d i s c u s s e d , amount o f g a s was e v o l v e d . no g r e a t During the e a r l y p a r t o f the f e r m e n t a t io n (3 r d to 9 t h d a y ) , p r i o r to th e a c t i v e g a s e v o l u t i o n , it is e v i d e n t from t h e g a s a n a l y s e s v a l u e s t h a t a n o t h e r g a s was p r e s e n t s i n c e t h e p e r c e n t ­ a g e s o f c a r b o n d i o x i d e and h y d r o g e n l a c k e d c o n s i d e r a b l e o f mak ing 100 per c e n t . T h i s was p r o b a b l y due t o n i t r o g e n f r o m t h e a i r b u b b l e s i n t h e cucumber t i s s u e w h i c h c o n s t i t u t e d a p o r t i o n o f t n e g a s s a m p le d u r i n g t h e u e r i o d o f s l o w g a s e v o l u t i o n from t h e v a t . soon as a c t iv e evolu tion started , However, it is noted th at as th e p e r c e n ta g e o f carbon d io x id e r o se s h a r p l y t o a b o v e 90 p e r c e n t and s t a y e d i n t h a t r a n g e u n t i l a b o u t t h e ** O F**tO U N T -129- ^fOO-- '5Zoa 8/07 / AtBOAA £ ~ £ £ S * \£ /V T A T / O H S A T V A R I O U S S ' A L T C O A /C £ A / T R .A T / O A / S /2S Si too too X o> * ?■JT 60 k o > SO Uj 40 40 40 60 rs 50 1 1.1 ' 1 1.0 t 1 1 21.0 . ' 1 1 2,*+30.0 7*0 1 , 600.0 910.0 1 , 620.0 420.0 1 , 000.0 1 1, 790.0 900.0 1 *+00.0 900.0 | 260.0 3*+o.o l *+.0 50.0 • 1 , 000.0 * I | 3 , 000.0 t • 330.0 • 0* I *+20.0 2.0 0* 520.0 1 1 t 320.0 * | 1 1 156.0 ' ! r 125.0 ' | 87.0 , 30.0 ' j f 9-0 1 0* I 560.0 | 2 , 000.0 | 2.8 1 * 1 1 1 0* 1.4 310.0 ' l 2 , 000.0 1 1 0* I 1 1.1 0* ! t 1.1 ■ I 1 1 1 1 1 6.0 1 1 , j t 0.1 , 7 .0 t 1 1 1 . t I• 3 .3 380*0 < 0.6 0.3 1.6 210.0 1 0* 1 1 1 0* 1 1 1 0.6 ! 0.6 ’ 1 0* ? 11 0.2 0.2 0 .9 1 t 0.1 ' 1.0 * 1 , 150.0 1 , 2.0 1 '| 1 25 0.2 3 .8 1 1 1 t 0.1 0*2 t ■ t \ 1 0 .3 1 1.0 Vat 6 0.5 1 1 1 ; 19 ! 23 ' 2*+ 0.6 1 1 *+00.0 Vat *+ 1 1 60° s a l . 1 Vat 3 t Vat 9 . M------------------*--------- ;-------- *. , th ousands thousands thousands , thousands , t h o u s a n d s , , per cc. per c c . per cc. , per cc. , per c c. , 1.0 1.0 *+0° s a l . l *+.0 | | . 6.0 'I , ! t 2 . 0 0* 1 ' 1 “ 136 - Table 1+3• 1 Gas Evolution from Fermentations in 20, Uo and 60° Salometer Brines. 20° S a l . ' i A P*e t ‘ Vat 2 t— .i t days •l i t e r s '1 1 . 5 | 2.2 2 .5 2 *5 ! 3,5 , h.o : ^.6 ! 5-5 2.1 1 6.7 ’• 1 1 6.9 1 3 .3 ^.3 • 20.1 1 1 ; 29.9 t 6• 8 't t 7.3 ' 66.8 76.6 ! 8 *3 t 't 7 . 0 9 .3 '* S . 5 i « 9.0 • 11.2 13.7 • 1^.0 16.8 ' 10.0 •10.5 i , 11.0 ' 1 8 .0 , 21.1 22.3 ',13.5 'lU.O 1 I ! 1.9 • 2.2 ' U.5 | 12.8 ' IS .2 | 0*2 t 1 1 1 t t 1 9-5 * 31.7 ■ 16.0 ? * ’ 23.3 > 31.0 t 1 * 1 1 I °*9 1 1 1 2.6 1 t t • S 5.3 1 1 ' 21.7 85-9 85.3 I ! 79.3 1 t 2S.4 30.1 ! 23.2 I I 1 1 1 1 • 12.0 • 13.0 1 • 77.8 1 2^.7 ' 11.5 !1! 2 . 5 ' ! 20.7 ! 9 .5 ^9.7 65.5 8.8 12.0 1 • 1+7 . 1+ t , 5S.g . 6 .5 7.5 1 . 1+ « 5.9 ! 1 1 1 1 * 11.6 1 1 1 g.O 29.0 3.^ 7 .9 6.2 ! • . ' 6.0 • 1.0 ; 1 1 » 7 .5 1 1 1 1 1 3 .8 5.0 5,5 6o ° S a l . t 1 t 7.6 : 1 1 1 i | *+.5 • 5.0 40° S a l. 1 Vat g 'V a t 10 * Vat 4 Vat 6 ' V at 3 Vat 9 ' —i—-----------t i .... . ■-—■■r f ---- —• -- t ■■— — > 'i i----------—-- 1 liters «l i t e r s t l i t e r s liter s •l i t e r s liters 1 0 .9 . 2.0 I 1 1 . ^ .5 ! 1 1 I 1 1 1 1 * 86.1 1 s 6.6 S9.3 7 *5 • 10.6 1 1 • 20.1 1 1 1 29.1 1 -137Table 1+3. Gas Evolution from Fermentations in 20, 1+0 and 60® Salometer Brines (Continued). i i 1 Age , i ....... 1 Vat 2 i -i- - ........ 20° S a l. ' J................ ....................... .....L 1 days 1' l i t e r s i r* ti^.5 i 1 1 I1 5 - 0 | . 22.0 ' Vat 1 'liters 1 I , !x7*5 ; 1 1 1 . t 1 1 1 1 1 '18.0 l .18.5 ' t1 11 1 !1 t | 1t , t 1 ' 1 i , , ! i , , I ' I 1 t 1 1 t 1 '15.5 t .17-0 l1^ 0 '19-5 ,20.0 * 2 0.5 < I ,21.0 t ,23..5 '22.0 ' I , 22•1 1i , ,22.5 ;23.o '23.5 1 , 2 5 .0 '25.5 f i2 6 .0 . 1 1 | , I ' 11 , , 1 ' 1■ 1, 1 22.2 ,'2 6 . 5 ; '27.0 I , 2 7 .5 I 1 1 1 1 t 1 1 1 1 t t I I » 1 1 , I f 1 1 1 « -------L. ' Vat 10 « ' liters I t 35-0 1 , 1+0 . 9 , 1 1+5.9 , 52.9 1 1 32.3 ' t 1 1 ! 1 V a t 1+ * V a t 6 • liters 89.1 60® S al. * 1 1 1 1 1 1 1 1 ; vat 3 : Vat 9 | 'lit e r s a1 I 22.2 ' t ' ' 92.4 ,' 95.0 ! 35-1 1 25.0 I ' 96. 8 1 t 1 1 1 1 1 « t ! f 36.6 ■ 1 1 t 1 1 1 , 1 1 t 28.0 1 I 32.5 . 1 t 33.3 ' I . 98.6 99.8 39.0 1 65.5 9 1 .6 93.1 1+3 . 0 ; 60. 5 • I 7 7 .^ ! ' | * 8 8 .3 ' | 96.5 ' 1 1 ' 1+9.5 , 102 .0 | 103 .1 ; 83.71 ' 1 03 .8 ! 9 5 *3 , 106 .9 t 1 < 1 1 5 .0 1 1 0 .3 ! , 1 121 .6 i 1 1 0 3.9 ', 1 2 1 . 5 ! 1 27 .2 1 0 3.9 1 123 .9 t t 1 1 t 1 , 128 .1 ' 1 1 1 1 t 13 1 .8 1 , 96 .3 * 32.4 • 1 ' ' ' 1 . ' , 97.7 99-1 | ' • ' ..-1---------- ---------------- i - I ' | . 6 8 .5 6 8 .9 't 'liters 1| 1 57.9 99-^ , 'lit e r s ; t1 1t I, . ' 30.5 ' S 1+0° S a l « ' f 69.5 1 3 2 .2 ' f I ' 1 1 ! 1 1 1 I 1 ' J 1 t ...... _ .... .. L Table 4 4 . 1 1 t Age \ I 11 1t 11 II t1 ! day s I! > 3 20° Sal • 1 1 Vat 8 ' co 2 * 3.3 1. > ii " 1 co2 e [ i ! * $ 65.2 2.0 ! 11 1"t t i I 66.9 1 1.9 t t 1 . 7 , 1 g a 1 78.2 ' 1.0 , 8.4 , 86.8 , 1.0 ' 9^ ,\ 1 9.4 t «t ! 1 1 1 \ \ 1 1 * ’ 55.0 ! t ' 8 3.0 9^.6 ; 0.2 1 1 1 1 ! 37.8 ! 51.7 47.6 51.3 4 s.1 ' 49.1 5 0 .4 X 1 49.0 50.5 ! 1 49.4 j * t 50 .9 36.1 ' 1 43.2 161.6 * 1 h2 t 1 55.2 4i.6 C02 't 1 47.3 ; 55.5 Vat 3 1I 5 1 .7 4o.6 I ’ 1 0.5 1 t ;! : h2 1 ' 1 0 .4 1' 9 2 . 8 1 |t > 11 >1 ; « * C02 X t t 1 I ! t1 1 5 0.9 ' | 0.7 . 0.7 1 « 1 1 88.4 1 11 , . 91.6 1 12c * h 2- ' 1 1 1 60° S a l . Vat 9 1' C02 H 2 1 i * ! 1 X X 1 \ t I 1 t t t \ X 11 I X X 11 1 t t1 I I 1 I I t 1 1 1 1 1 ! 6 .2 1 X 1 • 33.8 36.6 ' 32.2 1 X 50.6 ' ; 91.6 2.4 | 36.2 49.3 ; 49.4 4 6 . 2 1' 94.8 1.7 1 4 2 .9 52.6 ; 50.6 “ms. 5 ; I - F o o t n o t e s a t end o f t a b l e on n ex t p a g e . t 1 -13 s - 1 5 1 \ 6.5 ' co2 Vat 6 1 tI ’ 2 ! ! 6 ' ' Vat 4 h2 t t' Vat 10 ' I 1 . X X 4o° S a l . 1 i 1 « I A nalyses o f Gas from Ferm entations in 20, 40 and 60° Salometer B rin es. Table 4 4 . A nalyses o f Gas from Ferm entations in 20, 40 and 60° Salometer B r in e s. (C ontinued). ! 1! I t1 , Aee ,, t tT** ' t !! C02 1 , Hg 1 days ’ ' $ 9U.6 , 13 A ,, ’ lU It 1 15 t 16 * Vat 8 " t1 11 Vat 4 Vat 10 1' 'I1 1 Vat 6 h2 C02 1 Hg , | # , 0.4 "II 11 i * * 'I '* 1 $ 1 " 96.4 11 1.5 t1 1 II t1 t t 11 96 •2 0.9 !1 M 1 t \t 98.4 0.6 97.0 0.9 | 0.3 !! 93*7 0.6 0.8 89.4 1 1 11 COg 1I i d* 11 , 18 .. 1 ., 40° Sal. 1 $ h2 ' Vat 3 1 1 oo2 h2 * '1 $ $ * 1' t 1 . 81.8 16.0 I 49.2 48.7 1 t 1 1 1 1 ' 57.0 1 1 54.0 43.0 35*0 ' 57*8 3 8 .8 . ! 53.6 35* 2 ! 73.2 25*4 • 54.4 1 1 63.2 34.9 ' 95.9 1 3*2 ' 33.1 ' 96.c 2.9 1 ; 89.H 8 .2 ; 96.9 2 .5 ; * 97.7 1*7 » 93.2 3 .4 , ! 95.2 2.4 96*8 2 .2 1 1 92.4 3.7 ' 96.0 2.5 ' . 96.8 2.4 , 91*8 3.7 ! ’ 88.7 5 . 1 «1 ' 27.4 * 1 1 I tt r r 1 ,23 11 ,, 1 1 t r 1 2b 1 ,25 " t1 u 1) 11 l! *I I 1 t \ I t 1 26 1t 11 1 *» t ’ 94.2 1 3*4 \ " 1t 1 1 ,2 8 ,, I 1 1 1 , 94*2 3*4 \ \ | 15.6 1 |' 55.6 , 54.0 1 1• 11 " * i 47.6 1 1 r ' ' 4g.6 1 21 1 22 1 . 24.0 I 1 1 1 1 H2 ' 72.6 m 11 COg t ” 9^.7 I1 1t 11 ' 1 , t \ t " 1f 81.4 Vat 9 1 '19 1 « 20 I 1 * ( 63.2 COg 1 6o° Sal. I 1 1 t 1 1 t ! • 1 ■ I 1 56.6 43.2 1 | 43.8 , 32.4 | | 1 For Vat 8 o n ly ; a, 8 . 2 days; b, 9 - 1 d ays; c , 1 2 . 2 days; d, 1 8 .1 days. N o t e : The c a s e s where a n a l y s e s la c k c o n s id e r a b le o f t o t a l i n g 100 p e r c e n t c o r r e l a t e w it h p e r i o d s o f slo w gas e v o l u t i o n . During t h i s i n t e r v a l t h e p r e s e n c e o f a i r d i f f u s i n g from th e cucumber t i s s u e c o n s t i t u t e s a p o r t i o n o f t h e sam ple, th u s a c c o u n t in g f o r th e above d i f f e r e n c e . 1 \ 139- <17 20° Sal. ~iUoSUMMAHY AND CONCLUSIONS S t u d ie s upon th e m icroorganism s r e s p o n s i b l e f o r th e p r o d u c tio n o f h y d r o g e n i n cucu mb er f e r m e n t a t i o n s h a v e b e e n r e p o r t e d . m e n ta l work has d e a l t c h i e f l y w i t h th e i s o l a t i o n , The e x p e r i ­ i d e n t i f i c a t i o n and b i o c h e m i c a l s t u d i e s upon t h i s group o f organ ism s a s w e l l a s o b s e r v a t i o n s upon t h e i r t y p i c a l f e r m e n t a t io n s under commercial s a l t i n g c o n d i t i o n s . T w en ty —n i n e c u l t u r e s w e r e i s o l a t e d f r o m cucumber f e r m e n t a t i o n s a t b r i n e c o n c e n t r a t i o n s r a n g i n g f r o m 20 t o 60° s a l o m e t e r ( p e r c e n t s a t u r a t i o n w ith r e s p e c t to s a l t ) . 2,0 Of t h e a b o v e gr oup o f c u l t u r e s , w e r e i s o l a t e d d u r i n g t h e 1938 s e a s o n and n i n e d u r i n g t h e 1939 s e a s o n . The t w e n t y c u l t u r e s r e s p e c t to m orphological, ( 1 9 3 8 s e a s o n ) w ere s t u d i e d i n d e t a i l w i t h c u l t u r a l and p h y s i o l o g i c a l c h a r a c t e r i s t i c s and b a s e d u p o n t h i s i n v e s t i g a t i o n , A erobacter genus. study, th e organism s were p l a c e d i n the E i g h t e e n o f t h e 20 c u l t u r e s t h a t w e r e g i v e n d e t a i l e d r e v e a le d c h a r a c t e r i s t i c s in c lo s e r con form ity to th ose d escrib ed i n B e r g e y ' s Manual f o r A e r o b a c t e r c l o a c a e t h a n f o r t h o s e f o r A e r o b a c t e r aerogenes, the o n ly oth er s p e c ie s listed . The r e m a i n i n g two c u l t u r e s (H -13S and 233) a r e r e g a r d e d a s v a r i e t i e s o f A e r o b a c t e r c l o a c a e . A s a t is f a c t o r y apparatus, su ita b le for stud ying in d iv id u a l gase­ ous f e r m e n t a t io n s w it h r e s p e c t to gas p r o d u c tio n , o f e v o lu tio n has been d e scr ib e d . The a d v a n t a g e s o f t h i s paratus are; (a) s im p lic ity of con struction, o f h a n d l i n g and m a n i p u l a t i o n , (b) c o m p o s i t i o n and r a t e t y p e o f ap­ com pactness, and ( d ) i t s r e l a t i v e l y (c) ease s m a l l s i z e whi ch makes p o s s i b l e t h e f e r m e n t a t i o n o f a number o f s a m p l e s i n a l i m i t e d amount o f i n c u b a t o r s p a c e . -lH i- The p r e l i m i n a r y e x p e r i m e n t s d e a l i n g w i t h t h e f e r m e n t a t i o n o f d e x t r o s e a n d cucumber j u i c e b y s t r a i n H-lU3g i n d i c a t e d th e f o l l o w i n g : (a) That t h e g a s p ro d u ce d from t h e s e m edia p r o v e d t o b e composed s o l e l y o f h y d r o g e n an d c a r b o n d i o x i d e ; (b) t h a t t h e c o m p o s i t i o n o f t h e g a s fr o m t h e f e r m e n t a t i o n s depended upon th e carbon s o u r c e , that i s , the r a tio o f h y d r o g e n to ca rb o n d i o x i d e i n t h e g a s from t h e d e x t r o s e f e r m e n t a t i o n wa s 1 : 2 . 3 w h e r e a s , (c) f r o m t h e c uc um b er j u i c e f e r m e n t a t i o n i t t h a t t h e f e r m e n t a t i o n s f o r b o t h media were r a p i d , wa.s 1 : 5 * 0 ; th e major p o r t i o n o f t h e g a s b e i n g e v o l v e d d u r i n g t h e f i r s t 1+S t o 72 h o u r s . The d e x t r o s e f e r m e n t a t i o n s from th e s t o c k c u l t u r e co llectio n t h e amou nt o f g a s p r o d u c e d , e x c e p t i o n o f one s t r a i n , sim ilar decrease W it h t h e t h e g a s e v o l v e d f r o m f e r m e n t a t i o n s was th e hydrogen to carbon d io x id e r a t i o s b e in g be­ t w e e n 1 : 2 - 3 2 an d 1 : 2 . 5 9 . a d ifferen ce sh ow ed t h a t w i t h f i v e o f t h e g r o u p , 139 t o 1^8 c c . , wa s c o m p a r a b l e . H-13S, in com p osition , ( b u f f e r e d a t pH 5 * 1 5 ) of s e v e n s t r a i n s H ow e ve r , in th e case of the e x c e p tio n , (H-13S), i n f e r m e n t a t i o n b e h a v i o r wa s d e m o n s t r a t e d n o t o n l y b y a i n t h e amount o f g a s p r o d u c e d , but a l s o , by a s l i g h t increase i n th e p r o p o r tio n o f hydrogen found i n th e e v o lv e d gas ( l : l . ^ U ) . r u p l i c a t e fe r m e n ta tio n s o f d e x tr o s e by the type strain Quad­ (H-iU-38) showed no s i g n i f i c a n t d i f f e r e n c e s w i t h r e s p e c t t o g a s c o m p o s i t i o n w h i c h r a n g e d f r o m 1 : 2 . 3 5 t o 1 :2 .* + 9 , H2 t o C02 * resu lted in p r a c tic a lly A lso, t h e same amount o f e v o l v e d g a s , The f e r m e n t a t i o n o f d i f f e r e n t tem peratures (5°, 13°, the m ajority of the ferm en tation s 19°, lo ts IU 3 t o 150 c c . o f d e x tr o s e at e ig h t m aintained 2^ ° , 3 0 ° , 3 5 ° , ^ 0 ° and ^ 5 ° C . ) showed t h a t t h e optimum w as w i t h i n t h e 35 ° r a n g e f o r maximum g a s p r o d u c t i o n a n d r a t e of ev o lu tio n . The l o w e r an d h i g h e r l i m i t a t i o n s f o r t h e f e r m e n t a t i o n —lM-2w e r e 5 ° and. resp ectiv ely . F erm en ta tio n s m a in ta in ed e i t h e r above U0 ° o r so mewhat b e l o w 2^° t h e optimum r a n g e o f 35 ° w e r e c o n s i d e r a b l y r e t a r d e d and l e s s g a s was p r o d u c e d . Gas e v o l u t i o n a t i g ° was much s l o w e r t h a n a t U0 ° b u t a t t h e e n d o f t h e e i g h t day i n c u b a t i o n p e r i o d , t h e amount o f g a s w as a b o u t t h e s a m e . from a l l f e r m e n t a t i o n s The c o m p o s i t i o n o f t h e g a s e v o l v e d ( 13° t o U o ° ) was c o m p a r a b l e a s t o p e r c e n t a g e s o f hydrogen and carbon d io x id e found. The f e r m e n t a t i o n o f d e x t r o s e t o o k p l a c e o v e r a c o n s i d e r a b l e r a n g e w i t h r e s p e c t t o i n i t i a l pH a d j u s t m e n t However, (pH 5 - 0 5 t o 9 . 0 5 ) o f t h e medium. the ferm en tation o f d if fe r e n t f e r e d pH v a l u e s ( 3 * 6 , *+.25, 5*3, 6 .0 , lo t s of dextrose at 7*0, 7*55, S . 05, several buf­ and 8 . 8 5 ) r ®- v e a l e d t h a t pH 5*3 w a s t h e optimum a s d e m o n s t r a t e d b y ms.ximum g a s p r o d u c ­ t i o n and r a t e o f e v o l u t i o n . pH 3 * 6 r e s u l t e d i n no g r o w th i n t h e a c i d r a n g e a n d pH 8.85 s e e m e d t o a p p r o a c h t h e l i m i t o f a d e q u a t e g r o w t h i n t h e alk a lin e range. In g en e ra l, i t w as shown b y t h e g a s a n a l y s e s t h a t t h e p e r c e n t a g e s o f h y d ro g en and carbon d i o x i d e were s i m i l a r f o r a l l o f th e above f e r m e n t a t i o n s . D extrose ferm en ta tio n s (b u ffered at 5-15) t o w h i c h s a l t was a d d ed , t o t h e e x t e n t o f 5 , 10 , 15 , 20 and 25 p e r c e n t s a t u r a t i o n , a p rogressive decrease t in gas p ro d u ctio n as the s a l t c r e a se d above f i v e per cent s a t u r a t io n . In. t h i s dem onstrated con cen tration in­ series, sa lt concentra- t i o n s a s h i g h a s 20 a n d 25 p e r c e n t s a t u r a t i o n r e s u l t e d i n no g r ow th ; a p a r t o f t h e i n h i b i t o r y e f f e c t was due t o a l o w e r i n g o f t h e pH b e l o w t h e out imum ( 5 < 3 ) b y t h e a c t i o n o f s a l t on t h e b u f f e r e d b r o t h . S im ilar f e r ­ m e n t a t i o n s e m p l o y i n g u n b u f f e r e d c uc um b er j u i c e r e s u l t e d i n a d e q u a t e gr ow th w i t h m e a s u r a b l e g a s e v o l u t i o n i n t h e 20 p e r c e n t s a t u r a t e d l o t b u t not -1 ^ 3 i n t h e 25 p e r c e n t s a t u r a t e d l o t . T h e r e was no s i g n i f i c a n t d i f f e r e n c e s n o t e d a s t o g a s c o m p o s i t i o n "between f e r m e n t a t i o n s w i t h i n t h e same d e x ­ t r o s e an d cucumb er j u i c e s e r i e s , feren ce in the r a t io a l t h o u g h t h e r e was a c o n s i d e r a b l e d i f ­ o f h yd rogen t o carbon d i o x i d e found f o r t h e gas e v o l v e d f r o m t h e d e x t r o s e l o t s a s compared t o t h a t f r o m t h e cucumber ju ice lo ts. I t wa s d e m o n s t r a t e d t h a t c e r t a i n c h a n g e s o c c u r d u r i n g t h e h e a t i n g o f c uc um b er j u i c e b r o t h w h i c h r e t a r d s or d e l a y s t h e f e r m e n t a t i o n a s w e l l a s d e c r e a s e s t h e amount o f g a s e v o l v e d . This i n f l u e n c e r e s u l t e d i n a much d e l a y e d f e r m e n t a t i o n i n j u i c e r e c e i v i n g 100 mi n . h e a t i n g ( a t 15 l b . p r e s s u r e ) a s c om pa r e d t o a *40 m i n . h e a t i n g a l t h o u g h t h e f i n a l v o l u m e s o f g a s a f t e r one w e e k w e r e s i m i l a r . A com parative stud y o f the c o m p o s i t i o n o f t h e g a s e v o l v e d from t h e f e r m e n t a . t i o n o f 1*4 c a r b o n compounds r e v e a l e d t h a t t h e p r o p o r t i o n s • o f h y d r o g e n a n d c a r b o n d i o x i d e d e p e n d e d u p on t h e c a r b o n s o u r c e f e r m e n t e d . The f e r m e n t a t i o n o f 1- a r a b i n o s e , dextrose, d-g& lactose, lev u lo se, d-man- n o s e and s a c c h a r o s e y i e l d e d g a s compo se d o f a p p r o x i m a t e l y one vo lu m e o f h y d r o g e n and two v o l u m e s o f c a r b o n d i o x i d e 1- x y l o s e , rhamnose, m a l t o s e , raffin ose, (1:2). The f e r m e n t a t i o n o f d-m annitol, d - s o r b i t o l and s a l a c i n y i e l d e d g a s o f a p p r o x i m a t e l y e q u a l v o l u m e s o f h y d r o g e n and c a r ­ bon d i o x i d e (1:1). The f e r m e n t a t i o n o f l a c t o s e y i e l d e d g a s composed o f a p p r o x i m a t e l y two v o l u m e s o f h y d r o g e n an d one v o lu m e o f c a r b o n d i o x i d e (2:1). In c o n t r a s t to t h e r e s u l t s f o r t h e a b o v e compounds, the ferm en ta­ t i o n o f cucumber j u i c e y i e l d e d g a s compo sed o f a p p r o x i m a t e l y one volum e o f h yd rogen to f i v e volum es o f carbon d io x i d e (1:5)- The s t u d y o f t h e t y p i c a l f e r m e n t a t i o n s b r o u g h t a b o u t b y t h e A e r o - -ii+ i+ _ b a c t e r u n d e r s a l t i n g c o n d i t i o n s t y p i c a l o f t h e i n d u s t r y r e v e a l e d a num­ ber o f in t e r e s t in g o b serv a tio n s. i t was f o u n d t h a t t h e 60 ° G enerally, s a l o m e t e r s a l t i n g t r e a t m e n t shoivred t h e m o s t c o n s i s t e n t b e h a v i o r w i t h respect t o what i s term ed t h e t y p i c a l hydrogen f e r m e n t a t i o n . was o b s e r v e d t h a t t h e U-0 ® t r e a t m e n t may o r may n o t r e s u l t a c t i v e hydrogen fe r m e n ta tio n . Furthermore, A lso, it in the t y p ic a l , some f e r m e n t a t i o n s r e s u l t i n g f r o m t h e 20° t r e a t m e n t may h a v e s m a l l a m o u n t s o f h y d r o g e n i n t h e e v o l v e d gas w h ile in o t h e r s i t may b e c o m p l e t e l y a b s e n t . - The g a s e v o l u t i o n a s w e l l a s t h e c o m p o s i t i o n o f t h e g a s demon­ s tr a te d th a t t y p ic a l ferm en tation s vid ed in to two d i s t i n c t i n b o t h 1+0 an d 60® b r i n e s w e r e d i ­ gas e v o l u t i o n p h a s e s ; t h e f i r s t was b r o u g h t ab o u t b y t h e A e r o b a c t e r group and d u r i n g th e a c t i v e p e r i o d o f fe r m e n ta ­ tion , t h e g a s w as s i m i l a r carbon d io x id e , i n c o m p o s i t i o n w i t h r e s p e c t t o h y d r o g e n and t h e p r o p o r t i o n b e i n g a b o u t 1 : 1 ; t h e s e c o n d p h a s e was brought about by the y e a s t s , of p r a c tic a lly a l l d u r i n g w h i c h p e r i o d t h e g a s w as c om pos ed carbon d io x id e . I n t h e 1+0° f e r m e n t a t i o n , app roxim ately f o u r - f i f t h s o f the gas e v o l v e d was p r o d u c e d by t h e o rg a n is m s o f the A e r o b a c t e r group w h i l e t h e r e m a i n d e r was c o n t r i b u t e d b y t h e y e a s t s . s i t u a t i o n was so mewhat r e v e r s e d , In t h e 60 ° f e r m e n t a t i o n , the h e r e a p p r o x im a t e ly o n e - f o u r t h o f the g a s e v o l v e d was b r o u g h t a b o u t b y t h e A e r o b a c t e r w h i l e t h e maj or p o r t i o n ( t h r e e —f o u r t h s ) was p r o d u c e d b y t h e y e a s t s . A c o m p a r i s o n o f g a s e v o l u t i o n fr o m f e r m e n t a t i o n s i n 2 0, 60° b r i n e s r e v e s - l e d t h a t , sa lt in gen eral, the fer m en ta tio n s a t c o n c e n tr a tio n s resulted , in la r g e r q u a n t it ie s ko and the high er of evolved gas. The g a s f r o m ' • b l o a t e r s ” o r h o l l o w c u c u m b e r s , t h a t was f o r m e d d u r - -lin ­ in g th e a c t i v e phase o f the hydrogen fe r m en ta tio n , had p r a c t i c a l l y t h e same c o m p o s i t i o n w i t h r e s p e c t t o h y d r o g e n and c a r b o n d i o x i d e a s d i d t h e gas c o l l e c t e d from th e s u r f a c e o f the b r i n e . However, th is rela tio n ­ sh ip o n ly e x i s t e d d u rin g the a c t u a l form ation p e r io d o f the b l o a t e r s . T y p ic a l y e a s t ferm enta.tions r e s u l t e d in a l l b rin e treatm en ts (20, 1+0 a n d 6 0 ° ferent sa lt employed f o r s a l t i n g cucumbers. c o n c e n t r a t io n s d id not in the b r in e , n ite salom eter) The d i f ­ i n f l u e n c e t h e number o f y e a s t s f o u n d on ce f e r m e n t a t i o n was underway. 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