ACTION OF SPECIFIC MICRO-ORQANISMS ON NITRIC ACID. 373 XXVIK-The Action of some Bpec$c Micro. organisms on Nitric Acid. By PERCY F. FRANKLAND Ph.D. B.Sc. F.S.C. Associate Royal School of Mines. THAT nitric acid may be reduced to nitrous acid by the agency of bacteria appears to have been first shown in 1875 by Meusel (Ber. 8, 1215) who found that well-water containing nitrates and bacteria on standing for some days gave a definite reaction for nitrous acid, which was prevented by the addition of antiseptic substances such as salicylic carbolic or bmzoic acids. He also found that on adding nitrates and carbohydrates to water containing bacteria the nitrates soon became converted into nitrites. In 1882 Gayon and Dupetit (Ber. 15 1882 2736) describe the reduction of nitrates by " anaGrobic " micro-organisms.Again in the same year DBhBrain and Maqueniie (Bey. 15 1882 3081) ascribe the reduction of nifrates in the soil to the agency of the butyric ferment, the so-called BaciElus amylobacter. I n 1883 Gayon and Dupetit (Ber. 16 1883 '221 ; Conzpt. rend. 95, 1365) further describe the decomposition of nitrates by other anaiirobic micro- organisms, The reduction of nitrates by micro-organisms is also frequently referred to by Warington in his well-known researches on nitrification, and has been also confirmed and elaborated by Nunro (Trans. 1886, 632). Heraeus (ZeitscJir. f. Hygiene 1886 193) has also made experi-VOL. LIII. 2 374 FRANKLAND THE ACTION OF ments on the reduction of nitrates by micro-organisms and these appear to be the first in which pure cultivations obtained by the modern methods of separation and isolation which can alone guarantee the absolute purity of the cultures have been employed.To these experiments further reference will be made later on. With few exceptions however all the experiments referred to above have been made not with pure cultivations of well-characterised micro-organisms but with casual mixtures of microbes such as are obtained from soil sewage natiiral waters and the like. I n the course of investigations which I have now been carrying on for some years past. on the micro-organisms present in the atmosphere and in natural waters I hare had occasion to collect a number of micro-organisms from these sources to cultivate them in a state of purity and to characterise them in such detail that they may be readily identified by subsequent observers.It appeared to me there-fore very desirable that the action of these asrial and aquatic micro-organisms on nitrates should be carefully studied more especially as there has been some tendency to believe that nearly all microbes possess the property of decomposing nitrates if placed under suitable conditions. Method of Experiment. The nitric acid submitted to the action of the va,rious micro-organ-isms was in combination as calcium nitrate contained in a solution capable of nourishing the microbes. In the first instance solutions were employed in which the only nitrogen present was the nitric nitrogen of the calcium nitrate but in such solutions the growth of the micro-organisms was so uncertain that it was found necessary to introduce a small proportion of peptone although thereby somewhat complicating the composition of the nutritive liquid.The solution which ultimately appeared most suitable for the pur-pose had the following composition :-Potassium phosphate Calcium chloride (fused) Invert sugar Magnesium sulphate (cryst.). . Nitrogen (combined in the form of calcium nitrate) Peptone . 0.1 gram7 0.01 O.O2 ” I I’ > 0.5 ,, 0.65 , J in 1000 C.C. of dis-tilled water with 4 grams of pure ’ calcium cirbo-nate in suspen-sion. This solution was poured into sterilised bottles plugged with sterile cotton-wool so that each bottle was from three-fourths to five-sixths filled with liquid.The bottles so filled were then finally sterilised by steaming for one hour on four successive days SOME SPECIFIC MICRO-ORGANISMS ON NITRIC ACID. 37 5 The solution thus prepared gave no reaction either with Nessler's solution or with the sulphanilic acid and phenol test €or nitrous acid. Into these sterilised solutions the particular micro-organisms were inoculated with a platinum needle the cotton-wool stopper being singed in a Bunsen flame just before extracting it to permit of the introduction of the platinum needle the stopper being then imme-diately replaced when the inoculation was complete. Of the micro-organisms experimented with those derived from air have been fully described by me (Roy. Xoc. Phil. Trans. 1887 178 B, 255-287) whilst those derived from water will short,ly be similarly described.First Xem'es of Experz'meds. A series of bottles containing the above solution were inoculated in the way described with the various micro. organisms mentioned below ; the inoculated bottles were then placed in the incubator and kept at a temperature of 50" C. and submitted to examination after the specified intervals of time. In examining the solutions a number of pipettes which had been steam-sterilised were kept in readiness the cotton-wool plug of the bottle to be opened was singed in a Bunsen, then withdrawn with a pair of forceps and the sterile pipette intro-duced and by means of it a few cubic centimetres of the liquid taken out the cotton-wool plug being then immediately replaced. The results of this first examination (table p.376) showed at once that there exist very marked differences in the behaviour of different micro-organisms towards nitrates. Thus whilst some of the orga-nisms in question had in the course of tbree days converted a large amount of nitrate into nitrite others had given rise to the production of no nitrite at all whilst the growth of others again had resulted in the formation of only traces of nitrous acid. It is moreover noticeable that these differences are by no means dependent upon the greater or less vigour of the growth to which the several organisms give rise in this medium. Thus many of the organisms such as B. viscosus, nubilus subtilis produce strong visible growths in the solution, rendering the latter excessively turbid or producing flocculent deposits without giving rise to the formation of any nitrite.It is moreover very noticeable that all the organisms without exception which give rise to the production of an appreciable amount of nitrous acid are bacilli whilst not one of the micrococci examined gives rise to more than a doubtful indication of nitrite. Again it is very remarkable how differently two organisms like R. subtilis and B. cereus which in their microscopical and other mor-phological characters are only with difficulty dist.inguishable behave 2 c 376 FRASKLLAND THE ACTION OF Exanzination of h'olutiono after 3-4 Days' Growth at 30". Name of micro-organism. Bacillus ramwws . . B. uiolacew. B. v m i c u l a v i s B. Izu6ilus B. viscosus 3.aquatilis B. liquidus . B. arborescens . B. aurantiacus B. cereus B. subtilis . B. l m i s B. pest ;fer B. p&xxtecs B. prodigiosus. B. aurescens €3. aureus . B. Jluorescens . B. chlorinus B. citreus . B. profusus 3. pt?ymoq-pBus. . Sarcina aurantiaca. Sarcina lutea Sarcina lipuef aciens Streptococcus lipue-Xicrococcus rosaceus M. carnicolor . M. git9as M. albus M. candicaus M. chryseus . Blank experiment . . f aciens Appearance of solution. Liquid clear but abundant mem branous growths on sides ol bottle Liquid very slightly turbid nu. merous violet lumps on botton: of bottle Liquid very turbid but not muck deposit Liquid turbid not much deposit Liquid turbid not much deposii Liquid very tnrbid not much Liquid turbid nok much deposit No visible growth No visible growth Liquid not very turbid but con.Ditto Liquid turbid not much deposit Liquid very turbid not much Liquid very slightly turbid, Liquid turbid not much deposit Liquid not very turbid but some Liquid very turbid,and consider-Liquid very turbid not much Ditto Liquid very slightly turbid a Liquid slightly turbid not much Liquid scarcely turbid Liquid very turbid not much Ditto Liquid slightly turbid distinct Liquid very slightly turbid Liquid turbid not much deposit Ditto Ditto deposit siderable flocculent deposit deposit little or no deposit yellow deposit able yellow deposit deposit little yellow deposit deposit deposit deposit Liquid very slightly turbid Ditto Liquid almost perfectly clear Liquid perfectly clear Reaction with sulphanilic acid phenol, and ammonia.Very strong. Very strong. Strong. None. None. Very faint. Strong. None. None. Strong. None. None. Very strong. Very strong. Strong. Decided. Faint. Distinct. Distinct. Distinct . None. None. None. None. None. None. Very faint. None. None. None. None. None. None SOME SPECIFIC MICRO-ORGANISMS ON NITRIC ACID. 37 7 Examination of the same Solutions after 41 Days' Growth. Name of micro-organism. B. ramosus* . . . . B. violaceus* . . . . B. vermicularis" , B. nubi1u.P . B. viscosus . . . . B. aquatiZtP . . . . B. liquidus' . . . . , B . arborescens-B.aurantiacus" B. cereus* . . . . , B. subtilis* . . . . B. lawis* . . . . B. pestifer" . . . , B. pZicatus- . . . . B. prodigiosus + , B. aurescens- . . B. aurew- . . . . , B. YfEeCorescens - . B. chlorinus* . . . B. citreus- . . . . . B. profusus- . . . B. polymorphus -Sarcina auran-Sarcina Idea* Sarcina liquefaci-Streptococcus Ei-M. rosaceus- . . . tiaca -em* quefaciens" M. carnicolor- . M. gigas . . . . . . . M. alhus- . . . . . . M. candicans- . . M. chryseus + . . . .Blank exprnt,. - . . -Origin. Water 11 11 Y Y 1 , 9 9 9 9 9 1 9 1 Air 11 11 Y1 > ? 11 f Y Y 1 J I J 9 19 >1 >1 1 9 9 1 91 19 9 3 Y 9 ,Y >1 11 JJ -Appearance of solution. Liquid quite clear much flocculent deposit Liquid nearly clear.Con-siderable violet deposit Liquid elightly opalescent. Not much deposit Liquid almost clear Ditto Liquid slightly opalescent Liquid slightly turbid Liquid quite clear Liquid almost clear. Orange deposit Liquid nearly clear. Much flocculent deposit Ditto Liquid nearly clear Ditto Ditto Liquid slightly opalescent Liquid very turbid. Some yellow deposit Liquid very turbid. Con-siderable orange deposit Liquid almost clear Liquid opalescent. Some Liquid clear Liquid almost dear Liquid quite clear Liquid very turbid. Some yellow deposit Dit,to Liquid very turbid Liquid clear Liquid very opalescent. Liquid slightly opalescent. Liquid almost clear Liquid clear Liquid almost clear Ditto Liquid perfectly clear yellow deposit Little red deposit No red colour teaction with di-phenyl-amine.Strong 91 Y7 19 7 1 9 7 71 1? Y J 71 >1 7, 11 11 Y Y 1 , J Y 71 11 19 1 ) 9 1 1 7 >, Y Y 9 9 Y Y f9 > Y > I 2 1 1 ) J1 Reaction with sulphanilic acid phenol, and ammonia. Strong yellow-red. Strong yellow-red. Strong yellow-red. Straw colour. None. Very faint in-deed. Strong yellow-red. None. None. Strong yellow-red. None. None. Strong yellow-red, 39 9 1 Faint B trmw. Y Y Strong yellow. Straw. Strong yellow. Straw. None. None. None. None. None. Pale straw. Strong yellow. None. None.None. None. None 378 FRANKLASD THE ACTION OF towards nitrates ; thus whilst B. cereus reduces the nitrate power-fully B. subtilis yields no nitrite whatever and thus the two organisms become most sharply distinguishable on being submitted to this chemical test. The solutions tested a s above were now allowed to remain at the ordinary temperature of the laboratory (about 20") until they were again examined 41 days after their first inoculation (table p. 377). Thus the longer preservation of the solutions had produced no marked difference in the results. The solutions which gave a strong reaction for nitrous acid after three days' growth still gave the same, and those which gave no reaction in the former examination were still free from nitrite after 41 days excepting in the case of AI.carnicdor which though free from nitrite before now gave a dis-tinct reaction. Further experiments have shown that this organism, M. carnicolor does slightly reduce the nitrate and moreover in a more marked manner than the very similar M. rosaceus which in no case gave more than a very faint indication of nitrite. The fact that all the solutions gave strong reactions with diphenyl-ainine shows that i n no case had the oxidised nitrogen been destroyed, that is completely reduced to ammonia assimilated as organic nitro-gen or eliminated as free nitrogen or oxide of nitrogen. The solutions were again examined after another interval the total length of time since inoculation being now 137 days ; the reactions for nitrous acid were practically identical with those given in the last series and need not therefore be repeated.The solutions were, however on this occasion also tested for ammonia with Nessler's solution with which they were fouiid to exhibit great differences. Thus in the above solutions those which yielded a strong reaction with the Nessler test are marked* whilst those which yielded a distinct but not very strong one are marked + and those which yielded practically no reaction are marked -Second Series of Ezperirnents. In order to determine whether the results obtained in the former series of experiments were constant for the particular micro-organ-isms or merely accidental a number of similar solutions were inoculated with some of the micro-organisms which had produced the most striking results.After three days' growth in the incubator a t 30° these inoculated solutions were submitted to examination with Nessler's test with the diphenylamine test and by nieans of sulph-anilic acid phenol and ammonia. Thus : SOME SPECIFIC MICRO-ORGANISMS ON NITRIC ACID. 37 9 Examination of Xolutions after three Days' Growth at 30". Name of micro-organism. B. ram0su.s . . B. cereus . . . . B. subtilis . . . . B. viscosus . . . . B. chlorinw M. rosaceus . . . M. carnicolor . . Blank experi-ment Appearance of solu-tion. - - ~ Liquid quite clear but numerous tufts over sides and bottom of bottle Abundant flocculent matter and deposit Abundant flocculent deposit Liquid very opalescent, 1it.tle or no deposit Ditto Ditto Liquid very opalescent, very slight flocculent deposit Liquid quite clear Reactions with Diphenyl-smine._--Very deep blue Ditto Ditto Ditto Ditto Ditto Ditto Ditto Sulphanilic acid. Very strong yello w-red Ditto None None Strong chrome- yel-low Faint Chrome-y el-low colour None Nessler. Strong. Fairly strong. Ditto. None. Very strong. Slight. Slight. None. Thus in each case the previous results were confirmed. Quantitative Experiments. Having thus by the above experiments determined which micro-organisms give rise to the reduction of nitrates to nitrites a number of quantitdive experiments were now made in order to ascertain the manner in which the nitrogen was distributed in the forms of nitrate, nitrite and ammonia after the growth of the several organisms.The determinations of nitrate and nitrite were made on the lines laid down in my previous communication " On the Determination of Nitrous Acid," the nitric acid being estimated by the measurelrrent of the nitric oxide gas obtained by decomposition with mercury whilst the nitrous acid was estimated by measuring the nitrogen liberated by the action of urea and dilute sulphuric acid. For the purpose of these determinations 10 C.C. of the solutions were in each case taken and on this account the actual results obtained for this volume of solution are given below as well as those calculated to parts per 100,000. The accurate determination of ammonia in solutions of this kind is attended with great difficulty owing to the presence of basic product 380 FRANKLAND THE ACTION OF other than ammonia ; these give colorations with the Nessler-test which cannot be satisfactorily compared with those obtained with a standard solution of ammonium chloride owing to the difference in the colours.The estimations of ammonia cannot therefore lay claim to any great degree of accuracy but they serve to indicate certain broad differences in the action of the various micro-organisms. These determinations of ammonia were made by diluting a measured volume of the solution (the exact volume taken depending upon the propor-tion of ammonia present) with distilled water free from ammonia and then nesslerising. The first series of quantitative experiments was made with a num-ber of solutions which remained in the incubator at 30" for 26 days and then for nine days more at the ordinary temperature of the laboratory (about 16-18'] before they were submitted to examina-tion.With these solutions the following results were obtained. Blunk E::lperinzent.-The liquid was clear and transparent. (u.) 10 C.C. yielded 0.003342 gram nitrogen as nitrate. ( b . ) 10 , 0.003384 , 9 , (4 10 ) 0.003324 7 9 9 (d.) 10 , 0.003451 9 9 , Mean . . 0.003375 9 9 9 , or 33-75 parts of nitric nitrogen psr 100,000 parts of solution. Note.-This solution was prepared with twice the quantity of The solution was free from ammonia and nitrites. Bacillus vermicdnris.-The liquid was very opalescent with a fine deposit at the bottom it yielded strong reactions €or ammonia and nitrous acid.calcium nitrate mentioned on p. 374. 10 C.C. yielded 0.0012907 gram nitric nitrogen. 9 9 0.0021143 , nitrous nitrogen. 9 0.0034050 , nitrogen as nitrates and nitritee. Ammoniacal nitrogen in 10 C.C. = 0.000326 gram. 12.91 Parts per 100,000 Nitrous nitrogen . . . . . . 21.14 { Ammoniacal nitrogen . . 3.26 Nitric nitrogen . . . . . . . . BaciZEus pestifer.-The liquid gave very strong reactions for am-monia and nitrous acid. 10 C.C. yielded 0*001069 gram nitric nitrogen. 9 0.002285 , nitrous nitrogen. 3 ) 0.00335% , nitrogen as nitrates and nitrites SOME SPECIFIC MICRO-ORGANISMS ON NITRIC ACID. 381 Ammoniacal nitrogen in 10 C.C. = 0*000412 gram. Nitric nitrogen 10.69 Ammoniacal nitrogen . . 4.12 Parts per 100,000 22.85 Bacillus ramosus.-The liquid mas clear but contained abundance of characteristic tufted growths ; it gave strong reactions €or ammonia and nitrous acid.10 C.C. yielded 0.001668 gram nitric nitrogen. 3 0.001646 , nitrous nitrogen. Y 9 0.003314 , nitrogen as nitrates and nitrites. Ammoniacal nitrogen in 10 C.C. = 0*000309 gram. Nitric nitrogen 16.68 Ammoniacal nitrogen 3.09 Parts per 100,000 16.46 Bacillus prodigiosus.-The liquid was very opalescent but there was not much deposit ; it gave strong reactions for ammonia and nitrous acid. 10 C.C. yielded 0.0020355 gram nitric nitrogen. 9 0.0011931 , nitrous nitrogen. 79 0.0032286 , nitrogen as nitrates and nitrites. Ammoniacal nitrogen in 10 C.C. = OmOO0515 gram. Nitric nitrogen 20.36 Ammoniacal nitrogen .. 5.15 Parts per 100,000 11.93 Bacillus liquidus.-The liquid gave very strong reactions for am-monia and nitrous acid. 10 C.C. yielded 0*002042 gram nitric nitrogen. 9 0.001134 , nitrous nitrogen. 9 9 0.003176 , nitrogen as nitrates and nitrites. Ammoniacal nitrogen in 10 C.C. = 0*00068 gram. Nitric nitrogen 20.42 Parts per 100,000 11.34 Ammoniacal nitrogen . . 6.80 Bacillus pEicatus.-The liquid gave no ammonia reaction but n strong one for nitrous acid 382 FlZAKKLAND THE ACTION OF 10 C.C. yielded 0.002550 gram nitric nitrogen. ?9 0.000504 , nitrous nitrogen. 99 0.003054 , nitrogen as nitrates and nitrites. -Nitric nitrogen 25.50 Parts per 100,000 Nitrous nitrogen 5.04 { Ammoniacal nitrogen . 0 Bacillus jZuowscens.-The liquid gave practically no reaction for ammonia and not a strong one for nitrous acid.10 C.C. yielded 0.0032322 gram nitric nitrogen. 9 0.0000797 , nitrous nitrogen. 9 0.0033119 , nitrogen as nitrates and nitrites. Nitric nitrogen 32.32 Ammoniacal nitrogen . . 0 Parts per 100,000 0.80 Xnrcina Zutea.-The liquid was very opalescent with a fine yellowish It gave a very strong reaction for ammonia but none for deposit. nitrous acid. 10 C.C. yielded 0.0029928 gram nitric nitrogen. Ammoniacal nitrogen in 10 C.C. = 0.000371 gram. Nitric nitrogen 29.93 Ammoniacal nitrogen . . 3.71 Parts per 100,000 0 Bacillus apuatiZis.-The liquid was very opalescent with little or no I t gave a strong reaction for ammonia but none for nitrous deposit.acid. 10 C.C. yielded 0.002597 gram nitric nitrogen. Ammoniacal nitrogen in 10 C.C. = 0*000261 gram. Nitric nitrogen 25.97 { Ammoniacal nitrogen . 2.61 Parts per 100,000 Nitrous nitrogen 0 The above results may be conveniently summarised in the following table which also records the results of some determinations of am-monia made in solutions in which litkle or no nitrous acid had been produced by the action of the micro-organisms ; SOME SPECIFIC JIICRO-ORGANISMS ON ISITRIC ACID. 383 Quantiiative Results of Growth of Micro-organisms during 35 Days. Name of micro-organism. Blank experiment . . B. vermicularis . B. pestifer . B. ramosus B. l i p i d u s B. plicatus BJluorewens . Sarcina luiea . B. aquatilis Sarcina aurantiaca B.aurantiacus . B. aurescens . B. aureus B. viscosus B. prodigiosus Nitric nitrogen. 33 *75 12 -91 10 *69 16 '68 20.36 20.42 25-50 32 -32 29 -93 25 -97 Not determined Y j 91 32)-'63 Parts per 100,000. Nitrous nitrogen. -0 21 -14 22 -85 16 -46 11.93 11 -34 5.04 0.80 0 0 0 trace J 9 9 ) 0 Nitrogen as nitrates and nitrites. 33 '75 34 * 05 33 * 54 33 '14 33 '29 31 -76 30 *54 33'12 29 '93 25 -97 ----32 -63 Amrnoniacal nitrogen. 0 3 *26 4.12 3 a 0 9 5 *15 6'80 0 0 3.71 2 -61 0 - 3 4 2'06 0 *26 0 -82 0.52 From the above table it will be seen that in no case was the nitric acid of the original solution completely reduced and in those cases in which notable proportions of nitrous acid were produced the sum of the nitrous and nitric nitrogen was almost exactly equal to the nitric nitrogen in the original solution the ammonia in these cases being apparentlF derived from the decomposition of the peptone contained in the original solution.The peptone in the original solution amounted to 25 parts per 100,000 and it was found by combustion with soda-lime that the peptone employed contained 13.65 per cent. of uitrogen ; there would therefore be 3.41 parts of peptone-nitrogen per 100,000 which considering the impossibility of making accurate determiriations of ammonia in liquids of this kind does not differ very materially from even the largest proportions of ammoniacal nitrogen found in the solutions.There is in fact no evidence of the nitrate in the original solution being largely reduced to ammonia in any case. The following three experiments are of interest as showing how the reduction of the nitrate to nitrite may become complete and confirming the above opinion that none of the oxidised nitrogen is yeduced to ammonia. These experiments were made with a solution having the same composition as the one used in the above experi-ments only with less calcium nitrate in it. The solutions wer 384 FRANKLAND THE ACTION O F inoculated with Bacillus subtilis R. cereus and B. mmosus the nitric acid being also determined in a portion of t,he solution not inoculated. Blank Bayeriment.-This gave 19.30 parts nitric nitrogen per B. subti2is.-The solution was examined 70 days after inoculation.The nitric nitrogen only was determined as it gave no reaction for nitrous acid. 100,000. 10 C.C. gave 0.001950 gram nitric nitrogen or 19.50 parts of nitric B. cereus.-This also was examined 70 days after inoculation. 10 C.C. gave 0.001962 gram nitrous nitrogen or 19.62 parts nitrous B. ramsus.-This also was examined 70 days after inoculation. The 10 C.C. gave 0.001968 gram nitrous nitrogen or 19.68 parts nitrous nitrogen per 100,000. The nitrous nitrogen only was determined, nitrogen per 100,000. nitrous nitrogen only was determined. nitrogen per 100,000, Thus the solution in which the B. sicbtilis had flourished retained its nitrate quite unchanged although from numerom other experi-ments we know that a notable proportion of ammonia must have been formed and this must consequently have been derived from the organic nitrogen of the peptone.The whole of the original nitric nitrogen on the other hand was found as nitrous nitrogen in the solutions in which B. cereus and B. rurmsus had flourished so that the ammonia which numerous other experiments show to have been produced in these solutions, must have been derived from the organic nitrogen of the peptone. Experiments with Exclusion of Air. In all the experiments hitherto described the bottles containing the solutions were plugged with cotton-wool only so that more or less free circulation of air was possible. Experiments were now made in order to ascertain whether if access of air to the solutions was prevented materially different results would follow.For this purpose, immediately after inoculation the cotton-wool plugs were cut down so as to be flush with the neck of the bottle ; a quantity of melted wax was then allowed to drop on to the cut surface of the plug until the bottle was completely sealed and the surface of the wax was then further thickly coated with cerate. For these experiments a number of those micro-organisms wer SOME SPECIFIC MICRO-ORGANISMS ON NITRIC ACID. 385 taken which had in the previous experiments exerted no reducing action on the nitrate and which it was thought might possibly do SO when air was wholly excluded in this manner. The inoculated bottles were kept in the incubator at 30" for 14 days a,nd they then remained 21 days longer at the ordinary tem-perature of the laboratory (16-18") before examination.The solutions were tkus 35 days old when the following results were obtained :-BaciZZus subtiZis.-The liquid was quite clear but abundance of flocculent matter had collected on the bottom of the bottle. The Iiquid gave a strong reaction for ammonia but none for nitrous acid. 10 C.C. yielded 0.003349 gram nitric nitrogen. 7 ) 0.000168 , ammoniacal nitrogen. Parts per 100,000 Nitrous nitrogen 0 Nitric nitrogen 33.49 { Ammoniacal nitrogen . . 1.68 Thus just as in the previous experiments with this micro-organism, Bnc-ilZus Zavis.-The liquid was slightly opalescent ; some flaky The liquid gave a the nitric nitrogen remained wholly uiiaff ected by its growth. matter had collected at the bottom of the bottle.distinct reaction for ammonia but nolie for nitrous acid. 10 C.C. yielded 0.005379 gram nitric nitrogen. 71 , 0.000027 , ammoniacal nitrogen. Parts per 100,000 Nitrous nitrogen. 0 Nitric nitrogen 33.79 { Ammoniacal nitrogen . . 0.27 Thus as before the nitric nitrogen had remained wholly unaffected Sarcina Ziquefaciens.-The liquid had become quite clear but some The liquid by the growth of this organism. fine deposit had collected on the bottom of the bottle. gave a distinct reaction for ammonia but none for nitrous acid. 10 C.C. yielded 0.003360 gram nitric nitrogen. 9 , 0.000049 , ammoniacal nitrogen. Nitric nitrogen 33.60 { Ammoniacal nitrogen . . 0.49 Parts per 100,000 Nitrous nitrogen 0 In this case again the exclusion of air had not altered the result, the nitric nitrogen remaining quite unaffected by the growth of the organism 386 FRANRLANL) THE ACTION OF Sarcina aurantiaca.-The liquid was slightly opalescent with a con-It gave a strong reaction for siderable yellow flocculent deposit.ammonia but none for nitrous acid. 10 C.C. yielded 0.003420 gram nitric nitrogen. 9 2 , 0.000057 , ammoniacal nitrogen. Nitric nitrogen 34.20 { Ammoniacal nitrogen . . 0.57 Parts per 100,000 Nitrous nitrogeii 0 The nitric nitrogen had thus again remained quite unaffected. Micrococcus candicam-The liquid had become clear but some de-There was no reaction either for posit had collected at the bottom. ammonia or nitrous acid. 10 C.C. yielded 0.003330 gram nitric nitrogen. Nitric nitrogen 33.30 Parts per 100,000 Nitrous nitrogen O { Ammonincal nitrogen .0 In this case again the vigorous growth of the micro-organism had been without any effect on the nitrate. Bacillus aquatiZis.-The liquid was slightly opalescent with a small amount of deposit; it gave a strong reaction for ammonia but none for nitrous acid. (a.) 10 C.C. yielded 0.002621 gram nitric nitrogen. (b.1 9 , 0.002597 , ¶ ? Nitric nitrogen 26.09 c Ammoniacal nitrogen . . 1-34 Parts per 100,000 Nitrous nitrogen 0 The results obtained in this case coincide very closely indeed with those previously obtained when the air was not excluded and they show that this organism causes the disappearance of a considerable proportion of the nitric nitrogen without any corresponding formation oE nitrous acid or ammonia.Sirnilnr experiments were also made with two organisms which had previously been found to effect a powerful reduction of nitric to nitrous acid. Thus :-BaciZZus cerezcs.-The liquid had become quite clear but a consider-able amount of flocculent matter had collected on the bottom. The liquid gave vei-y strong reactions for ammonia and nitrous acid SOME SPECIFIC MICRO-ORGANISMS ON NITRIC ACID. 38 7 10 C.C. yielded 0.0016467 gram nitric nitrogen. !a 1 7 , 0.0013368 , nitrous nitrogen. (b.1 9 , 0.0013655 , 7 7, Y , 0.0002255 , ammoniacal nitrogen. Nitric nitrogen . . . . . . 16*47} = 29-98. Parts per 100,000 Nitrous nitrogen . . . . 13.51 { Ammoniacal nitrogen. 2.26 Bacillus ramosus.-The liquid had become quite clear but there were the characteristic streaming flocculi on the sides and bottom of the bottle.The solution gave very strong reactions for ammonia, and nitrous acid. 10 C.C. yielded 0.001724 gram nitric nitrogen. (4 3 , 0*001571 , nitrous nitrogen. (b.) 9 , 0.001561 , 9 9 9, 9 , 0.000309 , ammoniacal nitrogen, Nitric nitrogen . . . . . . 17'p4} = 32.90. Parts per 100,000 Nitrous nitrogen . . . . 15.66 { Ammoniacal nitrogen. 3.09 Thus in the case of both of these organisms the results are essentially similar whether the air is excluded or not the nitrate being powerfully reduced to nitrite whilst an appreciable quantity of aminoniais generated which at any rate in the case of B. ramosus, appears to be exclusively derived from the organic nitrogen of the peptone. Experimends with varying Proportions of Peptone and SzLgar.Of those organisms which powerfully reduce nitrates to nitrites, two representatives were taken-B. ramosus and B. pestifer-and with these further experiments were made with a view to ascertain whether the amount of nitrite formed in a given time was due to the proportion of either peptone or sugar or of both present in the solution. For this purpose five different solutions were employed as fol-lows :-Solution No. 1.-This was the same solution as tbat employed in the previous experiments and containing-Oe30 gram} in 1000 c.c. 0.25 Cane-sugar (inverted) . . . . . . . . Peptone ,, Solution Nos. 2 3 4 and 5.-These differed from No. 1 only in the proportions of sugar and peptone which they contained thus : 388 FRANKLAND THE ACTION OF 2.3. 4. 5 . in 1000 C.C. Cane-sugar (inverted). . 0.6 gram 2.4 0.6 2.4 { Peptone 0.25 , 0-25 1.0 1.0J These five solutions were inoculated with 3. ramosus and B. pestifel-respectively and kept for 19 days in the incubator at 32-33' before examination. The results obtained may be most conveniently snmmarised in the following table :-Parts per 100,000. Solution I. (Peptone 0.25 gram ; sugar 0.3 gram.) B. ramosus. B. pestifer. Nitric nitrogen 16.74 13.1 7 Nitrous nitrogen 16.08 22.27 Ammoniacal nitrogen 2.56 2.87 Solution 11. (Peptone 0.25 gram ; siigar 0.6 gram.) Nitric nitrogen 7.13 6.04 Nitrous nitrogen . . . . . . . . . . 27.02 28.72 Ammoniacal nitrdgen 2.26 1-64 Solutwn 111. (Peptone 0.25 gram ; sugar 2.4 gram.) Nitric nitrogen 11.71 4.15 Ammoniacal nitrogen 0.62 0.62 Nitrous nitrogen 21.18 29.09 Solutwn IV.(Peptone 1.0 gram ; sugar 0.6 gram.) Nitric nitrogen. . lost trace Ammoniacal nitrogen 6.15 3.69 Nitrous nitrogen . . . . . . . . . . 32.11 34.74 Solution '0. (Peptone 1.0 gram ; sugar 2.4 gram.) Nitric nitrogen 0 0 Nitrous nitrogen 32.60 33-98 Ammoniacal nitrogen 3.08 0.41 From this table it is apparent that as the proportion of organic matter in the shape of sugar and peptone is increased in these solutions so the amount of nitrate reduced to nitrite in a given time is also increased. Moreover the amount of nitrate reduced is far more dependeut 011 the proportion of peptone than on that of sugar SOME SPECIFIC MICRO-ORGANISMS ON NITRIC SCID.389 Thus solutions I1 and I11 contain the same proportion of peptone, whilst the sugar in 111 is four times as great as that in 11 yet not-withstanding the reduction to nitrite is much the same in both case, and with B. ramosus in fact the reduction is somewhat greater in solu-tion I1 than in 111. Again solution 1V contains f o u r times as much peptone as solution 11 whilst the proportion of sugar is the same in both; but whereas a considerable proportion of nitrate is left in solu-tion 11 it is practically completely reduced to nitrite in No. 1V ; or, in other words whilst the quadrupling of the peptone brings about complete reduction of the nitrate to nitrite (compare solutions I1 and IV) quadrupling the amount of sugar results in little or 110 increased reduction of nitrate.Another point brought out by these experiments is that the am-monia developed in these solutions is due to decomposition of the peptone and not to reduction of nitric or nitrous acids. Thus solu-tioris I 11 and IT1 all contain the same snznll proportion of peptone, and all yield comparatively small proportions of ammonia; it is, moreover particularly noticeable that solution 111 which contains four times as much sugar as 11 yields the smallest proportion of ammonia whilst solution I which contains the same amount of pep-toile but the least sugar yields the largest proportion of ammonia of the first three solutions ; whilst solution IT which contailis four times as much peptone as 11 with the same amount of s~igar yields the largest proportion of ammonia of all and solution V which con-tains the same amount of peptone as I V but four times the amount of sugar yields only a small proportion of ammonia.It appears there-fore that the maximum yield of ammonia is obtained when the pro-ycjrtion of peptone to sugar is high and the least when the proportion of peptone to sugar is low. Action of the various Micro-o?-ganisms o n ArnmoniTcat Nitrogen. I n order to ascertain whether any of these organisms possessed the power of oxidising ammoniacal nitrogen to nitrates and nitrites t h e j were severally inoculated into a solution of the following composi-tion :-Potassium phosphate. . . . . . . . Magnesiuni sulphate (cryst.). 0.02 ,, Calcium chloride (fused) a . . 0.01 ,, Ammonium chloride .. . . . . . . Cane-sugar (inverted) . . . . . . Peptone 0.1 gram] 1 in 1000 C.C. of dis-1 tilled water with } 4 grams of pure ” 1 calcium carbonate 79 I in suspension. , J 0.5 0.3 0-25 vor,. LIII. 2 390 ACTIOX OF MICliO - 0 RGAKISMS ON NITRIC ACID. The solutions were examined after 40 days' growth but in no case was anything more than a faint indication of nitrous acid obtainable with sulphanilic acid phenol and ammonia. It is worthy of notice that Heraeus (Zeitsch. f. Hygiene 1886 220) has experimented with three of the micro-organisms which I have had under observation viz. R. subfilis B. prodigiosus and B. ranzosus. On growing these in sterilised urine he found that B. subtiZis alone gave no nitrous acid reaction whilst the other two gave distinct reactions for nitrites; from this he concludes that 13.prodigiosus and B. ramosws possess oxidising powers and that R. suhtilis does not. My experiments however conclusively prove that both B. ~arnosus and B. prodigiosus exert a reducing action, whilst B. subtiZis does not ; and therefore that the nitrous acid reac-tions which he obtained in the case of the two former organisms, must obviously have been due to the reduction of the nitrate in the urine and not to oxidation of ammoniacnl nitrogen RS he supposes. That nitric nitrogen is an invariable constituent of human urine has been shown by Warington (Trans. 1884 669) and has i n fact been long known. The principal results arrived at in this investigation may be sum-marked as follows :-1.That there is a great difference in the power possessed by micro-organisms of reducing nitric acid. Of the 32 forms under examina-tion 16 or li were found to reduce nitric to nitrous acid more or less completely whilst 15 o r 16 were quite destit'ute of this power. 2. That this difference in reducing power may in certain cases be of great value in distinguishing between micro-organisms morpho-logically very similar. 3. The behaviour of the various micro-organisms in question was not altered in this respect by preventing access of air to the solu-tions in which they were cultivated. 4. I n no case did the reducing action lead t o the formation of any considerable amount of ammonia the development of ammonia in tdhe solutions being due principally if not wholly to the decomposi-tion of the peptone.5. In the case of two of the organisms possessing a strong reducing power it was found by more detailed experiments that the quantity of nitrate reduced to nitrite in a given time was depecdent on the proportion of organic matter-peptone and sugar-present in the solution the peptone exerting a far greater influence in this respect than the sugar. 6. I n these special experiments mentioned above it was found that the development of ammonia was dependent upon the peptone and sugar present the amount of ammonia formed being greatest wit ACTION OF ALCOHOLS ON ETHEREAL SALTS. 391 the highest proportion of peptone to sugar and least with the greatest proportion of sugar to peptone. 7. In nearly all cases in which partial or total reduct’on of nitrate to nitrite had taken place the sum of the nitrogen as nitrate and nitrite in the “ fermented ” solution was practically identical with the nitric nitrogen in the original ‘( unfermented ” solution whilst in those cases in which no reduction to nitrite took place the nitrate in the solution remained practically unaffected by the growth of the micro-organism. In one case however it was found that an organism, B. tquatilis which does not .reduce nitrates to nitrites caused by its growth the disappearance of a considerable proportion of nitric nitrogen this result being confirmed by a second experiment. 8. None of the organisms under examination were found capable of oxidising ammoniacnl nitrogen to nitrous or nitric acids when inti-o-duced in to a nutritive solution containing ammonium chloride