| | | | | | i ll SduVaGa * Iwo | nom Toa ee ee BE et td) Studies in Denitrification e ae &, Fred Edwards THESIS SPUNIFS JN DENTTRIFICATION BY ae S. FRED FDWARDS / 963 THESIS MEAT TPT II TR. Spt Pe POR mt ry 23 Ve Dams toN ees bt i beohaauly venitrification is, as tre term implies, a reducing process in which the soil nitrogen is eliminated in one form cr another by the hreak- ing dowm of the nitrate or ecmplex nitrogenous conpceunds, thereby beconine Icest to plant crowtnh. it is still to a sreat desree an open question in what form the nitro:en is lost, wnetrer as oxides of nitrosen, free nitroren or free aimicnia or in more than one of these forms. The denitrification phenomenon was first noticed in cultivated soil in 1862 by Goprel- sroeder (Technical c“‘yeology Lafar), and for a lons time it was rererded as a purely chenicel process. “he first reference to the arency of hacteria in the cnanges produced was made in 1875 by C. Feusel (Tech. “yc.) and the earliest ~ure cultures of such micro-organisms were made 36047 (2) AD jn 1862 ty U. Cayon and C. Punpetit (Tech. Myc.).. In 18&6 W. Heraeus (Tech. Myce.) made known several species of the denitrifying class, and two years later Percy Franklend and R. “arington (S“ranklarnd's Micro-crganisms in Water) found a jarce percentage of soil pacteria cupable of re- ducing nitrates to nitrites altihcugh they.do rot state whether their cultures possessed the power of further reduction. In later years scores cf workers have enter- ed the field, and, workins’‘upon different phases of the subject, have secured some very valuable results. The object cf the present investication was tc contribute sonethire toward a solution of the proklem by a detailed study of the denitrifvine proverties of three germs found commonly ata depth of two to six inches in different soils upon the collere farm, viz.; P. Subtilis, RB. “ercaterium, and R. “ycoides. A bricf outline of the investigation pur- sued follows:- 1. Action upon nitrate. ©. Action upon nitrite. 3. Action upon aranonium salt. (3) 4. Fffect of the presence of organic matter. 5. Influence of abundant oxyren surrly. 1. Action upon Yitrates. As has been stated, in 1868, *rankland and Yarineton found a large number cf soil fracteria capnahle of reducires nitrate to nitrite. “or treir work tneyv used a mineral solution which contained pesides the mineral elervents necessary for plant growth, a small amount of organic niatter in the form of pertone. In our studies an endeavor was made to determine quantitatively the reduction of nitrate to nitrite, and whether any further oroducts resulted. Tine culture soluticn used wes made up es follows:- Potassium nitrate 5. grams. Potassium phosphate wl PL bn:. Magnesium sulphate (eryst).02 " Caleium chloride (fused) .Ol " Glucose a) " Peptone (Wittes') 225 " Calcium Carhonate 5. " Distilled Water 1LO90, ec. em. 75 ¢. cm. “rlenmeyer flasks, plurred and steril- ized, received about °5 c. cm. each of the -_—~ w> ~~ culture soluticn and vere sterilized intermittent. ly in moist heat. The inoculaticns were made with .5 ©. cm. of cultures of the germs ina mineral solution containing a minimum of nitrate or orgenic matter, some of the flasks teing re- tained uninoculated for controls. The cultures were kept ina darkened reem at an acnroximately average temnrerature of 25° C. Determinations of nitrite and free armonia were made at the time of inoculaticn and at intervals thereafter. In all the work witn mineral soluticns beuillon tubes were inoculated with smell portiors from the culture and control flesks, to verify tne vitality of the cultures and stcrilit, of the controls. TABLE (7) Nitrites exvressed inn. Aiuionia exvressed inm. g. perc. mubtilis PB. Mepaterium mo. P ecides Uninoculated Centrol At xT a\9 O, “one rone one None inoculation Wwe Nol; “one wone Tone None B. two days NO, NH, “one .Ol21 /one None “one .QO4E “one None II. per Cc. cm. Cm. wT? WH, seven das . Ny, O, None None vwone TY. None hone N, 0; At sixty days NO; Trace Trace ~Cll Trace TR ag O048 0291 O194 None Lome Cn From a comparison cf the tables tne fact de- comes evident that most of the armnonia in the nitrate cultures results from tre neptone. TO reaction for nitrites was chtaineu until the cul- ures were old and then only a trace. Whether this vas due to spontaneous chemical changes i8 a question. the fact that tne control, which re- mained sterile, snowed a trace of nitrite would lead one to suspect that such was the case. The work was duplicated in order to verify the results “he same methods were e@iployed except tnat fewer tests were made of tne results. TABLE Jil. Nitrites expressed in m. g. per c. cm. %0,. Ammonia expressed inm. g. perc. ecm. VE,. At At inoculation forty-five davs. T 7 ~ KA mio, NH, NO, NH , FE, Subtilis Yone None None ,.02°1 R, Meraterinum “None Nene None .0194 PrP. “tyeoides None None None .0243 Uninoculated Control None None None Mone Here again the results show that the nitrites result from the reducticn of nitrate, but that at least a portion of the an ronia is the result of tne decomposition of the proteid molecule. These results agree with those of l’aassen (Cent. f. Takt. 1902.15. No. 5. S. 152) who grew 109 cormon bacterial species in mineral solution con- taining peptone, and who says in statins his re- sults;"Considerable aivtonia was also produced on account of the vigorous sentonization.” ©. Do the Miero-orcanisms Fave a Feducing Action upon hitrites? to determine whether the micro-organ- isis were capable of reducing nitrites, a nutrient solution was prevared as follows: Potassium nitrite oO frams Marnesium Sulphate 05 " Potassium pvhosrhate ol " Glucose 20 " Peptone A) " Calcium carbonate 5. " Pistilled water LOOQO. ce. cm. About 25 e. em. of this solution vas olaced in 75 ec. em. Frlenmever flasks and sterilized C10) intermittently in meijist heat. They were inoculat=- ¥ ed witn one-tenth c@. en. of Liquefied ¢elatine Sernit-d: D C , ~ ~ Cd fi ue M ct Nd én O° ©2 Pad! TAPRILE (OV. Tne anocunt of \)O,estimated in original cul- ture solution, equals .2329 m. g. per c. cm. Ke- sults inm. g. per c. em. ,0,. At At At At inccula- five eighteen forty-five tion days das aays B. Subtilis we eke Le as hb. “Megpateriun .Lk cee Le LR uo. » YCGides - as wil C77 O77 Uninociilated Control we wee ae ee - J dete y The Only consideration was tne aucunt of re- duction of tne nitrite, and in only one cage, B. Mycoides, was any decrease ir nitrites apparent. 3. Action of tne Organisms vwoon Armonia Cul*+ures were made in tne following solution toe aetermine, if any, the action of tne ferms upon armonium sulphate. Ammnoniun sulvrate i. gram Pvuvaswili purspuate 1. n hacnesium sulpnate O02 " Calcium cnicride .O1 " Glucose a) " Ferrous sulpnate 20 " Peptone .20 " Calciwn carocnate 5. " Mistilled water 1000, Cc. cm. Tne cultures were made as tefcre in 75 c. em. Erlemiever flasks, the inoculations he- ing made with a platinum loop from cultures cf the germs in bouillon. “essler determinations were made at intervals to discover, if any, tne decrease in the amount of amnonia, standard amnonium cnloride beins used Tur ecomnosriscn. Tuplicate tests were made at different pericds, the results cf which ecorressond. At no time wes any chxrin:te observed in tre amionia content, snow- ine taat the pacteria have not tne power cf s e-= ducing ammonium sulpnate, and iurtnermore tnat the salt prevents tne production of ammeonia Trou tiie pertone present. 4, The Fffect of tne Presence of Organic Matter upon Tenitrification. Yunnenan in 1698 (Tandw. Vers. Stat. 1698 J-TT. S. 65-1135), in experiments carried on witn a m.cro-organisiu wnicn ne nad isclaved From horse- dung, found tuat the amncunt cf aenitrificationr decreases 48 tie wiount of Grpanic metus increases, Whlen ohservaticn was also made by J-atz and Warner (Bandw. Vers. Stat. lagv-"LVTIT, £47) who staten tnat «as "numification" orcceeds the power of destroying nitrate decreases. On tne other hand, Pfeiffer (Deut. Landw. Tresse. 1é37-C11) states toat denitrification carn take piace only in tne presence of particles of stray, faeces or vege- table tissue whien act as food substance for tne a] tutzer and 2 aul (Tent. A denitrifying orrenisaiis. ~ ff. Bakt. LEA6- RV, S. 473-474) isolated two micro- orcanisis wiien gave very imerked nitvave reducing Dower When grovwi in a solution Cuntainin,; a Snail @lount of creanic matter. Maassen (loc. cit.) states tuat in a strictly inorganic sclution he Ohbtaincu ver, little reauction cf nitrate hy conmion bacteria. In cur studies alongs tris line five solutions vere used, differing only in the Nature of tne material furnisring the combined nitrocen to be acted upon, the cases of tne dir- ferent solutions beings as follows?’ Armionium nitrate ard Potassium nitrate, Asparagin, ctass- ZI nNibtrice, and Potassium nitrate and nitrite co:ibined in equal anounts. The other elsments necessary to plant grovztn were added in varicus salts but none of tne sclutions, except the aspar- agin, contained any organic matter. Tne cul- fLices were tested at tie end cf forty-five days for tne reduction products. In only one cr the soluss cons, ASSarasin, were satisfactory results Cuvained. there was practicall: no formation of nitrite or amvtonia, and upon plating in crdinary gelatine 10 was Found tnat while toe Inbero-fOr,anisus still possesscu vitality, cnerve was no viger of frowta., She cultures in Asvara- gin developed vigorously, and tnere was a very marked production of ammonia, the controls yield- yg no reaction for ammonia. A comparison of tne results obtained in tnese strictly mineral solutions with those in solutions containing a small amount of organic retter, leads tc tne con- clusion tnet tne vresence of at least a small amount of the latter is imperative in order to secure vigorous denitrification. ‘nat would he the effect of large amounts of organic matter is still an unsettled question. 5D. The Nffect upon Nenitrification of an Abundant Supply of sir. In a study of this question, Pehnerain in 1597, upon growing micro-organisiis from straw and tne solid excrement of animals, found tnat tne reducticn of nitrates was much more rapid in closed than in open flasks, although he does not sav whether the conditions were strictly an- aerobic. Taassen (loc. cit.) states, that while denitrificaticn is not inhibited by an- aerohic conditions it proceeds best in a limited supply of air. Pfeiffer in 1897 (Chem. Zeit. 21-1897) found that in a culture of B. Denitri- ficans t::e admitting of pure air did nct lessen the amount of denitrification. in LE965, Anpola {16} and Ulpiana (Techn. “ye.) isoleted from soil a miearc-organism which frew wel] both in the pre- sence and absence cf air. In our work one purvose was to so construct a niece ofavparatus that air micnt tre freely drawn through the culture, and yet that the dan- ser of contaminations he eliminated. Five 75 c. an. Frlenmever flasks were tiently fitted witii two-nole-rubber stoppers avd conrected by “eans of U tubes, one arm cf which extended to t bottom cf the flask. Two each of tne erd flasxs contained normel-fiftn sulphuric acid, the midatk one holdings tne culture solution. Thus when air was drawn throuvugn the avparatus the sulphuric acid in tne first two flasks would arscrb any ammonia from the air passing ‘through, as vell as any micro-crgranisnus whnicn misht be drawn in with tne current. The other two acid flasks were to catch any armonia that misnt be given off fror. S2é Culture and dravm over upon aeration. Aeret- ed and non-aerated control flest:s of acid were also wsed. “urtaermore, one series eacn of cultures sealed, and simply plugred with cotton wool were used, uninoculated controls Leing retained in each cxse; also cultures ant controls vere placed in an atmospnere entirely devoid of oxy-7en. Tne culture solution used wes rrankland's denitrify- ing seluticn , which is made up as fcllows:- Potassium nitrate 166 gram Potassium phosphate ol " varnesium sulphate 002 " Calcium Chloride oO1 " Glueese re) " Pentone 025 " Caleium cartcnuate D granis Distilled Water. COCO. ec. en. The inceculations were made frcm pure cultures in this solution and kent in the dark at an averate temperature of 25° C. Atter eleven davs determinations were made of nitrites and free ammonia in the cultures and controls. Tne acid flasks of the aeration apvaratus were titrated against normal-fifth sodium npdroxide, but sncewed no indication of absorption of anmnonia. Titrites expressed inim. ¢g. Cultures in Absence of Cxc zen TT: B. Subtilis B. mepgaterium B. liyeoides Uninoculated Control Cultures Sealed. &. Subtilis oS. égateriunm 2. Tareoides Uninoculated Ccntrol Cultures Unsealed Se SOubtilis r) rater ot ptf i- . .e | um rf B. Mycoide 2 Uninoculated TContrel Cultures Ticroughly Aerated Be. Subtilis B. :.ecateriun pb. Mycoides eninocilated Control per c. cm. Armenia expressed inm. g. per c. cm, nN” N, O3 one Trace Trace Hone oO 339 C440 None oe vone .0440 ~O495 ~C551 “one neon inst avy di “3 bone Trace .OO0EO “one C064 0080 0O240 Fone CL28 0244 9160 “one la@.om. sterile culture soiution contains ele64 i. g. 1, O, lc. em. sterile culture sclution ccntains JOL82 Mm. fF. aia. Tre results snow that tne reduction derends alineost entirely upen the presence cf at least a small amount of oxygen, and that, exceyvt in the case of B. !tycoides, a greater sun ol: of air induces a greater amount of reduction. CONCHL JUS Ilonbs . 1. The three micro-organisms under investicatiocn have the yower of acting upon ni- trates to reduce tnem to simpler substences, ni- trites, free armonia, and probably free nitregen. 2. The nitrites present in a culture scl- ution having nitrate for its basis result entire- ly from the nitrate. 3}. A portion of the annonia usually found in nitrate solutions containing organic matter, results from tne decomposition of this latter material. 4. For a vigorous development and activity of these micro-organisms, the presence of at “——~. Ad C2 least a trace of crganic matter is essential. 5. Mot all bacteria having the power of actins upon nitrates are cavable also of exerting a redvcing action upon nitrites. 6. Tne hacteria studied can not reduce armonium sulphate; furthermore, the ammonium sulphate checks the production of free ammonia from the pevtone in the solution. 7. These micro-oreunisms act more vigorous- ly in the presetce of an abundant supply of atmospucric oxygen. RWwUl Vol om" Whe 5 eS ™ we “eee ~ . ‘ . : , . 4 ve tf & , ¢ } ©. i. gf {or . : } be . * 4 x - é y ta , ’ tee tet - > ed we : a ‘ tw 4 “Tintin BEE E EER RR RE RE RE EEE EAR EEREREEEEEEHs