Arakawa, Satiyo

Itano, Arao

Okayama University Digital Information Repository

Nitrate Reduction by Azotobacter.

Okayama University Scientific Achievement Repository

Nitrate Reduction by Azotobacter. By Arao Itano and Satiyo Arakawa. [JI1tl. 19， 1932.] The ni七ratereduction by variou目白peci白目 ofAzoもobacterwas under句.kenin view of its application to the c1a自由ificationof Azotobacter and もoascerぬintheir ro1e in nature. Although there are numerou日reportson the niもrogenfixation by Azotobacter， comparative1y a few 1it，erature avai1ab1e on the nitrate reduction. STOKLARA.l.2) inv佃 tigatedconcern泊gth白 mechanismof reduction by Azotobacter， and noもedth叫 thepresence of c町'bohydratesand orga凶csa1句 isnec朗自aryto bring about the reduction proce自sand a1so it w帥 foundthat more nitrate is reduced even to ammonia in a sma11 quantity under aerobic 'than anaerobic condiもion. HILLS3) found也前Azo句bacterreduces nitr叫eto nitrite朗自hownby STOKLABA but， fai1ed to demonstrate the pre日enceof鉱nmonia，and further re∞gn包edthat Azotobacter assimi1ates the nitrate direct1y. BONAZzrU reported that Azo旬bacterbehave自制-th白 reducingbacteria in presence of nitraもe. KOSTYTBCHEW5) and his co-worker目前aもedthat it is probab1e， in出esoils， Azotobacter exi自旬阻tagonistica11y加the ni凶戸gb即 teriaby producing ammonia from nitrate. Whi1e the nitrat，e reduction is considered踊阻 imporも岨tf船 terin the dassmcation of bacteria担 genera1，it is not appliedもoAzoもobacterand no七recorded at a1泊 B凪 GEY'日Manua1of DeterIDInative Bacterio1ogyG). For these re朗 ons，it appe町sto be intere日tingand泊lporta品加 inve日もigateth泊 phaseof Azoもobacter目色udy.E玄perimental: 1. Culture media used : The following culture media were used ; 1. Syn出白紙cni七rateagar after the S. A. B.7) KN03 1.0g. K2HPO. 0.5 g. CaCl2 0.5g. G1ucose Eρ Agar 10.0g. 1，∞o cc.15.0g. Steri1ized in autoc1ave under 15 pound自 pres日町efor 15 minutes. In this medium，もhekind of niもrateand七hec町 bon日ourcewas changed as will be noted later. 282 A. ITANO IUld S.MlAKA.WA.: 2. Nitrate pepton日agar.Pepton白 5.0g.Meat extract 3.0 g. KN03 1.0 g. H20 1，0∞cc. Agar 15g. 3. AsH町、mannitagar (stated el自由wh白re.)n. Quantita.tive deもerminationof niもrite:GRIE8S' indicator metbod8) w佃 used，which is prepa.red and used as follows; Reag自ntA. Sulpha.nilic即 id(0.5% in 30% acetic舵id.)" B.α-naphthylamin (0.5% in 30% aceもicacid.) J ust before use， 2 c. of A and 1 c. of B町 emixed a.nd poured int<> the medium， and not自由自 pink加 redcolor pl'oduced after 30 minutes or on自hour，if nitri旬 pr佃 ent.m. Quanti凶 ivedetermination of a.mmonia. : ‘・fuNSEN'S indicator method9) was used as follows : Reagent A. Tbymol 2g. 2NjNaOH 10cc. E泊 90cc. " B. Bromine water 1oocc. H20 90cc. An equal amounもofA and B rea.gent is added to the medium. Then， u 阻lIDoni乱wa.日present，it turn日todark green color which will be cha.nged跡red-purple by shaking with ether and will co11ect in the ether layer. Besides this the NES8LER'S r白agentw制 useda1so with precaution sinc自由iste悦 producesa自国ilarcolor in presence of a.ldehyde. IV. C'l1ltures used: Twenty-six cultures in a1 were I1sed which are noted below : Cl1ltures; Azotobacter chro∞occum. 帯事211j Bo bh from ToTkhye o NImiもrpogr8ianl FUinzi叫veiomnitLya. boratory， 普3 From Wisconsin University. 3;)I仙附romOkayama Pref自cture回FFz2S ‘ :l " " Fukuoka " "・Ezl j " " Hir伺 hima " " . " " Kochi " " . " " Kagawa " " . Nitmte Reduotion by Azotobnoter. A.zotοbacter beijerincku. 最11 招~ Both from Tokyo Imperia.l University. 粉 FromWisconsin University. 011 02} Isola.ted from Oka.yama. Prefecture soils. 03J Azotoba.cter vinelandu. 神1l 抱 l> From Tokyo Imperial University. 粉 l神4J 最5 From Wisconsin University. 283 These cultures were grown on AsBBY'S mannit aga.r medium until ready tou自由， and their purity wa.s wa.tched c町 efullyunder micro自copeduring the e:xperiment. Experiment 1. To朗 certa.inthe nitrat白 reductionactivity by Azoもobacter，the organisms were grown <?n three different kinds of nitra.te media， namely nitraぬ meatextract a.gar， 1% and 3% manuit nitrate nutrient aga.r， and kept in a.n incuba.もora.t 28-ー筑1'0.，a.nd tested for the presence of nitrite. The r自由ultsa.re shown in Ta.ble L No. of Oulture. #1 ij 神2書3O. K， F， 0， #1 最2書3#1 #2 #3 神4Ta.ble 1. Nitra胎Reductionon血eSynthetic Agar Medium. …utr… 5da刊 I10旬 8. I 5必y8. I 10ω. I 5 days. I 10 dt>ys + ー 一 一 一+ ー + 一 一+ ー 。 。 。土 一 ー ー ー土 一 。 。 。+ 土 + 一 + + 土 。 。 。。 。 。 。 。+ 土 + 一 土+ 土 + 一 土ー ー ー 一 一ー ー 。 。 。+ ー + 一 一+ + 。 。 。Not伺: +， + intenBity of positi刊 reduotioll;土 doubtfu1; - no国duouon; 0 not ex戸rimented.一一。一。一。。一一一。一。284 A. bA.NO Wld S.AR.uUWA.: Table 1 indicate自由atmo凶 of出emhav自由自 nitra七ereducing action except Azot. vinelandii #1 and #2. In general， the manniιnitra加 nutrientag町 was加悦町白uitedfor the growth than the nitrate nutrient ag町 butfor the reduction 句自七thelat白rwa自better. The resul旬 wereobserved more markedly a.t the end of five da.y自由a.nten da.ys自othat出etest was a.pplied on the fourth or fifth da.y. Then the t伺も W闘 rep舗もedon th白 syn也eticnitrate medium and the following re自ult自wereobt&ined副 noもedin Table 11 : Ta.ble 11. Nitra句Reductionon也eSynthetic Aga.r Medium. Azotooocter chrooooooum. Azotobncter beijerincki. Azotooocter vinelandii. …叫川 Rednoti No. of onlture. Reduotion. 恥…韓1 + 韓1 土 韓1 一韓2 fi #2 + 韓2 ー#3 土 #3 + #3 付+0， + 。‘ 土 #4 fi O. + O. 土 神5 併+F， + O. 土F. + F. + F. + B， + H. + K， + K. 土0， 土0. + There且ultsshown in Table II substa'J.tiate those results which were obtained in the previous expe討ment.Experiment 11. The influence of v町iousnit:.raもes，no.mely ammonium nit:.ra.te， pota関 iumnitrate，日odiumnitro.te and ca.lcium ni佐a旬， w嗣 inv剖 tiga.tedby using the S戸 theticni回 teag町 andthe results are pr朗自旬din Table m: Nitmte Reduotion by A.zotoOOoter. 285 To.ble lli. ln.duence of Four Di宜erentNitra.tes. Azotoh凶terohrooooooum. AzotoOOoter凶 ijerinoki. Azotooooter vine1wdii. #1 #2 #2 神3 #1 #4 NB.NO. + t+ + + 一 t+ KNO. + + + + ー t+ NaNO. + + + + 一 fIt Ca (NO.)2 + + + + 一 柵A.s To.ble III indico.te自， 0.1 except Azot. vinel阻 dii軸1reぬ，inedthe reducing o.ction rego.rdle日目thekind of nitra.tes. ExperIlnent III. The infiuence of succes自iveculture on the ni仕o.t白mediumw朗 investigaおdby using the synthetic o.go.r m品ium組 dten successiveもra.nsferswere ma.de on e蹴 hfourth day， o.nd the reduction test w朗o.ppliedon倒 chculture. The results o.re noted in To.ble IV : To.ble IV. ln.duence of successive Tra.nsfers on Nitra.te Reducもion.No.of 山 oooter…。um'lAw…同erinokiL Azotoh同tervinel.ndi tmnsfe同. 韓1 骨2 韓1 I 韓2 神1 れ1. + t+ 土 + 一 t+ 2. + t+ + + 一 fIt 3. + + 土 + 一 fIt 4. + t+ 土 + ー fIt 5. + fIt + + 一 fIt 6. 柵 持+ 土 + 一 fIt 7. 柵 付+ 土 + 一 fIt 8. • + + + + 一 + 9. + + 土 + 一 t+ 10. + + 土 + ー t+ A.s To.ble IV indico.te日， o.pparently no influence i自 broughもo.boutby ten 8ucce日目ivetra.n自ferso.t four days intervals， and a1 but Awt. vinelandii continued not to reduce 出enitro.te自・ The other cultures seemed to weo.ken in reducing power o.fter the eighth tro.n日ferwhich mo.y be due to the graduo.l weo.kening in their grow也.286 A. ITANo nnd S. A:auuWA: Experiment IV. Ftom the preceding experimen旬， it w闘 found tho.t Azot. vinelo.ndu・昔1doe目not reduce the nitra.te while Azoも vinelo.nd話料 mo.nife自ted0. strong凶tra.旬reduction. Consequently it w制o.ttemptedto a.scer七o.inif the stra.in like #l mighもbe present in the s七ra.in#4 by cho.nce. Strain #4 was plo.旬douもo.ndten culもures'were prepared from different colonies appeared on the plo.te， and也ereduction te自twas co.rried out with o.n addition of different co.rbohydro.tes. The r倒ultso.re noted in Table V: To.ble V. Nitra七eReduction Test applied on various泊。，latedCulもurefrom Nitrate Reducing Stock Culture. No. of N飾，NO.i闘いtedou1tures. g1uoose. I fruoto同 叫同e.I gnlucto回 munnit. 1. + + t+ tI十 -If 2. + 側+ t+ -If 側+3. + 側十 t+ + + 4. + -If t+ fi十 + 5. t+ -If t+ 骨It 。6. 材+ -If 。 。 。7. 榊 。 。 。 。8. -If + 。 。 。9. 側+ 。 。 。 。10. + 。 。 。 。KNO. g1uo08e. 側+柵-If t+ t+ t+ 。-If 。。As To.ble V indico.t倒 tho.t0.1出。 i自ola.もedculもur倒 reducedthe nitra.もesshowing tho.t no possibility of盆nding0. non-reducing stro.in in the reducing sもockculture. Experimenも V.Them町kedinfl.uence by different c町'bon自ourceon the nitra.te reduction h制been well esto.blished but the旬stswere co.rried out on the synthetic niもra.teo.ga.r by using 21 different kind of compounds in a.1， namely 14 of sugo.rs， 2 of a.lcohol， 1 of a.lka.loid a.nd 4 kinds of orgo.nic臨l旬. Thωe compounds were a.1 of MERCK'S exωpもsodiummo.lo.te. The results a.re自hownin To.ble VI : Nitrl¥te Reduoti∞by Azotoo.oter. 287 T乱，ble VI. Different Carbon-自ourcesand NitraぬReduction.Azotooooter Azotobnot町 Azotooooter ohrαlO∞oum. beijerlnokii. vinela且di.仏rb∞・80urC田.最1Iが Igrowth. 神1 都2 growtb. 韓2 韓3 growth. XyJ伺 e. 土 土 (土) 一 一 (土} 一 + (+) Arubinose. 件 土 (+) + + (+) 一 主 (+) Rhamno回. 一 土 (土) 一 一 (土} 一 ++ (+) Glu∞回. ++ 叫+ (側) + + {榊) ー fi (榊)Fruotose. + ++ (榊) ++ ++ {榊) 一 側+ (榊)G泊、，ot佃 e. fi fi {榊). ++ fi (悦) 一 側4 {悦}M品目n08e. + + (+) 一 一 (企) 一 土 (土)Sucro開. 件 + (側) ++ ++ (柵) 一 fi (榊)Malぬ8e. 制 付+ (側) + + {柵) 一 (榊) (柵}L¥otoBe. + 土 (+) 一 一 (土) 一 + (+) R..鐙nosD. ++ ++ (++) + + (++1 一 fi {件)Siurob. fi 持+ {榊) 土 土 (+) 一 ++ (+) Dextrin. fi fi (術} ++ ++ (++1 一 fi (++1 lnulin. 骨+ 付+ (榊} 制 fi (++) 一 柑+ (件)Mぬnnit. 件 骨+ li!lt) + 土 {柵) ー 制 (i!It) Glyoe.riu. + + (+) 一 一 (土) ー 土 (側)Sulioin. 土 土 (土) 一 一 {士) 一 + (土)段泊ium-forn回te. 一 一 (土) 一 一 {土) 一 一 (土)Sodium-laotate. 土 土 {件) 土 土 (土) 土 土 {付)動dium・810oislte. fi 伺+ (++) fi fi (++1 + 柑+ (+1 政調iium-m，・，late. 制 付+ (++) 柵 "* (++) + 柵 (++) Note8 : (+) intenBity of growth;. (土)doubtful; in 加 Beof 8cdium Iaotate， the r創出WIlo8 not， definite 8ince it8 oontrol itself g晶vethe uitrite reaotion. Table VI indicates也叫也eniもratereduction is influenced ma.rkedly by the kind of carbon自upplied. One of the striking fac旬 foundis that Azot. vinelandii 昔1which has been found to be non-nitrate・reducingorganism up旬 thisexperi-ment， reduc吋也eniもratefor the first tinle in pr倒 enceof sodium succin叫eand sodium malate. Consequently it w嗣 foun~ 旬 be very impor恒叫句 considerthe carbon source in conjunc伝onwi也 thenitrate reduction tes旬.288 A. ITA.NO snd S. ABA.XA.WA.: Exper泊lent VI. The fo.te of nitrite produced o.s the re日ultof nitro.te reduction wa日investigatedto S8certain if the nitrite is reduced further to o.mmonio. or even to nitrogen go.s. or is S8similo.ted directly. Two po.r旬 ofper mi1lion of po凶闘iumnitrite wo.s o.dded in place of niもrateswith vo.rious co.rbon sources， and the tests were o.pplied o.s indicated in To.ble VII. To.ble VII. Fate of Ni色ratesin Syn出品icA.gar Medium. Cultures. 白rbon-回uroes.Fifth dぬy. lhrtie也NH. HN01 I NH. gluoose. 骨+ 一 骨+ 一Control. msnnit. tH- 一 制 一Azot.ohroc用O伺 um. glu∞se. + 一 土 一事1 回目nit. + 一 + 主Azot. bei骨j2exinoki. gluoose. + 一 + 一皿則mit + 一 + 一Azot. vinemndii. gl108e. 土 一 一 土神l msnnit. 土 一 ー 一g1uo08e. tH- 一 一 一神4mannit. 骨+ 一 一 一As To.ble VII indico.tes， Azotobo.cter use自由enitrite nitrogen o.nd grows very we11 although there wo.s 0.slighもva.rio.tion創出eco.rbon日ourcevo.ries， o.nd no・ammonio. wo.s detected. It is notewor出ytho.七 Azot.vinelo.ndii担師団imilo.tedpractico.lly 0.1 the nitrite on the :fifth do.y while制 culturetook longer time so tho.t the failure in detecting the nitrite in co.se of担 culturewhen the nitro.te reduction test wo.s o.pplied， might be due to this quick o.ssimilo.tion of nitrite. Again no production of free ni仕ogenw阻 observed.Summary. The nitro.te reduction te日to.nd the nitrite o.ssimilo.tion test together with the in:s.uence of various co.rbon source were invest唱叫edusing vo.rious strain oI A.zotobacter o.nd the results mo.y be summo.rized o.s fo11ow日:1. Azotobo.cter o.s 0. whole reduces the nitrate to nitrite but no de:finite experimental evidence wo.s obもa担edo.s to the o.mmonio. production in也eproce目白・2. Azotob即 ter朗自.imilo.testhe nitrates o.nd nitrite白畑出自国位ogensource. 、Nitmte RedUOtiOD by Azotooooter. 289 3. Glucose， fructose， galactose， sucrose， maltose， ra但nose，sta.rch， dextrin， inulin， mannit，自odiumsuccinate and sodium malate are all good for the growth of Azotobact白r，and出epresence of the冊目ubstancestimulate the nitrate reduction activiも，y.4. To ca町'yout the nitrat白 reducもiontest of Azotρbacter， itis advi日ableto use出e日:yntheticnitrate agar in presence of sodium succinate or日odiummalate. References : 1) J. STOKLABA U. E. VITECX， Zentmlbl. f. B.kt.， 11， 14: 102-118， 1905. 2) J. I:lTOXLA!lA， ibid. II， 21: 484-511， 1908. 3) T. L. HILLS， Jour. Agr. Res曲 roh，12: 183-230， 1918. 4) A. BoNAZZI. Jour. Bnot.， 6: 321-36!l， 1921. 5) S. K田TYTSCHEW，A. RYS:o:.u.TBCHUX U. O. BcHWEZOWA， Zeit. Physio. Chem.， 154: 1， 1926. 6) D. H. BElIGEY， 脇田u.lof Deterruinutive Booteriology， 1923. 7) Sooiety of Amer. B..oteri-lo酔 ts，M.由加1of Meth吋Bfor Pure Oulture 8tudy of &0ぬriao1923. 8) R. P. BABTHOLOMEW， Soil Soienoe， 25: 393， 1928. 9) P. A. HANBEN， Jour. Bact.， 19: 223， 1930. 

Nitrate Reduction by Azotobacter.

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