@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix skos: . vivo:departmentOrSchool "Land and Food Systems, Faculty of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Morgan, Joseph Francis"@en ; dcterms:issued "2011-11-15T20:09:47Z"@en, "1942"@en ; vivo:relatedDegree "Master of Science - MSc"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description "[No abstract submitted]"@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/38978?expand=metadata"@en ; skos:note "* STUDIES ON THE RESPIRATORY ENZYMES OF THE LACTIC ACID AND NITROGEN-FIXING BACTERIA by Joseph F r a n c i s Morgan A Thesis submitted•in P a r t i a l F u l f i l m e n t of The Requirements f o r the Degree of MASTER OF SCIENCE IN AGRICULTURE THE UNIVERSITY OF BRITISH COLUMBIA August, 1?42» PART I Contents - FINAL REPORT TO THE NATIONAL RESEARCH COUNCIL PART I I - STUDIES ON RESPIRATORY ENZYMES 1. The Dehydrogenase Enzymes of the Rhiz o b i a . 2. V a r i a t i o n i n Re s p i r a t o r y Enzymes of the Rhi z o b i a . 3 . Rhizobium t r i f o l i i and Zoning Phenomena. 4• The Dehydrogenase Enzymes of the L a c t i c A c i d B a c t e r i a . 5o Oxidative R e s p i r a t i o n of Strep, l a c t i s . 6. R e s p i r a t i o n and Fermentation by Strep* l a c t i s . 7• Influence of Adaptation upon the R e s p i r a -t i o n of Strep, l a c t i s . 8. Influence of Nitrogen Source on Fermentation and R e s p i r a t i o n * STUDIES OH THE RESPIRATORY ENZYMES OF THE LACTIC ACID AND NITROGEN-FIXING BACTERIA fines! Bepart of . VJork c a r r i e s out tiader a .National Research Council 'Bursarf i n A g r i c u l t u r a l B a c t e r i o l o g y -at '.til© . U a i v s r s i t y of B r i t i s h , Columbia Joseph f» Morgan Sua.*! I f 4 2 gable: of .Content's ' '\"*\"- • %,.. GENERAL SUI^&RY. » . . r , . 1 I I . 2MTE0DU0TX0N ..................... 4 I I I . EXPERIMENTAL' VTORK 8 ' A. L a c t i c A c i d S t r e p t o c o c c i 1# -Aerobic Oxidations ••»•«••••«•»•* 8 2, Fermentation anfi Oxidation ...... 11 3 , Peptone and Acid Production •... . 12 4, Adaptiire*»Gonetitutive Bnaymeo 16 B 8 R h i s o b i a !• Dehydrogenase Enzymes .« < 18 2, Aerobic and Anaerobic Oxidation » 20 3 . Substrain V a r i a t i o n 22 4«*Zoning Phenomena •••*•••......... 28 I f * DISCUSSIOB OF RESULTS ,*.»..•,....».'».»..., 32 V. CONCLUSION • ,,«*.,.•..•«.».•*•«....••.•*••.* 35 BIBLIOGRAPHY ,.. .... • |6 I . GENERAL SUMMARY. 5?ho problem under invest.Igation has been \"A Study of the Respiratory Enzymes of the L a c t i c Acid and -Hitffogen-'Fixing B a c t e r i a \" . This study has e n t a i l e d a c o n s i d e r a t i o n of both the aerobic and anaerobic r e s p i r a t o r y mechanisms of those two b a c t e r i a l groupso The dehydrogenase a c t i v i t y of s e v e r a l s t r a i n s of Rhisobiun t r i f o l i i upon s i x t y organic compounds has been deter-mined by the methylene blue reduction technique of Thunberg. The aerobic o x i d i s i n g a b i l i t y of both tho L a c t i c A c i d S t r e p t o -c o c c i and the N i t r o g e n - F i x i n g Rhizobia upon selected carbon sources has been determined q u a n t i t a t i v e l y by the Bareroft manometric technique. The mechanism of l a c t i c aeid production by trashed b a c t e r i a l c e l l s has been i n v e s t i g a t e d , employing the method introduced by Hegarty. The adaptive or c o n s t i t u t i v e ' character of the b a c t e r i a l enzymes concerned has also b©@a termined i n t h i s manner. An extensive i n v e s t i g a t i o n has beon conducted i n t o the problem of v a r i a t i o n i s r e s p i r a t o r y a c t i v i t y among s t r a i n s and. substrains of Rhlzobiuia t r l f o l i l * With t h i s species the i n f l u e n c e of l a b o r a t o r y media upon growth char-a c t o r s , r e s p i r a t o r y mechanism, and p h y s i o l o g i c a l nature hae also boon studied,, -The experimental r e s u l t s obtained show d e f i n i t e l y that the dehydrogenase a c t i v i t y of Rh. t r i f o l i i i s extremely v a r i a b l e w i t h i n s t r a i n s of the same speciec and even with the same strain- at d i f f e r e n t times. This v a r i a b i l i t y i n anaerobic r e s p i r a t i o n renders the methylene blue reduction t e s t of l i t t l e value as a b a s i c - f o r c l a s s i f i c a t i o n of these organisms* The oxygen uptake by trashed c e l l s of So, l a c t l s i n the presence of various carbohydrates has been found to be i r r e g u l a r i n manner and l i m i t e d i n degree. No r e l a t i o n s h i p was found to e x i s t between the anaerobic o x i d a t i o n , the aerobic o x i d a t i o n and the fermentation of carbohydrates by t h i s organism. L a c t i c a c i d production by washed b a c t e r i a l c e l l s was found to be dependent upon the presence of small amounts of a n i t r o g e n sourc©tS'Qoa as peptone, along with the carbohydrate, rather than upon the ' presence or absence of a v a i l a b l e oxygen. By the adaptive-c o n s t i t u t i v e enzyme method i t has been shown that the L a c t i c Acid S t r e p t o c o c c i u t i l i z e l a c t o s e as a carbohydrate source by f i r s t s p l i t t i n g the d i s a c c h a r i d e molecule i n t o i t s constituent monosaccharides, glucose and g a l a c t o s e . In i n v e s t i g a t i n g the r e s p i r a t i o n of the Rhisobium species i t has been found p o s s i b l e to separate a s i n g l e s t r a i n of t h i s organism Into a l a r g e number of substrains which d i f f e r from one another and from the parent s t r a i n i n both aerobic and anaerobic r e s p i r a t o r y a c t i v i t y . With t h i s species i t has also been shown that growth on laboratory media r e s u l t s i n a change i n the physiology of the organism, This change I s . s t r i k i n g l y i l l u s t r a t e d by the development of cleared sones about the colonies when the c u l t u r e i s p l a t e d on Wilson's Agar. As w e l l as c i r c u l a r cleared 0ones 8 secondary soaes of haaiiiess oj? deposition were also detected i n the medium at some distance from the c o l o n i e s . The appearance of these aoning pheomena i s associated with a change i n both aeroblo and anaerObla r e s p i r a t o r y a c t i v i t y of the c u l t u r e and a l s o with a change i n c o l o n i a l character. Passage of the c u l t u r e through s o i l e liminates the zoning' e f f e c t s and causes the c u l t u r e to r e v e r t to i t s normal form. I I . INTRODUCTION '» B i o l o g i c a l o x i d a t i o n or r e s p i r a t i o n has been defined (6) as the cum of a l l those processes i n the l i v i n g c e l l by which oxygen i s introduced into the system and carbon dioxide removed* In i t s broadest eeneo„ r e s p i r a t i o n may be said to involve a l l chemical r e a c t i o n s i n l i v i n g c e l l o \\~hich r e lease energy. The study of b a c t e r i a l r e s p i r a t i o n w i l l therefor© involve a consi d e r a t i o n o f both aerobic and anaerobic o x i d a t i v e processes* L i v i n g c e l l s obtain the energy f o r t h e i r v i t a l processes. throvxgh the o x i d a t i o n of compounds such as carbohydrates, with the ovo l u t i o n of carbon d i o x i d e and the formation of -water and various end products, The exact mechanism by which t h i s o xida-t i o n i o c a r r i e d out has never been completely e s t a b l i s h e d . I t i s b elieved to consi s t of an enzymic c a t a l y s i s , the enzymes concerned being termed \" r e s p i r a t o r y enzymes\" • In ..this process the f i r s t step i s believed to be the s p l i t t i n g o f f of a c t i v e hydrogen from the o x i d i s a b l e compound, through the agency of dehydrogenase enzymes. This hydrogon i s then passed over a complex s e r i e s of ox i d a t i o n - r e d u c t i o n systems w i t h l a the c o l l u n t i l i t i s f i n a l l y converted i n t o water 0 During t h i s passage the hydrogen gradually gives up i t s energy to the c e l l i n such a form that i t can be immediately u t i l i s e d * . The problem of b a c t e r i a l r e s p i r a t i o n has been e x t e n s i v e l y i n v e s t i g a t e d by J . H. Quastel and h i s co-workers at Cambridge U n i v e r s i t y ( 7 , 8, 9 ) . Sine© t h e i r pioneer trork, a good deal of research Into t h i s question has been carried, out „ mainly i s England. However, no systematic i n v e s t i g a t i o n i n t o the r e s p i r a t i o n of the L a c t i c Acid B a c t e r i a has yet been recorded. The r e s p i r a t i o n of the Rhlzobium species has been studied to a considerable extent by P. VJ. Wilson and the Wisconsin research workers ( l , 12, 14) • -The study of b a c t e r i a l r e s p i r a t i o n has been g r e a t l y advanced by the employment of the \" r e s t i n g c e l l \" technique. In t h i s method the organisms are employed as a suspension made up of c e l l s which have been washed f r e e of a l l t r a c e s of c u l t u r e medium by repeated c e n t r i f u g i n g . The use of \" r e s t i n g c e l l s \" has made i t -possible to d i s s o c i a t e the phenomena of growth and r e s p i r a t i o n and to carry out d e t a i l e d studies on the r e s p i r a t o r y mechanism f r e e from the complicating f a c t o r s of growth and m u l t i p i i c a t 1 o n • In the work reported upon herein the \" r e s t i n g c e l l \" technique has been employed almost e x c l u s i v e l y . The i n v e s t i g a t i o n of anaerobic o x i d a t i o n s Is most con-v e n i e n t l y c a r r i e d out by the method introduced by Thunberg ( 1 3 ) . In t h i s procedure the compound whose o x i d a t i o n i s to be te s t e d i s mixed w i t h a suspension of the organism being studied and a d i l u t e s o l u t i o n of methylene blue i s added. A i r I s then removed from the tube by means of a vacuum pump and o x i d a t i o n measured ae the time Required f o r d i s c o l o r i z a t i o n of the methf len©.. .bltiei • • Aerobic o x i d a t i o n by the \" r e s t i n g c o l l \" technique may be c a r r i e d out by s e v e r a l raanomotric procedures. The apparatus employed i n t h i s study has been the d i f f e r e n t i a l manometer ' • introduced by B a r e r o f t • For a d e t a i l e d d e s c r i p t i o n of t h i a apparatus the monograph by Dixon (2) should be consulted. Considerable confusion has been created i n the l i t e r a t u r e by the use of many d i f f e r e n t terms to describe the processes of b l o l o g i o a l o x i d a t i o n . In t h i s report the term \"dehydrogenation\" w i l l be used t o describe anaerobic o x i d a t i o n , while the term \"o x i d a t i o n \" w i l l be confined to the purely aerobic mechanism. The work herein reported on the Respiratory Ensymes of the L a c t i c Acid and Ni t r o g e n - F i x i n g B a c t e r i a represents an extension of work p r e v i o u s l y c a r r i e d out on the Dehydrogenase Enzymes of the L a c t i c A c i d B a c t e r i a (j>) and includes a study \"of the aerobic r e s p i r a t o r y a c t i v i t y of these groups of organisms. The dehydrogenase a c t i v i t y of twelve species of L a c t i c A c i d Strepto-c o c c i on s i x t y t e a t compounds, i n c l u d i n g carbohydrates, organic a c i d s , amino acids and a l c o h o l s , had already been determined. On account of the marked v a r i a b i l i t y i n dehydrogenase a c t i v i t y , I t had been found Impossible to use t h i s c h a r a c t e r i s t i c as a basis f o r the c l a s s i f i c a t i o n of t h i s group of microorganisms® In an attempt to e x p l a i n t h i s v a r i a b i l i t y i n o x i d a t i v e a b i l i t y studies have been c a r r i e d out on the a d a p t i v e - c o n s t i t u t i v e enzyme question and on the mechanism of l a c t i c a c i d production, employing the method es t a b l i s h e d by Hegarty (4-3 f o r t h i s type of i n v e s t i g a t i o n . In t h i s procedure two per cent of the carbohydrate to be t e s t e d i s added to a suspension of the organism under study and l a c t i c a c i d production i s then d e t e r -mined by t i t r a t i n g samples of the mixture at half-hourly-i n t e r v a l s over an eight-hour period. The determination of aerobic and anaerobic r e s p i r a t o r y enayrae c o n s t i t u t i o n has a l s o been appl i e d to the Nitrogen-F i x i n g R hizobia i n an attempt to f i n d a p h y s i o l o g i c a l b a s i s f o r the c l a s s i f i c a t i o n of t h i s group of organisms. This study has subsequently b©en extended to' an i n v e s t i g a t i o n of the problems of s t r a i n v a r i a b i l i t y w i t h i n t h i s species and the e f f e c t of l a b o r a t o r y media upon t h e i r physiology and r e s p i r a t o r y a c t i v i t y . This work has been c a r r i e d out at The U n i v e r s i t y of B r i t i s h Columbia i n the Departments of Dairying and Agronomy, I wieh to express my sincere thanks t o Dr. B. A. Eagles and Dr. D. G. L a i r d f o r t h e i r u n f a i l i n g i n t e r e s t and encouragement i n the prosecution of t h i s research. 112, EXPERIMENTAL WORK A. THE LACTIC 'ACID STREPTOCOCCI Respiratory studies on the L a c t i c Aolfi S treptococci have heen c a r r i e d out, employing the \"washed c e l l \" technique* The c u l t u r e medium used i n the preparation of the b a c t e r i a l suspensions has consisted of Casein -Digest Broth, prepared' ' a f t e r the manner of Orla Jensen (3) , containing 0.5?» T o t a l Nitrogen and enriched with 1 . 0 $ Dlfco \"Zeaet E x t r a c t , 0.5f° KgHPO^, and 0.5/° Glucose. The c u l t u r e s employed i n these studies have been: Sc. l a c t i s . S.A, 3 0, a t y p i c a l Sc. l a c t i s i s o l a t e d from butter possessing a caramel f l a v o u r (11) J Sc. l a c t i s A.T.C. 374, obtained from the N a t i o n a l Type Culture C o l l e c t i o n at Washington, D.C.5 Sc. l a c t i s EHBg 1, a starch-fermenting s t r a i n I s o l a t e d from a mature Kingston cheese. For comparative pur-poses studies have also been c a r r i e d out employing Bact• c o l l A.T.C. 4157 of tho American Type Culture C o l l e c t i o n . 1. Aerobic Oxidations The aerobic r e s p i r a t o r y a c t i v i t y of the L a c t i c Acid S t r e p t o c o c c i has been studied, employing the B a r c r o f t manometric technique. The values expressed g r a p h i c a l l y i n Figures 1 , 2 , 3 , 4 and 5 have been select e d as t y p i c a l of the experimental r e s u l t s obtained. These graphs portray the o x i d a t i v e a b i l i t y of Sc. l a c t i s A.T.C. j}74 upon c e r t a i n of the carbohydrates t e s t e d * Very s i m i l a r r e s u l t s have been obtained employing •So* l a e t i e S,A* ..JO-. In Figure 1 and Figure 3 oxygen uptake i n the presence of various carbohydrates i s shown g r a p h i c a l l y , Values are expressed as cubic m i l l i m e t e r s of oxygen u t i l i z e d per m i l l i g r a m of dry c e l l weight over a one-hour period* Control determinations have also been c a r r i e d out, i n Triileh oxygon uptake by the c e l l suspension has been measured i n the e n t i r e absence of c x i d i z a b l e substance* The value so obtained i s known as the endogenous r e s p i r a t i o n . The mechanism of endogenous r e s p i r a t i o n has not yet been e s t a b l i s h e d , but i t i s believed (14) to c o n s i s t of an o x i d a t i v e deamination of c e l l u l a r amino a c i d s , u t i l i z i n g the c e l l polysaccharide as an energy source. The s i g n i f i c a n c e of the appreciable aerobic endogenous r e s p i r a t i o n with the L a c t i c A c i d S t r e p t o c o c c i i s not c l e a r , since i t has already been shov;n (5) that these organisms possess no detectable anaerobic endogenous r e s p i r a t i o n , Prom the curves shown i n Figures 1 and 3 i t i s apparent that the oxygen uptake i s c h a r a c t e r i s t i c of the substance being oxidised,. Among the monosaccharides, glucose and fru c t o s e are s t r o n g l y o x i d i s e d , while galactose and arabinos© show an o x i d a t i o n almost i d e n t i c a l with that of the endogenous. The r e s u l t s tabulated i n f i g u r e 10 show that t h i s very s l i g h t 10 -ox i d a t i o n of galactose i s due to the presence of an adapt I T © r a t h e r than a c o n s t i t u t i v e easyme« .. Among the other carbohydrates, d e x t r i n i s e s p e c i a l l y r a p i d l y o x i d i z e d , while l a c t o s e and r a f f i n o s e are o x i d i s e d to a much l e s s e r extent. With a d o n l t o l and meleaitose, however, the o x i d a t i o n again i s almost n g l i g l b l e . In Figures ?. and 4 i s shown the carbon dioxide produc-t i o n from the carbohydrate sources employed i n Figures 1 and 5* In these graphs also there appears a s i g n i f i c a n t endogenous r e s p i r a t i o n . In general the curves f o r carbon d i o x i d e produc-t i o n correspond c l o s e l y to the curves f o r oxygen uptake. This i m p l i e s that the o x i d a t i o n tends to be c a r r i e d through completely to the carbon dioxide stage. In Figures £{a) and 5(b) the . r e l a t i o n s h i p between acygen uptake and carbon dioxide output i s shown by p l o t t i n g the Respir a t o r y Quotients, which are obtained by d i v i d i n g th© volume of oxygen taken up by the volume of carbon dioxide given o f f . In many cases the Respi r a t o r y Quotient i s a s t r a i g h t l i n e or tends toxvards a s t r a i g h t l i n e , as would normally be expected. In other cases, however, the Respiratory Quotients show a marked i r r e g u l a r i t y which can only be explained by assuming a sudden s h i f t i n c e l l metabolism to correspond with the sudden changes i n slope of these ourvee« • • ~ 2, Fermentation and Oxidation In Table 1 the comparative dohydrosanations, fermenta-t i o n s , and aerobic o x i d a t i o n s of these same carbohydrates by Be, l a c t i s A.T.C. 374 have been summarized, A study of t h i s t a b l e shows that there i s no apparent i n t e r r e l a t i o n s h i p between the processes of aerobic o x i d a t i o n , anaerobic o x i d a t i o n and fermentation. With, glucose „ fructose and lactose a l l throe processes are c a r r i e d out s t r o n g l y . With arabinose and a d o n l t o l a l l three processes are e i t h e r very weak or e n t i r e l y negative. With galactose and melesltose, however, aerobic and anaerobic o x i d a t i o n are weak, while a c i d production i s f a i r l y h igh. With r a f f i n o S e and d e x t r i n aerobic o x i d a t i o n i s strong, while both anaerobic o x i d a t i o n and a c i d production are very weak 0 From the r e s u l t s i n Table 1, i t i s apparent that an organism may be able to o x i d i s e a compound which i t cannot dohydrogenate and cannot ferment. I t may be able to dohydro-genate a compound which i t cannot o x i d i s e and cannot ferment• And i t may a l s o be able to ferment a compound which i t cannot o x i d i z e and cannot dehydrogenate. These observations stand l a d i r e c t c o n t r a d i c t i o n to the g e n e r a l l y accepted t h e o r i e s of b i o l o g i c a l o x i d a t i o n , which hold t h a t aerobic and anaerobic r e s p i r a t i o n are merely phases of the same general mechanism of which fermentation i s a measure of the end-producto. 5» Peptone and Acid Production The r e s u l t s reported In Table 1 showed c l e a r l y that l a c t i c a c i d production was dependent upon some f a c t o r other than aerobic or anaerobic o x i d a t i o n . I t was therefor© detlded to i n v e s t i g a t e the a d a p t i v e - c o n s t i t u t i v e enzyme question with the L a c t i c A c i d S t r e p t o c o c c i i n an e f f o r t to e x p l a i n the l a c k of c o r r e l a t i o n between fermentation and o x i d a t i o n , and al s o to determine what s p e c i f i c Influence was exerted by the nitrogen source, Hegarty (4, 10) introduced a method by which the adaptive or c o n s t i t u t i v e nature of r e s p i r a t o r y enzymes could be determined by t i t r a t i n g the l a c t i c a c i d produced from carbohydrates by a washed suspension of organisms. For a c i d production to take place, he found i t necessary to Include O.J$ peptone i n the buffer mixture. However, he d i d not i n v e s t i g a t e the part played by peptone i n a c i d production. The procedure as d e t a i l e d by Hegarty (4) was therefore c a r r i e d out and t^as extended to cover both aerobic and anaerobic fermentation. A b u f f e r mixture was prepared and varying amounts of peptone added. To the mixture was added 2% glucose and washed b a c t e r i a l c e l l s to form a i f . suspension. For the aerobic experiments samples were withdrawn at h a l f - h o u r l y i n t e r v a l s and t i t r a t e d with 0»1 N sodium hydroxide. The anaerobic experiments were c a r r i e d out i n a vacuum f l a s k under an atmosphere of nitrogen and* the apparatus so arranged t h a t f r e s h methylene blue could be added as reduction took place., Samples f o r t i t r a t i o n were drawn o f f by s u c t i o n . A l l samples were t i t r a t e d to pH 7 o 0 , using a Beckiaan pH meter. The r e s u l t s obtained are summarised l a Figures 7 9 8* 9 and 1 0 , l a a d d i t i o n to pH and aci d production, the oxygen uptake by these various mixtures was also determined. These r e s u l t s are recorded i n Figure H , Figure 7 shows the change i n pH under aerobic conditions during the course of t h i s experiment, while Figure 8 shows the change i n pH under anaerobic c o n d i t i o n s . The a d d i t i o n of peptone to the b u f f e r mixture has•caused a very marked decrease i n pH under both aerobic and anaerobic c o n d i t i o n s . When more than 0,25$ peptone has been added, however, the pH values a l l tend to reach a constant l e v e l at about pH 4,5* Figure 9 shows the i n f l u e n c e of peptone concentration upon l a c t i c a c i d production under aerobic c o n d i t i o n s , while Figure 10 shows the corresponding r e s u l t s obtained under anaerobic c o n d i t i o n s • From the values shown i n Figures 9 and 10 i t i s apparent that l a c t i c a c i d production has increased with i n c r e a s i n g peptone concentration. There i s , however, no tendency to reach a constant l e v e l , as was found with the pH value. This continued increase i n t i t r a t a b i e a e l d l t y i s due to the high 14 » b u f f e r i n g power of the phosphate mixture used, which has held the pH at 4.5 while a l l o w i n g the t i t rat-able a c i d i t y t o • increase r e g u l a r l y . In Figure 11 i s recorded the 'volume of oxygen taken up by these various r e a c t i o n mixtures. Under these conditions'-the amount of oxygen absorbed has been found to depend d i r e c t l y upon the cone eat r a t i o n of peptone. Tinea 0*125$ peptone i s added to the b u f f e r mixture, the oxygen uptake i s doubled! when i f . peptone i s added B the oxygen uptake i s increased s i x t i n e s , • From Figures 7, 8, 9, 10 and 11 the marked i n f l u e n c e of peptone i s apparent. The greatest e f f e c t appears to be exerted i n the oxygen uptake, where the s t i m u l a t i o n i s f a r greater than could be explained on the b a s i s of nitrogen content alone,. The a d d i t i o n of peptone has r e s u l t e d i n a marked decrease i s pH, a noticeable r i e e i n titratabl© a c i d i t y , and a very marked increase i n oxygen uptake, A s i m i l a r stimulation\"was found to' be exerted under both aerobic and anaerobic c o n d i t i o n s . Since the change i n pH i s not p r o p o r t i o n a l to peptone concentration. * but tends to reach a constant l e v e l f o r a l l concentrations, and since both oxygen uptake and l a c t i c a c i d production a*© p r o p o r t i o n a l to peptone concentration, i t seems probable that there i s a very close i n t e r r e l a t i o n s h i p bettreen ferment at l e a and oxygon uptake. This close i n t e r r e l a t i o n s h i p , however, i s not that postulated by the Glass t e a l Past©nj?-SIeywho£ theory i n ffnioh fermentation was stimulated by a decreased oxygen supply. The r e s u l t s recorded her© are r a t h e r the reverse of that theory, since i t has been -shown that increases oxygefc uptake i s associated with increased fermentation, These experiments have i n d i c a t e d that the mechanism of fermentation does not depend upon the presence or absence of oxygen, but depends rather upon the presence of a s u i t a b l e nitrogen source i n the r e a c t i n g mixture. 4. Adapt j ye-Pon'st i t l i t i v e • Snsyiae g. The a p p l i c a t i o n of Hegarty*8 method to the study .of Adaptive-Conetitutive enzymes i s shown i n Figure 1 2 . The washed ©ells were prepared from a glucose broth c u l t u r e . The c o l l and buffer mixture were made up as before, and 0 . 5 $ peptone added. To t h i s mixture was added 2$ of the carbo-hydrate to be t e s t e d . The mixture was incubated, and t i t r a t a b l e a c i d i t y determined as p r e v i o u s l y described. Where the organism possesses a c o n s t i t u t i v e enzyme c o n t r o l l i n g dchydrogenation of the carbohydrate, ac i d production w i l l ocour at once. However, where the organism possesses an adaptive enzyme f o r the carbo-hydrate t e s t e d , a considerable period of time w i l l elapse before a c i d Is produced i n any appreciable q u a n t i t y . This d i s t i n c t i o n I D c l e a r l y shown i n Figure 1 2 , where the organism possesses c o n s t i t u t i v e enzymes f o r glucose, mannose and f r u c t o s e , but-possesses an adaptive enzyme f o r galactose. I t i s believed that a c o n s t i t u t i v e enzyme e x i s t s as an i n t e g r a l part of the c e l l s t r u c t u r e and Is therefore always present i n the c e l l i n an a c t i v e form. An adaptive enzyme, on the other hand, i s b e l i e v e d to e x i s t i n the c e l l i n an i n a c t i v e state and to require s t i m u l a t i o n or adaptation through contact with the homologous substance before enzymlc a c t i v i t y oan be demonstrated. > 11 ~ In Figure. 1£ and Figure 2.4- i t i s shown that the organism possesses aa adapt I T® essyme f o r l a c t o s e , I t i s alee shown that growth i n the presence of l a c t o s e causes adaptation to both l a c t o s e and galactose* while growth l a galactose broth does not cause adaptation'to l a c t o s e , This i n d i c a t e s that the organism i s able to u t i l i z e l a c t o s e only through a pr e l i m i n a r y h y d r o l y s i s to galactose and glucose« ' I t i s also aotieeable that adaptation t© galactose i s not achieved by growing the orgaaisa i& galactose t>'a*oth, but i s achieved by growing the organism i n lactose broth. I t has not yet been p o s s i b l e to determine the cause of t h i s p e c u l i a r adaptation phettomeaoa* B* 'BSfZOBlA. ' • 1. Dehydrogenase Enzymes The dehydrogenase enzymes of the Rhlzobium species have been i n v e s t i g a t e d , employing f i v e s t r a i n s of the Red c l o v e r organism (Rhiao'biua t r i f o l l i ) and s i x t y ' t e s t compounds. The s t r a i n s employed were R.T. 22B, R.T, 224, H,T, 2 2 6 , R.T. 231 and R.T. 39-1. The f i r s t four s t r a i n s were stock cultures obtained from the U n i v e r s i t y of Wisconsin i n 1930. The f i f t h s t r a i n , R.T. 39-1, was i s o l a t e d from red c l o v e r nodules at The U n i v e r s i t y of B r i t i s h Columbia i n 1939. A l l s t r a i n s have been proved by c r o s s - i n o c u l a t i o n experiments. The r e s u l t s obtained i n the dohydrogenation t e s t s have been summarised i n Table 2„ A l l r e s u l t s have been c a l c u l a t e d on the basis Glucose s 100. The f i v e s t r a i n s of Rh. t r i f o l i i show a very marked v a r i a t i o n i n t h e i r dehydrogenase a c t i v i t y . This v a r i a t i o n i s so great that any attempt at arranging the Rhlzobia on the b a s i s of t h e i r anaerobic r e s p i r a t i o n would appear to be of l i t t l e value. Wilson (14) and Tarn and Wilson (12) i n t h e i r studies on the r e s p i r a t o r y enzymee of the Rhlzobia also encountered v a r i a t i o n i n dehydrogenase a c t i v i t y . However, they di d not attach any great s i g n i f i c a n c e to t h i s v a r i a t i o n , but considered the dehydrogenase a c t i v i t y to bo c h a r a c t e r i s t i c of each Rhlzobium species. The r e s u l t s reported upon herein completely disagree with those Of the Wisconsin•'workers, The marked v a r i a t i o n i n -anaerobic r e s p i r a t o r y a c t i v i t y w i t h i n f i v e s t r a i n s of the Rli. t r i f o l l i species makes I t evident that there can be-no s t a b l e species c h a r a c t e r i s t i c s , U n t i l such time as the basic f a c t o r s which govern r e s p i r a t o r y a c t i v i t y can be determined any attempt to e s t a b l i s h species c h a r a c t e r i s t i c s w i l l be f u t i l e . / - 20 -2* Aerobic and Anaerobic Oxidation. A systematic study of the r e s p i r a t o r y enzymes of the Rhizoblua species has been undertaken, employing a s t r a i n of the Red clover organism, Rh. t r l f o l i i 224* The study of the dehydrogenase enzymes of the Rhizobia, reported i n Table 1, had emphasized' the extreme v a r i a b i l i t y of anaerobic r e s p i r a t i o n w i t h i n t h i s species. St was t h e r e f o r e decided to conduct an i n v e s t i g a t i o n i n t o the causes of t h i s v a r i a b i l i t y by studying both the aerobic and anaerobic r e s p i r a t o r y enzymes of s t r a i n s and substrains w i t h i n t h i s group of organisms. The medium employed i n these etudies upon tho Rhizobia i s that recommended by Wilson (14)• St c o n s i s t s of a mineral s a l t s base to which are added 1$ yeast e x t r a c t , 0.1$ glucose, excess calcium carbonate, and 1.5$ agar. In preparing a c e l l suspension f o r r e s p i r a t o r y s t u d i e s , the c u l t u r e i s grown upon the surface of t h i s medium i n a large Roux f l a s k . A f t e r 48 hours' incubation at 30° C. the growth l o washed from the surface with M/JO phosphate bu f f e r of pH 7 . 2 , c e n t r i f u g e s , washed, and resuspended i n b u f f e r . The c e l l concentration i e made up to 2$ by volume and r e s p i r a t i o n experiments ere then c a r r i e d out at 37° C. Oxygen uptake and dehydrogenase a c t i v i t y were determined i n the presence of gluoose, mannltol, and sodium s u c c i n a t e , -Oxygen uptake and methylene blue r e d u c t i o n i n the t o t a l absence of carbon source were also noted. The r e s u l t s of t y p i c a l experiments ^re recorded i n Figures 15 and 16. In Figure IS the -curves f o r oxygen uptake i n the presence of glucose, mannitol, sodium succinate,, and water are reported. With glucose and mannitol the oxygen Uptake i s s i g n i f i c a n t l y high, while with sodium succinate I t i s very low. I t i s also noticeable that there i s an appreciable endogenous oxygen uptake* In Figure 16 the comparative aerobic and anaerobic r e s p i r a t o r y c o e f f i c i e n t s are graphed. In t h i s method of i n t e r -p r e t a t i o n , which i e used by the Wisconsin workers i n t h e i r studies on the Rhiaobia ( l , 12, 14), the reduction time with glucose and the volume of oxygen taken up i n the presence of . glucose arc taken as 100, and corresponding values with the other compounds are reduced to percentage of t h i s f i g u r e . This c a l c u l a t i o n makes i t p o s s i b l e to compare aerobic and anaerobic r e s p i r a t i o n by reducing both to the same b a s i s . - - From the data i n Figure 16 i t i s evident that the aerobic and anaerobic o x i d a t i o n of mannitol and sodium succinate occur x?ith varying degrees of i n t e n s i t y . I t Is a l s o n o t i c e a b l e that the endogenous r e s p i r a t i o n i s quite pronounced under aerobic c o n d i t i o n s , but i s e n t i r e l y absent under anaerobic c o n d i t i o n s . Since these r e s u l t s are c a l c u l a t e d on a comparative b a s i s , i t would appear most probable that d i f f e r e n t enzyme systems are Involved i n aerobic and anaerobie respiration.,, 3. Substrain V a r i a t i o n The problem of v a r i a t i o n i n r e s p i r a t o r y enzymes has been estended to a study of the substrains w i t h i n a given s t r a i n . The s t r a i n Rh. t r l f o l i i 224 was plated upon Wilson's Agar and 14 separate c o l o n i e s picked to form a s e r i e s of substr a i n s . The aerobic and anaerobic r e s p i r a t i o n of these substrains upon glucose, mannitol, sodium succinate, and water has been deter-mined as pr e v i o u s l y described. T y p i c a l r e s u l t s are summarized In Figures 17 and 18. In Figure 17 oxygen uptake i n the presence of mannitol by the mother c u l t u r e , R.T. 224, and eleven s u b s t r a i n s , i s shown g r a p h i c a l l y . There i s revealed a tremendous v a r i a t i o n i n o x i d i s i n g a b i l i t y among those s u b s t r a i n s , a v a r i a t i o n which i n coma eases Is as great as s i x hundred per cent. I t i s apparent that the mother c u l t u r e must have been extremely v a r i a b l e i n i t s r e s p i r a t o r y character and has been broken up i n t o sub-s t r a i n s possessing a very wide range of o x i d a t i v e a b i l i t y . Although previous xvorkers w i t h the Rhizobia have emphasized the extreme v a r i a b i l i t y i n p h y s i o l o g i c a l characters e x h i b i t e d by t h i s species, t h i s i s the f i r s t demonstration of i n s t a b i l i t y i n r e s p i r a t o r y a c t i v i t y w i t h any group of organisms. ' . In Figure 18 the comparative aerobic and anaerobic r e s p i r a t o r y c o e f f i c i e n t s when sodium succinate i s employed as the substrate are reported f o r t h i s same group of su b s t r a i n s . • - 23. -There i s again evident, an extreme v a r i a b i l i t y i n both aerobic and anaerobic -oxidative a b i l i t y . Although both aerobic and anaerobic o x i d a t i o n are v a r i a b l e , they vary Independently of. each other, The data recorded here,,, therefore\\ f u r n i s h added evidence that the aerobic and anaerobic- r e s p i r a t o r y ©nsyraee are d i s t i n c t l y d i f f e r e n t in characters as was i n d i c a t e d by the r e s u l t s d e t a i l e d i n figure-16» - 24 -In an attempt to determine the cause of the I n s t a b i l i t y of the Rh. t r l f o l i l 224 c u l t u r e i n r e s p i r a t o r y a c t i v i t y , the previous h i s t o r y of the c u l t u r e was i n v e s t i g a t e d , The s t r a i n employed i n these t e s t s was a stock lab o r a t o r y s t r a i n which had been cul t u r e d upon la b o r a t o r y media fox* more than a year* A fr e s h i s o l a t i o n of t h i s s t r a i n was now c a r r i e d out from the stock c u l t u r e , maintained i n s t e r i l e s o i l . A mass inoculum cultu r e was obtained, l a b e l l e d R.T. 2 24 E, plated and s i x Gxibstrains i s o l a t e d . The r e s p i r a t i o n of t h i s s e r i e s of c u l t u r e s was then determined. T y p i c a l r e s u l t s are recorded i n Figures 19 and 20. In Figure 19 the comparative aerobic and anaerobic r e s p i r a t o r y c o e f f i c i e n t s of these s t r a i n s i n the presence of sodium succinate are reported. There i s evident a marked decrease i n v a r i a b i l i t y i n both aerobic and anaerobic o x i d a t i v e a b i l i t y . Although t h i s v a r i a t i o n has been markedly decreased, the aerobic and anaerobic r e s p i r a t o r y enzymes appear to r e t a i n t h e i r i n d i v i d u a l character. V a r i a t i o n i n r e s p i r a t o r y a c t i v i t y appears to have been depressed, but the aerobic and anaerobic r e s p i r a t o r y enzymes are s t i l l f u n c t i o n i n g with d i f f e r e n t degrees of I n t e n s i t y , In Figure 20 the oxygen uptake by these f r e s h l y - I s o l a t e d s t r a i n s i n the presence of glucose are reported. I t i s apparent that the o x i d a t i v e a b i l i t y of t h i s s e r i e s of c u l t u r e s i s very - 25 -uniform* This uniformity In o x i d a t i v e a b i l i t y among f r e s h l y -i s o l a t e d substrains stands i n marked contrast to the extreme v a r i a b i l i t y encountered among the substrains :1Kelated from the o l d laboratory s t r a i n . I t i s therefore apparent that o u l t u r l n g on l a b o r a t o r y media f o r an extended period has modified the p h y s i o l o g i c a l character of the organism and has rendered i t extremely unstable i s r e s p i r a t o r y activity® - 26 -The change i n r e s p i r a t o r y a c t i v i t y undergone by the c u l t u r e as a r e s u l t of prolonged c u l t i v a t i o n upon laboratory media i s c l e a r l y shown In Figures 21 and 22, In these graphs the r e s p i r a t o r y a c t i v i t y of the o l d l a b o r a t o r y substrains i s compared with that of the f r e s h l y i s o l a t e d s t r a i n s . In Figure 21 the endogenous oxygen uptake of a l l the substrains i s shown g r a p h i c a l l y . I t i s apparent that i n the f r e s h l y i s o l a t e d s t r a i n s on the l e f t of the graph the endogenous r e s p i r a t i o n i s uniform i n character, while with the o l d l a b o r a -t o r y s u b s t r a i n s on the r i g h t the endogenous r e s p i r a t i o n i s extremely i r r e g u l a r . In Figure 2 2 the glucose o x i d a t i v e a b i l i t y of the f r e s h l y i s o l a t e d s t r a i n s i s uniform i n character and contrasts markedly with the v a r i a b i l i t y and i r r e g u l a r i t y e x h i b i t e d by the o l d l a b o r a t o r y s u b s t r a i n s . A f u r t h e r important change i n r e s p i r a t o r y a c t i v i t y was also noted. The f r e s h l y i s o l a t e d s t r a i n s were found to possess a very marked reducing a c t i v i t y upon methylene blue i n the ab-sence of carbon source. With the o l d l a b o r a t o r y s t r a i n s , on -the other hand, t h i s anaerobic endogenous r e s p i r a t i o n had almost e n t i r e l y disappeared. I t would appear that c u l t u r i n g the organism upon,laboratory media has r e s u l t e d i n a progressive diminution i n anaerobic endogenous r e s p i r a t i o n . - 27 -Therefore, the endogenous r e s p i r a t i o n e x h i b i t e d by a Rhizobium euit.ure w i l l be dependent upon the r e s p i r a t o r y enzyme aake*up of the c e l l as modified by the previous h i s t o r y of that c u l t u r e * - 28 -4. Zoning Phenomena .' Xsveati g a t l o n Into the e f f e c t of c u l t u r i n g the Rhlaobia upon l a b o r a t o r y media has l e d t o the observation of a f u r t h e r change i n p h y s i o l o g i c a l and c u l t u r a l c h a r a c t e r i s t i c s . Xt wee observed that p l a t i n g the o l d laboratory s t r a i n s or substrains upon Wilson's Agar l e d to the development of p e c u l i a r rough and feathery colonies of a d i s s o c i a t e d appearance. These colonies were surrounded by a c i r c u l a r zone i n which the suspended calcium carbonate was completely removed from the medium. Outside t h i s transparent zone there occurred a much smaller zone i n which the medium remained unchanged. Beyond t h i s eeoond zone there occurred a f u r t h e r region of concentration or p r e c i p i t a t i o n i n which the cloudiness and opacity of the medium was n o t i c e a b l y increased. I t was f u r t h e r e s t a b l i s h e d that these zoning phenomena did not appear w i t h c u l t u r e s f r e s h l y i s o l a t e d from the s o i l . Apparently, t h e r e f o r e , c u l t u r i n g on labo r a t o r y media has r e s u l t e d i n a change i n both c o l o n i a l type and c u l t u r a l char-a c t e r i s t i c s . Experiments were c a r r i e d out to determine the nature of these phenomena and the part played by the various c o n s t i t u e n t s present i n the medium. Media were therefore prepared with and without yeast e x t r a c t , glucose, g e l a t i n e and calcium carbonate. Brom thymol blue i n d i c a t o r was added to tr a c e the r e l a t i o n s h i p between a c i d production and disappearance of the calcium carbonate. These various media were then employed t o prepare plates from substrains which showed very a c t i v e sonine p r o p e r t i e s . • The r e s u l t s obtained showed d e f i n i t e l y that the primary zone of complete c l e a r i n g i s dependent upon the presence of glucose l a the medium. • -. E f f o r t s to determine the cause of the outer r i n g of deposition i n the medium have so f a r proved f r u i t l e s s . The appearance of t h i s phenomenon seems to be dependent upon th© presence i n the medium of the three f a c t o r s - glucose, yeast extract and calcium carbonate. When any one of these constituents was omitted the phenomenon f a i l e d to develop. This might i n d i c a t e that d e p o s i t i o n i s brought about through the p r e c i p i t a t i o n of a ealcium-proteinate complex from the yeast extract by a change i n the i s o - e l e c t r i c p o i n t . The r e l a t i o n s h i p of a c i d production to c l e a r i n g phenomenon appeared rather u n c e r t a i n . The calcium carbonate tended to f i x a c i d production w i t h i n the colony and prevent i t s d i s s o l u t i o n throughout the p l a t e . When no carbonate was present the a c i d d i f f u s e d r a p i d l y , but i n the absence of carbonate no zoning phenomena could be demonstrated. » 30 * It was also observed that g e l a t i n e exerted a profound e f f e c t upon 'Both colony type and c l e a r i n g phenomenon. When gel a t i n e replaced the yeast e x t r a c t i n the medium, complete c l e a r i n g zones developed but no deposition took 'place and the colonies a l l showed a t y p i c a l granulated appearance.. When ge l a t i n e was added to the medium containing yeast e x t r a c t , only incomplete zoning was observed and the colonies which developed were of a feathery d i s s o c i a t e d appearance. I t was fur t h e r observed that the zoning phenomenon w i l l develop only when the co l o n i c s on a p l a t e are r e l a t i v e l y few i n number. The presence of a largo number of colonies represses the zone formation, while on a very crowded p l a t e the phenomenon i s completely i n h i b i t e d . I t has not yet been pos s i b l e to postulate an explanation f o r t h i s a n t a g o n i s t i c action,, From the r e s u l t B obtained i n t h i s study of the r e s p i r a t i o n of the S h i z o b i a , i t appears probable that there e x i s t s a d e f i n i t e cycle i n r e s p i r a t o r y a c t i v i t y which the cul t u r e undergoes i n response to c u l t i v a t i o n upon laboratory, media. A c u l t u r e -when f r e s h l y i s o l a t e d from the s o i l possesses a watery transparent growth of c h a r a c t e r i s t i c appearance. A f t e r prolonged c u l t i v a t i o n upon Wilson's Agar, the c u l t u r e loses t h i s moist, g l i s t e n i n g c h a r a c t e r i s t i c and becomes dry and wrinkled i n appearance. The appearance of t h i s dry. wrinkled type of growth coincides w i t h the development of zoning phenomena about the colonies on Wilson's Agar. At the same time a marked change i n both aerobic and anaerobic r e s p i r a t i o n -takes place and the c u l t u r e becomes extremely unstable i n i t s o x i d a t i v e a b i l i t i e s . Passage of the c u l t u r e -. through s o i l n e u t r a l i z e s -these d i s s o c i a t i v e changes and restores the cultu r e to i t s o r i g i n a l c o n d i t i o n * * 31a The attached photograph i l l u s t r a t e s the zoning phenomena which develep when an o l d l a b o r a t o r y s t r a i n of Rlu t r i f o l i i 2 2 4 Is plated upon Wilson's Agar. The com-plete zone of c l e a r i n g which surrounds the colonies a B p e a r s black i n the p i c t u r e , while the- outer zone of deposition appears as a haziness i n the medium* Both, types of colonies are a l s o . c l e a r l y shown, the f i r s t a f a i r l y r e gular hard-centred type and the second a flat.,, spreading, v e i l - l i k e form. - 12 IV. . DISCUSSION OF RF.STJLTS The experimental work reported herein has consisted of an i n v e s t i g a t i o n i n t o the Respiratory Enzymes of the L a c t i c A c i d and Nitr o g e n - F i x i n g B a c t e r i a . In the. course of t h i s i n v e s t i g a t i o n , d e t a i l e d studies i n t o the mechanisms o f l a e t i o a c i d formation and of symbiotic nitrogen f i x a t i o n have been c a r r i e d out^ and i t i s f e l t that considerable progress has been made towards an understanding of the p h y s i o l o g i c a l bases of these fundamental processes. The aerobic and anaerobic r e s p i r a t i o n of the L a c t i c Acid S t r e p t o c o c c i have been e x t r e n s i v e l y studied i n an e f f o r t to evolve a p h y s i o l o g i c a l method f o r the c l a s s i f i c a t i o n of t h i s important group of micro-organisms. Experimental r e s u l t s i n d i c a t e that the lack of c o r r e l a t i o n between aerobic oxida-t i o n , anaerobic o x i d a t i o n and fermentation renders such a c l a s s i f i c a t i o n i m p r a c t i c a b l e . However, t h i s l a c k of c o r r e l a t i o n focuses a t t e n t i o n upon the mechanism of l a c t i c a c i d production, a mechanism which apparently i s a f u n c t i o n of n e i t h e r the aerobic nor the anaerobic r e s p i r a t o r y processes. I t has been f u r t h e r shown that l u e t i c a c i d fermentation i s governed not by the presence or absence of a v a i l a b l e oxygen, but by the presence of an a v a i l a b l e nitrogen source. These experimental r e s u l t s stand i n d i r e c t c o n t r a d i c t i o n to the Pasteur theory that fermentation c o n s i s t s In the formation of by-products of c e l l metabolism through the mechanism of anaerobic o x i d a t i o n . Since the Pasteur theory i s the b a s i s f o r many of the commercial fermentation processes c a r r i e d out by yeasts, moulds and b a c t e r i a , i t i s p o s s i b l e that these r e s u l t s may prove to have important i n d u s t r i a l a p p l i c a t i o n s * Studies i n t o the aerobic and anaerobic r e s p i r a t o r y mechanism of the n i t r o g e n - f i x i n g b a c t e r i a have r e s u l t e d i n the demonstration of a d e f i n i t e cycle i n r e s p i r a t o r y a c t i v i t y . I t has been c o n c l u s i v e l y shown'that c u l t u r i n g the organism upon laboratory media over an extended period causes the development of s t r i k i n g 'changes i n both p h y s i o l o g i c a l character and o x i d a t i v e a b i l i t y . I t i s therefore apparent that the aerobic and anaerobic r e s p i r a t o r y a c t i v i t y of an organism .are l a r g e l y determined by the previous h i a t o r y of the c u l t u r e t e s t e d , This i s a f u n d a m e n t a l p r i n c i p l e i n b a c t e r i a l r e s p i r a -t i o n whieh appears to have been completely overlooked i n previous s t u d i e s , Th© demonstration of v a r i a t i o n i n r e s p i r a t o r y a c t i v i t y i n response to c u l t u r i n g Upon labo r a t o r y media i s of. . p r a c t i c a l importance i n the problem of symbiotic nitrogen f i x a t i o n , a process which Is of .great a g r i c u l t u r a l importance i n Relation-to the maintenance of s o i l f e r t i l i t y . I t i s hoped that t h i s study of the r e s p i r a t o r y enzymes of the fiMzobla may contribute 34' -to a knowledge of the p h y s i o l o g i c a l process by 'which these microorganisms convert the &its?ogen of the atmosphere Into form a v a i l a b l e f o r plant use* ' - 35 -V, CONCLUSION The study of the L a c t i c Acid and Nit r o g e n - F i x i n g B a c t e r i a reported upon herein has opened up seve r a l f e r t i l e f i e l d s f o r f u r t h e r study. In p a r t i c u l a r the a p p l i c a t i o n of r e s p i r a t o r y onzyme studies to the a d a p t i v e - c o n s t i t u t i v e enzyme question and to the mechanism of l a c t i c a c i d production appears worthy of fu r t h e r study. Research i n t o these problems may have important i n d u s t r i a l a p p l i c a t i o n s i n the preparation of s t a r t e r c u l t u r e s and i n the r e g u l a t i o n and extension of i n d u s t r i a l fermentations c a r r i e d out .by microorganisms, 'The.' cause of v a r i a b i l i t y i n r e s p i r a t o r y enzymes and the i n f l u e n c e of the previous h i s t o r y of the c u l t u r e upon o x i d a t i v e a b i l i t y with the Rhizoblum species are fundamental questions which should bo f u r t h e r i n v e s t i g a t e d . The s o l u t i o n of these problems would be of d i r e c t value to the important a g r i c u l t u r a l process of symbiotic ni t r o g e n f i x a t i o n . - 36 -BIBLIOGRAPHY .1. B u r r i s , R»B a ana Wilson, P»W.'- Gold Spring Harbor Symposia on Quant i t at i ve Biology , 7* $49* 193?« 2 , Dixon, SI* - \"Hanometrlo Methods 1 1 « Cambridge P r e s s t 1934. 5. Eagles,, B SA, and Sadler, W. - Oan s 3*. Research 7& 364., 1932. 4. Hegarty, C.P. - Jour. Baet. 37s 145, 1939* 3. Morgan, - Bachelor 8s Thesis „ U n i v e r s i t y of B r i t i s h Columbia. ... . . . . . . 6. Oppenhelsier, 0.. and .Stem, It. G« ~ \" B i o l o g i c a l Oxidation\", Sordemaan, 19.39. • • -7.9 Quastel, 3T.H. and Whetham., M.X5e - Bioehem. 3\". 18: 519 0 1924. 8 . Quastel, J.Bt, and Whetham, M*D, - Blochem. J . 19: 3 2 0 s 1925® 9. Quastel, J.H. and Whet ham, M.D. - Bioehem. .T. 19: 645, 1925* 10. Rahn, 0., Hegarty, G . P . , D u e l l , R.E„ •« Jour. Baet. 3jj; 1 4 7 1 9 3 8 , -l i e Sadler, W, - Trans. Roy* See*. Can. Yolume 20, 1926« 1 2 . ' Tam, .R.K. and Wilson, P..W. - Jour* .Baot, 42: 529, 1941 13- Thunberg «• Slcand® A r c t , P h y s i o l . 40: 1, 1920. 14* Wilson* P.W=9 your. Baot. $$t 6 0 1 , 1938* Abstract The dehydrogenase enzyme a c t i v i t y of f i v e s t r a i n s of Rhizobium t r i f o l i i upon s i x t y t e s t compounds has been studied, employing the Thunberg technique. I t has been shown that the dehydrogenase a c t i v i t y of these s t r a i n s i s extremely v a r i a b l e , e s p e c i a l l y i n the a c t i v a t i o n of carbohydrates. The dehydrogenase a c t i v i t y of Rhizobium t r i f o l i i 224 has been tested at i n t e r v a l s over a period of one year, and considerable f l u c t u a t i o n i n r e s p i r a t o r y a c t i v i t y demonstrated. I t i s suggested that t h i s marked s t r a i n v a r i a t i o n may account f o r disagreement among the reported r e s u l t s o previous workers. I . INTRODUCTION The mechanism of r e s p i r a t o r y enzyme systems i n symbiotic nitrogen f i x a t i o n has been studied by sever a l i n v e s t i g a t o r s . Wilson (5) , working with R. t r i f o l i i , reported a d e t a i l e d study of the f a c t o r s i n f l u e n c i n g the preparation of \" r e s t i n g c e l l \" suspensions of t h i s species i n an e f f o r t to develop a technique s u i t a b l e f o r the study of the aerobic and anaerobic r e s p i r a t o r y a c t i v i t i e s of t h i s organism. Thome and Burr i s (4) compared the re s p i r a t o r y systems of \"nodular\" and \" c u l t u r e d \" r h i z o b i a and found no s i g n i f i c a n t d i f f e r e n c e . B u r r i s and Wilson ( l ) reported a survey of the aerobic r e s p i r a t o r y a c t i v i t y of ten s t r a i n s and f i v e species of the root nodule bacteria,. Tarn and Wilson (3) studied the dehydrogenase systems of R. t r i f o l i i and R. leguminosarum, employing some f o r t y t e s t compounds. The inf l u e n c e of i n h i b i t i n g agents upon the r e s p i r a t o r y enzyme systems of the Rhizobia has also been studied by B u r r i s and Wilson ( l ) and Tarn and Wilson ( 3 ) . In the study of r e s p i r a t o r y enzymes there has been a tendency to regard substrate a c t i v a t i o n as a constant species c h a r a c t e r i s t i c . However, Tarn and Wilson (3) found a s t r a i n variation,which was independent of sp e c i e s , i n the a c t i v a t i o n of c e r t a i n substrates. The present report deals with an i n v e s t i g a t i o n of the s t r a i n and species v a r i a t i o n among the red clover organisms. I I . EXPERIMENTAL METHODS The cult u r e s used i n these experiments consisted of f i v e s t r a i n s of Rhizobium t r i f o l i i . Of these c u l t u r e s , f o u r , namely RT 22B, RT 224, RT 226 and RT 231 , were obtained from the U n i v e r s i t y of Wisconsin i n 1930, while the f i f t h , RT 3 9 - 1 , was i s o l a t e d from red clover nodules at The U n i v e r s i t y of B r i t i s h Columbia i n 1 9 3 9 . The medium employed i n these studies was that recommended by Wilson ( 5 ) , c o n s i s t i n g of the mineral s a l t s of medium 79 (Fred, Baldwin, and McCoy, 1932) and enriched with 1.0$ D i f C o yeast e x t r a c t and 0.1$ glucose* 1 . 5 $ agar was used as the s o l i d i f y i n g agent. The b a c t e r i a l suspensions required f o r the \" r e s t i n g c e l l \" technique were obtained by growing the cu l t u r e s on the surface of the medium i n large Roux f l a s k s . A f t e r 48 hours* incubation at 28° C. the growth was washed from the surface of the agar with M/30 phosphate b u f f e r , centrifuged at 2,000 r.p.m., washed twice and f i n a l l y re-suspended i n phosphate b u f f e r . A l l suspensions were adjusted to a c e l l concentration of 2$ by volume, employing the Hopkins vaccine method (2). Suspensions of t h i s concentration were found to dehydrogenate glucose i n from 5 to 10 minutes, and not to reduce methylene blue endogenously i n l e s s than two hours. Dehydrogenase a c t i v i t y was determined by the Thunberg technique i n tubes containing 1 c.c. of \" r e s t i n g c e l l \" suspension, 2 c.c. of phosphate b u f f e r , 1 c.c. of 1:7,000 methylene blue, and 1 c.c. of M/20 substrate. The tubes were-evacuated f o r two minutes on a water pump. The reactions were c a r r i e d out at 40° C. and at a pH of 8.0, as recommended by Tarn and Wilson (3). Dehydrogenase a c t i v i t y was measured as time required to d e c o l o r i z e completely the methylene blue. The substrates included s i x t y t e s t compounds, e o n s i s t i n of carbohydrates, organic a c i d s , a l c o h o l s , and amines. A l l organic acids were adjusted to pH 7 . 0 with N/ i 0 sodium hydroxide* - 5 -I I I . . EXPERIMENTAL RESULTS A. VARIATION AMONG STRAINS OF R. TRIFOLII The dehydrogenase a c t i v i t i e s of f i v e s t r a i n s of Rhizobium m t r i f o l i i are presented i n Table 1. From the values recorded i n t h i s t a b l e i t i s apparent that there i s an extreme v a r i a b i l i t y i n substrate a c t i v a t i o n among the various s t r a i n s tested. This v a r i a b i l i t y i s most pronounced among the carbohydrate dehydrogenations, but i s also encountered among the a l c o h o l and organic a c i d a c t i v a t i o n s . 1. Carbohydrates Dehydrogenation of the hexoses i s characterized by unif o r m i t y of a c t i v i t y . With a l l f i v e s t r a i n s t e s t e d , glucose, mannose and fructose are r a p i d l y attacked, while galactose i s only slowly o x i d i z e d . With the pentoses, arabinose and xylose, one s t r a i n , RT 224, ox i d i z e s both compounds r e a d i l y ; a second s t r a i n , RT 251, attacks xylose with d i f f i c u l t y andarabinose not at a l l ; the remaining \"three s t r a i n s a c t i v a t e n e i t h e r compound. Among the disaccharides, sucrose, c e l l o b i o s e and trehalose are attacked by a l l f i v e s t r a i n s , while maltose and melibiose are a c t i v a t e d by four s t r a i n s . Lactose i s attacked by only two s t r a i n s and those with d i f f i c u l t y . Among the t r i s a c c h a r i d e s , r a f f i n o s e i s attacked by a l l s t r a i n s and melezitose by three s t r a i n s . Among the polysaccharides, a c t i v a t i o n i s c a r r i e d out by a l l s t r a i n s * The degree of v a r i a t i o n i n dehydrogenase a c t i v i t y upon c e r t a i n carbohydrates i s portrayed g r a p h i c a l l y i n Figure 1. The uniform a c t i v a t i o n c h a r a c t e r i s t i c of the hexoses i s contrasted with the v a r i a b i l i t y of that of the d i - , t r i - , and polysaccharides. Among these l a t t e r substrates, v a r i a t i o n s up to nine hundred per cent are encountered, while v a r i a t i o n s of four and f i v e hundred per cent are common. 2. Alcohols The dehydrogenase a c t i v i t y of R. t r i f o l i i s t r a i n s upon the higher alcohols i s markedly i r r e g u l a r . Of the compounds te s t e d , only mannitol i s a c t i v a t e d by a l l s t r a i n s employed. Gl y c e r o l and s o r b i t o l are .attacked by three s t r a i n s , while d u l c l t o l and i n o s i t o l are attacked by one. Ethylene g l y c o l and e r y t h r i t o l are not ac t i v a t e d by any of the s t r a i n s . S t r a i n v a r i a t i o n i n dehydrogenase a c t i v i t y upon these higher alcohols i s shown i n Figure 1 . There i s evident an extreme v a r i a b i l i t y i n the a c t i v a t i o n of mannitol and g l y c e r o l with the s t r a i n s employed i n these s t u d i e s * 3• Organic Acids Dehydrogenase a c t i v i t y upon organic acids was extremely l i m i t e d with the f i v e s t r a i n s of R. t r i f o l i i employed i n these s t u d i e s ; Sodium succinate was found to be the only organic a c i d a c t i v a t e d by a l l s t r a i n s t e s t e d . Sodium malate was attacked by two s t r a i n s , while sodium formate, sodium butyrate, and sodium l a c t a t e were attacked by one s t r a i n * A l l other organic a c i d s , i n c l u d i n g the majority of the mono-and d i c a r b o x y l i c lower f a t t y a c i d s , were not attacked by any s t r a i n , B. STRAIN VARIATION INFLUENCED BY TIME The dehydrogenase a c t i v i t y of R, t r i f o l i i 224, tested at four i n t e r v a l s over a period of one year, i s recorded i n Table 2, The values reported i n t h i s table show a marked v a r i a b i l i t y i n substrate a c t i v a t i o n w i t h i n t h i s one s t r a i n at d i f f e r e n t times. This v a r i a b i l i t y i s not l i m i t e d to any one group of substrates, but i s found equ a l l y among the carbohydrates, a l c o h o l s , and organic a c i d s . Among the carbohydrates the dehydrogenation of mannose and fructose remains f a i r l y constant. That of galactose, however, shows extreme v a r i a t i o n , the R e s p i r a t o r y C o e f f i c i e n t i n c r e a s i n g from a value of 3 to a value of 5 6 , Among the pentoses the dehydrogenase a c t i v i t y upon xylose decreases from a value of 133 to a value of 14, Among the other carbohydrates also there i s a considerable f l u c t u a t i o n i n dehydrogenase values, a f l u c t u a t i o n which appears to be c h a r a c t e r i s t i c of each i n d i v i d u a l carbohydrate and does not appear to show any d e f i n i t e tendency towards increased or decreased dehydrogenase a c t i v i t y on the part of the c u l t u r e i n general. A s i m i l a r v a r i a t i o n i n dehydrogenase a c t i v i t y i s found when the alcohols are used as substrates. I t i s noticeable that i n the case of mannitol, values of 84 or 17 may be obtained for the Respiratory C o e f f i c i e n t , depending upon the time of t e s t i n g ; and i n the ease of g l y c e r o l i t i s evident that a completely negative or very st r o n g l y p o s i t i v e t e s t may be obtained. With the organic acids t h i s f l u c t u a t i o n i s equa l l y pronounced. With the f i v e s t r a i n s of R. t r i f o l i i reported i n Table 1, sodium succinate was found to be the only organic a c i d attacked by a l l s t r a i n s . However, the s i g n i f i c a n c e of such a f i n d i n g may be questioned when i t can be shown that with one of these s t r a i n s the sodium succinate value may vary from 5 to 8 4 , depending upon when the t e s t i s c a r r i e d out. This f l u c t u a t i o n i n dehydrogenase a c t i v i t y upon the carbohydrates, a l c o h o l s , and organic acids i s shown g r a p h i c a l l y i n Figure 2« DISCUSSION The determination of dehydrogenase a c t i v i t y depends upon the rate of reduction of methylene blue. Measurement of t h i s reduction may be c a r r i e d out by v i s u a l approxima-t i o n s , using known concentrations of methylene blue. Tarn and Wilson (3) i n t h e i r studies on the dehydrogenase systems of R. t r i f o l i i and R. leguminosarum, employed a modified Thunberg method i n which the Evelyn e l e c t r i c photometer and s p e c i a l l y designed Thunberg tubes were used. This method was found to give more accurate and consistent r e s u l t s than the v i s u a l method gives. The values reported i n the present paper have been obtained by measuring the time required f o r complete d e c o l o r i z a t i o n of the methylene blue. A l l values were then converted to percentage of glucose reduction time and expressed as r e s p i r a t o r y c o e f f i c i e n t s . Since the r e s u l t s of Tarn and Wilson (3) have also been reported as r e s p i r a t o r y c o e f f i c i e n t s , c a l c u l a t e d from the slopes of the l i n e s as determined photo-m e t r i c a l l y , the values obtained by these two methods should be comparable. I n v e s t i g a t i o n of the dehydrogenase enzymes of the Rhizobia i s complicated by the occurrence of endogenous - 10 -r e s p i r a t i o n , as shown by the a b i l i t y of \" r e s t i n g c e l l \" suspensions to reduce methylene blue i n the absence of sub-s t r a t e . This endogenous r e s p i r a t i o n was a t t r i b u t e d by Wilson (5) to the elaboration and r e t e n t i o n of gummy mate r i a l which may then serve as a substrate f o r methylene blue reduction. The cu l t u r e s employed i n the present i n v e s t i g a t i o n , a f t e r prolonged c u l t i v a t i o n on yeast extract glucose agar, were found to possess a n e g l i g i b l e endogenous r e s p i r a t i o n , and proved unable to reduce methylene blue without substrate i n under two hours. I t was therefore possible to obtain clear-cut p o s i t i v e and negative dehydrogenase t e s t s from which the endogenous f a c t o r had been eliminated. These r e s u l t s are i n s t r i k i n g disagreement with those reported by Tarn and Wilson (3) , who were unable to reduce the endogenous r e s p i r a t i o n of t h e i r s t r a i n s below a r e s p i r a t o r y c o e f f i c i e n t of 3 8 , Comparison of the carbohydrate and alcohol dehydrogenations reported by Wilson (5) and Tarn and Wilson (3) with those reported herein reveals a d e f i n i t e l a c k of c o r r e l a t i o n . The values obtained by these i n v e s t i g a t o r s are markedly higher and more uniform than those obtained with the f i v e s t r a i n s con-sidered i n the present r e p o r t . I t i s p a r t i c u l a r l y noticeable that x ylose, arabinose, rhamnose, l a c t o s e , ethylene g l y c o l , e r y t h r i t o l , d u l c i t o l , i n o s i t o l and e t h y l a l c o h o l , which were found to be r e a d i l y attacked by the R, t r i f o l i i 202 and 209 s t r a i n s of Tarn and Wilson (3) , were attacked only s l i g h t l y or not at a l l by the f i v e s t r a i n s reported h e r e i n . - 11 -There appears to be a f u r t h e r s i g n i f i c a n t d i f f e r e n c e between these*two groups of R. t r i f o l i i s t r a i n s i n t h e i r dehydrogenation of organic a c i d s . Those s t r a i n s studied by Tarn and Wilson (3) were found to a c t i v a t e nearly a l l organic acids t e s t e d , while the f i v e s t r a i n s reported i n the present work were found to attack only sodium succinate and sodium malate and to be completely i n a c t i v e upon a l l those reported as p o s i t i v e by the other i n v e s t i g a t o r s . This l a c k of agreement among various i n v e s t i g a t o r s of the dehydrogenase enzyme systems of the Rhizobia may p o s s i b l y be explained by the occurrence of marked s t r a i n v a r i a t i o n w i t h i n t h i s species. I t has been shown i n the present paper that f i v e s t r a i n s of R. t r i f o l i i e x h i b i t s u f f i c i e n t l y d i s t i n c t i v e dehydrogenase a c t i v i t i e s to be regarded almost as separate species. I t has f u r t h e r been shown that the dehydrogenase a c t i v i t y of any s i n g l e s t r a i n of R. t r i f o l i i i s not a constant 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 but i s I t s e l f subject to extreme f l u c t u a t i o n . U n t i l such time as the f a c t o r s which a f f e c t t h i s s t r a i n v a r i a t i o n i n dehydrogenase enzyme a c t i v i t y have been f u r t h e r c l a r i f i e d , any attempt at arranging or c l a s s i f y i n g the Rhizobia upon t h e i r r e s p i r a t o r y enzyme character would appear to be of l i t t l e value. - 12 -REFERENCES B u r r i s , R. H. and Wilson, P. W. Cold Spring Harbour Symposia on Quantitative Biology, V o l , V I I , 1939« Hopkins, D. Jour. Amer. Med. Ass'n. 6 0 : 1615 , 1913. Tarn, R. K. and Wilson, P.W. Jour. Bact. 41: 529, 1941. Thome, D.W. and B u r r i s , R. H. Jour. Bact. 36: 261, 1 9 3 8 . Wilson, P.W. Jour. Bact. 35 : 6 0 1 , 1 9 3 8 . INTRODUCTION The f u n c t i o n of r e s p i r a t o r y enzyme systems i n the process of symbiotic nitrogen f i x a t i o n has been studied by s e v e r a l i n v e s t i g a t o r s . Walker, Anderson and Brown (9). employed the Warburg manometer to study oxygen uptake by R. leguminosarum and found that yeast extract g r e a t l y stimulated r e s p i r a t i o n . Neal and Walker (6) studied the oxygen uptake of R. m e l i l o t i and R. japoni cum i n the presence of various carbon sources. In both these i n v e s t i g a t i o n s , growing c u l t u r e s of the root nodule b a c t e r i a were employed. The study of the mechanism of r e s p i r a t o r y enzyme a c t i v i t y among the Rhizobia has been g r e a t l y f a c i l i t a t e d by the i n t r o d u c t i o n of the \" r e s t i n g c e l l \" technique. Wilson (11) reported an extensive i n v e s t i g a t i o n of the f a c t o r s i n f l u e n c i n g the preparation of suspensions of R. t r i f o l i i s u i t a b l e f o r aerobic and anaerobic r e s p i r a t o r y s t u d i e s . Thorne and B u r r i s (8) compared the r e s p i r a t o r y enzyme systems of \" c u l t u r e d \" and \"nodular\" r h i z o b i a , and found no s i g n i f i c a n t d i f f e r e n c e i n r e s p i r a t o r y mechanism. B u r r i s and Wilson (2) reported a survey of f i v e species and ten s t r a i n s of the root nodule b a c t e r i a . Tarn and Wilson (7) studied the dehydrogenase systems of R. t r i f o l i i and R. leguminosarum, employing some f o r t y t e s t compounds. The s e l e c t i v e i n h i b i t i o n of s p e c i f i c r e s p i r a t o r y enzyme systems of the Rhizobia has been studied by B u r r i s and Wilson (2) and by Tarn and Wilson (7:). Aside from the work of Thorne and B u r r i s (8) no attempt has yet been made to study the s t a b i l i t y of r e s p i r a t o r y enzyme systems of the Rhizobia i n response to changes i n environmental and c u l t u r a l c o n d i t i o n s . The work reported upon herein was undertaken with the object of determining the comparative r e s p i r a t o r y enzyme a c t i v i t y of substrains derived from a s o i l stock c u l t u r e and from a c u l t u r e c a r r i e d f o r a considerable period of time on laboratory media. EXPERIMENTAL METHODS The organism used i n these experiments was Rhizobium t r i f o l i i , Wisconsin s t r a i n 224, This culture had been maintained continuously upon l a b o r a t o r y media f o r a period of eleven years* At the end of t h i s time a d u p l i c a t e t r a n s f e r had been inoculated into s t e r i l e s o i l and main-tained as a stock c u l t u r e . Both the o l d laboratory c u l t u r e and the new s o i l stock culture (8 months i n s o i l ) were a v a i l a b l e f o r experimental study. Substrains were obtained by p l a t i n g these two mother cultures and p i c k i n g i s o l a t e d c o l o n i e s . The substrains derived from the l a b o r a t o r y s t r a i n have been l a b e l l e d s e r i e s B and C; those f r e s h l y i s o l a t e d from the s o i l culture have been l a b e l l e d s e r i e s E. Within t h i s l a t t e r s e r i e s , RT 224 E denotes the c u l t u r e r e - i s o l a t e d from the s o i l stock c u l t u r e by a mass sub-in o c u l a t i o n , while the remaining s t r a i n s of the E series represent substrains obtained by p l a t i n g t h i s mother cu l t u r e , RT 224 E. The c u l t u r e medium on which R« t r i f o l i i 224 had been ca r r i e d continuously was standard yeast water mannitol agar (4) . For r e s p i r a t o r y enzyme s t u d i e s , however, the s p e c i a l medium recommended by Wilson (11) was employed. This consisted of the mineral s a l t s of Medium 79 (4) enriched with ifo Difco yeast extract and O.lf. glucose, and contained 1*5% agar as the s o l i d i f y i n g agent© Resting c e l l suspensions f o r r e s p i r a t o r y studies were obtained by growing the organism on the surface of Wilson's medium (11) i n large Roux f l a s k s . A f t e r 48 hours' incuba-t i o n at 28 C. the growth was washed from the surface of the agar with M/30 phosphate buffer of pH 7 . 2 , centrifuged at 2000 r.p.m., washed tw i c e , and f i n a l l y resuspended i n phosphate b u f f e r . A l l suspensions were adjusted to a c e l l concentration of 1% by volume, employing the Hopkins vaccine tube method (5) • Dehydrogenase a c t i v i t y was determined by the Thunberg technique i n tubes containing 1 c.c. of r e s t i n g c e l l suspension, 2 c.c. of phosphate b u f f e r , 1 c.c. of 1:7,000 methylene blue, and 1 c.c. of M/20 substrate. A l l tubes were evacuated f o r 2 minutes on a water pump, and dehydrogenase a c t i v i t y measured as the time required f o r complete d e c o l o r i z a -t i o n of the methylene blue. The t e s t s were c a r r i e d out at 40° C. and at pH 8 o 0 , as recommended by Tarn and Wilson (7 ) • Aerobic o x i d a t i v e a b i l i t y was measured manometrically by the Bar c r o f t apparatus, as described by Dixon ( 3 ) . The cups used i n these experiments contained 1 c 0 c, of r e s t i n g c e l l suspension, 1 c.c. of M/20 substrate, and 1 c.c. of b u f f e r . Carbon dioxide was absorbed by f i l t e r paper soaked i n 20$ KOH held i n i n s e t tubes w i t h i n the cups. A l l t e s t s were ca r r i e d out at 37° C. and pH 7.2. The substrates employed i n these experiments consisted of glucose, mannitol and sodium succinate* Aerobic r e s p i r a t o r y a c t i v i t y has been expressed as cxibic m i l l i m e t e r s of oxygen taken up per m i l l i g r a m of c e l l dry weight per hour. This value i s known as the 0,02. C e l l dry weight was determined by drying 2 c.c. of the suspension at 100° 0. and deducting the weight of the s a l t s present i n the buffer s o l u t i o n . The s u i t a b i l i t y of the Q,02 value f o r use i n r e s p i r a t o r y studies with the Rhizobia has been questioned by Wilson (11), who showed that values of the Q,0g increased with increased concentration of yeast extract i n the growth medium. He assumed these increased Q-Og values to be due to decreased gum formation i n the culture and proposed the use of a new standard, the q0 2 value, defined as the oxygen uptake i n cubic m i l l i m e t e r s per hour per m i l l i g r a m nitrogen i n the c e l l s . This method of c a l c u l a t i o n can be v a l i d only i f the assumption that oxygen uptake v a r i e s d i r e c t l y with the nitrogen content of the c e l l s can be proved. Experiments c a r r i e d out upon fourteen substrains i s o l a t e d from the laboratory culture R« t r i f o l i i 224 showed d e f i n i t e l y that there i s considerable •variation i n nitrogen content among s t r a i n s and sub s t r a i n s . The nitrogen content of t h i s group of substrains ranged from 7,4?$ to 12,93$, Further, oxygen uptake d i d not appear to vary with the nitrogen content of the c e l l s , since those substrains w i t h high nitrogen contents did not e x h i b i t greater r e s p i r a t o r y a c t i v i t y than those substrains whose c e l l nitrogen value was low. I t was also noted that graphing oxygen uptakes on the basis of nitrogen content l e d to a markedly greater degree of v a r i a t i o n than when the dry weight basis was used® For these reasons i t was decided to ca l c u l a t e a l l r e s u l t s as QO2 values, based on dry weights® EXPERIMENTAL RESULTS The aerobic and anaerobic r e s p i r a t o r y a c t i v i t i e s of the substrains derived from the laboratory s t r a i n and from the s o i l stock c u l t u r e of R, t r i f o l i i 224 are presented i n Tables 1 and 2, In Table 1 are shown the comparative QOg values of these two groups of substrains when the glucose, mannitol, sodium succinate and endogenous o x i d a t i v e mechanisms are considered* In Table 2 the aerobic and anaerobic r e s p i r a t o r y c o e f f i c i e n t s of the mannitol, succinate and endogenous systems are d e t a i l e d . A l l f i g u r e s reported i n t h i s l a t t e r t a b l e have been ca l c u l a t e d as percentage of the glucose r e s p i r a t i o n , which i s given the value of 100, Results so obtained are termed \" r e s p i r a t o r y c o e f f i c i e n t s \" . Substrain V a r i a t i o n 1 The values reported i n Tables 1 and 2 show c l e a r l y that there i s a fundamental d i f f e r e n c e between the sub-s t r a i n s i s o l a t e d from the laboratory s t r a i n and the sub-s t r a i n s i s o l a t e d from the s o i l stock c u l t u r e . Substrains of the B and C s e r i e s , which have been derived from the \" c u l t u r e d \" s t r a i n , are characterized by an extreme f l u c t u a -t i o n i n r e s p i r a t o r y a c t i v i t y ; substrains of the E s e r i e s , which have been i s o l a t e d from the s o i l c u l t u r e , are char-a c t e r i z e d by a marked u n i f o r m i t y i n r e s p i r a t o r y a c t i v i t y . This e s s e n t i a l d i f f e r e n c e between the two groups of substrains i s apparent upon a l l substrates t e s t e d and holds true under both aerobic and anaerobic c o n d i t i o n s . In f i g u r e s 1 and 2 the c h a r a c t e r i s t i c d i s t i n c t i o n between these two groups of substrains i s portrayed g r a p h i c a l l y . In Figure 1 the anaerobic r e s p i r a t o r y c o e f f i c i e n t s of substrains of the E s e r i e s are compared to those of the B and C s e r i e s , with sodium succinate as the substrate. Within the E s e r i e s these r e s u l t s show a marked u n i f o r m i t y , with values ranging from 62 to 100. Among substrains of the B and C s e r i e s , on the other hand, there i s an extreme v a r i a t i o n i n r e s p i r a t o r y c o e f f i c i e n t s , and the values are found to range between 14 and 118• This u n i f o r m i t y among substrains of the E s e r i e s and v a r i a b i l i t y among substrains of the B and C s e r i e s i s fu r t h e r emphasized by the r e s u l t s i l l u s t r a t e d i n Figure 2• In t h i s graph the endogenous 0,02 values of the two groups of substrains are shown. Here again, the f r e s h l y - i s o l a t e d substrains show a regular oxygen uptake which varies between 20 and 35 cubic m i l l i m e t e r s per hour. With the \"cu l t u r e d \" s u b s t r a i n s , however, the endogenous oxygen uptake f l u c t u a t e s between values of 13 and 64 cubic m i l l i m e t e r s per hour© From, the values reported i n Tables 1 and 2 and the r e s u l t s graphed i n Figures 1 and 2, i t i s apparent that c u l t u r i n g i n s o i l has exerted a profound influence upon the physiology of the organism under study 0 Substrains i s o l a t e d from the s o i l culture e x h i b i t a u n i f o r m i t y i n r e s p i r a t o r y a c t i v i t y which i s i n marked contrast to the v a r i a b i l i t y of that of substrains derived from a \" c u l t u r e d \" s t r a i n . I t would appear, t h e r e f o r e , that c u l t u r i n g the organism i n s t e r i l e s o i l has s t a b i l i z e d the r e s p i r a t o r y enzyme mechanisms of R. t r i f o l i i 224* Endogenous R e s p i r a t i o n The r e s u l t s reported i n Tables 1 and 2 show d e f i n i t e l y that there i s a fundamental d i f f e r e n c e between the endogenous r e s p i r a t i o n of f r e s h l y - i s o l a t e d s t r a i n s and that of cultured s t r a i n s . This d i f f e r e n c e i s i l l u s t r a t e d i n Figures 2 and 3 » In Figure 2 the endogenous 0 , 0 2 of these two groups of substrains i s portrayed g r a p h i c a l l y . The endogenous r e s p i r a t i o n of the f r e s h l y - i s o l a t e d substrains i s markedly uniform, while that of the cultured substrains i s extremely v a r i a b l e . In s p i t e of t h i s i r r e g u l a r i t y , however, the average endogenous oxygen uptake of the cultured substrains ( 3 1 « 3 cu. num.) i s only very s l i g h t l y greater than that of the average of the f r e s h l y - i s o l a t e d substrains ( 2 5 . 5 cu. num.) I t would appear, t h e r e f o r e , t h a t , although c u l t u r i n g i n s o i l has s t a b i l i z e d the aerobic endogenous r e s p i r a t i o n , there has been e s s e n t i a l l y l i t t l e a l t e r a t i o n i n t h i s r e s p i r a t o r y mechanism. The anaerobic endogenous r e s p i r a t i o n of these two groups of substrains i s shown i n Figure 3 » This graph shows c l e a r l y that a fundamental d i f f e r e n c e e x i s t s between the anaerobic endogenous.respiration of the s o i l substrains and that of the cultured s u b s t r a i n s . The r e - i s o l a t e d substrains are - 10 -characterized by a very high rate of endogenous r e s p i r a t i o n , while that of' the cultured substrains i s almost n e g l i g i b l e . Among the f i f t e e n substrains of t h i s l a t t e r group, only one shows a high r a t e of endogenous r e s p i r a t i o n ; of the remaining fourteen s u b s t r a i n s , eight possess only very s l i g h t endogenous a c t i v i t y and f i v e have no endogenous r e s p i r a t i o n whatever. This would i n d i c a t e that c u l t u r i n g the organism, upon laboratory media has caused the anaerobic endogenous r e s p i r a t i o n to decrease from a very high value almost to the vanishing p o i n t . Since dehydrogenase studies on the Rhizobia are conditioned by a low endogenous r e s p i r a t i o n , the . importance of t h i s f i n d i n g i s apparent. Glucose Oxidation The o x i d i z i n g a b i l i t y upon glucose of these two groups of substrains i s portrayed i n Figure 4«s! Here again, there i s apparent a s i g n i f i c a n t d i f f e r e n c e between the substrains r e -i s o l a t e d from s o i l and those i s o l a t e d from the o l d laboratory c u l t u r e . The s o i l s ubstrains are c h a r a c t e r i z e d by a low and uniform r a t e of glucose o x i d a t i o n , while the cultured sub-s t r a i n s e x h i b i t a g r e a t l y increased and markedly i r r e g u l a r o x i d i z i n g a b i l i t y . Mannitol Oxidation The dehydrogenase a c t i v i t y of these two groups of sub-str a i n s upon mannitol i s shown i n Figure 3• I t i s apparent - 11 -that there i s l i t t l e d i f f e r e n c e between the s o i l s t r a i n s and the cultured s t r a i n s i n regard to t h e i r dehydrogenation of t h i s hexahydric a l c o h o l . The aerobic o x i d a t i o n of mannitol by these substrains i s portrayed i n Figure 6, In marked contrast to the s i m i l a r i t y i n dehydrogenase a c t i v i t y shown i n Figure 5» there i s revealed here a very s i g n i f i c a n t d i f f e r e n c e i n the o x i d i z i n g a b i l i t i e s of these two groups of s u b s t r a i n s . T]ie s t r a i n s r e - i s o l a t e d -from s o i l possess a very low and uniform oxidase a c t i v i t y , while the s t r a i n s i s o l a t e d from the laboratory c u l t u r e show a g r e a t l y increased and markedly i r r e g u l a r o x i d i z i n g ability© It would seem, t h e r e f o r e , that c u l t u r i n g on laboratory media has caused the mannitol o x i d i z i n g mechanism of t h i s organism to become increased i n strength and unstable i n character. Succinate Oxidation The aerobic and anaerobic r e s p i r a t o r y a c t i v i t y upon sodium succinate are graphed i n Figures I and 7» In Figure 1 i s shown the dehydrogenating a c t i v i t y upon succinate of these two s e r i e s of s u b s t r a i n s . The a c t i v a t i n g power of the r e - i s o l a t e d substrains i s very high and regular i n character, while that of the laboratory substrains i s very e r r a t i c i n behaviour. Among t h i s l a t t e r group, values ranging from 14 to 1 1 8 have been recorded. There i s no apparent tendency toward s t a b i l i z a t i o n w i t h i n t h i s group, and - 12 -the extreme f l u c t u a t i o n i n dehydrogenase a c t i v i t y can only be explained .as due to the inherent v a r i a b i l i t y and i n s t a b i l i t y i n r e s p i r a t o r y enzyme character of t h i s b a c t e r i a l species. The aerobic r e s p i r a t o r y c o e f f i c i e n t s of these substrains with succinate as the substrate are graphed i n Figure 7© The high a c t i v i t y ' a n d stable character of the succinate oxidase mechanism among the s o i l substrains are again emphasized. The cultured s t r a i n s , on the other hand, are characterized by an extreme i r r e g u l a r i t y , t h e m a j o r i t y possessing only a very l i m i t e d a c t i v i t y upon succinate. C u l t u r i n g on laboratory media has r e s u l t e d i n a decrease i n the oxidase a c t i v i t y of t h i s c u l t u r e towards succinates Aerobic and Anaerobic Mechanisms The aerobic and anaerobic r e s p i r a t o r y c o e f f i c i e n t s of four s t r a i n s selected at random are i n d i c a t e d i n Figure 8© The values recorded i n t h i s graph show d e f i n i t e l y that a c t i v a t i n g a b i l i t y under aerobic and anaerobic conditions does not maintain a constant r e l a t i o n s h i p . In the o x i d a t i o n of mannitol by s t r a i n RT 224 E the anaerobic a c t i v i t y i s appreciably greater than the aerobic, while i n the o x i d a t i o n of succinate the reverse holds t r u e . In the o x i d a t i o n of mannitol by s t r a i n RT 224, on the other,hand, the aerobic a c t i v i t y i s much greater than the anaerobic, and t h i s order - 13 -i s again reversed when succinate acta as the substrate. With the four s t r a i n s portrayed i n Figure 8 nearly a l l pos-s i b l e i n t e r r e l a t i o n s h i p s between the aerobic and anaerobic r e s p i r a t o r y a c t i v i t i e s can be demonstrated. This lack of d e f i n i t e r e l a t i o n s h i p can be shown i n the case of the mother c u l t u r e , s t r a i n RT 224, the s t a b i l i z e d s o i l c u l t u r e , s t r a i n RT 224 E, and any of the substrains i s o l a t e d from these two parent cultures® A l l these r e s u l t s i n d i c a t e that aerobic and anaerobic r e s p i r a t o r y a c t i v i t y are c a r r i e d out by separate and d i s t i n c t enzyme systems• DISCUSSION In studies on the r e s p i r a t o r y enzyme systems of b a c t e r i a the assumption has been made that the r e s p i r a t o r y a c t i v i t y of a c u l t u r e i s a constant and stable c h a r a c t e r i s t i c of the species under investigation® The work reported upon herein, however, demonstrates that there e x i s t s a s t r i k i n g and fundamental d i f f e r e n c e between the r e s p i r a t o r y enzyme systems of a s t r a i n of R. t r i f o l i i c a r r i e d continuously upon laboratory media and the same s t r a i n maintained as a stock c u l t u r e i n s t e r i l e s o i l s I t has been shown that substrains i s o l a t e d from a \"cultured\" s t r a i n e x h i b i t marked i r r e g u l a r i t i e s and v a r i a b i l i t y - 14 -i n t h e i r r e s p i r a t o r y a c t i v i t i e s . This substrain v a r i a t i o n shows that the r e s p i r a t o r y enzyme mechanisms of the s t r a i n cultured upon labo r a t o r y media have become extremely unstable i n character* A somewhat s i m i l a r i n s t a b i l i t y has been reported by Wilson, Hopkins and Fred (10) i n regard to s t r a i n v a r i a t i o n i n nitrogen f i x a t i o n by the Rhizobia. I t has f u r t h e r been shown by Almon and Baldwin ( l ) that various c u l t u r a l types d i s t i n c t from the t y p i c a l form of R. t r i f o l i i may be i s o l a t e d . I t would appear, t h e r e f o r e , that v a r i a t i o n i n c u l t u r a l character and n i t r o g e n - f i x i n g a b i l i t y of R. t r i f o l i i are accompanied by v a r i a t i o n i n r e s p i r a t o r y enzyme characters as wells In contrast to the marked i n s t a b i l i t y e x h i b i t e d by substrains from a \" c u l t u r e d \" s t r a i n , the r e s p i r a t o r y enzyme a c t i v i t i e s of substrains from a s o i l c u l t u r e have been shown to be extremely uniform and stable i n character. C u l t u r i n g i n s o i l f o r an eight-month period has r e s u l t e d i n fundamental changes i n r e s p i r a t o r y enzyme a c t i v i t y which are characterized by the development of marked s t a b i l i t y i n enzymic c o n s t i t u -t i o n . The mechanism by which s o i l exerts t h i s s t a b i l i z i n g i nfluence has not as yet been determined© This demonstration of a r e s p i r a t o r y enzyme di f f e r e n c e between \" c u l t u r e d \" s t r a i n s and \" s o i l \" s t r a i n s i s at .some variance w i t h the r e s u l t s reported by Thorne and B u r r i s ( 8 ) 0 - 15 -These workers i n v e s t i g a t e d the r e s p i r a t o r y enzyme mechanisms of \"nodular\" and \"cultured\" s t r a i n s of Rhizobia and found them to be e s s e n t i a l l y s i m i l a r * The previous h i s t o r y of t h e i r \" c u l t u r e d \" s t r a i n s was not described, however, and may have been responsible f o r the r e s u l t s obtained by them, Cu l t u r i n g upon la b o r a t o r y media has r e s u l t e d i n profound m o d i f i c a t i o n of the r e s p i r a t o r y enzyme systems of R, t r i f o l i i 224, Compared to that of the s t a b i l i z e d s o i l s t r a i n s , the r e s p i r a t o r y a c t i v i t y of the cultured s t r a i n s i s characterized by a tremendously decreased anaerobic endogenous r e s p i r a t i o n , a g r e a t l y increased oxidase a c t i v i t y upon glucose and mannitol, and a decreased oxidase a c t i v i t y upon succinates With the aerobic endogenous r e s p i r a t i o n , and the dehydrogenase a c t i v i t y upon mannitol and succinate, there has occurred l i t t l e demonstrable change. I t has g e n e r a l l y been assumed that the aerobic and anaerobic r e s p i r a t o r y a c t i v i t i e s of an organism upon any given substrate are c a r r i e d out through a common enzymic mechanism, i r r e s p e c t i v e of whether molecular oxygen or methylene blue functions as the f i n a l Hydrogen Acceptor, Wilson ( 1 1 ) , working with R, t r i f o l i i , found that the r e l a t i v e rate of reduction of methylene blue i n the presence of a given substrate was g e n e r a l l y lower than the rate of - 1 6 -oxidation of the same substrate. The rank of the substrates, however, as Hydrogen Donatorsy showed close agreement under aerobic and anaerobic conditions,. The r e s u l t s shown g r a p h i c a l l y i n Figure 8 i n d i c a t e that there i s no such close agreement among aerobic and anaerobic mechanisms with the s t r a i n s and substrains studied i n the present work, but r a t h e r a noticeable d i s s i m i l a r i t y i n a c t i v a t i n g a b i l i t y * This lack of agreement i s f u r t h e r emphasized when the r e s u l t s obtained from the various enzymic systems i n v e s t i g a t e d are compared* With the endogenous r e s p i r a t i o n i t has been shown that c u l t u r i n g upon laboratory media causes almost complete disappearance of the anaerobic reducing a c t i v i t y , while the aerobic a c t i v i t y has not been a l t e r e d . With the mannitol and succinate r e s p i r a t o r y mechanisms, c u l t u r i n g on lab o r a t o r y media has caused an increased o x i d a t i v b a b i l i t y upon mannitol and a decreased a c t i v i t y upon succinate, while i n neither case has the dehydrogenating mechanism been a l t e r e d . These r e s u l t s i n d i c a t e that aerobic and anaerobic o x i d a t i o n of any given substrate by an organism proceed through d i f f e r e n t enzymic mechanisms, or that, at any r a t e , these mechanisms are independently v a r i a b l e , < The r e s u l t s reported herein f u r n i s h d e f i n i t e evidence that the r e s p i r a t o r y enzyme character of the Rhizobia i s - 1 7 -extremely unstable and i s markedly influenced by environmental fa c t o r s „ I t may be assumed, the r e f o r e , that the r e s p i r a t o r y a c t i v i t y e x h i bited by a b a c t e r i a l c u l t u r e at any given time i s the r e s u l t a n t of the influences of the environmental and c u l t u r a l conditions to which the organism has p r e v i o u s l y been exposed® - 1 8 -' SUMMARY The aerobic and anaerobic r e s p i r a t o r y a c t i v i t y of s t r a i n s i s o l a t e d from a , s o i l c u l t u r e and from a laboratory culture of R. t r i f o l i i 224 have been compared. These s t r a i n s and substrains have been studied upon t h e i r endogenous, glucose, mannitol, and sodium succinate r e s p i r a t o r y mechanisms. I t has been shown that substrains i s o l a t e d from the laboratory c u l t u r e e x h i b i t an extreme v a r i a b i l i t y i n r e s p i r a t o r y a c t i v i t y upon a l l substrates t e s t e d . Substrains i s o l a t e d from the s o i l c u l t u r e , on the other hand, show a marked u n i f o r m i t y i n r e s p i r a t o r y a c t i v i t y . C u l t u r i n g i n s o i l has therefore been shown to s t a b i l i z e the r e s p i r a t o r y enzyme systems of R. t r i f o l i i 224© Cul t u r i n g on l a b o r a t o r y media has been proved to modify the r e s p i r a t o r y enzyme mechanisms of t h i s s t r a i n . The anaerobic endogenous r e s p i r a t i o n has been decreased from a very high value almost to the vanishing p o i n t , oxidase a c t i v i t y toward glucose and mannitol has been increased, that toward succinate has been decreased. Aerobic endogenous r e s p i r a t i o n and dehydrogenase a c t i v i t y upon mannitol and succinate have remained unchanged© - 19 -I t lias been shown that aerobic and anaerobic r e s p i r a t i o n upon any substrate are e i t h e r c a r r i e d out by separate enzyme systems or are independently variable© \\ - 2 0 -REFERENCES ' Almon, L. and Baldwin, I.L. Jour. Bact. 26: 229, 1933. B u r r i s , R.H0 and Wilson, P.W. Cold Spring Harbor Symposia on Quantitative Biology, \"Vols 7 : 3 4 9 , 1 9 3 9 * Dixon, M. \"Manometric Methods\", Cambrige U n i v e r s i t y Press, 1934. Fred, E.B. and Waksman, S„ \"Laboratory Manual of General Microbiology\", 19 28. Hopkins, Do Jour. Amer. Med. Ass'n 6 0 : 1615, 1913. Neal, O.Ro and Walker, R.H. Jour. Bact. 3 2 : 1 8 3 , 1 9 3 6 . Tarn, R.K. and Wilson, P.W. Jour. Bact. 42: 3 2 9 , 1941. Thorne, D.W. and B u r r i s , R.H. Jour. Bact. 3 9 : 1 8 7 , 1940. Walker, R.H., Anderson, D.H., Brown, P.E, S o i l Science 3 8 : 2 0 7 , 19 3 4 . Wilson, P.W., Hopkins, E.W., Fred, E.B. S o i l Science 3 2 : 231, 1931. Wilson, P.W. Jour. Bact. 3 3 : 6 0 1 , 1938 e INTRODUCTION The growth, physiology and n i t r o g e n - f i x i n g a b i l i t y of the root nodule b a c t e r i a have been extensively studied since t h e i r i s o l a t i o n i n 1886 by H e l l i e g e l and W i l l f a r t h . Colonies of these species possess a c h a r a c t e r i s t i c transparent, watery type of growth, which i s a t t r i b u t e d to the elaboration of gummy m a t e r i a l of a polysaccharide nature. While these organisms have been shown to u t i l i z e carbohydrate from t h e i r growth medium fo r the synthesis of c e l l u l a r polysaccharide the changes occuring w i t h i n the medium i t s e l f have received scant attention© During studies upon the r e s p i r a t o r y enzymes of Rhizobium t r i f o l i i , as reported p r e v i o u s l y (4), i t was observed that zones of c l e a r i n g and p r e c i p i t a t i o n appeared i n the medium around the c o l o n i e s . These zones appeared to be somewhat analogous to the Liesegang phenomenon. No previous reference to such growth characters has been reported with the R h i z o b i a , although Niven, Smiley, and Sherman ( 7 ) reported the formation of cleared zones around colonies of Strep. s a l i v a r i u s i n a carbonate medium. The work reported upon herein c o n s i s t s of a d e s c r i p t i o n of t h i s zoning phenomenon and an i n v e s t i g a t i o n i n t o the various f a c t o r s which determine i t s occurrence. EXPERIMENTAL METHODS The species employed most ext e n s i v e l y i n the t e s t s reported herein was Rhizobium t r i f o l i i , Wisconsin s t r a i n 224, In studies on the d i s t r i b u t i o n of the zoning phenomenon among st r a i n s and species of the Rhizobia a fu r t h e r group of Wisconsin s t r a i n s was employed, R T 2 2 B , RT205, R T 2 2 6 , RT227, R T 2 3 0 , RT231. A l l other species have been i s o l a t e d and i d e n t i f i e d at the U n i v e r s i t y of B r i t i s h Columbia and have been checked by c r o s s - i n o c u l a t i o n experiments. The medium employed was that recommended by Wilson (8) and c o n s i s t s of the mineral s a l t s of M.79(3) , with the ad d i t i o n of l.Of. Difco yeast e x t r a c t , 0.1/t glucose, O.J>% calcium car-bonate , and 1.5f° agar. This medium was prepared and s t e r i l i z e d i n f l a s k s containing approximately 2 0 0 cc. q u a n t i t i e s , and plates poured i n appropriate d i l u t i o n s . Before pouring, the agar was cooled nearly to the s o l i d i f y i n g point and was swir l e d v i g o r o u s l y i n order that the i n s o l u b l e calcium carbonate might be suspended uniformly throughout the medium. It was noted that p l a t e s prepared i n t h i s manner j e l l e d q u i c k l y and retained the calcium carbonate uniformly suspended 0 i n the medium. A l l plates were incubated at 30 C. The zones of complete c l e a r i n g appeared w i t h i n three days and reached a maximum at about seven days; the zones of de p o s i t i o n appeared at about ten days and reached a maximum at ten to f i f t e e n days. EXPERIMENTAL RESULTS A e D e s c r i p t i o n of Zoning Phenomena The zones or areas of complete c l e a r i n g and of deposition which appeared when Rhizobium t r i f o l i i . 224 was plated upon Wilson's Agar by the technique already described are ' S h o w n i n plates 1 to 6. 'In P l a t e 1 the r e l a t i o n s h i p of c o l o n i a l type to c l e a r i n g of the medium i s emphasized. This photograph was taken with the source of l i g h t thrown against the surface of t h e medium* The small colonies with i r r e g u l a r shapes, seen toward the top, centre and l e f t of the p l a t e , are surrounded by c i r c u l a r dark zones of complete, c l e a r i n g . These are subsurface colonies. The f l a t spreading colonies with the dark centres and white edges, seen toward the lower centre of the plate are surface colonies and apparently do not e x h i b i t c l e a r i n g of the medium. When the surface growth i s scraped away, however, the medium d i r e c t l y beneath .the colony i s seen to have been cleared . This plate was photographed a t s i x days p r i o r to the appearance of the secondary zones of deposi t i o n . A t h i r d c o l o n i a l type i l l u s t r a t e d on P l a t e s 2 and 3 appears as a very t h i n , spreading, feathery or v e i l - l i k e subsurface colony, which developed i n large numbers upon nearly every plate * This appeared to be a d i s s o c i a t e d form which i s , however, s i m i l a r to the more normal types i n zone development. The photograph as presented i n P l a t e 2 and a l l subsequent ones has been taken w i t h the source of l i g h t behind the plate and sh i n i n g through the medium. This emphasizes the develop-- 4 -ment of r i n g formation about the c o l o n i e s , rather than the colonies themselves. Pl a t e s 2 and 3 show c l e a r l y the inner zones of complete c l e a r i n g and the outer zones of deposition or darkening i n the medium around the c o l o n i e s . This e f f e c t i s p a r t i c u l a r l y n oticeable i n the areas between colonies growing f a i r l y close together. I t i s evident that surrounding the colonies there i s a c i r c u l a r region i n which the opaque medium has been completely cleared, the area i n most cases appearing dark on the photographs. Beyond t h i s dark zone there occurs a r i n g i n which the opaque medium remains untouched. Outside t h i s normal area, again, there occurs a t e r t i a r y zone or region of deposition i n which the opacity of the medium has been very n o t i c e a b l y i n t e n s i f i e d . With some c o l o n i e s , towards the lower part of the plates i n eaeh case, the primary zones of complete c l e a r i n g appear l i g h t and the colonies appear dark. -This i r r e g u l a r i t y i s due to the inherent d i f f i c u l t y of photographing these shadowy e f f e c t s . P l a t e 4 shows very c l e a r l y the r i n g formation which develops around i s o l a t e d c o l o n i e s . In the examples shown here the width of the various zones i s w e l l i l l u s t r a t e d . In P l a t e s 3 and 6 unusual arrangements of these zones are shown. In P l a t e 5 a very marked darkening of the medium i s apparent i n the region surrounding a group of co l o n i e s . The colonies themselves appear white, as do the c l o s e l y associated zones of complete c l e a r i n g . The deposition - 5 -which, occurs i s quite pronounced and takes place over a wide area at some distance from the colo n i e s . In P l a t e 6 a f u r t h e r example of t h i s heavy deposition i s shown. Growth has occurred i n a complete c i r c l e which i s surrounded by a very wide area of d e p o s i t i o n . A f u r t h e r area of p r e c i p i t a t i o n then occurs i n the medium enclosed by t h i s r i n g of growth. The examples shown i n Plates 1 to 6 i l l u s t r a t e i n every case the zoning phenomena which develop when the labo r a t o r y s t r a i n of Rhizobium t r i f o l i i 224 i s plated upon \"Wilson' s Agar. When, however, s t r a i n s of t h i s culture which have been f r e s h l y i s o l a t e d from s o i l are employed, there i s no e f f e c t whatever upon the medium. The organism develops as small, transparent colonies which resemble drops of water, and the medium remains untouched. The transparency of these colonies and the opacity of the medium made i t impossible to secure a photograph which would i l l u s t r a t e t h i s normal type of growth. However, the normal appearance of t h i s medium, even i n the presence of non-zoning s t r a i n s , i s w e l l i l l u s -t r a t e d i n the unchanged portions of Pl a t e s 1 and 4. I t i s apparent from the photographs presented that the type of growth developing on these pla t e s i s not t y p i c a l of Rhizobium t r i f o l i i . The s t r a i n employed, Rhizobium t r i f o l i i 224, had been cultured f o r over eleven years upon Laboratory media and had i n a d d i t i o n been c a r r i e d f o r s i x months on Wilson's (8), medium which as already noted was employed i n these s t u d i e s . This s t r a i n on the usual yeast water mannitol agar (M.79), p o s s i b l y as a r e s u l t of prolonged lab o r a t o r y • - 6 -c u l t i v a t i o n produced a dry, coarse and wrinkled type of growth which would i n d i c a t e a profound change i n the carbo-hydrate a s s i m i l a t o r y process. The i n f l u e n c e , which c u l t u r i n g upon laboratory media exerts upon r e s p i r a t o r y enzyme character, has already been reported (5). B* FACTORS INFLUENCING ZONE DEVELOPMENT.. . Since p r e l i m i n a r y studies i n d i c a t e that development of the zoning phenomenon i s associated with the presence of calcium carbonate, glucose and yeast extract i n the medium, an extensive i n v e s t i g a t i o n of the various f a c t o r s c o n t r i b u t i n g to the production of these phenomena was undertaken. The i n f l u e n c e of various carbohydrates, nitrogen sources, carbonates, and trace elements, as w e l l as the d i s t r i b u t i o n of t h i s phenomenon among species and s t r a i n s of the Rhizobia are reported herein. 1. R e l a t i v e Number of Colonies I t has been observed that with r e l a t i v e l y few colonies on a plate the zoning phenomena developed completely and r i n g formation was c l e a r , d i s t i n c t and extensive. However, as the number of colonies per plate increased the s i z e of the zones correspondingly diminished, u n t i l i n very crowded plates the e f f e c t had e n t i r e l y disappeared and the i n d i v i d u a l colonies were d i s t i n c t l y s mall. Since these r e s u l t s may suggest the e l a b o r a t i o n of i n h i b i t o r y substances under crowded conditions i t was decided to t e s t the p r o p e r t i e s of f i l t r a t e s of t h i s organism. A f l a s k of the usual medium was prepared without agar, inoculated with Rh. t r i f o l i i 224, and incubated f o r ten days at 30° C. The c u l t u r e was then passed through a S e i t z f i l t e r and a c l e a r s t e r i l e f i l t r a t e obtained. Due to the p o s s i b i l i t y that an acid i n h i b i t o r y substance might be n e u t r a l i z e d by the excess calcium carbonate, duplicate f i l t r a t e s were prepared from c u l t u r e s i n the basic l i q u i d medium with and without added carbonate. These f i l t r a t e s alone and i n a s s o c i a t i o n with growing cultures were tested to determine t h e i r i n f l u e n c e , i f any, upon the c l e a r i n g phenomenon. No effeGt was observed with these f i l t r a t e s * No explanation f o r the i n h i b i t i o n of zoning on densely seeded plates i s offered at t h i s time. I f c l e a r i n g phenomena are dependent upon the formation of a c i d from the glucose i n the medium i t seems reasonable to expect that more a c i d and consequently more c l e a r i n g would be produced i n a heavily-seeded than i n a l i g h t l y - s e e d e d p l a t e . Experiments i n which brom thymol blue- i n d i c a t o r was added to the medium to determine the r e l a t i o n s h i p of zone formation to acid production gave i n c o n c l u s i v e r e s u l t s . 2* Calcium Carbonate and Other S a l t s . Since p r e l i m i n a r y experiments had demonstrated that the zoning phenomenon was associated with three constituents of the medium - calcium carbonate, glucose and yeast e x t r a c t , i t appeared advisable to t e s t the e f f e c t of various calcium carbonates and other r e l a t e d s a l t s . Media were prepared c o n s i s t i n g o f mineral s a l t s , agar, glucose and yeast extract i n the usual proportions and to t h i s basic medium were added . - 8 -0 . 3 $ concentrations of the f o l l o w i n g s a l t s : Calcium carbonate ( s i x samples), calcium s u l f a t e , t r i c a l c i c phosphate, and magnesium carbonate. With these media poured plates were prepared i n s u i t a b l e d i l u t i o n s . The data reported i n Table 1 i n d i c a t e s that the primary zone of complete c l e a r i n g develops i n the presence of a l l the various calcium carbonates test e d . The secondary zone of d e p o s i t i o n , however, occurs only when one p a r t i c u l a r carbonate i s used. TABLE 1. Influence of Various Carbonates and Related S a l t s upon the Zoning Phenomenon. S a l t Added CaCoj - c o n t r o l CaCoj - S p e c i a l (Low i n A l k a l i s ) CaCoj - (4 Samples) CaS0 4 -C A j ( P 0 4 ) 2 MgCOj Growth C h a r a c t e r i s t i c s Both c l e a r i n g and deposition Growth, c l e a r i n g , no deposition C l e a r i n g , no deposition No growth Normal growth, no c l e a r i n g Growth, very s l i g h t c l e a r i n g This would i n d i c a t e that the zone of primary c l e a r i n g i s a general phenomenon eaused by acid production, while the secondary zone of deposition i s a s p e c i f i c , phenomenon dependent upon the presence of some impurity or group of i m p u r i t i e s oceuring i n the p a r t i c u l a r sample of calcium carbonate o r i g i n a l l y employed. The r e s u l t presented i n Table 1 suggest that the primary c l e a r i n g i s p o s s i b l y more l i n k e d with the carbonate r a d i c a l than with the calcium i o n , since some s l i g h t c l e a r i n g developed i n the presence of magnesium carbonate but not i n the presence of calcium phosphate, 3» E f f e c t of Trace Elements upon Zoning, In view of the r e s u l t s recorded i n Table 1 i t was decided to determine the e f f e c t of various trace elements upon the development of the primary and secondary zones. Accordingly the usual basic agar was prepared, containing 0. 3f« of a calcium carbonate which d i d not stimulate the formation of secondary zones. To 100 cc. q u a n t i t i e s of t h i s medium s a l t s of various elements were added to a concentration of 30 mgm. per 100 cc. These media were then plated i n s u i t a b l e d i l u t i o n s with Rh. t r i f o l i i 224. The experimental r e s u l t s recorded i n Table 2 show that small amounts of c e r t a i n trace elements exert a marked influence upon the c l e a r i n g phenomenon. The a d d i t i o n of calcium c h l o r i d e f o r instance, r e s u l t e d i n a marked increase i n the s i z e of cleared areas, while z i n c acetate e n t i r e l y - 10 - . prevented t h e i r appearance. Between these two extremes aluminum n i t r a t e , boric a c i d and l i t h i u m c h l o r i d e are observed to exercise no in f l u e n c e , while ammonium sulphate, barium chloride and manganese sulphate markedly depressed the degree of c l e a r i n g TABL1D 2 E f f e c t of Trace Elements Upon Clearing Phenomenon - Rh. t r i f o l i i 224. Sal t Added. Grov/th C h a r a c t e r i s t i e s AC(Noj)^ Primary c l e a r i n g , no deposition ( N H 4) 2 SO^ Primary c l e a r i n g markedly depressed BaCLg Primary c l e a r i n g s l i g h t l y depressed HjBOj Very l i t t l e e f f e c t upon c l e a r i n g CaClg Clearing zones very g r e a t l y extended No dep o s i t i o n . Medium granular CuSO^ Normal c l e a r i n g . Colonies have very noticeable dark, copper-colored centres. FeSO^ R e s t r i c t e d c l e a r i n g . Only surface colonies. PbAc2 Normal c l e a r i n g . The colonies are d i s t i n c t l y dark, denoting s u l f i d e formation. L i C l Normal c l e a r i n g MnS04 Very r e s t r i c t e d c l e a r i n g ZnAC2 No c l e a r i n g at a l l - 11 -Trace elements were also found to exert an influence upon f a c t o r s other than the c l e a r i n g phenomenon* When copper - sulphate was added to the medium normal c l e a r i n g developed but the colonies possessed dark-colored centres. This would i n d i c a t e that the organisms possessed the a b i l i t y to f i x copper or copper-protein complexes w i t h i n t h e i r c o l o n i e s . When ferrous sulphate was employed i n the growth medium a reducing p o t e n t i a l was set up and the colonies were able to develop only on the surface of the medium. This observation i s i n accord with the published r e s u l t s of A l l y n and Baldwin (1,2) r e l a t i v e to the influence of o x i d a t i o n - reduction p o t e n t i a l s on the growth of Rhizobia. When lead acetate was incorporated i n the medium normal c l e a r i n g occurred but the colonies were very noticeably darkened, denoting s u l f i d e formation. I n t e r e s t i n g l y , however, t h i s darkening of the colonies took place only on l i g h t l y -seeded p l a t e s ; when h e a v i l y seeded plates were examined the colonies were of normal appearance. There would, t h e r e f o r e , appear to be a close r e l a t i o n s h i p between s u l f i d e formation and the a c i d c l e a r i n g phenomenon. 4. Influence of Carbohydrates upon Zoning. Since preliminary experiments i n d i c a t e d that no zone formation of any sort developed i n the absence of glucose i t appeared d e s i r a b l e to t e s t the e f f e c t of various other carbohydrates sources i n the growth medium. Mineral s a l t s agar was therefore prepared as before, and to t h i s - 12 -basic medium various carbohydrates were added i n 0.1$ concentration. Poured p l a t e s were then prepared i n s u i t a b l e d i l u t i o n s . - , Reference to the experimental data as presented i n Table 3 d i s c l o s e s that d i f f e r e n t carbohydrate sources exert a marked influence upon the c l e a r i n g phenomenon. With the majority of these carbohydrates, c l e a r i n g develops to an extent comparable to that when glucose i s employed as the energy source. This i s e s p e c i a l l y noticeable with the monosaccharides, although with galactose the c l e a r i n g i s l e s s extensive. Of the pentoses, arabinose i s observed to produce complete c l e a r i n g , while that induced by xylose i s very uncomplete and r e s t r i c t e d . This r e s u l t i s the exact reverse of the dehydrogenation r e a c t i o n s , as reported by Morgan, L a i r d and Eagles ( 4 ) , who found that xylose was dehydrogenated r a p i d l y while arabinose was not attacked at a l l . The disaccharides\"except lactose and melibiose appear to be quite e f f e c t i v e ; l a c t o s e induces incomplete and r e s t r i c t e d c l e a r i n g while no zone formation occurs with melibiose. TABLE - 3 E f f e c t of Various Carbohydrate Sources upon Clearing Phenomenon Rh» t r i f o l i i 224. Carbon Source Growth C h a r a c t e r i s t i c s . Glucose - 1. Very complete zone formation. Mannose 2 * Very good zoning. Galactose 3. Good to f a i r c l e a r i n g . Fructose 4. Very good c l e a r i n g Arabinose 3. Very good c l e a r i n g . Xylose .6. • Clearing f a i r . Rhamnose 7 • Very go od c l e a r i n g . Methyl glucoside • 8. No c l e a r i n g . Sucrose 9. Very good c l e a r i n g . Cellobiose 10, Good to f a i r c l e a r i n g . Lactose 11. Clearing f a i r to poor. Maltose Very good c l e a r i n g . Trehalose 13. Clearing f a i r l y good. Melibiose 14. No c l e a r i n g . Raffinose 15.- No c l e a r i n g . M e l i z i t o s e 16.. Clearing d oubtful, s l i g h t trace„ Dextrin 17. Clearing only f a i r . Starch 18. Clearing good. S a l i c i n 19. Very good c l e a r i n g . G l y c e r o l 20. No c l e a r i n g . E r y t h r i t o l 21. No c l e a r i n g . A d o n i t o l 22* No c l e a r i n g . D u l c i t o l 23. No c l e a r i n g . Mannitol 24. Very good c l e a r i n g . S o r b i t o l 23. F a i r l y good c l e a r i n g . Sod. succinate 26. No c l e a r i n g . Sod* malate 27. No c l e a r i n g . . - 14 -A comparison of the r e s u l t s presented i n Table 3 with those on dehydrogenase a c t i v i t y , using the same s t r a i n of Rhizobia and the same carbohydrates ( 4 ) reveals the i n t e r e s t i n g fact that there i s l i t t l e c o r r e l a t i o n between these two processes. I t i s p a r t i c u l a r l y noticeable that there i s no zone formation when organic acids such as s u c c i n i c and malic are incorporated i n the medium, although these acids are quite r e a d i l y dehydrogenated. j>. Influence of Nitrogen Source upon Zoning. Since p r e l i m i n a r y experiments i n d i c a t e d that zone formation and deposition are associated i n part with the yeast extract contained i n the medium i t was decided to t e s t the influence of various other nitrogen sources i n t h i s regards Accordingly mineral sa l t s , a g a r was prepared as usual and 1.0$ concentrations of various nitrogen sources added. The calcium carbonate employed was the one which normally develops areas of complete c l e a r i n g but not areas of depos i t i o n . The data recorded i n Table 4 shows that the nitrogen source present i n the medium exerts a profound influence upon the development of both cleared zones and regions of d e p o s i t i o n . The most important r e s u l t to be observed i n t h i s connection i s the development of areas of deposition i n add i t i o n to c l e a r areas when e i t h e r beef extract or alamine were employed as nitrogen sources. Since the yeast extract c o n t r o l showed only areas of primary c l e a r i n g the mechanism of the formation of these secondary zones i s s t i l l obscure• - 15 -TABLE - 4. Influence of Various Nitrogen Sources upon Clearing and Zoning Phenomena Rh. t r i f o l i i 224, Nitrogen Source Edestin Bacto beef Gelatine Sod. Caseinate Proteose peptone Peptone Difco Peptone. - Witte Tryptone Yeast extract - Difco Yeast extract - Difco (pantothenic a c i d fr -ee) Yeast extract - o r l a J ens-en. Yeast water Beef extract Tyrosine Asparagine Alamine Glycine Urea Growth Ch a r a c t e r i s t i c s . . Growth f a i r , c l e a r i n g hazy and incomplete. Growth good, only s l i g h t c l e a r i n g Good growth, extensive c l e a r i n g Good growth with c l e a r i n g Clearing good, growth good* Good growth and c l e a r i n g Primary c l e a r i n g , good growth Growth good, primary c l e a r i n g Extremely good growth and c l e a r i n g Good growth, almost e n t i r e l y on the surface, incomplete c l e a r i n g . Very good growth, c l e a r i n g not extensive Very good growth, c l e a r i n g extensiv Clearing with areas of deposition No growth, no c l e a r i n g F a i r growth, good c l e a r i n g Good growth, with c l e a r i n g and p o s s i b l y deposition Very good growth, good c l e a r i n g Growth f a i r , no c l e a r i n g at a l l With the various nitrogen sources employed growth occurred i n a l l cases, except with t y r o s i n e , glycine and edestin. I t i s to be observed also that primary c l e a r i n g was obtained with almost every nitrogen source employed. Exceptions are observed with Bacto Beef and Orla-Jensen*s yeast e x t r a c t , where growth was good but c l e a r i n g very r e s t r i c t e d ; with pantothenic a c i d - f r e e yeast e x t r a c t , where growth was almost e n t i r e l y on the surface and c l e a r i n g incomplete; and with urea, where growth was f a i r l y good but c l e a r i n g was e n t i r e l y absent. 6• D i s t r i b u t i o n of Zoning Phenomenon. In view of the c l e a r i n g and zoning phenomena with Rh. t r i f o l i i 224, i t was decided to determine the extent to which these changes occur w i t h other species and s t r a i n s of Rhizobia. . Accordingly, a group of c u l t u r e s , representative of strains of Rh. t r i f o l i i , Rh. m e l i l o t i , Rh. p h a s e o l i , Rh. l u p i n i , Rh. leguminosarum, and the l o t u s and coropea organisms, were plated i n s u i t a b l e d i l u t i o n s upon the usual medium. The data as presented i n Table 5 i n d i c a t e s that the a b i l i t y to cause c l e a r i n g of the medium i s quite widely d i s t r i b u t e d among the various species of Rhizobia. This phenomenon was found to occur with a l l s t r a i n s of Rh. t r i f o l i i t e s t e d , but with varying degrees of completeness. I t was also observed to a marked degree with cultures of the l o t u s and coropea s t r a i n s . Among s t r a i n s of Rh. leguminosarum, Rh. l u p i n i and Rh. phaseoli c l e a r i n g was apparent, but to a very l i m i t e d extent. With Rh. m e l i l o t i , however, no c l e a r i n g was observed with any of the f i v e s t r a i n s t e s t e d . TABLE 5. D i s t r i b u t i o n of Clearing Phenomenon Among St r a i n s and Species of Rhizobia. Species or S t r a i n R. t r i f o l i i 224 R.T. 2 2 7 R.T. 2 3 0 R.T. 2 3 1 ' R.T. 2 0 5 R.T. 226 R.T. 40-1 R.T, 22B R.T. 3 9-1 R.T. 3 9 - 2 Rh. m e l i l o t i 3 9 - 1 R« mel. 40 - 1 R. mel. 40 - 2 R. mel. 41-2 R. mel. 42 - 1 R. phaseoli 42 - 1 R. l u p i n i 3 9 - 1 R. l u p i n i 42 - 2 R. leguminosarum 41 - 1 R. legumin. 41 - 2 R. lotus 42-2 R. coropea 42-1 Growth C h a r a c t e r i s t i c s . L i g h t growth, very pronounced c l e a r i n g . Medium growth, complete c l e a r i n g . Light growth, complete c l e a r i n g . Light growth, complete c l e a r i n g . Heavy gummy growth, only p a r t i a l c l e a r i n g . L i g h t growth, c l e a r i n g not extensive. Growth l i g h t , c l e a r i n g extensive. Growth medium, c l e a r i n g extensive. Growth l i g h t , complete c l e a r i n g • Growth l i g h t , complete c l e a r i n g . Gummy growth, some traces of c l e a r i n g . Gummy growth, no c l e a r i n g . Gummy growth, no c l e a r i n g * Gummy growth, no c l e a r i n g . Gummy growth, no c l e a r i n g . Gummy growth, s l i g h t trace of c l e a r i n g . L i g h t , watery growth, some c l e a r i n g * Medium growth, incomplete c l e a r i n g . Very gummy growth, p a r t i a l c l e a r i n g . Very gummy growth, some trace of c l e a r i n g . Medium growth, very complete c l e a r i n g . Very gummj'- growth, complete c l e a r i n g . DISCUSSION. From the photographs presented (Plates 1 to 6 i n c l ) i t i s apparent that the c o l o n i a l c h a r a c t e r i s t i c s of Rh. t r i f o l i i upon t h i s medium are extremely a t y p i c a l and suggestive of a rough or d i s s o c i a t e d form. I t would seem that c u l t u r i n g upon the p a r t i c u l a r medium employed has caused the production of two types of colonies, one of which i s extremely t h i n and of a v e i l - l i k e or feathery s t r u c t u r e . These c o l o n i a l types, however, are not stable i n character, since i t has been proved to be impossible to obtain f i x e d substrains even by repeatedly p l a t i n g and s e l e c t i v e l y p i c k i n g f o r c o l o n i a l type over an extended period. I t would appear that some f a c t o r , probably the high yeast content, has caused the production of these a t y p i c a l c o l o n i a l forms, which are the r e s u l t of a d i r e c t s t i m u l a t i o n by the medium i t s e l f and are consequently not of a stable nature. The c o l o n i a l type, moreover, has no e f f e c t whatever upon the development of the c l e a r i n g phenomenon, as i s shown c l e a r l y i n P l a t e s 2 and 3. The lack of c o r r e l a t i o n between zone formation and dehydrogenation with various carbohydrates i n d i c a t e s that the zoning phenomenon i s i n t i m a t e l y bound up with the a c i d production mechanism of the c e l l . I t would appear that there e x i s t s a lack of r e l a t i o n s h i p between the processes of a c i d production and r e s p i r a t i o n and i n t h i s respect i s somewhat s i m i l a r to that reported f o r the l a c t i c , acid s t r e p t o c o c c i by Morgan, Eagles and L a i r d (6). The r e p ression of zone formation and therefore a c i d production upon heavily-seeded plates i n d i c a t e s that the mechanism of a c i d production i s extremely s e n s i t i v e to environmental changes. F a i l u r e to secure a c t i v e f i l t r a t e s from cultures implies that the i n h i b i t o r y e f f e c t upon crowded plates i s caused by changes i n the physical-chemical nature of the medium i t s e l f rather than by the e l a b o r a t i o n of t o x i c chemical substances,, From the r e s u l t s obtained i n the study of the influence of various nitrogen sources i n the medium upon the c l e a r i n g phenomenon i t would appear that the Rhizobia are able to grow i n the presence of a wide range of nitrogenous compounds, ranging from simple amino acids up to complete p r o t e i n s . I t would appear too that c l e a r i n g of the medium i s a phenomenon independent of the growth requirements of the organism. Although c l e a r i n g does not appear to depend upon the presence of any s p e c i a l type of nitrogenous organic compound, a source more complex than urea i s required. There i s evident, too, a curious lack of consistency i n r e s u l t s when various yeast preparations are employed as the nitrogen source. This may i n d i c a t e that the c l e a r i n g and deposition phenomena are r e l a t e d i n some way to the accessory f a c t o r s of the Vitamin B complex. The r e s u l t s obtained from a study of the various s t r a i n s and species of Rhizobia show that the c l e a r i n g and presumably also the deposition phenomena are quite widely d i s t r i b u t e d . It i s n o t i c e a b l e , a l s o , that these phenomena are most completely developed by the Rh. t r i f o l i i s t r a i n s , which are ch a r a c t e r i z e d - 20 -by l i g h t growth and absence of gum formations, The Rh. m e l i l o t i s t r a i n s , which have a c h a r a c t e r i s t i c a l l y heavy and gummy growth, do not e x h i b i t t h i s c l e a r i n g of the medium. That there i s no consistent r e l a t i o n s h i p between gum production and zone formation i s shown by the s t r a i n s of the l o t u s and coropea organisms. These c u l t u r e s developed an extremely heavy gummy growth and also exhibited markedly complete primary zones of c l e a r i n g . The experimental studies reported herein have determined the influence of many f a c t o r s upon the zoning phenomena. Hovsrever, the mechanism of primary c l e a r i n g and, more p a r t i c u l a r l y , the zone of d e p o s i t i o n , remains obscure. Whereas the development of zones of complete c l e a r i n g may be a t t r i b u t e d to ac i d production from the carbohydrate i n the medium with the r e s u l t i n g d i s s o l u t i o n of the suspended calcium carbonate the formation of the ri n g s of deposition i s much more complicated. This deposition i s not a true darkening of the medium but i s rather a g r e a t l y increased opacity. I t i s demonstrable with c o l o r l e s s media, such as those containing g e l a t i n e as the nitrogen source. I t has been d e f i n i t e l y shown that for deposition to take place, three f a c t o r s are necessary, a fermentable carbohydrate source, a s u i t a b l e nitrogen source and calcium carbonate. I t i s important to note, moreover, that of a group of s i x carbonates t e s t e d , only one sample permitted true r i n g formation. This would i n d i c a t e the necessity f o r one or more tra c e elements or accessory f a c t o r s present as i m p u r i t i e s adsorbed on the calcium carbonate. - 21 -This view i s strengthened by the r e s u l t s obtained upon the ad d i t i o n of various trace elements to the medium as reported i n Table 2. . I t i s evident that both c l e a r i n g and deposition are markedly influenced by the presence of small amounts of various s a l t s . The most l o g i c a l explanation for the formation of the r i n g s of deposition would be the d i f f u s i o n from the colony of some u n i d e n t i f i e d substance, probably organic i n nature, which i s p r e c i p i t a t e d by the a c t i o n of calcium ions and catalysed by the presence of trace elements. This hypothesis, however, does not account for the r i n g of unchanged medium which intervenes between the primary zone of complete c l e a r i n g and the outer zone of deposition. Although the phenomenon of c l e a r i n g and deposition i n the media are i n t e r e s t i n g i n themselves, there i s the p o s s i b i l i t y that they may be r e l a t e d to the r e s p i r a t o r y enzyme character of the c e l l . I t has been demonstrated, with the s t r a i n employed i n these t e s t s , Rh. t r i f o l i i 224, that very extensive zones of c l e a r i n g and deposition are formed by the \"c u l t u r e d \" or \"laboratory\" s t r a i n . When, however, t h i s same s t r a i n i s f r e s h l y r e - i s o l a t e d from s o i l i t causes no change whatever i n the medium. The colonies now appear as t y p i c a l transparent watery forms. This observation i s i n accord with the previous report of Morgan, L a i r d and Eagles (5) that \"laboratory\" s t r a i n s of Rh. t r i f o l i i possess a s i g n i f i c a n t l y d i f f e r e n t type of r e s p i r a t i o n from f r e s h l y - i s o l a t e d \" s o i l \" c u l t u r e s . I t would appear, th e r e f o r e , that the development of «. 22 -these zoning phenomena i s i n d i c a t i v e of a d e f i n i t e i n s t a b i l i t y i n the r e s p i r a t o r y enzyme character of the culture which i s associated w i t h a fundamental change i n the carbohydrate mechanism of the c e l l . - 2? -SUMMARY9- , -The development of r i n g formations somewhat s i m i l a r to the Liesegang phenomenon has been demonstrated with Rhizobium t r i f o l i i . This zone formation i s described and i l l u s t r a t e d i n a s e r i e s of p l a t e s . The infl u e n c e of various f a c t o r s upon t h i s zone formation has been studied. Carbohydrates, nitrogen source, carbonates and trace elements are a l l shown to exert an in f l u e n c e . The occurrence of the zoning phenomenon has been surveyed using a representative group of twenty-two s t r a i n s and species of Rhizobia. The mechanism of zone formation i s discussed and r e l a t e d to r e s p i r a t o r y a c t i v i t y . - 24 -REFERENCES. 1. A l l y n , W.P.. and Baldwin, I.L. - Jour. Bact. 20:417, 1930 2. A l l y n , W.P. and Baldwin, I.L. - Jour. Bact. 2 3 : 3 6 9 , 1932 3 . Fred, E.B. and lakeman, S.- \"Laboratory Manual of General Microbiology\" 1928 4 . Morgan, J.F, L a i r d , D.G. Eagles, B.A. 3 . Morgan, J.F. Laird,' D.G. Eagles, B.A. 6. Morgan, J.F. L a i r d , D.G, Eagles, B.A. 7 . Niven, C.F. Smiley, K.L. Sherman, J,M. - Jour. Bact. 41: . 4 7 9 , 1941. 8 . Wilson, P.W. - Jour. Bact. 35: 6 0 1 , 1938 ABSTRACT Methods involved i n the preparation of r e s t i n g c e l l suspensions of the l a c t i c a c i d b a c t e r i a s u i t a b l e f o r dehydrogenase studies by the Thunberg technique have been in v e s t i g a t e d * The dehydrogenase a c t i v i t y of fourteen s t r a i n s of l a c t i c acid b a c t e r i a upon s i x t y t e s t compounds has been determined. V a r i a t i o n i n dehydrogenase a b i l i t y 'has been shown to ex i s t w i t h i n s t r a i n s of Strep, l a c t i s , Strep, cremoris and the Betacocci, and w i t h i n the same s t r a i n at d i f f e r e n t times. I t has been found possible to d i s t i n g u i s h the aerobic pseudo l a c t i c a c i d b a c t e r i a from the true l a c t i c acid strepto-cocci upon t h e i r r e s p i r a t o r y characters. The aerobic pseudo l a c t i c a c i d b a c t e r i a have been shown to possess an endogenous r e s p i r a t i o n which i s not exh i b i t e d by the l a c t i c a c i d s t r e p t o c i c e i . The feeble dehydrogenation of lactose by the l a c t i c s t r e p t o c o c c i , the r e l a t i o n s h i p of isomeric alcohols to dehydrogenase a b i l i t y , the i n h i b i t o r y e f f e c t of amines and the methyl group upon dehydrogenation, and f a i l u r e to dehydrogenate c i t r a t e have been discussed. The p o s s i b i l i t y of basing a c l a s s i f i c a t i o n of the l a c t i c acid s t r e p t o c o c c i upon r e s p i r a t o r y enzyme character has been considered* INTRODUCTION The f i r s t observations on the dehydrogenating a c t i v i t i e s of b a c t e r i a were recorded by Harden and Z i l v a ( 3 ) i n 1 9 1 5 , who noticed that washed suspensions of Bact. c o l i acquired the a b i l i t y to reduce methylene blue upon the a d d i t i o n of various i n a c t i v e reagents. The systematic i n v e s t i g a t i o n of these dehydrogenase enzymes was i n i t i a t e d by the i n t r o d u c t i o n of the anaerobic technique by Thunberg ( 1 8) i n 1 9 2 0 . The Thunberg method and \" r e s t i n g c e l l \" technique have been e x t e n s i v e l y applied to the study of the r e s p i r a t o r y enzyme systems of b a c t e r i a . Q,uast e l and Whetham ( 9 ) studied the e q u i l i b r i a e x i s t i n g between s u c c i n i c , fumaric, and malic acids i n the presence of \" r e s t i n g \" Bact. c o l i , and found a close a s s o c i a t i o n between chemical a c t i v i t y and p h y s i c a l structure of the organism. Q,uastel and Whetham (1 0) continued t h e i r study by t e s t i n g the dehydrogenase a c t i v i t y of Bact. c o l i upon a large number of substrates, i n c l u d i n g f a t t y a c i d s , d i b a s i c a c i d s , hydroxy a c i d s , polyhydric and monohydric alcoh o l s . In a f u r t h e r paper Quastel and Whetham ( l l ) reported dehydrogenations by Bact. c o l l i n the presence of various carbohydrates, and amino ac i d s . Kendall (6) inv e s t i g a t e d the dehydrogenase enzyme a c t i v i t i e s of Bact. e o l i and other related b a c t e r i a l species upon a v a r i e t y of substrates. The dehydrogenase enzymes of the L a c t i c Acid Strepto-cocci have not as yet been ex t e n s i v e l y i n v e s t i g a t e d . F a r r e l l (2) reported upon the r e s p i r a t o r y mechanism of 22 s t r a i n s of st r e p t o c o c c i , s t r a i n s which consisted mainly of pathogenic types but which a l s o included Strep, l a c t i s and Strep, f e c a l i s K a t a g i r i and Kltahara (j>) showed the presence of l a c t i c a c i d dehydrogenase among several species o f . l a c t i c a c i d b a c t e r i a . The work reported upon herein was undertaken with the object of obtaining more d e t a i l e d information upon the dehydrogenase enzyme systems of a l a r g e r number of species of l a c t i c a c i d b a c t e r i a , and of determining whether or not t h i s information might prove valuable i n t h e i r c l a s s i f i c a t i o n . EXPERIMENTAL METHODS 1• Cultures Organisms representative of c e r t a i n genera of the true l a c t i c and pseudo l a c t i c a c i d b a c t e r i a were selected f o r study These included: Strep, l a c t i s S.A. 30, a t y p i c a l Strep* l a c t i i s o l a t e d from cream possessing a caramel f l a v o r (12); Strep. l a c t i s A.T.C. 374, obtained from the National Type Culture C o l l e c t i o n at Washington, B.C.; Strep, l a c t i s EMB2 1 (14); Strep, cremoris HP (18); Strep, cremoris RW ( 1 8 ) ; Strep, cremoris EMBX 1 9 5 (14); Betacoccus EMB2 1 7 3 (14); Strep, citrovorus A.T.C. 797; Strep, paracitrovorus A.T.C. 7 9 8 ; Strep, bovis A.T.C. 6 0 5 8 ; Tetra. casei A.T.C. 391; Tetra. l i q u e f a c i e n s SM 3 ( 1 3 ) ; Bact. c o l i A.T.C. 4157, and Bact. aerogenes A.T.C. 2 1 1 . 2. Media Casein Digest Broth, prepared a f t e r the manner of Or l a -Jensen ( l ) , containing 0 . 5 $ T o t a l Nitrogen, and enriched with 1.of. Difco yeast e x t r a c t , 0 . 5 $ K 2 H P 0 4 and 0 . 5 $ glucose, served as the basic medium. The high percentage of yeast extract i s a s t r i k i n g feature of t h i s medium. The stimulatory e f f e c t of small q u a n t i t i e s of yeast extract upon the l a c t i c a c i d b a c t e r i a was f i r s t reported by Orla-Jensen ( 8 ) , who l a t e r showed that t h i s was due to the supplying of c e r t a i n accessory growth f a c t o r s r e l a t e d to the vitamin B complex. Wilson (19), i n a study of the r e s p i r a t o r y enzymes of the Rhi z o b i a , found that i n c r e a s i n g the yeast content of the medium from 0 . 2 5 $ to 1 . 0 $ r e s u l t e d i n c e l l suspensions which possessed markedly increased dehydrogenase a c t i v i t y . Above loOf. there was no f u r t h e r s t i m u l a t i o n . This e f f e c t was a t t r i b u t e d to a storing-up of e s s e n t i a l coenzyme f a c t o r s w i t h i n the c e l l s • ; Preparation of Suspensions For the determination of dehydrogenase a c t i v i t y , \" r e s t i n g c e l l \" suspensions were prepared from young broth cultures© After a s u i t a b l e incubation period at 30°. C. the c u l t u r e was centrifuged at 2,000 r.p.m. f o r 30 minutes i n flat-bottom centrifuge tubes, the supernatant l i q u i d poured from the sedimented c e l l s , and the c e l l s washed by mixing and then r e -ce n t r i f u g i n g with M/30 phosphate buffer of pH 7,2. A f t e r two washings w i t h buffer s o l u t i o n , the organisms were resuspended i n buffer and employed as \" r e s t i n g c e l l s \" . 4• Standardization of Suspensions In dehydrogenation reactions with b a c t e r i a l suspensions the v e l o c i t y of ox i d a t i o n i s pr o p o r t i o n a l to the concentration of organisms present. I t therefore becomes extremely important to standardize a l l suspensions before use. Two methods of st a n d a r d i z a t i o n have been employed by various workers: ( l ) the suspension i s dr i e d and weighed and converted i n t o milligrams of dry c e l l weight per cubic centimeter, and (2) the nitrogen content i s determined by the micro-Kjeldahl method and suspensions compared on the basis of milligrams of nitrogen per cubic centimeter. Both these methods a f f o r d a basis f o r comparison of r e s u l t s , but are open to serious o b j e c t i o n s . In the f i r s t place,.both the moisture content and the nitrogen content of the b a c t e r i a l c e l l are influenced by c u l t u r a l c o n d i t i o n s , and secondly, these procedures require considerable time before r e s u l t s are obtained. In order to avoid these d i f f i c u l t i e s the f o l l o w i n g method of standardization has been adopted. A 10-c.c. a l i q u o t portion of broth c u l t u r e i s placed i n a Hopkins vaccine tube (4) , centrifuged f o r 30 minutes, and the volume of c e l l sediment measured. From t h i s determination suspensions containing a d e f i n i t e percentage by volume of c e l l s are then prepared. With the organisms employed i n the t e s t s reported herein a concentration of i f . by volume was g e n e r a l l y found to be most convenient. P l a t e counts c a r r i e d out on suspensions of the same organism prepared at d i f f e r e n t times showed a v a r i a t i o n of l e s s than 10f.. The procedure f o r standardizing c e l l suspensions by t h i s method i s r a p i d , simple, and appears to be quite accurate. Suspensions prepared by t h i s volume method may also be con-verted to dry weight and nitrogen content bases with l i t t l e d i f f i c u l t y . Since dehydrogenase a c t i v i t y i s l i n k e d up with the structure of the b a c t e r i a l c e l l , i t i s to be expected that the period of growth i n cu l t u r e before harvesting w i l l i nfluence the a c t i v i t y of the b a c t e r i a l suspensions prepared therefrom. In order to determine the optimum incubation period, broth cultures of two s t r a i n s of Strep, l a c t i s and one s t r a i n of Strep, cremoris were incubated at 30° C, and samples removed from the mother c u l t u r e s at regular i n t e r v a l s . These samples were then plated, and the dehydrogenase a c t i v i t y upon glucose determined. The r e s u l t s obtained showed that dehydrogenase a c t i v i t y reached a maximum at about 24 hours, and then very slowly .declined, while maximum growth was not at t a i n e d u n t i l 48 to 72 hours. These f i n d i n g s i n d i c a t e d that suspensions prepared from c u l t u r e s i n the logarithmic growth phase were most a c t i v e . A l l suspensions employed i n these t e s t s were therefore prepared from c u l t u r e s grown from 18 to 24 hours* These r e s u l t s are i n accord with those of Wooldridg©, Knox and. Glass (20), who reported that 24-hour cultures of Bact, c o l l yielded the most a c t i v e suspensions f o r dehydrogenase s t u d i e s , 6, Methylene Blue Concentration The question of concentration of methylene blue i s of importance not only i n r e l a t i o n to the rate of reduction of the dye, but also i n r e l a t i o n to i t s t o x i c a c t i o n . - 7 -The t o x i c i t y of varying concentrations of methylene blue upon Bact, c o l i was determined by exposing suspensions of the organism i n Thunberg tubes f o r a one-hour period to methylene blue concentrations ranging from 1:10,000 to 1:50,000. After one hour the suspensions were plated and the number of vi a b l e c e l l s determined, A c r i t i c a l t o x i c concentration was found to occur between 1:15*000 and 1:20,000 methylene blue. Above 1:20,000 the t o x i c e f f e c t i s very s l i g h t , while below 1:15,000 the c e l l s r a p i d l y lose t h e i r v i a b i l i t y . The concentration selected f o r use was 1 :55,000 methylene blue. This was found to give a c l e a r and d i s t i n c t end-point eithout exerting any i n h i b i t o r y effects© The use of methylene blue i n dehydrogenase studies with the Streptococci has been questioned by F a r r e l l ( 2 ) , who found indigo t e t r a s u l f o n a t e to be much l e s s t o x i c . However, Sandiford and Wooldridge (15) and Wooldridge and Glass (21) showed c l e a r l y that dehydrogenase a c t i v i t y i s independent of v i a b i l i t y but i s associated equally with l i v i n g and dead c e l l s . P a r a l l e l experiments with the l a c t i c a c i d streptococci,employing methylene blue and indigo t e t r a s u l f o n a t e , showed no appreciable d i f f e r e n c e s . Resting c e l l suspensions of c e r t a i n b a c t e r i a l species possess the a b i l i t y to reduce methylene blue i n the absence of substrate:,. This type of reduction i s known as \"endogenous r e s p i r a t i o n \" and has been a t t r i b u t e d to an o x i d a t i v e deamination of c e l l u l a r amino acids i n which the b a c t e r i a l c e l l s u t i l i z e traces of polysaccharides or capsular ma t e r i a l as the energy source. Suspensions of the l a c t i c a c i d s t r e p t o c o c c i showed no reducing a c t i o n upon methylene blue i n the absence of substrate. When the more aerobic organisms of the pseudo l a c t i c group, namely Tetra. e a s e l , Tetra. l i q u e f a c i e n s , Bact. c o l i and Bact. aerogenes, were employed, the suspensions were observed to reduce methylene blue i n the absence of substrate. Repeated washing by c e n t r i f u g i n g with buffer f a i l e d to destroy the reducing a c t i v i t y , and i t was found necessary to aerate the suspensions, f o l l o w i n g the procedure of Q,uastel and Whetham (10). A f t e r 60 minutes * aeration the suspensions were found to have l o s t t h e i r reducing a c t i v i t y upon methylene blue. A l l suspensions of the aerobic organisms were therefore subjected to 60 minutes' ae r a t i o n before use. 8 » Thunberg Technique The dehydrogenase a c t i v i t y of the l a c t i c a c i d b a c t e r i a was determined i n modified Thunberg tubes containing 1 c.c. of -r e s t i n g c e l l suspension, 2 c.c. of M/30 phosphate buffer of pH 7 . 2 , 1 c.c. of 1:7 , 0 0 0 methylene blue and 1 c.c. of M/20 substrate. A l l tubes were evacuated f o r two minutes on a water pump. Tests were c a r r i e d out at 37,5° C. i n an e l e c t r i c a l l y c o n t r o l l e d water bath. Dehydrogenase a c t i v i t y was measured as time required f o r complete d e c o l o r i z a t i o n of the methylene blue. A l l t e s t s which d i d not reduce w i t h i n two hours were considered negative. Results obtained have been expressed as percentage of the reduction time of glucose, given the value of 1 0 0 . This value i s c a l l e d the r e s p i r a t o r y c o e f f i c i e n t . EXPERIMENTAL RESULTS The dehydrogenase a c t i v i t i e s of three s t r a i n s of Strep, l a c t i s and three s t r a i n s of Strep, cremoris are recorded i n Table 1. There i s evident a marked v a r i a t i o n i n dehydrogenase a b i l i t y among s t r a i n s w i t h i n these two species. This v a r i a -t i o n i s c l e a r l y shown i n the dehydrogenase reactions upon sucrose and t r e h a l o s e , r e a c t i o n s which y i e l d p o s i t i v e or negative values, depending upon the s t r a i n employed. The dehydrogenase enzymes of Strep, l a c t i s and Strep, cremoris appear to be very s i m i l a r i n character. Both species are able to dehydrogenate the four monosaccharides and the majority of the disaccharides and polysaccharides. They both • - 10 -f a i l , however, to o x i d i z e xylose, arabinose, melibiose, melezitose, rhamnose and methyl glucoside. Both species are further characterized by a low r e a c t i v i t y upon the monohydric and polyhydric alcohols and by a complete i n a b i l i t y to attack the s a l t s of organic a c i d s . An exception to the low r e a c t i v i t y upon alcohols i s found i n the case of Strep, cremoris RW, which has been found to possess a r e s p i r a t o r y c o e f f i c i e n t of 125 upon i s o propyl a l c o h o l and of 250 upon sec, b u t y l alcohol© This close s i m i l a r i t y i n dehydrogenase enzyme a c t i v i t y makes i t extremely d i f f i c u l t to attempt to d i s t i n g u i s h these two species by t h e i r r e s p i r a t o r y enzyme characters. The one test which might possess d i f f e r e n t i a l s i g n i f i c a n c e i s the dehydrogenation of r a f f i n o s e . A l l three s t r a i n s of Strep, cremoris attack t h i s compound, while a l l three s t r a i n s of Strep, l a c t i s proved unable to do so, The dehydrogenase a c t i v i t y of Strep, bovis and three s t r a i n s of Betacocci are tabulated i n Table 2, From the values recorded here there i s again evident a marked v a r i a t i o n i n dehydrogenase a b i l i t y among the three s t r a i n s of Betacocci studied. This v a r i a b i l i t y i s p a r t i c u l a r l y noticeable i n the dehydrogenation of d e x t r i n , s a l i e i n and r a f f i n o s e . Suspensions of Strep, bovis appear to possess very strong o x i d i z i n g mechanisms upon r a f f i n o s e and star c h , Dehydrogenation of d e x t r i n , however, i s appreciably weaker, while that of maltose i s very f e e b l e . - 11 -This would i n d i c a t e that the organism u t i l i z e s starch d i r e c t l y , without preliminary h y d r o l y s i s to the dex t r i n or maltose stages. The dehydrogenase a c t i v i t i e s of the pseudo l a c t i c acid \"bacteria, namely, two s t r a i n s of T e t r a c o c c i , Bact» c o l i , and Bact, aerogenes, are d e t a i l e d i n Table 3 , These species exhibit a very r a p i d o x i d i z i n g a b i l i t y upon nearly a l l carbo- -hydrates t e s t e d , Tetra. casei i s the only organism studied which showed a strong dehydrogenase a c t i v i t y upon the pentoses, xylose and arabinose. These species are also characterized by o x i d a t i o n of the hexahydric a l c o h o l s , mannitol and s o r b i t o l , and by dehydrogenase a c t i v i t y upon the s a l t s of c e r t a i n organic acids, p a r t i c u l a r l y formate, l a c t a t e , succinate, and malate. Oxidation of the monohydric alcohols i s appreciable, e s p e c i a l l y in the case of Tetra, l i q u e f a c i e n s , which shows a r e s p i r a t o r y c o e f f i c i e n t of 200 i n the presence of e t h y l , n propyl and a l l y l a l c ohols. The hydrogenation of formaldehyde and glutamine by both Bact. c o l i and Bact. aerogenes appears to be of some s i g n i f i c a n c e . The dehydrogenase reactions of Strep, l a c t i s S,A. 30 and Strep, l a c t i s A.T.C. 374 were redetermined 18 months a f t e r the previous t e s t s had been c a r r i e d out. The r e s u l t s of these two series of t e s t s are recorded i n Table 4, - 12 -The dehydrogenase a c t i v i t i e s of these two s t r a i n s appear to be subject 'to considerable v a r i a t i o n , depending upon when the culture i s t e s t e d . With many substrates there has been a marked increased i n dehydrogenase a b i l i t y , while with other substrates there has been a marked decrease. I t i s apparent, therefore, that t h i s v a r i a t i o n does not i n d i c a t e a d e f i n i t e tendency toward increased or decreased a c t i v i t y on the part of the c u l t u r e , but merely i l l u s t r a t e s the e s s e n t i a l i n s t a b i l i t y of the r e s p i r a t o r y enzyme systems of t h i s b a c t e r i a l species* DISCUSSION The r e s u l t s obtained from t h i s study of the dehydrogenase enzymes of the l a c t i c a c i d b a c t e r i a show c l e a r l y that fundamental d i f f e r e n c e s e x i s t between the r e s p i r a t o r y mechanisms of the true l a c t i c a c i d s t r e p t o c o c c i and the more aerobic pseudo l a c t i c a c i d cocci and rod forms. The true l a c t i c acid streptococci are characterized by a dehydrogenase a c t i v i t y which is r e s t r i c t e d almost e n t i r e l y to the carbohydrates and simpler monohydric a l c o h o l s . The pseudo l a c t i c a c i d b a c t e r i a , on the other hand, possess much greater dehydrogenase a c t i v i t y upon the carbohydrates and, i n a d d i t i o n , are able to attack mono-hydric and polyhydric alcohols as w e l l as the s a l t s of organic acids. A f u r t h e r d i f f e r e n c e i n r e s p i r a t o r y character between - 13 -these two groups i s shown by the endogenous r e s p i r a t i o n of the aerobic species. The dehydrogenating a b i l i t y of the l a c t i c acid s t r e p t o c o c c i upon carbohydrates i s quite v a r i e d , a l l four monosaccharides and the'majority of the d i - and polysaccharides being attacked. Among the monosaccharides, however, galactose i s oxidized much less r e a d i l y than glucose, fruetose or mannose, while among the disaccharides the dehydrogenating a c t i o n upon lactose i s very weako This low r e a c t i v i t y upon lactose i s rather s u r p r i s i n g , since the majority of these c u l t u r e s form s u f f i c i e n t l a c t i c acid to c l o t milk cultures i n from 18 to 48 hours. The a b i l i t y of organisms to dehydrogenats organic acids appears to be r e l a t e d to the acids produced during sugar fermentations. The aci d which accumulates i n the medium during fermentations c a r r i e d out by the l a c t i c a c i d cocci i s almost e n t i r e l y l a c t i c a c i d . Therefore these organisms should not be expected to possess an enzyme system capable of u t i l i z i n g l a c t a t e . Experimental r e s u l t s have confirmed t h i s hypothesis. In the case of the t e t r a c o c c i , however, where l a c t i c a c i d i s not the only a c i d accumulating i n the culture medium, dehydrogenation of l a c t a t e occurs. With Bact• c o l i and Bact. aerogenes, where a gaseous mixture of carbon dioxide and hydrogen i s produced, dehydrogenase enzymes f o r both l a c t a t e and formate are found. - 14 -The i n c l u s i o n of a large number of isomeric alcohols in these tests- afforded an opportunity to study stereochemical s p e c i f i c i t y of dehydrogenase a c t i o n . With the organisms tested here there appeared to be no such s p e c i f i c i t y , Tetra, casei was able to dehydrogenate iso propyl alcohol but not iso b u t y l a l c o h o l , n b u t y l a l c o h o l but not n propyl or n amyl alcohols, sec, amyl alcohol but not sec, b u t y l a l c o h o l * Similar e f f e c t s were observed with the other organisms. The most s t r i k i n g feature of the a l c o h o l dehydrogenation was the a b i l i t y shown by d i f f e r e n t s t r a i n s to s e l e c t i v e l y a c t i v a t e one or two a l c o h o l s with great r a p i d i t y . This a c t i v a t i o n i s apparently due e n t i r e l y to i n d i v i d u a l s t r a i n character. The majority of the organisms te s t e d proved able to dehydrogenate e t h y l a l c o h o l but not one was able to dehydrogenat ethyl amine. S u b s t i t u t i o n of an amino group f o r a hydroxyl group has apparently eliminated dehydrogenase a c t i v i t y . This observation agrees with the conception that the amines are f i n a l and i n most cases t o x i c products of b a c t e r i a l a c t i o n . None of the organisms t e s t e d showed any dehydrogenase a c t i v i t y upon potassium c i t r a t e . This was true of Strep, paracitrovorus as w e l l as of Bact, c o l l and Bact, aerogenes• The a b i l i t y of Bact, aerogenes to grow with c i t r a t e as the sole carbon source i s widely used to d i f f e r e n t i a t e these two organisms. U t i l i z a t i o n of c i t r a t e by Bact, aerogenes must - 15 -therefore proceed i n some way other than by dehydrogenation. The ' i n a b i l i t y of Strep, paracitrovorus to dehydrogenate c i t r a t e i s of i n t e r e s t because of the reported r e s u l t s of Slade.and Workman ( 1 6 ) , who showed that c e l l s of Strep, paracitrovorus grown i n c i t r a t e plus lactose were then able to ferment c i t r a t e . A f u r t h e r point of i n t e r e s t with the carbohydrate de-nydrogenations i s the possible i n h i b i t o r y e f f e c t of the methyl group. A l l the organisms tested were able to dehydrogenate glucose, while only one was able to dehydrogenate alpha methyl glucoside. Rhamnose, a methyl pentose, was not dehydrogenated by organisms which attacked xylose and arabinose. Several species were able to dehydrogenate e t h y l a l c o h o l , but never methyl a l c o h o l . These r e s u l t s a l l i n d i c a t e that the methyl group exerts an i n h i b i t o r y e f f e c t upon dehydrogenase a c t i v i t y . This observation agrees with that of Koser and Saunders (7) » who showed that methyl d e r i v a t i v e s of several of the commoner sugars were d i s t i n c t l y r e s i s t a n t to b a c t e r i a l a t t a c k . The dehydrogenase enzyme studies reported herein were carried out i n an attempt to e s t a b l i s h a basis for the c l a s s i f i c a t i o n of the l a c t i c a c i d s t r e p t o c o c c i . The r e s u l t s reported i n Tables 1 , 2 and 3 show c l e a r l y that no d e f i n i t e species c h a r a c t e r i s t i c s e x i s t i n r e s p i r a t o r y enzyme make-up. There i s apparent a very marked s t r a i n v a r i a t i o n w i t h i n a l l - 16 -the species studied* This s t r a i n v a r i a t i o n i s f u r t h e r emphasized by the r e s u l t s reported i n Table 4, which show that the dehydrogenase a c t i v i t y of a s i n g l e s t r a i n varies at d i f f e r e n t times. From the r e s u l t s of these studies i t i s apparent that c l a s s i f i c a t i o n of the l a c t i c acid s t r e p t o c o c c i upon t h e i r r e s p i r a t o r y enzyme character i s not f e a s i b l e . Such a c l a s s i f i c a t i o n cannot be attempted u n t i l such time as the factors i n f l u e n c i n g v a r i a t i o n i n dehydrogenase a c t i v i t y are further clarified© - 17, -. , REFERENCES 1. Eagles, B 0A.,and Sadler, W, Can. Jour* Res. 1: 364, 1932. 2. F a r r e l l , M.A. Jour. Bact. 29: 411, 1 9 3 5 . 3. Harden, A. and Z i l v a , S.S. Bioeliem. Jour. 9 : 3 7 9 , 1915. 4. Hopkins, D. Jour. Amer. Med. A Ss *n. 60: 1 6 1 5 , 1 9 1 3 . 5« K a t a g i r i , H. and K i t a h a r a , K, Bioeliem. Jour. 32: 1654, 1938 • 6. K e n d a l l , A, I . Jour. I n f . Dis. 47: 186, 1930. 7» Koser, S.A. and Saunders, F. Jour. Bact. 26: 475, 1933* 8 . Orla-Jensen, S. Jour. Bact. 12: 333, 1926. 9. Quastel, J.H. and Whetham, M.D. Biochem. Jour. 18: 519 , 19 24. 10. Quastel, J.H. and Whetham, M.D. Biochem. Jour. 19: 520, 1 9 2 5 . 11. Quastel, J.H. and Whetham, M.D. Biochem. Jour. 19: 645, 1 9 2 5 . 12. Sadler, W. Trans. Roy, Soc. Can. V o l , 20, 1926. 13. Sadler, W. S c i e n t i f i c A g r i c u l t u r e , V o l , K, No, 2, 1927, 14. Sadler, W,, Eagles, B.A. and Pendray, G. Can, Jour. Res, 7 3 7 0 , 1 9 3 2 . 15. Sandiford, B.R. and Wooldridge, W.R. Biochem. Jour, 25• 2172, 1931. 16. Slade, H.D. and Workman, C.H. Jour. Bact. 41: 19 , Ab S c 1942. 17. Thunberg Skand. Arch. P h y s i o l . 40: 1, 1920. 18. Whitehead, H.R. and Cox, G.A. Jour. Dairy R e s . 7: 556, 1936, 19. Wilson, P.W. Jour. Bact. 35: 601, 1938, 20. Wooldridge, W.R., Knox, R., and Class, V. Biochem.Jour• 30: 9 2 6 , 1936. 21. Wooldridge, W.R. and Glass, V. Biochem, Jour. 25: 2172, 1931, ABSTRACT The aerobic r e s p i r a t o r y a c t i v i t y of two s t r a i n s of Strep, l a c t i s has been studied upon t h i r t y substrates, c o n s i s t i n g of carbohydrates, alcohols and organic acids© These organisms have been shown to possess a varie d o x i d a t i v e a b i l i t y upon the substrates t e s t e d . I t has been shown that there e x i s t s a considerable v a r i a t i o n between the o x i d a t i v e systems of the two s t r a i n s studied* Strep, l a c t i s has been shown to e x h i b i t a consider-able endogenous oxygen uptake* The r e l a t i o n s h i p of t h i s endogenous r e s p i r a t i o n to the o x i d a t i v e systems and to the mechanism of r e s p i r a t i o n has been discussed. INTRODUCTION Although the dehydrogenating a c t i v i t y of many b a c t e r i a l species has been studied, the aerobic r e s p i r a t o r y mechanism has not yet been e x t e n s i v e l y investigated,, Cook and Stephenson ( 8 ) . have reported upon the oxida t i v e a b i l i t y of suspensions of Bact• c o l i . Lineweaver (15) studied the c h a r a c t e r i s t i c s of ox i d a t i o n by Azotobacter v i n e l a n d i i upon a v a r i e t y of substrates and found almost complete oxidation to carbon dioxide and water. Wilson (1 8) in v e s t i g a t e d the s u i t a b i l i t y of the Rhizobium species f o r r e s p i r a t o r y studies* Bernheim (4) determined the oxygen uptake of the tubercle b a c i l l u s i n the presence of various substrates and demonstrated a marked s t i m u l a t i o n i n the case of a l l aldehydes t e s t e d . The o x i d a t i v e a c t i v i t y of various species of co c c i has also been i n v e s t i g a t e d . Barron and Hastings ( l ) and Barron (2) showed that suspensions of gonococci were able t o oxid i z e alpha hydroxy and alpha ketonic acids to carbon dioxide and acids containing one l e s s carbon atom, e.g., l a c t i c or pyruvic a c i d to a c e t i c acid and carbon dioxide* The r e s p i r a t o r y mechanism of the stre p t o c o c c i was studied by F a r r e l l ( l l ) , employing twenty-two s t r a i n s , which consisted mainly of pathogenic and hemolytic types. Working with the hemolytic s t r e p t o c o c c i , Barron and Jacobs (3) obtained a v a r i a t i o n both i n the number and rates of oxidation of a large number of substrates. Studies upon the r e s p i r a t o r y mechanism of the l a c t i c acid b a c t e r i a have been r e l a t i v e l y few. Hunt (13) reported upon the oxygen uptake by l i v i n g c ultures and washed suspensions of the L a c t o b a c i l l i , emphasizing the e f f e c t of various i n h i b i t i n g agents upon c e l l r e s p i r a t i o n . F a r r e l l ( l l ) in h i s study of the r e s p i r a t o r y mechanism, of the s t r e p t o c o c c i , included three l a c t i c a c i d types among h i s various s t r a i n s . Hansen (12) studied the r e s p i r a t i o n of twelve species of the rod-shaped l a c t i c a c i d b a c t e r i a and found great di f f e r e n c e i n t h e i r r e s p i r a t o r y systems. The work reported upon herein was undertaken with the object of obtaining d e t a i l e d information upon the aerobic re s p i r a t o r y mechanism of the l a c t i c a c i d streptococci© EXPERIMENTAL METHODS The c u l t u r e s employed i n t h i s study were Strep, l a c t i s S.A. 3 0 ( 1 0 ) and Strep, l a c t i s No. 374- of the National Type Culture C o l l e c t i o n at Washington, D.C. Comparative t e s t s were also c a r r i e d out employing Strep, l a c t i s EMB2 1 ( 1 7 ) , and Bact. c o l i A.T.C. 4137. Casein Digest Broth, prepared a f t e r the manner of Orla-Jensen ( 1 0 ) , containing 0 . 5 $ T o t a l Nitrogen, and enriched with 1 $ Difco yeast e x t r a c t , 0 . 5 $ K 2 H P O 4 , and 0 . 5 $ glucose, served as the basic medium. Respiratory a c t i v i t y was determined by the use of \" r e s t i n g c e l l \" suspensions, prepared and standardized as d e t a i l e d by Morgan, Eagles, and L a i r d ( 1 6 ) . Oxygen uptake was measured manometrically i n the Barcroft apparatus, as described by Dixon ( 9 ) • The cups on t h i s apparatus contained a r e a c t i o n mixture c o n s i s t i n g of 1 c.c. of re s t i n g c e l l suspension, 1 c.c. of M/20 substrate, and 1 c.c. of M/30 phosphate b u f f e r . Carbon dioxide was absorbed i n f i l t e r paper soaked i n 2 0 $ potassium hydroxide held i n inset tubes w i t h i n the cups. A l l experiments were c a r r i e d out at 3 7 . 5 ° C. i n a t h e r m o s t a t i c a l l y c o n t r o l l e d water bath, and at a pH of 7 . 2 . - 4'-A l l r e s u l t s obtained have been recorded as Q0 2 and QC02 values* c a l c u l a t e d on the basis of c e l l dry weight. The Respiratory Quotients were derived by d i v i d i n g carbon dioxide output by oxygen uptakes EXPERIMENTAL RESULTS The aerobic r e s p i r a t o r y a c t i v i t y of two s t r a i n s of Strep, l a c t i s upon t h i r t y compounds i s recorded i n Table 1. Values are expressed as Q0 2, 0,002 and Respiratory Quotient at one hour. The r e s u l t s reported i n t h i s table show that these two s t r a i n s possess a v a r i e d o x i d a t i v e a c t i v i t y upon carbohydrates. Determination of carbon dioxide production and c a l c u l a t i o n of the Respiratory Quotients have shown t h a t , i n most cases, oxidation i s f a i r l y complete to carbon dioxide and water. The rat© of oxygen uptake against time i n the ease of Strep, l a c t i s S.A. 3 0 i s recorded g r a p h i c a l l y i n Figure 1© These r e s u l t s have been selected as t y p i c a l of the type of curve obtained when the oxygen uptake i s p l o t t e d against time over a one-hour period. I t i s apparent from t h i s graph that the rate of oxygen uptake i s f a i r l y regular and uniform i n characters f •jr.. ~ 5 « The comparative oxygen uptake of Strep, l a c t i s S.A. 3 0 upon ten substrates i s shown g r a p h i c a l l y i n Figure 2© •' Oxidation of the monosaccharides, as exemplified by flucose '< and galactose, i s quite high i n value. This i s also true of f mannose and f r u c t o s e . With the pentoses, xylose i s oxidized I guite r e a d i l y , while arabinose i s attacked but s l i g h t l y . A l l ; the disaccharides tested were oxidized r e a d i l y , with the f • • . • • • J- exception of melibiose. Among the t r i s a c c h a r i d e s , both i r a f f i n o s e and melezitose were attacked with ease. A l l the I polysaccharides t e s t e d were o x i d i z e d , with the exception of i n u l i n , where oxygen uptake was very s l i g h t . Among the • alcoho l s , o x i d a t i v e a c t i v i t y was high, except i n the case of I d u l c i t o l o When e t h y l a l c o h o l was the substrate, oxygen uptake li was appreciably greater than that of glucose. With e t h y l amine, \\ however, oxygen uptake was almost n e g l i g i b l e . Tests on the ' f \"• ••': • • • •' \" : a c t i v i t y of t h i s organism toward the s a l t s of organic acids {showed that a l l the compounds studied were r e a d i l y attacked. fr:'' ' • • ' • I; The aerobic r e s p i r a t o r y a c t i v i t y of Strep, l a c t i s A.T.C. 374 upon ten substrates i s shown i n Figure 3. Oxidation of the monosaccharides, glucose, mannose and f r u c t o s e , i s high, 1 but that of galactose i s low. With the pentoses, arabinose i i s oxidized r e a d i l y , but xylose hardly at a l l . Among the •ii disaccharides, sucrose, c e l l o b i o s e and la c t o s e are r e a d i l y attacked, while maltose, trehalose and melibiose are attacked : only weakly. With the polysaccharides the oxi d a t i o n of starch 1 ' I . . - 6 -and d e x t r i n i s * seen to b© markedly greater than even that of glucose, while the ox i d a t i o n of s a l i c i n and e s c u l i n i s low, and that of i n u l i n extremely s m a l l . With t h i s s t r a i n , a c t i v i t y upon the alcohols i s very low except i n the case of g l y c e r o l , With ethylamine o x i d a t i o n i s low. With organic acids the 0,0 2 is very nearly that found i n the o x i d a t i o n of glucose. The comparative o x i d a t i v e a c t i v i t y of Strep, l a c t i s •S,A, JO and Strep, l a c t i s A.T.C. 374 upon ten substrates i s shown g r a p h i c a l l y i n Figure 4 , These r e s u l t s show t h a t , except in the case of the endogenous, glucose and lactose r e s p i r a t o r y mechanisms, there e x i s t s a marked v a r i a t i o n i n the a c t i v i t y of these two s t r a i n s upon the majority of the compounds teste d . The existence of such a v a r i a t i o n i n o x i d a t i v e a c t i v i t y between two s t r a i n s of the same species would imply that aerobic r e s p i r a t o r y a c t i v i t y i s a s t r a i n , rather than a species, c h a r a c t e r i s t i c , -The Respiratory Quotients of Strep, l a c t i s A.T.C. 374 upon four substrates are shown g r a p h i c a l l y i n Figure 5, The curves shown i n t h i s graph are t y p i c a l of the r e s u l t s obtained upon various substrates with the two s t r a i n s employed. The R.Q.. i n the case of fructose i s shown to be a s t r a i g h t l i n o which very nearly approximates a value of l o 0 . With starch there i s demonstrated a decrease from a value of 0<>7 to 0 , 6 , while with l a c t o s e the Respiratory Quotient r i s e s from 0 O 6 to 0 , 8 . In the case of i n o s i t o l a graph i s obtained which is characterized by a very sharp dip i n the l i n e at JO minutes. This same type of curve has been noted with a d o n i t o l and d u l c i t o l with Strep, l a c t i s A.T.C. 3 7 4 and with arabinose in the case of both s t r a i n s . These r e s u l t s would i n d i c a t e , therefore, that where such a sharp break i n the graph of the Respiratory Quotient i s obtained, there must e x i s t a d e f i n i t e and sharp break i n the metabolism of the organism upon the substrate being t e s t e d . In Figure 6 the glucose o x i d a t i o n of Strep, l a c t i s S.A. 3 0 Strep, l a c t i s A.T.C. 374, and Strep, l a c t i s EMBg 1 i s compared with that of Bact. c o l i A.T.C. 4157. This graph shows the extreme d i f f e r e n c e i n o x i d a t i v e a c t i v i t y evidenced by aerobic organisms, such as Bact. c o l i , and the more anaerobic species, such as Strep, l a c t i s . DISCUSSION An important c h a r a c t e r i s t i c of the aerobic r e s p i r a t i o n of the l a c t i c a c i d s t r e p t o c o c c i i s the presence of an appreciable oxygen uptake i n the presence of added o x i d i z a b l e substrate. This \"endogenous r e s p i r a t i o n \" i n the case of the two s t r a i n s - 8 -studied herein amounts to f i f t y or s i x t y per cent of the oxygen uptake i n the presence of glucose. I t therefore acts as a complicating f a c t o r which may serve to mask true o x i d i z i n g a b i l i t y i n the case of substrates which are but weakly attacked. The endogenous oxygen uptake of a v a r i e t y of b a c t e r i a l species was studied by Callow ( 5 ) , who demonstrated a very appreciable uptake i n the case of aerobic organisms and a n e g l i g i b l e uptake i n the case of anaerobic species. Strep, l a c t i s was found to resemble the anaerobic rather than the aerobic species i n possessing only a very s l i g h t endogenous oxygen uptake. This work was confirmed by F a r r e l l ( l l ) , who found the endogenous oxygen uptake of twenty-two s t r a i n s of streptococci to be almost n e g l i g i b l e and c o r r e l a t e d t h i s fact with the absence of an indophenol-oxldase system i n these b a c t e r i a . The two s t r a i n s of Strep, l a c t i s reported herein have been found to possess an endogenous oxygen uptake which i s s i x or seven times the value reported by F a r r e l l . The mechanism of the endogenous r e s p i r a t i o n of b a c t e r i a i s s t i l l l a r g e l y unknown• Wilson (18) , studying the r e s p i r a -tion of the Rhizobia, found that ammonia was produced by resting c e l l suspensions,and postulated endogenous r e s p i r a t i o n to be an o x i d a t i v e deamination of c e l l u l a r amino acids i n •. - 9 -which c e l l p l l y s a c e h a r i d e was u t i l i s e d as an energy source, Ingram (14), studying the endogenous r e s p i r a t i o n of B 0 cereus, found that the grain-positive character and the high endogenous r e s p i r a t i o n were both associated with a high content of f a t i n the c e l l . The endogenous r e s p i r a t i o n of the l a c t i c a c i d s t r e p t o c o c c i , however, does not appear to p a r a l l e l that described by e i t h e r of these two workers, since repeated t e s t s upon r e s t i n g c e l l suspensions f a i l e d to demonstrate the production of ammonia, Furthermore, the l a c t i c a c i d s t r e p t o c o c c i do not produce s i g n i f i c a n t amounts of c e l l polysaccharide and are not considered to possess a high c e l l u l a r f a t content. I t has f u r t h e r been shown that oxygen uptake by r e s t i n g c e l l s of Strep, l a c t i s i s not c a r r i e d out through the f u n c t i o n i n g of a glutathione system, since repeated attempts f a i l e d to secure a p o s i t i v e n i t r o p r u s s i d e r e a c t i o n with these suspensions. The endogenous mechanism of the l a c t i c s t r eptococci i s s t i l l f u r t h e r complicated by the observation of Morgan, Eagles, and L a i r d (l6) t h a t , i n contrast to t h e i r appreciable aerobic endogenous oxygen uptake, these b a c t e r i a do not e x h i b i t any anaerobic endogenous r e s p i r a t i o n . The values of the Respiratory Q,uotents reported i n Table 1 are i n general very close to a f i g u r e of u n i t y . This i n d i c a t e s that i n the majority of cases o x i d a t i o n of these carbohydrates • - 10 -proceeds completely through to carbon dioxide and water. In some cases, however, appreciably lower values of the Respiratory Quotient have been recorded, as i n the case of l a c t o s e . This observation suggests t h a t , with these substrates, o x i d a t i o n i s incomplete and intermediate products may accumulate© An a l t e r n a t i v e suggestion has been advanced by C l i f t o n (6,7), who showed that with washed, c e l l s of Bact, c o l i the oxidation of many substrates was not c a r r i e d to completion, but a portion of the substrate was a s s i m i l a t e d by the c e l l as carbohydrate. This a s s i m i l a t o r y process could be blocked by adding sodium ozide or d i n i t r o p h e n o l . The values of the Respiratory Quotients reported i n Table 1 indicate that with several substrates considerably more carbon dioxide i s evolved than i s oxygen u t i l i z e d . This would suggest that i n such cases the f u r t h e r process of decarboxylation of some intermediately-formed compound may be t a k i n g place. The r e s u l t s reported herein show that Strep, l a c t i s e xhibits a strong o x i d i z i n g a b i l i t y upon a wide v a r i e t y of compounds, i n c l u d i n g carbohydrates, alcohols and organic a c i d s . The o x i d a t i o n of t h i s l a s t group appears to take place very strongly with a l l such compounds te s t e d . This i s rather su r p r i s i n g i n view of the report by Morgan, Eagles, and L a i r d (16) that the true l a c t i c a c i d s t r e p t o c o c c i possess no dehydrogenating a c t i v i t y upon the organic acids© - 11 -Comparison of the aerobic r e s p i r a t o r y a c t i v i t y of Strep, l a c t i s S.A, 3 0 with that of Strep, l a c t i s A.T.C. 374 shows that there i s a considerable v a r i a t i o n i n t h e i r o x i d i z i n g a b i l i t y upon several substrates. This v a r i a t i o n i n a c t i v i t y between two s t r a i n s of the same species implies that such t e s t s w i l l have l i t t l e taxonomic s i g n i f i c a n c e . - 12 REFERENCES 1. Barron, E.S.G. and Hastings, A.P. J 0 u r . B i o l , Chem. .- 91'. 7 3 , 1 9 3 2 , 2, Barron, E.S.G. Jour, B i o l , Chem. 113: 695 , 1936, 3, Barron, E.S.G, and Jacobs, H.R, _ Jour, Bact, 36: 433, 1 9 3 8 , 4, Bernheim, F. Jour, Bact, 41: 387, 1941, 5 . Callow, A,B, Biochem, Jour, 18: 507, 1924, 6. C l i f t o n , C.E. Enzymologia 4 : 246, 1937. 7« C l i f t o n , C.E. and Logan, W.A. Jour. Bact. 37: 3 2 3 , 1 9 3 9 , 8o Cook, R.P. and Stephenson, M. Biochem. Jour. 22: 1368 s 1928. 9 • Dixon, M. \"Manoinetric Methods\", Cambridge Univ. Press, 1934, 1 0 . Eagles, B.A. and Sadler, W. Can* Jour. Res. 7* 364, 1932. 11 . F a r r e l l , M.A. Jour, Bact, 29: 411, 1 9 3 5 . 1 2 . Hansen, P.A. Sent, fur Bact. 9 8 : 2 8 9 , 1938. 1 3 . Hunt, G.A, Jour. B a c t . 26: 341, 1 9 3 3 , 14. Ingram, M, Jour. Bact, 38: 5 9 9 , 1939, 15 . Lineweaver, Hans Jour. B i o l , Chem. 99? 5 7 5 , 1933. 16. Morgan, J.F., Eagles, B.A,, and L a i r d , D.G. 17. Sadler, W., Eagles, B.A., and Pendray, G. Can. Jour. Res, 7= 3 7 0 , 1 9 3 2 . 18. Wilson, P.W. Jour. Bact. 3 5 : 6 0 1 , 1938, ABSTRACT The aerobic o x i d a t i o n , dehydrogenation and l a c t i c a cid production from twenty-five carbohydrates by two s t r a i n s of Strep, l a c t i s have been st u d i e d o I t has been shown that there i s no c o r r e l a t i o n between the aerobic and anaerobic r e s p i r a t i o n of r e s t i n g c e l l suspensions and l a c t i c a c i d fermentation by growing cultures of these organisms. I t has been suggested that t h i s apparent d i f f e r e n c e between the mechanisms of r e s p i r a t i o n and fermentation may be due to the infl u e n c e of nitrogen sources i n the growth medium. INTRODUCTION The r e s p i r a t o r y enzyme systems of many b a c t e r i a l species have been i n v e s t i g a t e d , Q,uastel and Whetham ( l l , 12) studied the dehydrogenase a c t i v i t y of Bact. c o l i upon organic a c i d s , a l c o h o l s , amino acids and carbo-hydrates. Kendall (7) reported upon the dehydrogenase mechanisms of Bact. c o l i and a group of other c l o s e l y -r e l a t e d species. The aerobic oxidative mechanism of Bact. c o l i has been studied by Cook and Stephenson (2)• E a r r e l l (5) reported upon the r e s p i r a t o r y enzyme systems of the Stre p t o c o c c i under aerobic and anaerobic c o n d i t i o n s . Morgan, Eagles, and L a i r d (8) surveyed the dehydrogenase enzyme a c t i v i t y of fourteen s t r a i n s of l a c t i c acid b a c t e r i a , and l a t e r (?) reported the aerobic oxidase a b i l i t y of Strep, l a c t i S . Although aerobic and anaerobic r e s p i r a t o r y enzymes have been e x t e n s i v e l y i n v e s t i g a t e d , there has been l i t t l e attempt to r e l a t e r e s p i r a t o r y a c t i v i t y with fermentative a b i l i t y . K endall and Ishikawa (6), studying a large group of b a c t e r i a l species, reported that the reduction of methylene blue by r e s t i n g b a c t e r i a i n the presence of c e r t a i n carbo-hydrates was p r e c i s e l y p a r a l l e l e d by the fermentation of these same carbohydrates i n cult u r e media, Barron and Eriedemann (1) studied a group of b a c t e r i a which were unable to ferment glucose and demonstrated an appreciable oxidase a c t i v i t y upon t h i s carbohydrate. The work reported upon herein represents a comparison of the aerobic o x i d a t i o n , anaerobic oxidation and l a c t i c acid fermentation of Strep, l a c t i s upon twenty-five carbohydrates, EXPERIMENTAL METHODS The c u l t u r e s employed i n t h i s study were Strep, l a c t i s S.,A, JO ( 1 3 ) and Strep, l a c t i s A.T.C. 3 7 4 , the former i s o l a t e d from butter possessing a caramel f l a v o r , and the l a t t e r obtained from the N a t i o n a l Type Culture C o l l e c t i o n at Washington, D.C. Casein Digest Broth, prepared a f t e r the manner of Q r l a -Jensen ( 4 ) , served as the basic medium, Eor sugar fermenta-t i o n s , 2$ carbohydrate was incorporated, while f o r r e s p i r a t o r y studies the broth was enriched with 1 $ Difco yeast e x t r a c t , • 0 , 5 $ K 2 H P O 4 , and 0 , 5 $ glucose© Respiratory enzyme studies were c a r r i e d out employing \" r e s t i n g c e l l \" suspensions prepared and standardized as reported by Morgan, Eagles and L a i r d (8). Aerobic o x i d a t i v e a b i l i t y was measured manometrically with, the Barcroft apparatus, as d e t a i l e d by Dixon (3) . Dehydrogenase a c t i v i t y was determined by the Thunberg technique, f o l l o w i n g the procedure described by Morgan, Eagles and L a i r d (8) . Fermentation of carbohydrates was measured by t i t r a t i n g fourteen-day cu l t u r e s i n sugar broths with N/4 sodium hydroxide, using phenolphthalein as i n d i c a t o r . A f t e r subtracting that of the c o n t r o l , the remaining values were converted into grams of l a c t i c a c i d per liter® EXPERIMENTAL RESULTS The aerobic oxygen uptake, dehydrogenase a c t i v i t y and l a c t i c a c i d production by Strep* l a c t i s SSA„ 30 and Strep, l a c t i s A.T.C. 374 upon twenty-five carbohydrates are presented i n Table 1. Although these values f u r n i s h d e t a i l e d information about the r e s p i r a t o r y enzyme a c t i v i t y and fermentative a b i l i t y of these two s t r a i n s , d i s t i n c t l y d i f f e r e n t standards have been employed as the bases of these three types of determination, These r e s u l t s , t h e r e f o r e , cannot be studied comparatively u n t i l some common basis of i n t e r p r e t a t i o n has been evolved. The values of the Q,02 have accordingly been r e c a l c u l a t e d as percentage of the glucose o x i d a t i o n value taken as 100, while the l a c t i c acid production i n grams per l i t e r has l i k e w i s e been converted into percentage of the acid produced from glucose. Since dehydrogenase a c t i v i t y had already been expressed as percentage of glucose reduction time, a l l three determina-t i o n s are now based upon one general comparative standard, c a l l e d the Respiratory C o e f f i c i e n t s These r e c a l c u l a t e d values are presented i n Table 2. The data recorded i n Table 2 i n d i c a t e d e f i n i t e l y that there i s no c o r r e l a t i o n between aerobic r e s p i r a t i o n , anaerobic r e s p i r a t i o n and l a c t i c a c i d -production with the two s t r a i n s of Strep, l a c t i s studied. Further, there does not appear to be any r e l a t i o n s h i p between oxid a t i v e a b i l i t y and fermentation, dehydrogenase a c t i v i t y and fermentation, o even between the aerobic and anaerobic o x i d a t i v e mechanisms themselves. The l a c k of c o r r e l a t i o n between aerobic and anaerobic r e s p i r a t o r y enzymes and fermentation i s f u r t h e r emphasized by the r e s u l t s shown i n Figures 1 and 2. In these graphs the comparative enzymic a c t i v i t i e s of each s t r a i n of Strep. l a c t i s upon f i v e selected carbohydrates are portrayed. In Figure 1 the enzymic a c t i v i t y of Strep, l a c t i s S,A.30 upon galactose, sucrose, l a c t o s e , trehalose and mannitol i s shown. The reactions upon galactose and lactose are characterized by strong oxidase a c t i v i t y , weak dehydrogenase a c t i v i t y , and f a i r l y high l a c t i c a c i d pro-duction. This r e l a t i o n s h i p between the three enzymic systems i s the one most fr e q u e n t l y observed among the values reported i n Table 2. A r e v e r s a l of t h i s a c t i v i t y order i s observed i n the case of t r e h a l o s e , where acid production i s higher than oxidase a c t i v i t y , while dehydrogenase a c t i v i t y i s comparatively f e e b l e . With sucrose as. the substrate, an e n t i r e l y d i f f e r e n t p i c t u r e i s obtained. Dehydrogenase a c t i v i t y has become very powerful and i s appreciably greater than oxidase a b i l i t y , while l a c t i c a c i d production remains almost n e g l i g i b l e , Enzymic a c t i v i t y upon mannitol, on the other hand, i s characterized by an extremely high oxidative a b i l i t y , while l a c t i c a c i d production i s feeble and dehydrogenase a c t i v i t y i s e n t i r e l y absent. The enzymic a c t i v i t y of Strep. l a c t i s A.T.C. 374 upon galactose, sucrose, r a f f i n o s e , d e x t r i n and i n u l i n i s shown in Figure 2, With galactose there i s evident a moderate oxidase a b i l i t y , low dehydrogenase a c t i v i t y , and very high l a c t i c a c i d production. With i n u l i n , on the other hand, the exact reverse holds t r u e , oxidase a b i l i t y being low, - 6 -dehydrogenase a c t i v i t y high., and acid production e n t i r e l y l a c k i n g . When'sucrose serves as the substrate, dehydrogenase a c t i v i t y i s very strong, oxidase a b i l i t y f a i r l y strong, and acid production moderates With r a f f i n o s e there i s demonstrable a moderately strong oxidase mechanism, very s l i g h t l a c t i c acid production and no observable dehydrogenase a c t i v i t y . A somewhat s i m i l a r r e l a t i o n s h i p i s found i n the case of d e x t r i n , where there e x i s t s a tremendously strong oxidase a c t i v i t y , together with only feeble dehydrogenase a c t i o n and l a c t i c acid formation. The r e s u l t s recorded i n Table 2 and shown g r a p h i c a l l y i n Figures 1 and 2 o f f e r d e f i n i t e proof that no r e l a t i o n s h i p e x i s t s between the aerobic and anaerobic r e s p i r a t o r y processes and the mechanism of l a c t i c a c i d fermentations However , the fermentative end-products of b a c t e r i a l metabolism may be formed,there must ev i d e n t l y e x i s t some determining mechanism apart from the enzymic processes of aerobic and anaerobic r e s p i r a t i o n . V DISCUSSION • The r e l a t i o n s h i p between ox i d a t i o n and fermentation i s discussed i n d e t a i l by Oppenheimer and Stern (10): \"The time i s past when fermentation and oxidation were considered tq be two quite d i s t i n c t types of b i o l o g i c a l processes and when the enzymes ac t i v e i n fermentation, the zymases, and those a c t i v e i n v i t a l o x i d a t i o n , were treated as e n t i r e l y unrelated* Today we speak of one great system of enzymes c a t a l y z i n g the o v e r - a l l phenomenon of energy production i n the c e l l , termed desmolysis I f the anoxybiontic metabolism does not pass over i n t o o x y b i o s i s , c e r t a i n reactions take place leading to s t a b i l i z a t i o n of the a n a e r o b i c a l l y formed compounds, and l a c t i c a c i d or e t h y l a l c o h o l appear as the end-products e The c o r r e l a t i o n between fermentation and r e s p i r a t i o n , or rather between oxygen tension and fermentation, i s maintained by the s o - c a l l e d Pasteur-Meyerhof r e a c t i o n , the mechanism of which i s s t i l l l a r g e l y obscure. There i s one uniform mechanism operative i n both phases (fermentation and r e s p i r a t i o n ) of desmolysis, namely, the t r a n s f e r of metabolic hydrogen. In anaerobiosis i t terminates i n l a c t i c a c i d or alcohol and i n aerobiosis i t terminates i n water.\" This view of the complete i d e n t i t y of the two processes of o x i d a t i o n and fermentation i s also advanced by Szent-Gyorgyi (14 ) , who pointed out that both aerobic and anaerobic breakdown of sugars proceed through the same i n i t i a l steps, namely, the s p l i t t i n g of hexose-phosphate into t r i o s e - .8 -phosphate and the subsequent dehydrogenation of the r e s u l t i n g three-carbon-compounds. The di f f e r e n c e between these two processes comes i n only at the next.step.-. In oxid a t i o n , molecular oxygen acts as the f i n a l Hydrogen Acceptor of e l e c t r o n s , while i n fermentation the hydrogen i s accepted through an i n t e r n a l rearrangement of the molecules of the o x i d i z a b l e substance. The r e s u l t s reported herein are d i f f i c u l t to r e c o n c i l e with the accepted concept of the i d e n t i t y of the r e s p i r a t o r y and fermentative mechanisms. With the l a c t i c a c i d strepto-cocci i t has been shown that appreciable fermentation of a carbohydrate may occur even when both aerobic and anaerobic o x i d a t i v e mechanisms upon that carbohydrate are very f e e b l e . I t has also been shown that large q u a n t i t i e s of l a c t i c acid may be formed from a carbohydrate agaiast which the organism possesses a strong o x i d a t i v e but n e g l i g i b l e dehydrogenase a c t i v i t y . I t has f u r t h e r been shown that l i t t l e or no l a c t i c acid may be formed when the organism e x h i b i t s a strong o x i d i z i n g and feeble dehydrogenase a c t i v i t y , and even when the organism possesses both a strong o x i d i z i n g and a strong dehydrogenase a c t i v i t y upon a p a r t i c u l a r carbohydrate. These r e s u l t s suggest t h a t , with the l a c t i c a c i d s t r e p t o c o c c i , fermentation by growing cultures of these - 9 -organisms i s an enzymic process which appears to be separate and d i s t i n c t \" f r o m the aerobic and anaerobic r e s p i r a t o r y mechanisms e x h i b i t e d by r e s t i n g c e l l suspensions. The lack of c o r r e l a t i o n between these two metabolic processes may be due to the infl u e n c e of nitrogen source and accessory f a c t o r s contained i n the growth medium. The presence of small amounts of a nitrogen source has been found to exert a pronounced influence upon the r e s p i r a t o r y and fermentative mechanisms of r e s t i n g c e l l suspensions. The r e s u l t s of t h i s study w i l l be presented i n a subsequent paper. - 10 -REFERENCES lo Barron, E.S.G. and Friedemann, T.E. J . B j o l . Chem, 137: 573, 1941. 2» Cook, R.P. and Stephenson, M. Biochem. Jour. 22: 1368 1928. 3. Dixon, M. \"Manometric Methods\", Cambridge Univ e Press, 1934 0 4. Eagles, B.A., and Sadler, I , Can. Jour. Res. 7: 364, 1 9 3 2 . 5* E a r r e l l , M.A. Jour. B a c t . 29: 411, 1935, 6. K e n d a l l , A.I. and Ishikawa, M. J our. Inf e Di s. 44: 2 8 2 , 1929. 7. K e n d a l l , A.I. Jour. I n f . Dis. 47: 186, 1930 8. Morgan, J.F. , Eagles, B.A., L a i r d , D.G 9« Morgan, J.F., Eagles, B.A., L a i r d , D.G. 10. Oppenheimer, C. and Stern, K 0G. \" B i o l o g i c a l Oxidation\" Nordemann, 19 39s 11. Quastel, J.H. and Whetham, M.D. Biochem. Jour. 19: 5 2 0 , 1925. 1 2 . Quastel, J.H. and Whetham, M.D. Biochem. Jour. 19 : 645, 19 2 5 . 1 3 . Sadler, W. .Trans. Roy. Soc. Can. V o l . 20, 1926. 14. Szent-Gyorgyi, A.Y. \"Oxidation, Fermentation, Vitamins, Health and Disease\", Williams and W i l k i n s , 1939. ABSTRACT The infl u e n c e of carbohydrate adaptation upon the subsequent dehydrogenase a c t i v i t y and fermentative a b i l i t y of r e s t i n g c e l l suspensions has been studied with two s t r a i n s of Strep, l a c t i s . I t has been shown that dehydrogenase a c t i v i t y v a r i e s markedly i n response to carbohydrate changes i n the growth medium. D i r e c t adaptation by dehydrogenase enzymes and also more general eases of s t i m u l a t i o n and i n h i b i t i o n of dehydrogenation have been shown to occur. The adaptive and c o n s t i t u t i v e nature of the fermentative enzymes of Strep, l a c t i s has been determined. I t has been shown, by adaptive and c o n s t i t u t i v e enzymes, that fermentation of lactose by Strep, l a c t i s must occur through a pr e l i m i n a r y h y d r o l y s i s to glucose and galactose. The r e l a t i o n s h i p of these phenomena to the mechanism of adaptation has been discussed. INTRODUCTION The dehydrogenase a c t i v i t y of the L a c t i c Acid S t r e p t o c o c c i has not as yet been f u l l y i n v e s t i g a t e d . F a r r e l l (5) reported upon the r e s p i r a t o r y mechanism of 32 s t r a i n s of s t r e p t o c o c c i , i n c l u d i n g Strep, l a c t i s and Strep, f e c a l i e . K a t a g i r I and Kitahara (6) showed the presence of l a c t i c a s i d dehydrogenase among several species of l a c t i c a c i d b a c t e r i a . Wood and Gunsalus (14) studied the various f a c t o r s i n f l u e n c i n g the production of a c t i v e r e s t i n g c e i l suspensions of the Group B Streptococci Morgan, Eagles and L a i r d (7) surveyed the dehydrogenase a c t i v i t y of a group of fourteen s t r a i n s of l a c t i c a c i d s t r e p t o c o c c i . Hegarty (5),and Rahn, Hegarty and D u e l l (9), studied l a c t i c ac id pr oduct ion by washed c e l l suspensions of Strep, l a c t i s , T h e y demonstrated that the enzymes at t a c k i n g glucose, mannose and fructose were c o n s t i t u t i v e , while those a t t a c k i n g a l l other sugars were adaptive. The f i r s t observation of the phenomenon of adaptive and c o n s t i t u t i v e enzymes was made by Wor tmann (15) i n the case of starch h y d r o l y s i s by an unknown b a c t e r i a l species. The modern terminology of •'adaptive\" and \" c o n s t i t u t i v e \" enzymes was introduced by Karstrom, Virtanen and t h e i r associates (15). The influence of adaptation upon b a c t e r i a l enzymes was studied by 1 Stephenson and S t i c k l a n d (11) i n the case of the hydrogenlyases, Haines ( 4 ) with b a c t e r i a l gelatinase, Stephenson and Gale (12) with Bact, c o l i , and Quaste 1 (8) with M» l y s o d e i k t i c us. The work reported upon herein was undertaken with the object of determining the influence exerted by adaptive and c o n s t i t u t i v e enzymes upon the mechanisms of dehydrogen-a t i o n and l a c t i c a c i d fermentation by Strep. l a c t i s , , EXPERIMENTAL METHODS The c u l t u r e s employed i n t h i s study consisted of Strep, l a c t i s S.A. 30 (10), i s o l a t e d from butter posses-sing a caramel f l a v o r , and Strep, l a c t i s A TO 374, obtained from the National Type- Culture c o l l e c t i o n . Casein Digest Broth, prepared after the manner of Orla-Jensen (2), served as the basic medium and was en-r i c h e d with 0 . 5 $ K 2 H P O 4 and 1.0$ Difco yeast e x t r a c t . To t h i s basic medium various carbohydrates were added to a concentration of 0 . 5 $ . For the measurement of r e s p i r a t o r y enzyme a c t i v i t y and l a c t i c a c i d production \" r e s t i n g c e l l \" suspensions of Strep, l a c t i s were prepared and standardized as described by Morgan, Eagles, and L a i r d ( 7 ) , L a c t i c a c i d production was measured by t i t r a t i n g samples of a r e s t i n g c e l l , carbo-hydrate, b u f f e r , peptone mixture at regular i n t e r v a l s over a s i x hour period, f o l l o w i n g the method employed by Hegarty ( 5 ) . Dehydrogenase a c t i v i t y was determined by the Thunberg technique, under the experimental conditions previously described by Morgan, Eagles and L a i r d (7). EXPERIMENTAL RESULTS A. Adaptation and Dehydrogenation The carbohydrate dehydrogenating a c t i v i t y of r e s t i n g c e l l suspensions of Strep, lac t i s SA s o prepared from c e l l s grown i n the presence of various carbohydrates i s recorded i n Table 1. The r e s u l t s reported i n this table show that the presence of s p e c i f i c caibohydrates i n the growth medium has exerted a pronounced influence upon the subsequent de-hydrogenating a b i l i t y of r e s t i n g c e l l suspensions prepared therefrom. There i s apparent a marked v a r i a t i o n i n carbo-hydrate dehydrogenations i n response to change i n the carbon source of the growth medium© This v a r i a t i o n i s emphasized by the r e s u l t s shown i n Figure 1. In t h i s graph the r e l a t i v e dehydrogenase a c t i v i -t i e s upon arabinose, l a c t o s e , maltose, r a f f i n o s e and starch are compared, using r e s t i n g c e l l s obtained from glucose, l a c t o s e , s t a r c h and mannitol broths. The stimulatory e f f e c t of previous growth i n the homologous substrate i s shown i n the case of la c t o s e . Growth i n lactose broth has increased the dehydrogenase a c t i v i t y upon lactose fourteen time over the a c t i v i t y of c e l l s grown i n glucose broth. I t i s also noticeable that growth i n lactose broth has markedly lengthened the times required for dehydrogen-a t i o n , as recorded i n Table I , but at the same time has increased the strength of the r e l a t i v e reducing c o e f f i c i e n t s . The i n f l u e n c e of the carbohydrate i n the growth medium upon subsequent dehydrogenations i s very c l e a r l y shown i n the case of arabinose and r a f f i n o s e . With both these sugars dehydrogenation f a i l s to occur when c e l l s are obtained from glucose broth, but occurs very r e a d i l y when c e l l s are obtained from'lactose broth. The reverse, of t h i s phenomenon i s shown with maltose and s t a r c h . With these carbohydrates dehydrogenation takes place vith. c e l l s prepared from glucose l a c t o s e , or starch broth, but does not take place with c e l l s prepared from mannitol broth. The carbohydrate dehydrogenating a c t i v i t y of sus-pensions of Strep, l a c t i s A TO 374 prepared from c e l l s grown i n glucose, l a c t o s e , and mannitol broths i s recorded i n Table I I . The r e s u l t s shown i n t h i s table compare c l o s e l y with those recorded for Strep. l a c t i s SA 30 i n Table I . I t i s apparent with both these s t r a i n s that the carbohydrate i n the growth medium has markedly influenced the dehydrogenase a c t i v i t y of the subsequent r e s t i n g c e l l suspensions. This in f l u e n c e i s further i l l u s t r a t e d i n Figure 2, i n which the comparative dehydrogenations of mannose. galactose, l a c t o s e , mannitol and r a f f i n o s e are portrayed. The c l e a r e s t example of adaptation i s shown i n the ease of mannitol, suspensions obtained from mannitol broth dehydro genating mannitol twenty-seven times as r a p i d l y as c e l l s obtained from glucose broth. With the mannose dehydrogen ase, there appears to be l i t t l e e f f e c t exerted by the earb hydrate i n the growth medium. In the case of galactose dehydrogenase a c t i v i t y i s apparently increased by growing the c e l l s i n lac t o s e or mannitol broths. With r a f f i n o s e , dehydrogenation does not occur when the c e l l s have been prepared from glucose broth, but does occur when the c e l l s have been prepared from l a c t o s e or mannitol broths, B. A dap ta t i on and Fer men tat ion L a c t i c a c i d production from various carbohydrates by suspensions of Strep, l a c t i s SA 30 i s shown i n Table 3. These r e s u l t s have been obtained by t i t r a t i n g the acid produced by r e s t i n g c e l l suspensions i n phosphate buffer of pH. 7.2 containing 0.3$ peptone, to which has been added 2,0$ of the carbohydrate being tested ( 5). Where the organism possesses a co n s t i t u t i v e , enzyme c o n t r o l l i n g fermentation of the substrate ac i d production w i l l occur at once. However, where the organism possesses an adaptive enzyme for\" the carbohydrate under study, a considerable period of time w i l l elapse before a c i d i s produced i n any appreciable quantity. This d i s t i n c t i o n between adaptive and c o n s t i t u t i v e enzymes i s c l e a r l y shown i n Figure 3. In t h i s graph l a c t i c a c i d production from the four monosaccharides i s shown, employing r e s t i n g c e l l suspensions-obtained from glucose broth. I t i s apparent from the graph that Strep, l a c t i s SA 30 possesses c o n s t i t u t i v e enzymes for glucose, fructose and miannose, but an adaptive enzyme for galactose. The influence of adaptive and c o n e t i t u t i v e enzymes upon the fermentation of lactose by suspensions of Strep, l a c t i s SA 30 i s shown i n Figure 4. C e l l s obtained from glucose broth or galactose broth show p r a c t i c a l l y no fermentative a b i l i t y when placed i n the presence of l a c t o s e . When c e l l s which have been grown i n lactose broth, however, are placed i n la c t o s e there i s a r a p i d and heavy production of l a c t i c a c i d . This would in d i c a t e that growth i n l a c t o s e broth has stimulated the enzyme c o n t r o l l i n g l actose fermentation, which must therefore be an adaptive enzyme* The r e l a t i o n s h i p of adaptation to the fermentation of galactose by suspensions of Strep, l a c t i s 3A 30 i s shown i n f i g u r e 5. With, t h i s sugar i t would appear that growth i n lactose broth has stimulated the adaptive enzyme f e r -menting galactose, although growth i n galactose broth has not .caused a corresponding stimulation,, When Strep, l a c t i s ATC 374 was employed as the t e s t organism, rather than Strep, l a c t i s SA30, e s s e n t i a l l y s i m i l a r r e s u l t s were obtained. I t was found that, i n the fermentation of the monosaccharides, t h i s organism also possessed c o n s t i t u -t i v e enzymes f o r glucose, mannose and f r u c t o s e , and an adaptive enzyme f o r galactose. However, an important difference between these two s t r a i n s was observed i n the fermentation of l a c t o s e . The influence of adaptation upon the fermentation of t h i s disacchar.de by suspensions of Strep, l a c t i s ATC 374 i s shown i n Figure 6. I t i s apparent from t h i s graph that the organism possesses a c o n s t i t u t i v e enzyme f o r lactose fermentation, since c e l l s obtained from glucose broth strongly ferment l a c t o s e , and since growth i n lactose broth does not increase e i t h e r the r a t e or the amount of ac i d production from l a c t o s e . On the other hand, although lactose i s fermented by cell's obtained from glucose broth there i s l i t t l e fermentation by c e l l s obtained from galactose br o th . • A further i n t e r e s t i n g r e l a t i o n s h i p i s found i n Figure 7 which shows the influence of adaptation uponthe fermentation of galactose by suspensions of Strep . l a c t i s ATC 374. In t h i s graph l a c t i c a c i d production from galactose i s shown to occur when c e l l s are prepared from galactose broth or from l a c t o s e broth, but not when the c e l l s are obtained from glucose broth . This i s further proof that the fermentation of galactose i s c a r r i e d out by an adaptive enzyme. . However, i t would appear that t h i s adaptive enzyme for galactose fermentation has also been stimulated by growth i n l a c t o s e broth. DISCUSSION The r e s u l t s obtained with the two s t r a i n s of Strep, l a c t i s studied show c l e a r l y that the carbohydrate present i n the growth medium exerts a pronounced e f f e c t upon both the dehydrogenase a c t i v i t y and the fermentative a b i l i t y of r e s t i n g c e l l suspensions. The r e s u l t s recorded i n Tables 1 and 3 and portrayed i n Figures 1 and 3 make i t evident that there i s an extreme v a r i a t i o n i n dehydrogenase a c t i v i t y i n response to various carbohydrates i n the growth medium. I t i s apparent that growing these organisims i n lactose or mannitol broth very g r e a t l y increases the a b i l i t y of c e l l suspensions to de-hydrogenase these substrates. I t would appear, therefore, that these dehydrogenase enzymes,are adaptive i n character. A p a r t from these i n s t a n c e s of a d a p t a t i o n i n response to the presence of \"She homologous carbohydrate there occur other v a r i a t i o n s i n dehydrogenase a c t i v i t y which cannot be e x p l a i n e d on the b a s i s of simple a d a p t a t i o n . An i l l u s t r a -t i o n of such phenomena i s found i n the dehydrogenation of r a f f i n o s e . With both s t r a i n s of S t r e p , l a c t i s dehydrogena-t i o n of r a f f i n o s e does not occur i f the c e l l s have p r e v i o u s l y been grown In glucose b r o t h , but does occur i f the c e l l s are o b t a i n e d from media c o n t a i n i n g other c a r b o h y d r a t e s . I t would appear t h a t g l u c o s e i n the growth medium e x e r t s an i n h i b i t o r y e f f e c t upon the r a f f i n o s e dehydrogenase enzyme of subsequent su s p e n s i o n s . F u r t h e r examples of s i m i l a r phenomena are the i n a b i l i t y o f suspensions of S t r e p , l a c t i s SA 30 to dehydrogenat© maltose and s t a r c h i f the c e l l s have been grown i n m a n n i t o l b r o t h , and the appearance of a s t r o n g dehydrogenase a c t i v i t y upon arabinoe/e when the organism has been p r e v i o u s l y grown i n l a c t o s e b r o t h * These r e s u l t s do not appear to agree w i t h the o b s e r v a t i o n s o f Dubss ( 1 ) , who has emphasized the extreme s p e c i f i c i t y of a d a p t i v e enzymes. The r e s u l t s r e c o r d e d i n Table 3 and p o r t r a y e d i n F i g u r e s 3,4,5,6 and 7 emphasize the i n f l u e n c e of carbo-h y d r a t e a d a p t a t i o n upon the f e r m e n t a t i v e a b i l i t y of r e s t i n g c e l l s u s p e n s i o n s . I t has been shown t h a t both s t r a i n s of S t r e p , l a c t i s p ossess c o n s t i t u t i v e .enzymes f o r the fermenta-t i o n o f g l u c o s e , mannose and f r u c t o s e , but an a d a p t i v e -10-\"enzyme f o r the f e r m e n t a t i o n of g a l a c t o s e . T h i s had prev-i o u s l y been demonstrated with S t r e p , l a c t i s by Hegarty ( 5 ) . In c o n t r a d i c t i o n to h i s r e s u l t s , however', i t has been shown t h a t one of the s t r a i n s , S t r e p , l a c t i s ATC 374. possesses a c o n s t i t u t i v e enzyme for the f e r m e n t a t i o n of l a c t o s e . With both s t r a i n s of S t r e p , l a c t i s i t has a l s o been shown t h a t growth i n l a c t o s e b r o t h r e s u l t s ' i n the adapta-t i o n to both l a c t o s e and g a l a c t o s e , w h i l e growth i n g a l a c -tose b r o t h does not cause a d a p t a t i o n to l a c t o s e . This r e s u l t has been o b t a i n e d w i t h two s t r a i n s of S t r e p , l a c t i s , one of which has been shown to c o n t a i n an a d a p t i v e and one a c o n s t i t u t i v e enzyme f o r l a c t o s e . This would seem to f u r n i s h d e f i n i t e evidence t h a t w i t h S t r e p , l a c t i s the f e r m e n t a t i o n of l a c t o s e proceeds through a p r e l i m i n a r y h y d r o l y s i s to i t s c o n s t i t u e n t monosaccharides, g l u c o s e and g a l a c t o s e . W i t h S t r e p , l a c t i s SA 30 i t was a l s o n o t i c e d that a d a p t a t i o n to g a l a c t o s e was o b t a i n e d by growing the organism i n the presence of l a c t o s e , but not i n the presence of g a l a c t o s e . This would i n d i c a t e t h a t g a l a c t o s e formed through the h y d r o l y s i s of l a c t o s e i n the medium i s more a c t i v e i n c a u s i n g a d a p t a t i o n than g a l a c t o s e added as such to the medium. The r e l a t i o n s h i p of these r e s u l t s to the mechanism of a d a p t i v e and c o n s t i t u t i v e enzymes i s s t i l l obscure. I t i s b e l i e v e d t h a t a c o n s t i t u t i v e enzyme e x i s t s as an -11-i n t e g r a l part of the c e l l s t r u c t u r e and i s therefore always pre sent i n the c e l 1 i n an a c t i v e form. An adaptive enzyme on the other hand, i s b e l i e v e d to e x i s t i n the c e l l i n an i n a c t i v e state and to require s t i m u l a t i o n or adapta-t i o n through contact with the homologous substance before enzymic a c t i v i t y , can be demonstrated. This viewpoint, as advanced by Yirtanen (13), has been considered inadequate by Dubos (1), Quastel (8), and Haines (4), who emphasized the complex influences which enviromental f a c t o r s may exert on the enzymic c o n s t i t u t i o n of the m i c r o b i a l c e l l . The r e s u l t s reported herein with Stre p „ l a c t i s f u r n i s h f u r t h e r evidence of the important influence which the c o n s t i t u t i o n of the culture medium exerts upon the enzymic s t r u c t u r e of the b a c t e r i a l c e l l . -12-REFERENCES 1. Dubps, R.J. - Bacter. Rev. 4: 1, 19 40 2. Eagles, B. A. and Sadler, W. - Can. Jour. Res. 7:364 1933 3. F a r r e l l , M.A. - Jour. Bact. 29: 411, 1935. 4. Haines, R.B. - Biochem, Jour. 37: 466, 1933. 5. Hegarty, C P . - Jour Bact. 37: 145, 1939 . 6. K a t a g i r i , H. and Kitahara, K. - Biochem, Jour* 32: 1654, 1938. 7. Morgan, J.F., Eagles, B.A., L a i r d , D.G. - In p u b l i c a t i o n . 8. Quastel, J.Ho - Enzymologia 2:37, 193 7. 9. Rahn, 0;, Hegarty, C.P. , D u e l l , R.E. - Jour. ,Bact. 35: 547, 1938. 10. Sadler, W. - Trans. Roy. Soc. Canada, Vol.30, 1926. 11. Stephenson, M. and Stickla-nd, L.H. * Biochem, Jour. 26 : 712, 1932. 13, Stephenson, M. and Gale, E.F. - Biochem, Jour. 31: 1311, 1937. 13. Yirtanen, A.I. - Jour. Bact. 28: 447, 1934, 14. Wood, A.J. and Gunsalus, I.C. - Jour. Bact. 44: 333 1942. 15. W ortmann, J . - Quoted from Dubos, R.J. - Bact. Rev. 4:1, 1940 * ABSTRACT The s t i m u l a t i n g e f f e c t of f i f t e e n nitrogen sources upon the fermentation and r e s p i r a t i o n of glucose by washed c e l l suspensions of Strep, l a c t i s has been studied. I t has been shown that fermentation and r e s p i r a t i o n by c e l l s of Strep, l a c t i s are regulated, not by aerobic or anae-robic c o n d i t i o n s , but by the presence i n the buffer mixture of small amounts of various nitrogenous compounds. The r e l a -t i o n s h i p of t h i s phenomenon to the Pasteur e f f e c t i s discussed I t has been shown that aerobic and anaerobic l a c t i c acid formation are stimulated to approximately the same degree by these d i f f e r e n t nitrogen sources, but that the oxygen uptake i s stimulated i n a d i s t i n c t l y d i f f e r e n t manner. The r e l a t i v e s t i m u l a t o r y a c t i o n of d i f f e r e n t types of nitrogen sources has been compared, and the question of accessory f a c t o r s f o r fermentation and r e s p i r a t i o n discussed. Independent s t i m u l a t i o n of the r e s p i r a t o r y and fermen-t a t i v e mechanisms has been demonstrated, and the p o s s i b i l i t y of separate enzymic systems postulated. INTRODUCTION The fermentation reactions of many species of l a c t i c acid s t r e p t o c o c c i have been i n v e s t i g a t e d . Orla-Jensen (18) and Orlai-Jensen and Hansen (l?) studied l a c t i c acid production from carbohydrates by species and s t r a i n s of s t r e p t o c o c c i , i n c l u d i n g Strep, l a c t i s . They emphasized the v a r i a b i l i t y of such r e a c t i o n s and suggested the formation of sub-groups on t h i s b a s i s . Such sub-group formation, however, was opposed by Ayers, Johnson and Mudge (1), by Hammer and Baker (? ) , and by Sherman (24), who c l a s s i f i e d the true l a c t i c acid strep-t o c o c c i as Strep, l a c t i s and Strep, cremoris. The r e s p i r a t o r y enzyme a c t i v i t y of the l a c t i c acid strep-t o c o c c i upon a v a r i e t y of substrates has also been i n v e s t i g a t e d . F a r r e l l (8) reported upon the r e s p i r a t o r y mechanism of 22 s t r a i n s of s t r e p t o c o c c i i n c l u d i n g Strep. l a c t i s and Strep. cremoris. Morgan, Eagles and L a i r d (13) surveyed the dehyd-rogenase a c t i v i t y of fourteen s t r a i n s of l a c t i c a c i d b a c t e r i a and also (14) studied the oxidative a b i l i t y of two s t r a i n s of Strep, l a c t i s upon carbohydrate substrates. Although the l a c t i c a c i d production by growing cul t u r e s and the r e s p i r a t o r y a c t i v i t y of r e s t i n g c e l l suspensions of st r e p t o c o c c i have both been studied i n the presence of various carbohydrates, the r e l a t i o n s h i p of r e s p i r a t i o n to fermentation i s s t i l l l a r g e l y obscure with t h i s group of microorganisms. Rahn, Hegarty and Duell (20) and Hegarty (10) determined l a c t i a cid production by washed c e l l s of Strep, l a c t i s i n the presence of glucose. They found l a c t i c a c i d production to be dependent upon the presence of small amounts of peptone i n the buffer mixture. L a t e r , Rahn and Hegarty (21) showed that t h i s was due to the presence of accessory f a c t o r s , of which the most act ive were ascorbic a c i d and n i c o t i n i c a c i d . Morgan, Eagles and L a i r d (15) found no c o r r e l a t i o n between the amount of l a c t i c a c i d produced i n cult u r e and the aerobic and anae-rob i c r e s p i r a t o r y a c t i v i t y of suspensions of Strep, l a c t i s upon various carbohydrates. Smith and Sherman (25) studied the fermentation a b i l i t y of 151 cultures of s t r e p t o c o c c i and determined the percentage of l a c t i c a c i d produced from glucose They obtained almost complete u t i l i z a t i o n of the carbohydrate even i n the absence of a nitrogen source. The work reported upon herein was undertaken with the object of determining the influence which various nitrogen sources exert upon the r e s p i r a t i o n and fermentation of r e s t i n g c e l l suspensions of Strep, l a c t i s . I t was hoped that such an i n v e s t i g a t i o n would c l a r i f y the lack of r e l a t i o n s h i p between r e s p i r a t i o n and fermentation with t h i s group of organisms. EXPERIMENTAL METHODS. The c u l t u r e s employed i n t h i s study were Strep, l a c t i s S.A. JO (22), i s o l a t e d from butter possessing a caramel f l a v o r and Strep, l a c t i s ATC 374, obtained from the Na t i o n a l Type Culture C o l l e c t i o n at Washington, D. C. Casein Digest Broth, prepared a f t e r the manner of Orla-Jensen ( 7 ) , and enriched with 0.5% K 2 H P O 4 , 0.5% glucose and 1% Difco yeast e x t r a c t , served as the basic growth medium. Resting c e l l suspensions of Strep, l a c t i s were prepared from . 1 8 - 2 0 hour c u l t u r e s i n t h i s medium by c e n t r i f u g i n g , washing tw i c e , and resuspending i n phosphate buffer to give a c e l l concentration of 1% by volume, as measured by the Hopkins Vac-cine Tube method ( 1 J ) . These suspensions were found to con-t a i n 2 , 5 0 0 , 0 0 0 , 0 0 0 c e l l s per cu. ml. as measured by plate counts. L a c t i c a c i d production under aerobic conditions was measured by the procedure described by Rahn, Hegarty and Duell ( 2 0 ) . For anaerobic studies the procedure as o u t l i n e d by these workers was modified. The c e l l suspension, suspended i n a buffer mixture containing 2% glucose and 0 . 5 o f the nitrogen source being t e s t e d , was placed i n a 5 0 0 cc. suction f l a s k and the whole evacuated. A f t e r evacuation the contents of the f l a s k were adjusted back to atmospheric pressure with nitrogen and the f l a s k clamped o f f . Methylene blue was added as necessary through a dropping funnel and samples f o r t i t r a -t i o n were removed by sucti o n through a c a p i l l a r y tube. A l l samples from both aerobic and anaerobic experiments were t i t -r ated to pH 7 . 0 with K / 1 0 sodium hydroxide, employing a Beck-ma nn pH. meter. PH measurements were also recorded on a l l samples fo t i t r a t i o n and i t was found that the pH decreased i n a uniform manner as l a c t i c acid formation proceeded. In general, there-f o r e , these t e s t s have-been c a r r i e d out i n a highly-buffered phosphate s o l u t i o n under acid conditions. That such conditions should be optimum was in d i c a t e d by Niven and Smiley { 1 6 }, who showed that under a c i d conditions l a c t i c acid was the predomi-nating product of the fermentation of glucose by growing cult u r e s of Strep, l i q u e f a e i e n s , while under a l k a l i n e conditions formic a c i d and a c e t i c a c i d and e t h y l a l c o h o l would be produced i n large q u a n t i t i e s . R e s p i r a t i o n studies were c a r r i e d out manometrically with the Bareroft respirometer, as described by Dixon (6). The nitrogen sources employed i n t h i s study consisted of ammonium c h l o r i d e , ammonium s u l f a t e , sodium n i t r a t e , urea, u r i c a c i d , 1 g l y c i n e , 1 c y s t i n e , 1 asparagine, tryptone, pep-tone D i f c o , peptone Wltte, proteose peptone, sodium caseinate, beef e x t r a c t and yeast e x t r a c t D i f c o . P r e l i m i n a r y studies c a r r i e d out with suspensions of Strep«. l a c t i s S.A. 3 0 i n d i c a t e d that l a c t i c a c i d production increased d i r e c t l y with i n c r e a s i n g concentration of peptone. This r e s u l t i s i n agreement wi t h the work of Hirsch (II) who showed that a c i d production by washed c e l l s of E. c o l i i n the presence of glucose increased d i r e c t l y with increasing peptone concen-t r a t i o n . On the basis of t h i s preliminary study i t was decided to employ 0.5°/. concentrations of a l l nitrogen sources under c o n s i d e r a t i o n . Since i t was also observed that c e l l s of Strep, l a c t i s A.T.C. 3 7 4 gave a s l i g h t l y higher l a c t i c a c i d - 5 ~ production than those of Strep, l a c t i s S.A. 3 0 , r e s t i n g c e l l suspensions of the former s t r a i n were employed i n the subse-quent experiments. EXPERIMENTAL RESULTS.. The aerobic and anaerobic l a c t i c acid production and the oxygen uptake a f t e r f i v e hours i n the presence of various nitrogen sources are presented i n Table 1. A l l values ex-pressed i n t h i s t a b l e are c a l c u l a t e d on the common basis of 0 . 5 $ nitrogenous compound. The r e s u l t s recorded i n Table 1 show comparatively the degree of s t i m u l a t i o n i n response to the presence of these various nitrogen sources i n the r e a c t i o n mixture. I t i s noticeable that under aerobic conditions i n the absence of nitrogen source no l a c t i c a c i d i s formed from glucose f whereas under anaerobic conditions an appreciable amount i s elaborated. This format ion of l a c t i c a c i d from glucose i n the absence of nitrogen source i s i n agreement with the reported r e s u l t s of Smith and Sherman ( 2 5 ) . In contrast to the data presented i n the present paper, however, these workers obtained a s i m i l a r a c i d product ion under aerobic conditions as w e l l . L a c t i c a c i d formation under aerobic and anaerobic con-d i t ions i n response to the s t i m u l a t i o n of f i v e nitrogen sources i s shown g r a p h i c a l l y i n Figures l a and l b . In Figure l a i s portrayed the acid production under aerobic c o n d i t i o n s , while t h a t produced under anaerobic conditions i s shown i n Figure l b . From the r e s u l t s shown i n these graphs i t i s apparent that l a c t i c a c i d production i s very generally s i m i l a r under aerobic and anaerobic c o n d i t i o n s . I t i s noti c e a b l e , however, that a c i d production under aerobic conditions i s i n every case somewhat greater than the acid produced under anaerobic con-d i t i o n s . In general, the di f f e r e n c e i s not very marked and the curves f o r a c i d production under these two conditions are almost i d e n t i c a l . The only exception to t h i s r u l e i s to be noted i n the case of asparagine, where nearly twice as much acid i s produced under anaerobic as under aerobic conditions. No explanation has been found to account for t h i s i r r e g u l a r i t y . I t was also noticed that l a c t i c a c i d production under anaerobic conditions was stimulated e q u a l l y w e l l whether or not methylene blue was added to the re a c t i o n mixture to serve as a Hydrogen Acceptor. This would i n d i c a t e e i t h e r that l a c t i c a c i d production i s independent of the dehydrogenase enzyme system or that the Nitrogen Source i t s e l f functions as a Hydro-gen Acceptor which i s at l e a s t as e f f e c t i v e as the methylene blue. The st imulat ion of aerobic l a c t i c a c i d product ion from glucose under the in f l u e n c e of various nitrogen sources i s fur t h e r shown i n Figure 2, a l l values being c a l c u l a t e d on the basis of 0,51» nitrogenous compound. The r e s u l t s portrayed i n t h i s graph i n d i c a t e that there i s an equal s t i m u l a t i o n of fermentation whether urea, u r i c a c i d , ammonium c h l o r i d e , ammonium s u l f a t e , sodium n i t r a t e , g l y c i n e , asparagine, or beef extract serves as the nitrogen source. However, when tryptone, peptone, or proteo.se peptone serves as the nitrogenous compound there i s a markedly greater a c i d production. This s t i m u l a t i o n of fermentation by simple nitrogenous compounds i s i n some agreement with the work of Smythe ( 2 6 ) , who reported that various t i s s u e extracts stimu-l a t e the anaerobic fermentation of glucose by Baker's yeast, but that the a c t i v e agent i n these stimulations was ammonium c h l o r i d e . The stimulatory e f f e c t of nitrogenous compounds upon the r e s p i r a t i o n of washed c e l l suspensions of Strep, l a c t i s i s shown i n Figure 3• Here again, i t i s apparent that the more complex organic compounds, such as tryptone, beef e x t r a c t , and proteose peptone, have caused a f a r greater s t i m u l a t i o n of r e s p i r a t i o n than have the simpler compounds such as urea, g l y c i n e , and sodium n i t r a t e . Comparison of the r e s u l t s shown i n Figure 2 and 3 reveal that there i s a f a i r l y general agreement between the extent of a c i d production and the s t i m u l a t i o n of oxygen uptake. A very noticeable exception, however, i s found i n the case of beef e x t r a c t . This compound gives only a moderate s t i m u l a t i o n of a c i d production but a very marked s t i m u l a t i o n of oxygen uptake. In Figure 4 the r e l a t i v e s t i m u l a t i o n of aerobic and anaerobic l a c t i c a c i d production and r e s p i r a t i o n by various nitrogen sources i s shown. These r e s u l t s are based on the common value of 0.5$ nitrogenous compound. The comparative values shown i n Figure 4 i n d i c a t e t h a t , i n most cases, aerobic and anaerobic fermentation are stimu-l a t e d to the same extent by various nitrogen sources. Res-p i r a t i o n , however, i s stimulated to an e n t i r e l y d i f f e r e n t degree by these nitrogenous compounds. With the majority of the substances tested r e s p i r a t i o n i s stimulated to a much l e s s e r extent than fermentation. This appears to be true when the simpler chemical compounds are employed. With the amino acids and various enzymic digests other complicating f a c t o r s are encountered. In the case of c y s t i n e , r e s p i r a t i o n Is stimu-l a t e d more than a c i d production, while with asparagine anaerobic l a c t i c a c i d formation i s stimulated more than e i t h e r aerobic acid, production or r e s p i r a t i o n . S i m i l a r cases of greater s t i m u l a t i o n of r e s p i r a t i o n than fermentation are found with beef e x t r a c t , yeast e x t r a c t , and, to a l e s s e r extent, sodium caseinate. The reverse of t h i s e f f e c t , however, i s demonstrat-ed with Witte*s peptone. With t h i s compound both aerobic and anaerobic l a c t i c acid' production are high, but r e s p i r a t i o n i s low. I t would appear, th e r e f o r e , on the bases of these r e s u l t s , that the mechanism of r e s p i r a t i o n i s e i t h e r d i s t i n c t from the mechanism of fermentation or, at l e a s t , i s capable of indepen-dent s t i m u l a t i o n by various accessory f a c t o r s . The data presented i n Table 1 and shown g r a p h i c a l l y i n Figures 1, 2, 3 and 4 have been c a l c u l a t e d on the basis of 0.5$ nitrogen source added to the r e a c t i o n mixture. Since the nitrogen content of these compounds i s extremely v a r i a b l e i t - 9 -seemed, possible that rearranging the data on the basis of a common nitrogen content might give further information. Accordingly, the data as presented i n Table 1 have been r e c a l -culated to the basic value of 0.25 grams of nitrogen. These r e c a l c u l a t e d values are presented i n Table 2 . The r e s u l t s reported i n Table 2 are e n t i r e l y t h e o r e t i c a l and i n most cases are much higher than-could be obtained experi-mentally. Comparison of these r e s u l t s with those presented i n Table 1 reveals that there has been a very marked change i n the r e l a t i o n s h i p of the st i m u l a t o r y power of these various compounds. I t i s noticeable from the nitrogen contents recorded i n Table 2 and the r e l a t i v e s t i m u l a t i n g a c t i v i t y recorded i n Table 1 that the l e a s t s t i m u l a t i o n has been obtained from the compound with the highest nitrogen content, namely urea, while the greatest stimulatory a c t i o n has been achieved by the compounds with the lowest nitrogen contents, namely, beef extract and yeast e x t r a c t . R e c a l c u l a t i n g these values on the basis of a common nitrogen content w i l l therefore tend to emphasize the d i f f e r e n c e s between the stimulatory act i o n of these various types of nitrogenous compounds. S t i m u l a t i o n of aerobic l a c t i c a c i d production by these various nitrogen sources i s shown g r a p h i c a l l y i n Figure 5 , on the basis of 0 . 2 5 gm. Nitrogen. I t i s apparent that three l e v e l s of s t i m u l a t i o n e x i s t among these various compounds. I n , the f i r s t l e v e l a c i d production i s r e l a t i v e l y low, and the compounds f a l l i n g i n t o t h i s group are the simple ammonium s a l t s , - 10 -the amino acids and sodium caseinate. In the second l e v e l a c i d production i s much higher, and the nitrogenous compounds involved consist of the products prepared by enzymic h y d r o l y s i s , the peptone group. In the t h i r d l e v e l a c i d production i s very great and the two st i m u l a t i n g compounds are beef extract and yeast e x t r a c t , There would appear, therefore, to be a f a i r l y regular grouping based on the chemical nature of these com-pounds and t h e i r probable a c t i v a t o r content, which i s i n f a i r l y close agreement with the r e s u l t s shown i n Figure 2 . S t i m u l a t i o n of r e s p i r a t i o n by these same nitrogenous compounds i s shown g r a p h i c a l l y , i n Figure 6. There i s apparent a r e g u l a r increase from urea, with the lowest value, to beef e x t r a c t , with the highest value. In contrast to the r e s u l t s shown i n Figure j5 , there i s no grouping of these compounds on the basis of t h e i r chemical nature. I t i s also apparent that there i s a general s i m i l a r i t y i n t he appearance of the two sets of curves shown i n Figures 5 and 6, However, the s t i m u l a t i o n of r e s p i r a t i o n i s much more uniform than the s t i m u l a t i o n of acid product i o n . The s t i m u l a t o r y e f f e c t s when cystine i s employed as the nitrogen source are rather i r r e g u l a r . There i s a very marked immediate s t i m u l a t i o n of oxygen uptake such that a very high l e v e l i s reached w i t h i n two hours. A f t e r two hours, however, the oxygen uptake appears to reach a maximum value and the curve l e v e l s o f f . With l a c t i c a c i d production, on the other hand, the s t i m u l a t i o n i s regular and uniform and the curve r i s e s ~ 11 -s t e a d i l y to a t t a i n a maximum value at f i v e hours. I t would appear, th e r e f o r e , that s t i m u l a t i o n of r e s p i r a -t i o n b y c y s t i n e proceeds through a d i s t i n c t l y d i f f e r e n t mechan-ism than s t i m u l a t i o n of fermentation. These r e s u l t s are of i n t e r e s t i n view of the reported work of Chaix and Fromageot (3), who studied the a c t i v a t i n g a b i l i t y of various compounds upon the g l y c o l y t i c a c t i v i t y of Prop, pentosaceum, and found that the greatest stimulatory e f f e c t was obtained with various s u l f u r - c o n t a i n i n g compounds such as c y s t i n e , glutathione and th i o u r e a . They concluded that hydrogen s u l f i d e was the e s s e n t i a l accessory f a c t o r . When Witte's peptone i s employed as the nitrogen source the s t i m u l a t o r y e f f e c t s are again i r r e g u l a r . No l a c t i c a c i d i s produced during the f i r s t hour but during t h i s period there occurs an appreciable s t i m u l a t i o n of oxygen uptake. A f t e r t h i s one-hour period, however, acid product ion r i s e s with extreme r a p i d i t y while the oxygen uptake remains at a f a i r l y low l e v e l . I t would appear, t h e r e f o r e , that the two mechanisms of l a c t i c acid production and of r e s p i r a t i o n are stimulated i n an e n t i r e -l y d i f f e r e n t manner by the one nitrogen source. A somewhat s i m i l a r e f f e c t i s encountered with g l y c i n e and asparagine. These two compounds exert an i d e n t i c a l s t i m u l a t i o n upon r e s p i r a t i o n but a d i s t i n c t l y d i f f e r e n t s t i m u l a t o r y a c t i o n upon fermentation. The slow i n i t i a l s t i m u l a t i o n of l a c t i c a c i d formation by Witte's peptone as contrasted to that of Difco peptone and proteose peptone i s i n accord with the work of Sadler, Eagles, and Pendray {23) who found Witte - a peptone to be a t o t a l l y i n -adequate nitrogen source f o r the growth of the l a c t i c a c i d s t r e p t o c o c c i . - 12 -The t o t a l s t i m u l a t i o n of aerobic and anaerobic fermenta-t i o n and of r e s p i r a t i o n by these various nitrogen sources i s shown comparatively i n Figure 7. These r e s u l t s emphasize that aerobic and anaerobic l a c t i c a c i d production are generally stimulated to much the same degree by these d i f f e r e n t n i t r o g -nous compounds, but that r e s p i r a t i o n appears to be stimulated to an e n t i r e l y d i f f e r e n t degree. In general the r e s u l t s portrayed i n Figure 7 agree with those shown i n Figure 4. DISCUSSION The r e s u l t s presented herein show that fermentation and r e s p i r a t i o n by washed c e l l suspensions of Strep, l a c t i s are dependent not upon aerobic or' anaerobic conditions, but upon the presence of a nitrogen source. Washed c e l l s of Strep, l a c t i s suspended i n phosphate b u f f e r containing two percent glucose were found to form no l a c t i c acid under aerobic con-d i t i o n s , and only a very small amount under anaerobic condi-t i o n s , although a s l i g h t oxygen uptake could be demonstrated. The a d d i t i o n of a small amount (0»5$) of a group of n i t r o g e -nous compounds r e s u l t e d , i n the e l a b o r a t i o n of very large amounts of l a c t i c a c i d under both aerobic and anaerobic con-d i t i o n s , while at the same time ozygen uptake was very g r e a t l y stimulated. I t was also observed t h a t , while aerobic and anaerobic l a c t i c a c i d formation were generally stimulated to approximately the same extent by i n d i v i d u a l nitrogen sources, the s t i m u l a t i o n of r e s p i r a t i o n appeared to be d i s t i n c t l y d i f f e r e n t . I t was noticed that some nitrogenous compounds possessed the a b i l i t y -'13 -to stimulate acid production f a r more than r e s p i r a t i o n , while with other compounds the reverse held t r u e . These r e s u l t s i ndicate that r e s p i r a t i o n and fermentation are c a r r i e d out by d i f f e r e n t enzymic mechanisms, or, at l e a s t , by mechanisms which are independently responsive to various s t i m u l a t i n g agents. These r e s u l t s are i n agreement with the generally accepted t h e o r i e s of the r e l a t i o n s h i p of r e s p i r a t i o n to fermentation as d e t a i l e d by Oppenheimer and Stern ( 1 7 ) . \"There i s one uniform mechanism operative i n both phases ( r e s p i r a t i o n and fermentation) of desmolysis, namely, the t r a n s f e r of metabolic hydrogen. In anaerobiosis i t terminates i n l a c t i c a c i d and i n aerobiosis i t terminates i n water.\" This viewpoint i s rather d i f f i c u l t to r e c o n c i l e with the r e s u l t s reported i n the present work. I f the mechanism of both*processes i s e s s e n t i a l l y the same one would not expect to stimulate both r e s p i r a t i o n and aerobic fermentation at the same time nor would one expect to f i n d aerobic l a c t i c a c i d formation and anaerobic l a c t i c acid forma-t i o n stimulated equally by various nitrogen sources. The r e s u l t s obtained i n the present study also f a i l to agree with the c l a s s i c a l Pasteur and neo-Pasteur e f f e c t s as discussed by Burk and Lipman (2) and ( 1 2 ) . \"Most f a c u l t a t i v e organisms possess i n the Pasteur e f f e c t a regulatory device that enables them to use, as occasion demands, e i t h e r t h e i r aerobic or t h e i r anaerobic systems. By the operation of t h i s e f f e c t t h e i r fermentative apparatus i s blocked i n the presence of s u f f i c i e n t oxygen, and energy i s furnished almost e x c l u s i v e l y by the f a r more e f f i c i e n t and powerful r e s p i r a t o r y apparatus. - 14 -When oxygen i s l a c k i n g , however, the fermentation system i s brought into operation.\" With the washed c e l l suspensions of Strep, l a c t i s i t would appear that both r e s p i r a t i o n and fermen-t a t i o n are proceeding together under aerobic conditions and that- both processes can be independently stimulated by various nitrogenous compounds. It i s possible that much of t h i s lack of agreement a r i s e s from the use of r e s t i n g c e l l suspensions of a b a c t e r i a l species which i s predominantly anaerobic i n nature and which i s b e l i e v -ed to possess a very p r i m i t i v e type of metabolic mechanism which i s probably l a c k i n g i n the majority of the r e s p i r a t o r y en?yme and hydrogen transport systems possessed by the more aerobic b a c t e r i a and yeast. The mechanism by which the nitrogenous compounds studied exert t h e i r s t i m u l a t o r y a c t i o n i s s t i l l obscure. I t i s ex-tremely d i f f i c u l t to postulate a mechanism by which the r e s t i n g c e l l s of S t r e p - l a c t i s are able to u t i l i z e u r i c a c i d , which contains i t s nitrogen i n a r e l a t i v e l y stable and unavailable form, as an a c t i v a t o r f o r c e l l r e s p i r a t i o n and c e l l fermentation. I t i s also d i f f i c u l t to v i s u a l i z e the manner i n which urea, ammonium c h l o r i d e or sodium n i t r a t e exert t h e i r s t i m u l a t i n g e f f e c t s . That some f a c t o r other than simply nitrogen content of the a c t i v a t i n g substance must be involved i s apparent, since the compound with the highest nitrogen content, namely urea, gave the l e a s t s t i m u l a t i o n , and the compounds with the lowest nitrogen values, namely, beef and yeast e x t r a c t s , gave the greatest s t i m u l a t i o n . ' - 1 5 -From the r e s u l t s presented i n Table 1 i t i s noticeable that the f i g u r e s f o r anaerobic l a c t i c acid production remain quite constant, with a value i n the neighborhood of 0 . 6 percent, u n t i l tryptone and the various enzymic digests are reached. These compounds then show a markedly greater s t i m u l a t i o n which reaches a f a i r l y uniform l e v e l at about 1 . 0 percent. This may i n d i c a t e that substances of the peptone group possess a d d i t i o n a l a c t i v a t i n g compounds which have been elaborated during the enzymic processes e n t a i l e d i n t h e i r preparation. Such a d d i t i o n -a l a c t i v a t o r s would not be encountered with the pure s a l t s and amino acids employed, and t h e i r stimulatory power would con-sequently be lower. Such a hypothesis i s supported by the r e s u l t s shown g r a p h i c a l l y i n Figures 2 and 5 , which i n d i c a t e that the stimulatory a c t i o n of these compounds may be arranged i n groups which c l o s e l y p a r a l l e l the state of chemical p u r i t y of the compounds studied. The r e s u l t s reported herein are at marked variance with the published r e s u l t s of Smith and Sherman ( 2 5 ) . These workers measured the percentage glucose converted to l a c t i c acid by washed suspensions of the Str e p t o c o c c i i n a buffer s o l u t i o n without added ni t r o g e n source and obtained almost complete u t i l i z a t i o n of the glucose i n twelve hours. I t i s pos s i b l e that the a b i l i t y of washed c e l l s to u t i l i z e glucose i n the presence or absence of nitrogen source may be governed by the f u n c t i o n i n g of t h e i r a s s i m i l a t o r y rather than t h e i r r e s p i r a t o r y processes, as demonstrated by C l i f t o n ( 4 , 5 ) i n the case of E. c o l i and Ps c a l c o - a c e t i c i . - 1 6 -The r e s u l t s obtained i n t h i s study of the influence of various nitrogen sources upon fermentation and r e s p i r a t i o n by washed c e l l s f u r n i s h an explanation f o r the reported obser-vations of Morgan, Eagles, and L a i r d (15 ) . These workers found that no c o r r e l a t i o n existed between the dehydrogenation and o x i d a t i o n of carbohydrates by washed suspensions of Strep, l a c t i s and the fermentation of these same carbohydrates by the organism i n cu l t u r e media. I t i s apparent that the fermen-t a t i o n of carbohydrates by growing cultures i s governed by the influence which a c t i v a t i n g substances i n the medium exert upon the r e s p i r a t o r y and fermentative mechanisms of the c e l l , - 1 7 -REFERENCES* 1. Ayers, S. H., Johnson, W. T., and Mudge, C. S. - J. Inf. Dis. 3 4:29. 1 9 24. 2 . Burk, Dean - Cold Spring Harbour Symposia on Quantitative Biology, V o l . V I I , 1 9 3 9 . 3 . Chaix, P. and Fromageot, C. - Enzymologia 1 : 3 2 1 , 1 9 3 6 . 4. C l i f t o n , C. E. - Enzymologia 4: 24 6 , 1 9 3 7 . 5 . C l i f t o n , C. E. and Logan, W. A. - Jour. Bact. 3 7 : 5 2 3 , 1 9 3 9 . 6 . , Dixon, M. - \"Manometric Methods\", Cambridge Univ. P r e s s , 1 9 3 4 . 7. Eages, B. A. and Sadler, W. - Can. Jour. Res. 7 : 3 6 4 , 1 9 3 2 . 8. Earre11, M.A. - Jour. Bact. 2 9 : 411, 1 9 3 5 . 9. Hammer, B. W. and Baker, M.P. - Iowa Agr. Exp. Sta., Tech. B u l l . 2 3 2 , 1 9 2 6 . 10. Hegarty, C P . - Jour. Bact. 3 7 : 145 , 1 9 39 . 11. H i r s c h , J . - Enzymologia 4 : 9 4 , 1 9 3 9 . 1 2 . Lipman, F r i t z - \"A Symposium on Respiratory Enzymes\", U n i v e r s i t y of Wisconsin Press, 1942. 1 3 . Morgan, J . F. , Eagles, B.A., and L a i r d , D. G. - In P u b l i c a t i o n . 14. Morgan, J.F. , Eagles, B. A., and L a i r d , D. G. - In P u b l i c a t i on. 1 5 . Morgan, J . F., Eagles, B.A. , and L a i r d , D. G. - In P u b l i c a t i o n . 1 6 . Niven, C.F. and Smiley, K.L. - Jour. Bact. 4 4 : 2 6 0 , 1942. 17. Oppenheimer,•C, and Stern, E.G., \" B i o l o g i c a l Oxidation\", Nordemann, 1 9 3 9 . 1 8 . Orla-Jensen, S. - \"The.Lactic A c i d B a c t e r i a \" , Copenhagen,1919. 1 9 . Orla-Jensen, A.D. and Hansen, P.A. - Zent. Bact. I I Abt. 8 6 : 6 , 1 9 3 2 . 2 0 . Rahn, 0 . , Hegarty, C P . , and D u e l l , R.E. - Jour. Bact. 3 5 : 5 4 7 , 1 9 3 8 . - 18 -R e f e r e n c e s . (Cont'd. 2 1 . Rahn, 0 . , and Hegarty, C P . - P r o c . Soc. S x p l . B i o l . Med. 38: 218, 1938. 2 2 . S a d l e r , W. - Trans. Roy. Soc. Can. V o l . 2 0 , 1926. 2 3 . . S a d l e r , W. , E a g l e s , B.A., and Pendray, G. - Can. J o u r . Res. 7: 37 0, 19 3 2 . 24. Sherman, J.M. - Bact . Reviews 1 : 3 , 1937. 2 3 . Smith, P.A. , and Sherman, J.M. - Jo u r . Bact. 43:725,194-2. 2 6 . Smythe, C.V. - Enzymologia 6:9, 1939® Tabl© i CoBparative Oxidation, Dehydtogenation aad Fermentation of Carbohydrates with Sc. l a e t i s A.T.G, 374 COMPOUND AEROBIC OXIDATIOH DEHYDROGENATIOK; R.C. FERMENTATION GmoL,A« per l i t e r Glucose Strong Strong 100 7.4 Strong Fructose Strong 75 Strong 6.5 Strong Galactose Weak 8 Very Weak 4.5 F a i r l y Strong Arabinose Very Weak 1 Negative 1.8 Very S l i g h t Lactose F a i r l y Strotig 38 F a i r l y Strong 5.4 Strong Haffinose F a i r 0 Negative 1.1 Trace Melezitose Very Weak 0 Negative , 4.1 F a i r l y Strong Dextrin Very Strong 3 Trace 0.7 TracG Adonitol Very Weak 0 Negative 0.7 Trae© 2-• Table 2^' .. Dehydrogenase A c t i v i t y of Rli, t r i f o l i l . Red.Time Glneose Values ape Expressed as Respiratory Coe f f i c i e n t s ^^^^ lijae X Rh. t r i f o l i l = B.C. SUBSTRATE R.T, 231 R.T, 2 2 6 R.T, 224 R.T,39-1 R,T, 22B Glucose 100 100 100 100 Mannose 80 7 5 45 100 7 5 Galactose 20 0 5 6 5 Fructose 5 0 5 0 7 5 67 60 Arabinose 0 0 6 7 0 0 Xylose 16 0 133 0 0 Sucrose 55 100 57 100 20 Gelloblose 80 28 3 0 - 75 Lactose 0 0 0 35 5 Maltose 56 0 3 0 1 1 75 Trehalose 10 20 40 7 5 Melibiose 34 0 57 4 5 Raffinose 25 7 18 67 35 Melezitos© 0 . 0 67 30 5 0 • Mannitol 3 0 30 6 0 40 15 S o r b i t o l 1 50 0 0 7 16 3 Table 2 (cent.) SUBSTRATE R.T. 2 5 1 R.T. 226 R.T. 224 R.T . 3 9 - i R.T. 22B S a l i c i n 9 20 8 0 67 5 0 Dextrin 18 14 56 5 0 50 Starch • 18 55 8 0 67 42 Inu l i n 2 5 14 56 67 42 EsculiB 15 0 6 0 8 5 Rhamhose 0 0 0 0 0 Methyl glucoside 0 0 0 0 0 Glycerol 0 0 7 5 8 6 Sod. format® 0 0 0 5 0 Sod. lactate 28 0 0 0 0 Sod. succinate 7 10 6 1 0 Sod. acetate 0 0 0 0 0 Sod. malate 7 0 0 0 27 A l l y l alcohol 0 0 0 0 7 -4 - . Table 1 Relative Dehydrogenase A c t i v i t y of R. t r i f o l i i Strains RHIZOBIUM TRIFOLII STRAINS SUBSTRATES RT 2 2 B RT 224 RT 226 RT 2 3 1 RT 3 9 - 1 Glucose 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 Mannose 75 45 75 8 0 1 0 0 Galactose 3 5 0 20 6 Fructose 60 75 50 30 67 Arabinose 0 67 0 0 0 Xylose 0 1 3 3 0 16 0 Rhamnose 0 0 0 0 0 Sucrose 20 37 100 55 1 0 0 Cellobiose 75 30 28 8 0 4 5 Lactose 5 0 0 0 3 3 Maltose 75 30 0 56 11 Trehalose 5 40 20 1 0 7 Melibiose 5 37 0 34 4 Raffinose 35 18 7 25 67 Melezitose 50 67 0 0 50 Dextrin 50 56 14 18 50 Starch 42 84 33 18 67 Inulin 42 36 14 25 67 E s c u l i n 5 60 0 13 8 S a l i c i n 30 8 0 20 9 67 Methyl glucoside 0 0 0 0 0 • 5^ Table 1 (cont.) RHIZOBITJM TRIFOLII STRAINS SUBSTRATES RT 2 2B RT 224 RT 226 RT 2 3 1 RT 3 9 - 1 Ethylene glycol 0 0 0 0 0 Grlycerol 6 75 0 0 8 E r y t h r i t o l 0 0 0 0 0 Adonitol 0 28 0 0 0 Mannitol 15 60 30 30 20 S o r b i t o l 16 0 0 50 7 D u l c i t o l 0 3 0 0 0 I n o s i t o l 0 28 . 0 0 0 Sodium formate 0 0 0 0 5 Sodium acetate 0 0 0 0 0 Sodium propionate 0 0 0 0 0 Sodium butyrate 0 0 0 0 5 Sodium lactate 0 0 0 28 0 Sodium succinate 10 10 4 7 6 Sodium fumarate 0 0 0 0 0 Sodium malate 27 0 0 7 . 0 Ethyl alcohol 0 0 0 0 0 n Propyl alcohol. 0 0 0 0 0 A l l y l alcohol 7 0 0 0 0 Ethylamihe - J; 0 •;0 0 0 7 n Butylamine 0 6 . 0 0 0 • *A11 values expressed as percentage of reduction time with glucose Table 2 Dehydrogenase A c t i v i t y with Time RHIZOBIUM TRIFOLII 224 SUBTRATES F i r s t Tests 2 Months 8 Months 12 Months Glucose 1 0 0 1 0 0 1 0 0 1 0 0 Mannose 45 1 0 0 46 63 Galactose 5 33 25 56 Fructose 75 42 5 0 56 Arabinose 67 0 0 0 Xylose 135 30 14 32 Rhamnose 0 0 0 0 Sucrose 37 60 50 56 Cellobiose 30 75 4 6 63 Lactose 0 8 4 7 Maltose 3 0 ' 5 0 42 42 Trehalose 40 8 1 1 28 Melibiose 37 8 9 40 Raffinose 18 60 25 34 Melezitose 67 21 12 56 Dextrin 56 5 0 22 5 0 Starch 8 0 , 42 30 30 Inulin 36 18 30 10 E s c u l i n 60 60 45 32 S a l i c i n 8 0 37 56 56 Methyl glucoside • 0 0 0 0 Variation i n 7. Table 2 (cont.) RHIZOBIUM TRIFOLII 224 SUBSTRATES F i r s t Tests 2 Months 8 Months 12 Months Ethylene gly c o l 0 0 0 0 Glycerol 75 0 9 63 E r y t h r i t o l 0 0 0 0 Adonitol 28 0 0 0 Mannitol 60 18 17 84 Sor b i t o l 0 16 20 21 D u l c i t o l 3 0 0 0 In o s i t o l 28 4 0 0 Sodium formate 0 0 0 0 Sodium succinate 10 3 14 84 Sodium fumarate 0 6 0 9 Sodium male|iie 0 0 0 0 Sodium malate 0 38 0 14 Ethyl alcohol 0 0 20 10 n Propyl alcohol 0 0 0 11 n Butyl alcohol 0 0 15 0 iso Butyl alcohol 0 0 0 20 A l l y l alcohol 0 0 0 10 iso Butylamine 6 0 0 7 Formaldehyde t 0 0 20 0 Table 1 O2 Uptake in Cu.mra. per l i r . per mgm, dry weight w STRAIN Endogenous Glucose Mannitol Sodium succinate RT 224 E 3 4 . 7 9 3 . 6 - 40.5 1 0 9 . 2 ^1 2 8 . 3 5 4 . 2 4 6 . 7 6 2 . 6 ^2 24.7 6 5 . 5 3 5 . 3 5 6 . 6 ^5 2 7 . 7 5 7 . 8 29.7 8 3 . 5 ^6 2 2 . 4 5 0 . 6 1 3 . 9 • 6 0 . 8 E7 2 0 . 7 4 7 . 8 2 0 . 5 8 9 . 4 Es 2 0 . 2 5 7 . 3 25 .0 5 3 . 2 RT 224 2 7 . 2 5 8 . 9 6 8 . 6 19 .6 % 42 . 3 8 0 , 3 7 6 . 2 3 3 . 5 ^2 1 7 . 1 6 5 . 0 5 8 . 9 19.4 ° 1 2 8 . 8 1 2 6 . 0 4 3 . 7 42 .3 ° 2 6 4 . 3 2 3 2 . 4 2 1 7 . 7 4 5 . 1 ° 3 5 8 . 2 1 2 8 . 2 115.5 9 3 . 2 °4 2 3 . 1 1 3 8 . 6 1 3 5 . 6 3 2 . 8 ° 5 19 .6 1 2 9 . 4 9 7 . 4 2 8 . 8 C6 2 5 . 3 7 9 . 1 5 2 . 3 1 2 7 . 7 °7 2 9 . 9 8 0 , 6 8 3 . 2 3 5 . 1 1 3 . 0 3 0 . 8 3 1 . 7 2 2 . 1 3 0 . 8 1 7 9 . 1 1 3 2 . 8 4 7 . 6 ^ ^10 1 8 . 6 6 0 . 3 6 0 . 0 1 5 . 3 2 1 . 0 6 8 . 3 54.5 2 0 . 2 ° 1 2 41.9 1 6 7 . 8 1 8 5 . 6 31.9 Table 2 Aerobic and Anaerobic Respiratory Coefficients A a Anaerobic B = Aerobic Glucose r 1 0 0 Sub-s t r a i n Endogenous Mannitol Sodium succinate A B A B A B RT 224 E 58 37.6 75. 4 4 . 2 62 1 1 8 . 4 ^^1 82 ' 5 2 . 2 8 2 . 86.1 62 115 .4 7 0 37.7 8 0 • 8 0 8 6 . 4 88 • 4 7 . 9 75 - 88 144 . 4 H 61 . 4 4 . 2 8 0 27 . 4 80 1 2 0 . 1 ^7 60 4 3 . 3 66 42.8 66 1 8 7 . 0 ^8 90 35 . 2 100 4 3 . 6 1 0 0 92.8 RT 224 0 45 58 116 116 33 ^1 0 53 72 95 118 42 ^2 8 26 83 9 1 62 30 ^1 8 23 86 35 50 28 ^2 75 28 75 94 60 19 °3 6 45 63 90 36 73 C4 2 17 67 98 5 24 4 15 Al 75 14 22 H 20 32 86 66 30 1 6 1 '1 , 0 37 57 4 4 22 103 °8 0 42 70 103 7 0 72 C9 2 17 42 74 13 27 ° 1 0 5 31 7 0 99 60 .25 4 31 60 8 0 60 30 C12 0 25 / 80 110 1 0 0 19 /o. Table 1 Dehydrogenase A c t i v i t i e s of Strep, l a c t i s and Strep, cremoris* SUBSTRATE Sc. l a c t i s S.A .30 Sc. l a c t i s A.T.C .374 Sc. l a c t i s EMB2 1 Sc. cremoris HP Sc. cremoris EMBi 193 Sc. cremoris RW d-Glucose 100 1 0 0 100 1 0 0 1 0 0 1 0 0 d-Mannose 30 75 7 0 8 0 50 7 d-Galactose 20 8 1 0 0 20 25 0 d-Fructose 100 75 50 8 0 28 30 l-Arabinose 0 2 0 0 0 0 1-Xylose 0 2 0 0 0 0 Rhamnose 0 0 0 0 0 0 Sucrose 30 1 0 0 0 0 50 0 Cellobiose 50 1 0 0 32 4 8 50 37 Lactose 6 38 1 0 0 33 5 0 11 Maltose 5 30 28 40 33 22 Trehalose 6 37 0 0 0 23 Melibiose 0 0 0 0 0 0 Raffinose 0 0 0 8 0 ' 40 11 Melezitose 0 0 0 0 0 0 Dextrin 0 3 6 9 23 Starch 8 0 50 22 80 50 37 Inulin 20 50 0 56 20 0 Esculin 0 3 0 12 0 0 S a l i c i n 4 11 11 20 0 0 Methyl glucoside 0 0 0 0 0 0 • SUBSTRATE Sc. l a c t i s S.A .30 Sc. l a c t i s A.T.G .574 Sc. l a c t i s EMB2 1 Sc. cremoris HP Sc. cremoris EMBi 193 Sc. cremoris RW Ethylene g l y c o l 0 0 0 0 0 0 Glycerol 0 3 0 0 0 0 Adonitol 0 0 0 0 0 0 d-Mannitol 0 3 0 0 0 0 d-Sorbitol 0 3 0 0 0 0 D u l c i t o l 0 0 0 0 0 0 I n o s i t o l 0 0 0 0 0 0 Sod, formate 0 0 0 0 0 9 Sodo acetate 0 0 0 0 0 0 Sod, lactate 0 0 0 0 0 . 72 Sod, succinate 0 0 0 0 0 0 Ethyl alcohol 10 5 0 20 7 0 n-Propyl alcohol 8 0 0 0 0 10 n-Butyl alcohol 4 0 0 0 0 0 A l l y l alcohol 15 17 0 0 0 20 ^Recorded as percentage of the reduction time shown by each organism in the presence of glucose. Table 2 s Dehydrogenase A c t i v i t i e s of Strep, bovis and Betacocci SUBSTRATE Sc. bovis A.T.C. 6058 Betacoccus EM®2 17 3 SC. citrovorus A.T.C. 797 Sc. paracitrovorus A.T.C.798 d-Glucose 100 100 100 100 d-Mannose 100 60 66 38 d-Galactose 12 0 10 25 d-Fructose 45 66 60 46 l-Arabinose 0 0 0 0 1-Xylose 0 0 0 0 Rhamnose 0 0 0 0 Sucrose 89 7 2 50 16 Cellobiose 89 50 66 100 Lactose 55 6 10 12 Maltose 9 62 6 80 Trehalose 0 0 3 9 Melibiose 0 0 0 0 Raffinose 140 57 0 0 Melezitose 0 0 0 0 Dextrin 50 0 6 80. Starch 140 4 50 50 Inulin 50 12 0 0 E s c u l i n 78 0 0 0 ^ S a l i c i n . 9 40 4 0 Methyl glucoside 0 0 0 7 1^ • Table 2 (cont.) SUBSTRATE Sc. bovis A.T.C. 6 0 5 8 Betacoccus EMB2 175 Sc. citrovorus A.T.C.797 Sc. paracitrovorus A.T.C.798 Ethylene glycol 0 0 0 0 Glycerol 0 0 7 0 Adonitol 0 0 0 0 Mannitol 0 0 0 22 d-Sorbitol 0 0 0 0 d-Dulcitol 0 0 0 0 I n o s i t o l 0 0 0 0 Sod. formate 0 0 0 0 Sod. acetate 0 0 0 0 Sod. lactate 0 0 0 0 Sod. succinate 0 0 0 0 Ethyl alcohol 0 0 0 40 n-Propyl alcohol 0 0 0 0 n-Butyl alcohol 0 0 0 0 A l l y l alcohol 0 0 0 3 Recorded as percentage of the reduction time shown by each organism i n the presence of glucose. Table 3 Dehydrogenase A c t i v i t i e s of the Pseudo Lactic Acid Bacteria* SUBSTRATE Tc. casei A.T.C. 3 9 1 Tc. liquefaciens SM5 Bact. c o l i A.T.C. 4157 Bact. aerogenes A.T.C. 211 d-G-lucose 100 100 100 100 d-Mannose 20 65 8 0 75 d-Galactose 23 20 40 5 0 d-Fructose 33 100 67 44 l-Arabinose 25 0 12 0 1-Xylose 24 0 0 7 Rhamnose 0 0 20 0 Sucrose 8 0 100 57 85 Cellobiose 30 90 25 67 Lactose 66 33 30 50 Maltose 66 75 45 60 Trehalose 25 16 ' 67 75 Melibiose 45 . 0 10 9 Raffinose 8 0 90 45 50 Melezitose 12 16 11 0 Dextrin 50 89 30 80 Starch 40 0 36 68 Inulin 78 30 67 33 Escul i n 0 0 - -S a l i c i n 45 0 67 67 Methyl glucoside • 0 0 0 0 Table 3 (cont.) SUBSTRATE Tc. casei A.T.G. 391 Tc. liquefaciens SM5 Bacte c o l i A.T.C. 4157 Bact, aerogenes A.T.C. 211 Ethylene glycol 0 0 0 0 Glycerol 0 30 20 25 Adonitol 0 0 0 0 d-Mannitol 8 0 16 36 100 d-Sorbitol 13 0 20 60 D u l c i t o l 0 0 0 0 I n o s i t o l 0 0 0 0 Sod. formate 0 7 40 57 Sod, acetate 0 0 0 0 Sod, lactate 20 30 11 32 Sod, succinate 0 0 12 27 Sod, fumarate 0 0 10 0 Sod, malate 17 0 14 12 Ethy l alcohol 0 200 2 67 n-Propyl alcohol 0 200 3 24 n-Butyl alcohol 20 67 6 20 A l l y l alcohol 35 200 0 8 0 Formaldehyde 0 6 8 20 Glutamine 0 0 17 22 ^Recorded as percentage of the reduction time shown by each organism in the presence of glucose. 16,. Table 4 Dehydrogenase Reactions of Strep, l a c t i s at Different. Periods of Time SUBSTRATE Sc. l a c t i s SA 30 Sc. l a c t i s A.T.C. 374 Original Tests 18 mos. l a t e r Original Tests 18 mos. l a t e r d-Glucose 100 100 100 100 d-Mannose 30 100 75 100 d-Galactose 20 35 8 16 d-Fructose 100 80 75 100 l-Arabinose 0 0 2 0 1-Xylose 0 0 2 0 Rhamnose 0 0 0 0 Sucrose 30 4 100 8 Cellobiose 30 100 100 100 Lactose 6 20 38 37 Maltose 5 100 30 100 Trehalose 6 5 37 6 Melibiose 0 0 0 0 Raffinose 0 0 0 0 Melezitose 0 0 0 0 Dextrin 0 100 3 75 Starch 8 0 7 50 25 Inulin 20 100 50 42 Escul i n 0 5 3 27 S a l i c i n 4 12 11 37 Methyl glucoside • 0 0 0 0 Table 1 Aerobic Respiratory A c t i v i t y of Strep, l a c t i s Strep. l a c t i s S.A. 30 Strep. l a c t i s A.T.C. 374 SUBSTRATE Q.CO2 R.q. QO2 q c 0 2 R.Q. 1 hour 1 hour 1 hour 1 hour 1 hour 1 hour d-Glucose 2 6 . 6 2 6 , 6 1 . 0 24 . 2 1 9 . 4 . 8 0 d-Mannose 2 3 . 8 2 0 . 5 , 8 6 3 0 . 8 2 5 . 0 , 8 1 d-Galactose 2 1 . 7 2 1 . 0 . 9 6 7 . 6 4 . 7 . 7 3 d-Eructose 2 7 . 9 2 2 . 8 . 8 1 2 3 . 3 2 2 . 4 , 9 6 1-Xylose 1 7 . 8 14 . 9 . 8 3 7 . 5 5 . 5 . 7 3 l-Arabinose 8 . 9 5 . 9 . 6 6 1 0 . 2 9 . 2 . 9 0 Sucrose 2 1 . 8 24 .7 1 . 1 3 1 7 . 7 1 8 . 7 1 . 0 5 Cellobioae 2 3 . 8 1 6 , 8 . 7 0 25 . 2 2 2 . 3 . 8 8 Lactose 2 1 . 0 1 0 . 8 . 5 1 21.4 1 7 . 5 . 8 1 Maltose 22.4 22.4 1 . 0 1 3 . 0 1 1 . 0 . 8 4 Trehalose 1 8 . 0 1 3 . 9 . 7 7 14 , 9 14 . 0 . 9 4 Melibiose 1 2 , 0 1 6 . 4 1 . 3 6 1 3 . 0 1 2 . 0 . 9 2 Raffinose 1 8 , 7 2 0 . 2 1 . 0 8 14 , 0 1 3 . 1 . 9 3 Melezitose 2 3 . 8 2 8 . 2 1 . 1 8 8,4 9.4 1 . 1 0 Dextrin 2 3 . 2 2 3 . 1 . 9 1 3 7 . 1 3 0 . 4 . 8 2 Starch 1 7 . 1 1 7 . 1 1 . 0 44 . 9 2 6 , 4 . 5 9 Inulin 9 . 8 1 1 . 3 1 . 1 5 3 . 3 1 . 8 . 5 4 S a l i c i n 14 . 7 13 . 2 . 8 9 1 2 , 1 1 0 , 2 . 8 4 E s c u l i n 19o6 1 9 . 6 1 . 0 14 . 9 1 0 . 0 . 6 6 Alpha Methyl Glucoside 1 2 . 6 1 1 . 2 . 8 8 1 2 . 1 1 1 , 2 .92 Table 1 (cont.) Strep. l a c t i s S. A. 30 strep. l a c t i s A.T.C. 374 SUBSTRATE QO2 1 hour QCO2 1 hour R.q, . 1 hour QO2 1 hour qC0 2 1 hour R.ft. 1 hour Glycerol 1 8 . 2 21.2 1 . 1 6 2 7 . 3 2 1 . 5 . 7 9 Adonitol 7.0 5 . 3 . 7 8 10.2 10.2 1,00 d-Mannitol 24.9 20 .3 .82 8,4 4 . 5 . 5 3 d-Sorbitol 21.0 16 .6 . 7 9 11.2 , 10.2 .91 D u l c i t o l 11,2 17.1 1 . 5 2 8.4 6 . 4 . 7 6 I n o s i t o l 2 5 . 6 22.7 . 8 8 9 . 3 8 . 3 . 8 9 Ethyl alcohol 2 9 . 7 2 7 . 6 . 9 2 - - -Ethylamine 8 . 7 3 . 8 . 4 3 1 3 . 1 17.0 1 . 2 9 Sod. formate 2 6 . 6 25.2 .94 - - -Sod. lactate 1 9 . 2 1 9 . 2 1.00 - - -Sod. succinate 24.4 2 9 . 1 1.19 - - -Sod, malate - - - 24 .6 2 3 . 7 . 9 6 Sodi poti t a r t r a t e 2 3 . 8 1 8 . 7 . 7 8 Endogenous 12 .6 7 . 5 . 5 9 14 ,7 1 8 , 7 1 .27 Table 1 Respiration and Fermentation vjitb Strep, l a c t i s SUBSTRATE Sc. l a c t i s S.A, 30 Sc. l a c t i s A.T.C. 374 I QO2 ( Respiratory 3oef ficient Gm. L.A. per l i t e r E q 0 2 ^ iespiratory Joefficient Gm.L.A, per l i t e r d-Glucose 2 6 , 6 1 0 0 8 . 1 24 .2 1 0 0 7 . 4 d-Mannose 2 3 . 8 30 7 . 7 3 0 . 8 75 7 . 4 d-Galactose 2 1 , 7 .20 3 . 4 7 . 6 8 4 . 3 d-Fructose 2 7 . 9 1 0 0 7 . 4 2 3 . 3 75 6 . 3 1-Xylose 1 7 . 8 0 2 . 5 7 . 5 0 2 . 3 l-Arabinose 8 , 9 0 1 . 8 1 0 . 2 0 1 . 8 Sucrose 2 1 . 8 100 1 . 6 1 7 . 7 100 5 . 2 Cellobiose 2 3 . 8 50 2 . 7 2 5 . 2 1 0 0 6 . 3 Lactose 2 1 . 0 6 5 . 4 2 1 . 4 38 5 . 4 Maltose 2 2 , 4 5 3 . 6 1 3 . 0 30 5 . 0 Trehalose 1 8 . 0 6 7 . 0 14 .9 37 4 . 7 Melibiose 1 2 . 0 0 1 . 6 1 3 . 0 0 0 . 9 Raffinose 1 8 , 7 0 2 . 0 14 .0 0 1 . 1 Melezitose 2 3 . 8 0 2 . 0 8 . 4 0 4 . 1 S a l i c i n 14 .7 4;, 6 . 1 1 2 . 1 11 5 . 6 Dextrin 2 3 . 2 0 2 . 5 3 7 . 1 3 0 . 7 Starch 1 7 . 1 80 3 . 8 4 4 , 9 5 0 1 . 8 Inulin 9 . 8 20 0 . 9 3 . 3 50 0 Esculin 1 9 . 6 0 2 . 7 14 .9 3 2 , 0 Methyl glucoside 1 2 . 6 0 1.4 1 2 . 1 0 0 . 9 Glycerol 1 8 . 2 0 1 . 4 2 7 . 3 0 1 . 8 Adonitol 7 . 0 0 0 . 7 1 0 . 2 0 0 . 7 d-Mannitol 24 .9 0 0 . 7 8.4 3 2 . 7 ffllorbitol 2 1 . 0 0 1 . 1 1 1 . 2 3 0 . 7 D u l c i t o l 1 1 . 2 0 0 . 9 8 . 4 0 0 . 2 Endogenous 1 2 . 6 14 .7 Table 2 Comparative Respiratory and Fermentative A c t i v i t y of Strep, l a c t i s upon Carbohydrates w Sc. l a c t i s S. A. 30 Strep . l a c t i s A.T.C. 374 SUBSTRATE Aerobic R.C. Anaerobic R.C. Acid R.C. Aerobic R.C, Anaerobic R.C. Acid RiC. d-Glucose 100 100 100 100 100 100 d-Mannose 89 30 95 127 75 100 d-Galactose 8 1 20 66 .31 8 60 d-Fructose 104 100 91 96 75 87 1-Xylose 73 0 30 30 0 33 l-Arabinose 36 0 22 42 0 24 Sucrose 8 1 100 20 73 100 43 Cellobiose ,89 50 33 104 100 85 Lactose 78 6 66 88 38 72 Malto se 84 5 44 53 30 67 Trehalose 67 6 86 61 37 63 Melibiose 43 0 20 53 0 12 Raffinose 70 0 24 57 0 14 Melezitose 89 0 24 34 0 . 55 S a l i c i n 53 4 75 50 11 75 Dextrin 94 . 0 30 153 3 9 Starch 64 80 46 185 50 24 Inulin 36 20 10 14 50 0 Escu l i n 73 0 33 6 1 3 27 Methyl glucoside 47 0 17 50 0 12 Glycerol 68 0 17 0 24 Adonitol 26 0 8 42 0 9 d-Mannitol 93 0 8 34 3 36 ( i ^ o r b i t o l 78 0 13 46 3 9 D u l c i t o l 42 0 10 34 0 3 Endogenous 47 0 60 0 -TABLE I ' I f f e o t of Previous Adaptatlpn upon Carbohydrate Behydrogenations.^ Strep, l a c t i s SA 30. Dehydrogenation C e l l s Grown in Presence Of Substrate Glucoae Lactose Starch Manni to 1 Methyl Glucoside d Glucose 4 15 6 6 6 d Mannose 13 21 7 6. 12 d Galactose 20 28 16 20 d Fruotoae 4 17 7 7 8 1 Arabinose 0 23 120 0 0 Sucrose 4 27 7 10 9 Cellobiose 8 26 7 9 10 Lactose 64 18 18 27 33 Maltose 89 69 11 0 0 Trehalose 62 18 ,29 16 22 Raffinose 0 12 9 13 13 S a l i c i n 92 0 32 97 0 Dextrin 0 0 36 23 44 Starch 5 13 9 0 120 Inulin 21 37 25 6 9 d Mannitol 0 0 0 0 0 B A l l Values Expressed as Reduction Time i n Minutes; 3i a . •E&BLE II E f f e c t of Previous Adaptation on Carbohydrate D e h y d r o g e n a t i o n * S t r e p , l a c t i s A.T.C. 374. Dehydrogenation Substrate C e l l s Grown i n . Glucose C e l l s Grown in Lactose C e l l s Grown in Mannitol d^ Glucose 3 2 8 di Mannose. 4 3 10 d« Galactose 38 6 12 d. Fructose 4 5 10 Sucrose 3 4 11 Cellobiose 3 3 11 Lactose 8 5 12 Maltose 10 14 58 Trehalose 8 6 13 Raffinose 0 5 30 Sor b i t o l 90 56 31 Mannitol 90 21 10 Glycerol 110 8 36 S a l i c i n 88 20 64 Starch 6 4 9 Inulin 6 4 22 X A l l Values Expressed as Beduc tion Time i n Minutes^ TABLE III Lactic Acid Production from Carbohydrates by Suspensions Adapted to Glucose. of Strep, l a c t i s SA 30 Time C e l l s Grown i n Presence of Glucose in Hoxrrs Glucose Substrate Mannose Substrate Frutose Substrate Galactose Substrate Lactose Substrate. Mannitol Substrate 0.5 • 306 • 216 i270 •018 0 0 1.0 ,432 • 414 • 342 • 018 0 0 1.5 .684 .432 • 468 • 056 0 0 2.0- ,936 ^450 • 576 ;054 .036 0 2.5 .954 .540 .702 • 072 i054 0 3.0 .972 • 648 • 810 i072 i054 0 3.5 1.008 • 7ao • 846 .090 • 072 0 4.0 1.026 .774 • 900 .108 .072 0 4.5 1.092 ,810 i900 .108 .090 0 5.0 1.116 .864 .900 .108 .108 0 5. 5 1.142 .900 • 936 .124 , .126 0 6,0 1.260 .918 • 972 .124 il26 0 \\ TABLE I Stimulation of Acid Production and Oxygen Uptake by Various Nitrogen Sources upon Resting C e l l suspensions of Strep, l a c t i s A.T.C. 374 in Presence of Glucose, 3 Hours Q O2 Nitrogen Source* Gm. L.A. per 100 ml. Aerobic Anaerobic 3 hours Control - No Nitrog en 0 0 . 3 0 6 4 6 . 6 Ammonium Chloride 0 . 8 8 2 0 . 6 6 6 8 9 . 5 Ammonium Sulfate 1 . 0 0 8 0 . 6 6 6 6 4 . 3 Sodium Nitrate 0 . 8 4 6 0 . 6 3 0 6 0 . 6 Urea 0 . 7 7 4 0 . 5 9 4 5 2 . 1 Uric Acid 0 . 8 2 8 0 . 6 6 6 -Glycine 0 . 7 3 8 0 . 6 3 0 6 6 . 4 Cystine 0 . 8 8 2 0 . 6 8 4 159 .5 Asparagine 0 . 3 9 6 0 . 6 3 0 6 6 . 4 Tryptone-DifCO 1 . 1 3 4 0 . 9 0 0 1 7 9 . 0 Peptone-DifCO 1 . 1 8 8 0 . 9 7 2 1 7 0 . 3 Peptone-Witte's 1,044 1.044 7 5 . 5 Proteose Peptone 1 . 1 7 0 0 . 9 1 8 242 .6 Sodium Caseinate 0 . 6 3 0 0 . 5 7 6 1 0 1 . 0 Beef Extract 0 . 8 6 4 0 . 6 6 6 2 0 7 . 9 Yeast Extract-DifCO 1 . 2 0 6 0 . 8 2 8 1 8 7 . 4 •*• A l l nitrogen sources added to the resting c e l l - glucose mixture i n 0.3fo concentration. lis A TABLE 2. Acid Production and Oxygen Uptake Recalculated to Common Value of 0,25 gm. Nitrogen. Conversion % Lactic % Lactic Oxygen Nitrogen Source f. T.N. Factor ' Acid Acid Uptake Aerobic 3 Hrs. Anaerobic 5 Hrs. 3 Hrs. Ammonium Chloride 2 6 . 2 0 1 . 9 1 . 6 7 5 1 . 2 6 5 1 7 0 . 0 Ammonium Sulfate 2 1 . 2 0 2 . 3 2 . 3 1 8 1 . 5 3 1 147 .8 Sodium Nitrate 1 6 . 4 8 3 . 0 2 . 5 3 8 1-.890 1 8 1 . 8 Uric Acid 3 3 . 3 4 1 . 4 1.159 0 . 9 3 2 0 . 0 Urea 4 6 . 6 0 1 .07 0 . 8 2 8 0 . 6 3 5 5 5 . 7 Glycine 1 8 . 6 6 2 , 6 1 . 9 1 8 1 . 6 3 8 1 7 2 , 6 Cystine 1 1 . 6 0 4 . 3 3 . 7 9 2 2,941 6 8 5 . 8 Asparagine 1 8 . 6 7 2.6 . 1 . 0 2 9 1 . 6 3 8 1 7 2 . 6 Tryptone-DifCO 1 2 , 1 0 + 4 . 1 4 . 6 4 9 3 . 6 9 0 733 . 9 Peptone-DifCO 1 5 . 4 0 X 3 . 2 3 . 8 0 1 3 . 1 1 0 5 4 4 . 9 Peptone-Wltte 14,30* 3.5 3 . 6 5 4 3 . 6 5 4 2 6 4 . 2 Proteose Peptone 1 3 . 5 0 ^ 3 .7 4 . 3 2 9 3 . 3 9 6 8 9 7 . 6 Sodium Caseinate 1 3 . 3 6 3 . 7 2 . 3 3 1 2 .131 3 7 3 . 7 Beef Extract 7 . 7 0 7 . 1 6 , 1 3 4 4 , 7 2 8 1 4 7 6 . 0 Yeast Extract-Difc SO 8 ,43 5.9 7.115 4 . 8 8 3 1 1 0 5 . 6 Factor Required to Convert Results to Basic Value of 0 . 2 3 gm.N. ^ Values quoted from Eagles B.A. and Sadler, VJ. - Can. J.Res.7: 364 , 1 9 3 2 ® Value quoted from Difco Manual, 1 9 3 9 . Figure 1 Oiygen Uptake by Sc. l a c t i s A.T.C, 574 i n the Presence of Monosaccharides JO -I Figure 2 Carbon Dioxide Production by Sc. l a c t i s A.T.C. 374 i n the Presence of Uonosaccharides Fructose Glucose Endogenous Arabinose Galactose 15 30 45 Time i n Minutes 60 Figure 3 Oxygen Uptake by Sc. Lactis i n the Presence of Various Carbohydrates 2*. Dextrin Lactose Raffinose Adonitol Endogenous Melezitose 15 30 45 60 Time in Minutes Figure • Carbon Dioxide Production by Sc. l a e t i s A.T.C. 374. i n the Presence of Various Carbohydrates 3 0 ; Figure 5(a) Respiratory Quotients - Sc. l a c t i s A.T.C. 374 .4 -0 1 1 5 130 • 4 5 '60 Figure 6 Time in Minutes In Figure 6 i s shown the oxidation of glucose by several species of bacteria. This graph emphasizes the comparatively small oxidizing a b i l i t y of the La c t i c Acid Streptococci i n comparison with more aerobic species such as E, c o l i and Rhizobia, Figure 8 pH Ueasurementa - Anaerobic Sc. l a c t i s S,A. 30 7.0-6 . 5 6 . 0 . 5 . 5 P4 5 . 0 4 4 .5 4 . 0 . 3 . 5 No peptone 0 . 1 2 5 ^ peptone 1 . 0 ^ peptone 0.25> peptone 0 . 5 > peptone Figure 9 Influence of Peptone Concentration upon Aerobic Acid Production from Clueose by Sc. l a c t i s S.A, JO 1.0^ peptone 0,5^ peptone 0,251- peptone 0.125^ peptone Figure 10 Influence of Peptone Concentration upon Anaerobic Acid Production from Glucose by Sc. l a c t i s S.A. 30 o o o u o p. •p o H i 5 1 . 0 ^ peptone 0 . 5 ^ peptone 0.25yL peptone 0 . 1 2 5 ^ peptone No peptone Figure 11 Influence of Peptone Concentration upon Oxygen Uptake by Se* l a c t i s S.A. 30 in Presence of Glucose 200. o a « M « P 4 . 1^ o' +> ed U Pt at' 75-50-2 5 o o 3 o O P! 9 to O H O +> a a as ed C •o to o CO a> m o o m o bO o •cs a w o - p PI a CD e +3 a a • H o o O CO 0 Figure 17 Oxygen Uptake from Mannitol by Rh. t r i f o l i i 224 and Substrains 210 180 150 . 120 90 . 60 . 30 . R.T. B l ^1 ^2 >3 °4 ^5 7^ °11 ^12 224 Figure 18 Comparative Aerobic and Anaerobic Respiratory Coefficients on Sodium succinate - R.T, 224 and Substrains Anaerobic Aerobic 125 ra •p 0 o o • H u Vi (D O O >> U o +> (d u • H u o •*» m u P i « 125 100 75 . 50 25 R.T,224 E E S E , S 1 2 5 6 7 8^ 4--5 Figure 20 Glucose Oxidation by Freshly Isolated Strains of Rh. t r i f o l i i 224 100. 6 M 0) 4 3 P, to a o to >. M O 80-60. 40' 20 E 2 E 8 v3 Figure 21 Endogenous Oxygen Uptake with A n Substrains of Rh. t r i f o l i l 224 60. 50 « o 40. o M od p. p a « >. K O 30. 20-10-,E E, E. E. E. E, RT 224 B B, '10 '11 Figure 22 Glucose Oxidation by A l l Strains of Rh, t r i f o l i i 224 240 • • • • o d> M 0 43 P< P 0 O tiD >» K O 200 160 120-80 40 E El E. E, Er Es RT 2241 C6 Figure 1 Strain Variation i n Dehydrogenase A c t i v i t y 1. RT 22B 2 . RT 224 3 . RT 226 4. RT 231 3 . RT 3 9 - 1 1 2 3 4 3 1 2 5 4 3 1 2 3 4 5 1 23 4 5 1 2 3 4 5 Fructose Sucrose Cellobiose Raffinose Starch 1 1 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 S a l i c i n Mannitol Xylose Sod, malate Glycerol Figure 2 Variation i n Dehydrogenase A c t i v i t y with Time - R. t r i f o l i i 224 1 0 0 75 5 0 -25 0 1 . F i r s t Tests 2 . After 2 months 3 . After 8 months 4 . After 1 2 months 1 2 3 4 Mannose 1 2 3 4 Fructose 1 2 3 4 Xylose 1 2 3 4 Cellobiose 1 2 3 4 Raffinose 1 0 0 75 -50 25 ' 1 2 3 4 Glycerol 1 2 3 4 Mannitol 1 2 3 4 Sodium succinate 1 2 3 4 Sodium malate 1 2 3 4 E t h y l alcohol Figure 1 Dehydrogenase A c t i v i t y of Strains and Substrains of R. t r i f o l i i upon Sodium succinate 100-75-50-25-E E. E. E. E. E. E, Substrains 100 75 • 50 . 25 RT 224 B. B. 10 11 12 Substrains. Figure 2 Endogenous 0,0^ of Strains and Substrains of R. t r i f o l i i 224 CO 60-40 30 20 10' E E. E E E E E, Substrains CVJ o cf 60-50 • 40 • 3 0 -20 • 10 • RT 224 B, B„ C, C C c c. c c c c 1 2 1 2 3 5 6 7 8 9 10 11 12 Substrains Figure 3 Anaerobic Endogenous Respiration with Strains and Substrains of R, t r i f o l i i 224 100 . 73 • 30 25 E E. E. E. E, E. E, Substrains 100 75 • 50 25 RT 22 L i '10 '11 '12 Substrains Figure 4 Aerobic Glucose Oxidation of Strains and Substrains of R. t r i f o l i i 224 240 •-200 • 160 • 120 • 80 • 40 • E E, E, E. E, E< Substrains RT 224 B, '11 '12 Substrains Figure 5 Dehydrogenase A c t i v i t y upon Mannitol of Strains and Substrains of R. t r i f o l i i 224 100 75 50 . 25 • E E E E E 1 2 5 6 7 E, Substrains 100 • 75 50 • 25 • R 224 ^1 ^2 ^1 ^2 ^3 ^5 ^6 °7 °8 °9 °10 ° 1 ] Substrains Figure 6 Aerobic Oxidation of Mannitol by Strains and Substrains of R. t r i f o l i i 224 150 .T 100 . CM O & 50 E El E, 1. E, E, Ec Substrains 150 . CO o 100 50 RT 224 \"2 s \"7 =8 =9 ° 1 0 ° 1 1 12 Substrains Figure 7 Aerobic Respiratory Coefficients upon Sodium succinate of Strains and Substrains of R, t r i f o l i i 224 150-100. E E. E. E, E. E, E 8 Substrains 150-100. 50. RT 224 B„ C, C^ C, C/ c„ Cn c c G 1 2 1 2 3 5 6 7 8 9 10 11 12 Substrains Figure 8 Comparative Aerobic and Anaerobic Respiratory A c t i v i t y of Four Strains of R. t r i f o l i i 224 150 100 50 End, Mann. Sod. sueo, 150. 100 50 End. AT-E Mann. B Sod. succ. RT 224 E RT 224 150 100. 50. A~B\" End, Mann, Sod. succ. 150-100. 50, A B End. Mann, A B_ Sod. succ. RT 224 C, RT 224 C 12 Plate 1 i 5^ . Plate 3 Plata A. Plate 5. 45. Plate 6 Figure 1 Rate of Oxygen Uptake - Strep, l a c t i s S.A, 30 Time in Minutes O > a o o -O ft) CD 01 H -ti (B d-O O CO at) o CO CO o p: 4 (D ro Figure 5 Aerobic Respiratory A c t i v i t y of Strep, l a c t i s A.T.C. 374 40 • 35 • 30 25 • 20 • 15 -10 • 5 • 0 0 CO H fl © 0 0) 0 0 0 CO +> a> m m 4» 43 bD 0 0 m 0 0 0 ft d 0 0 cd 0 42 +» u r-i Pi 1-4 0 H a a a H > 0) 43 0 H w c» iA CO H CO Figure 4 Comparative Oxidative A c t i v i t y of Strep, l a c t i s S.A. JO and Strep, l a c t i s A.T.C. 374 A. Strep, l a c t i s S.A. 30 B. Strep, l a c t i s A.T.C. 374 40 • 35 • 30 • 25 20 15 10 A B O a o o XS A B o m o o H A B m o o (0 1-4 (d A B H A B o ^ l aj 43 CO A B o 43 A B 9 a ft e a H >. 43 Figure 5 Respiratory Quotients - Strep, l a c t i s A.T.C. 374 Figure 6 Comparative Glucose Oxidations by Strep, l a c t i s and Bact. c o l i 0 15 30 45 60 Time in Minutes Figure 1 Comparative Enzymic A c t i v i t y upon Carbohydrates with Strep, l a c t i s S.A. 30 A. Aerobic Respiratory Coefficients B. Anaerobic \" \" C. Acid GQ +> a © o o >1 u o 4 3 1^ •H p m (D 100 . 73 30 23 • A B C Galactose A B C Sucrose A B C Lactose A B C Trehalose A B C Mannitol CQ +3 fi O O >= fH O 4 3 CO u p4 © Figure 2 Comparative Enzymic A c t i v i t y of Strep, l a c t i s A.T.C. 374 150 125 100 . 75 . 50 • 25 A B C Galactose A B .C Sucrose A B C Raffinose A B C Dextrin A B C Inulin Figure 1 Influence of Previous Adaptation upon carbohydrate Dehydrogenation. Strep, l a c t i s SA. 30 A. Cells grown i n Glucose broth B. C e l l s grown i n lactose broth C. C e l l s grown in starch broth D. Cells grown in mannitol broth 100. 75. 50. 25. _ L _ A B C D Arab inose A B C D Lactose A B G D iltose A B C D Baffinose A B 0 D Starch Subs trat.e Expressed on Percentage of the Glucose Reduction Time. Figure S Influence of Previous Adaptation upon Carbohydrate Dehydrogenation. Strepi l a c t i s ATC 374 A. C e l l s grown in glucose broth B. C e l l s grown in lactose broth d. C e l l s grown in mannitol broth (D O o a o 3 a> 100 75 50 25 A B G Mannose A B C Galac to se A B C Lac to se A B C Mannitol A B C Raffinose Substrate K Expressed as Percentage of the Glucose Seduction Time. Time In Houra J^luene« A d a p ^ t l e a upon Wwmim.1mlsi.9iB. «f « i f' i t 4 ji m IS. Figure 5 Influence of Adaptation upon Fermentation of Galaotoee by Suspensions of Strep, l a o t i s SA. 30 Time i n Hours Figure 6 Influence of Adaptation upon Fermentation of Laotose t j Sttspensions of Strep, l a c t i s ATC 374. Time i n Hours 11. Figure 7 Influence of Adaptation upon Fermentation of Galactose by Suspensions of Strep, l a c t i a ATC.374 C e l l s from Galactose Broth 0 1 2 3 4 5 6 Time in Hours Figure 1 (a) Stimulation of Aerobic Production of Lactic Acid by Resting C e l l s of Strep, l a c t i s ATC 374 from Glucose Figure 1 (b) Stimulation of Anaerobic Production of Lactic Acid by Resting C e l l s of S t r e p , l a c t i s ATC 374 from Glucose, rH o o u P +3 d C5 1,03 0,73 Peptone-DifCO east Extract NH4 CI Asparagine Sod,caseinate 0,45 0,15 • .h FIGURE 2 S t i m u l a t i o n of Aerobio Production of L a c t i c A c i d by Suspensions of Strep, l a c t i s ATC 374 from Glucose. E f f e c t of Representative Nitrogen Sources / / Proteose ^ peptone ^ ^ ^ Tryptone / Beef E x t r a c t / ^ Urea ^^^^ G l y c i n e •1 *2 '3 '4 Time i n Hours FIGURE 3 Influence of various Nitrogen Sources i n Stimulating Respiration of Suspensions of Strep, l a c t i s ATC 314 with Glucose ^ P r o t e o s e peptone y ^ Beef Extract / Tryptone / / ^ // . Glycine // / _ . NaNO, 7 ' • ^ 1 1 ^rea •2 -3 Time i n Hours Figure 4 Belative Stimulation of Aerobic Lactic Acid Production, Anaerobic Lactic Acid Production and Heapiration by VariouB Nitrogen Sources, A, Aerobic Lactis Acid Production B, Anaerobic Lactic Acid Produc t ion 0, Oxygen Uptake 1.2-1,0 .6. .6. .4-.2. A B C Amm* Chloride A B C Amm. Sulfate A B C Urea A B C Cystine A B O Asparagin If % Figure 4 (Continued) Relative Stimulation of Aerobic Lactic Acid Production, Anaerobic Lactic Acid Production and Respiration by Various Nitrogen Sources* A, Aerobic Lactis Acid Production B, Anaerobic Lactic Acid Production C, Oxygen Uptake (0 w a o o OJ o 1.2. 1.0. .8. .6. .4. .2. A B C Peptone - Difco A B C Peptone • Witte's A B C Sod» Casei nat A B C Beef Extrac t A B O Yeast Extract •180 •150 • 120 . 90 . 60 • 30 o Figure 5-Stimulation of Aerobic Lactic Acid Production by Various Nitrogen Sources. Strep, l a c t i s Suspension i n the Presence of Glucose. ^ O o H P TJ •H O < o ft •p o cd Yeast Extract . Beef Extract Prot. peptone Cystine -Peptone - Witte Sod. caseinate Glycine N H 4 C I - Urea '1 '2 '3 - 4 Time in Hours A l l Values Calculated to the Common Basis 0 . 2 5 gm. Nitrogen, FIGURE 6 Stimulation of Respiration hj Various Nitrogen Sources. Strep, l a c t i s ATC J74 Suspension i n . Presence of Glucose. ^ 1 5 0 0 . Beef Extract Time in Hours X A l l Values Calculated to the Common Basis 0 , 2 5 gm. Nitrogen gIGUHE 7 R e l a t i v e Stim-alation of AerdDic L a c t i c A c i d Production, Anaerobic L a c t i c A c i d Production and R e s p i r a t i o n by Various Nitrogen Sources. A. Aerobic L a c t i c A c i d Production B. Anaerobic L a c t i c A c i d Production C. Oxygen Uptake A B C AB G Amm. c h l o r i d e Amm. s u l f a t e i l A B C Urea A B C A B C 1500 .1000 o I CD a t3 03 CD. 500 . 250 Glycine Cystine 91. gIGURE 7 _ ( c o n t r d j R e l a t i v e S t i m u l a t i o n of Aerobic L a c t i c I c i d Production, Anaerobic L a c t i c A c i d Production and R e s p i r a t i o n by Various Hitrogen Sources. A. Aerobic L a c t i c A c i d Production B. Anaerobic L a c t i c A c i d Production G. Oxygen Uptake a o o H CI nd • H O <^ o • H -P O 05 .625-.•37^-.250-.125-A B O Peptone L i fee A B C Pe pt one Wltte A B C Sod. daseinate A B C Beef E x t r a c t A B C Yeast E x t r a c t 1500 ,1250 1000 7^0 500 250 CfQ, CD P. CD, CD, "@en ; edm:hasType "Thesis/Dissertation"@en ; edm:isShownAt "10.14288/1.0105653"@en ; dcterms:language "eng"@en ; ns0:degreeDiscipline "Agricultural Economics"@en ; edm:provider "Vancouver : University of British Columbia Library"@en ; dcterms:publisher "University of British Columbia"@en ; dcterms:rights "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en ; ns0:scholarLevel "Graduate"@en ; dcterms:title "Studies on the respiratory enzymes of the lactic acid and nitrogen-fixing bacteria"@en ; dcterms:type "Text"@en ; ns0:identifierURI "http://hdl.handle.net/2429/38978"@en .