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Studies on the amino acid metabollism of bacteria responsible for the surface taint in butter Neilson, Nora Effie 1943

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;' "t H: 2> H V STUDIES ON THE AMINO ACID METABOLISM OP BACTERIA RESPONSIBLE FOR SURFACE TAINT IN BUTTER - b y -NORA EFFIE NEILSON Pi -\~U-a-St s S u b m i t t e d I n P a r t i a l F u l f i l l m e n t of t h e Requirements f o r the Degree o f i s t e r o f S c i e n c e i n A g r i c u l t u r e i n the Department o f D a i r y i n g / ft li. I^l OCTOBER, 1943. ! C 0 H T E B T S * 1 • 1 1 1 *** i Pag® General Introduction . . . 1 Part I - Studies on the De ami nation of Amino Acids . . 10 Historical . . . e 10 Experimental (With Tables, Figures and Discussion) 29 General Methods and Procedures 29 The Effect of pH on Ammonia Formation . » • « 52 The Effect of the Age of Culture on Ammonia Formation . . . . . . 37 The Effect of Aerobic and Anaerobic Conditions on Ammonia Formation «. 38 The Rate of Ammonia Formation . . . . . . . o 40 The Effect of the Presence of Carbohydrates i n the Medium on Ammonia Formation «,...<> 41 Fermentation of Carbohydrates i n Shake-Agar Cultures . . . . . « , 41 The Effect of the Presence of Carbohydrates in the Growth and Buffer Media on Ammonia Formation . . . » . < . . . . e 41b The Effect of Increasing the Buffering Capacity of the Cultures on Ammonia Formation and Subsequent Cell Multiplication . . . . . . . . . . . . . . . 49 The Breakdown of Arginine by Proteus iohthyosmius and Pseudomonas putrefaciens . e 56 Ammonia Formation from Non-Amino Acid Nitrogenous Compounds . 57 Discussion . 62 Page Part II - Studies on the Isolation and Identification of Odouriferous Compounds • 74 Historical . . •. . . • • 7 4 Experimental . . • 7 6 Discussion . . . • • » • • 8 ^ Summary and Conclusions . . . . . . . . . 84 Bibliography . . . . . . . . . • • 8 8 iksknowledgments . . . . <• • 92 - 1 • -INTRODUCTION S u r f a c e t a i n t i s the name g i v e n by Marker (48) t o a d e f e c t i n b u t t e r f i r s t observed and d e s c r i b e d b y him i n a l o t of p a s t e u r i z e d A l b e r t a creamery b u t t e r s o l d on the Vancouver market i n :1919. The d e f e c t i s c h a r a c t e r i z e d by a t y p i c a l odour d e s c r i b e d b y Thornton (71) as t h a t of " s w e a t y - f e e t " w hich develops a f t e r exposure to the a i r on samples of b u t t e r contaminated by one or more of a group of b a c t e r i a known a t p r e s e n t as " S u r f a c e T a i n t B a c t e r i a " . T h i s group has been shown by Campbell (10) t o c o n t a i n among ot h e r s two s p e c i e s of b a c t e r i a o r i g i n a l l y i s o l a t e d by Hammer (31) - P r o t e u s i c h t h y o s m i u s f o u n d i n a sample of e v a p o r a t e d m i l k h a v i n g a f i s h y odour and Pseudomonas p u t r e f a c i e n s f r o m s o - c a l l e d " p u t r i d " b u t t e r , s t r a i n s of which have more r e c e n t l y been i s o l a t e d f r o m samples of t y p i c a l s u r f a c e t a i n t b u t t e r b y T h o r n t o n et a l . The odour of s u r f a c e t a i n t has been shown, beyond doubt, t o be due t o b a c t e r i a l a c t i v i t y . L i t t l e i s known, however, about the c h e m i c a l o r i g i n and development of the d e f e c t i n b u t t e r . P r e s e n t e v i d e n c e i n d i c a t e s t h a t the "sweaty-f e e t " odour of t y p i c a l s u r f a c e t a i n t o r i g i n a t e s i n the p r o t e i n of b u t t e r . The mechanism of i t s e l a b o r a t i o n i s , as y e t , un-known; s e v e r a l hypotheses c o n c e r n i n g i t s f o r m a t i o n , however, have been advanced. O u t s t a n d i n g among t h e s e a r e the i n d o l e h y p o t h e s i s o f Campbell (9) and the d e c a r b o x y l a t i o n and deamin-a t i o n hypotheses of Campbell ( 1 0 ) . N e i l s o n (50) has shown t h a t the f a i l u r e t o d e t e c t o „ i n d o l e i n the s e r a of s u r f a c e t a i n t b u t t e r s i s p r a c t i c a l l y p r o o f p o s i t i v e t h a t i n d o l e e l a b o r a t i o n i s not e s s e n t i a l t o the p r o d u c t i o n of s u r f a c e t a i n t i n b u t t e r . The i n v e s t i g a t i o n s of Campbell (10) i n w h i c h e x p e r i -mental evidence c o u l d not be o b t a i n e d to support, the hypothe-s i s t h a t amines a r e formed d u r i n g the e l a b o r a t i o n of s u r f a c e t a i n t , I n d i c a t e t h a t d e c a r b o x y l a t i v e breakdown i s p r o b a b l y not a f a c t o r t o be c o n s i d e r e d i n t h i s s t u d y . The h y p o t h e s i s t h a t the f o r m a t i o n of hydroxy, k e t o and u n s a t u r a t e d a c i d s f r o m amino a c i d s by the d e a m i n a t i n g a c t i o n of s u r f a c e - t a i n t p r o d u c i n g b a c t e r i a was advanced by Campbell (10) as a p o s s i b l e e x p l a n a t i o n o f the mechanism r e s p o n s i b l e f o r the e l a b o r a t i o n of the " s w e a t y - f e e t " odour i n s u r f a c e t a i n t b u t t e r . The o b j e c t of the work r e p o r t e d upon h e r e i n has been t o determine th e v a l i d i t y of t h i s hypo-t h e s i s -The e a r l y s t u d i e s r e c o r d e d i n the l i t e r a t u r e con-c e r n i n g the n a t u r e of the m i c r o b i a l breakdown of amino a c i d s a r e of l i t t l e s c i e n t i f i c v a l u e because t h e y were c a r r i e d out w i t h mixed c u l t u r e s of p u t r e f a c t i v e m i c r o o r g a n i s m s on m i x t u r e s of p r o t e i n s and p r o t e i n d e c o m p o s i t i o n p r o d u c t s . These s t u d i e s were f o l l o w e d b y a p e r i o d of i n v e s t i g a t i o n s employing the a c t i o n of mixed c u l t u r e s on pure amino a c i d s , b u t the r e l a t i o n between the a c t i o n of any one o r g a n i s m and the p a r t i c u l a r type of p r o d u c t formed c o u l d not be e s t a b l i s h e d . The i n t r o -d u c t i o n by Harden (1901) and by E h r l i c h (1905) of p r o c e d u r e s f o r the s t u d y of the a c t i o n of pure c u l t u r e s of a s i n g l e micro-o r g a n i s m on i n d i v i d u a l pure amino a c i d s , i n i t i a t e d the modern ~ 3 -method of approach t o i n v e s t i g a t i o n s on amino a c i d c a t a b o l i s m -The e a r l y t h e o r i e s of d e a m i n a t i o n of Neuberg and L a n g s t e i n and of Embden p o s t u l a t e d t h a t the f o r m a t i o n of a l p h a -h ydroxy a c i d s w i t h the e l a b o r a t i o n o f ammonia r e s u l t e d f r o m the h y d r o l y t i c d e a m i n a t i o n of amino a c i d s . More r e c e n t e v i -dence, however, i n d i c a t e s t h a t d e a m i n a t i o n of amino a c i d s i s not l i m i t e d t o one type o f breakdown but may f o l l o w one of a number of courses dependent upon the s t r u c t u r e of the amino a c i d , the m i c r o o r g a n i s m employed and the c o m p o s i t i o n and con-d i t i o n of the r e a c t i n g medium ( 6 1 ) . The v a r i o u s paths of amino a c i d breakdown have been bro u g h t t o g e t h e r by Anderson (2) and h i s c h a r t of t h e i r a e r o b i c d e c o m p o s i t i o n i s p r e s e n t e d i n f i g u r e 1. The p r i m a r y a e r o b i c breakdown of amino a c i d s may f o l l o w one of two c o u r s e s , d e c a r b o x y l a t i o n o r d e a m i n a t i o n . As s t a t e d e a r l i e r , Campbell (10) was u n a b l e t o o b t a i n e v i d e n c e i n s u p p o r t of. the h y p o t h e s i s t h a t amines a r e formed d u r i n g the e l a b o r a t i o n o f s u r f a c e t a i n t , i n d i c a t i n g t h a t d e c a r b o x y l a -t i v e breakdown i s p r o b a b l y n ot a f a c t o r t o be c o n s i d e r e d i n t h i s s t u d y . Other s t u d i e s b y the same a u t h o r have shown t h a t a e r a t i o n i n c r e a s e s the development of s u r f a c e t a i n t i n b u t t e r , t h e r e b y s u g g e s t i n g t h a t the mechanism f o r the breakdown of amino a c i d s b y s u r f a c e t a i n t b a c t e r i a may be o x i d a t i v e i n n a t u r e . • The f i n d i n g s of N e i l s o n ( 5 0 ) s f r o m experiments i n w h i c h o r g a n i c compounds t h a t c o u l d r e s u l t f r o m the breakdown •Pi o ra U © W e tri •H O H «} o ri H" •ri 1 O o t3 © o •ri rQ O ?H < o 03 m o 03 O o s o o to c\3 o o o o « K o SI w o o o o o' •o o M o o o « S3 o o P4 o o o g o o O o o rH © o o •n •ri o a5 1 o W-P O CD o M O I • Gi O A. O Pi rH <> c3 03—' w — o o .8 8 . « ra-pt •m" o o ra + m 8 o ra o W. o o M o o to W o Pt ra o o o ra CV3 tj W. o o ra o <+H © o o O -P • © pt ^ © K o o o + ra Pt 03 O O • i ra > • P3 id a! rri O CO •ri ri © >» rH 43 o d ,ri ra • S' o © ri •ri rH © -ri © ,ri EH of c e r t a i n amino a c i d s , were d i s s o l v e d i n a u t o c l a v e d m i l k and the r e s p e c t i v e odours e m i t t e d "by them d e t e r a i i n e d b y Thornton's t e s t (71) add f u r t h e r e v idence to the h y p o t h e s i s t h a t the s u r -f a c e t a i n t odour i s i n t i m a t e l y r e l a t e d t o the d e c o m p o s i t i o n p r o d u c t s of p a r t i c u l a r amino a c i d s . The f i r s t s t e p i n the d e a m i n a t i o n of amino a c i d s appeal's t o be d e s a t u r a t i o n a t the a l p h a - b e t a - l i n k a g e w i t h the f o r m a t i o n of u n s a t u r a t e d a c i d s and the l i b e r a t i o n of ammonia. From t h i s s t a g e , the breakdown may proceed i n one or more of t h r e e d i r e c t i o n s . The f i r s t o f t h e s e i s h y d r o l y s i s g i v i n g a l p h a - h y d r o x y a c i d s . The odours e m i t t e d by the a l p h a - h y d r o x y a c i d s of c e r -t a i n amino a c i d s are known t o resemble c l o s e l y those e v o l v e d from p u t r e f a c t i v e m a t t e r and suggest t h e i r p o s s i b l e r e l a t i o n -s h i p to the s u r f a c e t a i n t odour i n b u t t e r . The second and t h i r d c o u r s e s of breakdown f r o m the u n s a t u r a t e d a c i d a r e o x i d a t i v e i n n a t u r e and r e s u l t i n the f o r m a t i o n o f the e n o l - f o r m s of the a l p h a - k e t o and b e t a - k e t o a c i d s r e s p e c t i v e l y . A g a i n t h e r e ^ i s the p o s s i b i l i t y t h a t the k e t o a c i d s are i n some way i n v o l v e d i n t h e e l a b o r a t i o n of the s u r f a c e t a i n t odour. D e c a r b o x y l a t i o n o f t h e s e k e t o a c i d s r e s u l t s i n the f o r m a t i o n of a r o m a t i c aldehydes whereas hydro-l y s i s causes the p r o d u c t i o n of l o w e r f a t t y a c i d s . These f a t t y a c i d s and aldehydes i n c o m b i n a t i o n w i t h hydroxy and k e t o a c i d s must a l s o be c o n s i d e r e d as p o s s i b l e s o u r c e s of the c h a r a c t e r -i s t i c " s w e a t y - f e e t " odour of s u r f a c e t a i n t b u t t e r . F u r t h e r e v i d e n c e i n s u p p o r t of the d e a m i n a t i o n hypo-t h e s i s i s c o n t a i n e d i n the r e p o r t s of D u n k l e y and Wolchow-- 5 .-The f i n d i n g s of D u nkley (12) i n d i c a t e t h a t the compounds formed i n the development of s u r f a c e t a i n t are a c i d i c i n n a t u r e . Wolchow (71) has suggested t h a t the p r o d u c t s of r e -d u c t i v e d e a m i n a t i o n may he concerned w i t h the e l a b o r a t i o n of s u r f a c e t a i n t i n b u t t e r . The s t r u c t u r e of the amino a c i d i s one of the p r i n -c i p a l f a c t o r s - d e t e r m i n i n g the n a t u r e of the d e a m i n a t i v e breakdown b r o u g h t about by the a c t i o n of m i c r o o r g a n i s m s . Anderson (2) concludes t h a t l o n g c h a i n amino a c i d s are more e a s i l y a t t a c k e d t h a n those c o n t a i n i n g r i n g s t r u c t u r e s , and t h a t the ease of a t t a c k i n c r e a s e s w i t h the l e n g t h of the c h a i n . The f i n d i n g s of N e i l s o n ( 5 0 ) , however, show c l e a r l y t h a t t h r e e s p e c i e s o f s u r f a c e - t a i n t p r o d u c i n g b a c t e r i a are a b l e t o open the i m i n a z o l r i n g of h i s t i d i n e and the p y r o l l i -d i n e r i n g of p r o l i n e i n c o n t r a s t w i t h t h e i r low ammonia-f o r m i n g a b i l i t y f r o m the s t r a i g h t c h a i n amino a c i d s - l e u c i n e , l y s i n e and m e t h i o n i n e ; and a t t a c k the s h o r t c h a i n amino a c i d s - g l y c i n e and b e t a a l a n i n e - t o a g r e a t e r degree t h a n the above mentioned l o n g e r c h a i n amino a c i d s . .The p o s i t i o n o f the amino group may a l s o p l a y an i m p o r t a n t r o l e not o n l y i n d e t e r m i n i n g the n a t u r e of the b r e a k -down but a l s o the ease w i t h which i t o c c u r s - The l i t e r a t u r e c o n t a i n s v e r y l i t t l e d a t a on t h i s p o i n t . I n the same i n v e s t i -g a t i o n ( 5 0 ) , the above a u t h o r o b t a i n e d e vidence t h a t the f u r t h e r the amino group was f r o m the a l p h a - p o s i t i o n , the more d i f f i c u l t i t was t o deaminate by the b a c t e r i a l s p e c i e s employed. The presence of c a r b o h y d r a t e s i n t h e r a c t i n g medium m o d i f i e s or changes the course of amino a c i d c a t a b o l i s m . A - 6 number of t h e o r i e s have been advanced-to e x p l a i n t h i s obser-vation.. K e n d a l l and h i s a s s o c i a t e s (39, 40 and 41) s t a t e t h a t the presence of c a r b o h y d r a t e " s p a r e s " p r o t e i n f r o m a t -t a c k b y s u b s t i t u t i n g a more r e a d i l y a s s i m i l a b l e f o r m of f o o d , b u t , as Stephenson (61) p o i n t s o u t , t h i s c l a i m r e s t s on a f a l s e comparison between t h e b a c t e r i a l p r o d u c t i o n of ammonia and the mammalian p r o d u c t i o n of u r e a . Urea i s a waste p r o -duct w h i c h cannot serve as a n i t r o g e n o u s f o o d f o r the a n i m a l i n w h i c h i t was produced, t h e r e f o r e i t s r i s e and f a l l i s a t r u e measure of p r o t e i n m e t a b o l i s m and when i n c r e a s e d c a r -b o h y d r a t e causes d e c r e a s e d p r o d u c t i o n of u r e a , the carbohy-d r a t e i s s a i d t o " s p a r e " the p r o t e i n . W i t h b a c t e r i a , however, w h i l e ammonia i s the c h i e f n i t r o g e n o u s p r o d u c t of the b a c t e r i a l d e c o m p o s i t i o n o f p r o t e i n , i t i s a l s o an e x c e l l e n t n i t r o g e n source f o r many s p e c i e s of b a c t e r i a . T h e r e f o r e i t s d i s a p p e a r -ance f r o m the c u l t u r e - media may be caused e i t h e r by d e c r e a s e d p r o d u c t i o n , o r , what i s more l i k e l y , by i n c r e a s e d u t i l i z a t i o n as a n i t r o g e n source f o r c e l l r e p r o d u c t i o n . R a i s t r i c k (57) i n an e x p l a n a t i o n of the e f f e c t of the presence of c a r b o h y d r a t e s concludes w i t h the b e l i e f t h a t c a r b o h y d r a t e , f a r f r o m h a v i n g a p r o t e i n - s p a r i n g e f f e c t , a c t u a l l y e n a b l e s the b a c t e r i a t o u t i l i z e more p r o t e i n or p r o -t e i n p r o d u c t s t h a n t h e y would i n the absence o f c a r b o h y d r a t e . Waksman and Lomanitz (68) p o i n t out t h a t a l i v i n g b e i n g d e r i v e s i t s energy f r o m the substance w h i c h i s more a v a i l a b l e to i t and w h i c h may be s p e c i f i c f o r the p a r t i c u l a r o rganism. ' - 7 ~ The s t u d i e s of a number of i n v e s t i g a t o r s whose f i n d -i n g s are g i v e n i n the h i s t o r i c a l s e c t i o n of t h i s t h e s i s show c l e a r l y t h a t the a d d i t i o n of a s p e c i f i c c a r b o h y d r a t e t o t h e r e a c t i n g medium w i l l change the course of amino a c i d b r e ak-down and g i v e d i f f e r e n t p r o d u c t s . A s a t i s f a c t o r y e x p l a n a t i o n of these r e s u l t s i s n o t , as y e t f o r t h c o m i n g . The f i r s t p a r t of the e x p e r i m e n t a l work of t h i s problem was devoted t o an i n v e s t i g a t i o n of the c o n d i t i o n s a f f e c t i n g the ammonia p r o d u c i n g a b i l i t i e s of two s p e c i e s of s u r f a c e - t a i n t p r o d u c i n g b a c t e r i a f r o m a s p e c i f i c group of amino a c i d s . These amino a c i d s - a r g i n i n e , a s p a r t i c a c i d , g l u t a m i c a c i d , h i s t i d i n e and p r o l i n e - were chosen f o r a number of r e a s o n s , the more i m p o r t a n t of w hich a r e : (1) Ammonia i s r e a d i l y l i b e r a t e d from them by s p e c i e s of s u r f a c e t a i n t b a c t e r i a . (2) The d e c o m p o s i t i o n p r o d u c t s of t h e s e f i v e amino a c i d s a r e r e l a t e d t o one a n o ther as can be seen i n f i g u r e 2. (3) C e r t a i n of t h e s e d e c o m p o s i t i o n p r o d u c t s have been shown t o emit odours t h a t c l o s e l y resemble the c h a r a c t e r i s t i c s u r f a c e t a i n t odour ( 5 0 ) . Prom the p o i n t of view of s t u d y i n g the d e a m i n a t i o n of amino a c i d s of d i f f e r e n t b a s i c s t r u c t u r e , the f i v e chosen r e p r e s e n t t h r e e s t r u c t u r a l g r o u p s . A r g i n i n e i s a complex s t r a i g h t c h a i n amino a c i d con-t a i n i n g f o u r n i t r o g e n s i n t h r e e d i f f e r e n t g r o u p i n g s . The guanido group a t t a c h e d to the d e l t a c arbon atom may be h y dro-l y s e d f r o m the r e m a i n i n g alpha-amino v a l e r i c a c i d - The - 8 -l i t e r a t u r e c o n t a i n s p r a c t i c a l l y no e vidence f o r t h i s c l e a v -age . The u s u a l method of decomposing a r g i n i n e i s t h rough the enzyme, a r g i n a s e , to g i v e urea and o r n i t h i n e ( a l p h a , d e l t a , diamino v a l e r i c a c i d ) . The u r e a may be s u b s e q u e n t l y hydro-l y s e d by the enzyme, u r e a s e , i n t o ammonia and c a r b o n d i o x i d e and the o r n i t h i n e d e a m i n i z e d a t e i t h e r the a l p h a or the d e l t a amino group or b o t h to g i v e v a l e r i c a c i d or one of i t s d e r i v a t i v e s • The next two amino a c i d s chosen f o r t h i s study, a s p a r t i c a c i d and g l u t a m i c a c i d , are mono-amino, d i c a r b o x y l i c a c i d s , the l a t t e r - c o n t a i n i n g one more m e t h y l group i n the c h a i n between the c a r b o x y l groups than the f o r m e r . The l a s t two amino a c i d s c o n t a i n f i v e - e l e m e n t r i n g s t r u c t u r e s . H i s t i d i n e or alpha-amino b e t a - i m i n a s o l p r o p i o n i c a c i d has two n i t r o g e n s i n the r i n g and one i n the s i d e c h a i n . P r o l i n e , on the o t h e r hand, i s not a t r u e amino a c i d , as i t s one n i t r o g e n i s p r e s e n t i n the r i n g as an imino g r o u p . As the f i r s t p a r t o f t h i s s t u d y d e v e l o p e d , i t soon became e v i d e n t t h a t c e r t a i n of the, chosen amino a c i d s were broke n down i n t o compounds g i v i n g odours s u g g e s t i v e of t h a t c h a r a c t e r i s t i c of s u r f a c e t a i n t . I t was d e c i d e d , t h e r e f o r e , to devote a p a r t of the e x p e r i m e n t a l s t u d y t o the a c t u a l i s o l a t i o n and, i f p o s s i b l e , the i d e n t i f i c a t i o n of these odour-f orming c ompounds. W i t h the u l t i m a t e o b j e c t i v e of d e t e r m i n i n g the n a t u r e of the compounds r e s p o n s i b l e f o r the e l a b o r a t i o n of the c h a r -a c t e r i s t i c " s w e a t y - f e e t " odour of s u r f a c e t a i n t b u t t e r , the f o l l o w i n g approaches were made to the problem: „ g >, (1) An I n v e s t i g a t i o n of the c o n d i t i o n s a f f e c t i n g ammonia f o r m a t i o n f rom a r g i n i n e , a s p a r t l c a c i d , g l u t a m i c a c i d , h i s t i d i n e , and p r o l i n e by two s p e c i e s of s u r f a c e - t a i n t p r o d u c i n g b a c t e r i a -(2) A s t u d y of the p r o d u c t i o n , i s o l a t i o n and i d e n t i f i c a t i o n of compounds e v o l v i n g odours i d e n t i c a l w i t h or i n t i m a t e l y r e l a t e d t o the c h a r a c t e r i s t i c s u r f a c e t a i n t odour. - 10-~ STUDIES ON TEE DEAMIMTIOH OF AMINO ACIDS. ij • . HISTORICAL: A s t u d y of the d e c o m p o s i t i o n of i n d i v i d u a l amino a c i d s by. pure c u l t u r e s of s i n g l e s t r a i n s of microorganisms has brought out many i n t e r e s t i n g f a c t s . The c o m p o s i t i o n of the medium, the c o n d i t i o n s of growth and the type of organism employed have be.en shown to e x e r t a marked i n f l u e n c e on the a c t i v i t y of the enzymes formed and on the type of d e a m i n a t i o n t h a t takes p l a c e . / H y d r o l y t i c d e a m i n a t i o n w i t h the f o r m a t i o n of the hydroxy a c i d may r e s u l t f r o m the a c t i o n of b a c t e r i a on amino a c i d s i n a medium c o n t a i n i n g a l t e r n a t i v e carbon and n i t r o g e n sources and from the a c t i v i t y of y e a s t i n a medium c o n t a i n i n g an i n v e r t sugar and no a l t e r n a t i v e n i t r o g e n s o u r c e . Schmidt, P e t e r s o n and F r e d (52) i s o l a t e d d - l e u c l c a c i d f r o m 1 - l e u c i n e employing Proteus v u l g a r i s . H i r a i (35) o b t a i n e d b e t a - i m i n a z o l l a c t i c a c i d f r om h i s t i d i n e u s i n g B. p r o t e u s . S a s a k i and Otsuka (60) i s o l a t e d the hy d r o x y "acids of 1 - p h e n y l a l a n i n e , 1 - t r y p t o p h a n and 1 - t y r o s i n e employing B. p r o t e u s , B- s u b t i l i s and B. c o l i . H i r a i ( 3 5 ) , u s i n g an a l t e r n a t i v e c arbon s o u r c e , g l y c e r o l , and an a l t e r n a t i v e n i t r o g e n s o u r c e , ammonium c a r -b onate, i s o l a t e d p-hydroxy b e t a - p h e n y l l a c t i c a c i d f r o m t y r o s i n e employing P r o t e u s v u l g a r i s . E h r l i c h and Jacob3en ( 1 9 ) , employing Oidium l a c t i s , o b t a i n e d the hydroxy a c i d s of 1-phenyla1anine, 1 - t r y p t o p h a n and 1 - t y r o s i n e . Woolf (77) i s o l a t e d the hydroxy a c i d of a s p a r t i c a c i d employing B. c o l i ! PART I-; - 11 -i n a medium w i t h no a l t e r n a t i v e carbon and n i t r o g e n source and c o n t a i n i n g t o l u e n e as a b a c t e r i a l growth i n h i b i t o r . H y d r o l y t i c d e a m i n a t i o n and d e c a r b o x y l a t i o n may r e -s u l t i n the f o r m a t i o n of an a l c o h o l w i t h one l e s s carbon atom than the c o r r e s p o n d i n g amino a c i d . T h i s type of breakdown i s caused p r i n c i p a l l y by y e a s t s and molds- E h r l i c h (13, 14, 15, 16, 17 and 1 8 ) , i n a s t u d y of the d e c o m p o s i t i o n of amino a c i d s b y y e a s t s , o b t a i n e d the c o r r e s p o n d i n g a l c o h o l s f r o m 1 - v a l i n e , 1 - l e u c i n e , d l - s e r i n e , 1 - h i s t i d i n e , 1 - p h e n y l a l a n i n e , 1 - t r y p t o -phan and 1 - t y r o s i n e . P r i n g s c h e i m ( 5 3 ) , employing a number of c u l t u r e s of molds, and one of y e a s t , i s o l a t e d the a l c o h o l f r o m 1 - l e u c i n e . R e d u c t i v e d e a m i n a t i o n r e s u l t i n g i n the p r o d u c t i o n of the s a t u r a t e d normal a c i d f r o m the c o r r e s p o n d i n g amino a c i d u s u a l l y occurs i n a medium c o n t a i n i n g no a l t e r n a t i v e carbon and n i t r o g e n s o u r c e s . B r a s c h ( 8 ) , employing B. p u t r i -f i c u s i n . s uch a medium, o b t a i n e d the c o r r e s p o n d i n g a c i d s f r o m g l y c i n e , 1 - a l a n i n e , d l - s e r i n e , 1-aspar t i c a c i d and 1 - t y r o s i n e , w h i l e Nawiasky ( 4 9 ) , u s i n g B. p r o t e u s , and B l a n c h e t i e r e ( 7 ) , u s i n g B. f l o u r e s c e n s , i s o l a t e d the a c i d f r o m a s p a r t i c a c i d . K o e s s l e r and Hanke (44) added g l y c e r o l t o the medium of B r a s c h and i s o l a t e d b e t a - i m i n a z o l p r o p i o n i c a c i d f r o m h i s t i -d i n e employing B. c o l l . Hopkins and Co l e (36) observed t h a t i n d o l e p r o p i o n i c a c i d was formed from t r y p t o p h a n i n a medium c o n t a i n i n g i n o r g a n i c s a l t s w i t h R o c h e l l e s a l t s and ammonium phosphate u s i n g B• c o l i . T r a e t t a - M o s c a (66) has shown t h a t p-hydroxy b e t a - p h e n y l p r o p i o n i c a c i d i s formed f r o m t y r o s i n e i n an i n o r g a n i c medium c o n t a i n i n g ammonium n i t r a t e by a - 12 -" b a c i l l u s r e s e m b l i n g B. pyocyaneus. K i y o k a v a (42) o b t a i n e d b e t a - i m i n a z o l p r o p i o n i c a c i d f rom h i s t i d i n e employing Oidium l a c t i s i n an i n o r g a n i c s a l t medium p l u s one p e r c e n t cane sugar. R e d u c t i v e d e a m i n a t i o n and d e c a r b o x y l a t i o n may r e s u l t i n the f o r m a t i o n of the normal; a c i d c o n t a i n i n g one carbon atom l e s s t h a n the c o r r e s p o n d i n g amino a c i d . The l i t e r a t u r e con-t a i n s o n l y one r e c o r d of t h i s type of breakdown brought about by the a c t i o n of pure c u l t u r e s on amino a c i d s - B r a s c h ( 8 ) , employing B. p u t r i f i c u s i s o l a t e d p r o p i o n i c a c i d f r o m 1-glutamic a c i d i n a medium of i n o r g a n i c salts» D e s a t u r a t i o n a t the a l p h a - b e t a l i n k a g e causes the f o r -m a t i o n of the c o r r e s p o n d i n g u n s a t u r a t e d a c i d w i t h the l i b e r a -t i o n of a m o l e c u l e of ammonia. R a i s t r i c k ' s i s o l a t i o n (55) of u r o c a n i c a c i d from 1 - h i s t i d i n e i n 1917 i s the f i r s t r e c o r d e d i n s t a n c e of the b a c t e r i a l c o n v e r s i o n of an amino a c i d i n t o an u n s a t u r a t e d a c i d . He employed the a c t i o n of pure c u l t u r e s o f f i v e s p e c i e s of i n t e s t i n a l b a c t e r i a on h i s t i d i n e i n R i n g e r ' s s o l u t i o n . H i r a i ( 3 5 ) , i n 1921, i s o l a t e d the a c r y l i c a c i d of - t y r o s i n e a f t e r t w e l v e days i n c u b a t i o n of B. p r o t e u s i n an i n o r -g a n i c s a l t medium c o n t a i n i n g no a l t e r n a t i v e carbon and n i t r o g e n s o u r c e s . Q u a s t e l and Woolf (54) o b t a i n e d f u m a r i c a c i d f r o m a s p a r t i c a c i d i n a phosphate b u f f e r medium c o n t a i n i n g t o l u e n e as a growth i n h i b i t o r . The o x i d a t i v e breakdown of amino a c i d s may r e s u l t i n the f o r m a t i o n o f a number of a c i d s c o n t a i n i n g a d e c r e a s i n g number of carbon atoms. The k e t o a c i d s formed f r o m the p r i m a r y o x i d a t i o n of the amino a c i d s have not been i s o l a t e d except i n r a r e c a s e s , b u t the a c i d s f r o m the subsequent o x i d a t i o n of the -13-k e t o a c i d s of c e r t a i n amino a c i d s have been o b t a i n e d and i d e n -t i f i e d . Nawiasky (49), employing B. p r o t e u s i n an i n o r g a n i c s a l t medium c o n t a i n i n g no a l t e r n a t i v e carbon and n i t r o g e n s o u r c e s , i s o l a t e d a c e t i c a c i d f rom 1 - a l a n i n e , i s o b u t y r i c , a c e t i c and f o r m i c a c i d s f r o m 1 - v a l i n e , a c e t i c a c i d f rom l - a s p a r t i c ' a c i d and s u c c i n i c a c i d f r o m 1 - g l u t a m i c a c i d -B r a s c h (8) i s o l a t e d f o r m i c a c i d f r o m d l - s e r i n e u s i n g B. p u t -r i f i c u s i n an i n o r g a n i c s a l t medium s i m i l a r t o t h a t of Nawias-ky . R a i s t r i c k (56) has shown t h a t organisms of the c o l i -typhosus group i n a medium of i n o r g a n i c s a l t s w i t h no a l t e r -n a t i v e carbon and v n i t r o g e n s o u r c e s , r u p t u r e the i m i n a z o l r i n g of h i s t i d i n e . Hopkins and Co l e (36) i s o l a t e d i n d o l e a c e t i c a c i d and i n d o l e f r o m the o x i d a t i v e breakdown of t r y p t o p h a n by B» c o l i i n an I n o r g a n i c s a l t medium c o n t a i n i n g R o c h e l l e s a l t s and ammonium phosphate. Woods (7 2 ) , Happold and Hoyle (32) and Woods (73) i s o l a t e d i n d o l e , employing, washed c e l l suspen-s i o n s of B. c o l i on t r y p t o p h a n i n b u f f e r s o l u t i o n . R a i s t r i c k and C l a r k e (57) o b t a i n e d e v i d e n c e f o r the r u p t u r e of t h e i n d o l e r i n g of t r y p t o p h a n by B. pyocyaneus and B. f l o u r e s c e n s i n an i n o r g a n i c s a l t medium. The o x i d a t i v e breakdown o f 1 - t y r o s i n e v a r i e s w i t h the c o m p o s i t i o n of the medium. T r a e t t a -Moaca (66) i s o l a t e d p-hydroxy b e n z o i c a c i d and p - c r e s o l f r o m t y r o s i n e employing B. pyocyaneus i n an i n o r g a n i c s a l t medium c o n t a i n i n g ammonium n i t r a t e . B e r t h e l o t (6) and R h e i n (58 and 59) o b t a i n e d e v i d e n c e f o r the f o r m a t i o n of p h e n o l by B. pheno-logenes f r o m t y r o s i n e i n an i n o r g a n i c s a l t medium c o n t a i n i n g ammonium l a c t a t e and asparagin» H i r a i ;(35) i s o l a t e d p-hydroxy b e t a - p h e n y l a c e t i c a c i d f r o m t y r o s i n e employing the same con--14-d i t i o n s as f o r the i s o l a t i o n o f p-hydroxy h e t a - p h e n y l a c r y l i c a c i d , except u s i n g f o r t y days' i n c u b a t i o n i n p l a c e of t w e l v e days. R a i s t r i c k and C l a r k e (57) found e v i d e n c e f o r t h e r u p -t u r e o f the r i n g or the removal of the hydroxy group from t h e r i n g of 1 - t y r o s i n e i n a medium of I n o r g a n i c s a l t s employing B. pyocyaneus and B. f l o u r e s c e n s , hut they were unable t o i d e n t i f y t h e p r o d u c t s formed* The n a t u r e o f t h e b a c t e r i a l breakdown of amino a c i d s may be determined by methods o t h e r than t h e a c t u a l i s o l a t i o n Of d e c o m p o s i t i o n p r o d u c t s employed i n the e a r l y i n v e s t i g a -t i o n s on t h i s s u b j e c t . Bernheim, Bernheim and Webster (4) measured the oxygen uptake and ca r b o n - d i o x i d e , output employing the Warburg r e s -p l r o m e t e r . They det e r m i n e d the ammonia formed t h r o u g h t h e accompanying d e a m i n a t i o n by d i s t i l l i n g i t from t h e c o n t e n t s o f t h e Warburg v e s s e l a t the c o m p l e t i o n of t h e experiment i n t o a N e s s l e r ' s s o l u t i o n . The ammonia p r e s e n t was t h e n de-t e r m i n e d c o l o r i m e t r i c a l l y . I n a s t u d y o f the o x i d a t i o n o f c e r t a i n amino a c i d s by P r o t e u s v u l g a r i s , employing the washed c e l l t e c h n i q u e f o r the p r e p a r a t i o n o f t h e i r b a c t e r i a l suspen-s i o n , t h e y found t h a t , at pH 7.6, g l y c i n e was t h e o n l y amino a c i d s t u d i e d t o be c o m p l e t e l y o x i d i z e d . L e u c i n e , p h e n y l a l a -n i n e and m e t h i o n i n e a re o x i d i z e d r a p i d l y and u t i l i z e one atom of oxygen p e r molecule of amino a c i d w h i l e s e r i n e , a l a n i n e and p r o l i n e u t i l i z e t h r e e , f o u r and seven atoms r e s p e c t i v e l y . The o x i d a t i o n o f t y r o s i n e and t r y p t o p h a n i s s l o w e r u s i n g two and t h r e e atoms o f oxygen r e s p e c t i v e l y . V a l i n e , i s o l e u c i n e , h y d r o x y p r o l i n e and h i s t i d i n e a re o x i d i z e d so s l o w l y t h a t no -15-d e f i n i t e uptakes of oxygen were obtained,, T h e i r work showed t h a t o n l y the n a t u r a l isomers were o x i d i z e d except i n t h e case o f a l a n i n e and s e r i n e , b o t h isomers of w h i c h were o x i -d i z e d , and f u r t h e r t h a t d e a m i n a t i o n corresponds w i t h the o x i d a t i o n except f o r v a l i n e . I n a l a t e r s t u d y (5) o f t h e o x i d a t i o n o f t h i r t e e n amino a c i d s by washed c e l l s u s p e n s i o n s o f B. pyocyaneus, they found t h a t t r y p t o p h a n and. m e t h i o n i n e were n o t a t t a c k e d , and t h a t t h e r e was c o n s i d e r a b l e v a r i a t i o n i n t h e o x i d a t i o n r a t e s and the e x t e n t t o w h i c h the a t t a c k e d amino a c i d s were o x i -d i z e d . They showed t h a t t h i s organism oxid-ized and deamina-t e d b o t h isomers of a l a n i n e , s e r i n e , t y r o s i n e and p r o l i n e ^ but o n l y the n a t u r a l isomers o f l e u c i n e , i s o l e u c i n e and h i s t i d i n e . They suggested t h a t t h e v a r i a t i o n i n t h e r a t e of o x i d a t i o n o f t h e amino a c i d s may mean t h a t t h e r e a r e s e p a r a t e c a t a l y s t s f o r each amino a c i d and t h a t t h e amino a c i d s are a t t a c k e d d i f f e r e n t l y by t h e d i f f e r e n t b a c t e r i a , o r , more p r o b a b l y , t h a t the r a t e o f f o r m a t i o n o f t h e enzyme-substrate complex i s de t e r m i n e d by the c h e m i c a l s t r u c t u r e o f t h e amino acid,, '' Woods and C l i f t o n ( 7 5 ) , i n the n e x t y e a r , employing t h e Warburg t e c h n i q u e and s u s p e n s i o n s o f C l o s t r i d i u m t a t a n o -morphum, found t h a t g l u t a m a t e , a s p a r t a t e , c y s t e i n e , c y s t i n e t y r o s i n e and m e t h i o n i n e y i e l d e d hydrogen and/or c a r b o n - d i o x i d e and were al m o s t c o m p l e t e l y deaminated. They also, observed t h a t h i s t i d i n e p roduced more t h a n one m o l e c u l e of ammonia p e r amino group p r e s e n t and c o n c l u d e d t h a t the i m i n a z o l r i n g had been opened. -16-I n a s t u d y .'of t h e metabolism of the amino a c i d s by C l o s t r i d i u m w e l c h i i , Woods and T r i m (76) observed t h a t t h i s organism a t t a c k s o n l y f i v e o f the twenty-one amino a c i d s s t u d i e d . S e r i n e gave hydrogen, c a r b o n - d i o x i d e and ammonia In e q u i m o l e c u l a r p r o p o r t i o n s and r e q u i r e d a coenzyme f o r i t s breakdown.* C y s t i n e / c y s t e i n e and t h r e o n i n e were a t t a c k e d s i m i l a r l y t o s e r i n e but the evidence f o r the need o f a coenzyme f a c t o r was i n c o m p l e t e . A r g i n i n e was a t t a c k e d t o g i v e ammonia and c a r b o n - d i o x i d e , the f i n a l y i e l d s of w h i c h v a r i e d w i t h the age o f the growth c u l t u r e . Hydrogen was not formed and a coenzyme was n o t r e q u i r e d . A r e c e n t i n v e s t i g a t i o n by N e i l s on- ( 5 0 ) , employing the Van.Slyke a e r a t i o n p r o c e d u r e f o r the d e t e r m i n a t i o n o f ammonia, i n d i c a t e d t h a t washed c e l l s u s pensions of P r o t e u s i c h t h y o s m i u s (Hammer.) a t t a c k e d b e t a - a l a n i n e , d - a r g i n i n e , g l y c i n e and 1 - p r o l i n e v i g o r o u s l y ; d l - a s p a r t i c a c i d , 1 - c y s t i n e , d - g l u t a m i c a c i d , 1 - h i s t i d i n e , d l - I s o l e u c i n e , 1 - l e u c i n e , d - l y s i n e and beta-amino b u t y r i c a c i d l e s s v i g o r o u s l y ; and m e t h i o n i n e , p h e n y l a l a n i n e , t r y p t o p h a n , t y r o s i n e , u r e a , d e l t a -amino v a l e r i c a c i d and e p s i l o n - a m i n o c a p r o i c a c i d o n l y s l i g h t l y . S i m i l a r s u s p e n s i o n s o f Pseudomonas p u t r e f a c i e n s (Hammer).attacked h i s t i d i n e and p r o l i n e v i g o r o u s l y ; a s p a r t i c a c i d and g l u t a m i c a c i d l e s s v i g o r o u s l y ; and b e t a - a l a n i n e , a r g i n i n e , c y s t i n e , i s o l e u c i n e , l e u c i n e , l y s i n e , m e t h i o n i n e , p h e n y l a l a n i n e , t r y p t o p h a n , t y r o s i n e , u r e a , beta-amino b u t y r i c a c i d , d e l t a amino v a l e r i c a c i d and e p s i l o n amino c a p r o i c a c i d o n l y s l i g h t l y . The mutual o x i d a t i o n and r e d u c t i o n by p a i r s o f amino -17-a c i d s has been s t u d i e d by S t i c k l a n d and Woods. S t i c k l a n d (63 and 64) observed t h a t washed c e l l s u s pensions o f C l o s t r i -dium sporogenes a c t i v a t e d c e r t a i n amino a c i d s as hydrogen-d o n a t o r s - a l a n i n e , v a l i n e and l e u c i n e , and a c t i v a t e d o t h e r s as h y d r o g e n - a c c e p t o r s - g l y c i n e , p r o l i n e and h y d r o x y p r o l i n e . The h y d r o g e n - a c c e p t i n g amino a c i d s are s u b j e c t e d t o r e d u c t i o n by the hudrogen-donating amino a c i d s which i n t u r n are o x i -d i z e d t o compounds s i m i l a r t o t h o s e r e s u l t i n g from o x i d a t i v e d e a m i n a t i o n . Woods (74) has observed t h a t 1 - c y s t e i n e may a c t as hydrogen-donator i n c o u p l e d r e a c t i o n s between amino a c i d s i n d u c e d by C l o s t r i d i u m sporogenes and t h a t , In a d d i t i o n , , i t i s p a r t i a l l y deaminated i n the absence o f o t h e r amino a c i d s . He a l s o has shown t h a t d - a r g i n i n e and d - o r n i t h i n e are a c t i -v a t e d as h y d r o g e n - a c c e p t o r s and are p a r t i a l l y deaminated i n the absence of h y d r o g e n - d o n a t o r s . When o r n i t h i n e r e a c t s w i t h t h e don at o r , a l a n i n e , i t accepts, two hydrogens and undergoes r e d u c t i v e d e a m i n a t i o n t o d e l t a - a m i n o n - v a l e r i c a c i d . Recent i n v e s t i g a t i o n s employing t h e r e s t i n g c e l l t e c h n i q u e o r enzymic e x t r a c t s have brought f o r t h c o n s i d e r a b l e d a t a on d e a m i n a t i o n and o n l y t h e l i t e r a t u r e c o n c e r n i n g the f i v e amino a c i d s s t u d i e d i n t h i s problem w i t h p a r t i c u l a r r e f e r e n c e to t h e i r b a c t e r i a l breakdown w i l l he r e v i e w e d i n d e t a i l . • The-study of t h e d e a m i n a t i o n of a s p a r t i c a c i d by washed c e l l s u s p e n s i o n s o f E. c o l i c a r r i e l out by Q u a s t a l and Woolf (54) r e v e a l e d t h a t the p r o d u c t i n the absence of any i n h i b i t o r i s s u c c i n i c a c i d , whereas, i f an i n h i b i t o r such as t o l u e n e i s p r e s e n t , the r a t e o f d e a m i n a t i o n i s not a f f e c t e d -18-i n the b e g i n n i n g but the p r o c e s s does not go t o c o m p l e t i o n , r e a c h i n g , i n s t e a d , an e q u i l i b r i u m m i x t u r e o f a s p a r t i c a c i d , f u m a r i c a c i d and ammonia. I n a s t u d y o f t h e f e r m e n t a t i o n o f g l u t a m i c a c i d by a s t r i c t l y a n a e r o b i c , s p o r e - f o r m i n g b a c t e r i u m , p r o b a b l y be-l o n g i n g to- t h e genus P e o t o c l o s t r i d i u m , B a r k e r (3) demonstra-t e d t h a t t h i s a c i d was decomposed p r a c t i c a l l y q u a n t i t a t i v e l y i n t o ammonia, c a r b o n - d i o x i d e , hydrogen, a c e t i c a c i d and b u t y r i c a c i d . A d l e r ( 1 ) , employing a deaminase enzyme e x t r a c t e d from E. c o l i , found t h a t g l u t a m i c a c i d , i n the presence o f coenzymes, i s decomposed i n t o k e t o g l u t a r i c a c i d and ammonia and s u g g ested t h a t the breakdown was t h r o u g h i m i n o g l u t a r i c a c i d t o k e t o g l u t a r i c a c i d w i t h t h e l i b e r a t i o n o f ammonia. The work o f K l e i n (43) on t h e o x i d a t i o n o f 1 - a s p a r t i c and 1 - g l u t a m i c a c i d s by Hemophilus p a r a i n f l u e n z a e showed t h a t b o t h a c i d s were o x i d i z e d t o a c e t i c a c i d , ammonia and carbon-d i o x i d e ; one m o l e c u l e o f a s p a r t i c a c i d r e q u i r i n g one m o l e c u l e o f oxygen and p r o d u c i n g two of c a r b o n - d i o x i d e , and one mole-c u l e o f g l u t a m i c a c i d r e q u i r i n g -two and o n e - h a l f m o l e c u l e s of oxygen and p r o d u c i n g t h r e e o f c a r b o n - d i o x i d e . By a s tudy of the m e t abolism of p o s s i b l e i n t e r m e d i a t e compounds, K l e i n has e s t a b l i s h e d t h a t t h e p r o b a b l e course of o x i d a t i o n of a s p a r t i c a c i d by t h i s o rganism proceeds t h r o u g h o x a l a c e t i c a c i d , p y r u v i c a c i d and • a c e t a l d e h y d e t o a c e t i c a c i d . Ammonia f o r m a t i o n from p r o l i n e or f r om h i s t i d i n e i n amount's g r e a t e r t h a n t h a t g i v e n by i t s amino group i n v o l v e s opening of a f i v e - e l e m e n t r i n g , the p y r o l l i d i n e i n the case -19-o f p r o l i n e and the I m i n a z o l f o r h i s t i d i n e , a f e a t e x t r e m e l y d i f f i c u l t t o a c c o m p l i s h I n • n o n - b i o l o g i c a l c h e m i s t r y . I n v e s -t i g a t i o n s i n b i o c h e m i s t r y , however, have shown t h a t c e r t a i n enzymes, p a r t i c u l a r l y those o f b a c t e r i a l o r i g i n , are capable o f opening these' r i n g s p r e p a r a t o r y to removing th e n i t r o g e n by d e a m i n a t i o n of t h e amino group formed. E l d b a c h e r and o t h e r s (20, 21 and 2 2 ) , I n s t u d i e s o f the i n t e r m e d i a t e metabolism o f h i s t i d i n e u s i n g the ensyme h i s t i d a s e o b t a i n e d from a n i m a l l i v e r , f o u n d t h a t one m o l e c u l e o f h i s t i d i n e gave two of ammonia, one of g l u t a m i c a c i d and p r o b a b l y one o f f o r m i c a c i d , t h e p r i m a r y a c t i o n o f the enzyme b e i n g t o open the I m i n a z o l n u c l e u s to form an a l p h a , d e l t a , diamino ,chain.. The i n v e s t i g a t i o n o f R a i s t r i c k (56) i n t o the a e r o b i c b a c t e r i a l d e c o m p o s i t i o n of h i s t i d i n e by B. paratyphosus A and B, B. f a e c a l i s a l c a l i g e n e d , B. pyocyaneus and B. p r o t e u s v u l -g a r i s r e v e a l e d t h a t the f i r s t f o u r of these organisms produced ammonia from t h e n i t r o g e n i n b o t h the side- c h a i n and the I m i n a z o l n u c l e u s , p r o v i n g t h a t t h e y are a b l e to open t h e I m i n a z o l r i n g ; whereas th e f i f t h organism formed ammonia o n l y from t h e s i d e c h a i n n i t r o g e n , showing i t s p r o b a b l e i n a b i l i t y t o s p l i t the r i n g . S t i c k l a n d ( 6 5 ) , t h r o u g h h i s experiments on the c o u p l e d o x i d a t i o n and r e d u c t i o n b y p a i r s o f amino a c i d s , ob-s e r v e d t h a t washed c e l l s u s p e n s i o n s o f C l o s t r i d i u m sporogenes r e d u c e d 1 - p r o l l n e , a t the expense o f t h e o x i d a t i o n o f 1 - a l a n i n e , to d e l t a - a m i n o n - v a l e r i c a c i d , b u t d i d not deami-n a t e t h i s p r o d u c t , whereas the a l a n i n e was deaminated d u r i n g -20-i t s o x i d a t i o n . W e il-Malherbe and Krebs ( 6 9 ) , employing k i d n e y t i s -sue, s t u d i e d the c o n v e r s i o n o f p r o l i n e i n t o g l u t a m i c a c i d and conclu d e d t h a t p r o l i n e i n t h e k i d n e y i s o x i d i z e d to g l u t a m i c a c i d which i n t u r n may be o x i d i z e d f u r t h e r t o a l p h a - k e t o g l u t a r i c a c i d w i t h the l i b e r a t i o n of ammonia, or may t a k e on more ammonia to form g l u t a m i n e . Krebs ( 4 6 ) , i n h i s work on the o x i d a t i o n of d-pro-l i n e by d-amino a c i d o x i d a s e , was a b l e t o i s o l a t e t h e 2, 4, d i n i t r o p h e n y l h y d r a z o n e of a l p h a - k e t o d e l t a - a m i n o v a l e r i c a c i d , thus d e m o n s t r a t i n g the opening o f t h e r i n g on t h e c a r b o x y l s i d e o f t h e n i t r o g e n i n c o n t r a s t w i t h the f o r m a t i o n of g l u -t a m i c a c i d when t h e o x i d a t i v e opening o f the r i n g occurs on the m e t h y l group s i d e o f t h e n i t r o g e n . The d e c o m p o s i t i o n o f a r g i n i n e , w h i c h c o n t a i n s one n i t r o g e n as an a l p h a amino group and t h r e e n i t r o g e n s i n t h e guanido group on t h e d e l t a carbon atom, may i n v o l v e a number of ensymes. Hun t e r and Dauphinee ( 3 8 ) , i n s t u d i e s on the a c t i o n o f a r g i n a s e e x t r a c t e d from l i v e r , have found t h a t t h i s ensyme, at pH 8.5 and room t e m p e r a t u r e , s p l i t s a r g i n i n e . i n t o u r e a and o r n i t h i n e t o the extent o f 99.1 p e r c e n t . The ur e a may s u b s e q u e n t l y be decomposed by the ensyme urease a t pH 6.8 and room temperature i n t o ammonia and c a r b o n - d i o x i d e . Vovchenko (67) i n v e s t i g a t e d the e f f e c t o f p r o d u c t s o f a r g i n i n e h y d r o l y s i s upon the a c t i o n of a r g i n a s e and found t h a t t h e presence o f o r n i t h i n e and/or u r e a i n h i b i t the a c t i o n o f the enzyme, the i n h i b i t i n g e f f e c t b e i n g s m a l l e r a t pH v a l u e s l o w e r than the o p t i m a l f o r the h y d r o l y s i s , pH 9.6, than -21-at h i g h e r v a l u e s . Tomota ( 6 5 ) , from a d e t e r m i n a t i o n o f t h e a c t i v i t y o f a r g i n a s e e x t r a c t e d from a number of b a c t e r i a l s p e c i e s , has shown t h a t e x t r a c t s from Staph, a u r e u s , Staph;, a l b u s , S t a p h , c i t r e u s and Bac. s u b t i l i s possess s t r o n g a r g i n a s e a c t i v i t y ; w h i l e t h e 6ne from S a r c i n a i s f a i r l y a c t i v e and th o s e from S t r e p t o c o c c i , Mycobacterium p h l e i , S a l m o n e l l a e n t e r i t i d i s , B. p r o t e u s , B. pyocyaneus, B. p r o d i g i o s u s and Mucosus capcu-l a t u s show weak and v a r i a b l e a c t i v i t y . The r e s u l t s o f an i n v e s t i g a t i o n i n t o the decomposi-t i o n of a r g i n i n e by gram p o s i t i v e c o c c i I n w h i c h two molecules o f ammonia and one o f o r n i t h i n e were produced from each mole-c u l e of a r g i n i n e have caused H i l l (34) t o conclude t h a t t h e ensyme i n v o l v e d was not a r g i n a s e but was a n o t h e r enzyme which he has named a r g i n i n e d i h y d r o l a s e . The reasons f o r h i s con-c l u s i o n a r e : (1) the s t r a i n s of s t r e p t o c o c c i used were shown not to p o s s e s s u r e a s e , (2) the urease of a s t o c k s t r a i n o f S t a p h y l o c c u s i s I n s u f f i c i e n t l y a c t i v e t o account f o r the r a t e o f breakdonw o f a r g i n i n e , and (3) i n a s t r a i n of S t a p h y l o -coccus t r a i n e d t o grow on an ammonia medium, t h e urease a c t i v i t y can be v a r i e d a t w i l l between v e r y wide l i m i t s , w h i l e t h e a r g i n i n e enzyme v a r i e s i n an i n v e r s e manner. T h i s r e c i p t o c a l r e l a t i o n s h i p s u g g e s t s • t h a t t h e enzymes may be r e l a t e d though n o t i d e n t i c a l . Woods (;74) has obse r v e d t h a t through t h e a c t i o n o f C l o s t r i d i u m sporogenes one m o l e c u l e of a r g i n i n e y i e l d s t h r e e m o l e c u l e s o f ammonia e i t h e r a l o n e o r as a hy d r o g e n - a c c e p t o r c o u p l e d w i t h a l a n i n e and s i m i l a r l y t h a t o r n i t h i n e g i v e s one -22-m o l e c u l e of ammonia and one o f d e l t a - a m i n o . v a l e r i c a c i d . The l i t e r a t u r e c o n c e r n i n g the f a c t o r s i n f l u e n c i n g b a c t e r i a l d e a m i n a t i o n i s e x t e n s i v e ; o n l y those f a c t o r s p e r t i -nent t o t h i s s tudy w i l l be r e v i e w e d , - the i n f l u e n c e o f t h e age of c u l t u r e , a e r o b i o s i s and a n a e r o b i o s i s , pH o f t h e growth and b u f f e r " m e d i a and pr e s e n s e o f c a r b o h y d r a t e s i n t h e growth and b u f f e r media. Stephenson and Gale have I n v e s t i g a t e d t h e i n f l u e n c e o f t hese f a c t o r s on the b a c t e r i a l d e a m i n a t i o n o f a number of amino a c i d s by washed c e l l s u s p e n s i o n s o f B a c t e r i u m c o l l . I n t h e i r f i r s t paper ( 6 2 ) , u s i n g g l y c i n e , d l - a l a n i n e and 1 - g l u t a m i c a c i d , t h e y f o u n d t h a t o n l y s l i g h t v a r i a t i o n i n t h e a c t i v i t y o f the su s p e n s i o n o c c u r r e d when the growth c u l t u r e was between e i g h t and twenty h o u r s , t h a t a n a e r o b i c c o n d i t i o n s d u r i n g growth i n h i b i t t h e p r o d u c t i o n o f the deaminase f o r g l y c i n e and a l a n i n e but f a v o u r i t f o r g l u t a m i c a c i d , and t h a t the e f f e c t of g l u c o s e o f the o x i d a t i v e d e a m i n a t i o n o f the t h r e e amino a c i d s i s t o i n h i b i t the f o r m a t i o n o f t h e enzyme * d u r i n g growth t o the e x t e n t o f n i n e t y - f i v e p e r c e n t . T h e i r paper (SO) on d l - s e r i n e brought out t h a t t h i s amino a c i d i s deaminated b o t h a e r o b i c a l l y and a n a e r o b i c a l l y , t h a t b o t h isomers a re a t t a c k e d though p r o b a b l y a t d i f f e r e n t r a t e s . The a c t i v i t y o f s e r i n e deaminase v a r i e s markedly w i t h t h e age of the growth c u l t u r e , a t t a i n i n g i t s h e i g h t a t el e v e n hours and s u b s e q u e n t l y f a l l i n g o f f , i s i n c r e a s e d by a n a e r o b i c growth c o n d i t i o n s and, l i k e the o t h e r deaminases, i s d e c r e a s e d n i n e t y - f i v e p e r c e n t o r more by t h e presence o f two p e r c e n t g l u c o s e i n t h e growth medium. The optimum pH f o r t h i s -23-deaminase was pH 8.0. The t h i r d paper by Gale (29) on a s p a r -t i c a c i d r e p o r t s t h a t t h e a c t i v i t y of t h e deaminase o f t h i s a c i d v a r i e s w i t h growth c o n d i t i o n s - a e r o b i c c o n d i t i o n s show-i n g about t w o - t h i r d s the a c t i v i t y o f a n a e r o b i c c o n d i t i o n s and two p e r c e n t g l u c o s e I n h i b i t i n g ' t h e a c t i v i t y about e i g h t y - f i v e p e r c e n t , and w i t h the age of t h e growth c u l t u r e b e i n g g r e a t e s t from f o u r t e e n t o e i g h t e e n h o u r s . T h i s l a t t e r v a r i a t i o n was e x p l a i n e d as b e i n g caused by an a l t e r a t i o n i n t h e c h e m i c a l c o n s t i t u t i o n of the growth medium f o l l o w i n g t h e m e t a b o l i c a c t i v i t y o f t h e c e l l s . The a c t i v i t y o f t h i s deaminase ranged f r o m pH 6.0 t o 8.0, b e i n g optimum a t pH 7.5. I n a s t u d y of t h e 1- and the d- amino a c i d o x i d a s e s e x t r a c t e d from, k i d n e y t i s s u e , Krebs ( 4 5 ) f o u n d t h a t t h e o p t i -mum pH curve o f t h e d-amino a c i d deaminase shows an optimum near pH 8.8 when used f o r t h e d e a m i n a t i o n of d l - a l a n i n e and d l - a s p a r t i c a c i d . The d e a m i n a t i o n o f 1 - a s p a r t i c a c i d shows an optimum at pH 7.4 but I t s t i l l r e t a i n s s i x t y p e r c e n t o f i t s a c t i v i t y a t pH 6.5. Krebs a l s o f o u n d t h a t a f i n a l con-c e n t r a t i o n o f M/200 a s p a r t l c a c i d gave the maximum.rate o f d e a m i n a t i o n . : Gale and Epps ( 3 0 ) , i n an i n v e s t i g a t i o n of t h e e f -f e c t o f t h e pH o f the medium d u r i n g growth on'the enzymic a c t i v i t i e s of E. c o l i and Mc. l y s o d e i k t i c u s , was a b l e t o divid.e the enzymes p r o d u c e d i n t o two groups. The f i r s t group c o n t a i n e d urease,- c a t a l a s e , h y d r o g e n l y a s e , e t c . and t h e second the amino a c i d enzymes. The d e c a r b o x y l a s e s were p r o -duced i n an a c i d medium and the deaminases i n ah a l k a l i n e medium and seemed, t h e r e f o r e , t o a c t as n e u t r a l i z a t i o n -24-mechanisms I n an e f f o r t t o b r i n g the p l i o f t h e medium n e a r e r n e u t r a l i t y . The q u e s t i o n of t h e e f f e c t of the presence o f f e r -mentable and non-fermentable c a r b o h y d r a t e s i n t h e growth and b u f f e r medium has caused c o n s i d e r a b l e c o n t r o v e r s y i n the l i t e r a t u r e s K e n d a l l and h i s a s s o c i a t e s (39, 40 and 41) i n a s e r i e s of papers from 1912 to 1922 s t u d i e d the p r o d u c t i o n o f ammonia by s e v e r a l b a c t e r i a l s p e c i e s , i n c l u d i n g B. p r o t e u s , g rowing i n p r o t e i n d i g e s t s and showed t h a t t h i s p r o d u c t i o n i s g r e a t l y checked and, i n some c a s e s , c o m p l e t e l y i n h i b i t e d by the p r e s e n c e o f g l u c o s e i n t h e growth medium. They I n t e r -p r e t e d t h i s as due t o a " s p a r i n g " a c t i o n e x e r t e d by the carbo-h y d r a t e on t h e deami n a t i o n o f p r o t e i n s , b e l i e v i n g t h a t i n t h e presence of a r e a d i l y a v a i l a b l e source of carbon and energy the organism decomposed l e s s n i t r o g e n o u s m a t e r i a l , R a i s t r i c k and C l a r k (57) p o i n t e d out t h a t ammonia Is not o n l y a p r o d u c t o f the d e c o m p o s i t i o n o f p r o t e i n s , b u t a l s o a sou r c e o f n i t r o g e n f o r growth, so t h a t w h i l e , I n the p r o t e i n d i g e s t medium the ammonia'produced i s i n excess o f t h a t r e q u i r e d f o r c e l l s y n t h e s i s , i t may be p o s s i b l e t h a t , i n the p r e s e n c e o f a d d i t i o n a l c a r b o h y d r a t e , t h i s ' e x c e s s i s used up i n I n c r e a s e d c e l l p r o d u c t i o n . To f u r t h e r i n v e s t i g a t e t h i s p o i n t , t h e y f o l l o w e d t h e growth o f B. pyocyaneus and B. f l o u r -escens i n s y n t h e t i c media c o n t a i n i n g t r y p t o p h a n or t y r o s i n e , w i t h o r w i t h o u t g l y c e r o l . They found t h a t i n . t h e absence o f g l y c e r o l t h e r e was a l a r g e amount o f ammonia w i t h v e r y l i t t l e s y n t h e s i z e d n i t r o g e n , but t h a t i n the p r e s e n c e o f g l y c e r o l t h e -25-r e v e r s e was t r u e and c o n c l u d e d w i t h t h e b e l i e f t h a t "carbo-h y d r a t e , f a r from h a v i n g a p r o t e i n - s p a r i n g e f f e c t , a c t u a l l y e n a b l e s the b a c t e r i a t o u t i l i z e more p r o t e i n or p r o t e i n p r o d u c t s t h a n t h e y would i n the absence of c a r b o h y d r a t e . " Waksman and Lomanltz (68) c a r r i e d out a d e t a i l e d i n v e s t i g a t i o n o f t h i s p o i n t u s i n g s p e c i e s o f b a c t e r i a , a c t i -nomyces and molds and came t o s i m i l a r c o n c l u s i o n s but p o i n t e d out t h a t "a l i v i n g b e i n g d e r i v e s i t s energy from a substance w h i c h i s most a v a i l a b l e t o i t and which may be s p e c i f i c f o r a p a r t i c u l a r organism." N i s i m u r a (51) s t u d i e d t h e e f f e c t of v a r i o u s carbo-h y d r a t e s on t h e f o r m a t i o n o f p-hydroxyphenyl l a c t i c a c i d ( I ) by a s t r a i n o f B. p r o t e u s , of p - h y d r o x y p h e n y l p r o p i o n i c a c i d ( I I ) by another two s t r a i n s of B, p r o t e u s and o f t y r a m i n e ( I I I ) by a s t r a i n of B. l a c t i s aerogenes from t y r o s i n e i n a S a s a k i p r o t e i n - f r e e n u t r i e n t s o l u t i o n . When g l u c o s e was added t o the s o l u t i o n , no I I was formed, f o r m a t i o n o f I was d i s t i n c t l y d e c r e a s e d but no e f f e c t was found on f o r m a t i o n o f I I I . A d d i t i o n o f l e v u l o s e or s u c r o s e t o t h e s o l u t i o n l o w e r e d the f o r m a t i o n o f I , I I and I I I . The a d d i t i o n of l a c t o s e ac-c e l e r a t e d the f o r m a t i o n o f I I by one s t r a i n o f B. p r o t e u s , i n h i b i t e d the f o r m a t i o n o f I by another s t r a i n o f B. p r o t e u s and o f I I I by B. l a c t i s aerogenes. A d d i t i o n of s t a r c h had no e f f e c t o f t h e f o r m a t i o n o f I but r e g a r d e d t h e f o r m a t i o n o f I I I s l i g h t l y and'of I I d i s t i n c t l y . I n the c o n t r o l experiments and those i n w h i c h c a r b o h y d r a t e s were added, th e pH of the n u t r i e n t s o l u t i o n was g e n e r a l l y s h i f t e d t o t h e a c i d s i d e . M a n n o z z i - T o r i k i and V e n d r a m i n i ( 4 7 ) , i n a s t u d y o f - 26 -the o x i d a t i o n o f amino a c i d s by B r u c e l l a a b o r t u s i n R i n g e r -phosphate s o l u t i o n , found t h a t t h i s organism c a t a l y s e d the o x i d a t i v e c leavage o f a l a n i n e , a s p a r a g i n , g l u t a m i c a c i d and c y s t e i n e and t h a t the presence of g l u c o s e had no c l e a v a g e on t h i s c l e a v a g e . Epps and Gale (23) compared t h e i n f l u e n c e o f the presence o f g l u c o s e d u r i n g growth on the enzymic a c t i v i t i e s o f E. c o l l w i t h the e f f e c t produced by f e r m e n t a t i o n a c i d s added t o the medium. They obser v e d t h a t the presence o f g l u c o s e i n the medium d u r i n g growth suppresses the f o r m a t i o n o f c e r t a i n enzyme-s, the degree o f i n h i b i t i o n b e i n g g r e a t e r than o r b e a r i n g no r e l a t i o n t o the e f f e c t produced by growth i n a medium a d j u s t e d to the f i n a l pH o f the g l u c o s e medium by f e r m e n t a t i o n a c i d s ; t h a t the n e u t r a l i z a t i o n o f f e r m e n t a t i o n a c i d d u r i n g growth i n g l u c o s e does not a l t e r the degree o f i n h i b i t i o n o f deaminase f o r m a t i o n produced by the g l u c o s e ; and t h a t the r e d u c t i o n i n the a c t i v i t y o f c e r t a i n enzymes as a r e s u l t o f growth i n g l u c o s e i s not a permanent change I n the enzyme c o n s t i t u t i o n o f the celLl as i t I s removed Immediately a f t e r growth t a k e s p l a c e i n the absence o f f e r m e n t a b l e c a r b o h y d r a t e . As a p a r t o f the i n v e s t i g a t i o n o f the mechanism f o r the p r o d u c t i o n o f i n d o l e from t r y p t o p h a n by B a c t e r i u m c o l i , the p r i m a r y s t e p o f w h i c h may be d e a m i n a t i o n , a number o f workers have s t u d i e d the e f f e c t o f the presence o f g l u c o s e i n the growth and b u f f e r medium. Happold and Hoyle (33) added g l u c o s e t o the growth medium and n o t i c e d t h a t the - 27 -i n h i b i t i o n o f the p r o d u c t i o n o f the tr y p t o p h a n a s e enzyme system does n o t occur from the s t a r t but shows a s h o r t l a t e n t p e r i o d f o l l o w e d by a complete i n h i b i t i o n w hich l a s t s o n l y u n t i l the f e r m e n t a t i o n o f the sugar I s completed. They a l s o showed t h a t the p r o d u c t s o f g l u c o s e f e r m e n t a t i o n have no i n h i b i t i n g a c t i o n . Happold and Hoyle (32) and P i l d e s (26) found t h a t washed c e l l s u s p e n s i o n s o f B. c o l i grown i n a t r y p t o p h a n -c o n t a i n i n g medium are t w e n t y - f i v e times more a c t i v e p r o d u c e r s o f i n d o l e from t r y p t o p h a n - p h o s p h a t e - b u f f e r s o l u t i o n s t h a n s i m i l a r s u s p e n s i o n s grown i n a medium c o m p l e t e l y l a c k i n g i n t r y p t o p h a n and t h a t t h i s e x c e s s a c t i v i t y i s e n t i r e l y I n h i b i t e d by the presence o f g l u c o s e i n the t r y p t o p h a n - b u f f e r s o l u t i o n . Evans, Handley and Happold (24) r e p o r t e d t h a t the t r y p t o p h a n a s e system f o r the p r o d u c t i o n o f i n d o l e doe s not e x i s t as such i n c e l l s o f B. c o l i w h i c h have been grown i n a medium c o n t a i n i n g g l u c o s e , b u t t h a t such c e l l s when f r e e d f r om g l u c o s e by washing w i l l r e - d e v e l o p the enzyme system when l e f t i n c o n t a c t w i t h t r y p t o p h a n . The f i n d i n g s o f N e i l s o n (50) i n a s t u d y o f the r e l a t i o n between i n d o l e p r o d u c t i o n and s u r f a c e t a i n t i n b u t t e r employing washed c e l l s u s p e n s i o n s o f P r o t e u s i c h -thyosmius c o n f i r m the o b s e r v a t i o n s o f Evans and o t h e r s . I t was found t h a t the presence o f one p e r c e n t g l u c o s e i n the agar growth medium had no i n f l u e n c e o f the q u a n t i t y o f i n d o l e formed by the s u s p e n s i o n i n a g l u c o s e - f r e e t r y p t o p h a n b u f f e r s o l u t i o n whereas the presence o f one p e r c e n t g l u c o s e - i n the - 28 -t r y p t o p h a n - b u f f e r m i x t u r e e x e r t e d an i n h i b i t i n g e f f e c t which was o n l y p a r t i a l when the c e l l s were grown on a g l u c o s e - f r e e agar but was complete when t h e y were grown on a g l u c o s e -c o n t a i n i n g a g a r . E vans, Handley and Happold ( 2 5 ) , i n t r y i n g t o work out a p o s s i b l e mechanism f o r the i n h i b i t i o n o f i n d o l e p r o -d u c t i o n by g l u c o s e i n c u l t u r e s o f B. c o l i , s t u d i e d the e f f e c t o f o t h e r f e r m e n t a b l e and non-fermentable sugars on t h i s i n h i b i t i o n . T h e i r experiments show t h a t g l y c o g e n , s t a r c h , d e x t r i n , s u c r o s e , d u l c i t o l , s a l i c i n , i n u l i n , p o t a s s i u m s a c c h a r a t e and hexosediphosphate are not fermented i n twenty-f o u r c u l t u r e s o f the organism and do not a f f e c t t h e p r o d u c t i o n o f i n d o l e when t r y p t o p h a n i s p r e s e n t i n the medium. On the o t h e r hand, complete i n h i b i t i o n o f i n d o l e p r o d u c t i o n i s ob-t a i n e d w i t h a r a b i n o s e , l a c t o s e , g l u c o s e , f r u c t o s e and m a n n i t o l , - sugars w h i c h are fermented by B. c o l i . The sugars w h i c h showed a c i d p r o d u c t i o n w i t h o u t marked i n h i b i t i o n o f i n d o l e p r o d u c t i o n were rhamnose, x y l o s e , s o r b i t o l , g a l a c t o s e , d - r i b o s e and mannose. f - 29 -EXPERIMENTAL The r e s u l t s o f e a r l i e r s t u d i e s (50) on the q u a n t i -t a t i v e d e t e r m i n a t i o n o f ammonia formed from i n d i v i d u a l amino a o i d s by s u r f a c e t a i n t b a c t e r i a gave support t o the t h e o r y t h a t d e a m i n a t i o n may be i n t i m a t e l y concerned w i t h the e l a b o r a t i o n o f the c h a r a c t e r i s t i c s u r f a c e t a i n t odour. T h e r e f o r e i t was d e c i d e d t h a t d e t a i l e d experiments on the c o n d i t i o n s r e q u i r e d f o r ammonia f o r m a t i o n from a number of amino a c i d s might b r i n g f o r t h f u r t h e r e vidence f o r the t h e o r y as w e l l as add t o our p r e s e n t knowledge of the m i c r o b i a l c a t a b o l i s m o f amino a c i d s . The r e s u l t s r e f e r r e d to above showed t h a t t h e r e was c o n s i d e r a b l e ammonia formed by s p e c i e s o f s u r f a c e t a i n t b a c t e r i a from the amino a c i d s a r g i n i n e , , a s p a r t i c a c i d , g l u t a m i c a c i d , h i s t i d i n e and p r o l i n e w h i c h are t h e o r e t i c a l l y , w i t h c o n s i d e r a b l e s u p p o r t i n g e v i d e n c e i n the l i t e r a t u r e , r e l a t e d t o one a n other t h r o u g h t h e i r d e c o m p o s i t i o n p r o d u c t s . W i t h th e o b j e c t o f i n v e s t i g a t i n g i n d e t a i l the c o n d i t i o n s o f ammonia f o r m a t i o n f rom the above f i v e amino a c i d s by two s p e c i e s o f s u r f a c e t a i n t p r o d u c i n g b a c t e r i a -P r o t e u s i c h t h y o s m i u s (Hammer) and Pseudomonas . p u t r e f a c i e n s (Hammer) - the f o l l o w i n g s e r i e s o f e x periments were c a r r i e d out The g e n e r a l e x p e r i m e n t a l procedure employed t h r o u g h -out the s t u d y was as f o l l o w s : 1. The organisms were I n o c u l a t e d from t r y p t i c c a s e i n d i g e s t b r o t h onto t r y p t i c c a s e i n d i g e s t - 30 -agar i n K o l l e f l a s k s and grown f o r a p e r i o d of 16 to 18 h o u r s . I n the case o f P r o t e u s i c h t h y o s m i u s , 30° G. was employed as the temperature of i n c u b a t i o n , and f o r Pseudomonas p u t r e f a c i e n s 23° C. The b a c t e r i a l c e l l s were t h e n washed o f f the agar w i t h M/30 phosphate b u f f e r , pH 7.4, (Sorensen's M/l5 phosphate b u f f e r d i l u t e d w i t h an e q u a l volume o f d i s -t i l l e d water) and c e n t r i f u g e d f o r a p p r o x i m a t e l y one hour i n f i f t y o r one hundred c u b i c c e n t i m e t e r c e n t r i -fuge t u b e s . The s u p e r n a t a n t was poured o f f a s e p t i c a l l y and the c e l l s t a k e n up and mixed t h o r o u g h l y w i t h about 30 cc o f M/30 phosphate b u f f e r and t h e n r e - c e n t r i f u g e d . The s u p e r n a t a n t was a g a i n poured o f f and the c e l l s t a k e n up and mixed t h o r o u g h l y w i t h about 50 cc o f M/30 b u f f e r or d i s t i l l e d w a t e r , depending on the n a t u r e o f the e x p e r i m e n t , ( v i d e i n f r a ) , p e r K o l l e f l a s k o f organisms and an a l i q u o t o f 5.0 cc t a k e n i n t o a Hopkins v a c c i n e tube t o determine the percentage b y volume of c e l l s p r e s e n t . A one p e r c e n t s u s p e n s i o n was used as an i n o c u l u m . The c u l t u r e s were then s e t up i n t e s t tubes con-t a i n i n g e i g h t c u b i c c e n t i m e t e r s of M/l5 b u f f e r , 1.0 cc of M/20 aqueous amino a c i d s o l u t i o n and 1.0 c c . o f one p e r c e n t washed c e l l s u s p e n s i o n . I n the experiments on the e f f e c t o f the presence o f c a r b o h y d r a t e , 1.0 cc o f a 5% aqueous s o l u t i o n o f the s p e c i f i c c a r b o h y d r a t e was - 31 -added and o n l y 7.0 cc o f b u f f e r were employed I n o r d e r t o m a i n t a i n the f i n a l volume a t 10 c c . The c u l t u r e s were i n c u b a t e d a t 30° C. f o r the r e q u i r e d number o f days. I n o r d e r t o overcome the d i f f i c u l t i e s r e s u l t i n g from the s t e r i l i z a t i o n o f a mixed medium, the n i t r o g e n s o u r c e , the carbon source and the b u f f e r were each s t e r i l i z e d s e p a r a t e l y and m i x e d - j u s t p r i o r t o i n o c u -l a t i o n . S t e r i l e equipment and m a t e r i a l s and a s e p t i c t e c h n i q u e were employed throughout the above p a r t o f the p r o c e d u r e . At the c o n c l u s i o n o f the i n c u b a t i o n p e r i o d , the pH o f the c u l t u r e s was t a k e n , when r e q u i r e d , w i t h a Beckman p o t e n t i o m e t e r , i n d u s t r i a l model, and t h e n the f r e e ammonia determined b y the Van S l y k e a e r a t i o n procedure u s i n g N/lOO s u l p h u r i c a c i d . The r e s u l t s are e x p r e s s e d as p e r c e n t o f the t o t a l n i t r o g e n o f amino a c i d I n c u l t u r e p r e s e n t as f r e e ammonia and are c o n v e r t e d fi?om c u b i c c e n t i m e t e r s o f N/lOO s u l p h u r i c a c i d t o p e r c e n t f r e e ammonia by the f o l l o w i n g f o r m u l a : .14 x cc o f N/100 H 2 S 0 4 x 100 = g f r e e ammonia No. o f N i n amino a c i d x .7 i n w h i ch: .14 = mg. o f n i t r o g e n e q u i v a l e n t t o 1.00 cc o f N/100 H 2 S 0 4 . .7 = mg. o f n i t r o g e n i n 1.0 cc o f M/20 ammonia or mono-nitrogen amino a c i d . - 32 -THE EFFECT OF pH ON AMMONIA FORMATION. The l i t e r a t u r e has shown t h a t the pH o f the b u f f e r medium c o n t a i n i n g the amino a c i d s a f f e c t s the amount o f ammonia t h a t i s l i b e r a t e d b y v a r i o u s s p e c i e s o f m i c r o -organisms . Employing the te c h n i q u e d e s c r i b e d above, the i n f l u e n c e o f pH on ammonia f o r m a t i o n from d - a r g i n i n e , d l - a s p a r a t i c a c i d , d - g l u t a m i c a c i d , 1 - h i s t i d i n e and 1 - p r o l i n e by P r o t e u s i c h t h y o s m i u s and Pseudomonas p u t r e f a c i e n s was det e r m i n e d . The f o l l o w i n g b u f f e r s o l u t i o n s made a f t e r the manner o f C l a r k (11) were employed: p h t h a l a t e b u f f e r - pH 4.5, 5.0, 5.5, and 6.0. phosphate b u f f e r - pH 6.3, 6.8, 7.3, and 7.9. b o r i c a c i d - K C I b u f f e r - pH 8.5, 9.0, 9.5, and 10.0. I n t h i s p a r t i c u l a r experiment ( I ) the c e l l s u s p e n s i o n s were, of n e c e s s i t y , made up i n d i s t i l l e d water r a t h e r t h a n phosphate b u f f e r o f pH 7.4. The c u l t u r e s were i n c u b a t e d a t 30° C. f o r f i v e days a f t e r w h i c h the f i n a l pH was t a k e n and the f r e e ammonia d e t e r m i n e d . The r e s u l t s are g i v e n i n t a b l e s 1 and 2, and i n t e r p r e t e d on f i g u r e s 3 t o 12 i n c l u s i v e . • The r e s u l t s o f experiment I show t h a t t h e r e i s a g e n e r a l optimum range i n pH f o r ammonia f o r m a t i o n f o r pH 6.0 t o pH 8.5. W i t h i n t h i s g e n e r a l range, the d i f f e r e n t amino a c i d s have an i n d i v i d u a l range w h i c h i s sometimes narrow and at o t h e r s r e l a t i v e l y w i d e . A r g i n i n e under the a c t i o n o f P r o t e u s i c h t h y o s m i u s has a range from pH 6.3 t o 8.0 w i t h a r a t h e r sharp optimum a t pH 7.4 whereas f o r Pseudomonas p u t r e f a c i e n s the range i s from 6.0 t o 8.8 w i t h no d e f i n i t e - 33 -optimum. A s p a r t i c a c i d l i b e r a t e s a r e l a t i v e l y c o n s t a n t amount of ammonia from pH 6.0 t o pH 8.5 f o r b o t h s p e c i e s o f b a c t e r i a employed. The r e s u l t s f o r g l u t a m i c a c i d are s i m i l a r t o those f o r a s p a r t i c a c i d except t h a t the range i s narrower and f u r t h e r t o ' t h e a l k a l i n e s i d e of the pH s c a l e ; pH 6.5 to pH 8.3 b e i n g the range f o r Pseudomonas p u t r e f a c i e n s and pH 6,8 t o pH 8.5 f o r P r o t e u s i c h t h y o s m i u s . H i s t i d i n e , on the o t h e r hand, has b o t h a narrow range and a sharp optimum w i t h i n the r a n g e . F o r P r o t e u s i c h t h y o s m i u s , h i s t i d i n e l i b e r a t e s ammonia i n q u a n t i t y f rom pH 7.3 t o pH 8.7 w i t h an optimum o f pH 8.0, w h i l e f o r Pseudomonas p u t r e f a c i e n s , the range i s from pH 7.0 t o pH 8.9 w i t h the optimum at pH 7.6. The r e s u l t s o b t a i n e d f o r p r o l i n e show two d i f f e r e n t graphs f o r the two s p e c i e s o f b a c t e r i a , P r o t e u s i c h t h y o s m i u s causes c o n s i d e r a b l e ammonia f o r m a t i o n f rom pH 7.4 t o pH 8.5 w i t h a v e r y sharp optimum a t pH 8,0. Pseudomonas p u t r e f a c i e n s s t i m u l a t e s the f o r m a t i o n o f a r e l a t i v e l y s t e a d y q u a n t i t y o f ammonia f r o m pH 6.0 t o pH 8.3 w i t h o u t a d e f i n i t e optimum. These f i n d i n g s are i n g e n e r a l agreement w i t h those o f p r e v i o u s workers i n t h a t the range f o r ammonia f o r m a t i o n i s above pH 6,0 and below pH 9.0 w i t h an optimum between pH 7.0 and pH 8.0. W i t h i n t h i s r e l a t i v e l y wide range , however, the optimum pH v a r i e s m a rkedly depending on the amino a c i d used and the s p e c i e s of m i c r o o r g a n i s m employed. From these r e s u l t s , i t was d e c i d e d t o use the f o l l o w i n g b u f f e r s f o r the d i f f e r e n t amino a c i d s . When - 34 -employing P r o t e u s i c h t h y o s m i u s t h e b u f f e r s were: a r g i n i n e -pit 7.5, a s p a r t i c a c i d - pH 6.5, g l u t a m i c a c i d - pH 8.0, h i s t i d i n e - pH 8.0 and p r o l i n e - pH 8.0. When employing Pseudomonas p u t r e f a c i e n s the b u f f e r s were: a r g i n i n e - pH 7.0, a s p a r t i c a c i d - pH 6.5, g l u t a m i c a c i d - pH 7.5, h i s t i d i n e -pH 7.5 a n d ' p r o l i n e - pH 7.0. I n o r d e r t o determine the constancy of t h e r e s u l t s r e c o r d e d i n t a b l e s 1 and 2, the experiment was r e p e a t e d ( I I ) a f t e r an e l e v e n month i n t e r v a l e mploying p h t h a l a t e b u f f e r at pH 4.6, Sorens.en's phosphate b u f f e r a t pH's 5.6, 6,6, and 7.6 and b o r i c a c i d - K G l b u f f e r at pH's 8.5 and 9.5. The e x p e r i -ment was s e t up i n t r i p l i c a t e and d e t e r m i n a t i o n s were c a r r i e d out a f t e r seven, f o u r t e e n and twenty-one.days i n c u b a t i o n r e s p e c t i v e l y . The r e s u l t s a r e r e c o r d e d i n t a b l e s 3 and 4 and shown g r a p h i c a l l y on f i g u r e s 3 t o 12 I n c l u s i v e . The f i n d i n g s o f experiment I I a r e i n g e n e r a l agree-ment w i t h t h o s e of experiment I i n t h a t the g e n e r a l optimum range i n pH f o r ammonia f o r m a t i o n f r o m the amino a c i d s s t u d i e d v a r i e s from pH 6.0 t o pH 8.5. There a r e , however, i n some i n s t a n c e s d i f f e r e n c e s i n t h e i n d i v i d u a l ranges of pH. The l o n g e r i n c u b a t i o n t i m e s show t h a t f o r some of t h e amino a c i d s the maximum amount of ammonia i s n o t formed aft-er f i v e days' i n c u b a t i o n . The pH range o f from pH 5.6 t o pH 7.6 f o r t h e ammonia formed from a r g i n i n e by P r o t e u s i c h t h y o s m i u s shows a s h i f t towards the a c i d s i d e . The c o m p a r a t i v e l y l a r g e quan-t i t y of ammonia produced, however, i n c r e a s e d the pH o f t h e s e c u l t u r e s s u g g e s t i n g t h a t the t r u e pH range i s p r o b a b l y h i g h -er than the I n i t i a l pH o f the c u l t u r e s i n d i c a t e s . An a c i d i t y The Effect of pH on Ammonia Production by Proteus ichthyosmius aid Pseudomonas putrefaciens. - Experiments 1 and I I Tables 1, 2, 3 and 4 Figures 3 to 12 inclusive Experiment 1 - 5 days incubation «=> violet Experiment II - 7 days incubation - blue 14 daysincubation - green 21 days incubation - red Effect of pE TABLE I. Proteus ichthyosmius i n i t i a l pH of culture nino acid 4.65 5.05 5.57 6.06 6.34 6.86 Arginine 1. 2 ® 0.5 5.5 42.0 53 • 5 68.5 68.5 Aspartic 1. 0.5 1.0 3 © 5 34.5 40.0 37.0 a.cid 2« 4.40 4.92 5.58 5.89 6.34 6S86 Glutamic i . 1.0 1.0 2.0 16,0 15,0 33.0 acid 2. 4.48 4.95 5.42 5.85 5.75 6.62 Histidine 1. 14.6 19,6 2. 6.15 6.64 Proline 1. 14.5 18.0 2. 6.26 6.65 i n i t i a l pl-l of culture 7,34 7.96 8.55 9.06 9.46 9.63 Arginine 2. 78.25 71.25 62.5 45,75 37,25 13,0 Aspartic 1. 35,0 36.0 36,0 29.0 25.0 17.5 acid 2. 7.38 7.96 8.35 8,95 9.34 9.55 Glutamic 1. 52.5 32.5 37,0 21.5 17.0 16,0 acid 2 « 7.14 7,75 8.05 8,72 9,05 9.20 Histidine 1. 28.3 38.0 34,6 25.6 18,6 18e3 2 e 7.17 7.60 8.05 8.65 8.97 9.15 Proline 1. 21.0 37,0 17.5 8.0 6,0 5 2. 7,18 7.86 8,20. 8.85 9.16 9.34 1. percent of total nitrogen of amino acid (in culture) present.as free ammonia. 2. pH of culture at time of ammonia determination. TABLE 2. Effect of pH Pseudomonas putrefaciens i n i t i a l pH of culture amino acid 4.65 5.05 5.57 6.06 6.54 7.05 Arginine X« 0.0 0«0 1.75 6.75 10.25 10.76 2. 4.65 5,10 5„57 6.24 6.65 7.14 Aspartic 1. 0.0 1.5 9.0 36.0' 40.0 34,5 acid 2. 4.50 4,95 5.45 5.96 6.46 6 ,98 Glutamic • 1. . 0.5 1.0 8.0 15.0 21.0 22,5 acid 2. 4.55 4.95 5.44 5,80 6.43 6.95 Histidine 1. IS a t3 17.3 32.0 2. 6.15 6.53 7.06 Proline 1. •'• 0.0 0.5' 11 o 5 26.0 29.0 29.0 2. 4.65 6.10 5.64 6.20 6.60 7.13 i n i t i a l pH of culture 7.60 8,25 8.42 8.85 9.25 9.57 Arginine 1 9 8.0 8.0 8.5 7.5 3.25 0.5 2. 7.72 8.30 8.38 8.78 9.15 9.45 Aspartic 1. 35.0 35.5 37.5 32.5 28.5 6.5 acid 2. 7.54 7.85 8.05 8.65 , 9.05 9.30 Glutamic 1. 23.0 21.0 12 • 5 10.5 4.5 3.0 acid 2. 7.46 7,84 8.05 8.60 9.10 9,35 Histidine 1. 40.3 32.6 25.3 31.6 24.0 13.6 2. 7.56 7.95 8.05 8.56 9,03 9.26 Proline 1. 28,0 22.5 8.5 8.0 1.5 1.5 2. 7.65 8.11 8.35 8.79 9.25 9.51 1.. percent of total nitrogen of amino acid in culture present as free ammonia 2. pH of culture at time of ammonia determination Effect of pH TABLE 5. Proteus ichthyosmius age of culture Arginine 7 days 14 days 21 days Aspartic Ac id_ .; 7 days 14 days 21 days Glutamic Acid 7 days ™~~ 14, days . 21 days i n i t i a l pH- of cultures 4.6 5.6 6*6 7.6 8.5 9.3 1. 2. 1. 2. 1. 2. 1. 2. 1. 2. ; l . 2. 1. 2. 1. 2. 1. 2. 5.5 , 10.0 4.70 14.0 4.75. 0.0 0.0 4.55 1.0 4.60 0.0 0.0 4.55, 1.0 4.65 74.75 . 67.5 84.0 6.45 82.75 6.55 48.0 32.0 5.95 20.0 14.0 5.60 23.0 5,75 84.75 7.05 82.0 7.05 44.0 40.0 6.65 39.0 6.70 18,0 33.0 6.65 52.0 6.75 68.5 70.25 8.35 60.75 8.30 45.0 40.0 7.60 37.0 7,70 19.0 33,0 7.60 55,0 7.75 45.75 41.5 45.0 8.50 43.0 8.55 48.0 25.0 8.25 20.0 8.40 21.0 26 .0 8,25 40.0 8.35 31.25 9.00 17,0 9,00 29.0 10,0 8.95 7.0 9.05 19.0 17.0 8,95 21.0 9.05 Histidine " 7 days 14 days 21 days Proline 7 days 14 days 21 days 1. i . 1. 2 a 1. 2. 1. 2, 1. 2. 0.3 0,0 4.70 1.0 4.75 0.0 5.0 4.75 2.0 4,75 15.6 4 21.0 5.80 25.3 6.00 15.0 30.0 6.00 49.0 6.15 33.3 36.0 6.70 35.6 6.70 19.0. 6.70 59^0 6.80 38.3 38.0 7.65 34,6 7.75 35.0 41.0 7.80 56. 0'-7.90 35,3 30.3 8.35 24.6 8.30 32.0 34.0 8.45 30,0 8,50 22.0 13.0 8.90 8.6 8.90 15.0 22.0 9.00 ,18.0 9.05 Effect of pH TABLE 4. Pseudomonas putrefaciens I n i t i a l pH of cultures Age of culture 4.6 5.6 6.6 7.6 8.5 9.3 Arginine 7 days 1. 0.0 14.5 13.0 12.75 10.5 3.75 2. - - - - -14 days 1. 0.0 35.0 36.0 32.75 17.5 1.5 4.70 6.25 6.80 7,90 8.40 9.05 21 days 1. 1.0 64.75 59.0 48.25 21.75 2.5 2. 4.75 6.45 6.95 8.05 8.50 9.05 Aspartic Acid 7 days 1. 2. 0.0 42,0 38.0 43.0 41.0 27,0 14 days 1. 0.0 35.0 37.0 34.0 29.0 16.0 2. 4.55 5.85 6.65 7.60 8.30 8.95 21 days 1. 1.0 36.0 - 31.0 23,0 8..0 2. 4.55 5,90 - 7.55 8.35 9,00 Glutamic Acid 7 days IL o 2. 0.0 26,0 20.0 16.0 16,0 11.0 14 days 1. o eo 30,0 24.0 24.0 28,0 15,0 3 • 4.60 5.95 6.65 7.60 8.20 8.95 21 days i . 2.0 45.0 57,0 35.0 27.0 10.0 4.60 6e15 6.75 7.65 8.35 9.05 Histidine 7 days i . 2. 0.0 47.6 '61.6 57.3 48.3 23.3 14 days 1. 0.3 73.0 72.3 64.3 42.6 16.6 2. 4.75 6?-30 6.85 7.85 8.25 8,90 21 days 1. 0.6 77.3 74.6 61.6 9.6 2. 4.75 6.35 6.85 7.95 8.30 8.95 Proline 7 days 1. 2. 0.0 45.0 39,0 32.0 15.0 3.0 14 days 1. 0.0 62.0 51.0 52.0 16.0 2.0 2. 4.75 6.10 6.75 7.75 8.40 9.10 21 days 1. 2.0 58.0 47.0 44.0 13.0 3.0 2. 4«75 6.15 6.75 7.80 8„45 9.10 Figure 4. Proteus ichthyosmius Figure 5. Aspartic Acid 501 10.0 Pseudomonas putrefaciens Figure 6 Aspartic Acid 10.0 Proteus ichthyosmius Figure 7. Glutamic Acid Pseudomonas put r e f a c i e n s Figure 8. Glutamic Acid Proteus iohthyosmius Figure 9. H i s t i d i n e 40^ 10.0 Proteus ichthyosmius Figure 11. Proline 601 50 • , — i ,— 4.0 5.0 i 1 1 i — 6.0 7.0 pH T 1 8.0 —> i 1 — 9.0 10.0 Pseudomonas putrefaciens Figure 12. P r o l i n e • - 35 -between pH 5.0 and pH 5.5 p e r m i t s the f o r m a t i o n of s m a l l amounts o f ammonia, as seen from the p r e v i o u s r e s u l t s . T h i s ammonia w i l l r a i s e the pH o f the r e a c t i n g medium t o a- more f a v o u r a b l e pH f o r i n c r e a s e d ammonia p r o d u c t i o n . The concen-t r a t i o n o f b u f f e r used was a p p a r e n t l y too weak t o c o n t r o l and absorb t h e I n c r e a s e i n h y d r o x y l i o n s and t h e r e b y m a i n t a i n the pH at i t s i n i t i a l l e v e l . The r e s u l t s g i v e n i n f i g u r e 4 f o r ammonia f o r m a t i o n from a r g i n i n e by Pseudomonas p u t r e f a c i e n s show t h a t the o p t i -mum range i n pH i s s h i f t e d s l i g h t l y towards t h e a c i d s i d e o f th e s c a l e and narrows as the t i m e o f i n c u b a t i o n o f t h e c u l -t u r e s i n c r e a s e s , and t h a t the q u a n t i t y of ammonia produced i n c r e a s e s w i t h t h e time o f i n c u b a t i o n . The r e s u l t s f o r t h e de a m i n a t i o n of a s p a r t i c a c i d are s i m i l a r f o r t h e two s p e c i e s of b a c t e r i a employed. The optimum pH range shows a w i d e n i n g on the a c i d end o f t h e range w i t h no change on the, a l k a l i n e end. As the time of i n c u b a t i o n i n c r e a s e s the q u a n t i t y o f f r e e ammonia decreases s l i g h t l y and the range i n pH narrows on the a l k a l i n e end. The organisms are p r o b a b l y u s i n g the ammonia as a n i t r o g e n source a l o n g w i t h the a c i d s from d e a m i n a t i o n as carbon sources f o r c e l l m u l t i p l i c a t i o n . F i g u r e s 7 and 8 f o r t h e breakdown o f g l u t a m i c a c i d w i t h the l i b e r a t i o n of ammonia are s i m i l a r i n some r e s p e c t s but d i f f e r e n t i n " o t h e r s . F o r P r o t e u s ichthyosmius,• t h e range In.pH i s from pH 5.6 t o pH 9.4 a f t e r seven days' i n c u -b a t i o n and narrows t o a range from, pH 6.6 t o pH 7 . 6 . a f t e r f o u r t e e n and twenty-one days' i n c u b a t i o n . The q u a n t i t y of - 36 -ammonia i n c r e a s e s r e g u l a r l y w i t h i n c r e a s e d i n c u b a t i o n t i m e . For Pseudomonas p u t r e f a c i e n s , the range i n pH a f t e r seven and f o u r t e e n days' i n c u b a t i o n i s from pH 5.6 t o pH 8.5 w i t h an optimum at pH 5.6 a f t e r the. seven-day i n c u b a t i o n . A f t e r " twenty-one days' i n c u b a t i o n , however, the range has narrowed down t o a s i n g l e optimum of pH 6.6. The f i n a l q u a n t i t y o f ammonia formed by Pseudomonas i s t h e same as f o r P r o t e u s but the r a t e o f f o r m a t i o n seems t o be s l o w e r between seven and f o u r t e e n .days than i t i s i n the f i r s t seven and the l a s t seven days o f i n c u b a t i o n . The curves g i v e n on f i g u r e s 9 and 10 f o r ammonia f o r m a t i o n from h i s t i d i n e by the two b a c t e r i a l s p e c i e s are e n t i r e l y d i f f e r e n t . Those f o r P r o t e u s Ichthyosmius from experiment I I show a w i d e r range i n pH compared w i t h t h a t found I n experiment I . The optimum range i s from pH-6.6. t o pH 8.8 w i t h a s l i g h t change towards a c i d i t y as t h e time o f . i n c u b a t i o n i n c r e a s e s , whereas p r e v i o u s l y t h e range was narrow-e r and d e f i n i t e l y on t h e a l k a l i n e s i d e of the pH s c a l e . The q u a n t i t y of ammonia remains c o n s t a n t as the t i m e o f Incubation i n c r e a s e s s h o w i n g t h a t the maximum amount has been formed a f t e r seven days' i n c u b a t i o n . The curves f o r Pseudomonas p u t r e f a c i e n s show a more pronounced s h i f t t o t h e a c i d s i d e of the pH s c a l e f o r optimum ammonia f o r m a t i o n from h i s t i d i n e . W h i l e the optimum, from experiment I was pH 7.6, t h a t from e x p e r i m e n t , I I i s pH 6.6 a f t e r seven days' I n c u b a t i o n and between pH 5.6 and pH 6.6. a f t e r f o u r t e e n days', b r i n g i n g the pH n e a r e r to the seven-day optimum. The i n c r e a s e i n ammonia, however, causes a s h i f t I n pH from pH 5.6 t o pH 6.5. The - 37 -maximum q u a n t i t y of ammonia I s not formed a f t e r seven days but i n c r e a s e s u n t i l f o u r t e e n days. The t o t a l q u a n t i t y f o r Pseudonomas p u t r e f a c i e n s i s about t w i c e t h a t f o r P r o t e u s i c h t h y o s m i u s . As i n the case of h i s t i d i n e , the optimum pH range f o r t h e f o r m a t i o n o f ammonia from p r o l i n e by the two bac-t e r i a l s p e c i e s a r e on d i f f e r e n t s i d e s of the n e u t r a l p o i n t of the p l l s c a l e . P r o t e u s i c h t h y o s m i u s b reaks down p r o l i n e b e s t between pH 7.0 and pH 8,0 except a f t e r twenty-one days' i n c u -b a t i o n when t h e optimum s h i f t s t o between pH 6.6 and pH 7.6. On the oth e r hand-, Pseudomonas p u t r e f a c i e n s p r e f e r s a d i s -t i n c t l y a c i d h y d r o g e n - i o n c o n c e n t r a t i o n o f from pH 5,6 t o pH 6.6. The f i n a l . p H f o r b o t h groups o f p r o l i n e c u l t u r e s i s r a i s e d from pH 5.6 t o pH 6.1. In g e n e r a l , where the s h i f t i n the optimum pH range i s towards a more a c i d pH, as the t i m e o f i n c u b a t i o n i n c r e a -s e s , t h e r e has been an i n c r e a s e i n the i n i t i a l pH o f t h e b u f f e r , p a r t i c u l a r l y a t pH 5.6, caused by t h e r e l a t i v e l y l a r g e q u a n t i t i e s of ammonia formed d u r i n g the e a r l y days o f i n c u b a t i o n . The E f f e c t o f the Age of C u l t u r e on Ammonia F o r m a t i o n . W i t h t h e o b j e c t o f d e t e r m i n i n g the e f f e c t o f the age o f the growth c u l t u r e upon t h e subsequent ammonia forma-t i o n from the f i v e amino a c i d s i n b u f f e r c u l t u r e s , an e x p e r i -ment ( I I I ) employing P r o t e u s i c h t h y o s m i u s was c a r r i e d o u t . S i x K o l l e f l a s k s were I n o c u l a t e d a t t h e same t i m e . One f l a s k was removed from the i n c u b a t o r a t t h e end o f t e n , t w e l v e , The Effect of the Age of the Growth Culture on Subsequent Ammonia Production by Proteus ichthyosmius« Experiment III . Table 5. Figure 13« Arginine Aspartic Acid Glutamic Acid Histidine Proline - red - green - blue - violet - orange TABLE 5. age of culture i n hours amino acid 10 12 14 16 18 20 Arginine -74.75. 76 . 2 5 69,0 74.0 69.75 67.0 Aspartic acid 50.0 52.0 50.5 50.5 50.5 51.0 Glutamic acid 23.0 22.0 21.0 21.5 21.0 22.0 Histidine 62.6 58.5 53.5 50.0 34.6 36.0 Proline 49.5 42.0 40.0 39.5 31.5 33.5 Figure 13. age of growth c u l t u r e i n hours - 58 -f o u r t e e n , s i x t e e n , e i g h t e e n and twenty hours i n c u b a t i o n r e s -p e c t i v e l y , the c o n t e n t s washed and c e n t r i f u g e d as u s u a l and the b u f f e r c u l t u r e s , thus p r e p a r e d , i n c u b a t e d a t 30° G. f o r f i v e days. The r e s u l t s a r e r e c o r d e d . i n t a b l e 5 and i n t e r -p r e t e d i n f i g u r e 13. These f i n d i n g s i n d i c a t e t h a t the age of the growth c u l t u r e , w i t h i n the l i m i t s employed, has l i t t l e or no e f f e c t on the ammonia f o r m a t i o n from a r g i n i n e , a s p a r t i c a c i d and g l u t a m i c a c i d , but t h a t I n c r e a s e i n t h e age of the growth , c u l t u r e , p a r t i c u l a r l y beyond s i x t e e n hours decreases the ammonia formed fr-om h i s t i d i n e and p r o l i n e by t h i s m i c r o -organism. E f f e c t of A e r o b i c and A n a e r o b i c C o n d i t i o n s on Ammonia F o r m a t i o n . Stephenson and Gale (28, 30 and 62) found t h a t con-d i t i o n s o f oxygen s u p p l y a f f e c t e d m a r k e d l y the d e a m i n a t i o n o f c e r t a i n amino a c i d s . I n o r d e r t o show the e f f e c t o f oxygen s u p p l y i n t h e growth and b u f f e r medium on ammonia f o r m a t i o n from the f i v e amino a c i d s by P r o t e u s i c h t h y o s m i u s , two experiments were u n d e r t a k e n . I n t h e f i r s t experiment ( I V ) , t h r e e c o n d i t i o n s o f growth medium were employed: (1) t h e u s u a l t r y p t i c c a s e i n d i g e s t agar I n a K o l l e f l a s k , (2) t r y p t i c c a s e i n d i g e s t b r o t h i n a Roux f l a s k , and (3) t r y p t i c c a s e i n d i g e s t b r o t h i n an E r l e n m e y e r f l a s k w i t h the a i r r e p l a c e d by n i t r o g e n . A f t e r .the i n c u b a t i o n p e r i o d o f e i g h t e e n h o u r s , the growth was c e n t r i f u g e d , washed, and s e t up as u s u a l i n t e s t tube c u l t u r e s . A f t e r f i v e days' i n c u b a t i o n , the f r e e ammonia of - 59 -t h e b u f f e r c u l t u r e s was determined. The r e s u l t s are g i v e n i n t a b l e 6 and f i g u r e 14. I n g e n e r a l , the growth on agar i n K o l l e f l a s k s gave the g r e a t e s t subsequent ammonia f o r m a t i o n . For the amino a c i d s , - a r g i n i n e , a s p a r t i c a c i d and p r o l i n e , - growth on agar gave h i g h e s t ammonia f o r m a t i o n , growth a e r o b i c a l l y i n b r o t h i n t e r m e d i a t e r e s u l t s , and growth a n a e r o b i c a l l y i n t h e presence of n i t r o g e n the l o w e s t ammonia f o r m a t i o n . For g l u -tamic a c i d , the a e r o b i c b r o t h procedure gave s l i g h t l y h i g h e r r e s u l t s t h a n the agar and t h e a n a e r o b i c b r o t h method s l i g h t l y l ower t h a n the agar.. F o r h i s t i d i n e , the agar grown- c u l t u r e and t h e a n a e r o b i c b r o t h grown c u l t u r e were p r a c t i c a l l y the same and g a v e , s l i g h t l y lower r e s u l t s than t h e a e r o b i c b r o t h . The o u t s t a n d i n g f i n d i n g o f t h i s experiment i s t h a t , f o r p r o -l i n e , the a g a r p r o c e d u r e f o r growing the c e l l s g i v e s about t w i c e t h e ammonia f o r m a t i o n as the a n a e r o b i c b r o t h procedure f o r growth, s u g g e s t i n g t h a t o x i d a t i o n may be a f a c t o r In the opening o f t h e p y r o l l i d l n e r i n g p r i o r t o t h e l i b e r a t i o n o f ammonia. • - ' y - • .In the second experiment ( V ) , o f t h i s group, th e c e l l s were grown as u s u a l on agar i n a K o l l e f l a s k and s e t up i n b u f f e r c u l t u r e s i n two ways: (1) a e r o b i c a l l y i n f i f t y c u b i c c e n t i m e t e r c e n t r i f u g e tubes which were shaken a t d a i l y i n t e r v a l s and (2) a n a e r o b i c a l l y i n t e s t tubes w i t h t h e a i r r e p l a c e d by n i t r o g e n . A f t e r f i v e days' i n c u b a t i o n the f r e e ammonia was measured and I s shown i n t a b l e 6 w i t h g r a p h i c i n t e r p r e t a t i o n i n f i g u r e 15. The c u l t u r e s p r o v i d e d w i t h the i n c r e a s e d oxygen s u p p l y i n t h e b u f f e r medium showed o n l y The Effect of Aerobic and Anaerobic Conditi ons on Ammonia Production by Proteus ichthyosmius. Experiment IV and Y Table 6 Figure 14 (Experiment IV) Agar - Aerobic - A Broth - Aerobic - B B r o t h - Anaerobic - C Figure 15 (Experiment V) Aerobic - A Anaerobic - B Arginine - red Aspartic Aoid - green Glutamic Acid - blue f Histidine - violet Proline - orange TABLE 6. Effect of Oxygen Supply in the Growth and Buffer Media, Growth agar- broth- . broth- - agar- agar-Conditions aerobic aerobic anaerobic aerobic aerobic Buffer Conditions arginine aspartic acid glutamic acid histidine proline aerobic aerobic aerobic aerobic anaerobic 75,75 67.25 61.75 68.75 66.0 50.0 43.0 39,0 45.0 42.0 21.0 25.0 15.5 27.5 26,0 37.6 40.8 38.3 34,0 33.3 47.0 41.0 24.0 34.5 17.5 Figure 14. A B C A B C A B C A B C A B C Figure 15. 70J 60 J 50 40 J 30 20 10 A B A B A B A B A B - 40 -s l i g h t i n c r e a s e i n ammonia f o r m a t i o n over the a n a e r o b i c b u f -f e r c u l t u r e s except i n the case o f p r o l i n e w h i c h formed o n l y h a l f the ammonia a n a e r o b i c a l l y t h a t i t d i d a e r o b i c a l l y . T h i s f a c t i s f u r t h e r e vidence f o r the h y p o t h e s i s t h a t the opening of the p y r o l l i d i n e r i n g I s an o x i d a t i o n r e a c t i o n . - The Sate of Ammonia F o r m a t i o n . The q u e s t i o n of how r a p i d l y the ammonia I s l i b e r a -t e d from the v a r i o u s amino a c i d s was p a r t i a l l y answered by an experiment (VI) on the r a t e o f ammonia f o r m a t i o n from the f i v e amino a c i d s by P r o t e u s i c h t h y o s m i u s . S i x s e t s o f b u f f e r c u l -t u r e s were p r e p a r e d and t h e ammonia determined at the end of two h o u r s , one, two, t h r e e , f o u r and f i v e days' i n c u b a t i o n r e s p e c t i v e l y . The r e s u l t s are r e c o r d e d I n t a b l e 7 and are shown g r a p h i c a l l y i n f i g u r e s 16 t o 20 i n c l u s i v e . T h i s experiment shows c l e a r l y t h a t the de a m i n a t i o n of a s p a r t i c a c i d t a k e s p l a c e w i t h i n t w e n t y - f o u r h o u r s , where-as t h e ammonia f o r m a t i o n from t h e o t h e r f o u r amino a c i d s i n c r e a s e s s t e a d i l y over the f i v e days o f i n c u b a t i o n . L a t e r experiments c a r r i e d out as c o n t r o l s i n the ca r b o h y d r a t e s e r i e s show t h a t t h e maximum ammonia f o r m a t i o n from g l u t a m i c a c i d , h i s t i d i n e and p r o l i n e by P r o t e u s i c h t h y -osmius i s n o t reached a f t e r o n l y f i v e days' i n c u b a t i o n , b u t c o n t i n u e s t o r i s e f o r f o u r t e e n days i n t h e case Of h i s t i d i n e , and f o r twenty-one days f o r g l u t a m i c a c i d and p r o l i n e . These f i n d i n g s have been c o m p i l e d and. a r e p r e s e n t e d i n f i g u r e s .above A p i c t u r e o f t h e ammonia f ormat i o n , from the f i v e amino a c i d s by Pseudomonas p u t r e f a c i e n s i s g i v e n i n the same The Rate of Ammonia Production by Proteus ichthyosmius and Pseudomonas putrefaciens. Experiment VI Table 7 Figures 16 to 20 inclusive Proteus ichthyosmius - blue (Experiment VI. - June 1942) Proteus ichthyosmius - orange (Experiment X. - October 1942) Proteus ichthyosmius - green (Experiment XII - June 1943) Pseudomonas putrefaciens - violet (Experiment XI - January 1943) Pseudomonas putrefaciens - red (Experiment XIII - July 1943) TABLE 7. Rate of Ammonia Production Proteus ichthyosmius age of buffer culture i n days amino acid 2 hrs. 1 2 3 4 5 Arginine 3. o 5 33.75 56.0 62.0 72.0 83.0 . Aspartic acid 3.0 49.5 50.5 48.0 49.5 50.0 G-lutamic acid 2.0 4.0 9.5 18,0 26.0 26.0 Histidine 0.6 4.3 12„0 27,6 39.3 43.0 Proline 3.0 4.0 8.5 14.5 29.0 52.0 Figure 16. Figure 17. Aspartic Acid 60-, days Figure 19. Histidine 70 H days Figure 20 7 3 - z days - 41 -series of figures compiled for Proteus ichthyosmius above. These figures w i l l be discussed i n the section t i t l e d "Discussion." Ef f e c t of the Presence of Carbohydrates i n the Medium on Ammonia Formation. 1. Fermentation of Carbohydrates i n Shake-Agar Cultures by Two Surface Taint Producing B a c t e r i a l Species. Prior to undertaking an extensive investigation of the effect of fermentable and non-fermentable carbohydrates on the ammonia producing^activities of the two ba c t e r i a l species from amino acids, i t i s necessary to know the sugar-fermenting a b i l i t i e s of Proteus ichthyosmius and Pseudomonas putrefaciens. In order to obtain these data, shake agar cultures prepared from nutrient agar and using brora-cresol-purple as the indicator were employed. In the f i r s t experi-ment (VII) a one-percent concentration of each of the following carbohydrates, added to the medium p r i o r to s t e r i l -i z a t i o n , were used: glycerol, xylose, arabinose, mannitol, laevulose, glucose, galactose, sucrose, maltose, lactose, rafflnose, i n u l i n , dextrin, s a l i c i n , and d u l c i t o l . The cultures were observed for acid and/or gas production after forty-eight hours' incubation at 30° C. and are recorded in table 8. The slight amount of acid produced from lactose by these microorganisms which are not supposed to ferment lactose at a l l , suggests that possibly s t e r i l i z i n g the carbon and nitrogen sourced together breaks down a small amount of the TABLE 8. Shake-Agar Sugar Per mentations (Carbon and Nitrogen Sotirces Sterilized Together) Carbohydrate Proteus ichthyosmius Pseudomonas putrefaciens acid alkaline gas acid alkaline gas glycerol 0 4-H- 0 + xylose 0 0 0 0 0 0 arabinose + 0 + 0 surface 0 mannitol +++ *o +++ -H-+ 0 laevulose •H-H-, 0 •++ +++ 0 ++ glucose ++++ 0 -H-+ H-H- 0 -H-f galactose 0 ++ +-H- 0 •H-sucrose -H-H- 0 -H-+ -HH- 0 ++ maltose •H-H- 0 +++ .0 ++ -lactose trace surface + it surface + raffinose trace surface + +-H- 0 + inulin 0 surface trape surface trace dextrin' 1 0 + ++ surface trace salioin •H-H- 0 -H- ++++ .0 ++ dulcitol 0 surface trace + surface trace - 41a -lactose Into i t s component sugars - glucose and galactose both of which are readily fermented. In order to eliminat t h i s p o s s i b i l i t y , the experiment (VIII) was repeated employing the technique of s t e r i l i z i n g the carbohydrate source separately and adding i t to the medium asoptically just prior to inoculation. The cultures were incubated at 30° Cc and observed at twenty-four, forty-eight and ninety-six hour intervals for a c i d i t y , a l k a l i n i t y and gas production. The findings are given i n tables 9 and 10. This experiment shows that Proteus ichthyosmius produces acid and gas from glycerol, mannitol, glucose, galactose, sucrose, maltose, dextrin and s a l i c i n and has no immediate action but goes slowly alkaline i n xylose, lactose, d u l c i t o l and the water control; whereas Pseudomonas putrefaciens produces acid and gas from" xylose mannitol, glucose, maltose and s a l i c i n , slowly produces acid with no gas from glycerol, and has no immediate reaction but slowly goes alkaline i n lactose, dextrin and the water control. The d i f f e r e n t i a t i n g sugars f o r these microorganisms are xylose and dextrin: Proteus ichthyosmius ferments dextrin but not xylose while Pseudomonas putrefaciens ferments xylose but not dextrin. TABLE 9. Shake-Agar Sugar Fermentations (Carbon and.Nitrogen' Sources, Sterilized Separately) Proteus ichthyosmius Carbohydrate Acid Alkaline Gas 24hr. 48hr, '96-hr. • 24hr, 48hr. 96,hr. 24hr. 48hr. 96hi glycerol -H- -H- .Uj,,l. ! 1T 0 0 0 -H-. -H -H-xylose .0 0 0 •H-H- •H-H- 0 0 0 mannitol ++-H- •H-H- ++++ sur. sur. sur. -H-f +++ -H-r glucose ++++ •++++• -H-H- \ 0 0 0 ++ ++ ++ galactose -H-H> •H-H- •H-H- 0 0 0 ,L|,-11 ++ - H sucrose . •H-H- -H-H- 0 0 0 •f-H- TIT maltose •H-H- -H-H- •H-H- 0 0 0 •H- •Hi "Hi" lactose 0 0 0 0 +-H-+ •H-H- G 0 • o. dextrin -H-H- +-H-+ • H - H G 0 0 -J—J. ++ s a l i c i n •-H-H- -H-H- •H-H- 0 , 0 0 •H- ++ dulcitol • 0 0 0 sur. -H-H- ++++ 0 0 •o mter (K) 0 0 0 sur. •HrHr •H-H- 0 0 0 sur. - surface TABLE 10. Shake-Agar Sugar Fermentations (Carbon and Nitrogen Sources Sterilized Separately) Carbohydrate glycerol xylose mannitol glucose galactose sucrose maltose lactose dextrin , s a l i c i n dulcitol water (K)' Acid Pseudomonas putrefaciens Alkaline Gas 24hr. 48hr. 96hr. 24hr., 48hr... 96hr. 24hr. 48hr. 96hr. trade + -H--H- ++ -H-H--H-H- -H-H- -H-H-++++ -H-H- -B-H-•H-H- ++++ -H-H--H-H- -H-H- -H-H-trace 0 . 0 trace trace 0 -H-H- . -H-H- ++++ 0 , 0 , 0 0 0 0 0 0 " sur. 0 sur. sur. 0 sur. sur. sur. sur. sur. sur. sur. sur. sur. sur. sur. + sur. sur. sur. + 0 0 + sur. sur. sur. -H-0 0 0 sur.. -H-H- ++++ sur. -H-H- -H-H-0 trace trace trace + + -H-H- -H-H- ++++ -H-H- -H-H- -H-H-++ 4~r 4~*i* *H~ -H- -H- -H-0. 0 0 trace trace trace -H- +-H- I 0 0. 0 0 0 0 sur. - surface - 41b -2. E f f e c t of the Presence o f Carbohydrates i n the Growth and Buffer Media on Ammonia Formation. The l i t e r a t u r e contains considerable data and a number of theories on the effect of fermentable and non-fermentable carbohydrates i n the growth, and buffer media on the subsequent a c t i v i t y of ammonia producing enzymes .from various species of microorganisms. With the object of investigating the eff e c t of a number of carbohydrates on the ammonia formation.- from arginine, aspartic acid, glutamic acid, h i s t i d i n e and proline by Proteus ichthy-osmius and Pseudomonas putrefaciens a series of experiments was undertaken. The object of the f i r s t experiment (IX) i n t h i s series was to determine the eff e c t of the presence of one percent glucose i n the growth medium on ammonia formation from arginine, aspartic acid and glutamic acid i n buffer media containing glucose, sucrose, maltose, lactose, g l y c e r o l , mannitol or water (control) respec-t i v e l y by Proteus ichthyosmius. The cultures were pre-pared as outlined i n the general procedure and incubated at 30° C. The ammonia from aspartic acid was determined after twenty-four hours' incubation, from glutamic acid after four days and from arginine after f i v e days. The results are shown i n table II and figures 21, 22 and 23. The glucose growth agar flask was incubated for forty-two hours before harvesting the b a c t e r i a l c e l l s and the growth was s t i l l only about twenty-five percent of that of the eighteen-hour non-glucose agar f l a s k . In general, the presence of glucose i n the growth medium only s l i g h t l y decreases the quantity of free ammonia i n the tubes. The amount of free ammonia from the two sugars fermented most rapidly by Proteus ichthyosmius is more than twice as great employing the c e l l s grown on glucose free agar as that using the c e l l s grown on glucose containing agar^ The volume of free ammonia from arginine i s greatest in the water control and the lactose containing cultures, less i n the maltose, g l y c e r o l and mannitol containing cultures and least in the glucose and sucrose containing cultures. The quantity of ammonia from aspartic acid i n the cultures containing glucose and sucrose i s the same for the c e l l s grown on the two types of agar whereas i n the culture containing lactose, i t i s about twenty-five percent greater employing c e l l s from glucose free agar than c e l l s from glucose containing agar. In addition, there is no marked difference i n the amount of ammonia present i n the cultures containing the various carbohydrates. The cultures containing lactose and the water control show a small quan-t i t y of ammonia that i s s l i g h t l y greater from c e l l s grown on glucose free agar than those from glucose containing agar The Effect of the Presence of Carbohydrates i n the Growth and Buffer Media cn Ammonia Production "by i Proteus ichthyosmius. Experiment - IX. Table - 11. Figures - 21, 22 and 23. Growth Medium: Glucose-Free Medium - straight line Glucose-Containing Medium - broken line . Buffer Medium; Water (control) - red Glucose - violet Sucrose - green Maltose - -brown Lactose - orange Glycerol - blue Mannitol - black TABLE 11. Effect of the Presence of Carbohydrates i n the Growth and Buffer Media Growth Buffer Aspartic Glutamic Medium Medium Arginine Acid Acid no glucose no sugar 62,5 38.5 17.0 glucose no sugar 54,0 34,5 10,5 no glucose glucose 28.75 35.5 2 © 5 glue os e glucos e 10.5 36.0 ©*o no glucose sucrose 32 ©5 3 9 © *5 2.0 glucose sucrose. 13.0 40.0 0.0 no glucose maltose 39.0 41,0 2 «t *3 glucos e maltose 34.0 33.0 0.0 no glucose lactose 59.25 43.5 9,0 glucose lactose 54.5 31.0 5.0 no glucose glycerol 46.0 44.5 5.0 glucose glycerol 40.5 39.0 , o.o no glucose mannitol 36.25 37.0 4.0 glucose mannitol 31.25 29,0 0.0 Figure 21. Arginine 50 4C 30 20 10 Figure 22. 40 J 304 ^ 3 20 4 104 Aspartic Acid I Figure 23. 20 Glutamic Acid 10 > - 4 3 -w h i l e t h e r e i s o n l y a s l i g h t amount o f ammonia from c u l t u r e s c o n t a i n i n g the r e m a i n i n g f i v e sugars when c e l l s from g l u c o s e f r e e agar are employed and no ammonia when those from g l u c o s e c o n t a i n i n g agar are used. W i t h the o b j e c t o f d e t e r m i n i n g the e f f e c t of the presence of f e r m e n t a b l e and non-fermentable c a r b o h y d r a t e s i n the b u f f e r medium upon the r a t e of ammonia f o r m a t i o n from the f i v e amino a c i d s by P r o t e u s i c h t h y o s m i u s and o f d e t e r m i n i n g the e x t e n t to which the organism may u t i l i z e ammonia as a n i t r o g e n source f o r c e l l r e p r o d u c t i o n , the second experiment . . . . (X) was u n d e r t a k e n . The e l e v e n c a r b o h y d r a t e s employed may be d i v i d e d i n t o t h r e e groups based on t h e i r r a p i d i t y o f a c i d p r o d u c t i o n b y P r o t e u s i c h t h y o s m i u s : Group 1 - composed o f g l u c o s e and su c r o s e a re r a p i d a c i d f o r m e r s , Group 2 - c o n t a i n i n g g l y c e r o l , m a n n i t o l , g a l a c t o s e , m a l t o s e , d e x t r i n and s a l i c i n produce a c i d b u t not so r a p i d l y as group one - a c i d p r o d u c t i o n from g l y c e r o l b e i n g much slower than t h a t from the o t h e r c a r b o h y d r a t e s .-in t h i s group, Group 5 - i n c l u d e s x y l o s e , l a c t o s e and d u l c i t o l w hich f a i l t o g i v e a c i d when a c t e d upon by P r o t e u s I c h t h y o s m i u s . D i s -t i l l e d w a ter was used as a c o n t r o l t o determine the q u a n t i t y of ammonia formed i n the absence o f a c a r b o h y d r a t e . The c u l t u r e s were s e t up i n seven groups and Incub a t e d at 30° C. f o r one, two, t h r e e , f o u r , s i x , e i g h t , and t e n days r e s p e c t -i v e l y . The, q u a n t i t y o f f r e e ammonia p r e s e n t i n each o f the c u l t u r e s a f t e r each p e r i o d o f i n c u b a t i o n i s r e c o r d e d i n The Effect of the Presence of Carbohydrate in the Buffer Medium on the Rate of Ammonia Production by Proteus ichthyosmius. Experiment X. Tables 12 to 16 inclusive Figures 24 to 28 inclusive C arb ohyd r ate s; Water (contol) red Glycerol - blue Xylose _ green Mannitol - broken blue Glucose - violet Galactose - broken green Sucrose - broken violet Maltose - broken orange Lactose orange Dextrin - bvo\m Salicin broken brown. Dulcitol broken red TABLE 12. Effeot of the Presence of Carbohydrates Proteus ichthyosmius Arginine age of culture i n days carbohydrate 1 2 3 4 6 8 10 glycerol 49.0 49.0 46.75 46.75 46.5 40,0 41.5 xylose 36.5 47.5 51,0 49.25 56.0 71.0 79.5 mannitol 36.0 38.75 3j§..75 36.75 34.5 _ 36.75 glucose 5.25 8.5 12.75 15.0 20.75 - 24 © 25 galactose 41.0 39.0 35,75 39.5 31,5 _ 36s0 sucrose 12.0 18.0 22 0 5 24,5 24.0 - 25.75 maltose 40.0 38.25 35.0 38.5 33,5 - 37.75 lactos e 49.5 51.75 54.25 61.5 59.5 - 74.0 dextrin 36.0 35 9 25 35.5 35.5 32«. 25 - 36.25 salic i n 42.0 4:2 © 25 40.75 41.5 39,5 - 43,25 dulcitol 47.0 56.25 59,0 62,0 64.5 - 77,0 water 45.5 58.0 57.0 56.5 58.25 78.5 TABLE 13. Effect of the Presence of Carbohydrates Proteus ichthyosmius Aspartic Acid age of culture in days 1 2 5 4 6 8 10 carbohydrate glycerol 45 45 43 43 42 43 39 xylose 49 47 47 49 46 48 48 mannitol 46 43 42 44 34 35 39 glucose 42 42 59 41 54 - 39 galactose 43 44 42 41 51 33 42 sucrose 44 45 42 43 33 33 42 maltose 40 38 52 37 27 28 S3 lactose . 49 48 41 44 37 41 48 dextrin 40 37 35 34 30 32 39 sa l i c i n 44 46 47 46 44 44 50 dulcitol 52 48 47 50 47 49 51 water 51 50 47 48 46 " 42 49 TABLE 14. Effect of the Presence of Carbohydrates Proteus ichthyosmius Glutamic Acid age of culture in days _1 2 3 4 6 8 10 carbohydrate glycerol 5 6 6 4 1 0 0 xylose :8- 8 9 13 17 21 33 mannitol 1 0 0 0 0 0 0 glucose 1 1 .0 0 0 0 0 galactos e 0 0 0 0 0 0 0 sucrose 2 0 0 0 0 0 0 maltose 1 > 0 0 0 0 0 0 lactose 8 10 10 12 17 18 21 dextrin 1 0 0 0 0 •o 0 s a l i c i n 3 1 0 ' 0 0 0 0 dulcitol 10 - 13 ' 16 26 32 37 water 10 11 13 20 28 - 27 42 TABLE 15. Effect of the Presence of Carbohydrates Proteus ichthyosmius Histidine age of culture in days __1 2 3 4 6 8 10 carbohydrate glycerol 3.6 8.0 13.3 21 » 3 21,3 20.6 22.0 xylose 3.3 9.5 29.3 31«3 32.6 35.0 35.0 mannitol 0.6 1.6 0.6 4.6 4.0 9.3 8.6 glucose 0 0 0 0.3 0.3 0.5 0.6 galactose 1.3 1.6 2.0 2 © 3 2 .6 3 © 3 3.6 sucrose 0 0 0 0.6 0.6 0.3 0.3 maltose 0.6 0.6 1 e3 1 9 3 2.0 3.0 4,0 lactose 2 .6 9.3 12.6 19.5 22.6 24.6 26.6 dextrin • 0 0.3 0.6 1»3 1.3 2.0 2,6 sa l i c i n 2.3 4.6 5,0 6.0 1.0 5.0 5.3 dulcitol 3.5 13.6 .21.3' 31,0 35.0 36.0 40.6 water 2.6 12.0 20.6 27.3 32 © 3 35.6 37,0 TABLE 16. Effect of the Presence of Carbohydrates Proteus ichthyosmius Proline 1 2 age 3 of culture 4 in days 6 8 10 carbohydrate glycerol 1 2 3 0 0 0 0 xylose 2 6 9 11 19 25 31 mannitol .0 0 *» 0 0 - 0 0 glucose 0 0 0 0 - 0 0 galactose 0 0 0 0 - 0 0 sucrose 0 0 0 2 0 0 0 maltose 0 0 0 0 . » 0 0 lactose 2 4 8 9 IS 10 14 dextrin 0 0 0 0 0 0 sa l i c i n 0 0 0 0 - 0 -dulcitol 2 7 12 21 28 31 -water • S 8 11 23 18 28 35 Figure 24. Proteus ichthyosmius Arginine 80 J Figure 25 Proteus ichthyosmius Aspartic Acid 60 1 days Figure 26 Proteus ichthyosmius Glutamic Acid 60 Figure 27 Proteus ichthyosmius Histidine 70 60 Figure 28 Proteus ichthyosmius Prolire 70 60 50 40 -I days - 4 4 -t a b l e s 12 to 16 I n c l u s i v e and i n t e r p r e t e d on f i g u r e s 24 to 28 i n c l u s i v e . The r e s u l t s show c l e a r l y t h a t f o r a r g i n i n e , g l u t a m i c a c i d , h i s t i d i n e and p r o l i n e , the q u a n t i t y of f r e e ammonia p r e s e n t i n the c u l t u r e s i s dependent i n most cases on the r a t e of a c i d p r o d u c t i o n from the r e s p e c t i v e c a r -b o h y d r a t e s . T h i s o b s e r v a t i o n can be seen most r e a d i l y i n the case o f a r g i n i n e , f i g u r e 24. X y l o s e , d u l c i t o l and l a c t o s e do not. show a c i d p r o d u c t i o n a f t e r t e n days i n c u b a t i o n and the q u a n t i t y o f f r e e ammonia f o r these c a r b o h y d r a t e s i s e q u a l t o t h a t from the water c o n t r o l , S a l i c i n , g l y c e r o l , m a l t o s e , m a n n i t o l , d e x t r i n and g a l a c t o s e e x h i b i t a c i d p r o -d u c t i o n i n 48 hours and do not show any i n c r e a s e i n f r e e ammonia a f t e r the f i r s t day. Glucose and sucrose are more r a p i d a c i d formers t h a n the second group o f c a r b o h y d r a t e s and do not demonstrate so g r e a t a q u a n t i t y o f f r e e ammonia from the b e g i n n i n g o f the experiment as e i t h e r group two o r t h r e e . No growth i n the c u l t u r e s c o n t a i n i n g the f i r s t group o f sugars was observed but i n those c o n t a i n i n g the second and t h i r d groups growth was p r e s e n t . Thus i t i s seen t h a t P r o t e u s i c h t h y o s m i u s appears t o have the a b i l i t y t o use- the p r o d u c t s o f carbohydrate breakdown as carbon sources and the ammonia as a n i t r o g e n source f o r c e l l m u l t i p l i c a t i o n i n the case o f the l a t t e r groups o f c a r b o h y d r a t e s r e s u l t i n g i n a decreas e d amount o f f r e e ammonia. When the f i g u r e s f o r g l u t a m i c a c i d , h i s t i d i n e and p r o l i n e ( f i g u r e s 26, 27 and 28) are c o n s i d e r e d the r e s u l t s - 45 -o b t a i n e d a r e . on the whole, s i m i l a r t o those r e c o r d e d i n the case of a r g i n i n e . I n s p e c t i o n of these f i g u r e s shows t h a t the t o t a l amount of ammonia formed i s c o n s i d e r a b l y l e s s but t h a t the i n f l u e n c e o f the s p e c i f i c c a r b o h y d r a t e s on the r e l a t i v e amount o f ammonia formed i s unchanged, p r a c t i c a l l y no ammonia b e i n g formed i n the c u l t u r e s c o n t a i n i n g the f i r s t and second groups of c a r b o h y d r a t e s . A s t r i k i n g d i f f e r e n c e between a r g i n i n e on the one hand and g l u t a m i c a c i d , h i s t i d i n e and p r o l i n e on the o t h e r hand i s t o be seen when the amounts of ammonia formed d u r i n g the f i r s t ' t w e n t y - f o u r hours i n c u b a t i o n are compared. T h i s d i f f e r e n c e i s u n d o u b t e d l y r e l a t e d to the c h e m i c a l s t r u c t u r e of the amino a c i d s and t o the mechanism o f breakdown po s s e s s e d by P r o t e u s i c h t h y o s m i u s . T h i s i n t e r e s t i n g r e l a t i o n s h i p w i l l be c o n s i d e r e d i n the f i n a l d i s c u s s i o n and w i l l be compared and. c o n t r a s t e d w i t h t h a t o f Pseudomonas p u t r e f a c i e n s . I n the case o f a s p a r t i c a c i d , a p i c t u r e d i s t i n c t from t h a t o b t a i n e d f o r the o t h e r ••four amino a c i d s employed i s t o be seen, f i g u r e 25, I t w i l l be n o t e d t h a t f o r t h i s amino a c i d , ammonia p r o d u c t i o n o c c u r s r a p i d l y , d e a m i n a t i o n of the n a t u r a l isomer of d l - a s p a r t i c a c i d b e i n g complete w i t h i n t w e n t y - f o u r h o u r s ' i n c u b a t i o n ; whereas i n the case of the o t h e r f o u r amino a c i d s , ammonia f o r m a t i o n t a k e s p l a c e at the same time as a c i d p r o d u c t i o n , a p o s t u l a t e which has been co n f i r m e d by l a t e r work, ( v i d e experiment X I I I ) . As the time o f i n c u b a t i o n i n c r e a s e s , i n the case of a s p a r t i c a c i d , the - 46 -l e v e l of f r e e ammonia i n the c u l t u r e s c o n t a i n i n g the f e r -mented carboh y d r a t e s decreases o n l y s l i g h t l y , w i t h a s l i g h t i n c r e a s e i n the number o f b a c t e r i a l c e l l s , w h i l e t h a t i n the c u l t u r e s o f the n o n - a c i d p r o d u c i n g group remains p r a c t i c a l l y unchanged. I n the presence of ample q u a n t i t i e s o f carbon and n i t r o g e n sources i n the c u l t u r e s , one would expect more a c t i v e c e l l m u l t i p l i c a t i o n w i t h a c o r r e s p o n d i n g l y g r e a t e r decrease i n the f r e e ammonia p r e s e n t . T h i s l a c k of a c t i v i t y suggests t h a t p o s s i b l y the c o n d i t i o n s were not optimum f o r the growth o f the organism. The experiment was r e p e a t e d , ( X I ) , employing Pseudomonas p u t r e f a c i e n s i n p l a c e o f P r o t e u s i c h t h y o s m i u s , and, i n o r d e r t o determine i f pH was the l i m i t i n g f a c t o r en-co u n t e r e d above, b r o m - c r e s o l - p u r p l e i n d i c a t o r was added t o the s e t o f c u l t u r e s t o be l e f t f o r the l o n g e s t p e r i o d o f i n -c u b a t i o n . A c o n t r o l s e t o f c u l t u r e s c o n t a i n i n g c a r b o h y d r a t e w i t h o u t an amino a c i d was set up t o determine the changes i n pH o f the c u l t u r e s i n the absence o f c e l l growth. The c u l t u r e s were i n c u b a t e d a t 30° C... A f t e r t w e n t y - f o u r h o u r s ' i n c u b a t i o n , the c u l t u r e s c o n t a i n i n g f e r m e n t a b l e sugars showed marked a c i d p r o d u c t i o n and i t was d e c i d e d to determine e l e c -tronic t r i c a l l y the f i n a l pH o f the c u l t u r e s p r i o r t o the d e t e r m i n a t i o n o f the ammonia c o n t e n t . Because o f u n u s u a l weather c o n d i t i o n s , the U n i v e r s i t y was c l o s e d f o r a number o f days p r e v e n t i n g the c a r r y i n g out of d e t e r m i n a t i o n s a t a l l i n t e r v a l s employed i n experiment X, one set o f c u l t u r e s o f n e c e s s i t y was not completed u n t i l an i n t e r v a l o f t w e n t y - f o u r - 47 -days' i n c u b a t i o n had e l a p s e d . I t was not p o s s i b l e t o complete the work u s i n g a s p a r t i c and g l u t a m i c a c i d s , but the r e s u l t s f o r the e a r l y days of i n c u b a t i o n i n d i c a t e t h a t i n the presence o f c e r t a i n c a r b o h y d r a t e s t h e r e i s ' marked a c i d p r o d u c t i o n and the pH o f these c u l t u r e s i s reduced below the l e v e l r e q u i r e d f o r b a c t e r i a l a c t i v i t y . These r e s u l t s a l s o show t h a t a s p a r t i c a c i d i s deaminated more s l o w l y by Pseudomonas p u t r e f a c i e n s than by P r o t e u s i c h t h y o s m i u s , w h i l e g l u t a m i c a c i d i s deamin-a t e d a t about the same r a t e by b o t h s p e c i e s o f b a c t e r i a . The r e s u l t s o b t a i n e d f o r a r g i n i n e , h i s t i d i n e , p r o l i n e and the water c o n t r o l - a r e g i v e n i n t a b l e s 17 t o 20 i n c l u s i v e and f i g u r e s 29 t o 35 i n c l u s i v e . The u n a v o i d a b l e d e l a y u n t i l the l a s t s e t o f c u l t u r e s was t w e n t y - f o u r days o l d when the ammonia content was d e t e r -mined shows t h a t i n the case o f a r g i n i n e and p r o l i n e the ammonia f o r m a t i o n by Pseudomonas p u t r e f a c i e n s i s not a t i t s maximum at t e n days i n c u b a t i o n . The c a r b o h y d r a t e s are d i v i d e d i n t o t h r e e groups based on t h e i r a c i d p r o d u c t i o n b y .Pseudomonas p u t r e f a c i e n s . The f i r s t group i n c l u d i n g l a c t o s e , d e x t r i n and d u l c i t o l does not produce a c i d i n shake-agar c u l t u r e s , the second c o n t a i n s g l y c e r o l w h i c h produces a c i d s l o w l y , w h i l e the t h i r d group made up of x y l o s e , g l u c o s e , m a n n i t o l , g a l a c t o s e , m a l t o s e , sucrose and s a l i c i n shows r a p i d a c i d p r o d u c t i o n . The pH d e t e r m i n a t i o n s on the c o n t r o l s e r i e s c o n t a i n i n g c a r b o h y d r a t e s o n l y , shown i n f i g u r e 35, a l s o c l e a r l y d i v i d e the carbo-h y d r a t e s i n t o the same t h r e e groups. The pH r e c o r d i n g f o r The Effect of the Presence of Carbohydrate. i n the Buffer Medium on the Bate of Ammonia Formation by Pseudomonas putrefaciens. Experiment XI, Tables 17 to 20 inclusive Figures 29 to 35 inclusive Carbohydrates Water (control) - red Glycerol Xylose Mannitol Glucose Galacto se Sucrose Maltose Lac to se Dextrin Salicin Dulcitol *•> blue green -broken blue - violet - broken green - broken violet - broken orange - orange - brown - broken brotvn - broken red TABLE,17. Effect of the Presence of Carbohydrates Pseudomonas putrefaciens age of culture in days ' 1 2 5 4 6 8 24 CAEBOBYDPA.TE ' glycerol 1. ©.25; 6.5 0.5 • 1.0 0.25 1.0 4:4;»25 2. 6.90 6.80 6.65 6,35 6.30 6.25 7.05 xylose 1. 0.25 0 0 0.25 0.25 4.25 45.75 2. 6.95 5,75 5,40 5.45 5.60 6.30 . 7,05 mannitol 1. 0.25. 0.5 0.75 0.75 1.5 11,25 42.25 2. ; 6..40., 5.55 i 5.45 5.30 6,05 6,45 . 7.05 glucose 1. 1.0 0.75 0,75 1.0 0.5 6.25 54.25 2. 5.75 5.05 5.10 5.00 5.10 5.95 •7.15 galactose 1. 0.75 1.0 ' 1,0 0.25 0.5 . 1,0 48.5 2. 5.80 5.25 5.55 5.20 5 e 25 5.80 7.15 sucrose 1. 1.5 ' 1.5 1,25 0.75 0.5 3,0 40.5 2. 5.80 5,45 5.45 5,50 5.75 5.50 7.05 mgltose 1. 0.5 0,5 0.25 0.5 0 5.75 45.5 2. 6,90 • 6,00: • 5,50 5.15 5.25 6.20 7.10 lactose 1. 1.0 3.0 2.75 6.25 6.5 15.25 42.75 2. 6,95 6.95 7.10 6.90 ; 6.85 6*80 6,05 dextrin, 1. 1.25 3.0 * . • f 5.0* 6.75 9.25 13.0 50.5 2. 6.95 6 .95 6.95 6.90 7.05 6.95 7.15 salacin 1. 0.25 0.75 0.75 1.25 1« 25 0.75 2,25 - 2. 6.55 5.40 5.30 - 5.20 5.40 5.10 5.15 dulcitol 1. 1.5 3.25 1.0 6.0 6.5 11,0 84.5 2. 7.00 7.00 7.05 6.95 7.05 7.05 7.55 water 1. 1.75 2.75 , 6.25 ' 5.5 7.5 8,5 78.75 2. "7.00 7.00 7.05 7.00 7.05 7,05 7.55 TABLE 18. Effect of the Presence of Carbohydrates Pseudomonas putrefaciens Histidine age of cul ture in days 1 2 4 9 24 Carbohydrate glycerol 1. 3.0 4.3 10.6 20.6 37.3 2. 7.10 6.75 6.60 6*20 6.55 xylose 3.0 3.3 4.3 8,3 10.6 2. 7.10 6.05 5.45 5.25 5.30 mannitol 1. 2*.0 3.6 4.3 6,6 16.6 2. 6.50 5.65 5.60 5.20 6,10 glucose 1. 0.3 1.0 1.6 3«3 16.0 2. 5.65 4,90 5.10 5.25 6.25 galactose X 0 - 1 e3 1.6 2,6 4.3 2. 5.80 5.05 5,30 5.30 5.15 sucrose 1 e - 2,0 4,0 8,6 20,3 ' 5.90 5,40 5.65 5,45 6,00 maltose . i . ' - 8.0 7.3 13.3 13.0 2. 7.05 5.80 5.30 4,90 4.90 lactose 1. 3.0 8.3, 14.6 45.0 54.6 2. 7,15 6.95 7.20 7,05 6.05 dextrin 1, 4.6 11.0 30.6 50.0 49.0 2 8 7.15 6.95 7.20 ,7.20 6.95 salic i n 1. 2-.0 3 © 3 5.0 6.0 8.3 2. 6.75 5.85 5.50 5.55 5.40 dulcitol 1. 5,6 12.0 37.3 51.0 55.6 2, 7.25 7.10 7.40 7.55 7.90 water 1. 6.0 10.6 39,6 57,0 53.6 2. 7.25 7.05 7.45 7.70 7,90 TABLE 19. Effect of the Presence of Carbohydrates Pse^ d^omonas putrefaciens Proline age of culture in days i 2 4 9 24 arbohydrate glycerol i . 0 1 6 32 2 ® 6.85 6.55 6.55 6,05 6,50 xylose i . . - 0 . 0 1 2 . 2. 6.80 5.95 5.55 . 5.05 5.00 mannitol 1. 0 0 2 5 2... 6.65 6.05 5.65. 5.35 5.05 glucose — 0 0 1 5 2, 6.05 5 © 25 5,20 5.20 5.00 galactose 1.. _ 0 0 _ 9 2. 6.05 5.35 5.40 5.20 6,25 sucrose 1.. - - 0 0 — 5 2. ' 6.15 5,65 . 5.55 5.75 5.75 maltose . 1 ® _ ' , 0 0 2 . 2,. 6.80 6.15 5,25 4,85 4.80 lactose 1. 3 10 • , 11 21 . 40 2. 6.95 6.65 6.95 6.55 5.80 dextrin 1.. 4 . 8 8 16 . 17 , 2. 6.85 7.35 6.95 6.80 6.85 sa l i c i n 1- 0 0 . 0 « 3 2. 6 .65 5.90 5.60 5»35 5.35 dulcitol 1 © 7 14 . 20 4 70 . 2. 6.95 6.80 7.15 7.45 7.05 water 1.. 5 12 25 39 . 71 . 2 «_ 6.95 6.80 7.15 7,10 7.05 TABLE 20. Fermentation of Carbohydrates i n the Absence of a Nitrogen Source. Pseudomonas putrefaciens Water (control) age of culture i n days 1 __2_ 4 _ 9 Carbohydrate glycerol ' . 7.40 7.10 7.25 6.70 xylose 7.25 6.65 6.55 5.15 mannitol 7.20 6*80 6„85 5.60 glucose 6.60 5.95 5.65 5.30 galactose 6.60 5.90 5.85 5.05 sucrose 6.65 5.95 6.05 5.65 maltose. 7,35 6.85 6.85 5.95 lactose 7.40 7.20 7.45 7,20 dextrin 7.40 7.1-5 7.40 7.25 sa l i c i n 7.10 6,55 6.45 5.50 dulcitol . 7.40 7.30 7.65 - 7.05 •water 7.40 7.35 7.60 7.45 (figures express final pH of cultures) Figure 29 Figure 30 Pseudomonas putrefaciens Histidine 70 60 -I te3 1 2 4 9 24 days Figure 31 Pseudomonas putrefaciens Arginine Figure 33 Pseudomonas putrefaciens P r o l i n e 1 2 4 9 24 days Figure 34 Pseudomonas putrefaciens Proline - 48 -f o r the f i r s t n i n e days of i n c u b a t i o n , as seen i n f i g u r e 31, 32, and 34, show t h a t the carboh y d r a t e s are fermented i n a s i m i l a r manner f o r the thr e e amino a c i d s , i n d i c a t i n g t h a t the p r o c e s s of f e r m e n t a t i o n i s independent of t h a t of ammonia f o r m a t i o n . These f i g u r e s a l s o show t h a t a c i d p r o d u c t i o n from l a c t o s e o c c u r s a f t e r n i n e days I n c u b a t i o n , s u g g e s t i n g t h a t the.breakdown o f t h i s sugar i s caused by an a d a p t i v e enzyme whereas the f e r m e n t a t i o n o f the more r a p i d l y a t t a c k e d sugars Is- caused by c o n s t i t u t i v e enzymes. Ammonia f o r m a t i o n from a r g i n i n e by Pseudomonas p u t r e f a c i e n s i n c r e a s e s s t e a d i l y u n t i l at l e a s t t w e n t y - f o u r days' I n c u b a t i o n , and as the ammonia content i n c r e a s e s the pH of the c u l t u r e s i n c r e a s e s except f o r s a l i c i n . I n t h i s c u l t u r e , a l t h o u g h the d e c l i n e i n pH i n d i c a t e s b a c t e r i a l a c t i v i t y , no ammonia i s formed as the i n c u b a t i o n p e r i o d proceeds nor i s t h e r e a subsequent i n c r e a s e i n pH. I t i s p o s s i b l e t h a t the f a i l u r e t o form ammonia i n the case o f s a l i c i n i s due t o the e l a b o r a t i o n o f t o x i c p r o d u c t s of f e r m e n t a t i o n p r e v e n t i n g f u r t h e r b a c t e r i a l a c t i v i t y . S a l i c i n , b e i n g a g l u c o s i d e o f glu c o s e and p h e n o l , w i l l , on h y d r o l y s i s to y i e l d g l u c o s e as a carbon s o u r c e , g i v e a h y d r o l y t i c r e s i d u e which has p h e n o l i c p r o p e r t i e s . A c i d i t y a l one c o u l d not be the reason f o r t h i s c e s s a t i o n of a c t i v i t y s i n c e the pH of the c u l t u r e c o n t a i n i n g g l u c o s e reached a lower l e v e l than t h a t of the one c o n t a i n i n g s a l i c i n . The maximum q u a n t i t y of ammonia produced from h i s t i d i n e by t h i s b a c t e r i a l s p e c i e s i s reacnea by n i n e days - 49 -i n c u b a t i o n i n the c u l t u r e s c o n t a i n i n g the n o n - a c i d p r o d u c i n g c a r b o h y d r a t e s . I n the c u l t u r e s c o n t a i n i n g g l y c e r o l , s u c r o s e , m a n n i t o l and g l u c o s e , the ammonia con t e n t i n c r e a s e s up to t w e n t y - f o u r days i n c u b a t i o n w i t h a c o r r e s p o n d i n g i n c r e a s e i n pH, as may be seen i n f i g u r e 32. The c u l t u r e s c o n t a i n i n g m a l t o s e , x y l o s e , s a l i c i n and g a l a c t o s e , on the o t h e r hand, do not i n c r e a s e i n pH or ammonia content a f t e r n i n e days i n c u b a t i o n i n d i c a t i n g t h a t a c t i v i t y has ceased i n these tubes at n i n e days. The r e s u l t s obtained, w i t h p r o l i n e show t h a t when the pH reaches too low a l e v e l , as i n the c u l t u r e s c o n t a i n i n g s a l i c i n , m a n n i t o l , g l u c o s e , x y l o s e , sucrose and m a l t o s e , a c t i v i t y decrease markedly and the b a c t e r i a cannot produce ammonia t o overcome t h e u n f a v o u r a b l e a c i d i t y as t h e y do i n the c u l t u r e s c o n t a i n i n g g a l a c t o s e . The c u l t u r e c o n t a i n i n g s a l i c i n d i d not r e a c h as low a pH as t h a t c o n t a i n i n g g a l a c t o s e , but i t d i d not show an i n c r e a s e i n ammonia s u g g e s t i n g , a g a i n , t h a t t h e r e i s something among the breakdown p r o d u c t s o f s a l i c i n t h a t i s not p r e s e n t I n those of g a l a c t o s e and t h a t i s capable o f p r e v e n t i n g f u r t h e r b a c t e r i a l a c t i v i t y . The q u e s t i o n t h a t a r i s e s from these r e s u l t s i s t h a t , i f the pH c o u l d be c o n t r o l l e d by i n c r e a s i n g the b u f f e r i n g power o f the c u l t u r e s , would ammonia be formed i n the presence o f a c i d - f o r m i n g c a r b o h y d r a t e s ? I n an attempt t o o b t a i n d a t a on t h i s aspect o f the problem, experiments employing f i v e key c a r b o h y d r a t e s i n the presence o f v a r y i n g b u f f e r c o n c e n t r a t i o n s ere u n d e r t a k e n employing P r o t e u s i c h t h y o s m i u s and Pseudomonas w - 50 -p u t r e f a c i e n s . The carbohydrates s e l e c t e d were g l u c o s e from which b o t h s p e c i e s of b a c t e r i a produce a c i d , g l y c e r o l --showing slow a c i d f o r m a t i o n by b o t h s p e c i e s , l a c t o s e -- from which no a c i d I s formed by e i t h e r s p e c i e s , x y l o s e -- from which Pseudomonas p u t r e f a c i e n s produces a c i d but P r o t e u s i c h t h y o s m i u s does n o t , and d e x t r i n -- from which P r o t e u s i c h t h y o s m i u s produces a c i d but Pseudomonas p u t r e f a c i e n s does n o t . A c o n t r o l of d i s t i l l e d water i n p l a c e of the carbohydrate was used to determine the ammonia f o r m a t i o n i n the absence of c a r b o h y d r a t e . I n the f i r s t e x p e r i m e n t , the a c t i o n o f P r o t e u s Ichthyosmius on a r g i n i n e i n the presence o f thr e e b u f f e r c o n c e n t r a t i o n s was s t u d i e d . The f i r s t s e t of c u l t u r e s had a f i n a l b u f f e r c o n c e n t r a t i o n the same as t h a t used i n p r e v i o u s e x p e r i m e n t s , M/20, the second had double the b u f f e r c o n c e n t r a -t i o n , M/lO, and the t h i r d had f o u r t i m e s the b u f f e r c o n c e n t r a -t i o n , M/5. Each s e t o f c u l t u r e s was pr e p a r e d i n s i x groups and i n c u b a t e d f o r one, t h r e e , s i x , t e n , f o u r t e e n , and twenty-one days r e s p e c t i v e l y at 30° C. The f i n a l pH of the c u l t u r e s and the f r e e ammonia co n t e n t are g i v e n i n t a b l e 21 and f i g u r e s 36 to 39 i n c l u s i v e . F o r the sake o f c l a r i t y i n p r e s e n t a t i o n , the r e s u l t s o b t a i n e d when M/lO b u f f e r was em-p l o y e d are not p o r t r a y e d . These r e s u l t s f e l l between those found f o r the l o w e r and h i g h e r b u f f e r c o n c e n t r a t i o n s . The r e s u l t s o f t h i s experiment show t h a t an i n c r e a s e o f f o u r times t h e b u f f e r c o n c e n t r a t i o n c o n t r o l s the pH o f the medium and p r e v e n t s the f e r m e n t a t i o n a c i d s from l o w e r i n g the • - 51 -pH t o a l e v e l u n f a v o u r a b l e to ammonia f o r m a t i o n . T h i s f a c t i s brought out most c l e a r l y i n the c u l t u r e s c o n t a i n i n g g l u c o s e . I n the presence o f M/20 b u f f e r c o n c e n t r a t i o n , the pH of t h i s c u l t u r e drops t o pH 5.3 and does not r i s e t o any c o n s i d e r a b l e e x t e n t w i t h the r e s u l t t h a t the q u a n t i t y o f f r e e ammonia i's s m a l l . When, however, the b u f f e r c o n c e n t r a t i o n i s i n c r e a s e d , the pH o f the gl u c o s e c u l t u r e s drops o n l y t o pH 6.5 and the q u a n t i t y o f ammonia i n c r e a s e s t o the same amount as t h a t i n the c u l t u r e s c o n t a i n i n g d e x t r i n and g l y c e r o l , the o t h e r c a r b o h y d r a t e s from w h i c h t h i s organism produces a c i d . I n the c u l t u r e s c o n t a i n i n g d e x t r i n , i n the presence of M/20 b u f f e r , the pH does not drop below pH 5.5 and because of the g r e a t e r ammonia f o r m a t i o n and the s l o w e r r a t e o f a c i d p r o d u c t i o n d u r i n g the f i r s t t h r e e days o f i n c u b a t i o n , as compared w i t h the g l u c o s e c o n t a i n i n g c u l t u r e s , the b a c t e r i a remain a c t i v e and co n t i n u e t o produce ammonia and, as a r e s u l t , r a i s e the pH of the medium p r a c t i c a l l y to i t s o r i g i n a l h y d r o g e n i o n c o n c e n t r a t i o n - pH 7.6. Whether the pH i s r a i s e d d i r e c t l y b y the i n c r e a s e d ammonia, or i n d i r e c t l y by the b a c t e r i a u s i n g the f e r m e n t a t i o n a c i d s as carbon sources w i t h the ammonia as a n i t r o g e n source f o r c e l l m u l t i p l i c a t i o n , i s a q u e s t i o n w h i c h cannot be answered a t p r e s e n t . Another i n t e r e s t i n g o b s e r v a t i o n from f i g u r e s 38 and 39 d e p i c t i n g the pH changes by P r o t e u s i c h t h y o s m i u s i s t h a t , i n the presence o f a r g i n i n e , l a c t o s e i s broke n down w i t h c o n s i d e r a b l e a c i d p r o d u c t i o n a f t e r t e n days' i n c u b a t i o n . The s p e c i f i c i n f l u e n c e of a r g i n i n e on a c i d p r o d u c t i o n from - 52 -l a c t o s e I s apparent when the a c t i o n of t h i s organism on l a c t o s e i n the presence of o t h e r amino a c i d s i s c o n s i d e r e d , ( v i d e i n f r a ) . I n o r d e r to o b t a i n more d a t a on the i n f l u e n c e of b u f f e r c o n c e n t r a t i o n on ammonia f o r m a t i o n , experiment X I I was extended t o i n c l u d e the a c t i o n of P r o t e u s i c h t h y o s m i u s on a s p a r t i c a c i d , g l u t a m i c a c i d , h i s t i d i n e and p r o l i n e , and of Pseudomonas p u t r e f a c i e n s on the same f i v e amino a c i d s i n the presence o f M/20 and M/5 b u f f e r c o n c e n t r a t i o n s employing each o f the f i v e c a r b o h y d r a t e s used above. The r e s u l t s o f t h i s experiment ( X I I I ) are r e c o r d e d i n t a b l e s 22 t o 30 i n -c l u s i v e and i n t e r p r e t e d i n f i g u r e s 40 to 75 i n c l u s i v e . I n g e n e r a l , the f i n d i n g s o f t h i s experiment show t h a t i n c r e a s i n g the c o n c e n t r a t i o n o f the b u f f e r p r e v e n t s the l o w e r i n g o f the pH o f the c u l t u r e s b y the f e r m e n t a t i o n a c i d s t o a l e v e l t h a t i s u n f a v o u r a b l e and, i n some cases, f a t a l t o f u r t h e r b a c t e r i a l a c t i v i t y . T h i s c o n t r o l o f the pH o f the c u l t u r e medium p e r m i t s the b a c t e r i a t o c o n t i n u e to e l a b o r a t e ammonia and, i n t h e presence o f a v a i l a b l e c a r b o h y d r a t e , t o use the ammonia f o r c e l l m u l t i p l i c a t i o n . A more d e t a i l e d e x a m i n a t i o n of the r e s u l t s b r i n g s f o r t h a number o f i n t e r e s t i n g o b s e r v a t i o n s , f o r some of which no adequate e x p l a n a t i o n can be advanced. I n c r e a s i n g the b u f f e r c o n c e n t r a t i o n markedly de-cr e a s e s the q u a n t i t y o f ammonia formed by Pseudomonas p u t r e -f a c i e n s from a r g i n i n e i n the presence of the n o n - a c i d producin, c a r b o h y d r a t e s -- l a c t o s e and d e x t r i n -- and o f the water The Effect of Increasing the Buffer Concentration i n the Presence of Carbohydrates on Ammonia Formation by Proteus ichthyosmius and Pseudomonas putrefaciens. Experiment XII and. XIII Tables 21 to SO inclusive Figures 36 to 75 inclusive C arb ohydrate s Water (control) - red Glycerol - blue Xylose - green , Glucpse - violet Lactose - orange Dextrin - brown TABLE 21. Proteus ichthyosmius Arginine Buffer Concentration M/20 age of culture in days carbohydrate 1 3 6 10 14 21 glycerol 1. 2. 24.5 7.65 40,75 7.45 40.75 7.30 33.7 " 6.90 34,0 7.15 32.0 7.25 xylose 1. 2. 22,5 7.65. 47.25 7.90 49.5 7.95 53.75 8.00 77.25 8.10 47.0 8.00 glucose 1. 2. 1.5 5.85 11.5 . 5.40 16.5 5.30 20.0 5.40 20.5 5.65 16.5 5,40 lactose X« 2. 32.5 7.75 49.0 7.90 54.75 7.75 59.75 7.65 54.25 7,35 39,75 6.15 dextrin 1. 2. ^29.5 5.90 30.75 5.55 25.5 6.25 30.0 6.70 32.5 7.05 •41.0 7.30 water 1. . 2. 28.5 7.75 50.25 .7.85 51.25 7.90 74.00 8.15 69.75 8.20 60.25 8.20 Buffer Concentration M/5 age of-culture i n days 1 .5 6 10 14 21 Carbohydrate glycerol 1. 31.0 .." . 38.0 36,25 . 30.5 27.5 40.75 2, 7.55 7.55 7.35 7.20 7.15 7.25 xylose 1, 3 X»5 ^4i.75 48,75 54.25 74.25 64.25 2. 7,55 7,65 7175 7.75 7,85 7.75 glucose. X 9 27.0 28.0 !%9.5 30.75 41.5 43.75 2. 6,50 6.75 . 7.05 7.15 7.25 7.25 lactose 1. 37.5 46.25 55.25 61.5 60.0 53.0 2. 7.65 7.65 7.65 7.65 7.50- 6,85 dextrin 1. 19.0 31.25 28.75 27,75 32.0 36.5 2 • 6.70 6.75 7. 05 7.25 7.10 7.30 water 1. 22.75 51.75 59.0 56.5 80.5 . 80,0 2. 7.65 7.70 7.75 7.75 7.75 7,90 1, percent of total nitrogen of amino acid in culture present as free ammonia, 2. pH of culture at time of ammonia determination. TABLE 22. Proteus ichthyosmius Aspartic Acid Buffer Concentration M/20 carbohydrate 1 age of culture i n days 3 6 - 10 14 21 glyceroJL . 1. 58-' 35 3l' ' 25. 23 21 2. . 7.15 7.00 6.90 6,65 6.50 6.35 xylose . 1. 40 37 37, 35, 38 39 2 9 7.25 7.25. , 7.35 7.45 7.55 7.60 glucose 1. 24. 28 24 18. 24 23: •2. 5.90 5.45 5.45 5.40 5.40 5.50 lactose 1. 40. 36 36. 31: 34. • 29-2. 7.20 7.25 7.35 7.25 7.40 . 7.35 dextrin 1. • '9 t 19 16.. ,-' 8 15.. 22 2, .5.90 5.60 5,45 5.55 5.-30 5.50 water •1. 37 44 41. 24 34, 44• 2. 7.25 7.35 7.35 .7.50 7.60 7.60 . Buffer Concentration M/5 age of culture in days carbohydrate 1 3. 6 10 14 21 glycerol 1. 31 ' 38 31 12 14' 26 ' 2. • 7.40- , 7.35 7.35 7.25 7.15 7.20 : xylose. 1. 21 41 : - ,40 22 • 31 ' 39 .* 2. 7.45 7.45 7.50 7,50 7.55 7.55 glucose 1. 16 9 -' 8 8 6 * 8 ' 2, 6.95 6,75 6.70 6.65 6,70 6e75 lactose 1.- 30 • 34 33 24 20 •  21 2. 7.45 7.45 7.45 7.45 7.45 7.40 dextrin IV 5 : 3 2 ' 3 3 " 1 2. 6.95 6.75 6,85 6.80 6 .95 6,95 water '1.'. 31 30 34 ' 33 ' 25 • 32 2. 7.45 7.45 7,45 7.50 7.5.5 7,55 1. percent of total nitrogen of stmino acid i n culture present , as free ammonia. 2. pH of culture at time of ammonia determination. TABLE 25. Proteus ichthyosmius Glutamic Acid Buffer Concentration M/20 age of culture in days carbohydrate 1 4 6 . 10 14 21 glycerol 1.: 3 4 4 •1 0 1 2. 7,25 7.15 7.15 6.90 6.75 6,55 xylose lv 4 11 15 26 25 41 2.- 7.25 ' 7.25 7.30 7.45 7.50 7.60 glucose 1. 2 2 2 1 0 1 2. 5,90 5.40 5,40 5.40 5.40 5.45 lactose 1. 4 10 10 11 9 11 2. 7,50 7.30 7.30 7.25 7.20 7.25 dextrin !*• 'v 2 1 2 1 0 0 2. 6 ..60 5.75 5.80 5.95 5.95 5,85 water 1. '5 15 14 21 27 42. 2. 7.25 7,30 7.55 7.40 7.45 7.65 Buffer Concentration I l/5 age of culture in days carbohydrate 1 4 ' 6 10 14 21 glycerol 1. 2 3 3 1 0 0 : '2*. 7.45 7.40 7.40 7,40 7,35 7.30 xylose 1. 4 J' • 8 14 7 . 26 2. 7.45' 7.45 7.45 7.45 7.55 7,55 glucose 1. 1 1 2 0 0 1 2. 7.05 6,70 . 6.75 6.65 6.75 6.75 lactose 1 9 4 5 6 . 2 1 1 2. 7.45 7.45 7,45 7.45 7,40 7.35 dextrin 1. 1 1 1 1 0 2 2. 7.35 6.85 6,75 6,95 6,95 7.05 water 1. 4 10 11 16 22 37 •2-. 7.45 7.50 7,45 7.50 7.55 7,60 1. percent of total nitrogen of amino acid i n culture present as free ammonia. 2. pH of c ulture at time of ammonia detenuination. TABLE 24. Proteus ichthyosmius Histidine Buffer Concentration M/20 . carbohydrate 1 4 6 10 14 21 glycerol 1. 2.0' 12.0 21.6 17.0 20.0 14.6 2. 7.30 7.10 7.05 6.75 6.55 6.25 xylose 1. 2.3 10.3 29.3 29.6 41.0 37.0 2. 7.35 7.35 7.35 7.45 7.60 7.55 glucose X 9 .6 .3 1.3 .6 1.0 6.6 2. 6.15 5.55 5.45 5.40 5.55 5.45 lactose 1. 2.3 7.6 19.6 26.6 32.0 25.3 2. 7.30 7.35 7-35 7.30 7.35 - 7.35 dextrin 1. . "< : .0.0^ 1.0 3.6 1.3 2.0 4.6 2. 6.35 5.60 6.05 5.65 5.60 5.85 water 1. 2.6 26.6 32,0 39.0 42.0 43.6 2. 7.30 7.40 7.35 7.45 • 7.60 7.65 Buffer Concentration M/5 age of culture i n davs carbohydrate 1 4. 6 10 "14 21 glycerol I. 2.0 •" . 21.0 26.0 23.6 20.0 16.3 2. 7.45 7.40 .7.40 7.,25 7.15 7.05 xylose 1. 2.0 '21 a'3. 31.6 35.0 37.0 38.3 2. 7.45 7.45 . 7,50 7.50 7.55 7.55 glucose 1, .3 3.3 ' ' ^3.6 10.6 8.6 9.0 2. 7.05 6.75 6.75 6.75 6.85 6.80 lactose 1. 3 .6 18.3 14.3 20.3, 28.0 22.0 2. 7.45 7.45 7.45 7.45 7.45 7.. 35 dextrin 1. .3 7.3 12.0 12.0 17,6 18.6 2. 6.95 6.80 6.90 6.80 6.95 7.05 water 1. 3.6 25.6 27.6 30.0 38.3 37.6 2. 7.45 7.50 7.50 7,45 7.55 7.55 1. 2. percent of total nitrogen of amino acid in culture present as free ammonia. pH of culture at" time of ammonia determination. TABLE 25. Proteus ichthyosmius Proline Buffer Concentration M/20 carbohydrate 1 age • • 4 -of culture in 6 10 days 14 21 glycerol , 1. 2 3 " 2 1 0 1 2. 7.55 7.35 7.25 7.10 6.90 6.35 xylose 1.' 4 ' 7 8 : 17 31 47 2. 7.60 7.55 7.60 7.60 7.70 7.75 glucose 1. ' 1 2 1 1 1 ' 2. 6.70 5.85 6.00 •5.95 6.05 5.60 lactose X • 3 7 8 9 4 2 2. 7.60 7.45 7 o 55 7.45 7.45 7.25 dextrin 1. .. .. T 2 1 i 1 0 1 2. 6.95 6.45 6.25 6.45 6.45 6.30 •water 1. 5 11 14 26 34 46 2. 7.65 7.55 7*65 7.65 7,70 7.75 Buffer Concentrati on M/5 age of culture i n days -. carbohydrate 1 ' 4 6 10 14 21 glycerol 1. . 1 3 2 2 0 1 2. 7.55 . 7.45 7.45 7.45 7.35 7.20 xylose 1. 3 (-. 5 ' A 8 v- • 16" 32 . 44 r 2. 7.55 7.55 7.55 7.55 7.65 7.*60 glucose 1. 1 1 2 0 0 1 ' 2. 7.25 7.00 6.95 6.75 6.85 6.85 lactose 1. 3 . 5 8 3 1 1 2. 7.55 7.50 7.50 7.45 7,45 7v25 dextrin . I. . 1. 1 2 1 1 2 2. 7.35 7.15 7.05 6.96 7.15 7,05 water 1. 4 9 14 16 40 43 2. 7.55 7.55 7.55 7.50 7.55 7."60 1. 2. percent of total nitrogen of amino acid in culture present fas. free ammonia pH of culture at time of ammonia determination. TABLE 26. Pseudomonas putrefaciens Arginine B.uffer Concentration M/20 age,of culture in days Carbohydrate 1 3 6 11 15 • 21 glycerol 1. 0.25 1.5 2,75 3.5 X o 2 5 8.5 2. 7.50 6.95 6.65 6.95 6,95 7.00 xylose 1. 0.25 0.25 0,0 2.25 1.75 3.25 2. 7.40 5.35 5,85 6.60 6.65 6.60 .glucose 1. 1.0 1.5 2»25 X e 25 4.5 8.25 2. . 6 . 0 5 5.10 5.10 6.55 6.70 6,70 lactose 1. 1.0 2.0 6.5 18.75 30.5 30,75 2 « -.'7» 5 0.v 7.45 7.45 7.15 6.75 5.90 dextrin X © 25 2.0 : 4.5 7.75 32.5 34.5 2, 7.45 7.50 7.25 7.30 7.60 7.40 water i . 1.0 5.5 12.5 19.5 30.5 32 e 75 2. 7.55 7.60 7.65 7.65 -7.85 7.90. Buffer Concentra t i on M/5 age of culture i n days Carbohydrate 1 6 11 15 21 glycerol 1, 0.0 0.5 1.0 0.75 2 © 5 3.25 2. 7.50 ( 7.25 7.15 7,05 7.15 xylose 1. 0.25 o .o ; 1.00 1.0 2.0 2. •7.50 7.00 6.90 7.05 7.15 7.25 glucose 1. '0.0 1.25 2.0 4.-75 4,5 4.5 2. 7,15 6,80 7,00 7.10 7**15 . 7.15 lactose I1. 0,5 1.5 1.5 4.25 9.5 14.75 2. 7.50 7.50 7.40 7.45 7.30 7.15 dextrin 1. 0.5 1.0 1.25 3.75 6.25 6.25 2. 7.45 7.50 7'. 45 7.45 7.50 7.50 water 1. 0.25 . 3 ,0 3.25 4,0 4.25 11.5 2. 7.50 7.55 7.55 7.55 7.60 7.60 1. percent of total nitrogen of amino acid in culture present as free ammonia. 2. pH of culture at time of ammonia determination TABLE 27. Pseudomonas putrefaciens Aspartic Acid Buffer Concentration M/20 age of culture i n days carbohydrate glycerol xylose glucose lactose dextrin .water 6 10 14 21 1. 2. 1. 2. 1. 2. 1. 2. 1. 2. 1. 2. 37 7.20 26 7.20 29 6.45 45 7.25 33 7.25 36 7.35 33 6.90 30 6.00 26 5.35 35 7.20 33 7.20 38 • 7.30 31 6.55 26 5.45 24 5.30 31 7.15 18 7.30 33 7.50 35 6,45 34 5.10 34 5.25 33 7.15 14 7.30 38 7.55 38 6.45 38 5.05 38 4.95 38 6.85 15 7.20 35 7.55 28 6.25 23. 5.55 24 5.20 27 6.80 10 7.20 29 7,65 Buffer Concentration M/5 carbohydrate glycerol Xylose glucose lactose dextrin water 1. 2. .1. 2. 1. 2. • 1. 2. 1. 2. 1. 2. 36 7.50 18 7.50 21 7.05 36 7.50 24 7.45 1 7.50 age of culture in days 6 10 14 21 32 7.35 22 7.05 15 6.70 38 7.45 30 7.45 28 7.45 25 7.15 14 Oil ' 6.75 18 7.45 12 7.45 24 7.50 7.05 6.70 18 6.85 29 7.35 10 • 7.40 38 7.55 17 6.90 17 6.70 23 6.80 29 7.25 9 7.40 35 7.55 14 7.05 14 6.85 15 6.95 20 . 7.25 6 7.35 40 7.55 1. .percent of total nitrogen of amino acid in culture present as free ammonia 2. pH of culture at time of ammonia determination TABLE 28. Pseudomonas putrefaciens ' Glutamic Acid Buffer Concentration M/20 age of culture i n days Carbohydrate 1 3 6 - io: . 14 21 glycerol 1. 1 3 3 6 9 13 2. 7 © 2 5 7.00 6,55 6.30 6.25 6.15 xylose 1, i 2 1 2 0 3 2. 7*25 5.55 5,25 3«2 5 5.15 5.05 glucose 1, 1 4 4 4 6 24 2. 6.50 5.10 5 © 2 5 5.25 5.15 6.10 lactose 1. 3 6 9 15 27 37 2 o 7.25 7.20 7.20 6.90 6.80 6.55 dextrin 1. 2 5 3 11 19 24 2. 7.25 7.20 7.10 7.15 7.10 7.15 mter 1. 4 11 17 26 41 62 2. 7,30 7.30 7.40 7.55 7.50 7,85 Buffer Concentration M/5 age of culture i n days carbohydrate 1' 3 6 10 14 21 glycerol 1. 1 3 0 1 2 0 2 e 7,50 7,45 7.25 7.00 6.95 6,95 xylose 1. 2 2 :• 1 4 7 20 2. 7,4-5 6.95 6j5 6.75 6.70 6B85 glucose 1. 1 3 2 14 19 27 2. 6.70 6.70 6.75 6.85 6.90 lactose 1. 3 8 5 14 18 39 2 9 7.50 7.40 7.45 7 © 3 5 7.20 7.15 dextrin 1. 2 5 5 10 18 32 2. 7.45 7,45 7.45 7.40 7.35 7,45 water 1. 4 11 12 51 37 62 2. 7,50 7.45 7.50 7.55 7.50 7.65 1. percent of total nitrogen of amino acid i n culture present as free ammonia. 2. pH of culture a t time of ammonia determination. TABLE 29. Pseudomonas putrefaciens Histidine Buffer Concentration H/20 carbohydrate 1 3 age of culture in 6 ' 10 days 14 20 glycerol. 6.6 25.6 29.6 36.0 41.3' 46.6 2. 7.30 6.95 6,40 6.35 .6.30 6.70 xylose 1. 6.6. 6.6 5.6 7.6 8.3 8.0 2. 7.25 5. 30 5.40 5.55 5.25 5.25 glucose 1. 3.6 4.6 3.3 7.3 4.6 5.6 2. 5.95 ' 5.15 5.15 5.50 5.20 5.35 lactose 1. 8.3 29.0 42.3 57.6 65.0 65.3 2. 7.35 7.30 7.25 7.20 6.95 . 6.65 dextrin 1* - .- % 7.3 36.6 49.3 54.3 57.3 58.3. 2. .7.35 7.35 7.15 7.25 7.20 7.05 water 1. 10.0 47.3 61.3 6.8.6 • 69.6 62.6 2. 7.40 7.45 7.65 7.80 7.90 , 8.10 Buffer Concentration M/5 l& of culture in days carb ohydrate , 1 3 6 . 10 14 ' 21 glycerol'. '1* , 6.0 26.6 40.6 43.6 •" 46.3' 41.0 2.- . 7.50. 7.35 7,05 6.90 6.85 • 7.00 xylose 1. 7.0 6.3 19.6, Si »3 39.3 40.6 7,45 6.65 ""•6.-70 6.-80 6.85 6.95 glucose i . 1,6 5,0 11.0 25.6 32.0. 40.6 2, 6,95 6.65 6.75 6.-85 6 .85 7.05 lactose X« 7.5 23.0 36.6 56.6 54,6 50.3 2. 7.50 7.45 7.45 7.45 7.25 7.20 dextrin 1. 6.6 31.6 39.6 53.0 50.6 44.0 2. 7,50 7.50 7.45 7.40 7,30 7,35 • water 1. 9.6 50.0 48.0 65.0 67,3 54.3 2 6 7.50 7.50 7.55 7,60 7.65 7.75 1. percent of total nitrogen of amino acid in culture present as free- ammonia. 2. pH of culture at time of ammonia determination. TABLE 30. Pseudomonas putrefacisns Buffer Concentration lf/20 carbohydrate age 1 ' of culture i n 3 6 '• days 10 14 21 glycerol 1. 2 1 •1 1 1 8 2. 7*55 7.10 6.50 6.35 6.35 6,95 xylose 1. 1 1 0 1 1 2 2. 7.25 6.30 . 5.45 5.05 5.40 .5.20 glucose 1. 0 1 0 2 2 0 , 2. 6., 80 5.90 5.65 5.05 5.85 6,25 lactose 1. 6 11 14 35 30 37 2. 7.55 7.45 7.30 7.05- 6.80 • 6.55 dextrin 1. 9 11 34 25 35 2. 7.55 7.40 7.30 7.30 7.05 7.15 water '. 1. 6 15 23 48 53 54 2- .7.65 7.60 7.65 7.65 7.70 7.95 Buffer Conc< antra t i on H/5 age of culture i n days carbohydrate . -*1' 3 6 10 14 21 glycerol 1• 2 2 0 1 0 1 2. 7.55 7.45 7.25 7.05. 7.00 7.00 xylos e 1. 1 iv • 1 / 1 o 7 2. ; 7.50 7.15 \ 7^.00 6.80 6.65 6.95 glucose 1. 0 .i 0 2 4 9 2. 7.35 7.05 6.90 6.80 6.85 7.05 lactose 1.' 4 9 19 ' 20 21 29 2. 7.55 7.50 7.45 7.30 7.20 7.05 dextrin 1. 3 10 12 24 20 18 7.55 7.50 7.45 7.45 7.55 7.40 water '"• 1. 10 20 32 35 43 2. 7.55 7.55 7.55 7.55 7.55 7.65 1. percent of total nitrogen of amino acid in culture present as free ammonia. 2. pH of culture at time of ammonia determination Figure 37 Proteus ichthyosmius Arginine M/5 Buffer Concentration 10 14 21 days Hgure 38 Proteus ichthyosmius M/20 Buffer Concentration Arginine 8.0 J 7.0 4 6.0 5.0 Figure 39 l/5 Buffer Concentration 8.0 A 7.0 6.0 days 10 14 Figure 40 Pseudomonas putrafaoiens M/20 B u f f e r Concentration Arginine 30 20 10 11 15 21 20 10 Figure 41 B u f f e r Concentration days Figure 42 Pseudomonas putrafaoiens M/20 Buffer Concentration Arginin© Figure 44 Probeu;- ichthyosmius Aspartic A c i d M/20 B u f f e r Concentration i — 10 days 14 21 Figure 4-5 Proteus ichthyosmius M/5 B u f f e r Concentration Asp a r t i c Acid Figure 46 Proteus ichthyosmius Aspartic Acid M/20 Buffer Concentration 8.0 7.0 li i X 6,0 5.0 1 — i — • 10 days — r — 14 21 8,0 4 Figure 47 i/5 Buffer Concentration 7.0 6.0 — i — 10 days 14 21 Figure 49 Pseudomonas putrefaciens M/5 Buffer Concentration Aspartic Acid Figure 50 Pseudomonas putrefaciens Aspartic Acid M/20 Buffer Concentration Figure 52 Proteus ichthyosmius Glutamic Acid M/20 Buffer Concentration Figure 53 Proteus ichthyosmius Glutamic Acid M/5 Buffer Concentration days Figure 56 Pseudomonas putrefaciens M/20 Buffer Concentration. Glutamic Acid Figure 57 Pseudomonas putrefaciens M/5 B u f f e r Concentration Glutamic Acid Figure 58 Pseudomonas putrafaciens Glutamic Acid M/20 B uf f e r G ore e n t r at ion. Figure 60 Proteus ichthyosmius M/20 Buffer Concentration. Histidine Proteus ichthyosmius Figure 61 M/5 Concentration Histidine Figure 6 2 Proteus ichthyosmius M/20 Buffer Concentration Histidine 8.0 -i Figure 63 M/5 Buffer Concentration 8.0 J days Figure 64 Pseudomonas putrefaciens M/ao Buffer G one out rati on Histidine 7 0 J i 1 r — 1 1 3 6 10 14 21 days Figure 65 Pseudomonas putrefaciens M/5 Buffer Concentration Histidine Figure 66 ( Pseudomonas putrefaciens Histidine M/20 Buffer Concentration Figure 68 Proteus ichthyosmius M/20 Buffer Concentration Proline days Figure 69 Proteus ichthyosmius M/5 Buffer Concentration Proline Figure 70 Proteus ichthyosmius 1 M/20 Buffer Concentration. Proline Figure 72 Pseudomonas putrefaciens M/20 Buffer Concentration Proline Figure 73 Pseudomonas putrefaciens M/5 Buffer Concentration Proline - 53 -c o n t r o l , f i g u r e s 40 and 41. There appears to be no adequate e x p l a n a t i o n o f t h i s f i n d i n g . The c u l t u r e s of t h i s s p e c i e s c o n t a i n i n g a r g i n i n e .show good growth i n the presence o f b o t h the a c i d - p r o d u c i n g and the n o n - a c i d - p r o d u c i n g c a r b o h y d r a t e s . T h i s suggests t h a t t h i s m i c r o o r g a n i s m i s capable o f u s i n g the n o n - a c i d - p r o d u c i n g c a r b o h y d r a t e s as carbon sources w i t h o u t r e s u l t i n g i n a c i d s as by - p r o d u c t s of the f e r m e n t a t i o n . I n the case o f l a c t o s e , a c i d p r o d u c t i o n i s i n d i c a t e d a f t e r e l e v e n days i n c u b a t i o n , f i g u r e s 42 and 43. I n the presence o f M/20 b u f f e r c o n c e n t r a t i o n ( f i g u r e 4 2 ) , the pH o f the c u l t u r e s c o n t a i n i n g g l u c o s e arid x y l o s e r i s e s f r om about pH 5.2 t o pH 6,6. a f t e r t h r e e days i n c u b a t i o n w i t h o u t showing an i n c r e a s e i n the f r e e ammonia content i n d i c a t i n g t h a t the b a c t e r i a are p o s s i b l y u s i n g the ammonia as r a p i d l y as i t i s formed'' as a n i t r o g e n source and the f e r m e n t a t i o n a c i d s as carbon sources t h e r e b y removing these a c i d s from the r e a c t i n g medium and p e r m i t t i n g the pH t o r i s e . An a l t e r n a t i v e ex-p l a n a t i o n i s t h a t the b a c t e r i a have some mechanism f o r the p r o d u c t i o n o f a l k a l i n e compounds''other than ammonia t o n e u t r a l i z e p a r t o f the a c i d i t y . I n the c u l t u r e s o f P r o t e u s Ichthyosmius i n a s p a r t i c a c i d , i n c r e a s e i n the b u f f e r c o n c e n t r a t i o n decreases the f r e e ammonia In those c u l t u r e s c o n t a i n i n g g l u c o s e and d e x t r i n but has p r a c t i c a l l y no e f f e c t on those c o n t a i n i n g x y l o s e , l a c t o s e and g l y c e r o l . The decrease I n the a c i d ^ p r o d u c i n g carbohydrate c u l t u r e s i s p r o b a b l y due t o the u t i l i z a t i o n o f the ammonia f o r c e l l m u l t i p l i c a t i o n . L a c t o s e i s not b r o k e n down w i t h a c i d - 54 -p r o d u c t i o n by P r o t e u s Ichthyosmius i n the presence o f a s p a r t i c a c i d ( v i d e supra a r g i n i n e ) w h i l e g l y c e r o l i s seen t o e x h i b i t slow a c i d p r o d u c t i o n . F i g u r e s 48 t o 51 i n c l u s i v e g r a p h i n g the r e s u l t s of the a c t i o n of Pseudomonas p u t r e f a c i e n s on a s p a r t i c a c i d r e v e a l two o u t s t a n d i n g p o i n t s . The f i r s t i s t h a t i n c r e a s e i n the b u f f e r c o n c e n t r a t i o n i n c r e a s e s the range i n ammonia con-t e n t o f the c u l t u r e s i n the presence of the two groups of c a r b o h y d r a t e s , a c i d - p r o d u c i n g and n o n - a c i d - p r o d u c i n g . The low c o n c e n t r a t i o n o f b u f f e r , M/20, I s i n s u f f i c i e n t t o m a i n t a i n t h e pH o f the c u l t u r e s c o n t a i n i n g x y l o s e and gl u c o s e w i t h the r e s u l t t h a t the a c i d i t y i s too g r e a t t o p e r m i t b a c t e r i a l a c t i v i t y and the ammonia i s not u t i l i z e d f o r c e l l r e p r o d u c t i o n . The i n c r e a s e d b u f f e r content m a i n t a i n s the pH l e v e l and the amount o f ammonia de c r e a s e s i n those c u l t u r e s w i t h f e r m e n t a b l e c a r b o h y d r a t e s . The second o b s e r v a t i o n i s t h a t the q u a n t i t y o f ammonia i n the c u l t u r e s w i t h d e x t r i n i n the presence o f b o t h b u f f e r c o n c e n t r a t i o n s decreases w i t h o u t any change i n pH. • T h i s s u g g e s t s , a g a i n , t h a t Pseudomonas p u t r e f a c i e n s i s capable of u t i l i z i n g d e x t r i n as a carbon source w i t h o u t f o r m i n g a c i d s . Ammonia f o r m a t i o n from g l u t a m i c a c i d by P r o t e u s i c h t h y o s m i u s t a k e s p l a c e s l o w l y over the t h r e e weeks o f i n c u b a t i o n . C onsequently, as the r e s u l t s showing o n l y s m a l l q u a n t i t i e s o f f r e e ammonia i n the c u l t u r e s c o n t a i n i n g t h e a c i d - p r o d u c i n g c a r b o h y d r a t e s i n d i c a t e , the b a c t e r i a are p r o b a b l y u s i n g the ammonia as r a p i d l y as i t I s formed as a n i t r o g e n source f o r c e l l m u l t i p l i c a t i o n . I n the c u l t u r e s c o n t a i n i n g a c i d - f o r m i n g c a r b o h y d r a t e s , I n c r e a s e i n the b u f f e r c o n c e n t r a t i o n s l i g h t l y d ecreases the ammonia content p r o b a b l y because the more f a v o u r a b l e hydrogen-ion c o n c e n t r a t i o n of the h i g h e r D u f f e r p e r m i t s more a c t i v e b a c t e r i a l r e p r o d u c t i o n * The r a t e o f ammonia f o r m a t i o n from g l u t a m i c a c i d by Pseudomonas p u t r e f a c i e n s f o r the f i r s t t e n days o f i n -c u b a t i o n i s about the same as t h a t f o r Prot e u s i c h t h y o s m i u s , b u t i n c r e a s e s a f t e r the t e n day i n t e r v a l t o such an e x t e n t t h a t ammonia f o r m a t i o n i s more r a p i d t h a n i t s u t i l i z a t i o n as a n i t r o g e n source by the b a c t e r i a and as a r e s u l t f r e e ammonia accumulates i n the c u l t u r e s . The o t h e r p o i n t o f note from f i g u r e s 56 and 58 i s the sudden i n c r e a s e i n a c t i v i t y i n t h e c u l t u r e c o n t a i n i n g g l u c o s e i n the presence o f the low b u f f e r , c o n c e n t r a t i o n a f t e r f o u r t e e n days i n c u b a t i o n as shown by the i n c r e a s e i n ammonia content and i n pH. I n s t u d i e s on the a c t i v i t y o f P r o t e u s i c h t h y o s m i u s on h i s t i d i n e , the o n l y e f f e c t o f the i n c r e a s e b u f f e r con-c e n t r a t i o n i n a d d i t i o n t o m a i n t a i n i n g the pH o f the c u l t u r e s i s t o I n c r e a s e the q u a n t i t y of ammonia i n the c u l t u r e s con-t a i n i n g d e x t r i n and g l u c o s e . T h i s i n c r e a s e I s , i n a l l p r o b a b i l i t y , the r e s u l t o f c o n t r o l l i n g the a c i d i t y t h e r e b y p r e v e n t i n g the decrease i n b a c t e r i a l a c t i v i t y t h a t t a k e s p l a c e when the pH drops too low. S i m i l a r r e s u l t s were o b t a i n e d f o r Pseudomonas p u t r e f a c i e n s i n h i s t i d i n e except t h a t x y l o s e and not d e x t r i n i s the c a r b o h y d r a t e w h i c h w i t h g l u c o s e shows i n c r e a s e d - 56 -ammonia content i n t h e i r r e s p e c t i v e c u l t u r e s when the b u f f e r c o n c e n t r a t i o n i s i n c r e a s e d f o u r f o l d . I t i s t o be r e c a l l e d t h a t a c i d p r o d u c t i o n from x y l o s e and n o n - a c i d p r o d u c t i o n from d e x t r i n d i s t i n g u i s h Pseudomonas p u t r e f a c i e n s from P r o t e u s i c h t h y o s m i u s . The c u l t u r e s o f P r o t e u s i c h t h y o s m i u s c o n t a i n i n g p r o l i n e show p r a c t i c a l l y no response i n the form o f i n c r e a s e d o r d e c r e a s e d ammonia f o r m a t i o n to the i n c r e a s e I n b u f f e r c o n c e n t r a t i o n . The pH of the M/20 b u f f e r c u l t u r e s c o n t a i n i n g the o t h e r f o u r amino a c i d s , s u g g e s t i n g t h a t the b a c t e r i a l a c t i v i t y does not decrease i n t h e case o f p r o l i n e t o the e x t e n t t h a t i t d i d w i t h the o t h e r amino a c i d s and t h a t c e l l m u l t i -p l i c a t i o n t a k e s p l a c e e q u a l l y w e l l I n the presence o f b o t h b u f f e r c o n c e n t r a t i o n s . i , , ( A s i m i l a r p i c t u r e was o b t a i n e d f o r the a c t i o n o f Pseudomonas p u t r e f a c i e n s on p r o l i n e e x c e p t t h a t the Increase i n b u f f e r c o n c e n t r a t i o n caused a marked decrease i n the r a t e o f ammonia a c c u m u l a t i o n a f t e r s i x days i n c u b a t i o n . The Breakdown o f A r g i n i n e by P r o t e u s i c h t h y o s m i u s and Pseudomonas p u t r e f a c i e n s . The d e c o m p o s i t i o n o f a r g i n i n e w i t h the c o n v e r s i o n o f s e v e n t y to e i g h t y p e r c e n t o f i t s t o t a l n i t r o g e n i n t o ammonia by P r o t e u s i c h t h y o s m i u s and about t h i r t y p e r c e n t by Pseudomonas p u t r e f a c i e n s , suggested t h a t the pathway o f the breakdown b y i n v e s t i g a t e d . I n o r d e r t o c a r r y out t h i s sugges-t i o n , the above two s p e c i e s o f b a c t e r i a were I n o c u l a t e d i n d i v i d u a l l y i n t o b u f f e r c u l t u r e s o f a r g i n i n e , o r n i t h i n e TABLE 31. Ammonia Formation from Arginine and i t s Decomposition Products. Proteus iohthysomius Compound cc. B/100.H2S04 % T. LT. into Arginine Ornithine Delta-Amino Valeric Acid Urea 12.85 13.95 7.0 7.0 0.5 0.55 1.0 1.0 64.25 69.75 70.0 70.0 10.0 11.0 10.0 10.0 Arginine Ornithine Delta-Amino Valeric Acid Urea Pseudomonas Putrefaciens 9.0 9.4 7.05 6*9 083 0.35 0.65 0.65 45,0 47.0 70.5 69.0 6.0 7.0 :6.5 6.5 ( p r e p a r e d as o u t l i n e d by Hunter ( 3 7 ) ) , delta-amino n - v a l e r i c a c i d and u r e a r e s p e c t i v e l y i n d u p l i c a t e and I n -cubated a t 30° G. f o r twenty-one days. The r e s u l t of t h i s experiment (XIV) are r e c o r d e d I n t a b l e 31. I t was f o u n d , I n the case o f P r o t e u s i c h t h y o s m i u s , t h a t the ammonia formed from a r g i n i n e was g r e a t e r than the t o t a l o f t h a t formed from o r n i t h i n e and u r e a , and t h a t t h a t formed from o r n i t h i n e was seven times g r e a t e r t h a n t h a t from d e l t a - a m i n o v a l e r i c a c i d . On the o t h e r hand, the ammonia formed by Pseudomonas p u t r e f a c i e n s from a r g i n i n e was o n l y s l i g h t l y more than t h a t from o r n i t h i n e and u r e a w h i l e t h a t formed from o r n i t h i n e was more than seven times g r e a t e r than t h a t from d e l t a - a m i n o v a l e r i c a c i d , '^ he r e s u l t s a l s o show c l e a r l y t h a t b o t h s p e c i e s o f b a c t e r i a employed have e q u a l a b i l i t y t o deaminate b o t h the a l p h a and the d e l t a animo groups Of the o r n i t h i n e used i n t h i s experiment, w h i l e n e i t h e s p e c i e s ' can form s i g n i f i c a n t q u a n t i t i e s o f ammonia from d e l t a - a m i n o v a l e r i c a c i d and u r e a . A d i s c u s s i o n o f the s i g n i f i c a n c e o f these r e s u l t s i n r e l a t i o n to the p r o b a b l e course o f breakdown o f a r g i n i n e w i l l be g i v e n l a t e r i n the t h e s i s . Ammonia F o r m a t i o n from Non-Amino A c i d N i t r o g e n o u s Compounds D u r i n g the course o f t h i s s t u d y , the q u e s t i o n arose as to whether amino o r imino groups o f n i t r o g e n o u s compounds o t h e r than'amino a c i d s c o u l d be c o n v e r t e d i n t o amminia by the s u r f a c e t a i n t p r o d u c i n g b a c t e r i a . I n o r d e r t o o b t a i n some d a t a on t h i s a s p e c t o f the problem, an experiment (XV) TABLE 32. Ammonia Formation from Amino-Group Containin, Compounds other than Amino Acids. Compound Prot. ichthyosmius Pseudo. putrefaciens 5 clays 2_2__days^  5 days 22 days Asparagin 6.80 4,95 6.80 5.45 glutamine (control 2.3 4,0 - not 4.35 subtracted) 4,1 5 ® 9 adenine 4.05 4,70 1.15 3 e 5 5 guanine 3.90 8.20 4.5 2.80 uracil „35 .8. . © 25 .75 uric acid .65 1.25 a 55 .85 nicotinic acid ,4 ,95 2.6 3.6 betaine ..45 ,65 .45 .75 urea .3 .8 .25 .6 Results expressed as cubic centimeters 83 Jl/lOO sulphuric acid equivalent to the ammonia formed in the culture from one cubic centimeter of M/20 solution of the nitrogenous compound. Application of the formula at the beginning of the Experimental section w i l l convert results to percent ammonia formed of the total nitrogen, (see discussion) - 58 - • employing the a c t i o n o f P r o t e u s i c h t h y o s m i u s and Pseudomonas p u t r e f a c i e n s on the f o l l o w i n g compounds was c a r r i e d out: a d e n i n e , a s p a r a g i n , b e t a i n e , g l u t a m i n e , guanine, n i c o t i n i c a c i d , u r a c i l , u r e a and u r i c a c i d . The experiment was s e t up as o u t l i n e d a t t h e b e g i n n i n g of t h i s s e c t i o n o f the t h e s i s w i t h the above n i t r o g e n o u s compounds r e p l a c i n g the amino a c i d s . C o n t r o l s c o n t a i n i n g t h e n i t r o g e n o u s compounds w i t h o u t i n o c u l a t i o n w i t h one o f the b a c t e r i a l s p e c i e s were employed t o determine the breakdown, i f any, o f the compounds by the a l k a l i n e a e r a t i o n o f the Van S l y k e p r o c e d u r e . The c u l t u r e s were se t up i n d u p l i c a t e , one §et b e i n g removed a f t e r i n c u -b a t i o n a t 30° C. f o r f i v e and twenty-two days respectively® The r e s u l t s a r e g i v e n i n t a b l e 32. The f i r s t p o i n t o f i n t e r e s t from these r e s u l t s i s t h a t g l u t a m i n e i s the o n l y one o f the compounds t e s t e d i n the c o n t r o l s e r i e s t h a t shows a s i g n i f i c a n t l i b e r a t i o n o f ammonia d u r i n g the a l k a l i n e a e r a t i o n . The q u a n t i t y l i b e r a t e d i s e q u i v a l e n t t o about f i f t y p e r c e n t of one n i t r o g e n . The n i t r o g e n group a f f e c t e d i s p r o b a b l y the amide group which decomposes r e a d i l y under s t r o n g l y a l k a l i n e c o n d i t i o n s . Because o f t h i s d e c o m p o s i t i o n , i t i s d i f f i c u l t t o determine i f the d e c o m p o s i t i o n o f the amide group i n the g l u t a m i n e c u l t u r e s c o n t a i n i n g the b a c t e r i a i s caused by the b a c t e r i a p r i o r t o a e r a t i o n o r t a k e s p l a c e c h e m i c a l l y d u r i n g the a c t u a l ammonia d e t e r m i n a t i o n . The amide group of aspara'gin d i d not decompose s i g n i f i c a n t l y d u r i n g the a l k a l i n e a e r a t i o n . T h i s s t r i k i n g - 59 -difference between the amides of aspartic acid and glutamic acid respectively w i l l be discussed more f u l l y later. The quantity of ammonia formed from asparagin by Proteus ichthosmius and Pseudomonas putrefaciens is equ ivalenl to between f i f t y and seventy percent of the t o t a l nitrogen. This finding shows c l e a r l y that these species are capable of s p l i t t i n g o f f both the amino and the amide groups of 1-asparagin. The results obtained for glutamine, on the other hand, do not necessarily indicate that both groups are attacked by these b a c t e r i a l species© The d i f f i c u l t i e s i n -herent i n the method fo r the determination of ammonia from glutamine by the Van Slyke procedure to which reference has previously been made, renders impossible a clear-cut inter-pretation of the figures obtained for the action of these micro organisms on this compound. No d e f i n i t e conclusion can be reached as to t h e i r action on the amide group of glutamine. There i s , however, some ammonia formed i n excess of the control but whether i t comes from the amino or the amide group cannot be determined. The object of studying the breakdown of adenine, guanine, u r i c a c i d and u r a c i l was to determine of Proteus ichthyosmius and Pseudomonas putrefaciens had the a b i l i t y to open the purine and pyrimidine rings with subsequent deamina-t i o n and formation of ammonia. U r a c i l (2, 6, dioxy pyrimidine) contains two imino groups. Uric acid (2, 6, 8, trlo x y purine) has four nitrogen groups, two i n the iminazol rin g and two i n the pyrimidine r i n g . Adenine (6 amino purine) has a free amino group i n additlon to the four - 60 -nitrogens in the purine ring, while guanine (2 amino, 6 oxy purine) has, i n addition to a free amino group, an oxy group. Whereas the amino group i n adenine i s i n position 6, that i n quanine i s i n position 2. The findings of the experiments employing these four compounds show c l e a r l y that the pyrimidihe r i n g of u r a c i l and the purine ring of uric acid are both opened to a slight extent with the formation of small quantities of ammonia and that the free amino group of both adenine and guanine i s deaminated. When the results for the twenty-two day period of incubation are considered, i t i.s seen that i n the case of Proteus ichthyosmius rupture of one of the rings i n guanine with the resultant formation of ammonia has also occurred. It would appear most l i k e l y that the formation of ammonia has resulted from cleavage of the iminazol nucleus and has arisen from either or both of the imino groups present i n t h i s r i n g . The r e s u l t s obtained when n i c o t i n i c acid was employed are of in t e r e s t . The findings show c l e a r l y that the pyridine r i n g is opened by each of the organisms studied, Pseudomonas putrefaciens possessing the more marked a b i l i t y to elaborate ammonia from this ring structure* The opening of f i v e d i s t i n c e nitrogen containing ring structures with subsequent deamination r e s u l t i n g i n the formation of ammonia has thus been demonstrated for these species of micro-organisms. These b a c t e r i a l species have previously been shown to attack the iminazol P p y r o l l i d l n e , pyrimidine and purine rings© The two other compounds studied, betaine (from - 61 -g l y c i n e ) and u r e a gave s m a l l quant i t l e d o f ammonia under the i n f l u e n c e o f t h e two b a c t e r i a l s p e c i e s employed. These r e s u l t s , however, were not s u f f i c i e n t l y s i g n i f i c a n t t o cause s p e c u l a t i o n . The f a i l u r e t o h y d r o l y s e u r e a to any extent by two organisms t h a t have been shown t o decompose a r g i n i n e r e a d i l y "is d i s c u s s e d elsewhere. - 62 -PART I . DISCUSSION The experiments reported upon herein constitute t h e f i r s t study of the conditions governing deamination by the two species of bacteria - Proteus ichthyosmius and Pseudo-monas putrefaciens - that has been reported. Similar studie employing different techniques and more rapidly acting bacteria such as Bacterium c o l i have been recorded. The technique followed i n previous studies, that using the Warburj apparatus, however, could not be followed in this study because the two species of surface taint bacteria employed are r e l a t i v e l y slow to attack the amino acids, sometimes requiring a number of days whereas Bacterium c o l i has the a b i l i t y to deaminate i n the matter of a few hours or less. The method of ammonia determination also d i f f e r e d i n t h i s study from most of the previous studies. Since the colorimetric determination of ammonia employing Nessler 9s reagent i s inhibited by the presence of certain nitrogeneous compounds, outstanding among which are h i s t i d i n e and tryptophan ( 5 0 ) , t h i s procedure, commonly used by other work-ers, could not be employed d i r e c t l y i n t h i s study. D i s t i l -l a t i o n of the ammonia from the culture followed by Nessleri-zation would have overcome this d i f f i c u l t y but such a proce-dure is too slow to permit completing the large number of determinations required i n the experiments described above. The alternative procedure that was chosen was the Van Slyke aeration procedure.. This procedure, although possibly not y i e l d i n g as accurate results as (may be obtained employing s Nessler's reagent, was found to aerate ninety-six to ninety-- 63 -eight percent of the ammonia added, i n a test series and had the (required^ advantage of permitting the carrying out of the large number of determinations required i n this study. The decomposition of arginine by Proteus ichthyos-mius has been shown to y i e l d seventy-five to eighty percent of i t s nitrogen as ammonia. This means that at least three, and in a l l p r o b a b i l i t y four, of the nitrogens of arginine are converted p a r t i a l l y or completely into ammonia by this species. I f the enzyme arginase i s produced by Proteus ichthyosmius, the products of hydrolysis by this enzyme would be ornithine and urea. The results of experiment XIV have shown that Proteus ichthyosmius converts seventy percent of the nitrogen of ornithine into ammonia but only ten percent of that of urea. These findings suggest that the elaboration of b a c t e r i a l urease for the s p l i t t i n g of urea may depend upon the p r i o r elaboration of arginase. The f a i l u r e to s p l i t urea to a s i g n i f i c a n t extent shows that t h i s species is unable to form urease i n the presence of urea alone. The results reported upon herein support the findings described by H i l l in work on the s p l i t t i n g of the arginine molecule and may lend credence to the theory put f o r t h by him with respect to the a c t i v i t y of arginine dihydrolase. The rate and quantity of ammonia formed from arginine by Pseudomonas putrefaciens i s not so great as that formed by Proteus"Ichthyosmius e Is would appear that the alaboration of the enzymes necessary for the breakdown of arginine i s much slower and not so complete in the case of Pseudomonas putrefaciens as compared with Proteus - 64 -ichthyosmius. The t o t a l quantity of ammonia formed by Pseudomonas putrefaciens from arginine i s , however, in excess of that formed by the same species from ornithine and urea, suggesting again that there is an associative action present in the alaboration of the different enzymes required f o r the complete" degradation of arginine. The breakdown of ornithine has been found to be similar for both b a c t e r i a l species - seventy percent of i t s nitrogen being converted into ammonia. This finding shows cl e a r l y that these b a c t e r i a l species have the a b i l i t y to att-ack both the. alpha and the deli^a amino groups of ornithing to a considerable extent. When, however, the results showing a l -most i n s i g n i f i c a n t quantities of ammonia formed from the deami nation of delta amino v a l e r i c acid (the compound formed by the deamination of the alpha amino group of ornithine) are con-sidered, i t can r e a d i l y be seen that the elaboration of the deaminase for attacking the delta amino group i s dependent upon the p r i o r elaboration of the alpha amino deaminase - a sequence of events similar to that found for the elaboration of ammonia from arginine. Aspartic and. glumatic acids are both dicarboxylic mono-amino acids d i f f e r i n g i n structure only by the presence of an additional GHg group in the case of glutamic acid. A comparison of the rates of ammonia formation from these two amino acids, however, reveals s t r i k i n g differences. The dl-aspartic acid employed was deaminated to the extent of f i f t y percent of i t s nitrogen within twenty-four hours by each of the two b a c t e r i a l species * A longer period of - 65 -incubation did not result in further formation of ammonia. Previous studies (50) have shown that the 1-isomer of t h i s amino acid is deaminated very rapidly suggesting that one hundred percent of the 1-compound in the dl-mixture i s attacked while the d-compound is not attacked. When d-glutamic acid i s considered, i t is found that deamination takes place slowly over the complete period of incubation - forty percent of the t o t a l nitrogen being converted into ammonia after twenty-one days incubation. One reason for t h i s slower rate of deamination when compared with that found i n the case of aspartic acid may be that the cVIsomer i s not readily attacked. It would be necessary to employ the 1~ or the dl-isomer i n order to make a direct comparison of the rates of deamination of these two amino acids• The conversion by Pseudomonas putrefaciens of about seventy percent of the nitrogen of h i s t i d i n e Into ammonia shows c l e a r l y that this b a c t e r i a l species has the a b i l i t y to open the iminazol ring and to deaminate the compound thus formed. Cleavage of the ri n g with the l i b e r a t i o n of one molecule of ammonia followed by oxidation, oxidative decarboxy-l a t i o n and deamination respectively may result i n the formation of glutamic acid (see figure 2)© In the experiments reported upon herein, 1-histidine was employed. I f the degradation of h i s t i d i n e takes place as suggested, l=glutamic acid should be formed. Providing the f i r s t two ammonia molecules in the degradation are released to the extent of approximately one hundred percent, about ten percent of the t h i r d molecule present i n the suggested glutamic acid are converted into «. 66 -ammonia showing a slower rate of breakdown than takes place when d-glutamic acid i s employed by i t s e l f . The cleavage of the iminazol ri n g by Proteus ichthy-osmius is not so rapid nor so complete as that found f o r Pseudomonas putrefaciens - about f o r t y percent of the t o t a l nitrogen being converted into ammonia. The r i n g Is d e f i n i t -ely opened, however, by Proteus Ichthyosmius since a maximum of thirty-three percent ammonia would be formed by deamination of the side chain amino group alone. The decomposition of proline with the l i b e r a t i o n of between for t y and f i f t y percent^ of i t s nitrogen as ammonia takes place equally well under the influence of the two bac-t e r i a l species employed. Proline is not a true amino acid but contains i t s nitrogen as an Imino group i n the p y r o l l i d i n e ring structure. Opening of this ring i s a prerequisite of ammonia formation from this compound at a l l times. The p y r o l l i -dine ring may be broken on either side of the imino group« Hydrolytic cleavage on the side next to the carboxyl group yiel d s alpha hydroxy delta amino v a l e r i c acid. If the deamination of this compound i s similar to that of delta amino v a l e r i c acid, i t is possible that this method, of opening the r i n g does not take place. Oxidative opening of the ring on the side of the imino group away from the carboxyl group would give glutamic acid. Weil-Malherbe and Krebs have concluded that proline is oxidized by kidney tissue to glutamic acid which i n turn may be further oxidized to alpha keto g l u t a r i c acid with the l i b e r a t i o n of one molecule of ammonia. Further evidence i n support of cleavage of the - 67 -p y r o l l i d i n e ring i n this manner may be obtained when the figures from the different experiments f o r glutamic acid and proline are compared. For example, figures 52 and 55 and figures 68 and 69 depicting the ammonia formation by Proteus ichthyosmius from glutamic acid and proline respectively show clearly" that the rate of formation and the f i n a l quantities . produced are highly comparable. The results obtained i n the experiments outlined above add further evidence to the hypothesis shown In figure 2 that the six amino acids studied are interrelated. The aIpha keto and alpha hydroxy apids of delta amino v a l e r i c acid and alpha keto glutarie acid are probably the links joining arginine, ornithine and proline to h i s t i d i n e , aspartic acid and glutamic acid. The quantities of ammonia formed during the decomposition of these s i x amino acids by the two b a c t e r i a l species are s u f f i c i e n t to suggest that their break-down proceeds through the number of oxidations and destinat-ions outlined. Arginine, under the influence of Proteus ichthyosmius shows the l i b e ration of four nitrogens i n the form of ammonia indicating the formation of alpha keto g l u t a r i e acid or one of i t s decomposition products. The action of Pseudomonas putrefaciens on arginine does not li b e r a t e so much ammonia as Proteus ichthyosmius but the decrease i s probably due to the f a i l u r e to hydrolyse urea since i t has been shown that this species is capable of l i b e r a t i n g seventy percent of the nitrogen of/ornithing i n the form of ammonia. The bre sled own of proline by both b a c t e r i a l species - 68 -also points to the formation of alpha keto glu t a r i c acid or one of i t s products. While the p o s s i b i l i t y of the course of breakdown from alpha keto delta-amino v a l e r i c acid proceeding by reductive deamination to the simpler compounds of v a l e r i c acid must be considered, the evidence obtained i n the expire-ments of the eff e c t of oxygen supply on ammonia formation, p a r t i c u l a r l y in the case of proline, strongly suggests that oxidative deamination rather than reductive deamination takes place. The experimental evidence obtained for the decom-po s i t i o n of h i s t i d i n e by Pseudomonas putrefaciens shows c l e a r l y that the three nitrogens are liberated in the form of ammonia adding further support to the hypothesis that this amino acid is degraded to alpha keto g l u t a r i c acid or lower. The findings i n the experiments employing Proteus ichthyos-mius oh h i s t i d i n e suggest that only two nitrogens are conver-ted into ammonia or that a smaller percentage of the h i s t i d i n e i s attacked with the formation of ammonia equivalent to the three nitrogens. The evidence is.stronger f o r the second alternative when i t is rec a l l e d that increase i n age of the growth culture of t h i s b a c t e r i a l species markedly decreases the quantity of ammonia formed from h i s t i d i n e . The younger growth cultures produced s u f f i c i e n t ammonia to indicate that deamination of the three nitrogen groups occurs. The direct oxidative deamination of glutamic acid gives alpha keto g l u t a r i c acid or one of i t s re l a t e d compounds. Aspartic acid when subjected to aimilar oxidative deamination yi e l d s oxalacetic acid which i s also formed from alpha keto - 69 -g l u t a r i e a c i d by o x i d a t i v e d e c a r b o x y l a t i o n . The ammonia formed from t h e s e two amino a c i d s by t h e b a c t e r i a l s p e c i e s s t u d i e d i n d i c a t e s t h a t they a r e deaminated c o m p l e t e l y i n the case o f t h e 1-isomer of d l - a s p a r t i c a c i d and p a r t i a l l y i n the case of d - g l u t a r a i c a c i d . The e x p e r i m e n t a l evidence o b t a i n e d f o r the i n f l u -ence of c a r b o h y d r a t e on ammonia f o r m a t i o n and i t s subsequent u t i l i z a t i o n as a n i t r o g e n source f o r c e l l m u l t i p l i c a t i o n i s considerable» In examining t h i s e v i d e n c e i t must be remem-b e r e d t h a t the f i g u r e s r e p r e s e n t t h e f r e e ammonia co n t e n t o f t h e c u l t u r e s which i s an e x p r e s s i o n of the r e s u l t a n t o f amino a c i d d e g r a d a t i o n and subsequent c e l l s y n t h e s i s . Thus i t can r e a d i l y be u n d e r s t o o d t h a t a d ecrease i n ammonia c o n t e n t does not n e c e s s a r i l y mean t h a t l e s s ammonia i s b e i n g formed but r a t h e r , i n the case of those c u l t u r e s showing c o n s i d e r a b l e b a c t e r i a l growth, t h a t t h e ammonia has been used f o r c e l l s y n t h e s i s . The f i n d i n g s o f experiment IX i n w h i c h the e f f e c t o f the presence of g l u c o s e i n the growth medium on subsequent ammonia f o r m a t i o n i s s t u d i e d , show c l e a r l y t h a t the organ-isms grown o f the g l u c o s e - c o n t a i n i n g agar are not so a c t i v e as t h o s e grown on the g l u c o s e - f r e e agar. This decrease i n a c t i v i t y may be due t o the g e n e r a l decrease i n a c t i v i t y o f •this organism - P r o t e u s i c h t h y o s m i u s - as e x h i b i t e d by the marked r e t a r d a t i o n i n the r a t e o f growth on t h e g l u c o s e -c o n t a i n i n g a g a r as compared t o t h e g l u c o s e - f r e e a g a r . Epps and Gale (23) i n an i n v e s t i g a t i o n t o f i n d an e x p l a n a t i o n f o r s i m i l a r f i n d i n g s o b t a i n e d employing E s c h e r i c h i a c o l i , observed . - 70 -that the degree of i n h i b i t i o n of deamination by the presence of glucose bore no r e l a t i o n to the effect produced by the addition of fermentation acids to the growth medium i n place of the glucose. They also showed that neutralization of the fermentation acids of glucose during growth does not a l t e r yhe degree of i n h i b i t i o n of subsequent deamination. Further investigation s i m i l a r to that outlined by Epps and Gale i s necessary before an adequate explanation of these findings can be advanced. In experiments of the effect of the presence of acid-producing and non-acid-producing carbohydrates i n the buffer medium upon ammonia formation, the findings have been cl e a r l y shown to be dependent upon both the carbohydrate and the amino acid employed. The fermentation of the carbo-hydrate with acid production and the breakdown of the amino acid with ammonia formation appear to be independent proces-ses * The rate at which these two processes take place, however, has been shown to materially affect the f i n a l out-come i n so far as the quantity of free ammonia i s concerned. When the results for the deamination rates of aspartic and glutamic acids are considered, this finding i s seen to be c l e a r l y demonstrated. While in the absence of carbohydrate, the f i n a l quantities of ammonia formed from these amino acids are approximately f i f t y and forty percent respectively, i n the presence of an acid-producing carbohydrate, considerable free ammonia results from aspartic acid and p r a c t i c a l l y no ammonia formation i s indicated i n the case of glutamic acid. The considerable quantities of free ammonia formed from - 71 -a s p a r t i c a c i d i n t h e presence o f f e r m e n t a b l e carbohydrate i s l a r g e l y dependent upon t h e f a c t t h a t the r a t e of ammonia f o r -m a t i o n from t h i s amino a c i d proceeds as r a p i d l y as does t h e f e r m e n t a t i o n o f t h e c a r b o h y d r a t e . I n the case o f g l u t a m i c a c i d , the f i n a l r e s u l t of p r a c t i c a l l y no ammonia p r e s e n t i n a c u l t u r e c o n t a i n i n g an a c i d - p r o d u c i n g carbohydrate may de-pend upon the speed o f a c i d p r o d u c t i o n o u t d i s t a n c i n g t h e f o r m a t i o n o f ammonia, r e s u l t i n g i n the l o w e r i n g o f t h e pH o f an i n a d e q u a t e l y b u f f e r e d c u l t u r e t o a l e v e l i n h l b i t i v e t o f u r t h e r b a c t e r i a l a c t i v i t y . On t h e o t h e r hand, w i t h an adequate b u f f e r , s u p p l y , the a v a i l a b l e c a r b o h y d r a t e may be used f o r purposes of c e l l m u l t i p l i c a t i o n u t i l i z i n g t he ammonia pr o d u c e d by de a m i n a t i o n as r a p i d l y as i t i s formed as a source o f n i t r o g e n . Whatever the e x p l a n a t i o n i n t h e case o f g l u t a m i c a c i d , p r a c t i c a l l y no f r e e ammonia i s t o be found i n the' c u l t u r e s c o n t a i n i n g a c i d - f o r m i n g c a r b o h y d r a t e s . The f i n d i n g s observed i n these experiments add f u r t h e r e v i d e n c e t o t h e h y p o t h e s i s o f R a i s t r i c k and C l a r k t h a t c a r b o h y d r a t e , f a r from h a v i n g a p r o t e i n - s p a r i n g e f f e c t , a c t u a l l y e n a b l e s t h e b a c t e r i a t o u t i l i z e more p r o t e i n o r p r o t e i n p r o d u c t s than they would i n t h e absence of carbo-h y d r a t e . The i d e a o f ammonia u t i l i z a t i o n as a n i t r o g e n s o u r c e i s a f a c t o r t h a t i s n o t encountered i n s t u d i e s employ-i n g B a c t e r i u m c o l i o r o t h e r r a p i d d e a m i n a t i n g s p e c i e s o f b a c t e r i a . The slowness o f the p r o c e s s i n t h e case o f the s u r f a c e t a i n t p r o d u c i n g b a c t e r i a and the consequent I n f l u e n c e o f t h e p o s s i b l e u t i l i z a t i o n o f ammonia makes more d i f f i c u l t t h e i n t e r p r e t a t i o n o f d a t a o b t a i n e d f o r the slowe r deaminat--72-I n g s p e c i e s . One o f the i m p o r t a i t problems t h a t a r i s e s from these o b s e r v a t i o n s i s t h e q u e s t i o n as t o t h e r e l a t i v e s u i t -a b i l i t y o f the methods o f s t u d y i n g dean i n a t i o n - the simple method u s i n g such b a c t e r i a l s p e c i e s as B a c t e r i u m c o l i and the Warburg apparatus o r the more c o m p l i c a t e d procedure employing the s l o w e r a c t i n g b a c t e r i a such as those used i n t h i s s t u d y . The m s w e r t o t h i s q u e s t i o n i s dependent upon the o b j e c t o f the s t u d y - whether t h e i n v e s t i g a t i o n i s p r i m a r i l y concerned w i t h the breakdown o f the amino a c i d o r w i t h the a c t i v i t y o f the p a r t i c u l a r b a c t e r i a l s p e c i e s , I n t h i s i n v e s t i g a t i o n , the more Important f a c t o r was t h e s t u d y of the a c t i o n o f two s p e c i e s o f b a c t e r i a known t o be among t h o s e r e s p o n s i b l e f o r s u r f a c e t a i n t i n b u t t e r on compounds t h a t might be the p r e -c u r s o r s o f the substance or substances formed i n the e l a b o r a -t i o n o f the s w e a t y - f e e t odour o f t y p i c a l s u r f a c e t a i n t • P r e v i o u s i n v e s t i g a t i o n s o f the e f f e c t o f the presence o f c a r b o h y d r a t e on de a m i n a t i o n have been p r i n c i p a l l y concerned w i t h the one c a r b o h y d r a t e ~ g l u c o s e • N i s i m u r a (51) used a number o f c a r b o h y d r a t e s , b u t l i m i t e d the amino a c i d s t u d i e d t o t y r o s i n e . The experiments r e p o r t e d upon h e r e i n were c a r r i e d o u t employing e l e v e n d i s t i n c t c a r b o h y d r a t e s , s u b s e q u e n t l y reduced t o f i v e key c arb ohydrat e s i n the e x p e r i -ments d e m o n s t r a t i n g the i n f l u e n c e o f v a r i a t i o n i n b u f f e r c o n c e n t r a t i o n . The f i n d i n g s show c l e a r l y t h a t the n a t u r e of the c a r b o h y d r a t e has a marked e f f e c t upon the r a t e and the f i n a l ammonia f o r m a t i o n . I n t h e absence o f adequate b u f f e r i n t h e medium, t h e pH of the c u l t u r e s containing the r a p i d - 73 -a c i d - p r o d u c i n g c a r b o h y d r a t e s i s r e a d i l y lowered t o a l e v e l u n f a v o r a b l e t o f u r t h e r b a c t e r i a l a c t i v i t y . I n c r e a s i n g the b u f f e r c a p a c i t y o f the c u l t u r e overcame t h i s d i f f i c u l t y and the anmonia f o r m a t i o n showed i t s e l f t o be p o s i t i v e o r n e g a t i v e depending upon the r e l a t i v e r a p i d i t y o f f e r m e n t a t i o n and d e a m i n a t i o n . The r a t e of a c i d p r o d u c t i o n from the v a r i o u s c a r b o h y d r a t e s has been shown t o v a r y c o n s i d e r a b l y - glucose and sucrose a p p e a r i n g t o be the most r a p i d l y fermented, w h i l e • g l y c e r o l i s one o f t h e s l o w e s t a c i d - f o r m e r s . As p r e v i o u s l y n o t e d , t h e a c i d p r o d u c t i o n from l a c t o s e a f t e r a f o u r t e e n day p e r i o d o f i n c u b a t i o n p a r t i c u l a r l y i n t h e presence o f a r g i n i n e i s an example of a carbohydrate t h a t p r o v i d e s a v a i l a b l e carbon s o u r c e s a f t e r the n i t r o g e n source has been formed i n l a r g e q u a n t i t i e s and p e r m i t s b a c t e r i a , m u l t i p l i c a t i o n t o proceed i n the l a t t e r days o f the t h r e e week p e r i o d o f i n c u b a t i o n . Thus as can r e a d i l y be seen from the f i n d i n g s o f these experiments c o n s i d e r a b l e d a t a has been added t o our knowledge o f amino a c i d breakdown. The study o f the e f f e c t o f the presence o f v a r i o u s c a r b o h y d r a t e s has I n c r e a s e d our i n f o r m a t i o n c o n c e r n i n g t h i s l i t t l e e x p l o r e d aspect o f amino a c i d d e c o m p o s i t i o n . The p r o v i s i o n o f an a v a i l a b l e carbo-h y d r a t e adds the c o m p l i c a t i o n o f b a c t e r i a l growth t o the a l r e a d y i n v o l v e d s t u d y o f the enzymes r e s p o n s i b l e f o r amino a c i d c a t a b o l i s m a i d subsequent ammonia f o r m a t i o n . PART IIo Studies on the Isolation and Identification of Odour i f ex* oug Compounds. HISTORICAL • . ' The problem of the nature of the compound or com-pounds formed during the development of surface taint i n butter has confronted investigators since studies of the defect were begun a number of years ago. Campbell(9) found that when indole, a derivative of tryptophan was added to milk, an odour strongly resembling that of surface taint was emitted. As already mentioned, however, Neilson (50) has shown that indole i s not found i n the sera of surface taint butters and therefore cannot be the cause of the odour of the defect. In the same study (50), a number of organic com-pounds related to the amino acids were added to milk and the odours emitted recorded. It was found that betaine, delta amino v a l e r i c acid (a derivative of both arginine and ornithine) and beta amino butyric acid gave odours reminis-cent of surface t a i n t . The following combinations of chem-i c a l s also emitted odours closely resembling the characteris-t i c "sweaty-feet" odour; betaine and delta amino val e r i c acid; betaine and beta amino butyric acid; betaine and i s o -v a l e r i c acid; betaine and para hydroxy phenylacetic acid; and betaine, i s - v a l e r i c acid and para hydroxy phenylacetic acid. The odour emitted from betaine i t s e l f was enhanced when the other compounds were also added to the milk. These findings suggest that the b a c t e r i a l synthesis of betaine may be con-cerned i n the development of surface taint i n butter. - 75 -' Wolochow, Thornton and Hood (71) of A l b e r t a , i n s t u d i e s on odour p r o d u c t i o n by Pseudomonas p u t r e f a c i e n s , one of the c a u s a t i v e agents o f s u r f a c e t a i n t i n b u t t e r , have found t h a t the " s w e a t y - f e e t " odour t h a t i s absent i n skim m i l k c u l t u r e s a t pH 7.6 becomes apparent when t h e m i l k c u l t u r e s o f the 'organism were a e r a t e d i n s u l p h u r i c a c i d s o l u t i o n but were n o t p r e s e n t when a e r a t e d i n sodium h y d r o x i d e s o l u t i o n . T h i s e v i d e n c e suggests t h a t the compound r e s p o n s i b l e f o r the odour i s a v o l a t i l e a c i d w h i c h i s p r e s e n t as a s a l t i n a l k a l i n e s o l u t i o n s and i s f r e e d as an a c i d a t l ower pH's. I n a l a t e r s t u d y , D tunkley, Hunter, Thornton and Hood (12) o b t a i n e d evidence i n d i c a t i n g t h a t t h e r e i s a d e f i n i t e c o n n e c t i o n between i s o v a l e r i c a c i d and the "sweaty-f e e t " odour. Under c e r t a i n unknown c o n d i t i o n s , the "sweaty-f e e t " odour seems t o a r i s e p o s s i b l y c h e m i c a l l y from i s o -v a l e r i c a c i d but i s d i s t i n c t l y d i f f e r e n t i n odour from t h i s a c i d . Wolochow, e t c , have e x p r e s s e d the o p i n i o n t h a t Pseudomonas p u t r e f a c i e n s may produce i n m i l k a substance w h i c h i s i n the reduced s t a t e and, i f odourous, i s p r e s e n t i n i n s u f f i c i e n t c o n c e n t r a t i o n to be d e t e c t e d by t h e sense of s m e l l . Exposure t o a i r o x i d i z e s t h i s substance t o a d e t e c -t a b l y odourous s t a t e . More s t r o n g l y o x i d i z i n g l e v e l s w i l l r e v e r s i b l y change the compound t o a non-odourous s t a t e . F u r t h e r support f o r t h i s o p i n i o n comes f r o m t h e f i n d i n g s o b t a i n e d f r o m commercial and e x p e r i m e n t a l l y produced s u r f a c e t a i n t b u t t e r i n the l a b o r a t o r i e s o f the Department of D a i r y -i n g a t the U n i v e r s i t y o f B r i t i s h C olumbia, The odour o f s u r f a c e t a i n t may be absent from a sample of d e f e c t i v e b u t t e r - 76 -when the bottle containing i t i s f i r s t opened, but i f the bottle be closed and re-examined about ten minutes l a t e r , the odour i s often present. Continued exposure has been found to decrease the concentration of the odour u n t i l i t i s no longer detectable, ' Posdick and Rapp ( 2 7 ) , i n the course of an i n v e s t i -gation on the degredation of glucose by Staphlococcus albus under aerobic conditions produced a reaction mixture of a p a r t i c u l a r l y f o u l odour which was not characteristic of any known product of fermentation. Subsequent extraction and p u r i f i c a t i o n l e f t a solution containing two organic compounds, one acidic i n nature and the other neutral. Neither compound could be isolat e d i n pure form without decomposition but i d e n t i f i c a t i o n tests indicated that the aci d i c compound was alpha-keto-gama-hydroxy v a l e r i c acid while the neutral one was the correspon-ding aldehyde. These compounds are closely related to t he amino acids arginine, ornithine, h i s t i d i n e , proline and glutamic acid, and may possibly be related to the characteris-t i c surface t a i n t odour. EXPERIMENTAL. In order to determine i f the surface t a i n t produc-ing bacteria are capable of forming odouriferous compounds from sodium pyruvate similar to those obtained by Fosdick aid Rapp the following experiment (XVI) was undertaken. Three 50 cc. flasks were set up i n duplicate and contained i n addition to the 8 cc. of phosphate buffer (M/l5 at pH 6,8) _ 77 -(1) 1.0 cc. of M/20 sodium pyruvate plus 1.0 cc. of ten percent washed c e l l suspension. (2) 0.1 cc. of M/20 sodium pyruvate plus 0.9 cc. of water plus 1.0 cc. of ten percent washed c e l l suspension. (3) 1.0 cc. of M/20 sodium pyruvate plus 1.0 cc. of one percent washed c e l l suspension respectively. One of the duplicates was Inoculated with a c e l l suspension of Proteus ichthyosmius and the other with that of Pseudomonas putrefaciens. A control containing 9 cc, of buffer plus 1 cc. of ten percent c e l l suspension was set up for each of the.two bacterial/*species. The flasks were incubated at 30° C . and the odours formed observed at d a i l y i n t e r v a l s . The flasks were observed to emit odours sugges-tive of surface t a i n t . The odours from the flasks contain-ing sodium pyruvate were more pronounced than t he controls containing b a c t e r i a l c e l l s only, although these also gave off putri d odours. With these results i n mind, an experiment was set up to f i n d out i f the compounds causing the odours produced by the two b a c t e r i a l species from sodium pyruvate could be isolated and i d e n t i f i e d . Two 250 cc. flasks containing 80 cc. M/l5 phosphate buffer (pH 6.8), 10 cc. M/20 sodium pyruvate and 10 cc« of a ten percent washed c e l l suspension of P. putrafaciens were prepared and incubated at 23° C. f o r 48 hours. The flasks were removed from the incubator and one of the flasks treated as followsj F i r s t the contents of the flask were centrifuged to remove the ba c t e r i a l c e l l s aa d the supernatant poured off - 78 -into a liquid-ether-extraction flask where i t was extracted f o r 16 hrs. The ether extract (1) was taken up i n water and gave a putrid odour. The residual l i q u i d from the extraction was taken down to pH 4.0 with 20% phosphoric acid, f i l t e r e d and the f i l t r a t e extracted with ether for another 16 hours. This extract (2) also gave a pu t r i d odour when taken up i n water. The second flask was taken down to pH 4.0 d i r e c t l y with phosphoric acid, f i l t e r e d and the f i l t r a t e extracted with ether f o r 16 hours• This extract (3) when mixed with water also gave-a putrid odour. An attempt was made to obtain a 2,4, d i n i t r o phenylhydrazone precipitate from these extracts but the quantities of material were too small to give anything more than a s l i g h t p r e c i p i t a t e . In the procedure of Dunkley, etc. the odouriferous compounds were separated from the remainder of the milk c u l -ture by steam d i s t i l l a t i o n of the a c i d i f i e d culture. This procedure would further hydrolyze, to a considerable degree, the products of the bacterial protein decomposition and i t would not be possible to determine which portion of the dis -t i l l a t e was from bacterial hydrolysis and which was from chemical hydrolysis. The hydroxy and keto acids of the lower f a t t y acids are usually ether soluble and therefore should be removed by ether extraction from a culture'contain-ing them. In order to obtain data on thi s hypothesis a flask containing 100 cc. of skim milk and 10 cc. of a ten percent washed c e l l suspension of Pseudomonas putrafaciens was pre-pared aid incubated at 23° C. for' twenty days. The protein - 79 -•• of the milk was then largely converted to soluble decomposit-ion products and emitted a f o u l , putrid odour. The contents of the flask were centrifuged and the pH of the supernatant taken. It was pH 5.9. The supernatant which was a bright yellow colour suggesting the presence of a fl a v i n e , was then extracted i n a l i q u i d ether extractor for 12 hours. The extract thus formed (1) was soluble i n water and gave of f a pungent f r u i t y odour. The residual f l u i d from t h i s extrac-t i o n was taken down to pH 4.0 with phosphoric acid and re-extracted with ether for a further 12 hours. This extract (2) was only sparingly soluble i n cold water and emitted a mixture of odours resembling those from well ripened Oka cheese. The extract (2) was treated further as follows: I t was tested f o r s o l u b i l i t y i n a number of s o l -vents and found to be s l i g h t l y soluble i n cold ether and hot water, soluble i n hot ether, and very soluble i n 95$ al cohol. The extract was f i n a l l y dissolved i n alcohol and f i l t e r e d . The f i l t r a t e was evaporated to dryness giving a yellowish white residue to which was added a small quantity of hot water. The residue clumped together i n the water and o i l e d -off into solution when the water was raised to b o i l i n g point. The solution was evaporated to dryness giving a yellow-brown o i l y residue which when dissolved i n hot ether c r y s t a l l i z e d out from the ether as white needles with a melting point of 175-177° C. The quantity of crystals was i n s u f f i c i e n t to permit further i d e n t i f i c a t i o n . ' With the object of obtaining information on the ether s o l u b i l i t y of the decomposition products of certain - 80 -amino a c i d s whose chemical s t r u c t u r e suggested t h a t odour-i f e r o u s compounds might r e s u l t from t h e i r breakdown a f u r t h e r experiment (XVII) was u n d e r t a k e n . The amino a c i d s employed were a r g i n i n e , h i s t i d i n e and p r o l i n e arid the b a c t e r i a used were P r o t e u s Ichthyosmius and Pseudomas p u t r e f a c i e n s . S i x f l a s k s c o n t a i n i n g 160 c c , o f M/l5 phosphate b u f f e r (pH 7,4), 20 c c , of M/20 Amino a c i d and 20 c c , o f one p e r c e n t washed c e l l s u s p e n s i o n were p r e p a r e d and i n c u b a t e d a t 30° C. E a c h f l a s k was t r e a t e d i n d i v i d u a l l y as f o l l o w s : F l a s k (1) c o n t a i n i n g P r o t e u s i c h t h y o s m i u s i n a r g i n i n e was removed from the> i n c u b a t o r a f t e r 7 days and the pH t a k e n e l e c t r o m e t r i c a l l y , I t was pH 8,0, The contents o f the f l a s k were taken down t o pH 4.0, w i t h p h o s p h o r i c a c i d and e x t r a c t e d w i t h e t h e r f o r f o u r h o u r s . The s m a l l amount o f e x t r a c t o b t a i n e d f a i l e d to g i v e o f f a p u t r i d odour and d i d . n o t d i s s o l v e r e a d i l y i n w a t e r . F l a s k (2) c o n t a i n i n g P r o t e u s i c h t h y o s m i u s i n h i s t i d i n e was t r e a t e d the same as f l a s k (1.) . The pH f o l l o w -i n g i n c u b a t i o n was pH 7,15. The e x t r a c t d i d not emit a p u t r i d odour. I n an attempt t o i s o l a t e the e t h e r i n s o l u b l e d e c o m p o s i t i o n p r o d u c t s o f h i s t i d i n e , the r e s i d u a l f l u i d f o l l o w i n g e t h e r e x t r a c t i o n was f i l t e r e d , r a i s e d to pH 10.0 w i t h sodium h y d r o x i d e and e v a p o r a t e d t o dryness i n vacuo. The r e s i d u e was t a k e n up i n 95% a l c o h o l and r e f l u x e d f o r about one h a l f h o u r . The a l c o h o l m i x t u r e was then f i l t e r e d and the f i l t r a t e e v a p o r a t e d t o d r y n e s s ! The r e s i d u e was t a k e n up i n a s m a l l q u a n t i t y o f d i s t i l l e d water and a few c c . of 19% p h o s p h o t u n g s t i c a c i d i n 5% s u l p h u r i c a c i d were added. The - 81 -heavy white precipitate formed was f i l t e r e d o f f , washed and dried carefully. PIask (3) containing Pseudomonas putrefaciens i n h i s t i d i n e was removed from the incubator after 14. days and treated after the same manner as flask (2). The pH after incubation was pH 7.55. A similar phosphotungstic p r e c i p i -tate was obtained. As a control check the phosphotungstic precipitate of h i s t i d i n e was prepared and found to resemble closely the precipitates obtained from flask (2) and (3). The compounds precipitated were basic i n nature otherwise they would not .have combined "with phosphotungstic acid. Flask (4) containing Pseudomonas putrefaciens i n the presence of proline was taken from the incubator after 14 days' incubation and treated the same as flask (1). The pH following incubation was pH 7.45. No putrid odours were observed i n the ether extract. Flask (5) containing Pseudomonas putrefaciens and arginine was permitted to incubate f o r 20 days, following which i t was treated the same as f l a s k (1). After incubation the pH was 7.75. The extract obtained emitted no putrid odours. Flask (6) containing Proteus ichthyosmius i n pro-l i n e had a pH of 7.45 after 20 days' incubation and when treated i n a similar manner to flask (1) gave an extract free from p u t r i d odours. DISCUSSION The findings obtained from the experiments des-cribed i n the above section serve to discourage certain lines. • - 82 -o f thought from the i n v e s t i g a t i o n i n t o the cause o f s u r f a c e t a i n t r a t h e r than t o g i v e p o s i t i v e evidence i n any one d i r e c t i o n . The i n a b i l i t y to i s o l a t e o d o u r i f e r o u s compounds from th e d e c o m p o s i t i o n p r o d u c t s o f a r g i n i n e , h i s t i d i n e and p r o l i n e - t h r e e amino a c i d s whose h y d r o l y t i c p r o d u c t s suggest odour f o r m a t i o n - i n d i c a t e s t h a t p o s s i b l y these a c i d s are not d i r e c t l y r e s p o n s i b l e f o r the e l a b o r a t i o n of t h e sweaty-feet odour of t y p i c a l s u r f a c e t a i n t . I t must be remembered, how-eve r , t h a t the t e c h n i q u e s employed i n t h i s i n v e s t i g a t i o n may not have p r o v i d e d t h e c o r r e c t c o n d i t i o n s o f p o t e n t i a l f o r t h e o m i t t a n c e of the odours. A l s o the odours from the compounds may have been masked by o t h e r c a c t o r s or substances, or the compounds themselves may n o t have been e t h e r s o l u b l e . This b r a n c h o f t h e i n v e s t i g a t i o n i n t o t h e cause of s u r f a c e t a i n t cannot be d i s p e n s e d w i t h as y e t because o f i n s u f f i c i e n t e v i -dence t o prove o r d i s p r o v e the h y p o t h e s i s t h a t the a c i d i c d e c o m p o s i t i o n p r o d u c t s o f c e r t a i n amino a c i d s may be r e s p o n -s i b l e f o r t h e s w e a t y - f e e t odour. The much sought a f t e r odour has been shown t o be p r e s e n t a t d i f f e r e n t times d u r i n g t h e c o u r s e o f t h e e x p e r i m e n t a l s e c t i o n of P a r t I i n c u l t u r e s con-t a i n i n g the amino a c i d s used above - a r g i n i n e , h i s t i d i n e and p r o l i n e . The e l a b o r a t i o n o f the t y p i c a l odour o f s u r f a c e t a i n t appears to be dependent upon a number o f p h y s i c a l , c h e m i c a l and b i o l o g i c a l f a c t o r s . The f i r s t o f t h e s e which may be p h y s i c a l or c h e m i c a l i s t h a t t h e odour i s n o t formed i n b u t t e r made from raw cream n o r i n raw m i l k I n o c u l a t e d w i t h s u r f a c e t a i n t p r o d u c i n g b a c t e r i a , but i s formed when the m i l k o r cream employed i s p a s t e u r i z e d . I t would appear t h a t t h e e l a b o r a t i o n o f s u r f a c e t a i n t i s dependent upon a s u b t l e change induced by the h e a t on t h e p r o t e i n complex o f the m i l k o r cream. A second f a c t o r i s t h a t t h e compound or compounds are not always p r e s e n t i n t h e o d o u r i f e r o u s s t a t e , but r e q u i r e a c e r t a i n o x i d a t i o n - r e d u c t i o n p o t e n t i a l b e f o r e t h e y emit the odour. E v i d e n c e f o r t h i s p o i n t comes, as p r e v i o u s l y mentioned, from th e appearance and disappearance of the s u r f a c e t a i n t • odour i n b u t t e r exposed t o the a i r . The o u t s t a n d i n g b i o l o g -i c a l f a c t o r i s t h a t a l l s p e c i e s o f p u t r e f a c t i v e b a c t e r i a do not cause s u r f a c e t a i n t i n b u t t e r . The number of s p e c i e s o f b a c t e r i a c a pable of e l a b o r a t i n g t h e d e f e c t has been shown t o be v e r y l i m i t e d - Pseudomonas p u t r e f a c i e n s b e i n g the major c a u s a t i v e agent and P r o t e u s i c h t h y o s m i u s a l s o p r o d u c i n g t h e d e f e c t but not so r e g u l a r l y . The source of t h e compound r e s p o n s i b l e f o r s u r f a c e t a i n t may not n e c e s s a r i l y be one o f the amino a c i d s s t u d i e d nor an amino a c i d a t a l l . The p r e c u r s o r o f t h e o d o u r i f e r o u s s ubstance may be a l a r g e r m o l e c u l e such as a simple p e p t i d e . E v i d e n c e /o'Lfy t h i s i d e a comes from the a b i l i t y t o i s o l a t e odour-f o r m i n g compounds from m i l k combined w i t h t h e i n a b i l i t y t o show the p r e s e n c e of such compounds among the d e c o m p o s i t i o n p r o d -u c t s o f c e r t a i n amino a c i d s . The d i f f i c u l t i e s encountered r e g a r d i n g t h e s t a b i l i t y o f t h e odour o r the substance respon-s i b l e f o r the odour f u r t h e r c o m p l i c a t e the problem of d e t e r -m i n i n g the cause of s u r f a c e t a i n t . C o n s i d e r a b l e i n v e s t i g a t i o n i s s t i l l r e q u i r e d b e f o r e more d e f i n i t e c o n c l u s i o n s r e g a r d i n g t h e s o u r c e of the s w e a t y - f e e t odour can be r e a c h e d . - 84 -SUMMARY AND CONCLUSIONS The problem under i n v e s t i g a t i o n has been o u t l i n e d and t h e f o l l o w i n g two approaches t o i t s s o l u t i o n have been made: 1. An i n v e s t i g a t i o n of the c o n d i t i o n s a f f e c t i n g ammonia f o r m a t i o n from a r g i n i n e , a s p a r t i c a c i d , g l u t a m i c a c i d , h i s t i d i n e and p r o l i n e by two s p e c i e s of s u r f a c e t a i n t p r o d u c i n g b a c t e r i a -P r o t e u s i c h t h y o s m i u s and Pseudomonas p u t r e f a c i e n s . 2. A s tudy of the p r o d u c t i o n , i s o l a t i o n and i d e n t i f i c a t i o n o f compounds e v o l v i n g odours i d e n t i c a l w i t h or i n t i m a t e l y r e l a t e d t o t h e c h a r a c t e r i s t i c s u r f a c e t a i n t odour. The l i t e r a t u r e c o n c e r n i n g the c o n d i t i o n s govern-i n g d e a m i n a t l g j i of amino a c i d s has been r e v e i w e d i n d e t a i l . The g e n e r a l methods and procedures employed have been o u t l i n e d . I t has been shown t h a t the g e n e r a l optimum range i n pH f o r t h e f o r m a t i o n o f ammonia from amino a c i d s i s from pH 6.0 t o pH 8.5 and t h a t the i n d i v i d u a l amino a c i d s v a r y w i t h i n t h i s range. I n c r e a s e i n t h e age of t h e growth c u l t u r e has been found t o d e c r ease the subsequent ammonia f o r m a t i o n from h i s t i d i n e and p r o l i n e b u t t o have no e f f e c t on t h e amount e l a b o r a t e d from a r g i n i n e , a s p a r t i c a c i d and g l u t a m i c a c i d . The c o n d i t i o n s o f oxygen s u p p l y d u r i n g growth and subsequent d e a m i n a t i o n d i d not m a t e r i a l l y a f f e c t t h e - 85 -ammonia f o r m a t i o n from a r g i n i n e , a s p a r t i c a c i d , g l u t a m i c a c i d and h i s t i d i n e hut d i d a f f e c t t h a t from p r o l i n e . A n a e r o b i c c o n d i t i o n s o f growth and deamination gave o n l y o n e - h a l f the q u a n t i t y o f ammonia formed under a e r o b i c c o n d i t i o n s * Graphs a r e p r e s e n t e d comparing the ammonia f o r m a t i o n from the f i v e amino a c i d s by Proteus Ichthyosmius and Pseudomonas p u t r e f a c i e n s at i n t e r v a l s o v er a p e r i o d o f one year» P r o t e u s i c h t h y o s m i u s has been shown t o produce a c i d and gas from g l y c e r o l , m a n n i t o l , g l u c o s e , g a l a c t o s e , s u c r o s e , m a l t o s e , d e x t r i n and s a l i c i n and t o have no immediate a c t i o n but s l o w l y t o become a l k a l i n e i n x y l o s e , l a c t o s e , d u l c i t o l and t h e c o n t r o l l a c k i n g a c a r b o h y d r a t e . Pseudomonas p u t r e f a c i e n s , on the o t h e r hand, produces a c i d and gas' from x y l o s e , m a n n i t o l , g l u c o s e , s u c r o s e , maltose and s a l i c i n , s l o w l y produces a c i d w i t h no gas from g l y c e r o l and has no immediate a c t i o n but s l o w l y becomes a l k a l i n e i n l a c t o s e , d e x t r i n , d u l c i t o l and the c o n t r o l w i t h o u t a carbo-h y d r a t e . The d i f f e r e n t l a t i n g c a r b o h y d r a t e s f o r t h e s e microorganisms are x y l o s e and d e x t r i n - P r o t e u s i c h t h y o s m i u s ferments d e x t r i n b u t not x y l o s e w h i l e Pseudomonas p u t r e f a c i e n s ferments x y l o s e but not d e x t r i n . The presence o f g l u c o s e i n t r y p t i c c a s e i n d i g e s t growth agar decreases t h e g e n e r a l r a t e of growth and sub-sequent a c t i v i t y o f the b a c t e r i a l c e l l s of P r o t e u s i c h t h y o s m i u s . The e f f e c t o f the presence of c a r b o h y d r a t e s i n the - 86 - . b u f f e r medium has been shown to be dependent l a r g e l y upon the r a t e o f f e r m e n t a t i o n and subsequent a c i d p r o d u c t i o n t h a t t a k e s p l a c e . The ca r b o h y d r a t e s themselves do n o t m a t e r i a l l y a f f e c t the ammonia f o r m a t i o n from the v a r i o u s amino a c i d s but the f e r m e n t a t i o n a c i d s lower t h e pH of the c u l t u r e s t o l e v e l u n f a v o u r a b l e t o f u r t h e r b a c t e r i a l a c t i v i t y . I t was found t h a t t h e a d d i t i o n o f adequate b u f f e r t o the c u l t u r e s c o n t r o l l e d the hydrogen-ion c o n c e n t r a t i o n t h e r e b y p e r m i t t i n g b a c t e r i a l a c t i v i t y t o c o n t i n u e . T h i s a c t i v i t y i n c l u d e d not o n l y f u r t h e r deamination but a l s o the u t i l i z a t i o n o f the a v a i l a b l e carbon sources from t h e fermented c a r b o h y d r a t e s a l o n g w i t h t h e ammonia as a n i t r o g e n source f o r c e l l m u l t i p l i c a t i o n . A s t u d y has been made o f ammonia f o r m a t i o n from a number of non-amlno a c i d n i t r o g e n o u s compounds. I t has been shown t h a t i n a d d i t i o n t o the i m i n a z o l and p y r o l l i d i n e r i n g s , the b a c t e r i a l s p e c i e s employed h e r e i n have t h e a b i l i t y t o open t h e p y r i m i d i n e , p u r i n e and p y r i d i n e r i n g s w i t h subsequent f o r m a t i o n o f ammonia. A d e t a i l e d d i s c u s s i o n o f the breakdown o f the s i x amino a c i d s - a r g i n i n e , o r n i t h i n e , a s p a r t i c a c i d , g l u t a m i c a c i d , h i s t i d i n e and p r o l i n e - by the two s p e c i e s of b a c t e r i a - P r o t e u s i c h t h y o s m i u s . a n d Pseudomonas p u t r e f a c i e n s -has been g i v e n . A comparison o f the approach made t o the problem of d e a m i n a t i o n i n t h i s i n v e s t i g a t i o n w i t h t h a t made by o t h e r workers and the f a c t o r s i n v o l v e d i s o u t l i n e d . W i t h the o b j e c t o f i s o l a t i n g and i d e n t i f y i n g t h e - 87 -o c p o u n a or e x p o u n d s r e s p o n s i b l e f o r the sweaty-feet odour o f s u r f a c e t a i n t , a s e r i e s of e x p o n e n t s was undertaken, ( P a r t I I ) . 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Jour. 23: 472 (1929) Bacteriological Rev. 4: 135 (1940) Ann. Rev. Biochem. 5: 247 (1936) Botanioal Rev. 8: 1 (1942) 92 -ACKNOWLEDGMENTS I w i s h t o express my s i n c e r e a p p r e c i a t i o n t o Dr. B. A. E a g l e s f o r h i s c o n t i n u e d guidance and k i n d l y encouragement throughout t h i s s t u d y . I a l s o express my g r a t e f u l thanks t o Miss L o i s Campbell and to Miss F l o r e n c e Tamboline f o r s u g g e s t i o n s and h e l p w i t h the e x p e r i m e n t a l work. I acknowledge w i t h g r a t i t u d e r e s e a r c h g r a n t s from the U n i v e r s i t y o f B r i t i s h Columbia w h i c h made p o s s i b l e t h e u n d e r t a k i n g o f p a r t o f t h i s s t u d y ; 

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