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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 I N BUTTER  - by NORA E F F I E NEILSON  Pi  -\~U-a-St  s  Submitted In P a r t i a l F u l f i l l m e n t  of the Requirements  f o r t h e Degree o f ister of Science i n Agriculture i n the Department o f D a i r y i n g  / ft li.  OCTOBER, 1 9 4 3 .  I^l  !  C 0 H T E B T S *  1  •  1  1  1  ***  i  Pag® General Introduction . . . Part I  -  1  Studies on the De ami nation of Amino Acids . .  Historical  . . . e  Experimental (With Tables, Figures and Discussion)  10 10 29  General Methods and Procedures  29  The E f f e c t of pH on Ammonia Formation . » • «  52  The E f f e c t of the Age of Culture on Ammonia Formation  . . . . . .  37  The E f f e c t of Aerobic and Anaerobic Conditions on Ammonia Formation «.  38  The Rate of Ammonia Formation . . . . . .  40  . o  The E f f e c t of the Presence of Carbohydrates i n the Medium on Ammonia Formation « , . . . < >  41  Fermentation of Carbohydrates i n Shake-Agar Cultures . ....«,  41  The E f f e c t of the Presence of Carbohydrates i n the Growth and Buffer Media on Ammonia Formation . . . » . < . ...e  41b  The E f f e c t of Increasing the Buffering Capacity of the Cultures on Ammonia Formation and Subsequent C e l l Multiplication . . . . . . . . . . . . . . .  49  The Breakdown of Arginine by Proteus iohthyosmius and Pseudomonas putrefaciens .  56  e  Ammonia Formation from Non-Amino Acid Nitrogenous Compounds . Discussion  57 .  62  Page  Part I I -  Studies on the 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 Odouriferous Compounds •  Historical  . . •. .  Experimental Discussion  iksknowledgments  •  . . • . . . •  Summary and Conclusions  Bibliography  . •  • »  .  . . . . .  . . . .  • •  . . . . . . . .  7  4  7  6  8  ^  84  . . . . • •  <•  74  •  8  8  92  - 1• INTRODUCTION Surface defect  taint  i n butter f i r s t  i s t h e name g i v e n b y M a r k e r o b s e r v e d and  (48)  d e s c r i b e d by him  o f p a s t e u r i z e d A l b e r t a c r e a m e r y b u t t e r s o l d on t h e m a r k e t i n :1919.  The  defect  i s c h a r a c t e r i z e d by  odour d e s c r i b e d b y T h o r n t o n  (71)  as  t h a t of  which develops a f t e r exposure to the contaminated by present  as  shown b y  one  in a lot  Vancouver  a  typical  "sweaty-feet"  a i r on s a m p l e s o f  Taint B a c t e r i a " . (10)  T h i s g r o u p has  t o c o n t a i n among o t h e r 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 b y ichthyosmius  butter  Hammer (31)  two -  been species  Proteus  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  f i s h y o d o u r and  a  o r more o f a g r o u p o f b a c t e r i a known a t  "Surface  Campbell  to  a  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 o f w h i c h have more r e c e n t l y b e e n 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  taint butter  by  T h o r n t o n et a l . The d o u b t , t o be  odour of s u r f a c e due  to b a c t e r i a l a c t i v i t y .  however, about the  chemical  defect  Present  i n butter.  o r i g i n and  The  i s known,  development of  m e c h a n i s m o f i t s e l a b o r a t i o n i s , as  have been a d v a n c e d .  (9) and  a t i o n hypotheses of Campbell (50)  has  the  protein un-  however,  the  the d e c a r b o x y l a t i o n  "sweaty-  yet,  i t s formation,  O u t s t a n d i n g among t h e s e a r e  of Campbell  Neilson  Little  t a i n t o r i g i n a t e s i n the  known; s e v e r a l h y p o t h e s e s c o n c e r n i n g  hypothesis  b e e n shown, b e y o n d  e v i d e n c e i n d i c a t e s t h a t the  f e e t " odour of t y p i c a l s u r f a c e of b u t t e r .  t a i n t has  indole  and  deamin-  (10).  shown t h a t t h e f a i l u r e  to  detect  o „ i n d o l e i n the s e r a  of s u r f a c e  taint butters  is practically  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 production  of s u r f a c e  The  i n v e s t i g a t i o n s of Campbell obtained  s i s t h a t amines a r e formed d u r i n g  not  Indicate  The and  considered  hypothesis  unsaturated  the  that decarboxylative  a f a c t o r t o be  that  (10)  i n which experi-  to support, the  hypothe-  e l a b o r a t i o n of  surface  breakdown i s p r o b a b l y  i n this  study.  the f o r m a t i o n  a c i d s f r o m amino a c i d s by  of h y d r o x y , the  keto  deaminating  a c t i o n 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 was  advanced  Campbell  mechanism  (10)  responsible i n surface  as a p o s s i b l e e x p l a n a t i o n  f o r the  e l a b o r a t i o n of the  taint butter.  u p o n h e r e i n has  the  taint i n butter.  m e n t a l e v i d e n c e c o u l d not be  taint,  e s s e n t i a l to  The  object  of the  "sweaty-feet"  of the work  been to determine the  by  odour  reported  v a l i d i t y of t h i s  hypo-  thesis The cerning are  early studies  r e c o r d e d i n the l i t e r a t u r e  t h e n a t u r e o f t h e m i c r o b i a l b r e a k d o w n o f amino  of l i t t l e  scientific  value  were f o l l o w e d b y  p r o t e i n decomposition products.  These  a p e r i o d of i n v e s t i g a t i o n s employing  a c t i o n o f m i x e d c u l t u r e s on p u r e a m i n o a c i d s , b u t between the  a c t i o n of any  one  o r g a n i s m and  t y p e o f p r o d u c t f o r m e d c o u l d n o t be d u c t i o n by for  the  H a r d e n (1901) and  acids  because t h e y were c a r r i e d  w i t h m i x e d c u l t u r e s o f 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 of p r o t e i n s and  con-  the  mixtures studies the relation  particular  established.  by E h r l i c h  the  out  (1905) o f  The  intro-  procedures  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 p u r e amino a c i d s , i n i t i a t e d  the  modern  ~ 3 method o f a p p r o a c h t o i n v e s t i g a t i o n s on amino a c i d  catabolism-  The e a r l y t h e o r i e s o f d e a m i n a t i o n o f N e u b e r g a n d L a n g s t e i n and o f Embden p o s t u l a t e d t h a t t h e f o r m a t i o n  of alpha-  h y d r o x y a c i d s w i t h t h e 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 t h e h y d r o l y t i c d e a m i n a t i o n o f amino a c i d s .  More r e c e n t  evi-  d e n c e , h o w e v e r , i n d i c a t e s t h a t d e a m i n a t i o n o f amino a c i d s i s not  l i m i t e d t o one t y p e  number o f c o u r s e s acid,  o f b r e a k d o w n b u t may f o l l o w one o f a  d e p e n d e n t u p o n t h e s t r u c t u r e o f t h e amino  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 o f t h e r e a c t i n g medium ( 6 1 ) . The v a r i o u s p a t h s o f amino a c i d b r e a k d o w n h a v e b e e n b r o u g h t t o g e t h e r b y A n d e r s o n (2) and h i s c h a r t o f 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 presented  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 o f amino a c i d s  f o l l o w one o f two c o u r s e s , As s t a t e d e a r l i e r , i n support  decarboxylation or  Campbell  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 ,  this  deamination.  (10) was u n a b l e t o o b t a i n e v i d e n c e  of. t h e h y p o t h e s i s  t i v e breakdown i s p r o b a b l y  may  indicating  that  decarboxyla-  n o t a f a c t o r t o be c o n s i d e r e d i n  study. O t h e r s t u d i e s b y t h e same a u t h o r  h a v e shown t h a t  aeration increases  t h e d e v e l o p m e n t of s u r f a c e  thereby  t h a t the mechanism f o r t h e breakdown of  suggesting  taint i n butter,  a m i n o 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 b e o x i d a t i v e i n nature.  • The f i n d i n g s o f N e i l s o n  which organic  ( 5 0 ) from experiments i n s  compounds t h a t c o u l d r e s u l t f r o m t h e b r e a k d o w n  •Pi  o ra U ©  W •H O  «}  e tri  H •ri  o ri  H"  1 O  o t3  © o •ri rQ O ?H  <  o 03 m  o 03 O o  o  so  c\3 o  to  SI  o o o « K o  w o  o  o o o'  •o o  «  M o o o  S3 o  o  o o o o  O  o o •n o  •ri  o W-P O CD  a5 1  g  Gi  O I •  ra  «  .8 8 ra-  .  pt  Pt  ra  +  K o o o  O A. O Pi •m" rH < > c3 03—' w — o  ra o o o  o ra  o  CV3 tj W. o  o o  ©  © ^  O -P • pt  M  o o  ©  rH  o o  o +H P4 < ©  03  i  O O  o o  ra + m 8 o ra o W.  •  P3  ra >•  o o  o o  M  to W o Pt  id a! rri O CO •ri  ri ©  >»  rH o  43  ra  d ,ri  o  • S'  © ri •ri  ©  rH -ri  ©  EH  ,ri  of  c e r t a i n a m i n o 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 test  e m i t t e d "by them d e t e r a i i n e d b y  Thornton's  (71) a d d 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 t h a t t h e s u r -  face taint  odour i s i n t i m a t e l y r e l a t e d t o the  decomposition  p r o d u c t s o f p a r t i c u l a r amino a c i d s . The  first  s t e p i n t h e d e a m i n a t i o n o f 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 t h e a l p h a - b e t a - l i n k a g e w i t h t h e f o r m a t i o n o f u n s a t u r a t e d a c i d s a n d t h e l i b e r a t i o n o f ammonia. F r o m t h i s s t a g e , t h e b r e a k d o w n may three  i n one  o r more o f  directions. The  acids.  proceed  The  first odours  of these i s h y d r o l y s i s g i v i n g  alpha-hydroxy  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  t a i n amino a c i d s a r e known t o r e s e m b l e f r o m p u t r e f a c t i v e m a t t e r and s u g g e s t  cer-  c l o s e l y those evolved  their possible  relation-  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  third  c o u r s e s of breakdown f r o m  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 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 acids respectively.  s u r f a c e t a i n t odour.  i n the  beta-keto  A g a i n t h e r e ^ i s the p o s s i b i l i t y  k e t o a c i d s a r e i n some way  that  the  i n v o l v e d i n the e l a b o r a t i o n of  the  D e c a r b o x y l a t i o n of these keto acids  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 lysis  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 .  a c i d s and a l d e h y d e s must a l s o b e istic  the  hydro-  These  fatty  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  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 -  " s w e a t y - f e e t " odour of s u r f a c e t a i n t  butter.  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 hypot h e s i s i s c o n t a i n e d i n t h e r e p o r t s o f D u n k l e y a n d Wolchow-  - 5 .The  f i n d i n g s o f D u n k l e y (12)  i n d i c a t e t h a t the  formed i n the development of s u r f a c e nature.  Wolchow (71) has  d u c t i v e d e a m i n a t i o n may surface taint The  taint  compounds  are a c i d i c  in  s u g g e s t e d t h a t the p r o d u c t s of r e -  he  concerned w i t h the  e l a b o r a t i o n of  i n butter. s t r u c t u r e of the  amino a c i d i s one  prin-  c i p a l f a c t o r s - determining  the n a t u r 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 that long  e a s i l y attacked t h a t the  The  that three able  deaminative  c h a i n amino a c i d s a r e more  than 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 ,  ease of a t t a c k i n c r e a s e s w i t h  chain.  of the  of t h e  f i n d i n g s of N e i l s o n species  t o open t h e  the l e n g t h of  ( 5 0 ) , h o w e v e r , show  of s u r f a c e - t a i n t producing  ability  i m i n a z o l r i n g o f h i s t i d i n e and  l y s i n e and  f r o m the  the  - g l y c i n e and  beta  a l a n i n e - to a greater  above mentioned l o n g e r .The important  only i n determining  contains  very l i t t l e  difficult  i t was The  modifies  data  on t h i s p o i n t .  ( 5 0 ) , the above a u t h o r o b t a i n e d the a m i n o  g r o u p was  or changes the  the  also play  The  I n the  an  of the  break-  literature same  evidence that  investithe  f r o m t h e a l p h a - p o s i t i o n , t h e more  to deaminate by  presence of  c h a i n amino a c i d s  the n a t u r e  a l s o the ease w i t h which i t occurs -  further  - leucine,  c h a i n amino a c i d s .  down b u t  gation  are  pyrolli-  degree t h a n  p o s i t i o n o f t h e a m i n o g r o u p may  r o l e not  clearly  ammonia-  s t r a i g h t c h a i n amino a c i d s  m e t h i o n i n e ; and a t t a c k t h e s h o r t  the  bacteria  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 forming  and  the b a c t e r i a l s p e c i e s  carbohydrates i n the  employed.  r a c t i n g medium  c o u r s e o f amino a c i d c a t a b o l i s m .  A  -6 number o f t h e o r i e s h a v e b e e n a d v a n c e d - t o e x p l a i n t h i s vation.. that  obser-  K e n d a l l and h i s a s s o c i a t e s ( 3 9 , 40 a n d 41) s t a t e  the presence  of carbohydrate  "spares" p r o t e i n from 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 o f f o o d , but,  as S t e p h e n s o n (61) p o i n t s o u t , t h i s  false and  comparison  b e t w e e n t h e b a c t e r i a l p r o d u c t i o n o f ammonia  t h e mammalian p r o d u c t i o n o f u r e a .  duct w h i c h i n which  cannot  i t was p r o d u c e d ,  causes  Urea i s a waste p r o -  serve as a n i t r o g e n o u s food f o r the animal t h e r e f o r e i t s r i s e and f a l l  t r u e measure o f p r o t e i n m e t a b o l i s m bohydrate  c l a i m r e s t s on a  decreased  a n d when i n c r e a s e d c a r -  p r o d u c t i o n of urea, the carbohy-  drate i s said to "spare" the p r o t e i n .  With b a c t e r i a ,  w h i l e ammonia i s t h e c h i e f n i t r o g e n o u s p r o d u c t decomposition  of protein,  is a  however,  of the b a c t e r i a l  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  s o u r c e f o r many s p e c i e s o f b a c t e r i a .  Therefore  a n c e f r o m t h e c u l t u r e - m e d i a may b e c a u 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 ,  i t s disappear-  e i t h e r by  by increased  decreased  utilization  as a n i t r o g e n s o u r c e f o r c e l l r e p r o d u c t i o n . Raistrick the presence  (57) i n a n e x p l a n a t i o n o f t h e e f f e c t o f  of carbohydrates  concludes  with the b e l i e f  that  carbohydrate, f a r from having 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 enables t e i n products  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 -  than t h e y would i n t h e absence o f c a r b o h y d r a t e .  Waksman a n d L o m a n i t z b e i n g d e r i v e s i t s energy  (68) p o i n t o u t t h a t a  from the substance  which  living  i s more  a v a i l a b l e t o i t a n d w h i c h may be s p e c i f i c f o r t h e p a r t i c u l a r organism.  ' - 7 The i n g s are  studies  g i v e n i n the  c l e a r l y that  the  down and  o f a number of i n v e s t i g a t o r s historical  addition  r e a c t i n g medium w i l l  p r o b l e m was  first  part  as of  d e v o t e d t o an the  of t h i s t h e s i s  yet the  A satisfactory explanation  e x p e r i m e n t a l work of  i n v e s t i g a t i o n of the  ammonia p r o d u c i n g a b i l i t i e s producing b a c t e r i a  amino a c i d s .  These amino a c i d s  g l u t a m i c a c i d , h i s t i d i n e and  - arginine,  proline  aspartic  The  Certain  - were c h o s e n f o r  a  are: by  bacteria.  are  related  seen i n f i g u r e  t o one  another  as  2.  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  b e e n shown t o e m i t o d o u r s t h a t  Prom t h e  of  d e c o m p o s i t i o n p r o d u c t s of these f i v e  can be  the  of  acid,  Ammonia i s r e a d i l y l i b e r a t e d f r o m them  amino a c i d s  of amino a c i d s  species  from a s p e c i f i c group  s p e c i e s of s u r f a c e t a i n t  (3)  this  conditions  o f two  r e a s o n s , t h e more i m p o r t a n t o f w h i c h  (2)  the  forthcoming.  surface-taint  (1)  show  c o u r s e o f amino a c i d b r e a k -  give different products.  The  number o f  section  whose f i n d -  of a s p e c i f i c carbohydrate to  change the  of t h e s e r e s u l t s i s n o t ,  affecting  ~  c l o s e l y resemble  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 point  o f v i e w of s t u d y i n g t h e  of d i f f e r e n t b a s i c  structure,  the  (50).  deamination five  chosen  represent three s t r u c t u r a l groups. Arginine  i s a c o m p l e x s t r a i g h t c h a i n amino a c i d  t a i n i n g four nitrogens i n three d i f f e r e n t groupings. guanido group a t t a c h e d to the l y s e d f r o m the  delta  c a r b o n a t o m may  remaining alpha-amino v a l e r i c a c i d -  con-  The be The  hydro-  - 8 literature age .  The  c o n t a i n s p r a c t i c a l l y no e v i d e n c e f o r t h i s  u s u a l method of d e c o m p o s i n g a r g i n i n e i s t h r o u g h  enzyme, a r g i n a s e ,  to g i v e urea  diamino v a l e r i c acid) . l y s e d by and  cleav-  The  and  o r n i t h i n e (alpha,  u r e a may  t h e enzyme, u r e a s e ,  be  delta,  subsequently  i n t o ammonia and  the  hydro-  carbondioxide  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 t h e a l p h a  or  the  d e l t a amino g r o u p o r b o t h t o g i v e v a l e r i c a c i d o r one  of i t s  derivatives• The  n e x t two  amino a c i d s c h o s e n f o r t h i s  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 , a r e mono-amino, d i c a r b o x y l i c  a c i d s , t h e l a t t e r - c o n t a i n i n g one c h a i n between the The  more m e t h y l g r o u p i n t h e  c a r b o x y l groups than the  last  two  study,  former.  amino a c i d s c o n t a i n f i v e - e l e m e n t  structures.  H i s t i d i n e or alpha-amino b e t a - i m i n a s o l  a c i d has  nitrogens  two  i n t h e r i n g and  one  ring  propionic  i n the s i d e  chain.  P r o l i n e , on t h e o t h e r h a n d , i s n o t a t r u e amino a c i d , as i t s one  nitrogen i s present As  the f i r s t  became e v i d e n t  part of t h i s  group.  study developed, i t soon  compounds g i v i n g o d o u r s s u g g e s t i v e  of s u r f a c e  taint.  I t was  to devote a p a r t of the e x p e r i m e n t a l i s o l a t i o n and,  imino  t h a t c e r t a i n o f t h e , c h o s e n amino a c i d s w e r e  b r o k e n down i n t o characteristic  i n t h e r i n g as a n  decided,  study to the  of  that  therefore, actual  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 o r m i n g 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 of the  compounds r e s p o n s i b l e f o r t h e  acteristic  "sweaty-feet"  e l a b o r a t i o n of the  odour of s u r f a c e  f o l l o w i n g a p p r o a c h e s were made t o t h e  the  taint butter,  problem:  nature charthe  „  (1)  g >,  An I n v e s t i g a t i o n of t h e c o n d i t i o n s ammonia f o r m a t i o n f r o m a r g i n i n e , glutamic acid, h i s t i d i n e , species  (2)  of s u r f a c e - t a i n t  A s t u d y of the p r o d u c t i o n ,  affecting  aspartlc  a n d p r o l i n e b y two producing b a c t e r i a 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 identical with  acid,  odours  or i n t i m a t e l y r e l a t e d to the  c h a r a c t e r i s t i c surface  taint  odour.  - 10-~ !  PART I-  ;  STUDIES ON TEE D E A M I M T I O H OF AMINO A C I D S . ij  • . HISTORICAL: A study of the decomposition of i n d i v i d u a l  amino  a c i d s by. p u r e c u l t u r e s o f s i n g l e s t r a i n s o f m i c r o o r g a n i s m s h a s b r o u g h t o u t 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 o f  t h e medium, t h e c o n d i t i o n s o f g r o w t h a n d t h e t y p e o f o r g a n i s m e m p l o y e d h a v e be.en shown t o e x e r t a m a r k e d i n f l u e n c e on t h e a c t i v i t y o f t h e enzymes f o r m e d a n d on t h e t y p e o f d e a m i n a t i o n that  takes p l a c e . / H y d r o l y t i c deamination with the formation of the  h y d r o x y a c i d may r e s u l t f r o m t h e a c t i o n o f 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  s o u r c e s a n d f r o m t h e a c t i v i t y o f 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 . P e t e r s o n a n d 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 employing Proteus v u l g a r i s .  Hirai  (60) i s o l a t e d  1-leucine  (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 o m h i s t i d i n e u s i n g B. p r o t e u s . Otsuka  Schmidt,  S a s a k i and  t h e h y d r o x y "acids o f 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 e m p l o y i n g B. p r o t e u s , Band B. c o l i .  Hirai  ( 3 5 ) , u s i n g an a l t e r n a t i v e  subtilis  carbon source,  g l y c e r o l , a n d a n 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 bonate, i s o l a t e d p-hydroxy beta-phenyl l a c t i c a c i d t y r o s i n e employing Proteus v u l g a r i s . (19), employing Oidium l a c t i s ,  from  E h r l i c h and Jacob3en  obtained the hydroxy acids of  1-phenyla1anine, 1-tryptophan and 1 - t y r o s i n e .  W o o l f (77)  i s o l a t e d t h e h y d r o x y a c i d o f a s p a r t i c a c i d e m p l o y i n g B. c o l i  - 11 i n a medium w i t h no a l t e r n a t i v e c a r b o n a n d n i t r o g e n 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 g r o w t h  source  inhibitor.  H y d r o l y t i c d e a m i n a t i o n a n d d e c a r b o x y l a t i o n may r e sult  i n the formation  o f a n a l c o h o l w i t h one l e s s c a r b o n atom  than the corresponding  amino a c i d .  caused p r i n c i p a l l y b y yeasts 16,  T h i s type  and m o l d s -  of breakdown i s  Ehrlich  17 and 1 8 ) , i n a s t u d y o f t h e d e c o m p o s i t i o n  by y e a s t s , obtained  the corresponding  ( 1 3 , 14, 1 5 , o f amino a c i d s  alcohols from 1-valine,  1-leucine, d l - s e r i n e , 1 - h i s t i d i n e , 1-phenylalanine, phan and 1 - t y r o s i n e .  Pringscheim  1-trypto-  ( 5 3 ) , e m p l o y i n g a number o f  c u l t u r e s o f molds, a n d one o f y e a s t ,  i s o l a t e d the a l c o h o l from  1-leucine. Reductive  deamination 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 from the corresponding 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  c a r b o n and n i t r o g e n s o u r c e s .  Brasch  f i c u s i n . s u c h a medium, o b t a i n e d glycine,  1-alanine,  amino  ( 8 ) , e m p l o y i n g B. p u t r i -  the corresponding  d l - s e r i n e , 1-aspar t i c  w h i l e N a w i a s k y ( 4 9 ) , u s i n g B. p r o t e u s ,  acids  from  a c i d and 1 - t y r o s i n e ,  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 t h e 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 a n d Hanke ( 4 4 ) a d d e d g l y c e r o l t o t h e medium o f Brasch  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  d i n e e m p l o y i n g B. c o l l .  Hopkins and Cole  (36) o b s e r v e d  i n d o l e p r o p i o n i c a c i d was f o r m e d f r o m t r y p t o p h a n containing inorganic salts with Rochelle p h o s p h a t e u s i n g B• c o l i . p-hydroxy beta-phenyl  Traetta-Mosca  histithat  i n a medium  s a l t s a n d ammonium ( 6 6 ) h a s shown t h a t  p r o p i o n i c a c i d i s formed from t y r o s i n e  i n a n 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 b y a  - 12 "bacillus resembling beta-iminazol lactis  B. p y o c y a n e u s .  Kiyokava  (42)  obtained  p r o p i o n i c a c i d from h i s t i d i n e employing  i n an i n o r g a n i c s a l t medium p l u s Reductive  i n the f o r m a t i o n  d e a m i n a t i o n and  of  t a i n s o n l y one  one  percent  d e c a r b o x y l a t i o n may  amino a c i d .  r e c o r d of t h i s  The  sugar.  result  c a r b o n atom  literature  type of breakdown brought  t h e a c t i o n o f p u r e c u l t u r e s on amino a c i d s -  e m p l o y i n g B. p u t r i f i c u s  Oidium  cane  t h e n o r m a l ; a c i d c o n t a i n i n g one  l e s s t h a n the c o r r e s p o n d i n g  by  -  Brasch  conabout (8),  i s o l a t e d p r o p i o n i c a c i d from 1-glutamic  a c i d i n a medium o f i n o r g a n i c salts» Desaturation m a t i o n of the  a t the a l p h a - b e t a  corresponding  unsaturated  t i o n o f a m o l e c u l e o f ammonia.  a c i d f r o m 1 - h i s t i d i n e i n 1917  instance  of the b a c t e r i a l  five  acid.  species  solution.  He  conversion  Hirai  and  libera(55)  recorded  employed the a c t i o n of pure c u l t u r e s  of  isolated  the a c r y l i c a c i d of  d a y s i n c u b a t i o n o f B.  Woolf  Ringer's  p r o t e u s i n an i n o r -  a l t e r n a t i v e c a r b o n and  (54)  obtained  fumaric  The the f o r m a t i o n  nitrogen  a c i d from  a s p a r t i c a c i d i n a p h o s p h a t e b u f f e r medium c o n t a i n i n g as a g r o w t h  of  an  ( 3 5 ) , i n 1921,  Quastel  for-  o f a n amino a c i d i n t o  s a l t medium c o n t a i n i n g no  sources.  i s the f i r s t  o f 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  - tyrosine a f t e r twelve ganic  a c i d w i t h the  Raistrick's isolation  urocanic  unsaturated  l i n k a g e causes the  toluene  inhibitor. o x i d a t i v e b r e a k d o w n o f a m i n o a c i d s may o f a number o f a c i d s  number o f c a r b o n a t o m s .  The  keto  containing a  result in  decreasing  a c i d s formed f r o m the  primary  o x i d a t i o n o f t h e amino a c i d s have n o t b e e n i s o l a t e d e x c e p t i n rare  cases, but  the a c i d s f r o m the  subsequent o x i d a t i o n of  the  -13k e t o a c i d s o f c e r t a i n amino a c i d s have b e e n o b t a i n e d tified.  Nawiasky  and i d e n -  ( 4 9 ) , e m p l o y i n g B. p r o t e u s i n a n 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 c a r b o n a n d n i t r o g e n sources,  i s o l a t e d a c e t i c a c i d from 1-alanine, i s o b u t y r i c ,  a c e t i c and f o r m i c  acids from 1-valine, a c e t i c a c i d from  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 from 1-glutamic a c i d Brasch  (8) i s o l a t e d f o r m i c  rificus ky.  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 -  i n a n 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 o f N a w i a s -  Raistrick  ( 5 6 ) has shown t h a t o r g a n i s m s o f t h e c o l i -  t y p h o s u s g r o u p i n a medium o f i n o r g a n i c s a l t s w i t h no a l t e r native  c a r b o n and n i t r o g e n s o u r c e s , v  of h i s t i d i n e .  Hopkins and C o l e  rupture  the i m i n a z o l  ring  (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 t h e o x i d a t i v e breakdown o f t r y p t o p h a n B» c o l i  i n an Inorganic  and  ammonium p h o s p h a t e .  and  Woods (73) i s o l a t e d  sions and  o f B. c o l i  Clarke  s a l t medium c o n t a i n i n g R o c h e l l e  salts  Woods ( 7 2 ) , H a p p o l d a n d H o y l e (32) i n d o l e , employing, washed c e l l  on t r y p t o p h a n  (57) o b t a i n e d  indole r i n g of tryptophan  i n buffer solution.  evidence f o r the r u p t u r e b y B. p y o c y a n e u s a n d B.  i n a n i n o r g a n i c s a l t medium. 1-tyrosine  by  suspen-  Raistrick  of t h e flourescens  The o x i d a t i v e b r e a k d o w n o f  v a r i e s w i t h the composition  Moaca (66) i s o l a t e d p - h y d r o x y b e n z o i c  o f t h e medium.  Traetta-  a c i d and p - c r e s o l  from  t y r o s i n e e m p l o y i n g B. p y o c y a n e u s i n a n 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 .  (58 a n d  59)  obtained  Berthelot  evidence f o r the formation  (6) and R h e i n  o f p h e n o l b y B. p h e n o -  l o g e n e s f r o m t y r o s i n e i n a n i n o r g a n i c s a l t medium ammonium l a c t a t e a n d asparagin»  Hirai  containing  ;(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 e m p l o y i n g t h e same c o n -  -14d i t i o n s as f o r t h e i s o l a t i o n o f p - h y d r o x y a c i d , except u s i n g f o r t y days' days.  Raistrick  heta-phenyl  acrylic  incubation i n place of twelve  and C l a r k e ( 5 7 ) f o u n d 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 f r o m t h e  r i n g o f 1 - t y r o s i n e i n a medium o f I n o r g a n i c s a l t s  employing  B. p y o c y a n e u s and B. f l o u r e s c e n s , h u t t h e y were u n a b l e t o i d e n t i f y the products The  formed*  n a t u r e o f t h e b a c t e r i a l b r e a k d o w n o f amino a c i d s  may be d e t e r m i n e d b y methods o t h e r t h a n t h e  actual  Of d e c o m p o s i t i o n p r o d u c t s e m p l o y e d i n t h e e a r l y  isolation  investiga-  t i o n s on t h i s s u b j e c t . Bernheim, Bernheim and Webster (4) measured t h e oxygen uptake  and c a r b o n - d i o x i d e , output e m p l o y i n g  plrometer.  They d e t e r m i n e d  t h e ammonia f o r m e d t h r o u g h t h e  accompanying deamination by d i s t i l l i n g of  t h e Warburg r e s -  i t from t h e contents  t h e Warburg v e s s e l a t t h e c o m p l e t i o n o f t h e e x p e r i m e n t  into a Nessler's solution.  The ammonia p r e s e n t was t h e n d e -  termined c o l o r i m e t r i c a l l y .  In a study of the oxidation o f  c e r t a i n amino a c i d s b y P r o t e u s v u l g a r i s , e m p l o y i n g  t h e washed  c e l l technique 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 f o u n d t h a t , a t 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 . n i n e and m e t h i o n i n e of  The  a r e o x i d i z e d r a p i d l y and u t i l i z e  oxygen p e r m o l e c u l e  and p r o l i n e u t i l i z e  Leucine, phenylalaone atom  o f amino a c i d w h i l e s e r i n e , a l a n i n 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 .  o x i d a t i o n o f t y r o s i n e a n d 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 o x y g e n r e s p e c t i v e l y .  Valine,  isoleucine,  h y d r o x y p r o l i n e a n d h i s t i d i n e a r e o x i d i z e d s o s l o w l y t h a t no  -15d e f i n i t e u p t a k e s o f o x y g e n were o b t a i n e d , ,  T h e i r work showed  t h a t o n l y t h e n a t u r a l i s o m e r s were o x i d i z e d e x c e p t i n t h e c a s e o f a l a n i n e and s e r i n e , b o t h i s o m e r s o f w h i c h were  oxi-  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 c o r r e s p o n d s w i t h t h e 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 b y washed c e l l  s u s p e n s i o n s o f B. p y o c y a n e u s ,  they  f o u n d 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 w e r e n o t a t t a c k e d , a n d that  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  and t h e e x t e n t t o w h i c h t h e a t t a c k e d amino a c i d s w e r e dized.  rates  oxi-  They showed t h a t t h i s o r g a n i s m o x i d - i z e d a n d d e a m i n a -  t e d both isomers of alanine,  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 , histidine.  i s o l e u c i n e and  They s u g g e s t e d 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 o f  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 e a c h a m i n o a c i d a n d t h a t t h e amino a c i d s a r e a t t a c k e d d i f f e r e n t l y b y t h e d i f f e r e n t b a c t e r i a , o r , more probably, 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 c o m p l e x i s d e t e r m i n e d b y t h e 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 a n d C l i f t o n  (75), i n the next year,  employing  t h e Warburg technique 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, f o u n d t h a t g l u t a m a t e , a s p a r t a t e ,  cysteine,  cystine  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 h y d r o g e n  and/or carbon-dioxide  and  They a l s o , o b s e r v e d  were a l m o s t c o m p l e t e l y d e a m i n a t e d .  t h a t h i s t i d i n e p r o d u c e d more t h a n one m o l e c u l e o f ammonia p e r amino group p r e s e n t a n d c o n c l u d e d t h a t t h e i m i n a z o l r i n g h a d been opened.  -16I n a s t u d y .'of t h e m e t a b o l i s m o f t h e amino a c i d s Clostridium  w e l c h i i , Woods and T r i m  organism attacks studied.  (76) observed t h a t  o n l y f i v e o f t h e t w e n t y - o n e amino  by  this  acids  S e r i n e g a v e h y d r o g e n , c a r b o n - d i o x i d e a n d ammonia I n  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 breakdown.*  Cystine/  s i m i l a r l y t o serine  cysteine  a coenzyme f o r i t s  a n d t h r e o n i n e were  but the evidence f o r the  coenzyme f a c t o r was i n c o m p l e t e .  Arginine  attacked  need of a  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 , t h e f i n a l y i e l d s o f w h i c h v a r i e d w i t h t h e age o f t h e growth c u l t u r e . f o r m e d and a coenzyme was n o t  H y d r o g e n was n o t  required.  A r e c e n t i n v e s t i g a t i o n b y N e i l s on- ( 5 0 ) , the  employing  Van.Slyke a e r a t i o n procedure f o r the determination of  ammonia, i n d i c a t e d ichthyosmius glycine  t h a t washed c e l l  (Hammer.) a t t a c k e d b e t a - a l a n i n e ,  and 1 - p r o l i n e  d-glutamic a c i d , d-lysine  suspensions of Proteus  vigorously;  d-arginine,  dl-aspartic acid,  1-histidine, dl-Isoleucine,  1-leucine,  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 ;  methionine, phenylalanine, tryptophan, tyrosine, 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 slightly.  1-cystine,  Similar  urea,  acid  s u s p e n s i o n s o f Pseudomonas  and delta-  only  putrefaciens  (Hammer).attacked h i s t i d i n e and p r o l i n e  vigorously;  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 ,  arginine,  lysine,  cystine,  isoleucine,  leucine,  aspartic  methionine,  p h e n y l a l a n i n e , tryptophan, t y r o s i n e , urea, beta-amino 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 only  butyric  amino c a p r o i c  acid  slightly. The m u t u a l 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  -17a c i d s has (63 and  been s t u d i e d by  64)  S t i c k l a n d and Woods.  o b s e r v e d t h a t washed c e l l  Stickland  suspensions of  Clostri-  dium s p o r o g e n e s a c t i v a t e d c e r t a i n amino a c i d s as h y d r o g e n 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  activated  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 The h y d r o g e n - a c c e p t i n g amino a c i d s a r e  others  hydroxyproline.  subjected  to  reduction  by t h e h u d r o g e n - d o n a t i n g amino a c i d s w h i c h i n t u r n a r e  oxi-  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 f r o m o x i d a t i v e deamination.  Woods (74) h a s  o b s e r v e d t h a t 1 - c y s t e i n e may  as h y d r o g e n - d o n a t o r i n c o u p l e d  r e a c t i o n s b e t w e e n amino  i n d u c e d by C l o s t r i d i u m s p o r o g e n e s and i s p a r t i a l l y deaminated i n the He  a l s o has  vated the  as h y d r o g e n - a c c e p t o r s and  and  acids  t h a t , In addition,, i t  absence o f o t h e r  shown t h a t d - a r g i n i n e  act  amino a c i d s .  d - o r n i t h i n e are  acti-  are p a r t i a l l y deaminated i n  absence of hydrogen-donators.  When o r n i t h i n e r e a c t s  t h e don a t o r , a l a n i n e , i t a c c e p t s , two  h y d r o g e n s and  with  undergoes  reductive deamination to delta-amino 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 the r e s t i n g technique data  o r enzymic e x t r a c t s have brought f o r t h  on d e a m i n a t i o n and  only the  t o t h e i r b a c t e r i a l b r e a k d o w n w i l l he  detail.  the  particular reviewed i n  • T h e - s t u d y of t h e  washed c e l l W o o l f (54)  deamination of a s p a r t i c a c i d  s u s p e n s i o n s o f E. revealed  coli  c a r r i e l out  is present,  the  by  by Q u a s t a l  t h a t the p r o d u c t i n the absence of  i n h i b i t o r i s s u c c i n i c a c i d , w h e r e a s , i f an toluene  considerable  l i t e r a t u r e concerning  f i v e amino a c i d s s t u d i e d i n t h i s p r o b l e m w i t h reference  cell  inhibitor  r a t e of d e a m i n a t i o n i s not  and  any  such  as  affected  -18i n the b e g i n n i n g but reaching, instead, f u m a r i c a c i d and  t h e p r o c e s s does n o t  an  equilibrium  go  to  completion,  mixture of a s p a r t i c  acid,  ammonia.  In a study of the  fermentation of glutamic a c i d  a s t r i c t l y anaerobic, spore-forming bacterium, probably longing ted that  to- t h e  genus P e o t o c l o s t r i d i u m ,  t h i s a c i d was  Barker  decomposed p r a c t i c a l l y  (3)  quantitatively  coli,  ( 1 ) , e m p l o y i n g a d e a m i n a s e enzyme  found that  glutamic acid,  i n the  c o e n z y m e s , i s decomposed i n t o k e t o g l u t a r i c and  suggested that  t h e b r e a k d o w n was  a c i d to k e t o g l u t a r i c The and  both acids  a c i d w i t h the  work o f K l e i n  1-glutamic acids  dioxide;  one  (43)  molecule of  iminoglutaric  the  ammonia.  oxidation  of1-aspartic  molecule  a c i d r e q u i r i n g one  of c a r b o n - d i o x i d e , and -two  and  a c i d by  mole-  By  a  study  i n t e r m e d i a t e compounds, K l e i n  p r o b a b l e course of o x i d a t i o n  t h i s organism proceeds through  a c i d and • a c e t a l d e h y d e t o  Ammonia f o r m a t i o n f r o m p r o l i n e amount's g r e a t e r t h a n t h a t  one  one-half molecules  producing three of carbon-dioxide.  t h a t the  that  aspartic  p r o d u c i n g two  established  acid, pyruvic  through  ammonia  carbon-  o f t h e m e t a b o l i s m of p o s s i b l e  aspartic  a c i d and  of  t o a c e t i c a c i d , ammonia and  cule of glutamic a c i d r e q u i r i n g o f o x y g e n and  presence  l i b e r a t i o n of  on  extracted  by H e m o p h i l u s p a r a i n f l u e n z a e showed  were o x i d i z e d  o f o x y g e n and  has  and  acid. Adler  f r o m E.  be-  demonstra-  i n t o ammonia, c a r b o n - d i o x i d e , h y d r o g e n , a c e t i c a c i d butyric  by  g i v e n by  opening of a f i v e - e l e m e n t r i n g ,  acetic  of  oxalacetic acid.  or f r o m h i s t i d i n e  i t s amino g r o u p  involves  the p y r o l l i d i n e i n the  case  in  -19o 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 , d i f f i c u l t to accomplish  a feat  extremely  In•non-biological chemistry.  t i g a t i o n s i n b i o c h e m i s t r y , h o w e v e r , h a v e shown t h a t enzymes, p a r t i c u l a r l y  those  and  others  ( 2 0 , 21 and  nitrogen  22), In studies of  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 h i s t i d a s e obtained from animal o f h i s t i d i n e gave two one  capable  o f t h e amino group f o r m e d .  Eldbacher  probably  certain  of b a c t e r i a l o r i g i n , are  o f opening these' r i n g s p r e p a r a t o r y to removing the by deamination  Inves-  liver,  o f ammonia, one  f o u n d t h a t one  Iminazol nucleus  molecule  of glutamic a c i d  of f o r m i c a c i d , the primary  b e i n g t o open the  ensyme  a c t i o n of the  t o f o r m an a l p h a ,  and enzyme  delta,  d i a m i n o ,chain.. The  i n v e s t i g a t i o n of R a i s t r i c k  b a c t e r i a l decomposition B,  B.  (56) i n t o t h e  o f h i s t i d i n e by B.  f a e c a l i s a l c a l i g e n e d , B.  aerobic  paratyphosus A  p y o c y a n e u s and B. p r o t e u s  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 ammonia f r o m t h e n i t r o g e n i n b o t h  and vul-  organisms produced  t h e s i d e - c h a i n and  Iminazol nucleus, p r o v i n g t h a t they are able  the  t o open t h e  I m i n a z o l r i n g ; w h e r e a s t h e f i f t h o r g a n i s m f o r m e d ammonia o n l y from the s i d e c h a i n n i t r o g e n , showing i t s probable to  split  the  inability  ring.  Stickland  ( 6 5 ) , t h r o u g h h i s e x p e r i m e n t s on  the  c o u p l e d o x i d a t i o n a n d r e d u c t i o n b y p a i r s o f amino a c i d s , 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  of C l o s t r i d i u m sporogenes  reduced 1 - p r o l l n e , at the  expense o f t h e o x i d a t i o n o f  1-alanine,  n - v a l e r i c acid, but  to delta-amino  nate t h i s product,  ob-  w h e r e a s t h e a l a n i n e was  d i d not  deami-  deaminated d u r i n g  -20its  oxidation. Weil-Malherbe  and Krebs  ( 6 9 ) , employing  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  kidney  tis-  i n t o g l u t a m i c a c i d and  concluded that p r o l i n e i n the kidney i s o x i d i z e d to glutamic 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 t h e l i b e r a t i o n o f ammonia, o r may t a k e on more ammonia t o f o r m Krebs  glutamine.  ( 4 6 ) , i n h i s work on t h e o x i d a t i o n o f d-pro-  l i n e b y 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, dinitrophenylhydrazone of alpha-keto delta-amino v a l e r i c thus d e m o n s t r a t i n g  the opening  acid,  o f t h e r i n g on t h e c a r b o x y l  side of 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 o p e n i n g o f t h e r i n g o c c u r s 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 nitrogen guanido  as an a l p h a amino group a n d t h r e e n i t r o g e n s i n t h e g r o u p on t h e d e l t a  of ensymes.  Hunter  action of arginase this  c a r b o n atom, may  i n v o l v e a number  and D a u p h i n e e ( 3 8 ) , i n s t u d i e s o n t h e e x t r a c t e d from l i v e r , have found t h a t  ensyme, a t pH 8.5 and room t e m p e r a t u r e ,  splits  arginine.  i n t o u r e a and o r n i t h i n e t o t h e e x t e n t o f 99.1 p e r c e n t . u r e a may  The  s u b s e q u e n t l y b e decomposed b y t h e ensyme u r e a s e a t  pH 6.8 a n d room t e m p e r a t u r e  i n t o ammonia a n d 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 t h e e f f e c t  of products  o f a r g i n i n e h y d r o l y s i s u p o n t h e a c t i o n o f a r g i n a s e and f o u n d 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 t h e enzyme, t h e 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 t h a n t h e o p t i m a l f o r t h e h y d r o l y s i s , pH 9.6,  than  -21at  higher values. Tomota ( 6 5 ) , f r o m 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 f r o m a number o f 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 f r o m S t a p h , a u r e u s , Staph;, a l b u s ,  Staph,  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 f r o m S a r c i n a i s f a i r l y S t r e p t o c o c c i , Mycobacterium B. p r o t e u s , B. p y o c y a n e u s ,  a c t i v e and t h o s e  p h l e i , Salmonella e n t e r i t i d i s , B. p r o d i g i o s u s and Mucosus  l a t u s show weak and v a r i a b l e  capcu-  activity.  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 t h e t i o n o f a r g i n i n e b y gram p o s i t i v e of  from  decomposi-  c o c c i I n w h i c h two m o l e c u l e s  ammonia a n d one o f o r n i t h i n e were p r o d u c e d f r o m each m o l e -  cule of a r g i n i n e have caused H i l l  (34) t o c o n c l u d e t h a t t h e  ensyme i n v o l v e d was n o t a r g i n a s e b u t was a n o t h e r enzyme h e h a s named a r g i n i n e d i h y d r o l a s e . clusion  are:  (1) t h e s t r a i n s  which  The r e a s o n s f o r h i s c o n -  o f s t r e p t o c o c c i u s e d were shown  not t o p o s s e s s u r e a s e , (2) t h e urease o f a s t o c k s t r a i n o f Staphyloccus 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 of  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 -  c o c c u s t r a i n e d t o grow on an ammonia medium, t h e u r e a s e activity  c a n be v a r i e d a t w i l l b e t w e e n v e r y w i d e  limits,  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 m a n n e r . reciptocal  This  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) h a s o b s e r v e d t h a t t h r o u g h t h e a c t i o n o f C l o s t r i d i u m sporogenes m o l e c u l e s o f ammonia  one m o l e c u l e o f a r g i n i n e y i e l d s t h r e e  e i t h e r a l o n e o r as a h y 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  -22molecule  o f 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 concerning the factors  b a c t e r i a l deamination nent age  to this  influencing  i s e x t e n s i v e ; only those f a c t o r s  perti-  s t u d y w i l l be r e v i e w e d , - t h e i n f l u e n c e o f t h e  o f c u l t u r e , a e r o b i o s i s a n d a n a e r o b i o s i s , pH o f t h e g r o w t h  and b u f f e r " m e d i a a n d p r e s e n s e  o f carbohydrates i n t h e growth  and b u f f e r m e d i a . 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 of  these f a c t o r s on the b a c t e r i a l deamination  amino a c i d s b y washed c e l l t h e i r f i r s t paper 1-glutamic  suspensions  o f a number o f  of Bacterium c o l l .  In  ( 6 2 ) , u s i n g g l y c i n e , d l - a l a n i n e and  a c i d , they found t h a t only s l i g h t v a r i a t i o n i n the  a c t i v i t y o f t h e s u s p e n s i o n o c c u r r e d when t h e g r o w t h  culture  was b e t w e e n e i g h t a n d t w e n t y 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 t h e deaminase f o r  g l y c i n e and a l a n i n e b u t f a v o u r i t f o r g l u t a m i c a c i d , a n d t h a t the e f f e c t  of glucose o f the o x i d a t i v e deamination  t h r e e amino a c i d s i s t o i n h i b i t d u r i n g growth paper  deaminated isomers  t h e f o r m a t i o n o f t h e enzyme *  t o the extent o f n i n e t y - f i v e percent.  (SO) o n d l - s e r i n e b r o u g h t  of the  Their  o u t t h a t t h i s amino a c i d i s  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  a r e 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 .  a c t i v i t y o f s e r i n e deaminase v a r i e s markedly the growth  The  w i t h t h e age o f  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 e l e v e n h o u r s and  subsequently f a l l i n g  o f f , i s i n c r e a s e d by anaerobic  c o n d i t i o n s and, l i k e  the other deaminases, i s decreased  n i n e t y - f i v e p e r c e n t o r more b y t h e p r e s e n c e g l u c o s e i n t h e g r o w t h medium.  growth  o f two p e r c e n t  The optimum pH f o r t h i s  -23d e a m i n a s e was  pH  8.0.  The the  t h i r d p a p e r by  t i c acid reports  that  acid varies with  growth conditions  i n g about t w o - t h i r d s the two  activity  with  the  activity  age  of the  e x p l a i n e d as b e i n g c a u s e d by c o n s t i t u t i o n of  the  a c t i v i t y of the  cells.  f r o m pH  6.0  t o 8.0,  The  pH  n e a r pH  growth c u l t u r e b e i n g  an optimum at pH activity  centration  7.4  a t pH  6.5.  1-  and  the  oxidases  ( 4 5 ) f o u n d that the  opti-  optimum  acid  and  shows  s i x t y percent  Krebs a l s o found that  a final  of con-  a c i d g a v e t h e maximum.rate  pH  Epps ( 3 0 ) ,  i n an  of  investigation  of the  ef-  o f t h e medium d u r i n g g r o w t h o n ' t h e e n z y m i c  activities  o f E.  divid.e the  enzymes p r o d u c e d i n t o two  coli  and  Mc.  c o n t a i n e d urease,- c a t a l a s e ,  medium and  amino a c i d  :  G a l e and  d u c e d i n an  d-  deamination of d l - a l a n i n e  deamination.  second the  metabolic  7.5.  It s t i l l retains  o f M/200 a s p a r t l c  f e c t of the  was  chemical  deamination of 1-aspartic  but  greatest  This l a t t e r v a r i a t i o n a l t e r a t i o n i n the  and  eighty-five  d-amino a c i d d e a m i n a s e shows an  The  show-  a c t i v i t y o f t h i s deaminase ranged  when u s e d f o r t h e  dl-aspartic acid.  its  a c t i v i t y about  from, k i d n e y t i s s u e , K r e b s  8.8  this  of anaerobic c o n d i t i o n s  b e i n g optimum a t pH  curve of the  aspar-  deaminase of  g r o w t h medium f o l l o w i n g t h e  In a study of the extracted  an  on  - aerobic conditions  from f o u r t e e n to eighteen h o u r s .  mum  of the  percent glucose I n h i b i t i n g ' t h e  p e r c e n t , and  G a l e (29)  l y s o d e i k t i c u s , was The  hydrogenlyase, etc.  amino a c i d enzymes. a c i d medium and  groups.  The  the  seemed, t h e r e f o r e ,  to  first and  group  the  d e c a r b o x y l a s e s were p r o -  d e a m i n a s e s i n ah  t o act  able  as  alkaline  neutralization  -24mechanisms I n an e f f o r t t o b r i n g t h e p l i o f t h e medium n e a r e r neutrality. The  question  of t h e e f f e c t of the presence o f f e r -  mentable and non-fermentable carbohydrates i n t h e growth and b u f f e r medium h a s c a u s e d c o n s i d e r a b l e  controversy  i n the  literatures Kendall  and h i s a s s o c i a t e s  ( 3 9 , 40 and 41) i n a  s e r i e s o f p a p e r s f r o m 1912 t o 1922 s t u d i e d t h e p r o d u c t i o n ammonia b y s e v e r a l b a c t e r i a l  species,  i n c l u d i n g B.  g r o w i n g i n p r o t e i n d i g e s t s a n d showed t h a t t h i s  of  proteus,  production  i s g r e a t l y c h e c k e d and, i n some c a s e s , c o m p l e t e l y  inhibited  by  They I n t e r -  t h e p r e s e n c e o f g l u c o s e i n t h e g r o w t h medium.  preted t h i s  as due t o a " s p a r i n g "  a c t i o n exerted  by t h e c a r b o -  hydrate on t h e deamination 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  o r g a n i s m 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  Is  not only  (57) p o i n t e d  out t h a t  ammonia  a product of the decomposition of p r o t e i n s , but  also a source of nitrogen  f o r growth, so t h a t w h i l e ,  In the  p r o t e i n d i g e s t medium t h e a m m o n i a ' p r o d u c e d i s i n e x c e s s o f that required f o r c e l l  synthesis,  i t may be p o s s i b l e t h a t , i n  the presence o f a d d i t i o n a l carbohydrate, this'excess up  i n Increased  point,  c e l l production.  i s used  To f u r t h e r i n v e s t i g a t e  t h e y f o l l o w e d t h e g r o w t h o f B. p y o c y a n e u s a n d B.  escens i n s y n t h e t i c media c o n t a i n i n g with or without g l y c e r o l .  They f o u n d t h a t  n i t r o g e n , but that  flour-  tryptophan or t y r o s i n e , in.the  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 synthesized  this  little  i n the presence of g l y c e r o l the  -25r 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  h y d r a t e , f a r from having a p r o t e i n - s p a r i n g e f f e c t ,  "carboactually  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 t h e y would i n the absence of Waksman a n d L o m a n l t z  carbohydrate."  (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 of this p o i n t using species of b a c t e r i a , nomyces and m o l d s and  came t o s i m i l a r c o n c l u s i o n s b u t p o i n t e d  o u t 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 e n e r g y  from a  which  be  i s most a v a i l a b l e t o i t and w h i c h may  particular  acti-  substance  specific for a  organism." Nisimura  (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 - h y d r o x y p h e n y l  lactic acid  b y a s t r a i n o f B. p r o t e u s , o f p - h y d r o x y p h e n y l  propionic acid  ( I I ) by  of  a n o t h e r two  strains  ( I I I ) by a s t r a i n o f B.  o f B,  lactis  p r o t e u s and  tyramine  aerogenes from t y r o s i n e i n a  Sasaki protein-free nutrient solution. a d d e d t o t h e s o l u t i o n , no I I was  When g l u c o s e  formed,  d i s t i n c t l y d e c r e a s e d b u t no e f f e c t was III.  The  c e l e r a t e d t h e f o r m a t i o n o f I I b y one  formation of I  o f I I I b y B.  l a c t i s aerogenes.  was  f o u n d on f o r m a t i o n o f lowered  a d d i t i o n of l a c t o s e  ac-  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 t h e f o r m a t i o n o f I by a n o t h e r s t r a i n o f B. and  was  A d d i t i o n of l e v u l o s e or sucrose to the s o l u t i o n  t h e f o r m a t i o n o f I , I I and I I I .  (I)  proteus  A d d i t i o n of s t a r c h had  no  e f f e c t of the f o r m a t i o n of I but regarded the f o r m a t i o n of 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 . and t h o s e i n w h i c h nutrient  I n the c o n t r o l  experiments  c a r b o h y d r a t e s w e r e a d d e d , t h e pH o f  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 to the a c i d  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  the  side.  (47), i n a study  of  - 26 t h e o x i d a t i o n o f amino a c i d s b y 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 l e a v a g e 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 t h e p r e s e n c e on t h i s  cleavage. E p p s and G a l e  presence  o f g l u c o s e h a d no c l e a v a g e  ( 2 3 ) compared t h e i n f l u e n c e o f t h e  o f g l u c o s e d u r i n g growth  on t h e e n z y m i c  activities  of E. c o l l w i t h the e f f e c t produced  by fermentation acids  added t o t h e medium.  that the presence of  They o b s e r v e d  g l u c o s e i n t h e medium d u r i n g g r o w t h  suppresses  the formation  o f c e r t a i n enzyme-s, t h e d e g r e e o f i n h i b i t i o n b e i n g g r e a t e r t h a n o r b e a r i n g no r e l a t i o n t o t h e e f f e c t p r o d u c e d  by  growth  i n a medium a d j u s t e d t o t h e f i n a l pH o f t h e 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 n o t a l t e r t h e d e g r e e 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 t h e r e d u c t i o n i n t h e a c t i v i t y o f c e r t a i n enzymes a s a r e s u l t o f growth  i n g l u c o s e i s n o t a p e r m a n e n t change I n  t h e enzyme c o n s t i t u t i o n o f t h e c e l L l a s i t I s removed Immediately  a f t e r growth  fermentable  carbohydrate.  t a k e s p l a c e i n t h e absence o f  As a p a r t o f t h e i n v e s t i g a t i o n o f t h e m e c h a n i s m f o r the p r o d u c t i o n o f i n d o l e from tryptophan by Bacterium  coli,  t h e 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 i n the growth  a n d b u f f e r medium.  Happold  of glucose  and H o y l e (33)  added g l u c o s e t o t h e g r o w t h medium a n d n o t i c e d t h a t t h e  - 27 i n h i b i t i o n o f t h e p r o d u c t i o n o f t h e t r y p t o p h a n a s e enzyme s y s t e m d o e s n o t o c c u r f r o m t h e s t a r t b u t 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 which only u n t i l  lasts  the f e r m e n t a t i o n of the sugar I s completed.  They  a l s o showed t h a t t h e 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 h a v e no i n h i b i t i n g  action.  H a p p o l d and H o y l e washed c e l l  (32) and P i l d e s  s u s p e n s i o n s o f B. c o l i  (26) f o u n d t h a t  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 a r e t w e n t y - f i v e t i m e s more a c t i v e p r o d u c e r s of  i n d o l e from tryptophan-phosphate-buffer s o l u t i o n s  than  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  Inhibited  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 . E v a n s , H a n d l e y and Happold  (24) r e p o r t e d t h a t t h e  t r y p t o p h a n a s e s y s t e m f o r t h e p r o d u c t i o n o f i n d o l e doe s n o t e x i s t as s u c h i n c e l l s o f B. c o l i w h i c h h a v e b e e n 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 s u c h c e l l s when f r e e d f r o m g l u c o s e b y w a s h i n g w i l l r e - d e v e l o p t h e enzyme when l e f t  system  i n contact with tryptophan. 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 t h e  r e l a t i o n b e t w e e n 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 e m p l o y i n g washed c e l l  suspensions of Proteusi c h -  t h y o s m i u s c o n f i r m t h e o b s e r v a t i o n s o f E v a n s and o t h e r s .  It  was f o u n d t h a t t h e p r e s e n c e o f one p e r c e n t g l u c o s e i n t h e a g a r g r o w t h medium h a d no i n f l u e n c e o f t h e 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 w h e r e a s t h e p r e s e n c e o f one p e r c e n t g l u c o s e - i n t h e  - 28 tryptophan-buffer mixture  e x e r t e d an i n h i b i t i n g  effect  which  was o n l y p a r t i a l when t h e c e l l s were grown on a g l u c o s e - f r e e a g a r b u t was c o m p l e t e when t h e y were grown on a g l u c o s e containing  agar. E v a n s , Handley and Happold ( 2 5 ) , i n t r y i n g  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 duction by glucose  i n c u l t u r e s o f B. c o l i ,  of other fermentable inhibition.  and n o n - f e r m e n t a b l e  Their experiments  and h e x o s e d i p h o s p h a t e  f o u r c u l t u r e s o f the organism o f i n d o l e when t r y p t o p h a n  of indole pros t u d i e d the e f f e c t  sugars  on  this  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 , sucrose, d u l c i t o l , s a l i c i n , saccharate  t o work  inulin,  potassium  are not fermented  i n twenty-  and do n o t a f f e c t t h e p r o d u c t i o n  i s present  other hand, complete i n h i b i t i o n  i n t h e medium.  On t h e  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 which are fermented  b y B. c o l i .  s u g a r s 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 m a r k e d o f i n d o l e p r o d u c t i o n were r h a m n o s e , x y l o s e , g a l a c t o s e , d - r i b o s e and mannose.  f  The inhibition  sorbitol,  - 29  -  EXPERIMENTAL The  r e s u l t s of e a r l i e r  tative determination aoids by  s t u d i e s (50)  be  i t was  decided  to the  i n t i m a t e l y concerned w i t h  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 Therefore  quanti-  o f ammonia f o r m e d f r o m i n d i v i d u a l  s u r f a c e t a i n t b a c t e r i a gave s u p p o r t  t h a t d e a m i n a t i o n may  on t h e  surface t a i n t  theory  the  odour.  t h a t d e t a i l e d e x p e r i m e n t s on  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  amino  the  f r o m a number o f  amino a c i d s m i g h t b r i n g f o r t h f u r t h e r e v i d e n c e f o r t h e as w e l l as add  to our present  theory  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 considerable  r e s u l t s r e f e r r e d t o a b o v e showed t h a t t h e r e ammonia f o r m e d b y  species of surface  was  taint  b a c t e r i a f r o m t h e 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 , glutamic  a c i d , h i s t i d i n e and  with considerable r e l a t e d t o one  supporting  p r o l i n e which are evidence i n the  theoretically,  literature,  another through t h e i r decomposition  products.  W i t h the object of i n v e s t i g a t i n g i n d e t a i l c o n d i t i o n s o f ammonia f o r m a t i o n f r o m t h e above f i v e a c i d s by Proteus  two  species of surface t a i n t producing  ichthyosmius  (Hammer) and  the amino  bacteria -  Pseudomonas . p u t r e f a c i e n s  (Hammer) - t h e f o l l o w i n g s e r i e s o f e x p e r i m e n t s were  carried  out The out  the  general experimental  s t u d y was 1.  The  p r o c e d u r e employed  through-  as f o l l o w s : o r g a n i s m s were I n o c u l a t e d f r o m  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  tryptic  casein digest  - 30  -  a g a r 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 o f 16 18 h o u r s . was  I n t h e c a s e o f P r o t e u s i c h t h y o s m i u s , 30°  e m p l o y e d as t h e t e m p e r a t u r e  Pseudomonas p u t r e f a c i e n s 23° The  bacterial  w i t h M/30  to  o f i n c u b a t i o n , and f o r  C.  c e l l s were t h e n washed o f f t h e  p h o s p h a t e b u f f e r , pH 7.4,  agar  (Sorensen's  M/l5  p h o s p h a t e 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 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  hour i n f i f t y  o r one  fuge  G.  hundred c u b i c c e n t i m e t e r  disone  centri-  tubes. The  cells  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  t a k e n up and m i x e d t h o r o u g h l y w i t h a b o u t 30  o f M/30 The  p h o s p h a t e b u f f e r and t h e n s u p e r n a t a n t was  cc  re-centrifuged.  again poured  o f f and  the  cells  t a k e n up and m i x e d t h o r o u g h l y w i t h a b o u t 50 c c o f  M/30  b u f f e r or d i s t i l l e d  of  the experiment, organisms  water, depending  (vide i n f r a ) , per K o l l e f l a s k  and an a l i q u o t o f 5.0  v a c c i n e tube t o d e t e r m i n e c e l l s present. an  on t h e n a t u r e  A one  of  cc t a k e n i n t o a  Hopkins  t h e p e r c e n t a g e b y volume o f  p e r c e n t s u s p e n s i o n was  used  as  inoculum. The  c u l t u r e s w e r e t h e n s e t up i n t e s t t u b e s  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 o f M/20  b u f f e r , 1.0  a q u e o u s amino a c i d s o l u t i o n and 1.0  p e r c e n t washed c e l l  suspension.  the e f f e c t of the presence  con-  cc. of  I n the experiments  o f c a r b o h y d r a t e , 1.0  a 5% aqueous s o l u t i o n o f t h e s p e c i f i c  cc one on  cc of  carbohydrate  was  - 31 added a n d o n l y 7.0 c c o f b u f f e r were e m p l o y e d I n o r d e r to maintain  t h e 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 days.  I n order  a t 30° C. f o r t h e r e q u i r e d number o f t o overcome t h e d i f f i c u l t i e s r e s u l t i n g  f r o m t h e s t e r i l i z a t i o n o f a m i x e d medium, t h e n i t r o g e n source,  t h e c a r b o n s o u r c e and t h e b u f f e r were e a c h  sterilized  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 to inocu-  lation. Sterile technique the  e q u i p m e n t and m a t e r i a l s and a s e p t i c  were e m p l o y e d t h r o u g h o u t t h e above p a r t o f  procedure. At  t h e c o n c l u s i o n o f t h e i n c u b a t i o n p e r i o d , t h e pH  o f t h e 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 t h e  f r e e ammonia d e t e r m i n e d b y t h e V a n S l y k e  aeration  p r o c e d u r e 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 a r e e x p r e s s e d as p e r c e n t 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 ammonia and a r e c o n v e r t e d  as f r e e  fi?om c u b i c c e n t i m e t e r s  N/lOO s u l p h u r i c a c i d t o p e r c e n t following  o f the t o t a l  of  f r e e ammonia b y t h e  formula:  .14 x c c o f N/100 H S 0 2  4  x 100  =  g  f  r  e  e  ammonia  No. o f N i n amino a c i d x .7 in  which: .14  =  mg. o f n i t r o g e n e q u i v a l e n t N/100 H S 0 . 2  .7  =  t o 1.00 c c o f  4  mg. o f n i t r o g e n i n 1.0 c c o f M/20 ammonia o r m o n o - n i t r o g e n amino a c i d .  - 32 THE EFFECT OF pH ON AMMONIA FORMATION. The  l i t e r a t u r e h a s shown t h a t t h e pH o f t h e b u f f e r  medium c o n t a i n i n g t h e amino a c i d s a f f e c t s t h e 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 organisms .  species  of micro-  Employing the technique described  i n f l u e n c e o f pH o n ammonia f o r m a t i o n  from  above, t h e  d-arginine,  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 a n d Pseudomonas p u t r e f a c i e n s was  determined.  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 t h e  manner o f C l a r k phthalate  ( 1 1 ) were e m p l o y e d : b u f f e r - pH 4.5, 5.0, 5.5, and 6.0.  p h o s p h a t e b u f f e r - pH 6.3, 6.8, 7.3, a n d 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, a n d 1 0 . 0 . In t h i s p a r t i c u l a r experiment  (I)the c e l l  o f n e c e s s i t y , made up i n d i s t i l l e d b u f f e r o f pH 7.4. five  water r a t h e r than phosphate  The c u l t u r e s w e r e i n c u b a t e d  a t 30° C. f o r  d a y s a f t e r w h i c h t h e f i n a l pH was t a k e n a n d t h e f r e e  ammonia d e t e r m i n e d . and  suspensions were,  The r e s u l t s a r e g i v e n  i n t a b l e s 1 a n d 2,  i n t e r p r e t e d o n 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 e x p e r i m e n t I show t h a t t h e r e i s a  general  optimum r a n g e i n pH f o r ammonia f o r m a t i o n  t o pH 8.5.  Within  t h i s general  f o r pH 6.0  r a n g e , t h e d i f f e r e n t amino  a c i d s h a v e a n i n d i v i d u a l r a n g e w h i c h i s sometimes n a r r o w a n d at others  r e l a t i v e l y wide.  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 h a s a r a n g e f r o m pH 6.3 t o 8.0 w i t h a rather  s h a r p o p t i m u m a t pH 7.4 w h e r e a s f o r Pseudomonas  putrefaciens  t h e r a n g e i s f r o m 6.0 t o 8.8 w i t h no d e f i n i t e  - 33 optimum.  Aspartic acid liberates a r e l a t i v e l y  amount o f ammonia f r o m pH 6.0 bacteria  t o pH 8.5  constant  f o r both species of  employed. The  r e s u l t s f o r glutamic a c i d are s i m i l a r to  f o r a s p a r t i c a c i d except further to'the alkaline 8.3  -  t h a t the range i s narrower s i d e o f the pH  s c a l e ; pH  those  and  6.5  to  pH  b e i n g t h e r a n g e f o r Pseudomonas p u t r e f a c i e n s and pH  t o pH  8.5  f o r Proteus  ichthyosmius.  h a n d , has b o t h a n a r r o w r a n g e and range.  For Proteus  pH 8.0,  H i s t i d i n e , on t h e  other  a s h a r p optimum w i t h i n t h e  ichthyosmius, h i s t i d i n e  ammonia i n q u a n t i t y f r o m pH 7.3  6,8  t o pH 8.7  liberates  w i t h an optimum o f  w h i l e f o r Pseudomonas p u t r e f a c i e n s , t h e r a n g e i s f r o m  pH 7.0  t o pH 8.9  w i t h t h e optimum a t pH  obtained f o r proline  show two  species of b a c t e r i a ,  Proteus  ammonia f o r m a t i o n f r o m pH optimum a t pH 8,0.  7.4  7.6.  The  results  d i f f e r e n t graphs f o r the ichthyosmius t o pH 8.5  two  causes c o n s i d e r a b l e  with a very  sharp  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  without  a definite  optimum.  These f i n d i n g s a r e 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 w o r k e r s i n t h a t t h e r a n g e f o r ammonia f o r m a t i o n i s above pH 7.0  6,0  and pH 8.0.  and b e l o w pH  9.0  w i t h an optimum b e t w e e n  W i t h i n t h i s r e l a t i v e l y wide range,  however,  t h e optimum pH v a r i e s m a r k e d l y d e p e n d i n g on t h e amino u s e d and  the s p e c i e s of microorganism F r o m t h e s e r e s u l t s , i t was  acid  employed. d e c i d e d t o use  f o l l o w i n g b u f f e r s f o r t h e d i f f e r e n t amino a c i d s .  pH  the  When  - 34  -  employing P r o t e u s i c h t h y o s m i u s the b u f f e r s were: pit 7.5,  a s p a r t i c a c i d - pH  h i s t i d i n e - pH  8.0  a s p a r t i c a c i d - pH  6.5,  pH 7.5  - pH  In order  pH  4.6,  a c i d - pH  8.0.  glutamic  a c i d - pH  7.5,  histidine  t o determine the 2,  the  e x p e r i m e n t was  repeated (II)  month i n t e r v a l e m p l o y i n g p h t h a l a t e  determinations  i n t r i p l i c a t e and  a f t e r seven, fourteen  and  9.5.  twenty-one.days  buffer  6,6, The  experi-  were c a r r i e d  incubation  general  r a n g e i n pH f o r ammonia f o r m a t i o n  amino  some i n s t a n c e s The  longer  acids  pH  agree-  acids  There are, however, i n  i n d i v i d u a l r a n g e s of  t h e maximum amount o f ammonia i s n o t The  and  optimum  i n c u b a t i o n t i m e s show t h a t f o r some o f t h e  r a n g e o f f r o m pH  pH. amino  formed aft-er f i v e 5.6  t o pH  7.6  for  ammonia f o r m e d f r o m a r g i n i n e b y 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 tity  from the 8.5.  d i f f e r e n c e s i n the  days' i n c u b a t i o n . the  t o pH  4  i n general  ment w i t h t h o s e o f e x p e r i m e n t I i n t h a t t h e  6.0  7.6  Inclusive.  f i n d i n g s of experiment I I are  s t u d i e d v a r i e s f r o m pH  at  and  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  shown g r a p h i c a l l y on f i g u r e s 3 t o 12 The  and  5.6,  ment was  The  -  constancy of the r e s u l t s  8.5  respectively.  7.0,  7.0.  a n d b o r i c a c i d - K G l b u f f e r a t pH's  out  8.0,  a r g i n i n e - pH  Sorens.en's p h o s p h a t e b u f f e r a t pH's  s e t up  -  When e m p l o y i n g  the b u f f e r s were:  r e c o r d e d i n t a b l e s 1 and a f t e r an e l e v e n  glutamic  and p r o l i n e - pH  Pseudomonas p u t r e f a c i e n s  and'proline  6.5,  arginine  acid side.  The  comparatively  o f ammonia p r o d u c e d , h o w e v e r , i n c r e a s e d  cultures suggesting er than the  t h a t the  I n i t i a l pH  t r u e pH  l a r g e quan-  t h e pH  of  these  range i s probably  high-  of the c u l t u r e s i n d i c a t e s .  An  acidity  The E f f e c t of pH on Ammonia Production by Proteus ichthyosmius  aid Pseudomonas putrefaciens.  - Experiments  Tables  Figures  1  and I I  1,  2,  3  3  to  12  Experiment  and  4  inclusive  1-5  days incubation «=> v i o l e t  Experiment II - 7 days incubation - blue 14 daysincubation  - green  21 days incubation - red  TABLE I. Proteus ichthyosmius Effect of pE  nino a c i d  4.65  i n i t i a l pH of culture 5.05 6.06 5.57 6.34  Arginine  1. 0.5 2®  5.5  Aspartic a.cid  1. 0.5 2« 4.40  1.0 4.92  Glutamic acid  i . 1.0 2. 4.48  1.0 4.95  Histidine  Proline  42.0  6.86  53 • 5  68.5  68.5  3©5 5.58  34.5 5.89  40.0 6.34  37.0 6 86  2.0 5.42  16,0 5.85  15,0 5.75  33.0 6.62  1. 2.  14.6 6.15  19,6 6.64  1. 2.  14.5 6.26  18.0 6.65  i n i t i a l pl-l of culture 7.96 8.55 9.06 9.46  9.63  7,34 Arginine  S  78.25  71.25  62.5  45,75  37,25  13,0  Aspartic acid  1. 35,0 2. 7.38  36.0 7.96  36,0 8.35  29.0 8,95  25.0 9.34  17.5 9.55  Glutamic acid  1. 52.5 2 « 7.14  32.5 7,75  37,0 8.05  21.5 8,72  17.0 9,05  16,0 9.20  Histidine  1. 28.3 2 e 7.17  38.0 7.60  34,6 8.05  25.6 8.65  18,6 8.97  18 3 9.15  1. 21.0 2. 7,18  37,0 7.86  17.5 8,20.  8.0 8.85  6,0 9.16  5 9.34  2.  Proline  e  1.  percent of t o t a l nitrogen of amino a c i d ( i n culture) present.as free ammonia.  2.  pH of culture a t time of ammonia determination.  TABLE 2. E f f e c t of pH  Aspartic acid Glutamic acid Histidine  i n i t i a l pH of culture 6.06 5.57 6.54  4.65  5.05  X« 2.  0.0  0«0 5,10  1.75 5„57  6.75 6.24  10.25 6.65  10.76  1.  0.0  2.  4.50  1.5 4,95  9.0 5.45  36.0' 5.96  40.0 6.46  34,5 6 ,98  • 1. . 0.5 4.55 2.  1.0 4.95  8.0 5.44  15.0 5,80  21.0 6.43  22,5 6.95  IS a t3 6.15  17.3 6.53  32.0 7.06  26.0 6.20  29.0 6.60  29.0 7.13  amino a c i d Arginine  Pseudomonas putrefaciens  4.65  1. 2.  Proline  1. •'• 2.  0.0 4.65  0.5'  6.10  11 o 5 5.64  7.05  7.14  i n i t i a l pH of culture  7.60  8,25  8.42  8.85  9.25  9.57  7.5 8.78  3.25 9.15  0.5 9.45  Arginine  19 2.  8.0 7.72  8.0 8.30  8.5 8.38  Aspartic acid  1. 2.  35.0 7.54  35.5 7.85  37.5 8.05  32.5 28.5 8.65 , 9.05  6.5 9.30  Glutamic acid  1. 2.  23.0 7.46  21.0 7,84  12 • 5 8.05  10.5 8.60  4.5 9.10  3.0 9,35  Histidine  1. 2.  40.3 7.56  32.6 7.95  25.3 8.05  31.6 8.56  24.0 9,03  13.6 9.26  Proline  1. 2.  28,0 7.65  22.5 8.11  8.5 8.35  8.0 8.79  1.5 9.25  1.5 9.51  1.. percent of t o t a l nitrogen of amino a c i d i n culture present as f r e e ammonia 2. pH of culture at time of ammonia determination  TABLE 5. Proteus ichthyosmius  E f f e c t of pH  age of culture Arginine 7 days  4.6 5.5 ,  i n i t i a l pH- of cultures 5.6 6*6 7.6 74.75 . 67.5  8.5  9.3  68.5  45.75  41.5  1. 2. 1. 2.  10.0 4.70  84.0 6.45  84.75 7.05  70.25 8.35  45.0 8.50  31.25 9.00  1. 2.  14.0 4.75.  82.75 6.55  82.0 7.05  60.75 8.30  43.0 8.55  17,0 9,00  1. 2.  0.0  48.0  44.0  45.0  48.0  29.0  14 days  1. 2.  0.0 4.55  40.0 6.65  40.0 7.60  25.0 8.25  10,0 8.95  21 days  ;l. 2.  1.0 4.60  32.0 5.95  39.0 6.70  37.0 7,70  20.0 8.40  7.0 9.05  1. 2.  0.0  20.0  18,0  19.0  21.0  19.0  1. 2.  0.0 4.55,  14.0 5.60  33.0 6.65  33,0 7.60  26 .0 8,25  17.0 8,95  1. 2.  1.0 4.65  23.0 5,75  52.0 6.75  55,0 7.75  40.0 8.35  21.0 9.05  1.  0.3  15.6  33.3  38.3  35,3  22.0  14 days  i.  0,0 4.70  4 21.0 5.80  36.0 6.70  38.0 7.65  30.3 8.35  13.0 8.90  21 days  1.  1.0 4.75  25.3 6.00  35.6 6.70  34,6 7.75  24.6 8.30  8.6 8.90  1. 2.  0.0  15.0  19.0.  35.0  32.0  15.0  1. 2,  5.0 4.75  30.0 6.00  6.70  41.0 7.80  34.0 8.45  22.0 9.00  1. 2.  2.0 4,75  49.0 6.15  59^0 6.80  56. 0'7.90  30,0 8,50  ,18.0 9.05  14 days  21 days Aspartic Ac id_ .; 7 days  Glutamic A c i d 7 days ™~~  14, days  21 days  Histidine " 7 days  .  2a Proline  7 days  14 days  21 days  TABLE 4. E f f e c t of pH  Age of culture Arginine 7 days  Pseudomonas putrefaciens 4.6 1. 2.  0.0  14 days  1.  0.0 4.70  21 days  1. 2.  I n i t i a l pH of cultures 5.6 6.6 7.6  9.3  13.0  12.75  35.0 6.25  36.0 6.80  32.75 7,90  17.5 8.40  1.5 9.05  1.0 4.75  64.75 6.45  59.0 6.95  48.25 8.05  21.75 8.50  2.5 9.05  1. 2.  0.0  42,0  38.0  43.0  41.0  27,0  14 days  1. 2.  0.0 4.55  35.0 5.85  37.0 6.65  34.0 7.60  29.0 8.30  16.0 8.95  21 days  1. 2.  1.0 4.55  36.0 5,90  -  31.0 7.55  23,0 8.35  8..0 9,00  IL o 2.  0.0  26,0  20.0  16.0  16,0  11.0  14 days  1. 3•  oo 4.60  30,0 5.95  24.0 6.65  24.0 7.60  28,0 8.20  15,0 8.95  21 days  i.  2.0 4.60  45.0 6 15  57,0 6.75  35.0 7.65  27.0 8.35  10.0 9.05  Aspartic A c i d 7 days  Glutamic A c i d 7 days  -  e  14.5  8.5  -  e  -  -  10.5  -  3.75  -  Histidine 7 days  i. 2.  0.0  47.6  '61.6  57.3  48.3  23.3  14 days  1. 2.  0.3 4.75  73.0 6 -30  72.3 6.85  64.3 7.85  42.6 8.25  16.6 8,90  1. 2.  0.6 4.75  77.3 6.35  74.6 6.85  61.6 7.95  8.30  9.6 8.95  7 days  1. 2.  0.0  45.0  39,0  32.0  15.0  3.0  14 days  1. 2.  0.0 4.75  62.0 6.10  51.0 6.75  52.0 7.75  16.0 8.40  2.0 9.10  21 days  1. 2.  2.0 4«75  58.0 6.15  47.0 6.75  44.0 7.80  13.0 8„45  3.0 9.10  21 days  ?  Proline  Figure 4.  F i g u r e 5. Proteus  ichthyosmius  Aspartic Acid  501  10.0  Figure 6 Pseudomonas p u t r e f a c i e n s  Aspartic Acid  10.0  Figure Proteus ichthyosmius  7. Glutamic A c i d  F i g u r e 8. Pseudomonas p u t r e f a c i e n s  Glutamic A c i d  Figure Proteus  iohthyosmius  9. Histidine  40^  10.0  Figure Proteus  11.  ichthyosmius  Proline  601  50  •  , — i ,—  4.0  5.0  i  1  1  6.0  i —  7.0 pH  T  1  8.0  —>  9.0  i  1—  10.0  Figure 12. Pseudomonas putrefaciens  Proline  • - 35 b e t w e e n pH 5.0 and pH 5.5 p e r m i t s  the formation  amounts o f ammonia, as s e e n f r o m t h e p r e v i o u s  of small  results.  This  ammonia w i l l r a i s e t h e pH o f t h e 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 .  t r a t i o n o f b u f f e r u s e d was a p p a r e n t l y absorb t h e Increase pH a t i t s i n i t i a l The  i n hydroxyl  The c o n c e n -  t o o weak t o c o n t r o l a n d  ions and t h e r e b y  maintain the  level.  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  formation  f r o m a r g i n i n e b y Pseudomonas p u t r e f a c i e n s show t h a t t h e o p t i mum r a n g e i n pH i s s h i f t e d s l i g h t l y t o w a r d s t h e a c i d s i d e o f t h e s c a l e a n d n a r r o w s as t h e 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 , a n d t h a t t h e q u a n t i t y o f ammonia p r o d u c e d increases 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 the deamination of a s p a r t i c a c i d  a r e s i m i l a r f o r t h e two s p e c i e s  o f b a c t e r i a employed.  The  optimum pH r a n g e shows a w i d e n i n g on t h e a c i d end o f t h e r a n g e w i t h no change on the, a l k a l i n e end. incubation increases  As t h e t i m e o f  t h e q u a n t i t y o f f r e e ammonia d e c r e a s e s  s l i g h t l y and t h e r a n g e i n pH n a r r o w s on t h e a l k a l i n e e n d . The  organisms are probably  source sources  u s i n g t h e ammonia as a n i t r o g e n  a l o n g w i t h t h e a c i d s from deamination as carbon forcell  multiplication.  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 with the l i b e r a t i o n but  acid  o f ammonia a r e s i m i l a r i n some r e s p e c t s  d i f f e r e n t i n "others.  For Proteus  ichthyosmius,• t h e  r a n g e I n . p H i s f r o m pH 5.6 t o pH 9.4 a f t e r s e v e n d a y s ' i n c u b a t i o n a n d n a r r o w s t o a r a n g e f r o m , pH 6.6 t o pH f o u r t e e n and t w e n t y - o n e d a y s ' i n c u b a t i o n .  7.6.after  The q u a n t i t y o f  - 36 ammonia i n c r e a s e s  regularly with increased  F o r Pseudomonas p u t r e f a c i e n s , fourteen  days' i n c u b a t i o n  optimum a t pH 5.6 twenty-one  incubation time.  t h e r a n g e i n pH a f t e r s e v e n  i s f r o m pH 5.6  a f t e r the. s e v e n - d a y  t o pH 8.5  with  incubation.  The  an  After"  days' i n c u b a t i o n , however, the range has  down t o a s i n g l e optimum o f pH 6.6.  and  narrowed  f i n a l quantity  of  ammonia f o r m e d b y Pseudomonas i s t h e same as f o r P r o t e u s b u t t h e 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 s e v e n f o u r t e e n .days t h a n i t i s i n t h e f i r s t s e v e n days o f  s e v e n and t h e  last  incubation.  The  c u r v e s 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 f r o m h i s t i d i n e b y t h e two b a c t e r i a l s p e c i e s entirely different.  found In experiment I . with a slight  incubation  increases,  e r and d e f i n i t e l y quantity  The  compared w i t h  that  optimum r a n g e i s f r o m pH-6.6. t o  change t o w a r d s a c i d i t y as t h e t i m e o f . w h e r e a s p r e v i o u s l y t h e r a n g e was  narrow-  on t h e a l k a l i n e s i d e o f t h e pH s c a l e .  o f ammonia r e m a i n s  The  c o n s t a n t as t h e 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 s h o w i n g t h a t t h e maximum amount h a s b e e n a f t e r seven days' i n c u b a t i o n . putrefaciens  are  Those f o r P r o t e u s Ichthyosmius from  e x p e r i m e n t I I show a w i d e r r a n g e i n pH  pH 8.8  and  The  show a more p r o n o u n c e d  formed  c u r v e s f o r Pseudomonas s h i f t to the a c i d  side  o f t h e pH s c a l e f o r optimum ammonia f o r m a t i o n f r o m h i s t i d i n e . W h i l e t h e optimum, f r o m e x p e r i m e n t I was pH 7.6, e x p e r i m e n t , I I i s pH 6.6 b e t w e e n pH 5.6  from  a f t e r s e v e n d a y s ' I n c u b a t i o n and  and pH 6.6.  pH n e a r e r t o t h e s e v e n - d a y however, causes a s h i f t  that  after fourteen optimum.  The  I n pH f r o m pH 5.6  days', b r i n g i n g t h e increase  i n ammonia,  t o pH 6.5.  The  - 37 maximum q u a n t i t y o f ammonia I s n o t f o r m e d a f t e r s e v e n d a y s but  increases  u n t i l f o u r t e e n days.  Pseudonomas p u t r e f a c i e n s  The t o t a l q u a n t i t y f o r  i s about t w i c e t h a t f o r  Proteus  ichthyosmius. As  i n t h e c a s e o f h i s t i d i n e , t h e optimum pH r a n g e  for the formation  o f ammonia f r o m p r o l i n e b y t h e two b a c -  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 o f t h e n e u t r a l p o i n t o f the p l l s c a l e .  Proteus  ichthyosmius  b r e a k s down p r o l i n e b e s t  b e t w e e n pH 7.0 a n d pH 8,0 e x c e p t a f t e r t w e n t y - o n e d a y s ' bation  incu-  when t h e optimum s h i f t s t o b e t w e e n pH 6.6 and pH 7.6.  On t h e o t h e r hand-, Pseudomonas p u t r e f a c i e n s tinctly pH 6.6.  a c i d hydrogen-ion concentration  prefers a dis-  o f f r o m pH 5,6 t o  The f i n a l . p H f o r b o t h g r o u p s 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 f r o m pH 5.6 t o pH 6.1. I n g e n e r a l , where t h e s h i f t  i n t h e optimum pH r a n g e  i s t o w a r d s a more a c i d pH, a s t h e 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 h a s b e e n a n i n c r e a s e i n t h e 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, c a u s e d b y 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 o f ammonia f o r m e d d u r i n g t h e e a r l y d a y s o f incubation. The  E f f e c t o f t h e Age o f C u l t u r e  on Ammonia  With the object of determining age  Formation.  the e f f e c t o f the  o f t h e g r o w t h c u l t u r e upon t h e s u b s e q u e n t ammonia f o r m a -  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 ) e m p l o y i n g P r o t e u s  ichthyosmius  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 was r e m o v e d f r o m t h e i n c u b a t o r  was c a r r i e d o u t .  a t t h e same t i m e .  a t t h e end o f t e n ,  One  flask  twelve,  The E f f e c t of the Age of the Growth Culture on Subsequent Ammonia Production by Proteus ichthyosmius«  Experiment Table  5.  Figure  13«  III .  Arginine  -  red  Aspartic Acid  -  green  Glutamic Acid  -  blue  Histidine  -  violet  Proline  -  orange  TABLE 5.  age of culture i n hours amino a c i d  10  12  14  16  18  20  69,0  74.0  69.75  67.0  Arginine  -74.75.  76 . 2 5  Aspartic a c i d  50.0  52.0  50.5  50.5  50.5  51.0  Glutamic a c i d  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  F i g u r e 13.  age of growth c u l t u r e i n hours  - 58 fourteen,  s i x t e e n , eighteen  p e c t i v e l y , the  -  and  twenty hours i n c u b a t i o n  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  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 f i v e days. preted  The  r e s u l t s are recorded.in  i n figure  and  G.  for  inter-  13.  c u l t u r e , w i t h i n the  limits  ammonia f o r m a t i o n  glutamic  a t 30°  t a b l e 5 and  These f i n d i n g s i n d i c a t e t h a t t h e age  on t h e  res-  a c i d , but  e m p l o y e d , has  o f the  little  growth  o r no  effect  from a r g i n i n e , a s p a r t i c a c i d  that Increase  i n t h e age  of the  growth  ,culture, p a r t i c u l a r l y beyond s i x t e e n hours decreases ammonia f o r m e d fr-om h i s t i d i n e a n d p r o l i n e b y  and  this  the  micro-  organism. E f f e c t of A e r o b i c Formation.  and  S t e p h e n s o n and d i t i o n s o f oxygen supply c e r t a i n amino a c i d s . supply  i n the  Anaerobic Conditions  G a l e (28,  and  62)  found that  con-  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  In order  g r o w t h and  30  on Ammonia  t o show t h e  e f f e c t of  b u f f e r medium on ammonia  oxygen  formation  f r o m t h e 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  e x p e r i m e n t s were u n d e r t a k e n . In the  first  experiment  ( I V ) , three  conditions  g r o w t h medium w e r e e m p l o y e d :  (1) t h e u s u a l  d i g e s t agar In 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  broth  i n a Roux f l a s k , and  a i r replaced  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 c e n t r i f u g e d , w a s h e d , and  cultures.  casein  digest  (3) t r y p t i c c a s e i n d i g e s t  i n an E r l e n m e y e r f l a s k w i t h t h e  was  tryptic  s e t up  by  broth  nitrogen.  h o u r s , the  as u s u a l  of  growth  i n t e s t tube  A f t e r f i v e d a y s ' i n c u b a t i o n , t h e f r e e ammonia o f  of  - 59 t h e b u f f e r c u l t u r e s was t a b l e 6 and  figure  -  determined.  The  r e s u l t s are given i n  14.  I n g e n e r a l , t h e g r o w t h on a g a r i n K o l l e f l a s k s t h e g r e a t e s t s u b s e q u e n t ammonia f o r m a t i o n .  For the  gave  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 , - g r o w t h  on  a g a r gave h i g h e s t ammonia f o r m a t i o n , g r o w t h a e r o b i c a l l y  in  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 the  p r e s e n c e 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 .  tamic  a c i d , the  a e r o b i c b r o t h p r o c e d u r e gave s l i g h t l y  r e s u l t s t h a n the agar and lower than the and  agar..  the anaerobic  b r o t h method  glu-  higher slightly  F o r h i s t i d i n e , t h e a g a r grown- c u l t u r e  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  same and g a v e , s l i g h t l y l o w e r The  For  the  r e s u l t s than the aerobic b r o t h .  outstanding f i n d i n g of t h i s experiment i s t h a t , f o r pro-  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 t w i c e t h e ammonia f o r m a t i o n as f o r growth, suggesting  about  the a n a e r o b i c b r o t h p r o c e d u r e  t h a t o x i d a t i o n may  opening o f the p y r o l l i d l n e  gives  be  a f a c t o r In  r i n g p r i o r to the l i b e r a t i o n  the of  ammonia. • - ' y - • .In the second experiment (V), of t h i s group, the c e l l s were grown as u s u a l on a g a r i n a K o l l e i n b u f f e r c u l t u r e s i n two cubic centimeter intervals  and  (2) a n a e r o b i c a l l y i n t e s t  up  daily  tubes w i t h t h e a i r  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  m e a s u r e d and  I s shown i n t a b l e 6 w i t h  i n t e r p r e t a t i o n i n f i g u r e 15. i n c r e a s e d oxygen supply  set  (1) a e r o b i c a l l y i n f i f t y  c e n t r i f u g e t u b e s w h i c h were shaken a t  r e p l a c e d by n i t r o g e n . ammonia was  ways:  f l a s k and  The  graphic  c u l t u r e s p r o v i d e d w i t h the  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  Broth  -  Anaerobic  -  C  (Experiment  V)  Figure  15  Aerobic  -  A  Anaerobic  -  B  Arginine  -  red  Aspartic Aoid  -  green  Glutamic A c i d f  -  blue  Histidine  -  violet  Proline  -  orange  TABLE 6.  E f f e c t o f Oxygen Supply i n the Growth and Buffer Media,  Growth Conditions  agaraerobic  broth- . aerobic  broth- agaranaerobic aerobic  agaraerobic  Buffer Conditions  aerobic  aerobic  aerobic  aerobic  anaerobic  arginine  75,75  67.25  61.75  68.75  66.0  aspartic acid  50.0  43.0  39,0  45.0  42.0  glutamic acid  21.0  25.0  15.5  27.5  26,0  histidine  37.6  40.8  38.3  34,0  33.3  proline  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 slight  increase  i n ammonia f o r m a t i o n  over the anaerobic  f e r c u l t u r e s except i n t h e case o f p r o l i n e which formed h a l f t h e 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 . f a c t i s f u r t h e r evidence f o r the hypothesis  bufonly This  that the opening  o f t h e 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 S a t e o f Ammonia The  question  t e d from the v a r i o u s  Formation.  o f how r a p i d l y t h e ammonia I s l i b e r a -  amino a c i d s was p a r t i a l l y a n s w e r e d b y an  e x p e r i m e n t ( V I ) on t h e r a t e o f ammonia f o r m a t i o n amino a c i d s b y P r o t e u s  ichthyosmius.  from t h e f i v e  S i x sets o f buffer  cul-  t u r e s were p r e p a r e d and t h e ammonia d e t e r m i n e d a t t h e end o f two  h o u r s , one, t w o , t h r e e ,  respectively.  f o u r and f i v e d a y s '  The r e s u l t s a r e r e c o r d e d  incubation  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 . This  e x p e r i m e n t shows c l e a r l y t h a t 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 takes  place w i t h i n twenty-four  as t h e ammonia f o r m a t i o n increases  h o u r s , where-  f r o m t h e o t h e r f o u r amino  acids  s t e a d i l y over t h e f i v e days o f i n c u b a t i o n . L a t e r e x p e r i m e n t s c a r r i e d o u t as c o n t r o l s i n t h e  carbohydrate  s e r i e s show t h a t t h e maximum ammonia  from glutamic  a c i d , h i s t i d i n e and p r o l i n e by Proteus  formation ichthy-  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 continues and  to rise  f o r fourteen  days i n t h e c a s e Of h i s t i d i n e ,  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 .  f i n d i n g s h a v e b e e n c o m p i l e d and. a r e p r e s e n t e d  These  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 , f r o m t h e f i v e amino a c i d s b y Pseudomonas p u t r e f a c i e n s  i s given  i n t h e same  The Rate of Ammonia Production by Proteus ichthyosmius and Pseudomonas putrefaciens.  Experiment  Table  Figures  VI  7  16 to 20 inclusive  Proteus ichthyosmius - blue (Experiment V I . - June 1942) Proteus ichthyosmius - orange (Experiment X. - October 1942) Proteus ichthyosmius - green (Experiment XII - June 1943) Pseudomonas putrefaciens - v i o l e t (Experiment XI - January 1943) Pseudomonas putrefaciens - red (Experiment XIII - J u l y 1943)  TABLE 7.  Rate of Ammonia Production  Proteus ichthyosmius  2 hrs.  age of buffer culture i n days 1 2 3 4  3. o 5  33.75  56.0  62.0  72.0  83.0 .  acid  3.0  49.5  50.5  48.0  49.5  50.0  G-lutamic a c i d  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  amino acid Arginine Aspartic  5  Figure 16.  Figure  17. Aspartic Acid  60-,  days  Figure 19. Histidine  70  H  days  Figure 20 7 3  days  -  z  - 41 s e r i e s of f i g u r e s compiled f o r Proteus ichthyosmius above. These f i g u r e s w i l l be d i s c u s s e d i n the s e c t i o n t i t l e d "Discussion." E f f e c t of the Presence of Carbohydrates i n the Medium on Ammonia Formation. 1.  Fermentation o f Carbohydrates i n Shake-Agar C u l t u r e s by Two Surface T a i n t Producing B a c t e r i a l Species. P r i o r to undertaking an extensive i n v e s t i g a t i o n o f  the e f f e c t of fermentable and non-fermentable  carbohydrates  on the ammonia p r o d u c i n g ^ a c t i v i t i e s of the two b a c t e r i a l species from amino a c i d s , i t i s necessary t o know the sugarfermenting a b i l i t i e s o f Proteus ichthyosmius and Pseudomonas putrefaciens.  I n order to o b t a i n these data, shake agar  c u l t u r e s prepared from n u t r i e n t agar and u s i n g brora-cresolpurple as the i n d i c a t o r were employed. ment  (VII) a one-percent  In the f i r s t  experi-  c o n c e n t r a t i o n of each o f the  f o l l o w i n g 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:  g l y c e r o l , x y l o s e , arabinose, mannitol,  l a e v u l o s e , g l u c o s e , g a l a c t o s e , sucrose, maltose,  lactose,  r a f f l n o s e , i n u l i n , d e x t r i n , s a l i c i n , and d u l c i t o l .  The  c u l t u r e s were observed f o r a c i d and/or gas production a f t e r f o r t y - e i g h t hours' i n c u b a t i o n a t 30° C. and are recorded i n t a b l e 8. The s l i g h t these microorganisms at a l l ,  amount o f a c i d produced from l a c t o s e by which are not supposed  t o ferment  lactose  suggests that p o s s i b l y s t e r i l i z i n g the carbon and  n i t r o g e n sourced together breaks down a small amount o f the  TABLE 8. Shake-Agar Sugar Per mentations (Carbon and Nitrogen Sotirces S t e r i l i z e d Together)  Carbohydrate  Proteus ichthyosmius  Pseudomonas putrefaciens  acid  acid  alkaline  glycerol  gas  0  alkaline  gas  4-H-  0  +  xylose  0  0  0  0  0  0  arabinose  +  0  +  0  surface  0  mannitol  +++  laevulose glucose  *o  +++  •H-H-,  0  •++  ++++  0 0  galactose  -H-+  0  +++  0  -H-+  H-H-  0  ++  +-H-  0  •H-  -HH-  0  ++  +++  .0  ++  ++ -H-f  sucrose  -H-H-  0  maltose  •H-H-  0  -lactose  trace  surface  +  it  surface  +  raffinose  trace  surface  +  +-H-  0  +  surface  trace  surface  trace  inulin  0  dextrin'  surface 1  -H-+  trape  0  +  salioin  •H-H-  0  -H-  dulcitol  0  surface  trace  ++ ++++ +  .0 surface  ++ trace  - 41a -  l a c t o s e Into i t s component sugars - glucose and galactose both of which are r e a d i l y fermented. t h i s p o s s i b i l i t y , the experiment employing  In order to e l i m i n a t  (VIII) was repeated  the technique of s t e r i l i z i n g the carbohydrate  source s e p a r a t e l y and adding i t to the medium just prior to inoculation.  asoptically  The c u l t u r e s were incubated  at 30° Cc and observed at twenty-four, f o r t y - e i g h t and n i n e t y - s i x hour i n t e r v a l s f o r a c i d i t y , a l k a l i n i t y and gas production.  The f i n d i n g s are given i n t a b l e s 9 and 10.  T h i s experiment shows that Proteus ichthyosmius produces  a c i d and gas from g l y c e r o l , mannitol, glucose,  g a l a c t o s e , sucrose, maltose, d e x t r i n and s a l i c i n and has no immediate a c t i o n but goes slowly 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 the water c o n t r o l ; whereas Pseudomonas p u t r e f a c i e n s produces a c i d and gas from" x y l o s e mannitol, glucose, maltose and s a l i c i n , slowly produces a c i d w i t h no gas from g l y c e r o l , and has no immediate r e a c t i o n but s l o w l y goes a l k a l i n e i n l a c t o s e , d e x t r i n and the water c o n t r o l . microorganisms  The d i f f e r e n t i a t i n g sugars f o r these  a r e x y l o s e and d e x t r i n :  Proteus  ichthyosmius ferments d e x t r i n but not x y l o s e while 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 .  TABLE 9. Shake-Agar Sugar Fermentations (Carbon and.Nitrogen' Sources, S t e r i l i z e d Separately)  Proteus ichthyosmius  Carbohydrate  Acid  Alkaline  Gas  24hr. 48hr, '96-hr. •• 24hr, 48hr. 96,hr. glycerol  -H-  -H-  .Uj,,l.  0  0  0  -H-.  -H  -H-  0  •H-H-  •H-H-  0  0  0  sur.  sur.  sur.  -H-f  +++  -H-r  ! 1T xylose  24hr. 48hr. 96hi  .0  0  mannitol  ++-H-  •H-H-  glucose  ++++ •++++• -H-H- \  0  0  0  ++  ++  ++  galactose  -H-H>  •H-H-  0  0  0  ,L11|,-  ++  -H  sucrose .  •H-H-  -H-H-  0  0  0  •f-H-  maltose  •H-H-  -H-H-  •H-H-  0  0  0  •H-  •Hi  lactose  0  0  0  0  G  0  dextrin  -H-H-  +-H-+  •H-H  G  0  0  salicin  •-H-H-  -H-H-  •H-H-  0,  0  0  dulcitol •  0  0  0  sur.  -H-H-  ++++  0  0  •o  m t e r (K)  0  0  0  sur.  •HrHr  •H-H-  0  0  0  sur.  ++++  •H-H-  - surface  +-H-+ •H-H-  TIT  -J—J. •H-  "Hi" • o.  ++ ++  TABLE 10. Shake-Agar Sugar Fermentations (Carbon and Nitrogen Sources S t e r i l i z e d Separately)  Pseudomonas putrefaciens Carbohydrate  Acid  Alkaline  24hr. 48hr. 96hr. glycerol xylose  trade -H-  +  -H-  ++ -H-H-  mannitol  Gas  24hr., 48hr... 96hr. 0  0  " sur.  24hr. 48hr. 96hr. 0  0  sur.  sur. trace  0  sur.  s u r . -H-H-  trace trace +  +  -H-H- ++++  glucose  -H-H-  -H-H- -H-H-  sur.  sur.  s u r . -H-H- -H-H- -H-H-  galactose  ++++  -H-H- -B-H-  sur.  sur.  sur.  sucrose  •H-H-  ++++ -H-H-  sur.  sur.  4~r  sur.  4~*i* *H~  +  maltose  -H-H-  lactose  trace  dextrin  ++  -H-H- -H-H-  0  trace trace  sur.  sur.  . 0 0  0  0  sur.  sur.  sur.  -H-  -H-  +  0.  0  +  -H-  0  s u r . trace trace trace -H-  , salicin  -H-H-. -H-H- ++++  -H-  0 dulcitol water (K)'  0 , 0  0 0  0  , 0 sur..  -H-H- ++++  sur.  -H-H- -H-H-  0  sur. - surface  +-H-  I  0  0.  0  0  0  0  0  - 41b -  2.  E f f e c t o f the Presence o f Carbohydrates i n the Growth and B u f f e r Media on Ammonia Formation. The l i t e r a t u r e contains considerable data and a  number o f t h e o r i e s on the e f f e c t o f fermentable and n o n fermentable carbohydrates i n t h e growth, and b u f f e r media on the subsequent a c t i v i t y of ammonia producing enzymes .from v a r i o u s species o f microorganisms.  With the object  o f i n v e s t i g a t i n g the e f f e c t o f a number o f carbohydrates on the ammonia formation.- from a r g i n i n e , a s p a r t i c a c i d , glutamic a c i d , h i s t i d i n e and p r o l i n e by Proteus i c h t h y osmius and Pseudomonas p u t r e f a c i e n s a s e r i e s o f experiments was undertaken. The o b j e c t of the f i r s t experiment  (IX) i n t h i s  s e r i e s was to determine the e f f e c t of the presence o f one percent glucose i n the growth medium on ammonia formation from a r g i n i n e , a s p a r t i c a c i d and glutamic a c i d i n b u f f e r media c o n t a i n i n g g l u c o s e , sucrose, maltose, l a c t o s e , g l y c e r o l , mannitol or water ( c o n t r o l ) r e s p e c t i v e l y by Proteus ichthyosmius.  The c u l t u r e s were p r e -  pared as o u t l i n e d i n the g e n e r a l procedure and incubated at 30° C.  The ammonia from a s p a r t i c a c i d was determined  a f t e r twenty-four hours' i n c u b a t i o n , from glutamic a c i d a f t e r f o u r days and from a r g i n i n e a f t e r f i v e days.  The  r e s u l t s are shown i n t a b l e I I and f i g u r e s 21, 22 and 23. The glucose growth agar f l a s k was incubated f o r forty-two hours before h a r v e s t i n g 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 o f glucose i n the growth medium only s l i g h t l y decreases the q u a n t i t y of f r e e ammonia i n the tubes.  The  amount o f f r e e ammonia from the two sugars fermented most r a p i d l y by Proteus ichthyosmius i s more than twice as great employing the c e l l s grown on glucose f r e e agar as that u s i n g the c e l l s grown on glucose c o n t a i n i n g agar^ The volume o f f r e e ammonia from a r g i n i n e i s g r e a t e s t i n the water c o n t r o l and the l a c t o s e c o n t a i n i n g c u l t u r e s , l e s s i n the maltose, g l y c e r o l and mannitol c o n t a i n i n g c u l t u r e s and l e a s t i n the glucose and sucrose c o n t a i n i n g c u l t u r e s . The q u a n t i t y o f ammonia from a s p a r t i c 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 glucose and sucrose i s the same f o r the c e l l s grown on the two types o f agar whereas i n the c u l t u r e c o n t a i n i n g l a c t o s e , i t i s about twenty-five percent g r e a t e r employing c e l l s from glucose f r e e agar than c e l l s from glucose c o n t a i n i n g agar.  In a d d i t i o n , there i s no  marked d i f f e r e n c e i n the amount of ammonia present i n the c u l t u r e s c o n t a i n i n g the v a r i o u s carbohydrates.  The c u l t u r e s  c o n t a i n i n g l a c t o s e and the water c o n t r o l show a small quant i t y of ammonia that i s s l i g h t l y g r e a t e r from c e l l s grown on glucose f r e e agar than those from glucose c o n t a i n i n g agar  The E f f e c t 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 l i n e  Glucose-Containing Medium - broken l i n e . 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 Medium  Buffer Medium  Arginine  Aspartic Acid  Glutamic Acid  no glucose glucose  no sugar no sugar  62,5 54,0  38.5 34,5  17.0 10,5  no glucose glue os e  glucose glucos e  28.75 10.5  35.5 36.0  2©5 ©*o  no glucose glucose  sucrose sucrose.  32 ©5 13.0  3 9 © *5 40.0  2.0 0.0  no glucose glucos e  maltose maltose  39.0 34.0  41,0 33.0  2 «t *3 0.0  no glucose glucose  lactose lactose  59.25 54.5  43.5 31.0  9,0 5.0  no glucose glucose  glycerol glycerol  46.0 40.5  44.5 39.0  no glucose glucose  mannitol mannitol  36.25 31.25  37.0 29,0  ,  5.0 o.o 4.0 0.0  Figure 21.  Arginine  50  4C  30  20  10  Figure  22. Aspartic Acid  40 J  304  ^  3  20 4  104  I  Figure  23. Glutamic A c i d  20  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 f r o m containing the remaining f i v e  cultures  s u g a r s when c e l l s f r o m  glucose  f r e e a g a r a r e e m p l o y e d a n d no ammonia when t h o s e f r o m  glucose  c o n t a i n i n g agar are used. With the o b j e c t o f determining the e f f e c t of the presence  o f f e r m e n t a b l e and n o n - f e r m e n t a b l e  carbohydrates i n  t h e b u f f e r medium u p o n t h e r a t e o f ammonia f o r m a t i o n f r o m t h e f i v e amino a c i d s b y 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 t o w h i c h  t h e o r g a n i s m 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 .  .  .  (X) was u n d e r t a k e n .  experiment  .  The e l e v e n c a r b o h y d r a t e s e m p l o y e d may  be d i v i d e d i n t o t h r e e g r o u p s b a s e d  on t h e i r r a p i d i t y o f a c i d  production by Proteus ichthyosmius: G r o u p 1 - composed o f g l u c o s e a n d s u c r o s e a r e r a p i d  acid  formers, 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 p r o d u c e a c i d b u t n o t so r a p i d l y a s g r o u p one  - a c i d p r o d u c t i o n f r o m g l y c e r o l b e i n g much s l o w e r  t h a t f r o m t h e o t h e r c a r b o h y d r a t e s .-in t h i s  than  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 h i c h  fail  t o g i v e a c i d when a c t e d u p o n b y P r o t e u s I c h t h y o s m i u s . t i l l e d w a t e r was u s e d a s a c o n t r o l t o d e t e r m i n e  the q u a n t i t y  o f ammonia f o r m e d i n t h e a b s e n c e o f a c a r b o h y d r a t e . c u l t u r e s were s e t up i n s e v e n  Dis-  The  g r o u p s a n d I n c u b a t e d a t 30° C.  f o r o n e , t w o , t h r e e , f o u r , s i x , e i g h t , and t e n d a y s r e s p e c t ively.  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 e a c h o f t h e  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 i n 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 Galactose  -  violet broken green  Maltose  - broken - broken  Lactose  orange  Sucrose  Dextrin  -  violet orange  bvo\m  Salicin  broken brown.  Dulcitol  broken red  TABLE 12.  Effeot of the Presence of Carbohydrates Proteus ichthyosmius  carbohydrate  1  Arginine  2  age of culture i n days 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  8.5  12.75  15.0  20.75  -  24 © 25 36 0  glucose  5.25  galactose  41.0  39.0  35,75  39.5  31,5  _  sucrose  12.0  18.0  22 0 5  24,5  24.0  -  maltose  40.0  38.25  35.0  38.5  33,5  lactos e  49.5  51.75  54.25  61.5  59.5  -  dextrin  36.0  35 9 25  35.5  35.5  32«. 25  -  36.25  salicin  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  s  25.75 37.75 74.0  78.5  TABLE 13.  E f f e c t of the Presence of Carbohydrates  Proteus ichthyosmius  Aspartic A c i d  age of culture i n days 4 6 8  1  2  5  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  48  41  44  37  41  48  10  carbohydrate  lactose  . 49  dextrin  40  37  35  34  30  32  39  salicin  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  _1  2  age of culture i n days 3 4 6  5  6  6  4  1  0  0  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  salicin  3  1  0  0  0  0  0  dulcitol  10  -  13  16  26  32  37  water  10  20  28  27  42  8  10  carbohydrate glycerol xylose  :  >  11  13  ' '  -  TABLE 15. E f f e c t of the Presence of Carbohydrates  Proteus ichthyosmius  Histidine  age of culture i n days __1  2  3  4  6  8  10  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  19 3  2.0  3.0  4,0  lactose  2 .6  9.3  12.6  19.5  22.6  24.6  26.6  0.3  0.6  1»3  1.3  2.0  2,6  carbohydrate  dextrin  • 0  salicin  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.  E f f e c t of the Presence of Carbohydrates  Proteus ichthyosmius  Proline  age of culture in days 3 4 6  1  2  glycerol  1  2  3  0  xylose  2  6  9  11  .0  0  0  0  glucose  0  0  0  0  galactose  0  0  0  0  sucrose  0  0  0  2  maltose  0  0  0  0  . »  lactose  2  4  8  9  IS  dextrin  0  0  0  0  salicin  0  0  0  0  dulcitol  2  7  12  water •  S  8  11  8  10  0  0  0  19  25  31  0  0  0  0  -  0  0  0  0  0  0  0  10  14  0  0  -  0  -  21  28  31  -  23  18  28  35  carbohydrate  mannitol  *»  -  Figure 24. Proteus ichthyosmius  80  J  Arginine  Figure 25  Proteus ichthyosmius  60  Aspartic Acid  1  days  Figure 26 Proteus ichthyosmius  60  Glutamic Acid  Figure 27  Proteus ichthyosmius 70  60  Histidine  Figure 28  Proteus ichthyosmius  Prolire  70  60  50  40  -I  days  -  4 4  -  t a b l e s 12 t o 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 t o 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 , glutamic  a c i d , h i s t i d i n e and p r o l i n e , t h e q u a n t i t y o f f r e e  ammonia p r e s e n t i n t h e c u l t u r e s i s d e p e n d e n t i n most c a s e s on t h e r a t e o f a c i d p r o d u c t i o n bohydrates. the  This  observation  from the respective  c a n be seen most r e a d i l y i n  case o f a r g i n i n e , f i g u r e 24.  Xylose,  l a c t o s e do not. show a c i d p r o d u c t i o n and  the quantity  duction  d u l c i t o l and  a f t e r t e n days  incubation  o f f r e e ammonia f o r t h e s e c a r b o h y d r a t e s i s  equal t o that from the water c o n t r o l , maltose, mannitol,  car-  Salicin, glycerol,  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 pro-  i n 48 h o u r s and do n o t show a n y i n c r e a s e  ammonia a f t e r t h e f i r s t  day.  i n free  G l u c o s e and s u c r o s e a r e 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 n o t d e m o n s t r a t e so g r e a t  a quantity  o f f r e e ammonia f r o m  t h e b e g i n n i n g o f t h e e x p e r i m e n t a s e i t h e r g r o u p two o r t h r e e . No g r o w t h i n t h e c u l t u r e s c o n t a i n i n g  the f i r s t  s u g a r s was o b s e r v e d b u t i n t h o s e c o n t a i n i n g t h i r d g r o u p s g r o w t h was p r e s e n t .  group o f  t h e second and  Thus i t i s s e e n  that  P r o t e u s i c h t h y o s m i u s a p p e a r s t o h a v e t h e a b i l i t y t o use- t h e p r o d u c t s o f c a r b o h y d r a t e b r e a k d o w n a s c a r b o n s o u r c e s and t h e ammonia a s 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 d e c r e a s e d amount o f f r e e ammonia. When t h e f i g u r e s f o r g l u t a m i c proline  a c i d , h i s t i d i n e and  ( f i g u r e s 2 6 , 27 a n d 28) a r e c o n s i d e r e d  the r e s u l t s  - 45 obtained  a r e . on  -  the w h o l e , s i m i l a r t o t h o s e r e c o r d e d i n the  case of a r g i n i n e .  Inspection  o f t h e s e f i g u r e s shows t h a t  the t o t a l amount o f ammonia f o r m e d i s c o n s i d e r a b l y t h a t the  i n f l u e n c e of the  less  s p e c i f i c c a r b o h y d r a t e s on  but  the  r e l a t i v e amount o f ammonia f o r m e d i s u n c h a n g e d , p r a c t i c a l l y no  ammonia b e i n g  and  second groups of A  one  formed i n the  glutamic  o t h e r h a n d i s t o be the  a c i d , h i s t i d i n e and  s e e n when t h e  b r e a k d o w n p o s s e s s e d by r e l a t i o n s h i p w i l l be w i l l be  compared.  chemical  t o the mechanism o f  Proteus ichthyosmius.  considered  the  amounts o f ammonia f o r m e d  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 amino a c i d s and  the  p r o l i n e on  f i r s t ' twenty-four hours i n c u b a t i o n are  s t r u c t u r e o f the  first  carbohydrates.  s t r i k i n g d i f f e r e n c e b e t w e e n a r g i n i n e on  h a n d and  during  c u l t u r e s c o n t a i n i n g the  This i n t e r e s t i n g  i n the f i n a l d i s c u s s i o n  and  compared and. c o n t r a s t e d w i t h t h a t o f Pseudomonas  putrefaciens. 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 from that obtained i s t o be  f o r t h e o t h e r ••four amino a c i d s  seen, f i g u r e 25,  It will  amino a c i d , ammonia p r o d u c t i o n  f o u r amino a c i d s , ammonia f o r m a t i o n a postulate  complete w i t h i n case of  i n the  the  takes place w h i c h has  c o n f i r m e d by l a t e r work, ( v i d e experiment X I I I ) . of incubation increases,  this  occurs r a p i d l y , deamination of  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  same t i m e as a c i d p r o d u c t i o n ,  employed  be n o t e d t h a t f o r  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  other  distinct  As  at  the  been the  case o f a s p a r t i c a c i d ,  time  the  - 46 l e v e l o f f r e e ammonia i n t h e c u l t u r e s c o n t a i n i n g t h e f e r mented c a r b o h y d r a t e s d e c r e a s e s 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 t h e 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 t h e 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 unchanged.  I n the p r e s e n c e  practically  o f ample q u a n t i t i e s o f c a r b o n and  n i t r o g e n s o u r c e s i n t h e c u l t u r e s , one w o u l d e x p e c t more active c e l l m u l t i p l i c a t i o n with a correspondingly greater decrease  i n t h e f r e e ammonia p r e s e n t .  This lack of a c t i v i t y  s u g g e s t s t h a t p o s s i b l y t h e c o n d i t i o n s were n o t optimum f o r the growth  of the organism. The  experiment  was r e p e a t e d , ( X I ) , e m p l o y i n g  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 d e t e r m i n e  i f pH was t h e l i m i t i n g f a c t o r e n -  c o u n t e r e d a b o v e , 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 t h e s e t o f c u l t u r e s t o be l e f t f o r t h e l o n g e s t p e r i o d o f i n cubation.  A c o n t r o l set of cultures c o n t a i n i n g carbohydrate  w i t h o u t an amino a c i d was s e t up t o d e t e r m i n e  t h e changes i n  pH o f t h e c u l t u r e s i n t h e a b s e n c e o f c e l l g r o w t h .  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 fermentable  sugars  m a r k e d a c i d p r o d u c t i o n a n d i t was d e c i d e d t o d e t e r m i n e  showed elec-  tronic t r i c a l l y t h e f i n a l pH o f t h e c u l t u r e s p r i o r t o t h e d e t e r m i n a t i o n o f t h e ammonia c o n t e n t .  Because o f unusual  w e a t h e r c o n d i t i o n s , t h e 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 t h e c a r r y i n g o u t o f 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 e m p l o y e d i n e x p e r i m e n t X, one s e t o f c u l t u r e s o f n e c e s s i t y was n o t c o m p l e t e d  u n t i l an  i n t e r v a l o f twenty-four  - 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 w o r k 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 , b u t  the  results  f o r t h e e a r l y days o f i n c u b a t i o n i n d i c a t e t h a t i n t h e of c e r t a i n carbohydrates the pH  t h e r e i s ' marked a c i d p r o d u c t i o n  of these c u l t u r e s i s reduced  for bacterial activity.  presence and  below the l e v e l r e q u i r e d  T h e s e 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 d e a m i n a t e d more s l o w l y b y Pseudomonas p u t r e f a c i e n s than by Proteus  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 a b o u t t h e same r a t e b y 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 obtained f o r arginine, h i s t i d i n e , proline  and  the w a t e r 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 f i g u r e s 29 t o 35 The was  inclusive.  unavoidable  twenty-four  and  delay u n t i l the l a s t  set of c u l t u r e s  d a y s o l d when t h e ammonia c o n t e n t was  m i n e d 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  deterthe  ammonia f o r m a t i o n b y Pseudomonas p u t r e f a c i e n s i s n o t a t i t s maximum a t t e n d a y s i n c u b a t i o n . The  carbohydrates  are d i v i d e d i n t o three  groups  b a s e d 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  first  g r o u p 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 which produces a c i d s l o w l y , w h i l e the t h i r d made up  of x y l o s e , glucose, mannitol, g a l a c t o s e , maltose,  s u c r o s e 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 .  d e t e r m i n a t i o n s on t h e c o n t r o l s e r i e s c o n t a i n i n g o n l y , shown i n f i g u r e 3 5 , hydrates  group  i n t o the  a l s o c l e a r l y d i v i d e the  same t h r e e g r o u p s .  The  The  pH  carbohydrates carbo-  pH r e c o r d i n g f o r  The E f f e c t 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  *•> blue green  Mannitol  -broken blue  Glucose  - violet  Galacto se  - broken green  Sucrose  - broken v i o l e t  Maltose  - broken orange  Lac to se  - orange  Dextrin  - brown  Salicin  - broken brotvn  Dulcitol  - broken red  TABLE,17. Effect o f the Presence of Carbohydrates Pseudomonas putrefaciens  age o f culture i n days '  1  2  CAEBOBYDPA.TE  5  4  6  8  24  '  glycerol  1. 2.  ©.25; 6.90  6.5 6.80  0.5 • 6.65  1.0 6,35  0.25 6.30  1.0 6.25  4:4;»25 7.05  xylose  1. 2.  0.25 6.95  0 5,75  0 5,40  0.25 5.45  0.25 5.60  4.25 6.30  45.75 . 7,05  mannitol  1. 0.25. 2. 6..40.,  0.5 5.55  0.75 5.45  0.75 5.30  1.5 6,05  11,25 6,45  42.25 . 7.05  ;  i  glucose  1. 2.  1.0 5.75  0.75 5.05  0,75 5.10  1.0 5.00  0.5 5.10  6.25 5.95  54.25 •7.15  galactose  1. 2.  0.75 5.80  1.0 5.25  ' 1,0 5.55  0.25 5.20  0.5 5 e 25  . 1,0 5.80  48.5 7.15  sucrose  1. 2.  1.5 ' 1.5 5.80 5,45  1,25 5.45  0.75 5,50  0.5 5.75  3,0 5.50  40.5 7.05  mgltose  1. 2.  0.5 6,90  0,5 • 6,00: •  0.25 5,50  0.5 5.15  0 5.25  5.75 6.20  45.5 7.10  lactose  1. 2.  1.0 6,95  3.0 6.95  2.75 7.10  6.25 6.90  6.5 ; 6.85  15.25 6*80  42.75 6,05  dextrin,  1. 2.  1.25 6.95  3.0 6 .95  5.0* 6.95  6.75 6.90  9.25 7.05  13.0 6.95  50.5 7.15  1. 0.25 - 2. 6.55  0.75 5.40  0.75 5.30  1.25 - 5.20  1« 25 5.40  0.75 5.10  2,25 5.15  3.25 7.00  1.0 7.05  6.0 6.95  6.5 7.05  11,0 7.05  84.5 7.55  2.75 , 7.00  6.25 ' 7.05  5.5 7.00  7.5 7.05  8,5 7,05  78.75 7.55  * . •f  salacin  dulcitol  1. 2.  water  1. 1.75 2. "7.00  1.5 7.00  TABLE 18.  E f f e c t of the Presence of Carbohydrates Pseudomonas putrefaciens  Histidine  age of cul ture i n days 4 9  1  2  24  1. 2.  3.0 7.10  4.3 6.75  10.6 6.60  20.6 6*20  37.3 6.55  2.  3.0 7.10  3.3 6.05  4.3 5.45  8,3 5.25  10.6 5.30  mannitol  1. 2.  2*.0 6.50  3.6 5.65  4.3 5.60  6,6 5.20  16.6 6,10  glucose  1. 2.  0.3 5.65  1.0 4,90  1.6 5.10  3«3 5.25  16.0 6.25  galactose  X0 2.  5.80  13 5.05  1.6 5,30  2,6 5.30  4.3 5.15  2,0 5,40  4,0 5.65  8,6 5,45  20,3 6,00  Carbohydrate glycerol  xylose  sucrose  -  -  1e '  maltose  5.90 ' -  e  i . 2.  7.05  8.0 5.80  7.3 5.30  13.3 4,90  13.0 4.90  lactose  1. 2.  3.0 7,15  8.3, 6.95  14.6 7.20  45.0 7,05  54.6 6.05  dextrin  1, 28  4.6 7.15  11.0 6.95  30.6 7.20  50.0 ,7.20  49.0 6.95  salicin  1. 2.  2-.0 6.75  3©3 5.85  5.0 5.50  6.0 5.55  8.3 5.40  dulcitol  1. 2,  5,6 7.25  12.0 7.10  37.3 7.40  51.0 7.55  55.6 7.90  water  1. 2.  6.0 7.25  10.6 7.05  39,6 7.45  57,0 7.70  53.6 7,90  .  TABLE 19.  Effect of the Presence of Carbohydrates Pse^^domonas putrefaciens  Proline  age of culture i n days 4 9 24  i  2  2®  i . 6.85  0 6.55  1 6.55  6 6,05  32 6,50  i.. 2.  6.80  0 . 5.95  0 5.55 .  1 5.05  2. 5.00  1. 2...  6.65  0 6.05  0 5.65.  2 5.35  5 5.05  0 5 © 25  0 5,20  1 5.20  5 5.00  0 5.35  0 5.40  _  5.20  9 6,25  0 5,65 .  0 5.55  5.75  5 5.75  0 6.15  0 5,25  4,85  2. 4.80  11 6.95  21 . 6.55  40 5.80  16 . 6.80  17 , 6.85  arbohydrate glycerol  xylose  mannitol  glucose  galactose  sucrose  maltose  -  —  2,  6.05  1.. 2.  6.05  1.. 2.  _  -' 6.15  —  .1® 2,.  6.80  lactose  1. 2.  3 6.95  dextrin  1.. 2.  4. 6.85  8 7.35  8 6.95  salicin  12.  0 6 .65  0. 5.90  0 5.60  5»35  3 5.35  dulcitol  1© 2.  7 6.95  14 . 6.80  20 7.15  4 7.45  70 . 7.05  water  1.. 2 «_  5 6.95  12 6.80  25 7.15  39 . 7,10  71 . 7.05  _',  10 • , 6.65  «  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  7.40  7.10  7.25  6.70  Carbohydrate glycerol  ' .  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  salicin  7.10  6,55  6.45  5.50  7.30  7.65  7.35  7.60  dulcitol •water  .  7.40 7.40  -  (figures express f i n a l pH of cultures)  7.05 7.45  Figure 29  Figure 30 Pseudomonas putrefaciens  Histidine  70  60  -I  te  3  1  2  4  9  24 days  Figure 31 Pseudomonas putrefaciens  Arginine  F i g u r e 33  Pseudomonas p u t r e f a c i e n s  1  2  4  Proline  9  24 days  Figure 34 Pseudomonas putrefaciens  Proline  - 48 for  the f i r s t  n i n e d a y s o f i n c u b a t i o n , as s e e n i n f i g u r e 3 1 ,  32,  and 34, show t h a t t h e c a r b o h y d r a t e s  are fermented i n a  s i m i l a r manner f o r t h e t h r e e amino a c i d s , i n d i c a t i n g  that  the p r o c e s s  of fermentation  formation.  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  lactose occurs  i s i n d e p e n d e n t o f t h a t o f ammonia  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  the.breakdown o f t h i s  s u g a r i s c a u s e d b y an a d a p t i v e  from  that enzyme  w h e r e a s t h e f e r m e n t a t i o n o f t h e more r a p i d l y a t t a c k e d  sugars  Is- c a u s e d b y c o n s t i t u t i v e e n z y m e s . Ammonia f o r m a t i o n f r o m a r g i n i n e b y Pseudomonas putrefaciens increases s t e a d i l y u n t i l at l e a s t d a y s ' I n c u b a t i o n , a n d a s t h e ammonia c o n t e n t of the c u l t u r e s i n c r e a s e s except although  for salicin.  twenty-four  i n c r e a s e s t h e pH In this  culture,  t h e 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 f o r m e d a s t h e i n c u b a t i o n p e r i o d p r o c e e d s n o r i s t h e r e a s u b s e q u e n t i n c r e a s e i n pH. failure  t o f o r m ammonia i n t h e c a s e o f s a l i c i n i s due t o t h e  e l a b o r a t i o n of t o x i c products further bacterial activity. glucose  I t i s p o s s i b l e that the  and p h e n o l , w i l l ,  of fermentation  preventing  S a l i c i n , being a glucoside of  on h y d r o l y s i s t o y i e l d g l u c o s e  as  a carbon source, give 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 properties.  A c i d i t y alone  cessation of a c t i v i t y glucose  reached  c o u l d n o t be t h e r e a s o n  for this  s i n c e t h e pH o f t h e c u l t u r e c o n t a i n i n g  a lower  level  t h a n t h a t o f t h e one c o n t a i n i n g  salicin. The  maximum q u a n t i t y o f ammonia p r o d u c e d f r o m  h i s t i d i n e by t h i s b a c t e r i a l  species i s reacnea by nine  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 non-acid carbohydrates. mannitol  I n the cultures containing g l y c e r o l ,  and g l u c o s e ,  twenty-four  producing  t h e ammonia c o n t e n t  i n c r e a s e s up t o  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  pH, a s may be s e e n i n f i g u r e 3 2 .  sucrose,  The c u l t u r e s  increase i n containing  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 n o t i n c r e a s e  i n pH o r ammonia c o n t e n t  after nine  days  i n c u b a t i o n i n d i c a t i n g that a c t i v i t y has ceased i n these at nine  tubes  days. The  r e s u l t s o b t a i n e d , w i t h p r o l i n e show t h a t when  t h e pH r e a c h e s t o o l o w a l e v e l , a s i n t h e c u l t u r e s s a l i c i n , mannitol,  glucose,  x y l o s e , sucrose  containing  and m a l t o s e ,  a c t i v i t y d e c r e a s e m a r k e d l y 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 the  cultures containing galactose.  s a l i c i n d i d not reach but  containing galactose,  i n ammonia s u g g e s t i n g ,  again,  i s s o m e t h i n g among t h e b r e a k d o w n p r o d u c t s o f  s a l i c i n that i s not present capable o f p r e v e n t i n g The if  The c u l t u r e  as l o w a pH a s t h a t c o n t a i n i n g  i t d i d n o t show a n i n c r e a s e  that there  a c i d i t y a s t h e y do i n  question  I n those of g a l a c t o s e  further bacterial  and t h a t i s  activity.  that a r i s e s from these r e s u l t s i s t h a t ,  t h e pH c o u l d be c o n t r o l l e d b y i n c r e a s i n g t h e b u f f e r i n g  power o f t h e c u l t u r e s , w o u l d ammonia be f o r m e d i n t h e p r e s e n c e of acid-forming  carbohydrates?  I n an attempt t o o b t a i n  data  on t h i s a s p e c t o f t h e p r o b l e m , e x p e r i m e n t s e m p l o y i n g f i v e k e y carbohydrates i n the presence o f v a r y i n g b u f f e r w ere u n d e r t a k e n e m p l o y i n g P r o t e u s  ichthyosmius  concentrations a n d Pseudomonas  - 50 putrefaciens.  The c a r b o h y d r a t e s s e l e c t e d were g l u c o s e  from which both species  o f b a c t e r i a p r o d u c e 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 both species,  l a c t o s e -- f r o m  w h i c h no a c i d I s f o r m e d b y e i t h e r s p e c i e s , x y l o s e w h i c h Pseudomonas p u t r e f a c i e n s  -- f r o m  produces a c i d but Proteus  i c h t h y o s m i u s does n o t , and d e x t r i n -- f r o m w h i c h P r o t e u s i c h t h y o s m i u s p r o d u c e s a c i d b u t Pseudomonas p u t r e f a c i e n s not.  A c o n t r o l of d i s t i l l e d water i n place  was u s e d t o d e t e r m i n e t h e ammonia f o r m a t i o n  does  of the carbohydrate i n the absence o f  carbohydrate. In the f i r s t experiment, the a c t i o n o f Proteus I c h t h y o s m i u s on a r g i n i n e concentrations f i n a l buffer  i n the presence o f three  was s t u d i e d . concentration  The f i r s t  buffer  s e t of c u l t u r e s had a  t h e same as t h a t u s e d i n p r e v i o u s  e x p e r i m e n t s , M/20, t h e s e c o n d h a d d o u b l e t h e b u f f e r  concentra-  t i o n , M/lO, and t h e t h i r d had f o u r t i m e s the b u f f e r  concentra-  t i o n , M/5.  E a c h s e t o f c u l t u r e s was p r e p a r e d i n s i x g r o u p s  and  i n c u b a t e d f o r one, t h r e e ,  one  d a y s r e s p e c t i v e l y a t 30° C.  and  t h e f r e e ammonia c o n t e n t a r e g i v e n  f i g u r e s 36 t o 39 i n c l u s i v e . presentation,  s i x , ten,fourteen,  i n t a b l e 21 and  F o r t h e sake o f c l a r i t y i n when M/lO b u f f e r was em-  These r e s u l t s f e l l b e t w e e n t h o s e  found f o r the l o w e r and h i g h e r The  The f i n a l pH o f t h e c u l t u r e s  the r e s u l t s obtained  ployed are not portrayed.  and t w e n t y -  buffer  concentrations.  r e s u l t s o f t h i s e x p e r i m e n t show t h a t a n  of f o u r times t h e b u f f e r concentration medium a n d p r e v e n t s t h e f e r m e n t a t i o n  increase  c o n t r o l s t h e pH o f t h e  acids from lowering the  • - 51 pH t o a l e v e l u n f a v o u r a b l e  t o ammonia f o r m a t i o n .  This  fact  i s b r o u g h t o u t most c l e a r l y i n t h e c u l t u r e s c o n t a i n i n g glucose.  I n t h e p r e s e n c e o f M/20 b u f f e r c o n c e n t r a t i o n , t h e  pH o f t h i s  c u l t u r e d r o p s t o pH 5.3 and does n o t r i s e t o any  considerable  extent w i t h the r e s u l t  ammonia i's s m a l l .  that the q u a n t i t y o f f r e e  When, h o w e v e r , t h e b u f f e r  i s i n c r e a s e d , t h e pH o f t h e g l u c o s e  concentration  c u l t u r e s drops o n l y t o  pH 6.5 a n d t h e q u a n t i t y o f ammonia i n c r e a s e s t o t h e same amount a s t h a t i n t h e 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 acid.  carbohydrates  from which t h i s organism produces  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  o f M/20 b u f f e r , t h e pH d o e s n o t d r o p b e l o w pH 5.5 and b e c a u s e o f t h e g r e a t e r ammonia f o r m a t i o n production during the f i r s t compared w i t h t h e g l u c o s e r e m a i n a c t i v e and c o n t i n u e  and t h e s l o w e r r a t e o f a c i d  t h r e e days o f i n c u b a t i o n , as  containing c u l t u r e s , the b a c t e r i a t o p r o d u c e ammonia a n d , as a  r e s u l t , r a i s e t h e pH o f t h e medium p r a c t i c a l l y t o 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.  W h e t h e r t h e pH  i s r a i s e d d i r e c t l y b y t h e i n c r e a s e d ammonia, o r 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 w i t h t h e ammonia a s a n i t r o g e n s o u r c e  a c i d s as c a r b o n for cell  sources  multiplication,  i s a q u e s t i o n w h i c h c a n n o t be a n s w e r e d a t p r e s e n t . A n o t h e r i n t e r e s t i n g o b s e r v a t i o n f r o m f i g u r e s 38 and  39 d e p i c t i n g t h e pH c h a n g e s b y P r o t e u s  ichthyosmius i s  t h a t , i n t h e p r e s e n c e o f a r g i n i n e , l a c t o s e i s b r o k e n down with considerable The  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 .  s p e c i f i c i n f l u e n c e o f 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 a p p a r e n t when t h e a c t i o n o f t h i s o r g a n i s m on l a c t o s e i n t h e p r e s e n c e o f o t h e r amino a c i d s (vide  is  considered,  infra). I n o r d e r t o o b t a i n more d a t a on t h e i n f l u e n c e o f  buffer  concentration  on ammonia f o r m a t i o n ,  was e x t e n d e d t o i n c l u d e on  experiment X I I  the a c t i o n of Proteus  a s p a r t i c a c i d , glutamic  o f Pseudomonas p u t r e f a c i e n s  ichthyosmius  a c i d , h i s t i d i n e a n d p r o l i n e , and on t h e same f i v e amino a c i d s i n  t h e p r e s e n c e o f M/20 a n d M/5 b u f f e r c o n c e n t r a t i o n s each o f the f i v e  c a r b o h y d r a t e s used above.  employing  The r e s u l t s o f  t h i s e x p e r i m e n t ( X I I I ) a r e 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 t o 75 i n c l u s i v e . In general, that  t h e f i n d i n g s o f t h i s e x p e r i m e n t show  increasing the concentration  lowering  of the buffer prevents the  o f t h e pH o f t h e c u l t u r e s b y t h e f e r m e n t a t i o n  to a l e v e l that  acids  i s u n f a v o u r a b l e a n d , i n some c a s e s , f a t a l t o  further bacterial activity.  This  c o n t r o l o f t h e pH o f t h e  c u l t u r e medium p e r m i t s t h e b a c t e r i a t o c o n t i n u e t o  elaborate  ammonia a n d , i n t h e p r e s e n c e 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  t h e 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 o f t h e r e s u l t 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 , no  adequate e x p l a n a t i o n Increasing  brings  f o r some o f w h i c h  c a n be a d v a n c e d .  the b u f f e r  concentration  m a r k e d l y de-  c r e a s e s t h e q u a n t i t y o f ammonia f o r m e d b y Pseudomonas p u t r e f a c i e n s from arginine  i n the presence of the non-acid  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 t h e w a t e r  The E f f e c t 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  carbohydrate  1  3  age of culture i n days 10 6 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  1  .5  age of-culture i n days 6 10 14  21  Carbohydrate glycerol  1. 2,  31.0 .." . 38.0 7.55 7.55  36,25 . 30.5 7.35 7.20  27.5 7.15  40.75 7.25  xylose  1, 2.  3 X»5 ^4i.75 7,55 7,65  48,75 7175  54.25 7.75  74.25 7,85  64.25 7.75  glucose.  X9 2.  27.0 6,50  28.0 %9.5 6.75 . 7.05  30.75 7.15  41.5 7.25  43.75 7.25  lactose  1. 2.  37.5 7.65  46.25 7.65  55.25 7.65  61.5 7.65  60.0 7.50-  53.0 6,85  dextrin  1. 2•  19.0 6.70  31.25 6.75  28.75 7. 05  27,75 7.25  32.0 7.10  36.5 7.30  water  1. 2.  22.75 7.65  51.75 7.70  59.0 7.75  56.5 7.75  80.5 7.75  . 80,0 7,90  !  1, percent of t o t a l nitrogen of amino acid i n culture present as free ammonia, 2. pH of culture at time of ammonia determination.  TABLE 22. Proteus ichthyosmius  Aspartic A c i d  Buffer Concentration M/20  carbohydrate  1  age of culture i n days 3 6 - 10 14  21  glyceroJL .  1. 2. .  58-' 7.15  35 7.00  3l' ' 6.90  25. 6,65  23 6.50  21 6.35  xylose .  1. 2  40 7.25  37 37, 7.25. , 7.35  35, 7.45  38 7.55  39 7.60  glucose  1. •2.  24. 5.90  28 5.45  24 5.45  18. 5.40  24 5.40  23: 5.50  lactose  1. 2.  40. 7.20  36 7.25  36. 7.35  31: 7.25  34. • 7.40 .  297.35  dextrin  1. 2,  • '9 t .5.90  19  16.. ,-' 5,45  8 5.55  15.. 5.-30  22 5.50  •1. 2.  37 7.25  24 .7.50  34, 7.60  44• 7.60  10  14  21  31 7.35  12 7.25  14' 7.15  26 ' 7.20  22 • 7,50  31 ' 7.55  39 .* 7.55  8 6.70  8 6.65  6* 6,70  8 ' 6 75  9  water  5.60 44 7.35  41. 7.35  . Buffer Concentration M/5 age of culture i n days carbohydrate glycerol  :  1 1. 2.  •  3.  31 ' 38 7.40- , 7.35  6  xylose.  1. 2.  21 7.45  41 - ,40 7.45 7.50  glucose  1. 2,  16 6.95  9 -' 6,75  :  e  lactose  1.2.  30 • 7.45  34 7.45  33 7.45  24 7.45  20 •• 7.45  21 7.40  dextrin  IV 2.  5 : 6.95  3 6.75  2 ' 6,85  3 6.80  3 " 6 .95  1 6,95  water  '1.'. 2.  31 7.45  30 7.45  34 ' 7,45  33 ' 7.50  25 • 7.5.5  32 7,55  1. percent of total nitrogen of stmino a c i d 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  carbohydrate  1  age of culture i n days 6 . 10 14 4  21  glycerol  1.: 2.  3 7,25  4 7.15  4 7.15  •1 6.90  0 6.75  1 6,55  xylose  lv 2.-  4 7.25 '  11 7.25  15 7.30  26 7.45  25 7.50  41 7.60  glucose  1. 2.  2 5,90  2 5.40  2 5,40  1 5.40  0 5.40  1 5.45  lactose  1. 2.  4 7,50  10 7.30  10 7.30  11 7.25  9 7.20  11 7.25  dextrin  !*•  water  6 ..60  1 5.75  2 5.80  1 5.95  0 5.95  0  2. 1. 2.  '5 7.25  15 7,30  14 7.55  21 7.40  27 7.45  42. 7.65  v  ' 2  5,85  Buffer Concentration I l/5  carbohydrate  1  age of culture i n days 6 10 14 4'  21  1. '2*.  2 7.45  3 7.40  3 7.40  1 7,40  0 7,35  0  xylose  1. 2.  4 J' • 7.45' 7.45  8 7.45  14 7.45  7 7.55  . 26 7,55  glucose  1. 2.  1 7.05  1 2 6,70 . 6.75  0 6.65  0 6.75  1 6.75  lactose  19 2.  4 7.45  5 7.45  6 . 7,45  2 7.45  1 7,40  1 7.35  dextrin  1. 2.  1 7.35  1 6.85  1 6,75  1 6,95  0 6,95  2 7.05  1. •2-.  4 7.45  10 7.50  11 7,45  16 7.50  22 7.55  37 7,60  glycerol  water  1. 2.  :  7.30  percent o f t o t a l nitrogen o f amino acid i n culture present as free ammonia. pH of c ulture a t time of ammonia detenuination.  TABLE 24. Proteus  ichthyosmius  Histidine Buffer Concentration M/20  . carbohydrate  1  4  6  10  14  21  glycerol  1. 2.  2.0' 7.30  12.0 7.10  21.6 7.05  17.0 6.75  20.0 6.55  14.6 6.25  xylose  1. 2.  2.3 7.35  10.3 7.35  29.3 7.35  29.6 7.45  41.0 7.60  37.0 7.55  glucose  X9 2.  .6 6.15  .3 5.55  1.3 5.45  .6 5.40  1.0 5.55  6.6 5.45  lactose  1. 2.  2.3 7.30  7.6 7.35  19.6 7-35  26.6 7.30  32.0 7.35 -  25.3 7.35  dextrin  1. . :"<.0.0^ 2. 6.35  1.0 5.60  3.6 6.05  1.3 5.65  2.0 5.60  4.6 5.85  water  1. 2.  26.6 7.40  32,0 7.35  39.0 42.0 7.45 • 7.60  43.6 7.65  2.6 7.30  Buffer Concentration M/5 carbohydrate  1  age of culture i n davs 4. 6 10 "14  glycerol  I. 2.  2.0 •" . 21.0 7.45 7.40  xylose  1. 2.  2.0 7.45  glucose  1, 2.  .3 7.05  lactose  1. 2.  3 .6 7.45  18.3 7.45  dextrin  1. 2.  .3 6.95  water  1. 2.  3.6 7.45  1. 2.  21  26.0 .7.40  23.6 7.,25  20.0 7.15  16.3 7.05  31.6 . 7,50  35.0 7.50  37.0 7.55  38.3 7.55  3.3 ' ' ^3.6 6.75 6.75  10.6 6.75  8.6 6.85  9.0 6.80  14.3 7.45  20.3, 7.45  28.0 7.45  22.0 7.. 35  7.3 6.80  12.0 6.90  12.0 6.80  17,6 6.95  18.6 7.05  25.6 7.50  27.6 7.50  30.0 7,45  38.3 7.55  37.6 7.55  '21 a'3. 7.45  percent of t o t a l nitrogen of amino a c i d i n 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 of culture i n days 6 10 14  • • 4-  glycerol ,  1. 2.  2 7.55  3" 7.35  2 7.25  xylose  1.' 2.  4' 7.60  7 7.55  glucose  1. ' 2.  6.70  lactose  X• 2.  dextrin  •water  1 7.10  0 6.90  1 6.35  8 7.60  17 7.60  31 7.70  47 7.75  1 5.85  2 6.00  1 •5.95  1 6.05  1 ' 5.60  3 7.60  7 7.45  8 7 o 55  9 7.45  4 7.45  2 7.25  1. 2.  ... .. T 2 6.95  1 6.45  i 6.25  1 6.45  0 6.45  1 6.30  1. 2.  5 7.65  11 7.55  14 7*65  26 7.65  34 7,70  46 7.75  Buffer  carbohydrate 1. . 2.  1 7.55  xylose  1. 2.  3 -. 5 ' 7.55 7.55  glucose  1. 2.  1 7.25  lactose  1. 2.  dextrin .  water  2.  :  Concentrati on M/5  age of culture i n days -. 4 6 10 14  1 '  glycerol  1.  21  3 . 7.45  21  2 7.45  2 7.45  0 7.35  1 7.20  8 v- • 7.55  16" 7.55  32 7.65  . 44 r 7.*60  1 7.00  2 6.95  0 6.75  0 6.85  1' 6.85  3 . 7.55  5 7.50  8 7.50  3 7.45  1 7,45  1 7v25  I. . 2.  1. 7.35  1 7.15  2 7.05  1 6.96  1 7.15  2 7,05  1. 2.  4 7.55  9 7.55  14 7.55  16 7.50  40 7.55  43 7."60  (  A  percent o f t o t a l nitrogen of amino a c i d i n culture present fas. free ammonia pH of culture a t time of ammonia determination.  TABLE 26. Pseudomonas putrefaciens  Arginine B.uffer Concentration M/20  Carbohydrate  1  3  age,of culture i n days 6 15 • 11  21  glycerol  1. 2.  0.25 7.50  1.5 6.95  2,75 6.65  3.5 6.95  X o25 6,95  8.5 7.00  xylose  1. 2.  0.25 7.40  0.25 5.35  0,0 5,85  2.25 6.60  1.75 6.65  3.25 6.60  .glucose  1.  1.0 .6.05  1.5 5.10  2»25 5.10  X e 25 6.55  4.5 6.70  8.25 6,70  -.'7» .v  2.0 7.45  6.5 7.45  18.75 7.15  30.5 6.75  30,75 5.90  2,  X © 25 7.45  2.0 : 7.50  4.5 7.25  7.75 7.30  32.5 7.60  34.5 7.40  i. 2.  1.0 7.55  5.5 7.60  12.5 7.65  19.5 7.65  30.5 -7.85  32 e 75 7.90.  2. lactose  1. 2«  dextrin  water  1.0 50  Buffer Concentra t i on M/5 age of culture i n days 6 11 15  1  Carbohydrate  0.0 7.50 (  0.5  2.  0.25 •7.50  glucose  1. 2.  lactose  dextrin  glycerol  1,  1.0 7.25  0.75 7.15  2©5 7,05  3.25 7.15  o.o 7.00  6.90  1.00 7.05  1.0 7.15  2.0 7.25  '0.0 7,15  1.25 6,80  2.0 7,00  4.-75 7.10  4,5 7**15  4.5 . 7.15  I. 2.  1  0,5 7.50  1.5 7.50  1.5 7.40  4.25 7.45  9.5 7.30  14.75 7.15  1. 2.  0.5 7.45  1.0 7.50  1.25 7'. 45  3.75 7.45  6.25 7.50  6.25 7.50  1. 2.  0.25 . 7.50  3 ,0 7.55  3.25 7.55  4,0 7.55  4.25 7.60  11.5 7.60  2. xylose  water  21  1.  ;  1. percent of total nitrogen of amino acid i n culture present as free ammonia. 2. pH of culture at time of ammonia determination  TABLE 27. Pseudomonas putrefaciens  Aspartic A c i d  Buffer Concentration M/20 age of culture i n days 6 10 14 21  carbohydrate glycerol  1. 2.  37 7.20  33 6.90  31 6.55  35 6,45  38 6.45  28 6.25  xylose  1. 2.  26 7.20  30 6.00  26 5.45  34 5.10  38 5.05  23. 5.55  glucose  1. 2.  29 6.45  26 5.35  24 5.30  34 5.25  38 4.95  24 5.20  lactose  1. 2.  45 7.25  35 7.20  31 7.15  33 7.15  38 6.85  27 6.80  dextrin  1. 2.  33 7.25  33 7.20  18 7.30  14 7.30  15 7.20  10 7.20  1. 2.  36 7.35  38 • 7.30  33 7.50  38 7.55  35 7.55  29 7,65  .water  Buffer Concentration M/5 age of culture i n days 6 10 14  carbohydrate glycerol  1. 2.  36 7.50  32 7.35  25 7.15  .1. 2.  18 7.50  22 7.05  14  glucose  1. 2.  21 7.05  15 6.70  lactose  • 1. 2.  36 7.50  dextrin  1. 2.  water  1. 2.  Xylose  21  7.05  17 6.90  14 7.05  6.70  17 6.70  14 6.85  Oil ' 6.75  18 6.85  23 6.80  15 6.95  38 7.45  18 7.45  29 7.35  29 7.25  20 . 7.25  24 7.45  30 7.45  12 7.45  10 • 7.40  9 7.40  6 7.35  1 7.50  28 7.45  24 7.50  38 7.55  35 7.55  40 7.55  1. .percent of t o t a l nitrogen of amino acid i n culture present as free ammonia 2. pH of culture a t time of ammonia determination  TABLE 28. Pseudomonas putrefaciens  '  Glutamic A c i d  Buffer Concentration M/20 age of culture i n days 1 3 6 - io: .  Carbohydrate glycerol  14  21  1 3 7 © 2 5 7.00  3 6,55  6 6.30  9 6.25  13 6.15  1, 2.  i 7*25  2 5.55  1 5,25  2 3«2 5  0 5.15  3 5.05  glucose  1, 2.  1 6.50  4 5.10  4 5 © 25  4 5.25  6 5.15  24 6.10  lactose  1.  3 7.25  6 7.20  9  7.20  15 6.90  27 6.80  37 6.55  2.  2 7.25  5 7.20  3 7.10  11 7.15  19 7.10  24 7.15  1. 2.  4 7,30  11 7.30  17 7.40  26 7.55  41 7.50  62 7,85  14  21  1.  2. xylose  2o dextrin  mter  1.  Buffer Concentration M/5 age of culture i n days 3 6 10 1'  carbohydrate glycerol  xylose  glucose  1. 2e  1 7,50  3 7,45  0 7.25  1 7.00  2 6.95  0 6,95  1. 2.  2 7,4-5  2 :• 6.95  1 6j5  4 6.75  7 6.70  20 6 85  1.  1  3  6.70  2 6.70  14 6.75  19 6.85  27 6.90  3 7.50  8 7.40  5 7.45  14 7 ©35  18 7.20  39 7.15  2. lactose  1. 2  9  B  dextrin  1. 2.  2 7.45  5 7,45  5 7.45  10 7.40  18 7.35  32 7,45  water  1. 2.  4 7,50  11 7.45  12 7.50  51 7.55  37 7.50  62 7.65  1.  percent of t o t a l nitrogen of amino a c i d 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  age of culture i n days 6 ' 10 14  1  3  2.  6.6 7.30  25.6 6.95  1. 2.  6.6. 7.25  6.6 5. 30  1. 2.  3.6 4.6 5.95 ' 5.15  lactose  1. 2.  8.3 7.35  dextrin  1* 2.  water  1. 2.  glycerol.  xylose  glucose  -  29.6 6,40  20  36.0 6.35  41.3' .6.30  46.6 6.70  5.6 5.40  7.6 5.55  8.3 5.25  8.0 5.25  3.3 5.15  7.3 5.50  4.6 5.20  5.6 5.35  29.0 7.30  42.3 7.25  57.6 7.20  65.0 6.95  65.3 . 6.65  .- % 36.6 7.3 .7.35 7.35  49.3 7.15  54.3 7.25  57.3 7.20  58.3. 7.05  10.0 7.40  61.3 7.65  6.8.6 • 69.6 7.80 7.90 ,  47.3 7.45  62.6 8.10  Buffer Concentration M/5  carb ohydrate glycerol'.  , 1  '1* , 2.-  .  3  6.0 26.6 7.50. 7.35  l& of culture i n days 6. 14 10 40.6 7,05  43.6 •" 46.3' 6.90 6.85  '  21  •  41.0 7.00  xylose  1.  7.0 7,45  6.3 6.65  19.6, ""•6.-70  S i »3 6.-80  39.3 6.85  40.6 6.95  glucose  i. 2,  1,6 6,95  5,0 6.65  11.0 6.75  25.6 6.-85  32.0. 6 .85  40.6 7.05  lactose  X« 2.  7.5 7.50  23.0 7.45  36.6 7.45  56.6 7.45  54,6 7.25  50.3 7.20  dextrin  1. 2.  6.6 7,50  31.6 7.50  39.6 7.45  53.0 7.40  50.6 7,30  44.0 7,35  1. 2  9.6 7.50  50.0 7.50  48.0 7.55  65.0 7,60  67,3 7.65  54.3 7.75  • water  6  1. 2.  percent of t o t a l nitrogen of amino a c i d i n culture present as free- ammonia. pH o f culture at time of ammonia determination.  TABLE 30. Pseudomonas putrefacisns Buffer Concentration lf/20  carbohydrate  1  age' of culture i n days 3 6 '• 10  14  21  glycerol  1. 2.  2 7*55  1 7.10  •1 6.50  1 6.35  1 6.35  8 6,95  xylose  1. 2.  1 7.25  1 0 6.30 . 5.45  1 5.05  1 5.40  2 .5.20  glucose  1. 2.  0 6., 80  1 5.90  0 5.65  2 5.05  2 5.85  0 , 6,25  lactose  1. 2.  6 7.55  11 7.45  14 7.30  35 7.05-  30 6.80 •  37 6.55  dextrin  1. 2.  7.55  9 7.40  11 7.30  34 7.30  25 7.05  35 7.15  '. 1. 2-  6 .7.65  15 7.60  23 7.65  48 7.65  53 7.70  54 7.95  14  21  1 7.05.  0 7.00  1 7.00  1 6.80  o 6.65  7 6.95  water  Buffer Conc<antra t i on H/5 age of culture i n days 3 6 . -*1' 10  carbohydrate glycerol  1• 2.  2 7.55  2 7.45  xylos e  1. 2.  1 ; 7.50  iv •  glucose  1. 2.  0 7.35  .i 7.05  0 6.90  2 6.80  4 6.85  9 7.05  lactose  1.' 2.  4 7.55  9 7.50  19 7.45  ' 20 7.30  21 7.20  29 7.05  dextrin  1.  3 7.55  10 7.50  12 7.45  24 7.45  20 7.55  18 7.40  water '"•  1. 2.  7.55  10 7.55  20 7.55  32 7.55  35 7.55  43 7.65  7.15  0 7.25 1  /  \ 7^.00  1. percent of t o t a l nitrogen of amino a c i d i n culture present as free ammonia. 2. pH of culture a t time of ammonia determination  Figure 37 Proteus ichthyosmius  Arginine M/5 Buffer Concentration  10 days  14  21  Hgure 38 Proteus  ichthyosmius  Arginine M/20  8.0  J  7.0  4  Buffer  Concentration  6.0  5.0  Figure 39 l/5 Buffer  8.0  Concentration  A  7.0  6.0 10 days  14  F i g u r e 40 Pseudomonas p u t r a f a o i e n s  Arginine  M/20 B u f f e r  Concentration  30  20  10  11  F i g u r e 41 Buffer 20  10  days  Concentration  15  21  Figure 42 Pseudomonas putrafaoiens M/20 Buffer Concentration  A ginin© r  Figure  44  Probeu;- i c h t h y o s m i u s  Aspartic M/20  Buffer  Acid  Concentration  i —  days  10  14  21  F i g u r e 4-5 Proteus  ichthyosmius  Aspartic Acid M/5  Buffer  Concentration  Figure 46 Proteus ichthyosmius  Aspartic Acid M/20  Buffer  Concentration  8.0  7.0  li  i X  6,0  5.0 1  —i—•  —r—  10  14  21  14  21  days  Figure i/5 Buffer  8,0  47 Concentration  4  7.0  6.0  —  i  —  10 days  Figure 49 Pseudomonas putrefaciens M/5 Buffer Concentration  Aspartic Acid  Figure 50 Pseudomonas putrefaciens M/20 Buffer Concentration  Aspartic Acid  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 p u t r e f a c i e n s M/5 B u f f e r  Glutamic A c i d Concentration  F i g u r e 58 Pseudomonas p u t r a f a c i e n s M/20 B uf fe r G ore e n t r at ion.  Glutamic A c i d  Figure 60 Proteus ichthyosmius  Histidine M/20  Buffer Concentration.  Figure Proteus  61  ichthyosmius  Histidine M/5  Concentration  Figure 6 2 Proteus  ichthyosmius  Histidine M/20  Buffer Concentration  8.0 -i  Figure 63 M/5  8.0  Buffer  J  days  Concentration  Figure 64 Pseudomonas putrefaciens M/ao  Histidine Buffer  G one out r a t i on  70J  i 3  r —  1  6  10 days  1  14  1 21  Figure 65 Pseudomonas putrefaciens M/5 Buffer Concentration  Histidine  Figure 66 Pseudomonas putrefaciens M/20 Buffer Concentration  ( Histidine  Figure 68 Proteus ichthyosmius M/20 Buffer Concentration  days  Proline  Figure 69 Proteus ichthyosmius M/5 Buffer Concentration  Proline  Figure 70 Proteus ichthyosmius 1  M/20  Proline Buffer Concentration.  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 4 1 .  There  explanation of this finding.  appears  t o be no  adequate  The c u l t u r e s o f 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 g r o w t h  i n the presence o f b o t h  t h e a c i d - p r o d u c i n g and t h e 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 . This suggests that t h i s microorganism  i s capable of 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 b y - p r o d u c t s o f t h e 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 e l e v e n d a y s i n c u b a t i o n , f i g u r e s 42 and 4 3 .  after  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 ) , t h e pH o f t h e cultures  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 o m about  5.2 t o pH 6,6. a f t e r t h r e e d a y s i n c u b a t i o n w i t h o u t  pH  showing  an i n c r e a s e i n t h e f r e e ammonia c o n t e n t i n d i c a t i n g t h a t t h e b a c t e r i a a r e p o s s i b l y u s i n g t h e ammonia a s r a p i d l y as i t i s formed'' as a n i t r o g e n s o u r c e and t h e f e r m e n t a t i o n a c i d s a s carbon sources thereby removing  these a c i d s from the r e a c t i n g  medium a n d p e r m i t t i n g t h e pH t o r i s e .  An a l t e r n a t i v e e x -  p l a n a t i o n i s t h a t t h e b a c t e r i a have some mechanism f o r t h e p r o d u c t i o n o f a l k a l i n e compounds''other t h a n 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 . In the c u l t u r e s o f Proteus 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 I n t h o s e 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 b u t  h a s p r a c t i c a l l y no e f f e c t o n t h o s e c o n t a i n i n g x y l o s e , and g l y c e r o l .  lactose  The d e c r e a s e I n 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  c u l t u r e s i s p r o b a b l y due t o t h e u t i l i z a t i o n o f t h e ammonia f o r c e l l multiplication.  L a c t o s e i s n o t b r o k e n down w i t h a c i d  - 54 production by Proteus acid  Ichthyosmius  i n the presence o f a s p a r t i c  ( 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  production. 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 o f  t h e a c t i o n o f Pseudomonas p u t r e f a c i e n s o n 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 that increase i n  t h e 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 t h e r a n g e i n ammonia c o n t e n t o f t h e c u l t u r e s i n t h e p r e s e n c e o f t h e two g r o u p s o f carbohydrates, low  acid-producing  and n o n - a c i d - p r o d u c i n g .  The  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 t h e 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 g l u c o s e w i t h t h e r e s u l t that the a c i d i t y i s too great t o permit  bacterial  a c t i v i t y a n d t h e ammonia i s n o t u t i l i z e d f o r c e l l The  i n c r e a s e d b u f f e r content maintains  amount o f ammonia d e c r e a s e s carbohydrates.  i n those  reproduction.  t h e pH l e v e l and t h e  cultures with  fermentable  The s e c o n d o b s e r v a t i o n i s t h a t t h e q u a n t i t y  o f ammonia i n t h e c u l t u r e s w i t h d e x t r i n i n t h e p r e s e n c e o f both b u f f e r concentrations decreases pH. • T h i s s u g g e s t s ,  without  a n y change i n  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  o f u t i l i z i n g d e x t r i n as a c a r b o n source  forming  acids.  without  Ammonia f o r m a t i o n f r o m g l u t a m i c a c i d b y  Proteus  ichthyosmius  t a k e s p l a c e s l o w l y o v e r t h e t h r e e weeks o f  incubation.  Consequently,  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 t h e c u l t u r e s c o n t a i n i n g t h e acid-producing carbohydrates  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 t h e ammonia as r a p i d l y a s i t I s f o r m e d a s a  n i t r o g e n source  forcell multiplication.  containing acid-forming  carbohydrates,  I n the c u l t u r e s  Increase  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 e c r e a s e s t h e ammonia c o n t e n t  probably  b e c a u s e t h e more f a v o u r a b l e 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 t h e higher  Duffer permits The  more a c t i v e b a c t e r i a l  r a t e o f ammonia f o r m a t i o n f r o m g l u t a m i c  b y Pseudomonas p u t r e f a c i e n s f o r t h e f i r s t cubation but  reproduction* acid  t e n days o f i n -  i s a b o u t t h e same a s t h a t f o r P r o t e u s  ichthyosmius,  i n c r e a s e s a f t e r t h e t e n d a y i n t e r v a l t o s u c h an e x t e n t  t h a t ammonia f o r m a t i o n a n i t r o g e n source  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 a s  b y t h e b a c t e r i a and a s a r e s u l t  ammonia a c c u m u l a t e s i n t h e c u l t u r e s .  free  The o t h e r p o i n t o f  n o t e f r o m f i g u r e s 56 and 58 i s t h e sudden i n c r e a s e i n a c t i v i t y i n the c u l t u r e containing glucose  i n the presence o f t h e 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 d a y s i n c u b a t i o n a s shown by t h e i n c r e a s e i n ammonia c o n t e n t  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 Proteus  ichthyosmius  on h i s t i d i n e , t h e o n l y e f f e c t o f t h e i n c r e a s e b u f f e r conc 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 t h e pH o f t h e c u l t u r e s i s to Increase  t h e q u a n t i t y o f ammonia i n t h e 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 u c o s e .  This increase I s , i n a l l  p r o b a b i l i t y , t h e 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  thereby  preventing  takes  the decrease i n b a c t e r i a l a c t i v i t y that  p l a c e when t h e pH d r o p s t o o l o w . 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 that xylose i s the carbohydrate  which w i t h glucose  and n o t d e x t r i n  shows i n c r e a s e d  - 56 ammonia c o n t e n t i n t h e i r r e s p e c t i v e concentration  c u l t u r e s when t h e b u f f e r  i s increased f o u r f o l d .  that acid production  from x y l o s e  I t i s t o be r e c a l l e d  and n o n - a c i d  production  f r o m 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 Proteus  from  ichthyosmius. 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  proline  containing  show p r a c t i c a l l y no r e s p o n s e i n t h e f o r m o f  o r d e c r e a s e d ammonia f o r m a t i o n concentration. the o t h e r  to the increase  In buffer  The pH o f t h e M/20 b u f f e r c u l t u r e s  f o u r amino a c i d s , s u g g e s t i n g  increased  containing  that the b a c t e r i a l  a c t i v i t y does not d e c r e a s e i n t h e case o f p r o l i n e t o t h e e x t e n t that i t d i d w i t h the other p l i c a t i o n takes place buffer  amino a c i d s a n d t h a t c e l l m u l t i -  e q u a l l y w e l l I n the presence o f both  concentrations. 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  Pseudomonas p u t r e f a c i e n s in buffer  concentration  f o r the a c t i o n of  on p r o l i n e e x c e p t t h a t t h e  Increase  caused a marked d e c r e a s e i n t h e 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 d a y s i n c u b a t i o n . The B r e a k d o w n o f A r g i n i n e and  Pseudomonas  by Proteus  ichthyosmius  putrefaciens.  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  of seventy to e i g h t y percent of i t s t o t a l nitrogen  conversion into  ammonia b y P r o t e u s i c h t h y o s m i u s and a b o u t t h i r t y p e r c e n t b y Pseudomonas p u t r e f a c i e n s ,  suggested that  the pathway o f t h e  breakdown b y i n v e s t i g a t e d .  I n order  t o carry out t h i s  t i o n , t h e above two s p e c i e s  o f b a c t e r i a were  sugges-  Inoculated  i n d i v i d u a l l y into buffer cultures of arginine, ornithine  TABLE 31. Ammonia Formation from Arginine and i t s Decomposition Products.  Proteus iohthysomius Compound  cc. B/100.H S0 2  Arginine  T  4  12.85 13.95  % T. L . into 64.25 69.75  Ornithine  7.0 7.0  70.0 70.0  Delta-Amino Valeric Acid  0.5 0.55  10.0 11.0  Urea  1.0 1.0  10.0 10.0  Pseudomonas  Putrefaciens  Arginine  9.0 9.4  45,0 47.0  Ornithine  7.05 6*9  70.5 69.0  Delta-Amino Valeric Acid  03 0.35  6.0 7.0  Urea  0.65 0.65  8  :  6.5 6.5  (prepared as o u t l i n e d b y Hunter  (37)),  delta-amino  n - v a l e r i c a c i d a n d 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 c u b a t e d a t 30° G. f o r t w e n t y - o n e experiment  days.  The r e s u l t o f t h i s  (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 t h e c a s e 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 t h e ammonia f o r m e d t o t a l o f t h a t formed formed  f r o m a r g i n i n e was g r e a t e r t h a n t h e  f r o m o r n i t h i n e and u r e a , and t h a t  that  f r o m o r n i t h i n e was s e v e n t i m e s g r e a t e r t h a n t h a t  delta-amino v a l e r i c a c i d .  from  On t h e o t h e r h a n d , t h e ammonia  f o r m e d b y Pseudomonas p u t r e f a c i e n s f r o m a r g i n i n e was o n l y s l i g h t l y more t h a n t h a t f r o m o r n i t h i n e a n d u r e a w h i l e t h a t formed  f r o m o r n i t h i n e was more t h a n s e v e n t i m e s g r e a t e r t h a n  t h a t from delta-amino 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 abilityto groups  deaminate  b o t h t h e a l p h a and t h e d e l t a animo  Of t h e o r n i t h i n e u s e d  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 ' c a n f o r m s i g n i f i c a n t q u a n t i t i e s o f ammonia f r o m 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 discussion of 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 probable c o u r s e o f b r e a k d o w n 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 t h e thesis. Ammonia F o r m a t i o n f r o m Non-Amino A c i d N i t r o g e n o u s  Compounds  D u r i n g t h e course o f t h i s study, the q u e s t i o n arose a s t o w h e t h e r amino o r i m i n o g r o u p s  o f n i t r o g e n o u s compounds  o t h e r t h a n ' a m i n o a c i d s c o u l d be c o n v e r t e d i n t o a m m i n i a b y the surface t a i n t producing b a c t e r i a .  I n order to obtain  some d a t a o n t h i s a s p e c t o f t h e p r o b l e m ,  an e x p e r i m e n t  (XV)  TABLE 32.  Ammonia Formation from Amino-Group Containin, Compounds other than Amino Acids.  Compound  Asparagin  Prot. ichthyosmius  Pseudo. putrefaciens  5 clays  5 days  6.80  2_2__days^  4,95  22 days  6.80  5.45  glutamine 4,0 4.35 (control 2.3 - not subtracted)  4,1  5®9  adenine  4.05  4,70  1.15  3 e55  guanine  3.90  8.20  4.5  2.80  uracil  „35  uric a c i d  .65  nicotinic acid  ,4  ,95  betaine  ..45  ,65  .45  .75  urea  .3  .8  .25  .6  .8. . 1.25  © 25  .75  a 55  .85  2.6  Results expressed as cubic centimeters 83 Jl/lOO sulphuric a c i d equivalent t o the ammonia formed i n 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 t o percent ammonia formed of the total nitrogen, (see discussion)  3.6  - 58 employing the putrefaciens  - •  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 on  the f o l l o w i n g compounds was  adenine, asparagin,  betaine,  a c i d , u r a c i l , u r e a and  Pseudomonas  carried  out:  glutamine, guanine, n i c o t i n i c  uric acid.  The  e x p e r i m e n t was  as o u t l i n e d a t t h e b e g i n n i n g o f t h i s s e c t i o n o f t h e with  the  acids.  above n i t r o g e n o u s compounds r e p l a c i n g t h e Controls  containing  i n o c u l a t i o n w i t h one  of the b a c t e r i a l species  a l k a l i n e a e r a t i o n o f t h e Van  The  C. f o r f i v e and  r e s u l t s are given The  i n table  f i r s t point  is  t h a t g l u t a m i n e i s the  in  the  equivalent  nitrogen  the  thesis amino  o f the  compounds b y  procedure.  §et  were e m p l o y e d  The  the  cultures  b e i n g removed a f t e r i n c u -  t w e n t y - t w o d a y s respectively® 32.  of i n t e r e s t from these r e s u l t s o n l y one  c o n t r o l series that  ammonia d u r i n g is  Slyke  i n d u p l i c a t e , one  b a t i o n a t 30°  up  t h e n i t r o g e n o u s compounds w i t h o u t  t o d e t e r m i n e t h e b r e a k d o w n , i f any,  were s e t up  set  of the  compounds t e s t e d  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  alkaline aeration.  The  quantity liberated  t o a b o u t f i f t y p e r c e n t o f one  group a f f e c t e d i s p r o b a b l y the  nitrogen.  The  amide g r o u p w h i c h  decomposes r e a d i l y u n d e r 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  of t h i s decomposition, i t i s d i f f i c u l t to determine i f the d e c o m p o s i t i o n of the containing  amide g r o u p i n t h e g l u t a m i n e  the b a c t e r i a i s caused by  a e r a t i o n or takes place determination.  The  s i g n i f i c a n t l y during  chemically  cultures  the b a c t e r i a p r i o r t o  during  the  a c t u a l ammonia  amide g r o u p o f aspara'gin d i d n o t the  alkaline aeration.  This  decompose  striking  - 59 d i f f e r e n c e between the amides of a s p a r t i c a c i d and a c i d r e s p e c t i v e l y w i l l be d i s c u s s e d more f u l l y  glutamic  later.  The q u a n t i t y o f ammonia formed from asparagin by Proteus ichthosmius and Pseudomonas p u t r e f a c i e n s i s equ i v a l e n l t o between f i f t y and seventy percent of the t o t a l n i t r o g e n . This f i n d i n g shows c l e a r l y that these species are capable of splitting  o f f both the amino and the amide groups of  1-asparagin.  The r e s u l t s obtained f o r glutamine,  on the  other hand, do not n e c e s s a r i l y i n d i c a t e that both groups are a t t a c k e d 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 f o r the determination of ammonia from glutamine by the  Van  Slyke procedure  t o which reference has  p r e v i o u s l y been made, renders impossible a c l e a r - c u t  inter-  p r e t a t i o n o f the f i g u r e s obtained f o r the a c t i o n of these micro organisms on t h i s compound.  No d e f i n i t e c o n c l u s i o n  can be reached as to t h e i r a c t i o n on the amide group of glutamine. of  There i s , however, some ammonia formed i n excess  the c o n t r o l but  whether i t comes from the amino or the  amide group cannot be The  determined.  o b j e c t o f studying the breakdown of  guanine, u r i c a c i d and u r a c i l was ichthyosmius  adenine,  to determine of Proteus  and Pseudomonas p u t r e f a c i e n s had the a b i l i t y  to  open the purine and p y r i m i d i n e r i n g s with subsequent deaminat i o n and formation of ammonia. p y r i m i d i n e ) c o n t a i n s two  U r a c i l (2, 6, dioxy  imino groups.  U r i c a c i d (2, 6,  t r l o x y p u r i n e ) has f o u r n i t r o g e n groups, two r i n g and two  i n the pyrimidine r i n g .  8,  i n the i m i n a z o l  Adenine (6 amino  p u r i n e ) has a f r e e amino group i n a d d i t l o n to the f o u r  - 60 nitrogens i n the purine r i n g , while guanine (2 amino, 6 oxy purine) has, i n a d d i t i o n to a f r e e amino group, an oxy group. Whereas the amino group i n adenine i s i n p o s i t i o n 6, that i n quanine i s i n p o s i t i o n 2.  The f i n d i n g s of the experiments  employing these f o u r compounds show c l e a r l y t h a t the p y r i m i d i h e r i n g of u r a c i l and the purine r i n g of u r i c  acid  are both opened to a s l i g h t extent w i t h the formation o f small q u a n t i t i e s of ammonia and that the f r e e amino group of both adenine and guanine i s deaminated. When the r e s u l t s f o r the twenty-two day p e r i o d of i n c u b a t i o n are c o n s i d e r e d , i t i.s seen t h a t i n the case of Proteus ichthyosmius  rupture of one of the r i n g s i n guanine  w i t h the r e s u l t a n t formation o f ammonia has a l s o occurred. I t would appear most l i k e l y t h a t the formation of ammonia has r e s u l t e d from cleavage o f the i m i n a z o l nucleus and has  arisen  from e i t h e r 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 a c i d employed are of i n t e r e s t .  was  The f i n d i n g s show c l e a r l y that the  p y r i d i n e r i n g i s opened by each of the organisms s t u d i e d , Pseudomonas p u t r e f a c i e n s p o s s e s s i n g the more marked a b i l i t y t o e l a b o r a t e ammonia from t h i s r i n g s t r u c t u r e *  The  opening  of f i v e d i s t i n c e nitrogen containing r i n g structures with subsequent deamination  r e s u l t i n g i n the formation of ammonia  has thus been demonstrated f o r these s p e c i e s of microorganisms.  These b a c t e r i a l species have p r e v i o u s l y been  shown to a t t a c k the i m i n a z o l  P  p y r o l l i d l n e , pyrimidine  and  purine rings© The two  other compounds s t u d i e d , betaine  (from  - 61 g l y c i n e ) a n d u r e a gave s m a l l q u a n t i t l e d o f ammonia u n d e r t h e 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 n o t 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 speculation.  The f a i l u r e t o h y d r o l y s e u r e a t o any e x t e n t b y  t w o o r g a n i s m s t h a t h a v e b e e n shown t o decompose r e a d i l y "is d i s c u s s e d e l s e w h e r e .  arginine  - 62 PART I .  DISCUSSION The experiments r e p o r t e d upon h e r e i n c o n s t i t u t e t h e  f i r s t study of the c o n d i t i o n s governing deamination by the two  species of b a c t e r i a - Proteus ichthyosmius and Pseudo-  monas p u t r e f a c i e n s - that has been r e p o r t e d .  Similar studie  employing d i f f e r e n t techniques and more r a p i d l y a c t i n g b a c t e r i a such as Bacterium c o l i have been recorded.  The  technique f o l l o w e d i n previous s t u d i e s , t h a t using the Warburj apparatus, however, could not be f o l l o w e d i n t h i s  study  because the two species of surface t a i n t b a c t e r i a employed are r e l a t i v e l y slow to a t t a c k the amino a c i d s , sometimes r e q u i r i n g 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 l e s s . The method o f ammonia determination also d i f f e r e d i n t h i s study from most of the previous s t u d i e s .  Since the 9  c o l o r i m e t r i c determination of ammonia employing N e s s l e r s reagent i s i n h i b i t e d by the presence o f c e r t a i n nitrogeneous compounds, outstanding among which are h i s t i d i n e tryptophan ers,  and  ( 5 0 ) , t h i s procedure, commonly used by other work-  could not be employed d i r e c t l y i n t h i s study.  Distil-  l a t i o n o f the ammonia from the c u l t u r e f o l l o w e d by N e s s l e r i z a t i o n would have overcome t h i s d i f f i c u l t y but such a procedure i s too slow t o permit completing the l a r g e number of determinations r e q u i r e d i n the experiments The a l t e r n a t i v e procedure that was a e r a t i o n procedure..  d e s c r i b e d above.  chosen was  the Van Slyke  This procedure, although p o s s i b l y not  y i e l d i n g as a c c u r a t e r e s u l t s as (may be obtained employing N e s s l e r ' s reagent, was  s  found to aerate n i n e t y - s i x t o n i n e t y -  - 63 eight percent  o f the ammonia added, i n a test s e r i e s and  had  the (required^ advantage of p e r m i t t i n g the c a r r y i n g out of the large number of determinations r e q u i r e d i n t h i s The  study.  decomposition of a r g i n i n e by Proteus  ichthyos-  mius has been shown to y i e l d s e v e n t y - f i v e to eighty percent i t s n i t r o g e n as ammonia.  This means that at l e a s t three,  i n a l l p r o b a b i l i t y f o u r , of the nitrogens  species.  i s produced by Proteus ichthyosmius,  the products of h y d r o l y s i s by t h i s enzyme would be and urea.  and  of a r g i n i n e are  converted p a r t i a l l y or completely i n t o ammonia by t h i s I f the enzyme arginase  of  ornithine  The r e s u l t s o f experiment XIV have shown that  Proteus ichthyosmius converts  seventy percent  of o r n i t h i n e i n t o ammonia but only ten percent  of the  nitrogen  of that of urea.  These f i n d i n g s suggest that the e l a b o r a t i o n o f b a c t e r i a l urease f o r the s p l i t t i n g of urea may e l a b o r a t i o n of a r g i n a s e . s i g n i f i c a n t extent  The  depend upon the  prior  f a i l u r e to s p l i t urea to a  shows that t h i s species i s unable t o form  urease i n the presence o f urea alone.  The r e s u l t s  reported  upon h e r e i n support the f i n d i n g s d e s c r i b e d by H i l l i n work on the s p l i t t i n g of the a r g i n i n e molecule and may  lend credence  t o the theory put f o r t h by him with r e s p e c t to the of a r g i n i n e The  activity  dihydrolase. rate and q u a n t i t y o f ammonia formed from  a r g i n i n e by Pseudomonas p u t r e f a c i e n s formed by Proteus"Ichthyosmius  e  i s not so great as t h a t  Is would appear that  the  a l a b o r a t i o n of the enzymes necessary f o r the breakdown of a r g i n i n e i s much slower and not Pseudomonas p u t r e f a c i e n s  as  so complete i n the  compared w i t h Proteus  case of  - 64 ichthyosmius.  The  t o t a l quantity of 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 s , however, i n excess  of t h a t formed by the same species from o r n i t h i n e and suggesting  again  urea,  that there i s an a s s o c i a t i v e a c t i o n  present  i n the a l a b o r a t i o n of the d i f f e r e n t enzymes r e q u i r e d f o r the complete" degradation of a r g i n i n e . The breakdown o f o r n i t h i n e has been found to be s i m i l a r f o r both b a c t e r i a l species - seventy percent n i t r o g e n being converted i n t o ammonia.  of i t s  This f i n d i n g shows  c l e a r l y t h a t these b a c t e r i a l s p e c i e s have the a b i l i t y to a t t ack both the. alpha and the deli^a amino groups of o r n i t h i n g to a considerable  extent.  When, however, the r e s u l t s showing a l -  most i n s i g n i f i c a n t q u a n t i t i e s of ammonia formed from the deami n a t i o n of d e l t a amino v a l e r i c a c i d (the compound formed by deamination of the alpha  amino group o f o r n i t h i n e ) are  the  con-  s i d e r e d , i t can r e a d i l y be seen that the e l a b o r a t i o n of  the  deaminase f o r a t t a c k i n g the d e l t a amino group i s dependent upon the p r i o r e l a b o r a t i o n of the alpha amino deaminase - a sequence o f events s i m i l a r to that found f o r the  elaboration  of ammonia from a r g i n i n e . A s p a r t i c and. glumatic  acids are both d i c a r b o x y l i c  mono-amino a c i d s d i f f e r i n g i n s t r u c t u r e only by the presence of an a d d i t i o n a l GHg  group i n the case of glutamic  comparison of the r a t e s of ammonia formation  acid.  from these  amino a c i d s , however, r e v e a l s s t r i k i n g d i f f e r e n c e s . d l - a s p a r t i c a c i d employed was f i f t y percent  A two  The  deaminated t o the extent  of  of i t s n i t r o g e n w i t h i n twenty-four hours by  each o f the two b a c t e r i a l species *  A longer p e r i o d of  - 65 i n c u b a t i o n d i d not r e s u l t  i n f u r t h e r formation of ammonia.  Previous s t u d i e s ( 5 0 ) have shown t h a t the 1-isomer of t h i s amino a c i d i s deaminated very r a p i d l y suggesting that one hundred percent of the 1-compound i n the dl-mixture i s attacked while the d-compound i s not attacked.  When d-glutamic  a c i d i s considered, i t i s found t h a t deamination  takes p l a c e  slowly over the complete p e r i o d of i n c u b a t i o n - f o r t y percent of the t o t a l n i t r o g e n being converted i n t o ammonia a f t e r twenty-one days i n c u b a t i o n . of deamination  One reason f o r t h i s slower r a t e  when compared w i t h t h a t found i n the case of  a s p a r t i c a c i d may  be that the cVIsomer i s not r e a d i l y attacked.  I t would be necessary t o employ the 1~ or the dl-isomer i n order t o make a d i r e c t comparison o f the r a t e s of of  deamination  these two amino a c i d s • The conversion by Pseudomonas p u t r e f a c i e n s o f about  seventy percent of the n i t r o g e n of h i s t i d i n e Into ammonia shows c l e a r l y t h a t t h i s b a c t e r i a l species has the a b i l i t y to open the i m i n a z o l r i n g and to deaminate the compound thus formed.  Cleavage  of  molecule  o f ammonia f o l l o w e d by o x i d a t i o n , o x i d a t i v e decarboxy-  l a t i o n and deamination of glutamic a c i d  the r i n g w i t h the l i b e r a t i o n of  r e s p e c t i v e l y may  (see f i g u r e 2)©  upon h e r e i n , 1 - h i s t i d i n e was  employed.  P r o v i d i n g the f i r s t two  i n the formation  In the experiments  h i s t i d i n e takes p l a c e as suggested, formed.  result  one  reported  I f the degradation of  l=glutamic a c i d should be  ammonia molecules  i n the  degradation are r e l e a s e d t o t h e extent of approximately hundred p e r c e n t , about ten percent of the t h i r d  one  molecule  present i n the suggested glutamic a c i d are converted i n t o  «. 66 ammonia showing a slower r a t e of breakdown than takes p l a c e when d-glutamic a c i d i s employed by The cleavage o f  itself.  the i m i n a z o l r i n g by Proteus  ichthy-  osmius i s not so r a p i d nor so complete as t h a t found f o r Pseudomonas p u t r e f a c i e n s - about f o r t y percent o f the t o t a l n i t r o g e n b e i n g converted i n t o ammonia.  The r i n g Is d e f i n i t -  e l y opened, however, by Proteus Ichthyosmius  s i n c e a maximum  of t h i r t y - t h r e e percent ammonia would be formed by  deamination  of the side chain amino group a l o n e . The decomposition of p r o l i n e w i t h the l i b e r a t i o n of between f o r t y and f i f t y percent^ of i t s n i t r o g e n as ammonia takes p l a c e e q u a l l y w e l l under the i n f l u e n c e o f the two terial  species employed.  bac-  P r o l i n e i s not a t r u e amino a c i d  but contains i t s n i t r o g e n as an Imino group i n the p y r o l l i d i n e ring structure.  Opening of t h i s r i n g i s a p r e r e q u i s i t e of  ammonia formation from t h i s compound a t a l l times. The dine r i n g may  pyrolli-  be broken on e i t h e r side of the imino group«  H y d r o l y t i c cleavage on the side next t o the c a r b o x y l group y i e l d s alpha hydroxy  d e l t a amino v a l e r i c a c i d .  I f the  deamination of t h i s compound i s s i m i l a r t o that of d e l t a amino v a l e r i c a c i d , i t i s p o s s i b l e that t h i s method, of opening the r i n g does not take p l a c e .  O x i d a t i v e opening of the r i n g  on the side of the imino group away from the c a r b o x y l group would g i v e glutamic a c i d .  Weil-Malherbe  and Krebs have  concluded t h a t p r o l i n e i s o x i d i z e d by kidney t i s s u e t o glutamic a c i d which i n t u r n may  be f u r t h e r o x i d i z e d t o alpha  keto 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 o f one molecule ammonia.  of  F u r t h e r evidence i n support of cleavage of the  - 67 p y r o l l i d i n e r i n g i n t h i s manner may  be obtained when the  f i g u r e s from the d i f f e r e n t experiments f o r glutamic a c i d and p r o l i n e are compared.  For example, f i g u r e s 52 and 55  and  f i g u r e s 68 and 69 d e p i c t i n g the ammonia formation by Proteus ichthyosmius from glutamic a c i d and p r o l i n e r e s p e c t i v e l y show c l e a r l y " t h a t the r a t e of formation and the f i n a l q u a n t i t i e s . produced are h i g h l y  comparable.  The r e s u l t s o b t a i n e d i n the experiments  outlined  above add f u r t h e r evidence t o the hypothesis shown In f i g u r e 2 t h a t the s i x amino a c i d s s t u d i e d are i n t e r r e l a t e d .  The  aIpha keto and alpha hydroxy apids of d e l t a amino v a l e r i c a c i d and alpha keto g l u t a r i e a c i d are probably the l i n k s j o i n i n g a r g i n i n e , o r n i t h i n e and p r o l i n e to h i s t i d i n e , a c i d and glutamic a c i d .  aspartic  The q u a n t i t i e s of ammonia formed  during the decomposition of these s i x 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 are s u f f i c i e n t t o suggest that t h e i r  two  break-  down proceeds through the number of o x i d a t i o n s and d e s t i n a t ions o u t l i n e d .  A r g i n i n e , under the i n f l u e n c e of Proteus  ichthyosmius shows the l i b e r a t i o n o f f o u r n i t r o g e n s i n the form of ammonia i n d i c a t i n g the f o r m a t i o n of alpha keto g l u t a r i e a c i d o r one of i t s decomposition products.  The  a c t i o n of Pseudomonas p u t r e f a c i e n s on a r g i n i n e does n o t l i b e r a t e so much ammonia as Proteus ichthyosmius but the decrease i s probably due t o the f a i l u r e t o h y d r o l y s e urea s i n c e i t has been shown that t h i s  species i s capable of  l i b e r a t i n g seventy p e r c e n t of the n i t r o g e n o f / o r n i t h i n g i n the form o f ammonia. The b r e sled own o f p r o l i n e by both b a c t e r i a l species  - 68 a l s o p o i n t s to the formation o f alpha keto g l u t a r i c a c i d or one o f i t s p r o d u c t s .  While the p o s s i b i l i t y of the course o f  breakdown from alpha keto delta-amino v a l e r i c a c i d proceeding by r e d u c t i v e deamination t o the simpler compounds of v a l e r i c a c i d must be considered, the evidence obtained i n the expirements of the e f f e c t of oxygen supply on ammonia formation, p a r t i c u l a r l y i n the case o f p r o l i n e , s t r o n g l y suggests that o x i d a t i v e deamination r a t h e r than r e d u c t i v e deamination takes place. The experimental evidence obtained f o r the decomp o s i t i o n of h i s t i d i n e by Pseudomonas p u t r e f a c i e n s shows c l e a r l y t h a t the t h r e e n i t r o g e n s are l i b e r a t e d i n the form of ammonia adding f u r t h e r support t o the hypothesis t h a t t h i s amino a c i d i s degraded t o alpha k e t o g l u t a r i c a c i d or lower. The  f i n d i n g s i n the  experiments employing Proteus i c h t h y o s -  mius oh h i s t i d i n e suggest that o n l y two n i t r o g e n s are convert e d i n t o ammonia o r that a s m a l l e r percentage of t h e h i s t i d i n e i s a t t a c k e d w i t h the f o r m a t i o n o f ammonia e q u i v a l e n t to the three n i t r o g e n s .  The evidence i s . s t r o n g e r f o r the second  a l t e r n a t i v e when i t i s r e c a l l e d that i n c r e a s e i n age of the growth c u l t u r e of t h i s b a c t e r i a l species markedly the q u a n t i t y of ammonia formed from h i s t i d i n e .  decreases The younger  growth c u l t u r e s produced s u f f i c i e n t ammonia t o i n d i c a t e that deamination o f the t h r e e n i t r o g e n groups occurs. The d i r e c t o x i d a t i v e deamination of glutamic a c i d g i v e s alpha k e t o g l u t a r i c a c i d or one of i t s r e l a t e d compounds. A s p a r t i c a c i d when s u b j e c t e d t o a i m i l a r o x i d a t i v e  deamination  y i e l d s o x a l a c e t i c a c i d which i s a l s o 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 . formed  from t h e s e two  The  ammonia  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 are deaminated  completely i n the  c a s e o f t h e 1 - i s o m e r o f d l - a s p a r t i c a c i d and p a r t i a l l y case of d-glutaraic The  i n the  acid.  experimental evidence obtained f o r the  influ-  e n c e o f 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 s u b s e q u e n t 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 f o r c e l l m u l t i p l i c a t i o n i s considerable»  I n e x a m i n i n g 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 t h e f i g u r e s r e p r e s e n t t h e f r e e ammonia c o n t e n t o f t h e c u l t u r e s w h i c h i s an e x p r e s s i o n o f t h e r e s u l t a n t a c i d d e g r a d a t i o n and subsequent  cell  synthesis.  o f amino  Thus i t c a n  r e a d i l y be u n d e r s t o o d t h a t a d e c r e a s e i n ammonia c o n t e n t does n o t 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 f o r m e d  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 g r o w t h , t h a t t h e ammonia has b e e n u s e d f o r c e l l synthesis. The f i n d i n g s o f e x p e r i m e n t of  the presence  IX i n which the  o f g l u c o s e i n t h e g r o w t h medium on  effect  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 t h e  organ-  i s m s grown o f t h e g l u c o s e - c o n t a i n i n g a g a r a r e n o t so as t h o s e grown on t h e g l u c o s e - f r e e a g a r . a c t i v i t y may •this o r g a n i s m  active  This decrease i n  be due t o t h e g e n e r a l d e c r e a s e i n a c t i v i t y - 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 b y  of the  m a r k e d r e t a r d a t i o n i n t h e r a t e o f g r o w t h 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 c o m p a r e d t o t h e g l u c o s e - f r e e a g a r . and  Epps  G a l e (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 obtained employing E s c h e r i c h i a c o l i ,  observed  .  - 70 -  that the degree o f i n h i b i t i o n o f deamination by the presence of glucose bore no r e l a t i o n t o the e f f e c t produced by the a d d i t i o n o f f e r m e n t a t i o n a c i d s to the growth medium i n p l a c e of the glucose.  They a l s o showed that n e u t r a l i z a t i o n o f the  fermentation a c i d s o f glucose d u r i n g growth does not a l t e r yhe degree o f i n h i b i t i o n o f subsequent  deamination.  Further  i n v e s t i g a t i o n s i m i l a r t o t h a t o u t l i n e d by Epps and Gale i s necessary b e f o r e an adequate explanation o f these f i n d i n g s can be advanced. In experiments  o f the e f f e c t of the presence of  acid-producing and non-acid-producing  carbohydrates i n the  b u f f e r medium upon ammonia formation, the f i n d i n g s have been c l e a r l y shown t o be dependent upon both the carbohydrate and the amino a c i d employed.  The fermentation of the carbo-  hydrate w i t h a c i d p r o d u c t i o n and the breakdown of the amino a c i d with ammonia formation appear t o be independent ses *  proces-  The r a t e a t which these two processes take p l a c e ,  however, has been shown t o m a t e r i a l l y a f f e c t the f i n a l outcome i n so f a r as the q u a n t i t y o f f r e e ammonia i s concerned. When the r e s u l t s f o r the deamination  r a t e s of a s p a r t i c and  glutamic a c i d s are considered, t h i s f i n d i n g i s seen t o be c l e a r l y demonstrated.  While  i n the absence of carbohydrate,  the f i n a l q u a n t i t i e s o f ammonia formed f r o m these amino a c i d s are approximately f i f t y and f o r t y percent r e s p e c t i v e l y , i n the presence o f an a c i d - p r o d u c i n g carbohydrate, c o n s i d e r a b l e f r e e ammonia r e s u l t s from a s p a r t i c a c i d and p r a c t i c a l l y no ammonia formation i s i n d i c a t e d i n t h e case of glutamic a c i d . The c o n s i d e r a b l e q u a n t i t i e s o f f r e e ammonia formed f r o m  - 71 a s p a r t i c a c i d i n t h e presence o f fermentable carbohydrate i s l a r g e l y d e p e n d e n t u p o n t h e f a c t t h a t t h e r a t e o f ammonia f o r m a t i o n f r o m t h i s amino a c i d p r o c e e d s fermentation o f the carbohydrate.  as r a p i d l y a s does t h e  I n the case o f g l u t a m i c  a c i d , t h e f i n a l r e s u l t o f 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 c a r b o h y d r a t e may d e p e n d upon t h e s p e e d 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 t h e 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 further bacterial activity.  On t h e o t h e r h a n d , 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 used f o rpurposes  c a r b o h y d r a t e may b e  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 the  ammonia p r o d u c e d b y d e a m i n a t i o n a s r a p i d l y a s i t i s f o r m e d a s a source o f n i t r o g e n .  Whatever t h e 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 n o f r e e ammonia i s t o be f o u n d 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 carbohydrates. The f i n d i n g s o b s e r v e d i n t h e s e e x p e r i m e n t s a d d 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 carbohydrate, f a r from having 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 carbohydrate.  The i d e a o f ammonia u t i l i z a t i o n a s a n i t r o g e n  source 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 Bacterium c o l i o r other r a p i d deaminating bacteria.  The slowness  species of  o f t h e p r o c e s s i n t h e case o f t h e  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 t h e c o n s e q u e n t  Influence  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 the i n t e r p r e t a t i o n o f data obtained f o r the slower  deaminat-  -72Ing s p e c i e s . One o f t h e i m p o r t a i t p r o b l e m s t h a t a r i s e s  from  t h e s e 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  suit-  a b i l i t y o f t h e methods o f s t u d y i n g dean i n a t i o n - t h e s i m p l e method u s i n g s u c h 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 t h e Warburg apparatus  o r t h e more c o m p l i c a t e d p r o c e d u r e  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 The  study.  m s w e r t o t h i s q u e s t i o n i s d e p e n d e n t upon t h e 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 t h e b r e a k d o w n o f t h e amino a c i d o r w i t h t h e 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 t h e more I m p o r t a n t  species,  In this  investigation,  f a c t o r was t h e s t u d y o f t h e 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 m i g h t be t h e p r e c u r s o r s o f the substance  o r substances formed i n the e l a b o r a -  t i o n o f t h e 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 • Previous i n v e s t i g a t i o n s o f the e f f e c t of the presence  o f c a r b o h y d r a t e on d e a m i n a t i o n h a v e b e e n p r i n c i p a l l y  c o n c e r n e d w i t h t h e 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)  u s e d 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 t h e amino a c i d studied to tyrosine.  The e x p e r i m e n t s  were c a r r i e d o u t e m p l o y i n g subsequently reduced  r e p o r t e d upon h e r e i n  eleven d i s t i n c t  carbohydrates,  t o f i v e k e y carb 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 t h e 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 concentration. of  The f i n d i n g s show c l e a r l y t h a t t h e n a t u r e  t h e c a r b o h y d r a t e h a s a marked e f f e c t upon t h e 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 o f t h e c u l t u r e s c o n t a i n i n g t h e r a p i d  - 73 acid-producing carbohydrates i s r e a d i l y lowered t o a l e v e l unfavorable to further bacterial  activity.  Increasing the  b u f f e r c a p a c i t y o f t h e c u l t u r e overcame t h i s d i f f i c u l t y and t h e a n m o n i a 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 d e p e n d i n g u p o n t h e 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 deamination.  The r a t e o f a c i d p r o d u c t i o n f r o m t h e v a r i o u s  c a r b o h y d r a t e s h a s b e e n shown t o v a r y c o n s i d e r a b l y - g l u c o s e and  s u c r o s e a p p e a r i n g t o be t h e most r a p i d l y f e r m e n t e d ,  • 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 .  while  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 f r o m 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 i s an example o f a c a r b o h y d r a t e  of arginine  that provides available  carbon  sources 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 p r o c e e d i n t h e l a t t e r d a y s o f t h e 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 c a n r e a d i l y be s e e n f r o m these experiments  c o n s i d e r a b l e d a t a has been added t o o u r  k n o w l e d g e o f amino a c i d b r e a k d o w n . o f the presence  the f i n d i n g s o f  The s t u d y o f t h e e f f e c t  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  information concerning t h i s l i t t l e acid decomposition.  e x p l o r e d a s p e c t o f amino  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 a d d s t h e c o m p l i c a t i o n o f b a c t e r i a l g r o w t h 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 t h e 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 s u b s e q u e n t ammonia f o r m a t i o n .  PART IIo  Studies on the 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 Odour i f ex* oug Compounds.  HISTORICAL • . ' The problem of the nature o f the compound or compounds formed during the development of surface t a i n t i n b u t t e r has  confronted i n v e s t i g a t o r s s i n c e studies of the  d e f e c t were begun a number o f years ago.  Campbell(9) found  that when i n d o l e , a d e r i v a t i v e of tryptophan was  added to  m i l k , an odour s t r o n g l y resembling t h a t o f surface t a i n t emitted.  As already mentioned, however, N e i l s o n (50)  was  has  shown that i n d o l e i s not found i n the s e r a of surface t a i n t b u t t e r s and t h e r e f o r e cannot be the cause of the odour of the d e f e c t . In the same study  (50), a number of organic com-  pounds r e l a t e d to the amino a c i d s were added to m i l k and odours emitted recorded.  I t was  the  found that b e t a i n e , d e l t a  amino v a l e r i c a c i d (a d e r i v a t i v e of both a r g i n i n e and o r n i t h i n e ) and beta amino b u t y r i c a c i d gave odours reminiscent o f s u r f a c e t a i n t .  The f o l l o w i n g combinations  i c a l s a l s o emitted odours c l o s e l y resembling the tic  "sweaty-feet"  of chemcharacteris-  odour; betaine and d e l t a amino v a l e r i c  a c i d ; b e t a i n e and b e t a amino b u t y r i c a c i d ; betaine and  iso-  v a l e r i c a c i d ; betaine and para hydroxy p h e n y l a c e t i c a c i d ;  and  b e t a i n e , i s - v a l e r i c a c i d and para hydroxy p h e n y l a c e t i c a c i d . The  odour emitted from b e t a i n e i t s e l f was  enhanced when the  other compounds were a l s o added t o the m i l k . suggest  These f i n d i n g s  t h a t the b a c t e r i a l s y n t h e s i s of b e t a i n e may  be con-  cerned i n the development of s u r f a c e t a i n t i n b u t t e r .  - 75  -'  Wolochow, T h o r n t o n and Hood (71) s t u d i e s on o d o u r p r o d u c t i o n of the  causative  found that the milk  of A l b e r t a , i n  b y Pseudomonas p u t r e f a c i e n s ,  agents o f surface  t a i n t i n b u t t e r , have  " s w e a t y - f e e t " odour t h a t i s absent i n skim  c u l t u r e s a t pH  7.6  becomes a p p a r e n t when t h e m i l k  o f t h e 'organism were 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 .  This evidence suggests t h a t the  compound r e s p o n s i b l e  for  o d o u r 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 s o l u t i o n s and  cultures  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  alkaline  i s f r e e d as an a c i d a t l o w e r  pH's. and  t  Hood (12)  obtained  definite  connection  evidence i n d i c a t i n g t h a t there b e t w e e n i s o v a l e r i c a c i d and  v a l e r i c a c i d but acid.  Wolochow, e t c , have e x p r e s s e d the  w h i c h i s i n the  "sweatyiso-  t o be  r e v e r s i b l y change the  a substance  i f odourous, i s present detected  by t h e  sense o f detec-  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 compound t o a n o n - o d o u r o u s s t a t e .  s u p p o r t f o r t h i s o p i n i o n comes f r o m t h e f r o m c o m m e r c i a l and  experimentally  findings  produced  surface  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 t h e D e p a r t m e n t o f i n g at the U n i v e r s i t y o f B r i t i s h Columbia, surface  t a i n t may  be  this  that  Exposure to a i r o x i d i z e s t h i s substance to a  t a b l y odourous s t a t e .  obtained  "sweaty-  from  opinion  produce i n m i l k  r e d u c e d s t a t e and,  i n i n s u f f i c i e n t concentration  Further  the  i s d i s t i n c t l y d i f f e r e n t i n odour f r o m  Pseudomonas p u t r e f a c i e n s may  smell.  is a  U n d e r c e r t a i n unknown c o n d i t i o n s , the  f e e t " o d o u r 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  the  in  I n a l a t e r s t u d y , D unkley, H u n t e r , Thornton  f e e t " odour.  one  The  odour  a b s e n t f r o m a sample o f d e f e c t i v e  Dairyof butter  - 76 when the b o t t l e c o n t a i n i n g i t i s f i r s t b o t t l e be c l o s e d and re-examined odour i s o f t e n p r e s e n t .  opened, but i f the  about t e n minutes l a t e r , the  Continued exposure has been found  to decrease the c o n c e n t r a t i o n of the odour u n t i l i t i s no longer d e t e c t a b l e , '  Posdick and Rapp ( 2 7 ) , i n the course of an i n v e s t i -  g a t i o n on the degredation of glucose by Staphlococcus albus under aerobic c o n d i t i o n s produced a r e a c t i o n mixture o f a p a r t i c u l a r l y f o u l odour which was not c h a r a c t e r i s t i c of any known product of f e r m e n t a t i o n . Subsequent e x t r a c t i o n and p u r i f i c a t i o n l e f t a s o l u t i o n c o n t a i n i n g two organic compounds, one a c i d i c i n nature and the other n e u t r a l .  N e i t h e r compound could be  i s o l a t e d i n pure form without decomposition but i d e n t i f i c a t i o n t e s t s i n d i c a t e d that the a c i d i c compound was alpha-keto-gamahydroxy v a l e r i c a c i d while the n e u t r a l one was the corresponding aldehyde.  These compounds are c l o s e l y r e l a t e d to t he  amino a c i d s a r g i n i n e , o r n i t h i n e , h i s t i d i n e , p r o l i n e and glutamic a c i d , and may p o s s i b l y be r e l a t e d to the c h a r a c t e r i s 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-  i n g b a c t e r i a are capable of forming odouriferous compounds from sodium pyruvate s i m i l a r to those obtained by F o s d i c k a i d Rapp the f o l l o w i n g experiment  ( X V I ) was undertaken.  Three  50 c c . f l a s k s were set up i n d u p l i c a t e and contained i n a d d i t i o n t o the 8 c c . o f phosphate b u f f e r (M/l5 at pH 6,8)  _ 77 (1) 1.0 c c . of M/20 sodium pyruvate plus 1.0 c c . o f t e n percent washed c e l l suspension. (2) 0.1 c c . o f M/20 sodium pyruvate p l u s 0.9 c c . of water p l u s 1.0 c c . of t e n percent washed c e l l suspension. (3) 1.0 c c . of M/20 sodium pyruvate p l u s 1.0 c c . of one percent washed c e l l suspension respectively. One of the d u p l i c a t e s was Inoculated with a c e l l suspension of Proteus ichthyosmius and the other w i t h t h a t of Pseudomonas p u t r e f a c i e n s .  A c o n t r o l c o n t a i n i n g 9 c c , of  b u f f e r p l u s 1 c c . o f t e n percent c e l l suspension was set up for  each o f the.two b a c t e r i a l / * s p e c i e s .  The f l a s k s were  incubated a t 30° C . and the odours formed observed at d a i l y intervals.  The f l a s k s were observed to emit odours  t i v e o f surface t a i n t . ing  sugges-  The odours from t h e f l a s k s c o n t a i n -  sodium pyruvate were more pronounced  than t he c o n t r o l s  c o n t a i n i n g b a c t e r i a l c e l l s o n l y , although these a l s o gave o f f putrid  odours.  With these r e s u l t s 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 s p e c i e s from sodium pyruvate could be i s o l a t e d and i d e n t i f i e d . phosphate  Two 250 c c . f l a s k s c o n t a i n i n g 80 c c . M / l 5  b u f f e r (pH 6.8), 10 c c . M/20 sodium pyruvate and  10 cc« o f a ten percent washed c e l l suspension of P. p u t r a f a c i e n s were prepared and incubated at 23° C . f o r 48 hours. of  The f l a s k s were removed from the incubator and one  the f l a s k s t r e a t e d as f o l l o w s j F i r s t t h e contents of the f l a s k were c e n t r i f u g e d  to  remove the b a c t e r i a l c e l l s aa d the supernatant poured o f f  - 78 i n t o a l i q u i d - e t h e r - e x t r a c t i o n f l a s k where i t was e x t r a c t e d f o r 16 hrs.  The ether e x t r a c t ( 1 ) was t a k e n up i n water and  gave a p u t r i d odour.  The r e s i d u a l l i q u i d from t h e e x t r a c t i o n  was taken down t o pH 4.0 w i t h 20% phosphoric a c i d , f i l t e r e d and the f i l t r a t e e x t r a c t e d w i t h ether f o r another 16 hours. This extract  (2) a l s o gave a p u t r i d odour when taken up i n  water. The second f l a s k was taken down t o pH 4.0 d i r e c t l y with phosphoric a c i d , f i l t e r e d and the f i l t r a t e e x t r a c t e d w i t h ether f o r 16 hours•  T h i s e x t r a c t (3) when mixed w i t h  water also gave-a p u t r i d odour.  An attempt was made to  o b t a i n a 2,4, d i n i t r o phenylhydrazone  p r e c i p i t a t e from these  e x t r a c t s b u t the q u a n t i t i e s of m a t e r i a l were too small to g i v e anything more than a s l i g h t p r e c i p i t a t e . In the procedure o f Dunkley,  e t c . the odouriferous  compounds were separated from t h e remainder o f the m i l k 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 c u l t u r e .  This  procedure would f u r t h e r h y d r o l y z e , t o a c o n s i d e r a b l e degree, the products o f the b a c t e r i a l p r o t e i n decomposition and i t would n o t be p o s s i b l e t o determine which p o r t i o n of the d i s t i l l a t e was from b a c t e r i a l h y d r o l y s i s and which was from chemical h y d r o l y s i s .  The hydroxy and keto acids o f the  lower f a t t y a c i d s are u s u a l l y ether s o l u b l e and t h e r e f o r e should be removed by e t h e r e x t r a c t i o n from a c u l t u r e ' c o n t a i n i n g them.  I n order t o o b t a i n d a t a on t h i s hypothesis a f l a s k  c o n t a i n i n g 100 c c . of skim m i l k and 10 c c . o f a t e n percent washed c e l l suspension o f Pseudomonas p u t r a f a c i e n s was prepared a i d incubated at 23° C. for' twenty days.  The p r o t e i n  - 79 -•• of the m i l k was then l a r g e l y converted to soluble decompositi o n products and emitted a f o u l , p u t r i d odour.  The contents  of the f l a s k were c e n t r i f u g e d and the pH o f the supernatant taken.  I t was pH 5.9.  yellow c o l o u r extracted  The supernatant which was a b r i g h t  suggesting the presence o f a f l a v i n e , was then  i n a l i q u i d ether e x t r a c t o r f o r 12 hours.  The  e x t r a c t thus formed (1) was soluble i n water and gave o f f a pungent f r u i t y odour.  The r e s i d u a l f l u i d from t h i s extrac-  t i o n was taken down t o pH 4.0 w i t h phosphoric a c i d and r e e x t r a c t e d w i t h ether  f o r a f u r t h e r 12 hours.  This  extract  (2) was only s p a r i n g l y s o l u b l e i n c o l d water and emitted a mixture of odours resembling those from w e l l ripened Oka cheese.  The e x t r a c t  (2) was t r e a t e d f u r t h e r as f o l l o w s :  I t was t e s t e d f o r s o l u b i l i t y i n a number o f s o l vents and found to be s l i g h t l y soluble i n c o l d ether and h o t water, s o l u b l e i n hot ether,  and very soluble i n 95$ al c o h o l .  The  e x t r a c t was f i n a l l y d i s s o l v e d i n a l c o h o l and f i l t e r e d .  The  f i l t r a t e was evaporated t o dryness g i v i n g a y e l l o w i s h  white r e s i d u e water.  to which was added a small q u a n t i t y o f h o t  The r e s i d u e  clumped together i n the water and o i l e d -  o f f i n t o s o l u t i o n when the water was r a i s e d to b o i l i n g p o i n t . The  s o l u t i o n was evaporated to dryness g i v i n g a yellow-brown  o i l y residue which when d i s s o l v e d i n hot ether  crystallized  out from the ether as white needles w i t h a m e l t i n g p o i n t of 175-177° C.  The q u a n t i t y o f c r y s t a l s was i n s u f f i c i e n t to  permit f u r t h e r i d e n t i f i c a t i o n . ' With the object o f o b t a i n i n g information ether  on the  s o l u b i l i t y o f the decomposition products of c e r t a i n  - 80 amino a c i d s whose c h e m i c a l  -  s t r u c t u r e suggested t h a t  odour-  i f e r o u s compounds m i g h t r e s u l t f r o m t h e i r b r e a k d o w n a f u r t h e r experiment  ( X V I I ) was  undertaken.  The  amino a c i d s e m p l o y e d  w e r e 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 t h e b a c t e r i a were P r o t e u s I c h t h y o s m i u s f l a s k s c o n t a i n i n g 160 20 c c , o f M/20 cell  and Pseudomas p u t r e f a c i e n s . S i x  c c , o f M / l 5 p h o s p h a t e b u f f e r (pH  7,4),  Amino a c i d and 20 c c , o f one p e r c e n t washed  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.  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 Flask  a r g i n i n e was  Each  follows:  (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  removed f r o m the> i n c u b a t o r a f t e r 7 d a y s and  pH t a k e n e l e c t r o m e t r i c a l l y , of  used  I t was  t h e f l a s k were t a k e n down t o pH  pH 8,0,  4.0,  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 . extract obtained f a i l e d  The  contents  w i t h phosphoric The  the  acid  s m a l l amount o f  t o g i v e o f f a p u t r i d o d o u r and d i d .  not d i s s o l v e r e a d i l y i n water. 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 ing  t r e a t e d t h e same as f l a s k  i n c u b a t i o n was  p u t r i d odour.  pH 7,15.  I n an a t t e m p t  (1.) .  The  pH  follow-  The e x t r a c t d i d n o t e m i t  a  to i s o l a t e the ether i n s o l u b l e  decomposition products o f h i s t i d i n e , the r e s i d u a l 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  filtered,  fluid  r a i s e d t o 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 d r y n e s s i n vacuo. The  r e s i d u e was  one h a l f h o u r . filtrate  t a k e n up i n 95% The  a l c o h o l m i x t u r e was  evaporated to dryness!  a small q u a n t i t y of d i s t i l l e d phosphotungstic  a l c o h o l and r e f l u x e d f o r a b o u t  a c i d i n 5%  The  then f i l t e r e d  r e s i d u e was  and  t a k e n up i n  w a t e r and a few c c . o f  s u l p h u r i c a c i d were added.  19% The  the  - 81 heavy white p r e c i p i t a t e formed was dried  f i l t e r e d o f f , washed and  carefully. PIask  (3) c o n t a i n i n g Pseudomonas p u t r e f a c i e n s i n  h i s t i d i n e was removed from the incubator a f t e r 14. days and t r e a t e d a f t e r the same manner as f l a s k ( 2 ) . i n c u b a t i o n was pH 7.55. t a t e was  obtained.  The pH a f t e r  A s i m i l a r phosphotungstic p r e c i p i -  As a c o n t r o l check the phosphotungstic  p r e c i p i t a t e of h i s t i d i n e was prepared and found t o resemble c l o s e l y the p r e c i p i t a t e s obtained from f l a s k  (2) and ( 3 ) .  The compounds p r e c i p i t a t e d were b a s i c i n nature otherwise they would not .have combined "with phosphotungstic a c i d . F l a s k (4) c o n t a i n i n g Pseudomonas p u t r e f a c i e n s i n the presence of p r o l i n e was  taken from the incubator a f t e r  14 days' i n c u b a t i o n and t r e a t e d the same as f l a s k (1). pH f o l l o w i n g i n c u b a t i o n was  pH 7.45.  The  No p u t r i d odours were  observed i n the e t h e r e x t r a c t . F l a s k (5) c o n t a i n i n g Pseudomonas p u t r e f a c i e n s and a r g i n i n e was p e r m i t t e d to incubate f o r 20 days, f o l l o w i n g which i t was the pH was  t r e a t e d the same as f l a s k ( 1 ) .  7.75.  After incubation  The e x t r a c t obtained emitted no p u t r i d  odours. F l a s k (6) c o n t a i n i n g Proteus ichthyosmius i n prol i n e had a pH of 7.45  a f t e r 20 days' i n c u b a t i o n and when  t r e a t e d i n a s i m i l a r manner to f l a s k (1) gave an e x t r a c t f r e e from p u t r i d  odours.  DISCUSSION The f i n d i n g s o b t a i n e d from the experiments  des-  c r i b e d i n the above s e c t i o n serve to discourage c e r t a i n 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  t a i n t r a t h e r t h a n t o g i v e p o s i t i v e e v i d e n c e i n any direction.  The  inability  from the decomposition  to i s o l a t e odouriferous  products  surface one  compounds  of arginine, histidine  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 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  acids are  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 odour of t y p i c a l s u r f a c e t a i n t . ever,  t h a t the techniques  not have p r o v i d e d the omittance may  suggest not  sweaty-feet  I t must be remembered, how-  employed i n t h i s  i n v e s t i g a t i o n may  c o r r e c t conditions of p o t e n t i a l for the  of the odours.  A l s o the  h a v e b e e n masked by o t h e r  compounds t h e m s e l v e s may  odours from the  compounds  c a c t o r s or s u b s t a n c e s ,  or  the  n o t have been e t h e r s o l u b l e .  This  b r a n c h of t h e i n v e s t i g a t i o n i n t o the cause of s u r f a c e  taint  c a n n o t be d i s p e n s e d  w i t h as y e t b e c a u s e o f i n s u f f i c i e n t  dence t o p r o v e o r d i s p r o v e decomposition  products  odour.  b e e n shown t o b e p r e s e n t  course  o f the experimental  t a i n i n g the  t h a t the  o f c e r t a i n amino a c i d s may  s i b l e f o r the sweaty-feet has  the h y p o t h e s i s  evi-  acidic be  respon-  The much s o u g h t a f t e r o d o u r  a t d i f f e r e n t times  during  the  s e c t i o n o f P a r t I i n c u l t u r e s con-  amino a c i d s u s e d above - a r g i n i n e , h i s t i d i n e  and  proline. The  e l a b o r a t i o n of the  t a i n t a p p e a r s t o be chemical may  be  and  t y p i c a l odour o f  surface  d e p e n d e n t upon a number o f p h y s i c a l ,  biological factors.  p h y s i c a l or chemical  The  first  of these  i s t h a t t h e odour i s not  which formed  i n b u t t e r made f r o m raw  cream n o r i n raw m i l k I n o c u l a t e d  with  surface t a i n t producing  b a c t e r i a , but  milk  i s f o r m e d when t h e  o r cream e m p l o y e d i s p a s t e u r i z e d .  I t would appear t h a t  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 s d e p e n d e n t upon a s u b t l e change i n d u c e d by t h e h e a t on t h e p r o t e i n complex o f t h e m i l k cream. not  A second f a c t o r i s that the  always present  i n the  compound o r compounds a r e  odouriferous  s t a t e , but  c e r t a i n oxidation-reduction p o t e n t i a l before odour.  t h e y emit  disappearance of the s u r f a c e  • odour i n b u t t e r exposed t o the a i r .  not  The  outstanding  cause s u r f a c e t a i n t  very  i n butter.  The  taint biolog-  number of s p e c i e s  defect but  not The  t a i n t may an  Proteus  ichthyosmius  the major  a l s o producing  s o u r c e o f t h e compound r e s p o n s i b l e f o r  amino a c i d a t a l l . be  The  o f the  amino a c i d s  precursor  of the  a l a r g e r m o l e c u l e s u c h as  surface studied  odouriferous  a simple  peptide.  E v i d e n c e /o'Lfy t h i s i d e a comes f r o m t h e a b i l i t y t o i s o l a t e forming  the  so r e g u l a r l y .  n o t n e c e s s a r i l y be one  s u b s t a n c e may  of  been shown t o  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  c a u s a t i v e a g e n t and  nor  the  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  b a c t e r i a c a p a b l e o f e l a b o r a t i n g t h e d e f e c t has be  require a  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,  f r o m t h e a p p e a r a n c e and  ical  or  compounds f r o m m i l k  combined w i t h t h e  inability  odourt o show  t h e p r e s e n c e o f s u c h compounds among the d e c o m p o s i t i o n p r o d ucts  o f c e r t a i n amino a c i d s .  regarding  the s t a b i l i t y  s i b l e f o r the m i n i n g the is s t i l l  The  difficulties  o f t h e odour o r the  odour f u r t h e r c o m p l i c a t e  cause of s u r f a c e  required before  the source of the  taint.  encountered  substance  the problem of  Considerable  deter-  investigation  more d e f i n i t e c o n c l u s i o n s  s w e a t y - f e e t o d o u r can be  respon-  reached.  regarding  - 84 SUMMARY AND The and  -  CONCLUSIONS  p r o b l e m u n d e r i n v e s t i g a t i o n has  the f o l l o w i n g  two  been  outlined  a p p r o a c h e s t o i t s s o l u t i o n have b e e n  made: 1.  An  i n v e s t i g a t i o n of the  conditions  ammonia f o r m a t i o n f r o m 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  affecting  aspartic  p r o l i n e by  acid, two  s p e c i e s of s u r f a c e t a i n t producing 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 2.  A s t u d y of t h e  Pseudomonas  production, i s o l a t i o n  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  putrefaciens. and odours  identical with  or i n t i m a t e l y r e l a t e d t o  characteristic  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  i n g deaminatlgji The  o f amino a c i d s  has  g e n e r a l methods and  -  the  conditions  govern-  been r e v e i w e d i n d e t a i l . p r o c e d u r e s employed have  been o u t l i n e d . I t has i n pH pH  6.0  f o r the t o pH  b e e n shown t h a t  the  g e n e r a l optimum r a n g e  f o r m a t i o n o f ammonia f r o m amino a c i d s 8.5  and  i s from  that  the  i n d i v i d u a l amino a c i d s  Increase i n the  age  of the  vary  w i t h i n t h i s range.  f o u n d t o d e c r e a s e the h i s t i d i n e and  and  been  s u b s e q u e n t ammonia f o r m a t i o n f r o m  p r o l i n e but  elaborated from a r g i n i n e , The  g r o w t h c u l t u r e has  conditions  t o h a v e no aspartic  e f f e c t on t h e  a c i d and  amount  glutamic  acid.  o f oxygen s u p p l y d u r i n g g r o w t h  subsequent deamination d i d not  materially affect  the  - 85  -  ammonia f o r m a t i o n f r o m 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 h u t d i d a f f e c t t h a t f r o m  proline.  A n a e r o b i c c o n d i t i o n s o f g r o w t h and d e a m i n a t i o n gave o n l y o n e - h a l f t h e q u a n t i t y o f ammonia f o r m e d u n d e r a e r o b i c conditions* Graphs a r e  p r e s e n t e d comparing  t h e ammonia  f o r m a t i o n f r o m t h e f i v e amino a c i d s by P r o t e u s  Ichthyosmius  and Pseudomonas p u t r e f a c i e n s a t i n t e r v a l s o v e r 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 h a s b e e n shown t o p r o d u c e a c i d and gas f r o m 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 a n d t o h a v e i m m e d i a t e a c t i o n b u t s l o w l y t o become a l k a l i n e  no  i n xylose,  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 t h e o t h e r h a n d , p r o d u c e s  acid  and gas' f r o m 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 , m a l t o s e and s a l i c i n , and h a s no  s l o w l y produces  a c i d w i t h no gas f r o m  glycerol  i m m e d i a t e a c t i o n b u t 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 carbohydrate.  The  microorganisms ferments  d i f f e r e n t l a t i n g carbohydrates f o r these are x y l o s e and d e x t r i n - P r o t e u s  d e x t r i n b u t n o t 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 The  dextrin.  presence o f glucose i n t r y p t i c c a s e i n d i g e s t  growth agar decreases the g e n e r a l r a t e of growth sequent  ichthyosmius  a c t i v i t y of the b a c t e r i a l c e l l s  and  sub-  of Proteus  ichthyosmius. The  e f f e c t o f the presence  of carbohydrates i n the  - 86 - . b u f f e r medium h a s b e e n shown t o be d e p e n d e n t l a r g e l y upon the  rate o f fermentation  that takes p l a c e .  and subsequent a c i d  production  The c a r b o h y d r a t e s t h e m s e l v e s do n o t  m a t e r i a l l y a f f e c t t h e ammonia f o r m a t i o n amino a c i d s b u t t h e f e r m e n t a t i o n cultures t o l e v e l unfavourable  from the v a r i o u s  a c i d s l o w e r t h e pH o f t h e  to further bacterial  activity.  I t was f o u n d t h a t t h e a d d i t i o n o f a d e q u a t e b u f f e r to the c u l t u r e s c o n t r o l l e d the hydrogen-ion thereby  permitting bacterial  concentration  a c t i v i t y t o continue.  This  a c t i v i t y included not only 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 f e r m e n t e d 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  multiplication.  A s t u d y h a s b e e n made o f ammonia f o r m a t i o n a number o f n o n - a m l n o a c i d n i t r o g e n o u s  compounds.  from I t has  b e e n shown t h a t i n a d d i t i o n t o t h e i m i n a z o l a n d p y r o l l i d i n e rings,  the b a c t e r i a l species  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 w i t h subsequent formation  and p y r i d i n e r i n g s  o f ammonia.  A d e t a i l e d d i s c u s s i o n o f t h e breakdown o f t h e 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 , acid, histidine  and p r o l i n e - b y  b a c t e r i a - Proteus h a s been  glutamic  t h e two s p e c i e s o f  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 -  given. A c o m p a r i s o n o f t h e a p p r o a c h made t o t h e p r o b l e m  o f 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 b y 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 t h e s w e a t y - f e e t odour o f surface t a i n t , a s e r i e s of e x p o n e n t s (Part I I ) .  was  undertaken,  While the f i n d i n g s of t h i s p a r t of the  investi-  g a t i o n d i d n o t y i e l d p o s i t i v e r e s u l t s , t h e y have p r o v i d e d d a t a on t h e n a t u r e o f  the decomposition products of the  amino a c i d s e m p l o y e d and h a v e s u g g e s t e d f u r t h e r s t u d i e s t h a t may be  undertaken.  88  BIB 1.  Adler  2.  Anderson, C. G.  3.  Barker, Ho A»  L I  OGRAPHY  - . Z e i t . Physiol. Chem. 255:14 (1938) -  "An Introduction t o Bacteriological Chemistry" (1938)  -  Enzymologia 2: 175 (1937)  -  Jour. B i o l . Chem. 110: 165 (1935)  -  Jour. Biol.-Chem. 114s 265 (1936)  6 * Berthelot, A.  -  C ?. Acad, S c i . 164: 196 (1917), v i a (61)  7.  -  C. R. Acad. 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Abstr. 31: 7906 (1937)  68o  Waksman, S. A. and Lomanitz, S.  -  Jour. Agric. Research 30: 263 (1925)  Weil-Malherbe Krebs, H. A.  -  Biochem. Jour. 29: 2077 (1935)  -  Thesis "Studies on Surface Taint Butter" (1940)  69.  35: 1812 (1941)  and  70.  Woloohow, H. •  71,  Woloehow, H. Thornton, H. R. and Hood, E.G.  ->  S c i . Agric. 22:277 (1942)  Wools, D. D .  -  Biochem. Jour. 29: 640 (1935)  -  Biochem. Jour. 29: 649 (1935)  -  Biochem. Jour. 30: 1934 (1936)  Woods, D. D. and C l i f t o n , C. E.  -  31: 1774 (1937)  Woods, D. D. and Trim  -  Biochem. Jour. 36: 501 (1942)  Woolf, B.  -  Biochem. Jour. 23: 472 (1929)  72, 73. 74. 75.  76.  77.  REVIEWS: Gale, E . F.  Bacteriological Rev. 4: 135 (1940)  Krebs, H. A.  Ann. Rev. Biochem.  Workman and Wood  Botanioal Rev.  5: 247 (1936)  8: 1 (1942)  92 -  ACKNOWLEDGMENTS  I w i s h t o e x p r e s s my s i n c e r e a p p r e c i a t i o n t o D r . B. A. E a g l e s f o r h i s c o n t i n u e d and k i n d l y encouragement t h r o u g h o u t I  guidance  this  study.  a l s o e x p r e s s my g r a t e f u l t h a n k s t o M i s s L o i s  Campbell and t o Miss F l o r e n c e Tamboline f o r s u g g e s t i o n s a n d h e l p w i t h t h e 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  which  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 study;  

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