UBC Theses and Dissertations

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UBC Theses and Dissertations

Action spectrum of Nitrobacter agilis Hill, Joan Emily 1968

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AN ACTION SPECTRUM OF NITROBACTER A G I L I S by JOAN E M I L Y H I L L B.Sc.  (Hons.), U n i v e r s i t y  of Toronto,  1966  A T H E S I S SUBMITTED IN P A R T I A L FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF MASTER OF  SCIENCE  i n the Department of PHYSICS  We a c c e p t t h i s t h e s i s a s c o n f o r m i n g to the required  standard  THE U N I V E R S I T Y OF B R I T I S H COLUMBIA September,  1968  OF  In p r e s e n t i n g  this thesis i n partial fulfillment for  an  advanced degree a t the U n i v e r s i t y of B r i t i s h C o l u m b i a , I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and extensive  study.  I f u r t h e r agree t h a t p e r m i s s i o n f o r  c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y purposes  may  be g r a n t e d by the Head of my Department o r by h i s r e p r e s e n t a tives.  I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n of  t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t written permission.  Department of The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada  this my  - i ABSTRACT  The p h y s i o l o g i c a l l i t e r a t u r e  on N i t r o b a c t e r i s r e v i e w e d  and a l i s t o f t h e u n s o l v e d problems p r e s e n t e d .  Modifications  t o t h e a c t i o n spectrum a p p a r a t u s b u i l t by BrOoks  (1967)  are d e s c r i b e d .  Nitrobacter  The a p p a r a t u s was then used w i t h  a g i l i s , ATCC no. 14123, t o o b t a i n an a c t i o n spectrum o f the r e l i e f o f c a r b o n monoxide i n h i b i t i o n by l i g h t .  The r e s u l t s  o f t h i s s t u d y i n d i c a t e t h a t cytochrome a^ i s a c t i v e as a terminal oxidase. principally  The p o s s i b i l i t y o f o t h e r cytochromes,  cytochrome £, a c t i n g as o x i d a s e s has n o t been  proven o r r u l e d o u t .  The r e s u l t s o f a s t u d y on t h e r a t e o f  oxygen uptake v e r s u s oxygen c o n c e n t r a t i o n  are also  the K ( 0 ) v a l u e s range from 0.021 t o 0.055 mM m  2  reportedJ  oxygen.  The a c t i o n spectrum r e p o r t e d here i s the. f i r s t one t o be d e t e r m i n e d on a c h e m o l i t h o t r o p i c  bacterium.  -111-  I would l i k e t o thank Dr. C. P. S. T a y l o r f o r h i s g u i d a n c e , a d v i c e and encouragement i n the c o u r s e of t h i s i n v e s t i g a t i o n and f o r h i s h e l p f u l c r i t i c i s m s i n the p r e p a r a t i o n of t h i s t h e s i s . F i n a n c i a l s u p p o r t from the N a t i o n a l Research and the U n i v e r s i t y of B r i t i s h Columbia acknowledged.  Council  i s gratefully  -iv-  TABLE OF CONTENTS Page INTRODUCTION Chapter I  ..  1  THE PHYSIOLOGY OF NITROBACTER  1-1  Nitrobacter  1-2  Conditions  2 f o r Growth  2.1  Growth F a c t o r s  2.2  E f f e c t of Light  2.3  N i t r i t e Requirement 2.3.1  1-3  2 ..  33  Oxygen E f f e c t on N i t r i t e Requirement  4  I n h i b i t o r S t u d i e s on whole C e l l s and  Cell-Free Extracts  3.1  I n h i b i t i o n by N i t r i t e  3.2  I n h i b i t i o n by I n o r g a n i c  5 6 Ions  7  3.2.1  I n h i b i t i o n by N i t r a t e  7  3.2.2  I n h i b i t i o n by Cyanate  7  3.2.3. I n h i b i t i o n by C h l o r a t e 1-4  8  O x i d a t i o n o f N i t r i t e by C e l l - F r e e Extracts  1-5  3  9  Spectroscopy of Nitrobacter  5.1  Absorption  Spectra  10  5.2  Difference  Spectra  11  5.3  C a r b o n Monoxide D i f f e r e n c e S p e c t r a 5.3.1  1-6 1-7  ...  12  Results with Nitrobacter  12  F l a v i n Involvement i n t h e Cytochrome Chain E n e r g y P r o d u c t i o n and E f f i c i e n c y  13 14  -vPage 1-8  The F i x a t i o n o f Carbon D i o x i d e  16  1-9  Unsoved Problems and C r i t i c i s m s  18  I - 10  Summary  20  Chapter I I  GROWTH METHODS  II-l  Medium and E x t e r n a l C o n d i t i o n s  II- 2  T e s t Methods  21  2.1 N i t r i t e  22  2.2 N i t r a t e  23  Chapter I I I III- l  INSTRUMENTATION AND SAMPLE PREPARATION The Oxygen  Electrode  1.1  Instrumentation  1.2  Calibration  1.3  Preparation  III-2  25 „  26  o f the Sample  26  The A c t i o n Spectrum A p p a r a t u s  27  2.1  Modifications  28  2.2  M o d i f i c a t i o n o f the Chopper  2.3  Modifications Detector  2.4  Sample P r e p a r a t i o n  C h a p t e r IV IV- 1  t o the R e f e r e n c e Beam ...  t o the Phase  30 Sensitive  31 31  CALIBRATION AND PERFORMANCE Relative  '  I n t e n s i t y a t the Top o f  the Cone IV-2  I n t e n s i t y Response o f the PSD  IV-3  Actinometer C a l i b r a t i o n s  33 .........  35 36  -viPage Chapter V  RESULTS AND ERRORS  V-l  Oxygen E l e c t r o d e R e s u l t s  V- 2  R e s u l t s from t h e A c t i o n  37 Spectrum  Apparatus  38  2.1  Procedure f o r Obtaining R e s u l t s  38  2.2 2.3  Results E r r o r s i n the R e l a t i v e E x t i n c t i o n Coefficient  41  2.4  E r r o r s i n t h e P o s i t i o n s o f the Peaks..  43  Chapter V I  42  DISCUSSION  VI- 1  Oxygen E l e c t r o d e R e s u l t s  44  VI-2  A c t i o n Spectrum R e s u l t s  45  VI-3  Summary o f C o n c l u s i o n s  49  BIBLIOGRAPHY  50  APPENDIX  53  -viiLIST OF FIGURES Following  Page:  1.  The Oxygen E l e c t r o d e  25  2.  C i r c u i t Diagram of Phase S e n s i t i v e D e t e c t o r . . .  27  3.  Power S u p p l y f o r the L i g h t Source .  29  4.  B l o c k Diagram o f A p p a r a t u s  28  5.  C i r c u i t Diagram f o r P h o t o m u l t i p l i e r Tube  33  6.  Relative Intensit y a t the t o p o f the Cone t o t h a t a t the Bottom I n t e n s i t y Response o f Lead S u l p h i d e and Phase S e n s i t i v e D e t e c t o r s  7.  35  8.  E f f e c t o f Oxygen C o n c e n t r a t i o n N i t r i t e Oxidation  9.  M i c h a e l i s - M e n t e n P l o t s f o r Data o f F i g . 8 ....  37  The E f f e c t o f L i g h t on the E l e c t r o d e C u r r e n t o f a Carbon Monoxide I n h i b i t e d Sample o f Nitrobacter  38  A T y p i c a l D e t e r m i n a t i o n o f the B a l a n c e P o i n t f o r L i g h t o f Wavelength, X and the Reference L i g h t , w  39  10.  11.  12.  on Rate o f  35  37  R e l a t i v e E x t i n c t i o n C o e f f i c i e n t s Obtained w i t h two P r e p a r a t i o n s  of N i t r o b a c t e r  41  13.  A c t i o n Spectrum o f N i t r o b a c t e r a g i l i s  41  14.  S p e c t r a l S e n s i t i v i t y o f P h o t o m u l t i p l i e r , Y ...  54  15.  S p e c t r a l S e n s i t i v i t y o f PSD, Q  54  - v i i i -  LIST OF TABLES F o l l o w i n g Page Table I Table I I  C a l i b r a t i o n V a l u e s a t the Top of the Cone  34  C a l i b r a t i o n o f Wavelength S e n s i t i v i t y o f Photomultiplier  34  T a b l e I I I Phase S e n s t i v e D e t e c t o r Wavelength Senstivity T a b l e IV Table V  K Values f o r Nitrobacter as S u b s t r a t e m  ••••  35  w i t h Oxygen  D a t a R e q u i r e d f o r the C a l c u l a t i o n Relative Extinction Coefficient  36 of the 52  -1INTRODUCTION  The work o f Brooks  (1967) has l e d t o an i n s t r u m e n t  based on t h a t o f C a s t o r and Chance (1955) which measures t h e r e v e r s a l by l i g h t o f t h e i n h i b i t i o n o f r e s p i r a t i o n by c a r b o n monoxide.  He suggested s e v e r a l improvements f o r  t h e a p p a r a t u s , some o f which have now been c a r r i e d o u t . The of  l i g h t chopper was r e p l a c e d by a t u n i n g f o r k and t h e path t h e r e f e r e n c e beam was changed.  These changes have  r e s u l t e d i n the e l i m i n a t i o n of the instrument a r t i f a c t s t h a t plagued B r o o k s . apparatus.  They a l s o a l l o w e a s i e r m a n i p u l a t i o n o f t h e  The changes and o t h e r s a s s o c i a t e d w i t h these a r e  o u t l i n e d i n Chapter I I . The improved  a p p a r a t u s was then  used i n a s t u d y o f t h e t e r m i n a l o x i d a s e o f N i t r o b a c t e r a g i l i s . A l o n g w i t h t h i s t h e l i t e r a t u r e on t h e p h y s i o l o g y of N i t r o b a c t e r was c r i t i c a l l y s u r v e y e d .  (Chapter I )  Oxygen uptake as a f u n c t i o n o f t h e oxygen c o n c e n t r a t i o n was s t u d i e d u s i n g a p o l a r o g r a p h i c t e c h n i q u e and t h e r e s u l t s were compared t o a s i m i l a r manometric s t u d y performed by B u t t and Lees  (1964).  CHAPTER I THE PHYSIOLOGY OF NITROBACTER  1-1.  Nitrobacter N i t r o b a c t e r i s a micro-organism of the f a m i l y  Nitrobacteriaceae  o f t h e o r d e r Pseudomonales.  I t i s prevalent  i n n a t u r e and i s u s u a l l y i s o l a t e d from s o i l by r e p e a t e d p l a t i n g on s i l i c a g e l o r agar p l a t e s . negative  The c e l l s a r e gram-  s h o r t r o d s 0.5 x 1.0 m i c r o n s .  F l a g e l l a have been  seen on N i t r o b a c t e r i n t h e e l e c t r o n m i c r o s c o p e .  1-2.  Conditions  f o r Growth  2.1 Growth  Factors  N i t r o b a c t e r f u n c t i o n s i n the c o n t r o l of the n i t r o g e n b a l a n c e o f t h e s o i l by t h e o x i d a t i o n o f n i t r i t e t o nitrate.  T h i s o x i d a t i o n i s i t s p r i m a r y and p r o b a b l y  s o u r c e o f energy.  I t i s autotrophic  d i o x i d e as i t s s o u r c e o f c a r b o n .  only  i n t h a t i t uses c a r b o n  N i t r o b a c t e r i s grown on a  m i n e r a l medium u s u a l l y c o n t a i n i n g K, Ca, Mg, Mn, and Fe (Lees and  Simpson, 1957).  Mg, F e , and phosphate a r e a l l e s s e n t i a l  but t h e r e q u i r e m e n t f o r o t h e r growth f a c t o r s has not been thoroughly  investigated.  a requirement f o r calcium.  Aleem and A l e x a n d e r (1958) r e p o r t Pure c u l t u r e s tend t o d i e o u t  i n d i c a t i n g t h a t t h e r e may be a r e q u i r e m e n t f o r o t h e r f a c t o r s o r t r a c e elements.  I t has been r e p o r t e d  that  growth biotin  -3i n c a t a l y t i c amounts s t i m u l a t e s the growth of N i t r o b a c t e r . ( K r u l w i c h and Funk, 2.2  1965)  E f f e c t of L i g h t Bock (1965) has shown t h a t l i g h t produces an  i n h i b i t i o n of n i t r i t e o x i d a t i o n i n N i t r o b a c t e r  winogradski.  M u l l e r - N e u g l i i c k and E n g e l (1961) showed t h a t l i g h t p r o c e e d s t o the p o i n t where no o x i d a t i o n o c c u r s .  inactivation Nitrobacter  a r e r e a c t i v a t e d i n the dark but i t i s s l o w e r the l o n g e r l i g h t has shone and  the g r e a t e r i t s i n t e n s i t y .  366-436 nanometers (nm) effect.  i s the cause of t h i s i n a c t i v i a t i o n . p o s s e s s e s no c a r o t e n o i d s .  winogradski  In other b a c t e r i a , carotenoids  photo-oxidation.  N i t r i t e Requirement  n i t r i t e concentration  optimal  f o r the growth of N i t r o b a c t e r .  found t h a t an i n i t i a l c o n c e n t r a t i o n  of 100  200jig n i t r i t e - N / m l when the f i r s t the f a s t e s t r a t e of growth.  had  300  \ig n i t r i t e - N / m l l e d t o a d e p r e s s i o n  and  i n i t i a l concentrations growth f o r one  week.  to  been used up r e s u l t e d  Initial  of 600  They  ug n i t r i t e - N / m l  w i t h subsequent a d d i t i o n s t o b r i n g the c o n c e n t r a t i o n  all cell  have  thus p r e v e n t  Gould and Lees (1960) have s t u d i e d the  in  no  photo-oxidation  Nitrobacter  been found t o have a p r o t e c t i v e mechanism and  2.3  light  i s most e f f e c t i v e , r e d l i g h t has  M u l l e r - N e u g l i i c k and E n g e l f e e l t h a t  d e s t r u c t i o n by  Blue  the  a d d i t i o n s of more than of the o x i d a t i o n r a t e  fig n i t r i t e - N / m l stopped  Subsequent s t u d i e s ( B u t t  and  -4-  L e e s , 1960, Boon and L a u d e l o u t , 1962) have c o n f i r m e d t h i s and have shown t h a t a t a t m o s p h e r i c c o n d i t i o n s — 2 0 %  o x y g e n — maximal  oxygen u p t a k e o c c u r s a t a n i t r i t e concentration  o f 16 mM  jig n i t r i t e - N / m l ) .  A t lower oxygen t e n s i o n s  occurs a t lower n i t r i t e c o n c e n t r a t i o n s .  (200  the maximal r a t e  G o u l d and Lees (1960)  have shown t h a t the r a t e o f n i t r i t e o x i d a t i o n i s l o g a r i t h m i c u n t i l 200 |ig n i t r i t e - N / m l have been o x i d i z e d , i . e . the l o g of the r a t e versus n i t r i t e c o n c e n t r a t i o n  i s a straight  line.  A l l growth c e a s e d when 2200 jig n i t r i t e - N/ml had been consumed. The maximal r a t e under these c o n d i t i o n s was 6 jig n i t r i t e - N / m l per hour.  D i a l y s i s o f n i t r a t e from the s o l u t i o n each day  r e s u l t e d i n the absence o f the s t a t i o n a r y phase even up t o the o x i d a t i o n o f 13,000 jig n i t r i t e - N / m l .  As a f u n c t i o n o f n i t r i t e  used t h e d r y w e i g h t o f c e l l s was s l i g h t l y case.  l a r g e r i n the  latter  The maximal o x i d a t i o n r a t e was 200 jig n i t r i t e - N / m l / d a y .  A e r a t i o n o f the c u l t u r e s i n c r e a s e d techniques simultaneously nitrite-N/ml/hr.  the r a t e , and use o f both  gave an o x i d a t i o n r a t e o f 100 jig  A f t e r the l o g a r i t h m i c phase the r a t e o f  o x i d a t i o n was l i n e a r a t a r a t e dependent on the a i r f l o w . 2.3.1 Oxygen E f f e c t on N i t r i t e  Requirement  B u t t and Lees (1964) c a l c u l a t e d the of n i t r i t e needed f o r maximal r a t e a t d i f f e r e n t p r e s s u r e s o f oxygen.  concentrations partial  The t h e o r e t i c a l c a l c u l a t i o n s a r e made  assuming t h a t a t e r n a r y complex o f n i t r i t e , the o x i d i z i n g enzyme, and an o x i d i z e d c a r r i e r i s formed w h i c h b r e a k s down, a f t e r r e a c t i o n , i n t o n i t r a t e , the enzyme, and reduced c a r r i e r .  -5The  c a r r i e r i s then o x i d i z e d by oxygen.  An i n a c t i v e complex  can be formed i f the enzyme r e a c t s w i t h two m o l e c u l e s of nitrite. %  On  the b a s i s of t h i s he o b t a i n s the f o l l o w i n g r e s u l t s :  i n gas phase 20 10 2.5  N i t r i t e Concentrations Theoretical  Experimental  9 mM 8 mM 5 mM  m  f o r Maximal Rate  15 mM 9 mM 6 mM  Boon and L a u d e l o u t c a l c u l a t e d the K  of the  terminal  m o x i d a s e o f the r e s p i r a t o r y c h a i n w i t h r e s p e c t oxygen by p l o t t i n g v v s v/S. or a p a r t i a l pressure  When the oxygen  m  i t follows f i r s t  16^M  concentration  the r e a c t i o n proceeds a t a  r a t e but when i t i s l e s s than K kinetics.  substrate  At 32°C, ^ ( o x y g e n ) was  of 1.5%.  i s much g r e a t e r than  t o the  constant order  Below the K ( o x y g e n ) the o x i d a t i o n proceeds much m  more s l o w l y w i t h i n c r e s i n g temperature due c h a r a c t e r i s t i c s of K  and m  t o the  temperature  the maximal r a t e V „ . max m 0  Aleem, Hoch and V a r n e r (1965) have shov/n t h a t i t i s not the oxygen from 0  but r a t h e r from water t h a t s e r v e s i n the  c o n v e r s i o n of NO2 t o NOg. 1-3 I n h i b i t o r S t u d i e s on Whole C e l l s and C e l l - F r e e E x t r a c t s A l l a u t h o r s are i n e s s e n t i a l agreement on.the matter presented to t h i s p o i n t . begin to disagree  and  subject  At t h i s p o i n t however t h e o r i e s  even some o b s e r v a t i o n s  are  contradictory.  -63.1  I n h i b i t i o n by  Nitrite  Boon and L a u d e l o u t have i n v e s t i g a t e d the e f f e c t s of n i t r i t e c o n c e n t r a t i o n ,  pH,  and  n i t r a t e concentration  the o x i d a t i o n of n i t r i t e , both i n whole c e l l s and extracts.  in cell-free  At f i r s t t h e y s u g g e s t t h a t a t h i g h e r  n i t r i t e i n h i b i t s i t s own  o x i d a t i o n by s u b s t r a t e  on  concentrations, inhibition.  However t h e y then go on t o suggest t h a t the e f f e c t i s a c t u a l l y due  t o u n d i s s o c i a t e d n i t r o u s a c i d which on the b a s i s of  k i n e t i c s i s a non-competitive i n h i b i t o r . growth o c c u r s a t a pH of 7.8.  On  Maximal N i t r o b a c t e r  the a c i d s i d e of t h i s ,  inhi-  b i t i o n by n i t r o u s a c i d e x p l a i n s most of the d e c l i n e i n growth rate.  On  the a l k a l i n e s i d e , Boon and L a u d e l o u t suggest t h a t  i n h i b i t i o n i s due  t o the a b s o r p t i o n  of h y d r o x i d e i o n s on  the  enzyme s i t e . These a u t h o r s f i n d t h a t the K  values  f o r n i t r i t e as  the  m  s u b s t r a t e are a l m o s t i d e n t i c a l f o r i n t a c t c e l l s and e x t r a c t s , being of K  m  values  1.6  and  2.2  mM  respectively.  The  cell-free similarity  p o i n t s t o an o x i d i z i n g system l o c a t e d on  the  o u t e r c e l l membrane as do u n p u b l i s h e d e l e c t r o n m i c r o s c o p e observations  on f r a c t i o n a t e d c e l l - f r e e e x t r a c t s r e p o r t e d  Boon e t a l .  However, Aleem and A l e x a n d e r (1958) u s i n g  by cell-  f r e e e x t r a c t s c o n t a i n i n g n i t r i t e o x i d a s e a c t i v i t y found  no  t o x i c i t y up t o c o n c e n t r a t i o n s  at  of 50 mM—a c o n c e n t r a t i o n  which whole c e l l s have a r a t e c l o s e t o z e r o . i n d i c a t e a high M i c h a e l i s constant p r o b a b l y of the o r d e r of 20 mM. found by Boon.  Their  f o r n i t r i t e as  data  substrate,  T h i s does not agree w i t h  that  -73.2  I n h i b i t i o n by I n o r g a n i c  Ions  B u t t and L e e s (1960) i n v e s t i g a t e d the e f f e c t s of several inorganic cells. acted  i o n s on the o x i d a t i o n o f n i t r i t e by whole  They found t h a t n i t r a t e , c y a n a t e , and similarly. 3.2.1  I n h i b i t i o n by N i t r a t e A certain concentration  33 mM but  arsenite a l l  n i t r a t e was  i n h i b i t o r y a t normal oxygen  t h i s same c o n c e n t r a t i o n  t r a t i o n stimulated  of the i o n ,  e.g.  concentrations  of i o n a t a lower oxygen concen-  the o x i d a t i o n r a t e .  i n v e s t i g a t i o n showed t h a t N i t r o b a c t e r  Boon and  Laudelout's  i n a grov/th medium  c o n t a i n i n g p r e c i p i t a t e d s a l t s e x h i b i t e d t h i s phenomenon but a medium f r e e from p r e c i p i t a t e , n i t r a t e a c t e d  as a s i m p l e  n o n - c o m p e t i t i v e i n h i b i t o r , showing no s t i m u l a t i o n a t oxygen c o n c e n t r a t i o n s .  low  Boon and L a u d e l o u t a t t r i b u t e the  s t i m u l a t o r y e f f e c t of n i t r a t e a t low oxygen t o an  ion  exchange e f f e c t .  N i t r a t e added t o the washed s u s p e n s i o n of  c e l l s and  p r e c i p i t a t e might r e l e a s e t r a c e elements  mineral  absorbed onto the m i n e r a l  particles.  i n h i b i t i o n of n i t r a t e (K^ = 180 mM) i n h i b i t i o n o b s e r v e d by Lees and 3.2.2  Their values for d i d not e x p l a i n  Simpson a t 100  B u t t and  the  I n h i b i t i o n by Cyanate  Lees i n 1964  on whole c e l l s and  the  mM.  U s i n g the same medium as t h e y had 1960,  in  done i n  i n v e s t i g a t e d the e f f e c t of cyanate  cell-free extracts.  They found t h a t cyanate  -8-  a p o w e r f u l i n h i b i t o r o f n i t r i t e o x i d a t i o n by whole c e l l s a t normal oxygen t e n s i o n s , had no e f f e c t whatever on t h e c e l l free extracts.  The t h e o r y  t h i s i s the f o l l o w i n g :  t h a t they propose t o account f o r  t h e r e i s a t r a n s p o r t system which  b r i n g s n i t r i t e from t h e medium t o t h e enzyme and t h a t cyanate i n t e r f e r e s w i t h t h i s enzyme, a l t h o u g h not w i t h t h e n i t r i t e oxidase.  I n t h i s way, a t normal oxygen t e n s i o n , a l l t h e n i t r i t e  t h a t would r e a c h t h e enzyme n o r m a l l y  would be o x i d i z e d .  i n t e r f e r e n c e o f t h e c y a n a t e p r e v e n t s n i t r i t e from the enzyme and s l o w s down the r a t e .  inhibition.  p r e v e n t s t h e i n h i b i t i o n by l o w e r i n g  reaching  A t lower oxygen concen-  t r a t i o n , t o o much n i t r i t e would n o r m a l l y r e s u l t i n g i n substrate  The  r e a c h t h e enzyme  A d d i t i o n o f cyanate the n i t r i t e  concentration  a t t h e enzyme, t h e r e b y s t i m u l a t i n g n i t r i t e o x i d a t i o n . a r e two p o s s i b l e problems w i t h t h i s t h e o r y .  There  The one i s the  p o s s i b i l i t y as s t a t e d by Boon t h a t t h e enzyme i s a l r e a d y on the o u t e r s u r f a c e o f t h e c e l l and t h a t n i t r i t e does not have t o d i f f u s e t h r o u g h t h e membrane.  The second i s t h a t i f these  ions are s u f f i c i e n t l y l i k e n i t r i t e t o a f f e c t the c a r r i e r , t h e n i t would seem p o s s i b l e t h a t they would a l s o a f f e c t the n i t r i t e oxidase.  Van G o o l and L a u d e l o u t (1965), i n c o n t r a s t ,  have e v i d e n c e t h a t c y a n a t e has a p p r o x i m a t e l y t h e same e f f e c t on whole c e l l s and c e l l - f r e e e x t r a c t s as t h e c o n c e n t r a t i o n for  50% i n h i b i t i o n i n t h e two c a s e s i s 2.5 and 7 mM. 3.2.3 I n h i b i t i o n by C h l o r a t e The e f f e c t s o f c h l o r a t e have been i n v e s t i g a t e d  -9by Lees and The  Simpson (1957) and  by Van  50% i n h i b i t i o n a f t e r 60 min  i s comparable w i t h t h a t of Van  G o o l and L a u d e l o u t  at 7 mM Gool.  C I O 3  The  found by Lees  incubation period .  must be s t a t e d s i n c e d e c o m p o s i t i o n p r o d u c t s of the destroy r a t e law  the cytochrome a c t i v i t y a c c o r d i n g (Lees and  (1965).  to a  inhibitor  first-order  Simpson).  Of a l l the i n h i b i t o r s s t u d i e d by Van G o o l and o n l y c h l o r a t e does not g i v e s i m i l a r v a l u e s and c e l l - f r e e e x t r a c t s .  The  Laudelout,  f o r whole c e l l s  s i m i l a r i t y o f the v a l u e s  would  seem t o a c c o r d w i t h a p e r i p h e r a l l o c a t i o n of the enzyme system causing 1-4  nitrite  Oxidation  oxidation.  of N i t r i t e by C e l l - F r e e E x t r a c t s  I have p r e v i o u s l y mentioned the use in studying Nitrobacter i n 1958  ( c . f . Sec.  1-3).  of c e l l - f r e e Aleem and  extracts  Alexander  were the f i r s t t o d i s r u p t c e l l s by s o n i f i c a t i o n and  t h e y found t h a t the e x t r a c t s c o n t a i n e d t h a t t h i s was  n i t r i t e oxidase a c t i v i t y ,  c o u p l e d t o oxygen uptake and  t h a t the  oxidized  n i t r i t e c o u l d a t a l l t i m e s be r e c o v e r e d as n i t r a t e i n d i c a t i n g t h a t t h e r e are no i n t e r m e d i a t e s .  As mentioned p r e v i o u s l y  t h e r e i s no n i t r i t e t o x i c i t y o b s e r v e d t o a c o n c e n t r a t i o n 50 mM.  Optimum c o n d i t i o n s f o r o x i d a t i o n r e q u i r e the  of i r o n and  a pH of 7.5  t o 8.0.  The  of  presence  n i t r i t e oxidase i s  i n h i b i t e d by low c y a n i d e c o n c e n t r a t i o n s .  A l o n g w i t h the i r o n  r e q u i r e m e n t t h i s l a t t e r f a c t s u g g e s t s a s i m i l a r i t y between the n i t r i t e o x i d i z i n g enzyme and  the cytochrome  oxidase.  F u r t h e r c e n t r i f u g a t i o n shows t h a t the n i t r i t e o x i d a s e i s  -10situated  i n the p a r t i c u l a t e  f r a c t i o n a t 144,000 x g.  1-5 S p e c t r o s c o p y o f N i t r o b a c t e r 5.1 A b s o r p t i o n S p e c t r a Lees and Simpson (1957) r e p o r t e d cytochrome a b s o r p t i o n peaks a t 589, 551, and 520-525 nm on a d d i t i o n o f n i t r i t e or d i t h i o n i t e .  Aleem and Nason  (1959) show t h a t  n i t r i t e o x i d i z i n g a c t i v i t y r e s i d e s s o l e l y i n a cytochrome containing p a r t i c l e . resulted  A d d i t i o n of n i t r i t e t o t h i s p a r t i c l e  i n a b s o r p t i o n peaks a p p e a r i n g a t 550 and 520 nm  r e p r e s e n t a t i v e o f t h e a and B peaks o f a c_ type cytochrome and i n t h e 585-590 and 438 nm r e g i o n s i n d i c a t i v e o f t h e a and y peaks o f and a t y p e cytochrome, p r o b a b l y a.]..  Added  dithionite  gave e s s e n t i a l l y s i m i l a r peaks b u t o f s e v e r a l f o l d g r e a t e r magnitude, and a l s o produced an a b s o r p t i o n maximum a t 415 nm c o r r e s p o n d i n g t o the y peak o f cytochrome c_. the  Production of  peaks was s p e c i f i c f o r n i t r i t e as s u b s t r a t e , s u c c i n a t e ,  DPNH, o r l a c t a t e f a i l i n g t o produce them.  Hemoglobin, c a t a l a s e  and p e r o x i d a s e were shown t o be absent from b o t h t h e p a r t i c u l a t e and s u p e r n a t a n t f r a c t i o n s . have been o b s e r v e d .  (Different  copper and i r o n  effects  Aleem and A l e x a n d e r (1958) showed t h a t  copper was i n h i b i t o r y , but i n 1959 Aleem and Nason s a y t h a t i t has no e f f e c t but t h a t i t enhances t h e non-enzymatic disappearance of n i t r i t e .  The i r o n r e q u i r e m e n t was shown but  i r o n a l s o r e d u c e s t h e cytochrome c_ l i k e component non-enzymatically.  -115.2 D i f f e r e n c e S p e c t r a A more r e c e n t s p e c t r o s c o p i c s t u d y was by Van G o o l and L a u d e l o u t  i n 1965  on N i t r o b a c t e r w i n o g r a d s k i .  D i f f e r e n c e s p e c t r a f o r i n t a c t c e l l suspensions at  523,  In  the zone o f 450 t o 490 nm the reduced  465 nm.  a t 419 and 439 nm, to 440 nm. extracts.  system had a h i g h e r  The minimum o c c u r r e d a t  In the S o r e t r e g i o n t h e r e were two which were f u s e d i n a t u r b i d c e l l  The  suspension  peak a t 597 f o r example, o c c u r r e d i n t h i s At low temperature  the h i g h e s t peak i n the v i s i b l e was 587, and 579 nm.  The  on c e l l - f r e e e x t r a c t s  split  i n t o t h r e e peaks,  peaks a t 609 and 450,  439 a r e i n d i c a t i v e o f JI type cytochromes.  594  Those a t 554,  and 523  i n d i c a t e a cytochrome <c w i t h the h i g h wavelength o f  the a peak, 554, The  peaks,  S l i g h t d i f f e r e n c e s were n o t i c e d w i t h c e l l - f r e e  system a t 594 nm.  and 419  last.  T h i s i s p o s s i b l y i n d i c a t i v e o f the b l e a c h i n g o f  f l a v i n components.  604,  showed maxima  554, and 597 nm w i t h a s h o u l d e r a t 609 on the  t r a n s m i t t a n c e than the o x i d i z e d .  at  performed  i n d i c a t i n g a b a c t e r i a l type cytochrome £.  f o l l o w i n g t a b l e i s a.summary o f t h i s and o t h e r s p e c t r o s c o p i c  d a t a t a k e n from the paper by Van G o o l and Author  Cytochromes «i  ±a & VanGool and L a u d e l o u t  Laudelout.  a  l a  609 450 594  Lees and Simpson  -  -  589  Aleem and Nason  -  -  586-590  Zavarzin  -  -  592  Cytochromes c_  c  aj^  Q  439 554 -  523  419  551 520-525  -  438 550 520 -  552  -  415  -125.3  Carbon Monoxide D i f f e r e n c e S p e c t r a A carbon monoxide d i f f e r e n c e spectrum  as the a b s o r p t i o n of the carbon monoxide-reduced minus the a b s o r p t i o n o f the reduced compound.  i s recorded compound  This  i n d i c a t e s what m o d i f i c a t i o n s of the cytochromes  spectrum  i n the organism  or e x t r a c t s have been caused by the a d d i t i o n of carbon monoxide. I t has been shown i n many organisms  t h a t carbon monoxide  complexes o n l y w i t h the t e r m i n a l compound i n the c h a i n , i n c o m p e t i t i o n w i t h oxygen.  cytochrome  I t thus prevents the  o x i d a t i o n of t h i s oxidase and of the o t h e r cytochromes the  c h a i n w i t h the r e s u l t  in  t h a t these compounds become reduced,  or more reduced as the case may  be, i n the presence of a  r e d u c i n g agent, i . e . supply of e l e c t r o n s .  On a d d i t i o n of  carbon monoxide one would thus expect to observe an i n c r e a s e d r e d u c t i o n of the cytochromes  g e n e r a l l y p l u s changes i n the  a b s o r p t i o n of the oxidase due  to i t s forming a complex with  carbon monoxide. A comparison  of the reduced and carbon monoxide-  reduced d i f f e r e n c e s p e c t r a o f t e n enables one to determine which of the cytochromes  are present and which are r e a c t i n g  w i t h carbon monoxide s i n c e the cytochromes b a s i s of t h e i r a b s o r p t i o n s p e c t r a .  are d e f i n e d on the  To date, f o u r types of  cytochromes  have been found to a c t as o x i d a s e s .  cytochromes  a^, a^, ag, and o_.  5.3.1  They are  R e s u l t s with N i t r o b a c t e r Van Gool and Laudelout have r e p o r t e d a  -13CO-reduced minus reduced d i f f e r e n c e spectrum of N i t r o b a c t e r . Peaks a t 523,  554,  and 608 nm  i n the v i s i b l e and 419  i n the  Soret r e g i o n occur a t the same p o s i t i o n s as those i n the reduced d i f f e r e n c e spectrum.  These peaks are due  r e d u c t i o n of the compounds—namely cytochrome which do not combine with carbon monoxide. in the  to i n c r e a s e d  & and c_—  Troughs were recorded  the carbon monoxide d i f f e r e n c e spectrum at 439 and 594  nm,  p o s i t i o n s of peaks i n the reduced d i f f e r e n c e spectrum.  These  troughs are i n d i c a t i v e of reduced a b s o r p t i o n i n the carbon monoxide sample w i t h r e s p e c t to the reduced sample. these wavelengths in  Since  do correspond to the peaks of cytochrome  the reduced d i f f e r e n c e spectrum,  i n d i c a t e s t h a t cytochrome  a^  t h e i r disappearance  has complexed  with carbon monoxide.  Other i n d i c a t i o n s of carbon monoxide b i n d i n g were a peak at 450 nm  i n the carbon monoxide d i f f e r e n c e spectrum of the  cell-  f r e e e x t r a c t and a peak at 426 nm and trough a t 440 i n the  cell  suspensions. The peak and trough of the cytochrome  a^ r e p o r t e d at  426 and 440 f o r i n t a c t c e l l s and 439 f o r c e l l - f r e e  extracts  correspond w e l l w i t h data r e p o r t e d by Chance (1953).  His  f i g u r e s f o r these p o s i t i o n s i n A. pasteurianum are 427 442 nm.  Chance a l s o r e p o r t s a band at 590 w h i l e Van  and L a u d e l o u t mention in  Gool  a trough c o r r e s p o n d i n g to the 594 peak  the reduced d i f f e r e n c e  1-6 F l a v i n Involvement  and  spectrum.  i n the Cytochrome Chain  I n h i b i t o r and d i f f e r e n c e s p e c t r a s t u d i e s by Van Gool  -14and  L a u d e l o u t p o i n t d e f i n i t e l y to the involvement of a f l a v i n  component, as there was  strong  r e v e r s a l of t h i s by FAD.  i n h i b i t i o n by q u i n a c r i n e  Zavarsin  (Lees,  1960)  has  t h a t i r o n a l o n g w i t h e i t h e r molybdate or tungstate n i t r i t e oxidation. and  L a u d e l o u t say  reported stimulate  T h i s simultaneous requirement f o r i r o n  molybdate l e d Z a v a r z i n  f l a v o p r o t e i n and  and  to p o s t u l a t e  that a molybdeno-  cytochrome a c t s e q u e n t i a l l y .  Van  Gool  and  t h a t the involvement of a f l a v i n would i n d i c a t e  t h a t the p r o c e s s o f n i t r i t e o x i d a t i o n must be p u l l e d over a thermodynamically u n f a v o u r a b l e 1-7  Energy P r o d u c t i o n The  step.  and E f f i c i e n c y  d r i v i n g f o r c e f o r the r e d u c t i o n  o x i d a t i o n of the i n o r g a n i c i o n by oxygen.  of carbon i s the The  of these organisms i s thus best expressed by inorganic nitrogen  o x i d i z e d to C0„  efficiency  the r a t i o  carbon a s s i m i l a t e d .  the N:C  1961).  microorganisms do not get a l l the energy p o t e n t i a l l y  a v a i l a b l e but o n l y a s m a l l p r o p o r t i o n e f f i c i e n c y of energy u t i l i z a t i o n . Baas-Becking and 50%  to 135:1  In  Nitrobacter The  r a t i o v a r i e s from 76:1  of  determined by  The  (Alexander  their  f r e e energy data of  the assumption that N i t r o b a c t e r  contains  carbon on a dry weight b a s i s i s used to show that  f r e e energy e f f i c i e n c y i s 30% N/ml  and  and  Lees,  15%  the  i n the presence of 200jig n i t r a t e -  i n the presence of 1000  p.g n i t r a t e - N / m l .  (Gould  1960)  Aleem and  Naspn i n 1960  have shown that  i s c o u p l e d to n i t r i t e o x i d a t i o n .  phosphorylation  Partially purified  nitrite  -15oxidase p a r t i c l e s c a t a l y z e d the formation  o f h i g h energy  phosphate bonds c o n c o m i t a n t w i t h t h e o x i d a t i o n o f n i t r i t e . ATP  i s formed when ADP i s s u p p l i e d as t h e a c c e p t o r .  o t h e r n u c l e o t i d e s were a l s o e f f e c t i v e . to uncouple phosphorylation  A l l t h e i r attempts  from o x i d a t i o n proved  a l t h o u g h aged n i t r i t e o x i d a s e p a r t i c l e s d i s p l a y e d rates of phosphorylation r e m a i n e d unchanged.  Several  unsuccessful decreased  while t h e i r rates of oxidation  Magnesium i o n s were found t o s t i m u l a t e  phosphorylation. I n 1963, Aleem, L e e s , and N i c h o l a s demonstrated t h a t t h e r e was a r e d u c t i o n o f NAD c o n c o m i t a n t w i t h t h e o x i d a t i o n o f added mammalian f e r r o c y t o c h r o m e e_. Aleem (1965) showed t h a t t h e hydrogen f o r NAD r e d u c t i o n was s u p p l i e d by water. Aleem e t a l (1963) s t a t e t h a t the r e d u c t i o n o f NAD i s dependent on ATP and t h a t t h e t h e o r e t i c a l r e q u i r e m e n t i s 4 ATP/NAD. 10-20:1.  I n c e l l f r e e systems t h e e x p e r i m e n t a l  v a l u e was  These a u t h o r s have proposed t h e f o l l o w i n g scheme  f o r the o v e r a l l metabolism of N i t r o b a c t e r . ATP Inorganic substrate  generated  cyt£ ^ reductase  > c y t . c_ flavoprotein  1  NAD(H) Such a m o d i f i e d  cytochrome oxidase  ^ 0  2  ATP required ATP + C 0 -^(CH 0) 2  2  scheme seems t o be n e c e s s a r y i n o r d e r  t o produce NADH ( a l t h o u g h  I do not agree t h a t Aleem e t a l  have proven t h i s i n t h e i r e x p e r i m e n t s ) .  The r e a s o n f o r t h i s  -16modified  scheme i s t h a t the E  G  f o r the n i t r a t e - n i t r i t e  i s +0.35 w h i l e t h a t f o r the NAD/NADH couple i s -0.32. energy would be r e q u i r e d the O x i d a t i o n  I t would seem probable that  might a l s o be a m o d i f i c a t i o n i n the f i r s t  +0.25.  Thus  t o couple these systems and r e s u l t i n  of n i t r i t e .  sequence s i n c e the  system  there  s t e p o f the proposed  of mammalian cytochrome c_ F e  + + +  /Fe  + +  is  I t c o u l d perhaps be as Lees (1962) suggests, namely  t h a t N i t r o b a c t e r may be compelled t o s y n t h e s i z e compound as adenyl n i t r i t e of the n i t r i t e the v a l u e s  are obtained Q  so as t o lower the redox p o t e n t i a l  (See  however Sec.  1-4)  o f these redox p o t e n t i a l s I f e l t  v a t i o n s should  couple E  couple.  some such  In c o n s i d e r i n g  that some r e s e r -  be borne i n mind: e s p e c i a l l y that these  values  i n " i n v i t r o " s i t u a t i o n s a t pH 7and the c y t . c_  value  used i s that f o r mammalian cytochrome c_. I t  i s p o s s i b l e and q u i t e probable that the values  may not apply  to the s i t u a t i o n i n i n t a c t N i t r o b a c t e r  In i n t a c t c e l l s ,  cells.  the o x i d a t i o n of n i t r i t e might proceed much more  favourably  t o produce energy due t o a change i n redox p o t e n t i a l r e s u l t i n g from the p h y s i c a l c o n f i g u r a t i o n s or d i s t o r t i o n s o f the molecules i n v o l v e d and from l o c a l pH and c o n c e n t r a t i o n 1-8  The F i x a t i o n o f Carbon The  energy obtained  changes.  Dioxide from the o x i d a t i o n o f n i t r i t e , i n  the form o f ATP and NADH i s used to f i x and reduce carbon dioxide.  Aleem i n 1965 performed an e x t e n s i v e  pathway o f carbon f i x a t i o n u s i n g r a d i o i s o t o p e s .  study on the The f i r s t  s t a b l e compounds t o be l a b e l l e d were phosphoglyceric  acid  -17and  aspartic acid.  A f t e r 10 sec exposure t o C*^0 , 60% of 2  the r a d i o a c t i v i t y appeared i n phosphoglycerate, 20% i n each a s p a r t i c a c i d and sugar phosphates, 4-6 % i n each malic  acid,  glutamic  cell-  a c i d and phosphoenolpyruvate.  f r e e e x t r a c t s the c a r b o x y l a t i n g  In d e a l i n g with  enzymes were l o c a l i z e d i n the  supernatant a f t e r 144,000 x g c e n t r i f u g a t i o n . catalyzed rapid f i x a t i o n of C 0  2  The supernatant  i n the presence o f added  r i b u l o s e - 1 , 5 - d i p h o s p h a t e o r r i b o s e monophosphate and ATP. The  l a b e l appeared i n phosphoglycerate.  Without added  C02-acceptor the s o l u b l e f r a c t i o n c a t a l y z e d r a p i d a s s i m i l a t i o n when s u p p l i e d with ATP and NADH. r e q u i r e d magnesium i o n s .  The c a r b o x y l a t i n g  Carboxydismutase  diphosphate d e c a r b o x y l a s e ) and other Benson, Kreb's and r e d u c t i v e l o c a l i z e d i n the supernatant. observations  enzymes  (D-ribulose-1,5-  enzymes o f the C a l v i n -  pentose phosphate c y c l e s were Aleem s t a t e s that the above  p o i n t t o the standard  autotrophic  pathway o f  CX>2 a s s i m i l a t i o n , namely:  co r-ibulose diphosphate  C0  2  ^phosphoglycerate NH  =»• o x a l o a c e t a t e K NAD TCA ; NADP cycle i  2  ^phosphoenolpyruvate  3  ^ a s p a r t i c acid  +  Malate Aleem a l s o g i v e s the p o s s i b l e pathways o f carbon metabolism. Bock and E n g e l (1966) have shown that i f N i t r o b a c t e r i s deprived  of carbon d i o x i d e , oxygen uptake slows down.  Under  -18-  a e r o b i c as w e l l as under a n a e r o b i c c o n d i t i o n s , t h e n i t r i f i e r s a r e a b l e t o f i x c a r b o n d i o x i d e a f t e r o x i d a t i o n has o c c u r r e d . The  s t r o n g e s t a c t i v i t y i n • t h i s r e s p e c t i s seen i n c e l l s  w h i c h have c a r r i e d o u t o x i d a t i o n s h o r t l y b e f o r e b e i n g to  allowed  f i x C0 . 2  1-9 U n s o l v e d  Problems and C r i t i c i s m s  There a r e s t i l l many problems w i t h r e g a r d t o t h e p h y s i o l o g y of N i t r o b a c t e r . (i)  Some o f these a r e l i s t e d below.  F i r s t t h e r e i s t h e problem o f a c a l c i u m Aleem and A l e x a n d e r  requirement.  (1958) mention t h a t c a l c i u m i s  r e q u i r e d f o r growth o f N i t r o b a c t e r .  Lees and  Simpson (1957) make no mention o f a c a l c i u m r e q u i r e ment y e t t h e medium used by these a u t h o r s  contains  calcium ions.  on t h e o t h e r  Boon and L a u d e l o u t  (1962)  hand, used a medium which c o n t a i n e d no c a l c i u m , (ii)  A second p o i n t a r i s i n g out o f the paper by Boon and L a u d e l o u t  concerns t h e i r suggestion that the  i n h i b i t i o n o f N i t r o b a c t e r a t h i g h pH i s due t o 0H~ ions.  That t h e c o n c e n t r a t i o n o f h y d r o x y l i o n s , when  p l a c e d i n t h e k i n e t i c e q u a t i o n y i e l d s a curve c l o s e  (iii)  to  the e x p e r i m e n t a l proves o n l y t h a t an i o n i z a t i o n  is  i n v o l v e d , n o t t h a t 0H~ i s the a c t i v e i n h i b i t o r ,  The e f f e c t s o f n i t r a t e on n i t r i t e o x i d a t i o n a r e s t i l l not c l e a r .  The i n h i b i t i o n observed  by Lees and  Simpson, and B u t t and Lees seems t o be v e r y from t h a t d e s c r i b e d by Boon and L a u d e l o u t  different  ( c f . 1-3.2.1)  -19-  ( i v ) The d i f f e r e n c e s i n t h e e f f e c t s o f n i t r a t e by these a u t h o r s c o u l d be d i s m i s s e d  as j u s t a d i f f e r e n c e i n  degree e x c e p t t h a t c y a n a t e , a second i n h i b i t o r i s r e p o r t e d as b e h a v i n g i n a c o n t r a d i c t o r y manner by the d i f f e r e n t a u t h o r s ( c f . Sec. 1-3.2.2). B u t t ' s and Lees's o b s e r v a t i o n s  Thus  on t h e e f f e c t s o f  n i t r a t e and c y a n a t e and t h e i r d i f f e r e n c e i n whole c e l l s and c e l l - f r e e e x t r a c t s i s i n a c c o r d w i t h proposal  that a c a r r i e r i s required to bring  i n t o the c e l l .  A l l o f the o b s e r v a t i o n s  their  nitrite  o f Boon  and L a u d e l o u t on i n h i b i t o r s and t h e i r u n p u b l i s h e d e l e c t r o n m i c r o s c o p e s t u d i e s on t h e o t h e r hand, p o i n t to the p a r t i c u l a t e oxidase f r a c t i o n being  l o c a t e d on  the o u t e r membrane o f the c e l l , (v) The f i n a l problem t o be mentioned here i s concerned w i t h ATP g e n e r a t i o n .  As s t a t e d i n Sec. 1-7, I do  not agree t h a t Aleem has proven t h a t NADH i s produced i n t h e manner i n d i c a t e d s i n c e l o a d i n g o f a cytochrome c h a i n w i t h ATP w i l l d r i v e i t backwards.  A scheme  such as he has proposed does seem t o be r e q u i r e d however, i n o r d e r  t o o x i d i z e n i t r i t e and produce  energy from t h i s o x i d a t i o n .  A d e c i s i v e experiment  t o p e r f o r m a t t h i s p o i n t would be t o u t i l i z e Chance's r a p i d f l o w t e c h n i q u e w i t h i n h i b i t o r s t o determine the o r d e r  o f r e d u c t i o n o f t h e compounds i n the  cytochrome c h a i n .  -201-10 Summary There i s much c o n t r a d i c t o r y evidence  concerning  N i t r o b a c t e r e s p e c i a l l y i n r e l a t i o n t o i n h i b i t o r s t u d i e s and the l o c a t i o n o f the n i t r i t e  oxidase p a r t i c l e  membrane or i n s i d e the c e l l ) .  (on the outer  T h i s c o u l d be due t o the use  of d i f f e r e n t s t r a i n s , t o d i f f e r e n t growth c o n d i t i o n s - e s p e c i a l l y media, o r t o d i f f e r e n t e x p e r i m e n t a l technique. e s t a b l i s h e d however that N i t r o b a c t e r  I t seems w e l l  oxidizes n i t r i t e to  n i t r a t e and t h a t t h i s o x i d a t i o n s u p p l i e s energy i n the form of ATP and NADH f o r the f i x a t i o n of carbon d i o x i d e and the production  of m a t e r i a l f o r growth.  A cytochrome c h a i n and  f l a v o p r o t e i n a r e seen t o take p a r t i n the e l e c t r o n  transport  system. Water i s the source o f hydrogen f o r the r e d u c t i o n o f pyridine nucleotide Molecular  and o f oxygen i n the o x i d a t i o n o f n i t r i t e .  oxygen i s o n l y i n v o l v e d  i n the o x i d a t i o n o f the  t e r m i n a l oxidase i n the e l e c t r o n t r a n s p o r t c h a i n . dioxide  f i x a t i o n i s s i m i l a r t o that of other  Carbon  chemoautotrophic  b a c t e r i a i n that i t makes use o f the Calvin-Benson and r e d u c t i v e pentose phosphate pathways.  -21CHAPTER II GROWTH METHODS II-l  Medium and E x t e r n a l  Conditions  N i t r o b a c t e r a g i l i s from the American Type C u l t u r e C o l l e c t i o n No. 14123 were grown i n a medium i d e n t i c a l t o that To 1 0 0 1 . o f water were added  of Gould and Lees (1960). 30 gm K H 2 P O 4 ,  1 gm C a S 0 ,  of d i a l y z e d i r o n . 7.8,  0.5 gm M g C l , 0 . 0 1 gm M n C l 2 ,  4  2  T h i s was f i l t e r e d  Sodium n i t r i t e  (NaN0 ) 2  of 1 0 0 jig n i t r i t e - N / m l ( 8 m M ) . sodium n i t r i t e were p l a c e d sterilized  and 0 . 2 ml  and the pH a d j u s t e d t o  was added to make a 5 0 - 1 0 0 ml o f medium  concentration plus  i n 250 ml Erlenmeyer f l a s k s and  a f t e r which approximately 0 . 5 ml o f a d i l u t e  Nitrobacter  suspension were added.  i n the dark ( c f . 1 - 2 . 2 ) .  N i t r i t e was added when r e q u i r e d by  the a d d i t i o n o f a s t e r i l i z e d concentration  of n i t r i t e  The c u l t u r e s were grown  0 . 8 M NaNOg s o l u t i o n to b r i n g the  i n the c u l t u r e t o 1 0 mM.  At the onset of a decreased requirement f o r n i t r i t e , or j u s t p r i o r t o t h i s as e s t i m a t e d by the t u r b i d i t y o f the s o l u t i o n s , the c u l t u r e s were d i l u t e d two t o ten times, depending on the immediate requirement f o r concentrated  cultures.  Or  a l t e r n a t e l y , new c u l t u r e s were made by adding 2 - 5 ml of a concentrated  s o l u t i o n t o 1 0 0 ml of f r e s h medium.  In t h i s  manner a type o f continuous c u l t u r e method was employed p r o v i d i n g s t e a d i l y growing and f a i r l y concentrated at  a l l times.  cultures  Once c u l t u r e s reached the p o i n t where they  r e q u i r e d no more n i t r i t e  ( 3 - 8 wk), d i l u t i o n u s u a l l y proved  -22u n s u c c e s s f u l i n producing v i a b l e  cultures.  A e r a t i o n and n i t r a t e removal were not employed although such methods l e a d t o a prolonged growth phase (Gould and Lees, 1960).  The method o f d i l u t i n g c u l t u r e s as mentioned above  ensured  t h a t the n i t r a t e c o n c e n t r a t i o n d i d not become h i g h  enough t o prevent f u r t h e r growth.  The problem  a e r a t i o n was d e a l t with by keeping the c u l t u r e s  o f adequate relatively  s h a l l o w (about 2 cm) and a l l o w i n g a i r t o c i r c u l a t e  through  c o t t o n p l u g s i n the tops o f the f l a s k s . The c u l t u r e s used i n the experiments  were o x i d i z i n g  n i t r i t e a t a r a t e of 25-50 jig n i t r i t e - N / m l / d a y (2-4 mM  nitrite/  ml/day). II-2  T e s t Methods 2.1  Nitrite The  method employing  presence o f n i t r i t e was determined  s u l f a n i l i c a c i d and N,N dimethyl-l-naphthylamine  ( S n e l l and S n e l l , 1949). up.  using a  Two stock s o l u t i o n s were f i r s t made  S o l u t i o n A was made by d i s s o l v i n g 3.3 gm o f s u l f a n i l i c  a c i d i n 750 ml water by h e a t i n g . were then added to t h i s .  250 ml g l a c i a l a c e t i c  acid  S o l u t i o n B c o n t a i n e d 5.25 gm of  N,N d i m e t h y l - l - n a p h t h y l a m i n e  i n 1 l i t r e of 1:3 g l a c i a l  acetic  a c i d : methanol. For a q u a l i t a t i v e t e s t o f the presence of n i t r i t e volumes o f s o l u t i o n A and B were mixed.  To about  equal  1 ml o f t h i s  mixture a sample o f l e s s than 0.5 ml o f medium was added. Development o f c o l o u r (pink t o p u r p l e ) i n d i c a t e d the presence of  nitrite.  A c o l o u r l e s s s o l u t i o n was i n d i c a t i v e of the  -23absence of n i t r i t e . the n i t r i t e The  employed to determine  requirement of the c u l t u r e s .  best c o n d i t i o n s f o r a quantitative t e s t were found  to be as f o l l o w s . d i l u t e d to 100 was  T h i s method was  1 ml of the s o l u t i o n to be t e s t e d  ml with d i s t i l l e d water.  5 ml of s o l u t i o n A  added f o l l o w e d by 5 ml of s o l u t i o n B.  Ten minutes a f t e r  the a d d i t i o n of s o l u t i o n B a r e a d i n g on the K l e t t was  taken u s i n g a green f i l t e r .  The  was  colorimeter  t i m i n g here i s q u i t e  c r u c i a l as each a d d i t i o n a l 30 seconds r e s u l t s i n a change of 6-10%  i n the The  reading.  development of c o l o u r i s a l s o very s e n s i t i v e to  temperature and to  run one  the age  standard  of s o l u t i o n B.  Thus i t i s  each time a d e t e r m i n a t i o n  necessary  i s performed.  Since a p l o t of K l e t t r e a d i n g versus c o n c e n t r a t i o n i s p r o p o r t i o n a l i n the c o n c e n t r a t i o n range encountered standard  i s usually sufficient.  Solution B i s unstable two  one  weeks.  over p e r i o d s of time g r e a t e r  T h i s i s not of any  consequence f o r q u a l i t a t i v e  t e s t s as c o l o u r develops i n the presence of n i t r i t e not develop i n i t s absence. g i v e a deeper c o l o u r but  than  and  does  In both t e s t s o l d e r s o l u t i o n s  too o l d a s o l u t i o n r e s u l t s i n the  appearance of a p r e c i p i t a t e i n l e s s than ten minutes so that a quat&i t a t i v e t e s t has 2.2  no  validity.  Nitrate Nitrobacter converts  it  nitrite  to n i t r a t e so t h a t  i s d e s i r a b l e to t e s t f o r the p r o d u c t i o n  of n i t r a t e  to  -24ensure t h a t the N i t r o b a c t e r a r e the agents c a u s i n g the disappearance o f n i t r i t e .  T h i s i s done u s i n g the f o l l o w i n g  method employing a p h e n o l d i s u l f o n i c a c i d reagent Snell,  ( S n e l l and  1949). The  reagent was prepared by d i s s o l v i n g 25 gm o f c o l o u r l e s s  phenol i n 150 ml o f concentrated H^SO^.  T r a c e s of n i t r i c a c i d  i n the s u l p h u r i c a c i d were removed by a g i t a t i n g with 75 ml o f fuming s u l p h u r i c a c i d c o n t a i n i n g 13% f r e e t r o x i d e were then added.  mercury.  sulphur  T h i s was s t i r r e d and heated two  hours on a b o i l i n g water bath. 10 ml o f sample were evaporated t o dryness. 2 ml of the p h e n o l d i s u l f o n i c a c i d reagent were added r a p i d l y to the c e n t r e and the d i s h r o t a t e d t o ensure c o n t a c t o f the reagent with  a l l o f the r e s i d u e .  T h i s was allowed  to stand  f o r 10  minutes a t which time 15 ml of c o l d water were added to d i s s o l v e the r e s i d u e .  1:2 ammonium hydroxide:water was added  u n t i l the s o l u t i o n was s l i g h t l y a l k a l i n e . by the p r o d u c t i o n was  o f a deep yellow c o l o u r .  then made up to 50 ml and a r e a d i n g  c o l o r i m e t e r u s i n g a blue f i l t e r .  T h i s was accompanied The s o l u t i o n  taken on the K l e t t  C a l i b r a t i o n was o n l y  r e q u i r e d once as the c o l o u r developed i s u n a f f e c t e d conditions.  by e x t e r n a l  -25-  CHAPTER I I I INSTRUMENTATION AND SAMPLE PREPARATION I I I - l The Oxygen E l e c t r o d e 1.1  Instrumentation The  r a t e o f oxygen uptake and hence o f n i t r i t e  o x i d a t i o n a s a f u n c t i o n o f oxygen c o n c e n t r a t i o n c a n be o b t a i n e d u s i n g an oxygen e l e c t r o d e .  This c o n s i s t s of a platinum  e l e c t r o d e p o l a r i z e d a t -0.6 t o -0.9 v o l t s w i t h r e s p e c t t o a Ag/AgCl e l e c t r o d e p l a c e d i n the same s o l u t i o n .  At t h i s  p o t e n t i a l i n t h e s o l u t i o n s used, oxygen d i f f u s i n g t o t h e p l a t i n u m e l e c t r o d e i s s e l e c t i v e l y reduced.  The amount d i f f u s i n g t o  the e l e c t r o d e i s a f u n c t i o n o f the c o n c e n t r a t i o n and r e s u l t s i n a c u r r e n t i n the e x t e r n a l c i r c u i t p r o p o r t i o n a l t o the concentration.  The c u r r e n t i s measured by p a s s i n g i t through  a r e s i s t o r and m o n i t o r i n g  t h e v o l t a g e drop a c r o s s t h e r e s i s t o r  w i t h a H e a t h k i t Servo R e c o r d e r model EUW-20A. In t h e e x p e r i m e n t s w i t h N i t r o b a c t e r a s m a l l c y l i n d r i c a l chamber h o l d i n g 8 ml o f f l u i d was employed f o r measuring oxygen uptake ( F i g . 1 ) . A magnetic s t i r r e r was used t o prevent  t h e N i t r o b a c t e r from s e t t l i n g t o t h e bottom and t o  keep t h e medium mixed w i t h r e s p e c t t o t h e oxygen c o n c e n t r a t i o n . T h i s r e s u l t e d i n a lower d i f f u s i o n d i s t a n c e and a h i g h e r c u r r e n t than would be found i n a s t a t i o n a r y c a s e .  Equilibration  o f t h e oxygen c o n c e n t r a t i o n i n the medium w i t h t h a t i n a i r was p r e v e n t e d  by c l o s i n g t h e chamber w i t h a greased c o v e r  slip^  Greased cover slip Oxygenated Sample Pt  electrode  Ag/AgCl e l e c t r o d e  Stirring bar  SI Support  Magnetic Stirrer  T  y  Figure 1  The Oxygen E l e c t r o d e  -26T h i s a l l o w e d t h e use o f oxygen c o n c e n t r a t i o n s h i g h e r than t h a t in a i r . 1.2 C a l i b r a t i o n The  oxygen e l e c t r o d e was c a l i b r a t e d u s i n g a 0.1 M  sodium c h l o r i d e s o l u t i o n .  100% oxygen c o n c e n t r a t i o n was  d e t e r m i n e d by b u b b l i n g t h e s o l u t i o n w i t h oxygen f o r about two m i n u t e s b e f o r e p l a c i n g i n t h e a p p a r a t u s .  The s e n s i t i v i t y  was then s e t t o e n s u r e t h a t t h i s c o n c e n t r a t i o n o c c u r r e d a t the a p p r o p r i a t e p o i n t .  0% oxygen was d e t e r m i n e d by b u b b l i n g  the s o l u t i o n w i t h n i t r o g e n and 20% oxygen c o u l d be d e t e r m i n e d by a l l o w i n g the s o l u t i o n t o e q u i l i b r a t e w i t h a i r . 1.3 P r e p a r a t i o n o f t h e Sample 10 m l o f c u l t u r e were removed and bubbled w i t h oxygen f o r 1 min.  Enough N i t r o b a c t e r were then p l a c e d i n  the a p p a r a t u s t o p r e v e n t  t h e f o r m a t i o n o f an a i r space when  the o p e n i n g was c l o s e d w i t h the c o v e r s l i p . concentrated  F o r more  samples t h e N i t r o b a c t e r i n 50 ml o f c u l t u r e  were c e n t r i f u g e d a t t h e f a s t e s t speed on a c l i n i c a l c e n t r i f u g e for  15 min and r e s u s p e n d e d i n 10 ml o f 20 mM phosphate b u f f e r  (18 mM N a H P 0 2  4  p l u s 2 mM K H P 0 ) o r i n 10 ml o f t h e i r own 2  4  medium w i t h t h e a p p r o p r i a t e n i t r i t e c o n c e n t r a t i o n .  These  samples were a l s o b u b b l e d w i t h oxygen f o r 1 min b e f o r e  being  placed i n the apparatus. I I I - 2 The A c t i o n Spectrum A p p a r a t u s The  a c t i o n s p e c t r u m a p p a r a t u s d e v e l o p e d by B r o o k s (1967)  -27and m o d i f i e d  here i s based on an instrument d e s c r i b e d by  C a s t o r and Chance (1955).  I t i s a s e n s i t i v e instrument f o r  measuring the p h o t o c h e m i c a l l y uptake by carbon monoxide.  r e v e r s i b l e i n h i b i t i o n o f oxygen  The e s s e n t i a l f e a t u r e s o f t h i s  equipment a r e 1) a l i g h t source and monochromator, 2) an i n t e n s i t y measuring d e v i c e , and 3) a chamber and r e s p i r a t i o n r a t e sensor.  These f e a t u r e s a r e d e s c r i b e d by Brooks while  f i c a t i o n s t o the r e f e r e n c e apparatus a r e d e s c r i b e d  beam and i n t e n s i t y measuring  i n l a t e r s e c t i o n s of t h i s t h e s i s .  In s h o r t , the l i g h t source p r o v i d e s beam and the r e f e r e n c e spectrum.  modi-  both the monochromatic  beam r e q u i r e d to o b t a i n the a c t i o n  To measure the i n t e n s i t y o f e i t h e r beam, the l i g h t ,  chopped by a mechanical d e v i c e , photodetector  f a l l s on a l e a d  sulphide  and the s i g n a l from t h i s i s passed through a  phase s e n s i t i v e d e t e c t o r 150 A microvoltmeter  (PSD, F i g . 2 ) . A K e i t h l e y model  ammeter i s used as a n u l l device  a bucking v o l t a g e s u p p l i e d from an e x t e r n a l c i r c u i t . chamber p r o v i d e s  with A closed  f o r the use of a c o n t r o l l e d atmosphere of  carbon monoxide and oxygen i n the experiments. sensor c o n s i s t s o f a platinum t o -0.9 v o l t s with r e s p e c t  The r a t e  e l e c t r o d e p o l a r i z e d at -0.6  t o a Ag/AgCl e l e c t r o d e p l a c e d i n  a drop o f c e l l suspension.  T h i s drop i s h e l d between the two  e l e c t r o d e s and a cover s l i p  forming the bottom o f the chamber.  The  c u r r e n t measurements are made by bucking out the e l e c t r o d e  c u r r e n t u s i n g the K e i t h l e y microvoltmeter  ammeter and an  E s t e r l i n e Angus r e c o r d i n g milliammeter as n u l l Unlike  detectors.  the oxygen e l e c t r o d e , the purpose i n the a c t i o n  + 300 V  BIAS VOLTAGE  BUCKING VOLTAGE  BUCKING CURRENT  A L L CAP. V A L U E S IN M I C R O F A R A D S  E L E C T R O D E OFFQ, 2 ^ P S D o o © SWITCH P O S I T I O N S  200  1.1B  FIGURE  2  Circuit  Diagram  of  Phase  Sensitive  Detector  -28spectrum a p p a r a t u s i s t o s e t up a s t e a d y s t a t e produced by the o p p o s i n g e f f e c t s of d i f f u s i o n o f oxygen from the s u r f a c e o f t h e d r o p t o the p l a t i n u m m i c r o e l e c t r o d e and by the o r g a n i s m i n the drop.  respiration  S m a l l changes i n r e s p i r a t i o n  r a t e w i l l a l t e r the s t e a d y s t a t e w i t h a consequent change i n c u r r e n t i n the e x t e r n a l  circuit.  2.1 M o d i f i c a t i o n s t o the R e f e r e n c e Beam B r o o k s (1967) suggested s e v e r a l improvements  to  reduce e r r o r and e x t e n d the range of the a c t i o n spectrum apparatus.  S e v e r a l o f t h e s e have been c a r r i e d o u t .  The method of b r i n g i n g the comparison beam from the lamp t o the sample was a l t e r e d .  To do t h i s the l i g h t s o u r c e used,  was changed from the 8v-50w Ace b u l b t o a 24v-150w P h i l l i p s q u a r t z i o d i n e lamp (model no. 7158).  The l a t t e r b u l b has a  q u a r t z e n v e l o p e but has no r e f l e c t i v e c o a t i n g as the former. T h i s makes p r o v i s i o n f o r t a k i n g a beam o f l i g h t from the back o f t h e b u l b as w e l l as the f r o n t but a t the same time reduces the i n t e n s i t y o f b o t h beams.  Some i n c r e a s e i n i n t e n s i t y  was  t h e n p r o v i d e d by u s i n g the h i g h e r power lamp. The c u r r e n t and v o l t a g e a r e p r o v i d e d by a Sorenson and an E i c o power s u p p l y p l a c e d i n s e r i e s .  S t a b i l i t y i s a c h i e v e d by  p l a c i n g two t w e l v e v o l t b a t t e r i e s i n p a r a l l e l w i t h the power s u p p l y . ( F i g . 3)  The power u n i t s a r e a d j u s t e d t o ensure t h a t  t h e r e i s no c u r r e n t p a s s i n g through the b a t t e r i e s .  Monochromator Filter Holder Intensity Control  Waveleng Control  Voltage Regulated Source Figure 4 B l o c k Diagram o f Apparatus  -29-  lamp  SORENSON  EICO  Batteries Fig.  3  Power Supply f o r the L i g h t  Source  The beam taken from the one s i d e o f the lamp passes through the monochromator as d e s c r i b e d by Brooks.  The second  beam f o l l o w s a path o p t i c a l l y e q u i v a l e n t t o that o f the monochromatic beam. the of  Condensing l e n s e s image the l i g h t  back of the lamp onto a s l i t which i s i n the f o c a l plane a convex  convex  lens.  The c o l l i m a t e d beam i s condensed  l e n s and imaged a t the e x i t s l i t  The l e n s e s a r e housed the  from  by a second  of the monochromator.  i n a tube 2 " i n diameter which  carries  l i g h t out the back of the lamp, around the monochromator  box and a l l o w s i t t o e n t e r the monochromator a t a p o s i t i o n p e r p e n d i c u l a r t o , but j u s t below the e x i t s l i t . p l a c e d on a t r a c k can be s l i d  A small mirror  i n t o p o s i t i o n to r e f l e c t the  r e f e r e n c e beam onto the e x i t s l i t .  (Fig. 4)  The t r a c k  c o n s i s t s o f two s m a l l rods such t h a t when the m i r r o r i s moved out  o f the path o f the monochromatic beam, t h i s beam passes  between these r o d s and i s f o c u s e d on the s l i t . - t h a t the e x i t s l i t the  advantage  The r e s u l t i s  i s a t a l l times f i l l e d w i t h l i g h t , having  as o u t l i n e d by Brooks, that no t r a n s i e n t  effects  -30are produced i n t h e sample. The  l o u v e r s were moved from t h e monochromator h o u s i n g  t o an e q u i v a l e n t p o s i t i o n i n t h e c o l l i m a t e d p o r t i o n o f t h e r e f e r e n c e beam thereby  reducing  t h e amount o f s c a t t e r e d  i n c l u d e d i n t h e monochromatic beam.  Any f i l t e r s  light  required  i n t h e r e f e r e n c e beam c o u l d be p l a c e d i n t h e l o u v e r  housing.  2.2 M o d i f i c a t i o n o f t h e Chopper The  above changes n e c e s s i t a t e d t h e i n t r o d u c t i o n o f  a f u r t h e r improvement.  The o n l y p h y s i c a l l y i d e n t i c a l path  f o l l o w e d by t h e two beams i s t h a t between t h e s l i d i n g and  t h e sample.  mirror  S i n c e the i n t e n s i t y o f both beams i s r e q u i r e d  for determination  o f t h e a c t i o n spectrum, t h e chopper and  d e t e c t i n g system must be so p l a c e d t h a t measurements can be made on b o t h beams. must be s m a l l .  Because o f space l i m i t a t i o n s t h e chopper  A m a g n e t i c a l l y d r i v e n tuning f o r k suggested  by B r o o k s meets t h i s r e q u i r e m e n t .  The l i g h t c h o p p i n g wheel  was thus d i s c a r d e d and r e p l a c e d by a B u l o v a l i g h t chopper o p e r a t e d on 28 v o l t s , 20 m i l l i a m p s from an E i c o DC power The  source.  n a t u r a l f r e q u e n c y o f t h e t u n i n g f o r k i s 400 Hz. F i g . 4 d e p i c t s t h e arrangement used f o r o b t a i n i n g i n t e n -  s i t y measurements.  A small spherical mirror intercepts a  p o r t i o n , about 10%, o f t h e l i g h t e n t e r i n g t h e cone and p a r t l y focuses  t h e l o u v e r s o r t h e g r a t i n g on t h e PbS, l e a d s u l p h i d e ,  detector. PbS  Thus a c r o s s s e c t i o n o f the beam i s sampled by t h e  d e t e c t o r and a l t h o u g h  t h i s does n o t remove i n h o m o g e n e i t i e s  i n t h e beam i t does a l l o w a 1:1 c o r r e s p o n d e n c e o f PbS r e a d i n g s  -31with i n t e n s i t y a t the t o p o f the cone. of  the beam i s removed by the scrambling  consequently  The inhomogeneity e f f e c t o f the cone and  i s not t r a n s f e r r e d to the sample.  2.3 M o d i f i c a t i o n s t o the Phase S e n s i t i v e D e t e c t o r In order t o measure the 400 Hz s i g n a l , the phase s e n s i t i v e d e t e c t o r , PSD, r e q u i r e d m o d i f i c a t i o n . ( F i g . 2) rejection f i l t e r was removed.  The  i n the feedback l o o p o f the f i r s t a m p l i f i e r  The purpose o f t h i s f i l t e r was t o a c t as a band  pass so t h a t the second a m p l i f i e r would not become overloaded with noise s i g n a l s .  The o v e r l o a d i n g o f the second a m p l i f i e r  was not a problem and the r e j e c t i o n f i l t e r phase s h i f t s .  i n t r o d u c e d unwanted  The feedback l o o p of the second a m p l i f i e r was  changed t o i n t r o d u c e a more uniform  gain c o n t r o l .  A 0.01 m i c r o f a r a d c a p a c i t o r was added a c r o s s the r e f e r e n c e i n p u t i n order t o round o f f the square wave s u p p l i e d by the t u n i n g f o r k .  A 0.047 m i c r o f a r a d c a p a c i t o r was p l a c e d  a c r o s s the secondary o f the s i g n a l transformer  t o c o r r e c t the  phase o f the s i g n a l . 2.4 Sample P r e p a r a t i o n f o r the A c t i o n Spectrum Apparatus For use i n the a c t i o n spectrum apparatus 50 ml of c u l t u r e were c e n t r i f u g e d on the h i g h e s t speed o f a c l i n i c a l c e n t r i f u g e f o r 15-20 min.  A l l but 2-3 ml o f medium was then  removed and the N i t r o b a c t e r resuspended by shaking. r e s u l t s were o b t a i n e d i f the sample was allowed 30 min before use.  Best  t o stand f o r  At the end of t h i s time 0.2-0.5 ml of the  -32concentrated  suspension  were p l a c e d i n the s y r i n g e and a  drop i n s e r t e d between the e l e c t r o d e s .  A mixture o f C0:02 i n  the r a t i o 4:1 s a t u r a t e d with water vapour was sucked i n t o the chamber by a 50 ml s y r i n g e .  Some samples d i d not r e c o v e r  c e n t r i f u g a t i o n and had t o be d i s c a r d e d .  from  Those t h a t showed  a c t i v i t y remained a l i v e i n the apparatus a maximum o f 90 min. In the a i r , the c o n c e n t r a t e d  suspensions l a s t e d up t o 3 hours  with the r e s u l t t h a t s e v e r a l samples c o u l d be obtained a single centrifugation.  from  - 3 3 -  CHAPTER IV CALIBRATION AND  • /  :. • ..=.  PERFORMANCE  IV-1 R e l a t i v e I n t e n s i t y a t t h e Top o f t h e Cone S i n c e t h e i n t e n s i t y o f l i g h t i s measured a t t h e bottom o f t h e cone a c a l i b r a t i o n i s r e q u i r e d t o d e t e r m i n e t h e a c t u a l i n t e n s i t y o f l i g h t f a l l i n g on t h e sample. i m p o r t a n t f o r t h e monochromatic  T h i s i s most  beam as t h e t r a n s m i s s i o n o f  the cone w i t h w a v e l e n g t h i s v a r i a b l e .  The c o m p o s i t i o n o f t h e  r e f e r e n c e beam i s always c o n s t a n t , o n l y t h e i n t e n s i t y Thus i n d e t e r m i n i n g t h e a c t i o n spectrum t h e f a c t o r  varies.  relating  the i n t e n s i t y o f l i g h t a t t h e t o p o f t h e cone t o t h a t a t t h e bottom f o r t h e r e f e r e n c e l i g h t i s not r e q u i r e d as i t c a n c e l s from t h e e q u a t i o n d e t e r m i n i n g t h e r e l a t i v e e x t i n c t i o n  coeffi-  cent. The method o f c a r r y i n g o u t t h i s c a l i b r a t i o n i s a s f o l l o w s . An RCA 5819, S-9 r e s p o n s e , p h o t o m u l t i p l i e r tube was p l a c e d a t t h e t o p o f t h e cone t o g a t h e r a l l o f t h e l i g h t . diagram f o r t h e arrangement  i s shown i n F i g . 5.  The c i r c u i t  A Keithley  240 A H i g h V o l t a g e Power S u p p l y was used t o b i a s t h e tube a t 600 v o l t s .  The o u t p u t c u r r e n t was r e a d from an Avometer.  At  no time was i t p o s s i b l e t o d i s t i n g u i s h a d a r k c u r r e n t on t h e 50 jiamp s c a l e o f t h e Avometer i n d i c a t i n g a d a r k c u r r e n t much l e s s than 1 (jiamp. At each wavelength t h e anode c u r r e n t from t h e p h o t o m u l t i p l i e r and t h e v o l t a g e from t h e phase s e n s i t i v e d e t e c t o r  C1,C2,C3, 16jif RH,  100K  R1-R10, 47K  Figure 5  C i r c u i t Diagram f o r P h o t o m u l t i p l i e r  Tube  -34were r e c o r d e d .  The l a t t e r was c o r r e c t e d f o r temperature  d e v i a t i o n from 24°C and to 10 times g a i n as d e s c r i b e d by Brooks.  Temperature  measurements were taken from a c a l i b r a t e d  thermocouple a t t a c h e d to the PbS d e t e c t o r .  The r e s u l t s of  t h i s c a l i b r a t i o n are t a b u l a t e d i n T a b l e I. It for  was  then n e c e s s a r y to c a l i b r a t e the p h o t o m u l t i p l i e r  i t s wavelength s e n s i t i v i t y .  For t h i s a c a l i b r a t e d  Eppley Thermopile s e r i a l no. 5094 was used. for  The  calibration  t h i s i s 0.054 m i c r o v o l t s / m i c r o w a t t / square cm.  At each  wavelength the t h e r m o p i l e and photomultiplier were p l a c e d in  the monochromatic  cone removed.  l i g h t emerging from the s l i t  w i t h the  T h e i r s e n s i t i v e elements were p l a c e d a t the  same d i s t a n c e from the s l i t  and i n the same mean p o s i t i o n .  The exposed area of the p h o t o m u l t i p l i e r was 0.0576 cm , the  same o r d e r of magnitude  the  cone  (0.0256 cm  ).  as the output area a t the top of  Repeated  (7-10) measurements of the  change i n v o l t a g e from the t h e r m o p i l e were taken and the average of these i s r e p o r t e d i n T a b l e I I . the  The c u r r e n t  from  p h o t o m u l t i p l i e r i s a l s o an average over s e v e r a l r e a d i n g s .  I suspect that most o f the 1% v a r i a t i o n i n p h o t o m u l t i p l i e r r e a d i n g s i s due to f l u c t u a t i o n s i n the output of the lamp. A s i m i l a r c a l i b r a t i o n t o determine the wavelength s e n s i t i v i t y of the PbS d e t e c t o r was made. for  Since r e a d i n g s  t h i s c a l i b r a t i o n were not taken a t the same time i t i s  p o s s i b l e o n l y to compare r a t i o s of i n t e n s i t i e s . d i f f e r e n t days, the output of the lamp may  Thus on  v a r y as much as  20%, yet the r a t i o of i n t e n s i t y a t a wavelength, X, to t h a t  TABLE I Calibration Values at the Top of the Cone Wavelength  Photomultiplier Current  PSD Voltage 24°C, lOx gain  nm  liA  mv  400 410 420 430 440  60 86 113 144 178  6.70 8.07 9.38 7.82 12.1  450 460 470 480 490  215 261 305 334 358  13.6 15.7 17.5 18.5 19.6  500 510 520 530 540  371 380 405 400 373  20.6 22.5 24.9 25.7 27.6  550 560 570 580 590  339 304 260 215 162  27.7 28.7 31.2 31.5 33.6  600 610 620 630 640  108 72 48 32.8 24.5  33.7 34.5 34.6 40.5 42.8  TABLE I I C a l i b r a t i o n o f Wavelength S e n s i t i v i t y o f Photomultiplier Wavelength nm  Photomultiplier Current |iA  Spectra Sensitiv |iw/|j.A  400 410 420 430 440  60 78 99 122 143  1.21*.04 1.44±.02 1.65±.02 1.85±.02 2.09±.09  .0215 .0197 .0168 .0162 .0156  450 460 '470 480 490  165 194 212 222 227  2.30±.04 2.69±.04 2.91±.05 3.05±.10 3.16±.04  • .0149 .0148 .;0146 .0147 .0149  500 510 520 530 540  22 226 235 218 195  3.37±.15 3.49±.13 4.01±.13 4.09±.09 4.22±.18  .0158 .0165 .0182 .0200 .0231  550 560 570 580 590  175 149 125 100 74  4.30±.08 4.38±.20 4.54±.08 4.66±.20 4.81±.ll  .0268 .0313 .0387 .0497 . 0694  4.84±.17 4.87±.17 4.96±.17 4.95±.13 5.16±.20  .102 .1565 .238 .350 .491  600 610 620 630 640 *  Thermopile Voltage liV  See Appendix  50.5 33.2 22.2 15.1 11.2  -35a t 550  nm  v a r i e s l e s s than 10% and  A b s o l u t e and  u s u a l l y not more than  r e l a t i v e c o r r e c t i o n f a c t o r s f o r the PbS  are t a b u l a t e d  i n Table I I I .  a c c u r a t e t o ± 20%.  The  absolute  4%.  readings  c a l i b r a t i o n i s only  the r e l a t i v e c a l i b r a t i o n s t o ±  5%.  From the c a l i b r a t i o n s performed i t i s p o s s i b l e t o a r r i v e a t a c u r v e f o r the r e l a t i v e i n t e n s i t y a t the top of the cone t o t h a t a t the bottom,  (Fig. 6).  IT/IB  shown i n the same diagram was  A second c u r v e  c a l c u l a t e d from the r e l a t i v e  i n t e n s i t y a t the top of the cone d i v i d e d by the r e l a t i v e i n t e n s i t y as measured by the PSD,  IT/*PSD»  Since^the  only  t h e o r e t i c a l d i f f e r e n c e s i n the numbers used i n these c a l c u l a t i o n s s h o u l d be c o n s t a n t s r e l a t i n g a r e a s , the  curves  s h o u l d by i d e n t i c a l , such c o n s t a n t s c a n c e l l i n g i n the calculation. identical.  W i t h i n e x p e r i m e n t a l e r r o r these c u r v e s are (See Appendix f o r the c a l c u l a t i o n s l e a d i n g  to  these curves) IV-2  I n t e n s i t y Response of The  response o f the PSD  the r e f e r e n c e was  PSD to increasing i n t e n s i t y of.  beam i s shown i n F i g . 7.  This c a l i b r a t i o n  performed by p l a c i n g an i n t e r f e r e n c e f i l t e r i n the  reference  beam and  adding c a l i b r a t e d n e u t r a l d e n s i t y  The  r e s p o n s e of the PSD  PbS  detector  was  found t o m i r r o r t h a t of  as measured by an O s c i l l o s c o p e .  filters. the  Thus.the  l i n e a r i t y of r e s p o n s e w i t h i n t e n s i t y i s produced by the detector,  not  the e l e c t r o n i c s c o n n e c t e d w i t h the  PSD.  nonPbS  TABLE I I I  Phase  Wavelength, nm 400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600 610 620 630 640  * See  Appendix  S e n s i t i v e D e t e c t o r Wavelength Spectral  Sensitivity  S e n s i t i v i t y , Q*  jaw/mv 0.134 0.132 0.131 0.175 0.128 0.126 0.127 0.123 0.122 0.119 0.121 0.115 0.119 0.118 0.113 0.115 0.113 0.108 0.110 0.106 0.107 0.108 0.106 0.092 0.089  12,  33  m  |.||  > H  I OF  m  H  m H -<  0-8  0-7  0  05  6  _|  400  Figure 6  I  '  *  •  I  1  500 WAVELENGTH Relative  I n t e n s i t y at t h a t at the  t h e Top Bottom  1  I  I  600  of  t h e Cone  to  Hi  Figure 7  I n t e n s i t y Response of Lead S u l p h i d e Phase S e n s i t i v e  Relative  Intensity  Detectors  and  -36IV-3 Actinometer  Calibrations  An attempt was  made to perform a more a c c u r a t e  c a l i b r a t i o n of the i n t e n s i t y a t the top of the cone u s i n g a potassium  f e r r i o x a l a t e actinometer.  by Hatchard and Parker 450 nm  i f a 0.15  The  technique as d e s c r i b e d  (1956) i s good f o r wavelengths  M s o l u t i o n i s used.  In these  experiments  a s o l u t i o n t h i c k enough to absorb a l l of the l i g h t from the cone was  used.  emerging  I r r a d i a t i o n of the s o l u t i o n  c a r r i e d out f o r p e r i o d s up to t h r e e hours.  up to  was  Following this  p h e n a n t h r o l i n e monohydrate and b u f f e r were added and  optical  d e n s i t y d i f f e r e n c e r e a d i n g s taken between the sample and a blank. seldom  The o p t i c a l d e n s i t y of the sample t r e a t e d i n t h i s manner reached 0.1  and the d i f f e r e n c e between the two  u s u a l l y l e s s than 0.05.  The  was  low v a l u e s r e s u l t e d because  of  the low output of the tungsten lamp i n that s p e c t r a l r e g i o n . With such low v a l u e s i t was r e s u l t s , i n part because l i k e l y due  i m p o s s i b l e to get r e p r o d u c i b l e  of the s c a t t e r e d l i g h t and  also  to f l u c t u a t i o n s i n the lamp output over the l o n g  i n t e r v a l s of time r e q u i r e d f o r the exposure.  Thus i t  was  found t h a t i n t h i s s i t u a t i o n t h i s c h e m i c a l actinometer c o u l d not be  used.  -37CHAPTER V RESULTS AND ERRORS V - l Oxygen E l e c t r o d e R e s u l t s Samples f o r these e x p e r i m e n t s were p r e p a r e d Sec.  III-1.3.  as i n  A r e c o r d i n g o f oxygen c o n c e n t r a t i o n  versus  time was made over t h e p e r i o d r e q u i r e d f o r t h e N i t r o b a c t e r t o consume a l l o f t h e oxygen i n s o l u t i o n . and t h e r e c o r d e d  From t h e c a l i b r a t i o n s  c u r v e s o f oxygen c o n c e n t r a t i o n v e r s u s  time  the r a t e o f oxygen uptake v e r s u s oxygen c o n c e n t r a t i o n was calculated. ["^2] »  a n t  *  P l o t s o f v e l o c i t y , v, v e r s u s s u b s t r a t e c o n c e n t r a t i o n , l/  0 1  respectively.  v v  s  l/p2]  a  r  e  shown i n f i g u r e s 8 and 9  From these c u r v e s t h e M i c h a e l i s c o n s t a n t , K , ffl  f o r oxygen as s u b s t r a t e ranges from 0.021 t o 0.055 mM depending on t h e c o n c e n t r a t i o n o f n i t r i t e . recorded T a b l e IV  0  2  The r e s u l t s a r e  i n T a b l e IV. K  V a l u e s f o r N i t r o b a c t e r w i t h Oxygen as S u b s t r a t e  Preparation Procedure  K V a l u e s , mM O2 1/v v s 1/s v vs s m  F i g . ? o.r\d ^ Cur ve  2x c o n c e n t r a t i o n  0.032  0.032  5x c o n c e n t r a t i o n  0.040  0.043  1  5x c o n c e n t r a t i o n  0.021  0.022  2  5x c o n c e n t r a t i o n  0.055  0.055  3  Figure  8  E f f e c t o f Oxygen C o n c e n t r a t i o n Nitrite  Oxidation  on R a t e  of  "»  Figure 9  1  i  i  9  1  {  e-  Michaelis-Menten P l o t s f o r Data of F i g . 8  -38V-2  R e s u l t s from the A c t i o n Spectrum  Apparatus  2.1 Procedure f o r O b t a i n i n g R e s u l t s The prepared sample was d e s c r i b e d i n Sec. I I I - 2 . 4 .  p l a c e d i n the chamber as  The e l e c t r o d e s were p o l a r i z e d  and a f t e r a p e r i o d of about 15 min the c u r r e n t , r e c o r d e d on an E s t e r l i n e Angus r e c o r d i n g milliammeter, reached i t s e q u i l i b r i u m value. the  At t h i s time when l i g h t was  switched onto the  c u r r e n t throught the e l e c t r o d e s decreased to a  equilibrium value.  new  The reason f o r t h i s change i s t h a t the l i g h t  breaks up the carbon monoxide-oxidase the  sample,  oxidase to r e a c t w i t h oxygen.  complex, thus a l l o w i n g  T h i s r e s u l t s i n a lower  oxygen c o n c e n t r a t i o n i n the drop and a lower e l e c t r o d e c u r r e n t . The dark c u r r e n t v a l u e s ranged from 20-40 nA.  A change from  dark t o the r e f e r e n c e beam (with a heat f i l t e r , C o r n i n g a k l o no. 97, and a green band pass f i l t e r range) decreased the c u r r e n t 3-5 in  to l i m i t  nA.  the s p e c t r a l  A greater  sensitivity  the form of l a r g e r a b s o l u t e c u r r e n t changes was o b t a i n e d  w i t h lower v a l u e s of the dark c u r r e n t . were 1 mm  A l l s l i t widths  and the InA s c a l e was used f o r the c u r r e n t r e a d i n g s  with the remainder of the c u r r e n t bucked out by and e x t e r n a l circuit.  A t y p i c a l change from dark to l i g h t and v i c e v e r s a  i s shown i n f i g u r e  10.  The CO a c t i o n spectrum i s a p l o t of the r e l a t i v e extinction coefficient  %-\\/ £\2  °^  t  n  e  enzyme-CO compound  w i t h wavelength.  The r e f e r e n c e wavelength i s u s u a l l y  taken as 550 nm.  As shown by Brooks, the r a t i o i s g i v e n by  F i g u r e 10  The E f f e c t o f L i g h t on t h e E l e c t r o d e Carbon Monoxide I n h i b i t e d Sample o f  Current of a Nitrobacter  -39-  the  formula:  *X  °X  ^550  "  W  W  55Q  C  x  5  5  5  0  5  0  X  i s the i n t e n s i t y o f the comparison l i g h t which g i v e s the same photochemical e f f e c t s as the monochromatic l i g h t o f wavelength, X . C"550 i s the i n t e n s i t y o f comparison l i g h t which g i v e s the same photochemical e f f e c t as 550  nm.  i s the i n t e n s i t y o f X WgejQ i s the i n t e n s i t y o f 550 nm l i g h t . In p r a c t i c a l terms t h i s means t h a t s w i t c h i n g from the r e f e r e n c e beam with  i n t e n s i t y C ^ ^ Q t o the 550 nm monochromatic  beam produces no change i n the e l e c t r o d e c u r r e n t . The by f i r s t and  i n t e n s i t i e s which cause equal e f f e c t s were determined s h i n i n g the l i g h t o f wavelength, X , on the sample  a f t e r e q u i l i b r a t i o n s w i t c h i n g t o the r e f e r e n c e beam.  If  the c u r r e n t decreased, the r e f e r e n c e l i g h t was too i n t e n s e and  the l o u v e r s were a d j u s t e d .  T h i s o p e r a t i o n was  u n t i l no change was observed on s w i t c h i n g . usually a d r i f t  repeated  Since there was  i n the c u r r e n t , a decrease o r i n c r e a s e i n  c u r r e n t was i n d i c a t e d  by a change i n the s l o p e o f the d r i f t ;  The  r e s u l t i n g t r a c e o f c u r r e n t with time i s shown i n F i g .  The  e s t i m a t i o n of the balance  11.  p o i n t c o u l d o n l y be made w i t h i n  1 m i l l i v o l t o f the i n t e n s i t y o f the r e f e r e n c e l i g h t s i n c e the smalfest t u r n o f the l o u v e r s d u r i n g the o p e r a t i o n corresponded  Figure  11  A T y p i c a l Determination of  Balance Point The  for Light  Reference  the  o f W a v e l e n g t h , X,  Light,  u.  and  -40to a 1 mv i n t e n s i t y change. reference  The average i n t e n s i t y of the  beam i n these experiments was 67 mv.  uncertainty  Thus the  i n the balance p o i n t i s 1 i %. 2  Once the balance p o i n t was determined the i n t e n s i t i e s of the r e f e r e n c e  and monochromatic beams were measured and  the temperature o f the PbS d e t e c t o r the  taken.  The balance f o r  i n t e n s i t y measurements was made on the 10 mv s c a l e o f  the K e i t h l e y microvoltraeter-ammeter with the bucking s u p p l i e d by an e x t e r n a l c i r c u i t readings  were c o r r e c t e d  ( c f . F i g . 2 ) . The PbS  t o 24°C and 10 x gain on the PSD.  g a i n f a c t o r s were 2.57 f o r the three f o r the 30 times s c a l e . beam was c o r r e c t e d  voltage  The  times s c a l e and 0.324  The i n t e n s i t y o f the monochromatic  t o give the i n t e n s i t y at the top o f the  cone, i . e . c o r r e c t e d f o r PbS wavelength s e n s i t i v i t y and cone transmission. not  Since  the response of the PbS d e t e c t o r i s  proportional to the i n t e n s i t y f a l l i n g on i t a c o r r e c t i o n  to the i n t e n s i t y o f the comparison beam i s a l s o r e q u i r e d . A sample c a l c u l a t i o n of S  / £ . , . i s o u t l i n e d below f o r X 550  A=600 nm. DATA  w  PSD Readings (mv)  31.7  84.4  36.2  85.75  Temp, (mv)  0.96  0.96  1.00  1.00  Temp, c o r r e c t e d PSD r e a d i n g s *  30.9  82.3  37.2  88.1  550  C  550  W  CALCULATION ^ 600  S  53  88.1 x 30.9 x 550 37.2 x 82.3 x 600  600  C  600  -41=0.815 From F i g . 7, the c o r r e c t i o n f o r the l i n e a r i t y o f the PbS d e t e c t o r i s 1.02 i s 1.067.  and from F i g . 6, I / I T  p  s  D  f o r X = 600 nm  Therefore* 6 0  2  0.815 x 1.02 x 1.067  550  S  = 0.890  * See Appendix. 2.2 R e s u l t s The  r e s u l t i n g values of £ ^ / £  i n f i g u r e s 12 and 13. o b t a i n e d with  are d i s p l a y e d  The f i r s t , F i g . 12, shows the r e s u l t s  two p r e p a r a t i o n s o f N i t r o b a c t e r .  These r e s u l t s  were p i c k e d t o be shown i n t h i s manner because of t h e i r consistency.  R e s u l t s obtained  within preparations  were u s u a l l y more c o n s i s t e n t  than between p r e p a r a t i o n s .  graph, F i g . 13, i s a graph o f a l l those were c r e d i b l e .  self-  The second  p o i n t s which I f e l t  The reasons f o r i n c l u d i n g these p o i n t s and  not o t h e r s that I took are the f o l l o w i n g : i ) Once the N i t r o b a c t e r i n the chamber s t a r t e d t o die  the c u r r e n t i n c r e a s e d a t an a c c e l e r a t e d r a t e .  As t h i s happened, the d e t e r m i n a t i o n  o f the balance  p o i n t became more and more u n c e r t a i n . o b t a i n e d with a f a s t d r i f t  Points  were thus u s u a l l y  discarded. i i ) E r r a t i c response of some p r e p a r a t i o n s meant that balance  c o u l d only be o b t a i n e d  on the 3 nA  -42scale.  .  T h i s was l a t e r d i s c o v e r e d not t o be a  s e n s i t i v e enough d e t e r m i n a t i o n  and the p o i n t s  were  disregarded.  iii)  Some p r e p a r a t i o n s gave r i s e t o o n l y one  p o i n t on the graph o f £ c o i n c i d e n t with  /€  V S X. I f ,  t h i s the t o t a l change from dark  to l i g h t was l e s s than 2 nA, these also  p o i n t s were  discarded.  2.3 E r r o r s i n the R e l a t i v e E x t i n c t i o n C o e f f i c i e n t The  error i n £ , X  / £  i s the sum o f the 550  i n h e r e n t e r r o r s and u n c e r t a i n t i e s i n each o f the terms i n the equation. i ) The d e t e r m i n a t i o n accurate  to 1 ^  o f the balance  point i s  ( c f . Sec. V-2.1).  there are two such balances  Since  r e q u i r e d f o r determina-  t i o n o f a p o i n t the e r r o r i n t r o d u c e d i s 3%. i i ) Brooks shows that the c a l c u l a t i o n o f the tempe r a t u r e c o e f f i c i e n t and i t s a p p l i c a t i o n i n t r o d u c e s a 1% u n c e r t a i n t y i n the monochromatic  intensities  with much l e s s than 1% e r r o r i n the r e f e r e n c e beam. i i i ) V a r i a t i o n i n output  o f the lamp and i n the  g a i n l e v e l s of the PSD i n t r o d u c e a 5% u n c e r t a i n t y i n the v a l u e s o f the r e l a t i v e i n t e n s i t y a t the t o p of the cone d i v i d e d by those measured by the PbS and  phase s e n s i t i v e d e t e c t o r s .  -43The  accuracy of  €  / £ 550  is  thus  W 550 S 550  W ± x  6%  C  making the l i m i t of accuracy  ± 6%  550  + 3%  X  550 + 15%.  On the graph, F i g . 13, the e r r o r bars are the 80% c o n f i d e n c e l i m i t s c a l c u l a t e d a c c o r d i n g t o Wilson (1952, pg.239). They a r e l a r g e r than the 15% c a l c u l a t e d above, i n some The  reason f o r t h i s d i s c r e p a n c y l i e s i n the n e c e s s i t y  d i f f e r e n t samples o f N i t r o b a c t e r . i s a c c u r a t e to w i t h i n  Thus w h i l e each  15%, the p o i n t s  instances. of u s i n g  point  at the same wavelength  i n d i f f e r e n t samples o f b a c t e r i a may not have r e l a t i v e extinction c o e f f i c i e n t s within s i o n i n Sec. VI-2 i s r e l e v a n t  15% of one another.  The discus^-  t o t h i s apparent d i s c r e p a n c y .  2.4 E r r o r s i n the P o s i t i o n s o f the Peaks The  peaks a t 430 and 591 nm can be more  accurately  determined because o f t h e i r steepness. (See C a s t o r and Chance, 1955)  Thus the e r r o r i n these peaks i s - 2 nm o f which 1 nm  error i s introduced  i n the d e t e r m i n a t i o n o f the wavelength  d e l i v e r e d a t the e x i t s l i t  by the monochromator.  at 540-550 nm i s much broader and t h e r e f o r e Added t o t h i s the l i n e o f average p o i n t s introducing on  further estimation  errors.  The peak  harder t o o u t l i n e .  i s not continuous Thus the e r r o r  the two apparent peaks a t 541 and 550 nm i s - 5 nm.  limit  -44CHAPTER VI DISCUSSION VI-1  Oxygen E l e c t r o d e R e s u l t s The  v a r i a t i o n of  ^ ( O g ) has been r e p o r t e d and  V  mSLX  w e l l documented by Butt and Lees (1964). r e p o r t e d by these authors were found  The maximal r a t e s  to i n c r e a s e and then  decrease w i t h i n c r e a s i n g n i t r i t e c o n c e n t r a t i o n . was t r u e of the K ( 0 2 ) . m  2 and 7.5 %  They r e p o r t e d K  Og i n the gas phase.  (1.75 and 4.6 % 0  v a l u e s between  Comparable r e s u l t s were  o b t a i n e d i n these experiments with K and 0.055 mM  m  The same  m  values between 0.021  i n the gas phase).  Samples f o r which r a t e s were o b t a i n e d above 20% showed a decrease  i n r a t e with i n c r e a s i n g Og.  to the e s t a b l i s h m e n t  T h i s i s not due  o f the p o l a r i z a t i o n v o l t a g e which  s e v e r a l minutes t o e s t a b l i s h i n the sample chamber. p r o c e s s r e s u l t s i n an apparent  takes  This  i n c r e a s e i n the r a t e .  Thus the  e f f e c t s a r e p o s s i b l y one o f two: 1) a s u b s t r a t e i n h i b i t i o n , or 2) t h a t the c e l l s had not r e c o v e r e d from c e n t r i f u g a t i o n and e x h i b i t e d a lower r a t e u n t i l complete r e c o v e r y was  reached.  (Note the 30 min wait r e q u i r e d i n the a c t i o n spectrum experiments, difficult  Sec. III-2.4  )  T h i s l a t t e r e f f e c t i s very  t o d e a l w i t h s i n c e the e f f e c t s o f c e n t r i f u g a t i o n  vary from sample t o sample even though the p r e v i o u s  history  appears to have been i d e n t i c a l . Maximal r a t e s r e p o r t e d by Butt and Lees ranged from  -451.9  t o 2.7 micromoles  oxygen/vessel/hr where each  c o n t a i n e d 3 ml of suspension.  Values o b t a i n e d f o r 7 ml  of suspension i n these experiments jxmoles/vessel/hr.  vessel  ranged from 0.7  to 3  These r a t e s are lower but of the same order  of magnitude as those of Butt and Lees, the d i s c r e p a n c y due probably to a d i f f e r e n c e i n the c o n c e n t r a t i o n of c e l l s i n the suspension but perhaps  a l s o due  to the method of  measurement. VI-2 A c t i o n Spectrum R e s u l t s Comparison of the peak p o s i t i o n s o b t a i n e d here at  430,  540-50, and 591 nm w i t h those o b t a i n e d by the same methods on other organisms  (Castor and Chance, 1959)  show that  these peaks i n d i c a t e the presence of e i t h e r cytochrome o  i*3> peaks b e i n g r e p o r t e d at 427-8, 548 and 591-2  r  nm f o r  cytochrome a_^ and a t 430-2, 547-50 and 585-91 f o r cytochrome a . Q  A b s o r p t i o n s p e c t r a of N i t r o b a c t e r r e p o r t peaks at  586-94 and 438 showing an a_^ r a t h e r than an a^ ( c f . Sec 1-5.1  and  1-5.2)  reduced d i f f e r e n c e s p e c t r a  cytochrome,  Carbon monoxide reduced minus (Sec. 1-5.3.1) i m p l i c a t e d cytochrome  a i i n the b i n d i n g of CO w i t h peaks a t 450 and 426 and at 439 and 594 nm. no peak around at 590 nm A.  troughs  Although Van Gool and Laudelout r e p o r t  590, C a s t o r and Chance (1953) r e p o r t e d a peak  f o r cytochrome a j i n a CO d i f f e r e n c e spectrum  of  pasteurianum. None of the peaks shown i n t h i s a c t i o n spectrum  i s as  w e l l d e f i n e d as those shown by C a s t o r and Chance (1959)  -46They were a b l e to determine the p o i n t s on t h e i r s p e c t r a cons e c u t i v e l y and " e f f o r t s to r e t u r n l a t e r to a g i v e n wavelength showed a r e p r o d u c i b i l i t y of about ± 5%". r e s p o n s i v e organisms such as E_. c o l i .  T h i s was  with  N i t r o b a c t e r i s however  a much more s e n s i t i v e organism with a s h o r t l i f e span i n the apparatus.  Both of these e f f e c t s r e s u l t i n poorer r e p r o d u c i -  b i l i t y and n e c e s s i t a t e t r y i n g to o b t a i n an o u t l i n e of a peak w i t h one  s e t of N i t r o b a c t e r r a t h e r than t r y i n g to d e f i n e  the peak i n d e t a i l u s i n g many s e t s of N i t r o b a c t e r .  This  latter  approach u s u a l l y r e s u l t e d i n a p o o r l y d e f i n e d peak. Comparison of the r e l a t i v e e x t i n c t i o n  coefficients  of the peaks f o r cytochrome a^ bands of the a c t i o n spectrum of A. pasteurianum 2.35  (Castor and Chance, 1953)  and 21 f o r the 592 and 428  peaks.  Values of € ^/ £ 5 5 0  f o r the same peaks i n t h i s work were 1.68 r a t i o of these v a l u e s i s 0.112 f o r t h i s work.  The  the order of 10%,  g i v e s v a l u e s of  and 9.8.  The  f o r C a s t o r and Chance and  e r r o r i n the 0.112  t h a t i n the 0.168  d e t e r m i n a t i o n i s of  i s 30%.  v a l u e s are the same w i t h i n e x p e r i m e n t a l  0.168  Thus the  error.  two  The cause of  the d i f f e r e n c e i f i t i s s i g n i f i c a n t most l i k e l y l i e s i n the r o l e of cytochrome <>. A. pasteurianum  was  No diagram of the ft, 548 band f o r  g i v e n nor are there any o t h e r v a l u e s of  the r e l a t i v e e x t i n c t i o n c o e f f i c i e n t s f o r cytochrome a^ i n other organisms.  Thus i t i s not p o s s i b l e to make an e x t e n s i v e  comparison of these v a l u e s f o r s i g n i f i c a n t d i f f e r e n c e s . C a s t o r and Chance (1959) showed t h a t when two are present i n a c e l l ,  oxidases  e i t h e r pigment appears capable  of  -47c a t a l y z i n g most of the r e s p i r a t i o n .  T h i s experiment was  performed by i l l u m i n a t i n g the c e l l s with l i g h t which would only be absorbed by one pigment.  With t h i s l i g h t most of the  CO i n h i b i t i o n was r e l i e v e d .  (Horio and T a y l o r , 1965J  Thus  when two or more oxidases operate i n p a r a l l e l the a c t i o n spectrum may  or may  not r e f l e c t  t h i s f a c t , depending on  both the r e l a t i v e a c t i v i t i e s and the l i g h t s e n s i t i v i t i e s of the CO complexes.  Thus the p o s s i b l e presence of other cytochromes  can not be r u l e d out. at 540-550 nm.  T h i s i s e s p e c i a l l y e v i d e n t i n the peak  The displacement of t h i s peak to lower  wavelengths c o u l d be due to the presence of a second cytochrome with a peak a t or below 540 nm. cytochrome o_ peak at 535-7 nm.  The major candidate i s a The presence of cytochrome o_  would a l s o r e q u i r e an i n d i c a t i o n of peaks a t 416-7 and These are not e v i d e n t i n the spectrum d i s p l a y e d .  566-7.  I f c y t . <>  i s p r e s e n t , the reason f o r the absence of these two peaks c o u l d be one of two e f f e c t s  s  The f i r s t  are masked by the cytochrome a^.  i s that the peaks  The e f f e c t on the |J peak  would seem to i n d i c a t e the presence of cytochrome o_ i n l a r g e enough q u a n t i t i e s to a f f e c t i n the r e g i o n of 415-430 nm. be the answer.  the spectrum more d r a s t i c a l l y Thus masking does not seem to  The second p o s s i b l e e x p l a n a t i o n l i e s i n a  d i f f e r e n c e i n the c e l l s used to determine the p o i n t s . and Chance  (1959) showed d i f f e r e n t cytochrome oxidase  i n s t a t i o n a r y and l o g phase c e l l s .  Castor activities  Thus i t i s p o s s i b l e  that  c e l l s c o n t a i n i n g no cytochrome o_ or c o n t a i n i n g a cytochrome <3 with a very low a c t i v i t y were used to determine the a and y  -48peaks but that the S peak was p a r t i a l l y determined with c o n t a i n i n g and a c t i v e cytochrome o_.  Although  s p e c t r a give no h i n t s of cytochrome £, there r u l e i t out because the present  absorption i s no reason to  method i s much more s e n s i t i v e .  Thus i t can be s t a t e d that cytochrome a t e r m i n a l oxidase i n N i t r o b a c t e r a possibility  that other  in certain c e l l s . oxidase,  i s a c t i n g as  a g i l i s and that there i s  cytochromes may  a l s o a c t as o x i d a s e s  I f cytochrome o_ does a c t as a  a study of l o g phase c e l l s i s very  terminal  l i k e l y to show  t h i s as a l l of the types of organisms s t u d i e d by C a s t o r Chance  cells  and  (1955 and 1959) which showed some cytochrome jo a c t i v i t y  i n the s t a t i o n a r y phase showed only cytochrome o_ a c t i v i t y i n the l o g phase of growth. A c t i o n s p e c t r a r e s u l t s t a b l e d by C a s t o r  and Chance  (1959)  i m p l i c a t e cytochrome a^ as an oxidase i n f o u r b a c t e r i a , a l l heterotrophs.  They are A c e t o b a c t e r pasteurianum, A c e t o b a c t e r  peroxydans, Azotobacter v i n e l a n d i i , and s t a t i o n a r y phase Proteus v u l g a r i s .  A. v i n e l a n d i i a l s o showed cytochrome o  a c t i v i t y , P. v u l g a r i s a l s o showed cytochrome <> and  activity  i n the s t a t i o n a r y phase and showed o n l y cytochrome o_ a c t i v i t y i n the l o g phase.  To date, o n l y cytochrome a^ has not been  shown to be a c t i v e i n c e l l s with a c t i v e cytochrome a^. generalized  statement about the a s s o c i a t i o n s of o x i d a s e s  can not be made, however, u n t i l more organisms have been i n t h e i r d i f f e r e n t phases of growth. here i s the f i r s t bacterium.  A  recorded  studied  The spectrum r e p o r t e d  a c t i o n spectrum of a  chemolithotropic  -49VI-3  Summary  ( i ) A survey of the l i t e r a t u r e on N i t r o b a c t e r was performed and (iij  the unsolved  problems p o i n t e d o u t .  M o d i f i c a t i o n s to the a c t i o n spectrum apparatus a r e d e s c r i b e d and  the performance o f the apparatus  assessed.  ( i i i ) The K ( 0 ) Of N i t r o b a c t e r a g i l i s v a r i e s from 0.021 m  2  to 0.055 mM 0  2  depending on the n i t r i t e  concentration  i n agreement with the data o f Butt and Lees (1964). ( i v ) Cytochrome a^ a c t s as a t e r m i n a l oxidase  i n Nitrobacter  agilis. (v) P a r a l l e l p a r t i c i p a t i o n o f other cytochromes as oxidases i s not r u l e d out.  A d i s t o r t e d B band suggests the presence  of cytochrome o i n some c u l t u r e s .  -50BIBLI0GRAPHY 1.  Aleem, M. I. H., 1965, B i o c h i m i c a et B i o p h y s i c a A c t a 107, 14 Path of Carbon and A s s i m i l a t o r y Power i n Chemosynthetic B a c t e r i a 1. N i t r o b a c t e r a g i l i s  2.  Aleem, M. I. H. and M. Alexander, 1958, J . B a c t e r i o l o g y 76, 510 C e l l - f r e e N i t r i f i c a t i o n by N i t r o b a c t e r  3.  Aleem, M. I. H., E. Hoch and J . E. Varner, 1965, Nat. Acad. S c i . Proc. 54_, 869 Water as the Source of Oxidant and Reductant i n B a c t e r i a l Chemosynthesis  4.  Aleem, M. I. H., H. Lees and D. J . D. N i c h o l a s , 1963 Nature 200, 759 ATP Dependent Reduction of NAD by Ferro-cytochrome c_ i n Chemoautotrophic B a c t e r i a  5.  Aleem, M. I. H. and A. Nason, 1959, B. and B. Res. Comm. JL, 323 N i t r i t e Oxidase, A P a r t i c u l a t e Cytochrome E l e c t r o n T r a n s p o r t System from N i t r o b a c t e r  6.  Aleem, M. I. H. and A. Nason, 1960, N a t ' l Acad. S c i . Proc. 46, 763 P h o s p h o r y l a t i o n Coupled to N i t r i t e O x i d a t i o n by P a r t i c l e s from the Chemoautotroph, N i t r o b a c t e r a g i l i s  7.  Alexander, M., 1961, I n t r o d u c t i o n to S o i l M i c r o b i o l o g y Wiley and Sons, N. Y.  8.  Bock, E. , 1965, Arch. Mikr. 5_1. 18 V e r g l e i c h e n d e Untersuchungen uber d i e Wirkung s i c h t b a r e n L i c h t e s auf Nitrosomonas europaea und N i t r o b a c t e r winogradskyi  9.  Bock, E. and H. E n g e l , 1966, Arch. Mikr. 54, 191 Untersuchungen uber p o s t o x y d a t l v e C 0 - F i x i e r u n g b e i N i t r o b a c t e r winogradskyi Buch 2  10.  Boon, B. and H. L a u d e l o u t , 1962, Biochem. J . J35, 440 K i n e t i c s of N i t r i t e O x i d a t i o n by N i t r o b a c t e r winogradskyi  11. > Brooks, D. E., 1967, MSc T h e s i s , U n i v e r s i t y of B r i t i s h Columbia An A c t i o n Spectrum Apparatus 12.  13.  B u t t , W. D. and H. Lees, 1960, Biochem. J . 76, 425 The B i o c h e m i s t r y of the N i t r i f y i n g Organisms, P a r t 6 The E f f e c t of Oxygen C o n c e n t r a t i o n on N i t r i t e O x i d a t i o n i n the Presence of D i f f e r e n t I n o r g a n i c Ions B u t t , W. D. and H. Lees, 1964, Can. J . Biochem., 4_2, 1217 The B i o c h e m i s t r y of the N i t r i f y i n g Organisms, P a r t 8 The E f f e c t s of Oxygen T e n s i o n , N i t r i t e C o n c e n t r a t i o n and Cyanate C o n c e n t r a t i o n on N i t r i t e O x i d a t i o n by N i t r o b a c t e r  -5114.  C a s t o r , L. N. and B. Chance, 1955, J . B i o l . Chem. 217, 453 Photochemical A c t i o n Spectrum Of Carbon. Monoxide I n h i b i t e d R e s p i r a t i o n  15.  C a s t o r , L. N. and B.-Chance, 1959, J . B i o l Chem. 234, 1587 Photochemical D e t e r m i n a t i o n s of the Oxidases o f B a c t e r i a  16.  Chance, B. 1953, J . B i o l . Chem. 202, 383 The Carbon Monoxide Compounds of the Cytochrome Oxidases I . Difference Spectra  17.  Gould, G. W. and H. Lees, 1960, Can. J . M i c r o b i o l o g y , (5, 299 The I s o l a t i o n and C u l t u r e o f the N i t r i f y i n g Organisms P a r t I N i t r o b a c t e r  18.  Hatchard, C. G. and C. A. P a r k e r , 1956, Royal S o c i e t y of London, P r o c . A, 235, 518 A New S e n s i t i v e Chemical Actinometer I I . Potassium F e r r i o x a l a t e as a Standard Chemical Actinometer  19.  H o r i o , T. and C. P. S. T a y l o r , 1965, J . B i o l . Chem. 240, 1772 The Photochemical D e t e r m i n a t i o n o f an Oxidase of the P h o t o h e t e r o t r o p h , R h o d o s p i r i l i u m rubrum,. and the A c t i o n Spectrum of the I n h i b i t i o n of R e s p i r a t i o n by L i g h t  20.  K r u l w i c h , T. A. and H. B. Funk, 1965, J . Bact. 90, 729 S t i m u l a t i o n of N i t r o b a c t e r a g i l i s by B i o t i n  21.  Lees, H. 1955 The B i o c h e m i s t r y o f A u t o t r o p h i c B a c t e r i a Butterworths  22.  Lees, H. 1960, Ann. Rev. M i c r o b i o l o g y , 14, 83, Metabolism i n C h e m o l i t h o t r o p i c B a c t e r i a  23.  Lees, H. 1962, Bact. Revs. 26, 165 Symposium on Autotrophy IV Some Thoughts on the E n e r g e t i c s o f Chemosynthesis  24.  Lees, H. and J . R. Simpson, 1957, Biochem. 6j3, 297 The B i o c h e m i s t r y o f the N i t r i f y i n g Organisms, P a r t 5, N i t r i t e O x i d a t i o n by N i t r o b a c t e r .  25.  Muller-Neuglucit, M. and H. E n g e l , 1961, A r c h . Mikr. 39, 130 P h o t o i n a k t i v i e r u n g von N i t r o b a c t e r winogradskyi Buch  26.  S e e l e r , G. and H. E n g e l , 1959, A r c h . Mikr. 3^3. 387 Die I n a k t i v i e r u n g des Oxydationsvermogens von N i t r o b a c t e r winogradskyi Buch  Energy  v  -5227.  S n e l l , F. D. and C. T. S n e l l , 1949, C o l o r i m e t r i c Methods of A n a l y s i s , Van Nostrand, N. Y.  28.  Van Gool A. and H. Laudelout, 1965, Biochim. e t Biophys, A c t a , 113, 41 The Mechanism of N i t r i t e O x i d a t i o n by N i t r o b a c t e r winogradskyi  29.  Wilson, E. B., 1952, An I n t r o d u c t i o n Research, McGraw-Hill, N. Y.  to S c i e n t i f i c  -53APPENDIX C a l c u l a t i o n s Performed Apparatus  i n C a l i b r a t i n g the Action  In t h i s s e c t i o n a sample  Spectrum  c a l c u l a t i o n of the r e l a t i v e  i n t e n s i t y a t t h e t o p o f t h e cone i s performed f o r t h e w a v e l e n g t h  600 nm. Abbreviations: Pm, P h o t o m u l t i p l i e r  r e a d i n g , jxA  T p , T h e r m o p i l e r e a d i n g , |iV PSD, P h a s e (Pm)  S e n s i t i v e D e t e c t o r R e a d i n g , mV Photomultiplier wavelength, X  Top,A.  reading  a t t h e t o p o f t h e cone a t a  (Pn^Bott ^ '  Photomultiplier reading a t a wavelength, X  (Tp)  Thermopile reading a wavelength, X  B o t ;  TABLE  t  V  ^ '  a t t h e bottom o f t h e cone  a t the bottom  o f t h e cone a t  Data Required f o r the C a l c u l a t i o n of the R e l a t i v e Extinction Coefficient  jiA  PSD rav  Temp rav  550  339  28.4  .962  600  108  32.8  .990  X  nm  Area o f measurement ( s q . cm.)  (Pro)Top  .0256  Thermopile C a l i b r a t i o n :  p s D  .04  0.054 \iV/ jxw/ cm  <  P n ,  )Bott  |iA 175  50.5 .0576  < P>Bott T  jiV  4.30 4.84  -54Temperature C o r r e c t i o n : l o g S = -2.4 x 1 0 "  T  2  At 24°C the thermocouple r e a d i n g i s 0.98  mv and the  s l o p e of the c a l i b r a t i o n graph i s 24.4 C° per mv. log S  - l o g S! = -2.4  2  x 10"  2  x 24.4  Thus,  (0.98 - X)  where, X = thermocouple r e a d i n g a t T j 51 = PSD s i g n a l a t T x  5  = PSD  2  s i g n a l a t 24°C  A p p l i c a t i o n of t h i s formula r e a d i n g s , from 28.4  leads to a change  to 27.7 and from 32.8  i n the PSD  to 33.7  P h o t o m u l t i p i i e r C o r r e c t i o n f o r Wavelength  mv.  Sensitivity  The c o r r e c t i o n f a c t o r f o r the p h o t o m u l t i p l i e r r e a d i n g s at a wavelength, X, are given by Y below: ( T  Y =  p)Bott,X  ^  .0576  V  .054 |iV/|iw/ cm*  x (x>  Cm  2  s ( ra)Bott,X p  = =  .0268 HW/JJLA a t 550 nm .102 |xw/|iA a t 600 nm  PSD C o r r e c t i o n f o r Wavelength  A  H  A  ( c f Table  I I and F i g . 14)  Sensitivity  The c o r r e c t i o n f a c t o r , Q, f o r the wavelength  sensitivity  of the phase s e n s i t i v e d e t e c t o r i s given by:-.. Q =  < P>Bott,X ^  '°  T  ,054 iiV/iiW/cnT  PSD  =  0.115  [iw/mv a t 550  =  0.107  |xw/mv at 600 nm  4  X  C n ) 2  mv  nm ( c f Table  Since the phase s e n s i t i v e d e t e c t o r and  I I I and F i g . 15) thermopile  Figure  14  Spectral  Sensitivity  of P h o t o m u l t i p l i e r  Tube  Figure  15  Spectral Sensitivity Detector  140  -I  1  1  of Phase  1  1  Sensitive  T"  T  T  CO "D  m o  H  130  > r~ to m  120  CO  H  <  H. -< 1 10 o 10  0  3  <  •  090  •080  ' ^  400  1  8  8  «  s  500  *_  1  L  WAVELENGTH  a  »  600  «  •  680  -55r e a d i n g s are not taken a t the same p o s i t i o n there a r e f a c t o r s r e l a t i n g the d i f f e r e n c e s i n a r e a s , p o s i t i o n and e f f e c t s o f the s p h e r i c a l m i r r o r and t u n i n g f o r k which a r e n e c e s s a r y . i f numbers, by themselves all  a r e t o mean a n y t h i n g .  these  However, s i n c e  these f a c t o r s a r e constant w i t h wavelength, a r e l a t i v e  c a l i b r a t i o n with r e f e r e n c e t o a common wavelength, 550 nm, is  meaningful.  C a l c u l a t i o n of I  T  IB  I  T  / Ig  Relative  I n t e n s i t y a t the Top of the Cone  Relative  I n t e n s i t y a t the Bottom of the Cone x Y  x  x Y For  X  ( F i g . 6)  600  7  /  (Pm)  T o P ) 5  5o  /  (Pm)  B o t t ) 5  Y  x  50  x  Y  5 5 0  550  nm, 108/339 I  T  /  I  B  ~  = C a l c u l a t i o n of I  T  1.105  / IpsD  6  )  Relative  I n t e n s i t y a t the Top of the Cone  Relative  I n t e n s i t y a t Bottom of Cone as measured by the PSD  (Pm) < =  50.5/17.5  P S D  T o p > x  J ott,X B  x x  Y  x  ®\  / /  (Prc) (  P S E )  T o p > 5  5Q  )Bott,550  x  Y  5 5 Q  Q550  x  1.067 a t 600 nm  Thus as noted i n Sec. IV-1, the v a l u e s of I / I p s D T  and  I / I g d i f f e r by 4% and a r e , w i t h i n experimental T  error,  - 5 6 -  identical.  The number I / I p T  S D  = 1.067  means t h a t i f t h e  i n t e n s i t i e s o f t h e two beams a t t h e b o t t o m o f t h e c o n e a r e identical,  t h e 6 0 0 nm l i g h t  a s t h e 5 5 0 nm l i g h t W  550/WQQO  will  be 1 . 0 6 7  times as  a t the t o p of the cone.  i t i s necessary  t o m u l t i p l y by  intense  Thus t o c o r r e c t  1.067.  

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