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UBC Theses and Dissertations

Action spectrum of Nitrobacter agilis 1968

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AN ACTION SPECTRUM OF NITROBACTER AGILIS by JOAN EMILY HILL B.Sc. (Ho n s . ) , U n i v e r s i t y o f T o r o n t o , 1966 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department o f PHYSICS We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September, 1968 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l l m e n t f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that 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 reference and study. I f u r t h e r agree that permission f o r extensive copying 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 granted by the Head of my Department or by h i s representa- t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n 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 - 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 reviewed and a l i s t of the unsolved problems presented. M o d i f i c a t i o n s to the a c t i o n spectrum apparatus b u i l t by BrOoks (1967) are d e s c r i b e d . The apparatus was then used with N i t r o b a c t e r a g i l i s , ATCC no. 14123, to o b t a i n an a c t i o n spectrum of the r e l i e f of carbon 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 of t h i s study i n d i c a t e t h a t cytochrome a^ i s a c t i v e as a t e r m i n a l oxidase. The p o s s i b i l i t y of other cytochromes, p r i n c i p a l l y cytochrome £, a c t i n g as oxidases has not been proven or r u l e d out. The r e s u l t s of a study on the r a t e of oxygen uptake versus oxygen c o n c e n t r a t i o n are a l s o reportedJ the K m ( 0 2 ) values range from 0.021 to 0.055 mM oxygen. The a c t i o n spectrum reported here i s the. f i r s t one to be determined on a chemolithotropic bacterium. -111- I would l i k e to thank Dr. C. P. S. Taylor f o r h i s guidance, advice and encouragement i n the course 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 prepa- 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 support from the N a t i o n a l Research C o u n c i l and the U n i v e r s i t y of B r i t i s h Columbia i s g r a t e f u l l y acknowledged. - i v - TABLE OF CONTENTS Page INTRODUCTION . . 1 Chapter I THE PHYSIOLOGY OF NITROBACTER 1-1 N i t r o b a c t e r 2 1-2 C o n d i t i o n s f o r Growth 2.1 Growth F a c t o r s 2 2.2 E f f e c t of L i g h t . . 3 2.3 N i t r i t e Requirement 33 2.3.1 Oxygen E f f e c t on N i t r i t e Requirement 4 1-3 I n h i b i t o r Studies on whole C e l l s and C e l l - F r e e E x t r a c t s 5 3.1 I n h i b i t i o n by N i t r i t e 6 3.2 I n h i b i t i o n by Inorganic 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 Chlorate 8 1-4 O x i d a t i o n 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 9 1-5 Spectroscopy of N i t r o b a c t e r 5.1 A b s o r p t i o n Spectra 10 5.2 D i f f e r e n c e Spectra 11 5.3 Carbon Monoxide D i f f e r e n c e Spectra ... 12 5.3.1 R e s u l t s with N i t r o b a c t e r 12 1-6 F l a v i n Involvement i n the Cytochrome Chain 13 1-7 Energy Pro d u c t i o n and E f f i c i e n c y 14 -v- Page 1-8 The F i x a t i o n of Carbon Dioxide 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 I I - l Medium and E x t e r n a l C onditions 21 I I - 2 Test Methods 2.1 N i t r i t e 22 2.2 N i t r a t e 23 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 25 1.2 C a l i b r a t i o n „ 26 1.3 P r e p a r a t i o n of the Sample 26 I I I - 2 The A c t i o n Spectrum Apparatus 27 2.1 M o d i f i c a t i o n s to the Reference Beam ... 28 2.2 M o d i f i c a t i o n of the Chopper 30 2.3 M o d i f i c a t i o n s to the Phase S e n s i t i v e Detector 31 2.4 Sample P r e p a r a t i o n 31 Chapter IV CALIBRATION AND PERFORMANCE ' IV- 1 R e l a t i v e I n t e n s i t y at the Top of the Cone 33 IV-2 I n t e n s i t y Response of the PSD ......... 35 IV-3 Actinometer C a l i b r a t i o n s 36 - v i - Page Chapter V RESULTS AND ERRORS V - l Oxygen E l e c t r o d e R e s u l t s 37 V- 2 R e s u l t s from the A c t i o n Spectrum Apparatus 38 2.1 Procedure f o r Obtaining R e s u l t s 38 2.2 R e s u l t s 41 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 42 2.4 E r r o r s i n the P o s i t i o n s of the Peaks.. 43 Chapter VI DISCUSSION V I - 1 Oxygen E l e c t r o d e Results 44 VI-2 A c t i o n Spectrum R e s u l t s 45 VI-3 Summary of Conclusions 49 BIBLIOGRAPHY 50 APPENDIX 53 - v i i - LIST OF FIGURES F o l l o w i n g 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 Detector... 27 3. Power Supply f o r the L i g h t Source . 29 4. Block Diagram of Apparatus 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. R e l a t i v e I n t e n s i t y at the top of the Cone to that at the Bottom 35 7. I n t e n s i t y Response of Lead Sulphide and Phase S e n s i t i v e Detectors 35 8. E f f e c t of Oxygen Concentration on Rate of N i t r i t e O x i d a t i o n 37 9. Michaelis-Menten P l o t s f o r Data of F i g . 8 .... 37 10. The E f f e c t of L i g h t on the E l e c t r o d e Current of a Carbon Monoxide I n h i b i t e d Sample of N i t r o b a c t e r 38 11. A T y p i c a l Determination of the Balance P o i n t f o r L i g h t of Wavelength, X and the Reference L i g h t , w 39 12. 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 wi 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 of 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 of 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 of PSD, Q 54 - v i i i - LIST OF TABLES F o l l o w i n g Page Table I C a l i b r a t i o n Values at the Top of the Cone 34 Table I I C a l i b r a t i o n of Wavelength S e n s i t i v i t y of P h o t o m u l t i p l i e r 34 Table I I I Phase Senstive Detector Wavelength S e n s t i v i t y • • • • 35 Table IV K m Values f o r N i t r o b a c t e r with Oxygen as Substrate 36 Table V 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 E x t i n c t i o n C o e f f i c i e n t 52 -1- INTRODUCTION The work of Brooks (1967) has l e d to an instrument based on that of Castor and Chance (1955) which measures the r e v e r s a l by l i g h t 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 carbon monoxide. He suggested s e v e r a l improvements f o r the apparatus, some of which have now been c a r r i e d out. The l i g h t chopper was rep l a c e d by a tuning f o r k and the path of the reference 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 Brooks. They a l s o a l l o w e a s i e r manipulation of the apparatus. The changes and others a s s o c i a t e d w i t h these are o u t l i n e d i n Chapter I I . The improved apparatus was then used i n a study of the t e r m i n a l oxidase of N i t r o b a c t e r a g i l i s . Along w i t h t h i s the l i t e r a t u r e on the physiology of N i t r o b a c t e r was c r i t i c a l l y surveyed. (Chapter I) Oxygen uptake as a f u n c t i o n of the 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 polarographic technique and the r e s u l t s were compared to a s i m i l a r manometric study performed by Butt and Lees (1964). CHAPTER I THE PHYSIOLOGY OF NITROBACTER 1-1. N i t r o b a c t e r N i t r o b a c t e r i s a micro-organism of the f a m i l y N i t r o b a c t e r i a c e a e of the order Pseudomonales. I t i s prevalent i n nature and i s u s u a l l y i s o l a t e d from s o i l by repeated p l a t i n g on s i l i c a g e l or agar p l a t e s . The c e l l s are gram- negative short rods 0.5 x 1.0 microns. 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 the e l e c t r o n microscope. 1-2. C o n d i t i o n s f o r Growth 2.1 Growth F a c t o r s 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 balance of the s o i l by the o x i d a t i o n of n i t r i t e to n i t r a t e . T h i s o x i d a t i o n i s i t s primary and probably only source of energy. I t i s a u t o t r o p h i c i n that i t uses carbon d i o x i d e as i t s source of carbon. N i t r o b a c t e r i s grown on a mineral 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, Fe, and phosphate are a l l e s s e n t i a l but the requirement f o r other growth f a c t o r s has not been thoroughly i n v e s t i g a t e d . Aleem and Alexander (1958) rep o r t a requirement f o r calcium. Pure c u l t u r e s tend to die out i n d i c a t i n g that there may be a requirement f o r other growth f a c t o r s or t r a c e elements. I t has been reported that b i o t i n -3- i 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 . (Krulwich and Funk, 1965) 2.2 E f f e c t of L i g h t Bock (1965) has shown that 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. Muller-Neugliick and Engel (1961) showed that l i g h t i n a c t i v a t i o n proceeds to the p o i n t where no o x i d a t i o n occurs. N i t r o b a c t e r are r e a c t i v a t e d i n the dark but i t i s slower the longer the l i g h t has shone and the greater i t s i n t e n s i t y . Blue l i g h t 366-436 nanometers (nm) i s most e f f e c t i v e , red l i g h t has no e f f e c t . Muller-Neugliick and Engel f e e l that photo-oxidation i s the cause of t h i s i n a c t i v i a t i o n . N i t r o b a c t e r winogradski possesses no c a r o t e n o i d s . In other b a c t e r i a , carotenoids have been found to have a p r o t e c t i v e mechanism and thus prevent d e s t r u c t i o n by photo-oxidation. 2.3 N i t r i t e Requirement Gould and Lees (1960) have s t u d i e d the optimal n i t r i t e c o n c e n t r a t i o n f o r the growth of N i t r o b a c t e r . They found that an i n i t i a l c o n c e n t r a t i o n of 100 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 to b r i n g the c o n c e n t r a t i o n to 200jig n i t r i t e - N / m l when the f i r s t had been used up r e s u l t e d i n the f a s t e s t r a t e of growth. I n i t i a l a d d i t i o n s of more than 300 \ig n i t r i t e - N / m l l e d to a depression of the o x i d a t i o n r a t e and i n i t i a l c o n c e n t r a t i o n s of 600 fig n i t r i t e - N / m l stopped a l l c e l l growth f o r one week. Subsequent s t u d i e s (Butt and - 4 - Lees, 1960, Boon and Laudelout, 1962) have confirmed t h i s and have shown that at atmospheric c o n d i t i o n s — 2 0 % o x y g e n — maximal oxygen uptake occurs at a n i t r i t e concentration of 16 mM (200 jig n i t r i t e - N / m l ) . At lower oxygen tensions the maximal r a t e occurs at lower n i t r i t e c o n c e n t r a t i o n s . Gould and Lees (1960) have shown that the r a t e of 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 s t r a i g h t l i n e . A l l growth ceased 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 of 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 of the s t a t i o n a r y phase even up to the o x i d a t i o n of 13,000 jig n i t r i t e - N / m l . As a f u n c t i o n of n i t r i t e used the dry weight of c e l l s was s l i g h t l y l a r g e r i n the l a t t e r case. 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 of the c u l t u r e s increased the r a t e , and use of both techniques simultaneously gave an o x i d a t i o n r a t e of 100 jig n i t r i t e - N / m l / h r . A f t e r the l o g a r i t h m i c phase the r a t e of o x i d a t i o n was l i n e a r at a r a t e dependent on the a i r flow. 2.3.1 Oxygen E f f e c t on N i t r i t e Requirement Butt and Lees (1964) c a l c u l a t e d the concentrations of n i t r i t e needed f o r maximal r a t e at d i f f e r e n t p a r t i a l pressures of oxygen. The t h e o r e t i c a l c a l c u l a t i o n s are made assuming that a ternary complex of 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 which breaks 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 . -5- The 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 molecules of n i t r i t e . 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 N i t r i t e Concentrations f o r Maximal Rate phase T h e o r e t i c a l Experimental 20 9 mM 15 mM 10 8 mM 9 mM 2.5 m 5 mM 6 mM Boon and Laudelout c a l c u l a t e d the K of the t e r m i n a l m oxidase of the r e s p i r a t o r y chain with respect to the s u b s t r a t e oxygen by p l o t t i n g v vs v/S. At 32°C, ^ ( o x y g e n ) was 16^M or a p a r t i a l pressure of 1.5%. When the oxygen co n c e n t r a t i o n i s much greater than the r e a c t i o n proceeds at a constant r a t e but when i t i s l e s s than K m i t f o l l o w s f i r s t order k i n e t i c s . Below the K m(oxygen) the o x i d a t i o n proceeds much more s l o w l y w i t h i n c r e s i n g temperature due to the temperature c h a r a c t e r i s t i c s of K and the maximal r a t e V m 0 „ . m max Aleem, Hoch and Varner (1965) have shov/n that i t i s not the oxygen from 0 but r a t h e r from water that serves i n the conversion of NO2 to NOg. 1-3 I n h i b i t o r Studies 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 authors are i n e s s e n t i a l agreement on.the subject matter presented to t h i s p o i n t . At t h i s p o i n t however t h e o r i e s begin to disagree and even some observations are c o n t r a d i c t o r y . -6- 3.1 I n h i b i t i o n by N i t r i t e Boon and Laudelout 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 c o n c e n t r a t i o n on 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 i n c e l l - f r e e e x t r a c t s . At f i r s t they suggest that at higher c o n c e n t r a t i o n s , 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 i n h i b i t i o n . However they then go on to suggest that the e f f e c t i s a c t u a l l y due to 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 . Maximal N i t r o b a c t e r growth occurs at a pH of 7.8. On the a c i d s i d e of t h i s , i n h i - 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 r a t e . On the a l k a l i n e s i d e , Boon and Laudelout suggest that i n h i b i t i o n i s due to the absorption of hydroxide ions on the enzyme s i t e . These authors f i n d that 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 almost i d e n t i c a l f o r i n t a c t c e l l s and c e l l - f r e e e x t r a c t s , being 1.6 and 2.2 mM r e s p e c t i v e l y . The s i m i l a r i t y of K m values p o i n t s to an o x i d i z i n g system l o c a t e d on the outer c e l l membrane as do unpublished e l e c t r o n microscope 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 reported by Boon et a l . However, Aleem and Alexander (1958) using c e l l - 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 oxidase a c t i v i t y found no t o x i c i t y up to c o n c e n t r a t i o n s of 50 mM—a con c e n t r a t i o n at which whole c e l l s have a r a t e c l o s e to zero. Their data i n d i c a t e a high M i c h a e l i s constant f o r n i t r i t e as s u b s t r a t e , probably of the order of 20 mM. This does not agree with that found by Boon. -7- 3.2 I n h i b i t i o n by Inorganic Ions Butt and Lees (1960) i n v e s t i g a t e d the e f f e c t s of s e v e r a l i n o r g a n i c ions on the o x i d a t i o n of n i t r i t e by whole c e l l s . They found that n i t r a t e , cyanate, and a r s e n i t e a l l acted s i m i l a r l y . 3.2.1 I n h i b i t i o n by N i t r a t e A c e r t a i n c o n c e n t r a t i o n of the i o n , e.g. 33 mM n i t r a t e was i n h i b i t o r y at normal oxygen concentrations but t h i s same co n c e n t r a t i o n of ion at a lower oxygen concen- t r a t i o n s t i m u l a t e d the o x i d a t i o n r a t e . Boon and Laudelout's i n v e s t i g a t i o n showed that N i t r o b a c t e r 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 i n 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 acted as a simple non-competitive i n h i b i t o r , showing no s t i m u l a t i o n at low oxygen c o n c e n t r a t i o n s . Boon and Laudelout 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 at low oxygen to an ion exchange e f f e c t . N i t r a t e added to the washed suspension of c e l l s and mineral p r e c i p i t a t e might r e l e a s e trace elements absorbed onto the mineral p a r t i c l e s . T h e i r values f o r the i n h i b i t i o n of n i t r a t e (K^ = 180 mM) d i d not e x p l a i n the i n h i b i t i o n observed by Lees and Simpson at 100 mM. 3.2.2 I n h i b i t i o n by Cyanate Using the same medium as they had done i n 1960, Butt and Lees i n 1964 i n v e s t i g a t e d the e f f e c t of cyanate on whole c e l l s and c e l l - f r e e e x t r a c t s . They found that cyanate -8- a powerful i n h i b i t o r of n i t r i t e o x i d a t i o n by whole c e l l s at normal oxygen t e n s i o n s , had no e f f e c t whatever on the c e l l - f r e e e x t r a c t s . The theory that they propose to account f o r t h i s i s the f o l l o w i n g : there 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 the medium to the enzyme and that cyanate i n t e r f e r e s w i t h t h i s enzyme, although not wit h the n i t r i t e oxidase. In t h i s way, at normal oxygen t e n s i o n , a l l the n i t r i t e t h at would reach the enzyme normally would be o x i d i z e d . The i n t e r f e r e n c e of the cyanate prevents n i t r i t e from reaching the enzyme and slows down the r a t e . At lower oxygen concen- t r a t i o n , too much n i t r i t e would normally reach the enzyme r e s u l t i n g i n s u b s t r a t e i n h i b i t i o n . A d d i t i o n of cyanate prevents the i n h i b i t i o n by lowering the n i t r i t e c o n c e n t r a t i o n at the enzyme, thereby 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 . There are two p o s s i b l e problems w i t h t h i s theory. 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 that the enzyme i s already on the outer s u r f a c e of the c e l l and that n i t r i t e does not have to d i f f u s e through the membrane. The second i s that 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 to a f f e c t the c a r r i e r , then i t would seem p o s s i b l e that they would a l s o a f f e c t the n i t r i t e oxidase. Van Gool and Laudelout (1965), i n c o n t r a s t , have evidence that cyanate has approximately the 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 the co n c e n t r a t i o n f o r 50% i n h i b i t i o n i n the two cases i s 2.5 and 7 mM. 3.2.3 I n h i b i t i o n by Chlorate The e f f e c t s of c h l o r a t e have been i n v e s t i g a t e d -9- by Lees and Simpson (1957) and by Van Gool and Laudelout (1965). The 50% i n h i b i t i o n a f t e r 60 min at 7 mM C I O 3 found by Lees i s comparable wi t h that of Van Gool. The i n c u b a t i o n p e r i o d . must be s t a t e d s i n c e decomposition products of the i n h i b i t o r destroy the cytochrome a c t i v i t y according to a f i r s t - o r d e r r a t e law (Lees and 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 Gool and Laudelout, only c h l o r a t e does not give s i m i l a r values f o r whole c e l l s and c e l l - f r e e e x t r a c t s . The s i m i l a r i t y of the values would seem to accord 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 n i t r i t e o x i d a t i o n . 1-4 O x i d a t i o n 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 of c e l l - f r e e e x t r a c t s i n s tudying N i t r o b a c t e r ( c . f . Sec. 1 - 3 ) . Aleem and Alexander i n 1958 were the f i r s t to 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 they found that the e x t r a c t s contained n i t r i t e oxidase a c t i v i t y , that t h i s was coupled to oxygen uptake and that the o x i d i z e d n i t r i t e c o uld at a l l times be recovered as n i t r a t e i n d i c a t i n g that there 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 there i s no n i t r i t e t o x i c i t y observed to a c o n c e n t r a t i o n of 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 presence of i r o n and a pH of 7.5 to 8.0. The n i t r i t e oxidase i s i n h i b i t e d by low cyanide c o n c e n t r a t i o n s . Along w i t h the i r o n requirement t h i s l a t t e r f a c t suggests 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. Further c e n t r i f u g a t i o n shows that the n i t r i t e oxidase i s -10- s i t u a t e d i n the p a r t i c u l a t e f r a c t i o n at 144,000 x g. 1-5 Spectroscopy of N i t r o b a c t e r 5.1 Absorption Spectra Lees and Simpson (1957) report e d cytochrome a b s o r p t i o n peaks at 589, 551, and 520-525 nm on a d d i t i o n of n i t r i t e or d i t h i o n i t e . Aleem and Nason (1959) show that 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 c o n t a i n i n g p a r t i c l e . A d d i t i o n of n i t r i t e to t h i s p a r t i c l e r e s u l t e d i n abs o r p t i o n peaks appearing at 550 and 520 nm r e p r e s e n t a t i v e of the a and B peaks of a c_ type cytochrome and i n the 585-590 and 438 nm regions i n d i c a t i v e of the a and y peaks of and a type cytochrome, probably a.].. Added d i t h i o n i t e gave e s s e n t i a l l y s i m i l a r peaks but of s e v e r a l f o l d greater magnitude, and a l s o produced an absorption maximum at 415 nm corresponding to the y peak of cytochrome c_. Production of the 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, or l a c t a t e f a i l i n g to produce them. Hemoglobin, c a t a l a s e and peroxidase were shown to be absent from both the p a r t i c u l a t e and supernatant f r a c t i o n s . ( D i f f e r e n t copper and i r o n e f f e c t s have been observed. Aleem and Alexander (1958) showed that copper was i n h i b i t o r y , but i n 1959 Aleem and Nason say that i t has no e f f e c t but that i t enhances the non-enzymatic disappearance of n i t r i t e . The i r o n requirement was shown but i r o n a l s o reduces the cytochrome c_ l i k e component non-enzym- a t i c a l l y . -11- 5.2 D i f f e r e n c e Spectra A more recent s p e c t r o s c o p i c study was performed by Van Gool and Laudelout i n 1965 on N i t r o b a c t e r winogradski. 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 showed maxima at 523, 554, and 597 nm w i t h a shoulder at 609 on the l a s t . In the zone of 450 to 490 nm the reduced system had a higher transmittance than the o x i d i z e d . The minimum occurred at 465 nm. This i s p o s s i b l y i n d i c a t i v e of the b l e a c h i n g of f l a v i n components. In the Soret r e g i o n there were two peaks, at 419 and 439 nm, which were fused i n a t u r b i d c e l l suspension to 440 nm. 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 e x t r a c t s . The peak at 597 f o r example, occurred i n t h i s system at 594 nm. At low temperature on c e l l - f r e e e x t r a c t s the highest peak i n the v i s i b l e was s p l i t i n t o three peaks, at 604, 587, and 579 nm. The peaks at 609 and 450, 594 and 439 are i n d i c a t i v e of JI type cytochromes. Those at 554, 523 and 419 i n d i c a t e a cytochrome <c w i t h the high wavelength of the a peak, 554, i n d i c a t i n g a b a c t e r i a l type cytochrome £. The f o l l o w i n g t a b l e i s a.summary of t h i s and other s p e c t r o s c o p i c data taken from the paper by Van Gool and Laudelout. Author Cytochromes «i Cytochromes c_ ±a & a l a a j ^ cQ VanGool and Laudelout 609 450 594 439 554 523 419 Lees and Simpson - - 589 - 551 520-525 - Aleem and Nason - - 586-590 438 550 520 415 Z a v a r z i n - - 592 - 552 - -12- 5.3 Carbon Monoxide Difference Spectra A carbon monoxide difference spectrum i s recorded as the absorption of the carbon monoxide-reduced compound minus the absorption of the reduced compound. This spectrum indicates what modifications of the cytochromes i n the organism or extracts have been caused by the addition of carbon monoxide. It has been shown i n many organisms that carbon monoxide complexes only with the terminal compound i n the cytochrome chain, i n competition with oxygen. It thus prevents the oxidation of t h i s oxidase and of the other cytochromes i n the chain with the r e s u l t that these compounds become reduced, or more reduced as the case may be, i n the presence of a reducing agent, i . e . supply of electrons. On addition of carbon monoxide one would thus expect to observe an increased reduction of the cytochromes generally plus changes i n the absorption of the oxidase due to i t s forming a complex with carbon monoxide. A comparison of the reduced and carbon monoxide- reduced difference spectra often enables one to determine which of the cytochromes are present and which are reacting with carbon monoxide since the cytochromes are defined on the basis of t h e i r absorption spectra. To date, four types of cytochromes have been found to act as oxidases. They are cytochromes a^, a^, ag, and o_. 5.3.1 Results with Nitrobacter Van Gool and Laudelout have reported a -13- CO-reduced minus reduced difference spectrum of Nitrobacter. Peaks at 523, 554, and 608 nm i n the v i s i b l e and 419 i n the Soret region occur at the same positions as those i n the reduced difference spectrum. These peaks are due to increased reduction of the compounds—namely cytochrome & and c_— which do not combine with carbon monoxide. Troughs were recorded i n the carbon monoxide difference spectrum at 439 and 594 nm, the positions of peaks i n the reduced difference spectrum. These troughs are i n d i c a t i v e of reduced absorption i n the carbon monoxide sample with respect to the reduced sample. Since these wavelengths do correspond to the peaks of cytochrome a^ i n the reduced difference spectrum, th e i r disappearance indicates that cytochrome has complexed with carbon monoxide. Other i n d i c a t i o n s of carbon monoxide binding were a peak at 450 nm i n the carbon monoxide difference spectrum of the c e l l - free extract and a peak at 426 nm and trough at 440 i n the c e l l suspensions. The peak and trough of the cytochrome a^ reported at 426 and 440 for i n t a c t c e l l s and 439 for c e l l - f r e e extracts correspond well with data reported by Chance (1953). His figures for these positions i n A. pasteurianum are 427 and 442 nm. Chance also reports a band at 590 while Van Gool and Laudelout mention a trough corresponding to the 594 peak i n the reduced difference spectrum. 1-6 F l a v i n Involvement i n the Cytochrome Chain Inhibitor and difference spectra studies by Van Gool -14- and Laudelout point 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 i n h i b i t i o n by quinacrine and r e v e r s a l of t h i s by FAD. Zavarsin (Lees, 1960) has reported that i r o n along with eit h e r molybdate or tungstate stimulate n i t r i t e oxidation. This simultaneous requirement for iron and molybdate led Zavarzin to postulate that a molybdeno- fla v o p r o t e i n and cytochrome act sequentially. Van Gool and Laudelout say that the involvement of a f l a v i n would indicate that the process of n i t r i t e oxidation must be pulled over a thermodynamically unfavourable step. 1-7 Energy Production and E f f i c i e n c y The d r i v i n g force for the reduction of carbon i s the oxidation of the inorganic ion by oxygen. The e f f i c i e n c y of these organisms i s thus best expressed by the r a t i o of inorganic nitrogen oxidized to C0„ carbon assimilated. In Nitrobacter the N:C r a t i o varies from 76:1 to 135:1 (Alexander 1961). The 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 only a small proportion determined by t h e i r e f f i c i e n c y of energy u t i l i z a t i o n . The free energy data of Baas-Becking and the assumption that Nitrobacter contains 50% carbon on a dry weight basis i s used to show that the free energy e f f i c i e n c y i s 30% i n the presence of 200jig n i t r a t e - N/ml and 15% i n the presence of 1000 p.g nitrate-N/ml. (Gould and Lees, 1960) Aleem and Naspn i n 1960 have shown that phosphorylation i s coupled to n i t r i t e oxidation. P a r t i a l l y p u r i f i e d n i t r i t e -15- oxidase p a r t i c l e s c a t a l y z e d the formation of high energy phosphate bonds concomitant w i t h the o x i d a t i o n of n i t r i t e . ATP i s formed when ADP i s s u p p l i e d as the acceptor. Several other 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 . A l l t h e i r attempts to uncouple phosphorylation from o x i d a t i o n proved unsuccessful although aged n i t r i t e oxidase p a r t i c l e s d i s p l a y e d decreased r a t e s of phosphorylation while t h e i r r a t e s of o x i d a t i o n remained unchanged. Magnesium ions were found to s t i m u l a t e p h o s p h o r y l a t i o n . In 1963, Aleem, Lees, and Ni c h o l a s demonstrated that there was a r e d u c t i o n of NAD concomitant w i t h the o x i d a t i o n of added mammalian ferrocytochrome e_. Aleem (1965) showed th a t the 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 et a l (1963) s t a t e that the r e d u c t i o n of NAD i s dependent on ATP and that the t h e o r e t i c a l requirement i s 4 ATP/NAD. In c e l l f r e e systems the experimental value was 10-20:1. These authors have proposed the 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 generated Inorganic c y t £ ^ cytochrome su b s t r a t e reductase > c y t . c_ oxidase ^ 0 2 ATP f l a v o p r o t e i n r e q u i r e d 1 ATP + C 0 2 NAD(H) -^(CH 20) Such a modified scheme seems to be necessary i n order to produce NADH (although I do not agree th a t Aleem et a l have proven t h i s i n t h e i r experiments). The reason f o r t h i s -16- modified scheme i s that the E G for the n i t r a t e - n i t r i t e system i s +0.35 while that for the NAD/NADH couple i s -0.32. Thus energy would be required to couple these systems and r e s u l t i n the Oxidation of n i t r i t e . It would seem probable that there might also be a modification i n the f i r s t step of the proposed sequence since the of mammalian cytochrome c_ F e + + + / F e + + i s +0.25. It could perhaps be as Lees (1962) suggests, namely that Nitrobacter may be compelled to synthesize some such compound as adenyl n i t r i t e so as to lower the redox p o t e n t i a l of the n i t r i t e couple. (See however Sec. 1-4) In considering the values of these redox potentials I f e l t that some reser- vations should be borne i n mind: e s p e c i a l l y that these values are obtained i n " i n v i t r o " s i t u a t i o n s at pH 7and the cyt. c_ couple E Q value used i s that for mammalian cytochrome c_. It i s possible and quite probable that the values may not apply to the s i t u a t i o n i n in t a c t Nitrobacter c e l l s . In inta c t c e l l s , the oxidation of n i t r i t e might proceed much more favourably to produce energy due to a change i n redox pote n t i a l r e s u l t i n g from the physical configurations or d i s t o r t i o n s of the molecules involved and from l o c a l pH and concentration changes. 1-8 The Fi x a t i o n of Carbon Dioxide The energy obtained from the oxidation of n i t r i t e , i n the form of ATP and NADH i s used to f i x and reduce carbon dioxide. Aleem i n 1965 performed an extensive study on the pathway of carbon f i x a t i o n using radioisotopes. The f i r s t stable compounds to be l a b e l l e d were phosphoglyceric acid -17- and aspartic acid. After 10 sec exposure to C*^0 2, 60% of the r a d i o a c t i v i t y appeared i n phosphoglycerate, 20% i n each aspartic acid and sugar phosphates, 4-6 % i n each malic acid, glutamic acid and phosphoenolpyruvate. In dealing with c e l l - free extracts the carboxylating enzymes were l o c a l i z e d i n the supernatant a f t e r 144,000 x g centrifugation. The supernatant catalyzed rapid f i x a t i o n of C0 2 i n the presence of added ribulose-1,5-diphosphate or ribose monophosphate and ATP. The l a b e l appeared i n phosphoglycerate. Without added C02-acceptor the soluble f r a c t i o n catalyzed rapid a s s i m i l a t i o n when supplied with ATP and NADH. The carboxylating enzymes required magnesium ions. Carboxydismutase (D-ribulose-1,5- diphosphate decarboxylase) and other enzymes of the Ca l v i n - Benson, Kreb's and reductive pentose phosphate cycles were l o c a l i z e d i n the supernatant. Aleem states that the above observations point to the standard autotrophic pathway of CX>2 as s i m i l a t i o n , namely: co 2 C0 2 r-ibulose ^phospho- ^phosphoenol- diphosphate glycerate pyruvate NH3 =»• oxaloacetate ^ a s p a r t i c acid K TCA ; cycle i Malate NAD+ NADP Aleem also gives the possible pathways of carbon metabolism. Bock and Engel (1966) have shown that i f Nitrobacter i s deprived of carbon dioxide, oxygen uptake slows down. Under -18- a e r o b i c as w e l l as under anaerobic c o n d i t i o n s , the n i t r i f i e r s are able to f i x carbon d i o x i d e a f t e r o x i d a t i o n has occurred. The s t r o n g e s t a c t i v i t y i n • t h i s respect i s seen i n c e l l s which have c a r r i e d out o x i d a t i o n s h o r t l y before being allowed to f i x C0 2. 1-9 Unsolved Problems and C r i t i c i s m s There are s t i l l many problems w i t h regard to the physiology of N i t r o b a c t e r . Some of these are l i s t e d below. ( i ) F i r s t there i s the problem of a calcium requirement. Aleem and Alexander (1958) mention that calcium i s r e q u i r e d f o r growth of N i t r o b a c t e r . Lees and Simpson (1957) make no mention of a calcium r e q u i r e - ment yet the medium used by these authors c o n t a i n s calcium i o n s . Boon and Laudelout (1962) on the other hand, used a medium which contained no calcium, ( i i ) A second point a r i s i n g out of the paper by Boon and Laudelout concerns t h e i r suggestion that the i n h i b i t i o n of N i t r o b a c t e r at high pH i s due to 0H~ io n s . That the c o n c e n t r a t i o n of hydroxyl i o n s , when placed i n the k i n e t i c equation y i e l d s a curve c l o s e to the experimental proves only that an i o n i z a t i o n i s i n v o l v e d , not that 0H~ i s the a c t i v e i n h i b i t o r , ( i i i ) The e f f e c t s of n i t r a t e on n i t r i t e o x i d a t i o n are 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 Butt and Lees seems to be very d i f f e r e n t from that described by Boon and Laudelout ( c f . 1-3.2.1) -19- ( i v ) The d i f f e r e n c e s i n the e f f e c t s of n i t r a t e by these authors could be dismissed as j u s t a d i f f e r e n c e i n degree except that cyanate, a second i n h i b i t o r i s reported as behaving 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 authors ( c f . Sec. 1-3.2.2). Thus B u t t ' s and Lees's observations on the e f f e c t s of n i t r a t e and cyanate 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 accord w i t h t h e i r proposal that a c a r r i e r i s r e q u i r e d to b r i n g n i t r i t e i n t o the c e l l . A l l of the observations of Boon and Laudelout on i n h i b i t o r s and t h e i r unpublished e l e c t r o n microscope s t u d i e s on the other hand, point 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 outer membrane of the c e l l , (v) The f i n a l problem to be mentioned here i s concerned w i t h ATP generation. As s t a t e d i n Sec. 1-7, I do not agree that Aleem has proven that NADH i s produced i n the manner i n d i c a t e d s i n c e l o a d i n g of a cytochrome chain with ATP w i l l d r i v e i t backwards. A scheme such as he has proposed does seem to be r e q u i r e d however, i n order to 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 to perform at t h i s point would be to u t i l i z e Chance's r a p i d flow technique w i t h i n h i b i t o r s to determine the order of r e d u c t i o n of the compounds i n the cytochrome chain. -20- 1-10 Summary There i s much contradictory evidence concerning Nitrobacter e s p e c i a l l y i n r e l a t i o n to i n h i b i t o r studies and the location of the n i t r i t e oxidase p a r t i c l e (on the outer membrane or inside the c e l l ) . This could be due to the use of d i f f e r e n t s t r a i n s , to d i f f e r e n t growth conditions-especially media, or to d i f f e r e n t experimental technique. It seems well established however that Nitrobacter oxidizes n i t r i t e to n i t r a t e and that t h i s oxidation supplies energy i n the form of ATP and NADH for the f i x a t i o n of carbon dioxide and the production of material for growth. A cytochrome chain and flavoprotein are seen to take part i n the electron transport system. Water i s the source of hydrogen for the reduction of pyridine nucleotide and of oxygen i n the oxidation of n i t r i t e . Molecular oxygen i s only involved i n the oxidation of the terminal oxidase i n the electron transport chain. Carbon dioxide f i x a t i o n i s s i m i l a r to that of other chemoautotrophic bacteria i n that i t makes use of the Calvin-Benson and reductive pentose phosphate pathways. - 2 1 - CHAPTER II GROWTH METHODS I I - l Medium and External Conditions Nitrobacter a g i l i s from the American Type Culture 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 to that of Gould and Lees (1960). To 1 0 0 1 . of water were added 30 gm K H 2 P O 4 , 1 gm C a S 0 4 , 0.5 gm MgCl 2, 0 . 0 1 gm M n C l 2 , and 0 . 2 ml of dialyzed i r o n . This was f i l t e r e d and the pH adjusted to 7 . 8 , Sodium n i t r i t e ( N a N 0 2 ) was added to make a concentration of 1 0 0 jig nitrite-N/ml ( 8 m M ) . 5 0 - 1 0 0 ml of medium plus sodium n i t r i t e were placed i n 250 ml Erlenmeyer f l a s k s and s t e r i l i z e d a f t e r which approximately 0 . 5 ml of a d i l u t e Nitrobacter suspension were added. The cultures were grown in the dark (cf. 1 - 2 . 2 ) . N i t r i t e was added when required by the addition of a s t e r i l i z e d 0 . 8 M NaNOg soluti o n to bring the concentration of n i t r i t e i n the culture to 1 0 mM. At the onset of a decreased requirement for n i t r i t e , or ju s t p r i o r to t h i s as estimated by the t u r b i d i t y of the solutions, the cultures were d i l u t e d two to ten times, depending on the immediate requirement for concentrated cultures. Or al t e r n a t e l y , new cultures were made by adding 2-5 ml of a concentrated s o l u t i o n to 1 0 0 ml of fresh medium. In t h i s manner a type of continuous culture method was employed providing s t e a d i l y growing and f a i r l y concentrated cultures at a l l times. Once cultures reached the point where they required no more n i t r i t e ( 3 - 8 wk), d i l u t i o n usually proved -22- unsuccessful i n producing viable cultures. Aeration and n i t r a t e removal were not employed although such methods lead to a prolonged growth phase (Gould and Lees, 1960). The method of d i l u t i n g cultures as mentioned above ensured that the n i t r a t e concentration did not become high enough to prevent further growth. The problem of adequate aeration was dealt with by keeping the cultures r e l a t i v e l y shallow (about 2 cm) and allowing a i r to c i r c u l a t e through cotton plugs i n the tops of the f l a s k s . The cultures used i n the experiments were oxi d i z i n g n i t r i t e at a rate of 25-50 jig nitrite-N/ml/day (2-4 mM n i t r i t e / ml/day). I I - 2 Test Methods 2.1 N i t r i t e The presence of n i t r i t e was determined using a method employing s u l f a n i l i c acid and N,N dimethyl-l-naphthylamine (Snell and S n e l l , 1949). Two stock solutions were f i r s t made up. Solution A was made by d i s s o l v i n g 3.3 gm of s u l f a n i l i c a cid i n 750 ml water by heating. 250 ml g l a c i a l a c etic acid were then added to t h i s . Solution B contained 5.25 gm of N,N dimethyl-l-naphthylamine 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 test of the presence of n i t r i t e equal volumes of sol u t i o n A and B were mixed. To about 1 ml of t h i s mixture a sample of less than 0.5 ml of medium was added. Development of colour (pink to purple) indicated the presence of n i t r i t e . A colourless so l u t i o n was i n d i c a t i v e of the -23- absence of n i t r i t e . This method was employed to determine the n i t r i t e requirement of the cultures. The best conditions for a quantitative test were found to be as follows. 1 ml of the solution to be tested was d i l u t e d to 100 ml with d i s t i l l e d water. 5 ml of s o l u t i o n A was added followed by 5 ml of solution B. Ten minutes a f t e r the addition of solution B a reading on the K l e t t colorimeter was taken using a green f i l t e r . The timing here i s quite 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 reading. The development of colour i s also very s e n s i t i v e to temperature and the age of s o l u t i o n B. Thus i t i s necessary to run one standard each time a determination i s performed. Since a plot of K l e t t reading versus concentration i s proportional i n the concentration range encountered one standard i s usually s u f f i c i e n t . Solution B i s unstable over periods of time greater than two weeks. This i s not of any consequence for q u a l i t a t i v e tests as colour develops i n the presence of n i t r i t e and does not develop i n i t s absence. In both tests older solutions give a deeper colour but too old a solution 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 less than ten minutes so that a quat&i t a t i v e test has no v a l i d i t y . 2.2 Nitrate Nitrobacter converts n i t r i t e to n i t r a t e so that i t i s desirable to test for the production of n i t r a t e to -24- ensure that the Nitrobacter are the agents causing the disappearance of n i t r i t e . This i s done using the following method employing a phenoldisulfonic acid reagent (Snell and S n e l l , 1949). The reagent was prepared by d i s s o l v i n g 25 gm of colourless phenol i n 150 ml of concentrated H^SO^. Traces of n i t r i c acid i n the sulphuric acid were removed by a g i t a t i n g with mercury. 75 ml of fuming sulphuric acid containing 13% free sulphur troxide were then added. This 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 of sample were evaporated to dryness. 2 ml of the phenoldisulfonic acid reagent were added rapid l y to the centre and the dish rotated to ensure contact of the reagent with a l l of the residue. This was allowed to stand for 10 minutes at which time 15 ml of cold water were added to dissolve the residue. 1:2 ammonium hydroxide:water was added u n t i l the solution was s l i g h t l y a l k a l i n e . This was accompanied by the production of a deep yellow colour. The solution was then made up to 50 ml and a reading taken on the Kl e t t colorimeter using a blue f i l t e r . C a l i b r a t i o n was only required once as the colour developed i s unaffected by external conditions. - 2 5 - 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 of oxygen uptake and hence of n i t r i t e o x i d a t i o n as a f u n c t i o n of oxygen c o n c e n t r a t i o n can be obtained 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 at -0.6 to -0.9 v o l t s with respect to a Ag/AgCl e l e c t r o d e placed 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 the s o l u t i o n s used, oxygen d i f f u s i n g to the platinum 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 to the e l e c t r o d e i s a f u n c t i o n of the co 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 to the c o n c e n t r a t i o n . The current i s measured by passing i t through a r e s i s t o r and monitoring the voltage drop across the r e s i s t o r w i t h a Heathkit Servo Recorder model EUW-20A. In the experiments 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 of 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 to prevent the N i t r o b a c t e r from s e t t l i n g to the bottom and to keep the medium mixed wi t h respect to the 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 higher c u r r e n t than would be found i n a s t a t i o n a r y case. E q u i l i b r a t i o n of the oxygen c o n c e n t r a t i o n i n the medium wit h that i n a i r was prevented by c l o s i n g the chamber wi t h a greased cover s l i p ^ Pt electrode Ag/AgCl electrode SI Support Greased cover s l i p Oxygenated Sample S t i r r i n g bar Magnetic S t i r r e r T y Figure 1 The Oxygen Electrode -26- T h i s allowed the use of oxygen concentrations higher than that i n 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 determined by bub b l i n g the s o l u t i o n w i t h oxygen f o r about two minutes before p l a c i n g i n the apparatus. The s e n s i t i v i t y was then set to ensure that t h i s c o n c e n t r a t i o n occurred at the a p p r o p r i a t e p o i n t . 0% oxygen was determined by bubbling the s o l u t i o n w i t h n i t r o g e n and 20% oxygen could be determined by a l l o w i n g the s o l u t i o n to 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 of the Sample 10 ml of 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 placed i n the apparatus to prevent the formation of an a i r space when the opening was c l o s e d w i t h the cover s l i p . For more concentrated samples the N i t r o b a c t e r i n 50 ml of c u l t u r e were c e n t r i f u g e d at the 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 f o r 15 min and resuspended i n 10 ml of 20 mM phosphate b u f f e r (18 mM Na 2HP0 4 p l u s 2 mM KH 2P0 4) or i n 10 ml of t h e i r own medium wi t h the ap 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 bubbled w i t h oxygen f o r 1 min before being placed i n the apparatus. I I I - 2 The A c t i o n Spectrum Apparatus The a c t i o n spectrum apparatus developed by Brooks (1967) -27- and modified here i s based on an instrument described by Castor and Chance (1955). It i s a se n s i t i v e instrument for measuring the photochemically r e v e r s i b l e i n h i b i t i o n of oxygen uptake by carbon monoxide. The e s s e n t i a l features of t h i s equipment are 1) a l i g h t source and monochromator, 2) an i n t e n s i t y measuring device, and 3) a chamber and r e s p i r a t i o n rate sensor. These features are described by Brooks while modi- f i c a t i o n s to the reference beam and i n t e n s i t y measuring apparatus are described i n l a t e r sections of t h i s thesis. In short, the l i g h t source provides both the monochromatic beam and the reference beam required to obtain the action spectrum. To measure the i n t e n s i t y of either beam, the l i g h t , chopped by a mechanical device, f a l l s on a lead sulphide photodetector and the sign a l from t h i s i s passed through a phase s e n s i t i v e detector (PSD, F i g . 2). A Keithley model 150 A microvoltmeter ammeter i s used as a n u l l device with a bucking voltage supplied from an external c i r c u i t . A closed chamber provides for the use of a cont r o l l e d atmosphere of carbon monoxide and oxygen i n the experiments. The rate sensor consists of a platinum electrode polarized at -0.6 to -0.9 v o l t s with respect to a Ag/AgCl electrode placed i n a drop of c e l l suspension. This drop i s held between the two electrodes and a cover s l i p forming the bottom of the chamber. The current measurements are made by bucking out the electrode current using the Keithley microvoltmeter ammeter and an E s t e r l i n e Angus recording milliammeter as n u l l detectors. Unlike the oxygen electrode, the purpose i n the action + 300 V BUCKING VOLTAGE BIAS VOLTAGE 200 1.1B BUCKING CURRENT ALL CAP. VALUES IN MICROFARADS E L E C T R O D E O F F Q , 2 ^ P S D o o © S W I T C H P O S I T I O N S FIGURE 2 Circuit Diagram of Phase Sensit ive Detec tor -28- spectrum apparatus i s to set up a steady s t a t e produced by the opposing e f f e c t s of d i f f u s i o n of oxygen from the surface of the drop to the platinum microelectrode and r e s p i r a t i o n by the organism i n the drop. Small 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 steady 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 c i r c u i t . 2.1 M o d i f i c a t i o n s to the Reference Beam Brooks (1967) suggested s e v e r a l improvements to reduce e r r o r and extend the range of the a c t i o n spectrum apparatus. S e v e r a l of these have been c a r r i e d out. The method of b r i n g i n g the comparison beam from the lamp to the sample was a l t e r e d . To do t h i s the l i g h t source used, was changed from the 8v-50w Ace bulb to a 24v-150w P h i l l i p s q u a rtz i o d i n e lamp (model no. 7158). The l a t t e r bulb has a quartz envelope 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 of l i g h t from the back of the bulb as w e l l as the f r o n t but at the same time reduces the i n t e n s i t y of both beams. Some increase i n i n t e n s i t y was then provided by u s i n g the higher power lamp. The c u r r e n t and voltage are provided by a Sorenson and an E i c o power supply placed i n s e r i e s . S t a b i l i t y i s achieved by p l a c i n g two twelve 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 supply. ( F i g . 3) The power u n i t s are adjusted to ensure that there i s no c u r r e n t passing through the b a t t e r i e s . Waveleng C o n t r o l V o l t a g e R e g u l a t e d Source Monochromator F i l t e r H o l d e r I n t e n s i t y C o n t r o l F i g u r e 4 B l o c k Diagram o f A p p a r a t u s - 2 9 - lamp EICO SORENSON Batteries F i g . 3 Power Supply for the Light Source The beam taken from the one side of the lamp passes through the monochromator as described by Brooks. The second beam follows a path o p t i c a l l y equivalent to that of the monochromatic beam. Condensing lenses image the l i g h t from the back of the lamp onto a s l i t which i s i n the f o c a l plane of a convex lens. The collimated beam i s condensed by a second convex lens and imaged at the e x i t s l i t of the monochromator. The lenses are housed i n a tube 2 " i n diameter which c a r r i e s the l i g h t out the back of the lamp, around the monochromator box and allows i t to enter the monochromator at a position perpendicular to, but ju s t below the e x i t s l i t . A small mirror placed on a track can be s l i d into position to r e f l e c t the reference beam onto the e x i t s l i t . (Fig. 4 ) The track consists of two small rods such that when the mirror i s moved out of the path of the monochromatic beam, t h i s beam passes between these rods and i s focused on the s l i t . The re s u l t i s -that the e x i t s l i t i s at a l l times f i l l e d with l i g h t , having the advantage as outlined by Brooks, that no transient e f f e c t s -30- are produced i n the sample. The louvers were moved from the monochromator housing to an eq u i v a l e n t p o s i t i o n i n the c o l l i m a t e d p o r t i o n of the reference beam thereby reducing the amount of s c a t t e r e d l i g h t i n c l u d e d i n the monochromatic beam. Any f i l t e r s r e q u i r e d i n the reference beam could be placed i n the louver housing. 2.2 M o d i f i c a t i o n of the Chopper The above changes n e c e s s i t a t e d the i n t r o d u c t i o n of a f u r t h e r improvement. The only 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 the two beams i s that between the s l i d i n g m i r r o r and the sample. Since the i n t e n s i t y of both beams i s r e q u i r e d f o r determination of the a c t i o n spectrum, the chopper and d e t e c t i n g system must be so placed that measurements can be made on both beams. Because of space l i m i t a t i o n s the chopper must be s m a l l . 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 Brooks meets t h i s requirement. The l i g h t chopping wheel was thus discarded and repl a c e d by a Bulova l i g h t chopper operated on 28 v o l t s , 20 mi l l i a m p s from an E i c o DC power source. The n a t u r a l frequency of the tuning f o r k i s 400 Hz. F i g . 4 d e p i c t s the 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 s m a l l s p h e r i c a l m i r r o r i n t e r c e p t s a p o r t i o n , about 10%, of the l i g h t e n t e r i n g the cone and p a r t l y focuses the louvers or the g r a t i n g on the PbS, lead s u l p h i d e , d e t e c t o r . Thus a cro s s s e c t i o n of the beam i s sampled by the PbS d e t e c t o r and although t h i s does not remove inhomogeneities i n the beam i t does a l l o w a 1:1 correspondence of PbS readings -31- with i n t e n s i t y at the top of the cone. The inhomogeneity of the beam i s removed by the scrambling e f f e c t of the cone and consequently i s not transferred to the sample. 2.3 Modifications to the Phase Sensitive Detector In order to measure the 400 Hz s i g n a l , the phase se n s i t i v e detector, PSD, required modification. (Fig. 2) The r e j e c t i o n f i l t e r i n the feedback loop of the f i r s t amplifier was removed. The purpose of t h i s f i l t e r was to act as a band pass so that the second amplifier would not become overloaded with noise s i g n a l s . The overloading of the second amplifier was not a problem and the re j e c t i o n f i l t e r introduced unwanted phase s h i f t s . The feedback loop of the second amplifier was changed to introduce a more uniform gain control. A 0.01 microfarad capacitor was added across the reference input i n order to round o f f the square wave supplied by the tuning fork. A 0.047 microfarad capacitor was placed across the secondary of the sign a l transformer to correct the phase of the s i g n a l . 2.4 Sample Preparation for the Action Spectrum Apparatus For use i n the action spectrum apparatus 50 ml of culture were centrifuged on the highest speed of a c l i n i c a l centrifuge for 15-20 min. A l l but 2-3 ml of medium was then removed and the Nitrobacter resuspended by shaking. Best r e s u l t s were obtained i f the sample was allowed to stand for 30 min before use. At the end of t h i s time 0.2-0.5 ml of the -32- concentrated suspension were placed i n the syringe and a drop inserted between the electrodes. A mixture of C0:02 i n the r a t i o 4:1 saturated with water vapour was sucked into the chamber by a 50 ml syringe. Some samples did not recover from centrifugation and had to be discarded. Those that showed a c t i v i t y remained a l i v e i n the apparatus a maximum of 90 min. In the a i r , the concentrated suspensions lasted up to 3 hours with the r e s u l t that several samples could be obtained from a single centrifugation. - 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 at the Top of the Cone Since the i n t e n s i t y of l i g h t i s measured at the bottom of the cone a c a l i b r a t i o n i s r e q u i r e d to determine the a c t u a l i n t e n s i t y of l i g h t f a l l i n g on the sample. T h i s i s most important f o r the monochromatic beam as the t r a n s m i s s i o n of the cone with wavelength i s v a r i a b l e . The composition of the reference beam i s always constant, only the i n t e n s i t y v a r i e s . Thus i n determining the a c t i o n spectrum the f a c t o r r e l a t i n g the i n t e n s i t y of l i g h t at the top of the cone to that at the bottom f o r the reference 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 the equation determining 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 - cent. The method of c a r r y i n g out t h i s c a l i b r a t i o n i s as f o l l o w s . An RCA 5819, S-9 response, p h o t o m u l t i p l i e r tube was placed at the top of the cone to gather a l l of the l i g h t . The c i r c u i t diagram f o r the arrangement i s shown i n F i g . 5. A K e i t h l e y 240 A High Voltage Power Supply was used to b i a s the tube at 600 v o l t s . The output curre n t was read from an Avometer. At no time was i t p o s s i b l e to d i s t i n g u i s h a dark curre n t on the 50 jiamp s c a l e of the Avometer i n d i c a t i n g a dark c u r r e n t much l e s s than 1 (jiamp. At each wavelength the anode c u r r e n t from the photo- m u l t i p l i e r and the volt a g e from the phase s e n s i t i v e d e t e c t o r C1,C2,C3, 16jif R H , 100K R1-R10, 47K Figure 5 C i r c u i t Diagram for Photomultiplier Tube -34- were recorded. The l a t t e r was corrected for temperature deviation from 24°C and to 10 times gain as described by Brooks. Temperature measurements were taken from a c a l i b r a t e d thermocouple attached to the PbS detector. 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 tabulated i n Table I. It was then necessary to c a l i b r a t e the photomultiplier for 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. The c a l i b r a t i o n for t h i s i s 0.054 microvolts/microwatt/ square cm. At each wavelength the thermopile and photomultiplier were placed i n the monochromatic l i g h t emerging from the s l i t with the cone removed. Their s e n s i t i v e elements were placed at the same distance from the s l i t and i n the same mean pos i t i o n . The exposed area of the photomultiplier was 0.0576 cm , the same order of magnitude as the output area at the top of the cone (0.0256 cm ). Repeated (7-10) measurements of the change i n voltage from the thermopile were taken and the average of these i s reported i n Table I I . The current from the photomultiplier i s also an average over several readings. I suspect that most of the 1% v a r i a t i o n i n photomultiplier readings i s due to fl 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 to determine the wavelength s e n s i t i v i t y of the PbS detector was made. Since readings for t h i s c a l i b r a t i o n were not taken at the same time i t i s possible only to compare r a t i o s of i n t e n s i t i e s . Thus on d i f f e r e n t days, the output of the lamp may vary as much as 20%, yet the r a t i o of i n t e n s i t y at a wavelength, X, to that TABLE I Calibration Values at the Top of the Cone Wavelength Photomultiplier PSD Voltage Current 24°C, lOx gain nm liA mv 400 60 6.70 410 86 8.07 420 113 9.38 430 144 7.82 440 178 12.1 450 215 13.6 460 261 15.7 470 305 17.5 480 334 18.5 490 358 19.6 500 371 20.6 510 380 22.5 520 405 24.9 530 400 25.7 540 373 27.6 550 339 27.7 560 304 28.7 570 260 31.2 580 215 31.5 590 162 33.6 600 108 33.7 610 72 34.5 620 48 34.6 630 32.8 40.5 640 24.5 42.8 TABLE I I C a l i b r a t i o n of Wavelength S e n s i t i v i t y of P h o t o m u l t i p l i e r Wavelength P h o t o m u l t i p l i e r Thermopile Spectra Current Voltage S e n s i t i v nm |iA liV |iw/|j.A 400 60 1.21*.04 .0215 410 78 1.44±.02 .0197 420 99 1.65±.02 .0168 430 122 1.85±.02 .0162 440 143 2.09±.09 .0156 450 165 2.30±.04 • .0149 460 194 2.69±.04 .0148 '470 212 2.91±.05 .;0146 480 222 3.05±.10 .0147 490 227 3.16±.04 .0149 500 22 3.37±.15 .0158 510 226 3.49±.13 .0165 520 235 4.01±.13 .0182 530 218 4.09±.09 .0200 540 195 4.22±.18 .0231 550 175 4.30±.08 .0268 560 149 4.38±.20 .0313 570 125 4.54±.08 .0387 580 100 4.66±.20 .0497 590 74 4.81±.ll . 0694 600 50.5 4.84±.17 .102 610 33.2 4.87±.17 .1565 620 22.2 4.96±.17 .238 630 15.1 4.95±.13 .350 640 11.2 5.16±.20 .491 * See Appendix -35- at 550 nm v a r i e s l e s s than 10% and u s u a l l y not more than 4%. Absolute and 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 readings are t a b u l a t e d i n Table I I I . The absolute c a l i b r a t i o n i s only accurate to ± 20%. the r e l a t i v e c a l i b r a t i o n s to ± 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 to a r r i v e at a curve f o r the r e l a t i v e i n t e n s i t y at the top of the cone to that at the bottom, I T / I B ( F i g . 6). A second curve shown i n the same diagram was 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 at 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, I T / * P S D » 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 should be constants r e l a t i n g areas, the curves should by i d e n t i c a l , such constants c a n c e l l i n g i n the c a l c u l a t i o n . Within experimental e r r o r these curves are i d e n t i c a l . (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 PSD The response of the PSD t o i n c r e a s i n g i n t e n s i t y of. the reference beam i s shown i n F i g . 7. T h i s c a l i b r a t i o n was 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 f i l t e r s . The response of the PSD was found to m i r r o r t h a t of the PbS d e t e c t o r as measured by an O s c i l l o s c o p e . Thus.the non- l i n e a r i t y of response wi t h i n t e n s i t y i s produced by the PbS d e t e c t o r , not the e l e c t r o n i c s connected w i t h the PSD. TABLE I I I Phase S e n s i t i v e D e t e c t o r Wavelength S e n s i t i v i t y Wavelength, S p e c t r a l S e n s i t i v i t y , Q* nm jaw/mv 400 0.134 410 0.132 420 0.131 430 0 .175 440 0.128 450 0.126 460 0 .127 470 0.123 480 0 .122 490 0 .119 500 0.121 510 0 .115 520 0 .119 530 0 .118 540 0.113 550 0 .115 560 0.113 570 0 .108 580 0 .110 590 0.106 600 0 .107 610 0.108 620 0.106 630 0 .092 640 0 .089 * See Appendix 12, 33 m | . | | > H m I OF H m H -< 0-8 0-7 0 6 0 5 400 _| I ' * • I 1 1 I I Hi 500 600 W A V E L E N G T H F i g u r e 6 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 F i g u r e 7 I n t e n s i t y Response of 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 Relative Intensity -36- IV-3 Actinometer C a l i b r a t i o n s An attempt was made to perform a more accurate c a l i b r a t i o n of the i n t e n s i t y at the top of the cone using a potassium f e r r i o x a l a t e actinometer. The technique as described by Hatchard and Parker (1956) i s good for wavelengths up to 450 nm i f a 0.15 M solu t i o n i s used. In these experiments a solu t i o n thick enough to absorb a l l of the l i g h t emerging from the cone was used. I r r a d i a t i o n of the solution was c a r r i e d out for periods up to three hours. Following t h i s phenanthroline monohydrate and buffer were added and o p t i c a l density difference readings taken between the sample and a blank. The o p t i c a l density of the sample treated i n t h i s manner seldom reached 0.1 and the difference between the two was usually l e s s than 0.05. The low values resulted because of the low output of the tungsten lamp i n that spectral region. With such low values i t was impossible to get reproducible r e s u l t s , i n part because of the scattered l i g h t and also l i k e l y due to fl u c t u a t i o n s i n the lamp output over the long i n t e r v a l s of time required for the exposure. Thus i t was found that i n t h i s s i t u a t i o n t h i s chemical actinometer could not be used. -37- CHAPTER 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 experiments were prepared as i n Sec. I I I - 1 . 3 . A r e c o r d i n g of oxygen c o n c e n t r a t i o n versus time was made over the p e r i o d r e q u i r e d f o r the N i t r o b a c t e r to consume a l l of the oxygen i n s o l u t i o n . From the c a l i b r a t i o n s and the recorded curves of oxygen c o n c e n t r a t i o n versus time the r a t e of oxygen uptake versus oxygen c o n c e n t r a t i o n was c a l c u l a t e d . P l o t s of v e l o c i t y , v, versus s u b s t r a t e c o n c e n t r a t i o n , ["̂ 2] » a n t* 0 1 l / v v s l / p 2 ] a r e shown i n f i g u r e s 8 and 9 r e s p e c t i v e l y . From these curves the M i c h a e l i s constant, Kffl, f o r oxygen as s u b s t r a t e ranges from 0.021 to 0.055 mM 0 2 depending on the c o n c e n t r a t i o n of n i t r i t e . The r e s u l t s are recorded i n Table IV. Table IV K Values f o r N i t r o b a c t e r with Oxygen as Substrate P r e p a r a t i o n Procedure K m Values v vs s , mM O2 1/v vs 1/s 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 3 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 F i g u r e 8 E f f e c t o f Oxygen C o n c e n t r a t i o n on Rate o f N i t r i t e O x i d a t i o n "» 1 i i 9 1 { e- F i g u r e 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 of F i g . 8 -38- V-2 Results from the Action Spectrum Apparatus 2.1 Procedure for Obtaining Results The prepared sample was placed i n the chamber as described i n Sec. III-2.4. The electrodes were polarized and a f t e r a period of about 15 min the current,recorded on an E s t e r l i n e Angus recording milliammeter, reached i t s equilibrium value. At t h i s time when l i g h t was switched onto the sample, the current throught the electrodes decreased to a new equilibrium value. The reason for t h i s change i s that the l i g h t breaks up the carbon monoxide-oxidase complex, thus allowing the oxidase to react with oxygen. This r e s u l t s i n a lower oxygen concentration i n the drop and a lower electrode current. The dark current values ranged from 20-40 nA. A change from dark to the reference beam (with a heat f i l t e r , Corning aklo no. 97, and a green band pass f i l t e r to l i m i t the s p e c t r a l range) decreased the current 3-5 nA. A greater s e n s i t i v i t y i n the form of larger absolute current changes was obtained with lower values of the dark current. A l l s l i t widths were 1 mm and the InA scale was used for the current readings with the remainder of the current bucked out by and external c i r c u i t . A t y p i c a l change from dark to l i g h t and vice versa i s shown i n figure 10. The CO action spectrum i s a plot 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 %-\\/ £\2 °^ t n e enzyme-CO compound with wavelength. The reference wavelength i s usually taken as 550 nm. As shown by Brooks, the r a t i o i s given by F i g u r e 10 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 of N i t r o b a c t e r -39- the formula: *X °X W55Q 5 5 0 ^550 " Wx C 5 5 0 X i s the i n t e n s i t y of the comparison l i g h t which gives the same photochemical e f f e c t s as the monochromatic l i g h t of wavelength, X . C"550 i s the i n t e n s i t y of comparison l i g h t which gives the same photochemical e f f e c t as 550 nm. i s the i n t e n s i t y of X WgejQ i s the i n t e n s i t y of 550 nm l i g h t . In p r a c t i c a l terms t h i s means that switching from the reference beam with i n t e n s i t y C^^Q to the 550 nm monochromatic beam produces no change i n the electrode current. The i n t e n s i t i e s which cause equal e f f e c t s were determined by f i r s t shining the l i g h t of wavelength, X , on the sample and aft e r e q u i l i b r a t i o n switching to the reference beam. If the current decreased, the reference l i g h t was too intense and the louvers were adjusted. This operation was repeated u n t i l no change was observed on switching. Since there was usually a d r i f t i n the current, a decrease or increase i n current was indicated by a change i n the slope of the d r i f t ; The r e s u l t i n g trace of current with time i s shown i n F i g . 11. The estimation of the balance point could only be made within 1 m i l l i v o l t of the i n t e n s i t y of the reference l i g h t since the smalfest turn of the louvers during the operation corresponded F i g u r e 11 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 R e f e r e n c e L i g h t , u. -40- to a 1 mv i n t e n s i t y change. The average i n t e n s i t y of the reference beam i n these experiments was 67 mv. Thus the uncertainty i n the balance point i s 1 i %. 2 Once the balance point was determined the i n t e n s i t i e s of the reference and monochromatic beams were measured and the temperature of the PbS detector taken. The balance for the i n t e n s i t y measurements was made on the 10 mv scale of the Keithley microvoltraeter-ammeter with the bucking voltage supplied by an external c i r c u i t (cf. F i g . 2). The PbS readings were corrected to 24°C and 10 x gain on the PSD. The gain factors were 2.57 for the three times scale and 0.324 for the 30 times scale. The i n t e n s i t y of the monochromatic beam was corrected to give the i n t e n s i t y at the top of the cone, i . e . corrected for PbS wavelength s e n s i t i v i t y and cone transmission. Since the response of the PbS detector i s not proportional to the i n t e n s i t y f a l l i n g on i t a correction to the i n t e n s i t y of the comparison beam i s also required. - A sample c a l c u l a t i o n of S / £ . , . i s outlined below for X 550 A=600 nm. DATA w550 C550 W600 C600 PSD Readings (mv) 31.7 84.4 36.2 85.75 Temp, (mv) 0.96 0.96 1.00 1.00 Temp, corrected PSD readings * 30.9 82.3 37.2 88.1 CALCULATION ^ 600 88.1 x 30.9 x 550 S 5 3 37.2 x 82.3 x 600 -41- =0.815 From F i g . 7, the correction for the l i n e a r i t y of the PbS detector i s 1.02 and from F i g . 6, I T / I p s D for X = 600 nm i s 1.067. Therefore* 6 0 2 0.815 x 1.02 x 1.067 S 550 = 0.890 * See Appendix. 2.2 Results The r e s u l t i n g values of £ ^ / £ are displayed i n figures 12 and 13. The f i r s t , F i g . 12, shows the r e s u l t s obtained with two preparations of Nitrobacter. These r e s u l t s were picked to be shown i n t h i s manner because of t h e i r s e l f - consistency. Results obtained were usually more consistent within preparations than between preparations. The second graph, F i g . 13, i s a graph of a l l those points which I f e l t were cr e d i b l e . The reasons for including these points and not others that I took are the following: i ) Once the Nitrobacter in the chamber started to die the current increased at an accelerated rate. As t h i s happened, the determination of the balance point became more and more uncertain. Points obtained with a fast d r i f t were thus usually discarded. i i ) E r r a t i c response of some preparations meant that balance could only be obtained on the 3 nA   -42- . scale. This was l a t e r discovered not to be a sensit i v e enough determination and the points were disregarded. i i i ) Some preparations gave r i s e to only one point on the graph of £ / € VS X. I f , coincident with t h i s the t o t a l change from dark to l i g h t was less than 2 nA, these points were also discarded. 2.3 Errors i n the Relative 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 £ , / £ i s the sum of the X 550 inherent errors and uncertainties i n each of the terms i n the equation. i ) The determination of the balance point i s accurate to 1 ̂  (cf. Sec. V-2.1). Since there are two such balances required for determina- ti o n of a point the error introduced i s 3%. i i ) Brooks shows that the c a l c u l a t i o n of the temp- erature 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 introduces a 1% uncertainty i n the monochromatic i n t e n s i t i e s with much less than 1% error i n the reference beam. i i i ) V a r i a t i o n i n output of the lamp and i n the gain l e v e l s of the PSD introduce a 5% uncertainty i n the values of the r e l a t i v e i n t e n s i t y at the top of the cone divided by those measured by the PbS and phase sens i t i v e detectors. -43- The accuracy of € / £ 550 i s thus W 550 ± 6% 550 S 550 W x ± 6% C + 3% 550 X making the l i m i t of accuracy + 15%. On the graph, F i g . 13, the error bars are the 80% confidence l i m i t s calculated according to Wilson (1952, pg.239). They are larger than the 15% calculated above, i n some instances. The reason for t h i s discrepancy l i e s i n the necessity of using d i f f e r e n t samples of Nitrobacter. Thus while each point i s accurate to within 15%, the points at the same wavelength in d i f f e r e n t samples of bacteria may not have 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 within 15% of one another. The discus^- sion i n Sec. VI-2 i s relevant to th i s apparent discrepancy. 2.4 Errors i n the Positions of the Peaks The peaks at 430 and 591 nm can be more accurately determined because of t h e i r steepness. (See Castor and Chance, 1955) Thus the error i n these peaks i s - 2 nm of which 1 nm error i s introduced i n the determination of the wavelength delivered at the ex i t s l i t by the monochromator. The peak at 540-550 nm i s much broader and therefore harder to outline. Added to t h i s the l i n e of average points i s not continuous introducing further estimation errors. Thus the error l i m i t on the two apparent peaks at 541 and 550 nm i s - 5 nm. -44- CHAPTER VI DISCUSSION VI-1 Oxygen Electrode Results The v a r i a t i o n of VmSLX ^(Og) has been reported and well documented by Butt and Lees (1964). The maximal rates reported by these authors were found to increase and then decrease with increasing n i t r i t e concentration. The same was true of the K m(02). They reported K m values between 2 and 7.5 % Og i n the gas phase. Comparable r e s u l t s were obtained i n these experiments with K m values between 0.021 and 0.055 mM (1.75 and 4.6 % 0 i n the gas phase). Samples for which rates were obtained above 20% showed a decrease i n rate with increasing Og. This i s not due to the establishment of the p o l a r i z a t i o n voltage which takes several minutes to es t a b l i s h i n the sample chamber. This process r e s u l t s i n an apparent increase i n the rate. Thus the e f f e c t s are possibly one of two: 1) a substrate i n h i b i t i o n , or 2) that the c e l l s had not recovered from centrifugation and exhibited a lower rate u n t i l complete recovery was reached. (Note the 30 min wait required i n the action spectrum experiments, Sec. III-2.4 ) This l a t t e r e f f e c t i s very d i f f i c u l t to deal with since the e f f e c t s of centrifugation vary from sample to sample even though the previous hi s t o r y appears to have been i d e n t i c a l . Maximal rates reported by Butt and Lees ranged from -45- 1.9 to 2.7 micromoles oxygen/vessel/hr where each vessel contained 3 ml of suspension. Values obtained for 7 ml of suspension i n these experiments ranged from 0.7 to 3 jxmoles/vessel/hr. These rates are lower but of the same order of magnitude as those of Butt and Lees, the discrepancy due probably to a difference i n the concentration of c e l l s i n the suspension but perhaps also due to the method of measurement. VI-2 Action Spectrum Results Comparison of the peak positions obtained here at 430, 540-50, and 591 nm with those obtained by the same methods on other organisms (Castor and Chance, 1959) show that these peaks indicate the presence of either cytochrome o r i*3> peaks being reported at 427-8, 548 and 591-2 nm for cytochrome a_̂  and at 430-2, 547-50 and 585-91 for cytochrome a Q . Absorption spectra of Nitrobacter report peaks at 586-94 and 438 showing an a_̂  rather than an a^ cytochrome, (cf. Sec 1-5.1 and 1-5.2) Carbon monoxide reduced minus reduced difference spectra (Sec. 1-5.3.1) implicated cytochrome a i i n the binding of CO with peaks at 450 and 426 and troughs at 439 and 594 nm. Although Van Gool and Laudelout report no peak around 590, Castor and Chance (1953) reported a peak at 590 nm for cytochrome aj i n a CO difference spectrum of A. pasteurianum. None of the peaks shown i n t h i s action spectrum i s as well defined as those shown by Castor and Chance (1959) -46- They were able to determine the points on t h e i r spectra con- secutively and " e f f o r t s to return l a t e r to a given wavelength showed a r e p r o d u c i b i l i t y of about ± 5%". This was with responsive organisms such as E_. c o l i . Nitrobacter i s however a much more se n s i t i v e organism with a short 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 reproduci- b i l i t y and necessitate t r y i n g to obtain an outline of a peak with one set of Nitrobacter rather than t r y i n g to define the peak i n d e t a i l using many sets of Nitrobacter. This l a t t e r approach usually resulted i n a poorly defined peak. Comparison 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 of the peaks for cytochrome a^ bands of the action spectrum of A. pasteurianum (Castor and Chance, 1953) gives values of 2.35 and 21 for the 592 and 428 peaks. Values of € ^/ £ 5 5 0 for the same peaks i n t h i s work were 1.68 and 9.8. The r a t i o of these values i s 0.112 for Castor and Chance and 0.168 for t h i s work. The error i n the 0.112 determination i s of the order of 10%, that i n the 0.168 i s 30%. Thus the two values are the same within experimental error. The cause of the difference 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 <>. No diagram of the ft, 548 band for A. pasteurianum was given nor are there any other values 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 for cytochrome a^ i n other organisms. Thus i t i s not possible to make an extensive comparison of these values for s i g n i f i c a n t differences. Castor and Chance (1959) showed that when two oxidases are present i n a c e l l , e i t h e r pigment appears capable of -47- catalyzing most of the r e s p i r a t i o n . This experiment was performed by il 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 . Thus (Horio and Taylor, 1965J when two or more oxidases operate i n p a r a l l e l the action 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 possible presence of other cytochromes can not be ruled out. This i s e s p e c i a l l y evident i n the peak at 540-550 nm. The displacement of t h i s peak to lower wavelengths could be due to the presence of a second cytochrome with a peak at or below 540 nm. The major candidate i s a cytochrome o_ peak at 535-7 nm. The presence of cytochrome o_ would also require an i n d i c a t i o n of peaks at 416-7 and 566-7. These are not evident in the spectrum displayed. If cyt. <> i s present, the reason for the absence of these two peaks could be one of two e f f e c t s s The f i r s t i s that the peaks are masked by the cytochrome a^. The e f f e c t on the |J peak would seem to indicate the presence of cytochrome o_ i n large enough quantities to a f f e c t the spectrum more d r a s t i c a l l y i n the region of 415-430 nm. Thus masking does not seem to be the answer. The second possible explanation l i e s i n a difference i n the c e l l s used to determine the points. Castor and Chance (1959) showed d i f f e r e n t cytochrome oxidase a c t i v i t i e s i n stationary and log phase c e l l s . Thus i t i s possible that c e l l s containing no cytochrome o_ or containing a cytochrome <3 with a very low a c t i v i t y were used to determine the a and y -48- peaks but that the S peak was p a r t i a l l y determined with c e l l s containing and active cytochrome o_. Although absorption spectra give no hints of cytochrome £, there i s no reason to rule i t out because the present method i s much more s e n s i t i v e . Thus i t can be stated that cytochrome i s acting as a terminal oxidase i n Nitrobacter a g i l i s and that there i s a p o s s i b i l i t y that other cytochromes may also act as oxidases i n c e r t a i n c e l l s . If cytochrome o_ does act as a terminal oxidase, a study of log phase c e l l s i s very l i k e l y to show th i s as a l l of the types of organisms studied by Castor and Chance (1955 and 1959) which showed some cytochrome jo a c t i v i t y i n the stationary phase showed only cytochrome o_ a c t i v i t y i n the log phase of growth. Action spectra r e s u l t s tabled by Castor and Chance (1959) implicate cytochrome a^ as an oxidase i n four bacteria, a l l heterotrophs. They are Acetobacter pasteurianum, Acetobacter peroxydans, Azotobacter v i n e l a n d i i , and stationary phase Proteus v u l g a r i s . A. v i n e l a n d i i also showed cytochrome o a c t i v i t y , P. vulgaris also showed cytochrome <> and a c t i v i t y i n the stationary phase and showed only cytochrome o_ a c t i v i t y i n the log phase. To date, only cytochrome a^ has not been shown to be active i n c e l l s with active cytochrome a^. A generalized statement about the associations of oxidases can not be made, however, u n t i l more organisms have been studied i n t h e i r d i f f e r e n t phases of growth. The spectrum reported here i s the f i r s t recorded action spectrum of a chemolithotropic bacterium. -49- VI-3 Summary (i ) A survey of the l i t e r a t u r e on Nitrobacter was performed and the unsolved problems pointed out. ( i i j Modifications to the action spectrum apparatus are described and the performance of the apparatus assessed. ( i i i ) The K m ( 0 2 ) Of Nitrobacter a g i l i s varies from 0.021 to 0.055 mM 0 2 depending on the n i t r i t e concentration i n agreement with the data of Butt and Lees (1964). (iv) Cytochrome a^ acts as a terminal oxidase i n Nitrobacter a g i l i s . (v) P a r a l l e l p a r t i c i p a t i o n of other cytochromes as oxidases i s not ruled out. A dis t o r t e d B band suggests the presence of cytochrome o i n some cultures. -50- BIBLI0GRAPHY 1. Aleem, M. I. H., 1965, Biochimica et Biophysica Acta 107, 14 Path of Carbon and Assimilatory Power i n Chemosynthetic Bacteria 1. Nitrobacter a g i l i s 2. Aleem, M. I. H. and M. Alexander, 1958, J. Bacteriology 76, 510 C e l l - f r e e N i t r i f i c a t i o n by Nitrobacter 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 in B a c t e r i a l Chemosynthesis 4. Aleem, M. I. H., H. Lees and D. J. D. Nicholas, 1963 Nature 200, 759 ATP Dependent Reduction of NAD by Ferro-cytochrome c_ i n Chemoautotrophic Bacteria 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 Electron Transport System from Nitrobacter 6. Aleem, M. I. H. and A. Nason, 1960, Nat'l Acad. S c i . Proc. 46, 763 Phosphorylation Coupled to N i t r i t e Oxidation by P a r t i c l e s from the Chemoautotroph, Nitrobacter a g i l i s 7. Alexander, M., 1961, Introduction to S o i l Microbiology Wiley and Sons, N. Y. 8. Bock, E. , 1965, Arch. Mikr. 5_1. 18 Vergleichende Unter- suchungen uber die Wirkung sichtbaren Lichtes auf Nitrosomonas europaea und Nitrobacter winogradskyi 9. Bock, E. and H. Engel, 1966, Arch. Mikr. 54, 191 Unter- suchungen uber postoxydatlve C0 2-Fixierung bei Nitrobacter winogradskyi Buch 10. Boon, B. and H. Laudelout, 1962, Biochem. J. J35, 440 Kine t i c s of N i t r i t e Oxidation by Nitrobacter winogradskyi 11. > Brooks, D. E., 1967, MSc Thesis, University of B r i t i s h Columbia An Action Spectrum Apparatus 12. Butt, W. D. and H. Lees, 1960, Biochem. J . 76, 425 The Biochemistry of the N i t r i f y i n g Organisms, Part 6 The E f f e c t of Oxygen Concentration on N i t r i t e Oxidation in the Presence of D i f f e r e n t Inorganic Ions 13. Butt, W. D. and H. Lees, 1964, Can. J. Biochem., 4_2, 1217 The Biochemistry of the N i t r i f y i n g Organisms, Part 8 The E f f e c t s of Oxygen Tension, N i t r i t e Concentration and Cyanate Concentration on N i t r i t e Oxidation by Nitrobacter -51- 14. Castor, L. N. and B. Chance, 1955, J. B i o l . Chem. 217, 453 Photochemical Action Spectrum Of Carbon. Monoxide Inhibited Respiration 15. Castor, L. N. and B.-Chance, 1959, J. B i o l Chem. 234, 1587 Photochemical Determinations of the Oxidases of Bacteria 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. Microbiology, (5, 299 The Iso l a t i o n and Culture of the N i t r i f y i n g Organisms Part I Nitrobacter 18. Hatchard, C. G. and C. A. Parker, 1956, Royal Society of London, Proc. A, 235, 518 A New Sensitive Chemical Actinometer I I . Potassium Ferrioxalate as a Standard Chemical Actinometer 19. Horio, T. and C. P. S. Taylor, 1965, J. B i o l . Chem. 240, 1772 The Photochemical Determination of an Oxidase of the Photoheterotroph, Rhodospirilium rubrum,. and the Action Spectrum of the Inh i b i t i o n of Respiration by Light 20. Krulwich, T. A. and H. B. Funk, 1965, J . Bact. 90, 729 Stimulation of Nitrobacter a g i l i s by B i o t i n 21. Lees, H. 1955 The Biochemistry of Autotrophic Bacteria Butterworths 22. Lees, H. 1960, Ann. Rev. Microbiology, 14, 83, Energy Metabolism i n Chemolithotropic Bacteria 23. Lees, H. 1962, Bact. Revs. 26, 165 Symposium on Autotrophy IV Some Thoughts on the Energetics of Chemosynthesis 24. Lees, H. and J. R. Simpson, 1957, Biochem. 6j3, 297 The Biochemistry of the N i t r i f y i n g Organisms, Part 5, N i t r i t e Oxidation by Nitrobacter . 25. Muller-Neuglucit, M. and H. Engel, 1961, Arch. Mikr. 39, 130 Photoinaktivierung von Nitrobacter winogradskyi Buch v 26. Seeler, G. and H. Engel, 1959, Arch. Mikr. 3̂ 3. 387 Die Inaktivierung des Oxydationsvermogens von Nitrobacter winogradskyi Buch -52- 27. S n e l l , F. D. and C. T. S n e l l , 1949, Colorimetric Methods of Analysis, Van Nostrand, N. Y. 28. Van Gool A. and H. Laudelout, 1965, Biochim. et Biophys, Acta, 113, 41 The Mechanism of N i t r i t e Oxidation by Nitrobacter winogradskyi 29. Wilson, E. B., 1952, An Introduction to S c i e n t i f i c Research, McGraw-Hill, N. Y. -53- APPENDIX C a l c u l a t i o n s P e r f o r m e d i n C a l i b r a t i n g the A c t i o n Spectrum A p p a r a t u s In t h i s s e c t i o n a sample c a l c u l a t i o n 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 o f the cone i s performed f o r the wave l e n g t h 600 nm. A b b r e v i a t i o n s : Pm, P h o t o m u l t i p l i e r r e a d i n g , jxA Tp, T h e r m o p i l e r e a d i n g , |iV PSD, Phase 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 (Pm) P h o t o m u l t i p l i e r r e a d i n g a t the t o p o f the cone a t a Top,A. w a v e l e n g t h , X ( P n ^ B o t t ^ P h o t o m u l t i p l i e r r e a d i n g a t the bottom o f the cone ' a t a w a v e l e n g t h , X ( T p ) B o t ; t ^ T h e r m o p i l e r e a d i n g a t the bottom of the cone a t ' a w a v e l e n g t h , X TABLE V Data R e q u i r e d f o r the C a l c u l a t i o n o f 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 X nm (Pro)Top jiA PSD rav T e m p p s D rav < P n , ) B o t t |iA < TP>Bott jiV 550 339 28.4 .962 175 4.30 600 108 32.8 .990 50.5 4.84 A r e a o f measurement .0256 .04 .0576 ( s q . cm.) T h e r m o p i l e C a l i b r a t i o n : 0.054 \iV/ jxw/ cm -54- Temperature Correction: log S = -2.4 x 10" 2 T At 24°C the thermocouple reading i s 0.98 mv and the slope of the c a l i b r a t i o n graph i s 24.4 C° per mv. Thus, log S 2 - log S! = -2.4 x 10" 2 x 24.4 (0.98 - X) where, X = thermocouple reading at T j 51 = PSD sig n a l at T x 5 2 = PSD s i g n a l at 24°C Application of t h i s formula leads to a change i n the PSD readings, from 28.4 to 27.7 and from 32.8 to 33.7 mv. Photomultipiier Correction for Wavelength S e n s i t i v i t y The correction factor for the photomultiplier readings at a wavelength, X, are given by Y below: ( Tp)Bott,X ^ V .0576 Cm2 .054 |iV/|iw/ cm* x (x> s A ( pra)Bott,X H A Y = = .0268 HW/JJLA at 550 nm = .102 |xw/|iA at 600 nm (cf Table II and F i g . 14) PSD Correction for Wavelength S e n s i t i v i t y The correction factor, Q, for the wavelength s e n s i t i v i t y of the phase sens i t i v e detector i s given by:-.. <TP>Bott,X ^ ' ° 4 C n ) 2 Q = ,054 iiV/iiW/cnT PSD X mv = 0.115 [iw/mv at 550 nm = 0.107 |xw/mv at 600 nm (cf Table III and F i g . 15) Since the phase se n s i t i v e detector and thermopile F i g u r e 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 Tube F i g u r e 15 S p e c t r a l S e n s i t i v i t y o f Phase S e n s i t i v e D e t e c t o r 1 4 0 CO "D m o H 130 > r~ to m 120 CO H < H . -< 1 10 o 10 0 3 < • 0 9 0 •080 -I 1 1 1 1 T" T T ' ^ 1 8 8 « s *_ 1 L a » « • 4 0 0 5 0 0 6 0 0 680 WAVELENGTH -55- readings are not taken at the same position there are factors r e l a t i n g the differences i n areas, position and e f f e c t s of the spherical mirror and tuning fork which are necessary.if these numbers, by themselves are to mean anything. However, since a l l these factors are constant with wavelength, a r e l a t i v e c a l i b r a t i o n with reference to a common wavelength, 550 nm, i s meaningful. Calculation of I T / Ig (Fig. 6) I T Relative Intensity at the Top of the Cone I B Relative Intensity at the Bottom of the Cone x Yx / ( P m ) T o P ) 5 5 o x Y 5 5 0 x Y 7 / ( P m ) B o t t ) 5 5 0 x Y550 For X 600 nm, 108/339 I T / I B ~ 5 0 . 5 / 1 7 . 5 = 1.105 Calculation of I T / IpsD 6 ) Relative Intensity at the Top of the Cone Relative Intensity at Bottom of Cone as measured by the PSD ( P m ) T o p > x x Y x / ( P r c ) T o p > 5 5 Q x Y 5 5 Q < P S D J B o t t , X x ®\ / ( P S E ))Bott,550 x Q550 = 1.067 at 600 nm Thus as noted i n Sec. IV-1, the values of I T/IpsD and I T / I g d i f f e r by 4% and are, within experimental error, - 5 6 - i d e n t i c a l . The number I T / I p S D = 1 . 0 6 7 means t h a t i f the i n t e n s i t i e s o f the two beams a t the bottom o f t h e cone a r e i d e n t i c a l , the 6 0 0 nm l i g h t w i l l be 1 . 0 6 7 t i m e s as i n t e n s e as the 5 5 0 nm l i g h t a t the t o p of the cone. Thus t o c o r r e c t W 5 5 0 / W Q Q O i t i s n e c e s s a r y t o m u l t i p l y by 1 . 0 6 7 .

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