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UBC Theses and Dissertations

Studies of microbial hydrocarbon fermentations Guthrie, Donald James 1972

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STUDIES OF MICROBIAL HYDROCARBON FERMENTATIONS by DONALD JAMES GUTHRIE B . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Department o f Food S c i e n c e 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 t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September, 1972 In presenting 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 m e n t of the requirements for 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 fu 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 representatives. 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 for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Food Science The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8 , Canada ABSTRACT A two part study of hydrocarbon u t i l i z a t i o n by microorganisms was undertaken. In the f i r s t part i t was decided to attempt the genetic transformation of B a c i l l u s s u b t i l i s with respect to the a b i l i t y to grow on hydrocarbon carbon source. One hundred f o r t y - f o u r cultures of B a c i l l u s organisms were i s o l a t e d on a medium with r e f i n e d kerosene as the sole carbon source. Most of these cultures were found to be B. cereus (92 i s o l a t e s ) , B. lentus or B_. firmus (15 i s o l a t e s ) and B. megaterium (8 i s o l a t e s ) . Neither of the 2 s t r a i n s i d e n t i f i e d as B. s u b t i l i s were capable of s u f f i c i e n t growth on kerosene medium, even with complex supplements added, to warrant a transformation experiment. In the second part of this.study, a cu l t u r e which was c l a s s i f i e d as a member of the genus Arthrobacter was investigated f or i t s a b i l i t y to grow r a p i d l y on hydrocarbons. Dodecane was shown to be the n-alkane u t i l i z e d most r e a d i l y . The a c i d i t y produced by t h i s culture was not due to formation of f a t t y acids, other organic acids or amino acids and was a t t r i b u t e d to the a c i d i t y generated by u t i l i z a t i o n of the ammonium nitrogen source. V7hen grown i n a fermentor with pH c o n t r o l , the Arthrobacter spp. oxidized ammonia forming n i t r a t e and nitrogen oxide gases when the dodecane carbon source was exhausted. This i s the f i r s t time ammonia oxidation has been reported i n an n-alkane fermentation system. i v TABLE OF CONTENTS Page PART I. INVESTIGATION OF KEROSENE UTILIZING BACILLUS SUBTILIS STRAINS INTRODUCTION 1 LITERATURE REVIEW ' 3 MATERIALS AND METHODS 9 RESULTS AND DISCUSSION 13 PART I I . UTILIZATION OF N-ALKANES BY AN ARTHROBACTER SPP. INTRODUCTION 19 LITERATURE REVIEW C l a s s i f i c a t i o n 23 Mi c r o b i a l Hydrocarbon U t i l i z a t i o n 25 U t i l i z a t i o n of Hydrocarbons by Arthrobacter 28 Oxidation of Ammonia 28 MATERIALS AND METHODS Cha r a c t e r i z a t i o n of Hydrocarbon S p e c i f i c i t y 32 Cha r a c t e r i z a t i o n of Acid Production 34 Materi a l s Balance Experiments 36 RESULTS AND DISCUSSION C l a s s i f i c a t i o n 41 Cha r a c t e r i z a t i o n of Hydrocarbon S p e c i f i c i t y 42 Materi a l s Balance Experiment 57 CONCLUSIONS 65 BIBLIOGRAPHY 66 LIST OF TABLES Table Page I I d e n t i f i c a t i o n of B a c i l l u s s u b t i l i s Cultures 14 II Normal Alkanes i n Kerosene 45 v i LIST OF FIGURES Figure Page 1 P a r t i a l Key for B a c i l l u s Organisms.- 7 2 E f f e c t of Malt Extract on the Growth of B. S u b t i l i s 17 s t r a i n 1 i n Kerosene Medium. 3 E f f e c t of S o i l E x t r a c t on the Growth of B_. s u b t i l i s 17 s t r a i n 1 i n Kerosene Medium. 4 E f f e c t of Malt Extract on the Growth of B. s u b t i l i s 18 s t r a i n 2 i n Kerosene Medium. 5 E f f e c t of S o i l Extract on the Growth of B_. s u b t i l i s 18 s t r a i n 2 i n Kerosene Medium. 6 Equipment Set-up i n Fermentation Experiments. 38 7 C e l l s of Arthrobacter spp. Grown i n Glucose-Nutrient 43 Broth. 8 Gas Chromatographic Analysis of Fisher Odorless 44 Kerosene. 9 Oxygen Uptake of Arthrobacter Culture on Various 47 n-Alkane Carbon Sources. 10 Oxygen Uptake of Arthrobacter Culture as a 48 Function of n-Alkane Chain Length. 11 pH Drop i n Shake Flask Medium as a Function of 49 n-Alkane Chain Length. 12 Growth i n Shake Flask Culture as a Function of 49 n-Alkane Chain Length. 13 Growth Curve of Arthrobacter Culture on D i f f e r e n t 50 Hydrocarbon Carbon Sources. 14 pH Curve of Medium a f t e r Growth on D i f f e r e n t 51 Hydrocarbons. 15 Thin Layer Chromatogram of the A c i d i c L i p i d 53 Extra c t Sprayed with Bromcresol Green. 16 Thin Layer Chromatogram of the A c i d i c L i p i d 54 Extra c t , Charred with S u l f u r i c Acid. 17 Gas Chromatogram of Methylated Extract from 55 Kerosene Medium. v i i Figur.e Page 18 Thin Layer Chromatogram for the Separation of 56 Organic Acids. 19 Change i n Substrate Concentration and Growth 58 Parameters during Fermentation. 20 Carbon Balance during Fermentation. 60 21 Nitrogen Balance during Fermentation. 61 v i i i ACKNOWLEDGEMENTS I would l i k e to express my gratitude to Dr. P.M. Townsley, Associate Professor, Department of Food Science, for h i s encouragement and many h e l p f u l suggestions during the course of t h i s research and for h i s guidance during the preparation of t h i s t h e s i s . I wish also to thank Dr. W.D. Powrie, Dr. J.F. Richards and Professor T.L. Coulthard for serving on my research committee. This research was supported by the Department of Energy, Mines and Resources of the Government of Canada. PART I. INVESTIGATION OF KEROSENE UTILIZING BACILLUS SUBTILIS STRAINS INTRODUCTION Most i n d u s t r i a l and food fermentations use t r a d i t i o n a l carbohydrate based substrates as t h e i r carbon source. For example, much of the mono-sodium glutamate used i n food products i s produced by Corynebacterium  glutamicum (Micrococcus glutamicus) grown on crude starch hydrolysate or molasses (51). The c i t r i c a c i d fermentation uses sucrose as the substrate for A s p e r g i l l u s niger (81). Because there can be economic advantages i n terms of the raw material purchase p r i c e and i n the ease of product i s o -l a t i o n when using a petroleum f r a c t i o n or a hydrocarbon as the mi c r o b i a l carbon n u t r i e n t source, an increased emphasis has been placed on fermen-t a t i o n s of the hydrocarbon organisms. One approach to the development of hydrocarbon fermentations i s to i s o l a t e cultures which can u t i l i z e hydrocarbons and which produce or have the p o t e n t i a l to produce products when exposed to s p e c i f i c environmental or n u t r i t i o n a l conditions. Another approach to fermentation research i s to s e l e c t an organism which forms a us e f u l product from carbohydrate and to convert i t through genetic mani-p u l a t i o n so that the modified organism can u t i l i z e hydrocarbons f o r growth and product formation. In t h i s experiment i t was decided to use the l a t t e r concept. I t has been shown that c e r t a i n b a c t e r i a l organisms can accept into the c e l l deoxyribonucleic acid (DNA) from c l o s e l y r e l a t e d b a c t e r i a l s t r a i n s and incorporate t h i s DNA in t o t h e i r own genetic make-up. This process, termed "transformation" i n which an i n h e r i t a b l e character of a DNA molecule i s t r a n s f e r r e d , has been extensively studied for B a c i l l u s  s u b t i l i s (97). Because a number of ind u c i b l e enzyme systems have been t r a n s f o r m e d u s i n g B. s u b t i l i s ( 9 6 ) , i t was f e l t t h a t t h e enzyme o r enzymes f o r h y d r o c a r b o n o x i d a t i o n m i g h t a l s o be t r a n s f o r m a b l e . There a r e a number o f s t r a i n s o f t h e s p o r e f o r m i n g r o d , B. s u b t i l i s w h i c h a r e used t o make p r o d u c t s o f c o m m e r c i a l i n t e r e s t t o t h e f o o d i n d u s t r y , s u c h as amylase w h i c h i s used i n t h e b a k i n g i n d u s t r y and i n o s i n e w h i c h i s us e d as a f o o d f l a v o u r e n h a n c e r . S i n c e t h e p u r p o s e o f t h i s i n v e s t i g a t i o n was t o a t t e m p t t o demon-s t r a t e t h e t r a n s f o r m a t i o n o f t h e g e n e t i c c h a r a c t e r r e s p o n s i b l e f o r h y d r o c a r b o n u t i l i z a t i o n , a r e a d i l y d e t e c t a b l e p r o d u c t from t h e c a r b o -h y d r a t e u t i l i z i n g B. s u b t i l i s was d e s i r a b l e . Because o f t h e ease o f d e t e c t i n g a n t i b i o t i c s q u a n t i t a t i v e l y , a s t r a i n o f B_. s u b t i l i s (ATCC 14593) w h i c h p r o d u c e s t h e a n t i b i o t i c b a c i t r a c i n was s e l e c t e d f o r t h e f i r s t e x p e r i m e n t s . B a c i t r a c i n i s a p o l y p e p t i d e a n t i b i o t i c a c t i v e a g a i n s t many Gram p o s i t i v e and a few Gram n e g a t i v e o r g a n i s m s ( 4 8 ) . S i n c e b a c i t r a c i n can be p r o d u c e d by o r g a n i s m s grown on a c e t a t e c a r b o n s o u r c e ( 4 7 ) , i t i s p o s s i b l e t h a t a s t r a i n t r a n s f o r m e d t o h y d r o c a r b o n u t i l i z a t i o n w o u l d s t i l l f o rm a n t i b i o t i c s i n c e a c e t a t e i s t h e normal p r o d u c t o f t h e b a c t e r i a l d e g r a d a t i o n o f a l k a n e s . 3 LITERATURE REVIEW Transformation of B a c i l l u s s u b t i l i s Genetic transformation i n ba c t e r i a i s the uptake of di s s o l v e d deoxyribonucleic acid (DNA) by an organism and the incorporation of t h i s DNA into the genetic material of the organi sm. The occurrence of transformation i n a culture i s shown by a change i n the i n h e r i t a b l e c h a r a c t e r i s t i c s of that p a r t i c u l a r s t r a i n of organisms. Transformation has been extensively reviewed by Spizizen et. a l . (97), Schaeffer (86), and Ravin (84). I t was f i r s t reported i n the c l a s s i c studies of Avery et. a l . i n 1944 (12). They showed that the addition of high molecular weight DNA from a s t r a i n of Diplococcus pneumoniae which formed smooth Type III capsular polysaccharide to an a c t i v e l y growing c u l t u r e of rough Type II pneumococci r e s u l t e d i n a small proportion of the l a t t e r popu-l a t i o n being converted to Type I I I . The proof that t h i s change was h e r i t a b l e and that i t r e s u l t e d from the uptake of p u r i f i e d DNA, free from p r o t e i n and r i b o n u c l e i c acid (RNA) was one of the f i r s t conclusive demonstrations that DNA i s the material involved i n genetic inheritance. Since that time transformation has been shown to occur for many c h a r a c t e r i s t i c s i n species i n other genera, such as drug resistance and capsular polysaccharides i n Hemophilus influenzae ( 4 , 5 ) and D. pneumoniae (12*50)/ and n u t r i t i o n a l independence, i n d u c i b l e enzyme forming a b i l i t y and a n t i b i o t i c production i n B a c i l l u s s u b t i l i s (94,96, 18)• Although transformation occurs most r e a d i l y when both the DNA donor and r e c i p i e n t s t r a i n s are i n the same species, that i s , when there i s a high degree of homology between the DNA of the two s t r a i n s , i t can also occur between d i f f e r e n t species i n the genera Hemophilus (87), 4 B a c i l l u s (73), N e i s s e r i a , (21)and Rhizobium (15). Transformation between the genera Streptococcus and Diplococcus and between Streptococcus and Staphylococcus i n d i c a t e how c l o s e l y these organisms are r e l a t e d (19). Transformation i n the genus B a c i l l u s was f i r s t reported by Spizizen i n 1958 (94). He showed that three auxotrophic mutant s t r a i n s of B a c i l l u s s u b t i l i s could be converted to n u t r i t i o n a l independence by the a d d i t i o n of wild s t r a i n - DNA under s u i t a b l e conditions. S t r a i n 168, which required indole or tryptophan had a p a r t i c u l a r l y high rate of transformation when wild type DNA was added. Further i n v e s t i g a t i o n s by S p i z i z e n and h i s co-workers (95, 96, 8, 115, 116) using t h i s s t r a i n demon-strated the optimum conditions for transformation. I t was found that c e l l s d i s p l a y t h e i r greatest competence, or a b i l i t y to take up DNA, at a d e f i n i t e stage i n the growth c y c l e , i . e . i n the l a t e logarithmetic phase. This competent period i s r e l a t e d to changes i n the c e l l wall during presporulation. Competent c e l l s are produced by growing them four hours i n a glucose-ammonium-salts semi-starvation medium, then harvesting and resuspending them i n transformation medium f o r 90 minutes. There are c e r t a i n s p e c i f i c requirements for t h i s transformation medium. As well as a low concentration of glucose, ammonium ions and s a l t s , there i s a requirement for d i v a l e n t cations, i n p a r t i c u l a r barium, strontium, calcium or magnesium. These are thought to s t a b i l i z e the DNA by n e u t r a l i z i n g the charge on the DNA molecules and p o s s i b l y the net negative charge on the c e l l surface (116). Because z i n c , n i c k e l and e s p e c i a l l y cupric ions tend to i n h i b i t transformation, a c h e l a t i n g agent such as ethylenediaminetetraacetic a c i d or h i s t i d i n e i s added to prevent the l a t t e r d i v a l e n t metal i n t e r f e r e n c e . The optimum temperature and pH 5 ranges for transformation with B_. s u b t i l i s are 34 - 37°C and 6.9 -7.4 r e s p e c t i v e l y . The transformation r e a c t i o n i s p o s s i b l e with a DNA concentration as low as 10 ^ t g / m l . for B_. s u b t i l i s , but i t occurs with greatest frequency at concentrations above l^g/ml. A medium r i c h i n n u t r i e n t s , e s p e c i a l l y with added amino acids, reduces transformation by promoting c e l l wall synthesis. This i s thought to i n t e r f e r e with the binding and uptake of DNA. As well as i n t r a s p e c i f i c transformation of B_. s u b t i l i s , i n t e r s p e c i f i c transformation i n the genus B a c i l l u s has been reported by Marmur et. a l . (73). These workers c o r r e l a t e d the degree of homology i n the DNA base composition between species with the a b i l i t y to transform. In p a r t i c u -l a r , i t was shown that s i m i l a r i t y of o v e r a l l base composition, as i n d i -cated, by the percent quanine plus cytosine, i s a necessary but not s u f f i c i e n t condition for i n t e r s p e c i f i c transformation. B. natto, B. s u b t i l i s var. aterrimus, B. niger, B. s u b t i l i s var. niger and B. polymyxa had s i m i l a r base compositions to B_. s u b t i l i s and were capable of transforming the l a t t e r organism. The DNA from 16 other species of B a c i l l u s could not transform B_. s u b t i l i s . I t should be pointed out that most of these transformations reported by Marmur et. a l . (73) were not t r u l y i n t e r s p e c i f i c . According to a d e f i n i t i v e study of the taxonomy of the genus B a c i l l u s by Smith et. a l . (91), cultures i d e n t i f i e d p r e v i o u s l y as B_. natto were shown to be s t r a i n s of B_. s u b t i l i s and B_. niger was r e c l a s s i f i e d as B_. s u b t i l i s var. niger. Marmur's experiments merely confirm that these s t r a i n s and v a r i e t i e s are indeed very c l o s e l y r e l a t e d to B_. s u b t i l i s . B. polymyxa i s the only separate species which transformed with B_. s u b t i l i s and t h i s 6 was at a very low l e v e l (0.04%). Biswas and Sen (18) have also attempted i n t r a and i n t e r s p e c i f i c trans-formation of B a c i l l u s organisms, i n t h i s case with respect to a n t i b i o t i c production. Within the species B_. s u b t i l i s the a b i l i t y to produce b a c i t r a c i n was s u c c e s s f u l l y transformed to 3 out of 5 a n t i b i o t i c a l l y i n a c t i v e s t r a i n s . The a b i l i t y to form b a c i l y s i n and u n i d e n t i f i e d a n t i -b a c t e r i a l and ant i f u n g a l substances was s u c c e s s f u l l y t r a n s f e r r e d to about 40% of the other s t r a i n s . Attempts to transform 5 other species of B a c i l l u s with DNA from a n t i b i o t i c producing B_. s u b t i l i s s t r a i n s f a i l e d , i n agreement with Marmur's r e s u l t s (73). C l a s s i f i c a t i o n of B a c i l l u s s u b t i l i s The genus B a c i l l u s i s made up of the aerobic, endospore forming Gram p o s i t i v e rod shaped organisms. P r i o r to the work of Smith, Gordon, and Clark (90,91) and Knight and Proom (65), there was considerable confusion regarding the taxonomy of organisms within the, genus B a c i l l u s . Smith et. a l . (91) developed a systematic key f o r the c l a s s i f i c a t i o n of the i n d i v i d u a l species, which i s followed i n B e r g e y ' s Manual (20). They studied 1,134 cultures of which a l l but 20 were placed i n three groups according to t h e i r morphology and physiology. About 65 percent of the cultures belonged to the same group as 13. s u b t i l i s . Smith's scheme was modified i n a key prepared by Wolf and Barker (114). In t h e i r key, B a c i l l u s organisms are placed i n three groups according to spore and c e l l morphology. B_. s u b t i l i s i s placed i n Group I which i s made up of organisms whose spores are oval or c y l i n d r i c a l and the sporangia are not d e f i n i t e l y swollen. Part of the key i s shown i n Figure 1.. 7 Group I No growth at 60°C,pH6.0 1 I Growth at 60"C, pH6.0 B. coagulans No s i g n i f i c a n t growth i n glucose broth under anaerobic c o n d i t i o n s Good growth i n glucose broth under anaerobic c o n d i t i o n s L e c i t h i n a s e negative Small c e l l s ( 0.9 m) Non-vacuolated B. l i c h e n i f o r m i s L e c i t h i n a s e p o s i t i v e Large c e l l s ( 0.9 m) Vacuolated B. cereus and i t s v a r i a n t s " "j A c i d i n ammonium/glucose/ s a l t s medium Glucose n o n - i n h i b i t o r y r~ — Voges-Proskauer negative Large c e l l s ( 0.9 m) Vacuolated B. megaterium Voges-Proskauer p o s i t i v e Small c e l l s ( 0.9m) Non-vacuolated B. s u b t i l i s and i t s v a r i a n t s F i g u r e 1. P a r t i a l key f o r B a c i l l u s organisms. Growth of B a c i l l u s organisms on hydrocarbons There have been r e l a t i v e l y few reports of B a c i l l u s organisms growing on hydrocarbons i n the l i t e r a t u r e . Shah (89) has c i t e d the early papers of Tausson (104) and Sohngen (92 ) as examples of B a c i l l u s u t i l i z i n g hydrocarbons. However, as pointed out by Fuhs (37), the organisms i s o l a t e d i n these early studies were not spore formers and many of them were Gram negative. The only culture which f i t s the present day c l a s s i f i c a t i o n of B a c i l l u s i s the B_. phenanthrenicus of Tausson (103). This was a s t r a i n capable of u t i l i z i n g the aromatic hydrocarbon phenanthracene. Many members of the genus B a c i l l u s have been i s o l a t e d i n studies of the m i c r o b i a l d e t e r i o r a t i o n . o f - a i r c r a f t f u e l s . F e l i x and Cooney ( 3 4 ) reported the response of nine s t r a i n s of B a c i l l u s i s o l a t e d from a f u e l system. They found that vegetative c e l l s could survive f or only a l i m i t e d time i n a basal salts-JP-4 (a kerosene type fuel) medium and that the c e l l s could not grow at a l l . Spores of these s t r a i n s , under s i m i l a r conditions, could survive but not grow; any that did germinate soon died. I t was- concluded that B a c i l l u s organisms found i n f u e l systems were present because they formed long-surviving and widely d i s t r i b u t e d spores, not capable of growth i n those systems. In a study of amino acid producing organisms i s o l a t e d from s o i l , Shah et. a l . (89) found four s t r a i n s of B a c i l l u s which grew w e l l using kerosene as the carbon source. These organisms resembled B_. cereus var. mycoides (2 s t r a i n s ) , B_. sphaericus and B. s u b t i l i s . The B_. s u b t i l i s s t r a i n d i d not f i t a l l the c r i t e r i a of that species given i n Bergey's Manual and may have been a B_. pumilis. 9 MATERIALS AND METHODS I s o l a t i o n of B a c i l l u s spp. on kerosene carbon source Forty-eight s o i l sludge samples were c o l l e c t e d i n s t e r i l e t e s t tubes from an o i N l r e f i n e r y ' s waste dumping area. In order to i s o l a t e the aerobic spore forming b a c t e r i a which could grow with kerosene as carbon source, 10 to 15 ml of s t e r i l e d i s t i l l e d water and one drop of Tw'een 80 e m u l s i f i e r were added to each sample. The contents of each tube were mixed on a t e s t tube a g i t a t o r . Seven m i l l i l i t r e s of the aqueous layer were tra n s f e r r e d to s t e r i l e tubes and heated i n a water bath at 80°C. A f t e r 15 and 30 minutes incubation, 1.0 ml and 0.1 ml aliquots were withdrawn and spread on P e t r i p l a t e s containing the following hydro-carbon basal medium ( 7 0 ) : K 2HP0 4 (anhyd.) 2.50 g KH 2P0 4 1.75 g NH„C1 1.00 g MgCl 2.6H 20 0.10 g FeCl 2.4H 20 0.05 g CaCl 2.2H 20 0.01 g MnCl .4H 0 0.002 g Na 2S0 4 (anhyd.) . 0.05 g Yeast extract 0.10 g D i s t i l l e d water 1,000 ml pH • 6.8 Agar 15 g A f t e r the surface of each p l a t e had d r i e d , two drops of Fisher odorless kerosene, l o t number 783716, were spread over the surface. The p l a t e s were incubated at 30°C f o r 48 hours. A f t e r incubation, colonies were examined m i c r o s c o p i c a l l y , and those containing spore forming rod shaped organisms were transferred to hydro-carbon basal medium sla n t s for furth e r c l a s s i f i c a t i o n . C l a s s i f i c a t i o n of k e r o s e n e - u t i l i z i n g B a c i l l u s cultures Media: i ) Nutrient agar, pH6.0. Difco n u t r i e n t agar was adjusted to pH6.0 with d i l u t e s u l f u r i c acid, i i ) Glucose broth ( 9 1 ) . Tryptose 10 g K 2HP0 4 5 g Beef extract 3 g Yeast extr a c t 2 g Glucose 10 g D i s t i l l e d water 1,000 ml Glucose was steam s t e r i l i z e d separately as a 25% (w/v) s o l u t i o n at 15 p . s . i . (121°C) for 15 minutes and added a s e p t i c a l l y . i i i ) Glucose ammonium s a l t s . (NH 4) 2P0 4 1.0 g KC1 0.2 g MgS0 4 0.2 g Yeast extract 0.2 g Glucose ' 5.0 g Bromcresol purple 20 ml of a 0.4% s o l u t i o n D i s t i l l e d water 1,000 ml 11 The medium was dispensed i n t e s t tubes with Durham tubes added to measure gas production. Glucose was s t e r i l i z e d separately and added a s e p t i c a l l y . iv) Voges-Proskauer medium (for B a c i l l u s spp.). Proteose-peptone 7 g NaCl 5 g Glucose 5 g D i s t i l l e d water 1,000 ml Test procedures i ) Microscopic examination. Cultures grown on both hydrocarbon basal medium and n u t r i e n t agar were examined at medium power (400 x) with a phase contrast microscope. Organisms which had s u i t a b l e morphology-cylindrical spores i n non-swollen sporangia were Gram stained by standard procedures (27). i i ) Growth at 60°C, pH6.0. Cultures to be tested were streaked on n u t r i e n t agar, pH6.0, slants and incubated at 60°C. Slants were examined f o r growth a f t e r two and f i v e days. i i i ) Anaerobic growth. Glucose broth tubes were steamed to remove di s s o l v e d and gaseous oxygen, inoculated, capped with s t e r i l e melted vaspar (50% Vaseline and 50% p a r a f f i n ) and incubated at 30°C. A f t e r 7 and 14 days the tubes were checked f o r growth, iv) E f f e c t of glucose on growth. Glucose-ammonium-salts medium was inoculated with cultures and examined for growth, acid and gas production a f t e r 10 days. The i n h i b i t o r y e f f e c t of glucose was determined by comparing growth on nutrient agar s l a n t s to growth on nu t r i e n t agar sl a n t s with 10 g/1 glucose added, v) Voges-Proskauer t e s t . Five m i l l i l i t r e s of medium was inoculated and incubated f i v e days at 30°C. At that time 3.0 ml of 5% «-naphthol i n absolute ethanol was added to each tube, followed by 1.0 ml of 40% potassium hydroxide. The t e s t i s p o s i t i v e i f a brig h t pink colour appears i n f i v e minutes. In each c l a s s i f i c a t i o n step a cul t u r e of B a c i l l u s s u b t i l i s ATCC 14593 was included as a c o n t r o l . Growth studies of B a c i l l u s s u b t i l i s i s o l a t e s To improve the growth shown on kerosene basal medium sla n t s by B. s u b t i l i s i s o l a t e s , various supplements were added to the medium to t e s t f o r stimulatory e f f e c t s . Growth studies were c a r r i e d out with 100 ml of medium i n 250 ml Erlenmeyer f l a s k s agitated on a New Brunswick Gyrotory Shaker at 200 r.p.m. Growth was measured as c e l l dry weight a f t e r 60 hours incubation at 30°C. Dry weights were determined by ce n t r i f u g i n g c e l l s at 10,000 xG f o r 15 minutes, washing twice i n 0.05 M phosphate b u f f e r , pH6.8, resuspending i n d i s t i l l e d water and drying overnight at 100 - 105°C. The supplements used to stimulate growth were malt extract i n the range 0 to 1.0 g/1 and s o i l extract i n the range 0 to 100% of the medium. S o i l extract was prepared by b o i l i n g s o i l i n d i s t i l l e d water i n a 1:1 (w/v) r a t i o f o r eight minutes, decanting the aqueous la y e r through cheesecloth and c e n t r i f u g i n g at 15,000 X-G f o r 20 minutes. The cl e a r supernatant was used i n the medium with lg/1 NH^Cl and 1.5 g/1 KH PO added and adjusted to pH6.8 with d i l u t e hydrochloric acid. 13 RESULTS AND DISCUSSION I s o l a t i o n of B a c i l l u s spp. on kerosene The procedure for i s o l a t i n g spore-forming organisms which would grow on kerosene carbon source res u l t e d i n the i s o l a t i o n of 172 cul t u r e s . Microscopic examination showed that 28 cultures were non-sporeformers. These were made up of an assortment of rods, c o c c i and 1 yeast. These organisms appeared i n plates which had a large amount of sludge carry over from the s o i l sample, r e s u l t i n g i n pr o t e c t i o n from the heat treatment. Of the 144 sporeformers, 117 belonged to the Group I morphology c l a s s which includes B. s u b t i l i s . C l a s s i f i c a t i o n . The c l a s s i f i c a t i o n of the Group I cul t u r e s following the key of Wolf and Barker (114)gave the r e s u l t s shown i n Table I. There were no st r a i n s of B. coagulans present, as indicated by lack of growth at 60°C, i pH6.0. A l l of the s t r a i n s which grew well under anaerobic conditions were large c e l l s , about 2 x 5yx, and were probably B_. cereus and i t s va r i a n t s . The cultures which were i n h i b i t e d by glucose and could not use ammonium s a l t s as nitrogen source were made up of B_^  lentus and B_. firmus and were not examined f u r t h e r . Of the organisms which grew well i n glucose ammonium s a l t s , eight s t r a i n s had very large c e l l s (2 x 8/tand longer) and contained many vacuoles. These are s t r a i n s of B_. megaterium. Only two cultures showed the reactions and c h a r a c t e r i s t i c s of B_. s u b t i l i s . Both were Gram p o s i t i v e and had c e l l s about 0.8 x 2.0/c. Table I. I d e n t i f i c a t i o n of B a c i l l u s s u b t i l i s c u l t u r e s Test Number of cultures No growth at 60°C, pH6.0 117 No anaerobic growth i n glucose broth. 25 Acid i n glucose - NH^ - s a l t s medium. Glucose non-i n h i b i t o r y . 10 Voges-Proskauer p o s i t i v e . Small c e l l s , non-vacuolated. 2 15 Growth studies of B_. s u b t i l i s The s t r a i n s of B_. s u b t i l i s i s o l a t e d showed poor growth on kerosene basal medium s l a n t s - there was j u s t a t h i n f i l m of growth formed. In order to determine i f some unknown fa c t o r was needed by these s t r a i n s to promote growth on kerosene, d i f f e r e n t concentrations of malt and s o i l extract were added to the medium. The growth curves on these media are shown i n Figures 2,3,4 and 5. With malt extract, there i s not a s i g n i f i -cant d i f f e r e n c e between the growth i n the c o n t r o l and kerosene f l a s k s . The increased growth at higher malt extract concentration i s probably due to the u t i l i z a t i o n of i t s content of maltose and dextrins (about 80 - 90% carbohydrate) (93). S i m i l a r l y , there was no stimulation of growth on kerosene with the a d d i t i o n of s o i l extract. Increased con-centrations of s o i l extract appear to be s l i g h t l y i n h i b i t o r y . From these r e s u l t s i t i s concluded the s t r a i n s of B_. s u b t i l i s i s o l a t e d i n t h i s p r o j e c t were not capable of s u f f i c i e n t growth on kerosene to warrant a transformation experiment. The s t r a i n s i s o l a t e d behaved s i m i l a r l y to the B a c i l l u s species studied by F e l i x and Cooney (34), which could survive as spores but not a c t i v e l y grow i n a kerosene f u e l system. Some of the B a c i l l u s organisms i s o l a t e d i n the i n i t i a l experiments did grow well on kerosene. In p a r t i c u l a r , those which resembled B. cereus grew r a p i d l y to form large, spreading colonies on agar pla t e s and s l a n t s . These large colonies on the i n i t i a l i s o l a t i o n p l a t e s may have contributed metabolites and endproducts which the poorer growing organisms such as B^ s u b t i l i s could use. This would account for B. s u b t i l i s showing up on the p l a t e s but not being able to grow on the kerosene medium alone. Such a relationship could also exist in a natural environment. 17 0.2 cn £ 0.1 >-DC O 0 Figure 3. 2 5 SOIL 5 0 E X T R A C T 7 5 1 0 0 Effect of s o i l extract on the growth of B_. subtilis strain 1 i n kerosene medium. H= with kerosene © = without kerosene 18 SOIL EXTRACT 100 s u b t i l i s E f f e c t of s o i l e x t r a c t on the growth of B_. s t r a i n 2 i n kerosene medium. B= w i t h kerosene ® = without kerosene 19 PART I I . UTILIZATION OF N-ALKANES BY AN ARTHROBACTER SPP. INTRODUCTION The development of hydrocarbon fermentations has accelerated r a p i d l y i n recent years. Previously, hydrocarbon microbiology was studied as an unusual occurrence, with a p p l i c a t i o n mainly to o i l exploration and corr o s i o n associated with o i l products (118, 17). In the 1960's the i n d u s t r i a l p o t e n t i a l of microorganisms growing on hydrocarbons was recognized, and many i n t e r e s t i n g and valuable products can now be produced by hydrocarbon fermentation. Many of these products are important i n the food industry. The f i r s t food a p p l i c a t i o n of hydrocarbon fermentations was the suggestion of Champagnat and Llewelyn i n 1962 (23) that food and feed yeast could be produced from o i l . This group, working f o r B r i t i s h Petroleum Ltd. (BP) has studied two methods of producing feed yeasts: one, by growth of Candida l i p o l y t i c a i n a continuous fermentation using r e f i n e d n-paraffins as carbon source, and the other by growth of C. l i p o l y t i c a on gas o i l , which r e s u l t s i n simultaneous de-waxing (n-alkane removal) of the gas o i l (24, 71). Both processes appear to be s u c c e s s f u l . The p r o t e i n concentrate produced 'from hydrocarbons has been evaluated f o r n u t r i t i o n a l value and t o x i c o l o g i c a l safety. When mixed i n animal feeds the yeast concentrate i s completely acceptable, and can be used to replace or supplement other p r o t e i n sources. In the future i t i s hoped to produce human food grade p r o t e i n from hydro-carbon yeast that can be used i n textured p r o t e i n products, s i m i l a r to those now made with vegetable p r o t e i n . The production of feed yeast i s increasing r a p i d l y around the world: BP produces 4,000 tons/year from n-paraffins at Grangemouth, Scotland, and 16,000 tons per year from gas o i l at Lavera, France (9). As well as these p i l o t p l ants, BP has entered a j o i n t venture to produce up to 350,000 metric tons annually from n-paraffins at two plants i n I t a l y . Czech workers are completing research for the production of 100,000 ton/year (31, 32, 82) with simultaneous de-waxing of gas o i l . The Soviet Union i s planning to help a l l e v i a t e i t s shortage of c a t t l e feed with the production of one m i l l i o n tons/year of fodder yeast from n-paraffins by 1975 (11). The production of an acceptable p r o t e i n concentrate from sin g l e c e l l p r o t e i n f o r human consumption i s s t i l l a few years away. There are problems of p a l a t a b i l i t y and n u c l e i c acid t o x i c i t y to be overcome. There are many food products other than s i n g l e c e l l p r o t e i n which can be obtained from hydrocarbon grown microorganisms, as indicated i n the extensive review by Abbott and G l e d h i l l (1). Most of these food products are produced from organisms growing on n-alkanes. Two of the highest y i e l d i n g processes are glutamic acid and c i t r i c acid fermentations. Glutamic acid i s an important flavour enhancing food a d d i t i v e , and i s produced by Corynebacterium glutamicum (Micrococcus glutamicus) i n large quantity from carbohydrate, and by C. hydrocarboclastus growing on n-alkanes. Y i e l d s as high as 75 g/1 have been reported from the l a t t e r . This may be high enough to r i v a l the carbohydrate process i n d u s t r i a l l y . Lysine, threonine, phenylalanine (106), and tryptophane, which may have use as d i e t a r y supplements, have also been produced i n hydrocarbon fermentations. C i t r i c a c i d , a sequestrant and acidulant used i n the food industry has been reported at remarkably high y i e l d s . A mutant s t r a i n of the 21 yeast Candida l i p o l y t i c a which was unable to degrade c i t r i c acid produced 112 g/1 i n 3 days. This represents a y i e l d of 138%, considerably higher than on carbohydrate substrates. Although no d e t a i l e d information i s a v a i l a b l e , the large number of patents and high y i e l d s f o r c i t r i c acid from hydrocarbons makes i t s commercial production very l i k e l y i n the near future. Numerous other products with food a p p l i c a t i o n s are formed by micro-organisms growing on alkanes, such as flavour enhancing nucleotides (guanylic and i n o s i n i c a c i d s ) , vitamins and pigments. Lipase and oxygenase enzymes may also f i n d future uses. In order to be commercially f e a s i b l e , a hydrocarbon fermentation must be able to compete economically with the corresponding carbohydrate substrate fermentation. An important factor i n t h i s i s the lower cost of hydrocarbons: 2 to 4 cents/lb. f o r re f i n e d hydrocarbons versus 4 to 8 cents/lb. for r e f i n e d carbohydrates, and 0.25 cents/lb. f o r natural gas versus 1.5 - 2.5 cents/lb. f o r cereal grains (52). Because hydro-carbons are i n a more reduced form and contain no oxygen, a higher y i e l d on a weight basis can be obtained. For example, i t requires only ha l f as much hydrocarbon substrate as carbohydrate to give a c e r t a i n c e l l y i e l d . Product recovery i s easier from a hydrocarbon process. At the end of a fermentation the medium usu a l l y contains j u s t inorganic s a l t s , c e l l s , substrate and product, without the complex organic substances found i n crude carbohydrates such as molasses. The i m m i s c i b i l i t y of the o i l phase with the aqueous medium makes i t s removal much simpler and l e s s expensive. On the other hand, some of these properties add d i f f i c u l t i e s to hydrocarbon fermentations. Because of the reduced state of p a r a f f i n substrates, these processes require about three times as much oxygen as c o n v e n t i o n a l aerobic processes and r e s u l t i n much grea t e r heat p r o d u c t i o n . I m m i s c i b i l i t y of the aqueous and o i l phases c r e a t e s a need f o r much more mixing. These increased c o s t s of a e r a t i o n , heat removal and a g i t a t i o n must be balanced a g a i n s t the p o s s i b l e advantages of sub s t r a t e p r i c e , product y i e l d and ease of recovery i n a hydrocarbon process i n order to evaluate i t s f e a s i b i l i t y . The purpose of t h i s study i s t o c h a r a c t e r i z e the growth and p o s s i b l e product formation of an organism growing on n-alkanes. When i s o l a t e d , t h i s c u l t u r e showed abundant growth and produced a hig h l e v e l of a c i d i t y on hydrocarbon medium. This study i s an i n v e s t i g a t i o n of which hydrocarbons support the best growth and of the nature of the a c i d i t y produced by t h i s microorganism growing on n-alkanes. 23 LITERATURE REVIEW C l a s s i f i c a t i o n The c l a s s i f i c a t i o n of b a c t e r i a resembling the cu l t u r e used i n t h i s p r o j e c t , that i s , those organisms which show rod shaped or filamentous growth breaking up i n t o rods and c o c c i , i s very d i f f i c u l t . A number of genera of r e l a t e d organisms show t h i s type of growth at d i f f e r e n t stages of t h e i r l i f e c y c l e and on d i f f e r e n t types of media. These include members of the Nocardia, Mycobacteria and Arthrobacter. Much of the e a r l y work of Jensen (58), Conn and Dimmick (28) and Clark (26) was concerned with separation and c l a s s i f i c a t i o n of p a r a s i t i c and saprophytic forms of these organisms. Jensen l a i d much of the groundwork by d e f i n i n g the s o i l mycobacteria as the a c i d - f a s t , rod to filamentous forms, which d i v i d e i n t o c o c c i , while the corynebacteria were non-acid-fast and showed v a r i e d morphology, with i r r e g u l a r shaped rods and filaments, sometimes branched, which broke up i n t o small rods or c o c c i . Some s t r a i n s formed large coccoid c e l l s c a l l e d c y s t i t e s . Conn and Dimmick added to the study of these forms by broadening the d e f i n i t i o n of Mycobacterium to include p a r t i a l l y acid f a s t and branched forms. They proposed the name*Arthrobacter for a group of common s o i l organisms previously included i n the genus Corynebacterium. These organisms were characterized by a complicated morphological c y c l e which included rods, c o c c i , club-forms, branched and unbranched filaments. These cultures were Gram v a r i a b l e and non-acid-fast. This designation of Arthrobacter was supported by Clark and f i r s t appeared i n Bergey's Manual i n 1957 (20). The c l a s s i f i c a t i o n , morphology, and physiology of Arthrobacter has been extensively characterized since then by Stevenson and Lochhead's group 24 (25, 72, 98). The l i f e c y c l e of Arthrobacter s t a r t s with coccoid c e l l s which enlarge into pleomorphic rods which l a t e r d i v i d e into small rods and coccoid forms. On c e r t a i n media filaments and c y s t i t e s may be formed. The c y c l e i s u s u a l l y complete within 24 hours. The mycobacteria and nocardia have been investigated by Gordon and her co-workers (39, 40, 41). They give the c r i t e r i a of acid-fastness and non-mycelial filamentous growth, sometimes branching, which l a t e r breaks into rods and c o c c i , f o r the mycobacteria. The Nocardia are regarded as non-acid f a s t organisms with a d e f i n i t e mycelial growth. The mycelium i s u s u a l l y branching and may contain a e r i a l hyphae. More rudimentary s t r a i n s may fragment i n t o rods and coccoid forms. These d e s c r i p t i o n s d i f f e r from Bergey's Manual, i n which the mycobacteria can contain organisms which are highly branched and only s l i g h t l y acid f a s t , and the nocardia include some p a r t i a l l y a c i d f a s t forms which have a rudimentary mycelium which fragments r a p i d l y into rods and c o c c i . The d i f f i c u l t y i n c l a s s i f y i n g these organisms i s summed up i n a remark by H.L. Jensen, (42) 1 " I t has been customary to t a l k about Nocardia as representing a t r a n s i t i o n between Mycobacterium and Streptomyces, but there i s another group with which the nocardiae merge, the arthrobacters ... ... Like Dr. Gordon, who knows of no borderline between Nocardia and Streptomyces, I know of no borderline between Arthrobacter and Nocardia." 1. Gray, T.R.G. and D. Parkinson (ed.). The ecology of s o i l b a c t e r i a . U n i v e r s i t y of Toronto Press, Toronto, (1968), p. 323. 25 M i c r o b i a l hydrocarbon u t i l i z a t i o n Microorganisms have been known to grow on hydrocarbons since 1895, when Miyoshi observed the fungus B o t r y t i s cinerea growing on p a r a f f i n wax (83). The study of mi c r o b i a l hydrocarbon u t i l i z a t i o n i n the f i r s t h a l f of t h i s century was mainly concerned with which organisms could grow on hydrocarbons, which ones were attacked, and the use of petroleum organisms i n o i l prospecting. These early developments are discussed i n the reviews by ZoBell (117, 118) and Beerstecher (17). As summarized by Quayle (83), the s t r a i g h t chain alkanes of intermediate chain length (9 to 16 carbon atoms) are most e a s i l y u t i l i z e d by microorganisms as compared with branched chain alkanes and aromatics. This i s . a g e n e r a l i z a t i o n based on reports i n the l i t e r a t u r e and may not hold true f o r any p a r t i c u l a r organism. Most of the organisms capable of growth on hydrocarbons are common, s o i l types, inc l u d i n g b a c t e r i a , yeasts and fungi. The b a c t e r i a most often found i n i s o l a t i n g hydrocarbon users are i n the genera Brevibacterium, Corynebacterium, Mycobacterium, Nocardia, Streptomyces and Pseudomonas. Candida and Torula yeasts and A s p e r g i l l u s , Fusarium and P e n i c i l l i u m fungi frequently can grow on hydrocarbons as well (37, 35, 83). Although the n-alkane hydrocarbons most frequently support growth, many other types can be attacked by some organisms such as branched alkanes, o l e f i n s , saturated r i n g com-pounds and aromatics, inc l u d i n g the m u l t i - r i n g ones l i k e anthracene. Hydrocarbon containing products such as kerosene f u e l s , rubber, and asphalt are subject to m i c r o b i a l degradation. Much work i n the l a s t few years has been devoted to determining the pathways and mechanisms of n-alkane oxidation (35, 108, 75, 64). The 26 p r i n c i p l e pathway of microbial attack on n-alkanes s t a r t s with the a d d i t i o n of oxygen to the terminal methyl group, as f i r s t studied by Senez and Azoulay (66, 14) and T h i j s s e and van der Linden (105). They showed that species of Pseudomonas oxi d i z e an n-alkane, e.g. n-hexane, by the a d d i t i o n of one oxygen atom to the terminal methyl group g i v i n g the primary a l c o h o l . This was subsequently dehydrogenated to the corresponding aldehyde and then f a t t y a c i d , which was u t i l i z e d by the common^-oxidation pathway. These r e s u l t s have been confirmed i n many studies since. Leadbetter and Foster (67) and Stewart e t . a l . (100) showed that molecular oxygen i s incorporated d i r e c t l y i n t o the alcohol molecule by using i s o t o p i c a l l y l a b e l l e d oxygen with Pseudomonas and Micrococcus organisms r e s p e c t i v e l y . The enzymes involved have been p a r t i a l l y characterized. Hayaishi (45, 46) introduced the concept of mixed-function oxygenase (or hydroxylase or oxidase) enzymes which catalyse the f i r s t step of the oxidation. Studies using c e l l - f r e e extracts have shown that t h i s i n i t i a l enzymatic r e a c t i o n requires ferrous ions and molecular oxygen i n order to produce the corresponding a l c o h o l . The subsequent conversion of the alcohol to the corresponding a c i d are c a r r i e d out by nicotinamide adenine d i n u c l e o t i d e (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) req u i r i n g dehydrogenases (16, 13, 70). Recent i n v e s t i g a t i o n s have been concerned with the mechansism of the i n i t i a l o x idation step and the nature of the e l e c t r o n transport system involved between molecular oxygen and NADH^ (79, 80, 22). This methyl group oxidation pathway has been demonstrated i n Mycobacterium rhodochrous (36) and a thermophilic B a c i l l u s (44). Klug and Markovetz (63) and Lebault (69) have shown that Candida yeasts u t i l i z e n-alkanes by a s i m i l a r pathway. 0 -oxidation of the f a t t y a c i d i s not the only pathway of u t i l i z a t i o n of n-alkanes. Stewart et a l (100) found that a f t e r the terminal methyl group was oxidized by a s t r a i n of Micrococcus c e r i f i c a n s the alcohol was combined with a f a t t y a c i d to form high molecular weight e s t e r s . Both the alcohol and acid moieties i n the ester were derived from the n-alkane, f o r example n-hexadecane ( c^g) ^ec3 to formation of c e t y l palmitate (.C )• Some ba c t e r i a and yeasts form d i c a r b o x y l i c acids from alkanes (60, 6, 57). I t appears that the monocarboxylic a c i d i s formed f i r s t , followed by ^ - o x i d a t i o n of the other end group. This i s followed by y5-oxidation to acetate. Some s t r a i n s of Pseudomonas (36), Brevibacterium (109) and P e n i c i l l i u m accumulate secondary alcohols and ketones when grown on n-alkanes. In most cases these are products of side reactions and are not metabolized, f u r t h e r by the organism. An i n t e r e s t i n g aspect of hydrocarbon metabolism i s the phenomenon of co-oxidation, i n which a n o n - u t i l i z a b l e hydrocarbon i s oxidized by an organism which i s growing on an a s s i m i l a b l e hydrocarbon. This was f i r s t reported by Leadbetter and Foster (68) who found that Pseudomonas methanica grown on methane would simultaneously o x i d i z e ethane to ethanol, acetaldehyde and acetate, propane to acetone and propionic acid and butane to 2-butanone and b u t y r i c a c i d . This culture could not grow on ethane, propane or butane as sole carbon source. Davis and Raymond (29) showed that a Nocardia species growing on n-hexadecane or n-octadecane could co-oxidize the a l k y l side chains of aromatic and c y c l i c hydrocarbons to give c y c l i c carboxylic' acids. Since then dozens of co-oxidative transformations of aromatic compounds have been reported, most of them using Nocardia cultures (85). 28 U t i l i z a t i o n of hydrocarbons by Arthrobacter The f i r s t reports of hydrocarbon oxidation by members of the genus Arthrobacter were by K l e i n e t . a l . (61, 62). They found that Arthrobacter s t r a i n s grown on glucose, would co-oxidize n-hexadecane to the corresponding 2, 3, and 4 hexadecanones i n the r a t i o of 72:23:4%. The corresponding alcohols were intermediates i n the oxidation. The organisms were not able to grow on the n-alkane or ketones as the sole carbon source. Perry and Scheld i s o l a t e d an Arthrobacter organism which oxidized propane c o n s t i t u t i v e l y a f t e r growth on a-phthalate (78). A number of workers i n the Japanese fermentation industry have i s o l a t e d Arthrobacter s t r a i n s which grow r a p i d l y on n-alkane carbon sources. The group at the Kyowa Hakko Kogyo Co. Ltd. have developed a number of s t r a i n s which form i n t e r e s t i n g products, i n c l u d i n g trehalose l i p i d e m u l s i f i e r s (101), o r o t i c a c i d and o r o t i d i n e (59), and d e r i v a t i v e s of phenazine (102). These mutant s t r a i n s of A. paraffineus were grown on a basal s a l t s medium with a mixture of C-12 to C-14 n-alkanes added as carbon source. Many of these c u l t u r e s and s t r a i n s of A. simplex have been patented for the production from hydrocarbons of glutamic acid (up to 39 g/1), l y s i n e (10 g/1), threonine, tryptophan, c i t r i c acid (28 g/1 y i e l d ) , i n o s i n i c a c i d , polysaccharides, and many other amino acids and nucleotides (1). Oxidation of ammonia The oxidation of ammonia i s part of the process of n i t r i f i c a t i o n . N i t r i f i c a t i o n i s a general term used i n reference to the oxidation of organic forms of reduced nitrogen and ammonia by s o i l organisms leading 29 to the formation of n i t r i t e s , n i t r a t e s and sometimes gaseous oxides (2). The major groups of organisms responsible for n i t r i f i c a t i o n i n the s o i l are the Nitrosomonas and Nitrobacter autotrophic b a c t e r i a . Nitrosomonas are thought to o x i d i z e ammonia to n i t r i t e through the intermediates hydroxylamine and the unstable n i t r o x y l r a d i c a l (NOH) (110). Energy i n the form, of adenosine triphosphate (ATP) i s derived by coupling these oxidations with oxidative phosphorylation i n the electron transport system. Some of t h i s ATP i s used to produce reduced py r i d i n e nucleotides f o r the f i x a t i o n of carbon dioxide. S i m i l a r l y , n i t r o b a c t e r s use the oxidation of n i t r i t e to n i t r a t e to generate energy and reducing power for autotrophic growth. Some heterotrophic organisms are also capable of n i t r i f i c a t i o n , u s u a l l y at a much lower rate than the autotrophs. In a survey of heterotrophic n i t r i f i e r s , E ylar and Schmidt (33) i s o l a t e d 26 moderately active c u l t u r e s . Most of these were fungi i d e n t i f i e d as A s p e r g i l l u s  f l a v u s , with a few P e n i c i l l i u m and u n i d e n t i f i e d b a c t e r i a . One pathway which has been found f o r heterotrophic n i t r i f i c a t i o n involves organic intermediates and occurs i n A. f l a v u s (74) and P. atrovenetum (55). The immediate precursor of n i t r a t e and n i t r i t e i s 3-nitropropionic acid which i s formed from eitherp-alanine or a s p a r t i c a c i d . These organisms do not produce n i t r i t e and n i t r a t e u n t i l a c t i v e growth has almost ceased and do not depend on n i t r i f i c a t i o n f o r energy for growth. The r o l e of t h i s phenomenon has not yet been explained i n possible b i o s y n t h e t i c pathways. An inorganic pathway of n i t r i f i c a t i o n operates i n some heterotrophic organisms. Using a c e l l f r e e extract of A s p e r g i l l u s w e n t i i , Aleem e t . a l . 30 (3) demonstrated the oxidation of ammonium ions through the intermediates hydroxylamine and n i t r i t e to give n i t r a t e . This i s s i m i l a r to the pathway operating i n Nitrosomonas and Nitrobacter. The enzyme systems of both A. wentii and the autotrophs require copper ions and have s i m i l a r cytochrome systems. Aleem and h i s co-workers state that t h i s inorganic pathway for ammonia oxidation i s the only one i n heterotrophs and that 3-nitropropionic acid i s not involved. This disagreement over organic or inorganic intermediates has not been resolved. Heterotrophic n i t r i f i c a t i o n i n b a c t e r i a has been studied by Gunner (43) working with a s t r a i n of Arthrobacter globiformis i s o l a t e d from s o i l . A f t e r growth i n a basal s a l t s medium with succinate as carbon source, t h i s c u l t u r e oxidized ammonium ions when suspended i n a buffer s o l u t i o n without carbon source. The products of oxidation were small amounts of hydroxylamine and n i t r i t e , and a large amount of n i t r a t e . U n i d e n t i f i e d nitrogen oxide gases were also produced and were postulated to be intermediates i n the oxidation. Gaseous oxides have also been reported by Anderson (10) to be formed by an extract of Nitrosomonas c e l l s . N i t r i c oxide and n i t r o u s oxides were released from hydroxylamine under anaerobic conditions. Nitrous oxide was also formed non-enzymatica i n b o i l e d extracts. Another type of heterotrophic ammonia oxidation, which has not been recognized by workers i n the s o i l n i t r i f i c a t i o n area, i s the formation of n i t r i t e from ammonia by methane u t i l i z i n g b a c t e r i a . This was f i r s t reported by Hutton and ZoBell (53, 54) before Schmidt (88) observed the f i r s t heterotrophic n i t r i f i c a t i o n by A s p e r g i l l u s f l a v u s . Hutton and ZoBell showed that some methane grown b a c t e r i a produced n i t r i t e from 31 ammonia at a rate p r o p o r t i o n a l to the rate of methane consumption. These r e s u l t s have been confirmed by Whittenbury et. a l . ( I l l ) , who found that more than 100 cu l t u r e s of methane u t i l i z i n g b a c t e r i a could a l l convert ammonia to n i t r i t e . In these organisms, the ammonium ions compete with methane for the methane oxidase enzyme (113). I t i s postulated that methane oxidase may have developed from a mutation i n the ammonia oxidase enzyme of autotrophic b a c t e r i a . I f t h i s i s the case, an examination of the mechanism of methane oxidation may help elucidate the mechanism of ammonia oxidation. 32 MATERIALS AND METHODS Charac t e r i z a t i o n of hydrocarbon s p e c i f i c i t y C u l t u r a l conditions The Arthrobacter organism was grown i n the hydrocarbon basal medium described i n Part I f o r these experiments. F i f t y ml of medium i n 250 ml Erlenmeyer f l a s k s were steam s t e r i l i z e d at 121°C for 15 minutes before i n o c u l a t i o n . The hydrocarbons were s t e r i l i z e d separately and added a s e p t i c a l l y to give a concentration of 10 ml/1. A 2% (v/v) inoculum of an a c t i v e l y growing 24 hour shake f l a s k c u l t u r e was added to each f l a s k . Flasks were incubated on a New Brunswick Gyrotory shaker operating at 200 strokes per minute i n a 30°C incubator. Dry weights were determined as i n Part I. The pH of the medium was measured with a meter a f t e r the c e l l s had been removed by c e n t r i f u g a t i o n . A l l t r i a l s were performed i n du p l i c a t e . Analysis of kerosene The kerosene used i n these experiments was Fisher odorless kerosene, l o t No. 704351. The major n-alkane components of t h i s kerosene were i d e n t i f i e d using gas chromatography. These components were i d e n t i f i e d by comparing t h e i r r e t e n t i o n times with pure standards and by observing the change i n the areas of the n-alkane peaks when pure n-alkanes were added to the kerosene. The a n a l y s i s was done using a Becker Model 3810 gas .chromatograph equipped with a s i n g l e column a n a l y t i c a l head. The column was HI-EFF-8BP/ cyclohexandimethanol succinate. The u n i t was equipped with a flame 33 i o n i z a t i o n detector and nitrogen c a r r i e r gas, flow rate 40 ml/min. The temperature was programmed from 50 to 190°C at 12°C/min., and then h.eld isothermally f or 5 minutes. 0.5yc<l of sample was used with the i n j e c t i o n port temperature set at 220°C. The V i t a t r o n model UR402 recorder was equipped with an e l e c t r o n i c integrator to measure peak areas and give q u a n t i t a t i v e r e s u l t s . Analysis of kerosene from shake f l a s k s The organism was grown i n shake f l a s k s for 24, 48, 72 and 120 hour periods. On each p a i r of d u p l i c a t e s , the pH of the medium was measured. The r e s i d u a l kerosene was removed from the surface of each sample a f t e r c e n t r i f u g a t i o n , d r i e d over anhydrous sodium s u l f a t e , and analysed gas chromatographically. Uninoculated 5 day c o n t r o l samples were also run. Respirometry The oxygen uptake of i n t a c t c e l l s on d i f f e r e n t n-alkanes was measured using standard manometric techniques (107). The apparatus used was a G i l s o n D i f f e r e n t i a l Respirometer. The f l a s k s contained 1.0 ml of c e l l s suspended i n hydrocarbon basal medium, 2.0 ml of basal medium, 0.2 ml of 20% potassium hydroxide i n the center well and 50/d of n-alkane i n the side arm. The substrate was tipped i n t o the f l a s k a f t e r 10 minutes e q u i l i b r a t i o n i n the 30°C water bath. Growth on d i f f e r e n t n-alkanes The organism was grown i n shake f l a s k c u l t u r e using the pure n-alkanes present i n kerosene as the sole carbon source. A f t e r 42 hours the growth (dry weight) and pH drop of the media were measured. 34 Growth curve i n shake f l a s k s The dry weight and pH of the medium were determined during a 7 day growth period. The organism was grown on the two n-alkanes which supported the most growth, dodedecane and tridecane, and kerosene. C h a r a c t e r i z a t i o n of acid production F a t t y acid a n a l y s i s The Arthrobacter c u l t u r e was grown i n shake f l a s k for 48 hours on dodecane, tridecane and kerosene carbon sources. The f a t t y acids were extracted by adjusting 300 ml of culture f l u i d to pH 8.5 and c e n t r i f u g i n g 20 minutes at 10,000 XG to remove the c e l l s . The supernatant was adjusted to pH 1.5 with 6 N hydrochloric acid and extracted three times with 50 ml of d i e t h y l ether. The combined ether f r a c t i o n s were extracted twice with 40 ml of 5% sodium carbonate. The aqueous carbonate l a y e r was a c i d i f i e d to pH 1.5 with 6N hydrochloric acid and extracted twice with 15 ml of ether. The combined f i n a l ether extracts were d r i e d over anhydrous sodium s u l f a t e and evaporated to about 3 ml with a stream of dry nitrogen. These ether extracts of the a c i d i c l i p i d f r a c t i o n s from the c u l t u r e f l u i d were analysed by t h i n layer chromatography. The extracts were spotted on S i l i c a Gel G (0.25 mm thick) t h i n layer p l a t e s , and developed with petroleum ether (30 - 60°C B.P): ether: a c e t i c a c i d , (90:10:1, v/v) solvent system (38). Decanoic acid was run as a f a t t y a c i d standard and sebacic acid as a d i c a r b o x y l i c a c i d standard. Spots were v i s u a l l i z e d on the p l a t e s with two d i f f e r e n t spray reagents: 0.04% bromcresol green i n ethanol, and charring with 50% s u l f u r i c acid followed heating at 105°C 35 f o r 30 minutes. The e x t r a c t from the kerosene medium was further analysed using gas chromatography. The acids i n t h i s extract were methylated by b o i l i n g f or 2 minutes on a steam bath with 5 ml of boron t r i f l u o r i d e -methanol (14% w:v) reagent (112)• Ten ml of d i s t i l l e d water was added and the t o t a l was extracted with 5 ml of ether. The ether layer was d r i e d over anhydrous sodium s u l f a t e , followed by removal of the ether by evaporation under a stream of dry nitrogen with the v e s s e l held i n an i c e bath. The small amount of o i l y residue was i n j e c t e d into a Becker Model 3810 gas chromatograph equipped with HI-EFF-8BP column with nitrogen c a r r i e r gas. The temperature was programmed f o r l i n e a r increase from 120°C to 230°C at 12°C/min., and then held isothermally. Standard methyl esters of f a t t y acids were analysed .under the same conditions to give standard r e t e n t i o n times. Organic acid analysis C e l l s were grown on dodecane, tridecane, and kerosene i n shake f l a s k c u l t u r e and removed from the medium by c e n t r i f u g a t i o n . The c u l t u r e media were tested f o r the presence of organic acids by t h i n layer chromatography on S i l i c a Gel G (0.25 mm). Unused medium was run as a c o n t r o l , and adipic and t a r t a r i c acids were run as standards for com-parison. The p l a t e was developed with ethanol:water:25% ammonia, (100:12:16), d r i e d 10 minutes at 100°C and sprayed with 0.04% bromocresol green i n ethanol (adjusted to a s l i g h t blue c o l o u r a t i o n ) , i n order to show a c i d i c spots (yellow). 36 Amino, acid analysis The c u l t u r e f l u i d from c e l l s grown on dodecane, tridecane, and kerosene was analysed f o r amino acids by descending paper chromatography on Whatman No. 1 f i l t e r paper. Unused medium was spotted as a c o n t r o l , and a mixture of glutamic acid and l y s i n e as standards. The chromatogram was run with butanol:acetic acid:water (4:1:2), and spots v i s u a l i z e d by spraying with a standard ninhydrin s o l u t i o n . M a t e r i a l s balance experiments C u l t u r a l conditions For the materials balance experiments, the Arthrobacter s t r a i n was grown i n the hydrocarbon basal medium described previously, with the m o d i f i c a t i o n that 7 g/1 ammonium s u l f a t e was added. Dodecane was used as the carbon source i n a concentration of 10 ml/1 (7.49 g/1). The cyclone research fermentor of Dawson (30), equipped with pH and tempera-ture c o n t r o l was used i n t h i s study. The fermentor was s t e r i l i z e d at 121°C f o r 30 minutes before f i l l i n g with-1,500 ml of s t e r i l e medium. The pH electrode was s t e r i l i z e d separately i n 500 ppm sodium hypochlorite followed by r i n s i n g with s t e r i l e d i s t i l l e d water. An a c t i v e l y growing 5% inoculum, grown 24 hours on the shaker, was used. Medium was c i r c u l a t e d through the fermentor with a Cole-Parmer magnetic drive pump. The temperature was maintained at 30°C by c i r c u l a t i n g water through the jacketed side arm of the fermentor. The pH of the medium was kept at 6.85 - 0.05 by add i t i o n of s t e r i l e 0.5 N sodium hydroxide or 0.5 N hydrochloric acid by a Fermentation Design pH c o n t r o l u n i t connected to an Ingold s t e r i l i z a b l e electrode. The co n t r o l u n i t was equipped with p e r i s t a l t i c pumps for both acid and base and a continuous pH recorder. The add i t i o n of acid or base was monitored with a Rustrak recording ammeter connected to the c o n t r o l u n i t . S t e r i l e a i r was pumped through a port i n the side of the fermentor at a rate of 1.8 1/min. The e f f l u e n t a i r was bubbled through a c o l l e c t i n g v e s s e l i n order to trap s p e c i f i c gases. Samples v/ere removed a s e p t i c a l l y at s u i t a b l e times for analyses. The set up of the apparatus can be seen i n Figure 6. A n a l y t i c a l procedures Growth and a c i d i t y Growth was measured as dry weight. The a c i d i t y was c a l c u l a t e d over 1 hour i n t e r v a l s from the Rustrak recording ammeter and the volume of sodium hydroxide or hydrochloric acid added from the r e s e r v o i r s . Residual dodecane To a 10 ml sample of the fermentation broth was added 5 0 ^ of tridecane as an i n t e r n a l standard. Two ml of n-hexane was added and mixed i n to extract the hydrocarbons. The r e s u l t i n g emulsion was centrifuged and the hexane layer was removed and dri e d over anhydrous sodium s u l f a t e . The extract was analysed using the gas chromatograph and column described previously, with the temperature programmed isothermally at 100°C for 1.5 minutes, and then increased l i n e a r l y to 172°C at 12°C/min. The dodecane was determined q u a n t i t a t i v e l y by the r a t i o of i t s peak area to the area.of the tridecane as measured by the i n t e g r a t o r , using a c a l i b r a t i o n curve of peak area r a t i o versus weight r a t i o . Equipment set-up i n fermentation experiments. 1. r e c o r d i n g ammeter. 2. pH c o n t r o l u n i t . 3. a i r s t e r i l i z i n g f i l t e r . 4. cyclone fermentor. 5. pH e l e c t r o d e . 6. e f f l u e n t gas t r a p . 7. sodium hydroxide r e s e r v o i r . 8. medium c i r c u l a t i n g pump. 9. temperature c o n t r o l water j a c k e t . 10. temperature c o n t r o l water bath. 39 Dissolved bicarbonate A 3 ml sample of medium was placed i n a Warburg f l a s k with 0.5 ml of 3 N s u l f u r i c acid i n the side arm. The f l a s k was attached to a G i l s o n D i f f e r e n t i a l Respirometer, e q u i l i b r a t e d at 30°C and the acid tipped i n from the side arm. The volume of carbon dioxide gas released was read d i r e c t l y from the manometer. Under these conditions, 24.6yWl carbon dioxide represent 1 m i l l i m o l e . Carbon dioxide gas The carbon dioxide i n the e f f l u e n t a i r was trapped i n a v e s s e l containing 3 N potassium hydroxide. The a i r going into the fermentor was scrubbed free of carbon dioxide by passing through a s i m i l a r trap. The carbon dioxide retained i n the e f f l u e n t trap was measured by removing a sample, adjusting i t to pH 7-8 with 1 N hydrochloric acid and making a s u i t a b l e d i l u t i o n (usually 1:25) with CO^-free d i s t i l l e d water. The carbon dioxide was then determined i n the same way as dissol v e d bicarbonate, given above. C e l l carbon The amount of carbon i n the c e l l s was determined by c e n t r i f u g i n g the c e l l s from 10 ml of medium, washing twice i n phosphate bu f f e r (0.05 M, pH 7.0), and resuspending i n 5 ml of C0 2~free d i s t i l l e d water. The carbon content was measured by i n j e c t i n g the c e l l suspension, previously homogenized with a Potter homogenizer (77) , into a Beckman Model 915 T o t a l Organic Carbon Analyser. Oxalic acid s o l u t i o n was used to construct a c a l i b r a t i o n curve. 40 Nitrogen a n a l y s i s For c e l l nitrogen-, the c e l l s were centrifuged from the medium, washed twice i n phosphate bu f f e r (0.05 M, pH 7.0), and resuspended i n d i s t i l l e d water. Total nitrogen was determined on medium d i r e c t l y from the fermentor. Both c e l l and t o t a l nitrogen were determined by the micro-Kjeldahl method given i n A.O.A.C. (49)• Ammonia nitrogen was determined on the medium a f t e r c e n t r i f u g a t i o n by using the micro-Kje l d a h l method from the d i s t i l l a t i o n step onwards. N i t r a t e was determined by the phenoldisulfonic acid method and n i t r i t e by the naphthylamine-sulfanilic a c i d method (7). V o l a t i l e amines The e f f l u e n t a i r from the fermentor was checked for v o l a t i l e amines by bubbling the a i r through a trap containing 3 N s u l f u r i c acid. The a i r entering the fermentor was passed through a s i m i l a r trap. The e f f l u e n t trap was analysed f o r nitrogenous organic compounds by the micro-Kjeldahl method. Nitrogen oxide gases Gaseous nitrogen oxides i n the e f f l u e n t a i r were c o l l e c t e d i n a trap containing 175 ml of 5 percent potassium hydroxide and 25 ml of 3 percent hydrogen peroxide. The peroxide was added to convert a l l the nitrogen oxides c o l l e c t e d to n i t r a t e (43). Nit r a t e i n the trap was determined by the phenoldisulfonic a c i d method (7). 41 RESULTS AND DISCUSSION C l a s s i f i c a t i o n The c u l t u r e used i n t h i s study was i s o l a t e d from a water cooling tower, loco Refinery, Imperial O i l Ltd., by p l a t i n g on hydrocarbon basal medium with kerosene as the sole carbon source. This organism was chosen for i n v e s t i g a t i o n because i t produced a high a c i d i t y on the kerosene medium - the colour of an added pH i n d i c a t o r (bromcresol green) was changed wi t h i n 24 hours. Growth on agar l e d to small ( 1 - 2 mm.), c i r c u l a r , convex colonies with the margin e n t i r e . Streaks on agar slants r e s u l t e d i n f i l i f o r m growth. No pigment was produced; the colonies were opaque and white to off-white. Broth media showed a l i g h t t u r b i d i t y and sediment with l i t t l e surface growth. The optimum growth temperature was near 30°C, with no growth occurring at 37°C. Microscopic examination showed the c e l l s to be non-motile, without endospores or capsules being formed. They were not acid f a s t when stained by the Ziehl-Neelson method (27). The cul t u r e displayed a high degree of pleomorphism. In young c u l t u r e s there were numerous long rods and some filaments. Filaments were more common i n broth media than on s o l i d media. As the c u l t u r e aged, rods and filaments were observed to fragment i n t o shorter rods, u s u a l l y within 24 hours on high nutrient media. These rods slowly transformed i n t o coccoid shaped c e l l s which predominate i n o l d c u l t u r e s . The coccoid c e l l s were 0.8 - 1.0/t i n diameter, smaller rods were 1.0 x 3.0/*, with longer ones up to 10/^ , and some filaments were longer than 50/c The c u l t u r e was Gram variable-rods and filaments tended to be Gram p o s i t i v e , c o c c i tended to be Gram negative. The organism had a s i m i l a r morphology on kerosene medium but rod and filamentous forms l a s t e d longer, up to 4 or 5 days. Occasional swollen forms were also observed. These morphological features can be seen i n Figure 7, which shows filaments, some at d i f f e r e n t stages of fragmentation, d i f f e r e n t s i z e rods and coccoid forms. The morphological cycle of t h i s c u l t u r e f i t s the d e s c r i p t i o n s of Arthrobacter given by Mulder (76) and Stevenson (99). Most arthrobacters do not grow at 37°C and are Gram v a r i a b l e (20). Mycobacteria and nocardia can have s i m i l a r morphology, but the mycobacteria are acid f a s t while the nocardia are branched and u s u a l l y m y c e l i a l . The organisms i n both of these genera generally grow well at 37°C, are Gram p o s i t i v e , and fragment much l a t e r i n the growth c y c l e . For these reasons, the cu l t u r e examined i n t h i s study was i d e n t i f i e d as a species of Arthrobacter. C h a r a c t e r i z a t i o n of hydrocarbon s p e c i f i c i t y In order to determine which components of the kerosene were used by the organism, the main n-alkane f r a c t i o n s i n the Fis h e r ' s odorless kerosene were i d e n t i f i e d by comparing t h e i r r e t e n t i o n times i n the gas chromatograph with those of pure n-alkanes. The gas chromatogram which was obtained i s shown i n Figure 8. The q u a n t i t a t i v e a n a l y s i s of each component i s l i s t e d i n Table I I . I t can be seen that the major n-alkanes i n t h i s l o t of kerosene are undecane, dodecane and tridecane, with l e s s e r amounts of decane, tetradecane and pentadecane. Since 72.5% of the kerosene i s n-alkane, the remainder i s probably made up of branched chain alkanes and o l e f i n s . Because microorganisms are known to metabolize normal alkanes much more r e a d i l y than branched and o l e f i n i c hydrocarbons (108) i t was decided to measure the disappearance of the d i f f e r e n t n-alkanes during growth. The composition of the kerosene from a 5 day growth f l a s k , compared to 43 F i g u r e 7. C e l l s of A r t h r o b a c t e r spp. grown i n glucose-n u t r i e n t broth. 1. filamentous forms. 2 . swollen rods. 3. fragmented f i l a m e n t . 4. t y p i c a l rod. 5. t y p i c a l coccoid forms. 44 15 10 5 TIME min. Figure 8. Gas chromatographic analysis of Fisher odorless kerosene. C g= nonane; C 1 Q= decane; c 1 ] _ = undecane; C = dodecane; C 1 3= tridecane;.C 1 4= tetradecane; C = pentadecane; C = hexadecane. Table I I . Normal Alkanes i n Kerosene n-alkane Percent i n Kerosene Percent i n Control Percent a f t e r Growth nonane decane undecane dodecane tridecane tetradecane pentadecane hexadecane 0. 2 5.4 19.3 18. 0 16.8 9.5 3.1 0. 2 0 1.2 14.7 19.3 18.5 11.5 3.4 0 0 2.4 15.3 17.7 17.6 8.5 3.4 0 72.5 68.6 64.9 a s t e r i l e c o n t r o l , i s given i n Table I I . I t can be seen that the c o n t r o l f l a s k l o s t a s i g n i f i c a n t amount of decane and undecane due to evaporation. As well as the evaporation of decane and undecane, the growth f l a s k shows a drop i n dodecane, tridecane and tetradecane due to microbial oxidation. In order to get a more pre c i s e measurement of which n-alkanes the organism could use most r e a d i l y , the oxygen uptake of the c u l t u r e was measured i n the presence of the n-alkanes. The p l o t s i n Figures 9 and 10 i n d i c a t e that dodecane and tridecane are the most r a p i d l y oxidized by t h i s organism. Because t h i s study was concerned with the acid production of t h i s organism and not j u s t growth, the pH drop and dry weight were determined on the various n-alkane carbon sources. The r e s u l t s i n Figures 11 and 12 i n d i c a t e that as well as supporting the greatest amount of growth, dodecane and tridecane also lead to the greatest a c i d production. Therefore these hydrocarbons were selected for f u r t h e r i n v e s t i g a t i o n . The growth curves of t h i s Arthrobacter s t r a i n on dodecane, tridecane and kerosene (Figures 13 & 14) i n d i c a t e that dodecane i s u t i l i z e d most r a p i d l y f o r growth and a c i d production. The growth on tridecane may be slower to i n i t i a t e because the c u l t u r e must adapt to an uneven chain length i.e.p -oxidation of an odd carbon number substrate leading to propionate formation. The c u l t u r e probably requires a longer period to synthesize enzymes to metabolize t h i s l a t t e r substrate. From the abrupt h a l t i n growth i t can be seen that 4.5 i s the l i m i t i n g pH. The slower growth and acid production observed on kerosene Figure 9. Oxygen uptake of Arthrobacter c u l t u r e on various n-alkanes v= endogenous; ©= nonane,- A= decane; •= undecane; A= dodecane; 0= tridecane; B = tetradecane; V= hexadecane. 43 500 100 9 10 11 12 13 14 16 n-ALKANE CARBON NO. Figure 10. Oxygen uptake of Arthrobacter culture as a function of n-alkane chain length. 3.3 mg c e l l s (dry weight) per f l a s k . 49 6.5 6.0 £ 5.5 5.0 4.5 10 11 12 13 HYDROCARBON 14 K F i g u r e 11. pH d r o p i n shake f l a s k medium as a f u n c t i o n o f n - a l k a n e c h a i n l e n g t h . H y d r o c a r b o n numbers i n d i c a t e n - a l k a n e c h a i n l e n g t h ; K i n d i c a t e s k e r o s e n e . >-Qi Q 1.5 1.0 0.5 0 F i g u r e 12. 10 11 12 13 HYDROCARBON 14 Growth i n shake f l a s k c u l t u r e as a f u n c t i o n o f n - a l k a n e c h a i n l e n g t h . H y d r o c a r b o n numbers i n d i c a t e n - a l k a n e c h a i n l e n g t h ; K i n d i c a t e s k e r o s e n e . 50 1.5. 0 2 4 6 8 TIME days Figur-e 13. Growth curve of Arthrobacter culture on d i f f e r e n t hydrocarbon carbon sources. • = dodecane; 0= tridecane; A = kerosene. 51 7.0 TIME days Figure 14. pH curve of medium a f t e r growth hydrocarbons. • = dodecane; 0= tridecane; A= on d i f f e r e n t kerosene. substrate probably r e s u l t s from, the lower concentration of the more r e a d i l y metabolized alkanes i n the mixture. I t may also be due to i n h i b i t i o n by other hydrocarbons i n the kerosene. C h a r a c t e r i z a t i o n of acid production The t h i n layer chromatograms of the a c i d i c l i p i d e xtract from the medium of dodecane, tridecane and kerosene grown c e l l s are shown i n Figures 15 and 16. I t can be seen that there are no spots corresponding to mono or d i c a r b o x y l i c acids from the dodecane and tridecane extracts. The kerosene extract contained a small amount of f a t t y a c i d , and p o s s i b l y some d i c a r b o x y l i c acid. To analyse these f u r t h e r , t h i s extract was methylated and i n j e c t e d i n t o a gas chromatograph, g i v i n g the chromatogram shown i n Figure 17. Only two peaks could' be i d e n t i f i e d (other than ether) by t h e i r r e t e n t i o n times - the methyl esters of octanoic and nonanoic acids. Since t h i s i s a highly concentrated extract (>200 X) analysed at very high s e n s i t i v i t y (attenuation X50 on the gas chromatograph), none of these compounds i s present i n a s i g n i f i c a n t concentration. The other peaks found probably represent minor co-oxidation products from the other kerosene components and small amounts of ketones or aldehydes c a r r i e d through i n the e x t r a c t i o n . Since some organisms grown on n-alkane hydrocarbons produce organic acids such as c i t r i c and fumaric acids e x t r a c e l l u l a r l y (1), the presence of such compounds was tested by t h i n layer chromatography (Figure 18). The only spots which appeared i n the growth media were also present i n the unused medium. These are due to components i n the yeast extract used i n the medium. 53 SOLVENT FRONT f \ O o /*~N v y O O O O ORIGIN Figure 15. Thin layer chromatogram of the a c i d i c l i p i d extract sprayed with bromcresol green. 1. dodecane medium extract. 2. tridecane medium extract. 3. kerosene medium extract. 4. decanoic acid. 5. sebacic a c i d . 6. unused medium. Cross-hatched areas represent strongly a c i d i c spots. 54 SOLVENT FRONT o o O O O ORIGIN Figure 16. Thin.layer chromatogram of the a c i d i c l i p i d e x t r a c t , charred with s u l f u r i c a c i d . 1. sebacic a c i d . 2. decanoic a c i d . 3. kerosene medium extract. 4. dodecane medium extract. 5. tridecane medium extract. 6. unused medium. Cross-hatched areas represent darkly charred spots. 55 Figure 17. Gas chromatogram of methylated extract from kerosene medium. S = solvent (ether); C = methyl octanoate; o Cg = methyl nonanoate. SOLVENT FRONT O O O O ORIGIN Figure 18. Thin layer chromatogram f or the separation of organic acids. 1. t a r t a r i c (T) and ad i p i c (A) acid standards. 2. dodecane growth medium. 3. tridecane growth medium. 4. kerosene growth medium. 5. unused medium. A f t e r the p o s s i b i l i t y of f a t t y a c i d and organic a c i d formation had been eliminated, the presence of a c i d i c amino acids i n the growth medium was investigated. Paper chromatograms of the growth medium showed there were no ninhydrin p o s i t i v e compounds present. Before spraying, the chromatogram was viewed under u l t r a v i o l e t l i g h t to check for the presence of nucleotides. None were v i s i b l e . M a t e r i a l s balance experiment Because the a c i d i t y produced by t h i s c u l t u r e was not due to the formation of a c i d i c organic compounds, i t was most l i k e l y the r e s u l t of the " p h y s i o l o g i c a l a c i d i t y " of the ammonium s a l t s nitrogen source (56). This a c i d i t y r e s u l t s from the a s s i m i l a t i o n of ammonium ions by the organism for amino acid and nucleotide synthesis. As ammonium ions are taken up by the c e l l s hydrogen ions are released to maintain an i o n i c balance i n the medium. In order to determine i f t h i s was the o r i g i n of the a c i d i t y , the Arthrobacter s t r a i n was grown i n a fermentor with pH c o n t r o l . Higher nitrogen concentration was used i n the medium so that i t would not be l i m i t i n g . The changes i n the carbon and nitrogen components i n the system were followed during the course of the fermentation, along with growth and a c i d i t y . As can be seen i n Figure 19, the c e l l dry weight increases r a p i d l y a f t e r a lag of about 5 hours. A f t e r 24 hours,- the growth stopped abruptly and c e l l dry weight declined slowly a f t e r that. Carbon dioxide gas was produced most r a p i d l y during the a c t i v e growth stage of the c u l t u r e , with the highest rate occurring j u s t before the end of growth. 58 TIME hours F i g u r e 19. Change i n s u b s t r a t e c o n c e n t r a t i o n and growth parameters during fermentation. A= dodecane; 0= dry weight; 0= a c i d i t y ; •= carbon d i o x i d e . The concentration of di s s o l v e d bicarbonate d i d not reach a s i g n i f i c a n t l y high l e v e l . The dodecane i n the medium was u t i l i z e d r a p i d l y . I t was completely depleted a f t e r 24 hours. The exhaustion of the carbon and energy source at t h i s time caused the c u l t u r e to stop growth and metabolism. The o v e r a l l carbon balance f o r the system i s pl o t t e d i n Figure 20. The net l o s s of carbon during the growth phase was probably due to the i n e f f i c i e n c y of the trap f o r carbon dioxide gas. Most of the carbon dioxide escaped i n t o the atmosphere. More traps would be required to catch t h i s l o s t carbon dioxide but t h i s could not be done because the aerating pump was not powerful enough to force the a i r through more than one trap i n s e r i e s . The changes i n c e l l u l a r nitrogen, ammonia nitrogen i n the medium and t o t a l nitrogen i n the medium are shown i n Figure 21. The c e l l nitrogen and ammonia nitrogen curves follow the growth of the organisms up to about 19 hours. At t h i s time there i s a sudden drop i n the ammonia nitrogen and i n the t o t a l nitrogen of the system. This sudden lo s s of nitrogen coincides with an anomalous increase i n acid production. Up t i l l t h i s point the t i t r a t a b l e a c i d i t y i s pro p o r t i o n a l to nitrogen uptake and growth, but at 19 hours there i s a sharp increase i n the t i t r a t a b l e a c i d i t y , which continues u n t i l 24 hours. A f t e r the 24 hour point, the a c i d i t y drops r a p i d l y and loss of nitrogen ceases, which corresponds with the end of growth and exhaustion of the dodecane carbon + + source. The nitrogen l o s s amounts to 0.21 - 0.02 g/1, or 15 - 2 meq/1. The anomalous increase i n a c i d i t y i s about 14 meq/1 above the a c i d i t y due to growth alone. 60 8 0 10 20 30 40 TIME hours Figure 20. Carbon balance during fermentation. 0= c e l l carbon; ® = carbon dioxide carbon; • = dodecane carbon; H= t o t a l carbon. 2.0 0 10 20 30 40 TIME hours Figure 21. Nitrogen balance during fermentation. 0= c e l l nitrogen; © = ammonia nitrogen; •= t o t a l nitrogen. In order to account f o r t h i s l o s s of nitrogen from the system, the e f f l u e n t gases were passed through an a c i d trap to c o l l e c t v o l a t i l e nitrogen compounds-amines or ammonia. It was not expected that any would be found because the pH of the medium was below the pK of amines (pK =10-11) and ammonia (pK =10.25) and therefore these compounds would a a not be v o l a t i l e . No nitrogen compounds were found i n the a c i d trap. In view of the studies of Gunner (43) which showed that Arthrobacter  globiformis could o x i d i z e ammonium ions to nitrogen oxide gases and n i t r a t e and reports of ammonium ion oxidation by methane grown b a c t e r i a (54, 111), the p o s s i b i l i t y of t h i s oxidation occurring during the fermentation was investigated. The production of nitrogen oxide gases was checked by passing the e f f l u e n t gas from the fermentor through the trapping s o l u t i o n used by Gunner, and analysing i t for n i t r a t e . The r e s u l t s showed that the trap contained 66 mg of n i t r a t e nitrogen, equivalent to 44 mg/1 from the fermentation medium. This represents about- 21% of the nitrogen l o s t from the medium. The n i t r a t e nitrogen concentration i n the medium was found to be 37 mg/1, about 18% of the decrease i n the K j e l d a h l nitrogen. This f i g u r e for n i t r a t e nitrogen i s of questionable accuracy, since there may have been in t e r f e r e n c e i n the determination due to c h l o r i d e ions and organic matter. Because n i t r a t e i s not q u a n t i t a t i v e l y measured by the K j e l d a h l method,, i t would be part of the drop i n t o t a l nitrogen during the fermentation. These r e s u l t s show that t h i s Arthrobacter s t r a i n can o x i d i z e ammonium ions i n the medium. This o x i d a t i o n takes place i n the rapid log phase of a c t i v e growth of the c u l t u r e , when the dodecane carbon source i s 63 becoming depleted. This i s s i m i l a r to the system described by Gunner (43) i n which an Arthrobacter globiformis s t r a i n , which was grown on succinate carbon source, oxidized ammonium ions when suspended i n buffer without carbon nutr i e n t source. The oxidation of ammonium ions described here i s the f i r s t demonstration of t h i s phenomenon i n an n-alkane carbon source fermentation and r a i s e s a number of i n t e r e s t i n g questions about the pathways and mechanisms involved. The oxidation probably occurs v i a the inorganic heterotrophic pathway as demonstrated i n A. globiformis. This pathway i s the only one which has been shown to y i e l d gaseous products. There i s the p o s s i b i l i t y that the c u l t u r e uses t h i s pathway as a source of energy as the carbon source becomes exhausted. Since the i n i t i a l o x i d a t i o n of ammonium to hydroxylamine i s endergonic GaF'=+3.85 kcal/mole) (2) subsequent oxidation steps, f o r example the dehydrogenation of hydroxylamine to the postulated n i t r o x y l (NOH) intermediate would have to be coupled with an e l e c t r o n transport system to produce ATP. I t may be that the oxidation i s only p a r t i a l l y completed due to a lack of the required enzyme systems, leading to the non-enzymatic formation of nit r o u s oxide as demonstrated by Anderson (10). Even i f the c u l t u r e were capable of d e r i v i n g some energy from t h i s oxidation, i t cannot f i x carbon dioxide as occurs i n Nitrosomonas and Nitrobacter and therefore growth stops when the carbon source i s exhausted. This would occur at a stage of the r a p i d logarithmetic growth phase before energy and carbon storage reserve materials are formed. Although there are some s i m i l a r i t i e s between the growth of micro-organisms on methane and higher n-alkane carbon sources, i t i s not l i k e l y that the i n i t i a l ammonia oxidation step of t h i s Arthrobacter c u l t u r e i s the same as i n the methane u t i l i z i n g b a c t e r i a . In the l a t t e r organisms, ammonium ions are oxidized by the methane oxidase enzyme competitively with methane (113). This phenomenon i s probably due to the s i m i l a r i t y between the geometry of the CH^ molecule and NH* ion. Conversely, there i s l e s s s i m i l a r i t y between the ammonium ion and the terminal methyl group of an n-alkane. Also, methane oxidase i s now believed to operate by a mechansim which d i f f e r s from other hydrocarbon oxidase enzymes (113). The oxidation of ammonium ions can be regarded as a form of the co-oxidation r e a c t i o n which has been observed i n other hydrocarbon systems (68, 29, 85). Hydrocarbon co-oxidations are characterized by the transformation of a non-growth supporting hydrocarbon to an oxygenated product while the c u l t u r e grows on another substrate, e i t h e r hydrocarbon or non-hydrocarbon. This oxidation can be a side r e a c t i o n of the oxygenase enzyme for the growth supporting hydrocarbon, or i t can be catalysed by a separate i n d u c i b l e enzyme. Further research i s needed to determine the r e l a t i o n of ammonium ion oxidation to hydrocarbon fermentations. I t would be i n t e r e s t i n g to answer questions concerning how widespread t h i s r e a c t i o n i s i n n-alkane fermentations, what pathways and mechanisms are involved, and what value t h i s r e a c t i o n could be i n i n d u s t r i a l fermentations, for example, could an organism be found which would s e l e c t i v e l y o x i d i z e a substituted amino group to form a product? These problems require f u r t h e r i n v e s t i -gations to provide answers. CONCLUSIONS The c u l t u r e i s o l a t e d i n t h i s study was a member of the genus Arthrobacter. I t was capable of rapid growth and ac i d production on a number of hydrocarbon carbon sources i n a n u t r i e n t s a l t s medium. While capable of good growth on r e f i n e d kerosene carbon source, the Arthrobacter spp. grew best on the p u r i f i e d n-alkanes, dodecane and tridecane. The a c i d i t y generated by the c u l t u r e was not due to the formation of a c i d i c compounds such as f a t t y acids, organic acids or amino acids. I t was due to the p h y s i o l o g i c a l a c i d i t y of the ammonium ion nitrogen source - as ammonium ions are taken up by the c u l t u r e , a c i d i c inorganic anions are l e f t i n the medium. When grown under conditions of pH c o n t r o l and l i m i t e d hydrocarbon concentration, t h i s c u l t u r e oxidized ammonium ions to form n i t r a t e i n the medium and nitrogen oxide gases. This i s the f i r s t time t h i s oxidation has been observed i n an n-alkane carbon source system. 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