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Microbial oxidation of n-alkane hydrocarbons Liu, Dickson Lee Shen 1971

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MICROBIAL OXIDATION OF n-ALKANE HYDROCARBONS by DICKSON LEE 8HEN LIU B.S., Taiwan Chung-Hsing U n i v e r s i t y , 1962 M.S., U n i v e r s i t y c f B r i t i s h Columbia, 1966 A T H E S I S SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Food Science We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1971 In presenting t h i s t h e s i s In p a r t i a l f u l f i l m e n t of the requirements 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 t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree tha t 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 r e p r e s e n t a t i v e s . I t i s understood tha 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. DICKSON LEE SHEN LIU 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 D a t e Apy-I 30, /9 7/ ABSTRACT A four-part inves t i g a t i o n was described i n which f i r s t , the use of t h i o l i g n i n and lignosulfonate to remove a factor involved i n growth l i m i t a t i o n i n hydrocarbon fermenta-t i o n was. investigated; secondly, the performance of the Cyelone-fermenter i n hydrocarbon fermentation was evaluated; t h i r d l y , continuous hydrocarbon fermentation with reference to hydrocarbon, nitrogen, t h i o l i g n i n and d i l u t i o n rate was studied; and fo u r t h l y , the mechanism of n-decane oxidation Was studied manometrically and gas chromatographically. The addition of polymerized lignosulfonate and t h i o l i g n i n into the hydrocarbon fermentation media greatly, increased the fermentation rate and the y i e l d of biomass. Lignin i t s e l f did not appear to be decomposed during the fermentation. Resting c e l l studies of Pseudomonas desmolytica indicated that the oxygen consumption increased with the decreasing n-alkane carbon number and did not p a r a l l e l the production of t o t a l biomass. The greatest biomass occurred using n-undecane and decreased sharply with lower and higher n-alkanes. The i n d i v i d u a l n-alkanes i n kerosene were not degraded uniformly, the lower ones were used p r e f e r e n t i a l l y . T h i o l i g n i n not only increased the rate of u t i l i z a t i o n of these lower n-alkanes but also extended microbial a c c e p t a b i l i t y to the higher n-alkanes. Gas chromatographic analyses revealed that f i v e monocarboxylic f a t t y acids corresponding to Cp, C p, C- n, C__, and C. 'Were present" i n the fermentation f l u i The Cyclone-fermenter was found t o be very s u i t a b l e f o r hydrocarbon fermentation. The hydrocarbon, kerosene, was fermented w i t h a pure b a c t e r i a l c u l t u r e i n a continuous process f o r 250 hours without any n o t i c e a b l e change i n the c u l t u r e behavior. Moreover, the a d d i t i o n of v a r i o u s c u l t u r e medium i n g r e d i e n t s could be optimized to.produce maximum c e l l y i e l d or maximum a c i d production. Manometric and gas chromatographic s t u d i e s r e vealed th a t c e l l - f r e e e x t r a c t s of o x i d i z e d n-decane t o n-decanol and n-decanoic a c i d , whereas the. p a r t i a l l y p u r i f i e d hydrocarbon-o x i d i z i n g enzyme only o x i d i z e d n-decane to decanol. The n-decane-oxidizing enzyme could be p r e c i p i t a t e d by 30% (NH ) S0 U and had a narrow optimal pH around 7.0. The enzyme a l s o r e q u i r e d a d i a l y z e d , heat s t a b l e 60% (NH^^SO^ supernatant f r a c t i o n and NAD f o r maximum enzyme a c t i v i t y . Ferrous, manganous and calcium ions d i d not s t i m u l a t e the enzyme a c t i v i t y . I t seemed tha t the enzymatic a t t a c k on n-decane occurred p r i m a r i l y at the t e r m i n a l carbon atom and t h i s was manifested by the f a c t that n-decanol, n-decanal and n-decanoic a c i d supported good growth f o r S^^. - i v -TABLE OF CONTENTS Page LIST OF TABLES v i i LIST OF FIGURES v i i i ACKNOWLEDGEMENTS x i GENERAL INTRODUCTION 1 LITERATURE REVIEW 4 CHAPTER I. THIOLIGNINS AND LIGNOSULFONATES IN PETROLEUM FERMENTATION INTRODUCTION 9 MATERIALS AND METHODS 10 RESULTS AND DISCUSSION 12 CONCLUSIONS 29 CHAPTER I I . THE CYCLONE RESEARCH FERMENTER VS. THE ROTARY SHAKE CULTURE INTRODUCTION 32 MATERIALS AND METHODS 33 RESULTS AND DISCUSSION 35 CONCLUSIONS 45 CHAPTER I I I . CONTINUOUS PETROLEUM FERMENTATION INTRODUCTION 4 6 MATERIALS AND METHODS 47 RESULTS AND DISCUSSION 52 CONCLUSIONS . 6 0 - v -Page CHAPTER IV. ENZYMATIC STUDIES OF HYDROCARBON OXIDATION INTRODUCTION 6 4 MATERIALS AND METHODS 6 5 RESULTS AND DISCUSSION 6 7 CONCLUSIONS 87 GENERAL SUMMARY AND CONCLUSIONS 90 ;LITERATURE CITED 9 3 - v i -Table I II III IV LIST OF TABLES Page Effect of Indulin on the Acid Production by Various Hydrocarbon U t i l i z a t i o n Bacteria. 13 C l a s s i f i c a t i o n of Culture S ^ 14 The Ef f e c t of Polymerization of Lignin on the C e l l Y i e l d of Culture S ± 1 30 Eff e c t of Indulin C on the C e l l Y i e l d and Acid Production of Culture S u using n-Decane, n-Decanol, n-Decanal and n~Decanoic Acid as Substrate * 8 8 - v i i -LIST OF FIGURES Eff e c t of Various Amounts of Kerosene on the Rate of Oxygen Uptake Oxygen Uptake by Culture S n grown on Kerosene Medium or Kerosene-Indulin C Medium Ef f e c t of Indulin C or Tween-60 on Oxygen Uptake by Culture S;Q. E f f e c t of Indulin C or Tween-60 on C e l l Y i e l d of Culture S-Q.. Oxygen Uptake, using pure n-Alkanes Substrates The E f f e c t of Indulin C on the C e l l Y i e l d i n the Presence of Pure n-Alkane Substrates E f f e c t of Indulin C or Tween-60 on Acid Production The C e l l Y i e l d as a Function of Kerosene Concentration i n the Presence of Indulin C or Tween-6 0 The Acid Production as a Function of Kerosene Concentration i n the Presence of Indulin C or Tween-60 The Rate of C e l l Growth of under Various Growth Conditions The Rate of Acid Production of S^i under Various Growth Conditions The Rate of S n C e l l Growth and Acid Production i n Kerosene-Indulin Medium i n Cyclone Fermenter Gas Chromatographic Separation of the Major Components of Fisher Odorless Kerosene Gas Chromatographic Separation of the Major Hydrocarbon Components Present i n the Residual Kerosene in the Kerosene Culture a f t e r k Days Incubation on Rotary Shaker. F 5. gure Page 15 Gas Chromatographic Separation of the Major Hydrocarbon Components Present i n the Residual Kerosene in the Kerosene Culture aft e r 7 Days Incubation on a Rotary Shaker. 43 16 Gas Chromatographic Separation of the Major Hydrocarbon Components Present i n the Residual Ker^osene i n the Kerosene-Indulin Culture a f t e r 2 Days Incubation on a Rotary Shaker. 44 17 Schematic Diagram of the Continuous Hydrocarbon Fermentation Equipment. 49 18 Continuous Growth of S u on Indulin-Mineral •Medium with Kerosene as Sole Carbon Source. 51 19 The E f f e c t of Kerosene Flow Rate on C e l l Y i e l d and Acid Production. 54 2 0 Gas Chromatographic Separation of the Straight Chain Fatty Acid Methyl Esters found i n the Supernatant of the F i n a l Stage of Continuous Growth of Su on Indulin-Mineral Medium with Kerosene as Sole Carbon Source. 55 21 Continuous Growth of Su on Kerosene-Mineral Medium with Indulin C as Emulsifying Agent. 56 22 The E f f e c t of Indulin C Flow Rate on C e l l Y i e l d and Acid Production. • 57 23 Continuous Growth of S^i on Kerosene with Varying Indulin-Mineral Medium Flow Rate. 58 24 The E f f e c t of Indulin-Mineral Medium Flow Rate on C e l l Y i e l d and Acid Production. 59 25 Continuous Growth of S u on Kerosene-Indulin-Mineral Medium with NH^Cl as Sole Nitrogen Source. 61 26 The Eff e c t of NH^Cl Flow Rate on C e l l Y i e l d and Acid Production. 62 27a Oxygen Uptake of Cell-Free Extract of n-Decane-Indulin C Grown Ce l l s 68 27b Oxygen Uptake of Cell-Free Extract of n-Decane Grown c e l l s . 6 8 - ix -Figure Page 2 0 The Ef f e c t of NAD and NADP on the Oxvgen Uptake of The Cell-Free Extract of S^. * • 69 29 E f f e c t of pK on Oxygen Uptake.of the C e l l -Free Extract. 71 30 Ef f e c t of pH on the A c t i v i t y of the Cell-Free Extract. 7 2 31 Effect of the Concentration of MAD on the A c t i v i t y of the Cell-Free Extract 7 3 3 2 E f f e c t of the Concentration of NAD on the A c t i v i t y of the Cell-Free Extract. 74 33 Manometric Evidence.for the Enzymatic Oxidation of n-Decane ( C ^ Q ) . 7 5 34 Manometric Evidence f o r the Enzymatic Oxidation of n-Decane' 7 6 3 5 Manometric Evidence f o r the Enzymatic Oxidation. of n-Decane 7 7 3 6 E f f e c t of Heat Treatment on the A c t i v i t y of the Hydrocarbon-Oxidizing Enzyme 7 9 3 7 Manometric Evidence f o r the Enzymatic Oxidation of n-Decane 80 38 Manometric Evidence f o r the Enzymatic Oxidation of n-Decane 81 3 9 Gas Chromatographic I d e n t i f i c a t i o n of n-Decanol as the Product of Enzymatic Oxidation of n-Decane (CFX + NAD + C 1 Q ) . 82 40 Gas Chromatographic I d e n t i f i c a t i o n of n-Decanoic Acid as the Product of Enzymatic Oxidation of n-Decane (CFX'+ NAD + C 1 Q ) . 83 .41 Manometric Evidence f o r the Enzymatic Oxidation of n-Decane 84 42 Gas Chromatographic I d e n t i f i c a t i o n of n-Decanol as the Product of Enzymatic Oxidation of n-Decane (30%+ + 60% s + NAD + C 1 Q ) . 85 4 3 Gas Chromatographic Investigation of the Methylation Product from the Enzymatic Oxidation of n-Decane (30%+ + 60% s + NAD + C 1 Q ) . 86 - x -ACKNOWLEDGEMENTS The author wishes to express his deepest gratitude to Dr. P.M. Townsley, Associate Professor, Department of Food Science, University of B r i t i s h Columbia, under whose supervision t h i s project was undertaken, and f o r his guidance i n the preparation of t h i s t h e s i s . Sincere appreciation i s also extended to Dr. M. Shaw, Dean of A g r i c u l t u r a l Sciences; Dr. Wm. D. Powrie, Head of the Department of Food Science; Dr.J.F. Richards, Food Science. Department; Professor T.L. Coulthard, A g r i c u l t u r a l Engineering Department and Dr. L.E. Lowe, S o i l Science Department, the University of B r i t i s h Columbia. This research was supported by the Department of Energy, Mines and Resources, Government of Canada. GENERAL INTRODUCTION The requirement for protein i n the human diet has l e the food microbiologist to examine the p o s s i b i l i t y of producin protein from micro-organisms. However, the shortage and the r e l a t i v e l y high cost of carbohydrate substrates, such as cane sugar molasses and plant starchs, have reduced the commercial intere s t i n production of microbial protein. The general reluctance of humans to consume foods containing microbial protein and the t o x i c i t y of a diet containing large quantities of nucleic acid has prevented t h e i r general acceptance i n the d i e t . The p o s s i b i l i t y of u t i l i z i n g the hydrocarbons of mineral o i l as a carbon source for the c u l t i v a t i o n of yeasts wa suggested by Champagnat et a l . i n 1963 (4). Subsequent studies indicated that microbial protein is o l a t e d from c e l l s grown on hydrocarbon contained a protein n u t r i t i v e value equivalent to that found i n f i s h protein and i n dried milk (34). Currently, the annual world production of the carbon source, crude petroleum, i s approximately 1500 m i l l i o n tons, of which 40-50 m i l l i o n tons would be p a r a f f i n . This l a t t e r amount of p a r a f f i n would have the pote n t i a l to y i e l d 20 m i l l i o n tons of microbial protein. Petroleum hydrocarbons d i f f e r from those substrates already partly oxidized, such as carbohydrates, i n that they do not contain oxygen. The need to produce the same c e l l essentials regardless of the carbon source requires that the hydrocarbon u t i l i z i n g organism have a ready access to t h i s increased demand f o r oxygen. It has been estimated that three times more oxygen i s required in a hydrocarbon fermentation than i n carbohydrate fermentation. Active aerobic metabolism of hydrocarbons can be carried out i n less than 0.5 ppm dissolved oxygen in the medium provided i t i s replenished at a s u f f i c i e n t rate to avoid a l i m i t i n g condition (31). Another problem encountered with hydrocarbon fermentations i s the limited s o l u b i l i t y of the o i l i n the aqueous medium. In a t y p i c a l carbohydrate fermentation the substrate i s dissolved in grams-per-liter quantities. On the other hand, i n a hydrocarbon fermentation, the dissolved substrate concentration i n the aqueous medium could be considered to be less than 1 ppm To provide a greater concentration of the substrate to the organism, e s p e c i a l l y i n the ear^ly stages of the fermentation a high speed impeller fermenter containing large quantities of substrate, such as 50-100 g of gas o i l per l i t e r of medium, have been used (2H). The r e s u l t i n g created emulsion provides a s u f f i c i e n t dispersion of o i l droplets in the aqueous phase to encourage increased microbial growth. It i s reasonable to assume therefore that an understanding of the mechanism of hydrocarbon oxidation i s imperative i n the design of a hydro-carbon fermentation u n i t . Several metabolic studies on the fate of hydrocarbons i n b a c t e r i a l n u t r i t i o n have been published (9, 11, 12, 13, 17, 32). These hydrocarbon substrates include n-alkanes, isoalkanes, alkenes, cycloalkanes and aromatic compounds. For several reasons, although primarily to minimize the pote n t i a l health hazards, n-alkanes, such as kerosene, would appear to be the. favoured hydrocarbon source for the production of microbial proteins (15). The merits of batch and continuous culture fermentation technology have been studied extensively during the past decade. Many investigators consider the continuous process to have overwhelming advantages f o r microbial c e l l production i n terms of c e r t a i n s t r u c t u r a l design, economics and maintenance of logarithmic rate of c e l l growth. However1, i n petroleum fermentation, the culture characters, complicated by the presence of a two phase system and a high oxygen demand, are not well recognized. Hence i t would seem advisable i n t h i s thesis to study the mechanism of continuous hydrocarbon fermentation. The primary objectives of the present study are to elucidate the mechanism of the microbial oxidation of n-alkane hydrocarbons. Several features of the fermentation are described; such as the use of t h i o l i g n i n s and lignosulfonates to overcome the growth l i m i t a t i o n i n hydrocarbon fermentation; the performance of the cyclone research fermenter i n hydro-, carbon fermentation; the continuous hydrocarbon fermentation with reference to hydrocarbon, nitrogen, t h i o l i g n i n , and d i l u t i o n rate, and the mechanism of n-decane oxidation using manometric and gas chromatographic techniques. LITERATURE REVIEW For a long time, the hydrocarbons, e s p e c i a l l y the n-alkanes, have been regarded as chemically i n e r t substances with a low order of r e a c t i v i t y . However, t h i s r e l a t i v e chemical inertness has been shown to be more apparent than r e a l . By the early 1950's, there was s u f f i c i e n t evidence that u t i l i z a t i o n of hydrocarbon was wide spread among micro-organisms (10). U n t i l recently, most of these studies have been primarily d e s c r i p t i v e . The subjects investigated involved mainly organism and substrate s p e c i f i c i t y (9, 11, 12, 28). Comparatively l i t t l e attention was given to the mechanism involved i n either transforming hydrocarbon to other compounds or converting the hydrocarbon to c e l l u l a r material (34).. In the recent years, the general area of fermentation technology has expanded greatly. Fermentation i s now a well-defined unit process f o r the production of a s i g n i f i c a n t number of economically important products, such as wine and cheese. In most i n d u s t r i a l aerobic and anaerobic fermentations, the substrates required by the organisms are generally soluble i n water at concentrations well i n excess of the value at which they would become growth-limiting. However, i n systems where the main or the only source of carbon i s hydrocarbon of low s o l u b i l i t y i n water, s u f f i c i e n t hydrocarbon-water i n t e r f a c i a l area must be provided i n order to supply enough substrate to the organism (3). Most of the hydrocarbons have a s o l u b i l i t y of less than 1 ppm i n water. The s o l u b i l i t i e s of n-nonane and n-decane at 25°C were found to be as low as 220 and 52 parts 5. per b i l l i o n (22). Humphrey pointed out that the s o l u b i l i t y of the substrate and the oxygen demand of the fermentation were the two l i m i t i n g growth conditions encountered i n hydro-carbon fermentations (10). These two l a t t e r problems could a l t e r the economics and equipment requirement for i n d u s t r i a l considerations. In order to minimize the growth-limiting f a c t o r i n hydrocarbon fermentation, fermenters have been designed to increase the area of the oil-water interface by mechanical dispersion of small o i l droplets throughout the medium. From the l i t e r a t u r e , i t seemed that mechanical s t i r r i n g could not provide s u f f i c i e n t substrate in the aqueous phase to promote rapid microbial growth (3). Various new concepts have been incorporated into the design of hydrocarbon fermenters, f o r example the medium may be c i r c u l a t e d by means of a i r such as used i n the "closed gas recycle fermenter" (1). The ingenious cyclone, research fermenter designed by Dawson (5) has been -accepted i n t h i s thesis a f t e r an evaluation of the problems involved i n hydrocarbon fermentation (20). Liu and Townsley i n 197 0 showed that the addition of polymerized t h i o l i g n i n s and lignosulfonates into the hydrocarbon fermentation medium greatly increased the fermentation rate and the y i e l d of biomass (18). Moreover, the removal of the hydrocarbon as the growth-limiting factor i n fermentation su b s t a n t i a l l y reduced the requirement f o r o i l dispersion equipment, i . e . lower production cost (19). From the thermodynamic point of view, hydrocarbons appear to be a superb source of energy f o r microbial growth. The addition of each methylene group i n a normal, saturated p a r a f f i n increases the heat of combustion (oxidation) about 17 5 K cal/g-mole (34). But the fact remains that today carbohydrates and proteins are s t i l l the p r i n c i p a l sources of carbon i n commercial fermentation, processes. However, with an increasing rate i n world population, petroleum, i f i t remains at i t s present unit cost, w i l l very l i k e l y be the major carbon source f o r the i n d u s t r i a l fermentation industry of the future. The annual world production of crude petroleum i s about 1500 m i l l i o n tons, of which 40-50 m i l l i o n tons would be p a r a f f i n . This p a r a f f i n would have the potential to y i e l d 20 m i l l i o n tons of food protein (30). This food protein contribution to the people of the world would be very s i g n i f i c a n t . In large-scale fermentations, e s p e c i a l l y i n biomass production from hydrocarbon, the continuous process has been considered to have overwhelming advantages i n terms of economics and techniques (21) . However, i n practice some problems have been encountered due to the d i f f i c u l t y in pre-d i c t i n g continuous fermenter performance from batch fermentation data (6). By using pulp m i l l t h i o l i g n i n and a modified cyclone fermenter, i t was found possible to carry on a continuous hydrocarbon fermentation for more than 2 00 hours without any noticeable change i n the culture behavior (19). Comparatively speaking, more work has been done on microbial oxidation of n-alkanes than any other hydrocarbons. Microbial attack on n-alkanes occurs primarily at the terminal carbon atom to give successively an alcohol, an aldehyde and then a carboxylic acid. Further attack of the l a t t e r may occur by the f a m i l i a r 3-oxidation of f a t t y acids (10). Secondly, the oxidation may also occur at a penultimate methylene group (C^) to give a methyl ketone, and t h i r d l y , the oxidation may occur at both ends of the molecule to give an oc-w-dicarboxylic acid (27). L i t t l e research has been done on the microbial attack of isoalkanes and alkenes. Straight-chain alkanes are oxidized f a r more e a s i l y than branched ones. Alkanes with a branched a l k y l group larger than methyl or with multiple methyl branches are considered d i f f i c u l t to degrade (10). Microbial oxidation of aromatic compounds proceeds through a series of intermediates. The r i n g i s f i r s t oxidized to give a trans-dihydrodihydroxy compound, which i s then dehydrogenated to the corresponding dihydroxy compound (10). The enzyme, oxygenase, has been reported to be responsible f o r the r i n g cleavage (9). In the case of a single r i n g aromatic compound, c i s - c i s muconic acid has been found to be the usual intermediate. This muconic acid i s eventually oxidized v i a acet y l CoA and succinate i n the TCA cycle (25). Co-oxidation, the oxidation of a non-growth supporting hydrocarbon, i s a very i n t e r e s t i n g phenomenon i n hydrocarbon fermentation and c e r t a i n l y o f f e r s commercial p o s s i b i l i t i e s i n various i n d u s t r i e s . For example, retention of molecular configuration i s a feature of microbiological co-oxidation (10) which has been u t i l i z e d i n pharmaceutical industries for the manufacture of s p e c i f i c steroids. In general, the aromatics and cycloparaffins are more suitable compounds for co-oxidation 8. than n-alkanes, because the hydrocarbon bacteria appear to use n-alkanes f o r growth rather than f o r co-oxidation. CHAPTER I. THI0LIGN1NS AND LIGNOSULFONATES IN PETROLEUM FERMENTATION INTRODUCTION The rate of u t i l i z a t i o n of l i q u i d n-alkane hydro-carbons as substrates i n i n d u s t r i a l fermentation i s dependent on the ease of t h e i r a v a i l a b i l i t y to the microorganisms (3). The concentration of the hydrocarbons, which may exist at any one time i n the aqueous phase, i s primarily a function of the rate of removal of the dissolved hydrocarbon by the organisms and the rate of solution across the oil-water i n t e r f a c e . Assuming an adequate oxygen supply, the s o l u b i l i t y of the petroleum i n the aqueous medium i s the most probable growth-limiting factor for petroleum micro-organisms growing i n submerged culture. For example, the s o l u b i l i t i e s of n-nonane and n-decane at 25°C are 220 and 52 p a r t s / b i l , respectively (22). In order to minimize t h i s l i m i t i n g f a c t o r , fermenters have been designed to increase the area of the oil-water interface by mecahnical dispersion of small o i l droplets throughout the medium (3). Another pote n t i a l growth-limiting step i n petroleum fermentations which cannot be overlooked i s the o i l - c e l l surface i n t e r a c t i o n . In the l a t t e r case i t i s possible that an ingredient i n the medium which could associate i t s e l f with the c e l l wall or a l t e r the c e l l surface, and could change the rate of transfer across the c e l l membrane. This study describes the b a c t e r i a l growth-stimulating e f f e c t s of added pulp-mill t h i o l i g n i n s and lignosulfonates to a petroleum medium. MATERIALS AND METHODS Medium The following medium was designed to serve as an enrichment medium f o r the i n i t i a l i s o l a t i o n of bacteria from s o i l samples and as a general basal medium f o r the support of the i s o l a t e d hydrocarbon u t i l i z i n g bacteria. K 2HP0 4 (anhydrous) 2.50 g KH 2P0 U 1.75 g NH4C1 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 2-UH 20 0.002 g Na 2S0 4 (anhydrous) 0.05 g Yeast extract ; 0.10 g D i s t i l l e d H20 1000 ml Kerosene (Fisher, Odorless) 20 ml pH 6.8 Pure cultures of the bacteria were obtained by streaking the culture on the above medium s o l i d i f i e d with 1.5 percent agar. I s o l a t i o n of the Cultures Oil-saturated s o i l samples taken from l o c a l r e f i n e r y , approximately 5 g each, were added to 50 ml of kerosene-liquid medium contained i n 125-ml Erlenmeyer f l a s k s . The cultures were incubated at 3 0°C on a New Brunswick rotary shaker operated at 11. 220 str.okes/min. The cultures were transferred every week f c r 6 weeks and then vrere streaked on p l a t i n g medium for i s o l a t i o n i n pure culture. Preliminary i d e n t i f i c a t i o n of the i s o l a t e d organisms was determined by morphology and the gram-sta i n reaction. Stock aqueous solutions of the l i g n i n samples and of the surface active agent, Tween-6 0 were prepared at a concentration of 50 mg/ml and were steam-sterilized at 15 l b / sq. i n . f o r 15 min. Dry Weight The bacteria were centrifuged from 30 ml of culture suspension at 5,000 x g for 20 min, washed by resuspension and centrifugation i n 0.05M phosphate, pH 7.4 buffer, followed by two washes i n d i s t i l l e d water. The c e l l p e l l e t was taken up i n 5 ml of d i s t i l l e d water and dried f o r 16 hr at 100°C. Respiration Oxygen uptake by the i n t a c t c e l l s was measured at 3 0°C following conventional methods using the Gilson D i f f e r e n t i a l Respirometer. After a preincubation period of 5 min," the substrate was added from the side arm into the main chamber. The CO^ was absorbed i n 0.2 ml of 20 percent (w/v) KOH contained i n a Whatman No. 4 f i l t e r paper wick supported i n the center well . The f i n a l f l u i d volume was 3.2 ml. pH Measurements Following the removal of the b a c t e r i a l c e l l s by c e n t r i f u g a t i o n , the pH of the fermentation medium was measured d i r e c t l y with a pH meter. A l l the experiments were done i n 12. t r i p l i c a t e i n order to minimize e r r o r . RESULTS AND DISCUSSION The E f f e c t of Highly Polymerized T h i o l i g n i n s on A c i d Production i n Kerosene Fermentations.. ' Pure b a c t e r i a l c u l t u r e s i s o l a t e d by the kerosene enrichment procedure, r e p r e s e n t i n g a wide range of d i f f e r e n t colony types and c e l l morphology.,, were i n o c u l a t e d i n t o 50 ml a l i q u o t s of b a s a l media contained i n 12 5 ml Erlenmeyer f l a s k s . The c u l t u r e s then were subjected to four treatments. These were: (a) the a d d i t i o n of kerosene; (b) the a d d i t i o n of I n d u l i n C; (c) the a d d i t i o n of both kerosene and I n d u l i n C; and (d) no a d d i t i o n . The r e s u l t s are recorded i n Table I . The r e s u l t s i n d i c a t e t h a t the production and r e l e a s e of a c i d i n t o the kerosene c u l t u r e medium was augmented g r e a t l y by the a d d i t i o n of a small q u a n t i t y of I n d u l i n C. This s t i m u l a t i o n d i d not appear to be due t o the fermentation of the I n d u l i n C i t s e l f . The Oxygen Uptake by Cult u r e S ^ Grown on Kerosene Basal Medium. The c u l t u r e was chosen because of i t s a b i l i t y to grow r a p i d l y g i v i n g a measurable c e l l y i e l d i n kerosene-basal medium on short c u l t u r e i n c u b a t i o n s . This micro-organism ( S ^ ) was i s o l a t e d from s o i l samples taken at a l o c a l o i l r e f i n e r y and c l a s s i f i e d as Pseudomonas desmolytica according to Bergey's Manual of Determinative B a c t e r i o l o g y , 7th e d i t i o n , see Table I I . The oxygen uptake of S ^ c e l l s i n b a s a l medium co n t a i n i n g v a r y i n g amounts of added kerosene i s shown i n Figure 1. The a d d i t i o n of s m a l l , g r o w t h - l i m i t i n g amounts of TABLE I EFFECT OF INDULIN ON THE ACID PRODUCTION.BY VARIOUS HYDROCARBON UTILIZATION BACTERIA Cultures pH afte r 48 hr* Basal Medium + 1 ml Kerosene Basal Medium + 50 mg Basal Medium + 1 ml Kerosene + 50 mg Indulin C t t l n d u l i n C s l 6.6 6.8 5.1 y .6.7 6.8 4.8 3 6.6 6.8 4.3 S3 6.5 6.7 4.7 4 6.6 6.8 4.5 5 6.6 6.8 4.4 9 6.0 6.8 4.5 S9 6.6 6.8 5.1 11 6.4 6.8 4.8 S l l 6.5 6.7 3 . 9 12 \ 6.6 6.8 4.3 S12 5.9 6.8 4.5 S15 6.6 6.8 4 .7 Controlt 6.8 6.8 6.8 * The experiments were carr i e d out at 30°C on a rotary shaker at 220 strokes/min. Each Erlenmeyer flask contained 50 ml basal medium with a 1 percent inoculum. t No addition, contains basal medium plus b a c t e r i a l inoculum only. t t Indulin C i s a trade name of T h i o l i g n i n produced by Kraft process. ( 14. TABLE II CLASSIFICATION OF CULTURE S 11 Test Result 1 Morphology 2 Gram st a i n 3 V i o l e t red b i l e agar plate 4 B r i l l i a n t green b i l e broth 5 EMB agar plate 6 Methyl-red t e s t 7 Voges-Proskauer tes t 8 1% Tryptone broth 9 Koser c i t r a t e broth 10 Urea broth 11 Nitrate broth 12 Litmus milk 13 Lead acetate slant 14 Starch plate 15 Nutrient , g e l a t i n 16 Glucose shake Agar* 17 Lactose shake Agar* 15a M o t i l i t y rod (straight) negative 1 mm small trans-lucent colony -ve, no growth small transparent colony -ve, i . e . no acid produced -ve -ve, i . e . no indole formation very poor growth no growth +ve no change no blackening occurred i n 7 days -ve not l i q u i f i e d within 7. days surface a c i d , but no gas -ve polar flagellum occurring singly negative coliform bacteria negative coliform bacteria negative coliform bacteria -negative coliform bacteria negative E. c o l i or A. aerogenes S^i i s not the genus Proteus J>11 w i l l reduce n i t r a t e to n i t r i t e motile! Continued.... • 15 TABLE II (continued) Test. Result 18 Tryptone glucose good grov:th yeast broth 3 0°C 19 Tryptone glucose good growth yeast broth 37°C 20 Tryptone glucose yeast broth 4 2°C -ve or poor growth 21 Tryptone glucose abundant, white, g l i s -yeast agar slant tening 22 Tryptone glucose 2-3mm Colony, s l i g h t yeast agar plate yellowish no pigment or UV fluorescent no pigment or UV fluorescent 23 Tryptone glucose yeast broth containing 5% NaCl .' 24 Sucrose shake ' Agar* 25 Catalase 26 Cytochrome oxid-ase 27 Octane 28 Nonane 29 Decane 30 Undecane 31 Dodecane 32 Tridecane 33 Hexadecane 34 Naphthalene 3 5 Decanol no growth surface acid, but no gas + ve +ve poor growth f a i r growth good growth good growth good growth poor growth poor growth good growth good growth £>11 resembles Pseudomonas desmolytica Source: S o i l from l o c a l r e f i n e r y . Nutrient Agar plus 0.00 3% brom cr e s o l purple plus 1% test carbohydrate 16. 4©- 8 © 12© ISO f w i m . MJN Figure 1. Eff e c t of various amounts of kerosene on the rate of oxygen uptake. Each vessel contained 1.7 mg(dry wt) of kerosene-grown S]_]_ c e l l s , and 0.5 ml of 0.05M phosphate buffer (pH 7.0). Substrate added at 0 time. Kerosene vapor pressure was not corrected. Total volume 3.2 ml. o 0.5 ml basal medium and 0 u l kerosene x 50 u l kerosene; e 100 y l kerosene A 200 y l kerosene; o 500 y l kerosene 17. kerosene, 50ul and 100u3., r e s u l t s i n a lag in oxygen uptake, following the oxygen uptake of the endogenous c e l l s for 8 0 min before showing an increase i n oxygen consumption. The explanation f o r the long delay i n oxygen uptake in the presence of low concentrations of kerosene may be related to the time required f o r the small amount of o i l to become available to the b a c t e r i a l c e l l s i n the two phase system. Higher concentrations of kerosene, i . e . 500ul, r e s u l t s i n an immediate stimulation of oxygen consumption. Endogenous Oxygen Consumption of Ce l l s Grown on Kerosene-Indulin C Basal Medium. The question arose as to whether the endogenous oxygen consumption of the kerosene-grown c e l l s could be increased by growing the c e l l s i n a medium containing both l i g n i n and kerosene. This was thought to be a p o s s i b i l i t y a f t e r noting the acid production i n the Indulin C-kerosene medium. The r e s u l t s of t h i s experiment are shown i n Figure 2. Obviously the endogenous r e s p i r a t i o n of the Indulin C-kerosene c e l l s was increased greatly, i n d i c a t i n g the storage of oxidizable metabolites within the c e l l . Possibly r e l a t e d to t h i s increase i n oxidizable substrate within the c e l l was a very obvious decrease i n the c e l l sedimentation rate. This apparent decrease in c e l l density may indicate the storage of f a t - l i k e materials. During the shaking of the basal medium with the Indulin C-kerosene addition, a stable emulsion was formed. Therefore, Indulin C was acting as an emulsifying agent increasing 18. 4© 8© 120. TINE in mw Figure 2. Oxygen uptake by culture S}j grown on kerosene medium or kerosene-Indulin C medium. Total volume 3.2 ml. © -kerosene grown c e l l s , 5.8 mg (dry wt) per vessel A kerosene-Indulin C grown c e l l s , 4.3 mg(dry wt) per vessel. 19. the a v a i l a b i l i t y c f the hydrocarbon to the c e l l s and stimulating oxygen consumption, c e l l growth, and c e l l storage materials. Comparison of Indulin C with the Emulsifier Tween-60 In order to determine whether an emulsion other than that created by Indulin C would stimulate hydrocarbon oxidation, the surface active agent Tween-6 0 was added to the kerosene basal medium. The l^esults of t h i s experiment are shown i n Figure 3. Tween-60 not only stimulated the oxygen consumption of the kerosene-grown c e l l s but also increased the oxygen uptake when used alone as the sole substrate i n the absence of kerosene. An explanation of the Tween-stimulated increase i n oxygen uptake over that observed with Indulin C would be the u t i l i z a t i o n of the hydrocarbon portion of Tween-6 0 molecule which i s a f a t t y acid e s t e r i f i e d with a h e x i t o l anhydride. The Increase in C e l l Dry Weight i n Kerosene Fermentations i n the Presence of Indulin C or Tween-60. The use of petroleum as a carbon source f o r the production of s i n g l e - c e l l protein i s favored by high c e l l y i e l d . The following experiment was designed to determine the increase i n c e l l dry weight i n kerosene fermentation by adding either Indulin C or Tween-60 to the fermentation medium. I t was determined previously that culture can grow equally well on either kerosene or glucose carbon sources. Thus i f sugar was present as an impurity in Indulin C, growth of c e l l s , would r e s u l t . The addition of either Indulin C or Tween-60 to the kerosene medium greatly increased the c e l l dry weight 20. it 120 § 0 A O -Q——=0 ' -A -e-It TIME 'IN Figure 3. E f f e c t of Indulin C or Tween-60 on oxygen uptake by culture S]_]_. Each vessel contained 1.7 mg(dry wt) of kerosene grown c e l l s , 0.5 ml of 0.05 M phosphate buffer (pH 7.0), 0.5 ml basal medium and various substrates A l l materials were added at 0 time. Indulin C and Tween-60 were emulsified with kerosene by vigorous shaking before addition. -endogenous; x 100 y l kerosene; -1 mg Indulin C; A -0.25 mg Tween-60 -1 mg Indulin C + 5 y l kerosene; • -o -o-A 0.25 mg Tween-60 + 5 y l kerosene. (Figure 4). The slopes of the l a t t e r two curves when compared with Indulin C or Tween-6 0 used alone would indicate that two separate factors are responsible for increased c e l l dry vreight. These are a r e s u l t of the concentration of either Indulin C or Tween-6 0 and. were not observed when these two additives were used without kerosene. Kerosene i s composed of a great number of hydrocarbon species and thus prevents the location of the hydrocarbon p r e f e r e n t i a l l y u t i l i z e d by the culture. To simplify the metabolic events, gas chromatographically pure n-alkanes ranging from Cg to vrere added separately to the basal medium as the carbon sources. The corrected value f o r the oxygen uptake of a kerosene-grown c e l l suspension was determined by subtracting the pressure change between the Warburg vessels with and without a b a c t e r i a l inoculum (Figure 5). The lower hydrocarbon substrates resulted i n an increased oxygen uptake over the higher carbon substrates. These r e s u l t s are surprising when compared with the data given i n Figure 6. In t h i s experi-ment, c e l l dry weight was determined as a function of the carbon chain length i n the presence and absence of Indulin C on the basal medium. The presence of Indulin C i n a l l of the pure n-alkane basal media greatly increased the r e s u l t i n g c e l l dry weight. The dry weight increased dramatically, s t a r t i n g with the substrate n-alkane to a maximum vrith n-alkane and then decreased with increasing chain length to C K . 22 . F i g u r e 4. E f f e c t o f I n d u l i n C o r Tween-60 on c e l l y i e l d o f c u l t u r e S ^ . Each f l a s k c o n t a i n e d 50 ml o f b a s a l medium and 1 ml o f kerosene w i t h v a r y i n g amounts o f I n d u l i n C o r Tween-60. I n c u b a t i o n was 40 h r a t 30°C. o I n d u l i n C w i t h o u t k e r o s e n e ; © I n d u l i n C + kerosene • Kerosene A Tween-60 w i t h o u t kerosene A Tween-60 + kerosene 23. 40 8.0 120 IS© TlfeSE III u r n Figure 5. Oxygen uptake using pure n-alkanes substrates. Each f l a s k contained 1 ml of S^^ c e l l suspension (11.5 mg dry v;t) , 1 ml of 0.05M phosphate buffer (pH 7.0) and 100 ul of pure n-alkane. o — C 8 ; — - C g ; A — C 1 0 ; A — C u - C 1 6 j . x endogenous. 2 4 . l o t 6 7 8 9 10 ' II 12 13 14 I CAR©@^ NO © F S U B S T R A T E Figure 6. The eff e c t of Indulin C on . the c e l l y i e l d i n the presence of pure n-alkane substrates. . Each flask contained 50 ml of basal medium, 200 u l pure n-alkane with or without 50 mg Indulin C. The S u stock culture used for the inoculum had been exhausted of kerosene by repeated transfer and growth i n medium without substrate. Incubation was 30 hr at 30°C. © With Indulin C; o Without Indulin C. 25. The Change of A c i d i t y of the Growth Medium as the Result of the Addition of eithe r Indulin C or Tween-6 0. The addition of Indulin C or Tween-60 as an ingredient in a kerosene fermentation using the culture as the inoculum resulted i n an increase i n the f i n a l a c i d i t y of the medium (Figure 7 ) . In the presence of an excess of kerosene substrate a greater production of acid occurred a f t e r HO hr of culture incubation with Indulin C than with Tween-60. The reason f o r the production of acid with increasing concentrations of Indulin C i n a medium containing excess kerosene i s unknown. A Comparison of the Dry-Cell Y i e l d and the Acid Production as a Function of the Kerosene Concentration The addition of increasing concentrations of kerosene to carbon-depleted c e l l s i n either an Indulin C or Tween-60 basal medium lim i t e d growth up to the l e v e l of 2 ml of kerqsene/1 (Figure 8). Beyond t h i s concentration of kerosene, the c e l l dry weight increased slowly with increased concentra-tions of kerosene. The medium a c i d i t y following fermentation was found also to be a function of the kerosene concentration (Figure 9 ) . The Active B a c t e r i a l C e l l Stimulatory Fraction of Indulin C. A l l of the a c t i v i t y of commercial Indulin C responsible for the increased c e l l dry weight, oxygen consumption, and a c i d i t y was located i n the Indulin C fractionated by p r e c i p i -t a t i o n at pH 3.5. A l l of the commercial l i g n i n s tested for a c t i v i t y were of reduced a c t i v i t y unless they were both capable of p r e c i p i t a t i o n at pH 3.5 and could form a stable kerosene-basal 26. 3i» C t ©.4 0»6 0.8 1*0 INMJUN C OR TV? SEN @© i / l Figure 7. E f f e c t of I n d u l i n C or Tween-6 0 on a c i d production. Each Erlenmeyer f l a s k contained 50 ml of basal medium and 1 ml of kerosene with v a r y i n g amounts of I n d u l i n C or Tween-60. Incubation was 40 hr at 30°C. o I n d u l i n C without kei-'osene; © I n d u l i n C with kerosene; O Kerosene; A Tween-6 0 without kerosene; A Tween-60 with kerosene. 27. Figure 8. The c e l l y i e l d as a function of kerosene concentra-ti o n i n the presence of Indulin C or Tween-60. Each f l a s k contained 50 ml basal medium, 50 mg Indulin C or Tween-60, 0.5 percent kerosene depleted S ^ i culture and varying amounts of kerosene. Incubation was 18 hr at 30°C. E Kerosene con t r o l ; ' © Kerosene + Indulin C; . A Kerosene + Tween-60. 2 8 . Figure 9. The acid production as a function of kerosene concentration i n the presence of Indulin C or Tv7een-60. Each f l a s k contained 50 ml basal medium, 50 mg of Indulin C or Tween-60, 0.5 percent kerosene depleted S^ -. culture and varying amounts of kerosene. Incubation was 18 hr at 30°C. Kerosene c o n t r o l ; Kerosene + Indulin C; Kerosene + Tween-60. 29. medium emulsion. Lignins which were water soluble at pH 3.5 could be used i f they were f i r s t polymerized by steam heating in 6N NaOH at 15 lb/sq.- i n . for several hours. Following. .sodium hydroxide.treatment.the l a t t e r l i g n i n s could be . precipitated at pH 3.5 and could form stable kerosene-water •emulsions (Table I I I ) . CONCLUSIONS The addition of polymerized lignosulfonates and thiolignins* into hydrocarbon fermentation media greatly increases the fermentation rate and the y i e l d of biomass. Lignin i t s e l f does not appear to be decomposed during tfie fermentation, however, immediately upon the addition and vigorous mixing a stable oil-water emulsion i s formed. This emulsion increases the hydrocarbon-water medium i n t e r f a c i a l area thus overcoming the major factor l i m i t i n g microbial a c t i v i t y . Moreover, the removal of hydrocarbon as the growth-l i m i t i n g factor i n the fermentation greatly reduces the require ment fo r o i l dispersion equipment. The oxygen consumption by the B a c t e r i a l culture increased with the decreasing n-alkane carbon number and did not p a r a l l e l the production of t o t a l biomass. The greatest biomass occurred using the n-alkane C substrate and decreased sharply with lower and higher n-alkane carbon substrates.. T h i o l i g n i n may be added to stimulate the removal of o i l waste or s p i l l a g e by microbial fermentation. A number of t h i o l i g n i n s and lignosulfonates have been prepared i n the • 3 0 . TABLE III THE EFFECT OF POLYMERIZATION OF LIGNIN ON THE CELL YIELD OF CULTURE S,, C e l l Y i e l d Dry v;t (mg/1) Commercial Lig--. Polymerized Polymerized Lignin nin Lignin I* Lignin l i t Lignosol AXD 270 445 84 Lignosol DXD . 23 3 52 7 190 Lignosol FG 241 580 180 Lignosol SFX 144 669 .9 0 Lignosol XD 281 477 74 Raylig LA-74 160 481 223 Raylig LA-79 236 370 457 Raylig-TA 281 560 467 Indulin A 624 568 Indulin B . 516 Indulin C 534 Reax 8 5A 320 Reax 82 154 Polyfon H 515 Polyfon 0 474 Polyfon T 280 Kerosene as control 67 Each f l a s k contained 50 ml basal medium, 1 ml kerosene, and 50 mg of l i g n i n . The cultures were incubated on a rotary shaker at 30 6C f o r 24 hr. * 5 g of commercial l i g n i n was dissolved i n 15 ml of 7.5 percent NaOH and heated at 240°F (115°C) f o r 3 hr. The polymerized l i g n i n was p r e c i p i t a t e d with 6N HC1, centrifuged, washed twice i n water and dried at 100°C overnight. The polymerized l i g n i n was dissolved i n very d i l u t e NaOH and adjusted to the f i n a l pH 6.8 t Exactly the same as above except that 30 percent NaOH was used and the preparation heated at 240°F (115°C) for 20 hr. l a b o r a t o r y which enter, i n t o e i t h e r phase of the hydrocarbon-water system i n v a r y i n g amounts. I t i s suggested from these st u d i e s that the l i g n o s u l f o n a t e s and t h i o l i g n i n s > by-products of pulp manufacture, may be an inexpensive i n g r e d i e n t i n hydrocarbon-water and p o l l u t i o n c o n t r o l fermentations. 32 . CHAPTER I I . THE CYCLONE RESEARCH FERMENTER VS. THE ROTARY SHAKE CULTURE INTRODUCTION The rate c o n t r o l l i n g steps in hydrocarbon fermentation are the transfer of both the substrate and the oxygen to the organism ( 1 ) . In general the size of the o i l droplets and t h e i r dispersion throughout the medium are determined, es p e c i a l l y during early microbial growth phases i n p i l o t submerged fermentations, by increased media ag i t a t i o n through increased impeller speeds. The cost incurred with the increased impeller speeds and the associated engineering requirements i s an important economic consideration i n i n d u s t r i a l fermentation and in.petroleum waste treatment, The addition of polymerized lignosulfonates and t h i o l i g n i n s into the culture greatly increases the microbial oxygen consumption and the y i e l d of c e l l biomass i n laboratory gyratory shake cultures. However, one of the problems of the gyratory shake culture i n c e l l metabolic studies i s the accumulation of a considerable amount of c e l l material i n a peripheral band on the glass at the medium surface. To overcome t h i s l a t t e r problem and to increase the t o t a l medium volume and medium-air contact, experiments were conducted using the Cyclone fermenter designed by Dawson (5). The purpose of t h i s study i s to evaluate the performance of these two fermenter designs, gyratory and cyclone, f o r research i n hydrocarbon fermentation. M A T E R I A L S AND METHODS Organism Pseudomonas desmolytica ( S ^ ) previously i s o l a t e d from a s o i l sample was used i n t h i s experiment. Growth Medium The growth medium used i n t h i s experiment was e s s e n t i a l l y the same as described i n Chapter I . The medium was s t e r i l i z e d f or 20 minutes at 15 lbs/sq. i n . Kex^osene (Fisher, Odorless) and an aqueous solution of Indulin C (.50 mg/ml) were s t e r i l i z e d separately f o r 15 minutes at 15 Ib/sq. i n . and added a s e p t i c a l l y to the s t e r i l i z e d basal medium. F i n a l kerosene concentration, 2 0 m l / l i t e r ; Indulin C, 1 gm/liter. Inoculum An active 2% (v/v) b a c t e r i a l inoculum was obtained from the t h i r d consecutive culture transfer of the organism i n the logarithmic phase of growth i n a kerosene medium. Gyro-batch culture Duplicate cultures of Pseudomonas desmolytica S ^ were prepared i n 500 ml of kerosene-basal medium contained i n 1000 ml Erlenmeyer fl a s k s and incubated at 30°C on a New Brunswick rotary shaker. The shaker was operated at 220 strokes per minute i n a 1 i n . c i r c u l a r o r b i t . Aliquots of 3 0 ml of culture suspension were withdrawn a s e p t i c a l l y at the required time i n t e r v a l s . Cyclone-fermenter batch culture Twelve hundred m i l l i l i t e r s of kerosene-basal medium 34. was placed a s e p t i c a l l y into a s t e r i l e cyclone fermenter (5). The medium was inoculated with 2% inoculum and the temperature controlled at 30°C. S t e r i l e a i r was supplied to the fermenter at 1.8 liters/mir.. Aliquots of 30 ml of culture suspension were withdrawn a s e p t i c a l l y at the required time i n t e r v a l s . Dry Weight The bacteria were removed from 3 0 ml of culture by centrifugation at 20,000 x g for 20 minutes. The r e s u l t i n g p e l l e t was washed with 0.05M phosphate buffer (pH 7.4) and two aliquots of d i s t i l l e d water by re-suspension followed by centrifugation at 10,000 x g for 15 minutes. The c e l l p e l l e t , was taken up i n 5 ml of d i s t i l l e d water, dried f o r 16 hours at 105°C and weighed. pH Measurements Following the removal of the b a c t e r i a l c e l l s by c e n t r i f ugation the pH of the fermentation medium vras measured d i r e c t l y with a pH meter. Hydrocarbon U t i l i z a t i o n For the analysis of the r e s i d u a l hydrocarbon in the fermentation f l u i d , 10 ml of the cultures was centrifuged at 20,000 x g f o r 20 minutes and r e s u l t i n g supernatant and hydrocarbon layer removed and extracted with 3 ml of n-hexane. The hexane-supernatant f r a c t i o n was centrifuged at 5000 x g for 5 minutes and 1 ml of the upper (hexane) layer was transferred into a glass stoppered tube. A small quantity of anhydrous sodium sulfate was added to bind the trace amount of water. The composition of the hexane extract was examined by 35. d i r e c t i n j e c t i o n into a gas chromatographic apparatus. Gas chromatographic analyses were performed with a Becker model 409 a n a l y t i c a l gas chromatogram equipped v/ith dual flame i o n i z a t i o n detectors. The detectors and i n j e c t i o n ports were operated at a temperature 3 0°C higher than the column temperatures. The dual st a i n l e s s s t e e l columns (1.5 m x 0.46 cm O.D.) contained 2% 0V-1 l i q u i d phase on 70-80 mesh chromcsorb-G~AW-DMCH (Becker). RESULTS AND DISCUSSION The growth curves of the Pseudomonas desmolytica S ^ grown i n kerosene medium and i n kerosene-Indulin C medium i n a cyclone fermenter and i n shake fl a s k s are shown i n Figure 10. These r e s u l t s indicate that the cyclone-fermenter i s more suitable i n terms of c e l l y i e l d f o r hydrocarbon fermentation than the conventional rotary shaker. After 20 hours of incubation, the c e l l y i e l d of S ^ i n kerosene medium i n the cyclone fermenter has increased almost threefold over the c e l l y i e l d of the rotary shaker. The addition of Indulin to the kerosene medium further enhances the cyclone fermenter y i e l d to a value exceeding f i v e f o l d of the c e l l y i e l d obtained i n the rotary shaker. One explanation f o r the observed difference i n performance of the two fermenters i s that the fast culture flow rate experienced i n the. cyclone fermenter increases the oxygen a v a i l a b i l i t y and the oil-water i n t e r f a c i a l area. Another p o s s i b i l i t y i s that the bacteria remain and divide within the medium i n the cyclone fermenter whereas a considerable percentage of the bacteria are removed from a c t i v i t y by 36. Sot tad •znJ o ©.4 ®y ^ 80 M £ igure 10. The rate of c e l l growth of under various growth conditions. x----culture m kerosene medium on the shaker; © culture i n kerosene medium on the cyclone fermenter; A culture i n kerosene + Indulin C medium on the shaker; 0 culture in kerosene + Indulin C medium on the cyclone fermenter. The experimental conditions are as described in the text. 37. accumulating as a r i n g at the top of the culture i n the rotary shaker. The Time Course of Acid Production Under Various Growth Conditions Figure 11 indicates that pulp m i l l t h i o l i g n i n s stimulate acid production. The rate of acid production of culture S^^ in kerosene-Indulin medium in the cyclone fermenter i s very f a s t , i . e . within 13 hours the medium pH has dropped to 3.75. The Rate of C e l l Growth and Acid Production i n Kerosene-Indulin Medium i n Cyclone Fermenter. In order to obtain a more accurate picture of the r e l a t i o n s h i p between culture dry weight and acid production, i n kerosene-Indulin medium i n the cyclone fermenter, the growth curve and the acid production of culture was followed c l o s e l y giving the r e s u l t s shown in Figure 12. Obviously the fermentation i n the cyclone fermenter can be f i n i s h e d within 16 hours a f t e r inoculation. The increase i n the pH of the medium a f t e r 30 hours was due probably either to the loss of v o l a t i l e acid through culture aeration or to the u t i l i z a t i o n of the acids by the culture. Gas Chromatographic Separation of the Major Components of Fisher Odorless Kerosene. In order to determine the components of the kerosene substrate that are u t i l i z e d by the culture, the major n-alkanes of kerosene were determined as shown in Figure 13. Five major n-alkanes were i d e n t i f i e d through t h e i r retention time when compared with authentic compounds. The alkanes were decane, B — I I - — m \ 4< Figure 11. The rate of acid production of S u under various growth conditions, The experimental conditions and symbols are the same as shown i n Figure 10. CO 39. 10 .. 2© 3© 40 T IME IN HOOK Figure 12. The rate of S-Q c e l l growth and acid production i n kerosene-Indulin medium in cyclone fermenter. © c e l l dry weight; A~--pH Ml Figure 13. Gas chromatographic separation of the major components of Fisher odorless kerosene. Column 1.5 m x 4.6 mm O.D. stainle s s s t e e l (2% OV-I on 70-30 mesh Chromosorb G-AW-DMCH). Column temperature 50°-150°C at 4°C/min. Carrier gas: nitrogen at 30 ml/min. Cg = nonane; C]_Q = decane; C]_]_ = undecane ; C22 = dodecane ; C]_3 = tridecane; Ci^atetradecane.; C]_5 = pentadecane. o 41. undecane, dodecane, trideeane and tetradecane. The quantity of each compound vras estimated by determining the area under the respective curve. Gas Chromatographic Separation of the Major Hydrocarbon  Components Present i n the Residual Kerosene i n the Kerosene  Culture a f t e r 4 and 7 Days Incubation on Rotary Shaker. The r e s i d u a l kerosene components present i n the kerosene shake culture a f t e r 4 and 7 day incubations on the rotary shaker are shown i n Figure 14 and 15 res p e c t i v e l y . It i s i n t e r e s t i n g to note that only the lower n-alkanes of kerosene, i . e . nonane, decane and undecane vrere u t i l i z e d . The higher n-alkanes, i . e . dodecane, trideeane and tetradecane v/ere not used by the Pseudomonas desmolytica . Afte r 4 days of cultui^e incubation 90% of the nonane, 53% of the decane and 17% of the undecane were u t i l i z e d . After 7 days of culture incubation a l l of the nonane, 91% of the decane and 56% of the undecane were u t i l i z e d . Hence i t seemed that the i n d i v i d u a l n-alkanes present i n kerosene were degraded p r e f e r e n t i a l l y . This data i s i n agreement with Munk et a l . (24) and our previous report using t h i s Pseudomonas organism (18). Gas Chromatographic Separation of the Major Hydrocarbon  Components present i n the Residual Kerosene i n the Kerosene- Indulin Culture a f t e r 2 Days Incubation on a Rotary Shaker. The e f f e c t of Indulin on the f i n a l kerosene composition of a medium containing Indulin-kerosene a f t e r two days culture on a rotary shaker i s shown i n Figure 16. It may be important to note that a l l of the nonane, 74% of the decane, 9. j 5 10 IS 20 TIME \U M m Figure 14. Gas chromatographic separation of the major hydrocarbon components present i n the residual kerosene i n the kerosene culture a f t e r 4 days incubation on rotary shaker. The a n a l y t i c a l conditions and symbols are the same as shown in Figure 13. S^solvent, n-hexane. 'igure 15. Gas' chromatographic separation of the major hydrocarbon components present i n the residual kerosene i n the kerosene culture a f t e r 1 day; incubation on a rotary shaker. The experimental conditions and symbols are the same as shown i n Figure 14. 5 I© m 20 TIME w mm Figure 16. Gas chromatographic separation of the major hydrocarbon components present i n the residual kerosene i n the kerosene-Indulin culture a f t e r 2 days incubation on a rotary shaker. The experimental conditions and symbols are the same as shown i n Figure 14. 45. 52% of the undecane, 16% of the dodecane and 4% of the tr i d e c a n e were u t i l i z e d w i t h i n 2 days. When one compares the above r e s u l t s with those of Figure 14 and 15, i t can be seen t h a t the t h i o l i g n i n not only s t i m u l a t e s the u t i l i z a t i o n r a t e of nonane, decane and undecane but a l s o enables the c u l t u r e to u t i l i z e dodecane and t r i d e c a n e as w e l l . CONCLUSIONS The design of a research or a p i l o t fermenter f o r hydrocarbon s t u d i e s i s a very important c o n s i d e r a t i o n . This study has shown t h a t by using the inexpensive cyclone fermenter i n place of the standard gyratory shaker the growth r a t e , c e l l y i e l d and a c i d production can be improved c o n s i d e r a b l y . The a d d i t i o n of the t h i o l i g n i n i n t o the kerosene c u l t u r e s t i m u l a t e s the growth r a t e and the a c i d product formation i n both the gyratory and the cyclone fermenters. In the l a t t e r fermenter the presence of t h i o l i g n i n causes a c i d to be formed 24 hours e a r l i e r than i n a s i m i l a r medium i n a gyratory shaker. The i n d i v i d u a l n-alkanes i n kerosene are not degraded u n i f o r m l y , the lower n-alkanes being used p r e f e r e n t i a l l y . The a d d i t i o n of t h i o l i g n i n s increases not only the r a t e of u t i l i z a t i o n of these lower alkanes but a l s o extends m i c r o b i a l hydrocarbon a c c e p t a b i l i t y to the higher alkanes. 4 6 • CHAPTER I I I . CONTINUOUS PETROLEUM FERMENTATION INTRODUCTION The continuous flow of i n d u s t r i a l waste and domestic sewage into a b i o l o g i c a l treatment process has required a knowledge of microbial behavior i n order to determine the parameters needed f o r improved equipment design f o r maximal microbial a c t i v i t y . In. an i n d u s t r i a l fermentation where the products i n terms of c e l l y i e l d or metabolite p a r a l l e l microbial m u l t i p l i c a t i o n and where the contamination of these fermentations with foreign organisms can be minimized, continuous fermentation methods may have considerable advantage over batch fermentation. Hydrocarbon fermentations appear to be p a r t i c u l a r l y well suited to continuous fermentation not only because the substrates are not r e a d i l y u t i l i z e d by the majority of the po t e n t i a l contaminating organisms but also because the hydrocarbon addition and emulsi-f i c a t i o n process can be controll e d . In large-scale fermentation, e s p e c i a l l y i n biomass production from hydrocarbon, the continuous process has been considered to have overwhelming advantages i n terms of economics and techniques (21). However, i n practice some d i f f i c u l t i e s have been encountered due to the d i f f i c u l t y i n predicting continuous fermenter performance from batch fermentation data (6). In continuous hydrocarbon fermentation two liquid phases are involved i n the culture medium which requires that both phases be added and removed from the culture i n a constant proportion. Due to the low s o l u b i l i t y of hydrocarbon i n water the flow from the continuous fermenter may not represent the average concentration of hydrocarbon within the fermenter (7). At t h i s laboratory we have found that by using pulp m i l l t h i o l i g n i n s or lignosulfonates and a modified cyclone fermenter i t i s possible to carry on a continuous hydrocarbon fermentation f o r more than 200 hours. The purpose of t h i s Chapter i s to study continuous hydrocarbon fermentation with reference to hydrocarbon, nitrogen, t h i o l i g n i n and d i l u t i o n rate. MATERIALS AND METHODS Organism The bacterium used throughout these studies was Pseudomonas desmolytica S ^ as described previously i n Chapter I. Medium The growth medium consisted of: ( i ) Fisher odorless kerosene as the sole carbon source; ( i i ) the t h i o l i g n i n , Indulin C (1 g / l ) ; ( i i i ) the following mineral s a l t - d i s t i l l e d water solution ( g / l ) . KjHPO^ anhydrous 2.65, KH 2P0 4 1.65, MgCl 2'6H 20 0.10, FeCl 2-4H 20 0.05 , C a C l ^ ^ O 0.01, MnCl^U^O 0.002, Na 2S0 4 anhydrous 0.05, yeast extract 0.05 and (iv) the nitrogen source NH^Cl (1 g / l ) . The f i n a l pH of the medium was pH 6.9. In practice the stock solutions of Indulin C and NH^Cl were prepared at 50.g/l respectively. The constituents cf the medium were s t e r i l i z e d at 15 lbs/sq. i n . f o r 20 min. The fermenter and related equipment i n contact with the medium, except the pH electrode, were steam 48. s t e r i l i z e d as above. The pH electrode was s t e r i l i z e d by washing with 500 ppm sodium hypochlori.de for 10 minutes 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. Inoculum The culture inoculum was grown on a rotary shaker at 30°C i n 100 ml of the kerosene-Indulin-rnineral salt-ammonium chloride medium. Following 24 hours of incubation the culture was harvested by centrifugation and washed once with mineral s a l t s o l u t i o n . The p e l l e t was resuspended i n 100 ml of mineral s a l t solution and 60 ml removed f o r inoculation of the fermenter. Fermenter and Related Equipment The continuous cyclone fermenter and accessory equipment used i n these experiments can be seen i n Figure 17. The medium was c i r c u l a t e d with a Cole-Parmer magnetic drive pump. The temperature was controlled at 3 0°C by means of water c i r c u l a t i n g through the jacketted arm of the fermenter. The f i l t e r s t e r i l i z e d a i r (1.8 1/min) was bubbled through the t h i n f i l m of broth into the side of the fermenter. The pH of the medium during the fermentation was con t r o l l e d within 0.02 pH units of the set point (pH 6.8) by a glass pH electrode* placed i n the fermenter c i r c u l a t i o n system. On a signal from the pH recorder sodium hydroxide (0.15N) was added to the broth by means of a V i r t i s p e r i s t a l t i c pump. A record of the volume of sodium hydroxide added was obtained by recording the number of pump revolutions. * Leeds and Northrup Speedomax H Controller. 49. Figure 17. Schematic diagram of the continuous hydrocarbon fermentation equipment. 1. fermenter; 2. l i q u i d l e v e l c o n t r o l l e r ; 3. a i r pump; 4. a i r f i l t e r ; 5. pH electrode; 6 . pH control and recorder; 7. NaOH pump; 8. NaOH rese r v o i r ; 9. magnetic drive pump; 10. p e r i s t a l t i c pump; 11. product tank; 12. p e r i s t a l t i c pump; 13..medium r e s e r v o i r ; 14. p e r i s t a l t i c pump; 15. kerosene r e s e r v o i r ; 16. p e r i s t a l t i c pump; 17. l i g n i n r e s e r v o i r ; 18. a i r outlet f i l t e r ; 19. sampling ou t l e t ; 20. water jacket for temp, contro l . 21. condenser. 50. The l e v e l of the medium inside the fermenter' was maintained within + 5 ml by means of a glass l e v e l c o n t r o l l e r through which the excess fermentation f l u i d was withdrawn automatically through a p e r i s t a l t i c pump. The kerosene, mineral medium., Indulin C and ammonium chloride were added continuously and a s e p t i c a l l y by means of a Harvard model 1201, 12 speed, p e r i s t a l t i c pump. The a i r exhaust was released to the atmosphere a f t e r f i r s t passing through a f i l t e r s t e r i l i z e r . Treatment of the Samples Culture aliquots of 3 0 ml were withdrawn a s e p t i c a l l y from the fermenter with a s t e r i l e syringe and the dry weight determined according to the procedure previously described in Chapter I I . The aliquots were examined microscopically with a phase contrast microscope to check culture purity. In addition the culture was examined p e r i o d i c a l l y for t y p i c a l colony morphology and gram s t a i n . Gas Chromatographic Analyses Two hundred m i l l i l i t e r aliquots of culture f l u i d * from the f i n a l growth stage shown i n Figure 18, i . e . at a flow rate for kerosene of 2.6 ml/hr and for Indulin C-mineral s a l t medium, 34 ml/hr, were centrifuged at 13 ,000 x g for 20 minutes._ at 30°C. The Supernatant was a c i d i f i e d to pH 2 with 6N HC1, and extracted twice with 30 ml of d i e t h y l ether. The ether extracts were combined and extracted with 50 ml of 5% Na^CO^ solution. The aqueous carbonate layer was a c i d i f i e d to pH 2 and extracted twice with 10 ml ether. The ether extract was concentrated to 3 ml and methylated according to the procedure *• Total volume of culture f l u i d was 1.5 1. 51. ©@ 100 ISO g<9@ T I M E m HOUR Figure 18. Continuous growth of S-^ on Indulin-mineral medium with kerosene as sole cax^bon source. o dry vreight; A —-meq t o t a l NaOH consumption. Indulin-mineral medium containing NH^Cl, flow rate = 34 ml/hr; kerosene flow rate A =60 y l kerosene/hr; B = 120 ul/hr; C = 250 ul/hr; D = 520 pl/hr; E = 1350 y l / h r ; F = 2600 ul/hr. 52. of Wijngaarden (33). Gas chromatographic analyses were done on the methyl esters using a model 3 810 Becker Research Gas Chromatogram equipped with a dual flame i o n i z a t i o n detector. The dual 1.5 m x 0.46 cm O.D. st a i n l e s s s t e e l column contained 2% OV-1 l i q u i d phase on 70-80 mesh Chromosorb-G-AW-DMCH. RESULTS AND DISCUSSION Hydrocarbon Limiting The problem of studying petroleum fermentation at l i m i t i n g hydrocarbon concentrations can be overcome by adding small amounts of t h i o l i g n i n to the fermentation medium. Figure 18 shows the. time-course of c e l l growth and acid production of Pseudomonas desmolytica S ^ grown i n Indulin C-mineral medium containing 1 g/1 ammonium chloride and l i m i t i n g amounts of kerosene. In t h i s experiment the culture S ^ was grown i n the absence of kerosene f o r 24 hours during which time Indulin C-mineral medium was fed to the fermenter at a rate of 34 ml/hr. Kerosene was then added at the rate of 0.06 ml/hr and the time noted as zero hour i n Figure 18. Forty-eight hours l a t e r the kerosene flow rate was adjusted to 0.120 ml/hr as indicated i n the graph. Further adjustments were made p e r i o d i c a l l y u n t i l the kerosene flow rate was 2.6 ml/hr. The c e l l wet weight was found to be a convenient index to locate growth plateau regions. It i s to be noted from the curves shown i n Figure 18 that acid production p a r a l l e l s the growth rate as measured by dry weight. 53. Figure 19 shows the ef f e c t of the kerosene flow rate on the c e l l y i e l d and the acid production. When the concentration of the substrate was low the c e l l y i e l d was proportional to the kerosene flow rate. Maximum c e l l y i e l d was.obtained at flow rate of approximately 1.6 ml of kerosene per hour. The curve describing acid production followed i n general the curve of c e l l y i e l d . The fermentation supernatant from t h i s experiment was subject to gas chromatographic analyses. Five monocarboxylic f a t t y acids corresponding to Cg, Cg, C ^ Q , and were found, see Figure 20. n-decanoic acid was the major f a t t y acid. Indulin C Limitation In t h i s experiment the kerosene flow rate was 2,6 ml/hr, the mineral s a l t solution containing NH^Cl flow rate was 34 ml/hr and the Indulin C flow rate was varied from 0 to 219 mg/hr. The r e s u l t s of t h i s experiment are shown i n Figure 21 and Figure 22. Obviously the c e l l y i e l d of i s dependent on the flow rate of Indulin C. At low additions of Indulin C, a l i n e a r r e l a t i o n s h i p exists between the presence of the l i g n i n and the. c e l l dry weight. Both the c e l l y i e l d and the acid production began to plateau at an Indulin concentration of 40 mg Indulin C/hr. Fermenter Washout Rate ' In t h i s experiment the kerosene flow rate was 2.6 ml/hr and the flow rate of the Indulin C-mineral medium (containing NH^Cl) was varied from 0 to 330 ml/hr. The r e s u l t s are presented i n Figure 23 and Figure 24. It can be seen that the c e l l 5'4. I 2 3 &{£d@§gfc3I FLOW RATE tea8 / W?. Figure 19. The ef f e c t of kerosene flow-rate on c e l l y i e l d and acid production. A — -dry -meq weight; t o t a l NaOH consumption. Figure 20. ME IN &8IN Gas chromatographic separation of the stra i g h t chain f a t t y acid methyl esters found i n the supernatant of the f i n a l stage of continuous growth of Sn on Indulin-mineral medium with kerosene as sole carbon source.' C8=methyl octanoate; Cg=methyl nonanoate; C]_g = methyl decanoate; Cji^methyl undecanoate; Ci2=methyl dodecanoate; S = ether. Column 1.5m x 4.6 mm O.D. stainless s t e e l (2% OV-I on 70-80 mesh Chromosorb G-AW-DMCK). Column temp. 50°C--170°C at 6°C/min. Carrier gas: nitrogen at 3 0 ml/min. cn cn 56. S@ 1 0 © I S O 20O TIME- \ W HOUR Figure 21. Continuous growth of on kerosene-mineral medium with Indulin C as emulsifying agent. © dry vreight; A meq t o t a l NaOH consumption. . . Kerosene flow rate = 2.6 ml/hr. Mineral medium flow rate*= 34 ml/hr. Indulin C flow rate; A = 1.2 5 mg Indulin C/hr; B = 6.25 mg/hr; C = 12.5 mg/hr; D = 26 mg/hr; E = 67.5 mg/hr; F = 130 mg/hr; G = 219 mg/hr. 57. e@ too isio E©@ I N D U L I N C F L O W R A T E Figure 22. The eff e c t of Indulin C flow rate on c e l l y i e l d and acid production. ©---dry weight; A meq t o t a l NaOH consumption. 58. BQ 1 0 0 IS© TIME IU HOUR Figure 23. Continuous growth of S^-, on kerosene with varying Indulin-mineral medium flow rate. © d ry weight; A meq t o t a l NaOH consumption. Kerosene flow rate = 2.6 ml/hr. Indulin-mineral medium containing NHi^cl flow rate, A = 0 ml Indulin-mineral medium/hr; B = 9 rnl/hr; C = 17 ml/hr; D = 34 ml/hr; E = 65 ml/hr; F = 157 ml/hr; G = 330 ml/hr. 59. 20© UHmW FLOW Jt&TE ml/HF. Figure 24. The e f f e c t of Indulin C-mineral medium flow rate on c e l l y i e l d and acid production. « dry weight; A meq t o t a l NaOH consumption. 60. washout rate increased very r a p i d l y when the medium flow rate was increased beyond 34 ml/hr (Figure 2 3 ) . The acid production did not follow the c e l l y i e l d as the medium flow rate increased. At.higher medium flow rates c e l l y i e l d was noted to drop stea d i l y whereas acid y i e l d increased with increasing medium flow rates. Obviously there i s an optimum flow rate f o r the Indulin C-mineral medium i n the range of 14 0 to 160 ml/hr which favours maximum acid production but not maximum c e l l y i e l d . The reason for thi s observation i s unknown. Ammonium Chloride Limitation In t h i s experiment the kerosene flow rate was 2.6 ml/hr, the Indulin C-mineral s a l t solution flow rate was 34 ml/hr and the NH^Cl flow rate was varied from 0 to 313 mg/hr. The re s u l t s are shown i n Figure 25 and 26. It seems of in t e r e s t to compare Figure 2 5 v/ith Figure 21. In these tv/o graphs there would appear to be s i m i l a r i t i e s between c e l l growth and acid production. The acid production increases dramatically when the c e l l y i e l d exceeds 1.5 g/1. As shown i n Figure 26, at low NH^Cl le v e l s both the c e l l y i e l d and the acid production were approximately proportional, as the c e l l y i e l d increased so did acid production. At higher lev e l s of NH^Cl an i n h i b i t i o n of c e l l y i e l d was noted. The explanation for the NHj^Cl i n h i b i t o r y e f f e c t i s unknown. CONCLUSION Hydrocarbons can be fermented with a pure b a c t e r i a l culture i n a continuous process for long periods of time up to 250 hours without any noticeable change i n the culture behaviour. 61. e@ i©@ is© g@@ TIME IN H©U^ Figure 25. Continuous growth of S^ ]_ on kerosene-Indulin-mineral medium v;ith NH^Cl as sole nitrogen source. © dry weight; A meq t o t a l NaOH consumption. Kerosene flow rate = 2.6 ml/hr. Indulin-mineral medium without N H 4 C I flow rate = 3Uml/hr. N H 4 C I , a d d e d , A = 1.5 mg NHt-Cl/hr; B = 3.13 mg/hr; C = 6.25 mg/hr; D = 13 mg/hr; E = 33.8 mg/hr; F = 65 mg/hr; G = 120 mg/hr; H 313 mg/hr. 6 2. Figure 26. The eff e c t of NH^Cl flow rate on c e l l y i e l d and acid production. © dry weight; A meq t o t a l NaOH consumption. The addition of various culture medium ingredients can be optimized to produce maximum c e l l y i e l d or maximum acid production. V/ith the addition of the t h i o l i g n i n , Indulin C, i t has been found that the kerosene appear^'to become more r e a d i l y available to the organism and as a r e s u l t the organism growth rate and acid pr-oduction can be plotted as a function of kerosene concentration. The addition of Indulin C to the culture medium creates a stable emulsion which overcomes the problem of the continuous removal of a two phase system from the fermenter. Because the thiolignins can be removed by p r e c i p i t a t i o n or f l o c c u l a t i o n on standing i n culture f l u i d s of high a c i d i t y , the use of l i g n i n to improve the rate of hydrocarbon u t i l i z a t i o n i n petroleum waste treatments or synthetic processes i s suggested. CHAPTER IV. ENZYMATIC STUDIES OF HYDROCARBON OXIDATION INTRODUCTION. A number of Investigations have been done on the mechanism of enzymatic hydrocarbon oxidation. Baptist ejt a l . in 19 63 showed that the c e l l - f r e e extract of a pseudomonad grown on hexane would catalyze the oxidation of radioactive octane-14 14 C to octanoic-C" acid in the presence of pyridine nucleotide (NAD). n-Octanol and octaldehyde were also i d e n t i f i e d as products of octane oxidation (2). In 1966, Peterson et a i . separated c e l l - f r e e extracts of hexane-grown Pseudomonas  oleovorans into three components, i . e . rubredoxin, a diphos-phopyridine nucleotide-rubredoxin reductase and a w-hydroxylase. A l l these three proteins were required for the oxidation of n-octane to n-octanol i n the presence of reduced NAD, ferrous ions and molecular oxygen (26). Kusunose and colleagues presented further evidence that n-decane oxidation required three d i s t i n c t protein f r a c t i o n s of the c e l l - f r e e extract of Pseudomonas desmolytica grown on hexane-mineral s a l t medium and that f l a v i n adenine dinucleotide (FAD) was also required f o r maximal a c t i v i t y (14). One of the technical d i f f i c u l t i e s encountered i n t h i s _ f i e l d i s the very li m i t e d s o l u b i l i t y of the hydrocarbon substrate i n water. Hence the isotope tracer method was necessary heretofore for the detection of low l e v e l of the hydrocarbon metabolites. It i s the purpose of th i s study to demonstrate n-alkane oxidation using manometric and gas chromatographic techniques. MATERIALS AMD METHODS Organism and Growth Condition Culture S^ of Pseudomonas desmolytica were n-decane-Indulin C-mineral s a l t solution i n a cyclone as described i n Chapter I I I . •Preparation of Intact C e l l s and of Cell-Free Extract C e l l s were harvested by centrifugation at 15,000 x g fo r 30 min at 4°C, followed by washing twice with 0.05M phosphate buffer at pH 7.0. The r e s u l t i n g packed c e l l s (approximately 3 0 g, wet weight) v/ere suspended i n 3 volumes of 0.05M -3 phosphate buffer, pH 7.0, containing 10 M 2-mercaptoethanol and submitted to a pressure drop from 15000 lbs/sq. i n . with use of a French Press c e l l . The following operations were carri e d out at about U°C, unless otherwise stated. The preparation was centrifuged for 1 hour at 20,000 x g and the r e s u l t i n g c l e a r pale yellow supernatant c e l l - f r e e extract (CFX) was transferred to a clean beaker for further p u r i f i c a t i o n . P a r t i a l P u r i f i c a t i o n of the Hydrocarbon-Oxidizing Enzyme To the above cle a r supernatant, ammonium sulfate was added to give a f i n a l concentration equivalent to 30% saturation and the r e s u l t i n g preparation centrifuged at 20,000 x g f o r 30 min. The p r e c i p i t a t e was re-dissolved in 0.05 M phosphate buffer containing mercaptoethanol (called 30%+ f r a c t i o n ) . A dditional ammonium sulfate was added to the supernatant (called 30% s fr a c t i o n ) to achieve a 60% saturation. The pr e c i p i t a t e which occurred between 30-60% saturation was stored at -20°C (called 60%+ f r a c t i o n ) and the superantant 65 . grown on fermenter c a l l e d 60% s. The 30% p r e c i p i t a t e f r a c t i o n (30%+) and 60% superantant f r a c t i o n (50% s) were dialyzed i n 6 l i t e r s of 0.025M phosphate buffer, pH 7.0 containing mercaptoethanol for 16 hours. The protein concentration of the enzyme solution was determined by the biuret method (16). Determination of Enzyme A c t i v i t y The o v e r a l l oxygen consumption as determined i n a Gilson D i f f e r e n t i a l Respirometer was taken as a measure of the enzyme a c t i v i t y . The composition of the assaying preparation unless otherwise stated consisted of 50 p moles of phosphate buffer (pH 7.0), 2 p moles of NAD, dialyzed 3 0%+ f r a c t i o n , dialyzed 60% s f r a c t i o n , 50 p i of n-decane ( C 1 Q ) and 0.2 ml 20% KOK (in the center well of Warburg f l a s k for C0 2 absorption). Total volume of the reaction mixture was 3.2 ml. The reaction was stopped by adding 2 drops of 6 N l^SO^ to the f l a s k , followed by extracting with 2 ml of CHCl^. The r e s u l t i n g emulsion was broken by centrifugation and the CHC1„ layer (bottom) was dehydrated with a small amount of anhydrous sodium s u l f a t e . The CHCl^ extract was used either d i r e c t l y f o r gas chromatographic analysis or methylated according to the procedure of Wijngaarden (33) p r i o r to gas chromatography. Gas Chromatographic Analyses Gas chromatographic analyses were performed with a Becker Research Chromatograph Model 3810 equipped for flame i o n i z a t i o n detection. The detector and i n j e c t i o n port were operated at temperature 30°C higher than column temperature. The aluminum chromatograph column, measured 6 feet x 0.25 i n . O.D. 67. and contained 15% Carbowax 20 M on 80-100 mesh Chromsorb W. Nitrogen gas was used as a c a r r i e r gas at flow rate of 30 ml/min. Standard curves for detector response of each of the oxidation products were established by use of authentic compounds before and after each experiment. RESULTS AND DISCUSSION In view of the dangers inherent i n r e l y i n g s o l e l y on radioactive substrate and column chromatographic techniques for studying the mechanism of enzymatic oxidation of hydro-carbons, attempts were made to use the manometric technique to measure enzyme a c t i v i t y and to use the gas chromatographic technique to examine the end products in the reaction mixture. Several experiments were designed i n an attempt to increase the l e v e l of the hydrocarbon-oxidizing enzyme a c t i v i t y i n the ba c t e r i a l c e l l s . Figures 27a and 27b showed that the oxygen uptake of the c e l l - f r e e extract prepared from the n-decane-Indulin C grown c e l l s wets more active than the si m i l a r c e l l - f r e e extract prepared from c e l l s grown i n the absence of Indulin C. This would confirm our e a r l i e r report that Culture grown on kerosene-Indulin C medium had a much higher oxygen consumption rate than c e l l s grown on kerosene medium alone. The Indulin C treated c e l l s had a more active oxidative enzyme system than the c e l l s grown i n the absence of Indulin C (18). The E f f e c t of NAD, NADP and pH on the Enzyme A c t i v i t y Figure 28 shows the ef f e c t of NAD and NADP on the a c t i v i t y of the c e l l - f r e e extract preparation. Obviously, NAD i s superior to NADP i n stimulating oxygen consumption i n Figure 27a, Oxygen uptake of c e l l - f r e e extract of n-decane-Indulin C grown c e l l s . ©---CFX; A — - C F X + C 1 0 ; ® CFX+NAD+NADP;-x CFX+NAD+NADP+CIQ. Protein concentration = 18.5 mg/flask. @© fig© rm& m mm Figure 27b.. Oxygen uptake of c e l l - f r e e extract of n-decane grown c e l l s . The experimental condition and symbols are ,the same as shown i n Figure 28a. Protein = 15.7 mg/flask cn C O 69 . ©©•© ©@ 100- IS© g@@ TIMIE IN 148^ Figure 28. The e f f e c t of NAD and NADP on the oxygen uptake of the c e l l - f r e e extract of S^,-Protein concentration - 19 mg/flask. o CFX; A CFX + 2.5 y moles NADP; o CFX + 2.5 y moles NADP + 200 y l C 1 0 ; B CFX + 2.5 y moles NAD; x CFX + 2.5 y moles NAD +200 y l C l 0 or CFX + 2.5 y moles NADH + 200 y l C 1 0 . 70. t h i s assay system. It i s of i n t e r e s t to note that NAD can be replaced by NADH without any loss of enzyme a c t i v i t y . Figure 29 and 30 show the e f f e c t of pH on the enzyme a c t i v i t y . Because the hydrocarbon oxidizing enzyme would appear to have an optimal pH at 7.0, t h i s reaction medium pH was chosen i n the subsequent experiments. The e f f e c t s of the concentration of NAD on enzyme a c t i v i t y are shown in Figures 31 and 32. The enzyme a c t i v i t y was proportional to the amount of NAD added to the reaction mixture. Sligh t substrate i n h i b i t i o n was noted when the concentration of NAD reached a l e v e l of 3 u moles/flask. To avoid t h i s problem, 2 u moles of NAD/flas was a r b i t r a r i l y chosen in a l l assaying mixtures. P a r t i a l P u r i f i c a t i o n of n-Decane Oxidizing Enzyme For maximal enzyme a c t i v i t y , the system required the following constituents: 30%4-, 30% s, NAD and n-decane (Figure 33). The n-decane-oxidizing enzyme appeared to be present, i n the 30% p r e c i p i t a t e f r a c t i o n and not i n the 30% supernatant f r a c t i o n . Ferrous, manganous and calcium ions did not stimulate the enzyme a c t i v i t y (Figure 34). The 30% p r e c i p i t a t e f r a c t i o n remained active following d i a l y s i s , however, i t required the presence of the dialyzed 60% supernatant plus NAD and n-decane to give maximal a c t i v i t y (Figure 35). Since the 60% s had been dialyzed overnight and had a n e g l i g i b l e amount of biuret protein, the stimulating factor i n the 60% supernatant f r a c t i o n would appear to be a non-dialyzable and non-protein material i n nature. Hence heat treatment was applied to the dialyzed 60%. supernatant to check out whether 71. Figure 29.. Effect of pH on oxygen uptake of the c e l l - f r e e extract. Protein concentration = 18 mg/flask; NAD = 2 y moles/flask; C i 0 = 100 y l . Phosphate buffer (Na) and Tris-HCl buffer were used pH 5-7 and 7-10 respectively. A = pK 7.0; •B = 7.4; C = 6.5; D = 8.0; E = 6.0; F = 5.5; G = 8.5 or 9.5; H = 5.0. 72. 5 6 7 8 9 pH Figure 30. Effe c t of pH on the a c t i v i t y of the c e l l - f r e e J extract. The experimental conditions were the same as shown i n Figure 29. 73. 400 U l j5 300 3 O 200 100 o - ' * H 6 F E D C e — 0 S /// 50 TIME 100 IN ISO Figure 31. E f f e c t of the concentration of NAD on the a c t i v i t y of the c e l l - f r e e extract. , Protein concentration = 36 mg/flask; • .. C i o = 100 u l / f l a s k . A, CFX as control; B, CFX+C 1 0; C, 0.1 y mole NAD; D, 0.25 y mole NAD; E, 0.5 y mole NAD; F, 4 y moles NAD; G, 1 y mole NAD; H, 2 y moles NAD. 7 4. Figure 32. E f f e c t of the concentration of NAD on the a c t i v i t y of the c e l l - f r e e extract. The experimental conditions were the same as shown i n Figure 31. 75. 50 100 ISO 200 TIME IN MIN Figure 33. Manometric evidence f o r the enzymatic oxidation of ' n-decane (C-^Q ) . 30% + and 30% s had been dialyzed against 0.025 M phosphate buffer, pH 7.0 for 16 hours. Protein concentration; 30% + = 7.5 mg/flask. 30% s = 0.06 mg/flask. NAD = 2 u moles/flask; C i o = 50 u l / f l a s k ; NADH = 2 u moles/flask. o---30% or 30%. s; x---30%+ +30% s; m 30%+ +NAD; A 30%+ +30% s + NAD or 30%+ +30% s + NADH; © 3 0%+ +30% s + NAD + C ] Q or 30%+ +3 0% s + NADH + C i o or 30%+ + 30% s + NAD + C i o + 1 U mole FAD. 76. 50 100 150 200 T I M E I h i M\H Figure 34. Manometric evidence.for the enzymatic oxidation of n-decane. Each Warburg fl a s k contained 50 y moles phosphate buffer, pH 7.0, 2 y moles NAD, and 50 y l C 1 0 . Protein concentration: 30% + = 43 mg/ml. 30% s = 2.5 mg/ml. © 1 ml 30% s + 0.1 ml 3 0%+;. A---1 ml 30% s +0.3 ml 30%+; a 1 ml 30%+ + 0.1 ml 30% s; x 1 ml 30%+ +1 ml 30% s or 1 ml 30%+ + 1 ml 30%s + 1 y mole F e + + , or 1 ml 30%+ + 1 ml 30% s + l.y mole Mn + + or 1 ml 30%+ + 1 ml 30% s + 1 y mole C a + + . 77. 50 100 150 200 TIME IW Mm Figure 35. Manometric evidence f o r the enzymatic oxidation of n-decane. Protein concentration. 30%+ = 64 mg/ml; 30%s -4.3 mg/ml; 60% s = 0.0 7 mg. • 3 0%s or 6 0%s; A 30%+ , o 30%+ +30%s or 30%+ +60% s; @_.._30%+ +60% s + 2 y moles NAD; A---30%+ + 60% s + 2 y moles NAD + 50 y l C i o ; » 30%+ +30% s + '2 y moles NAD; x 30%+ +30% s + 2 y moles NAD + 50 y l C]o• 78. t h i s stimulating factor was heat stable (Figure 36). Surprisingly i t was found that t h i s factor withstood heat treatment at 80°C for 6 min without losing any stimulating a c t i v i t y . The p r e c i p i -tate between 30-60% ammonium sulfate had l i t t l e apparent function i n n-decane oxidation (Figure 37). Joined Manometric and Gas Chromatographic Techniques i n the  Studies of n-Decane Oxidation Figure 38, 39 and MO represent the manometric and gas chromatographic evidence f o r the enzymatic oxidation' of n-decane by the c e l l f r e e extract of culture S^. n-Decanol and n-decanoic acid were gas chromatographically i d e n t i f i e d as the end products. It i s important to note that CFX, NAD and n-decane are absolutely necessary f o r the oxidation of n-decane to n-decanol and n-decanoic acid. Baptist e_t a l . (2) reported that c e l l - f r e e extract of Pseudomonas oleavorans would oxidize radioactive o c t a n e - l - ^ C exclusively to octanoate-l-^C, however, i n the presence of carbonyl-binding agent, octanol and octanal were also i s o l a t e d i n addition to octanoate. Figures 41, 42 and 43 represent manometric and gas chromatographic evidence f o r the enzymatic oxidation of n-decane by the p a r t i a l l y p u r i f i e d n-decane-oxxdizing enzyme. It i s of p a r t i c u l a r i n t e r e s t that two d i s t i n c t f r a c t i o n s , i . e . 30% p r e c i p i t a t e and 60% supernatant are required f o r the oxidation of n-decane to n-decanol. In the l a t t e r case, i t may be int e r e s t i n g to note that n-decanol was found to be the sole end product, whereas i f crude c e l l - f r e e extract was used, n-decanol and n-decanoic acid v/ere found as the end products. 79. Figure 3 6 . E f f e c t of heat treatment on the a c t i v i t y of the hydrocarbon-oxidizing enzyme. In t h i s experiment dialyzed 6 0 % supernatant was heated i n 8 0 ° C water bath f o r 6 min and cooled down ra p i d l y i n i c e -water mixture. Protein concentration; 3 0 % + = 3 2 mg/ml; 6 0 % s = 0 . 0 3 5 mg/ml. 0 3 0 % + + 6 0 % s; © 3 0 % + + heated 6 0 % s + 2 u moles M A D + 5 0 u l C 1 0 ; h 3 0 % + + 6 0 % s + 2 u moles N A D + 5 0 ul Cio or 3 0 % + + 6 0 % s + 2 u moles N A D H + 5 0 u l C ^ Q . 1 5 0 20 40 60 80 TINE IN MIN Figure 37. Manometric evidence for the enzymatic oxidation of n-decane. Protein concentration: - 30%+ = 32 mg/ml; 60%+ = 16.5 mg/ml; 60% s = 0.035 mg/ml. A---30% + 60% s + 2 y moles N A D + 50 y l C 1 0 ; ©•- — 30%+ +60% s + 2 y moles' N A D + 50 y l C 1 0 + 5 y l 60%+; D 30%+ + 60% s + 2 y moles N A D + 50 y l C +50 y l 60% +; x 30%+ +60% s + 2 y moles N A D + 50 y l ^ C 1 0 + 500 y l 60%+. 8 1 . 1500 UJ 3 1000 O 5 0 0 20 40 TIME 60 80 IN MIN 100 Figure 38. Manometric evidence for the enzymatic oxidation of n-decane. Each Warburg f l a s k contained 50 y moles of phosphate buffer, 1 ml CFX (72 mg protein), and various components. The reaction was stopped with 2 drops -of 6N K^SOi^ as described i n the text and used l a t e r f o r gas chromatographic analysis as shown i n Figure 39. e CFX as cont r o l ; A---CFX + 2 y moles NAD; x CFX + 2 y moles NAD + 100 pi-Cio. 82 . 6 10 15 20 Figure 39. Gas chromatographic i d e n t i f i c a t i o n of n-decanol as the product of enzymatic oxidation of n-decane (CFX + NAD + C 1 0 ) . The end products from the experiment as shown in Figure 38 were used i n t h i s experiment. ,S chloroform as extract solvent. Column; 6 feet x 0.25 inch O.D. (15% Carbowax 20 M on 80-100 mesh Chromsorb VJ). Temp, isothermal at.200°C. Ca r r i e r gas: nitrogen flow rate — 30 ml/min. 83 . 10 LsJ OT o CO UJ 3 w 2 Q K O O UJ , n s METHYL MECAW9ATE-5 10 15 TIME IM IV? IM 20 Figure 40. Gas chromatographic i d e n t i f i c a t i o n of n-decanoic acid as the product of enzymatic oxidation of n-decane (CFX + N A D + C J Q ) . The end pi-'oduct from the experiment as shown i n Figure 38 was methylated and used i n th i s experiment. S = chloroform solvent. Column: 6 feet x 0.2 5 inch O.D. (15% Carbowax 20 M on 80-100 mesh Chromsorb W). Temp, isothermal at 200°C. C a r r i e r gas: nitrogen gas flow rate = 30 ml/min. 6 4 . 50 100 150 200 TI^E IN mm Figure 4 1 . Manometric evidence for the enzymatic oxidation of n-decane. Each Warburg fl a s k contained SO u moles of phosphate buffer, pH 7.0 and various components. Protein concentration: 30% = 84 mg/ml; 60% s = 0.08 mg/ml.-@ 60% s + 2 y moles NAD + 50 y l Cio; «* 30%+ + 60% s + 50 y l C 1 0;. A 30%+ +2 y moles NAD + 50 y l C 1 0; x 30%+ + 60% s + 2 y moles NAD + 50 y l C 85. 5 10 15 TIME IN MIN Figure 42. Gas chromatographic i d e n t i f i c a t i o n of n-decanol as the product of enzymatic oxidation of n-decane (30%+ +60% s + NAD + C 1 Q ) . The end product from the experiment as shown i n Figure Ul was used in th i s experiment. S = chloroform solvent. Column: 6 feet x 0.2 5 inch O.D. (15% Carbowax 20 M on 80-100 mesh Chromsorb V / ) . Temp, isothermal at 200°C. Nitrogen flow rate = 30 ml/min. 8 6 . Figure 43. Gas chromatographic invest i g a t i o n of the methylation product from the enzymatic oxidation of n-decane (30%+ + 60% s + N A D + C '). The end product from the experiment as shown i n Figure 4 2 was methylated and used in t h i s experiment. A l l other experiment conditions were the same as shown i n Figure 42. 87. In view of the above findings, attempts were made to esta b l i s h that n-decanol, n-decanal and n-decanoic acid were the intermediates of n-decane oxidation by Culture S . In Table IV the l a t t e r three substrates could be u t i l i z e d more r e a d i l y than n-decane by suggesting that they were the possible intermediates i n the oxidation of n-decane. The Indulin C stimulates the u t i l i z a t i o n of a l l four substrates suggesting that Indulin C may be involved i n the mechanism of oxidation of n-decane. CONCLUSION The c e l l - f r e e extract prepared from the n-decane-Indulin grown c e l l s were more active i n the oxidation of n-decane than the corresponding c e l l s that had grown on n-decane alone. The hydrocarbon-oxidizing enzyme displayed i t s greatest a c t i v i t y at pH 7.0 and could be precipitated by 30% ammonium su l f a t e . I t also required a heat stable, dialyzable 60% supernatant f r a c t i o n and NAD for maximum enzyme a c t i v i t y . Ferrous, manganous, and calcium ions did not stimulate the enzyme a c t i v i t y . Manometric and gas chromatographic analyses indicated that c e l l - f r e e extracts of Culture S ^ oxidized n-decane to n-decanol and n-decanoic acid, whereas p a r t i a l l y p u r i f i e d enzyme preparations only oxidized n-decane to n-decanol. n-^Decanol, n-decanal and n-decanoic acid supported good growth f o r Pseudomonas desmolytica S ^ suggesting that they could possibly be the intermediates i n the n-decane oxidation. A l l the above findings indicated that the i n i t i a l 88. TABLE IV EFFECT OF INDULIN C ON THE CELL YIELD AND ACID PRODUCTION OF CULTURE S USING n-DECANE, n-DECANOL, n-DECANAL and n-DECANOIC ACID AS SUBSTRATE C e l l Y i e l d Dry Wt. pH a f t e r 20 hr Decane' 0.103 6.76 Decane + Indulin C 0.860 6.43 Decanol 0.193 6.70 Decanol + Indulin C 1.192 5.77 Decanal 0.147 6.23 Decanal + Indulin C 1.360 5.43 Decanoic acid 0.214 6.50 Decanoic acid + Indulin C 1.520 6.41 Control 6 . 82 The experiments were carr i e d out at 3 0°C on a rotary shaker at 220 strokes/min. Each Erlenmeyer f l a s k contained 50 ml basal medium with a 1% inoculum. The substrates and Indulin C added to the fl a s k were at the l e v e l of 1.25 m moles and 50 mg per flask r e s p e c t i v e l y . o x i d a t i v e a t t a c k occurred at the t e r m i n a l methyl carbon, r e q u i r i n g both oxygen and NAD. 90. GENERAL SUMMARY AND CONCLUSIONS A series of experiments' were conducted to study the mechanisms and problems of hydrocarbon fermentation. From the r e s u l t s of these experiments the following conclusions can be drawn: 1. The addition of highly polymerized t h i o l i g n i n and ligno-sulfonate into hydrocarbon fermentation media greatly increases the fermentation rate and the y i e l d of biomass. This grov*7th stimulation i n the presence of l i g n i n may be due to an increase i n the oil-water interface caused by the formation of a stable oil-water medium emulsion. 2 . A number of t h i o l i g n i n s and lignosulfonates, prepared i n t h i s laboratory, increase the y i e l d of c e l l biomass. This stimulation of hydrocarbon u t i l i z a t i o n by l i g n i n has been suggested as a means of increasing the b i o l o g i c a l removal of o i l from o i l y waste water. 3. The oxygen consumption by the organism Pseudomonas desmolytica S ^ increases with the decreasing n-alkane carbon number and does not p a r a l l e l the production of t o t a l biomass. The greatest biomass occurs using n-undecane and decreases sharply with lower and higher n-alkane carbon substrates. 4. The cyclone fermenter i s a very adaptable research fermenter and suitable i n terms of c e l l y i e l d and acid production f o r hydrocarbon fermentation. The fermenter's unique performance i s due i n part to the fas t culture flow rate r e s u l t i n g i n an increase of a v a i l a b i l i t y of oxygen and of o i l to the micro-organisms. 5. The i n d i v i d u a l n-alkanss i n kerosene are not degraded u n i f o r m l y , the lower ones being used p r e f e r e n t i a l l y . The a d d i t i o n of t h i o l i g n i n increases not only the r a t e of u t i l i z a t i o n of these lower n-alkanes but a l s o extends m i c r o b i a l hydrocarbon a c c e p t a b i l i t y to the higher n-alkanes. 6. Hydrocarbons can be fermented with a pure b a c t e r i a l c u l t u r e i n a continuous process f o r long periods of time without any n o t i c e a b l e change i n the c u l t u r e behavior or p u r i t y . The a d d i t i o n of. t h i o l i g n i n , I n d u l i n C, t o the c u l t u r e medium overcomes the problem of the continuous.removal of a two phase system from the fermenter. 7 . With the a d d i t i o n of I n d u l i n C, i t i s p o s s i b l e t o operate w i t h the petroleum carbon source i n a l i m i t i n g c o n d i t i o n . As a r e s u l t the growth r a t e and a c i d production of the c u l t u r e can be p l o t t e d as a f u n c t i o n of the hydrocarbon c o n c e n t r a t i o n , a. c o n d i t i o n normally d i f f i c u l t to achieve i n the more conventional fermentation systems. 8 . The n-decane-oxidizing b a c t e r i a l enzyme can be f r a c t i o n a t e d , w i t h (NH^^SO^ from crude c e l l - f r e e e x t r a c t p r e p a r a t i o n . The enzyme which has a narrow optimal pH around 7.0, a l s o r e q u i r e s a d i a l y z e d heat s t a b l e 60% supernatant f r a c t i o n and NAD f o r maximal enzyme a c t i v i t y . The a d d i t i o n of f e r r o u s , manganous and calcium ions t o the enzyme p r e p a r a t i o n does not s t i m u l a t e the enzyme a c t i v i t y . 9. Crude c e l l - f r e e e x t r a c t s of P_. desmolytica o x i d i z e s n-decane t o n-decanol and n-decanoic a c i d , whereas p a r t i a l l y p u r i f i e d enzyme p r e p a r a t i o n o x i d i z e s n-decane to n-decanol on l y . 92, 10. n-Decanol, n-decanal and n-decanoic a c i d support good growth for. suggesting t h a t these are the intermediates i n the metabolism of n-decane o x i d a t i o n by P. desmolytica. LITERATURE CITED Atkinson, J.H. and Newth, F.H. 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