Structural Effects on Arthrobacter ATCC 25581 Methylene Hydroxylase Activity

AN ABSTRACT OF THE THESIS OF

STEVEN SHIN HAYASAKA for theMASTER OF SCIENCE (Name of student) (Degree)

1 ' in MICROBIOLOGY presented on 2k INV (Major) Darce) Title: STRUCTURAL EFFECTS ON ARTHROBACTER ATCC 25581 METHYLEN YDRS MCTIVITY Redacted for Privacy Abstract approved: Dr. D. A. Klein

Arthrobacter ATCC 25581, capable of subterminal oxidation of n-hexadecane to 2-, 3-, and 4- alcoholic and ketonic products, was examined for the ability of this methylene hydroxylase capability to be induced and repressed, and for structural relationships influencing methylene function oxidation. Induction was best carried out by use of n-alkanes from 10 to 1 6 carbons in length, and was especially strong with methylcyclohexane among cyclic compounds tested.Induction was not observed with related alcohols,1 -unsaturate compounds or by methoxy and ethoxy compounds tested.After induction, n-alkanes C14 and C16 carbons in length were transformed to the corresponding internal oxidation products; however, no activity was observed with shorter chain length even-carbon alkanes.Hexadecene-1 and all alcohols tested, including cyclododecanol, were transformed to corresponding ketonic or aldehydic products.Cyclic compounds tested, including cyclododecane, were not oxidized by induced cells, suggesting the role of a methyl group in orientation of the substrate for the methylene hydroxylation, but that the methyl function was not as critical after completion of the hydroxylation step, regardless of structural configuration.Acetate, a preferred growth substrate for this isolate, was able to repress induction of n-hexadecane methylene hydroxylase activity.

. Inducibility of methylene hydroxylase activity was confirmed by use of cell-free systems, using methylcyclohexane as an inducer. A stimulation of methylene hydroxylase activity by addition of reduced pyridine nucleotides and ferrous ion was indicated. Structural Effects on Arthrobacter ATCC 25581 Methylene Hydroxylase Activity

by Steven Shin Hayasaka

A THESIS submitted to Oregon State University

in partial fulfillment of the requirements for the degree of Master of Science

June 1972 APPROVED: Redacted for Privacy

Assistht 15rc;fessOr of Microbiology in charge of major

Redacted for Privacy

Chairman of Department of Microbiology

Redacted for Privacy

Dean of Gradate School

Date thesis is presented,,,,?7 ) Typed by Opal Grossnicklaus for Steven Shin Hayasaka ACKNOWLEDGEMENT

The author is deeply thankful for Dr. D. A. Klein's guidance and advice throughout this investigation. Thanks are also extended to my parents for their patience and support, Dr. R. Y. Morita and Dr. L. W. Parks for their helpful suggestions and to the staff and students of the Oregon State Univer- sity who have helped me throughout this study. This research was supported in part by Environmental Protec- tion Agency research grant R-EP-00278-03. TABLE OF CONTENTS Pa e

INTRODUCTION 1

LITERATURE REVIEW 2

Arthrobacter 2 Hydrocarbon Utilization 4 Transformation in the Soil 4 Oxygenas es 5 Terminal Oxidation 6 Diterminal Oxidation 6 Subterminal Oxidation 8 Anaerobic Dehydrogenation 10 Induction and Repression 11 Oxidizability and Molecular Structure 12

MATERIALS AND METHODS 14

Culture Conditions 14 Hydrocarbons 14 Growth Experiments 16 Gas - Liquid Chromatography 17 Preparation and Assay of Cell-Free Extracts 17 Effects of Alternate Carbon Sources on Hexadecane Transformation 20 Quantitative Estimation of Ketones 21 Compounds Effective as Inducers in Paraffin Adaption 23 Hydrocarbon Transformation in a Non-Growth System 24 Thin Layer Chromatography 25

RESULTS 26

Growth Studies 26 Inducibility of Cell-Free Hydroxylase Activity 29 Effects of Alternate Carbon Sources on Hexadecane Transformation 31 Compounds Effective as Inducers in Paraffin Adaption 33 Hydrocarbon Transformation in a Non-growth System 37

DISCUSSION 39

SUMMARY 44

BIBLIOGRAPHY 45 LIST OF TABLES Table Page

1. Composition of basal media, mineral salts solution and vitamin mixture. 15

2. Carbon and nitrogen sources for Arthrobacter ATCC 25581 aerobic growth. 28

3. Role of cofactors and coenzymes on 2-hexadecanol formation by methylcyclohexane-induced and uninduced Arthrobacter ATCC 25581. 30

4. Effects of alternate carbon sources on hexadecane transformation. 32

5. Structural effects on induction of methylene hydroxylase activity. 36

6. Induced Arthrobacter ATCC 25581 non-growth transformation activity. 38 LIST OF FIGURES Figure Page

1. GLC separation of residue oxidation products produced from n-hexadecane by Arthrobacter ATCC 25581. 18

2. Calibration curve for the quantitative estimation of hydrocarbon ketonic product by vapor phase chromatography. 22

3. Arthrobacter ATCC 25581 growth responses to vitamin and nitrogen additions. 27

4. Formation of 2-hexadecanone by induced and uninduced Arthrobacter ATCC 25581 cells. 34 STRUCTURAL EFFECTS ON ARTHROBACTER ATCC 25581 METHYLENE HYDROXYLASE ACTIVITY

INTRODUCTION

Members of the genus Arthrobacter have long been considered indigenous soil microorganisms.Their presence in high numbers has caused speculation on their importance in chemical transformations in the soil.Reactions carried out by Arthrobacter frequently involve minor changes in molecules which make the molecules more available for use by other members of the microbial flora. Among these reactions is that of hydroxylation of methylene functions, or sub-terminal saturated carbons. This study was initiated to elucidate the structural factors influencing induction and activity of methylene function-containing substrate transformations as might be found in the soil containing this type of microorganism.Hopefully, this study will provide a better understanding of interactions between microorganisms and methylene function compounds which might be found in natural envi- ronments. 2

LITERATURE REVIEW

Arthrobacter

In 1928, Conn (12) called attention to a bacterium which exhib- ited a unique pleomorphism.In spite of the fact that it resembled the genus Corynebacterium, Conn named this bacterium Bacterium globiforme.However, in 1947 Conn and Dimmick (14) proposed the name Arthrobacter for those bacteria which were characterized by a unique cycle of morphological change.This cycle involves a sphere rod morphogenesis.Spherically-shaped cells occur in the stationary growth phase, which upon inoculation into a proper medium elongate into various pleomorphic or rod-shaped cells.Spherical cells again form upon depletion of the medium by a process described as "frag- mentation by reductive division." Subsequently, other investigators (54, 55) have isolated bacterial species which were assigned to the genus Arthrobacter solely on the basis of morphological criteria.Attempts to characterize Arthrobacter by nutritional requirements and physiological behavior have not been fruitful (63, 72, 84). With the emphasis on a morphological classification, many studies on Arthrobacter have dealt with a possible explanation for its unique pleomorphism.These studies have mainly concentrated on determining possible differences in cell wall composition (24, 44, 3

45, 46, 47).As a consequence of rod-coccal morphology in

Arthrobacter,speculation regarding possible evolutionary advan- tages have been raised.Interest centered on Conn's conclusion (13) that the small cocci observed by Winogradsky (83) to be the most prominent member of the indigenous microflora were actually the coccoid stage of Arthrobacter.Further investigations have also confirmed that Arthrobacter is able to maintain itself as the most prominent group in nutrient poor soils (27, 61, 62).In addition, starvation studies with two Arthrobacter species have shown better starvation survival in a phosphate buffer than other bacteria tested (16, 85).Nevertheless, in terms of starvation survival no advantage was found for Arthrobacterto be in the rod or spherical form (16). Because of the abundance of Arthrobacter in soil, they may play a significant role in chemical transformations. As a member of the indigenous soil flora, presumably this role could involve the formation and decomposition of the soil organic fraction, often termed humus (1).Humus is known to originate from both plant debris entering the soil and from microorganisms within the soil. Thus, the chemical nature of the organic material would be complex, and investigations concerning its transformation could be of significance.However, the role of Arthrobacter in such chemical transformation remains to be elucidated. 4 Hydrocarbon Utilization

Transformation in the Soil

Perhaps one of the more general microbial transformations occurring in soil, and yet least understood in terms ofthe role of specific bacteria, is hydrocarbon transformation. Asmight be suspected, numerous hydrocarbons or their derivatives arecontinu- ously being added or synthesized within the soil by plants andmicro- organisms.Thus, hydrocarbon transformations have been considered of great significance in the terrestrial cycle ofcarbon (1).With such a wide occurrence of this material in the soil, it may notbe too surprising that the ability of microorganisms toattack hydrocarbon seems to be quite general rather than unique(86).For example, one study has shown that many genera of bacteriawhich were not selected for theirability to oxidize hydrocarbon could be adapted to hydrocarbon substrates (22).Similarly, rapid disappear- ance of many hydrocarbonswhen added to soil attest to this point (1). Among these substances utilized were paraffins, gasoline,mineral oils, kerosene, lubrication oil, asphalt, tar, rubber andinsecticides. Nevertheless, it must be pointed out that, although bacteriahave beer selectively isolated for their ability to utilize these substances,little is known about their individual importance in hydrocarbondissimila- tion in soil. 5

.0,_g_genases

Early studies on hydrocarbon oxidation, dating from 1895 when Miyoshi (8) reported the utilization of paraffin by Botyris cinerea, have been concerned with the detection of microbial hydrocarbon utilization.These papers have been reviewed by Beerstecher (8) and ZoBell (86).However, it was not until 1955, when Hayashi (30) and Mason (59) independently established that oxygen could be enzy- matically incorporated into various substrates, that a mechanism explaining the possible mode of hydrocarbon oxidation was made available. Enzymes which catalyze such reactions were termed oxygenases (31). From studies using appropriate oxygen isotopes, it now appears firmly established that oxygenases are the preferred hydroxylating agents in hydrocarbon oxidation (56, 71).The most common type of monooxygenase in paraffin oxidation are the "mixed-function" oxygenase type.These monooxygenases require various kinds of electron donors and may be schematically represented by the following equation:

S + 02 + HX SO + X + H20 2 where S represents the substrate and H denotes an electron donor 2X (29).In the terminal oxidation of hydrocarbon and long chain fatty 6 acids this scheme may be further complicated by an electron transport system involving a number of protein fractions.

Terminal Oxidation

A review of the literature indicates that the most common pathway of aliphatic hydrocarbon degradation involves initial hydroxylation at the terminal carbon atom by a "mixed-function" oxygenase (60, 79).Through a sequence of oxidations, this alcohol is next converted to the aldehyde and then to the acid via the presence of both an alcohol and aldehyde dehydrogenase.After this initial breach of the hydrocarbon molecule, the oxygenated substrate is broken down by p-oxygenation to acetate units involving metabolic pathways no longer unique to hydrocarbon degradation. Attempts to further characterize this initial hydroxylation have involved two alternate mechanisms which differ on the nature of the reductant in their respective electron donating system.The first of these mechanisms, involving alkane 1 - hydroxylation or fatty acid co-hydroxylation, are typified by a pseudomonad system (49, 66, 67). In this scheme, electrons are transferred by a rubredoxin-NADH reductase, probably a flavoprotein, from NADH to rubredoxin.The rubredoxin is then able to provide the reducing power enabling hydroxylation.This system may be distinguished by its electron transport system involving at least three proteins, its CO 7 insensitivity, and its apparent lack of cytochrome P450, a common functional component in mammalian mitochondrial and microsomal hydroxylation reactions (29).In contrast, the second mechanism, as represented by a Corynebacterium strain, possess at least two fractions, is CO-sensitive and contains cytochrome P-450 (10, 11). These studies have indicated that one protein fraction contains a flavoprotein and is functional for reducing cytochrome P-450 in the presence of NADH. The second fraction, containing the cytochrome P-450, is then able to participate in reducing the substrate. In addition to the initial alcohol product, the presence of the corresponding aldehyde and free fatty acid have also been identified as products of hydrocarbon oxidation in bacterial cell free extracts.

These findings have suggested that both a DPN-linked alcohol (6,7,

48) and aldehyde (4,7,33, 48) dehydrogenase were respectively required. Furthermore, a bacterial tetradecanal oxidizing extract requiring NAD, ATP, and CoA was found not only able to oxidize the aldehyde to fatty acid, but the acid was further degraded and acetic acid was identified (4).

Diterminal Oxidation

Less conclusive at this time is evidence for a second mode of attack on fatty acids.Besides the usual p-oxidation, it has been 8 suggested that fatty acids are further oxidized by a second hydroxylation resulting in w- or w -1 -hydroxylated fatty acids (35, 36, 76, 78). It has been claimed that such a pathway is important in the metabolism of alkan -1 -ols whose subsequent dehydrogenation to fatty acids is inhibited by various substituents (35).The relative chain length of alkanoic acids has also been suggested as a means determining whether p-oxidation could occur effectively, or whether w- or w -1- hydroxylation took place (36).Nevertheless, there is little evidence that further hydroxylation of fatty acids is catalyzed by enzymes differing from those which catalyze alkane terminal oxygenation (35,

78).The data suggest w-oxidation to the dioic acid as a minor and perhaps less essential pathway, in comparison with p-oxidation (36,

37).

Subterminal Oxidation

Paraffin oxidation is usually at the terminal methyl group, and the discovery of subterminal modes of attack on alkanes was of interest (2, 23, 25, 38, 39, 40, 51, 56, 58, 69, 81).Methyl ketone accumulation along with partial or complete absence of aldehydes, nevertheless, have long been known (23, 25).However, this phenomenon could be explained in the following three ways (25):1) the aldehyde produced, owing to its high reactivity, could immediately be transformed, 2) the ease of dehydrogenating the different alcohols may vary considerably, 9 3) the energy requirement of the organism may be met more efficiently by the dissimilation of the primary alcohol.Further, other studies suggesting that an attack on both methyl and methylene groups were occurring have relegated the subterminal oxidation to a minor role (36, 42).In other words, it seemed possible that methyl ketones may be formed by the same enzymes which normally catalyzed the terminal hydroxylation of alkanes.In accordance with this belief, other investigators (22, 51), working with short chained alkanes, have accounted for formation of both fatty acids and methyl ketones by assuming the intermediate formation of an alkyl free radical.It was postulated that with hydroxylating enzyme mediation, this radical could either react to give a terminally oxygenated product or be rearranged to the more stable secondary free radical, subsequently yielding a subterminal oxygenated product.Similarly, accounting for the hypothetical intermediate 1-alkene, another group (60) has suggested that the concurrent formation of 0,- and w -1 - hydroxy-acid may arise from the alternate ring fission of an intermediate terminal expoxide.Neither of these two mechanisms, therefore, show that subterminal oxidation plays a distinct role in hydrocarbon dissimilation. Evidence demonstrating that subterminal oxidation occurs preferentially has implicated methyl ketones and secondary alcohols as important intermediates in microbial oxidation of aliphatic 10 hydrocarbons (2,20, 23, 39, 40). A mechanism has also been proposed (18, 19, 20, 21) for the complete subterminal degradation of tridecanone by a pseudomonad strain.By the identification of key intermediates using whole cells and cell-free extracts,the following pathway has been proposed:

n-tridecane 2-tridecanol 2-tridecanone undecyl acetate 1 -undecanol + acetic acid

Anaerobic Dehydrogenation

Although it seems conclusive that oxidation of alkanes by microorganisms probably involves a direct hydroxylation, a French group has claimed that hydrocarbon oxidation involves the intermediate formation of a 1-alkene (5, 70).This group has proposed that an anaerobic pathway by which paraffins undergo dehydrogenation as the initial metabolic reaction.Through infrared analysis, they have demonstrated the formation of 1 -heptene under anaerobic conditions in cell-free "heptane dehydrogenase" preparations.Another group further claims that small amounts of 1 -hexadecene has been isolated from a variety of microorganisms grown on hexadecane (82).In both these studies, however, the argument for an olefin intermediate in hydrocarbon oxidation is weakened by the lack of evidence that further attack on the molecule occurs primarily at the double bond.In only 11 a few instances, where 1 - alkenes were initially added to agrowth medium, has it been suggested that a reaction occurring at the double bond formed the major product (9,43).Rather, the bulk of such studies have shown that the saturated end of 1 - alkenes were preferentially hydroxylated (2, 34, 36, 58, 74, 78).In addition, a number of investigators have found no correlation between an organism's ability to grow on n-alkanes and the corresponding 1 - alkene (57, 58, 68).Therefore, although it seems probable that a number of organisms have the ability to convert saturated alkanes to the corresponding 1 - alkene, there is little evidence that a subsequent oxidation occurs primarily at the double bond.These results do not suggest the existence of a fundamentally different dehydrogenation mechanism for aliphatic hydrocarbon dissilation.

Induction and Repression

It seems to be well established that the synthesis of many catabolic enzymes are subject to two control systems: induction and repression. At present, a variety of inducible and repressible microbial enzymatic systems have been analyzed (3).Nevertheless, there have been very few studies made on the adaptive enzyme systems involved in paraffin oxidation, although almost all of the bacterial paraffin systems studied have been found to be inducible (79).Fur- thermore, the work of Van Eyk and Bartels (80) has been the most 12 detailed noted in the literature concerned with hydrocarbon enzyme catabolite repression relationships, concerned with terminal oxidation systems.

Oxidizability and Molecular Structure

A number of studies have been made to establish a correlation between oxidizability and alkane structure.In reviews by Zo Bell (86) and Beerstecher (8),it was originally believed that branched alkanes were more readily degraded by microorganisms. However, with the advent of commercially available high-purity hydrocarbons the oppo- site view has been supported (79). Although branched paraffins show decreased oxidation rates, their use in steric studies has given insights into the specificity of these reactions.Using the bacterium Pseudomonas aeruginosa, Thysse and Van der Linden (73) studied the oxidation of 2-methylhexane. Their conclusion that 2-methylhexane was mainly degraded from the "long" chain was supported by the finding that 2-methylhexane-grown cells preferentially accumulated 2-methylhexanoic acid.Similarly, Jones (35) using the yeast, Torulopsis gropengi- esseri demonstrated that alkanoic acids and alkan -1 -ols which have methyl substituents close to the functional group gave w- and w-l- hydroxy- derivatives, while alkanoic acids and alkan -1 -ols with methyl substitutions at the w-1 position gave w-hydroxy derivatives. 13 Further, it was concluded that the action of an enzyme (or enzymes) was inhibited more by methyl substitution at intermediate positions along the alkyl chain than by methyl substitution near the site of hydroxylation.This was interpreted as suggesting that a long unsubstituted chain of methylene groups may play an important role in binding the substrate to the enzyme.Finally, chain length specificity was clearly evident from the observation that while methyl-p- dodecyloxypropionate gave the highest yield of w-hydroxy-ester, methyl-p-tridecyloxypropionate gave the highest yield of (A) - 1 - hydroxylated product (36).Thus, both w - and w -1 -hydroxylation of p-alkoxypropionic acid takes place most efficiently when the carboxy-group and the site of hydroxylation are separated by the same fixed distance. An interesting study by Harris (28) using Hirschfelder molecular models has also given an insight into the problem of enzyme specificity.These models showed that a folded molecular shape without an exposed methyl group could explain why decane is less readily oxidized than octane and dodecane. With respect to studies on stereospecificity, the branched paraffins DL-3-methylhexane and DL-3-methylheptane were shown to be oxidized non-stereospecifically (32, 77).However, as pointed out by Van der Linden et al. (79), the lack of stereospecificity should not be generalized to include other microorganisms or hydrocarbons. 14

MATERIALS AND METHODS

Culture Conditions

The Arthrobacter species ATCC 25581 used in this work was randomly isolated from a sample of Woodburn silty clay loam (40). This isolate was specifically selected for its inability to emulsify n-hexadecane without yeast extract in a mineral salts solution, and was subsequently found to form 2-, 3-, and 4-alcohols and ketones from n-hexadecane in a mineral salts solution.Cultures of this species were subcultured monthly and maintained on veal infusion slants at refrigerator temperature. To obtain an inoculum, except where indicated, cells were grown in 50 ml of basal medium A ( Table 1) containing 1% w/v of the carbon source to be subsequently used.The inoculum was then added at 5% v/v to 200 or 800 ml of the same medium contained in 1000 ml Erlenmeyer or Fernbach flasks respectively.All flasks were shaken at 200 rev/min on a New Brunswick rotary shaker at 30°C.

Hydrocarbons

The following were purchased from Humphrey Chemical Co., North Haven, Connecticut and were of the highest purity available: Table 1.Composition of basal media, mineral salts solution and vitamin mixture.

Ingredients Amounts* (g/1) Basal medium A Basal medium B Mineral salts solution

K2HPO4 1.6 0.8 1.6

KH2PO4 0.8 0.8 0.8 MgSO4 0.2 0.2 0.2 CaC1 0.002 0.002 0.002 FeSO4.7H 0.001 0.001 0.001 20 NaNO 2.0 4.0 3 NH4C1 1.0 Distilled water 1000 ml 1000 ml 1000 ml Vitamin mixture+ 1.0 ml Yeast extract 20.0 pH 7.0 7.0 7.0

Vitamin mixture (mg/ml) Biotin 0.1

B12 0.2 Thiamine hydrochloride 50.0 Ca D-pantothenate 50.0 Nicotinic acid 50.0 pH 6.8

Sterilized at 121° for 20 minutes I1 + Sterilized through a fritted glass filter 16 n-decane, n-hexadecane, 1 -hexadecene, n-octane. From K & K Laboratories, Inc., Hollywood, California: cyclododecanol, 2-decanol, 2-decanone, n-dodecane, 2-hexadecanol, 3-hexadecanone, 1-methylnaphthalene, n-tetradecane. From Eastman Kodak Co., Rochester, N. Y.: Benzene, cyclohexane, diethoxymethane, methylcyclohexane, p-xylene. From the Matheson Coleman and Bell Co., Los Angeles, California:1 -decanol,1 -hexadecanol.From the Aldrich Chemical Co., Inc., Milwaukee, Wisconsin:2, 3-butanediol, cyclododecane, 1, 3-diethoxy-2-propanol, 1, 2-dimethoxyethane, dimethoxymethane, 4-heptanone, 1, 5-hexadiene, 2-hexanone, Isoeugenol, 2-octanone, trans, trans, cis-1, 5, 9-cyclododecatriene, 1-undecanol, 2-undecanone, 6-undecanone. From the Oregon State Univ. chemistry dept.: 2-butanone, ethanol (95%), n-hexane.

Growth Experiments

Inocula were grown on veal infusion agar slants for 24 hours. Growth was added to 2 ml of mineral salts solution (Table 1) so that a slight turbidity was evident. One drop of inoculum was then added to test tubes containing 6 ml of basal medium A. Carbon sources were sterilized separately from the basal medium either by autoclaving or filtering through a fritted glass filter.Water-soluble carbon sources were tested at 0. 5% w/v. For liquid and solid insoluble carbon sources, one drop or 0.025 17 gm respectively was used. All tubes were shaken at 30°C on a reciprocating shaker at 150 Cycles /min.Growth was read after five days using a Spectronic 20 (420 nm).

Gas-Liquid Chromatography

Gas chromatography analysis was carried out by use of a Aero- graph Hy-FI III Model 1 200 incorporating a high temperature flame- ionization detector.The unit was equipped with a 1/8 inch by 10 ft (0.32 cm by 3 m) column containing 10% of FFAP liquid phase (Varian- Aerograph, Walnut Creek, Calif. ) on 100 /120 mesh Chromosorb W (Varian-Aerograph) to detect C16 alcoholic and ketonic products (40). Injector, column oven, and detector thermal regions were set at 2800, 210°, and 270°C. respectively.The carrier gas argon, and detector gases hydrogen and air had flow rates of 25, 25, and 250 ml/ min respectively. This system allowed for the detection of the 2-, 3-, and 4-hexadecanols and their corresponding ketones (Figure 1).

Preparation and Assay of Cell-Free Extracts

The organism was grown on 1% disodium succinate basal medium A for 3 days at 30°C.The cells were then centrifuged and resuspended in 0. 5% disodium succinate basal medium A with or without 18

Figure 1.GLC separation of residue oxidation products produced from n-hexadecane by Arthrobacter ATCC 2SS81. A = n-hexadecane; B = 4-hexedecanone; C = 3-hexadecanone; D = 2-hexadecanone; E = 3-hexadecanol; F = 2-hexadecanol. 19 0. 3% methylcyclohexane or n-dodecane inducer.After induction for 8 hours with rotary shaking, cells were then harvested by centrifugat- ing at 5,000 x g for 20 minutes.The cells were washed 3 times with 0.05 M tris-HC1 buffer (pH,----7. 4) and resuspended in the same buffer. All subsequent operations were carried out at no greater than 10°C. Cell-free extracts were prepared by using a Sonifier Cell Disruptor Model W185 (Heat Systems-Ultrasonic, Inc., Plainview, N. Y.) with a power output of 90 watts.After sonication, extracts were centrifuged for 10 minutes at 15, 000 x g.The cell-free extract supernatant fluid contained approximately 17 mg of protein per ml. Soluble protein was estimated by the Biuret Method (26) with a bovine serum albumin standard. The assay for 2-hexadecanol activity in cell-free extracts was carried out in 125 ml Erlenmeyer flasks.The reaction mixture of cell-free extract (10 ml) in 0.05 M tris -HC1 buffer contained 20 p.1 of n-hexadecane, 10 p. moles of NADH, NAD, NADPH or NADP, and 2 p. moles of FeSO4. 7H20.All flasks were shakened on a New Brunswick rotary shaker at 275 rev/min at 30°C.After one hour of incubation, 0.5 ml of concentrated HC1 was added.Using a 50 ml separatory funnel, the mixture was extracted two times with a total volume of 8 ml of diethyl ether.After ether evaporation, 2 p.1 samples were assayed by vapor phase chromatography. 20

Effects of Alternate Carbon Sources on Hexadecane Transformation

Cells were grown for 72 hours either on 200 ml of 1% "test carbon" or 2% yeast extract basal medium A.The "test carbon" sources consisting of sodium acetate, disodium succinate, sodium pyruvate, sodium lactate and glucose were autoclaved separately before addition to the basal medium. The cells were then centrifuged and washed three times in phosphate buffer (pH= 7).All centrifuga- tions were done at 5, 000 x g for 20 minutes unless otherwise stated. Each set of "test carbon" grown cells was resuspended in 100 ml of phosphate buffer containing either 0. Q61 M of the same carbon source used for growth and 2 ml of hexadecane or only 2 ml of hexadecane. Cells suspended with 0.061 M glucose, however, were previously grown up on 1% sodium acetate basal medium A since glucose could not be utilized as a sole carbon and energy source (41).All flasks were then shaken on the rotary shaker and 10 ml samples were removed after 4 and 8 hours. The 10 ml samples were extracted with 1 ml of diethyl ether into durham tubes.After the diethyl ether was evaporated off,1 41 of hydrocarbon sample was analyzed by gas-liquid chromatography. 21

Quantitative Estimation of Ketones

Ketones and alcohols formed during the induction experiment were quantitated by addition of 1 - hexadecanol as an internal standard. Before this internal standard could be utilized, however, two criteria had to be met.First, the GLC detector response toward 1-hexadecanol and the hydrocarbon related ketone must be a linear function of concentration.Second, the extent to which the internal standard and ketonic compound is extracted should be constant.If this is true, by performing the identical extracting procedure as would be used in the induction experiment, extraction losses could also be accounted for. This was done by adding 2 ml of hexadecane plus various con- centrations of 1 -hexa,decanol and 3-hexadecanone to flasks containing 100 ml of mineral salts solution.After shaking thoroughly, a 10 ml sample was removed and extracted with 1 ml of diethyl ether in a screw top test tube. A calibration curve was then obtained by chromatographing these samples and plotting peak area ratios against molar ratios.As shown in Figure 2, the above criteria was achieved. Quantitation of ketonic product was calculated using the equa-

tion

(I.S.) (2-one) (0.74) (1-ol) 22

1 2 3 4 S

Molar Ratio1-Hexadecanol/ 3-Hexadecanone

Figure 2.Calibration curve for the quantitative estimation of hydrocarbon ketonic product by vapor phase chromatography. 23 where X = moles of ketone product; I. S. = moles of internalstandard; 2-one = peak area of ketone; 1 -ol = peak area of internal standard; and 0.74 = relative detector response per mole of internal standard (obtained from the slope of the plot in Figure 2).

Compounds Effective as Inducers in Paraffin Adaption

Cells grown for 48 hours on 1% disodium succinate basal medium A were centrifuged and resuspended in 0. 5% disodium succinate basal medium A containing 2.9 x10-4or 5 x10-3moles of inducer.After inducing the cells for 8 hours on a rotary shaker, the cells were again centrifuged and washed three times with mineral salts solution.Upon adjusting the cell concentration to approximately 1 x10-3 gm/ml dry weight, the cellswere resuspended in baffled flasks containing 2 ml of hexadecane and 100 ml of mineral salts solution.Cells were then shaken again on the rotary shaker at 200 rev/min at 30°C.After 2 or 4 hours incubation, 2 ml of concentrated HC1 and 4 x10-4gm of 1 - hexadecanol (internal standard) were added to the flask and then a 10 ml sample was removed. The 10 ml samples were extracted and assayed using the procedure previously outlined. Because of the possibility that this procedure may allow for the selection of a constitutive hydrocarbon transformation strain of 24 Arthrobacter,1 ml of a previously induced cell suspension was resuspended in a fresh 1% disodium succinate basal medium A containing no hydrocarbon. The procedure previously outlined for obtaining non-induced cells was then followed.If selection for a constitutive strain had taken place, the GLC analysis should indicate induced rather than non-induced cell transformation kinetics.

Hydrocarbon Transformation in a Non-Growth System

After culturing cells in 2% yeast extract basal medium B on a rotary shaker for 48 hours, the cells were centrifuged and washed three times in phosphate buffer (pH=7).The cells were then resuspended in 100 ml phosphate buffer at two times their concentration in the growth medium.Liquid hydrocarbon was added to the flasks in 1.5 ml quantities while solid hydrocarbon, dissolved in a minimal amount of 95% ethanol, was added in 0.06 gm quantities.Controls consisted of flasks containing 100 ml of phosphate buffer with either a hydrocarbon sample or washed cells.After shaking the flasks for 48 hours on a rotary shaker, the entire contents were extracted three times with a total volumn of 60 ml of diethyl ether.Samples were analyzed using thin layer chromatography. 25

Thin Layer Chromatography

TLC plates were prepared by layering 0.250 mm of a 50% w/v water slurry of Silica Gel G (American Optical Corp. #08075) on 20 cm x 5 cm glass plates.The plates were then allowed to dry overnight before activating them for 1 hour at 105°C. Solvent systems used for separating the various reaction products consisted of benzene; benzene/chloroform (1:1); benzene/ ethanol (9 8 :2) and butyl alcohol saturated with 1.5N NH4OH. Residual hydrocarbon products, dissolved in a minimal amount of 95% ethanol, were applied to the TLC plates in 10-20 41 volumn spots and dried with forced air.The solvent front was allowed to transverse 10 centimeters from where the sample was spotted before removal from the tank.After the solvents were evaporated from the plate, the following spraying reagents were used in separate analysis:

1)concentrated sulfuric acid-rafter spraying, the plates were dried at 105°C until charred spots were visualized.

2)2,4 DNPH (HCl- yo. 4% w/v solution of 2,4 Dinitrophenol- hydrazine in 2N HC1 to detect carbonyl compounds.

3)Bromo Cresol Green- - detection of carboxyl compounds. 26

RESULTS

Growth Studies

The Arthrobacter species ATCC 25581 described in this work gave optimal growth on inorganic nitrogen and various carbonsub- strates when provided with a vitamin mixture.That the vitamin mixture could not serve as a sole carbon and energy source was confirmed by checking for growth in a suitable medium containing vitamins but lacking a carbon source.Although no attempt was made to identify the specific vitamin(s) which stimulated growth, the need for such factor( s) in obtaining maximum growth is evident in the growth curves for cells grown on a 1% acetate mineral salts medium( Fig- ure 3). Since the Arthrobacter species was selectively isolated for its inability to emulsify n-hexadecane without yeast extract, a study of the organism's metabolic capabilities was thought to be of value (Table 2).Both ammonium and nitrate ions were found to be utilized as sources of nitrogen.Of the various hydrocarbons tested, only the long chained C16 and C14 hydrocarbons were utilized as sole carbon and energy sources.In contrast, shorter chained n-alkanes and their alcoholic and ketonic isomers were not observed to support growth.In media in which a single amino acid was added as a source 27

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100

Hours

Figure 3.Arthrobacter ATCC 25581 growth responses to vitamin and nitrogen additions. (A) 1% NHCl; (B) 1% NHCl + vitamin mixture; (C) 1% NaNO3; . (D) 1% NaNO+ 4 4 3 vitamin mixture Table 2.Carbon and nitrogen sources for Arthrobacter AT CC 25581 aerobic growth. Sources of Compounds supporting growth Compounds not supporting growth O. D. (420 nm) Compounds 5 days Compounds Carbon Hydrocarbon Hydrocarbons Sugars Amino Acids H exadecane 0.35 Dodecane Inulin Glycine 3 -Hexadecanone 0.37 Decane methyl-D-glucoside Valine 2-Hexadecanone 0.33 Octane D(+)Trehalose Dihydrate Leucine 1-Hexadecanol 0.55 Hexane D( +)Melezitose Isoleucine 1- Hexadecene 0. 10 1, 5-Hexadiene Amygdalin Serine T etradecane 0.24 6-Undecanone L( +)Sorbose Threonine Miscellaneous 2-Undecanone R(+)Raffinose Aspartic Acid 2-Decanone Disodium Succinate 2.5 -methyl-D-Mannose Asparagine.-H 0 2-Octanone D(+)Xylose Sodium Acetate 2.76 Glutamic Acid 4-Heptanone Sodium Lactate 3.0 3 -D(+)Cellubiose Lysine 2-Hexanone Sodium Pyruvate 2. 6 D(-)Arabinose Arginine 2-Butanone cr -D(+)Melibiose Phenylalanine Amino Acids 1-Undecanol L(+)Rhamnose Tyrosine Histidine 0.48 1-Decanol Saccharose Tryptophan Alanine 0.40 2-Decanol D(+)Mannose Cysteine Cyclododecane Cystine Nitrogen NH4 Cl, Na NO3 Sugar Alcohols Cyclododecanol Methionine Mannitol Cyclodecane Proline Adonitol Cyclohexane Hydroxyproline Methylcyclohexane Ducitol i-Erytritol Purines Miscellaneous D-Sorbitol Adenine Creatine H2O Phenolics Adenosine Sodium Formate Guanine Ferulic Acid Urea Xanthine Genistic Acid Sodium Citrate p-Hydroxycinamic Acid Pyrimi din e s Sodium Oxalate Glycerol Hydrocinamic Acid Cytosine Cytidine Uracil 29 of carbon and energy, only histidine and alanine of the 22 amino acids tested were utilized.Although no hexoses were found to support growth, various TCA-Glyoxylate cycle precursors and intermediates were metabolized.The observation that sodium citrate was not utilized, however, may indicate the lack of a transport enzyme rather than an inability to metabolize this compound. Other compounds tested where growth was not observed included purines, pyrimidines and various phenolics. One interesting observation noted in this study, was that cells grown on disodium succinate or sodium acetate produced a water soluble red pigment as compared to cells grown on other carbon sources.Because yeast extract, pyruvate and lactate gave a greater yield of cells, it would seem that much of the carbon from succinate and acetate media was being shunted off into pigment formation.

Inducibility of Cell-Free Hydroxylase Activity

It was necessary to confirm that the Arthrobacter species was inducible for paraffin transformation.Cell-free extracts from induced and uninduced cells were prepared and assayed with various cofactors and coenzymes (Table 3).The results of this study indicate that only induced cells accumulate the reaction product, 2-hexadecanol.The role of cofactors and coenzymes does not seem to be significant in crude extracts of this bacterium. DPNH 30

Table 3. Role of cofactors and coenzymes on 2- hexadecanol formation by methylcyclohexane- induced and uninduced Arthrobacter ATCC 25581.

Induced cells - Peak height - Methylcyclohexane 2-hexadecanol, arb. units

DPNH & FeSO4 20

DPN & FeSO4 15

TPNH & FeSO4 18

TPN & FeSO4 13

FeSO4 15

No additions 15

Heat-treated (100°C-2 min) 0

Uninduced cells - succinate

DPNH & FeSO4 1 31 and TPNH gave only a slight stimulation to 2-hexadecanol formation.It seems likely that cofactors and coenzymes may already be present in the crude extract.

Effects of Alternate Carbon Sources on Hexadecane Transformation

Other investigators, working with the same Arthrobacter isolate, have shown that glucose stimulated hexadecane transformation in a basal salts medium (40).However, glucose was also observed to be a non-metabolizable sole carbon source for this isolate (40, 41).Therefore, studies were conducted to see what effect utilizable carbon sources would have on the transformation of n-hexadecane by resting cells. When washed yeast extract basal medium A grown cells were resuspended in a phosphate buffer with or without a utilizable "test carbon" source, in all cases hexadecane transformation appeared to be stimulated.Similarly, glucose also stimulated this transformation. In contrast, cells which were originally grown in "test carbon" basal medium A and then resuspended with the same "test carbon" in phosphate buffer showed a somewhat different effect.As shown in Table 4, sodium lactate, disodium succinate, sodium pyruvate, and glucose had little effect on repressing hydrocarbon transformation. On the other hand, sodium acetate repressed transformation 80%. 32

Table 4.Effect of alternate carbon sources on hexadecane transformation.

Test carbon source Test carbon / Controla (0.061M) Yeastextractb Testcarbonb grown cells growncells

Sodium acetate 1.7 0.20

Disodium succinate 2.5 0.74

Sodium lactate 3.3 1.80

Sodium pyruvate 3.8 1.15

Glucose 2. 6 1.13

aRatio of the amount of 2-hexadecanone formed by cells suspended in a phosphate buffer (pH=7) containing n-hexadecane and a "test carbon" source to that formed when the cells were suspended in a phosphate buffer with only hexadecane. bCells were grown either on yeast extract or on a "test carbon" basal medium. A cell concentration of approximately 1 x 10-3 g /ml dry weight was used. 33 It was believed that a critical differencebetween yeast extract and "test carbon" grown cells was that the former wereperhaps induced for paraffin transformation. One couldtherefore envision that for induced cells, m-RNA for hexadecane methylenehydroxylase enzymes would be formed and itstranslation could be stimulated by the "test carbon" sources.Nevertheless, methylcyclohexane- induced cells were also found to be repressedby acetate. As will be shown later, methylcyclohexane was found tobe a non-metabolizable inducer for hexadecane oxidation.Another explanation for the stimulatory effect shown, might be attributed tothe medium in which the cells were grown.It is possible that a yeast extract basal medium allowed amino acid pool accumulation.Resting cells would then be able to use the amino acids along withthe "test carbon" source for growth.

Compounds Effective as Inducers in Paraffin Adaption

A number of compounds were tested fortheir ability to induce paraffin oxidation.Typical induced and uninduced kinetics forthe compounds tested are shown in Figure 4.As can be seen, 2-hexadecanone from induced cells could bedetected by 2 hours.In contrast, approximately 4 hours wasrequired before this product

was detected for uninducedcells. A four hour reading was therefore chosen to quantitate the induction efficiency. 34

10 in 9

41) 8

7 4)

Cd >4 6

C 5

1 2 3 4 5 6 7 8 9 10 11 12 Hours

Figure 4. Formation of 2-hexadecanone by induced and uninduced Arthrobacter ATCC 25581 cells. 35 With n-alkanes and various corresponding ketonic compounds (Table 5), induction of paraffin oxidation began at C straight chained 10 compounds.In contrast, all hydrocarbon alcohols tested, regardless

of chain length, failed to stimulate induction.Surprisingly,1 -hexadecene was not observed to function as a strong inducer. Of the analogues of n-alkanes containing oxygen instead of a methylene group, none induced paraffin oxidation.In this connection, it is interesting that in another study (80), one of these non-inducing compounds, 1, 2-dimethoxyethane, was found to be an excellent inducer for Pseudomonas aeruginosa. Among the various cycloalkanes and cycloalkenes tested, only methylcyclohexane induced paraffin adaption.This may indicate, as observed by other workers (40), that a methyl group may be required for enzyme specificity.In contrast, it should be noted that p-xylene was not observed to function as an inducer. Of the compounds which induced paraffin adaption, hexadecane and tetradecane were able to support good growth.The only other

compound observed to support growth was 1 -hexadecene.However, growth was sparse and delayed.This is perhaps related to the minimal inducing power observed with this compound. To check the possibility that the induction procedure may permit the selection of a constitutive hydrocarbon transformation mutant, an induced cell inoculum was used to obtain uninduced cells as outlined 36

a Table 5.Structural effects on induction of methylene hydroxylase activity

Compound 2- Hexadecanone yield Utilized Ix 10-7 molar) for growth

Non-induced 0.2 n-Hexadecane 3 . 5 n-Tetradecane 2.7 n-Dodecane 3 . 5 n-Decane 2.8 n-Octane* 0.4 n- Hexane* 0

1-Hexadecene 0.5 1, 5-Hexadiene* 0.5

6-Undecanone 2.8 2-Undecanone 2. 6 2-Decanone 2.5 2 -Octanone* O. S 4 - Heptanone 0.7 2-Hexanone* 0.7 2 - Butanone* 0.5

1-Undecanol 0 1-Decanol 0 2 -Decanol 0.8 2, 3 - Butanedi ol* 0.3 ND Ethanol* 0.3 ND

Dim ethoxymethane* 0.4 ND 1, 2 Dimethoxyethane* 0.3 ND 1, 3 Diethoxy-2-propanol* 0. 6 ND Diethoxym ethane* 0.3 ND

Trans, trans, cis-1, 5, 9 - cyclododec atriene 0.4 ND Cyclohexane* 0. 5 Methylcyclohexane* 2.8 1- Methylnaphtha lene 0 ND p- Xylene 0 ND

aCompounds tested at 2.9 x 10-4 molar except for those marked with an asterisk, which were also tested at 5 x 10-3 molar b (+)=growth observed; ( -)'no growth observed; ND=not determined 37 in Materials and Methods.The results of this work indicated that induced cells may be converted back to uninduced ones.

Hydrocarbon Transformation in a Non-Growth System

In comparison with the induction experiment, the n-alkanes which are oxidized to ketones start at C14 rather than C (Table 6). 10 Oxidation of residual n-dodecane and decane from test samples with KMnO4, to convert any alcohols possibly formed into ketones, also indicated that no oxidation of these compounds had taken place.Simi- larly, various cyclic compounds, including cyclohexane and cyclododecane were not transformed to alcoholic or ketonic products. This included methylcyclohexane, a compound found to be an excellent inducer of n-hexadecane methylene hydroxylase activity.Never- theless, if related straight chained and cyclic compounds already contained alcoholic functions, these were transformed to the corresponding ketonic products. 38

Table 6.Induced Arthrobacter AT CC 25581 non-growth transformati on activity.

Compounds Ketones Rf detected solvent system* 1 2 3 n-Hexadecane 0.32 0.53 n-Tetradecane 0. 26 n-Dodecane - n-Decane n-Octane - n-Hexane -

1-Hexadecene 0.06 0.41

1-Hexadecanol 0. 60

2-Hexadecanol 0.30

Cyclododecanol 0.42

Cyclodecanol 0.43

1-Dec anol 0.76

2-Decanol 0.70

Cyclododecane

Cyclohexane

Methylcyclohexane p-Xylene

Benzene

* Solvent 1. Benzene Solvent 2.Benzene: Chloroform (1:1) Solvent 3.Butyl alcohol (NH4OH) Detection:2, 4 DNPH ( 2N HC1) 39

DISCUSSION

It has been suggested that the genus Arthrobacter may play an indigenous role in the soil.This would seem to be complicated by the vitamin requirement shown by the Arthrobacter species used in this work.Nevertheless, it has been demonstrated by investigators that a relatively high proportion of indigenous soil bacteria require one or more vitamins for growth (1, 55).Furthermore, it has been well established that B-vitamins as well as other microbial growth factors are present in soil (53, 55).One could therefore envision soil as a habitat for such vitamin-requiring bacteria. The unique characteristic of the organism used in this study is that its primary mode of n-alkane oxidation is subterminal. Although other workers have previously identified methyl ketones as reaction products with other microorganisms, they were delegated to minor side reactions since the primary products which accumulated were the corresponding 1 - alcohol and fatty acid.In this study, therefore, the observation that internal alcohols and ketones were the main products which accumulated would suggest that these were primary intermediates in the aliphatic hydrocarbon degradation.Similarly, 2-alcohol accumulation in cell-free extracts without 1 - alcohol being observed would indicate that attack is predominately subterminal. Further, by the use of structurally related compounds, a number of 40

cyclic hydrocarbon compounds and n-alkanes were not transformed to either alcoholic or ketonic products.However, if the related substrates containing alcoholic functions were used, the cells could transform these to the corresponding ketones.These studies would therefore indicate that hydroxylation is a limiting steric step in subterminal oxidation. Such results substantiate the study of Fonken et al. (17), where hydroxylation also appeared to be a rate limiting step in cyclic hydrocarbon oxidation.Such a scheme would also be in agreement with the postulated pathway proposed by Forney and Markovetz (18, 20) for long chain methyl ketone degradation. Growth studies using various TCA intermediates and precursors including acetate as sole carbon sources also indicated the importance of the TCA and glyoxylate pathways to the Arthrobacter species used in this study.The apparent inability of this organism to utilize any of the sugars tested further augments the importance of TCA and glyoxylate amphibolic sequences. Regulation of paraffin oxidation can be postulated to occur at the point where further degradation is no longer unique to hydrocarbon metabolism.In this regard, the repressive effect of acetate on hydrocarbon transformation warrants further consideration.This effect seems to be rather specific when compared with results observed with the other carbon sources tested.For instance, acetate shows the strongest repression in relation to the growth which 41 it supports.Acetate effects on paraffin adaption may besimilar to the so-called glucose-effect. Whether this effectfunctions either directly at the level of transcription or translation orindirectly on a supposed transport systemremains to be investigated. Although the induction system used in this work demonstrated methylene hydroxylase induction, a number of shortcomings were inherent.It should be made clear that the differingsolubilities and vapor pressures of the varioushydrocarbons used made it extremely difficult to carry out quantitative studies on inductionkinetics.In this regard, only hydrocarbons which were liquid at room temperature could be used.Solid compounds at room temperature, when added to the system, did not give the induction kineticsshown in Figure 5.This might be attributed to the very slow aqueousdissolu- tion rate of these compounds.Furthermore, the addition of these compounds in various solvents to increase dissolution mayhave had a deleterious effect onthe cells.Perhaps hydrocarbon alcohols and 1 - hexadecene did not cause induction due to possibletoxic effects, as observed in other studies (28,50, 75, 79), perhaps due to deter- gent properties as postulated for saturated andunsaturated fatty acids (15, 65, 75). Nevertheless, these studies do give an insight intomolecular configuration requirements for induction of methylenehydroxylase activity with this organism. From the observationthat 42 methylcyclohexane caused induction while the unmethylated cyclohexane did not, the requirement for amethyl group would be indicated. However, as other methyl group containing compounds, such as p-xylene and 1 - methyl naphthalene did not cause induction, indicates further structural requirements may be required. One such requirement might be a dependence on chain length or solubility properties. Such a dependence is even more evident in the oxidation and growth experiments where the ability to utilize n-alkanes and the corresponding hydrocarbon ketones decreased rapidly as the chain length decreased below that of n-hexadecane. Further, the observation that the corresponding ketones also caused induction would indicate that for this organism induction may not be sequential, in contrast to induction studies dealing with acetone oxidation (52, 56).Nevertheless, two basic differences which exist between this study and others may account for such discrepancies. First, the Arthrobacter species used in this study hydroxylates n-alkanes subterminally.In other studies, a subterminal hydroxylation was either delegated to a minor role or took place by terminal oxidation.Second, this Arthrobacter species optimally degraded long-chain methyl ketones, while other studies have mainly dealt with short- to medium-chain length alkanes. As has been suggested by Lukins and Foster (56), the mechanisms for long chain alkane degradation may differ from those involved in short chain alkane 43 transformation. This study has indicated a rather narrow range of hydrocarbons that can be oxidized or used for growth for this particular bacterium, indicating that molecular specificity in paraffin oxidation is rather stringent.Exceptions, however, are known. The bacterium used by Qoyama and Foster (64) for instance, not only oxidized n-alkanes ranging from C1 -C2 but also branched chained and cyclic hydrocarbon substrates. In contrast, Van Eyk and Bartels (80) as well as this study have indicated that molecular specificity for induction can be somewhat less stringent.Whether this indicates the hydrocarbon distribution found in any given habitat or simply a lack of specificity can only be speculated upon. This study indicates that methylene hydroxylase activity in Arthrobacter is controlled by inducer, substrate, and repressor structures, which can be postulated to function in nature.Further work should be undertaken to extend this study to natural environ- ments, hopefully to allow a better understanding of mechanisms by which organic compounds are transformed in natural systems. 44

SUMMARY

Induction of subterminal paraffin oxidation enzymes in whole cells of Arthrobacter species ATCC 25581 was investigated.Cell- free extracts indicated that whole cells could reliably reflect the synthesis of the first inducible enz-yrne(s) in n-alkane oxidation.Induc- tion and oxidation were found significantly repressed by sodium acetate, which might be related to acetate's possible role as an intermediate in paraffin oxidation, acting as an end product repressor.Sodium lactate, disodium succinate and sodium pyruvate were non-repressive carbon sources. Oxidation of n-alkanes was shown to be dependent on chain length.In comparison, molecular specificity required for induction was found to be less stringent.Methylcyclo- hexane and various medium length n-alkanes (C10 and C12) which caused induction were not observed to be oxidized.These structural studies also indicated that hydroxylation was a limiting steric step in subterminal oxidation, and that after hydroxylation, structural considerations for oxidation of alcohols to ketonic products are of lesser importance. 45

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