A Study of Methane-Oxidizing and Methanol-Oxidizing Bacteria." (1964)

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1964 A Study of Methane-Oxidizing and Methanol- Oxidizing Bacteria. Peter Konrad Stocks Louisiana State University and Agricultural & Mechanical College

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STOCKS, Peter Konrad, 1927- A STUDY OF METHANE- AND METHANOL- OXIDIZING BACTERIA.

Louisiana State University, Ph.D., 1964 Bacteriology

University Microfilms, Inc., Ann Arbor, Michigan A STUDY OF METHANE- AND METHANOL-OXIDIZING BACTERIA

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy

The Department of Bacteriology

by Peter Konrad Stocks B.S., Millsaps College, 1959 M .S., University of Southern Mississippi, 1961 „ . August, 1964 PLEASE NOTE: Figure pages are not original copy. They tend to ’’curl". Filmed in the best possible way. University Microfilms, Inc. ACKNOWLEDG MENT

The writer wishes to express his sincere gratitude to Dr. C. S.

McCleskey for his suggestions and criticism during this investigation and in the preparation of the dissertation.

He gratefully acknowledges the help and understanding extended to him by Drs. A. R. Colmer, A. D. Larson, M .D. Socolofsky, A. F.

Novak and J. A. Liuzzo.

He especially wishes to thank Dr. A. R. Colmer for his help in the preparation of the photographic material.

The writer also owes a great debt of gratitude to his friends and fellow graduate students for their inspiration, aid and criticism during the course of this study.

He also wishes to thank the Office of Naval Research for the financial support of this investigation.

This dissertation is dedicated to my family whose love and encouragement made this work possible.

ii TABLE OF CONTENTS

Page

I ACKNOWLEDGMENT...... IT-

II LIST OF TABLES ...... vi

III LIST OF FIGURES...... viii

IV ABSTRACT...... x.

V INTRODUCTION...... 1

VI REVIEW OF LITERATURE...... 3

VII MATERIALS AND METHODS...... 17

A. The Methane-Oxidizing Bacteria...... 17

C u ltu re s ...... 17

M e d ia...... 20

Isolation procedures...... 25

Source of samples ...... 25

Culture apparatus ...... 25

Isolation technique...... 29

Purity tests...... 31

Inhibitory agents as aids in the isolation of methane oxidizers...... 31

Morphological studies...... 33

S t a i n s...... 33

Photomicrography...... 33

Physiological and nutritional studies...... 33

iii Page

Carbon sources...... 34

Nitrogen sources...... 34

Serological studies...... 35

B. The Pink-Pigmented Methanol-Oxidizing Bacteria. . . 36

C u ltu re s ...... 36

M e d ia...... 36

Morphological studies...... 41

S t a i n s...... 41

Assay for poly-B-hydroxybutyric ...... ac id 41

Physiological and biochemical studies...... 42

Serological studies...... 46

VIII RESULTS AND DISCUSSION...... 47

A. The M ethane-Oxidizing B acteria ...... 47

Enrichment and isolation of the methane oxidizers . 47

Studies with inhibitory agents...... 49

Isolation by agglutination...... 55

Discussion of enrichment and isolation procedures. 57

Morphological characteristics...... 59

Cultural characteristics ...... 61

Physiological characteristics...... 63

The effect of methanol on the growth of the methane oxidizers...... 68

iv Page

The effect of NaCl on the growth of the ------methane oxidizers...... 70

The effect of various dyes on the growth of the methane oxidizers...... 75

Serological relationships...... 77

Discussion of results obtained with methane - oxidizing bacteria...... 77

B. The Pink-Pigmented Methanol Oxidizers...... 80

Isolation...... 80

Characteristics of pink-pigmented methanol- o x id iz e r s...... 83

Morphological characteristics...... 83

Cultural characteristics ...... 83

Biochemical and physiological characteristics . . . 88

Studies with antibiotics...... 95

Serological studies...... 98

Discussion of the pink-pigmented methanol- oxidizing bacteria...... 101

IX SUMMARY...... 105

X LITERATURE C IT E D ...... 108

XI VITA...... 113

v LIST OF TABLES

Page

1 S ources of-methanegQxldlzlna cultures ...... 18

2 Sources of organisms used in comparative studies . . . . 19

3 The composition of additional mineral salts solutions employed in this investigation...... 22

4 Sources of pink-pigmented methanol oxidizers...... 37

5 Pink-pigmented methanol oxidizers obtained from other investigators...... 38

6 The effect of various antibiotics on the growth of the methane oxidizers...... 51

7 The effect of various antibiotics in suppressing the growth of contaminants in methane enrichments 54

8 Utilization of hydrocarbons as sole carbon source by methane-oxidizing bacteria...... , ...... 65

9 Substrates tested for their ability to support the growth of methane-oxidizing bacteria...... 67

10 The availability of various nitrogen compounds for growth and methane consumption by methane o x id iz e r s...... 69

11 The effect of methanol on the growth of the methane- oxidizing bacteria...... 71

12 The effect of sodium chloride on the growth of the methane oxidizers in Brown's medium...... 73

13 The effect of various dyes on the growth 'of a methane oxidizer (strain PBG)...... 76

14 Serological relationships among methane oxidizers and morphologically related organism...... s 78

vi TABLE Page

15 Biochemical reactions of pink-pigmented methanol oxidizers ...... 90

16 Growth of the pink-pigmented methanol oxidizers on various carbon substrates...... 93

17 Availability of various nitrogen sources for growth of methanol oxidizers...... 96

18 The effect of various antibiotics on the growth of methanol oxidizers...... 97

19 Serological relationships among pink-pigmented methanol oxidizers...... 100

vii LIST OF FIGURES

FIGURE Page

1 Diagram of the apparatus used for measuring and mixing the g a se s...... 24

2 Sohngen unit used for measuring gas consumption 26

3 Method of introducing gases into reactor bottle of Sohngen u n i t ...... 28

4 Method of introducing inoculum into the reactor bottle of Sohngen u n it...... 28

5 Bottles used in the enrichment and growth of the methane oxidizers...... 30

6 Pink-pigmented colonies with microcolony of the methane-oxidizing bacteria...... 48

7 Mineral salts agar plates showing enhancement of methane oxidizers around sensitivity discs . 52

8 Flagellated cell of Methanomonas methanooxldans..... 60

9 Vacuoles in cells of Methanomonas methanooxldans.... 60

10 Cells of Methanomonas methanooxldans with bud like projections...... 62

11 Rosette formation of Methanomonas methanooxldans . . . . 62

12 Colonies of Methanomonas methanooxldans growing on mineral salts agar under methane...... 64

13 Colonies of Hvphomicrobium vulgare on mineral salts methanol agar...... 64

14 The effect of methanol on the growth of the methane- oxidizing bacteria...... 72

15 The effect of sodium chloride on the growth of the methane oxidizers in Brown's liquid medium...... 74

viii FIGURE Page

16 A pink-pigmented colony mixed with a methane- oxidizing colony on mineral salts agar under methane. . . 82

17 Electronmicrograph of flagellated cell of pink- pigmented methanol-oxidizing organism...... 84

18 Fat globules in cells of the pink-pigmented organisms as revealed by Sudan Black B...... 85

19 Absorption spectrum of crotonic acid...... 86

20 Colonies of pink-pigmented methanol oxidizers on mineral salts methanol ag a r...... 87

21 Reddish-pink pellicles of pink-pigmented methanol oxidizers...... 89

22 Pink-pigmented methanol-oxidizing bacteria on mineral salts agar with sodium oxalate as sole carbon source. . . 92

23 The effect of sensitivity discs on the growth of the pink-pigmented methanol oxidizers on mineral salts agar...... 89

ix ABSTRACT

Methane-oxidizing bacteria were isolated from coal mine water, rumen of a fistulated cow and oil field soil and compared with an isolate of Methanomonas methanooxldans of Brown and Strawinski.

The investigation also included the identification of pink-pigmented bacteria constantly found in the enrichment cultures of methane- oxidizing bacteria.

Initial enrichments were carried out in Sohngen units to confirm the presence of methane-oxidizing bacteria by consumption of methane.

Isolations were made on mineral salts agar plates under methane, employing surface-streaked plates and the dilution to extinction procedure.

The methane-oxidizing isolates are apparently identical in morphological and physiological characteristics with Methanomonas methanooxidans Brown and Strawinski. They are Gram-negative, monotrichously flagellated, non-sporeforming rods. The cells are highly vacuolated and some are much enlarged at one end, causing the other end to appear as a bud-like projection suggestive of the genus

Hvphomicrobium. Another striking feature is the common occurrence of rosettes similar to those observed in Agrobacterium. Phvllobacterium,

Chromobacterium and Rhizobium.

x The organisms developed only minute colonies under the most

favorable conditions. On mineral salts agar under methane the colonies

were about 0.05 to 0.1 mm in diameter after 3 weeks of incubation at \ 28 C. There was no discernable pigmentation of the colonies.

All the strains isolated utilized methane as the only carbon

source; ethane, propane and butane were not utilized, nor were any

of the common carbon substrates. Methanol was the only carbon

source other than methane which permitted growth of the cultures.

Nitrogen requirements were satisfied by either nitrates,

ammonium salts, peptone or certain amino acids.

Methanol, sodium chloride, calcium chloride and certain dyes

enhanced the growth of the organisms in the mineral salts medium of

Jayasuriya (1955).

Serologically, strains PBG and RUM were related to the strain

of Brown; the strain isolated from soil did not reveal any antigenic

relationship with other strains.

Pink-pigmented bacteria observed in methane enrichment cultures

were readily isolated on conventional peptone agar and on mineral salts

agar. In peptone broth at concentrations below 0.5 per cent growth was greatly delayed.

All the pink-pigmented organisms were Gram-negative, polar-

flagellated, non-sporeforming rods. Cells of all the strains contained

poly-B-hydroxybutyric acid . xi All the strains produced coral pink to red colonies; pigmentation

was greater in the presence of methanol and increased with prolonged

incubation. On mineral salts methanol agar colonies were small (about

0.2 mm after 5 days), circular, entire and butyrous. All the strains

were catalase positive, reduced nitrate to nitrite, grew in 10 per cent

methanol and in the presence of 1 per cent sodium chloride. All the

isolates grew ..on oxalate agar and in mineral salts broth with either

methanol, glycerol, formate, fumarate, succinate or benzene.

M ethane, ethanet propane and butane were not utilized by any of the

strains. Some of the strains were able to grow in the presence of

fructose, others grew in the presence of ribose. Glucose, galactose,

mannose, sucrose, lactose and sorbose were not utilized by any of the strains. Other carbon sources were attacked by some of the strains.

Some serological relationships were found among the pink- pigmented methanol oxidizers; no antigenic relationship was detected between strains of the pink-pigmented organisms and the methane- oxidizing bacteria.

The pink-pigmented methanol oxidizers isolated in this study, and the previously described organisms, Pseudomonas AMI (Peel and

Quayle), Pseudomonas methanica (Harrington and Kallio), Protamino- bacter ruber (den Dooren de Jong) and Pseudomonas PRL-W4 (Kaneda and Roxburgh) are closely related and are sufficiently like Vibrio extorauens (Bassalik) Bhat and Barker to be considered strains of that species. xii INTRODUCTION

Interest in the microbial oxidation of the hydrocarbon gases has centered chiefly on methane, and has been stimulated by three con siderations * Methane was being constantly produced in considerable amounts throughout the world in the anaerobic degradation of organic matter, and yet only traces were found in the atmosphere. Research was initiated to find organisms which utilized methane. Evidence was soon obtained by Sohngen (1906) and Kaserer (1906) that methane could be used as a carbon and energy source by bacteria.

Others have been interested in the microbial oxidation of the hydrocarbon gases because they have considered them as cheap sub strates for the synthesis of various chemical substances. Taggart

(1946) described a process for the production of a variety of chemicals using the gases from oil wells and refineries as substrates for micro organism s.

Another group of workers has been interested in the possibility of using microorganisms that oxidize the hydrocarbon gases as aids in finding underground deposits of oil and gas. Mogilevskii in 1940

(Brown, 1958) and Strawinski (1954) described microbiological methods for the location of petroleum deposits in the earth.

Although interest in the bacterial oxidation of hydrocarbon gases has extended over a period of nearly sixty years, there is still

1 2 uncertainty as to whether one or more species of bacteria is involved in the oxidation of methane, and doubt has been expressed concerning the purity of the methane-oxidizing cultures that have been described.

It now seems probable that methanol-oxidizing bacteria/ which are always present in crude cultures of methane-oxidizing cultures/ may have confused the description of the methane oxidizers.

The purpose of this investigation was to isolate additional strains of methane oxidizers for comparison with the Methanomonas methanooxidans of Brown and Strawinski (1957) and other cultures of methane-oxidizing bacteria described in the literature. An addi tional purpose of this study was to determine the identity of the pink- pigmented organisms constantly found in enrichment cultures of methane- oxidizing bacteria. REVIEW OF LITERATURE

A. Methane-Oxidizing Bacteria

The absence of methane in the atmosphere was first noted by

Urbain in 1901 (Brown, 1958). Urbain postulated that methane was

oxidized by ozone in the atmosphere or by green plants, or possibly

by bacteria. Sohngen (1906) was the first investigator to isolate

methane-oxidizing bacteria. Initially he suspected that plants could «• oxidize methane. However, careful washing of the plant material

decreased the absorption of methane and he concluded that micro

organisms must be the responsible agents. This investigator deter

mined a balance for the bio-oxidation of methane by bacteria: from

132 mm^ methane and 238 mm^ oxygen the organisms produced

117.5 mm^ carbon dioxide and 8 mg organic carbon. He concluded

that oxidation proceeded largely according to the overall equation

CH4 + 2 0 2 ------> CO2 + 2 H20

In units devised by Sohngen for culturing methane-oxidizing bacteria

he observed the consumption of methane by a change in the gas

volume. He noted that the organisms, after stationary incubation in these units, formed a reddish-brown pellicle.

S&hngen isolated the methane-oxidizing organisms on a washed agar mineral salts medium in an atmosphere of 1/3 methane and 2/3 air. According to his description, the organism was a short, thick rod with red or pink pigmentation, motile only in very young cultures, and with polar flagellation. In old cultures the organism . resembled a micrococcus. He called the organism Bacillus

methanlcus.

Kaserer (1906) reported the possibility of methane absorption by microorganisms and concluded that the absence of large amounts of methane gas in the atmosphere was due to the activity of these organisms. The methods he employed were rather crude and he never isolated or described bacteria capable of oxidizing methane.

In Italy, Giglioli and Masoni (1909) investigated the incidence of methane-oxidizing bacteria in sewage mud, river sediments and soil taken at different depths from plowed fields and alfalfa fields.

They found that methane-oxidizing bacteria were not abundant on the surface (0 to 30 cm depth) of tilled fields and of meadows, but were found in greater numbers at depths of 30 to 60 cm. Methane- oxidizing bacteria were found to abound in stable manure and in sewage.

The fact that methane-oxidizing organisms were found in soils stimulated investigators to look for them in other places. Harrison and Aiyer (1914) reported th at a bacterium was responsible for the oxidation of methane at the surface of rice swamp soils. The organism was identified as Bacterium fluorescens liquefaciens.

Munz (1915), using the procedures described by Sfihngen

(1906), isolated an organism which he called Bakterium methanicum. In addition to methane# this organism readily utilized acetate#

butyrate# tartrate# glycerol, mannitol# glucose, sucrose and peptone

as sole energy sources. According to ZoBell (1950)# the methane-

oxidizing cultures of Munz frequently contained Pseudomonas

fluorescens and Pseudomonas aeruginosa, but since pure cultures of

these organisms could not utilize methane, Munz concluded that they

grew at the expense of the organic matter synthesized by the methane-

utilizing bacteria. Munz doubted that the species of methane-oxidizing

organism with which he worked was the same as that described by

Sohngen (1906).

Hasemann (1927) isolated a methane-oxidizing organism from garden soil, using illuminating gas as carbon source. Based on

macroscopic and microscopic observations, the isolated organism was considered by Hasemann to be identical to the Bacillus methanicus described by Sohngen.

Tausz and Donath (1930) described two hydrocarbon-oxidizing bacteria. One, Bacterium aliphaticum liauefaciens, did not utilize methane, ethane or butane, but readily attacked pentane, hexane, heptane, octane and decane. The other organism attacked methane and all the higher members of the series up to and including paraffin oil. The unsaturated hydrocarbons-, propylene, butylene and possibly acetylene, but not the cyclic hydrocarbons benzene and cyclohexane, were also utilized. They concluded that, in general, the ease with which a hydrocarbon is attacked increases with the length of the carbon chain, so that when a member of the series is attacked by a given organism, it may be assumed that all higher members will be attacked by the same organism.

Subbota (1947), in contrast to Tausz and Donath (1930) stated that some bacteria oxidize methane and are not capable of utilizing the heavier hydrocarbons. Some bacteria consumed only propane.

While prospecting, Subbota analyzed soil samples not only for methane oxidizers but also for propane oxidizers and cellulose- decomposing bacteria. The cellulose-decomposing organisms pro duced methane which influenced the abundance of methane-oxidizing bacteria. He concluded that the oxidation of methane and propane is accomplished by organisms "working together."

Bokova, Kuznetsova and Kuznetsov (1947) attempted to deter mine the specificity of bacteria for individual hydrocarbons. They found that methane, pentane, hexane and heptane were decomposed by Methanomonas methanica and that ethane and propane were utilized by distinctly different bacteria.

Hutton and ZoBell (1949) found methane-oxidizing bacteria in marine sediments collected off the coast of California, in the Gulf of Mexico, in the Atlantic Ocean and in surface soil from oil and gas fields in California, Louisiana, Oklahoma and Texas. The organisms were partially described by these investigators, but not identified. Some of them used only methane as sole carbon source, others methane, ethane and propane. None of them grew on nutrient agar or gelatin in the absence of the gaseous hydrocarbons, Among the organisms able to use methane as sole carbon and energy source it was found that the "partial pressures" producing the most rapid utilization of methane were carbon dioxide 10, oxygen 40, and methane 50 per cent.

In the same year Nechaeva (1949) reported the isolation of two organisms from the sediments in methane tanks. These organisms were capable of utilizing methane, propane and various other organic carbon compounds. One of these organisms was named Mycobacterium methanicum; the other was identified as Mycobacterium flavum, but due to its adaptive ability to utilize methane in the absence of other organic carbon sources, it was designated M. flavum var. methanicum.

Morphologically the two mycobacteria were almost indistinguishable from one another. They were wedge-shaped or bent rods of various sizes, from 0.8 to 5.0 by 0.5 to 0.7 /a. The cells distributed them selves in pairs at an angle. Both organisms were non-motile and Gram- positive. Presumably the organisms were acid fast since they were classified as a species of Mycobacterium, but the author never men tioned acid-fastness in the paper. On inorganic agar media in an atmosphere consisting of a mixture of methane and air, the colonies were transparent, flat, dull, dry, wrinkled and colorless, often in the 8 shape of rosettes. The same characteristic of growth was observed on protein and potato media with agar. The addition of glucose or mannitol to peptone agar increased the growth. On potato slices and starch agar the surface growth was almost transparent. Of the hydrocarbons tested,

M. methanicum utilized only methane and propane, while .M.. flavum var. methanicum utilized methane,propane and heptane.

A pink-pigmented methane-utilizing organism was isolated by

Dworkin and Foster (1956). Their inoculum consisted of triturated leaves and stems of the aquatic plant Elodea obtained from a shallow fresh water pond. Incubation was under stationary conditions in an atmosphere of 40 per cent methane and 60 per cent air. After 7 to 10 days of incubation of the primary enrichment culture, there appeared a distinct pinkish pellicle on the surface of the medium, as described by Sohngen (1906). Serial dilutions of a cell suspension prepared from pink colonies were streaked until pure cultures were obtained.

The criteria adopted for purity were: homogeneous morphology as determined by microscopic examination of living and stained cell preparations, absence of non-pink colonies on streak plates incubated in methane-air, and absence of growth on nutrient agar in the absence of methane. The organism was a Gram-negative rod; the cells usually occurred singly, but sometimes in pairs or in chains up to four cells in length. The cells, when stained with basic dyes, were character ized by the presence of unevenly staining intracellular material giving the cells a mottled appearance. The cells possessed a single,polar flagellum and were highly motile in young and old liquid cultures.

Colonies on agar usually were pink. The colonies had a mucoid appearance, and a tacky consistency. After one to two weeks of incubation they became difficult to disrupt with a needle. Growth in liquid media shaken during incubation was either dispersed or clumpy.

In stationary cultures, the freshly isolated organism grew either as a thin, reddish-pink membrane on the surface of the medium, or as a pink turbidity throughout the medium with accumulation of sedimented cells as the culture aged. These investigators considered their organism to be nearly identical with Bacillus methanlcus of Sohngen

(1906). It agreed with the description of B. methanicus in all major taxonomic features, i.e. morphology, flagellation, methane utilization, pink-pigmentation and occasional pellicle formation. Dworkin and

Foster objected to the physiological classification of Orla-Jensen

(1909) which placed the Sohngen organism in the new genus

Methanomonas. and proposed that it be placed in the genus

Pseudomonas.

Leadbetter and Foster (1957) reported that they were unable to confirm the earlier findings of Dworkin and Foster (1956) with regard to a growth factor requirement of P. methanica and that the organism was now able to grow prototrophically. Leadbetter and Foster (1958) isolated about thirty methane- dependent organisms which were all morphologically indistinguishable/ polar flagellated/ Gram-negative pseudomonads. They differed with respect to pigmentation; strains were pink, brown, yellow or unpig- mented. Each of the organisms was obligately dependent on methane or methanol for growth, being unable to utilize any of the conventional hydrocarbon and non-hydrocarbon substrates. These investigators con cluded that the obvious physiological homogeneity indicated they were one species comprising four varieties: the pink P.. methanlca (Sohngen); a yellow P.. methanica var. fulva; a brownJP. methanica var. fusca; and non-pigmented P.. methanica var. incolorata.

Brown (1958) reported the isolation of methane-dependent cultures from a variety of sources. He considered his isolates to be different from Methanomonas methanica (Bacillus methanicus of Sohngen), and named it Methanomonas methanooxidans. The organism was described as a non-sporeforming rod, 1.5 to 3.0p. in length and 1.0 /j in width.

It stained unevenly with the Gram stain and was non-acid fast. The organism was motile by means of a single^polar flagellum. The methane oxidizer did not grow on nutrient agar in the absence or in the presence of methane. In the presence of methane the organism formed only micro colonies on mineral salts agar after 3 weeks of incubation. Variations of the oxygen content of the gas mixture from less than one to thirty per cent failed to increase colony size. A wide variety of solid media 11 containing various organic adjuncts, including vitamins, amino acids, and complex organic compounds also failed to enhance colony ______size.

Growing cultures of this organism consumed methane and oxygen in a ratio of 1.0:1.1 as determined by chemical analysis of the gas. The presence of intermediates in the oxidative pathway determined by chemical methods indicated that methane oxidation by that organism proceeded as follows: '•

CH4------> CHgOH------> HCHO------> HCOOH » C02

Methanol served as sole carbon and energy source, but growth was less abundant than with methane.

Johnson and Temple (1962) reported a pink-pigmented methane- dependent organism which they regarded to be identical with the organism described by Dworkin and Foster (1956) and Leadbetter and Foster (1958).

It was a Gram-negative rod, motile by means of a single, polar flagellum.

With stationary incubation it formed a pellicle. According to these investigators it varied from the Dworkin and Foster organism in its pH requirements and phosphate tolerance.

While most of the work reported so far was concerned with the isolation or description of hydrocarbon-oxidizing bacteria, a few workers investigated the possibility of the use of these organisms for industrial purposes. Taggart (1946) described a process for the production of chemicals from the gaseous materials from oil wells and refineries by Bacillus methanicus. Yurovskii, Kopilash and Mangubi 12

(1939) reported that in preliminary field tests up to 96 per cent of the methane in the atmosphere of coal mines was destroyed by strategically located cultures of Methanomonas m e th a n ic a______.

B. Pink-Pigmented Bacteria Associated with

Methane-Oxidizing Cultures

Ever since Sohngen reported the presence of a reddish-brown methane-oxidizing organism which he named Bacillus methanicus. pink- pigmented organisms have been observed either as methane-dependent bacteria (Dworkin and Foster, 1956; Leadbetter and Foster, 1958;

Johnson and Temple, 1962) or in association with crude cultures of methane-oxidizing bacteria (Brown, 1958; Holmes, 19,62). Brown

(1958) concluded that Methanomonas methanooxidans could be entrapped in the slime of some of the pink cultures and escape detection. Holmes

(1962) concluded that pink-pigmented pseudomonads which do not utilize methane are found in aquatic methane enrichments. Her data confirmed the conclusion of Brown (1958) that pink cultures free of

Methanomonas methanooxidans do not oxidize methane. Brown (1958) and Holmes (1962) reported that pink-pigmented organisms isolated by them from methane enrichment cultures were able to grow in the culture filtrates of Methanomonas methanooxidans.

Pink-pigmented bacteria capable of utilizing methanol as sole carbon and energy source were initially reported by Bassalik (1913). 13

The organism described by this investigator was isolated from the excreta of an earthworm that had ingested plant material containing crystals of calcium oxalate; later the organism was shown to be present in garden soil also. This organism was named Bacillus extorquens and described as a polar-flagellated, Gram-negative, slightly bent, non-sporulating rod. It formed a rose-red to blood red pigment. Growth was slow on ordinary nutrient agar and gelatin medium, but was rapid and abundant on synthetic media containing one of the following compounds as sole carbon source; oxalate, glyoxalate, malonate, succinate, fumarate, maleate, formate, methanol, glycerol, sorbitol, mannitol and glucose.

Bhat and Barker (1948) isolated a non-pigmented, vibrio-like organism from a soil suspension streaked on mineral-salts agar with oxalate as the only carbon source. They classified it as Vibrio oxalitlcus and considered it to be closely related to Bassalik's

Bacillus extorquens. These investigators concluded that the latter organism is also a vibrio and should be called Vibrio extorquens to fit into the modern system of.bacterial taxonomy. Janota (1950) called the organism Pseudomonas extorquens.

In 1927 den Dooren de Jong described the isolation of a pink- pigmented organism capable of utilizing methylamine. When grown in liquid media in an atmosphere of methylamine under stationary conditions this organism formed a red pellicle. In view of the fact 14 that this organism occurred quite frequently in media containing the

lower amines and showed a pink to red pellicle, he classified it

provisionally as Protaminobacter-ruber-^ This investigator considered

as belonging to the genus Protaminobacter all those organisms which

are non-sporeforming, non-motile, Gram-negative rods capable of

degrading lower alkylamines. An additional characteristic of this

new genus was its slight to moderate growth on peptone agar. Pigment

formation was less distinct on peptone agar than on mineral salts agar with methylamine as the only carbon source. This investigator did not test carbon sources other than the lower alkylamines.

Weaver, Samuels and Sherago (1938) isolated an organism from

sewage which proved to have characteristics identical with those of

Protaminobacter ruber den Dooren de Jong, except that the organism was actively motile with a single/ polar flagellum. These investigators

recommended that the description of P.. ruber be changed in accordance with their findings.

Slepecky and Doetsch (1954) isolated by enrichment techniques

organisms capable of using amines as the sole source of carbon and

energy. Of twenty-three isolates studied, only one resembled a known

Protaminobacter species. These investigators questioned the validity

of the genus Protaminobacter.

Kaneda and Roxburgh (1959) reported the isolation of a biotin-

dependent, pink-pigmented pseudomonad from methanol enrichment !5 cultures (Pseudomonas PRL-W4). They observed marked similarities between their organism and the Pseudomonas methanica of Dworkin and~Fostei (195~fe) . Botlr organisms"po s sessed systems- for the oxida tion of methanol/ formaldehyde and formate, but methane was not utilized by the organism of Kaneda and Roxburgh.

Harrington and Kallio (1960) obtained a methanol-utilizing pseudomonad by streaking a soil suspension directly on mineral salts agar plates with methanol as sole carbon source. The isolate was described as a small Gram-negative non-motile rod. The organism had a pink pigment and individual cells were sudanophilic. Colonies on solid media corresponded to the description of Pseudomonas methanica colonies as reported by Dworkin and Foster (1956).

Methanol was the only carbon source utilized. Other growth sub strates tested (citrate, peptone, yeast extract and higher alcohols) did not support growth. The investigators concluded that the methanol- utilizing organism was a strain of Pseudomonas methanica. They sug gested that the failure of the organism to utilize methane may have been a consequence of its cultivation for over a year in methanol media.

Kallio and Harrington (1960) reported that the sudanophilic granules of Pseudomonas methanica were composed largely of poly-B- hydroxybutyric acid and a small amount of monopalmitin.

A pink-pigmented, Gram-negative, monotrichously flagellated rod was identified by Peel and Quayle (1961) as a species of Pseudomonas and was referred to as Pseudomonas AMI. This organism was able to grow on methylamine, methanol, formate and formamide.

Methane and formaldehyde did not serve as growth substrates, nor did the organism grow autotrophically at the expense of oxidation of hydrogen. Poly-B-hydroxybutyric acid was found by these investi gators in methanol-grown cells of Pseudomonas AMI. MATERIALS AND METHODS

A. The Methane-Oxidizing Bacteria

Cultures

The cultures employed in this study included that of Brown and

Strawinski (1958) as described by Strawinski and Brown (1957) and five new isolates obtained from widely different sources (Table 1).

For comparative studies, Hvphomicrobium vulgare. Agrobacterlum tumefaciens, Agrobacterium radiobacter, Agrobacterium stellulatum and

Rhizobium leguminosarum were also used (Table 2).

Stock cultures of the methane oxidizers were transferred once a month to Sohngen units and incubated under shake conditions at room temperature (25-28 C) for four days. Ten ml portions of these cultures were removed aseptically, tested for purity and stored in 125 ml bottles under methane as described below. Cultures stored in this way were viable after 3 years. Hvphomicrobium stock cultures were grown in

Mevius' mineral salts solution (Table 3) with methanol as the only carbon source (0.1 per cent, v/v). Agrobacterium tumefaciens and

A. radiobacter were propagated on tryptone glucose extract agar

(Difco) slants. Five per cent (w/v) sodium chloride was added to the same medium in order to grow Agrobactefium stellulatum. The

Agrobacterium species were transferred every month. Rhizobium

17 18’

Tabie-1. Sourees-

Strain Isolated Obtained Reference designation from from

Brown Oil field soil near Dr. L. R. Brown (1) (2) Baton Rouge, La. (3)

PBG Coal mine water from original isolation Bottrop, Germany

RUM Rumen of a fistulated cow original isolation

Soil Oil field soil near original isolation Baton Rouge, La.

BS* Beach sand from original isolation Gulfport, Miss.

OP* Oxidation pond, LSU original isolation Agr. Exp. Station, Baton Rouge, La.

(1) Brown (1958) (2) Brown and Strawinski (1958) (3) Strawinski and Brown (1957) * Isolated too late to be included in physiological studies. 19

Table 2. Sources of organisms used in comparative studies.

Name of organisms______Obtained from

Hvphomicrobium vulgare Dr. R. Naveke, Botanisches Institut der Techn.Hochsch. Braunschweig, West-Germany

Agrobacterium tumefaciens Dr. C. Stapp Braunschweig Agrobacterium radiobacter Magnitorwall 5 West-Germany Agrobacterium stellulatum

Rhizobium lequmiinos arum American Type Culture Collection #10314 20 lequminosarum was grown at room temperature on slants of the medium recommended by the American Type Culture Collection (see below).

They were subcultured every four weeks. The storage of all the cultures was at refrigerator temperature (4-6 C).

Media

Cultures of the methane oxidizers were routinely propagated in the mineral salts solution of Jayasuriya (1955) with methane as the only carbon and energy source. The mineral salts solution was made up in 10-liter batches with distilled water and contained the following ingredients:

Chemicals Grams/liter

kh 2p o 4 1.4

Na2HP04 2.1

(n h 4)2s o 4 0.5

MgS04 *7H20 0.2

FeS04 *7H20 0.005

MnS04 *5H20 0.002

CaCl2 0.01

The pH was adjusted to 6.3-6.6 with HC1 or NaOH and the solu tion was allowed to stand several days for the precipitate to settle.

Only the clear supernatant was used. Tests to determine the suit ability of various carbon and nitrogen compounds for growth were carried out with this mineral salts solution. For comparative purposes 21

some studies were made with the media of Kaserer (1906), Mevius

(1953) and Brown (1958). These media are described in Table 3.

Mineral salts agar was prepared by the addition of 2 per cent

Difco agar to the mineral salts solution. Unless otherwise indicated,

sterilization was accomplished at 121 C for 20 minutes.

Silica gel plates were prepared with colloidal silica gel as

described by Brown (1958). Into each petri plate was pipetted 12 ml

of a suspension prepared by mixing 1000 ml of a salts solution

(KN03, 7.5 g; K2HP04*3H20 , 3.7 g; MgS04*7H20, 1.5 g; FeCl3-6H20,

0.38 g) with 500 ml of Ludox 15 (E. I. duPont de Nemours and Co.).

The pH of the mixture was adjusted to 7.0 before use. The plates were

autoclaved at 121 C for 12 minutes during which solidification and

sterilization were effected. Silica gel plates prepared in this fashion

had a completely smooth surface and were streaked as readily as agar

p la te s .

The medium recommended by the American Type Culture Collection

for the growth of Rhizobium lequminosarum had the following composi-

position: yeast extract, 0.5 g; soil extract, 100 ml; mannitol, 5.0 g;

Bacto-agar, 7.5 g; pH, 7.4. The soil extract was prepared by placing

77.0 g of African Violet soil (obtained from a local market) and 0.2 g of

NaCOg into 200 ml of distilled water. After autoclaving for 1 hour, the

extract was filtered through filter paper (Reeve Angel No. 202) and

added to the medium in the amount listed above. The complete medium was autoclaved at 121 C for 15 minutes. Table 3. The composition of additional mineral salts solutions employed in this investigation.

Kaserer (1906) Mevius (1953) Brown (1958)

j . Chemicals Grams/liter Chemicals Grams/liter Chemicals Grams /lite r k 2h p o 4 0.5 kh 2p o 4 2.7 K2HP04 0.5

1.0 kn o 3 1.0 kno 3 1.0 NH.C14 MgS04 0.2 MgS04 *7H20 0.25 MgSO4 *7H20 0.2

FeCl3 trace CaCl2*6H20 0.20 FeCl3*6H20 0.05

FeS04 *7H20 0.028 NaCl 0.2 23

A, special medium for Agrobacterium tumefaciens and A. radiobacter was prepared according to Stapp and Bortels (1931). Five hundred grams of finely minced carrots were boiled in 1000 ml of tap water for 1 hour.

After cooling, the carrot juice was filtered and diluted to 3000 ml with tap water. The following salts were added per liter of medium: 0.1 g

FeS0 4 * 7H2 0 and 0.1 g MnSO/j^HgO. Agar plates of this medium were prepared by adding 2 per cent Difco agar.

The hydrocarbon gases employed (Matheson Company, East-

Rutherford, New Jersey) were of the highest purity commercially avail able: methane, C. P. grade, 99.0 per cent pure; ethane, 95.0 per cent; n-propane, instrument grade, 99.9 per cent; n-butane, instrument grade, 99.9 per cent; oxygen, extra dry grade, 99.6 per cent; carbon dioxide, bone dry grade, 99.9 per cent; hydrogen; extra dry grade,

99.9 per cent pure. The gases were stored in separate containers and mixed as required for use in a large graduated cylinder by liquid displacement (Fig. 1).

The gas mixture used routinely in this investigation was com posed of approximately 65 volumes of methane, 30 volumes of oxygen and 5 volumes of carbon dioxide (hereafter referred to as the methane gas mixture). Other gas mixtures employed were: 65 volumes of ethane, n-propane or n-butane, 30 volumes of oxygen and 5 volumes of carbon dioxide; 50 volumes.of hydrogen, 40 volumes of oxygen and

10 volumes of carbon dioxide. 23

A special medium for Agrobacterium tumefaciens and A. radiobacter was prepared according to Stapp and Bortels (1931). Five hundred grams of finely minced carrots were boiled in 1000 ml of tap water for 1 hour.

After cooling, the carrot juice was filtered and diluted to 3000 ml with tap water. The following salts were added per liter of medium: 0.1 g

FeS0 4 * 7H2 0 and 0.1 g MnSC^^HgO. Agar plates of this medium were prepared by adding 2 per cent Difco agar.

The hydrocarbon gases employed (Matheson Company, East-

99.9 per cent pure. The gases were stored in separate containers and mixed as required for use in a large graduated cylinder by liquid displacement (Fig. 1).

10 volumes of carbon dioxide. Figure 1. Diagram of the apparatus used for measuring and mixing the gases. 24

RESERVOIR

OAS MEASURING CYLINDER (vol. LSNtori)

CLAMPS AT A,B,C,D.

NEEDLE FILTER — 5-fe. -

C 0 o 25

Isolation procedures

Source of samples. Soil and water samples were obtained from various locations, paying particular.attention to locations which would foster the enrichment of methane-oxidizing bacteria in a natural environ ment. Ten grams of soil were added to 90 ml of water and thoroughly mixed in a Waring Blendor at moderate speed for one minute. The coarser particles were allowed to settle to the bottom and the super natant was filtered through a Buchner funnel containing three layers of gauze. The filtrate was centrifuged in order to concentrate the organisms in 5 ml of water. After centrifuging and resuspending the organisms three more times, the suspension was used as the initial inoculum. Water samples (100 ml) were allowed to stand for awhile to allow suspended particles to settle to the bottom and then were concentrated by centrifugation.

Culture apparatus. The presence of methane-oxidizing organ isms was initially determined with modified Sohngen units, using

195 ml prescription bottles, as described by Hutton and ZoBell (1949), but with some modifications in procedures. The bottles of the Sohngen units, hereafter referred to as reactor and reservoir bottles, were filled to the 110 ml mark with the appropriate medium and fitted as shown in

Figure 2. The openings leading to the air were protected from con tamination by cotton filters. After cooling, vacuum was applied to the reactor bottle until the medium from the reservoir filled the reactor; Figure 2. Sohngen unit used for measuring gas consumption. Left, reactor bottle: right, reservoir bottle.

27 the outlet of the reactor was then closed with a clamp. The gas

mixture was introduced through a sterile filter into the reactor bottle by a needle inserted through the rubber hose just below the clamp

(Fig, 3). The gas was allowed to displace the medium to the 20 ml graduation mark on the bottle. All Sdhngen units were allowed to stand

at least a day to insure that there was no leakage.

The inoculum (2.5 ml; 5% v/v) was then injected into the reactor with a hypodermic syringe (Fig. 4) as follows: the rubber tube below the clamp was sterilized with 70 per cent alcohol or merthiolate and the needle was inserted through the rubber and down into the inlet tube of the reactor bottle. To insure the removal of all the inoculum from the inlet tube, vacuum was applied to the reservoir bottle and the

clamp on the reactor was momentarily released. The vacuum was then

disconnected from the reservoir and the clamp on the reactor was re leased until the medium in the reactor reached the 50 ml mark. At this

point the reactor was sealed by placing the clamp on the inlet tube below the needle puncture.

All the manipulations were carried out in an inoculating hood.

Incubation was at room temperature (25-28 C) on New Brunswick rotary

shakers (Model V, New Brunswick Scientific Company, New Brunswick,

N. J.). The shakers were adjusted to rotate at approximately 180

rev/m in.

The inoculated Sohngen units were incubated until gas Figure 3. Method of introducing gases into reactor bottle of SiShngen unit.

Figure 4. Method of introducing the inoculum into the reactor bottle of Sohngen unit.

29 consumption was nearly complete, as indicated by the volume of medium drawn back into the reactor vessel-fronrthe reservoir. Since the composition and volume of the initial gas mixture was known it could readily be determined that methane was consumed without having to resort to chemical analysis. This was known when the volume of medium drawn back into the reactor had surpassed that which could be accounted for by the depletion of all the oxygen, carbon dioxide and other contaminating gases that were present.

Usually most of the gas was consumed.

Isolation technique. From methane-consuming enrichment cultures 5 ml were withdrawn, washed three times by centrifiguation and resuspended in 5 ml sterile phosphate buffer. Flint glass 4 oz bottles (E. A. Sargent, No. S—8275) which contained 10 ml of sterile mineral salts solution, were inoculated with 0.2 ml of the washed cell suspension. The bottles were closed with serum stoppers

(Fig. 5) and flushed with the gas mixture using one needle to admit the gas and another to allow the gas to escape. Incubation was at room temperature on a rotary shaker.

As soon as slight turbidity appeared, a portion of the culture was withdrawn, the cells were washed and resuspended as before, and fresh cultures were started. After the fifth transfer showed the slightest turbidity, the culture was withdrawn, the cells washed, resuspended in mineral salts solution (MSS), and serial dilutions Figure 5. Bottles (125 ml) used in the enrichment and growth of the methane oxidizers. Left, before autoclaving; right, after autoclaving with serum stoppers in place.

(10-1 to 10"10) prepared in MSS. One-tenth ml of each dilution was transferred to 3 separate bottles containing 10 ml of sterile medium to which the gas mixture was added. Each dilution was also streaked on tryptone glucose extract agar (TGE) and on mineral salts agar, the former for incubation in air to detect non-methane-dependent organisms, the latter for incubation under the methane gas mixture in a desiccator.

Bottles which showed growth under methane and colonies which appeared on the mineral salts agar plates under methane were used to inoculate

Sohngen units to confirm the presence of methane-oxidizing bacteria.

A stereoscopic microscope was used in the picking of colonies.

Purity tests. Brown (1958) found that Methanomonas methano oxidans did not grow in the absence of methane or methanol. Conse quently, Sohngen units showing consumption of methane were tested for purity by streaking TGE agar plates for incubation in air at room temperature. Any culture showing growth on these plates after incuba tion for at least 72 hours was discarded as contaminated or subjected to purification procedures.

Inhibitory agents as aids in the isolation of methane oxidizers.

Agar plates were prepared by adding 2 per cent agar to the mineral salts solution. The plates were allowed to dry for 1 hour at 37 C, then 0.1 ml of a cell suspension was smeared on the plates with a sterile, bent glass rod. The following antibiotic sensitivity discs were placed on the plates (Baltimore Biological Laboratory, Baltimore,

M d.): 32

Agent Concentration

Aureomycin 5 >ig Carbomycin 2 P* Erythromycin 2 jig Chloromycetin 30 jig Tetracycline 5 /ig Neomycin 5 W Streptomycin 10 p g Dihydrostreptomycin 2 jig

Sulfadiazine 1 mg Triple Sulfa 0.25 mg

Polymyxin B 50 units Penicillin 2 units Penicillin 10 units Bacitracin 2 units

The plates were incubated at 25-28 C in a desiccator which was flushed with the methane gas mixture.

Stock solutions of the following antibiotics were prepared in phos phate buffer (pH 7.0) and sterilized by filtration: streptomycin, 800

/ig/ml; chloramphenicol, 1000 jag/ml; Aureomycin, 1000 ^ig/ml; bacitracin, 38.5 units/ml. Serial dilutions of the antibiotics were prepared in mineral salts solutions in 125 ml bottles and inoculated with 0.1 ml of a suspension of methane-grown cells. The bottles were closed with serum stoppers, flushed with the methane gas mixture and incubated at room temperature on a rotary shaker.

Certain dyes, known to exert bacteriostatic effects, were incor porated into liquid mineral salts solutions. Stock solutions of the following dyes were prepared in concentrations of 0.01 g/ml: 33 methylene blue, crystal violet, congo red and neutral red. They were sterilized by filtration and serial dilutions were made in mineral salts solutions dispensed in 125 ml bottles. Inoculation and incubation were carried out as described above. After five days of incubation, the cultures were filtered through tared Millipore filters (MF type HA;

Millipore Filter Corporation, Bedford, M ass.), to determine the dry weight of the cells. The filters containing the cells were dried to constant weight in an oven at 85 C.

Morphological studies

Stains. Unless otherwise indicated, the stains used in the identification procedures were prepared according to the Committee on Bacteriological Technic, Society of American Bacteriologists (1957).

Flagella stains were prepared according to the method of Bailey (1929) as modified by Fisher and Conn (1942). Spore stains were made with the method of Schaeffer and Fulton (1933).

Photomicrography. The black and white photographs were taken with Panatomic-X or Contrast Process film* the colored photographs were made with Kodachrome II (Eastman Kodak Company, Rochester,

N. Y.). Photomicrographs were taken with a compound microscope

(Leitz Ortholux).

Physiological and nutritional studies

Tests to determine the ability of the organisms to utilize 34 various carbon and nitrogen sources other than the hydrocarbon gases were conducted as described below.

Carbon sources. Stock solutions of carbon compounds were

adjusted to pH 7.0, sterilized by filtration, and added to 10 ml sterile

mineral salts medium in 125 ml screw-capped bottles to give concentra tions varying from as little as 0.001 per cent for formaldehyde and phenol, to 1.0 per cent for the carbohydrates. Cells of methane-grown

cultures were washed three times in sterile phosphate buffer (pH 7.0)

prior to use as inocula. The density of the cell suspension was about

9x10® cells/ml. An inoculum of 0.1 ml was added to each of three bottles; a fourth bottle served as uninoculated control. Incubation was at room temperature for six weeks on a rotary shaker at about

180 rev/min. All bottles showing turbidity were tested for purity by

streaking TGE agar plates as described previously.

Nitrogen sources. With the Jayasuriya mineral salts medium

less ammonium sulfate, nitrogen compounds were tested in concen trations ranging from 0.01 per cent to 0.20 per cent. The pH of the

stock solutions was adjusted to 7.0. The inorganic compounds were

added to the medium prior to autoclaving; all organic nitrogen com

pounds were sterilized by filtration. The sterile medium was dis

pensed in 10 ml amounts in 125 ml bottles, closed with serum stoppers

and the bottles were flushed with the methane gas mixture which served

as carbon and energy source. Tests were carried out in triplicate, a 35 fourth bottle served as uninoculated control and another control bottle contained inoculum but no methane gas. Incubation was at room temperature. Cultures which showed growth through two transfers in a substrate were tested for purity, then used to inoculate duplicate

Sohngen units containing the same medium to test for methane con sumption.

Serological studies

For serological studies, cells of the methane oxidizers were grown in the Jay.dsuriya mineral salts solution in Sohngen units until gas con sumption ceased. The cultures were stored at 5 C until their^urity was determined. Cells of Hyphomicrobium vulgare were grown in Mevius' medium with methanol as carbon source. Agrobacterium tumefaciens and A. radiobacter were grown in nutrient broth (Difco). Rhlzobium leguminosarum was cultured in the medium recommended by the American

Type Culture Collection with the agar omitted. The cells were centri fuged, washed twice in physiological saline and resuspended in formolized saline.

Cell suspensions corresponding to the McFarland No. 3 standard were injected intraperitoneally into rabbits, 1 ml of antigen every two days for two weeks. Sera were collected by cardiac puncture seven days after the last injection.

Tube agglutination tests were incubated at 5 0 C in a water bath 36 for 24 hours; readings were made at 2 hours, 24 hours, and finally after an additional day of incubation at 5 C.

B. The Pink^Pigmented Methanol-Oxidizing Bacteria

Cultures

The sources of the pink-pigmented methanol-oxidizing bacteria isolated during this investigation are shown in Table 4.

Several other pink-pigmented methanol oxidizers which have been described in the literature were kindly supplied by other workers, as indicated in Table 5.

Media

The mineral salts solutions employed were the same as those for the studies with methane oxidizers, except that the medium of

Brown (1958) was used routinely (Table 3). The growth of the pink- pigmented methanol oxidizers was compared on the media of Mevius

(1953) and Jayasuriya (1955). The pH of all mineral salts media was

7.0. Sterilization, unless otherwise indicated, was accomplished in the autoclave at 121 C for 20 minutes. 37

Table 4. Sources of pink-pigmented methanol oxidizers.

Laboratory designation Source of isolation

AS-P Methane enrichment culture from oil field soil

PBG-P Methane enrichment culture from coal mine in West Germany

Me Contaminant in a culture of Methanomonas methanooxidans

DOR Methane enrichment culture from Dr. D. Holmes

Rumen Methane enrichment culture from the rumen of a fistulated cow

SEW Methane enrichment culture from sewage

S, T, U, V Contaminants of methane-oxidizing cultures 38

Tabie-S-,—Pink-pigmented methanol oxidizers obtained from other investigators.

Laboratory Name of organisms Reference desiq nation

KAL Pseudomonas methanica Harrington and Kallio, (1960)

VX Vibrio extorauens Peel and Quayle, (1961)

P. ruber Protaminobacter ruber Ibid. deh Dooren de Jong

AMI Pseudomonas AMI Ibid.

JT Pseudomonas methanica Johnson and Temple, (1962) 39

The following media were used for biochemical and physiological tests and were prepared as indicated.

Spirit Blue Agar. Modified (Starr. 1941)

Bacto-tryptone 10 g Bacto-yeast extract 5 g Bacto-agar 30 g 20% Wesson oil emulsion 25 ml 0.3% alcoholic solution of spirit blue (National Aniline) 50 ml Distilled water to make 1000 ml pH 6.9 ’«.» * Wesson oil emulsion was prepared by homogenizing thoroughly in a Waring Blendor: 100 ml of fresh Wesson oil, 10 g finely powdered gum arabic and 400 ml of warm, distilled water.

Carbohydrate Medium

Bacto-peptone 5 g Bacto-beef extract 3 g Sodium chloride 5' g Carbohydrate 10 g Andrade's indicator 10 ml Distilled water 1000 ml pH adjusted to 7 .0

DNase Test Acar (Difco #0632. Dehydrated)

Bacto-tryptose 20 g Desoxyribonucleic acid 2 g Sodium chloride 5 g Bacto-agar 15 g Distilled water 1000 ml pH 6.8

Urea Broth (Difco #B272. Dehydrated) Bacto-yeast extract 0.1 g Monopotassium phosphate 9.1 g Disodium phosphate 9.5 g Urea 20.0 g Phenol red 0.01 g Distilled water 1000 ml pH 6.8 40

The urea test medium was sterilized by filtration through a sterile Coor's No. 3 bacteriological filter and added to sterile screw-capped tubes. The tubes were incubated at 37 C for 24 hours prior to inocula tion to check for contamination.

MR-VP Medium (Difco #B 16, Dehydrated)

Buffered peptone 7 g Dextrose 5 g Dipotassium phosphate 5 g pH 6.9

Trvptone Broth (Difco #B 123, Dehydrated)

Bacto-tryptone 10 g Distilled water 1000 ml pH 7.2

Peptone Iron Agar (Difco #B 89, Dehydrated)

Bacto-peptone 15 g Proteose peptone, Difco ' 5 g Ferric ammonium citrate 0.5 g Dipotassium phosphate 1 g Sodium thiosulfate 0.08 g Bacto-agar 15 g Distilled water 1000 ml pH 6.7

Oxalate Agar (Bhat and Barker, 1948)

Sodium oxalate 2 g Ammonium sulfate 0.5 g Magnesium sulfate 0.1 g Ferrous sulfate 0.02 g Calcium sulfate 0.01 g Phenol red 0.03 ml Yeast extract 1 g Calcium chloride, 0.1 M 20 ml Distilled water 1000 ml pH 7.0

The calcium chloride was autoclaved separately and added to the medium prior to pouring the plates. Care was taken to disperse the medium evenly. 41

Morphological studies

Stains. The Gram reaction was determined according to

Hucker's modification (1922). To demonstrate capsules a method was employed which consisted essentially of making a negative stain according to Benians (Williams, 1959). The acid alcohol was saturated with acid fuchsin. The sudanophilic characteristic of the organisms was demonstrated by Burdon's method (1949). For the detection of spores the method of Schaeffer and Fulton was used (1933). Acid fastness was determined by the Ziehl-Neelsen method. Motility was observed with the light microscope using hanging drop mounts of liquid cultures. Flagella stains were made according to the method of Bailey

(1929) as modified by Fisher and Conn (1942).

Assay for polv-B-hydroxvbutvric acid

Poly-B-hydroxybutyric acid was extracted and assayed according to the method described by Law and Slepecky (1961).

Cells were grown in the mineral salts solution of Brown (1958).

Five-hundred ml of this medium, containing 0.5 per cent (v/v) methanol were dispensed in 2-liter flasks. The inoculum consisted of 5 ml of a heavy suspension of the organisms. The flasks were incubated at room temperature on a rotary shaker.

After five days of growth 100 ml of the cell suspension were removed, the cells harvested by centrifugation and washed twice in sterile phosphate buffer (pH 7.0). The cells were treated with 10 ml 42 portions of chloroform-methanol (1:1) until no more pigments could be extracted (Peel and Quayle# 1961).

For the extraction of the poly-B-hydroxybutyric acid the de- pigmented cells were centrifuged in polypropylene centrifuge tubes

(IvanSorvall# Inc.; Norwalk# Conn.; no. 254). Any plasticizers which adhered to the tubes were removed by washing with ethanol and hot chloroform prior to use. The cells were suspended in 20 ml of com mercial sodium hypochlorite solution and incubated for 4 hours at 37 C.

The lipid granules were sedimented by centrifugation# washed with distilled water and further washed with acetone and 95 per cent ethanol.

Finally# the poly-B-hydroxybutyric acid was extracted with three small portions of boiling chloroform. The extract was filtered and placed under a hood for evaporation of the chloroform. The residue was suspended in 5 ml of concentrated sulfuric acid and heated to boiling for 10 minutes in a water bath. This converted the poly-B-hydroxybutyric acid to crotonic acid. The solution was cooled# and after thorough mixing, a sample was measured at 300 to 200 m^i against a sulfuric acid blank.

For the spectrophotometric assay a Beckman DB Spectrophotometer was used.

Physiological and biochemical studies

Oxidase activity (Kovac# 1956). A 6 cm square piece of filter paper (Whatman No. 1) was placed in a petri dish. Two or three drops of a 1 per cent solution of tetramethyl-para-phenylenediamine- 43 dihydrochloride were placed in the center of the paper. Three day old colonies of the organisms which had grown on mineral salts-methanol agar were removed and placed on the filter paper. With this test a positive reaction is indicated by a dark purple to black color that develops in 5 to 10 seconds.

Catalase activity. The production of catalase was determined by flooding five day old cultures grown on mineral salts-methanol agar with three per cent hydrogen peroxide and observing for the evolution of oxygen.

Urease activity. Urease test medium was aseptically distributed in 2 ml amounts into sterile screw-capped tubes (13 x 125). The inocu lated tubes were observed for growth and color change at intervals for a period of three weeks.

Lipase activity. Fat hydrolysis was determined as suggested by

Starr (1941). Lipolysis was considered positive if a blue zone appeared around the colonies within three weeks.

Deoxyribonuclease activity. DNase test agar (Difco) was used according to the method described by Jeffries, Holtman and Guse

(1957). The plates were heavily inoculated in a streak across the center of the plate. After four days of incubation, the plates were flooded with IN HC1 solution. DNA hydrolysis was indicated by a clear zone around the streak.

Tyrosinase activity. Mineral salts agar plates supplemented 44 with 0.5 per cent 1-tyrosine were heavily inoculated in a streak.

Care was taken in the preparation of the plates to obtain uniform distribution of the tyrosine crystals. Tyrosine decomposition was recorded as positive if the crystals disappeared around the streak of growth.

Cellulase activity. The ability to decompose cellulose was determined by growing the cultures in mineral salts broth dispensed in screw-capped tubes (13 x 125) in which filter paper strips were placed before sterilization. Final observations for digestion of cellulose were made after incubation for 6 weeks.

Proteolytic activity. The ability to hydrolyze gelatin was tested in nutrient gelatin in tubes. Final observations were made after incubation for 3 weeks at room temperature.

To determine hydrolysis of casein a medium was prepared by mixing 500 ml of 3 per cent agar in water with 500 ml of 10 per cent

skim milk. Plates were poured and inoculated by streaking across the

center of the plate.

Proteolysis of milk was determined by growing the organisms in

skim milk. Additional information was obtained on the ability of the organism to induce pH changes by the incorporation of litmus into the

skim milk.

Diastatic activity. Petri plates prepared with nutrient agar

supplemented with 0.5 per cent starch were heavily inoculated by 45

making a streak across the agar. Starch was considered hydrolyzed if, at the end of seven days, a colorless zone appeared around the

colonies after the addition of Gram's iodine.

Paraffin utilization. The mineral salts medium of Brown (1958) was supplemented with approximately 1 per cent paraffin. Since the

paraffin solidified on the surface of the liquid mineral salts medium

upon cooling, making the medium anaerobic, it was broken up with a

sterile needle before inoculation. The tubes were incubated for 6 weeks

before making final readings. The presence of growth would indicate

utilization of paraffin.

Oxalate utilization. The oxalate agar of Bhat and Barker

(1948) was heavily streaked across the center of the plates. Incuba

tion was at room temperature. The ability to utilize oxalate was

indicated by a clear zone around the inoculum and a change of color

of the indicator from yellow to red.

Other biochemical tests. The tests for nitrate reduction, indole

production and the M. R.-V. P. tests were carried out according to

the Committee on Bacteriological Technic, Society of American

Bacteriologists (1957).

Carbon and nitrogen sources. The mineral salts solution of

Brown (1958) was used throughout this study. The procedures were

the same as described under the section entitled "The Methane-

Oxidizing Bacteria." The inocula consisted of methanol-grown cells which had been washed three times in sterile phosphate buffer

prior to use. The ability of the organisms to grow in the presence of the various nitrogen sources was carried out with 0.5 per cent

methanol as carbon source.

Serological studies

The techniques and procedures used in this study were the same

as those given under the section entitled "The Methane-Oxidizing

Bacteria," except that the cells were grown in 0.5 per cent mineral

salts-methanol medium. RESULTS AND DISCUSSION

A. The Methane-Oxidizing Bacteria

Enrichment and isolation of the methane oxidizers

When the fifth consecutive enrichment culture showed slight turbidity and was plated out in decimal dilutions, it was found that on TGE agar plates there were no colonies prepared with dilutions —8 —9 —10 10 , 10 and 10 . The lower dilutions showed many colonies, some of which were pink.

The mineral salts agar plates which were incubated under methane for two weeks had small (0.1 to 1.0 mm) pink colonies in the lower dilutions and no macroscopic growth on plates of dilutions

10-7 , 10~® and 10“^. When the plates were examined with the micro scope, the microcolonies described by Brown (1958) were observed.

The microcolonies were present in large numbers in the lower dilu tions , intermingled with the larger pink colonies (Fig. 6). To insure that a pure culture of the methane-oxidizing bacteria was obtained, a fine needle was used to transfer some of the microcolonies into

5 ml of sterile mineral salts medium in 125 ml bottles. The bottles were closed and the methane gas mixture was introduced according to the procedures described in the methods section. When turbidity developed (after 3 weeks), purity tests were carried out and after it

47 Figure 6. Pink-pigmented colonies with microcolony of the methane-oxidizing bacteria.

i 48

S'M 48 49 had been ascertained that no growth occurred on TGE# a Sohngen unit was inoculated with three ml of the culture. All of the isolates con sumed methane as indicated by the volume change that took place in the reactor .bottle of the Sohngen unit. All of the cultures obtained from these microcolony isolations appeared to be identical morpho logically and culturally.

i Using the isolation procedures described above, pure cultures of methane oxidizers were obtained from oil field soil, water from a coal mine in Bottrop, West Germany and from the rumen of a fistulated cow.

Studies with inhibitory agents. Brown (1958) described the diffi culties involved in isolating the methane oxidizers. This was mainly due to the slow growth of the organisms which gave faster growing contaminants ample opportunity to outgrow the methane oxidizers.

Furthermore, the formation of only microcolonies by these methane- oxidizing bacteria made it difficult to detect these organisms on solid substrates. The addition of organic adjuncts, the change of the in organic components of the media or a change in the composition of the gas mixture used did not seem to enhance the growth of the organisms.

According to Brown (1958), Holmes (1962) and investigations carried out in this study, more than four months are required to isolate a pure culture of the methane-oxidizing bacteria.

In an attempt to reduce the time required for the isolation of 50 these organisms, various antibiotics were tested for possible use in enrichment and plating media. The effects of these antibiotics were first determined in mineral salts broth on a pure culture of the methane oxidizers (strain Soil). The results are given in Table 6. Streptomycin in all concentrations inhibited the growth of the organisms. Chloram phenicol in concentrations up to 30pg/ml allowed growth of the bacteria, but coneentrations above pg/m6 lretarded growth as

measured by turbidimetric methods. Aureomycin in concentrations

above 6 pg/ml completely inhibited the organisms. Bacitracin in concentrations of 19.25 units/ml did not prevent growth.

To further study the effects of antibiotics on the methane oxidizers, 0.1 ml of a cell suspension was spread with a glass rod on mineral salts agar plates, and various sensitivity discs were placed on the plates. None of the antibiotics tested inhibited the growth of the methane oxidizers. Some of these antibiotics actually enhanced the growth of these bacteria. Increased growth in the form of a circle

around the sensitivity discs was observed with the following: dihy

drostreptomycin, 2 ^ig; penicillin, 10 units; Aureomycin, 5 jug;

carbomycin,2 pg; polymyxin B, 50 units; streptomycin, 10 ug and triple sulfa, 0.25 mg (Fig. 7). The bacteria developed evenly over the entire plate, but close to the discs a concentrated ring of organisms was observed, about 0.5 to 1.0 cm away from the discs.

t Table 6. The effect of various antibiotics on the growth of methane oxidizers.

Streptomycin Growth* Chloramphenicol Growth Aureomycin Growth Bacitracin Growth >ig/ml ug/ml >ig/ml units/m l

80 - 100 - 100 - 19.25 +

40 - 50 - 50 - 15.40 +

32 - 40 - 40 - 11.55 +

24 - 30 + 30 - 7.70 +

16 - 20 20 - 3.85 +

8 - 10 + 10 - 3.08 +

6.4 - 8 + 8 - 2.31 +

4.8 - • 6 ++ 6 + 1.54 +

3.2 - 4 ++ 4 ++ 0.77 +

1.6 - 2 ++ 2 ++

* no growth; +, moderate growth; ++, abundant growth. Figure 7. Mineral salts agar plates showing enhance ment of methane oxidizers around sensitivity discs. (Aureomycin, penicillin and streptomycin). 52 53

In view of these findings, methane enrichment cultures were started in Sohngen units. These units were inoculated with cell sus pensions prepared from a sample of soil obtained from the same area from which a methane-oxidizing culture had been isolated previously.

The Sohngen units contained the following concentrations of antibiotics:

Sohngen unit A Aureomycin, 6 pg/ml

Sohngen unit B Bacitracin, 19.25 units/ml

Sohngen unit C Chloramphenicol, 6 ^ig/ml

Control No antibiotics

All units were set up in triplicate and incubated on a shaker and rotated at approximately 180 rev/min. As soon as 75 ml of gas were consumed by a unit, plate counts were carried out on TGE in air to determine the number of contaminants in each unit. The plates were incubated for 72 hours. Results are shown in Table 7. The control unit which did not contain any inhibitory agent had a lower number of contaminants than the Sohngen units containing Aureomycin, bacitracin or.chloramphenicol. With regard to the antibiotics it is improbable that the organisms utilize them as substrates for energy and growth; how ever, it is possible that they acted as some sort of growth factor, or else in a detoxifying capacity.

Experiments were also conducted with antibiotic sensitivity discs. The discs were placed on mineral salts agar plates after 0.1 ml of various dilutions of enrichment cultures were spread over the Table 7. The effect of various antibiotics in suppressing the growth of contaminants in methane enrichments.

Sohngen unit Antibiotic Test Count/ml xlO® Mean

Aureomycin, 1 7.2 8 6 jig/m l 2 4.7 5.5 x 10 3 4.6

B Bacitracin, 1 5.6 19.25 units/ml 2 7.2 7.2 x 10 8 3 8.9

Chloramphenicol 1 8.7 6 jig/m l 2 4.5 6.4 x 10 8 3 5.9

Control No antibiotics 1 2.3 8 2 3.9 2.7 x 10 3 1.9

tn 55

surface. After incubation under methane for 10 days, there was no

evidence of inhibition of the contaminants by any of the antibiotics.

Isolation bv agglutination. The ultimate goal of an enrichment technique is to obtain the desired organisms in predominant numbers in a culture. It was shown by Brown (1958) however, that the methane oxidizers produce metabolic by-products capable of supporting rela tively large numbers of contaminants. Results obtained with anti biotics indicated that they increased the contaminants. It was then

decided to attempt separation of the methane-oxidizing bacteria by

specific serum agglutination.

A 10 ml portion w as removed from a completed Sohngen unit which was known to be contaminated with a pink-pigmented organism.

The sample was centrifuged and resuspended in saline to a volume

of 5 ml. A plate count on.TGE agar in air revealed 5 x 10^ organisms/ml.

Five-tenth ml of heat-inactivated antiserum for this particular methane

oxidizer (strain PBG) was added to the cell suspension. Following

agglutination in the cold, the suspension was shaken lightly and the

aggregates allowed to settle to the bottom of the tube. The super

natant was removed with a sterile pipette and the suspension was

made up to the original volume with sterile saline. This was re

peated five times. A plate count done on TGE incubated in air

revealed only 2.7 x 10® organisms/ml.'

The supernatant was removed again and sterile distilled water was added to the aggregates. The organisms were incubated at 37 C 56 for 12 hours, after which they were centrifuged, the supernatant dis carded, and fresh distilled water added to the aggregates. Before each centrifugation, the tubes were vigorously shaken. This was repeated three times at intervals of 12 hours and after the third time a marked decrease in the size of the aggregates was observed.

Complete dissociation of the aggregates was finally accomplished by adding acetate buffer (pH 4.0) to the suspension and incubating at

37 C for 5 hours. The cells were centrifuged, the buffer discarded and replaced by sterile saline. Serial dilutions of the suspension were streaked on mineral salts agar and the plates were incubated under methane. Upon observation with a microscope, the plates inoculated with the cell suspension of dilution 10”1 was completely overgrown by the pink contaminants, but only isolated colonies were observed on dilution plates 10"^ and 10"^. Plates streaked with higher dilutions were free of any contaminants. Characteristic microcolonies were observed on all of these plates, the number of colonies decreasing with higher dilutions. These colonies had the characteristic colonial morphology of the methane oxidizers and upon transfer into Sohngen units showed gas consumption. The bacteria from completed Sohngen units were tested for purity and were found free of contaminants.

Following this procedure and using antiserum prepared with strain RUM, isolates were obtained from a soil sample from beach sand (Gulfport, M iss.) and a sample from an oxidation pond (LSU 57

Agricultural Experiment Station, Baton Rouge, La.). While attempts to isolate methane oxidizers by the dilution to extinction method usually required 3 to 4 months, pure methane-oxidizing bacteria from these two samples were obtained in 4 weeks.

Discussion of enrichment and isolation procedures

According to previous workers, the isolation of methane-oxidizing bacteria has been very difficult. Hutton and ZoBell (1949) reported that from 82 colonies which they transferred from washed agar plates into methane only 2 were able to utilize this hydrocarbon. When colonies were transferred from silica gel plates, only 12 out of 68 were able to use methane. According to Brown (1958), it was Hutton's opinion that the difficulty in isolating methane oxidizers is due to their slow rate of growth.

Tortorich (1955) and Strawinski and Tortorich (1955) concluded that the clumping of the organisms in enrichment culture with entrap ment of contaminants was the reason for their early failure to isolate methane-oxidizing bacteria. To prevent the clump formation of these organisms they tried Tween 80 in their enrichment medium.

Dworkin and Foster (1956) investigated various growth factors as aids in the enrichment and isolation of Pseudomonas methanica.

They concluded that the presence of agar extract and calcium pantothenate enhanced the growth of P. methanica. A selective enrichment technique is absolutely necessary to carry out a dilution to extinction, since it is an indispensible feature of this method that the desired organisms be present in predominant numbers. The addition of such compounds as Tween 80, as suggested by Tortorich (1955), or agar extract and calcium pantothenate as re ported by Dworkin and Foster (1956) may upset this enrichment tech nique since these substances, (like the antibiotics tested) may enhance the growth of contaminants. The problem is further complicated by the fact that the methane oxidizers themselves produce substances capable of supporting relatively large numbers of contaminants (Brown, 1958;

Holmes, 1962).

Some earlier workers reported "methane oxidizing" bacteria without proof that their isolates actually consumed methane. Munz

(1915) considered as a criterion for methane consumption the increase in organic matter in culture media. Nechaeva (1949) apparently de termined the quantity of oxygen consumed and calculated the amount of methane utilized on the basis that two molecules of oxygen were required to oxidize one molecule of methane.

A generally accepted method for isolating a pure culture of a desired organism is to streak the surface of some solid substrate directly from an enrichment culture. However, as noted by Brown

(1958), classical examples of the necessity of deviating from this method are found in the isolation of the nitrifying bacteria and in the 59 original isolation of Thiobacillus thiooxidans. Brown (1958) likewise found it necessary to deviate from the usual methods. He found the inability of the methane oxidizers to form macrocolonies on the solid media prevented the use of the conventional streak plate method. He, therefore, utilized a dilution to extinction procedure. This method was also employed in this investigation.

An obvious advantage of the "isolation by agglutination" method described above is the more rapid elimination of most of the contaminating bacteria. However, this method can not be employed unless pure cul tures are available for the production of antiserum. After an initial isolation by the dilution to extinction method, the agglutination pro cedure could be employed to facilitate the isolation of more organisms of the same serotype.

Morphological characteristics

The isolates from coal mine water, rumen of a fistulated cow, and soil are morphologically indistinguishable from Methanomonas methanooxidans of Brown (1958). The organism is a non-sporeforming rod, 1.0 by 1.5 to 4.0^i, and motile by means of a single,polar flagellum (Fig. 8). It is Gram-negative, but stains unevenly because of vacuolation of the cells; it is not acid-fast. The frequent occurrence of vacuolated and club-shaped cells is characteristic of this organism.

The vacuoles do not take the usual stains and apparently are not fat bodies since they are not stained by Sudan black (Fig. 9). Some of Figure 8. Flagellated cell of Methanomonas methanooxldans. Fisher and Conn's modification of the Bailey stain. Magnification ca. 4000 X.

Figure 9. Vacuoles in cells of Methanomonas methanooxidans. Cells grown in mineral salts-methanol medium. Crystal violet stain. Magnification ca. 4000 X. 60

T 61 the cells are much enlarged at one end, causing the other end to

appear as a bud-like projection, suggestive of the genus Hvphomicrobium

(Fig. 10). However, the long, slender filaments characteristic of

Hvphomicrobium vulqare described by Kingma-Boltjes (1936) have not

been observed in these cultures.

Another striking morphological feature of these organisms is the

common occurrence of the cells in rosettes, resembling the spokes of

a wheel (Fig. 11). These structures are similar to those observed in

Agrobacterium. Phvllobacterium. Rhizobium and Chromobacterium by

various workers (Kndsel 1962, 1963). The characteristic of rosette

or star formation is most obvious when there are three to six radially

arranged cells, but the rosettes may attain sufficient size to cause

large aggregates, and the appearance of a fine-grained, sandy pre

cipitate in mineral salts methane broth. This sandy-grained pre

cipitate was also observed in liquid cultures of H. vulqare.

Cultural characteristics

The methane oxidizers develop only minute colonies under the

most favorable conditions thus far devised for their cultivation. On

mineral salts agar the colonies are about 0.05 to 0.1 mm in diameter

after incubation for three weeks. The size of the colonies is not

appreciably increased with longer incubation. Colonial development

and growth is not enhanced on silica gel plates. There is no pig

mentation of the colonies. Changes in the composition of the methane Figure 10 . Cells of Methanomonas methanooxidans with bud- like projections. Mineral salts agar under methane. Magnification ca. 3000 X. (Unstained)

Figure 1 . Rosette formation of Methanomonas methanooxidans. Mineral salts agar under methane. Magnification 1000 X. (Unstained) S f e .

^ * * r & • % s - • . m • ■'' * 1 1 • J ^ \> 63 gas mixture and the addition of various organic adjuncts (vitamins, amino acids, peptone, carbohydrates) do not appreciably increase colony size.

The colonies of the various strains on mineral salts agar under methane are indistinguishable; the margins are somewhat irregular and the structure is granular in appearance (Fig. 12). The colonies bear some resemblance to those of Hvphomicrobium vulqare, but the latter are considerably larger and have entire margins (Fig. 13). In liquid mineral salts methane medium under shake conditions the organisms produce good growth in 4 to 8 days with moderate turbidity and white sandy sediment. The pink flaky or flocculent growth re ported by Dworkin and Foster (1956) for P. methanica has not been observed with these cultures.

Physiological characteristics

All the strains isolated by the method described by Brown (1958) utilized methane as the sole carbon source. The two strains isolated by the method of agglutination-dilution to extinction utilized methane, were unable to grow on TGE in air, cross-reacted immunologically with antiserum prepared against strain RUM, and their morphological and cultural characteristics were identical to the above strains; however, physiological characteristics were not tested. None of the isolates consumed ethane, n-propane, n-butane, n-hexane, n-heptane or n-octane (Table 8). There was no growth when the organisms were Figure 12. Colonies of M_. methanooxidansgrowing on mineral salts agar under methane. Magnification ca. 450 X.

Figure 13. Colonies of Hvphomicrobium vulqare on mineral salts methanol agar. Magnification ca. 450 X.

Table 8. Utilization of hydrocarbons as sole carbon source by methane-oxidizing bacteria.

Substrate Brown PBG RUM Soil

Methane +++* +++ +++ +++

Ethane -- - - n-Propane - - -- n-Butane - - - - n-Hexane - - - n-H eptane - - -- n-O ctane - - --

* +++, abundant growth; no growth after 28 days of incubation. 66 supplied with a mixture of hydrogen and carbon dioxide, or hydrogen, carbon dioxide and oxygen.

Other compounds tested as carbon sources are listed in Table 9.

The organisms grew in mineral salts medium with methanol, but less abundantly than with methane, and no growth occurred in concentrations above 2 per cent.

Only Pseudomonas methanica. the methane-oxidizing organism isolated by Dworkin and Foster (1956) had similar characteristics with i regard to the utilization of carbon sources. The organism isolated by

Munz (1915), Bakterium methanicum. grew with acetate, butyrate, tartrate, glycerol, mannitol, glucose, sucrose and peptone, besides methane. The methane oxidizer reported by Tausz and Donath (1930) utilized hydrocarbons from methane to paraffin oil, hydrogen, and also propylene and butylene. Bukova et al. (1947) found that methane, pentane, hexane and heptane were decomposed by Methanomonas methanica. Hutton and ZoBell (1949) isolated organisms, some of which utilized only methane, and others utilized methane, ethane and propane. The organisms isolated by Nechaeva (1949) were capable of utilizing methane, propane and various other organic carbon sub strates . Mycobacterium methanicum utilized only methane and propane, while M. flavum var. methanicum was said to utilize methane, propane and heptane.

Various substances were tested to determine their availability 67

Substrates tested for their ability to support the growth of methane-oxidizing bacteria*.

Substrates Concentrations (%) Growth

Methanol 0.05 0.15 + Ethanol 0.05 0.15 - n-Propanol 0.05 0.15 - n-Butanol 0.05 0.15 - Formate 0.01 0.10 - Acetate 0.01 0.10 - Propionate 0.01 0.10 - Butyrate 0.01 0.10 - Xylose 0.10 1.00 - G lucose 0.10 1.00 - Lactose 0.10 1.00 - Sucrose 0.10 . 1.00 - M altose 0.10 1.00 - G lycerol 0.05 0.10 - Formaldehyde 0.001 0.10 - Methylamine 0.01 0.10 - Acetone 0.01 0.10 - Benzene 0.01 0.10 - Phenol 0.001 0.10 ~

♦Strains Brown, PBG, RUM and Soil. 68

as nitrogen sources for the methane-oxidizing bacteria. Results obtained with the various compounds are shown in Table 10. They

are in agreement with the nitrogen sources utilized by other methane- oxidizing cultures, as reported by earlier investigators. The nitrogen

sources utilized by the organism isolated by Munz (1915) were ammonium

salts, nitrates, peptone, leucine and asparagine. The organisms isolated from marine sediments by Hutton and ZoBell (1949) utilized

ammonium salts, nitrates and certain amino acids. The organisms

of Nechaeva (1949) were grown on ammonium nitrate or potassium nitrate, but were said not to reduce nitrate to nitrite. The nitrogen requirements

of Pseudomonas methanica Dworkin and Foster (1956) were satisfied by

ammonium salts and nitrates. Nitrite and asparagine could not be utilized.

All the organisms isolated in this study reduced nitrate to nitrite.

The effect of methanol on the growth of the methane oxidizers.

Methanol was found to be the only carbon source, besides methane, to support the growth of the organisms. Attempts were made to deter

mine how methanol would affect the growth of the organisms either when supplied as sole carbon source or in the presence of methane.

Ten ml of mineral salts medium were dispensed in a series of 125 ml

bottles. After sterilization and cooling, some bottles received 0.5

per cent methanol only, others 0.5 per cent methanol plus methane.

Control bottles contained only methane as carbon source or no carbon 69

Table_Krr~The availability of various nitrogen compounds for growth and methane consumption by methane oxidizers?

Nitrogen source Concentration (%) Growth Gas consumption

Ammonium sulfate 0.05 0.20 + + Ammonium chloride 0.05 0.20 + + Ammonium nitrate 0.05 + + Potassium nitrite 0.05 0.10 -- Potassium nitrate 0.05 0.20 + + Glycine 0.10 -- dl-Valine 0.10 - - DL-Alanine 0.10 -- L(-)Leucine 0.10 + + L-Glutamate 0.05 + + DL-Aspartate .0.05 0.20 - - L-Asparagine 0.05 0.20 - - - l(+)Arginine 0.05 0.20 + + DL-Methionine 0.05 0.20 -- L-Cysteine 0.10 + + L(-)Cystine 0.05 0.20 -- dl -Phenylalanine 0.05 0.20 - - Methylamine 0.05 0.20 - - Urea 0.01 0.05 0.20 - - Peptone 0.05 0.20 0.50 + +

*Strains Brown, PBG, RUM and Soil. 70 source at all. Inoculation, addition of the gas mixture where applicable, and incubation were carried out as described in the methods section. Readings were made every 3 days with a Spectronic

20 spectrophotometer (Bausch and Lomb) at a wave length of 560 mjj.

The instrument was standardized with mineral salts medium serving as a blank. Results are shown in Table 11 and Fig. 14.

It was already noted that the organisms grew less abundantly when methanol was supplied as only carbon source. The growth of the organisms appeared to be slightly accelerated when some methanol was present in the mineral salts methane medium. This may be due to the increased solubility of the methane gas in the presence of alcohol.

The effect of NaCl on the growth of the methane oxidizers.

Brown (1958) observed that the addition of 0.02 per cent NaCl to his mineral salts medium resulted in somewhat larger colonies. The medium routinely used in this investigation (Jayasuriya, 1955) did not contain any NaCl but the colonial development was apparently as good as on the medium of Brown. The medium of M^vius (1955) was unsatisfactory as a plating medium because of the presence of precipitates which made detection of microcolonies difficult. It was quite satisfactory as a liquid medium. • Since no studies were made by Brown as to the effect of cations on the growth of the organisms in liquid culture, tests were set up with and without NaCl, using Brown's medium.

Methane was used as the carbon source. Results are presented in

Table 12 and Fig, 15. 71

Table 11. The effect of methanol on the growth of the methane oxidizing bacteria.

Time in days Substrate* 0 3 6 9 12 15 18 21

Per cent transmittancy**

CH. 95 91 74 61 8 9 8 4 99 c h 4 + c h 3o h 99 94 91 28 22 14 8 8

CH3OH 99 97 93 93 89 50 51 50

* Three bottle cultures of each medium.

**Per cent transmittancy values are averages of three cultures. Figure 14. The effect of methanol on the growth of the methane-oxidizing bacteria. Data from Table 11.

• ------• CH3OH o —o c h 4 A------A CH3OH+CH4 * TRANSMITTANCY 60 Oo A o « o N o

S IS

"J N> 73

Table 12. The effect of sodium chloride on the growth of the methane oxidizers in Brown's medium.

Per cent transmittancy Time in days with 0.02% NaCl without NaCl

0 100 100

3 98 99

6 90 96

9 26 91

12 13 45

15 10 33

18 9 32

21 8 32 Figure 15. The effect of sodium chloride, on the growth of the methane oxidizers in Brown's liquid medium. Data from Table 12.

0*02 per cent NaCl no NaCl % % TRANSMITTANCY to 40 60 90 0 8 90 70 0 8 IE N DAYS IN TIME 75

Brown (1958) thought that water contained some toxic factor which was only partially removed by distillation, but was neutralized at least in part by the addition of 0.02 per cent NaCl. It was observed in this investigation that the absence of NaCl in liquid medium had an apparent effect on the growth of the organisms. However, substituting

Brown's medium with the medium of Jayasuriya, which contained 0.001 per cent CaCl2 / indicated that calcium chloride was as effective as sodium chloride in promoting growth in liquid cultures under methane.

Potassium chloride in concentrations of 0.02 per cent was unsatisfactory and apparently could not replace NaCl or CaC^ as a stimulating agent.

It is not known at the present time whether the enhancement of the growth of the methane-oxidizing bacteria in liquid culture by these salts was due to the chloride ions present, or was effected by the catio n s.

The effect of various dves on the growth of the methane oxidizers. Limited experiments were conducted with dyes in the hope that the enrichment medium for methane oxidizers could be made more effective. The ease of isolating these organisms would be greatly increased if the methane-oxidizing bacteria were stimulated or un affected by concentration of dyes which would inhibit the contaminants.

The results of these tests are presented in Table 13.

All the dyes enhanced the growth of the methane oxidizers in some dilutions. Congo red was markedly stimulating even in 0.1 per 76

Table 13. The effect of various dyes on the growth of a methane oxidizer (strain PBG).

Concentration Methylene Crystal Congo Neutral mg/ml blue violet red red

Gain in dry weight*

1 0.0 0.0 6.1 0.0

10"1 0.0 0.0 6.9 0.0 CM o 1 H 2.0 1.7 8.4 6.7 i CO i—* o 4.7 3.4 8.2 6.3

10“4 5.2 4.2 5.7 2.1

10"5 7.8 2.1 4.2 1.4

Control** 0.6 0.6 0.6 0.6

* Weight in mg after incubation for 5 days (average of three cultures).

**Culture of medium without dye. 77 cent concentration; other dyes inhibited growth0.01 at per cent concentration. The mechanism of the stimulation by the dyes is not understood. Additional studies need to be made with these substances.

Serological relationships

Serological studies revealed that the strain of Brown (1958) was antigenically related to the strain isolated from the water of a coal mine in West Germany and to the strain isolated from the rumen of a fistulated cow.

Interestingly enough, however, it showed no relationship to the

Soil strain, though both were isolated from soil in the same vicinity.

Since there are certain morphological and cultural similarities between the methane oxidizers and species of Agrobacterium, Rhizobium and

Hvphomicrobium. these cultures were tested in the antisera prepared with the methane oxidizers, and the methane-oxidizing bacteria were tested in sera prepared against the other species. No antigenic rela tionships were indicated between these species and the methane oxidizers (Table 14).

Discussion of results obtained with methane-oxidizing bacteria

The methane-oxidizing organisms studied in this investigation appear to differ in some important respects from all those previously reported. Table 14. Serological relationships among methane oxidizers and morphologically related organisms.

Antiaens Antiserum

Strain Brown PBG RUM SOIL A. turn. A .rad . H. vul.

Brown 160 160 160 -

PBG 320 320 320 ---

RUM 160 160 320 - - -

SOIL - - 320 - --

Aarobacterium tumefaciens - - 5120 640 -

Aarobacterium radiobacter - -- 640 5120 -

Rhizobium leauminosarum ------

HvDhomicrobium yulaare mm mm 640 79

The organism described by Sohngen (1906) produced a reddish- brown pellicle on liquid media under methane and grew on common media in the absence of methane. The methane oxidizers of Hutton and ZoBell (1949) and of some earlier workers (Munz, 1915;Ayer,

1920; Hasemann, 1927; Tausz and Donath, 1930) were inadequately described for detailed comparison with the isolates studied in this investigation, but none of them were reported to form only micro colonies and to form rosettes. Some of the methane-oxidizing bacteria described in the literature resembled those in this investigation in their dependency on methane and methanol. The organisms of Nechaeva

(1949), Mycobacterium methanicum and M. flavum var. methanicum were unlike the methane oxidizers described in this investigation in that they were non-motile, Gram-positive and utilized various carbon substrates in addition to methane. The formation of rosettes has not been reported in other methane oxidizers, except that Nechaeva (1949) noted rosette structure in colonies of Mycobacterium methanicum.

Rosette or star formation has been reported in all the genera of the family Rhizobiaceae Conn and also in the genera Pseudomonas and Xanthomonas of the Pseudomonadaceae Winslow et al. Morpho logically, the methane oxidizers studied in the present investigation are indistinguishable from Agrobacterium tumefaciens, A. radiobacter and A. stellulatum. The methane oxidizers resemble these species also in Gram reaction and in motility by means of one or possibly more 80 flagella. Culturally and physiologically, however, these organisms do not resemble any species of the family Rhizobiaceae. . Serological

studies did not reveal any antigenic relationship between species of

Aarobacterium. Rhlzoblum, Hvphomicroblum and the methane-oxidizing bacteria.

According to Dworkin and Foster (1956) the taxonomic status of the genus Methanomonas should undergo critical review. Creation of the genus Methanomonas,bv Orla-Jensen (1909) was part of his proposal to give physiological characteristics a dominant role in bacterial classi fication . Dworkin and Foster stated that this extreme position has never been accepted. However, Beroev’s Manual of Determinative

Bacteriology (Breed. Murray and Smith, 1957) lists many generic taxa based on physiology.

Methanomonas is now a recognized genus (Breed, Murray and

Smith, 1957). The organisms in this study, though apparently distinct from other methane oxidizers, are sufficiently like that of Sohngen to be considered a member of the same genus. The differences are great enough however, to justify a separate species.

B . The Pink-Pigmented Methanol Oxidizers

Isolation

Various soil and aquatic samples obtained from different locations

and enriched for methane-oxidizing bacteria invariably contained 81 pin-pigmented organisms. These organisms were usually abundant after the first or second enrichment, sometimes giving the medium a reddish-pink appearance. When the enrichment bottles were allowed to stand for awhile, a pink sediment was usually observed. In

stationary methane enrichments these organisms sometimes formed a pink or red pellicle on the surface of the medium.

In dilution to extinction experiments the mineral salts agar plates under methane and the TGE plates which had been incubated in air invariably contained pink-pigmented colonies. Sometimes colonies which were colorless on mineral salts medium under methane became pink when further incubated in the presence of methanol vapors.

Brown (1958) reported that various nonmethane-oxidizing bacteria were able to grow on the metabolic by-products of methane oxidation, and that the methane oxidizers frequently became entrapped in the slime about the cells of these organisms. This relationship makes it difficult to separate the two types of organisms, for colonies of the pink- pigmented bacteria may contain the methane oxidizers also (Fig. 16).

The inability of the methane oxidizers to grow on TGE in the absence of methane, while the pink-pigmented organisms grew slowly on this

medium, afforded one method for obtaining pure cultures of the latter, but not of the former.

To minimize the possibility that methane oxidizers were carried over, the pink organisms were transferred on TGE agar five times. Figure 16. A pink-pigmerited colony (lower) mixed with a methane-oxidizing colony (upper) on mineral salts agar under methane. Unstained. Magnification 1000 X. 82 83

The absence of methane-oxidizing organisms was finally determined by inoculating the pink-pigmented bacteria into Sohngen units under methane, and incubating them for6 weeks on a shaker. If no gas was consumed during this time the organisms were considered to be free of methane-oxidizing bacteria.

Characteristics of pink-pigmented methanol oxidizers

Morphological characteristics. Morphologically all the strains were similar, although variations in size were observed, ranging from

0.5 by 1.0 to 0.8 by 3.0 all were Gram-negative, non-sporeforming rods. Curved rods sometimes occurred in species normally composed of straight rods and the movement of motile cells in a culture was vibrio like in some cases but not in all. They were motile by means of a single polar flagellum (Fig. 17). No demonstrable capsule was ob served. All the strains studied in this investigation were subanophilic, as revealed by Burdon's fat stain (Fig. 18). Extraction of these fat bodies, carried out according to the method described by Law and

Slepecky (1961), demonstrated the presence of poly-B-hydroxybutyric acid. A characteristic peak was observed at 235 mji (Fig. 19).

Cultural characteristics. All strains produced coral pink to red colonies, with deeper pigmentation at the center of the colonies and increasing in intensity with age. On mineral salts methanol agar-7

colonies were small (about 0.2 mm after 5 days), circular, entire

and butyrous (Fig. 20). The addition of 0.1 per cent yeast extract Figure 17 . Electronmicrograph of flagellated cell of pink-pigmented methanol-oxidizing organism. Magnification 13,500 X. m Sm i Figure 18, Fat globules in cells of the pink-pigmented organisms as revealed by Sudan Black B. Burdon's method. Magnification 5000 X.

Figure 19. Absorption spectrum between 300 and 200 m^i of crotonic acid formed from the poly-B- hydroxybutyric acid extracted from all strains of the pink-pigmented methanol oxidizers. 86

0.4

0.1

300 80 60 40 20 200 WAVE LENGTH Figure 20. Colonies of pink-pigmented methanol oxidizers on mineral salts methanol agar. Magnification 10 X. 87

/ . ••.v#• • H• * • • •• •

•I 88

(Difco) did not enhance growth or colony size, but seemed to stimulate pigmentation of the organisms. In stationary cultures with methanol as carbon source a reddish-pink pellicle was observed (Fig . 21). A nutri tional dependency on yeast extract was not demonstrable; organisms have been propagated for almost 3 years on methanol agar slants without the addition of any organic nitrogen or ancillary growth factors.

Biochemical and phvsioloftical characteristics

The biochemical and various other tests carried out with the pink- pigmented organisms are indicated in Table 15.

All the pink-pigmented organisms studied were catalase positive and oxidase negative. Fat, tyrosine and urea were not hydrolyzed after

3 w eeks.

Deoxyribonucleic acid was not attacked by any of the organisms, and cellulose was not decomposed by any of the strains after6 weeks of incubation.

Gelatin and casein were not hydrolyzed. Litmus milk was

slightly alkaline after 3 weeks but no rennet curd was formed nor did peptonization occur.

Starch was not hydrolyzed by any of the strains.

No growth occurred in the mineral salts medium with paraffin as

sole carbon source during a 6 weeks period of incubation.

All strains grew well on oxalate agar prepared according to the

method of Bhat and Barker (1948). Growth of the organisms was Figure 21. Reddish-pink pellicles of pink-pigmented methanol oxidizers in mineral salts methanol medium under stationary incubation.

Table 15. Biochemical reactions of pink-pigmented methanol oxidizers.

Test AS-P PBG-P Me DOR JT RUMEN SEW S T xy V KAL VX P. rub. AMI

Casein Tyrosine Starch G elatin h 2s Indol Acetoin n o3 + + + • + + + + + + + + + + + + C ellulose DNase Urease C atalase + + + + + + + + + + + + + + + Lipase ------Oxidase Growth in Methanol 10% + + + + + + + + + + + + + + + NaCl 1% + + + + + + + + + + + + + + + 2% —

+ indicates growth or positive reaction; no visible growth or negative reaction. 91 indicated fry a clearing of the medium in the vicinity of the Inoculum and a change of color of the indicator (phenol red) from light yellow to red (Fig. 22).

Nitrate was reduced to nitrite during seven days of incubation.

The tests for indole production, methyl red reaction, and < - the formation of acetylmethylacrbinol were negative after one week of incubation. No hydrogen sulfide was produced after incubation in peptone-iron agar for 10 days.

No acid or gas was produced from glucose, sucrose, lactose, arabinose, maltose, xylose, glycerol, sorbitol, mannitol, inositol and inulin.

The growth of all strains was inhibited by 2 per cent but not by

1 per cent concentration of NaCl.

All the strains grew in mineral salts broth with 10 per cent methanol but growth was delayed in concentrations of methanol higher than 1 per cent.

None of the strains consumed methane, ethane, n-propane or n-butane.

The results of tests with various other carbon sources are shown in Table 16. All the strains showed abundant growth in mineral salts broth with methanol or glycerol as the sole source of carbon.

Protaminobacter ruber grew moderately in formate, while all the other strains grew profusely with this substrate. It was a characteristic of Figure 22. Pink-pigmerited methanol-oxidizing bacteria on mineral salts agar plates with sodium oxalate as sole carbon source. Upper row, left to right; V. extorauens. P. ruber. P. AMI; lower row, left to right; JP* methanica (strain KAL), strain PBG-P, P. methanica (strain JT). 92 Table 16. Growth of pink-pigmented methanol oxidizers on various carbon substrates.

Substrate AS-P PBG-P Me DOR JT RUMEN SEW S U V KAL VX P. rub. AMI n-Hexane - - - - + ------n-Heptane + n-Octane Benzene + + + + + + + + + + + + + + + Toluene Paraffin Methanol ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++• ++ ++ Ethanol + + + + + + + + + + + + + . - + Propanol + - + + + + + + + + + + + - + Butanol + - - - + + + + + + - - + + + Formate ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + ++ Acetate + + -+ + + + + + + + + + Propionate - + + - Butyrate I + + Lactate Succinate + + + + + + + + + + + + + + + C itrate + + + + -- - - - ' - - - + + + Fumarate + + + + + + + + + + + + + + + Glycerol •H- ++ ++ ++ -H- -H- -H- ++ ++ ++ ++ ++ ++ ++ ++ Sorbitol •* Mannitol Inositol G lucose G alactose

<£> CO Table 16 (Continued)

Substrate AS-P PBG-P Me DOR JT RUMEN SEW ST u V KAL vx P. rub. AMI

Mannose . Fructose + -- - ++ ++ ++ ++ -H- ++ ++ ++ + + + Sucrose ------Lactose M altose Ribose + + + + + + + - + + + + + - + Sorbose Xylose + - + Glycine ------_ . . • • Alanine ------+ - - + - - - Valine - -- + -- - - / -- + --- Leucine - - -• ---- r- -- + - - - Aspartic acid + + + + + + + + T+ - - + + ' + + Asparagine + + + + + + + + - -- + + + + Methionine - Cysteine Methylamine + + + + . + + + + " ""

++ indicates good growth; +, slight growth; no .growth. 95 all the strains that growth was delayed in a solution containing peptone in a concentration below 0.5 per cent as only carbon source. Higher concentrations of peptone allowed good growth. All the strains grew moderately well with benzene as sole carbon source.____

All the strains grew with peptone, ammonium salts, nitrate, alanine, asparagine, aspartic acid or leucine as nitrogen source.

Other organic nitrogen compounds afforded growth of some strains

(Table 17). It is of interest to note that Pseudomonas methanica of

Dworkin and Foster (1956) did not grow when asparagine was supplied as the only nitrogen source.

Studies with antibiotics

In procedures for the isolation of methane oxidizers it was noted that the pink-pigmented organisms invariably appeared as contaminants.

The effect of various antibiotics on the growth of the pink-pigmented bacteria was determined in the hope that the antibiotics would prevent their growth in methane enrichment cultures. If successful, this would greatly simplify the isolation of Methanomonas methanooxidans. The results of these tests are given in Table 18. All concentrations of streptomycin, bacitracin and chloramphenicol were ineffective in inhibiting the growth of the organisms. There seemed to be some inhibition with streptomycin in all concentrations used during the first

4 days of incubation, but resistant organisms always developed after this time and abundant growth was observed after 14 days. The Table 17. Availability of various nitrogen sources for growth of methanol oxidizers

N sources AS-P PBG-P Me DOR JT RUMEN SEW S T UV KAL VX P . rub. AMI

(n h4 )2s o 4 + + + + + + + + + + + + + + + n h 4c i + + + + + + + + + + + + + + + n h4 n o3 + + + + + + + + + + + + +. + + k n o 2 KNO3 + + + + + + + + + + + + + + + Glycine + + + + + + + + + + + + + + ' + Valine + + + + + + + + + + + + + + + Alanine + + + + + + • + + + + + + Asparagine + + + + + + + + + + + + + + + Aspartic acid + + + + • + + + + + + + + + + + I ------Phenylalanine ■ - - + + + . + - - Leucine + + + + + + + + . + + + + + + + Methionine - - + ------. ■ - -- C ysteine + Cystine -- -- + + + ------Hippurate + + + + + + + + + + + + + + + Uric acid + + + + + + + + + + + + + + + Methylamine

Urea + + + + + + , + + + + + + +

+ , growth; no growth

CO cn Table 18. The effect of various antibiotics on the growth of methanol oxidizers.

Streptomycin Growth* Chloramphenicol Growth Aureomycin Growth BacitraOin Growth /ig/m l /ig/m l /ig /m l u n its / ml

80 + 100 + 100 - 19.25 . +++

40 + 50 + 50 - 15.40 +++

32 + 40 + 40 - 11.55 +++

24 + 30 + 30 7.70 +++

16 + 20 •H- 20 - 3.85 -H-+

8 + 10 -H- 10 - 3.08 +++

6.4 + 8 ++ 8 - 2.31 +++

4.8 + 6 ++ 6 - 1.54 +++

3.2 + 4 ++ 4 + 0.77 +++

1.6 + 2 ++ 2 +

♦Results recorded 5 days after incubation. 98 organisms were more sensitive to the action of Aureomycin; no growth

occurred in 10 days with6 jig/ml of this antibiotic. However, after

longer incubation growth occurred in all concentrations of the antibiotic

below 20 jag/ml. Concentrations of Aureomycin greater than 20 fig/ml

completely inhibited the organisms even after incubation for 4 weeks.

Tests were also carried out with antibiotic sensitivity discs

on mineral salts agar plates with cultures spread over the agar surface. « Of the antibiotics tested, only streptomycin pg),(10 Aureomycin (5pq)

and tetracycline pg) (5 inhibited the growth of the organisms (Fig. 23).

According to this experiment it can be concluded that attempts

to facilitate the isolation of the methane-oxidizing bacteria by in

hibiting the growth of the pink-pigmented methanol oxidizers with

various antibiotics were without success. The two species are

either sensitive to the same antibiotics or the methanol oxidizers

develop resistant strains which invariably outgrow the slow growing

methane oxidizers.

Serological studies

The serological studies indicated that the strains of pink-

pigmented methanol oxidizers are not antigenically homogeneous

(Table 19). However. Vibrio extorauens was agglutinated at low titer

with antiserum prepared against strain AS-P, and Protaminobacter ruber

was agglutinated by antisera for strain PBG-P and strain S. There was

no agglutination when any of the pink-pigmented methanol oxidizers Figure 23. The effect of sensitivity discs on the growth of the pink-pigmented methanol oxidizers on mineral salts agar. Shown in photograph are: streptomycin, 10jag; tetracycline, 5 /ig; bacitracin, 2 units; penicillin, 10 u n its.

100

Table 19. Serological relationships among pink-pigmented methanol oxidizers.

Antigens Anti serum______

Strain______AS-P PBG-P______S______T______KAL

AS-P 640. -

PBG-P - 5120 - - -

Me 320

S - - 5120 - 80

T - 5120 80

KAL - 320 - 1280

VX 160

P. rub. - 20 1280

AMI - - - - 101 were tested in antiserum for Methanomonas methanooxidans strains

Brown, PBG, RUM and SOIL.

"Discussion of the pink-pigmented methanol-oxidizing bacteria . __

All the pink-pigmented methanol oxidizers were readily isolated

on conventional peptone agar, though growth was slow. In peptone

broth at concentrations below 0.5 per cent, growth was greatly

delayed. This is in agreement with the findings of Bassalik (1913)

who reported the slow growth of Bacillus extorauens on ordinary

nutrient agar and gelatin. Den Dooren de Jong (1927) reported that

one of the characteristics of his pink-pigmented organisms was the

slow growth on peptone agar. Harrington and Kallio (1960) reported

that their organisms did not utilize peptone as the only carbon source.

Of the few compounds tested by these investigators, only methanol

was utilized as carbon source. Since their organism was similar

-in morphological and cultural characteristics to Pseudomonas

methanica of Dworkin and Foster (1956), they called their organism

P.. methanica. although it was not able to utilize methane. The

organism of Johnson and Temple (1962) was reported as being able

to utilize methane and methanol, but no other conventional carbon

source. The organism used in the present study, designated JT,

was isolated from the mixed culture obtained from Mr. Johnson;

it does not utilize methane. 102

The pink-pigmented methanol-oxidizing bacteria included in this investigation comprised a group of organisms similar in morpho

logical, cultural and physiological characteristics. All were Gram-

negative7~motile^non-s poref orming rods. Cells of all the strains

contained poly-B-hydroxybutyric acid. This is in agreement with the

findings by Kallio and Harrington (1960) and Peel and Quayle (1961)

for their cultures.

Growth of these bacteria in liquid media under stationary con

ditions usually resulted in the formation of a pink pellicle; under

shake conditions the media sometimes showed pink flocculent growth,

but at other times the growth was homogeneous. Why the growth was

not always the same in appearance is not known, but may be related

to conditions which influence slime production by the organisms.

Dworkin and Foster (1956) concluded that cultures containing a large

enough amount of slime assumed a turbid, viscous character, while

those forming only enough slime to coat the cells resulted in clumped

or agglutinated growth.

It is interesting to note that all the strains tested utilized

oxalate, glycerol, methanol, formate and fumarate, as did the Gram-

negative, pink-pigmented organism described by Bassalik (1913),

which was isolated on a calcium oxalate medium. The similarity of

the pink-pigmented organisms is revealed also in their resistance to

antibiotics. 103

Serological studies indicated that there are some antigenic rela tionships among the pink-pigmented methanol oxidizers. Not all the strains# however# were found to be antigenically related. With regard to these differences It should be considered that antigenic differences among members of a species often occur# as in Escherichia coll.

To be distinguished from the methanol oxidizers employed in this study# which do not utilize methane# but merely grow in the favor able environment created by the oxidation of methane# is the bacterium described by Dworkin and Foster (1956) and Leadbetter and Foster

(1958# 1960) as pink-pigmented and methane dependent. This organism has not been encountered during these studies. Also the Pseudomonas

methanica of Dworkin and Foster# which oxidizes methane# should not be confused with Pseudomonas methanica of Harrington and Kallio (1960)# which is a typical pink-pigmented methanol oxidizer# as shown in this investigation.

The large number of pink-pigmented bacteria isolated from methane

oxidizing enrichment cultures by Brown (1958) and Holmes (1962) were

no doubt methanol oxidizers# though they were not tested on this sub

strate. Brown (1958) observed that contaminants readily grew in large

numbers on the by-products formed during the oxidation of methane.

Probably some of these contaminants were methanol oxidizers.

The pink-pigmented methanol oxidizers included in this study#

and Pseudomonas PRL-W4 of Kaneda and Roxburgh (1959) are sufficiently 104 alike to be considered one species. The first published description of an organism of this group was apparently that of Bacillus extorquens by Bassalik (1913). This species was placed in the genus Pseudomonas

Migula by Janota (1950), and in the genus Vibrio Muller by Bhat and

Barker (1948). According to Beroev's Manual of Determinative

Bacteriology (Breed. Murray and Smith, 1957) the borderline between the genera Pseudomonas and Vibrio is not sharp, since curved rods sometimes occur in species which are normally straight rods. The pink- pigmented methanol oxidizers are not clearly vibrios, though some curved rods are observed. The movement of motile cells in a culture was vibrio like in some cases but not in all. Although these organisms are not typically vibrios, it is suggested they be considered strains of Vibrio extorguens Bassalik (Breed, Murray and Smith, 1957). SUMMARY

The morphological, cultural, physiological and immunological

- characteristics of methane-oxidizing bacteria isolated from coal mine

water, rumen of a fistulated cow and oil field soil were compared with

an isolate of Methanomonas methanooxidans of Brown and Strawinski.

The investigation also included the identification of pink-pigmented

bacteria constantly found in the enrichment cultures of methane-

oxidizing bacteria.

Initial enrichments were carried out in Sohngen units to confirm

the presence of methane-oxidizing bacteria by consumption of methane.

Isolations were made on mineral salts agar plates under methane,

employing surface-streaked plates and the dilution to extinction pro

cedure .

Methane-oxidizing isolates are apparently identical in morpho

logical and physiological characteristics with Methanomonas methano

oxidans Brown and Strawinski. They are Gram-negative , monotrichously

flagellated, non-sporeforming rods. Cells are highly vacuolated and

some are much enlarged at one end, causing the other end to appear as

a bud-like projection suggestive of the genus Hvphomicrobium. Another

striking feature is the common occurrence of rosettes similar to those

observed in Aqrobacterium, Phyllobacterium, Chromobacterium and

Rhizobium. 106

The organisms developed only minute colonies under the most

favorable conditions. On mineral salts agar under methane, the

.colonies were about 0.05 to 0.1 mm in diameter after 3 weeks of

incubation. There was no discemable pigmentation of the colonies.

All the strains utilized methane as the sole carbon source; ethane,

propane and butane were not utilized. Methanol was the only carbon

source other than methane which permitted growth of the cultures.

Nitrogen requirements were satisfied by either nitrates,

ammonium salts, peptone or certain amino acids.

Methanol, sodium chloride, calcium chloride and certain dyes

enhanced the growth of the organisms in the mineral salts media of

Brown (1958) and Jayasuriya (1953) with methane as chief carbon source.

Serologically, strains PBG and RUM were related to strain Brown;

the strain isolated from soil did not reveal any antigenic relationship

with other strains.

Pink-pigmented bacteria observed in methane enrichment cul

tures were readily isolated on conventional peptone agar and on

mineral salts agar. In peptone broth at concentrations below 0.5

per cent growth was greatly delayed.

All the pink-pigmented isolates |||e Gram-negative, polar-

flagellated, non-sporefprming rods. Cells of all the strains contain

poly-B-hydroxybutyric acid.

All the strains produced coral pink to red colonies; pigmentation

was greater in the presence of methanol and increased with prolonged 107

incubation. On mineral salts methanol agar, colonies were small

(about 0.2 mm after 5 days), circular, entire and butyrous. There was no dependency for any growth factor. All the strains were

catalase positive, reduced nitrate to nitrite, grew in10 per cent

methanol and in the presence of 1 per cent sodium chloride. All the

isolates grew on oxalate agar and in mineral salts broth with either

methanol, glycerol, formate, fumarate, succinate, or benzene. i Methane, ethane, propane and butane were not utilized by any of

the strains. Some of the strains utilized fructose as sole carbon

source, ribose supported growth of others. Glucose was not utilized

by any of the strains. Other carbon sources were attacked by some

strain s.

Some antigenic relationships were found among the pink-

pigmented methanol oxidizers; no serological relationship was

detected between strains of the pink-pigmented organisms and the

methane-oxidizing bacteria.

The pink-pigmented methanol oxidizers isolated in this stydy,

and the previously described organisms. Pseudomonas AMI, P..

methanica (strain KAL), Protaminobacter ruber den Dooren de Jong,

are closely related and are sufficiently like Vibrio extorquens

(Bassalik) Bhat and Barker (1948) to be considered strains of that

sp ecies. LITERATURE CITED

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Peter Konrad Stocks was born December 3, 1927 in Bottrop, W est-

Germariy. From January, 1944 to May, 1945 he served in the German navy and was a prisoner of war until September, 1945 . He graduated from the Oberschule fur Jungen in 1949. In 1954 he emigrated to the

United States of America where he married Margie Louise Hinton in

1957. They have one son.

In 1959 he received his bachelor of Science degree from Millsaps

College, Jackson, Mississippi. He received his Master of Science degree in 1961 from the University of Southern Mississippi. Stocks is a member of the American Society for Microbiology and the Society of

Sigma Xi. He is a candidate for the degree of Doctor of Philosophy in

Bacteriology at Louisiana State University in Baton Rouge, Louisiana.

113 EXAMINATION AND THESIS REPORT

Candidate: Peter Konrad Stocks

Major Field: Bacteriology

Title of Thesis: A Study of Methane- and Methanol-Oxidizing Bacteria.

Approved: I VteQloJ* Major Professor and Chairmanr

Dean of the Graduate School

EXAMINING COMMITTEE:

- f t «

Date of Examination:

July 28, 1964