This dissertation has been microfilmed exactly as received 67-2418 BRAIDS, Olin Capron, 1938- A STUDY OF THE COMPONENTS OF THE LIPID FRACTION OF RIFLE PEAT. The Ohio State University, Ph.D., 1966 Agriculture, soil science

University Microfilms, Inc., Ann Arbor, Michigan A STUDY OF THE COMPONENTS OF THE LIPID FRACTION OF RIFLE PEAT

DISSERTATION presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio S tate U niversity

Rjr Olln Capron Braids, B. A., M« S.

*******

The Ohio State University 1966

Approved by

Adviser Department of Agronoay ACKNOWLEDGMENTS

The a"th o r i s indebted to P ro fesso rs Frank L. Himes and Robert

H. M iller, The Ohio State University, for their guidance, encouragement and help with this research. He is also appreciative of the assistance given at various times by other professors and graduate students in the

Department of Agronomy. Finally, he wishes to thank his wife, Elaine, for her patience and encouragement throughout the course of this pro­ j e c t .

i i VITA

A pril 29s 1938 Born, Providence, Rhode Island i 960 ..... B. A ., U n iv ersity o f Mew Hampshire, Durham, New Hampshire

1960-1963. . . Research Assistant, Department of Agronomy, Uni­ v e r s ity o f New Hampshire, Durham, New Hampshire

1963 ..... M. S., University of New Hampshire, Durham, Mew Hampshire

I 963-I 966. . . Research Assistant, Department of Agronomy, The Ohio Agricultural Research and Development Center, Columbus, Ohio

PUBLICATIONS

"F ats, Waxes and Resins in S o il," Robert H. M iller and O lin C. B raid s, Chapter 6, Handbuch der Bodenkunde, Springer-Verlag, Berlin, in press.

FIELDS OF STUDY

Major Field; Agronomy - Soil Organic Matter Chemistry

Studies in Soil Chemistry. Professors E. 0. McLean and F. L. Himes

Studies in Soil Microbiology, professor R. H. Miller

Studies in Statistical Analysis. Professor D. A. Ray

Studies in Chemistry. Professors M. L. Wolfrom, D. H. Busch, S. G. Shore, M. S. Newman, L. A. Paquette, and H. A. Schechter

i i i CONTENTS

Page

ACKNOWLEDGMENTS...... '**■

VITA ...... ^

TABLES ...... y i

ILLUSTRATIONS...... v i i

INTROLTICTION ...... 1

LITERATURE REVIEW...... 3

Introduction O rigin and decom position o f s o il lip id s O rigin Decomposition E x tractio n Components

MATERIALS AND METHODS...... 20

Incubation of soil columns Proximate analysis Two percent hydrochloric acid extraction Eighty percent sulfuric acid extraction Total and radiocarbon analysis Peat fat and wax extractions Thin layer chromatography Preparation of plates Spotting and developing Spraying Recovery Methylation with diazomethane Methylation with methanol and acid catalyst Gas-liquid chromatography Instrumentation Column p rep aratio n Sample preparation and introduction Sample collection Infrared analysis Ultraviolet analysis

iv Fage

RESULTS AND DISCUSSION...... 2.1

Preliminary experiments Experiments with C-14- labelled compounds

SUMMARY AND CONCLUSIONS...... 7M

BIBLIOGRAPHY...... 7.6

v TABLES

Table Page

1. Comparison of some physical and chemical properties of various peat waxes ...... 13

2. X-ray diffraction d-spacings for carbazole and two cryst­ alline materials extracted from Rifle peat ...... ^0

3. Carbon dioxide and radiocarbon loss from soils incubated after treatment with C-1A labelled fatty acid and glyceride 53

Yields for the benzene-methanol extract of the 0-2 inch lay layer of Rifle peat from incubated soil columns...... 58

5. Radioactivity data for benzene-methanol extracts from soil columns incubated with C-1A labelled palmitic acid and glyceryl tripalm itate...... 59

6. Radioactivity data for materials separated by TLC...... 81

7. Retention times in seconds for separated components of the crude extracts of incubated Rifle peat. Column temperature 295° C isothermal ...... 33

8. Retention times and activities for compounds separated by GLC from methylated extracts of incubated peat. Column temperature 295° C iso th erm al...... 85

9. Sample numbers, calculated carbon numbers, and retention times for samples collected for infrared analysis from the gas chromatograph...... 86

10. Total carbon expressed as percent organic carbon for Rifle peat after extraction with benzene-methanol, 2$ hydrochlor­ ic a c id , and 80$ sulfuric acid ...... ' ...... 72

11. Radiocarbon content of Rifle peat after extraction with benzene-methanol, 2$ hydrochloric acid, and 80$ s u lfu ric acid expressed as percent of C-l^ added...... '73

v i ILLUSTRATIONS

Figure Page

1. Solvent fractionation flow diagram for a benzene-methanol extract of Rifle peat...... * . 2k

2. Infrared spectra of Fraction I (top) and Fraction III (bottom). See Fig. 1...... 33

3. Infrared spectra of combined Fractions II and IV (top, .spe Fig. 1) and fatty acid mixture primarily octacosanoic a c i d ...... 3k

k . Infrared spectra of compounds separated by TLCi Top — material with an Rf of 0.97 in PE:ether from plate 2. Bottom — material with an Rf of 0.99 in pyridine:acetone from plate 8 ...... 36

5. Infrared spectra of carbazole extracted from Rifle peat (top) arid pure carbazole (bottom)...... k2

6. Ultraviolet spectra of carbazole extracted from peat (top) and pure carbazole (bottom) ...... k3

7. Infrared spectra of behenic acid (top) and methyl stear­ ate (bottom)...... k k

8. Infrared spectra of materials separated by TLC. Top — material with an Rf of 0.95 in methanol:pyridine from plate 10. Bottom — material with an Rf of 0.99 in ether from plate 9 ...... k6

9. Infrared spectra of compounds separated by TLC. Top — material with an Rf of 0.25 in pyridine:methanol from plate 10. Bottom — material with an Rf of 0.25 in ether: cello solve from plate11 ...... k8

10. Infrared spectrum of dioctylphthalate extracted from Tygon t u b i n g...... 50

11. Carbon dioxide and radiocarbon loss from incubated soil column no. 1 ...... 52

v i i Figure Page

12. Carbon dioxide and radiocarbon loss from incubated soil column no. 2 ...... 53

'3 . Carbon dioxide and radiocarbon loss from incubated soil column no. 4 ...... 54

14. Carbon dioxide loss from soil column no. 7...... 55 15. Infrared spectra of gas chromatographic fractions 5 and 12 (top) and gas chromatographic fractions6 and 13 (b o tto m )...... 68

16. Infrared spectra of gas chromatographic fractions 7 and 14 (top) and 8 and 15 ( b o tto m ) ...... 69

v i i i INTRODUCTION

Although soil organic matter constitutes only a small percentage of most soils, the influence it exerts on the physico-chemical proper­ ties of soil is disproportionately great. Workers over the years in this laboratory and elsewhere have investigated humic acids, polysac­ charides, and phenolic compounds and derivatives in order to gain more insight into the composition of soil organic matter.

With the increasing use of organic soils for commercial farming, the problems of peat and muck management received the attention of soil organic matter chemists. One particular problem which has been investi­ gated is that of subsidence. Drainage of muck soils brings about more rapid oxidation and humification reactions which lead to loss of soil and a re s u ltin g economic lo s s .

Stotzky (1956) and Hayes (i 960) utilized radioactive plant tis­ sue, labelled with C ^, in incubation experiments to follow the decompo­ sition pathways of added plant tissue in muck soils. In the earlier study, it was concluded that under laboratory conditions the organic matter content of Rifle peat could be increased with addition of plant tissue. Hayes measured the rate of decomposition of added rye tissue and loss of organic matter in drainage waters as part of the assessment of subsidence rate in Rifle peat.

The incubated soils of Hayes were extracted (Chahal, 19&3) with various reagents to bring the organic matter into solution for further fractionation. The greatest amount of radioactivity was found in the lipid (benzene-methanol extractable) fraction which contained 40.3 per­ cent of the residual

Since Chahal*s data showed that a significant part of the added plant tissue upon decomposition constitutes the lipid fraction of soil organic matter, the present study of this fraction was initiated. The objectives of this investigation were the identification of components of soil lipids and determination of the path of decomposition of lipid compounds added to th e s o il. LITERATURE REVIEW

Introduction

Soil fats, waxes and resins (often called bitumens or simply lip­ ids) are probably the least studied of the components of soil organic matter. Soil organic matter chemists have largely ignored these mate­ rials in preference to studies on the true humic materials. This neg­ lect is probably due to the fact that bitumens comprise but a small part of the total organic matter of mineral soils (1-5$)* However, in organic soils the values may be as high as 10 - 20$.

Knowledge about soil lipids has been derived from a number of specific unrelated interests. The possible commercial use of peat wax as a substitute for Montanwax (a commercially valuable wax obtained from lignite) has been the impetus for much of the research. Addi­ tional research has been done by coal and petroleum chemists interested in peat as a precursor of coal and petroleum. Peat wax also has been used in the Soviet Union as a mold material for molten metals.

Except for the investigations of Schreiner, Shorey and associ­ ates of organic substances in soils which might affect soil fertility,

agronomic interest in lipids as soil components has not developed exten­

sively. Since their studies, which included numerous reports on the

isolation and identification of fats, waxes and resinous substances from

soil, relatively few studies have been conducted on the effect of these materials on the nature and properties of soils. Occasional reports in the literature since that tim , however, implicate these hydrophobic materials as influencing soil behavior in a number of interesting ways.

Bitumens form a convenient analytical group rather than a struc­

tural group having only the common property of being variably, soluble in numerous organic solvents (e.g. benzene, methanol, ethanol, ether, petro­

leum ether, acetone, chloroform, gasoline etc.) or mixtures of these

so lv en ts. The r e s in s are more p o lar than f a ts and waxes and th u s show

greater solubility in methanol or ethanol, a property often used to

separate the groups. Structurally these lipid materials are very diverse, ranging from the relatively simple materials, such as fatty

acids and glycerol, to complex compounds such as chlorophyll, complex

terpenes, sterols, and polynuclear hydrocarbons.

Widespread usage of pesticides and the extensive urbanization of large areas of the world within the past decade have caused extensive

casual contamination of soils with hydrocarbons and other chemical com­

pounds extractable with organic solvents. Future chemical investiga­

tions of soil lipids must carefully consider the possible unnatural

origin of chemical compounds which would appear as components of the

soil lipid fraction.

Origin and decomposition of soil lipids

Origin. Soil lipid materials accumulate from two sources. One

is the decomposition products of plant tissue and the other the meta­

bolic products and tissue residues of soil microorganisms. Of the two,

the plant material provides the greater part of the soil lipids.

Plant residues contain both saponifiable and unsaponifiable fats

and waxes. The saponifiable fraction includes fatty acids and fatty acid esters including triglycerides, phospholipids, and glycolipids.

The unsaponif ia b le m a te ria ls include long chain alco h o ls and .hydro*- carbons, terpenes, sterols, quinones, and phloroglucinol derivatives

(Robinson, 1963)* Many of the sterols and terpenoid alcohols exist in the plant as the aglycon units of glycosides or as esters of fatty acids. Plant resins containing triterpenoid acids are frequently asso­ ciated with polysaccharide gums in gum resins.

Compounds which have been id e n tifie d and l i s t e d in ''Components''

(page lif ) could conceivably all be derived from plant material unmodi­ fied or slightly modified by microorganisms. More investigations are necessary to delineate the proportion of plant vs. microbial products found in. s o il lip id s .

There is little information in the literature about the contri­ bution of microorganisms to the soil lipids. The only reference con­ necting soil microorganisms to the production of an identified lipid substance is that of Schreiner and Lathrop (1911). They reported that the occurrence of dihydroxystearic acid coincided with the growth of fungi. Lipid materials are constituents of microbial cells and are necessary for the integrity and function of the cell. It would be expected that these lipids would be released into the soil upon death and lysis of the cells. The lipids themselves include fatty acids, waxes, and complex lipids containing sugars and proteins similar to those in plants.

Yeasts including the soil inhabiting yeasts of the genera

Liponyces (Starkey, 19^6; Kononenko, 1958), Cryptococcus (Pedersen,

1958), and Rhodotorula (DiMenna, 1958) are capable of efficient conversion of carbohydrates into lipids. In all but the last of these, the lipids are incorporated intracellularly as large fat globules.

The f a t commonly accounts fo rW-kOfi of the dry weight of the cells with a maximum of 50-63$ reported (Starkey, 19^6). DiMenna (1958) isolated a strain of Khodotorula graminis from the aerial parts of pasture grasses which yielded copious amounts of extracellular lipids in culture. Deinema and Landheer (i 960) and Deinema ( I 96I) analyzed the components of the intra- and extracellular lipids and found that the extracellular fat of R. graminis was of a different composition than the intracellular fat. With gas chromatography, groups of peaks were noted around palmitic acid, stearic acid, and before behenic acid.

The contribution from bacteria appears to be primarily from intracellular lipids which are released after death of the cells.

Bacterial cells contain 5-10$ lipid on the average, which is slightly less than the average for fungi of 10-25$. There are important dif­ ferences between the lipid components of bacteria and fungi. Fungi universally contain both free and bound steroids, whereas evidence has not yet been found for true steroids in bacterial cells (Foster,

19**-9). Many species of bacteria contain a unique energy storage mat­ erial, poly-beta-hydroxybutyrate, not found in fungi. The quantity of poly-beta-hydroxybutyrate within the bacterial cell varies widely, but may constitute as much as 50$ of the dry weight. It could be assumed that this compound would be present in the soil, but no evidence for its existence has been reported in the literature. Jarvis and John­ son (19^9) and Hauser and Kamovsky (195*0 have isolated a glycolipid from th e medium in which Pseudomonas aeruginosa was c u ltu re d . This

e x tra c e llu la r lip id was composed of two moles o f L-rhamnose and n-

beta-hydroxydecanoid acid. Hauser and Karnovsky reported yields of

the crystalline compound as high as 3*16 g/l of medium.

It is readily apparent that more research conducted toward iso­

lating the metabolic products of microorganisms from the soil would help to make an assessment of this contribution more quantitative.

Decomposition. The extent of the lipid decomposition in a soil

depends upon the type of lipid and upon environmental conditions which

affect the microbial population, since it is the microbial population

which is primarily responsible for losses. The balance of the decom­

position and additive processes such as residue additions and microbial

synthesis determine the lipid content of a given soil. Where condi­

tions are not optimal for microbial activity, an increase in soil lip­

ids can be expected.

plant materials are decomposed at different rates dependent up­

on the plant species and general soil conditions. This is understand­

able as there are many types of organic compounds which made up the

plant tissue with a great range of resistance to decomposition. The

lipid fraction and the lignins are generally thought of as the most

resistant to microbial attack. Specifically, the plants vary greatly

in lip id content (Waksman, 1928) and the lipids vary in their resis­

tance to decomposition. The variation is due to the different assoc­

iations, i.e. glycolipids, glycerides, proteolipids etc., arid the

nature of the plant lipids both dependent on the age and species of

plant. Teriney and Waksman (1929) found that the ether soluble frac­ tion of composted com stalks and alfalfa tissue was more readily de­ composed than the ether soluble fraction of rye straw, m a study of the decomposition of green fodder, straw, spruce needles, beech leaves, and peat, Springer and Lehner (1952)^ £ reP°rted that the rate of aer­ obic decomposition over a four year period was in the order, green fodder > straw > le a v e s> n eed les> p eat m ull> highmoor p e a t. The same sequence was observed for the decomposition of the constituent fats, oils, resins, chlorophyll, waxes * tannins, and polysaccharides. Under anaerobic conditions, the rate was less; but the order of decomposi­ tion remained the same. Tsybul'kin and Bel'kevich (196*0 conducted a study of the benzene extractable substances of some plant species and peat bitumens. Extracts of Sphagnum, Schenchyeria, and Eriophorum plants grown on peat were correlated with extracts of peat derived from these plants. The amount of waxes and paraffins extractable with benzene increased in the plants in the order Schenchyeria Erio­ phorum Sphagnum. The content of peat bitumens derived from the plants increased in the same order. Tenney and Waksman (1930) ob­ served that lowmoor peats formed from herbacious (Cladium, Carex,

Phragmites) and woody plants are low in ether soluble components, while highmoor peats derived from sphagnum mosses are very high in ether soluble materials. Moreover, almost one third of the organic matter of pollen peats formed from pond weeds, pollen, plankton, etc. was eth e r and alcohol so lu b le.

The accumulation of soil lipids is influenced by the environ­ mental conditions. Studies on plant decomposition in soil by Tenney and Waksman (1929» 1930) and S pringer and Lehner (1952)^ g demon­ strated that the lipid substances had essentially disappeared under aerobic conditions after one year, whereas under anaerobic conditions the rate of loss was much less. The waterlogged conditions of organic

soils and concommittent anaerobic conditions are considered respons­ ible for the greater accumulation of lipids in these soils than in relatively well drained mineral soils.

Another factor which influences the decomposition of soil fats

and waxes is pH. Waksman and Hutchings (1935) compared the ether and

alcohol soluble fractions from soils which had been limed for25 years vs. untreated soils. Liming decreased the ether soluble fraction to

0.7# as opposed to 5*7# in the untreated soil. Resinous material in

the alcohol soluble fraction remained unchanged. Turfitt (19^3»

19^ i ) , in an investigation of the factors affecting the accumulation of sterols in different soil types, noted that conditions of high

acidity, water-logging, and lack of aeration individually or collect­

ively inhibited decomposition of sterols. A single genus of actino- mycetes, Proactinomyces (Nocardia), was responsible for decomposing

added cholesterol in all of the soils studied. The Proactinomyces

were abundant in fertile areas, but were infrequently present under

conditions of extreme acidity, water-logging, and lack of aeration.

In addition to the foregoing investigations of the overall de­

composition of the lipid materials in soil, some studies have been

made on the microbial transformations of specific lipid materials.

Most of the investigations have been made in solution culture under

laboratory conditions. It would be difficult to assay the performance

of the microorganisms in question in soil because of the lack of con­

trol over microbial interactions. There is little information on the 10 decomposition of specific groups of lipids in soil, but the informa­ tion available w ill be included.

There have been a number of investigations regarding the decom­ position of fatty acids by bacteria. In most of the studies, the mechanism for aerobic oxidation was that of beta-oxidation, Ivler et al. (1955), Randles (1950), Silliker and Rittenberg (1951, 1952). The latter obtained soil isolates by enrichment with stearic acid which proved to be gram-negative rods, apparently of the genus Pseudomonas.

Studies with these isolates and with strains of Pseudomonas, Escher­ ichia coli, Bacillus, Neisseria, and Serratia indicated that the abil­ ity to oxidize fatty acids and use them as a sole source of carbon is a common characteristic of aerobic bacteria. The enzymes concerned were constitutive in all strains except Serratia marcescens and Bac­ illus brevis which required an adaptive period prior to oxidation.

Bishop and S t i l l ( I 96I), however, found a strain of Serratia marces­ cens which did possess a constitutive enzyme for fatty acid oxidation.

In a study using 2,4-dinitrophenol as a blocking agent, Silliker and

Rittenberg (1952) found evidence against the beta-oxidation mechanism in a strain of Serratia marcescens.

The literature pertaining to decomposition of fatty acids by actinomycetes is limited. Webley (195*0 and Webley et al. (1955) in laboratory experiments found one species, Nocardia opaea, which was able to oxidize a number of different fatty acids. Evidence was cited for beta-oxidation as the oxidative mechanism. Adelson et al. (1957) reported that a strain of Streptonwces griseus from the soil was able 11 to utilize a variety of lipids including stearic and palmitic acids, and c o n stitu e n ts of m yelin.

In another specific area of lipid reaction in the soil, Turfitt

(19^3, 19^ i s2» 19^7) conducted several experiments dealing with decom­ position of steroids in the soil. He found that steroid decomposition was accomplished almost exclusively by a soil actinomycete, Proactino­ myces (Nocardia) erythropolis. (Other species of bacteria and actino- mycetes were observed with the Proactinomyces, but were considered to be inactive because of poor growth on cholesterol.) Certain gram-neg­ ative bacteria could, however, utilize sterols in which the C-17 side chain was modified or lacking, pro actinomyc e s species were relatively abundant in areas of good soil fertility , but were present infrequent­ ly under conditions of extreme acidity, water logging and lack of aer­ ation. Interestingly, an accumulation of sterols in the soil also occurred under these conditions. All of the Proactinomyces species tested completely metabolized cholesterol by oxidation to cholesten- one followed by ring cleavage either in mineral media or in the soil.

No mechanism was given for the cleavage of the ring system. Schatz

(19^9)» contrary to Turfitt, found only motile, gram-negative, rod­ shaped eubacteria in cholesterol enrichment cultures of soil. In all, nineteen isolates capable of utilizing cholesterol or its esters were obtained. In contrast, ergosterol was toxic to all but one of the bacteroal isolates. Comparative studies with Nocardia erythropolis and one bacterial isolate showed that the bacterium metabolized more steroids than the Nocardia. With the possibility of biological con­ tamination from sterol residues as an impetus, Gregors-Hansen (196k) 12 studied the decomposition of diethylstilboestrol (DES) in soil. From experiments with DES-monoethyl-l-C^\ it was determined that from 1.6-

16$ of the added was recovered as C ^ 0 2 in three months. After six months, as much as 12-28$ was recovered. Determination of remain­ ing in the soil after the experiments showed an increase in the water- extractable fraction compared to that found shortly after addition of

DES. It was further noted that in sterile soils enzymes present were capable of attacking DES.

Soil microorganisms have the capacity to degrade a miscellany of other lipid materials. Among those capable of decomposition by one or more soil microorganisms are aliphatic hydrocarbons and long chain ali­ phatic alcohols (Webley, 195^» Krause and Lang, 1965)} phospholipids

(lecithin and cephalin), inositol lipid, and complex proteolipids

(Adeleon et al. , 1957)* the polynuclear hydrocarbon phenanthrene (Rog- off and Wender, 1951)} and a variety of fatty acid esters (Krause and

Lang, 1965).

E x tractio n

Extraction of lipids from soils is accomplished by using single organic solvents or mixtures of organic solvents. Lipids, by defini­ tion, are soluble in these ‘'fat solvents," but their extraction from soil is often complicated by numerous factors. Discussions by Hance and Anderson (1963)^ on extraction of lipid materials from mineral soil s o ils and Howard and Hamer ( i 960) for organic soils effectively point out the problems involved. These factors are discussed in the follow­ ing paragraphs.

The choice of solvent influences the type and yield of the ex­ tract. Non-polar solvents such as petroleum ether, ether, benzene, etc. tend to extract non-polar lipid material, e.g., long chain fatty acids, fatty esters and some paraffinic materials. Polar solvents such as alcohols extract the so-called asphalts and resins. Alcohols have also been used successfully to extract heavy metal salts of fatty acids from the soil (Wander, 1949) • Lipids which might occur as lipoprotein and glycolipid complexes are not soluble in non-polar solvents, but can be broken with the inclusion of alcohols as part of the extracting mix­ ture. Unfortunately, breaking the lipid complexes prevents the inves­ tigation of them in situ.

Kwiatkowski ( 1963)2 extracted peat soil with ether, benzene, petroleum ether, chloroform, and trichloroethylene. The extract yields varied from 8.4-15.3$ for ether and trichloroethylene with the other solvents giving intermediate yields. The composition of the extracts also varied, petroleum ether was found to extract small amounts of free acids and large amounts of esters, whereas the ether and benzene extracts were rich in acids. Chloroform and trichloroethylene extract­ ed colored compounds and hydrocarbons.

The best extractant for peat bitumens found by Katkowski and

Karosik (195*0 was a 1:1 mixture of alcohol and benzene. This gave a maximum yield of 24.9$. Pure benzene, gasoline, and carbon tetrachlor­

ide yielded 14.0, 9.0, and 10.3$, respectively. Reilly et al. (1939)

extracted peat soil with pure organic solvents and azeotrope-like mix­

tures. Their data agree with that of Katkowski and Karosik. An an­ hydrous mixture of petroleum and alcohol yielded 10-14$, a petroleum-

alcoho1 -water mixture yielded 11 - 15 $, and pure petroleum spirits yield- 14 ed 6-8 fo. The melting points of the extracts were not significantly d iff e r e n t.

Pretreatment of the soil is another factor in determining yields of soil lipids. Hance and Anderson (1963)2 found that the solubility, of soil lipids was greater in field moist soils than in dried soils, however, the difference was eliminated by pretreatment with an HClrHF mixture. They attributed the decreased solubility of lipids on drying o f s o il to adsorption by* clay m inerals. Howard and Hamer (960 i ) also refer to numerous reports on decreased yields of peat wax after oven drying prior to extraction by an apparent polymerization process. This information indicated that a more efficient extraction of soil lipids would be obtained when performed on field moist or acid pretreated soil samples. Pretreatment of the soil with hot mineral acid (Feustel and

Byers, 1930) or dilute acid (Stadnikov and Wahner, 1931) 9 before ex­ traction with organic solvents, has been shown to increase the yield of lipid materials. The effectiveness of the hot acid pretreatment was attributed to the release of “combined” fats and fatty acids while the dilute acid treatment removed metal cations responsible for formation of insoluble salts with fatty acids.

Components

Investigators of the soil lipid material have over the years separated and identified a number of individual compounds which make up this fraction of the soil organic matter. The compounds discussed here are present in untreated extracts or those which have undergone only saponification or esterification reactions. In the Soviet Union es­ pecially, peat soils have been treated on an industrial scale to pro- duce gases, waxes, and tars. The products from these degredative reac­ tions are not included in this discussion.

The following acids have been identified structurally. Schrein­ er and Shorey (1910 ) found beta-mono-hydroxy stearic acid in an ethanol extract of soil humic acid. They also (1908) identified dihydroxy-

s te a ric (9, 10 -dihydroxyoctadecanoic) acid from a sodium hydroxide ex­ tract of soil. They reported some of the adverse effects that thjse

acid s may e x e rt on p la n ts when grown in so lu tio n c u ltu re . S chreiner

and Shorey (1910) also extracted lignoceric (tetracosanoic, Cg^H^gC^)

acid from soil. Pentacosanoic acid, £>2^ 5$ 2 * was sePara-ted from th e resin fraction of a peat bitumen extract by Rakowski and Edelstein

(1932) and R e illy and Wilson (19*10). T itov (1932), R e illy and Wilson

(19*1-0), and Roginskaya (1936) reported carboceric acid (heptacosanoic

acid, as a component of peat bitumens. Montanic acid (octa-

cosanoic acid, £2$ $ $ 2) was iso la te d from p e a t by Ryan and D illon

(1909) and Roginskaya (1936). Aschan (1921) reported humoceric acid

(Cj^H^jjjO^ as a component in peat.

Several alcohols have been identified after saponification of

the lipid extract from organic soils or soil humic acids. Zalozieki

and Hausman (1907) reported a alco h o l, m.p. 12*1-130° C and an

alcohol melting above 160° C. Ceryl alcohol, 2CgH^j,0 was is o ­

lated by Reilly and Wilson (19*1-0). An alcohol C^H^gO, m.p. 86.5° C

was reported by Titov (1932). Schreiner and Shorey (19ll)2 found gly­

cerol C-^HgO'j after saponification of an ethanol extract of soil humic

acid .

Hydrocarbons found in the bitumen fraction of soil organic mat- ter fall into two categories, those which are aromatic and those which belong to the paraffin series. Blumer (1961) has reported the occur*- rence of phenanthrene C^H^q , fluoranthene pyrene C^H^q , and

3s4-benzpyrene ^2(^X2 ^ benzene e x tra c t o f s o il. Bergmann e t a l .

(196*0 and Gilliland et al. (I960) have isolated perylene C20H12 from peat bitumens. Kern (19*1-7) reported chrysene as a component of soil organic matter. All of these compounds are polynuclear aromatic hydro­ carbons, and at least one (3,^-benzpyrene) has carcinogenic properties.

Schreiner and Shorey (1911)2 identified hentriacontane, C^H^, one of the paraffins, as a component of an ethanol extract of soil.

Others, Meinschein and Kenny (1957)» Kwiatkowski (I963), Butler et al.

(1964), Hildebrand et al. ( 1963) , and V olarovich and Gusev ( i 960) have mentioned the presence of hydrocarbons of the paraffin series in the lipid fraction of soil; but have not enumerated any of the individual compounds.

Two sterols which are abundant in plant tissue have been report­

ed as components of the soil bitumens. One, beta-sitosterol, found by

B el’kevich e t a l . ( 1963) , Ives and O’N e ill (1956 )9 and Ikan and Kashman

(I 963) has the formula £2. ^ 5$ me^ s 137-137.5° C. The o th e r, beta-sitostanol m.p. 137° C, was reported by Ives and O’Neill

(1958) and Ikan and Kashman (1963). S ch rein er and Shorey (1909) ex­

tracted what they termed ”agrosterol” Cq ^H/^O , m.p. 237° C. Neither

the formula nor the melting point is typical of other known sterols,

and it is doubtful that it actually was a sterol.

In recent years, there have been several terpene compounds ex­

tracted from peat soils. The terpenes are relatively common components

of some plant species, so it is not surprising to find them in partial- 1? ly decomposed plant tissue. McLean et al. (1958) and Ikan and Kashman

(I 963) found friedelin, C^qH^qO, m.p. 265-6° C and McLean found fried-

elanol-3-ol, m,P* 301.4° C in a light petroleum extract of

peat. Ives and O'Neill (1958) reported taraxerol (Skimmiol) C^qH^q0 5 m.p. 282-3° C, taraxerone (skimmione) C^qH^O, m.p. 240° C, and alpha-

amyrin C^qH^o0 * m* P* 186-7° C.

Plant pigments tend to be somewhat resistent to decomposition

and have been identified in the organic solvent extractable material

of soil. Johnson and Thiessen (1934) identified beta-carotene C^qH^,

xanthophyll C^gH^Og* and chlorophyllC ^ in ether extracts of

soils. Butler et al. (1964) found a red pigment in the extracts from

colored patches in lateritic and podzolic soils. It has the formula

C20H4O5CI6 and is theorized to be a hexachloro-polynuclear quinone.

There is evidence (Hance and Anderson, 1963)2 that phospholipids

are present in the soil. These investigators reported the isolation

o f glycerophosphate, ch o lin e, andethanolamine from an organic solvent

extract of soil. It was concluded that phosphatidyl choline was one of

the predominant lipid components.

Several investigators have reported physical and chemical data

for crude wax extracts of peat. The data are presented in Table 1 in­

cluding the appropriate references. Table 1. Comparison of some physical and chemical properties of various peat waxes.

Wax type $wax ^paraffin °jo resin ^asphalt Acid val. Ester val. Sap. no.

Crude 52.5 20. 4 24.6 — 50.1 75-2 125-2

D e-resin- if ie d 88.5 5.6 5-5 — 51-4 113-1 61.7

P u rifie d 92.8 6 .k 0 .8 — 116.3 168.4 52.2 ( 1 )

German 74-82 — 14 — 33 ^3 76 montan

Devon 73-83 -- 40 -- 30 45 75 lig n ite

E nglish 63-68 — — — 50 69 119 p e a t wax

Scottish 64-73 — 23 — 48 65 113 p e a t wax (2)

Petroleum -- — — -- 35 5^ 93 e x tra c t

Petroleum/ -- — -- 42 84 126 alco h o l e x t. ( 3)

Montan 73-3 73-9 ■f

Table 1. (Contd.)

Wax type $wax ^paraffin $resin ^asphalt Acid val. Ester val. Sap. no. I no.6 m.p. °c

Montanin 56.8 1.0 57-9 95-7 (*)

Crude — — 22 12 **•5 75 120 7 65-70

De-asphalted — -- 19 3 ^5 75 120 9 61-66

De-resin- — — 3 1^ ko 95 135 11 65-72 if ie d

De-asphalted — — 3 3 30 95 125 10 63-71 & resinified (5)

(1) Bel'kevich (i 960) (2) Cawley and King (19^5) ( 3) Reilly and Bnlyn (19^0) (4) Ryan and B illo n (1909) (5) Cawley (19^8) (6) Actually Br values

\o MATERIALS AND METHODS

The purpose of this investigation was to characterize chemically the components of the lipid fraction of Rifle peat. Toward this end, various methods of extraction and analysis were tried. Techniques were perfected on untreated soil to be used with soils amended with carbon­ ic. Peat soil columns were incubated with carbon-1C labelled fatty acids and glyceride in order to provide further insight into the degra­ dation and distribution of lipids in the soil.

Incubation of soil columns

Samples of Rifle peat were collected at the Muck Substation of the Ohio Agricultural Research and Development Center on May 2, I96C apd sto red a t C° C u n til Ju ly 1, I 96C. At that time, 7 incubation col­ umns of plastic pipe (3” ID X 12" long), fitted with fiberglass mats, were each filled with 717 g- of peat (bulk density 0.52 g/cc). Water was added to the point of saturation and free water drained from each column. The soil columns were mounted on a wooden rack in a growth chamber and incubated a t 27° C. The CO 2 evolved was trapped in 1 N sodium hydroxide following a procedure similar to Stotzky et al. (1958)*

1 Lit The soils were incubated 57 days before addition of C labelled compounds in order to establish equilibrium as measured by constant COg evolution. Further addition of water to maintain the moisture level had no affect on CO2 evolution, and it was not leached through the soil. Palmitic acid uniformly labelled, palmitic acid-C-1-14, and

20 21 glyceryl tripalmitate-C-1-14 samples1 were diluted to 25 ml. with ben­ zene. Five gram samples of moist soil from the surface were removed from columns 1, 2, 3» 4, and 6 and amended as follow s: Column 1 was amended w ith 20 ml. of uniformly labelled palmitic acid solution (80 uc), columns 2 and 6 were amended w ith 10 ml. of glyceryl tripalmitate-

C-l-14 solution (0.2 me each), columns 3 and 4 were amended with 10 ml. of palmitic acid-C-1-14 solution (0.2 me each). Benzene was removed from th e amended samples w ith heatin g in an a i r stream and the tre a te d soil was mixed with the top 2 inches of soil which had been removed from the column. The treated and mixed soil was returned to the columns and settled by tapping.

Carbon dioxide quantities were assayed by titration of the sod­ ium hydroxide and precipitation of barium carbonate. The barium car­ bonate was counted in a dioxane-water scintillation solution with a 2 3 Packard Tri-Carb liquid scintillation counter.

Proximate analysis

Two percent hydrochloric acid extraction. Subsamples (20 g.) from each of the incubated soil columns which had been previously ex­

tracted with benzene and methanol were used. They were refluxed with

400 ml. of 2'$ hydrochloric acid for 5 hours (Chahal et al., 1966). The

1 Radioactive samples were obtained from Nuclear Chicago Corp., Des P la in e s, 1 1 1 . Activities: Glyceryl tripalmitate and palmitic acid-C-1- 14 0.5 me, uniformly labelled palmitic acid 100 uc. 2 Packard Instrument Co., LaGrange, Illinois. 3 The author thanks Mr. Wesley Nelson for the CO2 a n a ly sis. 22 hot mixture was filtered and washed with water until a clear filtrate was obtained. The soil residues were dried at 105° C and analyzed for 1A to t a l C and C .

Eighty percent sulfuric acid extraction. Subsamples (5 g») from the residues of the 2$ sulfuric acid extractions were treated with 50 ml. of 80$ sulfuric acid at A° C for 2 hours. At that time, 750 ml. of water was added and the mixture refluxed for 5 hours. The hot mixture was filtered and washed until the filtrate was clear. The residue was dried at 105° C and analyzed for total C and C^.

Total and radiocarbon analysis

Samples (0.1000 g.) of extracted residues were placed in ceramic boats for dry combustion. The carbon train apparatus consisted of a furnace operated at 900° C followed by conc. sulfuric acid, manganese dioxide, and magnesium perchlorate traps. An oxygen stream was passed over the sample in the furnace and through the traps. Carbon dioxide for the total carbon analysis was trapped in Ascarite. For the 1C Ll analysis, 10 ml. portions of hydroxide of hyamine ^p^(diisobutyl-cres- oxyethoxyethyl)-dimethylbenzylammonium hydroxide] as a 1 M methanol solution were diluted with 5 ml* of toluene in a series of test tubes. 1A The gas stream was bubbled into these tubes to trap the C 03* Sodium hydroxide was used as a final trap.

A scintillation solution consisting of 6.6 g. PP0 (2,5-diphenyl- oxazole) and 0.A g. P0P0P [ 2 ,2-p-ph.enylenebis( 5-phenyloxazole) ] in 1 1. of toluene was prepared. A 5 ml. aliquot of the hyamine solution was added to 10 ml. of this solution in a polyethylene counting vial. The samples were counted in a Packard Tri-Carb liquid scintillation counter (Rapkin, 1962). 23

Peat fat and wax extractions

Bulk samples were obtained from the Ap horizon of Rifle peat

(Hayes and Mortensen, 1963) at the Muck Substation of the Ohio Agricul­ tural Research and Development Center. The soil was mixed, air dried, and sto red in a bin a t room tem perature. The f a t and wax fra c tio n was extracted from two 1 kg. subsamples in the following manner. Each sub­ sample was relfuxed with 3 1* of benzene and methanol (10:1 v/v) for approximately 10 hours. The resulting mixture was filtered while hot and re-extracted two more times in the same manner.

The crude wax extract was fractionated into four fractions on the basis of its solubility in petroleum ehter and 95$ ethanol (Fig. 1):

Fraction I, soluble in petroleum ether and insoluble in ethanol; II, soluble in petroleum ether and soluble in ethanol; H I, insoluble in petroleum ether and insoluble in ethanol; IV, insoluble in petroleum ether and soluble in ethanol.

In subsequent studies, instead of refluxing, fresh solvent was passed through peat in order to attain greater efficiency of extraction.

As a result of contamination from Tygon tubing used in the initial ex­ traction, an all-glass extraction apparatus was assembled. A one-neck

1 1. round bottom flask was fitted with a connecting adapter with a 75° side arm. A vacuum takeoff adapter was fitted to a one-neck, 500 ml* round bottom flask in which the soil samples were placed. The two adapters were connected to form the extraction apparatus. An azeotrop- ic mixture of benzene and methanol (5*5s^s5» v/v) was boiled in the large flask and the vapors passed into the smaller flask where they con­ densed. The bubbling of vapor into the condensed liquid in the sample flask tended to keep the peat samples agitated. The vacuum connection PEAT WAX FRACTIONATION

PEAT

Benzene : MeOH 10:1

CRUDE WAX

INSOLUBLE!SOLUBLE

Add petroleum ether

INSOLUBLE SOLUBLE

Add hot ethanol Add hot ethanol and cool. and cool.

SOLUBLE IN INSOLUBLE IN SOLUBLE IN INSOLUBLE IN COLD COLD ETHANOL COLD COLD ETHANOL ETHANOL III ETHANOL I IV m. p. 82-85°C II m. p. 69-73°C

Figure 1. Solvent fractionation flow diagram for a benzene-methanol extract of Rifle peat. 25

of the adapter acted as an overflow from which the extractant mixture was filtered into a 500 ml. Erlenmeyer receiving flask. At the conclu­

sion of the extraction, most of the solvent was removed on a rotary film

evaporator at 50° C, and that remaining was removed with an air stream

at ambient temperature.

Thin layer chromatography

Lipid m a te ria ls have been shown (Mangold, 1961; Blank, 196^-;

Randerath, I 963) to be readily separated by means of thin layer chroma­

tography (TLC). Moreover, the time required for development of plates

is measured in minutes as opposed to hours for paper chromatography.

Preparation of plates. Thin layer plates(8x8 in.) were coated \ with Silica Gel G (250 u) using a spreader assembly. After spreading,

the plates were dried at room temperature for 5-10 minutes to allow the

coating to set. They were then activated by heating in an oven to 110-

115° C for at least an hour. For preparative TLC using thicker coat­

ings, the spreader was modified with the addition of a layer of plastic

electrical tape applied to the bottom.

Spotting and developing. Before spotting, the coating on the

plate was scored horizontally at the 10 cm. mark by use of the supplied

template. It was also scored vertically to provide columns for the sol­

vent to move free of interference. Samples to be chromatographed were

dissolved in chloroform or similar organic solvent. Standard capillary

j Model A-200 Chromatofilm Assembly, Research Specialties Company, Richmond, Calif. tubes (1.5-2.0 x 100 mm.) which were drawn out to a smaller diameter were used to spot the sample on the plate.

Solvents used for developing were decided upon in accordance

with the eluotropic series. A number of single organic solvents and

solvent mixtures were used. These are given in the "Results and Dis­

cussion" section (page6l) along with the information on the separations

they effected.

Spraying. Several spryas including iodine, antimony pentachlor-

ide, Rhodamine B, and 2,7'-dichlorofluorescein were tested on the de­

veloped places. The 2,7*-dichlorofluorescein appeared to be the most

satisfactory for locating separated spots. A Mineralite ultraviolet

lamp was used to scan the plates.

Recovery. After spraying the thin layer plate and scanning it

with TJV light to locate separated spots, the absorbent in these areas

was scraped off of the plate into a small sample tube. Chloroform (3-5

ml.) was added, the mixture shaken, and then filtered into another

small sample tube. The solvent was removed with an air stream and the

residue saved for further use. Chloroform and ether were effective in

eluting separated materials from the adsorbent and spray reagent (2,7-

dichlorofluorescein) giving uncontaminated samples. For C^ monitoring,

spots were scraped and placed directly in scintillation solution (Sny­

der, 1962).

Methylation with diazomethane. Ethanol (95$, 25 ml.) was added

to a solution of5 g. of potassium hydroxide in 8 ml. of water in a 100

ml. distilling flask fitted with a dropping funnel and downward conden­

ser. Two 125 ml. Erlenmeyer receiving flasks each containing 15 ml. of 27 ether were located, one at the exit of the condenser and the other con­ nected in series with the first. The condenser tube and inlet tip were

both located below the ether surface. Since diazomethane is both toxic

and explosive, the entire apparatus was located in a fume hood and was

free from ground glass joints except for the stopcock on the dropping

funnel. The flask containing the alkali solution was heated in a water

bath a t 65-70° C. A saturated solution (150 ml.) of Dupont EXR-101

(70# N,N‘ -dinitroso, N,N1 -dimethyl terephthalamide in mineral oil) was

introduced dropwise from the dropping funnel into the alkali solution.

The rate was adjusted so that the rate of addition approximated the

rate of distillation. The preparation of diazomethane in this manner

required ljt hours. The diazomethane-ether solutions were combined and 1 divided evenly among the experimental samples.

The experimental samples were kept in an ice bath until reaction

was complete as indicated by the loss of color in behenic acid,

B®k©nic acid was included as a standard and was the only non­

colored sample. The reaction mixture was decanted from the insoluble

material of each sample and the solvent was removed by evaporation.

These samples were treated with diazomethane a total of three times.

Gas chromatographic measurements indicated the behenic acid to be 60.3#

methylated by this procedure.

Methylation with methanol and acid catalyst. About 0.1 g. of

sample was added to a mixture of U0 ml. methanol and 3 drops conc. sul-

1 Procedure modified from that given in a brochure on "Diazaid*' by the Aldrich Chemical Co., Milwaukee, tfis. 28 furic acid. After 2k hours of refluxing, the mixture was cooled and saturated sodium carbonate was added dropwise until the solution was basic. The reaction mixture was transferred to a separatory funnel,

150 ml. of water was added, then it was extracted three times with 30 ml. of ether. Drying of the ether extract was accomplished with 25 ml. of saturated sodium chloride and filtering through anhydrous magnesium sulfate. Most of the solvent was removed by distillation and the re­ mainder with an air stream.

Gas-liquid chromatography

Instrumentation. A Barber Coleman Series 5000^ dual column gas chromatograph with a Packard Tri-Carb Gas Chromatography Fraction Col­ lector was employed for analysis. Although the chromatograph was out­ fitted with electron capture and flame ionization detectors, only the flame ionization detectors were used. Column temperatures were pro­ grammed as needed for separation. Kuelmans (1957) discusses the theory and practice of gas chromatography.

Column preparation. Pyrex 6 ft. x 5 mm* U-shaped columns were packed with 20$ Silicone Rubber SE-52 on 60/80 mesh Cnromosorb W HMDS

(hexamethyldisilazane) treated. A 5*10 g. sample of SE-52 rubber was dissolved in about 250 ml. of ether. To this was added 25*5 g« °f

Chromosorb W. The mixture was stirred periodically to aid coating and evaporation of the solvent. When air-dry, the adsorbent was placed in a vacuum desiccator and evacuated with a vacuum pump overnight. The adsorbent was then packed uniformly in the glass columns with the aid

•I ■‘•Barber Coleman Co., Rockford, 1 1 1 . 29 of an electric vibrator. After packing, the columns were cured at 295°

C fo r 2b hours before use.

Sample preparation and introduction. A Hamilton M icroliter syr­

inge (10 ul. capacity) was used to introduce the dissolved samples into

the columns. Nitrogen was used as a carrier gas for non-radio active

samples and argon was used for radioactive samples. All samples with

the exception of those from the incubated soil columns were measured

qualitatively.

Sample collection. The Packard fraction collector was fitted to

the gas effluent connection of a 5il gas stream splitter. The larger

volume of gas was directed to the fraction collector, the smaller vol­

ume to the detector. The fraction collector held 50 vials which were

changed by a manually actuated mechanism. Anthracene filled vials were

used for collection. The samples were cued from the mass peaks to in­

clude the peaks and effluent to the next peak. After collection, the

vials were placed in metal holders and counted in a Packard Tri-Carb

liquid scintillation counter. One problem inherent in this approach was

that radioactive materials present in too small a mass for recording,

but detectable by scintillation counting were included.

The vials used for radio as say were modified for use with infra­

red analysis by removing anthracene, inverting the filters, and replac­

ing them 1 cm. from the top of the vials. Powdered potassium bromide

(300-400 mg.) was placed in the vials which were capped until used.

Samples were collected on the potassium bromide cued from the mass peak

on the recorder. Infrared analysis

Methods of sample preparation and measurement are covered by Rao

(1963), pp. 62-97. The routine analyses were made with the use of a potassium bromide disk formed as described including the use of a

Wig-L-Bug amalgamator, Carver Model B press, and the evacuated die. The instrument used was a Perkin-Elmer Model 27 Infrared Spectrophotome­ te r .^

Samples collected from the gas chromatograph were placed directly into the die and pressed. Equivalent samples were later combined by grinding the disks together and re-pressing.

Lipid samples which had an oily consistancy were run by the - J ' sandwich technique using NaCl plates. A pure NaCl plate was placed in the reference beam.

Ultraviolet analysis

A Perkin-Elmer Ultraviolet-Visible Model 202 Spectrophotometer was used for analysis of soil lipid samples where applicable. The samples were measured as ether solutions in a 1 cm. Suprasil cuvet. A discussion of the interpretation of ultraviolet spectra can be found in

J a ff e and Orchin ( I 962).

•1 Perkin-Elmer Corporation, Norwalk, Conn. RESULTS AND DISCUSSION

Preliminary experiments

Extractions and fractionations included here were done as

preliminary steps to the analysis of soil incubated with labelled

lipid compounds. These analyses served to develop efficient extraction

techniques and background information on the lipid components of Rifle

p eat.

One kg. samples of Rifle peat were extracted under reflux as

previously described on page23 . One sample yielded 15.4 and the other

15.2 g. of crude lip id e x tra c t (1.54 and 1 . 52$ on a dry weight basis

respectively). The crude extracts were fractionated according to their

solubilities in organic solvents as shown in Fig. 1.

Fraction I (m.p. 69-73° C), insoluble in cold ethanol, was a

light tan color and granular. The infrared spectrum for Fraction I is

shown in Fig. 2. Absorption bands were assinged as follows: 2.9 P- -

free OH possibly alcoholic and acidic from both types of compounds, 3.40

and 3.47 ji = aliphatic C-H stretching, 5.70 and 5.80 p. = carbonyl 0=0 bands split perhaps because of mixed ketonic, acidic or ester functions;

6.80 }i = C-H bending of CH^-C groups, 8.50 p = ester C-0 stretching, 13.90 p = poly-CH2 greater than four units. Since this fraction was a mixture of compounds (TLC evidence), the spectrum did not have the fine structure

it would have had with pure material. From the evidence, the fraction was considered to be composed of aliphatic, predominantly saturated, long chain fatty acid esters and fatty alcohols or acids or both.

Fraction I when developed on thin layer plates with petroleum ether: ether (8:2) produced two spots with Rf values of 0.9^ and 0.99 and some poorly resolved material near the origin.

Fraction III, insoluble in petroleum ether and cold ethanol, melted at 82-5° C and was light tan in color and granular. The infrared spectrum for Fraction III is also shown in Fig. 2. Absorption bands were assigned as follows: 3*^0 and 3*50 P = aliphatic C-H stretching,

5.80 p = carbonyl C=0, 6.80 p = C-H bending of CI 13-C groups, 8.60 p =

C-0 stretching probably ester, 13.95 P = poly-CH2 greater than four units. Fraction III was also composed of a mixture of compounds (TLC evidence). The assigned functional group absorption peaks probably be­ longed to more than one type of compound. For example, the rather broad carbonyl absorption was very likely due to a mixture of ketonic and ester carbonyl groups which were too close to be resolved. Fraction III prdduced spots with Rf values of 0.25 and 0.97 when developed with ben­ zene: ethanol (9: 1 ) on thin layer plates.

Fractions II and IV, both soluble in cold ethanol, were dark- colored and had a gummy consistency which prevented melting point determinations. Preliminary chromatography on thin layer plates indi- ' cated that the fractions were similar, so they were combined for further analysis. The infrared spectrum for combined Fractions II and IV is shown in Fig. 3* Absorption bands were assigned as follows: 2.9 P = 3 5 6 7 8 9 10 11 12 13 Wavelength (microns) Figure 2. Infrared spectra of Fraction I (top) and Fraction III (bottom). See Figure 1.

r 3 5 6 7 8 9 10 11 12 13 14 Wavelength (microns)

Figure 3. Infrared spectra of combined Fractions II and IV (top, see Fig. 1) and fatty acid mixture primarily octacosanoic add. 3 5 free OH, 5.75 P = carbonyl C=0, 6.80 p. = asymmetrical C-H bending bf a CH^-C group, 7,25 p = symmetrical C-H bending of a CH3-C group, 8.55 p = C-0 stretching of an ester, 13.90 p = poly-CH2 greater than four units. These data suggest that both fractions contained long chain saturated aliphatic alcohols and esters. When developed with ether: petroleum ether:acetone (2: 8:1 ) on thin layer plates, the two frac­ tions produced a spot with an Rf of 0 . 5^ which fluoresced purple after spraying with 2, 7-dichlorofluorescein. With petroleum ether: eth e r (8:2) two spots with Rf values of0.15 and 0.97 (which fluor­ esced purple and bright yellow respectively) were produced.

The infrared spectrum for the material recovered from the spot w ith Rf value 0.97 i s shown in Fig. 4 . The absorption bands were a s­ signed as follows: 2.9 P = free alcoholic OH, 3.**0 and 3.^7 P = aliphatic C-H stretching, 5*85 P = carbonyl C=0, but only trace; 6.00 and 7.25 P = asymmetrical and symmetrical C-H bending of a CHg-C group respectively, 8.95 P = C-0 stretching of alcohol group. The band a t 8.95 u is typical for a saturated secondary alcohol, and other absorption bands support this structure. Evidence indicates a long chain alcohol, although the methylene absorption 13 ( - 1 ^ p) was poorly defined. Titiv (1932), Reilly and Wilson (19^0), and Zalozieki and

Hausman (1907) have reported long chain aliphatic alcohols as compo­ nents of the lipid fraction of peat.

Fractions II and IV and caprylic acid (CgH^gOg) included as a standard for comparison were developed on TLC plates with petroleum ether:benzene:chloroformjmethanol:ether (8:2:1:1:1). These fractions Wavelength (microns) Figure Infrared spectra of compounds separated by TLC. Top--material with an Rf of 0.97 In PEJether from plate 2. Bottom—material with an Rf of 0.99 in pyridine:acetone from plate 8. V ji ) O n 37 produced spots with values of 0.18 (light yellow), 0.72 (purple), and 0.97 (bright yellow). The caprylic acid had an Rf value of 0.52

(light yellow) and did not match any of the other components. An at­ tempt to improve resolution of TLC using other solvents was unsuccess­ f u l.

Evaporation of the solvent from the chloroform solution of com­ bined samples II and IV used for TLC resulted in the crystallization of

a colorless component; A sample of the crystalline material was chrom­

atographed on a thin layer plate producing a spot with a purple color reaction and an R^ value equal to that for the purple spot from frac­ tio n s H and IV. A t o t a l o f 62 mg. o f th e c r y s ta llin e compound was r e ­

covered from the peat extract fractions.

Another crystalline material was successfully isolated from the mixture of Fractions II and IV. These crystals had a spheroid form in­

stead of the rhombohedral form of the white crystals and were straw-

colored. They appeared more like a microcrystalline wax.

The two crystalline components of the peat extract were identi­

fied as carbazole (white crystals) and a mixture of fatty acids predom­

inantly octacosanoic acid (C22H44O2, straw-colored crystals) by mass

and nuclear magnetic resonance (NMR) spectrometry. Verification of

structure was obtained by infrared and ultraviolet spectroscopy, and

x-ray diffraction analysis. This evidence is reviewed below. The two c r y s ta llin e samples were sen t o u t fo r MR and mass spectrometric analysis."^ Because of a small sample size and the presence of impurities, the MR spectrum of the carbazole was poor.

It did, however, suggest a heterocyclic structure. The MR spectrum for the fatty acid had a sharp peak at 8.? PPM and a smaller less distinct peak at 9.1 PPM. The downfield peak was due to methylene

absorption and the upfield peak was due to methyl absorption.

The relative intensities indicated a long chain compound.

The mass spectrum of the carbazole had a parent peak at 167

(C^HgN) indicative of the molecular weight. A peak at 140 indicated . loss of HCN. This evidence pointed to carbazole as the compound

in question. The melting points were also compatible: observed

242-2.5° C, reported 245° C (Stecher, i 960).

The mass spectrum of the fatty acid showed parent peaks at

396 (^26^52^2^ * ^28® 56^2 ^9 ^ ^52 (C^QHgQOg). Some odd-carbon- number fatty acid peaks were also present corresponding to C^H^gOg,

C27H54O2, and ^ 29^ 58^2 ^ H'tuch smaller concentrations than the former even-carbon-numbered acids. Octacosanoic acid was the major

component making up about twice as much as the next most important

component. The th eo ry and p ra c tic e of MR and mass spectrom etry i s

covered in a practical way by Silverstein and Bassler (1963).

2 The author thanks Dr. Richard Schwendinger, then at the Mellon Institute, Pittsburg, Pa. for the MR and mass spectrometric analyses. A Varian A-60 MR spectrometer and an Associated Electronic Industries MS-9 mass spectrometer were used for the analyses. .39

A few tenths of a milligram of each of the crystal samples were

ground and placed in capillary tubes for x-ray powder diffraction analy­

sis. The d-spacing data is presented in Table 2. After the soil-derived

carbazole sample had been identified, the’-pure carbazole sample was measured as a further check. The two sets of values agree quite well.

Further criteria for verifying the identity of the crystalline materials were developed by infra-red analysis of the extracted materials

and appropriate standards. The infrared spectra shown in' Jig. 5 are for

carbazole extracted from peat and a sample of pure carbazole. With the

exception of small bands at 3-35 and $.80 yx due to impooiities, the spec­

tra were identical. The infrared spectrum of the extracted fatty acid

c ry s ta ls i s shown in F ig. 3* The absorption bands were assigned as fol­

lows: 2.9 = OH, probably carboxyl; 3.40 and 3.^7 V- = aliphatic C-H

stretching, 5*83 Ji = carbonyl C=0 of carboxylic acid, 6.80 ji = C-H bend­

ing of a CH^-C group, 13.72 and 13.9 U = poly-CH2 gr®ater than four units, all characteristic of a long chain fatty acid. The infrared

spectrum of behenic acid (C 22H44P2) f ° r com parative purposes i s shown

in Fig. 7. It has a great similarity to that of the soil-derived sample.

Since carbazole was aromatic, ultraviolet spectra of extracted

and pure carbazole were compared (Fig. 6). Although these spectra were not as diagnostic of structure as were the IR spectra, they were indica­

tive of a polynuclear aromatic molecule. It was also evident that the

two spectra were identical.

Thin layer chromatography was employed extensively to determine

structures of non-crystalline components of peat lipid extracts. The

components discussed below are from th e columns amended w ith C 1 ll , b u t 4o

Table 2. X-ray diffraction d-spacings for carbazole and two crystalline materials extracted from Rifle peat.

Carbazole______Aromatic crystals______Aliphatic crystals

9.4-7 9.50 4.220

4.78 4.80 3.742

4.51 4.49 2.991

4.27 4.29 2.496

3.83 3.82 2.239 3.32 3.38

3.17 3.19

2.86 2.87

2.47 2.46

2.36 2.37

1.98 2.03

1.92 1.93

1.8? 1.84 Figure 5« Infrared spectra of carbazole extracted from Rifle peat (top) and pure carbazole (bottom).

4 1 3 ^ 5 6 7 8 9 10 11 12 13 U ^ Wavelength (microns) *3

200 250 300 350 Wavelength (millimicrons) Figure 6. Ultraviolet spectra of carbazole extracted from peat (top) and pure carbazole (bottom). Wavelength (microns)

Figure 7* Infrared spectra of behenic acid (top) and methyl stearate (bottom). were not assayed for activity. Components separated by TLC and as- 1^ sayed for activity are described in the section dealing -with C experi­ ments .

It was noticed that with ether as the develping solvent, all of the toluene soluble fractions as well as most of the other benzene- methanol soil extracts produced a single spot with an Rf value of 0.99 with some material remaining at the origin. The separated material fluoresced brightly under UV light after being sprayed with dichloro- fluorescein and had a melting point of 76-8° C. M aterial was accumu­ lated from preparative plates and an infrared spectrum was made (Fig. 8) .

The absorption bands were assigned as follows: 3.^2 and 3.50 P = ali­ phatic C-H stretching, 5*75 and 5*85 P - carbonyl C=0, probably ketonic and ester absorption overlapping; 6.15 P = olefin C=C stretching, 6.85 p. = asymmetrical C-H bending of a CH^-C group, 7.97 P = C-0 stre tc h in g of ester, 12.55 P = C-H out-of-plane bending of R2C=CHR group, 13.77 P = poly-CH2 gr ®ater than fo u r u n its . The spectrum showed evidence o f an unsaturated ketonic ester.

It was noted that even polar solvents such as alcohols and ace­ tone in the solvent systems for TLC were ineffective in separating the materials which remained at the origin. Application of still more polar solvent combinations such as pyridine:methanol 1 (: 1 ) and ether:methyl cellosolve 1 ( :1 ) were successful in separating this dark-colored mat­ erial into two spots with Rf*s of0.25 and 0. 95.

The IR spectrum for the dark-colored spot at Rf 0.95 is shown in

Fig. 8. The absorption bands were assigned as follows: 2.9 p = free OH,

3.^2 and 3»50 P = aliphatic C-H stretching, 5*80 P = carbonyl C=0, 6.15 53 6 ? 8 9 10 11 12 13 14 Wavelength (microns) Figure 8. Infrared spectra of materials separated by TLC* Top—material with an Bf of 0.95 in methanol:pyridine from plate 10* Bottom—material with an Rf of 0*99 a 1 ether from plate 9* p = olefin C=C absorption, 6.85 and 7.27 p = asymmetrical and symmetrical

C-H bending of a CH^-C group, 12.50 p = C-H out-of-plane bending of k

R2C=CHR group, 13*95 P = poly-CH2 greater than four units. The evidence pointed to an unsaturated long chain carboxylic acid structure. As pre­ viously indicated several investigators including Titov (1932), Reilly and t/ilso n (19*10), and Ryan and D illo n (1909) have reported long chain fatty acids as components of the lipid fraction of peat.

The IR spectra for the materials with Rf values of 0.25 are shown in Fig. 9. Even though the R^ values were equal, the IR spectra indi­ cate that the different solvents separated different compounds. The absorption bands for the material separated with methanol:pyridine (1 :1 ) were assigned as follows: 2.90 p = free OH, 3«**0 p = aliphatic C-H

s tre tc h in g , 5*85 P = carbonyl C=0, 6.9 and 7.25 p = asymmetrical and

symmetrical C-H bending of a CH-j-C group, 10.55 P = OH out-of-plane bending of carboxyl. Since the spectrum was made of a chloroform solu­ tion, the region above 13 P was blanked out and affords no additional information. This spectrum was characteristic of a fatty acid. The in­ frared spectrum for the other material also had the hydroxyl absorption typical of carboxylic acids, but showed absorption typical of an unsat­ u rated compound ( 6.05 p). In addition to unsaturation, the split car­ bonyl indicated a keto-acid as no ester absorption was present to ac­

count for the carbonyl.

Two products from a reflux extraction of peat preliminary to 1A those with C were analyzed by gas liquid chromatography (GLC). One

fraction was precipitated from petroleum ether by partial evaporation

(F-III) and the other precipitated from hot petroleum ether upon cooling 5 6 7 8 9 10 11 12 13 Wavelength (microns)

Figure 9* Infrared spectra of compounds separated by TLC. Top-material with an Rj. of 0.25 in pyridine methanol from plate 10. Bottom—material with an R» of 0,25 in ether jcellosolve from plate 11. 49

(F-IV). Portions (0.10 g.) of these samples were methylated three times with diazomethanein ether solution using the procedure in “Materials and

Methods-.I*

The gas chromatograph was programmed for five minutes isothermal time at 150° C and a 5°/min. temperature increase up to 295° C. An 8 p.1 sample of F-IV in benzene was injected. Four peaks were produced with

R^ values of 101.25, 405, 911 »25, and 1462.5 seconds.

As no peaks were observed before methylation, i t could be assumed that four different long chain fatty acids were present in each sample.

Because the operating parameters were different from those of standard esters, approximate carbon numbers could not be calculated.

Reference was made in “M ate rials and Methods*1 to improved y ie ld s of crude wax obtainable with a chromatographic column extraction system

(range 1.7-2.25$)• There were, however, certain problems associated wit with this technique. One such problem involved material extracted from the top of the column precipitating in the lower cooler portion of the column. This problem was aggravated by this precipitate which along with the normal compaction of the peat impeded the flow of solvent caus­ ing further cooling of the lower portion of the column. In addition, it

should be pointed out that Tygon tubing must be absent from an extraction

system of this type which utilizes organic solvents. Initially a short length of Tygon tubing was incorporated in the apparatus between the boiling flask and column. The benzene-methanol solvent extracted plas- ticizer (dioctylphthalate) from this tubing which contaminated crude wax

extracts. The infrared spectrum for dioctylphthalate is shown in Fig. . 10 . I

Wavelength (microns) Figure 10. Infrared spectrum of dioctylphthalate extracted from Tygon tubing.

o 1 h, Experiments w ith C la b e lle d compounds 1 4 Carbon dio x id e evolved from th e C amended p e a t samples during • lij, incubation was measured periodically for total C and C . Figs. 11, 12,

13, and 14 show p lo ts of CO2 evolution vs. time for each treatment: col­ umn 1 - palmitic acid-U-C-14, column 2 - glyceryl tripalmitate-C-1-14, column 4 - palmitic acid-C-1-14, and column 7 - check. Duplicate treat­ ments agreed so well that only one curve for each is included. It should be noted that the scale is broken on the graphs and the curves begin at the 400 hour mark. This was necessary because the early data was invalidated by faulty preparation of the sodium hydroxide used in t i t r a t i o n . The la b e lle d compounds were added a t 1366 hours and in ­ cubated until 2400 hours. Slopes of the curves decreased after 800 hours indicating equilibrium.

Cumulative values for total C and C^ lost as CO2 after amend- 14 ment with the labelled sources are listed in Table 3« Total C loss from the palmitic acid treated soils was about ten times greater than from glyceride treated soils. To determine whether unreacted acid or glyceride remained in the soil which might account for the difference in 14 C loss, the extracts from both soils were chromatographed on thin layer plates. The small mass of material added to the soil as labelled material made it impossible to detect decomposition products by spray reaction or other chemical means and made radioactivity assay the only method for analysis. There was no detectable radioactivity from the soil extracts which corresponded with co-chromatographed palmitic acid.

In contrast, a spot in the chromatogram of the soil extract from the glyceride treated soil which corresponded to that of glyceryl tripalm- iue1. Carbon dioxideand radiocarbon Figure loss from incubated11. soil column 1.no. Mg C EVOLVED 10 10 ,3 800 o 1200 HOURS 139 1600 2000 hours 883 1177 52 iao1* Carbon dioxideand radiocarbon12*Figaro loss from Incubated soil Mg C EVOLVED column 2.no. 10 10 400 800 o 1200 HOURS 3 237 139 1600 52 k k 2000 hours 762 1076 53 iue1. Carbon dioxide and radiocarbonFigure loss 13. from incubated soil Mg C EVOLVED 10 column no. k. 1200 o HOURS 139 2000 hours 8 1177 883 5* iu«3 Cro doie os fo Icbtd l oun o 7. no. column il o s Incubated from ss lo dioxide Carbon Figur«13. Mg C EVOLVED 10 800 1200 HOURS 1600 2000 2400 55 56

Table 3» Carbon dioxide and radiocarbon loss from soils incubated after treatment with C-14 labelled fatty acid and glyceride.

Total, mg.'C1'^ Treatment T o tal ir.g, C $ of added C^ as CC>2 x 10 -^ •

Palmitic acid-U- c-14 500.8 53.6 69.1

Glyceryl tripalm- itate-C-1-14 465.6 97.9 7.1

Palmitic acid-C- l-iij. 487.6 323.2 66.5 palmitic acid-C- 1 - 1 ^ 494.2 319.5 65.7

Check 35^.6 — —

Glyceryl tripalm- itate-C-1-14 511.2 87.9 6.3

Check 494.4 — — 5?- itate was radioactive. This evidence from TLC supported the previous evidence from C02 evolution that only part of the glyceride and most or all of the palmitic acid had decomposed.

The all-glass extraction apparatus was used to extract the soil 1 L samples incubated with C labelled lipid compounds. Since preliminary extractions of the columns indicated no extractable radioactivity lower than 2'* from the surface, all subsequent extractions included only the top 2" of soil. Chahal (1966) reported the movement of a small amount of radioactivity into the soils below the layer of incorporation, but the soils in his experiments were periodically leached with water.

Table U gives the yield for crude wax from extracts of Rifle peat incubated with labelled compounds. The yields of crude wax ranged between 1 .5 and 2. 25$.

After extraction, the crude wax from the incubated soil columns was fractionated according to solubility in toluene. Toluene was chosen as a solvent so the material extracted for liquid scintillation counting would be soluble in the toluene base scintillation solution. Most of ' . the crude wax was soluble in toluene as shown in Table ^ and contained

essentially all of the extracted C1 Ll as insoluble samples showed no act­

ivity in scintillation solution. A portion of the toluene soluble frac­

tions of each extract from the incubated columns were made to standard

volume (0.1000 g/50 m l.) and analyzed f o r a c tiv ity .

The radioactivity data for the toluene soluble material of the

benzene-methanol extracts is presented in Table 5* The extracts from

s o il amended w ith g ly cerid e accounted f o r 6 .2 and 6. 8fo of the added car- 58 Table 4. Yields for the benzene-methanol extract of the 0-2 inch layer of Rifle peat from incubated soil columns. -

Column Sample w t. Crude e x t . ' % Yield Toluene sol- Toluene in- g. wt. g. uble frac- soluble ______tio n g.______fraction g.

P alm itic acid-U- C-14 81.5 1.837 2.25 1.694 0.143

G lyceryl trip a lm i- tate -C -1 - 14 74.1 1.375 1.80 0.863 0.181 P alm itic acid-C- 1-14 64.7 0.975 1.50 0.890 0.085 P alm itic acid-C- 1- 11* 7 4.0 1.461 1.97 1.290 0.162 G lyceryl trip a lm i- tate -C -1 - 14 92.4 1.960 2.12 1.660 0.261 59

Table 5* Radioactivity data for benzene-methanol extracts from soil columns incubated with C-14 labelled palmitic acid and glyceryl tri- p alm ita te .

Treatm ent cpm/mg fo o f added $ o f C-14 crude C-14 rem aining e x tra c t in s o il

Palmitic acid-U- C-14 1315 3.7 11.9 Glyceryl tripalm- itate-C-1-14 6060 6.8 7.3

Palmitic acid-C- 1-14 3566 4.0 11.9 palmitic acid-C- 1-14 3050 3 > 10.0

Glyceryl tripalm- itate-C-1-14 5^59 6.2 6.5 60 bon, whereas the extracts from the palmitic acid amended soils ranged between 3 .4 and 4 .0 $ . The p alm itic acid amended s o ils , however, con- tained more of the residual C 1 Ll after extraction.

To more thoroughly assess the efficiency of various extractants and in an attempt to extract more labelled material, the peat sample from Column 3 was extracted with trichloroethylene after extraction with benzene-methanol. The use of this solvent was based on a report by

Kwiatknwski ( 1963)2 that the highest yield in a series of extractions was with trichloroethylene. The yield of crude wax after trichloro­ ethylene extraction was 0.247 g. (0.38$) as opposed to a 1.5$ yield for the inital extraction with benzene-methanol. In addition, this extrac­ tion removed 0.24$ of the added and suggested that alternate use of solvents might allow for further recovery.

After obtaining the structural information from components of the lipid extracts from incubated peat by means of TLC, further application of this method was made to separate samples for radioactivity measure­ ments. Table 6 lists the separated spots and corresponding activities.

The activities given for plates 1 and 2 were from composite material of the same taken from the thin layer chromatograms of all incubated samples. The other activities were determined individually for compon­ ents of extracts from each treatment. Itshould be noted that spots with

values of 0.80 from toluene insoluble fractions of soil extracts were not radioactive.

Only one of the components separated and radioassayed was also recovered for IR analysis. This was dark colored material (R^. 0.99) separated with acetone:pyridine (1:1). The infrared spectrum is shown 61 Table 6. Radioactivity data for materials separated by TLC.

Sample Rf cpm Solvent

P la te 1 Toluene soluble CgHg:CH^OH (10:1) C o l's 1-6 • 99 320 C o l's 1-6 • 30 902

P la te 2 Toluene soluble CgHg*. CH^OH (10:3) C o l's 1-6 .99 732 P la te 3 Toluene soluble Pyridine:acetone (l:l) Col. 1 •99 99 followed by C5H5:ace­ Col. 2 • 99 593 tone: CHoOH (7 :3 :1 ) Col. 3 •99 219 Col. k •99 ikQ Col. 6 •99 b25 Toluene insoluble Col. 1 .80 0 Col. 2 .80 0 Col. 3 .80 0 Col. b .80 0 Col. 6 .80 0

P la te b Toluene soluble . CgHg:acetone:CHLOH Col. 1 • 99 0 (8: 2: 1 ) Col. 2 • 99 220 Col. 3 •99 295 Col. b •99 139 Col. 6 •99 187 62 in F ig . k . Absorption bands were assigned as follows: 2.85 P- = free

alcoholic OH, 3.38 and 3.^6 ji = aliphatic C-H stretching, 5.72 p = car­ bonyl C=0, 6.08 p = olefin C=C stretching, 6.82 p = asymmetrical C-H bending of a CH^-C group, 8.60 p = C-OH stretching of alcohol, 13.90 P = poly-CH2 greater than four units. This infrared data suggested a long

chain unsaturated fatty alcohol structure. There was some carbonyl ab­

sorption, but no other bands to suggest an ester or carboxylic acid

group.

After having obtained successful separations with TLC, gas liqu­

id chromatography was utilized. GLC possesses a much greater resolving

power -man TLC and therefore each peak produced could be attributed to

a sin g le compound.

Samples (0.1-0.2 g.) of the crude wax extracts from the incubated soil columns were methylated with methanol and acid catalyst, made to 10

ml. volume with benzene, and injected into the gas chromatograph. Table

7 lists the retention times (R^) for peaks corresponding to separated

compounds from the incubated soil extracts. It can be seen that a num­

ber of components were common to several of the extracts including both

even- and odd-carbon numbered compounds. Occurrence of these odd-num­

bered components was not completely unexpected since mass spectral data

had previously indicated some odd-carbon number fatty acids to be pre­

sent. These results were particularly interesting since odd-numbered

fatty acids are seldom found in natural products. Altogether 2k com­

pounds were separated from five column extracts ranging to C^q. Because

the column temperature was limited to 295° C, the high molecular weight Table 7. Retention times in seconds for separated components of the crude extracts of incubated Rifle peat. Column temperature 295° C isothermal.

Rt C no. R(. C no. R^ C no. Rt C no. R^ C no. Column 1 Column 2 Column 3 Column 4 Column 6 Palmitic Glyceryl tri­ Palmitic Palmitic Glyceryl tri- acid- U-C-14 palmitate-C- acid-C-1-l4 acid-C- l-l4 palmitate-C- 1-14 l - l 4 101.25 135.0 135.0 157.5 15 225.0 17 225.0 17 236.25 281.25 18 281.25 18 281.25 18 247.5 427-5 20 427-5 20 427-5 20 540.0 21 540.0 21 607.5 607.5 607.5 877.5 945.5 956.25 24 1068.75 1170.0 25 1170.0 '25 H7O.O 25 1361.25 1383.75 1687.5 1687.5 1755.0 27 2160.0 28 2205.0 28 2700.0 29 2700.0 29 3442.5 CT\ 4320.0 30 4320.0 30 4320.0 30 6<* compounds with long R^’s became diluted with carrier gas which caused broadening of the peaks. A different stationary phase and higher temp­ eratures might improve separation of components.

After these preliminary separations by GLC, chromatograms were made for the purpose of collection and radioassay. Components were col­ lected as described in "Materials and Methods" and the values and ac­ tivities are listed in Table 8. With the exception of column 2, the most active component had an R^ value of 135 seconds. This came close to a calculated compound. It could be that some of the palmitic acid and glyceride was only slightly changed and still retained the ori­ ginal carbon skeleton. The table lists only active peaks, and values differ from those in Table 7 because the carrier gas flow in this separ­ ation was increased. Components from the glyceride treated soil ex­ tracts contained the highest activity, and extracts of columns 3 and k contained the fewest active compounds. In all 13 radioactive components were resolved -which proved that the incorporated compounds have -under­ gone extensive degredative reactions. The difference in the number of components between acid and glyceride amended soils indicated that the degredative pathways may be different.

In order to obtain structural information for separated compo­ nents, the fraction collection vials were modified for use with KBr.

Bennett (1966) found glass wool-filled collection vials to be quantita­ tive, and it was assumed that KBr would be equivalent to glass wool.

R^. values for peaks collected on KBr for infrared analysis and collec­ tion numbers are listed in Table 9* The temperature was programmed from

260-295° C for better resolution of more volatile components. Samples

1-A and 9-11 were present in too small an amount to produce usable in- Table 8• Retention, times and activities for compounds separated by GLC from methylated extracts of incubated peat. Column temperature 295° C isothermal.

cpm cpm cpm cpm cpm Rt Rt Rt Rt Column 1 Column 2 Column 3 Column 4 Column 6 P alm itic Glyceryl tri- P alm itic P alm itic Glyceryl tri- acid-U-C-l4 palmitate-C- ecid-C-l-l4 acid-C-l-l4 palmitate-C- l - l 4 1-14

33-75, 135 230 405 26 135 4l6 135 403 135 1377

213-75 61 720 840 213-75 68 213-75 48 213-75 132

337-5 35 900 52 540 l6 337-5 35

540 21 1226.25 26 540,585 29

843-75 16 2205 24 1440 21

1350 16 2160 29

,0"

.1 Table 9. Sample numbers, calculated carbon numbers, and retention times for samples collected for infrared analysis from the gas chroma­ tograph.

Sample no. Rj. C no Sample no. R-j. C no.

Column 6 Column 3

1 78.75 9 202.5 16

2 326.25 10 236.25 17

3 438.75 20 11 427.5 20

4 618.75 22 12 = 51 618.75 22

5 663.75 22 13 = 6 900.00 24

6 900.00 24 14 = 7. 1485.00 26

7 1338.75 15 = 8 2430.00 27 8 1980.00 27

1 The R^ ;values for equivalent samples are different because of different temperature programs. f reared spectra. Samples with identical IR spectra were combined in

order to increase the concentration and improve the spectra. The

values fall in the same range as the radioactive samples, but tempera­

ture programming was changed and the values aren't directly comparable,

so it is impossible to correlate the structures and activities.

The in fra re d spectrum fo r fra c tio n s 5 and 12 i s shown in F ig. 15*

Absorptionbands were assigned as follows: 2.88 p. = free alcoholic OH,

3.42 and 3*5 P = aliphatic C-H stretching, 5*78 p = carbonyl C=0, 6.15 p = o le fin C=C s tre tc h in g , 6.85 and 7.27 P = asym m etrical and symme­

trical C-H bending of a CH^-C group respectively, 7*90 P = C-0 stretch­

ing of an ester with the alcohol group propyl or larger, 8.95 or 9*35 P

= C=0 stretching of alcohol and the other may be another ester band,

12.5 P = C-H out-of-plane bending of a R2C=CHR group, 13.85 p = poly-

CH2 greater than four units. This compound was an unsaturated, hydroxy

ester. Since the material was not a methyl ester, it undoubtedly was

present in the soil in the ester rather than acid form.

The in fra re d sp ectra fo r fra c tio n s 6 and 13, 7 and 14, and 8 and

15 are shown in Figs. 15 and 16. Absorption bands were assigned as fol­

lows: 2.87 p = free alcoholic OH, 3*42 and 3.5 P = aliphatic C-H

s tre tc h in g , 5*75 P = carbonyl C=0, 6.15 p = o le f in ic C=C stre tc h in g ,

6.85 and 7.27 p = asymmetrical and symmetrical C-H bending of a CH^-C

group respectively, 7.97 P = C-0 stretching of methyl ester, 9.80 p =

C-0.stretching of alcohol, 12.5 P = C-H out-of-plane bending of a

R2C=CHR group, 13.80 and 14.0 p = poly-CH2 greater than four units, All

of these compounds were methyl esters of unsaturated hydroxy acids. The 53 6 7 8 9 10 11 12 13 Wavelength (microns)

Figure 15 . Infrared spectra of gas chromatographic fractions 5 and 12 (top) and gas chromatographic fra c tio n s 6 and 13 (bottom ). J n i g 3 5 6 7 8 10 11 12 139 Wavelength (microns) Figure 16. Infrared spectra of gas chromatographic fractions 7 and 14 (top) and 8 and 15 (bottom), o\ NO 70 only differences seem to be in the number of carbon atoms and perhaps degree of skeletal carbon branching as indicated in the fine ’'finger­ p r in t” s tru c tu re between 8 and 8.75 P*

It was a little surprising to find that all of the collected com­ pounds had an alcoholic functional group. However, beta-oxidation has been shown to be a microbial oxidative reaction (Ivler et al., 1 955?

Randles, 1950; and Silliker and Rittenberg, 1951> 1952) and may account for the prevalence of hydroxy acids. A number of hydroxy fatty acids have been isolated from the extracellular lipids of yeasts of the phyllo- sphere and Schreiner and Shorey (1908, 1910) have reported hydroxy- stearic acid as a soil lipid fraction component.

Another structural feature to note is the apparent branched chain at the double bond position. The absorption at 12.5 P is characteristic f o r an R2C=CHR structure. The evidence from the TLC and GLC analysis indicates that the lipid compounds added to the peat were being oxidized to hydroxy and unsaturated compounds. Both radio- and structural analysis support this idea.

Proximate analysis

Incubated soils after extraction with benzene-methanol were sub­ jected to partial proximate analysis to gain further knowledge of the distribution of the radiocarbon. This included a 2$ hydrochloric acid extraction which according to Chahal (I963) removed h em icellu lo ses and other polysaccharides in hydrolyzed form, and an 80$ sulfuric acid ex­ traction which is considered to remove cellulose and lignin. Samples subjected to acid extraction were analyzed for total C and C1^ by combustion analysis both before and after extraction. The results for total C areppresented in Table 10 as percent organic carbon.

The samples extracted with acid were approximately the same with aver­ ages differing only by 1.15$« The difference between acid extracted samples and samples not so extracted lies in the removal of materials low in carbon content, i.e. cellulose, hemicellulose, and sugars, while leaving materials high in organic carbon such as humic and fulvic acids and other more humified materials. 14 The results for the C combustion analysis are presented in

Table 11. The consistency between treatments on the same samples was poor. This was true especially of the samples from the peat treated with palmitic acid-U-C-14 (Col. 1) .and one of the samples treated with palmi­ tic acid-C-1-14 (Col. 3) • The values appear to be too high for the ben- zene-methanol extracted and sulfuric acid extracted sample from column 1 1 h as 69.1 $ of the C was lost in CC>2 evolution. The value for column 3 extracted with sulfuric acid was much lower than the other samples.

These discrepancies might be explained by sampling errors since combus­ tion samples from a ' somewhat heterogeneous m aterial such as peat were only 0.1 g. and represented from l/50th to 1/900th of the total sample.

The results show that the 2$ hydrochloric acid extraction removed more 14 C from th e p eat amended w ith p alm itic acid-C -1-14 than from th e glyc­ eride amended soils. The 80$ sulfuric acid extraction, however, removed

4 ft between 15 and 20$ of the C from the glyceride amended peat. This evi- dence indicated• • that more C *1 f i from glyceride was in the cellulose-lignin fraction than in the carbohydrate fraction. 72 J, Table 10. Total carbon expressed as percent organic carbon for Rifle peat after extraction with benzene-methanol, 2$ hydrochloric acid, and 80$.sulfuric acid.

Treatment C6H6:CH30H 2$ HCL 80$ H2S0^

Palmitic acid-U- C-14. 40.99 49.36 53.04

Glyceryl tripalm- itate- C-l-14 41.59 51.68 52.88 Palmitic acid-C- 1-14 41.53 52.17 52.88

Palmitic acid-C- 1-14 42.35 52.17 51.5? Glyceryl tripalm- itate-C-1-14 44.61 ' __1 52.14

Chock 42.32 Average 42.23 51.35 52.50 ^"Erroneous measurement. f t

Table 11. Radiocarbon content of Rifle peat after extraction with benz­ ene-methanol, 2°jo hydrochloric acid, and 80$ sulfuric dpid expressed as percent of C-l4 added.

Treatment C5H5: CH3OH 2°jo HCL 80$: H^SO^

Palmitic acid-U- C-l4 40.2 20.2 51.5

Glyceryl -tripalm- itate-C-l-l4 25.6 20-5 6.96

Palmitic acid-C- 1-14 22.7 9-63 0.21

Palmitic acid-C- 1-14 23.5 7-74 11.5

Glyceryl tripalm- itate-C-l-l4 22-7 28.4 8.50

* * SUMMARY AND CONCLUSIONS

The purpose of this study was to identify as completely as pos­ sible the components of the lipid fraction of Rifle peat and to trace

4 f t the decomposition products of C labelled lipid compounds added to

Rifle peat.

Rifle peat was extracted with benzene-methanol to obtain the crude lipid fraction. Long chain fatty acids C25 through C30 were iden­ t i f i e d along w ith carbazole (C-j^H^N). Long chain f a tty acids have p re ­ viously been reported in this fraction of soil organic matter, but there was no previous report in the literature of carbazole being proaetit in soils. At present, without further study, we cannot explain the origin of carbazole. It has been isolated from coal and its occurrence in peat may substantiate the theory of peat being a precursor to coal.

Other long chain saturated and unsaturated alcohols, acids, and esters were also recovered from the peat lipids. These were not identi­ fied completely, but some unsaturated esters with gas chromatographic evidence were as large as C^q .

Palmitic acid-U-C-14, palmitic acid-C-1-14, and glyceryl tripalm- itate-C-1-14 were added to Rifle peat and then incubated. Carbon dioxide evolution accounted for about 6. 6$ of added C of g ly cerid e and 66$ of added C 14 of palmitic acid. Evidence was also found for incomplete gly­ ceride decomposition.

74 75 Carbon-1^ was found incorporated in compounds separated chromato- graphically and assayed for radioactivity. This provided evidence that decomposition products from the incubation were identical with or very similar to those already present in the soil. From chromatographic evi­ dence also, it appeared that the decomposition products were in a higher state of oxidation as evidenced by hydroxyl units and unsaturation.

The analytical methods of thin layer chromatography and gas liqu­ id chromatography were especially useful and efficient in accomplishing separations of the components of the peat lipid fraction. Combining the chromatographic methods with modern instrumental techniques for structur­ al identification such as infrared and ultraviolet spectroscopy, NIIR and mass spectrometry and liquid scintillation counting provided a great deal of information from a relatively small sample. BIBLIOGRAPHY

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