Fatty acids of bacterial origin in contemporary marine sediments

Fatty acids of bacterial origin in contemporary marine sediments G. J. PERRY,J. K. VOLKMAN* and R. B. JOHNS Department of Organic Chemistry, University of Melbourne, Parkville, Vie. 3052, Australia and H. J. BAVOR JR Department of Microbiology, University of Melbourne, Parkville, Vie. 3052, Australia (Received 16 November 1978; accepred in revised form 22 June 1979)

Abstract-Contributions by bacteria to recent sediments have been recognized as one important source of input for the extractable lipids. It has, however, proved difficult so far to conclusively relate the components identified to the contributing bacteria. This fact is primarily related to the lack of information on both the lipid chemistry of marine bacteria, and of detailed structures of the sedimentary lipids. In this paper a study of the fatty acids from a tropical marine sediment selected because of its high biomass content is reported, and relationships between the sedimentary extracts of the surface layer to fatty acid components of bacteria cultured from the sediment sample are detailed. By selecting specific structural features, a group of fatty acids have been identified as valid markers for bacteria in this en~ronment: these include iso- and anteiso-branched chain acids; lo-methylpalmitic acid; ~clopropyl 17:0 and 19:0 acids of which V19:O (11,12) is unique to bacteria; cis-vaccenic acid; and the 15: 1, 17: 1 w6 and ~8 isomers especially when these occur in pairs; iso A7-15:l and iso A9-17:l are branched unsaturated acids apparently unique to bacteria. Tram-monoene fatty acids are likely to be a direct bacterial input, and the hydroxy acids identified are probably of bacterial cell wall origin. This study, whilst emphasizing the necessity for detailed structural information on fatty acids in order to use them validly as biological markers, considerably extends the range of fatty acids as markers of bacterial input to contemporary sediments.

THE LIPID composition of recently deposited sediments is a result of the input of biological material and its utilization by microorganisms, together with the direct contribution from microorganisms contributing to the sediment biomass. The rapid alterations which lipids can undergo during the early stages of deposition which is referred to as biological diagenesis, is considered to be brought about largely by microbial activity in the upper layers of recent sediments. (POLTZ, 1972; FARRINGT~N and QUINN, 1973; JOHNWN and CALDER, 1973; MATSUDA and KOYAMA, 1977.) Microorganisms may bring about this modification in two distinct ways: actual biodegradation within the micr~rg~isms and chemical degradation as a consequence of the immediate environment created by their activity (EGLINTON,1973). In addition there is the possibility of some abiological chemical reactions such as dechelation and hydrolysis. Sedimentary microorganisms are held responsible for the decomposition of lignins, carbohydrates, proteins (KUZNETXN, 1975) and lipids, which may serve as the primary source of energy for heterotrophic sedimentary bacteria (AYERS, 1961). A number of organic compounds which are believed to provide evidence * Present address: Organic Geochemistry University, U.K.

Unit, Bristol

for microbial activity have been identified in recent sediments (MORRISONand BICK, 1966; EGLINTONet al., 1968; VAN DORSSELAER et al., 1974), and dicarboxylic acids have been proposed as indicators of microbial activity at the time of deposition (JOHNSand ONDER, 1975). Other lines of evidence for microbial activity which may be brought to bear include, the high lipolytic activity of some sediments (RONALD, 1946; F~R~NG~N and QUINN, 1973); and the rapid conversion of [‘4Cjlabeiled oleic acid into a series of saturated acids in an estuarine sediment (RHEAD et af., 1971; GASKELLet al., 1976). A number

of studies have

compared sediment parameters such as extractable adenine nucleotides, muramic acid, respiratory activity and heterotrophic potentid with rates of lipid synthesis from r4C or [3zP]labelled precursors and have shown that lipid biosynthesis also reflects microbial mediated activity (WHRE et al., 1977; KING et al., 1977). The observed relative abundances for many fatty acids in recent sediments are the result of several factors, such as microbial utilization and/or alteration of deposited fatty acids generally derived from primary production (vegetation and phytoplankton~ and hence may be u~avo~able substrates for microorganisms or, are acids produced as a result of diagenetic changes (SCHUTZ and QUINN, 1973; CRANWELL, 1976). However, because microorganisms are

1715

1716

G. J. PERRY.J. K. VOLKMAK. R. B. JOHNSand H. J. BAV~RJR

capable of incorporating directly between 2@40’>,,of the organic matter which they utilize (OPPENHEIMER 1960; DEMEYERet al., 1978) cellular lipids of microorganisms, particularly bacteria, must be recognized as another important source of sedimentary fatty acids. A number of s~imentary fatty acids, particul~ly branched and cyclic alkanoic acids have been considered to be bacterial in origin (LEO and PARKER. 1966; CRANWELL,1973; 1974). More recent evidence has indicated that phytanic acid detected in Dead Sea sediments is most likely derived from extremely halophihc bacteria (ANDERSONet al., 1977). Boon et al. (1975)postulated consumption of algal lipids by bacteria in a diatoma~eous ooze and replacement of aigat acids by bacterial acids in an early stage of burial. The replacement of acids derived from allochthonous material by bacterial fatty acids is likely to occur in many, if not all recent marine sediments. However despite the number of reports (LEO and PARKER,1966; CWPER and BLUMER,1968; CRANWLL, 1973; 1974) of fatty acids of inferred bacterial origin in sediments and the number of structural types proposed as bacterial markers, there is still no definitive work to show the extent and nature of bacterial contributions to marine sediments. In this paper we report the fatty acid composition of a surface marine sediment from a tropical, coral island environment of the Low Isles, North Queensland. Australia (JOHNSet al., 1977) and compare it to fatty acid distributions of aerobic and anaerobic populations of microorganisms cultured from this surface sediment. The extent to which particular fatty acids can be used as bacterial markers is discussed, and by determining and judiciously comparing the exact molecular structures of sedimentary and bacterial fatty acids, this study identifies sedimentary fatty acids which are of bacterial origin.

phur oxidizmg bacteria) isolated and grown in Beijerinck medium (RODINA.1972). LI Y--Predominant yeast isolate-isolated as described by CROW et al. (1976) and grown in modified Zobell’s 2216E medium. LIAN-- Total anaerobic heterotrophs were grown in the modified Zobells 22f6E medium with addition of O.l%, thioglycollate (Difco). Cultures were incubated at 25°C in stationary flasks with no air space, but loosely capped to allow gas escape.

Sediment samples were extracted by saponification with methanolic KOH (pH 12) as described previously (JOHNS and ONDER,1975). Bacterial cells were harvested by centrifugation at 9OOOrpm for 30min (Beckman J 21) and washed twice with OS?,, saline. Isolation of bacterial fatty acids was performed according to methods previously described (JOHNSet af., 1977). Culture medium blanks were extracted and checked for fatty acid content.

Fatty acids from both sediment and bacteria were methylated using 147; (w/v) BF,/MeOH (METCALF and SCHhllTZ,1961) prior to chromatographic analysis. Sedimentary fatty acid methyl esters were separated into monocarboxylic esters (Rf 0.521, a,w-dicarboxylic esters (Rf 0.35) and total hydroxylated esters (Rf 0.11) by thin layer chromatography (t.1.c.)on silica GFzs4 (Merck) developed in a n-heptane/diethyl ether/methanol (40: 10: 1, v/v/v). Bands were visuahzed under U.V.light after spraying plates with 2’,7’-dichlorofluorescein. Monocarboxylic esters from bacteria and the sediment were further separated by argentation t.1.c. [silica GF,,, impregnated with 5% (w/v) AgNOj] into saturated esters (Rf 0.52), monounsaturated esters (Rf 0.42), diunsaturated esters (Rf 0.29), triunsaturated esters (Rf 0.17) and polyunsaturated esters (Rf < 0.05) by development with n-heptane/diethyi ether/methanol (90: lO:l, v/v/v). Adequate separation of positional and geometrical isomers of monoun~turated esters was achieved on sihca GFzs4 [5:! (w/v) AgNO,] with saturated esters (Rf 0.68), trans-monounsaturated esters (Rf 0.61), cis Al I-monounsaturated esters (Rf 0.56), cis-A9139monounsaturated esters (Rf 0.52) and diunsaturated esters (Rf 0.26) by double development in toluene/n-heptane (70:30, v/v) (MORRIS et al., 1967). Isolation of discrete bands of fatty acid esters with particular double bond positions is possible using these argentation t.1.c. techniques. EXPERIMENTAL Hydrogenation of unsaturated esters was performed in methanol with Adams catalyst (Pfaltz and Bauer) in a Parr Snmples hydrogenator at 20 psi for about 12 hr. Gas-liquid chromatography (g.1.c.)of esters was performed on a glass support Sediment samples were collected from a mangrove coated open tubular column (SCOT) coated with SE30 swamp region on the Low Isles, North Queensland (60 m x 0.5 mm I.D., S.G.E. Australia) and a stainless steel (16”23’S, 145”34’E)in the Great Barrier Reef. Sediments are wall coated open tubular column (WCOT) coated with uncompacted with a moisture content of 65%. and recent, BDS (46.7 m x 0.25 mm I.D., Perkin-Elmer) in a Perkinhence geochemical parameters of heat and pressure would Elmer 900 gas chromatograph. Both of these high resolunot be affecting them. US-Surface scraping. top 3 mm tion capillary columns with a high number of effective from a site chosen to minimize mangrove input. theoretical plates [Neff (SE301 = 52,000 and Netf Bacterial cultures were obtained from the oxidized surface sediment by the methods previously described (JOHNS (BDS) = 39,000] were capable of separating ~sitional and geometrical isomers of monounsaturated fatty acids. Satuet a!., 1977) using the media outlined below. rated, cis monounsaturated and polyunsaturated fatty acid LIZ-total aerobic heterotrophs grown on modified Zobell’s 2216E medium (OPPENHEIMER and ZOBELL, 1952: methyl esters were identified by coinjection with authentic standards, when available. Double bond positions were LITCHFIELD et al., 1975) made with a mixture of three parts aged seawater and one part distilled water. Nutrient levels determined by use of equivalent chain length (ECL) values (JAMIESON, 1970; ACKMAN,1972), separation factors (ACKwere modified by incorporation of 194 glucose and reducMAN and CAST~LI.,1967; ACKMAN,1972) and relative retention to 0.05% Peptone (Difco) and 0.01% Yeast Extract tion time (RRT) measurements (ACKMANet at., 1967; ACIC(D&o) in order to maximize bacterial recovery and to MAN 1969). By plotting log RRT against chain length and minimize the problem of in~rporation of exogenous Iipid joining points for fatty acids of common w value and the material (KANEDA,1977). same number of double bonds, but different chain lengths, LIZ-I and LIZ-~-TWO predominant isolates from the a series of parallel lines is obtained (ACKMAN, 1969; ACKtotal aerobic heterotrophs grown as above. LIMB-Thiobacillus species (A chemo-autotrophic sul- MAN, 1972). Using this plotting procedure isomer identifica-

Fatty acids of bacterial origin in contemporary tion was possible and, combined with the other techniques mentioned above including argentation t.1.c. double bond positions could be determined with certainty. The identification of tram geometry for a number of

monounsaturated acids was based on the isolation of a

trans band from argentation t.i.c. of the total monocarboxylic acids (LEES and KORN,1966; ACKMAN et a!., 1972)and comparison of RRT’s and ECL’s with authentic standards (Alltech Associates, Analabs). and literature values (ACKMANand CASTELL,1967; ACKMANet al., 1972). Double bond positions for these trans fatty acids were determined by comparison to literature retention times (BDS column) (ACKMAN and CASTELL,1967; ACKMANet al., 1972) and separation factors (SE30 column) derived from separations of cis isomers for a particular chain length. These techniques have previously been used successfully to identify trans fatty acids formed as a result of hydrogenation of oils and fats (ACKMAN et al., 1972). The presence of cyclopropane fatty acids was confirmed by catalytic hydrogenation with Adams catalyst and a few drops of acetic acid (MCCLOSKEY and LAW,1967). RESULTS

marine sediments

1717

rarely reported in sediments (VOLKMANand JOHNS, 1977). The saturated acids are dominated by palmitic acid (16:0) which is the major sedimentary fatty acid. Double bond positions for the majority of cis-unsaturated acids have been determined and are shown in Table 1 along with a series of deans-monounsaturated acids (Ci4-C1s). Branched chain fatty acids (both saturated and monounsaturated) are abundant, representing 15% of the total monocarboxylic fatty acids. Two cyclopropanoid fatty acids V17:O (9, 10) and V19:O (11, 12) were identified in significant quantities. a,w-Dicarboxylic acids in the range Cs-Cz6 identified in this sediment are shown in Table 2. Dicarboxyhc acids account for 9.4% of the total non-hydroxylat~ fatty acids. 4,8,12-T~methyltrid~~oic acid (4,8,12 TMTD) was the only significant isoprenoid acid detected, pristanic and phytanic acids were present in trace amounts only (cO.l%). (ii) Populations of sedimentary microorganisms

(i) Sedimentary fatty acids

A preliminary fatty acid analysis for a similar sediment sample from this environment has previously been reported (JOHNSet al. 1977) however this new analysis by the utilization of capillary g.1.c. techniques reports greater structural and isomer details of the components. The monocarboxylic fatty acids (Table 1) consist of straight chain saturated acids Cl&&,

straight chain monounsaturated acids C,&& ad a number of ~lyunsaturated fatty acids which are

The populations for a number of microbial physiological types enumerated in the Low Isles surface sediment, are given in Table 3. Aerobic heterotrophs predominate in this surface sediment sample but anaerobic heterotroph numbers increase with depth and increasingly negative Eh (unpublished results). Similar microbial distributions have been previously reported in a number of studies (VANDERIQSTand DUTKA, 1971; LITCHFIE~,D et al., 1975). Bacterial populations in sediments are determined

Table i. Monocarboxylic fatty acids from Low Isles Sediment (LIS) Acid 9:o lo:o is0 1l:O 1l:O is0 12:o 12:o is0 13:0 anteiso 13:O 13:o is0 14:0 14:o 4,8,!2 TMTDd is0 15:O ante& 15:O ls:o is0 16:O 16:O 1OMe 16:O is0 17:O anteiso 17:O 17:o v17:o (9,lO) is0 18:0 18:0

Percentage TR 0.4 0.4 0.3 0.2 2.7 0.6 0.2 0.9 1.8 10.0 0.2 4.3 2.7 4.7 1.4 21.0 1.2 1.1 0.8 1.3 0.8 0.1 1.7

Acid 19:o v19:o (11,12) 20:o 21:o 22:o 24~0 12:lb 13: lf06” 14: lw7 14: 1ws trans 14: 1 iso 87-15: 1 1S:loS 1S:lw6 tram 15: lb is0 16: lb 16: 109 16: iw7 16: 1~5 trans 16: 1~13 tram 16: 101 trans 16: lw5 iso A9-17:l

Percentage TR 1.4 0.2 0.1 0.1 0.1 0.4 0.1 0.3 ::: Z 0;9 :: 0.2 12.0 1.2 g 0:1 0.3

Acid

Percentage

ante&o 17:l” 17: 1~8” 17:1&j 18:1&J 18:107 18:105 trans 18: 109 trans 18: 107 20: lwll & w9 14:2b 16:207 16:2cu4 16:3r.u4 17:2

0.2 1.2 0.5 1.9 5.6 0.2 0.1 0.3 TR 0.6 0.3

18:2&j 18:306 18:ku3 18:4#3 20:4w6 20:503 22:Sr06 22:6w3 Unjdentifi~

2.4 0.5

0.5

0.6 0.1

1.8

TR Components less than 0.1%. a Double bond positions (0) are numbered from the methyl end, all subsequent bonds are methylene interrupted. b Double bond positions unknown. ’ Includes some trans 17: 1. A4,8,l2-Trimethyltridecanoic acid.

1718

G. J. PERRY, J. K. VOLKMAN,R. B. JOHNSand H. J. BAVORJR

Table 2. G(,ODicarboxylic fatty acids from Low Isles Sediment (LIS) Acid

Percentage

Acid

Percentage

8:O 9:o lo:o 11:o 12:o 14:o 16:0 17:o 18:0

4.4 19.1 1.8 0.4 3.1 2.8 33.7 I.2 10.7

19:o 20:o 21 :o 22:o 23:0 24:0 25:0 26:0 Unidentified

0.7 2.4 0.7 5.8 1.0 10.1 0.6 0.9 0.6

Table

3. Microorganism populations Sediment”

in

Low

Organisms

Population (Counts per gram of dry sediment)

Aerobic heterotrophs Proteolytic@ Yeasts and Fungi” Hexadecane utilizer? Cellulose utilizer? Anaerobic heterotrophs

1.9 x lo9 1.8 X 10s 2.5 x lo4 2.x X 105 > lo6 1.8 x 10h

Isles

ASurface sediment O- I cm. b Media described by BAVORand MILLIS (1976). by such factors as organic carbon, total nitrogen and sediment grain size (DALE, 1974), with numbers typically ranging from 105-10’ bacteria per gram of sediment (OPPENHEIMER, 1960; DALE, 1974; ZOBELL, 1975). A conservative estimate of the numbers of bacteria in this surface sediment would be 2 x lo9 organisms per gram of dry sediment (Table 3). This large number of organisms may be a reflection of the high level of organic matter which is characteristic of mangrove sediments (WOOD, 1965). If one assumes that a gram wet weight of bacteria contains about lOI* organisms (OGINSKY and UMBREIT, 1959) and that bacterial cells are about 80% water, then 2 x IO9 bacteria represent a biomass of 400 pg per gram of dry sediment. The lipid input of this biomass may range from 10 to 15% (some yeasts may contain 50% lipid)

of dry weight depending upon the microbial which are present (AIBA et al., 1973). (iii) Fatty u-ids ~$srdimentary microorganisms

The fatty acid composition of a number of bacterial cultures and a yeast from the Low Isles sediment were examined for comparison to sedimentary fatty acids. These analyses in most cases contain more detail than previous reports in the literature (of which there are few for marine bacteria) especially with regard to minor components and isomers of monounsaturated fatty acids. Table 4 shows a detailed fatty acid analysis of the total aerobic heterotrophs (LIZ). A series of normal and branched chain fi-hydroxy acids (Table 5) was

Table 4. Fatty acid composition of total aerobic heterotrophs Isles Sediment (LIZ) Acid

RRT”

Percentage

9:o lo:o is0 11:0 ll:o 11:lb 12:o 12: 107 is0 13:0 anteiso 13 :0 13:o 13: lw8 is0 14:0 14:o 14: 107 14: 105 is0 15:0 anteiso 15:0 iso A7-15:l 15:o 15: lw8 15: 106 iso 16:0

0.034 0.053 0.064 0.077 0.088 0.110 0.122 0.136 0.145 0.159 0.173 0.196 0.230 0.261 0.274 0.284 0.301 0.309 0.334 0.371 0.383 0.409

0.3 0.6 0.1 0.3 0.1 1.5 1.7 0.1 TR 0.3 0.4 0.3 1.8 0.8 0.2 1.4

species

Acid

from the Low

RRT”

Percentage

iso A9-16: 1 16:0 trans-16: 1~7‘ 16: 1~7 lOMe-16:O 16: 1015 is0 17:0 anteiso 17:0 iso A9-17: 1 anteiso 17: lh 17:o 17: 1(0X 17: 1~6 iso 18:0 18:O

0.443 0.48 1 0.530 0.543 0.545 0.565 0.593 0.626 0.652

0. I 13.8 0.7 27.6 0.8 TR 0.3 0.2 0.1 TR 6.5 13.1 I.1 0.1 0.4,

I .4

18:lw9

0.4 4.8 2.6 0.3 1.0

IOMe-18:0 trans-18: 107’ 18: 1~7 19: 108 v19:o (9,lO) v19:o (11,12) Unidentified

1.OY 1.10 1.11 1.12 1.57 1.57 1.60

0.694 0.767 0.795 0.855 1.00

TR Components less than 0.1”;. a Relative retention times (18:0 = 1.00) on capillary BDS at 17O.C. b Double bond positions unknown. ’ Tram acids only tentatively identified.

2.0 0. I TR 10.5 0.5 0.2 0.3 0.6

Fatty acids of bacterial origin in contemporary Table 5. Hydroxy-fatty acids from Low Isles Aerobic Heterotrophs (LIZ) Hydroxy Acid

Relative percentage

#?-OH-l&O iso+OH- 11: 0

13.2 2.7 0.8 21.3 4.7 34.8 TR TR 1.4 1.8 TR 1.9 0.4 0.4 1.4 0.8 0.8 TR 1.0 12.6

anteiso+OH-1

I:0

B-OH-1 1:O iso+OH-12:0 B-OH-12:O iso+OH-13:0 anteiso+OH-13:O &OH-13:O iso-B-OH-14:O /?-OH-14:O iso-fl-OH-15:O ante&-B-OH-15:O j?-OH-15:O iso-B-OH-16:0 /Y-OH-16:O iso-j-OH-17:O ante&-#?-OH-17:O fi-OH-17:O Unidenti~ed

TR Components less than 0.1%

Straight-chain also identified in this culture. j?-hydroxy acids occur in bacterial lipids as intermediates in the anaerobic biosynthesis of unsaturated acids (KATES, 1966) and as cell wall constituents (HANCOCEet al., 1970; BRAUN and HANTIE, 1974). The biosynthesis and function of branched chain /I-hydroxy fatty acids is unknown, however a similar series has recently been reported in Desulphovibrio desulfuricans @ON

et al., 1%‘).

Double bond positions of the even chain monounsaturated acids (Table 4) are all compatible with known biosynthetic pathways (BLOCH,1969). Heptadecenoic acid (17: 1~8) and other odd chain monounsaturated acids which,~llectively represent over 18% of the total fatty acids are not often reported in bacteria. However, of the few reports that deal specifically with fatty acids of marine bacteria, several note odd chain monounsaturated acids (BLUMERet al., 1969; OLIVER and COLWELL, 1973) and KUNIMOX) (1975) has suggested that high levels of odd chain acids may be a distinguishing feature of marine strains. The mixtures of isomers (036 and w8) for these odd chain monounsaturated acids found in the aerobic heterotrophs are consistent with biosynthesis from propionate via the anaerobic pathway (SCHLENK, 1972). Two isomers of the Cl,-cyclopropane fatty acid were detected in LIZ (Table 4) and the predominance of the 11, 12 isomer is expected due to the relatively high Ievel of 18: 107 from which it is biosynthesized (Lru and HOFMAN,1972). A series of branched chain unsaturated acids (iso A7-15:1, iso A9-16:1, iso A9-17:l and ante&o 17:l) was detected in the aerobic heterotrophs (Table 4). Branched chain unsaturated acids, although uncommon in bacterial lipids are known to occur in members of the genus Bacillus G.C.A. 43/l&B

marine sediments

1719

1971, 1977), in Desulphovibrio desulfuricans et al., 1977) and iso All-17:l was identified in the marine bacterium Flexibacter polymorphus (JOHNS

(KANEDA, (&ON

and PERRY,1977). The biosyn~esis and function of these acids has not been studied, however a rational biosynthetic pathway in bacteria from amino acid precursors via an anaerobic pathway is possible. Fatty acid compositions of two predominant colonies (LIZ-l and LIZ-2) cultured from the total aerobic heterotrophs (LIZ) are shown in Table 6. These two fatty acid profiles differ markedly in the proportions of branched chain and un~turated acids. The fatty acids of LIZ-2, dominated by anteiso 15:O (45%) and anteiso 17:0 (23Ok)are similar to those of Bacillus subtilus (KANEDA 1973), which is often a dominant organism in marine and estuarine sediments (WOOD, 1965). Fatty acids of the total anaerobic heterotrophs (LIAN, Table 7) are similar to those of the total aerobic heterotrophs (Table 4) with 16:lw7, 16:0 and 18 : 1~07predominating. A series of ~rans-monounsaturated fatty acids was detected in both the anaerobes (LIAN) and the aerobes (LIZ). These trans acids appear to. be genuine components of the bacterial extracts, howeve; further work is proceeding to establish their precise molecular structure and distribution. Although not common in bacteria, the presence of trans acids has been demonstrated in rumen bacteria where they are produced by biohydrog~ation of 18:2cu6 (KEMPet al., 1975). The fatty acids of a predominant yeast (LIY) cultured from this sediment are presented in Table 6. Unexpectedly it was found that almost 88% of the total fatty acids were branched, with iso- and anteiso15:0 clearly predominating. Also shown in Table 6 are fatty acids from a Thiobacillus sp. isolate (LITB), the lipids of which contain significant quantities of cyclopropane fatty acids (V17:O and V19:O). DISCUSSION This sediment sample from the Low Isles was a surface scraping (Top 3 mm) selected to not only take advantage of a high biomass content but also selected to minimize eucaryotic plant input (in particular mangrove root material) and to be su~ciently oxic to yield a high aerobic bacterial count. The entry of macroscopic marine organisms to this sediment is restricted by the shingle rampart surrounding the Isles (JACKSON,1971). However as the sediment is under direct tidal influence it is expected that marine detritus would contribute to the organic content of the sediment. Planktonic organisms exert significant influence on the fatty acid composition of this surface sediment particularly to the Cj6, C,,, CzO and Cz2 polyunsaturated fatty acids. Fatty acid analysis of a plankton tow (~RRY, 1977) conducted off the Isles revealed significant levels of 20:4w6, 20:503, 22:5w3 and 22:6w3 and the presence of 16:2u4 and 16:2w7 which are indicative of the presence of diatoms

G. J. PERRY. J. K. VOL.KMAN,R. B. JOHNS and H. J. BAVOH JR

1720

Table

6. Fatty acid compositton of sedimentary organisms from the Low Isles Sediment

Acid ._____i__~ 12:O is0 13:O anteiso 13:O 13:o is0 14:O 14:0 is0 15:0 ante&o 15:O 15:o 15 : 1d3 15: It06 iso 16:O 16:O 16: lto7 is0 17:0 ante&o 17:O 17:o v17:o (9. IO) 17: It08 17: 106 is0 1X:0 18:O I8 : I tu9 18: 1~7 V19:O (11. 12) Unidentified

LIZ-lh

Percentare LIZ-i

-

of total acids LITB’

0.5

0.2 0.1 0.1

0.5

0.5

0.6 0.3

5.0

0.7 5.X 44.6 0.6

4.8 0.3 0.3 1.4

9.x 3.2 0.9 5.5 23.4 0.4

0.2 19.0 8.1”

I.5 0.3 0.5 7.0 5.5 I.2 1.5 Il.1 25.0” 0.5 0.3 7.1 7’ 5 __.. 2.3 0.2 1.3 0.7 7.1

0.3 2.1 1.0 0.9

0.1

.._

1.0 6.6

13.1 I.5 14.5” 4.4 5.1

micro-

LIY 0.7 I.2 O? 0 I 3.6 0.7 28.7 34.6 1.4

7.9 4.7 1.3 4.0 X.0 o..l

1.5 0.6 0.6

’ Truns component also present. hAlso found 12: 1 (i.i’-;), 13.1 (0.7?;,), 14:l (0.80f0) and 19 : I(118 (0.77”). ‘ Also found 18:2o6 (7.1”;,): 19:O (0.6”$ and 20:0 (0.5”,,),

Table

7. Fatty

Acid

acids of total anaerobic heterotrophs (LIAN) Acid

Percentage

1o:o is.0 1t:O 12:o 12: It07 is0 13:O anteiso 13:0 is0 13:l” 13:o 13:108 is0 14:0 14:o 14: lw7 14: lw5 is0 15:0 anteiso 15 : 0

0.1 0.2 3.7 1.3 0.2 TR 0.2 0.4 0.3 0.4 3.9 0.3 0.1 4.0 1.5

iso A%15: 1 ante& 15. I” 15:o . is0 16:O 16:O trans-16: lcu?

0.4 0.1 2.6 0.9 17.9 1.5

TR Components less than O.lY,, BDouble bond position unknown. ’ Trans acids only tentatively identi

from Low Isles Sediment

Percentage

16: 107 16: lw5 lOMe-16:O is0 17:O anteiso 17:0 iso A9-17: 1 anteiso 17: 1” 17:o 17 : 1w8 17: lW6 v17:o (9,lO) iso 18.0 18:O

18.8 0.7 2.4 0,s 0.3 0.3 0.1 2.6 2.7 I.2 1.5 0.4 2.7

18:109 IOMe-18:O trans-18: 1~7~ 18: 107 19:o 19: lo8 v19:o (11,12) 20:o Unidentified

3.6 0.2 0.1 17.3 0.1 0.5 3.3 0.1 0.9

ed.

Fatty acids of bacterial origin in contemporary marine sediments (VOLKMAN and Jams,

1977). Mangrove vegetation appears to be the only significant higher plant input to this sediment and contributes particularly to the CIs ~l~n~~rated acids and the di~~boxylic fatty acids (VO~KMAN,1977; PRRRY1977). The contribution of organisms other than bacteria to this sediment and an analysis of the changes fatty acids undergo with depth will be discussed in a separate paper. A bacterial biomass as high as 1Omg per gram of sediment is indicative of a significant contribution of bacterial lipids to sedimentary fatty acids. The relative importance of this input from aerobic or anaerobic (or facultative anaerobic) mi~oorg~isms is difficult to assess, however the data presented in Table 3 suggests that aerobes predominantly contribute to the bacterial lipid content in this surface layer. To ascertain the wide range of fatty acids of bacterial origin found in this sediment, we have developed an approach whereby the fatty acid profiles of mi~oorg~isms cultured from sediment inoculums are compared to sedimentary fatty acids. Ideally one would seek biological markers for bacteria in the extracts. However, the extent to which particular acids can be used as bacterial markers depends on their uniqueness to bacteria, and this in turn depends on their distribution in other organisms. Probably the first organic compounds to be used as markers for bacterial contribution to sediments were the iso- and antei~br~ched chain fatty acids (LEO and Pm=, 1966; CoopER and BLUMER,1968). These acids are commonly encountered in bacterial lipids and a similar range was identified in the sediment (Table 1) and the bacterial cultures (Tables 4 and 7). These branched chain acids have been reported in other organisms including fungi (HARTMANet al., 1960; TYRRELL,1968), molluscs (ACKMANet al., 1971) and marine phytopl~kton (Crrun~~s and RILEY, 1969). Although not detectable in mangrove vegetation at the Low Isles (ONDER, 1976) these branched acids were identified in significant amounts in plankton (PERRY,1977) and a yeast (Table 6) isolated from the sediment. The fact that these acids are generally found in lower concentrations in other organisms than in bacteria, make them useful indicators of bacterial lipid contribution. Internaily branched acids such as l~methyl 16:O have been reported from a lake sediment, where they were thought to be of microbial origin (CRANWELL 1973,1974). lo-Methyl 16:0 was identified in the Low Isles surface sediment (Table 1) and this acid along with lO-methyl 18:0 has been found in a deeper Low Isles sediment sample. Both of these branched acids which are often reported in bacterial lipids were identified in the aerobic heterotrophs (Table 4) and in the anaerobic heterotrophs (Table 7). No 9-methyl branched acids were detected in this sediment, indicating that an abiological origin via cleavage of cyclopropane fatty acids is probably not occurring to any significant extent. Lipids of a number of filamentous fungi are also known to contain lo-methyl branched

1721

acids (CERNIGLIAand Pnaay, 1974). The importance of fungi in marine sediments is difficult to estimate; certainly in sediments at the Low Isles their numbers are lower than bacteria (Table 3) but it has been suggested that marine fungi may be more important than has generally been accepted (AHEARNet al., 1970; MEYERSet al., 1971; LITCHHELD and FL~ODGA~, 1975) and as noted above, the lipid content of some yeasts and fungi may be as high as 50% of dry weight. These mid-chain branched acids are not unique to bacteria, and hence cannot be considered as exclusively bacterial markers but they certainly are indicators of microbial ~ntribution to sediments. Cyclopropane fatty acids (VCt, and VCi9) have occassionally been reported in sediments (CRANWELL, 1974, 1976; ONDER,1976) but the position of the cyclopropyl ring has not often been determined. CRANWELL (1973) has however, identified both cis-11,12 and cis-9,lO isomers of VCi9 in a lake sediment. In this Low Isles sediment (Table 1) VI7:O (9,lO) and V19:O (11,12) were identified, whilst in the total aerobic heterotrophs, V19:O (11,12) was identified with a small amount of the 9,10 isomer (Table 4). Significant quantities of V17:O (9,lO) were found in the total anaerobic heterotrophs (Table 7) and in the Thiobacillus culture (LITB, Table 6). The most likely source of these sedimentary cyclopropanoid acids is from bacteriai lipids, in which they are often reported, particularly V19:O (11,12) which appears to be unique to bacterial lipids. As some plants contain cyclopropane acids, including V19:O (9,10) (YANOet al., 1972) it is essential to determine the position of the cyclopropyl ring before firm conclusions can be made about their origin in sediments. The more detailed our understanding of the chemical, and particularly stereochemical features of an organic substrate can become, the more likely it is that it can validly be used as a biological marker. Sedimentary monounsaturated fatty acids is one good case in point where a knowledge of isomers now shows that a hitherto unpromising lipid fraction can be useful in determining source organisms, Although isomer separation techniques have been known for some time, organic geochemists have rarely applied them to sedimentary extracts. Double bond positions for the majority of unsaturated acids in the Low Isles sediment are given in Table 1 with cts-vaccenic acid (18: 107) being the major C,s-monounsaturated acid to occur. In the majority of studies concerning sedimentary lipids, cis-vaccenic acid has not been separated from oleic acid (18: 1019) and other isomers which may be present, and the total of these has been simply reported as 18: 1, or worse, assumed to be entirely oleic acid. (F~ING~N and QUINN, 1973; JOHNSONand CALDER,1973; ONDER, 1976). Recently Eglinton and co-workers (BROOKS et al., 1976) appreciated that the 18: 1 found in sediments may include both oleic acid (18: 109), a common component of animals and higher plants, and cis-vaccenic acid (18: 1~7) an abundant bacterial constituent. During

II22

G. J. PERRY.J. K. VOLKMAN.R. B. JOHNSand H.J. BAVOK JR

characterization of mono-unsaturated acids from an estuarine sediment cis-vaccenic acid was detected in small quantities but no indication of its origin was given (VAN VLEETand QUINN, 1976). MATSUDAand KOYAMA(1977) reported cis-vaccenic acid in lacustrine sediment and we have recently identified this acid in a sandy intertidal marine sediment at Corner Inlet, Victoria. (VOLKMAN and JOHNS,1977.) High levels of cis-vaccenic acid were found in the aerobic heterotrophs (Table 4), the anaerobic heterotrophs (Table 7) and a Thiobacilhs species (Table 6) as well as in the Low Isles sediment (Table 1). Due to the high relative levels of cis-vaccenic acid in both the sediment and the bacterial cultures, and its virtual absence in mangroves, in this particular environment a bacterial origin seems certain. However, although cis-vaccenic acid is the dominant C1 ,-alkenoic acid in bacterial lipids, it has also been reported in other marine organisms including plankton (PERRY, 1977), eucaryotic algae (JOHNSet al., 1979), molluscs (ACKMANand HOOPER,1973, PERRY 1977) and fish (ACKMANet al., 1967) It appears that this acid has a wide distribution, as it is often detected in organisms which contain oleic acid (18: 1~9) and where isomer separation has been possible. Like iso- and anteisobranched acids cis-vaccenic acid appears to be present in higher relative concentrations in bacteria than in other organisms and hence in its own right is a useful biological marker for the occurrence of bacteria. It should be noted at this point that the mode of energy metabolism used by a microorganism does not determine the pathway of fatty acid biosynthesis (ERWIN and BLOCH,1964). In fact most photosynthetic bacteria use the anaerobic pathway regardless of whether they are grown aerobically or anaerobically (WOODet al., 1965). Hence the identification of unsaturated fatty acids presumably produced via an anaerobic pathway (~7 and w5 isomers particularly) in aerobically cultured bacteria is not unexpected (Table 4). Unsaturated acids like 18: 1 have been shown to be rapidly lost from many sediments both by labelling studies (RHEADet al., 1971) and by the observed depletion of unsaturated acids with depth (FARRINGTON and QUINN, 1973; JOHNSONand CALDER,1973; JOHNS et ul., 1978). However monounsaturated acids are often reported in sediments and have even been found in ancient sediments (PARKER,1969) suggesting that some degree of preservation can occur. Unsaturated acids derived from deposition of dead or senescius algae and higher plant material (including mangroves) are more susceptible to degradation and/or utilization than intracellular bacterial unsaturated acids like 18:lw7 which are protected from the sedimentary environment. Thus the unsaturated acids in sediments may partly be derived from the lipids of in situ sedimentary bacteria, particularly in sediments where 18: 107 can be identified. An odd carbon number series of monounsaturated fatty acids 13: 1-17: 1 have been identified in the Low Isles sediment (Table 1) and in both the aerobic and

anaerobic heterotrophs (Tables 4 and 7). For the Cl5 and Cl7 monoenoic acids, the same two isomers, w6 and w8 are present in both the sediment and bacteria. The aerobic heterotrophs contain a high proportion of 17: 1~8 (13%) and this is the predominant odd chain monoenoic acid in the sediment. There are a number of reports of odd chain monoenoic acids including 15: 1 and 17 : 1 in sediments (PARKER,1967; GASKELLet al., 1975; BRINKS et al., 1976) however double bond positions have not been specified. MATSUDAand KOYAMA(1977) identified a range of odd chain monoenoic acids in a lacustrine sediment, but did not speculate as to their origin. Although the abundance of the w6 and 08 isomers of 15: 1 and 17: 1 in other organisms at the Low Isles is not known, their presence in bacteria represents a major potential source for these acids in this sediment, particularly as pairs of isomers of monoenoic acids are indicative of the anaerobic pathway of biosynthesis, which is peculiar to bacteria. These odd chain monoenoic acids appear to be valuable markers for bacterial lipid input to this sediment, and taken as isomer pairs appear to be strong markers for bacterial input more generally. Branched chain unsaturated acids iso 15: 1 and iso 17: 1 have been found in a diatomaceous ooze where they were thought to be of bacterial origin (BOONet al., 1975) and recently three isomers of branched 17: 1 (A7, A9 and Al 1) have been reported from a lacustrine sediment MATSUDAand KOYAMA 1977). In the Low Isles sediment iso A7-15: 1 and iso A9-17: 1 are the two most abundant acids of this class identified (Table 1). In both the aerobic and anaerobic heterotrophs (Tables 4 and 7) these two acids were also found. The close correspondence between the sediment and the bacteria for this unusual class of acids suggests that they may provide excellent markers of bacterial contribution to sediments. Although the isomers mentioned above appear to be unique to bacteria, a number of other isomers have been identified in mammalian skin lipids (DOWNING, 1976), hence once again the need to determine the exact position of the double bond is emphasized. Our data show that these branched unsaturated acids are more widely distributed in bacteria than previously believed, hence the sedimentary occurrence of these acids cannot be exclusively equated with the presence of Desulfouibrio spp as suggested by Boon et al. (1978). All the monounsaturated fatty acids mentioned in the preceeding discussion were of cis configuration, however tram fatty acids were also identified in the sediment (Table 1) and in the bacterial cultures (Table 4 and 7) from the Low Isles. Two tram acids (16:l and 18:l) have been reported in an estuarine sediment where they were believed to be the result of a direct input or isomerization of cis-monounsaturated acids in the sediment (VAN VLEETand QUINN 1976). As mentioned earlier trans-monounsaturated acids are produced in some bacteria by biohydrogenation of 18:2w6 and it is possible that a similar

Fatty acids of bacteria1 or&in in contemporary

marine sediments

1723

process is operative in sediments, particularly anaerobic sediments @oON et al., 1978). However because

analyses of marine bacteria in the literature, which have generally not been performed in sufficient detail to allow useful comparison with sedimentary data. truns fatty acids were identified in the bacterial cul2. The technique of culturing mixed bacterial coltures examined here and the same but limited carbon number isomers as detected in the sediment appear to onies in low nutrient media can be useful in indicating be present, a direct input from bacterial lipids appears ~diment~y fatty acids which may be of bacterial origin. probable particularly since abiological diagenetic processes are unlikely to be operative in our environ3. Many sedimentary fatty acids previously thought ment. Truns-unsaturated acids may prove to be useful to be of bacterial origin do occur in sedimentary bacbacterial markers, and work is under way to establish teria. the precise identity of these acids in bacterial and 4. A wide range of ~diment~y fatty acids appear sedimentary lipids. ‘Duns 16: 1013 which is an importo be derived from bacterial lipids and a number of tant constituent of photosynthetic eucaryotes (WOOD, these can be considered as bacterial markers. 1974; JOHNSet al., 1979) is believed to arise in this 5. The exact molecular structure of many sedimensurface sediment from Phytoplankton (PERRY,1977). tary fatty acids must be known before firm decisions can be made about their origin. Hydroxy acids in this sediment although not reported in this paper, have been investigated briefly, and the presence of a number of branched chain Acknowledgements-The authors thank the Australian Research Grants Committee; G. J. PERRYand J. K. VOLK&OH (including iso- and anteiso-15:O and 17:0) have been demonstrated (VOLKMAN,1977). Hydroxy acids, MAN acknowledge Commonwealth Postgraduate Research Awards. Thanks are also due to Dr J. BAULD for culturing particularly iso- and anteiso$ hydroxy acids which the Thiobacillus isolates. appear to be limited to bacterial lipids, were detected in the aerobic heterotrophs (Table 5) and may provide REFERENCES valuable markers of bacterial input to sediments. ACKMANR. G. (1969) Gas-Liquid Chromatography of fatty Although no a,w-dicarboxylic acids were identified acids and esters In Methods in Enzymology Vol. 14, (ed. in the bacterial extracts reported here, it is believed J. M. Lowenstein), pp. 329-381 Academic Press. that some bacterial contribution via oxidative proACKMANR. G. (1972) The analysis of fattv acids and cesses to the sedimentary dicarboxylic acids (Table 2) related materials by Gas Liquid Chromatography. In may be occurring. Progress in the Chemistry of Fats and Other Lioids Vol. 12, (ed. R. T. Holman), pp. 165284 Per8amon Press. When attempting to identify sedimentary fatty ACKMANR. G. and CAS~LL J. D. (1967) A generalized acids of bacterial origin one must ideally obtain bacstudy of the effect of structure and other factors in Gpen terial cultures which closely represent the types and Tubular Gas Chromatography of fatty acids in a polyesdistribution of bacterial species in a sediment. This is ter liquid phase with particular reference to monoethylenic fatty acid isomers. J. Gas Chromat. S, 489-496. virtually impossible in practice. In addition to this ACKMANR. G. and Hooprat S. N. (1973) Non-methyleneproblem, bacterial fatty acid content can be both interrupted fatty acids in lipids of shallow water marine qualitatively and quanti~tively affected by variables invertebrates. A comparison of two molhtscs (&ttorina such as culture medium pH, temperature and age of iittorea and Lunafia triseriata) with the sand shrimp cells when harvested (KATESet al., 1961; KATE+, 1964; (Crangon septemspinosus). Comp. Biochem. Physiol. 46B, 153-165. O’LEAW 1962, OLI= and ~OLWELL,1973). However the fatty acid pattern of particular organisms can act ACKMANR. G., Smos J. C. and JANGAARDP. M. (1967) A quantitation problem in the open tubular gas chromaas a marker or a ‘fingerprint’ if the organisms are tography of fatty acid esters from cod liver lipids. Lipids grown without exogenous direct fatty acid precursors 2,251-257. present. A simple medium with insignificant levels of ACK~UN R. G., I&PER S. N. and KE P. J. (1971) The distribution of saturated and isoprenoid fatty acids in chain initiator precursors should meet the condition the lipids of three species of molluscs Litt~ri~ littarea, of total dependence on de now synthesis which is a Crussostrea uirginica and Venus mercenaris. Comp. Biorequisite to the use of this technique (KANEDA, 1977). them. Physiol. 39J& 579-587. It is believed that culturing mixed coionies and pure ACKMAN R. G., Hoom S. N. and HINGLEY J. (1972) cultures in this type of medium and extracting their Isomer subfractionation effects in the study of longer chain monoethyiene fatty acids in hydrogenated oils. f. fatty acids can indicate the types of fatty acids which Chromat. Sci. 10, 430-436. the bacteria have the potential to biosynthesise and AHEARND. G., MEMRS S. P. and YARROW D. (1970) Pichia this can assist the assignment of particular sedimensparrinae n. from Louisiana marshland habitats. Ant. tary acids to a bacterial origin. This type of study may Van ~~we~oek 36,503-508. be useful in assessing bacterial input to other classes AIBA S., HUMPHREYA. E. and M~LLISN. F. (1973) Biochemical Engineering, 2nd edn, University of Tokyo of organic compounds in recent sediments and it is Press. believed that the results presented here for bacterial ANDERSON R., KATE~ M., BAEDECKER M. J., KAPLAN I. R. marker fatty acids can be general&d to other sediand ACKMANR. G. (1977) The stereoisomeric composimentary environments. tion of phytanyl chains in lipids of Dead Sea sediments. Geochim. Cosmochim. Acta 41, 1381-1390.

SUMMARY AND CONCLUSIONS 1. This work points to the inadequacy of fatty acid

AYERSW. A. (1961) Oxidation of fats and fatty acids by marine bacteria. Bacterial. Proc. 61, 60. BAVOR H. J. and MIUIS N. F. (1976) Bacterioiogicai

1124

G.

J. PERRY,J. K. VOLKMAN,R. B. JOHNSand H. J. BAVORJR

Studies in Westernport Bay, Environmental Studies Series. Ministry for Conservation, Victoria. BLOCHK. (1969) Enzymatic synthesis of monounsaturated fatty acids. Ace. Chem. Res. 2, 192-202. BLUMERM., CHASET. and WATSONS. W. (1969) Fatty acids in the lipids of marine and terrestrial nitrifying bacteria. J. Bacterial. 99, 366-370. BOONJ. J., DE LEEUW J. W. and SCHENCKP. A. (1975) Organic geochemistry of Walvis Bay diatomaceous ooze-I. Occurrence and significance of the fatty acids. Geochim. Cosmochim. Acta 39, 1559-1565. BOCINJ. J., DE LEEUWJ. W. and BURLINGAME A. L. (1978) Organic geochemistry of Walvis Bay diatomaceous ooze-III. Structural analysis of the monoenoic and polycyclic fatty acids. Geochim. Cosmochim. Acta 42, 63144. BOONJ. J., DE LEEUWJ. W., V.d. HOEKG. J. and VOSJAN J. H. (1977) Significance and taxonomic value of iso and anteiso monoenoic fatty acids and branched b-hydroxy acids in Desulphouibrio desu@uricans. J. Bacterial. 129, 1183-1191. BRAUNV. and HANTKEK. (1974) Biochemistry of bacterial cell envelopes. Ann. Rev. Biochem. 43, 89-121. BROOKSP. W., EGLINTONG., GASKELLS. J., MCHUGH D. J., MAXWELLJ. R. and PHILP R. P. (1976) Lipids of recent sediments. Part 1. Straight chain hydrocarbons and carboxylic acids of some temperate lacustrine and sub-tropical lagoonal/tidal flat sediments. Chem. Geol. 18, 21-38. CERNIGLIA C. E. and PERRYJ. J. (1974) Effect of substrate on the fatty acid composition of hydrocarbon utilizing filamentous fungi. J. Bacterial. 118, 844-847. CHUECASL. and RILEYJ. P. (1969) Component fatty acids of the total lipids of some marine phytoplankton. J. Mar. Biol. Ass. U.K. 49, 97-116. COPPER W. J. and BLUMERM. (1968) Linear, iso and anteiso fatty acids in recent sediments of the North Atlantic. Deep-Sea Res. 15, 535-540. CRANWELLP. A. (1973) Branched chain and cyclopropenoid acids in a recent sediment. Chem. Geol. 11, 307-3 12. CRANWELL P. A. (1974) Monocarboxylic acids in lake sediments: indicators derived from terrestrial and aquatic biota of paleoenvironmental trophic levels. Chem. Geol. 14, 1-14. CRANWELLP. A. (1976) Decomposition of aquatic biota and sediment formation: organic compounds in detritus resulting from microbial attack on ihe alga Ceratium hirundinella. Freshwater Biol. 6, 4148. CROW S. A., COOK W. L., AHEARND. G. and BOURQIJIN A. W., (1976) Microbial populations in coastal surface slicks. In Proceedings of the 3rd International Biodegradation Symposium (ed. J. M. Sharpley and A. M. Kaplan), pp. 9398. Applied Science Publications. DALE N. (1974) Bacteria in intertidal sediments: factors related ‘to their distribution. Limnol. Oceanogr. 19, 509-518.

DEMEYERD. I., HENDERSONC. and PRINS R. A., (1978) Relative significance of exogenous and de novo synthesized fatty acids in the formation of rumen microbial lipids in vitro. Appl. Enuiron. Microbial. 35, 24-31. DOWNINGD. T. (1976) Mammalian waxes In Chemistry and Biochemistry of Natural Waxes (ed. P. E. Kolattukudy), pp. 17-48, Elsevier. EGLINTON G., HUNNEMAN D. H. ANDDOURAGHI-ZADEH K. (1968) Gas-chromatoaraDhic-mass suectrometric studies bf long chain hydrox‘; a’cids. II. Thd hydroxy acids and fatty acids of a So00 year old lacustrine sediment. Tetrahedron 24, 5929-5941.

ECLINTONG. (1973) Chemical fossils: a combined organic eeochemical and environmental aDDrOaCh. Pure Aool. 11 Chem. 34,61 l-632. ERWINJ. and BLOCHK. (1964) Biosynthesis of unsaturated fatty acids in microorganisms. Science 143, 100~1012. 1.

FARRINC~TON J. W. and QUINN J. G. (1973) Biogeochemistry of fatty acids in recent sediments from Narragansett Bay, Rhode Island. Geochim. Cosmochim. Acta 31, 259-268.

GASKELLS. J., MORRISR. J., EGLINTONG. and CALVERTS. E. (1975) The geochemistry of a recent sediment off North West Africa. An assessment of source of input and early diagenesis. Deep-Sea Res. 22, 777-789. GASKELLS. J., RHEADM. M., BROOKSP. W. and EGLINTON G. (1976) Diagenesis of oleic acid in an estuarine sediment. Chem. Geol. 17, 319-324. HANCOCK1. C., HUMPHREYSG. 0. and MEADOWP. M. (1970) Characterization of the hydroxy acids of Pseudomonas aeruginosa 8602. Biochim. biophys. Acta 202, 389-391.

HARTMANL., HAWKEJ. C., MORICE1. M. and SHORLAND F. B. (1960) The component fatty acids of Sporidesmium bakeri lipids. Biochem. J. 75, 274278. JACKWN K. S. (1971) M.Sc. Thesis, University of Melbourne. JAMIEXING. R. (1970) Structure determination of fatty esters by Gas Liquid Chromatography. In Topics in Lipid Chemistry Vol. 1, (ed. F. D. Gunstone), pp. 107- 159. Logos Press. JOHNSR. B. and ONDER0. M. (1975) Biological diagenesis: dicarboxylic acids in recent sediments. Geochim. Cosmochim.

Acta 39, 129-136.

JOHNSR. B. and PERRY G. J. (1977) Lipids of the marine bacterium Flexibacter polymorphus. Arch. Microbial. 114, 267-271.

JOHNSR. B., PERRYG. J. and JACKSONK. S. (1977) Contribution of bacterial lipids to recent marine sediments. Estuarine Coastal Mar. Sci. 5, 521-529. JOHNS R. B., VOLKMAN J. K. and GILLANF. T. (1978) Kero-

gen precursors: chemical and biological alteration of lipids in the sedimentary surface layer. The APEA Journal. pp. 157-160. JOHNSR. B., NICHOLSP. D. and PERRY G. J. (1979) Fatty acid composition of some marine algae from Australian waters. Phytochemistry 18, 799-802. JOHNWN R. W. and CALDERJ. A. (1973) Early diagenesis of fatty acids and hydrocarbons in a salt marsh environment. Geochim. Cosmochim. Acta 37, 1943-1955. KANEDAT. (1971) Factors affecting the relative ratio of fatty acids in Bacillus cereus. Can. J. Microbial. 17, 269.-275.

KANEDAT. (1973) Biosynthesis of branched long chain fatty acids from the related short chain fi-keto acid substrates by a cell free system of Bacillus subtilus Can. J. Microbial. 19, 87-96.

KANEDAT. (1977) Fatty acids of the genus Bacillus: an example of branched-chain preference. Bacterial. Rev. 41, 391418. KATESM. (1964) Bacterial lipids. Adu. Lipid Res. 3, 17-90. KATESM. (1966) Biosynthesis of lipids in microorganisms. Ann. Rev. Microbioi. 20, 1%44. KATESM., SEHGALS. N. and GIBBONSN. E. (1961) The lipid composition of Micrococcus halodenitrijicans as influenced by salt concentrations. Can. J. Microbial. 7. 427435.

KEMPP., WHITER. W. and LANDERD. J. (1975) The hydrogenation of unsaturated fatty acids by five bacterial isolates from sheep rumen, including a new species. J. Gen. Microbial. 90, 100-l 14. KING J. D., WHITED. C. and TAYLORC. W. (1977) Use of lipid composition and metabolism to examine structure and activity of estuarine detrital microflora. Appl. Environ. Microbial. 33, 1177-1183. KUNIMOT~M. (1975) Lipids of marine bacteria. II. Lipid composition of bacteria of marine type. Bull. Fat. Fish. Hokaido Univ. 25, 342-350.

KUZNET%XS. I. (1975) The role of microorganisms in the formation of lake bottom deposits and their diagenesis. Soil Sci. 119, 81-88.

Fatty acids of bacterial origin in contemporary LEO R. G. and PARKERP. L. (1966) Branched chain fatty acids in sediments. Science 1‘52, 649650. LEESA. M. and KORN E. D. (1966) Senaration of nositional

isomers of long-chain unsaturated fatty acid methyl esters by thin-layer chromato~aphy. The presence of mono-nuns di-cis isomers in commercial linolenic acid. Biochim. Biophys. Actu 116,4033406. LITCHFIELD C. D. and FLOODGATE G. D. (1975) Biochemistry and microbiology of some Irish sea sediments. II. Bacteriological analysis. Mar. Biol. 30, 97-103. LITCHFIELD C. D., RAKEJ. B., ZINDULISJ., WATANABE R. T. and SmIN D. J. (1975) Optimization of procedures for the recovery of heterotrophic bacteria from marine sediments. Microb. EC&. 1, 219-233. Ltu T. Y. and HOFMANN.K. (1962) Cyclopropane ring biosynthesis. Biochemistry 1, 189-191. MATSUDAH. and KOYAMAT. (1977) Positional isomer composition of monounsaturated fatty acids from a lacustrine sediment. Geochim. Cosmochim. Acta 41, 341-345. MCCLOSKEYJ. A. and LAW J. H. (1967) Ring location in cyclopropane fatty acid esters by a mass spectrometric method. Lipids 2, 225-230. METCALFEL. D. and SCHMITZA. A. (1961) Rapid preparation of fatty acid esters for gas Chromato~raRhic _ _ analysis. Anaf. Chem. 33, 363-364. MEYERSS. P.. AHEARND. G. and MILESP. C. (1971) Characterization of yeasts in Barataria Bay. Louisiana State Univ. Coastal Studies Bull. No. 6, 7-15. MORRISL. J.. WHARRYD. M. and HAMMONDE. W. (1967) Chromatographic behaviour of isomeric long-chain ali: phatic compounds. II. Argentation thin-layer chromatography of isomeric octadecenoates. J. Chromat. 37, 69-76. MORRISN R. I. and BICK W. (1966) Long chain methyl ketones in soils. Chem. Ind. 596-597. OGINSKIE. L. and UMBREITW. W. (1959) An introduction to Bacterial Physiology, 2nd edn, Freeman. Q’LEARYW. M. (1962) Fatty acids of bacteria. Bacterial. Rev. 26, 421447.

OLIVERJ. D. and aLWELL R. R. (1973) Extractable lipids of gram negative marine bacteria. Fatty acid Composition. Int. J. Syst. Bacterial. 23, 442458. ONDER 0. M. (1976) Ph.D. Thesis University of Melbourne. OPP~N~EI~R C. H. and ZOBELLC. E. (19523 The erowth and variability of sixty-three species of marine bacteria as influenced by hydrostatic pressure. J. Mar. Res. 11, 10-18. OPPENHEIMER C. H. (1960) Bacterial activity in sediments of shallow marine bays. Geochim. Cosmochim. Acta 19, 244-260. PARKERP. L. (1967) Fatty acids in recent sediments. Cancrib. Mar. Sci. 12, 135-142. PARKERP. L. (1969) Fatty acids and alcohols. In Organic Geoc~emisrr~ feds G. Eglinton and M. J. J. Mu~hy~ pp. 357-373. Springer Verlag. PERRY G. J. (1977) Ph.D. Thesis, University of Melbourne.

marine sediments

1725

POLTZV. J. (1972) Investigations on the occurrence and decomposition of fats and fatty acids in lakes. Arch. Hvdrobiol. Supal. 40, 315-399. RHE~D M. M., ~GLINTOON G., DRAFFANG. H. and ENGLANDP. J. (1971) Conversion of oieic acid to saturated fatty acids in Severn estuary sediments. Nature 232, 327-330. RODINAA. G. (1972) Methods in Aquatic Microbiology, pp. 344347. Univ. Park Press Baltimore. ROSENFELD W. D. (1946) Lipolytic activities of anaerobic bacteria. Archs Biochem. 11, 145-154. SCHLENKH. (1972) Odd numbered and new essential fatty acids. Federation Proc. 31, 14uT1435. SCHULTZD. M. and QUINN J. G. (1973) Fatty acid composition of organic detritus from Spartina a~terni~oru. Estuarine Coastal Mar. Sci. 1, 177-190. TYRRELLD. (1968) The fatty acid composition of some Entomophthoraceae. II. The occurrence of branched chain fatty acids in Conidiobolus denaesporus. Lipids 3, 368-372.

VANDERPOST J. M. and DUTKA B. J. (1971) Bacteriological study of Kingston Basin sediments. Proc. Conf Great Lakes Res., Internat. Assoc. Great Lakes Rex, 14, 1377156. VAN D~RSSELAERA., ENSMINGER A., SPYCKERELLE C., DA.+ TILLUNGM., SIESKINDO., ARPINOP., ALBRECTP., OURISSONG.. BR~OICSP. W., GASKELLS. J.. KIM~LE B. J.. PHILP R. P., MAXWELLJ. R. and ECLII&N G. (1974) Degraded and extended hopane derivatives (C,, to C9s) as ubiauitous neochemical markers. Tetrahedron Lett. 14, 1346-1352. VAN VLEET E. S. and QUINN J. G. (1976) Characterization

of monounsaturated fatty acids from an estuarine sediment. Nature 262, 126128. VOLKMANJ. K. (1977) Ph.D. Thesis, University of Melbourne. VOLKMANJ. K. and JOHNS R. B. (1977) The geochemical significance of positional isomers of unsaturated acids from an intertidal zone sediment. Nature 267, 693-694. WHITE D. C., BOBBIER. J., MORRNN S. J., O~STERHOFD.

K., TAYLORC. W. and MEETERD. A. (1977) Determination of microbial activity of estuarine detritus by relative rates of lipid biosynthesis. Limnol. Oceanogr. 22. 10891099. WOODE. J. F. (1965) ~ar~ne ~icrobjo~ Ecology. Chapman & Hall. Woof B. J. B. (1974) Fatty acids and saponifiable lipids. In Botanical Monographs, Vol. 10, (ed. W. D. P. Stewart). Blackwells. WC~~DB. J. B., NICHOLSB. W. and JAMESA. T. (1965) The lipids and fatty acid metabolism of photosynthetic bacteria. Biochim. Biophys. Acta 106, 261-273. YANO I., NICHOLSB. W., MORRIS L. J. and JAMES A. T. (1972) The distribution of cyclopropane and cyclopropene fatty acids in higher plants. Lipids 7, 30-34. ZOBELLC, E. (1975) MicrobiaI activities and their interdependency with environmental conditions in submerged soils. Soil Set. 119, l-2.