The relationship between fatty acid distributions and bacterial respiratory types in contemporary marine sediments

Estuarine,

Coastal

and

Shelf

Science

(1983)

16,

173-189

The Relationship Between Fatty Acid Distributions and Bacterial Respiratory Types in Contemporary Marine Sediments

R. J. Parkes and J. Taylor Dunstaflnage Marine Research Argyll, Scotland, PA34 4AD, Received

22

April

1982

Keywords: bacteria;

Laboratory, U.K.

and in revised

P.O.

form

Box

z August

fatty acids; sediments;

3, Oban,

1982

chromatography;

Scottish

coast Bacterial communities of different respiratory types were isolated from a marine sediment in a multiple chemostat system in an attempt to obtain bacterial cultures representative of the sediment. The fatty acid distribution of a mixed culture of sulphate-reducing bacteria isolated from this system showed a good correlation with the lipid distribution of the zone of maximum sulphate-reduction activity within the sediment. Both distributions had significant concentrations of C14 : o, iso and anteiso C15 : o, C16 : o, C16 : 107 and CI~ : 107. This was in contrast to the lipid profile of Desulfovibrio deszdfuricans obtained by batch enrichment, which was dominated by iso C I 7 : I 07 fatty acids and correlated poorly with the sediment. The bacterial community cultured from the sediment was further differentiated according to respiratory types (aerobic, facultative aerobic and facultative anaerobic) by growth in a chemostat under defined conditions. The fatty acid distributions of these communities were sufficiently different to enable the various respiratory types to be recognized by their particular fatty acid composition. Cyclopropyl fatty acids (~17 and ~19) were present in significant levels only in the aerobic bacteria, while the facultative aerobes had significantly higher CI~ : 1017 than the other cultures and the facultative anaerobic community was the only culture to have significant amounts of CIZ : o. The fatty acid distribution of Loch Eil sediment over the range C12-19 seemed to be predominantly of bacterial origin and were relatively abundant to a depth of 6 cm. The concentration of cyclopropyl fatty acids was highest in the oxidized surface sediments and decreased with depth as anaerobic conditions began to dominate, indicating that these fatty acids may only be indicative of aerobic sedimentary bacteria, In contrast to the fatty acids characteristic of bacteria, those fatty acids in the range Czo-30 attributed to a terrestrial input were found in relatively constant concentrations over the whole o-12 cm depth profile.

Introduction Kecent sediments play a vital role in the mineralization reactions that are an essential part of the cycles for nitrogen, phosphate, carbon, sulphur and other nutrients and also in the early stage diagenesis of detritus entering the geosphere. These reactions are mediated by a diverse mixture of both aerobic and anaerobic microorganisms. The individual anaerobic bacteria 173 0272~7714/83/020173+

18

$03.00/o

@ 1983 Academic Press Inc. (London)

Limited

R. J. Parkes H J. Taylor

‘74

involved in this mineralization processtend to have restricted metabolic potential and hence completemineralization is brought about by associationsof physiologically different types of microorganisms(Jorgensen,1980) operating asmicrobial communities. The activity of these communitieswithin marine sedimentshas beenshown to be vertically stratified, reflecting a changefrom aerobic to anaerobic conditions and associatedwith the availability of different electron acceptors(e.g. O,, NO,-, SOt2- and CO,; Sorensenet al., r,979). However, there is little information on the bacteria involved in these communities reflecting different respiratory activities. Microscopic analysis of the sediment can provide data on bacterial distribution but morphology gives very little indication of the types of organismspresent. Culture techniques using selective media can provide information concerning the types of bacteria present, but can both distort and underestimate the in situ population (see Discussion). Analysesof lipids, especiallyfatty acids,have beenof great value in understandingbacterial phylogenic and taxonomic classifications(Lechevalier, 1977). Fatty acidshave alsobeen used in an attempt to study the contribution of bacterial lipids to sedimentary lipids (Perry et al., 1979; v;.n Vleet & Quinn, 1979), the role of bacteria in metabolizing allochthonous lipids (Gaskell et al., 1976), and the successionalchangesin microbiota associatedwith detritus (Cranwell, 1976a, b; King et al., 1977). They may therefore be useful in investigating the complex community interactions within marine sediments. Bobbie & White (1980) have recently usedfatty acid analysisin this context to assess the relative contribution of eucaryotes and procaryotes to the benthic microbial community. In this study we have attempted to usefatty acid analysisto distinguish between different types of procaryotes within a marine sediment. Bacterial communitiesof different respiratory types were segregatedin a multiple chemostatsystemin an attempt to overcome someof the problems associatedwith more traditional culture techniquesand the fatty acid distributions obtained were comparedwith those of a marine sediment. Experimental Sampling

Sediment cores approximately 60 cm in length were obtained, using a Gravity Corer, from Station E24 in the deep basin of Loch Eil, an enclosedsealoch on the Scottish west coast. Loch Eil is approximately IO km long by 1.5 km wide and reaching 70 m at its deepestpoint [seePearson(1981) for further description of general site and Stations E24 and LYI]. The cores for fatty acid analysis were frozen, sectioned at z-cm intervals from 0-12 cm and stored at -25 “C prior to analysis.Sedimentcoresfor microbiological analysiswere obtained using a Craib corer (Craib, 1965) with 15 mm diameter holes drilled down the side of the core barrel to allow sedimentto be removed by syringe with minimal exposure to air. Cores were analysedon the day of collection. Isolation

of bacterial

communities

Bacteria were isolated in four continuous culture vesselsconnected in series(Plate I). Each vesselconsistedof a quickfit vessel(I 1) and top, with stainlesssteel tubing for temperature control, gassing,sampling,medium outflow, and a magnetic stirrer for mixing. Connections between vesselswere made with flexible neoprene tubing. Gas flow was controlled by rotameters and medium flow by peristaltic pumps (Watson Marlow Ltd., Falmouth, U.K.). Gaseswere sterilized by filtration (Microflow Ltd., Hants, U.K.) and medium by autoclaving (120

“C).

Plate growth

I. Multiple vessels.

chemostat

system.

A,

medium

pump;

B, medium

in;

C, medium

out;

I, 2, 3 and

4,

Fatty acid distributions

and bacterial respiratory

types

‘75

Medium composition (in g 1-l) was glucose I, cellulose 0.1, yeast extract 0.5, NH,NO, 0.001, NH&l 0.5, K,HPO, 1.4, KH,PO, 0.35, EDTA disodium salt 0.067, MgC1,.6H,O 7, FeS0,.7H,O 0.05, NaCl 20.1, Resazurin I ml of a 0.1% w/v solution and I ml of trace elements solution. Trace elements solution contained (in g l-l), NaHCO, 2, MnS0,.4H,O 12, ZnS0,.7H,O 7.2, (NH,),Mo,0,,.4H,O 0.04, CuSO,.sH,O 1.23, Al,(S0,),.16H~0 0.2, CoCl,.6H,O 2.4, FeSO,.7HaO 7, H,BO, 0.31, CaCl, 2.2 and EDTA disodium salt 7.5. The pH of the medium was adjusted to 7.2 with 6-N NaOH prior to autoclaving. Aerobic medium was pumped into Pot I at a rate of 3 ml h-l and the volume of Pot I was set at 650 ml. From a second reservoir Na,SO, (2.15 g 1-l) was pumped into Pot I at a rate of 2 ml h-l in order to provide a sulphate concentration of approximately 6 ITIM. The combined flow rate into the multiple vessel system was 5 ml h-l giving a dilution rate of 0.008 h-l in Pot I (650 ml) and 0.006 h-l in the remaining three pots (900 ml working volume). Nitrogen (oxygen-free) was passed over the headspace of all the vessels at a rate of 60 ml min- l and air was pumped into the culture of Pot I at a rate of I ml min- l. Pot I was autoclaved with 650 ml of complete medium (including 6 mM SO,) and allowed to cool in air. Pot 2 was autoclaved with 900 ml of complete medium but cooled under nitrogen. The other vessels were autoclaved without medium. The pots were all connected and flow from both reservoirs started, then Pots I and 2 were inoculated each with 2 ml of sediment from the surface (o-5 cm) of the deep basin of Loch Eil. Pots 3 and 4 were allowed to fill up slowly from the previous vessels and, when at working volumes, were also inoculated with sediment. This was in order to try and overcome possible substrateaccelerated death of bacterial isolates. Viable counts were obtained by growing bacteria in the complete medium plus 2”, (w/v) agar either in air or in an anaerobic cabinet. Direct counts were conducted using a Helber Counting Chamber with phase contrast microscopy. Bacterial communities from Pot I were grown up for lipid analysis in a 2.5-l chemostat (L.H. Engineering, Stoke Poges) with pH control (between 6.0 and 6.5), with identical medium to that used in the multiple vessel system and identical dilution rates. Three different bacterial communities were obtained from Pot I by growing these organisms under different and (iii) anaerobically; these conditions: (i) aerobically, ( ii ) aerobically then anaerobically were termed aerobic, facultative aerobic and facultative anaerobic bacteria, respectively. In order to enable selection of the desired bacterial types, two pot volume changes were allowed before the culture was harvested for lipid analysis. Only the bacteria in the growth vessel and not the collection vessel were analysed. All incubations were conducted at 25 T. The mixed culture of sulphate-reducing bacteria was obtained from the collecting vessel at the end of the series where significant concentrations of sulphide were produced and there was sufficient biomass for lipid analysis. Similar concentrations of sulphide were also present in Pots 3 and 4 of the series but as the prevailing culture conditions for these were not known (see Discussion) they could not be grown to sufficient biomass for lipid analysis. DesuIfoaibrio desulfuricans were isolated from the surface sediment (o-5 cm) of the deep basin of Loch Eil using Postgates liquid medium B, further purification and indentification was conducted as described by Postgate (1979) except that the ‘roll-tube’ technique of Hungate (1969) was used for growth on solid medium. Activity of @hate-reducing bacteria Undisturbed sediment sub-cores (5 ml) were obtained from different depths in the sediment through the pre-drilled holes in the core tube using a s-ml syringe from which the luer fitting had been removed. The end of the syringe was then stoppered with a suba-seal and

176

R. J. Parkes & J. Taylor

20 pl of 35S-S0,2- (equivalent to I pCi) injected evenly through the sub-core by slowly withdrawing the syringe during injection. The syringes were then incubated for 24 h at zg “C. Activity wasstopped by freezing the sub-coreswhich were stored frozen until subsequent analysis. The 35S-S2- produced during the incubation was analysed in a similar manner to that describedby Jorgensen(1978) and counted in a scintillation counter using an external standard for quench correction. A parallel core was sectionedand squeezedunder nitrogen through a membrane filter and sulphate (Howarth, 1978) and sulphide (Cline, 1969) analysed.The rate of sulphate reduction was calculated using the formula quoted by Jorgensen(1978). Eh measurements

Eh was measuredusing a combined reference and platinum electrode which was slowly pushedinto the intact sediment core at known depths (Pearson& Stanley, 1979). Extraction

techniques

All solvents were redistilled in glass.Glasswarewas chromic acid-cleaned,rinsed five times in doubly distilled water and dried at I IO “C. Solvent-extracted materialswere usedthroughout subsequent procedures. Preparative thin layer chromatography (TLC) plates 20 x 20 cm and 0.5 cm thick were pre-eluted with ethyl acetate. Bacterial cultures were harvested by centrifugation (M.S.E. High Speed) at 34 ooog for 20 min and the lipids extracted by a modified Bligh and Dyer procedure (Boon et al., 1977~). The lipids were recovered in the chloroform phase,and the fatty acidsreleasedby refluxing for 3 h with 2-M KOH in methanol (50 ml). After cooling, methanol wasremoved under vacuum, 40 ml of distilled water added and the mixture extracted with three 3o-ml portions of methylene chloride to remove non-acidic components. The fatty acids were obtained from the aqueouslayer by acidification to pH 2 with concentrated KC1 and extracted with three 3o-ml portions of methylene chloride. After removal of the solvent under vacuum, the acids were methylated with BF,/CH,OH (Metcalfe & Schmitz, 1961). The monocarboxylic acid methyl esters(R, = 0.56-0.69) were purified by TLC on silica gel (Merck 7731) eluting with hexane : diethyl ether (9 : I v/v). Sediment samples were freeze-dried and soxhlet extracted for 48 h with benzene : methanol (I : I, 500 ml). The solvent was concentrated to approximately IOO ml under vacuum and saponifiedby refluxing for 2 h in 2-M KOH in methanol (200 ml). After cooling, water (200 ml) was added and the neutral lipids extracted with four Iso-ml portions of chloroform. The remaining aqueousphasewasacidified to pH 2 with concentrated HCl and the fatty acids extracted with four roe-ml portions of chloroform. The combined extracts were washedwith two so-ml portions of water and evaporated under vacuum to yield the crude fatty acids. To ensurecomplete recovery of extracted sedimentary acids the neutral fraction waswashedwith dilute HCl and subjectedto column chromatography on silica gel (Merck 9385) impregnated with KOH (McCarthy & Duthie, 1962). The small amount of fatty acids isolated in this way was combined with the major fatty acid isolate, methylated and thin-layer chromatographedas previously described. Fatty acid nomenclature

The length of the carbon chain is indicated by a number following the symbol C for carbon. The degreeof unsaturation is given by a number separatedby a colon from the chain length. The position of unsaturation is defined by the number of carbon units from the methyl end of the chain by the symbol w followed by a number (e.g. C16 : 1w7 is a straight chain fatty

Fatty acid distributions

and bacterial

respiratory

types

177

acid of 16 carbon units with one double bond (monoenoic)situated sevencarbon units from the methyl end). Methyl branching occurring one carbon unit from the methyl chain end is designatediso and two carbon units from the methyl chain end anteiso. The presenceof a cyclopropane ring in the fatty acid is indicated by the symbol V. Fatty acid analysis

Fatty acids were analysedastheir methyl estersby gas-liquid chromatography (GLC) using a glass capillary MEGA OV-I column (25 m, O.I-0.15 pm, Erba Science) and MEGA carbowax zoM (25 m, 0.1-0.15 urn, Erba Science) in a Carlo Erba FTV 4160 gaschromatograph with hydrogen carrier gas at a flow rate of 2.5 ml min-‘. The OV-I column was programmed from 70-290 “C at 4 “C min-’ and the carbowax 2oM column from 70-210 “C at a similar rate. Flame ionization detection wasusedand quantitation obtained by integrating peak areasusing a computing integrator (Hewlett Packard 1360).Multiple injection of fatty acid standards gave a 99% confidence for the percentage peak areaswith a &0*033% variation. Mass spectral data were obtained by a computer-linked gaschromatograph-mass spectrometer (GC-MS) using a Finnigan 4000 instrument under standard conditions (Volkman et al., 1981). The fatty acids were identified from their GLC retention times and where possible coinjection with standards(Applied ScienceLaboratories) on both OV-I and carbowax 2oM columns, together with massspectral fragmentation patterns. The massspectral resultswere in accordancewith data published by Ryhage& Stenhagen(1963) and Douglaset al. (1971). Iso and anteisomethyl branchesin the saturatedmethyl estersshowedenhancedintensity of fragments {M+-43} and {M+-57}, with the anteisoisomersshowing additional fragment ions {M+-61) and {M+-79) and intensity reversalof fragments {M+-29) and {M+-31). The straight chain monoenoic and cyclopropyl methyl esters were characterized by their parent molecular ion and high intensity fragment ions at {M+-32}, {M+-74) and @I+-I 16). The latter also showed enhanced abundance of the parent ion relative to {M+-32) (Campbell & Naworal, 1969) and was separablefrom its monoenoic C17 : I isomer by AgNO, TLC. Iso and anteisoC17 : I methyl estersare characterizedby fragment ions m/e 227, 195, 177 and 213, 181, 163 respectively (Boon et al., 19773) with comparable fragments in the massspectra of iso and anteiso CI~ : I and Cx3 : I. roMe-Cr6 : o was identified from its molecular ion and characteristic fragment ions m/e 167, 171, 172, 173 and 199 (Cranwell, 1973; Apon & Nicolaides, 1975). The triterpenoid acidswere identified from their GLC retention times and massfragmentogramsm/e 191, 249, 263, 277 (Boon et al., 1978; Quirk, 1980). Results Characteristics

of bacterial

communities

isolated frcm Loch Eil sediments

The main characteristicsof the communitiesobtained from Pot I are shownin Table I. The bacteria initially isolated in Pot I had approximately equal proportions of both aerobesand anaerobes.When the community was grown under aerobic conditions in the chemostat, a community was selectedthat was dominated by aerobic bacteria (95%); anaerobicbacteria representedonly a small part of the total community. The total bacterial count alsoincreased markedly, probably indicating that the organic substratecould be converted more efficiently into cell biomassunder full aerobic conditions, and that in Pot I a significant amount of the carbon input was incompletely oxidized providing growth substratesfor the next vesselin

178

R. J. Parkes

& J.

Taylor

I. Characteristics

TABLE

of bacterial

communities

from

Pot

I of the

multiple

chemostat Bacterial

__-______ -___-___ Total counts x IO’ ml-r Total viable counts x IO’ ml-’ % of viable aerobic % of viable anaerobic

TABLE 2. Fatty sediment

acid

Pot I

Aerobic

23

161

20.8 59 41

composition

of bacterial

Percentage

Acid

Aerobic

-

of total

Fat. aerobic

-~

communities Facultative aerobic 4o

32’5

20.9 9.5

28.3 48

24 44

5

52

56

cultures

acids

0'12 0’01

-

-

0.14

0’71

tr

tr

14:107 14 :o

0’20

0.13

3’57

is015 :I anteiso I5 : I is0 15 : 0 anteiso 13 : 0 15 :o iso 16 : o

0.17

I.82 0’07

0’02

0’02

4’15 4‘53

I’47 2’73 0.56 0’30

is0 I3 : I anteiso 13 : I is0 I3 : o anteiso I3 : 0 is0 I4 : 0 ‘4 : 103

1’15 0‘22

16:1o7 16:x05 16:o

27’45 2'01 22.04

is0 17 : I anteiso 17 : I is0 I7 : o anteiso 17 : 0 V 17 :o 17

:1o8 :o iso 18 :o 18:109 18:107 18:rog 18 :o

‘7

“The

important

fatty

0'09 -

I .67 0.03

0.92 7'30 I'39 0'09 2.31 I.80 0'23 0.71

0.31 3’59 0.19 0'10

3’54 13’41 ’ ‘44 0’41

0.26

0’02

0.79 0’35 9.60 0.26 0’14 0.13 0.18

O’I3 I .80

0’10

9.76

0'45

0.16

0.24

tr tr tr -

23’58 0.98 2.06

3.96 2.14 0.43

2.36

49'73 -

32.07 3’53 18.26

19'74 3'04 0.38

I.10

O‘II 0’25

0.09

0.32

tr

I.13 I ‘85

acid concentrations

-

tr

1.06

unknown V19:o 19 :o

0’47

19‘07 3’32 26.88 0.36

12’94 0.27 6.68

from

Fat. Sulphateanaerobic reducing ____-__5.61 0.68 0'07

:o

isolated

in bacteria’

0.15 -

I2

Facultative anaerobic

0’57 0.42

tr 0.23

tr 0'54 14'37 0.57 0.68

0’20 -

are shown

0'54 -

0'13

in bold

type.

Loch

Eil

Fatty acid distributions

and buctekl

respiratory

types

179

(d)

%

30

20

Ii L-Li i

IO

‘8

14

i i

16

I !

i

;

i

i I8

14 Carbon

16

18

number

Figure I. Fatty acid distributions of mixed bacterial cultures. (a) Aerobic bacteria, (b) facultative aerobic bacteria, (c) facultative anaerobic bacteria and (d) sulphatereducing bacteria. -, Saturated fatty acids; - . - ., mono-unsaturated; - - -, branched; . . ., cyclypropyl

the series.When the sameculture wasgrown under anaerobicconditions to obtain facultative aerobestotal counts decreasedand a community developed from the original aerobic culture with approximately equal proportions of the bacteria being able to grow under aerobic and anaerobic conditions. When the community in Pot I was grown under anaerobic conditions a community developed in which 56% of the bacteria grew only anaerobically. Direct counts under these conditions were then lower than those of either the aerobic or facultative aerobic bacteria, although higher than in Pot I. The number of total viable bacteria remained remarkably constant in all the cultures despite the proportion of this total able to grow under aerobic or anaerobicconditions changing considerably.

Fatty acid composition

of the isolated bacterial

communities

The fatty acid composition of the four mixed cultures of bacterial respiratory types isolated from Loch Eil sediment are presented in Table 2. In each casethe acids fall in the CIZ-19 range, commonly found in bacteria (Kates, 1964; Shaw, 1974), with even-chain saturated and mono-unsaturated acidspredominating, particularly C16 : o, C16 : 1w7 and C18 : I 07, which account for more than 60% of the total fatty acids in all four cultures. Monounsaturated acids in all the cultures, including the aerobic bacteria, exhibited positional

R. J. Parke,

180

isomers

indicative

@ J.

of biosynthesis

Taylor

by the anaerobic

metabolic

pathway

(Erwin

& Bloch,

1964). The major compositional differences found between the fatty acid profiles of the mixed bacterial cultures examined are highlighted in Figure I. Aerobic and facultative aerobic cultures contain approximately equal amounts of saturated and mono-unsaturated C16 together with significant quantities of cyclopropyl C17 and C19 fatty acids. In contrast, the 16

(0) 35

26

37

33

(cl

(b) 14

6 24

16

L---

Ic 3

I

200

150

100 Figure sediment

z. Gas-liquid chromatograms of fatty acid (E24, 4-6 cm), (b) sulphate-reducing desulfuricans. Peak numbers refer to Table 4.

methyl bacteria

esters and

from

(c)

(a)

Loch

Desulfowibrio

Eil

Fatty

acid

distributions

and bacterial respiratory

181

types

facultative anaerobicand sulphate-reducing cultures contained approximately twice asmuch C16 : 1w7 as saturated C16 : o fatty acid and only trace amounts of C17 and C19 cyclopropyl acids. Branched chain anteisoCI~ : o fatty acid was in comparatively high abundance only in the sulphate-reducing culture. A gas-liquid chromatogram of the fatty acid methyl estersobtained from the mixed culture of sulphate-reducing bacteria is shown in Figure 2(b) (peak numbers refer to Table 4). Fatty acid camposition of the sulphate-reducing bacteria (D. desulfuricans) isolated from Loch Eil sediment The fatty acid composition of a pure culture of D. desulfuricans isolated from Loch Eil

sediment was analysedfor comparisonwith the mixed culture of sulphate-reducing bacteria isolated from the samesediment. Figure 2(c) shows the gas-liquid chromatogram of the extractable fatty acids. Their relative abundancesare presented in Table 3. The observed fatty acid distribution closely resemblesthat of a D. desulfuricans strain isolated from the Dutch Wadden Sea (Vosjan, 1975) and analysed by Boon et al. (1977a). The most striking feature of the fatty acid distribution of this bacterium was the unusual abundanceof monoenoic branched chain fatty acids with iso c17 : I 07 being the major component. 3. Fatty isolated

TABLE furicans)

Acid -______‘4 :o is0 15 anteiso is0 15 anteiso 15

acid composition of the sulphate-reducing from Loch Eil sediment Percentage”

(D.

desul-

Percentage

--~ I.92

: 1w7 15 : *CO7 :0 15 : 0

2’53 0.18

is0 17 : 0 anteiso 17 : 0 17

:o

18 18

: 1~09 : 1017 18 :o is0 19 : 1w7

0‘02 0.40 O’II

16 : 1017 16 :o

0.56 4’47 43.08 4’72

is0 17 : 107 anteiso 17 : 107 important

Sulphate-reducing

fatty

:o

iso 18 : 1~7 iso 18 : o

‘5’97 “5’

iso 16 :107 iso 16 : o

“The

Acid

bacterium

anteiso I 9 : I (07 is0 19 : 0

acid concentration

activity within

19

:o

is shown

12.33 4.06 0.31 0.19 0’17 0’32 2.38 2.85 1'09 0'15 0.52 0'10

in bold

type.

Loch Eil sediment

The sulphate-reducing activity increasedwith depth and reached a maximum at about 6 cm (Figure 3). After this maximum the activity rapidly decreased,until at 12 cm it was at a similar level to that observed at a site of low organic carbon input, site LYI-0.23 pg SOd2- ml day-r (R. J. Parkes, unpublished data). This maximum in anaerobic sulphate reduction occurred after the Eh had decreasedbelow -t-100 mV (Table 5) and coincided with an increasein the level of free sulphide. Fatty acid pro$les of Loch Eil sediment

A gaschromatogramof the fatty acid methyl estersof the 4-6 cm sedimentsectionfrom Loch Eil is shown in Figure 2(a), and was representative of the fatty acid composition found throughout the 0-12 cm depth of the sediment. The percentagedistributions of these fatty acids are listed in Table 4 and consistsof saturated straight chain acids C12-35, straight chain monoenoic acidsand a number of poIyenoic straight chain acids. The distribution was bimodal over the CIZ-35 range with maxima at C16 and C24 [Figure 2(a)].

182

R. J. Parkes

H J.

Taylor

pg

Figure

3. Profile

TABLE

4. Fatty

I2

:o

I5 : 0

16:107

16 :Iq+iso 16 :o 16 :o is0 17 : I +unknown IO Me C16 : o is0 17 : 0 anteiso 17 : o+isop. vx7:o 17:o 18 :2ti+18 18 : 1~7 18 : Iwg+isop.

from

Loch

activity

Peak no.b

C19

II 12

I3 I4 I5 16

I7 18

0.15

0.70 :3w3

0’52

C2o “The %ee

23 24

1.87 2.74 important fatty Figure 3(a).

I7k x7a, 178,

Percentage”

18 :o

7’97 0’20

: 5~3 + unknown unknown : 0 :o :o

33 :o 21kC31 ZIP-C32 34 :o 21bC32 35 :o

concentrations

are shown

0.67 0.62 0.87 7’73 1’01

7.01

I .80 IO.39 I.84 8.26 1.17 6.47 0’99 3’74 0.66

Hop. Hop. Hop.

25 acid

of Loch

Eil (E24).

cm depth)

23 :o 24 :o 25 :o 26 :o 27 :o 28 :o 29 : 0 30 : 0 31 :o 32 :o

9 IO

basin

V 19 :o 19 :o

20 21 22

I9 20 21 22

(4-6

Acid

20

0.30 I.54 2.47 0.73 0.31 2.16 0.46 10.28 0’09 0.23 0.28 0.73

3.75

in the deep

Eil sediment

2.65

is0 x.5 : 0 anteiso I5 :o unknown

acids

0’93 0’04 0’04 0.04 0’03 0.06 0.38

13 : I anteiso I3 : I is0 13 : 0 anteiso 13 : 0 I3 :o is0 14 : 0 I4 :o unknown

day-’

2.5

of sulphate-reducing

Percentage”

is0

ml-’

I .25

0

Acid

SOi-

in bold

2’45 0.46 0.30 0.28 0.86 3’15 0’31 -

Peak no. b 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 -

type.

Branched chain saturated and mono-unsaturated fatty acids accounted for 6% of the total, with iso and anteiso CI~ : o dominating. v C17 : o and v C19 : o were identified in small amounts, while significant quantities of 17 PH, 21 PH bishomohopanoic acid was found together with minor quantities of its 17aH, 21 PH isomer and 17 PH, 21 PH homohopanoic acid analogue. The origin of these hopanoic acids is still uncertain. Possible precursors exist in both higher plants and in procaryotes (Ourisson et al., 1979). The total fatty acid concentrations over the 0-12 cm depth studied are listed in Table 5 and show a maximum concentration at 2-4 cm of 360 pg g-l dry weight which gradually

Eh (mV

+106 + 53 + 35 + 35 + 34 -

Depth (cm)

o-2 2-4 4-6 6-8 8-10 IO-12

:9 67

I34 I36 120

Z CIZ-19

Total acids

312 360 307 291 262 270

5. Sediment

TABLE

I78 223 I87 200 I96 202

Z Czo-30

profile

:o

7’45 6.42 4’90

12.71 10.87 12.84

pg g-l

brCxg dry

:0

3’20 2’49 2.34 1.56 I’3I 1.07

weight

c15

1.06 0.98 0.48 0.28 0'21 0.18

V C17

: o

17.21 17.21

2.5’51

33.69 34.06 33’07

C16

Cx6

15.82 12.08 6.95 3’57 2.28 2’39

-____

: 107

3’97 4.36 5 ‘49 4.78 4’90 4.58

c1g:o

brCrg

:o

I2 II 27 27 30 28

V CI7

brCIg

:o

0.47 0’35 0.21 0.14 0.13 0’14

C16:o

(2x6

: 107

Q

184

R. J. Parkes @ J. Taylor

decreaseswith depth and falls within the range reported for several marine sediments (Brown et al., 1972; van Vleet & Quinn, 1979). The high molecular weight fatty acids (> Czo) were found at relatively constant levels throughout the profile, while the low molecular weight acids (< C20) decreasedin abundancebelow the 6-cm horizon. The concentration profiles of several individual fatty acids commonly found in bacteria, C16 : o CI~ : o, brCrg, C16 : 1017 and VC17 are also presentedin Table 5, together with ratios of brCrg : o/C15 : o, brCrg : o&C17 and C16 : ro7/C16 : o. Discussion In attempting to identify the sedimentary fatty acids of bacterial origin, it is imperative to obtain cultures that closely represent the types and distribution of bacterial speciesin a sediment. Many workers have relied on the enrichment of pure cultures of bacteria on high carbon agar plates, or on mixed populations from such plates (Johnset al., 1977; Perry et al., 1979). Other workers have identified sedimentary fatty acids of bacterial origin by subtle manipulation of the environment in order to changethe benthic microbial community in a predictable manner (Bobbie & White, 1980; White et al., 1980), or from our knowledge of bacterial lipids, in general, selecting those unique to bacteria (Leo & Parker, 1966; White et al., 1979). The selectivity of cultural medium for obtaining sedimentary bacteria however, must distort the in situ bacterial populations and a large proportion of the in situ bacteria do not grow on rich agar media (Jannasch& Jones, 1959; Gray et al., 1968). The use of batch enrichment systems(e.g. agar plate, shakeflask) themselvescauseselection over and above that determined by the substrates and other medium conditions (e.g. pH, temperature). Batch systems tend to enrich organisms with characteristic growth parameters (opportunistic/zymogenous organisms) or with nutritional requirements (auxotrophs) and tend to lead to the isolation of pure cultures (Parkes, 1982). It hasbeenargued that the types of organisms enriched in batch systems are unrepresentative of microorganismsactually active in the environment where autochthonous/scavenging organisms may dominate (Jannasch, 1974; Veldkamp, 1977).Furthermore, it is becomingapparent that most microbial activity in the environment results from the action of a mixture of interacting organisms (Bull, 1980) and that microorganismsable to grow in isolation may not be representative of the natural microbial population. Manipulation of the natural environment by using inhibitors to select for specific microorganismsovercomesthese culturing difficulties but has its own limitations and problems (Yetka & Wiebe, 1974). The use of a chemostat overcomesmany of the culturing difficulties allowing autochthonous microorganisms(Veldkamp, 1977) and mixed microbial communities to be reproducibly isolated (Senior et al., 1976). The useof a number of chemostatsin series reduces the problems associatedwith selection of appropriate culture conditions, as only the input conditions in the first vessel(Pot I) have to be determined. Growth of the mixed microbial community in Pot I defines both type and concentration of substrates entering Pot 2 and to a certain extent the prevailing environmental conditions in that vessel (e.g. dissolved oxygen, Eh, pH and various nutrient concentrations) and so on down the series. Thus enrichment conditions after Pot I are set by the metabolism of previous microbial communities as within a consolidatedsediment. This results in a redox gradient acrossthe multiple chemostatsystem which may allow the microbial succession(in terms of electron acceptors) existing with depth in a sediment (Fenchel & Jorgensen, 1977) to be modelled in the laboratory. It also provides realistic enrichment conditions for bacterial communities.

Fatty acid distributions

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The usefulnessof this enrichment technique is indicated by the similarity between the fatty acid distribution of the mixed sulphate-reducing bacterial community that developed in the end vesselof the seriesand those in the Cxa-19 range of the 4-6 cm region of the sedimentwhich wasshownto be the areaof maximum sulphate-reduction activity [Tables 2, 4; Figure 2(a), (b)]. Both distributions show large concentrations of normal C14 : 0, C16 : o and CIS : o acids together with significant quantities of the characteristic bacterial iso and anteiso CI~ : o acids. However, C16 : 1w7 and C18 : 1w7 acids, major constituents of the mixed sulphate-reducing culture were found in lesserrelative amounts in the sedimentary distribution. This perhapssuggeststhat bacterial lipids extracted from the sedimentoriginate from lysed aswell as viable bacteria (seediscussionof sedimentary profile). In contrast, the lipid profile of the D. desulfuricans isolated from the deep basin of Loch Eil by batch enrichment is dominated by iso C17 : I 07 fatty acid and correlatespoorly with both the sediment and the mixed community of sulphate-reducing bacteria (Table 3, Figure 2). It may well be that the different speciesof sulphate-reducing bacteria recently isolated by Widdel (1980) may play an important role in the sediment and in the mixed community of sulphatereducing bacteria, and hence explain the poor correlation between the lipids of the Desulfowibrio speciesand the sediment. This possibility is being investigated. The bacterial community that developed in Pot I was chosenfor further study as all the prevailing culture conditions were known. The community was separated into three respiratory types by growth in defined conditions. All the isolates(Table 2) contained large amounts (>60%) of straight chain saturated and unsaturated acids in the range C12-19, indicative of Gram-negative bacteria (Kates, 1964). The lipid profiles of the isolatesbear some similarity to the mixed cultures reported by Perry et al. (1979), C14 : o, brCIg : 0, C16 : 0, CI~ : o, C16 : 107 and C18 : 107 being present in significant concentrations and considered to be major fatty acids in bacteria (Oliver & Colwell, 1973). In the aerobic communities significant levels of cyclopropyl fatty acids (~17 and ~19) are present, but absent in the facultative anaerobic, and in the mixed community of sulphate-reducing bacteria. This suggeststhat cyclopropyl fatty acids may be more indicative of aerobic than of anaerobic bacteria. The facultative anaerobiccommunity wasthe only culture to have significant amounts of C12 : o fatty acid. This was also found in total anaerobic heterotrophs from Low Isles Sediment [grown under non-rigorous anaerobicconditions (Perry et al., 1979)], together with relatively high amounts of C14 : o, which was also true for our culture. The facultative aerobesdiffered from the other cultures by having significantly higher amountsof CIS : I 07 fatty acid. Similarly high concentrations of this fatty acid were alsofound in marine sediment isolatesby Oliver & Colwell(1973). The aerobic community had the highest concentration of CI~ : o fatty acid and similarly high concentrations of this acid were found in an obligate aerobic bacterium (Thiabacillus sp.) by Perry et al, (1979) together with significant concentrations of ~Cr7 and vC19 cyclopropyl fatty acids. These different respiratory communities therefore could be differentiated by their fatty acid distribution, although they had originally been enriched under similar conditions. The possibility of recognising fatty acids from different bacterial respiratory types in a sedimentary environment, however, is complicated by the presencein the sediment of fatty acids derived from terrestrial and marine sources. But bacteria in general (O’Leary, 1962; Kates, 1964; Shaw, 1974) and the current isolatesin particular do contain a number of characteristic acids which may be used as biological markers in sedimentsenabling their biomasscontribution to be assessed. Branched chain iso and anteiso CI~ : o acidshave been usedeXtenSiVely in this respect(Cooper & Blumer, 1968; Cranwell, 1973 ; Perry et al., 1979).

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These acids are rare in eucaryotic organismsbut are found extensively in bacteria (Oliver & Colwell, 1973; Shaw, 1974)and were observed in large amountsin all the bacterial communities isolated. Their distribution with depth in the Loch Eil sediment is shown in Table 5, where high abundancesto a depth of 6 cm suggesteda high bacterial biomassover the top o-6 cm section of this sediment. The reduction in this bacterial biomarker below the 6-cm horizon correlates with the decreasein sulphate-reducing activity (Figure 3), and since methanogenesiswas not observed in the sediment (Drake & Parkes, unpublished observations), the end of sulphate-reducing activity probably indicates the limit of bacterial input to the sediment. Cyclopropyl fatty acids(~17 and Vrg) have alsobeen proposed asbacterial biomarkers in sediments(Cranwell, 1973; Perry et al., rg7g), and have been reported asmajor constituents in a large number of Gram-negative eubacteria, lactobacilli and clostridia (Kates, rg64), but are rare in eucaryotes, having been found in a few protozoa (Meyer & Holz, 1966) and in someterrestrial plants (Yano et al., 1972). How-ever, analysisof the bacterial lipids isolated in this study indicates that cyclopropyl fatty acidswere only characteristic of aerobic bacteria. This is supported by the concentration profile of cyclopropyl C17 in Loch Eil sediment (Table 5) which was observed at maximum levels in the more oxidized surface layers and decreasedwith depth where more anoxic conditions prevail [seeEh values, Table 5. An Eh value of +IOO mV indicates oxidizing conditions buffered by the presenceof NO,- (Graetz et al., 1973); oxygen is probably alsopresent but at low concentrations. At this redox level, which is the boundary between oxidizing and reducing conditions, small changesin Eh can represent significant changesin bacterial metabolism, e.g. Jones (1979) demonstrated that anaerobicrespiration increasedrapidly when the Eh decreasedfrom +IOO to $50 mV]. The abrupt change at 4-6-cm in the ratio of brCr5 : o/VC17 which, if our proposalsare correct, is a measureof the proportion of aerobic bacteria correlateswell with the peak in anaerobic sulphate-reducing activity (Figure 3) and hencewith a changein dominancefrom aerobic to anaerobic metabolism. The increase in the ratio of brCr5 : o/C15 : o with depth observed in the sediment (Table 5) suggestsa higher eucaryotic contribution to the surface lipids (Bobbie & White, 1980). However, from the evidence available it was not possibleto distinguish between the contribution of indigenous benthic eucaryotesand that of marine detritus. High levels of brCr5 : o and CIS : I ~7 acids with smaller amounts of other characteristic bacterial fatty acids (branched chain saturated and w7 mono-unsaturated Cr3, C14, Cr7 and C18 acids) identified in the sedimentary fatty acid distributions, together with the generally high correlation between the fatty acid distribution of the bacterial cultures and those of the sediment in the Crz-rg range and the absenceof significant concentrations of lipid markers for eucaryotic organisms(e.g. C18 : I wg and polyenoic Cr8 and CZOacids), suggeststhat the sedimentary acids in the Crz-rg range found in Loch Eil sediment are predominantly bacterial in origin. This view is supported by the total Cx2-19 acid concentration profile in the sediment (Table 5) which closely parallels that of bacterial biomarker branch CI~ : o acids. Interestingly, the CIZ-19 fatty acid concentration profile contrasts with that of high molecular weight acidsin the Czo-30 range, widely attributed ascharacterizing a terrestrial input to sediments(Gaskell et al., 1975), which were found at relatively constant concentrations (Table 5) over the whole o--I2-cm depth profile of the sediment.The explanation for this may be attributed to the resistanceto degradation of theselonger chain acids by bacterial enzymes (Green & Allmann, 1968). Similar differences in concentration between allochthonous and autochthonous derived fatty acidswere observed by Matsuda & Koyama (1977) in a freshwater lake sediment.

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187

Finally, the unsaturated acids CI~ : I 07 and Cx8 : I 07, unlike their saturated analogues, exhibit concentration profiles (Table 5) that do not parallel those of the characteristic bacterial brCrg : o acids. Their concentration was found to decrease with depth from the surface, resulting in a decrease in the ratio of unsaturated/saturated acid (e.g. C16 : rw7/C16 : o, Table 5). Since most of the acidsin the C12-19 range found in the sediment are consistent with a bacterial origin, a similar source appearslikely for the unsaturated acids. However, both dead and living bacteria will contribute to the bacterial lipids within a sediment, and, of the fatty acids contributed by dead bacteria, the unsaturated acids will be degradedmore rapidly than saturated fatty acids, possibly explaining the decreasein unsaturated fatty acids with depth. Similar decreasesin the ratio of unsaturated to saturated fatty acids with depth have been found by other workers (Farrington & Quinn, 1973; Johnson& Calder, 1973). Acknowledgements The authors would like to acknowledgethe technical assistanceof Will Buckingham and David McGuinn and thank Drs M. Droop and P. A. Cranwell for reading the manuscript and providing many helpful comments.Mass spectral data were kindly supplied by Professor G. Eglinton and Mrs A. Gower, Organic Geochemistry Unit, University of Bristol. References Apon,

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