Polar lipid ester-linked fatty acid composition of Lake Vechten seston: an ecological application of lipid analysis

FEMS Microbiology Ecology 38 (1986) 381-396 Published by Elsevier

381

FEC 00094

Polar lipid ester-linked fatty acid composition of Lake Vechten seston: an ecological application of lipid analysis (Fatty acid; mesotrophic monomictic lake; organic geochemistry; capillary GC-MS)

H.L. Fredrickson a,*, T.E. Cappenberg a and J.W. de Leeuw b a Limnological Institute, Vijverhof Laboratory, Rijl~straatweg 6, 3631 A C Nieuwersluis, and b Delft University of Technology, Department of Chemistry and Chemical Engineering, Organic Geochemistry Unit, De Vries van Heystplantsoen 2, 2628 RZ Delft, The Netherlands Received 4 June 1986 Revision received 7 October 1986 Accepted 8 October 1986

1. SUMMARY In order to relate the benthic lipid composition to possible sources in the water column, the sestonic communities of a monomictic lake were profiled using their saponifiable polar lipid fatty acids, which were identified by capillary gas chromatography-mass spectrometry (GC-MS). The epilimnion, dominated by the dinoflagellate alga Ceratium hirundella, was characterized by C20:5 and C22:6 polyunsaturated fatty acids. The photic anoxic metalimnion supported a radically different community, dominated by photosynthetic sulfur-oxidizing bacteria ( Chromatium and Chloronema spp.) and a Synechococcus-like cyanobacterium, and was characterized by high concentrations of C16 and Cls monounsaturated fatty acids. The fatty acid compositions of the hypolimnetic seston and the sediment were qualitatively similar to that of the metalimnion. Methylbranched acids, commonly found in eubacteria, increased with depth in the water column. The concentrations of several unusual fatty acids found

* To whom correspondence should be addressed.

in Desulfovibrio spp. Desulfobacter spp. and Desulfotomaculum spp. were inversely related to sulfate concentration in the metalimnion. After the water column mixed in the winter, steep gradients of respiratory terminal electron acceptors developed in the surface sediment and were reflected in the polar lipid fatty acid compositions. The results show that fatty acids derived from the membranes of epilimnetic phytoplankton were efficiently metabolized in the oxic portion of the water column. The fatty acids synthesized by prokaryotic microorganisms at and below the oxycline dominated the sediment. The polar lipid fatty acid composition of the sediment showed seasonal changes which can be associated with concentrations of terminal electron acceptors of microbial respiration, and thus with physiologically distinct bacterial groups.

2. INTRODUCTION Organic geochemical studies have been performed on a number of oligotrophic [1] and eutrophic lakes [2,3] and current research into the organic compounds extractable from lake sedi-

0168-6496/87/$03.50 © 1987 Federation of European Microbiological Societies

382

ments has been summarized [4,5]. Organic compounds extracted from lake sediments can provide information on the trophic status [6-8] as well as the source of sedimentary organic carbon [9-12]. However, detailed ecological interpretation of biogeochemical data is limited by a lack of information on the changes lipids undergo between the time of cell death and their possible incorporation into the sediment [5]. Recent studies have shown that microbial succession [13] and biological particle reworking [14] can greatly affect the total lipid composition of particles as they sink to the benthos. In order to relate lipids present in the benthos to their possible sources in the overlying water column, we have determined the polar lipid fatty acid compositions of sestonic particles and surface sediments in a much studied lacustrine environment. Lake Vechten is a small (4.7 ha) slightly eutrophic monomictic lake, 10 m deep, located in The Netherlands (52°04'N and 5°05'E). A complete physical description of the lake can be found elsewhere [15]. The lake has been the subject of intensive limnological study for the past 20 years, and this research has been summarized recently [161. Summer thermal stratification prevents the water column from mixing and effectively partitions it into two physically distinct compartments. Most of the lake's carbon is fixed by epilimnetic phytoplarukton [17]. This primary production is efficiently cropped by zooplankters, principally

Diaphanosoma brachyrum and Ceriodaphnia pulchella [18] and undergoes aerobic mineralization by heterotrophic bacteria [19]. Depletion of respiratory terminal electron acceptors (oxygen, nitrate and sulfate) in the deeper water layers results in a chemocline in the photic zone of the lake and limits microbial physiological activities. Water in the region of the thermocline/chemocline is colored red. Pigment analysis has shown that this is due to the presence of the photosynthetic purple sulfur-oxidizing bacteria Chromatium and Thiocapsa spp., which coexist with the green sulfur bacteria Chlorobium spp. and a Synechococcus-like cyanobacterium [20]. Although physically distinct from one another,

these microbial communities are related by energy flow and nutrient recycling. Consumption of epilimnetic phytoplankton results in a rain of particles through the water column to the sediment surface. For example, during the 151-day stratification period in 1979, 5.9 kg dry weight of material sedimented to the 8 m horizon of the lake providing energy and nutrients to the benthos [21]. The sediment in turn generates carbon dioxide, methane, ammonia and reduced sulfur compounds which re-enter the water column via diffusion and bubble formation [22,23]. In this study, the sestonic microbial communities which mediate the majority of carbon cycling in Lake Vechten were characterized by the esterlinked fatty acids of their polar lipid. This approach was chosen because it can provide quantitative data on extant in situ microbial communities free of the biases which can be introduced by selective growth of microbes on nutritive media. Membrane lipids with restricted phylogenetic distributions and fast environmental turnover times can be used as measures of microbial community biomass and community structure [24]. Phospholipids are the principal molecular component of most biological membranes. Their synthesis is genetically regulated, and upon cell death, hydro, lysis of their phosphodiester linkage is rapid [25]. Fatty acid biosynthetic pathways reflect microbial evolution, and thus fatty acid compositions are of taxonomic value. Surveys of the fatty acid compositions of bacteria [27], photosynthetic bacteria [27,28] and eukaryotic plankton [29-34] have been published. Thus, examination of phospholipids provides a means of examining living biota. The utility of polar lipid fatty acid analysis in profiling sedimentary microbial communities has been demonstrated recently using sediments enriched with selected nutrients [35]. Here we show that the polar lipid fatty acid profile of Lake Vechten surface sediment: (1) differs from the total lipid fatty acid profile of the same sample; (2) strongly resembles the polar lipid profile of the metalimnetic photosynthetic prokaryotic community when the lake is stratified; and (3) changes rapidly at holomixus when oxygen, nitrate and sulfate are made available to the sediment. We interpret these results in light of lipid

383

analyses of axenic microbial cultures of genera which inhabit the lake and discuss these results in relation to pertinent physico-chemical parameters of the water column and sediment, and biological processes which influence the lipid composition of the benthos.

3. MATERIALS AND METHODS

3.1. Materials Glass-distilled solvents were obtained from Merck (Amsterdam, The Netherlands). Fatty acid methyl ester standards (FAME, 99% pure) were obtained from Applied Science (State College, PA, U.S.A.). The purple and green photosynthetic sulfuroxidizing bacteria Thiocapsa roseopersicina (Chromatiaceae) and Chlorobium phaeobacterioides (Chlorobiaceae) were obtained from Dr. H. van Gemerden (Groningen, The Netherlands) and grown in batch culture [36]. Stephanodiscus hantzschii (Bacillariophyceae), Chlorella oulgaris (Chlorophyta), and a Synechococcus-type microorganism (Cyanophyceae) were isolated from Lake Vechten and grown in continuous culture under conditions which simulated those found in the lake [20]. Formalin-killed cultures were harvested by continuous centrifugation and extracted as described below. D. desulfuricans ssp. aestuarii (ATCC17900) was grown in a mineral salts marine medium containing lactate and reduced with sodium sulfide. A freshwater sulfate-reducing bacterium was isolated from Lake Vechten sediment using a freshwater mineral salts medium containing lactate and reduced with sodium thiosulfate.

ous flow rotor (M.S.E., Sussex, U.K.) at a flow rate of 80 ml- min-1. Sediment cores were collected from the eastern basin of the lake at a depth of 10 m with a modified Jenkin mud sampler. Cores were returned to the laboratory within 2 h of collection, subcored with a 30-mm-diameter plexiglass tube, extruded, sectioned into 2-cm slices, and immediately extracted. Dry weight per unit volume was determined on replicate samples, which were lyophilized.

3.3. Lipid analysis Lipids were extracted from samples by a modified Bligh and Dyer [37] procedure (Fig. 1). The total lipid fraction thus obtained was thoroughly dried by 3 consecutive chloroform evaporations and weighed. It was dissolved in chloroform and quantitatively loaded onto a 1-g Unisil column 1 cm in diameter (100-200 mesh, Clarkson Chemical, Williamsport, PA, U.S.A.). Sequential 10-ml elutions with chloroform, acetone and methanol

I Wet SampLe I Btigh - Dyer extraction I

I Chtoroform

I Aqueous Hethanol ]

phase t {$i021

'

1

Hethanol, etuate I 1) Saponification 2) Extraction

J Chtomform etuate

~Partition ,

3.2. Sample handling Water samples (10 1) were collected from Lake Vechten at 9:00 a.m. on September 15, 1984, using a Friedinger water sampler. The samples were stored on ice in tightly capped 10-1 carboys and returned to the laboratory within 2 h of collection. The use of this method precluded the sampling of a water layer less than 1 m thick. The water was centrifuged (20000 × g) at 2°C using a continu-

I Ne trat I !./MeOH 2) TLC ,

I °H-F"NE I GC GC-NS Fig. 1. Row diagram of the analytical procedure used to recover lipids from cultures and environmental samples (CK~, gas liquid chromatography; TLC, thin-layer chromatography).

384 produced 3 lipid fractions with increasing polarity; non-polar lipid (NL), polar lipid fraction I (PLI) and polar lipid fraction II (PLII), respectively. Solvents were removed under a stream of nitrogen and the separate lipid fractions were weighed. The PLII fraction was dissolved in 0.2 ml of chloroform and hydrolyzed for 24 h at 20°C in 0.5 ml of a solution composed of 1 M aqueous potassium hydroxide and methanol (1:4, v/v). Lipids were recovered in chloroform after the pH of the aqueous phase was lowered to 6 with 1 M HCI and dried under N 2. Non-saponifiable lipid was removed by dissolving this dry lipid residue in 1 ml of petroleum ether, vigorously mixing with 2 ml of aqueous 1 M potassium hydroxide, and centrifuging. T h e aqueous base was extracted sequentially with 2 additional volumes of petroleum ether. The fatty acids were recovered from the aqueous base by lowering the pH to 6 with HC1, extracting them into chloroform and drying them under N 2. The acids were methylated for 2 h at 100°C in 0.5 ml of a solution containing chloroform, concentrated HC1 and methanol (1 : 1 : 10, v / v / v ) . After cooling, 2 ml of chloroform and 2 ml of distilled water were added, the solution was mixed and the FAME were recovered in chloroform. FAME (R F = 0.6) were separated from hydroxylated fatty acid methyl esters (OHFAME, R F = 0.2) by thin-layer chromatography on Whatman K6 silica gel plates developed in petroleum ether, diethyl ether and glacial acetic acid (80 : 20 : 1). The recovery efficiency of phospholipid fatty acids through the analytical procedure was determined by standard additions of L-a-phosphatidylcholine dimyristoyl (1 /tg) to 5 sediment samples. It was found to be 93% with a standard error of 5% ( N = 5). It is therefore assumed that ester-linked fatty acids from lipids with polarities similar to phosphatidylcholine extractable from environmental samples will be efficiently reCovered in the PLII fraction with this procedure and precisely measured. Capillary gas chromatography (GC) analyses were performed on a Carlo Erba Model 4160 equipped with an on-column injector, a 25-m CPSil 5 fused silica capillary column (Chrompack, Middelburg, The Netherlands) and a flame ioniza-

tion detector. Helium was used as a carrier gas at an average linear velocity of 40 cm. s -1. The initial oven temperature was 40°C. At injection the oven temperature rose ballistically to 130°C and was then programmed to increase at 3°C • m i n -1 until a final temperature of 320°C. All gas chromatographic analyses were repeated using the same chromatographic system except that a 50 m CP-Sil 88 fused silica column was used with hydrogen (33 cm. s -1) serving as a carrier gas. Peaks eluting from the capillary column were tentatively identified by co-injection with standards and by calculating retention indices as equivalent carbon length (ECL) and by comparison with published values. Polyunsaturated fatty acid standards were prepared from cod liver oil using methods previously described [38]. Retention times (RT) of individual peaks in a trace were corrected so that the RT of 1 6 : 0 was constant from trace to trace, and relative RT (RRT) were thus generated. The RRT of the straight-chain saturated acids containing 13-20 carbons were plotted against carbon number and fitted with a linear function (r = 0.999974, A = -31.492, B = 2.968). The ECL value for 16 : 0 was set at 16.00, and all other values were calculated from their respective RRT through this function. All response factors for relating integration area to mg of FAME were assumed to be equal to Cxs:0. Mass spectral (MS) analyses were performed on a Hewlett-Packard Mass Selective Detector (HP 5970B) directly coupled to an HP 5890 capillary gas chromatograph without an interface. The chromatographic conditions were the same except a splitless injection was used with an isolation time of 2 min.

3.4. Sulfate analysis Sulfate was separated from other anions by high pressure liquid chromatography on an ion-exchange column and quantified by indirect photometric detection [39]. 3.5. Dissolved oxygen and temperature Dissolved oxygen and temperature were deterrnined by lowering a calibrated sensor (Orbisphere, Switzerland) into the lake on a graduated cable.

385

3.6. Particulate organic carbon The organic carbon content of lyophilized seston and sediment samples was determined on a Carlo Erba Elemental Analyzer (model 1106). O i

0 10 ........................

20

?

1,o..

0

. . . .

. . . .

,5

10 K

. . . .

20 ,

. . . .

. . . .

30

i . . . , l

[

zt / i

4. RESULTS The points of collection of the 10-1 seston samples are related to the temperature, oxygen and sulfate profiles of the lake in Fig. 2. The thermocÀine occurred at 5.5 m and effectively prevented water column mixing. Terminal electron acceptors for respiration, i.e., oxygen, nitrate (data not shown) and sulfate, are rapidly consumed at the thermocline and result in the diffusion gradients shown. The depletion of these terminal electron acceptors limits microbial physiological activities and hence the microbial community composition. The lipid content of Lake Vechten seston and sediment is related to bulk carbon measures in Table 1. The results are expressed on a volume basis so that comparison between the water column and sediment can be made simply. However,

40

TEMP.(*C) . - - . DO (rag/t) . - - . SO4 {mglt) o--,.

. . . .

-S

-S

E ~ 5-S

-S

10

-S

Fig. 2. Depth profiles of some pertinent physicochemical parameters of Lake Vechten during the autumn stratification in relation to the depths of the 10-1 seston samples (DO, dissolved oxygen; S, seston sample).

Table 1 Bulk carbon measures of Lake Vechten seston and sediment during stratification Depth

Dry weight

Water column ( p g . c m -3) 2.0 m 41.55 4.0 m 47.58 6.0 m 58.97 8.0 m 187.53 10.0 m 283.01 Sediment c (rag.era-3) 0 - 2 cm 136.22 2 - 4 cm 201.78 4 - 6 cm 235.90

Particulate a organic carbon (% dry weight)

27 39 36 13 10 6.9 6.3 5.8

Lipid (% particulate organic carbon) b Total

Nonpolar

PLII

7.8 1.6 3.4 5.7 6.2

4.5 0.5 1.0 2.8 1.9

0.9 0.3 0.5 0.8 0.7

92 24 45 77 71

11 7.6 6.6

7.4 4.6 4.2

1.2 0.6 0.1

142 38 5

Total PLII fatty acids ( × 1 0 -3 )

a Particulate organic carbon expressed as percentage of the total dry weight of the sample. b Lipid content expressed as a percentage of the absolute concentration of particulate organic carbon. c Repficate analyses ( N ffi 5) were performed on the sediment. The values represent means. The standard error of the mean never exceeded 15% (1-way analysis of variance).

386

notice must be taken of the 1000-fold change in concentration between the water and sediment (/xg. cm -3 to m g - c m - 3 ) . The concentrations of seston and total organic carbon increased with depth through the water column and these trends continued into the first 6 cm of the sediment. The total lipid content of the sestonic particles, as well as that of all constituent lipid fractions, reached a minimum at 4 m. The total lipid concentration of the 10-m water sample was twice that of the 2-m sample. The concentration of N L relative to the total lipid was highest in the epilimnion. This N L fraction contained alkanes, storage lipids (i.e., wax esters and triacylglycerols), degradation products of polar lipids and photosynthetic pigments. The N L fraction of seston collected from below the epilimnion contained large amounts of elemental sulfur. The PLI lipid fraction showed concentration maxima at 2 and 6 m. This lipid fraction contained glycolipids, which are abundant in the photosynthetic apparatus of prokaryotic and eukaryotic phytoplankton. However, these lipids lack the phosphodiester linkage, and their environmental turnover rates have not been determined. Therefore, the result of the detailed analysis of this lipid fraction is beyond the scope of this paper. The sediment collected from the 10 m depth of the lake during autumnal stratification proved to be horizontally homogeneous. However, the mass of sediment per unit volume almost doubled through the first 6 cm as a result of compaction. The total organic carbon content per volume increased by 30% but the total lipid content remained almost constant. The concentration of the PLII fraction decreased by a factor of 10, while the mass of material remaining unrecovered on the silica column doubled. The PLII ester-linked fatty acid composition of the epilimnetic seston was markedly different from that of the seston collected at greater depths (Fig. 3 and Table 2). The epilimnetic microbial community was characterized by C18, C20, and C22 mono-, di- and polyunsaturated fatty acids. The most abundant acids were C22:6 (Table 2) (51), C16:0 (20), C18:1~,9 (34) and C20:5 (41). The components which elute between C26:0 and C27:0 on the non-polar capillary column (63,64) elute between

C30:0 and C32:0 on the polar capillary column, suggesting that they are C28 polyunsaturated fatty acids, although their precise structures have not yet been determined. The water sample taken at 4 m contained essentially the same fatty acids as in the 2-m sample, although at approximately half the concentrations. However, low concentrations of iC15:0 and a C15:0 became apparent at 4 m. The PLII fatty acid composition of seston from the red-colored water at 6 m was profoundly different from that of the overlying water. The concentration of the C22:6 fatty acid (51) was reduced to 15% of that found at 2 m. Palmitoleic acid (C16:1~7c) (35) was the major fatty acid comprising 35% of the total epilimnetic PLII fatty acids. Iso and anteiso methyl-branched acids increased in concentration, and C18:1,~7 (35) became more abundant than Cx8:l,~9 (34). This fatty acid composition showed little qualitative change with depth in the water column below the thermocline, but the total concentrations of fatty acids increased to a maximum at the sediment-water interface. The fatty acid composition of the sediment (Fig. 4A) strongly resembled that of the anoxic water column (Fig. 3C) and did not change qualitatively with depth (0-6 cm) during the period when the lake was stratified. However, the total concentration of these fatty acids in the deeper sediments was only 5% of that of the surface sediment. A portion of the total lipid collected from the top 2 cm of lake sediment in September was saponified before being fractionated into the polarity classes (Table 3). The total lipid fatty acid profile was characterized by saturated straight even-carbon-numbered chains ranging from C14 to C34 (Table 3). When compared to the PLII fatty acid profile of the same extract, the monounsaturated acid C16:1 in the total lipid profile decreased in abundance relative to C16:0 , and C18:1w9 (34) was more abundant than C18:1,o7 (35). C18:2o~6 (32), C18:3,o3 (33) and several polyunsaturated C2o, C22 , C24 and C26 acids were present but C2o:5 (41) and C22:6 (51) were detectable only in low concentrations. The PLII fatty acid compositions of surface sediments collected when the lake was undergoing holomixus were markedly different from those

387

Seston 2 m

A 20 34

41

63

~5 ~8

_1

B

Seston

6m

20

35

L C

~6

Seston

8 m

20

35

4

ECL

14

78

15

16

1~

1~

19

20

21

22

2"3

2~

25

26

Fig. 3. Capillary GC traces of polar lipid ester-linked fatty acids of Lake Vechten seston from 2 m (A), 6 m (B) and 8 m (C).

388 Table 2 The major ester-linked polar lipid fatty acid contents of Lake Vechten seston during the summer stratification FAME, fatty acid methyl esters, are abbreviated as follows; 16 : l~7t represents a 16 carbon fatty acid with 1 double bond located between the 7th and 8th carbon from the aliphatic end of the molecule. 't' indicates the double bond is in the tr a n s configuration. 'i' represents an iso methyl branch, 'a' represents anteiso branching and ' b ' indicates that the point of methyl branching is not certain. 'c' indicates the presence of a cyclopropane ring. Structures were assigned based on ECL values on 2 columns and mass fragmentography. ECL, equivalent carbon lengths, were calculated using the retention times (25 m CP Sil 5) of the saturated straight-chain fatty acids to derive a linear function (A = - 31.492, B = 2.9683, r = 0.99997). The ECL of 16 : 0 was set to 16.00 and all other ECL values were calculated through the equation. ND, not detected; TR, trace. Component No. a

Content ( p g - c m - 3 ) at indicated water depths (m) FAME

ECL

2.0

4.0

6.0

8.0

1 2 3 4

i14:0 14 : 1 14 : 1 14:0

13.68 13.81 13.89 13.99

ND ND ND ND

ND ND ND 41

ND ND 8 110

16 ND 25 413

52 14 56 687

5 6 7 8 9 10

i15 : 1 a15 : 1 i15 : 0 a15 : 0 15 : 1 15 : 0

14.34 14.39 14.68 14.74 14.81 14.99

ND ND ND ND ND 10

ND ND 13 12 ND 20

ND ND 57 39 16 78

24 19 399 285 139 294

56 31 699 570 290 344

11 12 14 16 17 18 20

ND ND 13 166 16 20 2160

ND ND

ND ND

i16 : 0 16 : 1~o7 16 : loa7t 16 : 1 16 : 0

15.40 15.48 15.66 15.70 15.76 15.84 16.00

8 322 8 42 1040

7 3434 180 183 1617

14 19 96 7026 ND 936 2643

34 27 108 7617 ND 1162 2 889

21 22 23 24 25 27 28

i17 : 1 a17 : 1 b17 : 0 i17 : 0 al,7 : 0 17 : 1 17 : 0

16.35 16.40 16.42 16.68 16.74 16.84 17.02

ND ND ND 8 ND ND 10

ND ND ND 14 27 5 17

102 8 21 47 88 59 79

586 32 101 125 263 166 144

707 60 223 131 318 197 138

17.39 17.44 17.50 17.59 17.63 17.72 17.76 17.81 18.02

241 581 11 204 81 1467 221 68 180

91 198 7 105 63 431 198 9 166

97 128 104 117 115 377 1089 7 179

60 130 77 263 272 387 1290 6 296

54 154 50 268 279 418 1363 10 255

19.17 19.21 19.40 19.42 19.54 19.89 19.99

256 703 ND 10 ND 150 39

130 344 ND 20 ND 31 14

121 313 ND 38 72 19 11

264 297 23 45 64 5 12

226 276 30 16 5 8 10

20.26

ND

ND

74

57

34

29 30 31 32 33 34 35 36 38 40 41 42 43 44 45 46 47

18 : 3co6 18 : 4~03 18 : 2aJ6 18 : 3~3 18 : 1~9 18 : lw7 18 : l~07t 18 : 0

20 : 5

20 : 1 20 : 0

10.0

389 Table 2 (continued) Component

Content (pg. c m - 3 ) at indicated water depths (m)

No. a

FAME

51 52 53 54 56 57

22 : 6 22 : 4

58 60 61 63 64

22 : 0

24 : 0

ECL

2.0

4.0

6.0

21.00 21.09 21.18 21.22 21.83 21.91

2 390 116 32 37 35 25

962 31 14 11 48 13

350 21 11 7 34 16

22.40 23.32 23.58 25.64 25.67

59 26 73 532 193

26 8 16 52 21

7 7 16 27 13

8.0

10.0

101 6 ND ND 42 20

21 ND ND ND 25 15

ND

ND ND 11 5 ND

2 13 ND ND

a Reference n u m b e r s of major components refer to Figs. 3 and 4.

collected when the lake was stratified (Table 2; Fig. 4 A and B). The latter resembled the microbial community at the thermocline but the former contained much less Ct6:t. The oxic sediments of April contained increased concentrations of C14:t, Ct5:1 and straight chain even carbon numbered acids. The April sediment showed a strong vertical stratification pattern which corresponded to steep oxygen, ell, nitrate and sulfate gradients in the top 2 cm. The interfacial sediment (approx. 0-1 cm) was enriched in C t 4 : t , i C t s : 0 , i C t 5 : t , aCts:t , iCt7:0 and aC17:1 relative to the fatty acid composition of the entire 0-2 cm sediment sample. To relate the saponifiable PLII fatty acid composition of Lake Vechten seston to lipids of microorganisms which inhabit the lake, axenic cultures of pertinent microorganisms were analyzed using the same methods. Algal species which had been previously isolated from the lake, S. hantzschii and C. vulgaris, contained unsaturated fatty acids (Table 4) characteristic of eukaryotic algae. However, the absence from the epilimnetic seston of the predominant fatty acids in these algae, C22 polyunsaturated fatty acids of Stephanodiscus (tentatively identified as ECL 20.7) and the unusual C16:t,0t3t and polyunsaturated C18 acids of Chlorella indicate that these algae were not abundant in the epilimnion at the time of sampling. Microscopic observations showed that Ceratium hirundinella was the most abundant alga during the sampling period. Unfortunately, this

alga is not easily cultured in the laboratory and as a result there are few reports of its lipid composition. The photosynthetic prokaryotes examined did not Contain polyunsaturated fatty acids (Table 4). The Synechococcus-like cyanobacterium isolated from the lake lacked iso and anteiso methylbranched fatty acids and contained almost exclusively C16:x and Cx6:0 (86%). The purple sulfur bacterium Thiocapsa roseopersicina also contained CI6:I and C16:0 as major fatty acids but in addition contained C18:10,7 (35%). Chlorobium phaeobacterioides, isolated from a marine environment, differed from the other photosynthetic prokaryotes examined in that it contained C15 (9%) and CI7 (38%) carbon acids. Both of the colored sulfur bacteria contained significant amounts of methyl branched acids. The sulfate-reducing bacteria examined, D. desulfuricans ssp. aestuarii and a lactate-oxidizing sulfate-reducing bacterium isolated from the lake, were similar to one another in their high content of iso and anteiso methylbranched C15 and C17 carbon saturated and unsaturated fatty acids.

5. DISCUSSION Analysis of the organic carbon composition of Lake Vechten seston provides insights into the

390 Table 3 The major ester-linked total and polar lipid fatty acid content of Lake Vechten surface sedimentary layers during summer stratification and Spring holomixus Abbreviations are explained in Table 2 Component No.

Mean fatty acid content in indicated sediment layer (ng. c m - 3)

FAME

ECL

September 1984

April 1985

Total lipid

Polar lipid

Polar lipid

0 - 2 cm

0 - 2 cm

2-4 cm

4 - 6 cm

0 - 2 cm

0-1 cm

1 3 4

i14 : 0 14 : 1 14 : 0

13.68 13.89 13.99

140 305 1843

96 14 407

40 ND 210

ND ND 25

67 130 586

ND 29 47

5 6 7 8 9 10

i15 : 1 a15 : 1 i15 : 0 a15 : 0 15 : 1 15 : 0

14.34 14.39 14.68 14.74 14.81 14.99

97 100 660 690 65 396

39 49 356 482 46 99

8 17 156 269 14 37

TR TR 17 189 ND 4

11 18 340 428 106 146

TR TR 173 15 21 10

11 12 14 16 17 20

i16 : 0 16 : 1~7 16 : 1 16 : 0

15.48 15.55 15.66 15.70 15.76 16.00

146 330 602 6 982 618 8 284

31 66 82 4423 152 2115

4 13 57 953 28 718

ND 6 5 92 10 101

TR TR 98 88 202 2120

TR TR 23 TR 56 734

21 22 24 25 27 28

i17 : 1 a17 : 1 i17 : 0 a17 : 0 c17 : 0 17 : 0

16.35 16.40 16.68 16.74 16.84 17.02

75 70 304 338 83 229

26 23 63 136 96 55

18 15 72 96 44 33

10 TR 7 12 TR 6

142 112 287 110 41 86

127 46 177 32 26 8

32 33 34 35 36 38

18 : 2~6 18 : 3~3 18 : 1~9 18 : 1~7 phytanic 18 : 0

17.59 17.63 17.72 17.76 17.86 18.02

839 456 3 943 1773 149 1522

316 244 633 1050 ND 164

136 90 357 375 ND 110

21 12 58 51 ND 40

46 ND 200 200 36 268

ND ND ND ND 15 240

19.54 19.67 19.99

110 120 844

15 20 67

4 10 32

ND TR 17

ND 36 241

ND 10 TR

154 163 79 281 844

44 39 32 13 34

TR TR TR 16 32

TR TR TR 7 4

236 250 ND 262 404

13 TR ND ND ND

44 45 46

20:1 20 : 0

48 50 51 55 57

22 : 6 22 : 0

20.70 20.80 21.00 21.65 21.91

59

23 : 0

22.72

140

5

8

TR

96

ND

60 61

24 : 0

23.32 23.58

2 041 2 722

18 26

3 27

ND 3

TR 381

TR TR

62

26 : 0

25.21

2 338

15

18

TR

275

TR

391 Table 3 (continued) Mean fatty acid content in indicated sediment layer (ng. c m - 3 )

Component No.

FAME

63 64 65 66 67 68

ECL

25.64 25.67 28:0 30:0 32:0 34:0

N u m b e r of replicate samples analyzed

September 1984

April 1985

Total lipid

Polar lipid

Polar lipid

0 - 2 cm

0 - 2 cm

2 - 4 cm

4 - 6 cm

0 - 2 cm

0 - 1 cm

2700 1313

ND 15

ND 18

ND TR

TR TR

TR TR

2581 921 260 862

8 ND ND ND

12 ND ND ND

TR ND ND ND

206 67 ND ND

TR ND ND ND

3

3

3

lake's sestonic microbial community compositions, trophic structure and sediment formation. The rapid sedimentation rate during the period in which the lake is stratified is evidenced by the mass of sestonic particles in the water column. Although the total mass of particles increases with depth in the water column, the percentage of organic carbon (extractable and combustible) comprising the sestonic mass decreases with depth. In the epilimnion, this is indicative of biological particle reworking, which recycles readily metabolizable organic compounds and results in sestonic particles comprised of recalcitrant substances. In the hypolimnion a large portion of the sestonic mass is probably composed of reduced mineral salts of nitrogen and sulfur which were deposited as the result of anaerobic microbial respiration. The polar lipid concentration per volume of lake water showed that membrane biomass had maxima in the epilimnion and sediment-water interface and a minimum at 4 m. Saponification of this lipid and analysis of the resulting fatty acid methyl esters revealed that the microbial community above the thermocline was markedly different from that found in the deeper water and sediment. The fatty acid composition of the epilimnetic seston is indicative of phototrophic eukaryotes. Even-carbon-number fatty acids were predominant, with modes at C16 and C22. Half the acids were polyunsaturated, with C20:s and C22:6 comprising 30% of the total polar fatty acids. The

3

3

1

C20:5 acid is widely distributed through photoeukaryotic taxa, but C22:6 has a far more limited distribution and is usually found as the major phospholipid fatty acid in the unicellular flagellated algae, division Pyrrophyta, class Dinophyceae [32]. Previous observations have revealed that Ceratium hirundinella, a member of the Dinophyceae, was the principal alga in the epilimnion at the time of sampling (3.5 × 105 cells, l -x) [40]. No information on the fatty acid composition of pure cultures of Ceratium spp. could be found in the literature. However, the fatty acid composition of the epilimnetic seston strongly resembles the fatty acid compositions reported for other members of the Dinophyceae [28,41] and is similar to the fatty acids identified in analysis of a dense bloom of this organism collected from the British Lake District [42]. The last peaks to elute from the capillary column (Fig. 3A) comprise a considerable portion of the PLII fatty acids, and have been tentatively identified as C2s polyunsaturated acids. Long-chain polyunsaturated fatty acids have previously been reported in species of Cryptomonas and Chlorella, but the origin of the acids reported in this paper remains to be determined. Some polyunsaturated fatty acids were conspicuous by their absence. Polyunsaturated C16 fatty acids which are found in most members of the Bacillariophyceae (diatoms) were not detectable in the epilimnetic seston. The unusual acid Cxr:x~,x3t, which is abundant in Chlorella vulgaris isolated

392

Sediment Stratified

O-2cm Lake

2O

2o

Sediment 0-2cm Nonstratified L a k e

4

liT..

3. Sediment

C

~o

7

~i

Nonst

0 - 1 cm

ratified

Lake

24

2

38

D.

desulfuricans

'iL L ECL

. I~.

15

16

17

18

19

2'0

2'I

Fig. 4. Capillary GC traces of polar lipid ester-linked fatty acids of (A), lake sediment (0-2 cm) 9/84; (B) lake sediment (0-2 cm) 4/85; (C) lake sediment (0-1 cm) 4/85; (D) and D. desulfuricans ssp. aestuarii.

from Lake Vechten and most other Chlorophyceae species examined, was also absent. This is indicative of the absence of these species from the epilimnion at the time of sampling. The water above the thermocline freely mixes but the concentration of polyunsaturated algal fatty acids was greatly reduced in the seston collected at 4 m. This is probably the result of the phototactic migration of the phytoplankters up to the shallow water layers where sufficient insolation can be intercepted for photosynthetic production, and to where they are followed by zooplankton. Efficient transfer of photoeukaryotic carbon to subsequent trophic levels is mediated by zooplankton [40] and heterotrophic bacteria [19], and is evidenced in these results by the lack of polyunsaturated fatty acids in the polar lipids in the deeper water layers and sediment. The concentrations of polar lipids at the photic chemocline in Lake Vechten are as high as those in the epilimnion, but the large differences in their fatty acid compositions indicate that their microbial community structures are very different. Previous microscopic observations and pigment analysis have shown that a Synechococcus-like cyanobacterium was the most populous member of this anoxic phototropic community and that it coexisted with the colored sulfur-oxidizing bacteria Chromatium, Thiocapasa and Chlorobium spp. [20]. The principal fatty acids of the metalimnetic seston, palmitoleic acid (C16:1~7), C16:0 and C18:1to7 are major fatty acids in these microbial taxa. Precise estimates of the biomass contributed by the metalimnetic species to the community can be made by measuring complex polar lipids which can be associated unambiguously with individual species [43] within the microbial consortium. This can be readily achieved by further chromatographic fractionation of the complex polar lipid components before saponification and fractionation of the fatty acids. Several unusual fatty acids have been isolated from sulfate-reducing bacteria and these acids may be used as markers for this microbial guild in the lake. The concentration of iC17:1, the principal fatty acid of Desulfooibrio [44], also present in Desulfotomaculum [45,46], increased with depth below the chemocline and was inversely related to

393

Table 4 Major saponifiable polar lipid fatty acid compositions of axenic cultures of species which represent genera of microorganisms which

inhabit Lake Vechten See Table 2 for explanation of abbreviations. Content (mg/g dry weight) in representative taxa a

Component

Chle

Chlo

Thi

ND

TR

TR

ND ND ND

ND ND ND

TR 0.59 2.74

TR TR 0.45

15.59 15.66 15.68 15.70 15.91 16.00

0.50 TR ND 8.44 ND 11.44

ND 0.74 0.57 0.78 6.05 14.08

ND 0.74 ND 7.30 ND 3.52

16.35 16.40 16.68 16.74 16.79 16.84 17.02

ND ND ND ND ND ND ND

ND ND ND ND ND ND ND

17.59 17.63 17.72 17.81 18.02 18.05

ND ND 1.19 3.05 0.72 0.36

20.70 20.76 21.91

No.

FAME

ECL

4

14: 0

13.99

7 8 10

i15 : 0 a15 : 0 15 : 0

14.34 14.39 14.99

13 14 15 16 19 20

16 : 3 i16 : 0 16 : 1 16 : lo~7 16 : l~13t 16 : 0

21 22 24 25 26 27 28

i17 : 1 a17 : 1 i17 : 0 a17 : 0 17 : 1 c17 : 0 17 : 0

32 33 34 35 38 39 48 49 57

18 : 2~6 18 : 3 t~ 3 18 : 1~9 18 : loJ7 18 : 0

22 : 0

Minor acids b Total acids

Ste

Des

SRB

TR

0.11

ND ND ND

0.66 0.12 TR

0.61 0.25 TR

ND 0.69 ND 11.43 ND 8.06

ND ND ND 4.92 ND 6.96

ND TR ND 0.70 ND 1.34

ND ND ND ND ND 1.48

ND ND ND ND 1.10 6.09 7.30

TR ND ND ND TR 1.43 1.96

ND ND ND ND TR ND TR

1.24 0.28 0.60 0.31 ND ND TR

1.01 0.31 1.54 0.25 ND ND TR

5.77 6.87 3.20 0.71 TR ND

ND ND ND 7.37 TR TR

ND ND ND 14.18 0.43 0.81

ND ND TR 0.82 0.20 0.15

ND ND ND 0.92 1.66 0.02

ND ND ND ND 1.84 ND

1.55 0.99 ND

ND ND 0.57

ND ND ND

ND ND ND

ND ND ND

ND ND ND

ND ND ND

4.80

2.72

1.41

1.54

0.41

0.28

0.15

34.20

41.32

38.16

40.98

13.68

8.11

7.37

0.64

Syn 0.22

Stephanodiscus; Chle, Chlorella; Chlo, Chlorobium; Thi, Thiocapsa; Syn, Synechococcus; Des, Desulfovibrio; SRB, a sulfate-reducing bacterium isolated from Lake Vechten. b The total of all acids whose individual relative concentrations were less than 1% of the total fatty acids in the gas chromatographic a Ste,

trace.

dissolved sulfate concentration. The predominant fatty acids of Desulfobacter (bE17:0) (47) and Desulfobulbus (C17:10,6) (48) showed similar trends but were present at lower concentrations. The polar lipid composition of the surface sediment changed after holomixus of the water colunto. During stratification the polar lipid fatty acid composition strongly resembled that of the

metalimnetic seston and did not change qualitatively with sediment depth. After holomixus introduces oxygen, nitrate and sulfate to the benthos, the polar lipid fatty acid composition of the surface sediment changes. These changes are restricted to a narrow band of interracial sediment less than 1 cm thick, in which the aforementioned electron acceptors are rapidly consumed, resulting in steep

394 c o n c e n t r a t i o n g r a d i e n t s [49]. T h e f a t t y acid c o m p o s i t i o n of the u p p e r m o s t layer s t r o n g l y r e s e m b l e s that of D. desulfuricans, especially in the relative a b u n d a n c e s of the u n u s u a l iC17:a a n d aCl7:l acids, which have been f o u n d in a n u m b e r of lactateo x i d i z i n g s u l f a t e - r e d u c i n g b a c t e r i a [45]. T h e s u l f a t e - r e d u c i n g b a c t e r i u m which was isolated f r o m L a k e Vechten s e d i m e n t also c o n t a i n e d these u n u s u a l b r a n c h e d m o n o u n s a t u r a t e d acids. S a p o n i f i c a t i o n of the total l i p i d e x t r a c t e d f r o m s e d i m e n t p r o d u c e d a fatty acid profile which differed from that of the P L I I alone. T h e p o l a r l i p i d f a t t y acids are a v a r i a b l e subset of a larger relative c o n s t a n t p o o l of f a t t y acids in the sediment. In a s u b s e q u e n t p u b l i c a t i o n we will show that the m a j o r i t y of e x t r a c t a b l e s e d i m e n t a r y f a t t y acids are either free or esterified to less p o l a r molecules. T h e results of this s t u d y d e m o n s t r a t e the utility of lipid analysis in ecological studies. Conversely, we e m p h a s i z e the need for an ecological basis to p r o p e r l y r e c o n s t r u c t the c i r c u m s t a n c e s in the p a s t u n d e r which s e d i m e n t s have b e e n deposited. T h e p r e s e n t s t u d y has focused on fatty acid analysis of the total s a p o n i f i e d c o m p l e x p o l a r lipids. H o w ever, m o r e d e t a i l e d i n f o r m a t i o n o n m i c r o b i a l c o m m u n i t y structure m a y b e o b t a i n e d b y the structural d e t e r m i n a t i o n o f i n t a c t c o m p l e x p o l a r lipid which can be u n a m b i g u o u s l y associated with specific m i c r o b i a l taxa. This w o u l d be p a r t i c u l a r l y useful in the e x a m i n a t i o n of a r c h a e b a c t e r i a , which lack ester-linked f a t t y acids. M e t h a n o g e n s obviously p l a y a m a j o r role in c a r b o n cycling in the lake, b u t have e s c a p e d e x a m i n a t i o n in the c u r r e n t study.

ACKNOWLEDGEMENTS

T h e a u t h o r s wish to t h a n k H. van G e m e r d e n ( D e p t . of M i c r o b i o l o g y , T h e U n i v e r s i t y of G r o ningen) a n d C.L.M. Steenbergen (The L i m n o l o g i cal Institute, Nieuwersluis) for b a c t e r i a l cultures, P.J.D. Sakkers ( H e w l e t t - P a c k a r d , A m s t e l v e e n ) for m a s s s p e c t r o m e t r i c analysis, H. G o o s s e n s a n d P.H. Best for their critical view o f the m a n u s c r i p t , W.I.C. R i j p s t r a for d r a w i n g the figures a n d C.C.C. K r o o n for t y p i n g the m a n u s c r i p t .

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