Chemical Geology, 95 (1992) 347-360 Elsevier Science Publishers B.V., Amsterdam 347 [1] Biogeochemistry of hot spring environments 3. Apolar and polar lipids in the biologically active layers of a cyanobacterial mat Y. Bing Zenga,~, David M. Ward b, Simon C. Brassellc and Geoffrey Eglintona aOrganic Geochemistry Unit, School of Chemistry, University of Bristol, Bristol BS8 1TS, UK bDepartment of Microbiology, Montana State University, Bozeman, MT 59717, USA CDepartrnent of Geology, Stanford University, Stanford, CA 94305-2115, USA (Received August 14, 1990; revised and accepted August 2, 1991 ) ABSTRACT Zeng, Y.B., Ward, D.M., Brassell, S.C. and Eglinton, G., 1992. Biogeochemistry of hot spring environments, 3. Apolar and polar lipids in the biologically active layers ofa cyanobacterial mat. Chem. Geol., 95: 347-360. The apolar lipid, glycolipid and phospholipid components of the biologically active top 5-mm surface layers of a hot spring cyanobacterial mat were investigated. Most of the major components could be associated with bacteria isolated from the mat; the vertical distribution of lipids followed the known or presumed vertical distribution of these organisms. For example, hydrocarbons (e.g., 7-methylheptadecane), phytadienes (methanolysis products from chlorophyll a) and polar lipid fatty acids typical of mat-forming cyanobacteria maximize in the top 0-1 mm and decrease in concentration with depth. Wax esters and octadecanol (produced upon methanolysis of bacteriochlorophyll cs) typical of Chloroflexus aurantiacus maximized in the 1-2- and 2-4-mm intervals. Long-chain diols derived mainly from glycolipids and typical of the aerobic heterotroph Thermomicrobium roseum maximize in the 1-2- and 2-4-mm intervals. 1-O-Alkylglycerols derived from polar lipids and typical of anaerobic fermentative or sulphate-reducing bacteria, increase in concentration with depth and maximize in deeper layers. The relative abundances of lipids appear to reflect the trophic structure of the microbial community. I. Introduction This paper is a continuation of our collaborative investigation of lipid biomarkers in hot spring microbial mats as model systems in which community composition is simplified and relatively well defined (Ward et al., 1985, 1989 ). In earlier papers of the series we investigated the apolar lipids of the cyanobacterial mat in Octopus Spring, Yellowstone National Park (Dobson et al., 1988), as well as polar lipids of this and other hot spring microbial mats of varying degrees of community com"Present address: Institute for Water Sciences, Western Michigan University, Kalamazoo, MI 49008-5150, USA. plexity (Zeng et al., 1992 in this issue; in this paper referred to as Part 2). Much of our previous work has compared the composition of extractable lipids or polar lipid components in bulk mat samples. Here, we concentrate on the Octopus Spring cyanobacterial mat, well-characterized with respect to many of the microorganisms which are thought to be involved in photosynthetic formation and subsequent decomposition of the mat. Many of these microorganisms have been obtained in pure culture and their lipid compositions have been investigated (Ward et al., 1989). The relationship of cultivated to uncultivated mat inhabitants of similar phylogeny is becoming increasingly understood (Ward et al., 1990, 1992). Pro- 0009-2541/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved. 348 Y.B. ZENG ET AL. duction and decomposition of this mat occur principally within the top 5 m m (Ward et al., 1987 ). We investigated this zone of biological activity at depth intervals relevant to the distribution of microorganisms and the reactions they catalyze in order to learn whether the vertical distribution of mat inhabitants was reflected in the distribution of the lipids they are likely to synthesize. 2. Methods Octopus Spring is located in the Lower Geyser Basin of Yellowstone National Park, ~ 150 m SSE of Great Fountain Geyser. Samples were removed from a 52-55°C site along the southernmost effluent channel using a stainless-steel coring tube (44-mm diameter). Using a spatula one core was immediately sectioned along natural laminae into the top green layer ( ~ 01 mm; 160 mg dry weight), a reddish underlayer ( ~ 1-2 mm; 84 mg dry weight), a deepred coloured layer ( ~ 2 - 4 mm; 95 mg dry weight) and a brown-green layer ( ~ 4-5 mm; 41 mg dry weight). Samples were immediately frozen on dry ice for transit, lyophilized upon return to the laboratory and kept frozen except for a few days in transit to the U.K. All solvents were redistilled and all glassware and materials (including sampling materials and containers) were solvent-rinsed before use. The samples were ground to powder with a mortar and pestle before extraction using a modification of the Bligh and Dyer (1959) method (see Part 2). The total lipid extract was separated into apolar lipid, glycolipid and phospholipid fractions by column chromatography (see Part 2 ) and their weights were determined after solvent evaporation. Following addition of internal standards, glycolipid and phospholipid fractions were subjected to methanolysis, derivatized with N,Obis (trimethylsilyl)trifluoroacetamide (BSTFA) and analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) as previously described (see Part 2 ). 3. Results 3. I. Lipid class composition Concentrations of the various fractions, and the total concentrations of wax ester components and polar lipid fatty acid methyl ester (FAME) methanolysis products (estimated TABLE 1 Compound classes of extractable lipids Compound class Concentration in #g g - 1 dry mat (% of total extracts ) 0-1 mm 1-2 mm 2-4mm 4-5 mm Apolarlipids*~ - Wax esters.2 10,959 ( 1 8 . 0 % ) 7,462 26,027 ( 3 6 . 5 % ) 20,294 28,235 ( 4 1 . 3 % ) 16,254 19,231 ( 2 7 . 8 % ) 8,600 Glycolipids*~ - FAME's .2 31,507 ( 5 1 . 7 % ) 15,389 28,767 ( 4 0 . 4 % ) 7,806 27,059 ( 3 9 . 7 % ) 6,795 23,077 ( 3 3 . 3 % ) 3,629 Phospholipids.1 - FAME's .2 18,493 ( 3 0 . 3 % ) 8.325 16,438 ( 2 3 . 1 % ) 4,219 12,941 ( 1 9 . 0 % ) 3,177 26,923 ( 3 8 . 9 % ) 4,597 Total extracts .3 60,959 (100%) 71,233 (100%) 68,235 (100%) 69,231 (100%) *~Concentration determined by gravimetric method. *:Concentration obtained by summation of GC quantitation of individual methanolysis products (Tables 2-4 ). *3Sum of apolar lipids, glycolipids and phospholipids. BIOCHEMISTRYOF HOT SPRING ENVIRONMENTS,3 349 0-1mm 1.3 Y 2 20 1 -2mm 1 3 , lB 8 . A x 2O 2-4mm 18 4 - 5 mrn 320 ._, ' 11 8 1.| . _ . . . . . ., I I ' ' ' ' 1 ' ' ' ' 10 19 20 I 30 I I 1 ~ I 40 I 5O 60 RETENTION TIME (minutes) Fig. 1. Gas chromatograms of apolar lipid fractions of the biologicallyactive layers of the 52-55°C Octopus Spring cyanobacterial mat. Assignmentsand abundances of major components are given in Table 2. Minor constituents include: 15, 23, 24 = n, n-C29, -C37 and -C38 wax esters, respectively; il, i2 = internal standards (n-C23 aikane and 5a (H)-cholestane, respectively). All carboxyland hydroxylgroups were present as the TMS esters and ethers, respectively.Unlabelled peaks represent components which could not be unambiguouslyassigned from their mass spectra. from GC analysis), are reported in Table 1. The glycolipid fractions (and their c o m p o n e n t FAME's) were most a b u n d a n t in the top layer and decreased in concentration with depth. The phospholipid fractions ( a n d their c o m p o n e n t FAME's) showed a similar pattern, with the exception that concentrations were higher in the 4 - 5 - m m layer. Apolar lipids and their principal components, wax esters, m a x i m i z e d in the 1-2- and 2 - 4 - m m subsurface intervals, where they comprised ~ 4 0 % o f t h e total lipid extracts. 3.2. A p o ~ r l ~ i d s Gas chromatograms of apolar lipid fractions are shown in Fig. 1 and principal components are quantified in Table 2. In all samples wax esters were the d o m i n a n t components. Several series o f wax ester homologs were detected, 350 Y.B. ZENGET AL. TABLE 2 Concentration of major compounds in the neutral lipid fractions Peak Compound label (Fig. 1 ) Hydrocarbons: I 2 3 4 Alcohols: 8 I1 Wax esters: n,n-Chain: 16 17 18 19 20 21 22 Concentration (gg g-~ dry mat) 0-1 mm 1-2 mm 2-4 mm 4-5 mm 248 136 270 51 281 110 37 43 142 42 12 194 200 97 7 131 30 13 144 54 161 54 82 83 80 255 1,326 1,194 2,633 682 329 204 714 3,650 3,491 6,220 1,659 683 197 542 3,165 2,627 4,822 1,035 517 111 317 1,523 1,190 1,639 322 193 C35 29 112 157 223 91 124 517 658 867 312 112 524 519 825 211 164 651 514 793 126 C32 tr. C34 11 47 101 73 142 128 239 n-CtTalkane n-Ctsalkane 7Me heptadecane phyt-l-ene n-Cl7:o i-C 17:o C3o C31 C32 C33 C34 C35 C36 /,n-Chain: 26 27 C31 28 C33 C34 29 30 /,/-Chain: 31 33 C32 tr.=trace. with straight-chain (n,n-) ester components predominating over branched ones. Most individual wax esters showed peak concentration in the 1-2- or 2-4-mm depth interval. Hydrocarbon fractions contained predominantly n-C17 and n-C18 alkanes and 7Me-heptadecane in the 0-1-mm top layer, n-C17 w a s also predominant in deeper layers, whereas 7Me-heptadecane decreased dramatically. Phyt-l-ene, also a major hydrocarbon component, increased with depth. Free alcohols ranging from C~5 to C,8 (maximizing at C~7) were present as minor components, n-Alkan-1-01s predominated over iso-alkan-l-ols. Phytol, which was previously detected in a whole mat sample (see Part 2 ), was below detection in the upper layers we investigated. Similarly, bishomohopan-32-ol, a minor component in the whole mat sample (see Part 2), was not detected in the individual layers. 3.3. Glycolipid fraction constituents Gas chromatograms of the glycolipid fraction methanolysis products are presented in Fig. 2. Major components of this fraction are quantified in Table 3. FAME's were abundant in the methanolysis products of the glycolipid fractions in all lay- 351 BIOCHEMISTRY OF HOT SPRING ENVIRONMENTS, 3 O-lmm 33 i 5 i 20 2 6 7 20 1 -2mm 2 ~7 ¢, 30 5 ? • 2 21 2O 2-4ram 2 I 26 8 6 • 11 16 2 17 1O 28 .L. 4-5ram 26 ' I 10 ' ' ' I 20 ' ' ' I ' 30 ' ' I I ' 40 ' 1 1 1 1 1 1 1 1 1 50 60 RETENTION TIME (minutes) Fig. 2. Gas chromatograms of methanolysis products of glycolipid fractions obtained from the biologically active layers of the 52-55 °C Octopus Spring cyanobacterial mat. Assignments and abundances of major components are given in Table 3. Minor constituents include: 22 = br-C22 alkane- 1,2-diol; 23, 24 = n-C16 and -Cl 7 l-O-alkylglycerol, respectively; 28 = C, 5,C~ 5 1, 2-di-O-dialkylglycerol; 30 = n-C ~7:0alcohol; 33 = phytadienes; il, i2 = internal standards ( t/-C23 alkane and 5a (H)-cholestane, respectively ). All hydroxyl groups were analyzed as the TMS derivatives. ers. I n d i v i d u a l F A M E ' s s h o w e d different vertical distributions. T h e top layer was d o m i n a t e d by C16:0, C18:0, Cl6:l, C18:1 a n d cyclopropyl-C~9 F A M E ' s , a n d these decreased in c o n c e n t r a t i o n with depth. O t h e r F A M E ' s present in relatively high c o n c e n t r a t i o n in the top layer, such as Cis:0, C~7:0 a n d Cls:l FAME's, showed m a x i m u m c o n c e n t r a t i o n s in the 1-2or 2 - 4 - m m layers, B r a n c h e d F A M E ' s were relatively low in c o n c e n t r a t i o n in all cases, except for i-Cls:O which occurred only in traces in the top layer, a n d increased in c o n c e n t r a t i o n in subsurface layers. CI9-C21 straight-chain a n d m o n o m e t h y - 352 Y.B. ZENGET AL. TABLE 3 Concentration of major methanolysis products of glycolipid fractions Peak label (Fig. 2) Compound(s) Concentration (#g g - 1 dry mat) 0-1 mm 1-2 mm 2-4 mm 4-5 mm Fatty acid methyl esters: Normal chain: I Ci4:o 2 C15:o 3 Ct6:o C|7:o 4 5 Ct8:o 6 Cls:l 7 Cl6:l(S) 8 C18:~(s) 85 866 5,714 497 1,311 304 832 2,783 163 1,550 2,148 594 357 476 669 795 180 1,170 1,902 633 218 248 341 778 74 322 817 339 268 62 76 870 Cyclopropyl: 9 2,574 460 57 121 99 132 75 92 tr. 43 75 I11 106 113 143 494 49 91 106 115 110 268 425 199 153 734 372 295 330 228 180 105 41 tr. 131 1,574 138 229 3,001 267 180 2,206 186 46 355 tr. 166 80 145 877 253 419 159 170 400 185 300 150 180 Cl9 Mono-methyl branched chain: 10 i-Cis:o I1 i-Cl6:o 12 i-Ci7:o 13 a-Cl7:o 14 br-C 16:0 15 br-C~7:0 Alkane- 1,2-diols: Normal chain: 16 Cl9 17 C2o 18 C21 Mono-methyl branched chain: 19 Cl9 20 C2o 21 C21 1-O-alkylglycerols: Normal chain: 25 C~8 Mono-methyl branched chain: 26 Cl7 27 C18 Chlorophyll derivatives: 31 n-C~8 alcohol 32 phytadienes tr. tr. tr. tr. tr. - tr. 36 - tr. 120 1,500 (s) = sum of all isomers; tr. = trace; - - = below detection by GC and GC-MS. lated alkane-l,2-diols, identified from their c h a r a c t e r i s t i c m a s s s p e c t r a ( s e e P a r t 2 ), w e r e major components in all layers, especially in the 1-2- and 2-4-ram intervals. Diols exhib- ited maximum concentration at the 1-2-mm depth interval, where the major component, a C2o b r a n c h e d d i o l , e x c e e d e d c o n c e n t r a t i o n s o f individual FAME's. BIOCHEMISTRY OF HOT SPRING ENVIRONMENTS, 3 353 0 - 1 mm $ 2 1 1 -2mm 5 2-4mm ,. ~ 11~ ] 8I 9 16 IT Lr/1825 4-Smm t? 7 IIlll 10 I ' 2O ' '' I ' ' 30 RETENTION '' I 40 TIME ' ' ' ' I ' 50 ' ' ' I ' 60 (minutes) Fig. 3. Gas chromatograms of methanolysis products of phospholipid fractions obtained from the biologically active layers of the 52-55°C cyanobacterial mat in Octopus Spring. Assignments and abundances of major components are given in Tables 3 and 4. Minor constituents are assigned in Fig. 2. All hydroxyl groups were analyzed as the TMS derivatives. Similarly, 1-O-alkylglycerols were also identified from their characteristic mass spectra (see Part 2 ). A l-O-alkylglycerol with a possible methyl branched CI7 alkyl group was present in all samples and increased with depth to become a major product of methanolysis of the glycolipid fraction in the 2-4-ram interval. Other CI7 and CIS straight-chain or branched 1-O-alkylglycerols were absent or present in only trace amounts in the 0-1- and 1-2-ram layers, but they also increased in deeper layers. Other products of methanolysis of the glycolipid fraction may have been derived from chlorophyll pigments which coeluted with the glycolipid fraction on column separation. These included phytadienes, present mainly in 354 Y.B. ZENG ET AL. TABLE 4 Concentration of major methanolysis products of phospholipid fractions Peak Compound(s) label (Fig. 3) Concentration (#g g- 1dry mat) 0-1 mm 1-2 mm 2-4 mm 4-5 mm 49 273 2,800 230 1,429 199 1,075 52 399 1,361 300 567 135 308 54 250 996 289 503 58 136 75 197 1,459 340 1,171 77 219 872 341 108 211 771 258 205 tr. 345 105 193 30 184 89 183 184 123 84 230 239 24 15 17 90 54 53 41 35 36 29 26 28 94 7 306 24 180 20 180 19 tr. tr. 15 46 72 183 99 248 Fatty acid methyl esters: Normal chain: I 2 3 4 5 7 8 C14:o Cls:o C|6:o ClT:O Cls:o C16:1(s) CIs:I(S) Cyclopropyl: 9 Ci9 Mono-methyl branched chain: i - e l 5:0 I0 11 12 I5 i-C16:o i-CI7:o br-C 17:o Alkane- I, 2-diols: Normal chain: 16 17 18 Ct9 C2o C21 Mono-methyl branched chain: 20 21 25 26 C2o C21 1-O-alkylglycerols: n-C~s br-C l 7 (s) =sum of all isomers; tr. =trace. the top layer and decreasing with depth, and nC i 7 and n-C~8 alcohols, present mainly in the 1-2-, 2-4- and 4-5-mm layers and more abundant in subsurface layers. A C~5,C~5 1,2-di-O-dialkylglycerol, identified from its mass spectrum (see Part 2), was detected as a minor component in the 0-1-, 12- and 2-4-mm samples. 3.4. Phospholipidfraction constituents Apart from the low abundance or absence of alcohols and phytadienes, the products of methanolysis of the phospholipid fraction were similar in composition and depth distribution to those of methanolysis of the glycolipid fraction (Fig. 3; Table 4). The predominant products were FAME's. In comparison to the glycolipid FAME's there was a lower relative concentration of C16:l FAME and cyclopropylC19 FAME and a higher relative concentration of i-Cls:o FAME. Also, monomethyl FAME's were more abundant in the 0-1-mm layer and decreased in concentration with depth. Diols were much less abundant in the phospholipid fraction than in the glycolipid fraction, but BIOCHEMISTRY OF HOT SPRING ENVIRONMENTS, 3 showed a vertical profile similar to that of diols derived from the glycolipid fraction, maximizing in the l - 2 - m m layer, l-O-Alkylglycerol ethers were also less abundant in the phospholipid fraction, but, as in the glycolipid fraction, maximized in the deeper layers. 4. Discussion 4.1. Correlations between vertical distribution of lipids and bacteria There appear to be three distinct classes of vertical distribution of lipids in the Octopus Spring cyanobacterial mat bioactive zone, as illustrated in Fig. 4. These distributions can be interpreted in light of the many component bacteria whose lipids have been studied, taking into account what is known of the vertical distributions of these organisms and the turnover oflipids which is likely to occur upon burial in the mat. The major polar lipid FAME's, C16:0 , C 1 8 : o and C18:1, maximize in the 0-1-mm uppermost layer and their concentrations decrease with depth. These are the major polar lipid fatty acids of the cyanobacterial isolate which is thought to play a role in formation of hot spring mats, Synechococcus lividus, when grown at 55°C (Miller, 1976; Fork et al., 1979). These are not particularly distinctive polar lipid fatty acids. They are, for instance, the major total cellular fatty acids of the other cultivated mat phototroph, the photosynthetic green nonsulfur bacterium Chloroflexus aurantiacus (Kenyon and Gray, 1974; Knudsen et al., 1982 ), and the aerobic heterotrophic mat isolate Isophaera pallida (Giovannoni et al.,1987 ). Organisms such as these are thought to inhabit the 0-1-mm interval (Doemel and Brock, 1977 ). Based on the vertical distributions of chlorophyll a (Bauld and Brock, 1973), oxygenic photosynthesis (Revsbech and Ward, 1984) and S. lividus-shaped cells (Doemel and Brock, 1977 ), cyanobacteria are restricted to the top 0-1-mm interval. Presumably they consume all 355 consume all the light available for oxygenic photosynthesis very close to the mat surface. which may be more diagnostic of cyanobacteria also maximize in the 0-1-mm interval and decrease in concentration with depth below the 0-1-mm layer. Phytadienes are presumably derived during the methanolysis of cyanobacterial chlorophyll a in the glycolipid fraction. This inference is supported by the comparative prominence of phytadienes and n-octadecanol as methanolysis products of glycolipids from cyanobacterial- and Chloroflexus-dominated mats, respectively (see Part 2). 7Meheptadecane is also often attributed to cyanobacterial sources (Han et al., 1968; Gelpi et al., 1970; Blumer et al., 1971; Shiea et al., 1990). Based on the vertical distribution of bacteriochlorophylls (Bauld and Brock, 1973), Chloroflexus aurantiacus is thought to be more abundant in the 1- or 2-mm undermat beneath the cyanobacteria-dominated top layer, where it receives infrared light suitable for its photosynthesis. Perhaps the most diagnostic lipid biomarkers for this organism are the C28-C38 wax esters it produces (Edmunds, 1982; Knudsen et al., 1982; Shiea et al., 1991 ) as a significant proportion of its lipids ( Beyer et al., 1983 ). Compounds of this type maximized in the 1-2- and 2-4-mm depth intervals, coincident with the distribution of C. aurantiacus. This bacterium produces a unique bacteriochlorophyll which esterifies mainly n-octadecanol (Gloe and Risch, 1978). It is thus consistent that the n-octadecanol released during methanolysis of the glycolipid fraction, possibly derived from this bacteriochlorophyll, is most abundant in subsurface layers. Long-chain diols of the type found in this study have only been reported in the hot spring isolate Thermomicrobium roseum, an aerobic heterotrophic bacterium (Pond et al., 1986). The mat diols were dominated by the branched C2o component, the most abundant diol of T. roseum cultured at 60 °C (Pond and Langworthy, 1987). It is certainly possible that other mat inhabitants might also produce diols, but 356 Y.B. ZENG ET AL A !1-C16:0 F A M E _n-Cl8:l FAME phytadienes 7MeC17 mg/g TOC: mcj/g TO<~ ug/g TOG rng/g TO<:; o~~~ o; i -! | 3 i i! _n,_n-C~ wax ester rng/g TO<3 ~_ oi br-C2o dioi n-Cog alcohol rncj/g TOC ug/g TOG o --m 3 c.- ~ --m c~ L C 5" 3 L ~-%7 FAME ug/g "[OC br-Cl7 m o n o e t h e r ug/g TO(] l I I I °i ' ? ? 5" c~- il= Fig. 4. Vertical distributions of lipids in the biologically active layers of the 52-55°C cyanobacterial mat of Octopus Spring: (A) compounds which maximize at the surface and decrease in concentration with depth; (B) compounds which maximize in intermediate depths; and (C) compounds which increase in concentration with depth and maximize in deeper layers. 3 57 BIOCHEMISTRY OF HOT SPRING ENVIRONMENTS, 3 their presence in the 0-1-mm layer and maximization in the l - 2 - m m layer is consistent with the aerobic physiology of T. roseum and the greater abundance of oxygen in the layers near the active zone of oxygenic photosynthesis (Revsbech and Ward, 1984). Branched-chain FAME components of the glycolipid and phospholipid fractions showed two distribution patterns with depth. The major phospholipid branched FAME's, i-Cls:0, Cl6:O and -Cl7:O FAME's, were most abundant in the 0 - l - m m layer and decreased in concentration with depth. These are characteristic components of the phospholipids of two mat heterotrophic isolates which are aerobic, Thermus aquaticus (Ray et al., 1971a, b), or facultatively aerobic, Bacillus stearothermophilus (Card et al., 1969; Card, 1973). A branched C17:o phospholipid FAME and most of the branched glycolipid FAME's showed an increase in concentration with depth, with highest concentrations in the 2-4- or 4-5-ram layers. This might indicate a source organism of different, possibly anaerobic, physiology. In this regard, it is interesting that the anaerobic fermentative mat isolate, Clostridium thermosulfurogenes, is known to produce i-C17 and C~5 fatty acyl chains (Langworthy and Pond, 1986). Interestingly, the C3o dicarboxylic acid with "head-to-head" condensed iso-C~5 fatty acids, which comprises a major proportion of this organism's lipids (Langworthy and Pond, 1986), was not detected in the mat. 1-O-alkylglycerols maximized in the deeper layers of the mat. Two mat isolates are known to produce glycerol monoethers with alkyl moieties comparable to those of the major mat monoethers. One of these is the anaerobic fermenter, C. thermosulfurogenes (Langworthy and Pond, 1986); the other is the anaerobic sulphate reducer Thermodesulfobacterium commune (Langworthy et al., 1983). The latter produces mainly glycerol diethers which were not found in abundance in the mat layers, implying that the former organism is the more likely source of the monoethers found. 1,2-Di-O-dialkylglycerols typical of sulphate-reducing and methanogenic bacteria, which were observed in a bulk mat sample (see Part 2 ), were not detected in the individual mat layers. This is presumably due to the high trophic status and, thus, very low abundance of these organisms and their distinctive lipids. The vertical distribution of ether-linked isoprenoid lipids in the Octopus Spring 55 °C cyanobacterial mat was investigated previously (Ward et al., 1985, 1987). Phytanyl and biphytanyl ethers characteristic of the only methanogenic bacterium isolated from the mat, Methanobacterium thermoautotrophicum (Tornabene and Langworthy, 1979; Tornabene et al., 1978), were low in the 0-3-mm interval, but maximized in the 3-6-mm and deeper layers, correlating with the obligately anaerobic nature of this organism. Our analysis was done on samples collected during a mid-day period of high light intensity. As this mat undergoes diurnal change in light and oxygen distribution (Revsbech and Ward, 1984) which might influence repositioning of organisms in the mat, the lipid distribution might be more dynamic than indicated by our results. For instance, Doemel and Brock (1977) have suggested that Chloroflexus migrates upward by positive aerotaxis at night - a possible mechanism driving upward mat accretion. Thus, the vertical distribution of Chloroflexus lipids (e.g., wax esters, octadecanol derived from bacteriochlorophyll c~) might maximize in the 0-1-mm interval after a period of darkness. 4.2. Lipid abundance and trophic structure As we have previously observed (Ward et al., 1989; and Part 2) there seems to be a correlation between lipid patterns and abundances and the expected abundance of organisms occupying different trophic levels in this community. The vertical distribution of lipids representative of organisms occupying different trophic levels also follows the expected distri- 358 Y.B. ZENG ET AL. bution of such organisms relative to light and oxygen gradients in the mat. Lipids in upper layers of the mat which are likely to represent inputs of phototrophic microorganisms (base of the food chain, e.g., major polar lipid FAME's, wax esters) are in greatest abundance. Lipids which occur in upper or middle mat layers and which may reflect the inputs of aerobic heterotrophic mat decomposers (e.g., diols, some branched polar lipid FAME's), and lipids which maximize in deeper layers and characterize anaerobic fermentative bacteria (e.g., 1-O-alkylglycerols, some branched FAME's) (middle of the food chain) are secondary in abundance. The least abundant lipids are those maximizing in the deepest layers and which reflect the inputs of methanogenic bacteria ( ~ 10 and ~ 2 5 pg g-1 biphytane and phytane, respectively, released during ether cleavage of the 3 - 6 - m m layer, see Ward et al., 1987). This type of organism terminates the consortium of microorganisms which carries out anaerobic decomposition of the mat (top of the food chain). nonisoprenoid glycerol diethers. Its presence in top layers of the mat suggests that it may originate from a phototrophic or aerobic microorganism. Isopentadecane has been released during ether cleavage of Messel oil shale kerogen of Germany (Chappe et al., 1980) and from polymeric organic matter subfractions of a sealoch sediment in Scotland (Eglinton, 1983). At present, it is not known whether the diether pentadecyl groups found in the mat are branched. Lipids of unassigned origin might reflect inputs of known mat isolates which are not expressed in pure cultures due to some difference between culture and natural environment. However, there is evidence from other lipid analyses (Ward et al., 1985), and more recently from 16S rRNA sequence analysis (Ward et al., 1990, 1992) that this mat contains numerous uncultivated community members which could be the sources of some of these lipid components. 4.3. Lipids of unknown origin The major lipid components of the 52-55 ° C Octopus Spring cyanobacterial mat are typical of many bacteria which have been isolated from this community and seem to reflect the known or predicted distribution of these types of microorganisms within the mat vertical profile. 7-Methylheptadecane, phytadienes derived from glycolipid fraction components (probably chlorophyll a ) and major polar lipid FAME's are typical of mat-forming cyanobacteria. These show a m a x i m u m in the top 0-1 m m and decrease in concentration with depth. Wax esters and octadecanol (presumably derived from bacteriochlorophyll cs in the glycolipid fraction) are characteristic of the photosynthetic green nonsulfur bacterium, C. aurantiacus, and maximize in the 1-2- and 24-mm intervals. Long-chain diols derived mainly from the glycolipid fraction are typical of the aerobic heterotrophic thermophile T. roscum and maximize in the 1-2-mm interval. Several lipids we observed are not known to be produced by the many bacteria which have been isolated from the Octopus Spring cyanobacterial mat. Cyclopropyl-Cl9 FAME, derived from both glycolipid and phospholipid fractions, is a major component of the 0 - 1 - m m layer and decreases in concentration with depth, suggesting a possible link with source organisms having either phototrophic or aerobic metabolism. Branched-chain wax esters are not known to be produced by C. aurantiacus. However, their similarity in carbon number and depth distribution to the straight-chain wax esters probably synthesized by this organism suggests a c o m m o n origin. The C l ~,C 15 1,2-di-O-dialkylglyceryl diether is not known to be produced by T. commune, the only mat inhabitant known to synthesize 5. Conclusions BIOCHEMISTRY OF HOT SPRING ENVIRONMENTS, 3 S o m e b r a n c h e d fatty acids a n d 1-O-alkylglycerols d e r i v e d f r o m p o l a r lipid f r a c t i o n s are typical o f a n a e r o b i c f e r m e n t a t i v e isolates, such as C. thermosulfurogenes. T h e s e increase in c o n c e n t r a t i o n with d e p t h a n d m a x i m i z e in d e e p e r layers. 1,2-Di-O-dialkylglycerols typical o f the u n i q u e s u l p h a t e r e d u c e r , T. comm u n e , or the m e t h a n o g e n , M. thermoautotrop h i c u m , isolated f r o m this m a t are below d e t e c t i o n in p o l a r lipid f r a c t i o n s o f i n d i v i d u a l layers. H o w e v e r , the vertical d i s t r i b u t i o n o f p h y t a n e a n d b i p h y t a n e d e r i v e d f r o m an e t h e r cleavage r e a c t i o n suggests t h a t m e t h a n o g e n i c b a c t e r i a also increase in a b u n d a n c e in d e e p e r layers. T h e a b u n d a n c e o f the v a r i o u s lipid c o m p o n e n t s seems to reflect the t r o p h i c s t r u c t u r e o f the c o m m u n i t y with lipids c h a r a c t e r i s t i c o f p h o t o t r o p h s p r e d o m i n a t i n g o v e r lipids characteristic o f h e t e r o t r o p h s , w h i c h in t u r n pred o m i n a t e o v e r lipids c h a r a c t e r i s t i c o f b a c t e r i a t e r m i n a t i n g the a n a e r o b i c f o o d chain. S o m e lipids, notably, cyclopropyl-C19 F A M E a n d C ls,Cls-di-O-dialkylglycerol ( m e t h a n o lysis p r o d u c t s o f p o l a r lipid f r a c t i o n s ) a n d b r a n c h e d wax esters (in a p o l a r lipid fract i o n s ) , are n o t k n o w n to be p r o d u c e d b y bacteria isolated f r o m this m a t a n d r e m a i n o f unc e r t a i n origin. Acknowledgements Y.B.Z. was s u p p o r t e d b y S E D C a n d the R o y a l Society o f L o n d o n . G C - M S facilities were p r o v i d e d by the U . K . 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