Methanogenic Antarctic Lake Ecosystem as. Determined by Phospholipid Analyses. C. A. Mancuso, l p. D. Franzmann, 1 H. R. Burton, z and P. D. Nichols 3.
Microb Ecol (1990) 19:73-95
MICROBIAL ECOLOGY @ Springer-VerlagNew York Inc. 1990
Microbial Community Structure and Biomass Estimates of a Methanogenic Antarctic Lake Ecosystem as Determined by Phospholipid Analyses C. A. Mancuso, l p. D. F r a n z m a n n , 1 H. R. Burton, z and P. D. Nichols 3 ~Australian Collection of Antarctic Microorganisms, Department of Agricultural Science, University of Tasmania, Box 252C, Hobart, Tasmania 7000; 2Antarctic Division, Channel Highway, Kingston, Tasmania 7150; and 3CSIRO Division of Oceanography, GPO Box 1538, Hobart, Tasmania 7001, Australia
Abstract. Phospholipid analyses were p e r f o r m e d on water c o l u m n particulate and sediment samples from Ace Lake, a m e r o m i c t i c lake in the Vestfold Hills, Antarctica, to estimate the viable microbial biomass and COmmunity structure in the lake. In the water column, methanogenic bacterial phospholipids were present below 17 m in depth at concentrations which c o n v e r t e d to a biomass o f between 1 and 7 x 108 cells/liter. Methanogenic biomass in the sediment ranged from 17.7 x 109 cells/g dry weight of sediment at the surface to 0.1 x 109 cells/g dry weight at 2 m in depth. This relatively high methanogenic biomass implies that current microbial degradation o f organic carbon in Ace Lake sediments m a y occur at extremely slow rates. Total microbial biomass increased f r o m 4.4 x l 0 s cells/ liter at 2 m in depth to 19.4 x 108 cells/liter at 23 m, near the b o t t o m o f the water column. Total nonarchaebacterial biomass decreased from 4.2 • 109 cells/g dry weight in the surface sediment (1/4 the biomass o f methanogens) to 0.06 x 108 cells/g dry weight at 2 m in depth in the sediment. Phospholipid fatty acid profiles showed that microeukaryotes were the major microbial group present in the o x y l i m n i o n o f the lake, while bacteria d o m i n a t e d the lower, anoxic zone. Sulfate-reducing bacteria (SRB) comprised 25% o f the microbial population at 23 m in depth in the water column particulates and were present in the surface sediment but to a lesser extent. Biomass estimates and c o m m u n i t y structure o f the Ace Lake ecosystem are discussed in relation to previously measured metabolic rates for this and other antarctic and t e m p e r a t e ecosystems. This is the first instance, to our knowledge, in which the viable biomass o f methanogenic and SRB have been estimated for an antarctic microbial c o m m u n i t y . Introduction The lakes o f the Vestfold hills, an area o f approximately 400 k m 2 (near the Australian antarctic station, Davis) are natural m i c r o c o s m s characterized by a Wide range o f physical and chemical features [7, 17], which provide microor-
74
C.A. Mancusoet al.
ganisms with a variety of environmental extremes in salinity, Eh, light, and temperature [24, 25]. Ace Lake, which has been described in the past by Burton and others [2, 6-8], was chosen as the subject of the present study. It is surrounded by low hills and covered by ice nearly all year. It is completely isolated from seawater incursions and is meromictic as a pronounced salinity gradient from the upper to the lower waters of the lake produces permanent stratification. The anoxic bottom layers are depleted in sulfate and extremely high in sulfide [17]. The dissolved methane concentration in the bottom waters approaches saturation [6]. Organic geochemical studies of the organic-rich lake sediments recently have provided evidence for the existence of methanogenic bacteria, as well as other microbes [48, 49]. Methanogenic bacteria are ubiquitous in most anaerobic sediments [57], where they perform the terminal step in the mineralization of organic carbon [26]. Information on the structure of the microbial community, including methanogens, in Ace Lake should provide some insight into the cycling of carbon in this antarctic lake system and, by extrapolation, in antarctic marine sediments. Phospholipids can be used to identify viable members of microbial communities in nature and to quantify cell biomass [1, 42, 52-54]. White et al. [55] manipulated oxygen concentration and nutrient levels in estuarine sediments to change microbial mass. Phospholipids extracted from subsamples over a 5-day period were shown to correlate linearly with extractible ATP (r = 0.84) and with rate of DNA synthesis (r = 0.99). The recovery of ~4C-labeled lipids from sediments was quantitative [55]. Pulse-chase experiments have shown active metabolism of sedimentary phospholipids indicating that these chemical markers provide good estimates for viable microbiota [55]. The use of membrane phospholipids to estimate viable cell biomass has also been extensively validated for subsurface aquifer sediments [1]. The advantages of these biochemical procedures have been reviewed by White [52]. The cell membranes of methanogenic archaebacteria are unique and consist of lipids formed with ether linkages and isoprenoid branching [28, 46]. Identification and quantification of these phospholipid-derived ether lipids (PLEL) from anoxic environments provide a means by which to estimate the methanogenic bacterial component of the microbial community [30, 35]. Certain ester-linked phospholipid-derived fatty acids (PLFA) isolated from nonarchaebacterial cell membranes can serve as unique signatures for known taxa [19, 29] and can allow identification of other eubacterial components in environmental samples [19, 52]. Together these methods were applied to the water column and sediments of Ace Lake to complement other investigations (i.e., cell number by direct microscopic counts and studies on the rates of methanogenesis by incorporation of radiolabelled metabolic substrates) (P.D. Franzmann, manuscript submitted for publication). Information gained from studies of this microcosm can be applied to more complex systems which include a methanogenic component. Information on the importance of methanogenic populations in antarctic lake ecosystems is currently unavailable. We undertook this study to understand better, and to describe more accurately, the microbial component of the biological community of Ace Lake.
Microbial Ecology of Ace Lake, Antarctica
Materials
75
and Methods
Sample Site Ace Lake is situated at 68~ 78~ 'E on a narrow section of the Long Penisula in the Vestfold Hills, approximately 8 km from the Australian antarctic base, Davis. This site has been described in detail previously [6]. During sample collection (November 1987 to January 1988), the lake was covered by a 1.7-m thick layer of ice. Water samples and sediment cores were obtained through holes 22.5 cm in diameter drilled using a Jiffy drill (Feldmann Engineering, Wisconsin). All samples were collected above the deepest part of the lake (24.75 m) so that the maximum n u m b e r of horizontal gradients in the water column were accessible.
Physieochemical Parameters In situ temperature was measured with a Yeo-Kal Model 606 conductivity and temperature detector (CSIRO, Hobart, Australia). Salinity (+ 1%o) was measured with a hand refractometer (ATAGO, Japan). The following parameters were measured as cited: sulfate concentration [45], sulfide coneentration [13], and oxygen concentration [44]. Samples collected for methane concentration determination had 5.0 ml of 2.0% CdCI2 (to precipitate sulfide) added to the Winkler bottles prior to Sealing at the sampling site. Methane was extracted from water samples by the syringe technique of Martens and Val Klump [31 ] and quantified by the method of Culbertson et al. [ 10]. Methane was quantified by gas chromatography (12-inch Haysep Q column; column isothermal at 50~ detection by thermal conductivity with detector temperature at 200~ and a current of 250 mA, helium carrier gas flow rate at 30 ml/min, Varian 3700 gas chromatograph).
Sediment Coring Cores of Ace Lake sediment were collected using a modified version of a Zullig piston corer (J. Ferris, personal communication). After the 2-m core (3.5 cm i.d. PVC pipe) was retrieved from the lake, the pipe was kept in a vertical position and the ends were sealed with parafilm and aluminum foil for transportation back to Davis station by helicopter. There it was cut into 1-m lengths, the ends were sealed again, and the cores were kept at -200C until time of core extrusion and analysis.
SeaStar in situ Water Sampler A programmable automatic in situ water sampler (SeaStar Model 8300, Instruments Ltd., Sidney, 9.C.) was employed to obtain samples of the particulate matter at various depths in the lake. This instrument is capable of collecting water samples from a very narrow depth range [20]. Particulate samples were collected sequentially from the surface down, thereby minimizing or eliminating disturbance and intermixing of these stratified layers. The instrument was lowered by a cable to Predetermined depths, through a hole in the ice. The cable was suspended from a tripod and secured there for the duration of the sampling period. Water was filtered through premuffied (12 hours, 450~ glass fiber filters (#8, 142 m m in diameter, Schleicher and Schuell, Dassel, FRG) at the rate of 150 ml/min for between 30 rain and 4 hours. After the appropriate length of time, the apparatus was raised to the surface where the filter was removed from the filter assembly with methanol.rinsed forceps. The filter was wrapped in aluminum foil and stored frozen (-20~ until analysis.
76
C . A . Mancuso et al.
Extruding the Sediment Core To facilitate the removal of the sediment core from the PVC tubing, the core in the tubing was left to stand at room temperature I hour. The core was then held horizontally, and the sediment was extruded from the bottom end into a semicircular piece of tubing (cut lengthwise), 1 m in length, and lined with methanol-washed aluminum foil. The liquid material (0.5 ml) at the top of the core was considered to be part of the water-sediment interface. This sample, the "0 cm sample," formed the reference point from which all other depths in the core were determined. The core was then cut into 0.5- to 2-cm slices which were placed in tared beakers and frozen until time of extraction. Small portions of the sediment at various depths in the core were placed in additional tared beakers and oven dried for measurement of sediment pore-water content. The sediment was observed to be well stratified into horizontal horizons of distinct color and texture.
Extraction and Fractionation of Lipids Due to logistical problems and time constraints associated with sampling in Antarctica, one set of water column particulate samples and one core were available for analysis. Sediment samples and filters were solvent extracted using the modified Bligh and Dyer procedure as described previously [21, 55]. The lipid was frozen until further analysis. Lipids from the sediment and water column particulates were fractionated into neutral lipids, glycolipids, and phospholipids by column chromatography on Unisil silicic acid (Clarkson Chemical Co., Pennsylvania), according to procedures described previously [24].
Diacylphospholipids A mild alkaline methanolysis was applied to the phospholipid fraction of the lipids to release and methylate the ester-linked fatty acids by methods previously described [55]. The phospholipidderived fatty acid methyl esters (PLFAME) were purified by thin layer chromatography (TLC). The plates of silica gel K6, size 20 cm • 20 cm x 250 #m (Whatman, Alltech Pty. Ltd., NSW, Australia) were precleaned in hexane-diethyl ether-acetic acid (70/30/1, vol/vol/vol). A C~9 fatty acid methyl ester (FAME) standard was applied to the end lanes of the TLC plate, and the sample was spotted in the middle lane. After development in the solvent system described above, the end lanes were sprayed with 1,2,dichlorofluoroscene (0.1% in water/methanol, 1/1, vol/vol) for visualization of the FAME standard under UV light. The 2-cm wide band corresponding to the standard FAME spot was scraped from the sample lane and the silica gel was collected into Pasteur pipettes plugged with glass wool preextracted with chloroform-methanol (1/1, vol/vol). PLFAME were eluted from the silica gel with 10 ml chloroform. The PLEL in the samples still existed as polar phospholipids and were, therefore, still on the sample origin o f the TLC plate. A band 2 cm in width was scraped from the origin of the TLC plates, and the PLEL were eluted from the silica gel with 10 ml chloroform-methanol (1/2, vol/vol). The PLFAME and PLEL samples were dried under a stream of nitrogen and stored frozen until further analysis.
Hydrolysis of PLEL PLEL were hydrolyzed in 2 ml methanol--chloroform-concentratedHCI (10/1 / 1, vol/vol/vol) at 100*C overnight (12 to 16 hours) to remove the polar phosphate group(s). After the addition of 2 ml water, the resultant glycerol ether lipids were extracted with 2 ml hexane--chloroform (4/1, vol/ vol). The organic layer was transferred to a second test tube and an additional 2 ml solvent was added and the extraction was repeated. The pooled organic layers were dried under a stream of nitrogen, and the samples were frozen until HPLC analysis.
Microbial Ecology of Ace Lake, Antarctica
77
HPLC Analysis of PLEL Diether and tetraether PLEL were separated and quantified by a normal phase HPLC system consisting of a Rbeodyne R7125 injector (California) fitted with a 20 #1 sample loop, a Waters M45 pump (Massachusetts), a Spherisorb $5 amino column (250 x 4.5 ram, SGE Pty. Ltd., Victoria, Australia) heated to 35~ in a TC 1900 temperature controller (ICI Instruments, Victoria, Australia). The lipids were detected by an Erma ERC 7511 refractive index detector (Erma Instruments, Japan), the signal was processed with a Milton Roy CI- 10B integrator, the ehromatogram was printed on a LDC/Milton Roy chart recorder (Florida), and the data was stored for reprocessing by a Commodore 1001 disc drive (NSW, Australia). The solvent system (hexane-n-propanol, 99/1, vol/vol) was pumped at a rate of 0.5 ml/min. To all Samples, 1.8 ~zg 1,2 di-O-hexadecyl-rac-glycerol (Sigma Chemical Co., Missouri) was added as an internal standard. Retention times for the diether and tetraether lipids were established using lipids isolated from Methanobacteriumthermoautotrophicum strain Hveragerdi identified as described previously [30]. Fractions containing methanogen PLEL were collected from "the HPLC eluent in small vials, the solvent was evaporated under a stream of nitrogen, and the samples were frozen until gas chromatographic (GC) or Fourier transform/infrared (FT/IR) speetrophotometric analysis.
GC of PLFAME and PLEL PLFAME samples, containing methylnonadecanoate (19:0) as an internal standard, were dissolved m chloroform. GC analyses were performed with a Hewlett Packard (HP) 5890 GC equipped with a 50 m x 0.20 m m i.d. cross-linked methyl silicone fused-silica capillary column and a flame ionization detector. Samples were injected at 50~ in the splitless mode with a 0.5-rain venting time. Quantitative recovery was checked with an n-alkane mixture (n-Cjzto n-C30 ). After 1 min, the oven temperature was programmed from 50 to 150~ at 30~ then at 3~ to 310~ HYdrogen was used as a carrier gas, and the injector and detector were maintained at 310~ Identification of diether PLEL from sediment and water column particulate samples was confirmed further by capillary GC and GC-mass spectrometry (GC-MS). Conditions for GC of the d/ether PLEL were similar to those above except that the oven temperature was programmed from 50 to 250oc at 30~ then at 4~ to a final temperature of 310~ The injector and detector were maintained at 310~ Tentative peak identification, prior to GC-MS analysis, was based on comparison of retention times with those obtained for authentic and laboratory standards (Alltech, NSW, Australia) and previously identified compounds as in the case ofarchaebacteria diether lipid [30], Peak areas were quantified with chromatography software (DAPA Scientific Software, Western Australia) operated Using an IBM-XT personal computer.
GC-MS Analyses GC-MS analyses of the PLFAME samples were performed according to the procedures of Nichols et al. [38].
Determination of Double-Bond Configuration PLFAME monounsaturated double-bond position and geometry were determined using the dinaethyl-disulfide (DMDS) procedure described previously by Nichols et al. [34].
78
C.A. Mancuso et al.
Fatty Acid Nomenclature Fatty acids are designated by the total number of carbon atoms: number of double bonds, followed by the position of the double bond from the w, or terminal methyl end of the molecule in monounsaturated fatty acids. In the polyunsaturated fatty acids (PUFA), ~ is followed by the position of the first double bond from the terminal methyl end of the molecule. Other double bonds are methylene interrupted. The suffixes "c" and "t" indicate cis and trans geometry. The prefixes "i" and "a" refer to iso and anteiso branching; "br" indicates the type of branching is undetermined. Other methyl branching is indicated as position of the additional methyl carbon from the carboxylic end (i.e., 10-Methyl 16:0). Cyclopropane fatty acids are designated with the prefix "cy."
FT/IR HPLC fractions containing PLEL were collected and were examined by FT/IR spectroscopy for structural confirmation using a Digilab FTS-206 FT/IR spectrometer fitted with a microscope accessory. Samples containing PLEL were dissolved in a minimum volume of hexane and were then spotted onto a calcium fluoride disc in preparation for analysis. All other procedures were as previously described by Mancuso et al. [30].
Results and D i s c u s s i o n A c e l a k e is a p e r e n n i a l l y m e r o m i c t i c l a k e w h i c h is c o v e r e d b y ice f o r at l e a s t 9 m o n t h s o f t h e y e a r [25]. T e m p e r a t u r e v a r i a t i o n s r e i n f o r c e t h i s s t r a t i f i c a t i o n and range from -0.1~ a t t h e l a k e s u r f a c e t o 11 t o 14~ at 9 m a n d t h e n d e c r e a s e to 1.7~ at t h e s e d i m e n t - w a t e r i n t e r f a c e (P. D. F r a n z m a n n , m a n u s c r i p t s u b m i t t e d for p u b l i c a t i o n ) . T h e w a t e r c o l u m n w a s d i v i d e d i n t o t h r e e z o n e s b a s e d o n o x y g e n c o n c e n t r a t i o n [25]. T h e u p p e r o x y l i m n i o n is s e p a r a t e d f r o m t h e l o w e r a n o x y l i m n i o n b y t h e o x y c l i n e w h i c h o c c u r s b e t w e e n 1 1 a n d 12 m (Fig. 1). S u l f a t e c o n c e n t r a t i o n i n c r e a s e s f r o m 1 m m o l / l i t e r at t h e w a t e r s u r f a c e to 9 m m o l / l i t e r at 10 m a n d t h e n d e c r e a s e s to 0.7 m m o l / l i t e r a t 19 m . Sulfide, in c o n t r a s t , i n c r e a s e s f r o m 0 m m o l / l i t e r a t 12 m t o 8 m m o l / l i t e r at 24 m . M e t h a n e first o c c u r s at a d e p t h o f 12 m , a n d b e l o w 20 m , t h e w a t e r c o l u m n is e s s e n t i a l l y s a t u r a t e d w i t h m e t h a n e [6]. H y d r o g e n gas w a s n o t d e t e c t e d in A c e L a k e (P. D. F r a n z m a n n , m a n u s c r i p t s u b m i t t e d for p u b l i c a t i o n ) .
Methanogen Signature Lipids The measure of archaebacterial PLEL isolated from water column particulates w a s l i m i t e d b y the s e n s i t i v i t y o f the r e f r a c t i v e i n d e x d e t e c t o r o f t h e H P L C s y s t e m u s e d in t h i s a n a l y s i s . T h e l o w e r l i m i t o f d e t e c t i o n w a s 0.2 #g for t h i s analysis. Particulate samples taken from the anoxic region of the Ace Lake w a t e r c o l u m n (i.e., 17, 20, a n d 23 m ) w e r e a n a l y z e d for t h e p r e s e n c e o f e t h e r l i p i d s . O n l y d i e t h e r l i p i d s w e r e d e t e c t e d in t h e s e s a m p l e s . A m o u n t s o f d i e t h e r P L E L r a n g e d f r o m < 0 . 0 3 ~ g / l i t e r a t 17 m to 0.2 ~tg/liter at 20 m d o w n to 0 . 0 4 # g / l i t e r at 23 m. T h e s e v a l u e s w e r e c o n f i r m e d b y c a p i l l a r y G C ( T a b l e 1). T h e i d e n t i t y o f p h o s p h o l i p i d - d e r i v e d d i e t h e r l i p i d s in t h e s e d i m e n t w a s also
Microbial Ecologyof Ace Lake, Antarctica
79
Oxygen ( m m o | I tiler ) 0 --
0.2 i
0.4 I
0.6 I
0.8 ,
I
I .0 9
I
S u l f a t e , M e t h a n e , S u l f i d e (retool I I/~er ) 10 0 5
2O 25 FiR. 1. Profiles of concentrations (rnmol/liter) of oxygen (~)), methane (I~), sulfide (11), and sulfate (m) with depth in Ace Lake, December 1987.
established as described above for water column particulate samples. A comPonent tentatively indentified as a tetraether lipid was detected in the sediments based on HPLC retention data. Insufficient material was available for structural confirmation by mass spectrometry of the intact tetraether or by FT/IR analysis. As structural verification of the tetraether was not obtained, these peaks were not included in the biomass calculations. The amount of PLEL found at various depths in the sediment is presented in Table 1. The diether lipid decreased from 4.87 #g/g dry weight at the sediment-water interface (the top of the core) to 0.04 ug/g dry weight at 2 m. The detection ofdiether PLEL in the water column particulates and sediment indicates the presence of intact, viable archaebacteria in Ace Lake [35]. Since the lake is neither hypersaline nor hot and acidic, halophilic and thermoacidophilic archaebacteria, which also possess PLEL, could not colonize the ecosystem [28]. Archaebacterial methanogens are well suited to the anaerobic bottom waters and sediment of Ace Lake. Methane is present in the water COlumn at 11 m (0.01 mmol/liter) and increases with depth (5.5 retool/liter at 24 m, Fig. 1). The biomarkers for methanogenic bacteria, PLEL, were detected at and below 17 m in water column particulate samples. These data are supported by other studies on the ecology of Ace Lake [6, 8], which showed active methane production below 18 m in the water column. Phytane and 2,6,10,15,19-pentamethyleicosane, two hydrocarbon markers for raethanogenic bacteria, were in abundance above 35 cm in the sediment but their concentration decreased below this depth [48]. It is interesting to note the similarity between the depth profile ofmethanogen isoprenoid hydrocarbon markers [48] and that of the PLEL (Table 1).
80
C . A . Mancuso et al.
Table l. Concentrations of methanogenic diether lipids and methanogenic biomass estimates for Ace Lake water column particulate and sediment samples
Depth in the water column (m)
Phospholipid diethers (ug/liter)
Methanogenic biomass~ [cell number ( • 10~)/liter]
17 20 23
I00,
150'
A
200 Cell number ( 9 I0 7) / I~ dxy weight sedJmp-at 0 0 i
I0 i
[
20
~
I
30 ,
'
40 '
501oo. I~
15o
B 200
50 ',
'
Fig. 3. (A) Profiles of cell numbers of nonarchaebacteria (m) determined from total PLFA in Ace Lake sediment. (B) Profiles of cell numbers for Desulfovibrio spp (m) determined from i 17: lw7e and for Desulfobacter spp. (~) determined from 10Mel6:0 in Ace Lake sediment.
peak in the algal b i o m a s s at the oxycline d e m o n s t r a t e d b y previous workers [5, 49] was, therefore, not o b s e r v e d in the present analysis. By c o m p a r i s o n o f the P U F A a n d b r a n c h e d P L F A , it is possible to note that the relative a b u n dances o f m i c r o e u k a r y o t e s , which include p h y t o p l a n k t o n , declined through the aerobic zone a n d decreased abruptly below the oxycline as indicated b y the trace a m o u n t s o f the P U F A signatures below the oxycline. The relative changes in P U F A and b r a n c h e d P L F A with d e p t h are an indication o f the m i c r o e u k a r y o t i c and bacterial c o m p o n e n t s o f the s e d i m e n t microbial c o m m u n i t y (Fig. 5). T h e average relative a b u n d a n c e o f b r a n c h e d P L F A was between 10 a n d 15% o f the total. In contrast, P U F A were generally above 30 c m and the p r o p o r t i o n o f these fatty acids decreased f r o m approximately 10 to 15% o f the total in the u p p e r s e d i m e n t to 2 to 3% o f the total PLFA at 25 a n d 30 cm, respectively. T h e eukaryotic signatures 20:5w3 a n d 22: 6w3 m a d e up a p p r o x i m a t e l y 4 to 7% o f the P U F A a b o v e 2 cm. T h e d i a t o m markers 20:4w6 and 20:50;3 a c c o u n t e d for up to 2% o f the p h o s p h o l i p i d P U F A above 2 era. V o l k m a n et al. noted that C h l o r o b i u m p i g m e n t s were a b u n d a n t at a depth o f 23 m, well below the photic zone, a n d this suggested that intact
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Microbial Ecology of Ace Lake, Antarctica
91
bacterial component of the community [35], is divided by the relative abundance of 16:0, a PLFA found ubiquitously in most organisms, the result gives an indication of the proportion of the bacteria in the water column particulates at each depth. This ratio (Table 2) increases from 0.3 near the surface to 4.1 at 23 m, implying a greater than 10-fold increase in the relative abundance of bacterial signatures through the water column. This is in agreement with the decrease in the relative abundance of the microeukaryotic component of the COmmunity with depth in the water column as indicated by relative abundances ~ The ratio ofiso and anteiso 15:0 to 16:0 in sediment samples ranges from
