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
<0.03 b 0.2 0.04
<1.0 7.4 1.3
Sediment depth (cm)
#g/g dry weight sediment
Cell number ( x 109)/g dry weight sediment
0 0.5 1 2 4 8 12 17 20 25 30 35 40 50 75 100 125 147 175 198
4.9 0.5 3.1 0,5 0.2 0.5 0,7 0.3 0.3 0.1 0.1 0.04 0.1 0.0 1.1 0.1 1.3 0.1 0.6 0.04
17.7 1.9 11.4 1.7 0.6 1.9 2.6 1.1 1.0 0.5 O.3 0.2 0.3 0.0 3.8 0.5 4.7 0.2 2.1 0.1
a Conversion factor: 4.24 • 10-J9 mol ether lipid/methanogenic bacterial cell b Diether lipid concentration at lower limit of detection
Methanogenic Biomass Estimates The biomass of viable methanogenic bacteria in Ace Lake was determined using two conversion factors. There are approximately 2.5 umol PLEL/g dry weight of methanogenic bacteria. This value was determined in studies on pure cultures of methanogenic bacteria having varying proportions of diether and tetraether PLEL and obtained from various sources, grown under various conditions and analyzed using the same procedure used in this study [35]. Given that total lipids comprise approximately 4% of the dry weight ofarchaebacterial cells and polar lipids account for nearly 85% of these lipids [27, 28], it is a reasonable conversion factor. Phosphate-containing lipids make up approximately half of the polar lipids [28]. Therefore, 1.7% of the dry weight of an
Microbial Ecologyof Ace Lake,Antarctica
81
archaebacterial cell is composed of PLEL. This value is in close agreement with the average of 2.5 txmol PLEL/g dry weight methanogenic bacterial cells (I .6% PLEL/g dry weight) used in this study. Several studies have determined that the average weight of a bacterial cell is approximately 1.7 • 10 -13 g/cell (5.9 x 1012 cells/g dry weight of cells [I, 54]). Thus, it can be calculated that one rnethanogenic bacterial cell contains 4.24 x 10 -~9 mol diether and/or tetraether PLEL. Application of these two conversion factors to concentrations of PLEL in the water column particulate samples shows that a viable rnethanogenic archaebacterial population of approximately 1 x 108 cells/liter occurred at 17 m. Their number increased to 7.4 x 108 cells/liter at 20 m and then decreased to 1.3 x 108 cells/liter at 23 m (Table 1). The biomass of methanogens in the sediment was considerably higher than in the water column. Approximately 17.7 x 109 cells/g dry weight sediment Were present at the water-sediment interface (Table 1). The cell number decreased to approximately one tenth of this value over the next 2 cm (1.7 x 109 cells/g dry weight) and then gradually diminished to 0.1 • 109 cells/g dry Weight at 200 cm. Three biomass peaks at 12, 75, and 125 cm corresponded to depths in the core at which the sediment was darker in color than the SUrrounding regions and sponge-like in texture. These lipid-rich regions deep lr~ the sediment of Ace Lake may have contained sufficient amounts of organic Carbon to sustain an increased microbial population as indicated by the elevated Concentrations of methanogenic bacterial signature lipids. A methanogenic bacterial biornass of 6.8 x 10 ~ cells/g dry weight has been measured in anaerobic sewage sludge samples [35]. Comparisons with methanogenic biomass in Ace Lake sediment show that the methanogenic population ~n Ace Lake surface sediments and at several other depths are approximately J/4~as dense as that of anaerobic sewage sludge which is known to be rich in rnethanogenic bacteria [57]. Methanogenic biomass in a methane-generating, Water hyacinth-fed anaerobic digestor was estimated at 8.5 x 10 ~o cells/g dry Weight digestor material [35]. This biomass value is more similar to that determined for Ace Lake sediments and suggests that Ace Lake may have an anaerobic sediment microbial community enriched in methanogenic bacteria. Rates of methanogenesis from t4C-labeled precursors were measured in Ace Lake during the 87/88 sampling season (P. D. Franzmann, manuscript submitted for publication). The highest rate of methanogenesis in the water column Was 2.5 umol/kg/day at I~ at 20 m, which was the depth of the maximum Concentration ofmethanogenic bacteria in the water column. This rate, although the highest detected for Ace Lake, is low when compared to other eutrophic anaerobic aquatic environments (P. D. Franzmann, manuscript submitted for Publication). The highest rate of methanogenesis measured in Ace Lake sediment was 0.46 urnol/kg/day at 12 era, which also corresponds with a subsurface maximum in methanogenic bacterial biomass. Although the methanogenic bacterial biomass approached that of an anaerobic digestor at this depth in the sediment, the methane production rate measured at this depth was well below the average rate of rnethanogenesis (61 mmol/kg/day) measured for an anaerobic waste
82
C.A. Mancusoet al.
digestor [32]. In the anaerobic sediment of Lake Mendota, Wisconsin, workers found rates of methanogenesis of 633 ~mol/kg/day at 10~ were accompanied by a biomass of 105 methanogens/g dry weight sediment [58]. The low activity yet high methanogenic hiomass estimated for Ace Lake sediment and water column particulates may be partially explained by the low (1 ~ temperature in the lower water column and sediment of the lake. To date, no methanogen has been isolated which produces methane optimally at less than 25~ [3]. In studies of the methanogenic community of Lake Mendota, methanogenesis was severely temperature-limited [58]. The maximum annual in situ temperature (23~ in Lake Mendota was far below the temperature optimum (35 to 42~ for methanogenesis, although the predominant methanogenic population was metabolically active between 4 and 45~ Rates of methanogenesis measured in lakes on antarctic maritime islands, which displayed consistently low temperatures (annual range 1 to 3~ showed optima in excess of 30~ although these temperatures were never experienced in the field [ 12]. Methanogenic bacterial populations which inhabit the water column and sediment of Ace Lake may show similar trends for optimum methane production at higher than in situ temperatures (P. D. Franzmann, manuscript submitted for publication). Alternatively, psychrophilic bacteria may exhibit strategies similar to those observed in macrofauna which are adapted to permanently cold antarctic marine environments [33, 41]. Under these extremes, population dynamics of the benthic macrofauna are controlled by low metabolic rates, reduced individual energy requirements, and greatly increased standing crops [9]. The age of Ace Lake sediment from a depth of 200 cm in a sediment core was estimated to be 5,000 years with carbon isotope dating techniques (M. Bird, personal communication). The high methanogenic biomass measured at depth in Ace Lake sediment implies that the current microbial degradation of organic carbon in these sediments could be occurring very slowly. Studies of rates of methanogenesis (P. D. Franzmann, manuscript submitted for publication) support this theory. Seasonal differences in the biomass ofphytoplankton which occur in the upper water column of Ace Lake have been observed [5]. The intermittent descent, on a seasonal scale, of organic material into the depths of the lake may also cause seasonal variations in rates of metabolism in microorganisms, which supply the precursors for methanogenesis. Annual rate studies, in addition to manipulations of pure methanogenic cultures and enrichments (when such microbes have been isolated and are available) from Ace Lake, may provide some insight into the adaptation of the microbial community to low temperature.
Nonarchaebacterial Biomass Estimates Forty-eight individual PLFA were identified in the water column particulate samples using capillary GC and GC-MS (Table 2). Total PLFA isolated from the water column particulate samples ranged in concentration from 1.9 #g/liter at 2 m to 8.4 #g/liter at 23 m, showing an increase with depth. Amounts of
Microbial Ecology of Ace Lake, Antarctica
83
individual P L F A isolated from water c o l u m n particulates are expressed as a percentage o f the total P L F A for each depth (Table 2). Concentrations o f total P L F A in water c o l u m n particulates were used to calculate a p p r o x i m a t e cell n u m b e r s o f nonarchaebacterial microbes in the water COlumn and sediment. The following approximations were used: the average bacterium, the size o f E. coli contains 100 #mol P L F A / g dry weight [1, 54] and 1 g o f bacteria is equivalent to 5.9 x 10 '2 cells [1, 54], which gives 1.7 x 10-'7 mol PLFA/bacterial cell. Using these conversion factors, nonarchaebacterial water c o l u m n microbes, after an initial decline from 4.4 • 108 cells/liter at 2 m to 2.2 • 108 cells/liter at 5 m, increased steadily to 19.4 • 108 cells/ liter at 23 m (Fig. 2). Total P L F A isolated from Ace Lake sediment also showed a similar decreasing trend with increasing depth (Table 3). P L F A decreased from 18.4/xg/g dry Weight at 0.5 cm to 4.6/zg/g dry weight o v e r the next 2 cm.. T h e level o f P L F A then generally decreased to a m i n i m u m o f 0.03 ~g/g dry weight at a p p r o x i m a t e l y 2m. When the biomass conversion factors for nonarchaebacteria were applied to these data, microbial biomass was calculated to be greatest at 0.5 cm depth (42.3 • 108 cells/g dry weight, Fig. 3A) and then decreased by a factor o f 4 OVer the next 2 cm. Biomass then decreased m o r e gradually to a m i n i m u m o f 0.06 • 108 cells/g dry weight at 2 m, with sharp increases to 7.5 x l0 s at 12 cm and 10.2 • 108 at 75 cm. A somewhat smaller increase to 2.4 • 108 was observed at 125 cm. T h e relatively higher biomass in the surface sediments and the peaks in biomass at three depths corresponded to elevated m e t h a n o genic biomass d e t e r m i n e d from P L E L concentrations (Fig. 1). N o n a r c h a e b a c terial microbes were, however, only one fourth as a b u n d a n t as the methanogenic bacteria in the sediment. These data suggest that the methanogens are substrate limited as they are d e p e n d e n t on the eubacterial metabolism to p r o v i d e their energy source. Total bacterial (nonarchaebacterial and methanogenic) phospholipid-based biomass approximations c o m p a r e d favorably with those estimates obtained using acridine-orange direct microscopic counts (AODC) on water c o l u m n particulate samples. Counts were p e r f o r m e d according to the m e t h o d o f Zimm e r m a n n [59]. For example, at a water c o l u m n depth o f 2 m and 20 m, A O D C of 7.5 x 108 and 81.0 • 108 cells/liter corresponded to phospholipid-based estimates o f 4.4 • 108 and 13.3 x 108 cells/liter, respectively. T h e similarity of these findings supports the use o f phospholipids as a biomass assessment tool for this e n v i r o n m e n t .
Signature PLFA of SRB The presence o f 1 0 M e l 6 : 0 P L F A without an a b u n d a n c e o f other 10 methylbranched P L F A is a signature for the SRB genus, Desulfobacter [16, 43]. This fatty acid contributes approximately 27% to the total P L F A o f this organism [16]. Similarly, i l 7: lw7c has been identified as a signature for Desulfovibrio Spp. and comprises 30% o f the P L F A o f this group o f SRB [11]. Calculations
84
C.A.
T a b l e 2.
Composition
of PLFA
in Ace Lake water column Percentage
Fatty acid
2 m"
5 m
particulate
samples
composition
10 m
17 m
20 m
23 m
brl 3:0
--~'
tr'
0.2
tr
tr
tr
i14:0
0.5
tr
0,3
1.2
1.2
2.5
14:16o7c 14:0
. 4.0
1.8
2.0
0.1 3.2
.
.
. 4.6
--
.
brl 5:0
tr
tr
tr
tr
0.8
1.1
i15:0
2.7
2.8
2.8
2.7
3.1
4.1
a15:0
4.4
3.7
11.4
21.7
21.4
24.8
15:1 '~
--
--
2.3
0.7
--
0.5
15:0
4.4
--
4.0
1.4
0.9
0.9
brl7:0
1.6
--
0.2
4.5
2.2
1.3
--
--
tr
--
--
--
16:2'; i16:0
1.0
--
0.2
1.7
1.6
16:16o9c
1.4
4.4
0.3
0.7
0.6
2.0
16:16o7c
8.7
12.4
27,8
14,5
18.8
17.8
16:lto9t
--
--
0.6
0.9
5.6
3.1
16:16o5c
1.0
--
2.9
5.3
6,7
7.0 7.1
16:0
2.0
26.7
26.0
17.8
8.4
6.7
it7:16o7c
--
--
0.7
0.4
0.7
1.2
a17:16o7c 10Mel6:0
---
---
tr 1.5
0.6 2,3
0.4 5.8
tr 4.2
i17:0 a17:0
0.3 0.5
---
0.3 0.9
0.6 4.4
-3.7
0.3 3.3
+ 17:16o8"
17:16o6c
--
--
2.1
1.1
0.7
0.4
cyl7:0
--
--
2.1
0.7
0.8
0.4
cyl7:0
--
--
tr
1.7
1.6
--
17:0
0.3
--
--
tr
tr
--
b r 18:1 'j
tr
tr
tr
--
tr
tr
10MelT:0 CI8PUFA
. 5.8
tr
tr
d
.
. 1.2
. .
.
.
.
18:2w6
1.5
2.1
0.7
--
0.9
0.1
18:36o3
3.4
3.2
1.1
2.5
1.6
0.9
18:16o9c 18:1w7c + phytanic
3.8 8.3
3.8 22.8
1.1 9.3
3.1 11.4
4.0 7.6
4.7 6.1 tr
acid"
18 16o7t
--
--
tr
tr
tr
18:1w5c
--
--
tr
tr
tr
tr
18:0
1.8
3.6
1.8
2.0
1.8
2,1
b r l 9 : l ~;
--
--
tr
3.1
tr
tr
10Mel8:0
--
--
--
tr
tr
tr
19:1
--
--
--
tr
tr
tr
a 19:0
--
--
tr
tr
tr
tr
cyl9:0
--
--
3.1
1.3
--
--
20:46o6
--
--
tr
tr
tr
--
20:56o3
5.9
5.1
tr
tr
tr
tr
20:1'; 22:56o6
---
---
-tr
tr --
---
---
22:6~3
12.5
tr
d
9.5
tr
--
tr
Other
1.2
--
--
tr
tr
tr
Total #g/liter filtered
1.9
0.9
2.3
5.0
5.8
8.4
(i15:0 + a15:0)/16:0
0.3
0.2
0.8
2.9
3.6
4.1
1 6 : 1 6 o 9 t / l 6:1o~9c
0.0
0.0
1.7
1.3
8.7
1.6
(Footnotes to table on next page,)
Mancuso
e t al.
Microbial Ecology of Ace Lake, Antarctica
Cell number (x I0 8) ! liter 5 I0 15
0 0
85
,
,
,,,
a
~
20 i
5
tD
25 Profiles o f cell numbers for nonarchaebacteria (r~) determined from total' PLFA, for Desulfovibrio spp. (iP) determined from i 17'.1 toTc, and mr Desulfobacter spp. ~ determined from 10Mel6:0 in Ace Lake water column particulates. Fig. 2.
Using these a p p r o x i m a t i o n s do not take into account contributions o f these c o m p o n e n t s f r o m other organisms. Similarly, changes in P L F A profiles due to differences in nutrient or growth conditions m a y contribute to the o b s e r v e d differences. Nonetheless, we believe, in spite o f these considerations, that such an a p p r o a c h at this stage will highlight P L F A differences in t e r m s o f the contributing microbial c o m m u n i t y . A s s u m i n g that 10Me 16:0 a n d i 17" I ~7c are d e r i v e d solely f r o m Desulfobacter and Desulfovibrio, respectively, the percentage values discussed a b o v e were Used to estimate the c o n t r i b u t i o n o f these bacteria to the w a t e r c o l u m n particulate and s e d i m e n t m i c r o b i o t a . E m p l o y i n g the s a m e c o n v e r s i o n factors used above, m e m b e r s o f the genus Desulfobacter, while n o t present in the oxygenated Upper water c o l u m n (Fig. 2), increased in n u m b e r f r o m 0.5 • l0 s cell/liter at l0 m to 4.8 • 108 cells/liter at 23 m, where it accounted for a p p r o x i m a t e l y 25% o f the total nonarchaebacterial population. M e m b e r s o f the genus Desulfovibrio were less a b u n d a n t and increased f r o m 0.1 x l0 s cells/liter at 10 m to 0.9 • 10 s ceils/liter at 23 m. M e m b e r s o f the genus Desulfobacter were also detected in the top 30 c m of the s e d i m e n t profile (Fig. 3B). N u m b e r s o f this group decreased f r o m 4.6 x l0 s at 0.5 c m to 0.2 • l0 s at 30 cm. M e m b e r s of the genus Desulfovibrio were present at only trace levels in the sediment. Sulfate concentrations decreased with d e p t h in the water c o l u m n and b e c a m e limiting for sulfate reduction ( < 1 retool/liter [40]) below 18 m. T h e increase
" Depth in the water column (m) ~'(~) not detected *(tr) trace amount (<0.1%) of total PLFA *'Unable to confirm double bond position(s) due to insufficient material "Compounds co-eluted on capillary GC; unable to quantify indi'~iduaUy
T a b l e 3.
Composition
of PLFA
in Ace Lake sediment
Fatty
samples
Percentage
acid
0.5 cm"
brl 3:0
t r ~'
1 cm
2 cm
--'
.
composition
4 cm .
8 cm .
i14:0
1.4
--
--
1.8
1,3
14:1
--
--
--
tr
.
14:0
7.1
5.9
6.8
9.5
i15:0 al5:0
3 12.4
l 6.6
tr 4.9
3 l 1.5
tr 1.3
--
--
1.6
1.2
0.8
--
1.7
--
tr
i16:0
1.9
16:1w9c"
0.4
16:1w7c 16:lw9t
7.2 1.2
15:1
~7c
u
15:0 CI6PUFA 16:2
d
't
16:1w5c
1.2
16:0
26.7
12 c m
.
.
17 c m
20 cm
. --
.
.
1.7
1.3
.
7
7.3
9.2
11.2
2.1 8.5
1.7 7
2.7 9.5
2.4 7.8
tr
tr
1.4
tr --
tr 1.6
tr
2 --
1.9
2.2
tr
0
--
tr
tr
--
1.5 tr
8.2
1.5
2.1
1.6
--
1.2
1.4
--
tr
--
0.8
--
0.3
0.5
4.2 --
7.2 tr
7.8 --
5.2 1.7
4.3 1.2
4.4 2.1
4 1.5
tr
--
34.9
37.5
39.2
1.6
1,3 36
-35.4
1.1
1.6
1
29.8
38.9 tr
i 17:1~7c
0.6
--
--
--
tr
tr
tr
10Mel6:0
1.9
1.2
tr
0.5
1
tr
0,7
tr
i 17:0
--
tr
--
--
tr
tr
tr
tr
a17:0 +
2.3
1.6
tr
2.6
1.8
tr
1.8
1.3
cyl7:0
17:1~08 /
0.4
tr
--
tr
--
--
0.6
tr
17:lw6
0.3
tr
--
tr
--
--
0.7
tr
17:0
0.8
tr
--
tr
--
--
tr
--
0.9
tr
2.6
tr
--
--
1.3
1.3
18:2w6 18:3w3
2.4 1.9
2.5 1,7
2.7 2.7
1.2 1.2
2 2
3.6 2.8
7.8 --
2.5 2.1
18:1w9c
9.3
14.7
7.7
9,5
17.3
--
9.3
18:1w7c
.
3,2
4.9
16.5
CI8PUFA
Phytanic
d
acid
10.3 .
.
6.5
4,2
. 4. l
1.7
3,2
6.5
--
5.1 --
18:0
4.5
10,5
3.2
6.4
4.4
4.1
4.1
3.8
brl9: l d
tr
--
--
tr
tr
--
--
--
10Mel8:0
tr
tr
--
tr
1.2
1.7
tr
--
cy 19:0
tr
tr
--
--
tr
--
tr
--
19:1 '~
--
tr
--
--
tr
--
0
--
20:4w6
tr
.
1
--
20:5w3 20:1 d
1.6 tr
2 .
0 0
tr --
20: lw9c
tr
tr
2.8
tr
tr
tr
1.9
tr
20:0
tr
tr
--
--
tr
--
tr
1.3
22:0
tr
.
.
tr
tr
22:5w6 22:6w3
2.3 --
. 3.6
.
0 tr
-tr
.
.
.
2.8 .
tr .
3.0
.
.
.
.
.
.
tr
tr
.
. . tr
1.2
tr
24:0
tr
tr
--
tr
1.7
tr
tr
0.6
26:0
tr
--
--
tr
tr
--
tr
tr
g dry weight sediment
18.4
13.2
4.6
3.8
6.2
3.3
4.6
5.1
015:0 + a15:0)/ 16:0
0.6
0.2
0.1
0.4
0,3
0.3
0.4
0.3
16: l o J 9 t / I 6: loJ9c
2.8
__4,
_
_
2.3
--
8
2.8
T o t a l izg P L F A /
" Depth in the sediment (cm); concentration of total PLFA was detected at trace amounts (0 cm) sample l, (tr) t r a c e a m o u n t ( < 0 . 1 % ) o f t o t a l P L F A ' (--) not detected d Unable to confirm double bond position(s) due to insufficient material for analysis
in surface
T a b l e 3.
Continued Percentage composition
25 cm
30 cm
35 cm
tr
tr
1.1
2.3
tr
tr
8.5
9.6
2.7
3
9.6 2
40 cm
50 cm
75 cm
100 cm
125 c m
--
tr
tr
tr
tr
tr
3.5
2.9
1.4
tr
-
tr
--
tr
tr
.
13.1
15.6
11.6
6.6
9.6
--
15.4
6.6
9.3
4.2
6.4
1 1.4
4.5
2.5
4.9
2.2
2.5
tr
10.2
12.7
10.5
13.5
4
8.3
tr
tr 2.6
tr 3.4
tr 4.1
. 1.8
t .8
tr
1.2
tr 1.9
-tr
tr
tr
tr
--
tr
--
--
--
0
--
tr
--
0
--
0
tr
--
0.2
0
--
1.2
0.9
1.9
tr
--
1
--
1.9
--
tr
0
--
1.4
1.4
2.7
.
1.5
.
1.6
.
.
.
.
ll.l .
14.2 .
t
.
175 c m
198 c m
0.3
tr
tr
0.2
1
tr
tr
--
.
7 .
147 cm
.
0.9
3.7
2.6
3.1
2.5
1.5
tr
tr
0.3
1.3
--
1.8
6.9
4
3.1
1
1.6
tr
--
0.9
4.2
--
39.9
tr 40.1
tr
tr
1.2
tr 47
tr 40.3
tr 43.7
tr 64.7
tr 57.6
. 81
tr
tr
tr
tr
--
0.9
tr
tr
tr
tr
tr
tr
tr
tr
tr
1.8
1.5
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
0.7
tr
tr
tr
tr
.
66.4
. 52. I
tr
--
tr
--
--
tr
1.3
--
tr
--
tr
tr
tr
--
1
--
tr
tr
tr
tr
--
0.8
tr
tr
tr
tr
tr
--
--
tr
tr
tr
tr
tr
--
tr
tr
tr
tr
tr
tr
--
tr
--
tr
--
2.1
1.6
tr
tr
tr
--
tr
3.6
--
tr
1.3
tr
tr
tr
tr
tr
--
tr
--
--
tr
tr
--
tr .
.
.
.
.
82.2 --
8.5
6.2
3.3
2.2
1.5
3.1
4.2
tr
0.7
4.8
tr
6.6
4.4
2.6
3.7
2.4 .
--
--
1.5
-1.8
tr
_
0.6 .
--
3.9
4
3.7
tr
5.8
4.9
tr
tr
--
--
4.8
0.7
.
.
. 4.3
.
2.7
. --
tr
--
--
tr
-tr
tr .
tr
.
1.8
-1.5
tr tr
. tr
tr
tr
tr
tr
3.6
tr
tr tr
tr tr
tr tr
tr tr
0.5 tr
tr
tr
tr
tr
.
1
tr
tr
tr
tr
tr
--
--
tr
tr
tr
tr
tr
--
tr
2
1.4
1.7
1.8
1.5
4.4
0.9
0.3 0.7
0.3 .
.
0.4 .
.
. .
.
.
.
. .
.
.
.
.
. .
.
tr
. 10.1
. --
--
4.6
tr
tr
--
tr
tr
--
tr tr
---
---
0.2
--
--
0.3
1.6
--
0.3
tr
--
--
tr
--
--
I
0.9
0.2
.
0.6 .
. . .
.
0.4 .
. .
.
0.2 .
.
0.2 .
.
.
.
0.2 .
.
.
0.1 .
0.2
0.03
<0.1
.
16:1o~10c a n d 1 6 : l w l 0 t w e r e a l s o d e t e c t e d a s m i n o r c o m p o n e n t s i n t h e 8 c m s a m p l e f o l l o w i n g G C - M S a n a l y s i s o f the D M D S a d d u c t s /(2O m p o u n d s c o - e l u t e d o n c a p i l l a r y G C ; u n a b l e t o q u a n t i f y i n d i v i d u a l l y "
(--) 16:1o~9c n o t d e t e c t e d
88
c.A. Mancuso et al.
of SRB biomass with depth, despite the lack o f an available electron acceptor, m a y be the result o f the settling o f cells down through the stagnant water o f the lake as electron acceptors were at rate-limiting concentrations. It is possible that the SRB do not use sulfate as an electron acceptor but are involved in interspecies hydrogen transfer as proposed by Bryant [4]. F r a n z m a n n et al. (manuscript submitted for publication) reported negligible sulfate reduction rates in the surface sediment o f Ace Lake. It is also possible that SRB detected in these sediments are virtually inactive. Similar rates m a y occur in the lower water c o l u m n and m a y help to explain the SRB biomass increase observed in this study at depths where sulfate reduction is not metabolically feasible.
Phospholipid PUFA Signatures Phospholipid P U F A are characteristic markers for microeukaryotes [14, 35, 50]. PUFA content and branched PLFA, which serve as bacterial signatures [18, 39], were totaled for each depth in the water c o l u m n (Fig. 4). P U F A contributed 25 to 30% to the total P L F A above 5 m in the water column, whereas branched fatty acids m a d e up only 7 to 12% at these depths. F r o m a depth o f 10 m to the lake b o t t o m , branched fatty acids increased in relative percentage to between 25 to 45% in c o m p a r i s o n to P U F A which represented only 2 to 4% o f the total P L F A below 10 m. Specifically, 20:5c03, a eukaryotic b i o m a r k e r [35, 56], decreased from approximately 5% o f the total P L F A in the upper water c o l u m n d o w n to trace a m o u n t s ( < 0 . 1 % o f the total PLFA) at I 0 m and below (Table 2). The relative abundance o f 22:6w3, which has been found in sea-ice diatoms [36] and other algae, decreased from approximately 10% in the upper water c o l u m n to trace a m o u n t s below 10 m. A c o m m o n microeukaryotic m a r k e r 20:4~06 [43], occurred in trace a m o u n t s at depths o f 10, 17, and 20 m. Essentially, biomarkers for aerobic eukaryotes were restricted to the oxic zone o f the lake. In a 1979 study o f the annual cycling o f p h y t o p l a n k t o n in Ace Lake, Burch [5] observed a p h y t o p l a n k t o n c o m m u n i t y o f four flagellates restricted to the oxic zone o f the lake from the surface to 10 m. Active growth occurred during the spring and summer, and peak p h y t o p l a n k t o n biomass production was attributed to Pyramimonas gelidicola which occurred at the interface o f the oxic and anoxic zones (8 to 10 m). In an analysis o f the pigment and total lipids o f Ace Lake water c o l u m n particulates and surface sediment [49], pigments and lipids o f P. gelidicola were most a b u n d a n t at the base o f the oxic zone during the 1984 season. These and other lipids and pigments were in discrete horizontal bands through the aerobic layer o f the water c o l u m n and indicated that vertical zonation o f the p h y t o p l a n k t o n c o m m u n i t y occurred. T h e P U F A data presented here also support this finding. Over the past 4 years, the rise in water level o f Ace Lake has resulted in the oxic zone increasing from 9 m to a depth o f 11 m. The SeaStar in situ sampler used in the present study sampled water from a precise depth ( + 2 to 3 cm) unlike the K e m m e r e r bottle used for sample collection in the previous studies which sampled at depths o f _ 50 cm. T h e depths sampled in the present study did not include a sample at the current oxycline, 12 m (Fig. 1). The discrete
Microbial Ecology of Ace Lake, Antarctica
89
Cell n u m b e r ( 9 I0 8 ) / g dry weight sedJmP-nt 0
I0 9
O-
[]
ra,u
5'084
20
I
.
30
I
I
--
40
50
I
I
~
[]
>
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
oql ~u!luosaadoa '0:g I 3 o saotuos~, o s ! o l u e p u g os! j o s o o u g p u n q g OAI.113|&Ioql 3 o t u n s a q l JI "[6s '8 I] u!~.uo u! Ig!aologq 3~llgO!ls.~olagagqo aae V d q d p o q o u e a 0
sP!dt.7 a,mlvu&s VdTd palwmws parlaUn.~ff "SHOO a!lo,Qmtno loelu! ~ u ! l u o t u ! p o s aoj deal g sg la~ osle Al~tu s l u a t u ! p a s aql l'~ql slso$$ns aouap!Ao V A F I d P.td.qoqdsoqd "[617] u t u n l o o aa:tgax a q l u! aoq~.~q tuoaj l n o polllas p e q SllOa lg,Uoloeq o.tl~qlu.~solotld 9luatu!pos o~leq aoV u! Vd'-Id lglOl aql jo so~uluoaaods e qldop ql!,~ (~) sp!a~ ,~ID3Jpaqoueaq pug ([B) V::IFId30 soougpunqe oA!l~[oau! a~uuqa aql jo solUOad "S "~hI OOZ
OS!
oo~
OS
....- - r I
,__
i
0~'
(vq) sp!ae ,s
Og
OI
OZ
0
9salslno!laed uumloo aole,,a a)le'l oaV u] Vdqd lglOl oq130 so~eluaaaod se qldop ql!,~ paqoueaq pu~ ([~) Yzlfld jo sasuepunqg oA!llgloau! o~ueqo aql Jo soI~O.ld "t' "~!z[ gz
":":'.7:. Z~Z::. Z~L.? .:&.~L:"::'.:~Z.:'Zr s
.':" -"'":" i
OS
O~
O~
OZ
01
0
"I~ lo osnau~lAI"V "D
06
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 <0.1 to 0.6 and, unlike the water column, seems to show no trend with increasing depth in the sediment (Table 3).
Monounsaturated PLFA Signatures Bacterial input into the sediment can be associated with 18: l w7c [18, 21, 35]. In the water column, this PLFA was not resolved from phytanic acid on the GC column used. In the sediment, 18:1w7c was not present in significant COncentration above 8 cm in the sediment (Table 3), but was present at up to 3% of the total PLFA in sediment just below this depth. The abundance increased to 16.5% at 17 cm and then decreased to 2.4% at 75 cm. Phytanic acid Was found above 12 cm in the sediment, where it contributed less than 7% to the total PLFA present in the sediment. It has been reported that phytanic acid is incorporated by bacteria into membranes after being transformed biologically from phytol [51]. The presence of phytanic acid PLFA in the membranes derived from bacteria, implies bacterial activity at depth in the ancient anaerobic sediment of Ace Lake.
Trans Monounsaturated PLFA
Cis isomers of monounsaturated PLFA are common microbial cellular comPOnents [23]. High abundances of trans isomers have been recently detected in cultures of Pseudomonas atlantica, Vibrio cholerae, and environmental en.richments, and have been associated with strategies for survival during physIological stress in these organisms [21-23, 34]. The trans/cis (t/c) ratio for most bacterial cultures and sediments was determined to be less than 0.1 [18, 21, 36, 39]. Ratios of greater than 1 have been determined during starvation. It has been suggested that the t/c ratio of monoenoic PLFA may be useful as an index of stress for the determination of the nutritional status of bacteria in aquatic environments [22]. In this study, high proportions of 16:1 wgt were detected in both water column Particulates and sediments. The t/c ratio for 16:1o)9 from water column particulates is shown in Table 2. The ratio which was <0.1 at depths of 2 m and 5 m, increased to 1.7 at 10 m and to 1.3 at 17 m. The ratio increased abruptly to 8.7 at 20 m and finally decreased to 1.6 at 23 m. In Ace Lake sediment, the t/c ratio for 16:1 w9 was generally greater than one down through the sediment (Table 3). The trans isomer of 16:1 w7 was not detected in the sediment. In recent analyses of the PLFA derived from sewage sludge, it was suggested
92
C. A. Mancuso et al.
Table 4. Microbial community structure of Ace Lake Component Methanogens
Desulfobacter spp.
Desulfovibrio spp. Total Nonarchaebacterial microorganisms Bacteria
Microeukaryotes
Presence in water column > 1 x 108 cells/liter below 17 m; peak to 7 x 108 cells/liter at 20 m Increased from 0.5 • 108 cells/liter at 1 0 m t o 2 5 % ofnonarchaebacterial population at 23 m (5 • 108 cells/liter) Found below 10 m at 1/~the abundance of Desulfobacter spp. Increased with depth (4 • 108 cells/liter at 2 m to 19 • 10Xcells/liter at 23 m) Increased in relative abundance of total PLFA with depth Restricted to the oxic zone (<10 m)
Presence in sediment
Signature
18 x 109 cells/g dry weight at surface to ~2 x 109 cells/g dry weight at 2 m Present only in upper sediment (<30 cm) 4 x 108 to 0.2 x 10~ cells/g dry weight
Ether-linked isoprenoidbranched phospholipids 10Mel6:0 phospholipid fatty acids (PLFA)
Present only at trace levels in upper sediment (<20 cm) Decreased with increasing depth (42 • 108 cells/g dry weight at 0.5 cm to 0.06 x 108 cells/g dry weight at 2 m) Increased in relative abundance of total PLFA with depth Present at low levels in upper sediment (< 20 cm)
i17: Iw7c PLFA
Total PLFA
Branched PLFA
Phospholipid PUFA
t h a t Ct8 P U F A p r e s e n t i n the sludge were b i o h y d r o g e n a t e d to p r o d u c e 18: lw7t a n d i n c o r p o r a t e d i n t o b a c t e r i a l m e m b r a n e s [37]. T h e b i o h y d r o g e n a t i o n o f l i n oleic acid (18:2w6) to 18: l w 9 t is well d o c u m e n t e d for a n a e r o b e s o f t h e g e n u s Clostridium a n d E u b a c t e r i u m [15, 47]. T h e i n p u t o f t h e trans P L F A 16: l w9t f r o m b i o t r a n s f o r m a t i o n s o f P U F A f r o m the w a t e r c o l u m n is h i g h l y u n l i k e l y in the Ace Lake s y s t e m . C~6 P U F A , w h i c h w o u l d act as the b i o l o g i c a l s u b s t r a t e for this t r a n s f o r m a t i o n , were f o u n d i n m i n o r a m o u n t s i n the w a t e r c o l u m n ( T a b l e 2) a n d c o n t r i b u t e d o n l y m i n i m a l l y to the t o t a l P L F A o f the s e d i m e n t ( T a b l e 3). T h e high a b u n d a n c e o f trans P L F A i n A c e Lake w a t e r c o l u m n p a r t i c u l a t e s a n d s e d i m e n t is, therefore, m o r e likely to b e the r e s u l t o f p h y s iological stresses, i n c l u d i n g s u c c e s s i v e d e p l e t i o n o f a v a i l a b l e e l e c t r o n a c c e p t o r s w i t h d e p t h , w h i c h are i m p o s e d o n the m i c r o b i a l c o m m u n i t y i n h a b i t i n g the l o w e r w a t e r c o l u m n a n d s e d i m e n t o f the lake. I n this study, p h o s p h o l i p i d a n a l y s e s h a v e b e e n p e r f o r m e d o n w a t e r c o l u m n p a r t i c u l a t e a n d s e d i m e n t s a m p l e s f r o m A c e Lake, A n t a r c t i c a . P h o s p h o l i p i d d e r i v e d e t h e r - l i n k e d i s o p r e n o i d - b r a n c h e d l i p i d s were d e t e c t e d as s i g n a t u r e s o f v i a b l e m e t h a n o g e n i c a r c h a e b a c t e r i a . P h o s p h o l i p i d fatty a c i d profiles p r o v i d e d i n f o r m a t i o n o n the v i a b l e n o n a r c h a e b a c t e r i a l c o n s t i t u e n t s o f t h e lake e n v i r o n m e n t , i n c l u d i n g SRB. T h e s e b i o c h e m i c a l m a r k e r s were u s e d to e s t i m a t e the v i a b l e m i c r o b i a l b i o m a s s . T h i s i n f o r m a t i o n , s u m m a r i z e d i n T a b l e 4, t o g e t h e r
Microbial Ecology of Ace Lake, Antarctica
93
with physiological and physicochemical data from previous studies provides insight into the structure of the microbial community of Ace Lake as an antarctic lake e c o s y s t e m .
Acknowledgments. The authors wish to thank Dr. N. Roberts for his excellent field assistance, as Well as members of the Australian Antarctic Program serving at Davis Station during the 1987/ 88 summer season for expert logistic support. Drs. E. C. V. Butler and J. K, Volkman of the CSIRO bivision of Oceanography and Dr. T. A. McMeekin of the Department of Agricultural Science at the University of Tasmania are gratefully acknowledged for many helpful discussions, in addition to the use of their laboratory space and equipment. Dr, A. MeEwan is thanked for permitting access to the facilities at the CSIRO Division of Oceanography. This work was supported by grants from the Antarctic Science Advisory Committee and from the Australian Research Council. The manuscript was improved by the comments and suggestions of two anonymous reviewers.
References 1. Balkwill DL, Leach FR, Wilson JT, McNabb JF, White DC (1988) Equivalence of microbial biomass measures based on membrane lipid and cell wall components, adenosine triphosphate, and direct counts in subsurface aquifer sediments. Microb Ecol 16:73-84 2. Bayly IAE, Burton HR (1987) Distribution of Paralapidocera antarctica in Ace Lake. Aust J Mar Freshw Res 38:537-543. 3, Boone DR, Whitman WB (1988) Proposal of minimum standards for describing new taxa of rnethanogenic bacteria. Int J Sys Bacteriol 38:212-219. 4. Bryant MP, Wolin EA, Wolin M J, Wolfe RS (1967) Methanobacillus omelianskii, a symbiotiie association of two species of bacteria. Arch Microbiol 59:20-31 5. Burch MD (1988) Annual cycle ofphytoplankton in Ace Lake, an ice covered, saline meromictic lake. In: Ferris JM, Burton HR, Johnstone GW, Bayly IAE (eds) Biology of the Vestfold Hills, Antarctica. Kluwer Academic Publishers, Dordrecht, pp 59-75 6. Burton HR (1980) Methane in a saline Antarctic lake. In: Trudinger PA, Walter MR (eds) Biogeochemistry of ancient and modern environments. Australian Academy of Science, Canberra, pp 243-251 7. Burton HR (198 I) Chemistry, physics and evolution of antarctic saline lakes. Hydrobiologia 82:339-362 8. Burton H R, Barker RJ (1979) Sulfur chemistry and microbiological fractionation of sulfur isotopes in a saline Antarctic lake. Geomicrobiology J 1:329-340 9. Clarke A (1979) On living in cold water: k-strategies in Antarctic benthos. Mar Bio155:111-119 10. Culbertson CW, Zehnder AJB, Oremland RS (1981) Anaerobic oxidation of acetylene by estuarine sediments and enrichment cultures. Appl Environ Microbiol 41:396--403 11. Edlund A, Nichols PD, Roffey R, White DC (1985) Extractable and lipopolysaccharide fatty acid and hydroxy acid profiles from Desulfovibrio species. J Lipid Res 26:982-988 12. Ellis-Evans JC (1984) Methane in maritime antarctic freshwater lakes. Polar Biol 3:63-71 13. Ellman GL (1959) Tissue sulphyldryl groups. Arch Biochem Biophys 82:70-74 14. Erwin JA (1973) Comparative biochemistry of fatty acids in eucaryotic microorganisms. In: Erwin JA (ed) Lipids and biomembranes ofeucaryotic microorganisms. Academic Press, New York, pp 41-143 15. Eyssen H, Verhulst A (1984) Biotransformation oflinoleic acid and bile acids by Eubacterium lentum. Appl Environ Microbiol 47:39-43 16. Dowling NJE, Widdel F, White DC (1986) Phospholipid ester-linked fatty acid biomarkers of acetate-oxidizing sulfate-reducers and other sulfide-forming bacteria. J Gen Microbiol 132: 1815-1825 17. Franzmann PD, Skyring GW, Burton HR, Deprez PP (1987) Sulfate reduction rates and some aspects of the limnology of four lakes and a fjord in the Vestfold Hills, Antarctica. In: Ferris JM, Burton HR, Johnstone GW, Bayly IAE (eds) Biology of the Vestfold Hills, Antarctica. Kluwer Academic Publishers, Dordrecht, pp 25-33
94
C.A. Mancuso et al.
18. Gillan FT, Hogg RW (1984) A method for the estimation of bacterial biomass and community structure in mangrove-associated sediments. J Microbial Methods 2:275-293 19. Goodfellow M, Minnikin DE (1985) Introduction to chemosystematics. In: Goodfellow M, Minnikin DE (eds) Chemical methods in bacterial systematics. Soc Appl Bact Tech Ser No 20, Academic Press, London, pp 1-15 20. Green DR, Stull JR, Heesen TC (1986) Determination of chlorinated hydrocarbons in coastal waters using a moored in situ sampler and transplanted live mussels. Mar Pollut Bull 17:324329 21. Guckert JB, Antworth CP, Nichols PD, White DC (1985) Phospholipid, ester-linked fatty acid profiles as reproducible assays for changes in procaryotic community structure of estuarine sediments. FEMS Microbiol Ecol 31 : 147-158 22. Guckert JB, Hood MA, White DC (1986) Phospholipid ester-linked fatty acid profile changes during nutrient deprivation of Vibrio cholerae: Increase in the trans/cis ratio and proportions of cyclopropyl fatty acids. Appl Environ Microbiol 52:794-801 23. Guckert JB, Ringelberg DB, White DC (1987) Biosynthesis of trans fatty acids from acetate in the bacterium Pseudomonas atlantica. Can J Microbiol 33:748-754 24. Hand RM (1980) Bacterial populations of two saline antarctic lakes. In: Trudinger PA, Walter MR (eds) Biogeochcmistry of ancient and modern environments. Australian Academy Science Press, Canberra, pp 123-129 25. Hand RM, Burton HR (1981) Microbial ecology of an Antarctic saline meromictic lake. Hydrobiologia 82:363-- 374 26. Hanselmann KW (1986) Microbially mediated processes in environmental chemistry. Chimia 40:146-- 159 27. Langworthy TA (1985) Lipids ofarchaebacteria. In: Woese CR, Wolfe RS (eds) Archaebacteria. Academic Press, New York, pp 459-497 28. Langworthy TA, Tornabene TG, Holzer G (1982) Lipids of archaebacteria. Zbl Bakt Hyg 1 Abt Orig C 3:228-244 29. Lechevalier MP (1977) Lipids in bacterial t a x o n o m y - - A taxonomists view. Crit Rev Microbiol 5:t09-210 30. Mancuso CA, Nichols PD, White DC (1986) A method for the separation and characterization of archaebacterial signature ether lipids. J Lipid Res 27:49-56 31. Martens CS, Van Klump J (1980) Biogeochemical cycling in an organic-rich coastal marine basin-I. Methane sediment-water exchange processes. Geochim Cosmochim Acta 44:471-490 32. Molnar L, Bartha I (1988) High solids anaerobic fermentation for biogas and compost production. Biomass 16:173-182 33. Morita RY (1975) Psychrophilic bacteria. Bacteriol Rev 39:144-167 34. Nichols PD, Guckert JB, White DC (1986) Determination of monounsaturated fatty acid double-bond position and geometry for microbial monocultures and complex consortia by capillary GC-MS of their dimethyl disulfide adducts. J Microbiol Methods 5:49-55 35. Nichols PD, Mancuso CA, White DC (1987) Measurement ofmethanotroph and methanogen signature phospholipids for use in assessment of biomass and community structure in model systems. Org Geochem 11:451-461 36. Nichols PD, Palmisano AC, Smith GA, White DC (1986) Lipids of the antarctic sea ice diatom Nitzschia cylindrus. Phytochemistry 25:1649-1653 37. Nichols PD, Volkman JK (1988) Lipid assays in mariculture and environmental studies. Australian Mar Sci Assoc, Silver Jubilee Conference Commemorative Vol, Wavelength Press, Chippendale, NSW, pp 198-202 38. Nichols PD, Volkman JK, Palmisano AC, Smith GA, White DC (1988) Occurrence of an isoprenoid C.,5 diunsaturated alkene and high neutral lipid content in antarctic sea ice diatom communities. J Phycol 24:90-96 39. Perry G J, Volkman JK, Johns RB, Bavor HJ (1979) Fatty acids of bacterial origin in contemporary marine sediments. Geochim Cosmochim Acta 43:1715-1725 40. Postgate JR (1951) The reduction of sulfur compounds by Desulfovibrio desulfuricans. J Gen Microbiol 5:725-738
Microbial Ecology of Ace Lake, Antarctica
95
41. Reichardt W (1988) Impact of the antarctic benthic fauna on the enrichment of biopolymer degrading psychrophilic bacteria. Microb Ecol 15:311-321 42. Ringelberg DB, Davis JD, Smith GA, Pfiffner SM, Nichols PD, Nickels JS, Henson JM, Wilson JT, Yates M, Kampbell DH, Read HW, Stocksdale TT, White DC (1988) Validation of signature phospholipid fatty acid biomarkers for alkane-utilizingbacteria in soils and subsurface aquifer materials. FEMS Microbiol Ecol 62:39-50 43. Smith GA, Nichols PD, White DC (1986) Fatty acid composition and microbial activity of benthic marine sediments from McMurdo Sound, Antarctica. FEMS Microb Ecol 38:219-231 44. Strickland JDH, Parsons TR (1972) A Practical handbook of seawater analysis; 2nd ed. Bull Fish Res Bd Can 167:3 I0 45. Tabatabai MA 0974) Determination of sulfate in water samples. Sulfur Inst J 10:11-12 46. Tornabene TG, Langworthy TA (t979) Diphytanyl and dibiphytanyl glycerol ether lipids of methanogenic archaebacteria. Science 203:51-53 47. Verhulst A, Semjen G, Meerts U, Janssen G, Parmentier S, Asselberghs S, Van Hespen H, Eyssen H (1985) Biohydrogenation of linoleic acid by Clostridium sporogenes, Clostridium 9 bifermentans, Clostridium sordellii and Bacteroides sp. FEMS Microb Ecol 31:255-259 48. Volkman JK, Allen DI, Stevenson PL, Burton HR (1985) Bacterial and algal hydrocarbons in sediments from a saline Antarctic lake, Ace Lake. Org Geochem 10:671-681 49. Volkman JK, Burton HR, Everitt DA, Allen DI (1988) Pigment and lipid compositions of algal and bacterial communities in Ace Lake, Vestfold Hills, Antarctica. In: Ferris JM, Burton HR, Johnstone GW, Bayly IAE (eds) Biology of the Vestfold Hills, Antarctica. Kiuwer Academic Publishers, Dordrecht, pp 41-57 50. Volkman JK, Johns RB, Gillan FT, Perry GJ, Bavor HJ (1980) Microbial lipids of an intertidal sediment-- 1. Fatty acids and hydrocarbons. Geoehim Cosmochim Aeta 44:1133-1143 51. Volkman JK, Maxwell JR (1986) Acyclic isoprenoids as biological markers. In: Johns RB (ed) Biological markers in the sedimentary record, methods in geochemistry and geophysics. Elsevier Science Publishers, The Netherlands, pp 1-42 52. White DC (1983) Analysis of microorganisms in terms of quantity and activity in natural environments. In: Slater JH, Whiltenbury R, Wimpenny JWT (eds) Microbes in their natural environments. Soc Gen Microbiol Ltd, Cambridge University Press, Cambridge, pp 38-66 53. White DC (1986) Quantitative physiochemical characterization of bacterial habitats. In: Poindexter JS, Leadbetter ER (eds) Bacteria in nature, vol 2. Plenum Press, New York, pp 177203 54. White DC, Bobbie RJ, Herron JS, King JD, Morrison SJ (1979) Biochemical measurements of microbial mass and activity from environmental samples. In: Costerton JW, Colwell RR (eds) Native aquatic bacteria: Enumeration, activity and ecology ASTM STP 695. Am Soc for Testing and Materials, Philadelphia, pp 69-81 55. White DC, Davis WM, Nickels JS, King JD, Bobbie ILl (1979) Determination of the sedimentary microbial biomass by extractible lipid phosphate. Oecologia 40:51-62 56. White DC, Smith GA, Nichols PD, Stanton GR, Palmisano AC (1985) The lipid composition and microbial activity of selected antarctic benthic marine sediments and organisms: A mechanism for monitoring and comparing microbial populations. Antarctic J U S 130-132 57. Zeikus JG (1977) The biology of methanogenic bacteria. Bacteriol Rev 41:514-541 58. Zeikus JG, Winfrey MR (1978) Temperature limitation of methanogenesis in aquatic sediments. Appl Environ Microbiol 31:99-107 59. Zimmermann R (1977) Estimation of bacterial number and biomass by epifluorescence microscopy and scanning electron microscopy. In: Reinheimer G (ed) Microbial ecology of a brackish water environment. Springer-Verlag, Heidelberg, pp 103-120
