Microb Ecol (1987) 14:203-217
MICROBIAL ECOLOGY
(~ Springer-VerlagNew York Inc. 1987
Dynamics of Microbial Biomass and Activity in Five Habitats of the Okefenokee Swamp Ecosystem Mary Ann Moran, A. E. Maccubbin,* Ronald Benner, and Robert E. Hodson Institute of Ecology and Department of Microbiology,University of Georgia, Athens, Georgia 30602, USA Abstract. A variety o f freshwater marsh and swamp habitats are found interspersed in a mosaic pattern throughout the Okefenokee Swamp, Georgia, USA. We examined spatial and temporal patterns in standing stocks and activity in the microbial community o f five habitats within this heterogeneous ecosystem. Standing stock dynamics were studied by measuring microbial biomass (ATP) and bacterial numbers (AODC) in both water and sediments over a 14 month period. Abundance varied temporally, being generally lower in winter months than in spring and summer months. However, a large proportion o f the measured variability was not correlated with temporal patterns in temperature or with bulk nutrient levels. Spatial variability was characteristic o f the Okefenokee at a variety of large and small scales. Habitat-level heterogeneity was evident when microbial standing stocks and activity (measured as [t4C]lignocellulose mineralization) were compared across the five communities, although abundance differences among sites were restricted to nonwinter months when microbial biomass was high. Spatial variation within habitats was also found; patches o f surface sediment with differing microbial activity or abundance were measured at scales from 30 cm to 150 m. Introduction The Okefenokee Swamp is a highly heterogeneous ecosystem, in which plants and animals are patchy in distribution and organized into many distinct habitats. Pine islands, tree and shrub swamps, emergent and floating-leaved macrophyte marshes, and small lakes are interspersed in mosaic fashion throughout this vast, acidic wetland. Macroorganisms of the swamp are heterogeneous on a temporal scale as well; plant communities undergo long-term directional changes along successional sequences [ 13, 15], which, for example, may begin with barren peat masses and culminate in hardwood-dominated islands. Plant communities also undergo short-term changes in response to hydrologic variations, such as a shift from floating-leaved vegetation to an emergent community following a period of exceptionally low water levels [ 17]. * P r e s e n t address: Department of ExperimentalBiology,RoswellPark Memorial Institute, Buffalo, New York 14263, USA.
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Our previous work provides evidence that microorganisms, too, may be spatially and temporally heterogeneous in their distributions. The Okefenokee has relatively high and strongly seasonal rates oi'bacterial secondary production in the water column, the changes in which appear to be coupled to both temperature and patterns of primary production [29]. Rates of microbial decomposition of the large store of detrital lignocellulose, the complex vascular plant structural polymer comprised of lignin and polysaccharides, are also seasonal, with a twofold decrease in rates of lignocellulose mineralization during winter months compared to summer [5]. The seasonal depression of this largely bacterial degradative process is directly attributable to low temperatures rather than to shifts in the functional or species composition o f the microbial community [5, 6]. In the Okefenokee Swamp, as in other detritus-based wetlands, microorganisms are important in mediating the mineralization and repackaging of the detrital material which forms the base of many swamp food webs [30, 33]. In this blackwater system, primary production is dominated by vascular plants, so that structural plant polymers account for a large fraction of available organic matter. Direct assimilation of plant detritus by animals is minimal [35], however, microorganisms use the lignocellulosic detritus, as well as highly dilute dissolved organic matter (DOM) of plant and animal origin. Subsequently, detritivores feed on high-quality microbial biomass produced at the expense of low-quality particulate and dissolved carbon sources; thus microorganisms potentially form an important link between primary producers and animal consumers [30, 33]. Those components of the partially degraded vascular plant detritus which are not converted to microbial biomass and do not enter aquatic food webs contribute to peat accumulation; microbial activity also plays a role in regulating this important organic geochemical process. Given the patchy distribution of plants and detritivorous animals and the acknowledged importance of heterotrophic microorganisms to the overall trophodynamics of the Okefenokee ecosystem, we recognized an absence of information on the distribution patterns of the microbial community. A study was thus undertaken to determine patterns in microbial standing stocks and activity on structural and temporal scales. We chose simple, widely used indices to examine temporal trends in microbial biomass, spatial variation in biomass and activity among community types, and spatial variation in biomass and activity within individual Okefenokee communities. Total microbial biomass was measured using the ATP technique [21 ] in which the firefy luciferase assay is used to determine the concentrations of adenosine triphosphate as an index of live biomass. Shortcomings of the procedure are recognized [2, 1 l, 14, 23]; however, the technique is sensitive, relatively simple, and includes all microbial groups (bacteria, fungi, protozoa, meiofauna, and algae). Bacterial biomass determinations were made using the acridine orange direct count method (AODC), in which cells stained with a fluorescent dye are counted via epifluorescence microscopy [20]. Although bacteria are included in ATP biomass determinations, their potential importance in biological and biogeochemical processes warrants separate enumeration. Mineralization ofradiolabeled plant lignocelluloses was followed as an index of microbial transformation rates of this abundant vascular plant structural polymer. Heterotrophic activity mea-
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surements are most commonly made by following rates of uptake of labile dissolved carbon compounds such as glucose or amino acids; however [14C]lignocelluloses prepared from the plants indigenous to a particular wetland ecosystem have the advantage of representing the complex and refractory nature of substrates commonly encountered by microorganisms. As vascular plants are a dominant source of primary production in wetland ecosystems, [14C]lignocellulose degradation serves as an index of a major pathway in particulate organic matter transformations.
Methods
Site Descriptions Five sites representing diverse community types in the Okefenokee were chosen for our investigation (Fig. 1): (1) MizeU Prairie (MZP), a marsh or "prairie," is an emergent wetland strongly dominated by a single species of sedge, Carex walteriana. Sphagnum spp., Utricularia spp., and Lachnanthes caroliana are found interspersed with sedge stems; Panicum hemotomon occurs at the prairie border. Standing water is present on Mizell Prairie during the winter months. The prairie's standing water dries down each summer, although the sediment surface remains saturated. Mizell Prairie has an average water depth o f 27 cm when flooded. (2) Little Copter Prairie (LCP), an aquatic maerophyte marsh, is characterized by white water lily, Nymphaea odorata; Utricularia spp., Eriocaulon compressum, and Orontium aquaticum are also important plant species, and tufts o f Lachnanthes caroliana can be found at lower densities. Little Copter Prairie, and other aquatic macrophyte prairies o f this type, are characterized by deeper water than is typical o f the emergent sedge prairies, and summer dry-down does not normally occur. Average water depth is 43 cm. (3) The Rookery (R) is an aquatic macrophyte prairie adjacent to a presently abandoned white ibis (Eudocimus albus) rookery active from 1970 through 1981. Yellow water lily (Nuphar luteum) and water milfoil (Myriophyllum heterophyllum) dominate the prairie. Average water depth at the Rookery is 68 cm. (4) Rookery Control (RC). Vegetation and hydrology o f this site are comparable to those of the Rookery area, but there is no history of bird use. (5) Buzzards Roost Lake (BRL) is a small (40,000 m 2) lake occurring as a depression in an emergent marsh community.
Sample Collection Samples for biomass measurements were collected approximately every 6 weeks from June 1982 through July 1983. At three sites (BRL, R, and RC), samples were collected within a 10 x 10 m plot selected randomly during each sampling period. On each sample date and at each site, three 20 ml water samples were preserved with 0.22 um filtered Formalin (5% Formalin final concentration) for subsequent AODC. Three sediment samples (0.1 ml) from each site, diluted with 20 ml of 0.22 t~m filtered water and preserved with Formalin, were also collected for AODC. Estimation of bacterial density in preserved samples was carried out within 10 days of collection. Triplicate water samples (100 ml) and sediment samples (2.0 ml) were collected in sterile Whirl-Pak bags for ATP determination, and the analyses were conducted the same day. Prior to ATP analysis, the water samples were filtered through a 110 #m mesh to remove large debris. In all instances, sediment samples were collected from the flocculent upper layer of aerobic sediment, no deeper than 2 cm. At the remaining two Okefenokee sites (MZP and LCP), three l0 • 10 m permanent plots were established, chosen at random along a 150 m transect. Water and sediment collections were taken from each of the plots as outlined above. This more extensive sampling scheme permitted us to examine within-site patterns in microbial biomass in addition to our among-site comparisons. As
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Mizell Prairie
,Rookery Control
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Fig. 1. Okefenokee Swamp site locations.
the MZP site dried down during the late summer and early fall months, water samples were not collected from June through November 1982, and in July 1983. At two sites (R and RC), sampling was not initiated until July ! 982. Sediment ATP samples were not collected at MZP and LCP in August and November 1983; AODC samples were not collected at R and RC in April 1983. At the time of sample collection, surface water temperature at aU five sites was measured. Inorganic nutrient concentrations in the surface waters were also determined for each site at each sample date. Phosphate, ammonia, and nitrate plus nitrite concentrations were determined with a Technicon Autoanalyzer II, using background corrections for the highly colored water o f the Okefenokee. Samples for lignocellulose degradation rate determinations were collected on three dates in 1985 at MZP (February, July, and September), and on one date at LCP (September). A total of nine surface sediment samples were collected at each site: three samples were collected 30 cm apart from each o f three 0.5 x 0.5 m plots. The plots were located 30 m apart on the prairie. Microbial Biomass Measurements Particulate ATP concentrations in the water column were determined by passing the prescreened water samples (25-100 ml) through a 0.2 pm Nuclepore filter, followed by extraction of the filter
Okefenokee Microbial Community Dynamics
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in 5 ml boiling sodium bicarbonate buffer (0.1 M, pH 8.5) for 2 min. For determination of ATP in the sediment, 1 ml sediment samples were extracted with 16 ml boiling sodium bicarbonate buffer for 2 min [4, 28]. A replicate 1 ml sediment sample was used to determine extraction efficiency by addition of a 10 ttg ATP standard just prior to extraction. ATP was assayed by the firefly luciferin-luciferase method [21, 28]. A subsample of each sediment was dried at 60~ to determine the percentage of water in each sediment sample.
Bacterial N u m b e r s in W a t e r a n d S e d i m e n t s Bacteria in water and sediments were counted using epifluorescence microscopy [20]. A portion of each water sample (from 0.5-2.0 ml) was filtered onto a previously stained (Irgalan black) 0.2 tzm Nuclepore filter. Acridine orange (0.01%) was placed on the filtered sample, allowed to stain for 2 min, and then removed by gentle vacuum. One filter was prepared from each water sample (three from each plot), and 20 half-fields were counted from each filter. Sediment samples (0.1 ml) were first made into slurries (1:200 or 1:250; volume sediment:volume water) then treated as described above. ATP and bacterial numbers data were log-transformed for all statistical analyses.
fl4C] Lignocellulose
Mineralization M e a s u r e m e n t s
Microbial decomposition of lignocellulosic carbon was measured by following the evolution of ~4CO2 from ['4C]lignocellulose prepared from the sedge Carex walteriana (February and July experiments) or redroot (Lachnanthes caroliana) (September experiments). Lignocellulose comprises 84% and 53% of the total biomass of C. walteriana and L. caroliana, respectively. Details of our methodology for preparation of uniformly labeled plant material and extraction of [14C]lignocellulosefrom other plant components are given elsewhere [7]. Slurries of swamp sediment containing the natural microbial populations were prepared by diluting sediment samples 1:50 with filter-sterilized water from the site. Triplicate incubations of 10 ml of the slurries and 10 mg of the labeled lignocellulose were conducted in microcosms for 10 days at 25~ (February and July) or 30~ (September). Microcosms were aerated with sterile, humidified air for 15 rain every 2 days. During each aeration period, mineralization of lignocellulose was monitored by trapping the evolved 14CO2 from the aeration outflow in a series of two vials containing liquid scintillation cocktail, followed by quantification in a Beckman LS 9000 spectrometer. Because of the low pH of Okefenokee water and sediments, the efficiency of CO2 trapping by aeration was high, averaging over 90%. Thus each microcosm could be sampled periodically to monitor cumulative CO2 production over a 10 day period.
Results and D i s c u s s i o n T e m p o r a l Trends M e a n b a c t e r i a l n u m b e r s (all sites a v e r a g e d ) v a r i e d 1 5 - f o l d i n t h e s u r f a c e s e d i m e n t s o f t h e O k e f e n o k e e S w a m p , f r o m a l o w o f 1.5 x 109 cells g - l d r y w e i g h t s e d i m e n t i n N o v e m b e r 1982 t o a h i g h o f 23.0 x 109 cells g - i s e d i m e n t in J u l y 1983. A v e r a g e b a c t e r i a l n u m b e r s d i f f e r e d s i g n i f i c a n t l y a m o n g s a m p l e d a t e s ( t w o - w a y A N O V A , P < 0.0 i), a n d a g e n e r a l p a t t e r n o f l o w e r n u m b e r s i n w i n t e r m o n t h s a n d h i g h e r n u m b e r s in w a r m m o n t h s is e v i d e n t (Fig. 2), a l t h o u g h J a n u a r y 1983 s a m p l e s c o n t a i n e d h i g h b a c t e r i a l n u m b e r s r e l a t i v e to o t h e r w i n t e r m o n t h s . A l t h o u g h s u b s t a n t i a l v a r i a b i l i t y exists, m e a n s e d i m e n t b a c t e r i a l n u m b e r s at all sites h a v e a w e a k p o s i t i v e c o r r e l a t i o n w i t h t e m p e r a t u r e at t i m e o f
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9
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Fig. 2. Annual pattern of bacterial numbers in sediments of Mizell Prairie (E3), Little Comer Prairie (O), Rookery (O), Rookery Control (z~),and Buzzards Roost Lake (m) sites from June 1982 to July 1983. Each point represents the mean of nine (MZP, LCP) or three (R, RC, BRL) replicate samples.
collection (Pearson P r o d u c t M o m e n t r = 0.275, P < 0.05, n = 50). Inorganic nutrient levels in the overlying w a t e r at each site were variable b u t n o t seasonal in nature (Fig. 3). T h e r e was no correlation between nutrient concentrations a n d m e a n bacterial a b u n d a n c e , with the single exception o f a positive relationship between s e d i m e n t bacterial n u m b e r s a n d a m m o n i a c o n c e n t r a t i o n at Mizell Prairie (r = 0.917, P < 0.05, n = 5). In the water c o l u m n , the bacterial c o m m u n i t y (all sites averaged) exhibited t e m p o r a l patterns similar to those o f the s e d i m e n t c o m m u n i t y . A significant date effect was evident ( P < 0.01), a n d again bacterial n u m b e r s generally were higher during the w a r m m o n t h s (Fig. 4). Average bacterial n u m b e r s ranged f r o m a low o f 0.64 • 106 cells m1-1 in J a n u a r y 1983 to a high o f 3.1 x 106 ceils m1-1 in July 1983. M e a n bacterial n u m b e r s h a d a w e a k but significant positive correlation with t e m p e r a t u r e (r = 0.446, P < 0.01, n = 45), but no correlation with inorganic nutrient concentrations. F o r s o m e sites, bacterial n u m b e r s in the s u m m e r m o n t h s o f 1983 were twofold greater t h a n they were in s u m m e r 1982, p r o v i d i n g evidence o f y e a r - t o - y e a r v a r i a t i o n s s u p e r i m p o s e d on seasonal v a r i a t i o n s in m i c r o b i a l standing stocks. Large y e a r - t o - y e a r differences in a b u n d a n c e h a v e also been f o u n d for plant a n d a n i m a l p o p u l a t i o n s in the O k e f e n o k e e ecosystem, m o s t likely due to the strong controlling influence o f annual variations in h y d r o l o g y on the biological c o m m u n i t i e s [ 10,17]. A T P m e a s u r e m e n t s o f the s e d i m e n t m i c r o b i a l c o m m u n i t y give an index o f total m i c r o b i a l b i o m a s s , which, in this ecosystem, is c o m p r i s e d p r i m a r i l y o f b i o m a s s o f bacteria, fungi, p r o t o z o a , m e i o f a u n a , a n d benthic algae. M e a n concentrations o f A T P (all sites averaged) ranged f r o m 2.1 #g A T P g-~ d r y surface s e d i m e n t in July 1983 to 8.4/~g g-~ in April 1983 (Fig. 5). A T P levels v a r i e d a m o n g s a m p l e s t a k e n at different t i m e s (1~ < 0.01); however, m e a n A T P values were n o t correlated with either bulk inorganic nutrient levels in the overlying w a t e r or t e m p e r a t u r e at t i m e o f collection. Consistent with these results, A T P
Okefenokee Microbial Community Dynamics
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3O
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levels in sediments at four Okefenokee sites, including M Z P and three other sites not sampled in the present study, were f o u n d by M u r r a y and H o d s o n [28] to vary widely with no apparent seasonal or t e m p e r a t u r e correlations. Linkage between a seasonally varying bacterial population and the total sediment microbial c o m m u n i t y is thus not evident, even though bacteria are often considered a m a j o r food source o f the m i c r o f a u n a and m e i o f a u n a o f aquatic ecosystems. In the water column, average (all sites together) A T P concentration was lowest in January 1983, at 0.60 #g l - i ; the highest average A T P concentration was 2.4 gg 1-~ in June 1983 (Fig. 6). Statistical analysis showed a significant date effect on A T P levels (two-way A N O V A ; P < 0.01), and a low but significant positive correlation o f m e a n A T P level with t e m p e r a t u r e (r = 0.379, P < 0.01, n = 47). At the L P C site, m e a n A T P levels were positively correlated with phosphate concentration (r = 0.726, P < 0.05, n = 10), although inorganic
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Table 1. Overall means (and standard errors) of microbial biomass (ATP) and bacterial numbers (AODC) in water and sediments of five Okcfenokee communities
Site
Microbial biomass sediment~ (#g ATP g-~)
MZP LCP R RC BRL
5.43 (0.39) 3.01 (0.20) 4.40 (0.90) 3.84 (1.01) 2.93 (0.39)
~ = bn = cn = an =
Microbial biomass waterb (#g ATP liter-t)
Bacterial numbers sedimentc (cells • 109 g-l)
Bacterial numbers watera (cells x 106 ml-1)
1.98 (0.44) 0.83 (0.05) 1.15 (0.09) 1.27 (0.10) 1.16 (0.08)
8.10 (0.54) 16.08 (1.50) 9.07 (2.73) 5.87 (1.23) 7.17 (1.13)
1.61 (0.09) 1.28 (0.07) 1.59 (0.15) 1.65 (0.15) 2.31 (0.19)
81 (MZP, LCP), n = 30 (R, RC), n = 33 (BRL) 45 (MZP), n = 99 (LCP), n = 30 (R, RC), n = 33 (BRL) 99 (MZP, LCP), n = 27 (R, RC), n = 30 (BRL) 45 (MZP), n = 99 (LCP), n = 27 (R, RC), n = 33 (BRL)
nutrient and A T P levels were otherwise n o t related. A T P concentrations at M Z P were extremely high at the June sampling date, the last sample date before s u m m e r dry-down. M u r r a y and H o d s o n [28] f o u n d similar peaks in A T P as Okefenokee water levels decreased in the s u m m e r o f 1981. They attributed high A T P levels to the concentration o f microbial biomass in the remaining pools o f water. Seasonality in microbial standing stocks has been d o c u m e n t e d in a variety o f aquatic ecosystems. W i n t e r m i n i m a in bacterial n u m b e r s have been f o u n d in salt m a r s h water and sediments [24, 34, 37], in intertidal sediments [12], and in sea and lake water [18, 19, 22, 26], although in some cases bacterial n u m b e r s have been f o u n d to r e m a i n constant t h r o u g h o u t the year [9, 16]. A T P levels in aquatic ecosystems also usually follow a seasonal pattern, with high concentrations in s u m m e r or fall a n d low concentrations in winter [8, 34]; exceptions to A T P seasonality have also been reported [9]. Seasonal patterns in microbial a b u n d a n c e have been attributed to D O C and P O C availability [16, 19, 24, 34] and to temperature-related decreases in microbial metabolic activity, including cell growth and division [ 12, 19, 36]. Wright and Coffin [38] suggest that bacterial n u m b e r s are controlled by low temperature and substrate availability in the winter m o n t h s and by grazing losses during the s u m m e r , resulting in seasonality in bacterial n u m b e r s within a prescribed range. In the Okefenokee, microbial a b u n d a n c e clearly has seasonal aspects; however, substantial variability in standing stocks is left unexplained by seasonal p h e n o m ena. W e suggest that the remaining, and superficially r a n d o m , variability is due to the response o f microbial populations to spatial patchiness o f the environment.
Among-Site Spatial Patchiness A m o n g the five Okefenokee c o m m u n i t i e s sampled, we f o u n d significant differences in sediment a n d water bacterial n u m b e r s and in sediment and water
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ATP levels (two-way ANOVA, P < 0.01 for all tests). The ranking of sites was not consistent across the four abundance measures: bacterial numbers were not correlated with ATP concentrations, and sediment abundance data were unrelated to water column data (Table I). For example, LCP had the highest average number of bacterial cells in sediments, the lowest average number of bacteria in the water column, the lowest average ATP concentration in the water, and a median level of ATP in sediments. The magnitude of differences in standing stocks among sites appears to be strongly influenced by the date of sample collection, as indicated by a significant site by date interaction in both ATP and AODC measurements on sediment and water samples (two-way ANOVA, P < 0.01 for all tests). During colder months in the Okefenokee, microbial standing stocks in the five sites are largely indistinguishable. Differences become evident in the summer, possibly because release from temperature limitation increases the importance of other growthcontrolling factors which may be more site-specific, including organic substrate availability (quantity and quality of both POC and DOC), predation pressure, pH, and oxygen saturation. Between-site spatial variability in microbial degradative activity was examined by comparing rates of mineralization of [14C]lignocellulose prepared from Lachnanthes caroliana in sediment samples from MZP and LCP. Mean percent mineralization was significantly higher in LCP sediments (Mann-Whitney Test, P < 0.01), although pH and temperature at the two sites were not different. LCP activity was greater than MZP by a factor of 1.7 (Table 2, September data).
Within-Site Spatial Patchiness Replicate samples within sites allowed us to look for small-scale patterns in microbial community structure and activity. Bacterial numbers in the water column of LCP and bacterial numbers in MZP sediment were significantly different among plots (plot by date two-way ANOVA, P < 0.01; Table 3). Spatial patchiness has been found previously for bacterial numbers in water samples collected from 10 cm apart [1] to 20 m apart [31], and even in subsamples of a well-mixed 1 liter sample [25]; patchiness in sediments has also been previously found [27]. However, all these studies examined heterogeneity at a single point in time. We found patchiness to persist over a 14 month period, possibly due to association of bacteria with macrophytes or other stationary sources of organic carbon. The lack of among-plot differences in the other microbial biomass parameters may reflect either the absence of structuring, or possibly structuring at a scale smaller than our sampling scheme could resolve. For example, in a within-site analysis of variations in ATP levels at Chesser Prairie, an Okefenokee community similar vegetatively to LCP, Murray and Hodson [28] used 1 • 1 m plots rather than the large 10 x 10 m plots used in this study. They found ATP levels to vary substantially among the plots (more than 2-fold), while sediment samples collected from within each plot were similar in ATP content ([28] and R. Murray, personal communication).
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Cumulativepercentmineralization(andstandarderror)of[~4C]lignocellulosefromCarex walteriana (February and July experiments) or Lachnanthes caroliana (September experiments)
Table 2.
during a 10 day incubation
Mizell Prairie July ~.c
Mizell Prairie Sept~,~
Little Cooter Prairie Septb
Plot
Sample
Mizell Prairie Feb. ~.c
A A A
1 2 3
2.77 (0.41) a 2.16 (0.25) 4.36 (0.91)
1.12 (0.04) 0.94 (0.07) 1.12 (0.09)
3.05 (0.07) a 2.57 (0.03) 3.65 (0.10)
6.68 (1.18) 4.84 (0.68) 5.79 (0.07)
B B B
1 2 3
1.40 (0.13) a 3.51 (1.82) 1.96 (0.16)
0.79 (0.07) 0.87 (0.02) 0.94 (0.06)
3.95 (0.25) 3.26 (0.78) 3.98 (0.44)
6.29 (0.16) a 5.41 (0.06) 5.76 (0.07)
C C C
1 2 3
1.77 (0.06) 1.86 (0.20) 1.51 (0.19)
0.74 (0.04) 0.75 (0.05) 0.72 (0.03)
3.76 (0.09) 3.09 (0.21) 3,43 (0.58)
5.99 (0.63) 5.34 (1.44) 4.91 (0.22)
Plots (0.25 m 2 area) were separated by 30 m. Samples within plots were collected 30 cm apart arl~4
~n-3 c Plots differ significantly in mineralization rates (Kruskal-Wallis Test, P < 0.05) a Samples within the plot differ significantly in mineralization rates (Kruskal-Wallis Test, P < 0.05)
Table 3. Overall mean (and standard error) of microbial standing stocks from permanent plots at Mizell Prairie and Little Cooter Prairie
Site
Microbial biomass Microbial sedimenta biomass water ~ (t~g ATP g-~) (~g ATP liter -~)
Bacterial numbers sedimentr (cells • 109 g-~)
Bacterial numbers waterd (cells x 106 ml-')
Mizell Plot 1 Plot 2 Plot 3
5.08 (0.59) 6.03 (0.85) 5.17 (0.54)
2.26 (0.93) 1.50 (0.56) 2.21 (0.83)
8.27 (0.89)" 6.88 (0.75) 9.18 (1.i0)
Little Cooter Plot 1 3.40 (0.37) Plot 2 3.14 (0.40) Plot 3 2.47 (0.19)
0.81 (0.06) 0.85 (0.10) 0.82 (0.09)
17.33 (3.03) 17.66 (2.80) 13.26 (1.77)
1.58 (0.18) 1.61 (0.17) 1.63 (0.14) 1.08 (0.09) e 1.29 (0.10) 1.50 (0.16)
a n = 27 bn = 15 (MZP), n = 36 (LCP) cn=36 an = 18 (MZP), n = 36 (LCP) e Plots within a site differ significantly (two-way ANOVA, P < 0.01)
Considering the possibility that our sampling scheme failed to resolve smallscale patchiness in microbial abundance, the sampling procedure used for measuring within-site variability in sediment microbial activity was designed at a smaller scale; we compared lignocellulose mineralization rates among small plots separated by 30 m, and among samples collected 30 cm apart from within each of these plots. Variability among plots was significant at MZP for all three
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dates (Kruskal-Wallis Test, P < 0.05), although at LCP rates did not differ among plots for the single September sample (Table 2). Within plots, significant differences in microbial activity were also found, with four cases of within-plot differences out of 12 total plots sampled over all dates (Table 2). Patchiness within Okefenokee microbial communities thus exists on many scales, most likely reflecting the patchiness of physical and chemical factors influencing microbial activity. However, the magnitude of differences in lignocellulose mineralization activity between patches was not great, perhaps as expected for this relatively abundant and refractory carbon source. The lowest activity plot within a site averaged 38% of the highest activity plot, while the lowest activity sample within a plot averaged 77% of the highest activity sample. In the Okefenokee Swamp ecosystem, where macroorganisms are visibly patchy in distribution and organized within a variety of community types, we have attempted to characterize the patterns in the microorganism communities. Although the structure of these microscopic communities is not easily studied, we were able to look at several biomass and activity measures to determine the presence or absence of distinct spatial and temporal patterns. Slow sheet movement of water throughout the Okefenokee interconnects the varied habitats and potentially homogenizes the concentrations of nutrients, DOC, and POC. Thus an initial inspection might suggest that microbial communities, in contrast to plant communities, would be more homogeneous. Alternatively, the Okefenokee may provide a mosaic of habitats for microorganisms, either correlating with the patterns in macroorganism distribution, or perhaps at scales smaller than those perceived on the macroorganism level, or both. We have indeed found both spatial and temporal heterogeneity to be characteristic of the microbial communities. Temporal variation in abundance of microorganisms, for example, was as great as 15-fold during the course of a year for average bacterial numbers in the sediments. This seasonality in bacterial standing stocks in the Okefenokee parallels (although with changes of different amplitude) the previously determined pattems of seasonality in bacterial production [29] and in rates of degradation of lignocellulose [5], a process known to be largely mediated by the bacterial community [6]. Spatial structuring of the microbial community, both in abundance and activity, was evident as well. Differences found among the varied habitats sampled in this study demonstrate that, as expected, microbial communities can be structured at scales correlating with macroorganism distribution. However, spatial patchiness was also quite evident within superficially homogeneous plant communities. Although differences within habitats were generally smaller compared to between-habitat comparisons (Tables 2 and 3), significant heterogeneity in the microbial community was found at very small scales, for example, sediment microbial activity varied among microsites within a 0.25 m 2 area. The patchiness in the sediment microbial community, significant in the upper sediment layers, might be expected to diminish with depth, particularly beneath the root zone. In the water column, spatial patchiness was probably a consequence of macroorganism distribution and low rates of water movement. The picture emerging for the microorganisms of the Okefenokee Swamp is that of a dynamic community, on both temporal and spatial scales. This char-
Okefenokee Microbial Community Dynamics
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acterization invites speculation as to the factors controlling m i c r o b i a l p o p u lations a n d producing the patterns o f v a r i a t i o n observed. O n e o b v i o u s factor is t e m p e r a t u r e , as this correlates positively with m i c r o b i a l a b u n d a n c e a n d has p r e v i o u s l y been s h o w n to be i m p o r t a n t in d e t e r m i n i n g microbial activity [5] a n d bacterial p r o d u c t i o n [29] in the Okefenokee. Y e t t e m p e r a t u r e alone c a n n o t explain o b s e r v e d spatial v a r i a t i o n n o r the restricted limits within which the m i c r o b i a l p o p u l a t i o n s a n d activity vary. A m o n g - s i t e differences in the m i c r o bial c o m m u n i t y suggest the i m p o r t a n c e o f o t h e r controls which are associated or correlated with m a c r o o r g a n i s m c o m m u n i t i e s , possibly c a r b o n availability or quality. W e know, for example, t h a t lignocellulose c o m p r i s e s 84% o f Carex walteriana, the d o m i n a n t plant in the M Z P c o m m u n i t y , while the w a t e r lily d o m i n a t i n g L C P is only 55% lignocellulose, a n d thus is a potentially superior carbon source. In addition, however, s o m e controls on m i c r o b i a l p o p u l a t i o n s m u s t operate at the very small scales o f v a r i a t i o n f o u n d in this study. Smallscale heterogeneity in c a r b o n availability a n d quality is the m o s t likely influence, as exudates f r o m live plants a n d d e a d particulate plant material will create a m o s a i c o f c a r b o n sources in b o t h the water c o l u m n a n d sediments. T h e influence o f grazing on bacterial or total m i c r o b i a l p o p u l a t i o n s also m u s t be considered, especially in light o f evidence f r o m o t h e r systems that p r o t o z o a n grazers can control the u p p e r limits o f bacterial a b u n d a n c e [3, 32, 38]. G r a z i n g m a y act to d a m p e n spatial a n d t e m p o r a l patchiness o f the O k e f e n o k e e m i c r o b i a l c o m m u n i t i e s b y cropping m i c r o o r g a n i s m s to constant levels. Sampling protocols should address potential heterogeneity in m i c r o b i a l c o m munities. We found that b o t h replicate s a m p l e s at each s a m p l i n g date a n d multiple sampling dates were necessary to u n c o v e r the spatial a n d t e m p o r a l heterogeneity o f the Okefenokee. In studies o f c a r b o n a n d energy flow through the microbial c o m p o n e n t o f ecosystems, fluctuations in the m i c r o b i a l c o m m u n i t y need to be well characterized. A s a m p l i n g regime which was any less resolved spatially or t e m p o r a l l y would not h a v e adequately described activity or b i o m a s s o f the m i c r o o r g a n i s m s in this ecosystem.
Acknowledgments. This work was carried out within the Okefenokee Swamp National Wildlife Refuge, and we appreciate the cooperation of John Schroer and the staff of the U.S. Fish and Wildlife Service. B. J. Freeman generously provided information and expertise on the Okefenokee ecosystem, and William J. Wiebe provided valuable comments on the manuscript. National Science Foundation Grants BSR 81-14823 and BSR 82-15587 were the sources of funding for this work. This study is Okefenokee Empirical Series contribution No. 65.
References 1. Ashby RE, Rhodes-Roberts ME (1976) The use of analysis of variance to examine the variations between samples of marine bacterial populations. J Appl Bacteriol 41:439--451 2. Azam F, Hodson RE (1977) Dissolved ATP in the sea and utilization by marine bacteria. Nature 267:696-698 3. Azam F, Fenchel T, Field JG, Gray JS, Meyer-Reil LA, Thingstad F (1983) The ecological role of water-column microbes in the sea. Mar Ecol Prog Ser 10:257-263 4. Bancroft K, Paul EA, Wiebe WJ (1976) The extraction and measurement of adenosine triphosphate from marine sediments. Limnol Oceanogr 21:473-480
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5. Benner R, Maccubbin AE, Hodson RE (1986) Temporal relationship between the deposition and microbial degradation of lignocellulosic detritus in a Georgia salt marsh and the Okefenokee Swamp. Microb Ecol 12:291-298 6. Benner R, Moran MA, Hodson RE (1986) Biogeochemical cycling of lignocellulosic carbon in marine and freshwater ecosystems: relative contributions of procaryotes and eucaryotes. Limnol Oceanogr 31:89-100 7. Benner R, Newell SY, Maccubbin AE, Hodson RE (1984) Relative contributions of bacteria and fungi to rates of degradation of lignocellnlosic detritus in salt-marsh sediments. Appl Environ Microbiol 48:36-40 8. Christian RR, Bancroft K, Wiebe WJ (1975) Distribution of microbial adenosine triphosphate in salt marsh sediments at Sapelo Island, Georgia. Soil Sci 119:89-97 9. Chrzanowski TH (1985) Seasonality, abundance, and biomass of bacteria in a southwestern reservoir. Hydrobiol 127:117-123 10. Conti RS, Gunther PP (1984) Relations ofphenology and seed germination to the distribution of dominant plants in Okefenokee Swamp. In: Cohen AD, Casagrande DJ, Andrejko MJ, Best GR (eds) The Okefenokee Swamp: its natural history, geology, and geochemistry. Wetland Surveys, New Mexico, pp 144-167 11. Cunningham HW, Wetzel RG (1978) Fulvic acid interferences on ATP determinations in sediments. Limnol Oceanogr 23:166-173 12. DeFlaun MF, Mayer LM (1983) Relationship between bacteria and grain surfaces in intertidal sediments. Limnol Oceanogr 28:873-881 13. Duever MJ, Riopelle LA (1982) Successional sequences and rates on tree islands in the Okefenokee Swamp. Am Midl Natur 110:186-193 14. Fairbanks BC, Woods LE, Bryant RJ, Elliot ET, Cole CV, Coleman DC (1984) Limitations of ATP estimates of microbial biomass. Soil Biol Biochem 16:549-558 15. Glasser JE (1984) Successional trends on tree islands in the Okefenokee Swamp as determined by interspecific association analysis. Am Midl Natur 113:287-293 16. Goulder R (1986) Seasonal variation in the abundance and heterotrophic activity of suspended bacteria in two lowland rivers. Freshwat Biol 16:21-37 17. Greening HS, Gerritsen J (in press) Variation in aquatic macrophyte community structure and biomass dynamics following drought in blackwater marshes of Okefenokee Swamp, Georgia, USA. Aquat Bot 18. Hagstrom A (1984) Aquatic bacteria: measurements and significance of growth. In: Klug M J, Reddy CA (eds) Current perspectives in microbial ecology. American Society for Microbiology, pp 495-501 19. Hessen DO (1985) The relation between bacterial carbon and dissolved humic compounds in oligotrophic lakes. FEMS Microbiol Ecol 31:215-223 20. Hobbie JE, Daley RJ, Jasper S (1977) Use of Nuclepore filters for counting bacteria by epifluorescence microscopy. Appl Environ Microbiol 33:1225-1228 21. Holm-Hansen O (1973) Determination of total microbial biomass by measurements of adenosine triphosphate. In" Stevenson LH, Colwell R R (eds) Estuarine microbial ecology. University of South Carolina Press, Columbia, pp 73-89 22. Indrebo G, Pengerud B, Dundas J (1979) Microbial activities in a permanently stratified estuary I. Primary production and sulfate reduction. Mar Biol 51:295-304 23. Karl DM (1980) Cellular nucleotide measurements and applications in microbial ecology. Microbiol Rev 44:739-796 24. Kirchman D, Peterson B, Juers D (1984) Bacterial growth and tidal variation in bacterial abundance in the Great Sippewissett salt marsh. Mar Ecol Prog Set 19:247-259 25. Kirchman D, Sigda J, Kapuscinski R, Mitchell R (1982) Statistical analysis of the direct count method for enumerating bacteria. Appl Environ Microbiol 44:376-382 26. Meyer-Reil L-A, Dawson R, Lubizeit G, Tiedge H (1978) Fluctuations and interactions of bacterial activity in sandy beach sediments and overlying waters. Mar Biol 48:161-171 27. Montagna PA (1982) Sampling design and enumeration statistics for bacteria extracted from marine sediments. Appl Environ Microbiol 43:1366-1372
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28. Murray RE, Hodson RE (1984) Microbial biomass and utilization of dissolved organic matter in the Okefenokee Swamp ecosystem. Appl Environ Microbiol 47:685-692 29. Murray RE, Hodson RE (1985) Annual cycle of bacterial secondary production in five aquatic habitats of the Okefenokee Swamp ecosystem. Appl Environ Microbiol 49:650-655 30. Odum EP, de la Cruz AA (1967) Particulate organic detritus in a Georgia salt marsh-estuarine ecosystem. In: LauffGH (ed) Estuaries. AAAS Publication no. 83, Washington, DC, pp 383-388 31. Palmer FE, Methot RD, Staley J T (1976) Patchiness in the distribution of planktonic heterotrophic bacteria in lakes. Appl Environ Microbiol 31:1003-1005 32. Pomeroy LR (1985) The microbial food web of the southeastern U.S. continental shelf. In: Atkinson LP, Menzel DW, Bush KA (eds) Oceanography of the southeastern continental shelf. A m Geophy Union, Washington, DC, pp 118-129 33. Pomeroy LR, Wiegert R G (1981) The ecology of a salt marsh. Springer-Verlag New York, Inc, New York 34. Rublee PA (1982) Seasonal distribution of bacteria in salt marsh sediments in North Carolina. Est Coast Shelf Sci 15:67-74 35. Schoenberg SA, Maccubbin AE, Hodson RE (1984) Cellulose digestion by freshwater microcrustacea. Limnol Oceanogr 29:1132-1136 36. Stevenson LH (1978) A case for bacterial dormancy in aquatic systems. Microbial Ecol 4: 127-133 37. Wilson CA, Stevenson LH (1980) The dynamics of the bacterial population associated with a salt marsh. J Exp Mar Biol Ecol 48:123-138 38. Wright RT, Coffin RB (1983) Planktonic bacteria in estuaries and coastal waters of northern Massachusetts: spatial and temporal distribution. Mar Ecol Prog Ser 11:205-216
MICROBIAL ECOLOGY
(~ Springer-VerlagNew York Inc. 1987
Dynamics of Microbial Biomass and Activity in Five Habitats of the Okefenokee Swamp Ecosystem Mary Ann Moran, A. E. Maccubbin,* Ronald Benner, and Robert E. Hodson Institute of Ecology and Department of Microbiology,University of Georgia, Athens, Georgia 30602, USA Abstract. A variety o f freshwater marsh and swamp habitats are found interspersed in a mosaic pattern throughout the Okefenokee Swamp, Georgia, USA. We examined spatial and temporal patterns in standing stocks and activity in the microbial community o f five habitats within this heterogeneous ecosystem. Standing stock dynamics were studied by measuring microbial biomass (ATP) and bacterial numbers (AODC) in both water and sediments over a 14 month period. Abundance varied temporally, being generally lower in winter months than in spring and summer months. However, a large proportion o f the measured variability was not correlated with temporal patterns in temperature or with bulk nutrient levels. Spatial variability was characteristic o f the Okefenokee at a variety of large and small scales. Habitat-level heterogeneity was evident when microbial standing stocks and activity (measured as [t4C]lignocellulose mineralization) were compared across the five communities, although abundance differences among sites were restricted to nonwinter months when microbial biomass was high. Spatial variation within habitats was also found; patches o f surface sediment with differing microbial activity or abundance were measured at scales from 30 cm to 150 m. Introduction The Okefenokee Swamp is a highly heterogeneous ecosystem, in which plants and animals are patchy in distribution and organized into many distinct habitats. Pine islands, tree and shrub swamps, emergent and floating-leaved macrophyte marshes, and small lakes are interspersed in mosaic fashion throughout this vast, acidic wetland. Macroorganisms of the swamp are heterogeneous on a temporal scale as well; plant communities undergo long-term directional changes along successional sequences [ 13, 15], which, for example, may begin with barren peat masses and culminate in hardwood-dominated islands. Plant communities also undergo short-term changes in response to hydrologic variations, such as a shift from floating-leaved vegetation to an emergent community following a period of exceptionally low water levels [ 17]. * P r e s e n t address: Department of ExperimentalBiology,RoswellPark Memorial Institute, Buffalo, New York 14263, USA.
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Our previous work provides evidence that microorganisms, too, may be spatially and temporally heterogeneous in their distributions. The Okefenokee has relatively high and strongly seasonal rates oi'bacterial secondary production in the water column, the changes in which appear to be coupled to both temperature and patterns of primary production [29]. Rates of microbial decomposition of the large store of detrital lignocellulose, the complex vascular plant structural polymer comprised of lignin and polysaccharides, are also seasonal, with a twofold decrease in rates of lignocellulose mineralization during winter months compared to summer [5]. The seasonal depression of this largely bacterial degradative process is directly attributable to low temperatures rather than to shifts in the functional or species composition o f the microbial community [5, 6]. In the Okefenokee Swamp, as in other detritus-based wetlands, microorganisms are important in mediating the mineralization and repackaging of the detrital material which forms the base of many swamp food webs [30, 33]. In this blackwater system, primary production is dominated by vascular plants, so that structural plant polymers account for a large fraction of available organic matter. Direct assimilation of plant detritus by animals is minimal [35], however, microorganisms use the lignocellulosic detritus, as well as highly dilute dissolved organic matter (DOM) of plant and animal origin. Subsequently, detritivores feed on high-quality microbial biomass produced at the expense of low-quality particulate and dissolved carbon sources; thus microorganisms potentially form an important link between primary producers and animal consumers [30, 33]. Those components of the partially degraded vascular plant detritus which are not converted to microbial biomass and do not enter aquatic food webs contribute to peat accumulation; microbial activity also plays a role in regulating this important organic geochemical process. Given the patchy distribution of plants and detritivorous animals and the acknowledged importance of heterotrophic microorganisms to the overall trophodynamics of the Okefenokee ecosystem, we recognized an absence of information on the distribution patterns of the microbial community. A study was thus undertaken to determine patterns in microbial standing stocks and activity on structural and temporal scales. We chose simple, widely used indices to examine temporal trends in microbial biomass, spatial variation in biomass and activity among community types, and spatial variation in biomass and activity within individual Okefenokee communities. Total microbial biomass was measured using the ATP technique [21 ] in which the firefy luciferase assay is used to determine the concentrations of adenosine triphosphate as an index of live biomass. Shortcomings of the procedure are recognized [2, 1 l, 14, 23]; however, the technique is sensitive, relatively simple, and includes all microbial groups (bacteria, fungi, protozoa, meiofauna, and algae). Bacterial biomass determinations were made using the acridine orange direct count method (AODC), in which cells stained with a fluorescent dye are counted via epifluorescence microscopy [20]. Although bacteria are included in ATP biomass determinations, their potential importance in biological and biogeochemical processes warrants separate enumeration. Mineralization ofradiolabeled plant lignocelluloses was followed as an index of microbial transformation rates of this abundant vascular plant structural polymer. Heterotrophic activity mea-
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surements are most commonly made by following rates of uptake of labile dissolved carbon compounds such as glucose or amino acids; however [14C]lignocelluloses prepared from the plants indigenous to a particular wetland ecosystem have the advantage of representing the complex and refractory nature of substrates commonly encountered by microorganisms. As vascular plants are a dominant source of primary production in wetland ecosystems, [14C]lignocellulose degradation serves as an index of a major pathway in particulate organic matter transformations.
Methods
Site Descriptions Five sites representing diverse community types in the Okefenokee were chosen for our investigation (Fig. 1): (1) MizeU Prairie (MZP), a marsh or "prairie," is an emergent wetland strongly dominated by a single species of sedge, Carex walteriana. Sphagnum spp., Utricularia spp., and Lachnanthes caroliana are found interspersed with sedge stems; Panicum hemotomon occurs at the prairie border. Standing water is present on Mizell Prairie during the winter months. The prairie's standing water dries down each summer, although the sediment surface remains saturated. Mizell Prairie has an average water depth o f 27 cm when flooded. (2) Little Copter Prairie (LCP), an aquatic maerophyte marsh, is characterized by white water lily, Nymphaea odorata; Utricularia spp., Eriocaulon compressum, and Orontium aquaticum are also important plant species, and tufts o f Lachnanthes caroliana can be found at lower densities. Little Copter Prairie, and other aquatic macrophyte prairies o f this type, are characterized by deeper water than is typical o f the emergent sedge prairies, and summer dry-down does not normally occur. Average water depth is 43 cm. (3) The Rookery (R) is an aquatic macrophyte prairie adjacent to a presently abandoned white ibis (Eudocimus albus) rookery active from 1970 through 1981. Yellow water lily (Nuphar luteum) and water milfoil (Myriophyllum heterophyllum) dominate the prairie. Average water depth at the Rookery is 68 cm. (4) Rookery Control (RC). Vegetation and hydrology o f this site are comparable to those of the Rookery area, but there is no history of bird use. (5) Buzzards Roost Lake (BRL) is a small (40,000 m 2) lake occurring as a depression in an emergent marsh community.
Sample Collection Samples for biomass measurements were collected approximately every 6 weeks from June 1982 through July 1983. At three sites (BRL, R, and RC), samples were collected within a 10 x 10 m plot selected randomly during each sampling period. On each sample date and at each site, three 20 ml water samples were preserved with 0.22 um filtered Formalin (5% Formalin final concentration) for subsequent AODC. Three sediment samples (0.1 ml) from each site, diluted with 20 ml of 0.22 t~m filtered water and preserved with Formalin, were also collected for AODC. Estimation of bacterial density in preserved samples was carried out within 10 days of collection. Triplicate water samples (100 ml) and sediment samples (2.0 ml) were collected in sterile Whirl-Pak bags for ATP determination, and the analyses were conducted the same day. Prior to ATP analysis, the water samples were filtered through a 110 #m mesh to remove large debris. In all instances, sediment samples were collected from the flocculent upper layer of aerobic sediment, no deeper than 2 cm. At the remaining two Okefenokee sites (MZP and LCP), three l0 • 10 m permanent plots were established, chosen at random along a 150 m transect. Water and sediment collections were taken from each of the plots as outlined above. This more extensive sampling scheme permitted us to examine within-site patterns in microbial biomass in addition to our among-site comparisons. As
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Mizell Prairie
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the MZP site dried down during the late summer and early fall months, water samples were not collected from June through November 1982, and in July 1983. At two sites (R and RC), sampling was not initiated until July ! 982. Sediment ATP samples were not collected at MZP and LCP in August and November 1983; AODC samples were not collected at R and RC in April 1983. At the time of sample collection, surface water temperature at aU five sites was measured. Inorganic nutrient concentrations in the surface waters were also determined for each site at each sample date. Phosphate, ammonia, and nitrate plus nitrite concentrations were determined with a Technicon Autoanalyzer II, using background corrections for the highly colored water o f the Okefenokee. Samples for lignocellulose degradation rate determinations were collected on three dates in 1985 at MZP (February, July, and September), and on one date at LCP (September). A total of nine surface sediment samples were collected at each site: three samples were collected 30 cm apart from each o f three 0.5 x 0.5 m plots. The plots were located 30 m apart on the prairie. Microbial Biomass Measurements Particulate ATP concentrations in the water column were determined by passing the prescreened water samples (25-100 ml) through a 0.2 pm Nuclepore filter, followed by extraction of the filter
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in 5 ml boiling sodium bicarbonate buffer (0.1 M, pH 8.5) for 2 min. For determination of ATP in the sediment, 1 ml sediment samples were extracted with 16 ml boiling sodium bicarbonate buffer for 2 min [4, 28]. A replicate 1 ml sediment sample was used to determine extraction efficiency by addition of a 10 ttg ATP standard just prior to extraction. ATP was assayed by the firefly luciferin-luciferase method [21, 28]. A subsample of each sediment was dried at 60~ to determine the percentage of water in each sediment sample.
Bacterial N u m b e r s in W a t e r a n d S e d i m e n t s Bacteria in water and sediments were counted using epifluorescence microscopy [20]. A portion of each water sample (from 0.5-2.0 ml) was filtered onto a previously stained (Irgalan black) 0.2 tzm Nuclepore filter. Acridine orange (0.01%) was placed on the filtered sample, allowed to stain for 2 min, and then removed by gentle vacuum. One filter was prepared from each water sample (three from each plot), and 20 half-fields were counted from each filter. Sediment samples (0.1 ml) were first made into slurries (1:200 or 1:250; volume sediment:volume water) then treated as described above. ATP and bacterial numbers data were log-transformed for all statistical analyses.
fl4C] Lignocellulose
Mineralization M e a s u r e m e n t s
Microbial decomposition of lignocellulosic carbon was measured by following the evolution of ~4CO2 from ['4C]lignocellulose prepared from the sedge Carex walteriana (February and July experiments) or redroot (Lachnanthes caroliana) (September experiments). Lignocellulose comprises 84% and 53% of the total biomass of C. walteriana and L. caroliana, respectively. Details of our methodology for preparation of uniformly labeled plant material and extraction of [14C]lignocellulosefrom other plant components are given elsewhere [7]. Slurries of swamp sediment containing the natural microbial populations were prepared by diluting sediment samples 1:50 with filter-sterilized water from the site. Triplicate incubations of 10 ml of the slurries and 10 mg of the labeled lignocellulose were conducted in microcosms for 10 days at 25~ (February and July) or 30~ (September). Microcosms were aerated with sterile, humidified air for 15 rain every 2 days. During each aeration period, mineralization of lignocellulose was monitored by trapping the evolved 14CO2 from the aeration outflow in a series of two vials containing liquid scintillation cocktail, followed by quantification in a Beckman LS 9000 spectrometer. Because of the low pH of Okefenokee water and sediments, the efficiency of CO2 trapping by aeration was high, averaging over 90%. Thus each microcosm could be sampled periodically to monitor cumulative CO2 production over a 10 day period.
Results and D i s c u s s i o n T e m p o r a l Trends M e a n b a c t e r i a l n u m b e r s (all sites a v e r a g e d ) v a r i e d 1 5 - f o l d i n t h e s u r f a c e s e d i m e n t s o f t h e O k e f e n o k e e S w a m p , f r o m a l o w o f 1.5 x 109 cells g - l d r y w e i g h t s e d i m e n t i n N o v e m b e r 1982 t o a h i g h o f 23.0 x 109 cells g - i s e d i m e n t in J u l y 1983. A v e r a g e b a c t e r i a l n u m b e r s d i f f e r e d s i g n i f i c a n t l y a m o n g s a m p l e d a t e s ( t w o - w a y A N O V A , P < 0.0 i), a n d a g e n e r a l p a t t e r n o f l o w e r n u m b e r s i n w i n t e r m o n t h s a n d h i g h e r n u m b e r s in w a r m m o n t h s is e v i d e n t (Fig. 2), a l t h o u g h J a n u a r y 1983 s a m p l e s c o n t a i n e d h i g h b a c t e r i a l n u m b e r s r e l a t i v e to o t h e r w i n t e r m o n t h s . A l t h o u g h s u b s t a n t i a l v a r i a b i l i t y exists, m e a n s e d i m e n t b a c t e r i a l n u m b e r s at all sites h a v e a w e a k p o s i t i v e c o r r e l a t i o n w i t h t e m p e r a t u r e at t i m e o f
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Fig. 2. Annual pattern of bacterial numbers in sediments of Mizell Prairie (E3), Little Comer Prairie (O), Rookery (O), Rookery Control (z~),and Buzzards Roost Lake (m) sites from June 1982 to July 1983. Each point represents the mean of nine (MZP, LCP) or three (R, RC, BRL) replicate samples.
collection (Pearson P r o d u c t M o m e n t r = 0.275, P < 0.05, n = 50). Inorganic nutrient levels in the overlying w a t e r at each site were variable b u t n o t seasonal in nature (Fig. 3). T h e r e was no correlation between nutrient concentrations a n d m e a n bacterial a b u n d a n c e , with the single exception o f a positive relationship between s e d i m e n t bacterial n u m b e r s a n d a m m o n i a c o n c e n t r a t i o n at Mizell Prairie (r = 0.917, P < 0.05, n = 5). In the water c o l u m n , the bacterial c o m m u n i t y (all sites averaged) exhibited t e m p o r a l patterns similar to those o f the s e d i m e n t c o m m u n i t y . A significant date effect was evident ( P < 0.01), a n d again bacterial n u m b e r s generally were higher during the w a r m m o n t h s (Fig. 4). Average bacterial n u m b e r s ranged f r o m a low o f 0.64 • 106 cells m1-1 in J a n u a r y 1983 to a high o f 3.1 x 106 ceils m1-1 in July 1983. M e a n bacterial n u m b e r s h a d a w e a k but significant positive correlation with t e m p e r a t u r e (r = 0.446, P < 0.01, n = 45), but no correlation with inorganic nutrient concentrations. F o r s o m e sites, bacterial n u m b e r s in the s u m m e r m o n t h s o f 1983 were twofold greater t h a n they were in s u m m e r 1982, p r o v i d i n g evidence o f y e a r - t o - y e a r v a r i a t i o n s s u p e r i m p o s e d on seasonal v a r i a t i o n s in m i c r o b i a l standing stocks. Large y e a r - t o - y e a r differences in a b u n d a n c e h a v e also been f o u n d for plant a n d a n i m a l p o p u l a t i o n s in the O k e f e n o k e e ecosystem, m o s t likely due to the strong controlling influence o f annual variations in h y d r o l o g y on the biological c o m m u n i t i e s [ 10,17]. A T P m e a s u r e m e n t s o f the s e d i m e n t m i c r o b i a l c o m m u n i t y give an index o f total m i c r o b i a l b i o m a s s , which, in this ecosystem, is c o m p r i s e d p r i m a r i l y o f b i o m a s s o f bacteria, fungi, p r o t o z o a , m e i o f a u n a , a n d benthic algae. M e a n concentrations o f A T P (all sites averaged) ranged f r o m 2.1 #g A T P g-~ d r y surface s e d i m e n t in July 1983 to 8.4/~g g-~ in April 1983 (Fig. 5). A T P levels v a r i e d a m o n g s a m p l e s t a k e n at different t i m e s (1~ < 0.01); however, m e a n A T P values were n o t correlated with either bulk inorganic nutrient levels in the overlying w a t e r or t e m p e r a t u r e at t i m e o f collection. Consistent with these results, A T P
Okefenokee Microbial Community Dynamics
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3O
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20" z
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14 ~.12 ~10
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4
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levels in sediments at four Okefenokee sites, including M Z P and three other sites not sampled in the present study, were f o u n d by M u r r a y and H o d s o n [28] to vary widely with no apparent seasonal or t e m p e r a t u r e correlations. Linkage between a seasonally varying bacterial population and the total sediment microbial c o m m u n i t y is thus not evident, even though bacteria are often considered a m a j o r food source o f the m i c r o f a u n a and m e i o f a u n a o f aquatic ecosystems. In the water column, average (all sites together) A T P concentration was lowest in January 1983, at 0.60 #g l - i ; the highest average A T P concentration was 2.4 gg 1-~ in June 1983 (Fig. 6). Statistical analysis showed a significant date effect on A T P levels (two-way A N O V A ; P < 0.01), and a low but significant positive correlation o f m e a n A T P level with t e m p e r a t u r e (r = 0.379, P < 0.01, n = 47). At the L P C site, m e a n A T P levels were positively correlated with phosphate concentration (r = 0.726, P < 0.05, n = 10), although inorganic
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Table 1. Overall means (and standard errors) of microbial biomass (ATP) and bacterial numbers (AODC) in water and sediments of five Okcfenokee communities
Site
Microbial biomass sediment~ (#g ATP g-~)
MZP LCP R RC BRL
5.43 (0.39) 3.01 (0.20) 4.40 (0.90) 3.84 (1.01) 2.93 (0.39)
~ = bn = cn = an =
Microbial biomass waterb (#g ATP liter-t)
Bacterial numbers sedimentc (cells • 109 g-l)
Bacterial numbers watera (cells x 106 ml-1)
1.98 (0.44) 0.83 (0.05) 1.15 (0.09) 1.27 (0.10) 1.16 (0.08)
8.10 (0.54) 16.08 (1.50) 9.07 (2.73) 5.87 (1.23) 7.17 (1.13)
1.61 (0.09) 1.28 (0.07) 1.59 (0.15) 1.65 (0.15) 2.31 (0.19)
81 (MZP, LCP), n = 30 (R, RC), n = 33 (BRL) 45 (MZP), n = 99 (LCP), n = 30 (R, RC), n = 33 (BRL) 99 (MZP, LCP), n = 27 (R, RC), n = 30 (BRL) 45 (MZP), n = 99 (LCP), n = 27 (R, RC), n = 33 (BRL)
nutrient and A T P levels were otherwise n o t related. A T P concentrations at M Z P were extremely high at the June sampling date, the last sample date before s u m m e r dry-down. M u r r a y and H o d s o n [28] f o u n d similar peaks in A T P as Okefenokee water levels decreased in the s u m m e r o f 1981. They attributed high A T P levels to the concentration o f microbial biomass in the remaining pools o f water. Seasonality in microbial standing stocks has been d o c u m e n t e d in a variety o f aquatic ecosystems. W i n t e r m i n i m a in bacterial n u m b e r s have been f o u n d in salt m a r s h water and sediments [24, 34, 37], in intertidal sediments [12], and in sea and lake water [18, 19, 22, 26], although in some cases bacterial n u m b e r s have been f o u n d to r e m a i n constant t h r o u g h o u t the year [9, 16]. A T P levels in aquatic ecosystems also usually follow a seasonal pattern, with high concentrations in s u m m e r or fall a n d low concentrations in winter [8, 34]; exceptions to A T P seasonality have also been reported [9]. Seasonal patterns in microbial a b u n d a n c e have been attributed to D O C and P O C availability [16, 19, 24, 34] and to temperature-related decreases in microbial metabolic activity, including cell growth and division [ 12, 19, 36]. Wright and Coffin [38] suggest that bacterial n u m b e r s are controlled by low temperature and substrate availability in the winter m o n t h s and by grazing losses during the s u m m e r , resulting in seasonality in bacterial n u m b e r s within a prescribed range. In the Okefenokee, microbial a b u n d a n c e clearly has seasonal aspects; however, substantial variability in standing stocks is left unexplained by seasonal p h e n o m ena. W e suggest that the remaining, and superficially r a n d o m , variability is due to the response o f microbial populations to spatial patchiness o f the environment.
Among-Site Spatial Patchiness A m o n g the five Okefenokee c o m m u n i t i e s sampled, we f o u n d significant differences in sediment a n d water bacterial n u m b e r s and in sediment and water
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ATP levels (two-way ANOVA, P < 0.01 for all tests). The ranking of sites was not consistent across the four abundance measures: bacterial numbers were not correlated with ATP concentrations, and sediment abundance data were unrelated to water column data (Table I). For example, LCP had the highest average number of bacterial cells in sediments, the lowest average number of bacteria in the water column, the lowest average ATP concentration in the water, and a median level of ATP in sediments. The magnitude of differences in standing stocks among sites appears to be strongly influenced by the date of sample collection, as indicated by a significant site by date interaction in both ATP and AODC measurements on sediment and water samples (two-way ANOVA, P < 0.01 for all tests). During colder months in the Okefenokee, microbial standing stocks in the five sites are largely indistinguishable. Differences become evident in the summer, possibly because release from temperature limitation increases the importance of other growthcontrolling factors which may be more site-specific, including organic substrate availability (quantity and quality of both POC and DOC), predation pressure, pH, and oxygen saturation. Between-site spatial variability in microbial degradative activity was examined by comparing rates of mineralization of [14C]lignocellulose prepared from Lachnanthes caroliana in sediment samples from MZP and LCP. Mean percent mineralization was significantly higher in LCP sediments (Mann-Whitney Test, P < 0.01), although pH and temperature at the two sites were not different. LCP activity was greater than MZP by a factor of 1.7 (Table 2, September data).
Within-Site Spatial Patchiness Replicate samples within sites allowed us to look for small-scale patterns in microbial community structure and activity. Bacterial numbers in the water column of LCP and bacterial numbers in MZP sediment were significantly different among plots (plot by date two-way ANOVA, P < 0.01; Table 3). Spatial patchiness has been found previously for bacterial numbers in water samples collected from 10 cm apart [1] to 20 m apart [31], and even in subsamples of a well-mixed 1 liter sample [25]; patchiness in sediments has also been previously found [27]. However, all these studies examined heterogeneity at a single point in time. We found patchiness to persist over a 14 month period, possibly due to association of bacteria with macrophytes or other stationary sources of organic carbon. The lack of among-plot differences in the other microbial biomass parameters may reflect either the absence of structuring, or possibly structuring at a scale smaller than our sampling scheme could resolve. For example, in a within-site analysis of variations in ATP levels at Chesser Prairie, an Okefenokee community similar vegetatively to LCP, Murray and Hodson [28] used 1 • 1 m plots rather than the large 10 x 10 m plots used in this study. They found ATP levels to vary substantially among the plots (more than 2-fold), while sediment samples collected from within each plot were similar in ATP content ([28] and R. Murray, personal communication).
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Cumulativepercentmineralization(andstandarderror)of[~4C]lignocellulosefromCarex walteriana (February and July experiments) or Lachnanthes caroliana (September experiments)
Table 2.
during a 10 day incubation
Mizell Prairie July ~.c
Mizell Prairie Sept~,~
Little Cooter Prairie Septb
Plot
Sample
Mizell Prairie Feb. ~.c
A A A
1 2 3
2.77 (0.41) a 2.16 (0.25) 4.36 (0.91)
1.12 (0.04) 0.94 (0.07) 1.12 (0.09)
3.05 (0.07) a 2.57 (0.03) 3.65 (0.10)
6.68 (1.18) 4.84 (0.68) 5.79 (0.07)
B B B
1 2 3
1.40 (0.13) a 3.51 (1.82) 1.96 (0.16)
0.79 (0.07) 0.87 (0.02) 0.94 (0.06)
3.95 (0.25) 3.26 (0.78) 3.98 (0.44)
6.29 (0.16) a 5.41 (0.06) 5.76 (0.07)
C C C
1 2 3
1.77 (0.06) 1.86 (0.20) 1.51 (0.19)
0.74 (0.04) 0.75 (0.05) 0.72 (0.03)
3.76 (0.09) 3.09 (0.21) 3,43 (0.58)
5.99 (0.63) 5.34 (1.44) 4.91 (0.22)
Plots (0.25 m 2 area) were separated by 30 m. Samples within plots were collected 30 cm apart arl~4
~n-3 c Plots differ significantly in mineralization rates (Kruskal-Wallis Test, P < 0.05) a Samples within the plot differ significantly in mineralization rates (Kruskal-Wallis Test, P < 0.05)
Table 3. Overall mean (and standard error) of microbial standing stocks from permanent plots at Mizell Prairie and Little Cooter Prairie
Site
Microbial biomass Microbial sedimenta biomass water ~ (t~g ATP g-~) (~g ATP liter -~)
Bacterial numbers sedimentr (cells • 109 g-~)
Bacterial numbers waterd (cells x 106 ml-')
Mizell Plot 1 Plot 2 Plot 3
5.08 (0.59) 6.03 (0.85) 5.17 (0.54)
2.26 (0.93) 1.50 (0.56) 2.21 (0.83)
8.27 (0.89)" 6.88 (0.75) 9.18 (1.i0)
Little Cooter Plot 1 3.40 (0.37) Plot 2 3.14 (0.40) Plot 3 2.47 (0.19)
0.81 (0.06) 0.85 (0.10) 0.82 (0.09)
17.33 (3.03) 17.66 (2.80) 13.26 (1.77)
1.58 (0.18) 1.61 (0.17) 1.63 (0.14) 1.08 (0.09) e 1.29 (0.10) 1.50 (0.16)
a n = 27 bn = 15 (MZP), n = 36 (LCP) cn=36 an = 18 (MZP), n = 36 (LCP) e Plots within a site differ significantly (two-way ANOVA, P < 0.01)
Considering the possibility that our sampling scheme failed to resolve smallscale patchiness in microbial abundance, the sampling procedure used for measuring within-site variability in sediment microbial activity was designed at a smaller scale; we compared lignocellulose mineralization rates among small plots separated by 30 m, and among samples collected 30 cm apart from within each of these plots. Variability among plots was significant at MZP for all three
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dates (Kruskal-Wallis Test, P < 0.05), although at LCP rates did not differ among plots for the single September sample (Table 2). Within plots, significant differences in microbial activity were also found, with four cases of within-plot differences out of 12 total plots sampled over all dates (Table 2). Patchiness within Okefenokee microbial communities thus exists on many scales, most likely reflecting the patchiness of physical and chemical factors influencing microbial activity. However, the magnitude of differences in lignocellulose mineralization activity between patches was not great, perhaps as expected for this relatively abundant and refractory carbon source. The lowest activity plot within a site averaged 38% of the highest activity plot, while the lowest activity sample within a plot averaged 77% of the highest activity sample. In the Okefenokee Swamp ecosystem, where macroorganisms are visibly patchy in distribution and organized within a variety of community types, we have attempted to characterize the patterns in the microorganism communities. Although the structure of these microscopic communities is not easily studied, we were able to look at several biomass and activity measures to determine the presence or absence of distinct spatial and temporal patterns. Slow sheet movement of water throughout the Okefenokee interconnects the varied habitats and potentially homogenizes the concentrations of nutrients, DOC, and POC. Thus an initial inspection might suggest that microbial communities, in contrast to plant communities, would be more homogeneous. Alternatively, the Okefenokee may provide a mosaic of habitats for microorganisms, either correlating with the patterns in macroorganism distribution, or perhaps at scales smaller than those perceived on the macroorganism level, or both. We have indeed found both spatial and temporal heterogeneity to be characteristic of the microbial communities. Temporal variation in abundance of microorganisms, for example, was as great as 15-fold during the course of a year for average bacterial numbers in the sediments. This seasonality in bacterial standing stocks in the Okefenokee parallels (although with changes of different amplitude) the previously determined pattems of seasonality in bacterial production [29] and in rates of degradation of lignocellulose [5], a process known to be largely mediated by the bacterial community [6]. Spatial structuring of the microbial community, both in abundance and activity, was evident as well. Differences found among the varied habitats sampled in this study demonstrate that, as expected, microbial communities can be structured at scales correlating with macroorganism distribution. However, spatial patchiness was also quite evident within superficially homogeneous plant communities. Although differences within habitats were generally smaller compared to between-habitat comparisons (Tables 2 and 3), significant heterogeneity in the microbial community was found at very small scales, for example, sediment microbial activity varied among microsites within a 0.25 m 2 area. The patchiness in the sediment microbial community, significant in the upper sediment layers, might be expected to diminish with depth, particularly beneath the root zone. In the water column, spatial patchiness was probably a consequence of macroorganism distribution and low rates of water movement. The picture emerging for the microorganisms of the Okefenokee Swamp is that of a dynamic community, on both temporal and spatial scales. This char-
Okefenokee Microbial Community Dynamics
215
acterization invites speculation as to the factors controlling m i c r o b i a l p o p u lations a n d producing the patterns o f v a r i a t i o n observed. O n e o b v i o u s factor is t e m p e r a t u r e , as this correlates positively with m i c r o b i a l a b u n d a n c e a n d has p r e v i o u s l y been s h o w n to be i m p o r t a n t in d e t e r m i n i n g microbial activity [5] a n d bacterial p r o d u c t i o n [29] in the Okefenokee. Y e t t e m p e r a t u r e alone c a n n o t explain o b s e r v e d spatial v a r i a t i o n n o r the restricted limits within which the m i c r o b i a l p o p u l a t i o n s a n d activity vary. A m o n g - s i t e differences in the m i c r o bial c o m m u n i t y suggest the i m p o r t a n c e o f o t h e r controls which are associated or correlated with m a c r o o r g a n i s m c o m m u n i t i e s , possibly c a r b o n availability or quality. W e know, for example, t h a t lignocellulose c o m p r i s e s 84% o f Carex walteriana, the d o m i n a n t plant in the M Z P c o m m u n i t y , while the w a t e r lily d o m i n a t i n g L C P is only 55% lignocellulose, a n d thus is a potentially superior carbon source. In addition, however, s o m e controls on m i c r o b i a l p o p u l a t i o n s m u s t operate at the very small scales o f v a r i a t i o n f o u n d in this study. Smallscale heterogeneity in c a r b o n availability a n d quality is the m o s t likely influence, as exudates f r o m live plants a n d d e a d particulate plant material will create a m o s a i c o f c a r b o n sources in b o t h the water c o l u m n a n d sediments. T h e influence o f grazing on bacterial or total m i c r o b i a l p o p u l a t i o n s also m u s t be considered, especially in light o f evidence f r o m o t h e r systems that p r o t o z o a n grazers can control the u p p e r limits o f bacterial a b u n d a n c e [3, 32, 38]. G r a z i n g m a y act to d a m p e n spatial a n d t e m p o r a l patchiness o f the O k e f e n o k e e m i c r o b i a l c o m m u n i t i e s b y cropping m i c r o o r g a n i s m s to constant levels. Sampling protocols should address potential heterogeneity in m i c r o b i a l c o m munities. We found that b o t h replicate s a m p l e s at each s a m p l i n g date a n d multiple sampling dates were necessary to u n c o v e r the spatial a n d t e m p o r a l heterogeneity o f the Okefenokee. In studies o f c a r b o n a n d energy flow through the microbial c o m p o n e n t o f ecosystems, fluctuations in the m i c r o b i a l c o m m u n i t y need to be well characterized. A s a m p l i n g regime which was any less resolved spatially or t e m p o r a l l y would not h a v e adequately described activity or b i o m a s s o f the m i c r o o r g a n i s m s in this ecosystem.
Acknowledgments. This work was carried out within the Okefenokee Swamp National Wildlife Refuge, and we appreciate the cooperation of John Schroer and the staff of the U.S. Fish and Wildlife Service. B. J. Freeman generously provided information and expertise on the Okefenokee ecosystem, and William J. Wiebe provided valuable comments on the manuscript. National Science Foundation Grants BSR 81-14823 and BSR 82-15587 were the sources of funding for this work. This study is Okefenokee Empirical Series contribution No. 65.
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