Temperate Salt Marsh - Applied and Environmental Microbiology

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1976, p. 959-968 Copyright © 1976 American Society for Microbiology

Vol. 31, No. 6 Printed in U.S.A.

Carbon Metabolism in Model Microbial Systems from Temperate Salt Marsh

a

ROBERT D. FALLON' AND FREDERIC K. PFAENDER* Curriculum in Marine Sciences and Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina 27514 Received for publication 7 November 1975

The metabolism of a saltwater leachate of '4C-labeled Spartina alterniflora examined in laboratory systems using mixed, salt marsh microbial communities and, by addition of appropriate antibiotics, communities with bacteria or eukaryotes inhibited. Label uptake was more rapid in the systems with bacteria alone and with the mixed microbial community than with fungi alone. Mineralization of the added label was more extensive in the mixed and bacterial systems, whereas the fungi appear more efficient at converting the label into particulate biomass. Particulate biomass production efficiencies ranged from a high of 0.82 for the fungal system to 0.21 in the mixed community, with the bacterial system giving an intermediate value of 0.54. The presence of protozoa and microcrustaceans in the mixed system appears to account for an increase in the mineralization of the label assimilated. Additional experiments with whole labeled Spartina, a leachate from Spartina, and the Spartina after leaching revealed that the seawater-soluble portions of the plant were attacked most rapidly by the microbial community. was

The heterotrophic microbial community in a temperature salt marsh consists of many diverse populations. Studies conducted over the last 20 years have concentrated on numerically and taxonomically assessing these populations. Bacterial populations have been examined in a number of studies (3, 5, 13). Bacillus, Serratia, Flavobacterium, Achromobacter, and Pseudomonas are the most commonly isolated heterotrophic groups from surface sediments and waters within the marsh. Over 27 species of fungi have been found associated with the marsh grass Spartina alterniflora in Rhode Island (10-12), whereas many other species have been found in Louisiana marshes (24, 25). Many protozoan and micrometazoan species are also thought to be important in decomposition processes in the marsh. Species of ciliated and flagellated protozoans present in the marshes have been reviewed by Lackey (22) and Fenchel (7). Studies by Gessner et al. (12) have shown nematodes and mites present on Spartina culms, apparently feeding on fungal mycelia. Many microcrustacean species are also present in the marsh waters, presumably ingesting plant detrital material. Much of the primary production occurring in the salt marsh takes place in the vast stands of S. alterniflora. The trophic relationships of the Present address: Department of Bacteriology, University of Wisconsin, Madison, Wis. 53706.

organisms using this production and the specific compounds involved have not been well studied. Apparently, little of the plant material is used directly by herbivores within the marsh (29). Some of the marsh grass appears to be used as a substrate for microbial colonization, with the microbial species in turn serving as food sources for many macroconsumers (17, 26). The relative significance of soluble versus particulate materials as substrates has not been established. Soluble carbon compounds can be added to the salt marsh in several ways. Gallagher et al. (9) have recently shown that a few percent of the photosynthate produced by S. alterniflora is leached from the living plant by the rising tide. There is also a considerable loss of carbon as dissolved organic matter (DOM) from the plant material as it dies and undergoes conversion to detritus. The amount of DOM produced by the marsh plants at any one time is unknown and may be small, but over the growing season it may constitute a significant source of nutrients for the microbial community. Although some taxonomic studies have provided important information, the heterotrophic microbial community is, as yet, poorly defined functionally. The present study attempts to gain a better understanding of the functional aspects of carbon metabolism by this community. Using antibiotics to selectively inhibit 959

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FALLON AND PFAENDER

portions of the microbial community, the metabolism of a '4C-labeled DOM leached from S. alterniflora was observed in model systems containing these populations and compared to the patterns observed in the intact, mixed community. In a second experiment, the mineralization of whole Spartina, Spartina leachate, and leached Spartina by the intact microbial community was observed. These experiments yield information addressed to three questions: (i) Which groups of microorganisms are potentially most important in mineralization and/or uptake of dissolved organics produced from Spartina? (ii) How efficient are the different populations and the microbial community as a whole in converting the dissolved organic carbon into new particulate biomass? (iii) Do protozoans and microcrustaceans increase rates of mineralization of the DOM over the rates observed in the presence of bacteria or fungi alone? MATERIALS AND METHODS Labeled Spartina. A '4C-labeled leachate was obtained by boiling 14C-labeled S. alterniflora in artificial seawater, 35 ppt (23) for 10 min, and removing particulates by filtration. This treatment was used to speed the normal leaching process. S. alterniflora plants had been labeled using a growth chamber technique described elsewhere (6). The leachate had an approximate activity of 8.1 ,uCi/g (dry weight). Data on the uniformity of labeling of the various organic molecules that comprise the leachate were not obtained, although label was found in proteinnucleic acid, organic acid, lipid, and fiber fractions of the plant (6). Specific activity (microcuries per gram, dry weight) did vary in different plant fractions, and uniform labeling of the leachate is, therefore, doubtful. Apparatus. The apparatus used in the microbial metabolism experiments is shown in Fig. 1. Salt water, collected from an S. alterniflora marsh near Beaufort, N.C., with the attending microbial populations, was placed in 2-liter polyethylene bottles for all experiments. All bottles were kept at 15 C and aerated continuously. Exhaust gases were passed through CO2 traps. The traps consisted of two scintillation vials; the first contained 10 ml of ethanolamine-ethanol (2:3, vol/vol), which trapped all CO2 in the exhaust gases. This was followed by a vial containing 8 ml of amyl alcohol, which trapped any material lost as an aerosol from the first bottle. At flow rates used, approximately 300 ml of air per min, this system was shown to trap 100% of the CO2 entering it. Air was supplied by a vacuum-pressure pump. Before entering the bottles, the air first passed through two oil-soaked cartridges in the pump and a filter of sterile cotton in the manifold. Intermittent checks with Trypticase soy agar (BBL) plates indicated that the air entering the bottles remained sterile. Using the manifold, air could be supplied to as many as 24 bottles simultaneously.

APPL. ENVIRON. MICROBIOL.

All parts of the apparatus used in the experiments, except for the gas traps and air pump, were autoclaved at the beginning of the experiment. The polyethylene bottles were washed with Cidex, a germicidal solution, rinsed with sterile water, and dried for 3 days at 80 C. Aseptic technique was used during all transfers. Community fractionation experiments. At the start of the fractionation experiments, 12 bottles were set up. There were three replicates for each of the three experimental systems and a group of three control bottles. To each of the bottles, 1 liter of marsh water inoculum was added. An additional inoculum of decaying (unlabeled) S. alterniflora culms, which had been pulverized in a sterile blender, was also added to each bottle as a slurry containing approximately 190 mg (dry weight) of particulate material. To obtain a system containing only bacteria (A), 1.0 g of actidione was added to each of the three replicate bottles. Penicillin (2 x 10" U), streptomycin (1.1 g), and chloramphenicol (0.2 g) were added to each of the bottles in the system to contain only eukaryotes (B). No antibiotics were added to the mixed community (C) or sterile control (D) bottles. Aeration was then begun and proceeded for 72 h. During this 72-h period, before the addition of the labeled Spartina leachate, antibiotics were present INPUT HOSE

OUTLET FILTER (COTTON FILLED)

ETHANOLAMINE AMYL ALCOHOL ETHANOL IV, GAS TRAPS -

2 LITER BOTTLE TO CONTAIN MICROBIAL SYSTEMS

5M NaOH (j (CO2 COLLECTOR)

GAS TRAPS a OUTLET Fl LTER 2 LITER. BOTTLE

FIG. 1. Apparatus used in the microbial metabolism experiments, with blow up of sample bottle and gas trap system.

VOL. 31, 1976

CARBON METABOLISM IN MICROBIAL SYSTEMS

at twice the concentrations found necessary to eliminate target organisms in preliminary experiments. This delay period allowed the elimination of the target organisms before the metabolism of the labeled leachate was allowed to proceed. At the end of this 72-h period, 300 ml of labeled Spartina leachate, containing 21.6 jACi of 14C, was added to each of the bottles. Sterile artificial seawater was added to bring the final volume to 1,900 ml in all the bottles. In the control bottles 200 ml of formalin was substituted for part of the seawater, resulting in a final concentration of 4% (vol/vol) formaldehyde. Aeration was then resumed. Gas-trapping vials were changed daily and counted within 48 h. Material within the bottles was sampled for microbial counts and analysis with a hypodermic syringe to avoid loss of respired gases. For the bottles in systems A and B, samples were taken from the bottles on days 2, 5, and 8, whereas systems C and D were sampled on days 3, 6, and 9. Samples were filtered through 0.45-,um Gelman membrane filters. Two 0.5-ml subsamples of the filtrate were taken for scintillation counting. Clumping due to fungal mycelium and pulverized plant material resulted in a nonhomogeneous distribution of solid material in the incubation bottles; therefore, the mass balance for 14C was made from the 14C02 and dissolved 14C counts. Counts for the particulate 14C fraction were determined by difference from the original amount of label added to the bottles. During the experiment, samples were taken for plate counts of bacteria and fungi and for microscopic counts of protozoans and microcrustaceans. On the first and last days of the experiment, two of the three bottles in each experimental system were sampled for plate counting. Bacterial counts were done in triplicate on marine agar 2216 (Difco) plates, and fungal counts were done on triplicate Sabouraud agar + 1.5% NaCl plates containing chloramphenicol (0.2 g/liter) to inhibit bacterial development. Plates were incubated at room temperature. For enumeration of protozoans and microcrustaceans, four replicate counts were done for two of three bottles in each of the three experimental systems on a Palmer nanoplankton counter. Plate counts were taken on days 2, 5, and 8 for all bottles in the A and B systems to insure that the desired target organisms were still being inhibited. Plant fraction mineralization experiments. Plant fraction experiments were done using the experimental apparatus and inoculum described above. No antibiotics or 72-h delay period was used in this experiment, since a mixed community was to be used in all systems. Three different substrates were used: (i) system E, containing leached Spartina, consisting of pulverized plant material from which all saltwater-soluble material had been extracted by boiling it in artificial seawater for 10 min; (ii) system F, whole Spartina, simply the dried, pulverized plant; and (iii) system G, Spartina leachate prepared in the same manner as that used in the first experiment. Each of the three systems (E, F, and G) had triplicate experimental bottles and two sterile control bottles. Sterile, radiolabeled substrate was

961

added to each of the bottles using aseptic techniques. To the three bottles in system E, 1.475 g (dry weight) of leached Spartina, containing approximately 10.4 ,uCi of 14C, was added. Bottles in system F received 1.475 g (dry weight) of whole Spartina, containing approximately 9.7 ,uCi of 14C. A 75-ml aliquot of the Spartina leachate, containing approximately 10.5 ,uCi of 14C, was added to the bottles in system G. lUsing techniques described above, samples were taken for plate counts for bacteria and fungi on days 0, 2, 5, and 7. Quadruplicate counts for protozoans and microcrustaceans were done using a Palmer nanoplankton counter on days 0 and 7. Gas-trapping vials were changed every 24 h. Samples for soluble counts were taken on the last day (day 7) of the experiment. Scintillation counting. All radioactive counting was done using a toluene-based scintillation fluid containing: 666 ml of toluene, 333 ml of Triton X-100, 4.0 g of PPO (2,5-diphenyloxazole), and 0.05 g of POPOP [1,4-bis-2-(5-phenyloxazolyl)benzene] per liter. Counting was done on a Packard 3302 spectrometer. All samples were counted to 20,000 counts or 20 min. Efficiency correction was done using the channel ratios method of quench correction (4, 18). Calculations are based on microcuries of 14C in each fraction. All results are stated as arithmetic means + standard error. RESULTS Community fractionation experiments. Results of the metabolism experiments should be viewed in light of observations of plate counts and microscopic counts (Tables 1 and 2). Due to difficulties involved in the identification of protozoan species, the microscopic counts presented may not reflect absolute numbers but, rather, demonstrate comparative numbers in the different systems. Plate counts should also be considered only for comparative purposes. Three observations are important. First, eukaryotic species, except fungi, had disappeared from the system receiving antibiotics to inhibit bacteria (B) by day 0. Second, bacteria were detected in the B system bottles on day 5 of the experiment. Third, in the mixed community of the mineralization experiment with whole Spartina fractions (Table 2) microcrustaceans were undetected, and the protozoans were considerably reduced in comparison to the mixed (C) system in the community fractionation experiments. Uptake of the label was rapid in all systems (Fig. 2). On day 2 of the experiment, in the bacterial (A) system, an average of 68 + 1.4% of the label originally present in DOM had been incorporated into particulate matter, whereas in the fungal (B) bottles only 41 + 0.9% of the labeled DOM had been converted to particulate material. On day 3 in the mixed (C) system, an average of 51 + 1.4% of the label was present as

962


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particulate matter. The patterns of label uptake into particulate matter may indicate two dissimilar trends. In the bacterial (A) and mixed (C) systems, maximum label incorporation had occurred by day 2 or 3 of the experiment and declined thereafter. In contrast, the fungal bottles demonstrated a continual increase in label incorporation, still increasing on the last sampling day. The percentage of label remaining in the dissolved fraction (Fig. 3) showed a rapid initial decline in the bacterial (A) and mixed (C) systems, with no significant trend during the remainder of the experiment. The fungal (B) system showed a less rapid decline, which continued over the experimental period. The curve for '4CO2 produced from labeled DOM appears in Fig. 4. The bacterial (A) and mixed (C) systems rapidly converted the labeled DOM to '4CO2, whereas fungal (B) systems demonstrated a lag period followed by a much lower level of '4C02 evolution. Because the amount of '4CO2 evolved during any sampling interval may depend on the amount of labeled material available for mineralization, corrections have been made for daily losses of label as '4CO2 from the bottles. Figure 5 represents daily '4C02 evolution as a percentage of

the label available at the end of the preceding 24-h period. Averaged for the whole experimental period, rates of '4CO evolution from the bacterial bottles were 60% of that in the mixed (C) system, whereas rates for the fungal (B) system were 15% of that in the mixed system. Using values from Fig. 2, 3, and 4, and correcting for the control (D) system, efficiency values for the conversion of labeled DOM to particulte biomass can be calculated (Table 3). Two types of efficiencies have been calculated; production efficiency demonstrates how much of the metabolized label has been converted to particulate biomass, whereas conversion efficiency shows how much of the substrate originally added has been converted to particulate biomass. The fungal system (B) appeared to be the most efficient at incorporating the labeled material into biomass. As defined above, mean production efficiency was 0.82 ± 0.03 and mean conversion efficiency was 0.56 + 0.1 for the fungal system, which did have some bacterial contamination (Table 1). The bacterial system (A) yielded a mean production efficiency of 0.54 ± 0.08 and a mean conversion efficiency of 0.43 ± 0.01. A mean production efficiency of only 0.21 ± 0.03, with a conversion efficiency of 0. 16 + 0.02, was calculated for the mixed commu-

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FALLON AND PFAENDER

DISCUSSION The community fractionation experiments indicated that bacteria present in the marsh waters and on decaying S. alterniflora not only responded to and had assimilated labeled DOM leached from S. alterniflora more rapidly than D 80 the fungi present (Fig. 2), but also mineralized 0 the material more rapidly (Fig. 4 and 5). How0 ever, after 8 days, the fungal system had incor-j porated more of the label into new particulate Z 60 biomass than the bacterial system. A rapid assimilation, similar to the other two systems, (C) also seen in the mixed (C) community syswas 0 tem. Here again, as in the bacterial (A) system, Uthe labeled substrate was rapidly metabolized. rate of daily 14CO2 evolution in the mixed The BATRIA (A) 00 (C) community was the most rapid of all three systems (Fig. 4 and 5). Bacterial and mixed N systems incorporate label into cells initially 0 o~20(Fig. 2) and, subsequently, when DOM levels become low (Fig. 3), may begin using intracellular carbon pools and storage compounds, which results in a decline in the amount of particulate label in the A and C systems after 2 0 2 3 4 5 6 7 8 9 and 3 days, respectively. Since the daily rate of DAY 14CO2 evolution reached a maximum on day 2 FIG. 4. Cumulative percentage of the label, origi- (Fig. 5) for both the A and C systems, it appears nally added as dissolved organic matter, present as that the maximum percentage of label in the 14CO2. particulate fraction may have occurred at about the time the first particulate samples were nity (C), indicating that a significantly lower taken at the end of days 2 and 3. Thus, even percentage of both the carbon added and the C,) 20- BACTERIA (A) carbon metabolized is expressed as new micro15bial biomass. Plant fraction mineralization experiments. C,) 10cr This experiment was intended to demonstrate 0 cli MEAN the rate of mineralization of different fractions 5100

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of the labeled S. alterniflora. The daily "4CO2 evolved, as a percentage of the label available

at the end of the previous 24 h, is shown in Fig. 6. The data indicate that the Spartina leachate

and the whole Spartina were mineralized much more rapidly than the Spartina material from which the solubles had been leached. Average daily rates of '4CO2 evolution for the Spartina (G) leachate and the whole Spartina (F) were 3.6 + 0.1 and 3.6 + 0.8%/day, respectively. These rates were approximately 15 times the rate of 0.23 + 0.02%/day observed in the

leached Spartina system. This indicates that

the saltwater-soluble portions of the S. alterniflora are the most rapidly mineralized, as might be expected. It should also be noted that system G, with a rate of 3.6 + 0.2%/day, evolved '4CO2 at a much slower rate than the similar type system (C) in the community fractionation experiment, which had a daily rate of 14CO2 evolution of 9.2 + 0.5%/day.

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VOL. 31, 1976


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TABLE 3. Values for the percentage of label in the particulate '4C, dissolved '4C, and "CO2 fractions from the community fractionation experiments, accompanied by production and conversion efficiencies for experimental systems A (bacterial), B (fungal), C (mixed), and G (mixed) System

Bacterial (A) Day 2 Day 5 Day 8 Fungal (B) Day 2 Day 5 Day 8 Mixed (C) Day 3 Day 6 Day 9 Mixed (G)

Production efficiency"

Conversion effi-

12.0 ± 1.2 7.3 ± 0.3 6.6 ± 0.1

0.54 ± 0.02

0.43 ± 0.01

0.25 ± 0.2 6.7 ± 1.5 11.9 ± 1.6

58.5 ± 0.8 27.7 ± 4.0 19.5 ± 0.4

0.82 ± 0.03

0.56 ± 0.1

27.4 ± 2.4 49.4 ± 2.8 59.0 ± 3.4

12.4 ± 0.5 13.1 ± 0.5 11.5 ± 0.9

0.21 ± 0.03 0.63 ± 0.03

0.16 ± 0.02 0.38 ± 0.02

Particulate 14C

14CO2

Dissolved 14C

55.1 ± 1.1 44.8 ± 0.6 42.9 ± 0.6

20.2 + 4.5 35.3 ± 1.6 37.8 ± 1.7

28.7 ± 0.6 52.8 ± 2.5 55.9 ± 0.9 38.0 ± 1.1 21.2 ± 1.3 16.0 ± 1.0

Production efficiency = [particulate '4C/(particulate 14C + 14C02)] "Conversion efficiency = percent particulate '4C/100.

I1.0 1 LEACHED SPARTINA (E)

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the carbon assimilated was subsequently lost from the particulate fraction by mineralization to CO. This hypothesis is supported by the observation that the decrease in percentage of ()M 0.5label in the dissolved state occurred more rap0 in the mixed (C) system than in the fungal idly -X- MEAN (B) system (Fig. 3). w cr Rapid assimilation of simple organic moleacules has been demonstrated in marine waters o 0O in the field (19, 20, 30). Size fractionation by 0 10' WHOLE SPARTINA (F) filtration showed that much of the metabolic z w activity was associated with particles of 2 ,um or less, the bulk of the activity being credited < 5to bacteria. Paerl (27), using autoradiographic z techniques, has made similar observations. w LLI The hypothesis that bacteria are responsible cn w for the major part of heterotrophic metabolism a: in salt marsh-estuarine systems is supported acU by the present experiment. The mixed microSPARTINA LEACHATE (G) bial community associated with S. alterniflora z detritus, containing many species of micro0 metazoans and protozoans, appeared to be Chighly diverse. Even so, bacterial assimilation 0 (A) was as rapid as that seen in the mixed 0-t 5community, and "4CO.2 evolved by the bacteria ----MEAN CNJ could potentially account for 62% of that 0 evolved by the mixed community (Fig. 5). This 1,. 0 was true even without the presence of con1 2 3 4 5 6 7 sumer species in the bacterial system, which would keep bactera growing in the log phase. DAY Although bacteria appear to be most imporFIG. 6. Daily "CO.2 evolution as a percentage of the label available at the end of the preceding 24 h. tant in the mineralization process, the presence Note scale differences. of protozoans and microinvertebrates did increase the rate of mineralization of DOM (Fig. though the mixed (C) community appears in 4 and 5). In this experiment, the daily evolution Fig. 2 to have an initial rate of label assimila- of "4CO2 is, on the average, 38% greater in the tion slower than that of the fungal (B) system, mixed (C) system than in the bacterial (A) in reality it may have been faster, but much of system. A similar pattern has been observed by cn

I CM

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FALLON AND PFAENDER

other workers. The presence of protozoans and microinvertebrates was shown to increase the rate of mineralization of PO from particulate S. alterniflora (21) and that of carbon from particulate plant detritus (8, 15). In the present experiment such an effect has been noted upon a dissolved substrate. Usually, two explanations are offered for such increases in rates of mineralization. First, consumption of the bacteria by protozoans and microinvertebrates causes bacterial populations to turn over more rapidly. Thus, more substrate material is consumed per unit of time. Second, the presence of a more diverse community in the mixed (C) system adds additional levels of transfer of material and energy, making the whole system less efficient. Fenchel (7) has shown that protozoans associated with marine detritus form a very complex community within themselves. Not only do some species prey upon bacteria, but they also prey upon each other and consume dissolved organic material. Both factors, increased complexity and increased rates of turnover, could cause the mixed community to be less efficient in converting substrate into new biomass, with more lost as CO., per unit of time. This decrease in efficiency for a complex community can be seen in Table 3. The bacterial (A) and fungal (B) systems had values larger than the mixed (C) system for both production and conversion efficiencies. It should be kept in mind, however, that productivity by bacterial and fungal populations within this mixed community could be similar to that seen in the individual A and B systems. These factors may also explain the differences in observed efficiencies for the two mixed communities tested in this study, system C in the community fractionation experiments and system G in the plant fraction mineralization experiments. Data in Tables 1 and 2 show that protozoan and microcrustacean numbers were much reduced in the mixed community of the second experiment. The decrease in predator species may have resulted in a reduction in the bacterial turnover and material transfers per unit of time, resulting in increased efficiency in converting labeled DOM into new particulate biomass in system G, as shown by the considerably higher efficiencies in Table 3. The lower efficiency of biomass production and higher rate of mineralization in mixed communities has significant implications for nutrient cycling in salt marsh ecosystems. The rapid utilization and turnover of plant compounds by the microbial community can contribute to the richness of the estuarine environment by increasing the amount of nutrients available to plant species.


Efficiency values, as defined earlier, are presented for the conversion of soluble label into particulate label. The production efficiency calculation is based on the assumption that no label is re-excreted as soluble material and all assimilated label is converted to particulate material or CO. Thus, carbon flow is unidirectional from soluble pools to particulate material or '4CO2. This efficiency represents the maximum value for the system. However, the assumption of no label re-excretion is probably not valid for most microbial populations, since excretion of waste products and extracellular enzymes does occur. Thus, conversion efficiency is calculated as an indication of minimum efficiency. This value represents the proportion of the label originally present as soluble material that has been converted to particulate material. Therefore, these two values represent the upper and lower limits for label assimilation in this system. A review of heterotrophic growth effliciencies by Payne (28) shows a range of 0.56 to 0.60 as the commonly observed range for values of production efficiency under laboratory conditions. These values are derived from model systems using bacterial or yeast cultures, both unispecific and in groups of three to four species. The value obtained for the bacterial (A) system, 0.54 + 0.02, approaches the range stated by Payne (28). The fungal system (B) production efficiency, 0.82 + 0.03, is much above this range, which may be the result of considerable quantities of labeled carbon being incorporated into fungal spores, where it would not be immediately available for use in energy production and concomitant conversion to CO. There exist only two other studies concerned with how efficiently marsh microbial species convert S. alterniflora material to new biomass. Burkholder and Bornside (3), using unispecific bacterial cultures, with an S. alterniflora leachate, obtained conversion efficiencies of only 0.20. In other words, after a 3-day incubation, only 20% of the material added as substrate had been converted to bacterial biomass. From the data presented by Burkholder and Bornside (3) it appears that these cultures had reached steadystate conditions when the conversion efficiency value of 0.20 was calculated. The lack of uniform labeling of the DOM used in the experiments reported in this paper makes direct comparison of efficiency values difficult; however, a conversion efficiency of 0.43 was obtained in the bacterial (A) system. The higher value in the present experiment may be partially explained by the presence of a heterogeneous bacterial population. Such a population, because of its

VOL. 31, 1976


heterogeneity, may be able to utilize parts of the substrate that went unused in an unispecific culture of the type Burkholder and Bornside (3) used. Gosselink and Kirby (14), using a mixed microbial community obtained from Louisiana coastal marshes, with S. alterniflora detritus as a substrate, obtained production efficiency values that ranged from 0.24 to 0.63 in 30-day experiments. Values obtained for production efficiency in a mixed microbial community in the present study showed a similar range of 0.21 + 0.03 in system C to 0.63 + 0.30 in system G. The shorter period of the present experiment 08 to 9 days) could have caused the lower value seen in the C system, if label incorporation had not reached a maximum. However, as Fig. 2 shows, the maximum value for label incorporation had probably been reached when efficiency values were calculated, and the amount of label in the '4CO2 fraction was remaining relatively constant (Fig. 4 and 5). Thus, a longer experimental period would have been likely to result in a slighly lower production efficiency value. Results from the plant fraction mineralization experiments (Fig. 6) indicate that, under aerobic conditions, soluble material from S. alterniflora was mineralized more rapidly than nonsoluble material. This is as might be expected, since metabolism of nonsoluble substrates by the microorganisms requires that the material be first broken down by extracellular enzymes. Over a long experimental period, maximum daily rates of 14CO2 evolution in the leached Spartina (E) system might eventually approach the maximum rates observed in the whole Spartina (F) and Spartina leachate (G) systems. The fact that aerobic breakdown of cellulosic material by bacteria is very slow (2) also contributed to the lower mineralization rate in the leached Spartina (E) system. As slow as the mineralization was in the leached Spartina (E) system, it is interesting to note that the average daily rate of mineralization from this system at 15 C, 0.23 + 0.02%/day (Fig. 6), is similar to rates observed in the field for the decomposition of S. alterniflora detritus. Odum and de la Cruz (26), in a 2-month experiment using litter bags, observed an average daily weight loss of 0.30%/day, whereas Burkholder and Bornside (3), in a similar experiment, obtained a value of 0.28%/day. The percentage of the daily photosynthate of S. alterniflora growing in marshes that is excreted as DOM is approximately 5% (9); the amount contributed from the decay and conversion to detritus of dead S. alterniflora is unknown. The results of the present study in a

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model ecosystem indicate that the marsh microbial community may be able to rapidly utilize such material. Although the overall percentage of the annual production that may follow such a pathway is open to speculation and in need of further research, it is very likely quite important in the development of the aerobic microbial community. This community. in turn, as demonstrated by a number of studies (1, 8, 15-17), may act as the base of a food web important to many marsh-estuarine species. ACKNOWLEDGMENTS This investigation was supported by the University of North Carolina, University Research Council and the Curriculum in Marine Science. We also wish to thank Dirk Frankenberg for his thoughtful suggestions regarding the manuscript. LITERATURE CITED 1. Adams, S. M., and J. W. Angelovic. 1970. Assimilation of detritus and its associated bacteria by three species of estuarine animals. Chesapeake Sci. 11:249-254. 2. Brock, T. D. 1966. Principles of microbial ecology. Prentice-Hall, Englewood Cliffs, N.J. 3. Burkholder, P. R., and G. H. Bornside. 1957. Decomposition of marsh grass by aerobic marine bacteria. Bull. Torrey Bot. Club 84:366-383. 4. Bush, E. T. 1963. General applicability of the channel ratios method of measuring liquid scintillation counting efficiency. Anal. Chem. 35:1024-1029. 5. Day, J. W., Jr., W. G. Smith, P. R. Wagner, and W. C. Stowe. 1973. Community structure and carbon budget of a salt marsh and shallow bay estuarine system in Louisiana. Center for Wetland Resources, Louisiana State University, Baton Rouge. 6. Fallon, R. D., and F. K. Pfaender. 1976. Production and fractionation of 14CO2 labeled smooth cordgrass, Spartina alterniflora. Chesapeake Sci., in press. 7. Fenchel, T. 1969. The ecology of marine microbenthos. IV. Structure and function of the benthic ecosystem, its chemical and physical factors and the microfauna communities with special reference to the ciliated protozoa. Ophelia 6:1-182. 8. Fenchel, T. 1970. Studies on the decomposition of organic detritus derived from turtle grass Thalassia

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