Biological Dinitrogen Fixation (Acetylene Reduction) - Europe PMC

APPID AND ENVIuRoNmETAL MICROBIOLOGY, Mar. 1978, p. 567-575

Vol. a35, No. 3

0099-2240/78/0035-0567$02.00/0 Copyright 0 1978 American Society for Microbiology

Printed in U.S.A.

Biological Dinitrogen Fixation (Acetylene Reduction) Associated with Florida Mangroves D. A. ZUBERERt AND W. S.

SILVER*

Department ofBiology, University of South Florida, Tampa, Florida 33620

Received for publication 11 July 1977

Biological dinitrogen fixation in mangrove communities of the Tampa Bay region of South Florida was investigated using the acetylene reduction technique. Low rates of acetylene reduction (0.01 to 1.84 nmol of C2H4/g [wet weight] per h) were associated with plant-free sediments, while plant-associated sediments gave rise to slightly higher rates. Activity in sediments increased greatly upon the addition of various carbon sources, indicating an energy limitation for nitrogenase (C2H2) activity. In situ determinations of dinitrogen fixation in sediments also indicated low rates and exhibited a similar response to glucose amendment. Litter from the green macroalga, Ulva spp., mangrove leaves, and sea grass also gave rise to ignificant rates of acetylene reduction. Higher rates of nitrogenase activity (15 to 53 nmol of C2H4/g [wet weight] per h were associated with washed excised roots of three Florida mangrove species [Rhizophora mangle L., Avicennia germiuana (L) Stern, and Laguncularia racemosa Gaertn.] as well as with isolated root systems of intact plants (11 to 58 itg of N/g [dry weight] per h). Following a short lag period, root-associated activity was linear and did not exhibit a marked response to glucose amendment. It appears that dinitrogen-fixing bacteria in the mangrove rhizoplane are able to use root exudates and/or sloughed cell debris as energy sources for dinitrogen fixation. Mangroves represent unique and ecologically coastal waters are often nutrient limited, particimportant coastal habitats throughout much of ularly in terms of combined nitrogen (19), the the tropical and subtropical world. It is esti- high productivity of these areas is somewhat mated that 75% of these areas are lined with paradoxical. mangrove vegetation. Davis (4) defined "manInvestigations of biological nitrogen fixation grove" as a general term applied to plants which in the highly productive mangrove communities live in muddy, loose, wet soils in tropical tidal of the subtropics are few in number. Rodina (17) waters. According to Macnae (12), mangroves reported the presence of several types of diazotrophs in soil (sediment) samples of mangrove are trees or shrubs that grow between the highwater mark of spring tides and a level close to thickets taken from the coast of the Gulf of but above mean sea level. They are circumtrop- Tonkin, Vietnam, and later analyzed in the ical on sheltered shores and often grow along U.S.S.R. Kimball and Teas (10) observed low the banks of rivers as far inland as the tide levels of N2 fiXation (C2H2) in sediments from penetrates. Chapman (3) described silt, sand, what they defined as five types of mangrove peat, and coral reefs as mangrove habitats. Man- communities in southern Florida, and another groves are frequently seen as pioneer vegetation report concerned such activity in decaying vegin many coastal areas. Three species of man- etation (6). Similarly, Zuberer and Silver (20) grove, Rhizophora mangle L., Avicennia ger- observed N2 fixation (C2H2) in mangrove sediminans (L) Stern, and Laguncularia racemosa ments of the Tampa Bay region of the Florida Gaertn., are common in coastal regions of South Gulf Coast. This paper reports the results of further efFlorida. The high productivity of the mangrove community and its importance in the estuarine forts to assess the role of biological nitrogen food web have been well documented (E. S. fixation in the establishment and/or mainteHeald, Ph.D. dissertation, University of Miami, nance of Florida mangrove communities. Miami, Fla., 1969; 13). In view of the fact that (This work was submitted by D.A.Z. in partial fulfillment of the requirements for the Ph.D. t Present address: Department of Microbiology and Celi degree, University of South Florida, Tampa, Science, University of Florida, Gainesville, FL 32611. 1976.) 567

568

ZUBERER AND SILVER

MATERIALS AND METHODS Study sites. The principle area investigated was a recently colonized (within the past 10 years) dredgefill site adjacent to Whiskey Stump Key on the southeastern edge of Hilisborough Bay (Fig. 1). This site was chosen because it represented an emerging mangrove community in a nutrient-poor (at least in terms of combined nitrogen) sandy sediment. All three Florida mangrove species [R. mangle L. (red mangrove), A. germinans (L.) Stern (black mangrove), and L. racemosa Gaertn. (white mangrove)] were present in various stages of development. The entire stand was completely inundated during high tides and completely exposed during low tides. Two additional areas studied were Cockroach Bay and Fish Creek in upper Old Tampa Bay (Fig. 1). Cockroach Bay represented an extensive mature mangrove community containing many islands comprised mainly of R. mangle. This area allowed us to obtain a variety of sediment types ranging from sandy muds to mangrove peat. The Fish Creek site represented a mangrove stand containing all three mangrove species in an area of Tampa Bay subject to a greater degree of nutrient enrichment than the other two sites. Acetylene reduction assay. The acetylene reduction assay for nitrogenase activity was conducted by commonly accepted methods (9). Throughout these studies, samples designated aerobic were incubated under 0.2 atmosphere acetylene in air, and samples designated anaerobic were incubated under 0.05 atmosphere acetylene in argon. Anaerobic systems were flushed with argon prior to gassing with acetylene. All

APPL. ENVIRON. MICROBIOL.

systems were prepared so as to attain an initial internal pressure of 1.0 atmosphere. Analysis for ethylene was accomplished by gas chromatography using a Varian Aerograph 1440 gas chromatograph equipped with a hydrogen flame ionization detector. Instrument operating conditions were as follows: a stainless-steel column (152 by 0.63 cm ID) packed with Poropak N, 100 to 120 mesh at 90°C, injector and detector temperature of 140°C, N2 carrier gas at a flow rate of 60 ml/min, H2 flow rate of 30 ml/min, and air flow rate of 300 ml/min. Conversion of nanomoles of ethylene produced to nanomoles of nitrogen fixed was made assuming the theoretical ratio of 3to 1. Sediment sampling and assay procedures. Sediment samples were taken by coring in plant-free areas at mean low water and mean tide and from the root zone by coring immediately adjacent to shoots of the three mangrove species. Core samples 1.5 cm in diameter by 5.0 cm in length were obtained with a coring device equipped with a plunger to expel the core. Three such cores were pooled in 125-ml Erlenmeyer flasks with perforated screw tops sealed with a rubber septum for gas sampling. Sediments were routinely assayed anaerobically, since early trials (20) revealed that the bulk of the activity occurred under these conditions. No preincubation period (5) was employed, and unamended samples were incubated for a sufficient length of time to establish the rates of acetylene reduction activity in the absence of added substrate. Following the establishment of the unamended rates, glucose was added to an approximate final concentration of 2.0 mg of C per ml of sediment slurry, and the

TAMPA BAY REGON

HILLSBOROUGH BAY GULF OF MEXICO

N A

FIG. 1. Sampling sites in the Tampa Bay region of South Florida and its location on the Florida gulf coast

(insert).

VOL. 35, 1978

assay was continued. Other substrates tested for their ability to stimulate nitrogenase activity included lactose, mannitol, sodium acetate, calcium lactate, sodium succinate, glycolic acid, L-mahc acid, and DLaspartic acid. The effects of incubation temperature and depth within the core on acetylene reduction activity were also investigated. Habitat water was assayed regularly to determine how much, if any, nitrogenase activity was accounted for by the water itself when it was used to prepare samples for acetylene reduction assay. Assay of litter and other plant materials. Mangrove leaf litter as well as allochthonous materials periodically imported to the habitats which might serve as potential carbon sources for diazotrophic and other heterotrophic bacteria were assayed. The primary source of litter in the mangrove community consisted of leaves of the red mangrove. Other materials assayed included pieces of the green macroalga, Ulva spp., and sea grass litter. Root system studies. Young plants or seedlings of the three mangrove species were harvested from field sites with the root-sediment ball intact and returned to the laboratory. The adherent sediment was removed by rinsing the root systems in serial baths of fresh habitat water or one-half-strength artificial sea water (Utility Marine Mix, Utility Chemical Co., Paterson, N.J.). Washed roots were cut into shorter segments and placed in flasks for assay. The roots were fully immersed in 10 to 20 ml of habitat water which had previously been sparged with argon for anaerobic incubations, whereas unsparged habitat water was used for aerobic incubations. In addition, washed root systems of intact plants of Laguncularia were assayed using the method described by Burris

(1).

In situ determinations of nitrogenase activity. In situ determinations of nitrogenase activity were conducted using the apparatus diagramed in Fig. 2. The apparatus was tested both with and without a perforated central aluminum cylinder (Fig. 2B) designed to facilitate diffusion of C2H2 into the thixotropic sediments. Internal head space was quantitated by measuring the distance from the slightly compressed sediment surface to a mark on the inside of the column, at which point the rubber stopper rested when fully seated. Bacterial populations. Selected bacterial groups enumerated during this investigation included the following: aerobic and facultatively anaerobic or anaerobic bacteria, aerobic and facultative diazotrophs, and sulfate-reducing diazotrophs. Though not enumerated, purple photosynthetic bacteria were isolated from enrichment cultures using a nitrogen-free modification of Larsen medium (18). Enumeration of these selected populations was performed by standard plate count (pour plate and spread plate) or by most probable number techniques using appropriate media. When most-probable-number enumeration techniques were employed in nitrogen-free media, tubes at the highest positive dilutions were subjected to acetylene reduction assay to verify the presence of diazotrophs. Total aerobic and anaerobic (under 100% N2) plate counts were conducted on tryptic soy agar (Difco) prepared with one-half-strength artificial sea water to compare numbers of hetero-

N2 FIXATION IN MANGROVES

569

.5 3 1CM B-

C-

4.8CM

32CM

A PVC CONDU T

FIG. 2. Apparatus designed for the measurement of nitrogenase activity in situ. (A) Polyvinyl chloride conduit outer cylinder; (B) perforated aluminum inner cylinder to facilitate gas exchange within the enclosed core; (C) solid aluminum rod used during the insertion of the inner cylinder to keep it free of sediment.

trophic bacteria able to grow on this medium against numbers capable of growth on a nitrogen-free sea water medium containing the following (g/liter of onehalf-strength artificial sea water): glucose, 10.0; fructose, 10.0; FeCl3 6 H20, 0.005; yeast extract, 0.05; agar, 20.0. Following autoclaving, 10 ml of a trace element solution containing (mg/liter) ZnSO4 7 H20 (1100.0), MnSO4 H20 (500.0), CoS04 (2.5), H.3BO3 (2.5), Na2MoO4 (100.0), and CuS04 5 H20 (0.35) and 1 ml of a vitamin solution containing (mg/100 ml) thiamine (100.0), B12 (0.2), and biotin (0.1) were added. Sulfatereducing bacteria were enumerated in tightly sealed screw-top tubes containing the following medium (g/liter of one-half-strength artificial sea water): sodium lactate, 3.0; ascorbic acid, 0.1; sodium thioglycolate, 0.2; ferrous sulfate, 0.2; and vitamins and trace elements as above.

RESULTS Ethylene production by mangrove-associated sediments. In general, preamendment C2H2 reduction activities were low (Fig. 3), the mean ranging from 0.21 to 0.91 nmol of ethylene/g of wet sediment per h. There was always a very marked response to the addition of carbon (Fig. 3), and after a lag of 12 to 24 h, rates of acetylene reduction became linear and eventually declined (not shown on curve). Plant-associated sediments generally exhibited higher

570

ZUBERER AND SILVER


acetate were more effective under aerobic conditions. A greater lag period before acetylene reduction became linear was observed in aerobic systems, especially for sugars. However, in the aerobic flasks, the final level of ethylene production was equivalent to, or greater than, that in the anaerobic flasks. Further substrate studies are summarized in Table 2. Glucose and lactose stimulated the greatest increases in C2H2 reduction, followed by DL-malic acid. Malic acid, like other tricarboxylic acid cycle intermediates, was HOURS

FIG. 3. Effect of the addition of glucose (2 mg of C/ml, final concentration) to mangrove sediments from the Whiskey Stump Key site. Note the lag period (12 to 24 h) after which nitrogenase activity (Cd2l) becomes linear. (U) Sediment associated with Laguncularia; (A) sediment associated with Rhizophora; (0) sediment associated with Avicennia; (0) plantfree mean tidal sediment; (*) plant-free mean lowwater sediment.

activities than "plant-free" sediments, as demonstrated by the Laguncularia-associated sediments from the mean high water level in the community (Fig. 3). Occasional samples of mean low water and mean tidal plant-free sediments gave rise to unexpectedly high rates of acetylene reduction. These rates were always correlated with the presence of organic material (usually leaf fragments) in the core samples. In all ofthe sediment samples tested, variation among replicates was unpredictable and frequently quite high, with only occasional replicates showing a very close similarity. Sediments from the Fish Creek and Cockroach Bay sites exhibited preamendment rates similar to those of the Whiskey Stump Key site. Two notable exceptions, however, are the rates recorded for a peaty sediment slurry and for surface litter collected at Cockroach Bay on two separate occasions (Table 1). Acetylene reduction activity was inhibited upon the addition of saturated ammonium sulfate into the sediment systems (data not shown), substantiating that C2H4 evolution was mediated by nitrogenase. No significant endogenous ethylene was detected in sediments incubated in the absence of acetylene under aerobic or anaerobic conditions. Effects of substrates, temperature, and core depth on acetylene reduction. Studies conducted to determine the effects of various carbon sources on nitrogenase activity (C2H2) indicated that, of the substrates tested, glucose and mannitol were most stimulatory (Fig. 4), and that this activity was greatest under anaerobic conditions. However, lactate, succinate, and

TABLE 1. Nitrogenase activity (Cjl,) from mangrove communities in the Tampa Bay region of South Florida

nmmol of CsH4/g

Collection site and sample type

(wet wt) per ha

Fish Creek Anoxic sulfide mud .. 0.17 Mean tidal sediment .......... 0.13 Mean low-water sediment 0.16 Prop-root-associated sedimentb 0.19 Pneumatophore-associated sedimentc ... 0.06 Cockroach Bay Anoxic sulfide mud .. 1.26 Mean low-water sediment 0.25 Prop-root-associated sediment 0.36 Peatty sediment slurry . 34.37 Litter materialsd 17.75 aAll values are means of three replicate samples. b Specialized "breathing root" of the red mangrove. c Breathing root of the black mangrove. d Mangrove leaf and sea grass litter. .

E

460

-C 13

40

-

E3J

AIR

E:J

ARGON

360

-"

2" 26E 1.I

A 02 03 None

E

g |

N

|

° :-:.2

F

6

Mann

Gluc

Lact

Fucc

Mann

Gluc

Lact

Succ

0o4

I Acet

AMENDMENT

FIG. 4. Effect ofseveral carbon and energy sources on mangrove sediment slurries. Data are expressed as the change in nanomoles of ethylene per gram of wet weight of sediment per hour in a 25-h period following amendment. (A) Endogenous acetylene reduction, (B) mannitol, (C) glucose, (D) calcium lactate, (E) sodium succinate, (F) sodium acetate. AU substrates were added at near neutral pH at a level of approximately 2 mg of C/ml, final concentration.

TABLE 2. Effect of selected substrates on

nitrogenase activity (C2H2) Change in nmol of ethylene postamendment Substratea

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VOL. 35, 1978

Aerobic

Anaerobic

24.0k h 48.0 h 24.0 h 48.0 h Glucose 36.78 296.49 47.60 173.10 2.30 49.00 Lactose 0.10 12.30 0.06 0.63 Glycolic acid 0.09 0.29 Malic acid 7.50 14.58 1.36 2.55 0.31 1.62 Aspartic acid 0.57 0.98 2.00 Endogenous (no 2.48 0.29 1.02 amendment) All substrates were added to approximately 2 mg of C/ml of slurry, final concentration. 'Time elapsed after substrate addition.

more effective under aerobic conditions in promoting C2H2 reduction. Glycolic acid and aspartic acid had little or no effect on C2H2 reduction. Nitrogenase activity (C2H2) occurred maximally at 28°C and declined toward 37°C. The sediment system exhibited a Qlo value of 2.3 to 3.0 in the range of 18 to 280C, and some activity was observed at 80C, as low a temperature as would likely be encountered in the area studied. The rate of nitrogen fixation was relatively uniform throughout the 6-cm core samples analyzed. Acetylene reduction activity of habitat waters assayed in the same manner as sediments was negligible (data not shown). Only in a few instances, after prolonged incubation in the dark following glucose supplementation, was any activity observed. C2H2 reduction associated with litter and other plant materials. Litter from the green macroalga, Ulva spp., which washed up into the community in large quantities in late fall, was collected and assayed. As much as 6.45 nmol of ethylene per g (wet weight) per h were produced by this litter prior to the addition of glucose. These rates are in some cases comparable to those observed for amended sediments. The observed activity was greatest under anaerobic conditions, while aerobic systems responded only after a considerable lag following carbon

supplementation. Mangrove leaf litter exhibited high rates of acetylene reduction. Rates as high as 38 nmol of ethylene per g (wet weight) per h (anaerobic) and 35 nmol (aerobic) were recorded, with no appreciable response to carbon supplementation. Aerobic and anaerobic rates were not significantly different, and activities under both conditions commenced without a substantial lag period. At Cockroach Bay, surface litter consisting of

mangrove leaves (R. mangle) and sea grass material transported into the site exhibited high rates of acetylene reduction (16.75 nmol of ethylene per g [wet weight] per h) under anaerobic conditions. Activity associated with this material was linear and proceeded without a substantial lag period, indicating that the litter materials were well colonized by diazotrophs. Root-associated acetylene reduction. Considerably greater acetylene reduction was associated with washed root systems than with sediments (including plant-associated sediments). Roots of L. racemosa consistently showed the highest rates (10 to 53 nmol of ethylene per g [wet weight] per h), whereas A. germinans was intermediate (13 to 16 nmol of ethylene per g [wet weight] per h), and R. mangle was generally the lowest (5 to 15 nmol of ethylene per g [wet weight] per h). In general, nitrogenase activity proceeded at a nearly linear rate following a short lag (probably due to experimental manipulations of the root tissues), and there was little, if any, response to the addition of glucose (Fig. 5). A wide variation among replicates of root tissue samples was also observed. In addition, considerable nitrogenase activity (1.4 x 103 to 11.0 x 103 nmol of C2H4 per g [dry weight] per h) was associated with intact submerged root systems. Although these plants were incubated in light and dark conditions, it is unclear which resulted in the

3*0*-LAGUNCULARIA *-RHIZOPHORA A a v-AVICENNIA I

co U. 2.0

NL5w -j 0

O GLUCOSE

0.5 0 0

24

48 72 HOURS

96

FIG. 5. Nitrogen fixation (C2H5) associated with washed, excised mangrove roots from the Whiskey Stump Key collection site. Note the shortened lag period (samples averaged 2.5 to 5.0 nmol of C2H4/g [wet weight] per h at 8.5 h) and the relative insensitivity to glucose amendment. (U) Roots of L. racemosa; (@) roots of R. mangle; (A) roots of A. germinans.

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ZUBERER AND SILVER

greatest activity. With the exception of one rep-

licate among the series assayed in the light, there was little difference between the means of the two series. In situ determinations of nitrogenase activity. Results of in situ studies using the apparatus in Fig. 2 but lacking the central cylinder indicated that low rates of nitrogenase activity (99 to 659 ,ug of N/m2 per day) occurred within the isolated sediment cores. Enclosed cores amended with 2 ml of 1 M glucose after 48 h of incubation time showed a response to amendment quite similar to that of cores incubated in the laboratory. Further, this response was uniform, and there was good agreement among replicates of different sediment types (Table 3). Rates of acetylene reduction averaged 2.5 to 8 times higher in cores containing the inner cylinder. Thus, the inner cylinder provided a means of acetylene either reaching greater depths within the core or of attaining higher concentrations in zones where its reduction occurred most actively. Rates obtained with this technique were equivalent to 1.2 to 8.8 mg of N/m2 per day. Microbial populations. Microbial counts for selected physiological groups of bacteria are summarized in Table 4. Aerobic and anaerobic (probably including facultative anaerobes) counts are not appreciably different from one another and were approximately 1,000 times more numerous than facultative N2-fixing heterotrophs. A substantial population of N2-fixing, sulfate-reducing bacteria was present in all of the sediments. N2 fixation by this population was confirmed by subjecting tubes from the highest positive dilution of the most-probable-

number series to acetylene reduction. These cultures contained a preponderance of highly motile vibrios, presumably members of the genus Desulfovibrio. Purple photosynthetic bacteria were observed at the sediment surface in the littoral zone on several occasions. Although no attempts were made to enumerate this group, cultures were obtained using a nitrogen-free modification of Larsen medium (18). The dominant organism obtained resembled members of the genus Chromatium, being large oval cells with definite intracellular sulfur granules. Spiral-shaped cells were also observed in some N-free cultures. Cultures overwhelmingly dominated by the Chromatium-like organisms gave positive acetylene reduction tests. A bacterium capable of reducing C2H2 was obtained from roots of L. racemosa. The isolate TABLE 3. Nitrogen fixation (C2H;) associated with intact cores incubated in situ and amended with 2 ml of 1 M glucose at 48 h Tim 'MOI Of C2H4/core Time Sample

(h)

A

B

C

Anoxic sulfide mud

0

0.0

0.0

0.0

24

48b 72

Sandy

sedi-

0

Mean'

0.071 0.018 0.018 0.035 ± 0.030 0.178 0.039 0.041 0.086 ± 0.079 2.030 2.420 1.740 2.060 ± 0.266 0.0

0.0

0.0

ment 0.015 0.018 0.014 0.016 ± 0.002 0.027 0.032 0.031 0.030 ± 0.002 72 0.974 0.443 0.744 0.720 ± 0.266 aMean of three replicates ± one standard deviation. 24

486

'Glucose added.

TABLE 4. Counts of selected bacterial populations from plant-associated and plant-free mangrove sediments

Sediment type

Organisms enumerated used and medium

Cell no./g (wet wt) 7/23/74

Rhizophora associated sediment

Aerobic heterotrophse

8/1/74 2.6 x 10" 8.6 x 105 2.7 x 103

Anaerobic heterotrophsa N2-fixing heterotrophsb 2.9 x 103 Sulfate-reducing bacteriac 9.3 x lO Plant-free sediment N2-fixing heterotrophs 2.2 x 103 4.4 x 103 Sulfate-reducing bacteria 4.3 x 103 Avicennia associated sediment 5.0 x 103 8.2 x 103 N2-fixing heterotrophs Sulfate-reducing bacteria 9.3 x 105 Plant-free sediment N2-fixing heterotrophs 8.9 x 103 4.4 x 103 Sulfate-reducing bacteria 9.3 x 105 Habitat water N2-fixing heterotrophs 10.0-20.0 10.0-20.0 Sulfate-reducing bacteria 460.0 a These counts were intended only to indicate differences in the sizes of the populations appearing on plates incubated in air and under 100% N2. Under these conditions, both series could be expected to contain facultative organisms. b c

Plate count on N-free 50% sea water medium Most-probable-number counts in N-free sea water lactate medium

VOL. 35, 1978

was transferred repeatedly on a nitrogen-free medium containing fructose and glucose with trace quantities of yeast extract. On this nitrogen-free medium, small (1 to 2 mm), flat, white colonies were observed after 48 h of incubation under 100% nitrogen or in air. Young cultures (18 h) consisted of gram-negative, highly motile, small, slightly curved rods. Older cultures took on a more curved to spiral appearance and showed the presence of inclusions within the cells. Electron microscopical examination of negatively stained 18-h cultures indicated that the bacterium was a small curved rod with a single polar flagellum. (Details of these observations will be presented elsewhere.)

DISCUSSION Nitrogenase activity (C2H2) was associated with many different components of the mangrove ecosystem. These included: sediments, mangrove root systems, mangrove leaf litter, and litter from macroalgae and sea grasses, as well as low activity associated with fresh, healthy mangrove leaves (data not presented here). The waterlogged anoxic sediments of the mangrove community provided favorable conditions for several groups of diazotrophic bacteria. Sediment activities with few exceptions were low prior to the addition of various carbon sources. Slightly elevated rates exhibited by plant-associated sediments were probably due to the presence of plant-derived organic matter (i.e., exudates, sloughed cell debris, root fragments) within the cores. The marked response to substrate addition is indicative that nitrogen fixation in the mangrove sediment system is subject to energy limitation as it is in salt marshes (7, 8). The relatively long lag periods observed (12 to 24 h) following carbon amendment suggest that the enhanced activity is the result of population increases and/or nitrogenase synthesis, not the result of an immediate increase in the supply of adenosine 5'-triphosphate necessary to "drive" preformed nitrogenase.

Established mangrove vegetation serves as an excellent trap for detritus and litter materials transported by tidal action. The high rates of nitrogenase activity associated with leaf litter (primarily of red mangroves) in various stages of decomposition indicated that it serves as an energy source for nitrogen fixation. A recent report by Gotto and Taylor (6) is in agreement with our earlier unpublished findings. Further studies indicated that decomposing fragments of Ulva spp. and sea grass litter (Thalassia testudinum, Syringodium filiforme, and Dtplanthera wrightii) also served as energy sources for diazotrophic bacteria. It is not known, however,


573

whether the diazotrophs utilize these materials directly as energy sources or cross-feed on metabolites released by non-nitrogen-fixing components of the degradative microflora. The occurrence of nitrogen fixation associated with healthy growing sea grasses is well established (2, 15, E. J. Babiarz, Jr., M.S. thesis, University of South Florida, Tampa, Fla., 1976). In situ determinations of nitrogenase activity in intact, isolated sediment cores indicated low but consistent rates. A response to glucose amendment in field-incubated cores confirmed that nitrogenase activity is energy limited under natural conditions as well as in the laboratory. Cores penetrated with a perforated aluminum cylinder gave rise to ethylene production 2.5 to 8 times greater than cores lacking the cylinder. Apparently acetylene penetrates to greater depths, reaching more "active sites," or attains greater concentrations in the areas where the bacteria are located. With another apparatus (not shown here) we were able to demonstrate that in cores lacking the inner cylinder, C2H2 penetrated to a maximum depth of about 4.5 cm after 48 h. In cores penetrated by the cylinder, C2H2 was detected (although in reduced concentrations) at depths approximating 20 cm. It was also possible to monitor methanogenesis in the sediment cores using this method and, like nitrogen fixation, greater rates of methanogenesis were observed in cores containing the inner cylinder. This system provided an improved method for conducting studies where physiological processes may be limited by the diffusion of experimental gaseous substrates within the core. It is to be emphasized, however, that even with the central cylinder, acetylene reduction could be limited at lower depths within the core due to a failure to attain substrate concentrations sufficient to saturate nitrogenase. Although it is also possible that the central cylinder may give rise to overestimations of rates due to an artificially increased access to the experimental substrate (C2H2), N2 is generally present in concentrations near that of overlying water in marine sediments (16). Further studies along these lines should prove rewarding in that valid in situ determinations of various microbial processes (particularly aspects of the nitrogen cycle) are essential to prepare reliable nutrient budgets for ecosystems. Data obtained using excised, washed root systems and roots of intact plants demonstrated the presence of an effective diazotrophic rhizoplane microflora. The lack of an appreciable response to glucose addition suggests that the bacteria are well suited to growth and nitrogen fixation utilizing root exudates and/or sloughed cell debris. The fact that samples incubated aerobically

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and anaerobically were not greatly different suggests again that the diazotrophs were facultatively anaerobic or favored a greatly reduced P02, as do Spirillum lipoferum (Azospirillum brasilense) (14) and Azotobacter paspali (5). It is difficult to arrive at a precise figure for the contribution made by biological N2 fixation to the overall nitrogen budget of the mangrove community. Results of this study indicate that the process is rather ubiquitous within these habitats and that it may contribute significantly to the well-being of the community. Ofparticular importance are the root-associated diazotrophs. Their contribution of combined nitrogen for plant growth in nutrient-deficient areas may be highly significant. There is some evidence that the rates of root-associated N2 fixation observed in this study are sufficient to supply much of the nitrogen requirement for plant growth in South Florida mangrove communities. Taking into consideration the percent nitrogen in mangrove tissues (1.5 to 2.0% N) (13) and a mean tree growth rate of 1.74 g/m2 per day in Florida mangroves (11), approximately 26 mg of N/m2 per day is required. Sediment-associated nitrogen fixation, as estimated from some in situ determinations, could supply about 30% of the requisite nitrogen, and root-associated activity could supply substantially more. The intertidal nature of mangrove vegetation subjects it to a variety of forms of stress. Alternate submergence and exposure and rooting in predominantly anoxic saline sediments are but a few of these. It is possible that these stresses bring about or maintain changes at the root surface or in the pattern of exudation which predispose them to colonization by nitrogen-fixing bacteria, thereby establishing an associativesymbiosis between these intertidal halophytes and diazotrophic bacteria. It is not known whether there is any species specifically selected by any of the three mangrove species, but roots from L. racemosa consistently exhibited the highest rates of nitrogen fixation. A systematic investigation of diazotrophic bacteria associated with each of the three mangrove species would appear worthwhile and is currently under investigation. The results of these studies and those of other workers indicate that N2 fixation in mangrove communities is quite widespread, and this study indicates that the level of activity is of significance to the well-being of the community, especially in areas subject to nitrogen limitation. The data indicate the presence of many different types of diazotrophic bacteria, only a few of which were considered within the scope of this study. More precise information as to the species

and numbers of diazotrophs in the diverse microhabitats of the mangrove ecosystem are needed. It is hoped that this study will provide some baseline information to other investigators working in this unique ecotone between marine and terrestrial systems. ACKNOWLEDGMENTS We wish to express our sincere thanks to Dieter Pukatzki (formerly of the Mathematics Department at the University of South Florida) and Peter W. Lyons of the Central Florida Regional Data Center for their invaluable assistance in designing and implementing the computerized data analysis and plotting used throughout these studies.

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