Nitrogen Fixation (Acetylene Reduction) in a Salt Marsh Amended with Sewage Sludge and Organic Carbon and Nitrogen Compounds1

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1977, p Copyright X) 1977 Americn Society for Microbiology Vol. 33, No. 4 Printed in U.S.A. Nitrogen Fixtion (Acetylene Reduction) in Slt Mrsh Amended with Sewge Sludge nd Orgnic Crbon nd Nitrogen Compounds1 ROGER B. HANSON2 The University of Georgi Mrine Institute, Spelo Islnd, Georgi Received for publiction 26 October 1976 Sesonl distribution of nitrogen fixtion by Sprtin lterniflor epiphytes nd in surfce nd soil smples ws investigted in Georgi slt mrsh which ws mended with sewge sludge or with glucose nd/or mmonium nitrte. There ws no significnt difference between the rtes of fixtion in the unmended nd sewge sludge plots. Additionl perturbtion experiments suggested tht nitrogen ddition indirectly stimultes nitrogen fixtion by enhncing Sprtin production nd root exudtion. Glucose dditions, on the other hnd, suppressed nitrogen fixtioxi on long-term bsis. It is suggested tht the microbil popultion in the soil out-competed the plnts for the vilble nitrogen nd in turn suppressed plnt production nd possibly root exudtion. A comprison of nitrogen fixtion in clipped nd unclipped Sprtin plots substntited the suggestion tht root exudtion probbly supports nitrogen fixtion. Fixtion in the clipped plots ws significntly lower (P < 0.05) thn the rtes in the unclipped plots. Mrshlnds on the est cost of the United Sttes represent nerly 600 thousnd hectres (22). They hve mny functions, one of which is providing nursery nd hbitt for mny migrtory nd indigenous fish popultions. The economic importnce of mrshes is quite obvious. Therefore, future usge of these lnds must not disrupt the function of the vrious trophic levels which exist in the ecosystem. Gosselink et l. (7) proposed using the mrshes s potentil nturl tertiry tretment plnt. However, studies of the effects of secondry sewge on mrsh production nd microbil ecosystem hve only recently been initited (see 3, 31, 32). The results from these studies indicte tht the growth of Sprtin lterniflor is stimulted. Other biologicl processes (e.g., nitrogen fixtion nd dentrifiction) re either stimulted or inhibited. To understnd the possible consequence of sewge sludge disposl on the slt mrsh metbolism, mny intricte nd connected biologicl nd chemicl processes require investigtion. In this pper the results of 12-month study on nitrogen fixtion in nturl nd sewge sludge-mended short S. lterniflor slt mrsh re exmined. I Contribution no. 333 of the University of Georgi Mrine Institute. 2 Present ddress: Skidwy Institute of Ocenogrphy, Svnnh, GA MATERIALS AND METHODS Sewge sludge sites. The study sites were in the Duplin River slt mrsh contiguous to Spelo Islnd, G. Four seprte blocks (100-M2 plots) were chosen in the short S. lterniflor mrsh for this study (see 4 nd 5 for description of the plnt zones round Spelo Islnd). Two of the blocks received ground-up dried sewge sludge twice monthly t rte of 200 g/m2, equivlent to 4.0 g of N/M2 (3). The sludge ws pplied by hnd. The other two control blocks were seprted from the sewge sludge plots by rod so s not to be influenced by the pplied sewge sludge. High-mrsh nutrient mendments. Four replicte blocks in the short Sprtin mrsh were divided into ten 0.1-nM2 plots. The plots were rndomized nd treted in 10 different wys. Sprtin in one-hlf of the plots in ech replicte ws clipped nd lwn edgings were pushed down into the mrsh to depth of 15 cm; the other hlf remined unclipped with no lwn edging. The lwn edgings were used to prevent ny lterl root movement into the experimentl plots from djcent zones. The clipped nd unclipped plots were then treted with: (i) glucose (50 g/m2 per month), (ii) mmonium nitrte (10 g/m2 per month), (iii) glucose plus mmonium nitrte, (iv) rhodmine WD, nd (v) no ddition. Beginning in Februry, the nutrients were pplied t rte of 250 ml (50 g of glucose/liter, 11.4 g of mmonium nitrte/liter) per 0.1 m2 once month for 5 months. The nutrients were injected into the plots (0 to 15 cm) with 50-ml syringe with l/hs-inch (1.6 mm) guge stinlesssteel tube (20 cm long). As control on nutrient injections, two plots (clipped nd unclipped) were 846

2 VOL. 33, 1977 injected with distilled wter. There ws no difference in slinities between the distilled wter controls nd the djcent mrsh t the end of the fifth month (Robert R. Christin, Drexel University, unpublished dt). The blocks were smpled in July, nd duplicte cores were tken from ech plot except the rhodmine plots. The 0- to 5-cm nd the 10- to 15- cm zones from ech core were cut in hlf verticlly nd plced in bottles for cetylene reduction ssy s described below. Cores from outside ech replicte were lso smpled nd run simultneously with the experimentl cores on ech smpling dy. Nitrogen fixtion: mrsh surfce. Rtes of fixtion were estimted in the short Sprtin mrsh by the cetylene reduction method (13). For in situ mesurement of surfce nitrogen fixtion, reltively low-volume (1,100 cc), lrge-surfce-re (0.043 i2) dome incubtor ws used. After clipping nd removing Sprtin plnts from m2 re of the mrsh, the incubtor ws pushed down into the soil. During the summer, two lyers of 1-mm mesh screen were plced over the incubtor to prevent temperture increse inside the incubtor. Tempertures never incresed by more thn 5 C. Acetylene nd ethne (used s n internl stndrd) were dded, yielding n tmosphere of 15% cetylene nd 0.1% ethne. After n equilibrium period of 45 to 60 min, three gs smples were removed for nlysis every hour for 4 h. Concentrtions of ethylene nd ethne were nlyzed by use of flme ioniztion detector gs chromtogrph with Porpk N column. The ethylene concentrtion ws normlized to the ethne in the incubtor. In control experiments, in situ ethylene nd ethne production were not found in the bsence of dded cetylene. After ech run, surfce sediment (0 to 1 cm) ws removed from beneth the dome incubtors nd exmined for totl crbon nd nitrogen. Sediment smples were dried t 65 C, ground in mortr nd pestle, sieved through 0.5-mm mesh screen, nd dried to constnt weight. Soil crbon content ws TABLE 1. AMENDMENT EFFECTS ON N2 FIXATION 847 nlyzed with Colemn model 29 nitrogen nlyzer. The percent crbon nd nitrogen vlues were used to clculte the C-N rtio (wt/wt). Soil depth profile. Three intct cores 2.6 cm in dimeter were tken to 25 cm. Slices 2 cm thick from selected depths were plced in smll incubtion chmbers with n internl gs spce of 50 cc. No disturbnce of core geometry ws cused by this procedure. Acetylene nd ethne were dded to yield the concentrtions described bove, nd the chmbers were incubted in the drk t in situ tempertures. Gs smples were nlyzed for ethylene nd ethne t 4- to 8-h intervls for 30 h. Ethylene production generlly becme liner fter n initil period of 10 h nd remined liner up to 36 h. Control jrs (sediment plus ethne) hd only trces of ethylene. Sediment cores were nlyzed for nitrogen s described bove. Sprtin plnts. Nitrogen fixtion by epiphytes on eril Sprtin (leves nd stems) ws determined on the culms clipped from the 0.05-M2 plot used for the surfce incubtor. The plnt mteril, both live nd ded, ws plced in wide-mouth jrs. The sme gs concentrtions (percentge of internl gs volume) were used s for the soil smples. The jrs were incubted under full sunlight in tube of wter (25 to 30 C). Gs smples were nlyzed every hour for 4 h, nd ethylene production ws clculted from the liner portion of the curve. In the control jrs without dded cetylene, plnts produced no ethylene over the incubtion period. RESULTS Mrsh surfce fixtion. Monthly rtes of nitrogen fixtion were mesured for the slt mrsh soil surfce (Tble 1). The results indicte temporl vritions, with low in situ rtes (1 to 10,umol of C2H4/m2 per h) during the winter nd high rtes (10 to mol/m2 per h) during the summer. There ws no significnt In situ nitrogen fixtion rtes (X + stndrd error) in control nd sewge sludge plots in the high mrsh, Spelo Islnd, G. Crbon nd nitrogen Surfce nitrogen fixtion (men % dry wt) Month in (,umol of C2H4/m2 per h) 1975 Control Sewge sludge Control Sewge sludge C N C-N C N C-N Jnury (3) 3.51 (2) NDb ND Februry (2) 7.37 (2) : :1 Mrch (4) (4) : :1 April (2) 12.7 (2) : :1 My (2) 98.2 (2) : :1 June (2) 15.4 (2) : :1 July... ND ND ND ND ND ND August (4) 12.2 (2) : :1 September (4) 11.8 ± 1.61 (4) : :1 October ± (4) (4) ND ND ND ND November (4) ± (4) : :1 December (4) 2.25 ± 0.78 (4) : :1 Crbon nd nitrogen vlues re mens of duplictes, nd the C-N rtios re bsed on dry weights. Vlues in prentheses re replicte numbers. b Not determined.

3 848 HAKNSON AAPPL. ENVIRON. MICROBIOL. difference (P > 0.05) between the rtes mesured in the sewge sludge plots nd the control plots. The integrted nnul rte for the sewge sludge plots ws slightly higher (5.64 mmol/m2 per h), but not significntly higher (P > 0.05) thn the control plots (4.09 mmol/m2 per h). The crbon nd nitrogen content for the surfce soil in the control plots rnged from 3.74 to 5.41% nd 0.28 to 0.73%, respectively (Tble 1), with men C-N (wt/wt) rtio of 10.7:1 (rnge, 6.6:1 to 13.7:1). On the other hnd, crbon nd nitrogen content for the surfce soil in the sewge sludge plots rnged from 6.93 to 11.5% nd 0.63 to 1.23%, respectively, with men C-N rtio of 9.2:1 (rnge, 6.6:1 to 11.4:1). Sprtin epiphytes. Epiphytic nitrogen fixtion in the sewge sludge plots nd the control plots ws mesured from Mrch to December 1975 (Tble 2). The results indicte tht high TABLE 2. Nitrogen fixtion ssocited with highmrsh Sprtin lterniflor Epiphytic nitrogen fixtion Month in (Amol of C2H4/m2 per h) 1975 Control plots Sewge sludge plots Jnurẏ... NDb ND Februry... ND ND Mrch (4) (4) April (2) 7.93 ± 2.44 (4) My (2) ND June (2) 1.83 (2) July... ND ND August (2) 20.5 (2) September (2) 16.1 (2) October ± 2.81 (4) ± (3) November ± 0.81 (4) ± (4) December ± 2.06 (4) 4.99 ± 1.25 (4) The men rte ± the stndrd error is given, with the number of replictes in prentheses. b Not determined. fixtion rtes occurred during the lter prt of the growing seson (August nd September) in both res. As ws found for the surfce fixtion, there ws no significnt (P > 0.05) difference between the rtes mesured in the control nd sewge sludge plots. The integrted nnul rtes for the control nd sewge sludge plots were 2.13 nd 2.32 mmol of C2H4/m2 per h, respectively, bout one-hlf of the surfce vlues. Soil depth profile. Subsurfce nitrogen fixtion (0 to 21 cm) ws mesured over 7-month period (Tble 3). The rtes were integrted over depth, nd the estimted monthly rtes were used to clculte the nnul rtes. Vlues for the control nd sewge sludge plots were 228 nd 252 mmol of C2H,/m2 per h, respectively. Agin, there ws no significnt difference between tretments. The distribution of nitrogen fixtion over depth ws investigted. The monthly rtes were normlized on grm (dry weight) per hour nd milligrm of N per hour bsis, nd the men monthly rtes were plotted long with the men monthly percent N in the soil profile (Fig. l nd b). The results clerly indicte definite zone of ctivity t 5 to 7 cm. Below 5 to 7 cm the ctivity decresed rpidly. The distribution of ctivity pproximtes the root density distribution in the short Sprtin mrsh (8). The verge coefficient of vrition for the normlized dt (grms, dry weight) ws 92 ± 20% (stndrd devition), which reflects both sesonl vrition nd heterogeneity in the system. Two different ctivity ptterns were found for the control (Fig. l) nd sewge sludge (Fig. lb) plots when the rtes were normlized on milligrm of N bsis. Significntly more (P < 0.05) nitrogen ws fixed t 5 to 7 cm in the sewge sludge plots thn in the control plots. TABLE 3. Nitrogen fixtion soil depth profile in the high mrsh Nitrogen fixtion Tretment Depth (jamol of C2H4/m2 per h) (cm) September October November December Jnury Mrch Control plots ± ± t ± ± t ± Integrted , , Sewge sludge plots ± ± ± ± ± ± 1.58 Integrted , , The vlues re X + stndrd error.

4 VOL. 33, 1977 IL E F NITROGEN FIXATION 1o o I. \ MEAN % NITROGEN NITROGEN FIXATION lb / MEAN % NITROGEN FIG. 1. Men monthly nitrogen fixtion rte versus depth in the control () nd in the sewge sludge plots (b). The men rtes were normlized to nnomoles of C2H4 per grm per hour () nd nnomoles of C2H4 per milligrm ofn per hour (A) nd plotted with the men monthly percent N (0). The results suggest stimultion of nitrogen fixtion by root exudtion (see Discussion). In the surfce lyer the totl nitrogen content ws two to three times higher in the sewge sludge plots thn in the control plots (Fig. l nd b, respectively). The nitrogen content below 5 cm ws similr in both res. High-mrsh nutrient mendment. In ddition to the high-mrsh sewge sludge perturbtion experiments, severl Sprtin plots were either clipped or left unclipped nd the soil ws enriched with glucose nd/or mmonium nitrte. As shown in Fig. 2, nitrogen fixtion ws significntly (P < 0.05) influenced in the present Sprtin culms. In soil plots which were clipped (Fig. 2 nd 2b), fixtion ws significntly (P < 0.05) lower thn in plots which were not clipped (Fig. 2c nd 2d). There ws no significnt (P > 0.05) difference between tretments within either the clipped or the unclipped plots (Fig. 2). However, in the unclipped 0- to 5-cm zone (Fig. 2), mmonium nitrte ppered to stimulte nitrogen fixtion, wheres fixtion ws low in the plots enriched with glucose nd glucose plus mmonium nitrte (Fig. 2). On the other hnd, nitrogen fixtion within the 10- to 15-cm zone ws significntly greter (P < 0.05) in the mmonium nitrte-treted plot thn in the other plots (Fig. 2b) Ṅitrogen fixtion in the outside control ws AMENDMENT EFFECTS ON N2 FIXATION 849 lower only in the 0- to 5-cm zone (Fig. 2). This my hve been due to the injection holes which were produced when the vrious nutrient solutions were dded to the soil. In ny cse, the clipped nd unclipped nutrient tretments re directly comprble. The men coefficient of vrition for the entire dt set ws % (stndrd devition), gin indicting heterogeneity. Light nd drk nitrogen fixtion. Diel surfce nitrogen fixtion proceeded t reltively constnt rte except in res where heterocystic nitrogen-fixing blue-green lge were present (Tble 4). In those res, drk fixtion ws less thn 5% of the light fixtion. However, drk fixtion ws 70 to 100% of the light fixtion in the bsence of heterocystic lge. In Februry, Anben ptches covered extensive res in one mrsh, nd in Mrch Clothrix crust lyered mssive re in sndy mrsh. However, the reltively high fixtion generlly found ws not lwys due to heterocystic lge, s ws the cse in July. The dominnt lg ws n Oscilltori. It ws not possible to determine whether it ws fixing nitrogen or ws n ssocited bcteril popultion. The crbon nd nitrogen content of the upper 0 to 5 mm ws noted to be high when heterocystic nitrogen-fixing blue-green lge were present (Tble 4). DISCUSSION S. lterniflor hs been found to be nitrogen limited (2, 6, 19, 29, 30, 32). On the other hnd, + Unclipped Clipped 2. * c I 3E b O fi _ t, O/SC RC Glu NN GNN I 2d I RC. FhF]FIl -NN RC Glu NN GNN FIG. 2. Nitrogen fixtion rtes in unclipped (2 nd 2b) nd clipped (2c nd 2d) Sprtin lterniflor plots in two depth zones: 0 to 5 cm (2 nd 2c) nd 10 to 15 cm (2b nd 2d). Histogrm brs represent the men rtes ofeight cores from four replicted plots. Verticl brs re ± 1 stndrd error of the men. O/SC = Outside control; RC = replicte control; Glu = glucose; NN = mmonium nitrte; nd GNN = glucose nd mmonium nitrte.

5 850 HANSON TABLE 4. Light nd drk nitrogen fixtion in vrious mrshes contiguous to Spelo Islnd, G. Crbon nd nitrogen Blue-green l- C Surfce nitrogen (men % dry wt) ge present fixtion C N C-N Jnury Airport Mrsh None Dylight 1.62 NDb ND Night 1.64 (100) Februry Lughing Gull Anben Dylight :1 Mrsh Drk 48.2 (5) Mrch Cbrett Mrsh Clothrix Dylight :1 Drk 12.7 (2) Subsurfce :1 July Cbrett Mrsh Oscilltori Dylight :1 Drk 57.4 (72) Expressed s micromoles of C2H4 per squre meter per hour. Vlues in prentheses re the percentge of dylight fixtion rtes. phosphorus hs been shown not to be limiting production s consequence of the phosphorus reserves in mrsh soils (21). Although Sprtin nd benthic lge support the detritl food chin, the lrge microbil community hs been shown to be operting below its metbolic potentil (i.e., crbon limited) (9, 10, 35; R. R. Christin, Ph.D. thesis, University of Georgi, Athens). Vliel nd Tel (30), Vliel et l. (32), nd Chlmers et l. (3) found tht sewge sludge increses Sprtin nd lgl production s result of the reltively high nitrogen content of sewge (2 to 3%). On the other hnd, Vn Rlte et l. (33) reported tht nitrogen fixtion by blue-green lge in Msschusetts ws inhibited by high dosges (25.2 g/m2 per week) but not low dosges (8.4 g/m2 per week) offertilizer (10% N). Although they did not investigte rhizosphere nitrogen fixtion, the surfce fixtion dt from the low-dose plots gree with the nitrogen fixtion dt reported here. To investigte the enhncement of nitrogen fixtion by sewge sludge in the Georgi mrsh, reltively simple field experiment ws designed (see Fig. 2). In generl, the results indicte tht the energy source for nitrogen fixtion in the soil ws most likely Sprtin crbon: 1. Nitrogen fixtion ws significntly higher in the unclipped thn in the clipped soil plots. Thus, the removl ofsprtin shoots prevented ny crbon input vi the roots, other thn possibly the diffusion of surfce lgl crbon into the rhizosphere. In system similr to slt mrshes, it hs been determined tht nonsymbiotic nitrogen fixtion ws stimulted in the rhizosphere of rice plnts (17, 24, 36). 2. It ws found tht the monthly nitrogen APPL. ENVIRON. MICROBIOL. dditions significntly incresed plnt production over the nonmended plots (Christin, unpublished dt), substntiting other investigtions (2, 6, 19, 29, 30, 32). The incresed nitrogen fixtion ctivity in the nitrogenmended Sprtin plots ws pprently relted to incresed Sprtin productivity nd root exudtion. 3. Nitrogen fixtion ws enhnced in the unclipped mmonium nitrte-enriched plots. It is well known tht inorgnic nitrogen (> 25 mg/ liter hs n immedite inhibitory effect on bcteril nitrogen fixtion (10, 16, 18, 20). The immedite inhibitory effect of combined nitrogen on nitrogen fixtion hs no doubt decresed, vi chemicl or biologicl trnsformtions, between nitrogen mendments nd smpling of the mrsh for nitrogen fixtion ctivity. Therefore, the increse in Sprtin productivity from the nitrogen mendment outweighed the inhibitory effect of inorgnic nitrogen on nitrogen fixtion in the field. 4. It ws found tht ddition of glucose to the plots suppressed nitrogen fixtion in the soil. However, nitrogen fixtion in soil slurries ws immeditely stimulted by number of different crbohydrtes (10). This cn be explined if the glucose ws consumed long with the vilble soil nitrogen by the microbil community, thereby reducing the utilizble Sprtin nitrogen pools in the soil. As result, the plnts were further nitrogen stressed. Plnts in the glucose-mended plots ppered yellow, wheres plnts in the nitrogen-mended plots were green. Therefore, Sprtin production supported the energy requirements of heterotrophic nitrogen fixtion in Georgi slt mrsh soils. Bcteril nd blue-green lgl nitrogen fix-

6 VOL. 33, 1977 tion hs been reported in numerous other slt mrshes nd esturies (1, 8, 11, 12, 14, 15, 18, 26, 28, 33, 34). As ws reported here, most of these studies hve shown sesonl trends: highest rtes in lte spring to erly fll nd lowest rtes during the winter months, with the exception of the Rhodes River estury where fixtion ws out of phse with the period of rpid plnt growth (18). In ddition to temporl vritions, sptil vrition of nitrogen fixtion in soils nd sediments hs suggested tht most of the energy nd crbon compounds enter the system from the surfce; tht is, nitrogen fixtion ctivity decreses with depth (1, 14, 34). But, in the short Sprtin mrshes contiguous to Spelo Islnd, the highest ctivity hs been found primrily in the 5- to 7-cm depth zone. Mximl short Sprtin root density hs lso been reported between 0 nd 10 cm (5). These results suggest rhizosphere interctions. In ddition to the nitrogense ctivity found on nd in the mrsh soils, it hs lso been reported on mrsh plnt leves nd stems (8), s hs been found in this study. Plnt exudtes, which re collected between the Sprtin lef sheths nd stems, contin orgnic-rich metbolites to mintin heterotrophic nitrogen fixtion (25). Mny nonsymbiotic nitrogen fixers hve been reported, nd they exist nerly everywhere (27). Between Sprtin lef nd stem interfces in Georgi, photosynthetic bcteri hve been seen microscopiclly during the ltter prt of the growing seson (September nd October). Those tht grew in nitrogen-free enrichment medi resembled Rhodospeudomons nd Chloropseudomons species. These photosynthetic bcteri were lso present on the soil surfce long with nitrogen-fixing Clothrix nd Anben species. Clothrix nd Anben were lso present on the stnding ded Sprtin stems nd leves. Below the surfce, cetylene-reducing Desulfovibrio nd Clostridium species were enriched from soil smples in the high mrsh. Considerble effort hs been mde to estimte the reltive importnce of vrious nitrogen fluxes nd nitrogen stnding stocks in the mrshes of Spelo Islnd (9). As yet there re mny fluxes still unccountble (e.g., tidl nitrogen flux, nitrifiction, mmonifiction, mmoni voltiliztion, etc.). Mny of those fluxes nd stnding stocks mesured re only estimtes, but they re the best estimtes vilble. For exmple, the cetylene reduction method is the most sensitive nd inexpensive technique vilble for mesuring nitrogen fixtion potentil in vrious diverse ecosystems. However, 15N2 trcer experiments re required AMENDMENT EFFECTS ON N2 FIXATION 851 TABLE 5. Estimted nnul nitrogen fixtion in control nd sewge sludge-mended plots (experimentl), Spelo Islnd, G., 1975 Annul N2 fixtion (g of NS2m2) Smple Smple Control Coto plot lt Experimentl plot 10 h5 24 h 10 h 24 h Soil surfce Sprtin epiphytes Soil profile (0-22 cm) Totl Bsed on 3:1 (C2H4-N2) conversion fctor. b Fixtion period. for ccurte conversion of ethylene units to nitrogen units, since conversion fctors hve been shown to rnge from the theoreticl rtio of 3:1 to 18:1 (23). Nevertheless, extremely good estimtes cn be derived from the existing ethylene production dt (Eldor A. Pul, University of Ssktchewn, personl communiction). Therefore, rtes of ethylene production were converted to nnul potentil nitrogen fixtion rtes bsed on 3:1 conversion fctor (13) (Tble 5). The yerly nitrogen fixtion rte of 20 to 50 g of N2/m2 could significntly support utotrophic production (plnt nd lge) in the mrsh. Hines et l. (9) reported tht the nitrogen flux through mrsh utotrophs my be s high s 30 to 40 g of N/M2 per yer. In conclusion, sewge sludge pprently enhnces rhizosphere nitrogen fixtion in the soil by stimulting Sprtin production in the slt mrshes of Georgi. However, its effect on other processes my be inhibitory. For exmple, dentrifiction in Georgi mrshes ws inhibited by sewge sludge (3). ACKNOWLEDGMENTS This study ws supported by Ntionl Science Foundtion grnts DES A02, DES , nd GA 35806, by Office of Wter Resources reserch grnt A-057-GA, nd by the Spelo Islnd Reserch Foundtion, Spelo Islnd, G. Thnks re due to J. Snyder nd T. St. Onge for vluble technicl ssistnce. I would like to express my thnks to Berry Sherr, Evelyn Hines, Alice Chlmers, nd Robert Christin for discussions of their reserch in relted res. Specil thnks re due -to R. R. Christin nd W. J. Wiebe for vluble suggestions nd criticisms relted to the mnuscript. LITERATURE CITED 1. Brooks, R. H., Jr., P. L. Brezonik, H. D. Putnm, nd M. A. Keirn Nitrogen fixtion in n esturine environment: the Wccssss on the Florid Gulf Cost. Limnol. Ocenogr. 16:

7 852 HANSON 2. Broome, S. W., W. W. Woodhouse, Jr., nd E. D. Senec The reltionship of minerl nutrients to growth of Sprtin lterniflor in North Crolin. II. The effects of N, P, nd Fe fertilizers. Soil Sci. Soc. Am. Proc. 39: Chlmers, A. G., E. B. Hines, nd B. F. Sherr Cpcity of Sprtin slt mrsh to ssimilte nitrogen from secondrily treted sewge. Technicl Completion Report USDI/OWRT, Project no. A-057-GA. Environmentl Resources Center, Georgi Institute of Technology, Atlnt. 4. Christin, R. R., K. Bncroft, nd W. J. Wiebe Distribution of microbil denosine triphosphte in slt mrsh sediment t Spelo Islnd, Georgi. Soil Sci. 119: Gllgher, J. L Smpling mcro-orgnic mtter profiles in slt mrsh plnt root zones. Soil Sci. Soc. Am. Proc. 38: Gllgher, J. L Effect of n mmonium nitrte pulse on growth nd elementl composition of nturl stnds of Sprtin lterniflor nd Juncus roemerinus. Am. J. Bot. 62: Gosselink, J. C., E. P. Odum, nd R. M. Pope The vlue of the tidl mrsh. Publiction no. 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