Patterns of nitrogenase activity (acetylene reduction) associated with standing, decaying shoots of Spartina alterniflora

Estuarine, Coastal and Shelf Science (1992) 35, 127-140

Patterns of Nitrogenase Activity (Acetylene Reduction) Associated with Standing, Decaying Shoots of Spartina alterniflora

S. Y. N e w e l l ~, C. S. H o p k i n s o n b a n d L. A. S c o t t a ~Marine Institute, University of Georgia, Sapelo lsland, GA 31327, U.S.A., bThe Ecosystem Center, Marine Biological Laboratory, Woods Hole, M A 02543, U.S.A. Received 16 August 1991 and in revisedform I4 January 1991

Keywords: nitrogen fixation; acetylene reduction; saltmarsh; Spartina alterniflora; decomposition; clay deposition Samples of standing-dead cordgrass have been reported to yield consistently high rates ofnitrogenase activity (as acetylene [C2H2] reduction) per unit mass of sample (up to about 2 lamol ethylene [C2H4]. g - ~dry mass. day- l). We measured rates of C2H ", reduction over a range of conditions during the decomposition of standing cordgrass shoots. The degree to which standing, decaying leaves supported Cell ", reduction was correlated with the degree of coverage of the leaves b~¢ clay films (r 2= 0-41, P < 0'01). Leaves that had the heaviest films (ash contents >40°o) had rates of 102-104nmol C2H 4 p r o d u c e d . g -~ organic mass. day -l , whereas decaying leaves higher up the shoots with little or no clay coverage had rates < 5 x 101 nmol. g-l organic mass. day -I. Acetylene reduction was not limited to the clay layer on dead leaves; when living, green leaves and dead, decaying leaves bore equal clay coverages, the green leaves exhibited nitrogenase activity only 7°0 as high as that associated with the decaying leaves. One likely source of the high nitrogenase activity may be a fungal/bacterial consortium. T h e highest rates of C2H 2 reduction by the decaying cordgrass-leaf system (about 10-30~tmol C2H4.g -t organic mass. day-t) were equivalent to the highest rates reported for other decomposition systems, both natural (e.g. mangrove leaves) and anthropogenic (e.g. wheat straw plus selected N2-fixing inocula).

Introduction F i x a t i o n o f a t m o s p h e r i c d i n i t r o g e n b y d i a z o t r o p h s in s m o o t h - c o r d g r a s s (Spartina alterniflora Loisel.) s a l t m a r s h e s is a p o t e n t i a l l y i m p o r t a n t source o f n i t r o g e n to these e x c e p t i o n a l l y p r o d u c t i v e b u t n i t r o g e n l i m i t e d saline m e a d o w s ( C a s s e l m a n et al., 1981; H a n s o n , 1983). M a r s h N - f i x a t i o n m a y b e a factor c o n t r i b u t i n g to t h e o b s e r v e d n e t n i t r o g e n outflows (as d i s s o l v e d o r g a n i c N ) in tidal e x c h a n g e a n d d r a i n a g e (4"5g N . m -2 . y e a r - l : W h i t i n g et al., 1989; see also A z i z & N e d w e l l , 1986). H o w e v e r , in a r e c e n t review, H o w a r t h et al. (1988) a r g u e that d i n i t r o g e n fixation p r o b a b l y c o n s t i t u t e s a m i n o r i n p u t o f n i t r o g e n to m a r s h e c o s y s t e m s . T h i s a r g u m e n t is b a s e d on the fact t h a t l o n g i n c u b a t i o n s (>_ 24 h) c h a r a c t e r i s t i c a l l y e x h i b i t i n g lag phases were u s e d in o b t a i n i n g the 0272-7714/92/080127 + 14 $03.00/0

© 1992 Academic Press Limited

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higher rates reported (8-51 g N . m -2 . year-~); shorter incubations without lags gave much lower rates ( < 0.5-2 g N . m - 2 . year- 1). Rising fixation following lag phases could be a result of artificial conditions of laboratory incubation (Capone, 1988). The lower range of rates is consistent with calculations based on results of Whiting and Morris (1986), who used short-term, non-manipulative incubations (ca. 1.2 g N . m -2 . year-i). Definitive conclusions regarding the importance of dinitrogen fixation to marshecosystem nitrogen budgets must await clear resolution of the question of the validity of currently available nitrogen-fixation rates (as acetylene reduction) associated with the rhizosphere and root surfaces of smooth cordgrass. Three groups of investigators have reported that one type of cordgrass marsh sample consistently gave higher rates of dinitrogen fixation (as acetylene reduction) per mass of sample than did others: pieces of standing-dead shoots of cordgrass (Green & Edmisten, 1974; Casselman et aL, 1981; Thomson & Webb, 1984). At least for Casselman et al. (1981), these rates did not exhibit the suspicious lag phase seen in sediment incubations. Newell et al. (1989) reported that standing, decaying leaves of smooth cordgrass could exhibit a sharp upturn in nitrogen content and that this coincided with a sharp rise in clayfilm coverage of the leaves. The clay contained potentially nitrogen-fixing cyanobacteria. Deposition of saltmarsh clay (Frey & Basan, 1985) on cordgrass shoots during spring tides is a regularly occurring phenomenon in Georgia saltmarshes (Letzsch & Frey, 1980; Newell et al., 1988, and unpubl.). Clay films on leaves become more persistent as decomposition proceeds (Newell et al., 1989). In this paper we report the investigation of nitrogen fixation (as acetylene reduction) associated with a variety of types of decaying shoots of smooth cordgrass, with the objective of establishing whether the deposition of clay on the shoots enhances rates of nitrogenase activity. S u b s t r a t u m a n d sites

Leaves and stems of smooth cordgrass (Spartina alterniflora Loisel.) were collected from four saltmarsh sites adjacent to Sapelo Island (31°23'N; 8°17'W; site description: Wadsworth, 1980; Pomeroy & Wiegert, 1981; Hopkinson & Schubauer, 1984). The four sites varied in the relative content of short vs. intermediate and tall-form cordgrass, and presumably also frequency of tidal inundation. T h e sites were: (1) the Odum-Teal Boardwalk, South-End-Creek watershed; (2) Dike Road marsh (Newell et al., 1989), Doboy-Sound watershed; (3) Airport Marsh, Duplin-River watershed; (4) Post-OfficeCreek Marsh, Duplin-River watershed. Sites 1 and 2 are dominated by cordgrass shoots of intermediate height (ca. 0.5-1-0 m), Site 3 by short shoots ( < 0.5 m), and Site 4 by tall shoots (1.0-1.5 m). Materials and methods

We conducted eight experiments (Table 1), one with leaves tagged to indicate age from senescence and with the leaves rinsed to remove clay (Newell et al., 1989), and seven subsequent ones to test interactive effects of several factors with extent of clay loading. Tagged leaves, fall--winter

At Site 1, orange plastic tags were placed on senescent leaves of cordgrass in October 1988 as described by Newell et al. (1989); mean height of ligules of tagged leaves above the sediment was 18 cm (range 10-32 cm). Leaves were harvested at time zero and at 2, 4, 8,

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TABLE1. Summary of the acetylene-reductionexperiments"

Date October 88 April 89 May 89 June 89 June 89 July 89 July 89 July 89

Substratum

Rinsing

Marsh

Shoot

Experimental Focus

Lvs Lvs Lvs Lvs Lvs Lvs,Stm Lvs,Ftr Lvs,Stm

+ __+ + --

ID ID HD,ID,LD HD,ID HD,ID HD,ID HD,ID ID

IF IF SF,IF,TF TF TF IF,TF TF TF

Tagged series Low/high leaves Marsh variation Time course Clay removal Stem activity Clay alone Light/dark

~Abbreviations: Lvs, leaves; Stm, stems; Ftr, glass-fiber filter; HD, high density of drainage creeks (Site 4); ID, intermediate density (Sites 1 and 2); LD, low density (Site 3); SF, short form of cordgrass shoots; IF, intermediate height form; TF, tall form.

and 12 weeks after tagging. Leaves were rinsed in running tapwater with gentle rubbing for 0'5 min, to remove adherent clay and drained. A 15 cm length was cut from each leaf, beginning 3 cm distal to the ligule, and this was cut into three 5 cm sections. T h e three sections were placed in a plastic dish (three replicate dishes, nine sections total per sampling time) and 5 ml deionized water added. T h e dishes were covered and incubated at field temperature for 30 min. so that the leaf-microbial assemblage could adapt to watersaturated conditions. Leaf sections from each dish were drained and transferred to a 37 ml serum bottle for measurement of nitrogen fixation as indicated by acetylene reduction (Havelka et al., 1982). Fifty lal of deionized water was added to provide 100°o relative humidity, and the bottles sealed with serum stoppers. An 8 ml volume of headspace gas was withdrawn from each bottle and replaced with 7 ml acetylene (C2H2; freshly generated from calcium carbide) (see H a r d y et al., 1968; Patriquin, 1978; Roper, 1983). T h e bottles were incubated at 45 ° tilt from vertical to maximally expose the leaf sections to light (300 laE. m -z . s-1 photosynthetically available radiation [PAR]). Temperature of incubation was 20 °C (to find potential rates at one standard temperature), and duration was 29 h. T w o types of control bottles (4 replicates each) were prepared containing no sample or receiving no acetylene. At the end of the incubation period, headspace gas samples (250 ~tl) were withdrawn from the bottles and injected into a Varian 3400 gas chromatograph for measurement of ethylene concentrations. Chromatograph characteristics: injection port at 40 °C; column, Poropak N 80/100 mesh, 2 m x 3 mm, at 50 °C; carrier gas, He at 30 ml. min.-~; flameionization detection, at 100 °C; inboard data handling for internal peak integration. Calibration was performed by injecting air standards containing known concentrations of ethylene. Although carbon monoxide was not added to control bottles (for suppression of nitrogenase and detection of natural ethylene-production rates in the presence of acetylene: Hendrickson, 1990; see also Giller, 1987), the maximum concentration of ethylene present in acetylene-free controls after incubation was 0.2% of that in sample bottles. After gas measurements were completed, the serum bottles were opened and the leaves were dried in a microwave oven and ashed (450 °C, 4 h) for determination of organic and ash mass per sample (Newell et al., 1991). T h e statistical reference used in data analysis was Sokal and Rohlf (1981).

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Effect of rinsing on leaves, April T h r e e types of leaf were collected from intermediate-height shoots at Site 1: (1) highest standing-dead leaves, on fully dead shoots (note: leaves die in succession from bottom to top of shoot); (2) the most recently dead leaves, on living shoots; (3) the lowest dead leaf above the sediment on living shoots. A 5 cm section was cut from each leaf, between 3 and 8 cm distal to the ligule. H a l f of the sections were rinsed as described previously, and half were not rinsed, so that the clay film would be retained. All of the sections were soaked for 30min., half in seawater ( 2 0 g . 1-1 salinity), half in deionized water (simulating rainwater or tidal washing) in plastic bags under the incubator lights (300 laE. m -2 . s-1 PAR) at 25 °C. Six sections of each leaf type were placed together in 37 ml serum bottles (3 replicate bottles) for the acetylene-reduction assay (25 °C, 14 h).

Unrinsed leaves for three types of site × shoot, May T w o leaf types were collected at Sites 2, 3, and 4: (1) the lowest dead leaf on a living shoot, collapsed onto the sediment; (2) the second leaf up from the collapsed one. T h e s e two types of leaf were collected from short ( < 0 . 5 m), intermediate-height (0.5-1.0 m), and tall ( > 1.0 m) shoots at each site (exception: intermediate-height, sparsely distributed shoots were substituted at Site 4 for short shoots; short shoots were absent at Site 4). Leaves were soaked in saltwater before transfer to serum bottles (5 replicates) for the acetylene-reduction assay (25 °C, 16 h).

Effect of duration of incubation, June One leaf type was collected at Sites 2 and 4: the third dead leaf up on living, tall-form shoots. L e a f handling and incubation was as for the M a y samples, except that ethylene sampling was repeatedly performed, at approximately 3.5, 9.3, 13.6, and 21.0 h after acetylene injection. Rates of acetylene reduction were calculated based on incubation from time zero.

Effect of rinsing, June T h r e e leaf types were collected from tall-form shoots at Sites 2 and 4: (1) the lowest, dead, on-sediment leaf: (2) the second dead leaf up-shoot; (3) the fourth dead leaf up from the on-sediment leaf. L e a f handling and incubation were as for M a y samples, except that one-half of the leaves were rinsed in seawater to remove the clay film.

Wholly dead shoots, with clay, July Four types of shoot material were collected from the dominant height forms at Site 2 (intermediate height) and Site 4 (tall form): (1) standing-dead stems with no remaining leaf sheaths attached; (2) standing-dead stems with sheaths still attached; (3) lowermost dead leaves (mean height of ligule above sediment = 49 cm) (Site 4 only); (4) u p p e r m o s t dead leaves (mean height of ligule = 128 cm) (Site 4 only). Leaves were handled and incubated as for M a y samples. A 3 cm-long internode portion was taken from stems, at approximately 10 cm up from the sediment. Only one stem piece was used per incubation bottle (5 replicate bottles). This experiment was repeated in July with samples from Site 1, but 5 extra replicates were prepared for each type of sample. T h e s e extra replicates were entirely protected from light during soaking and incubation by wrapping the bottles with aluminium foil.

Patterns of nitrogenase activity

131

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40 o "o

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2. o

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2

4

6

8

I0

12

Time a f t e r ragging (weeks)

Figure 1. Rates of acetylene (C2Hz) reduction to ethylene (C2H4)per g organic mass (mean + 1 SD; n = 3; 29 h incubation) for rinsed, standing leaves of Spartina alterniflora that were tagged in place at the yellow-green, senescent stage. At the 2-week point, all leaves had turned brown. Ethylene production was not detectable at time zero. Plot excludes one outlier at week 2 (78 nmol CzH4 . g i organic mass. day *).

Clay film + organic substratum, July T w o types o f tall-form leaf sample, and one clay-only sample were collected at Sites 1 and 4: (1) m a t u r e green leaves with an obvious clay film; (2) standing-dead leaves (not t o u c h i n g sediment) with a clay fil~a; (3) the u p p e r m o s t m m of clay sediment, spread onto a tared glass-fiber strip ( W h a t m a n G F / F ) . Leaves were handled and incubated as for M a y samples. Glass-fiber strips were saturated with seawater rather than soaked, and incubated (clay-film toward light) as for leaves. F o r this experiment, acetylene reduction was calculated per g d r y clay, in addition to per g organic mass. Clay mass on leaves was taken as the ash mass (after 450 °C muffling) m i n u s the intrinsic leaf ash (from Newell et al., 1989).

Results

Tagged leaves By the second week after tagging, all the leaves had t u r n e d y e l l o w - b r o w n to brown. Little or no acetylene reduction ( < 5 n m o l ethylene p r o d u c e d , g - ~ organic m a s s . d a y - ~) was f o u n d over the first four weeks after tagging (Figure 1). O v e r the period 4-12 weeks after tagging, the m e a n acetylene-reduction rate rose to 43 n m o l C 2 H 4 . g-~ organic m a s s . d a y - L C o n t r a r y to expectation, no substantial clay film developed on these leaves ( m a x i m u m ash content o f 15%).

Effect of rinsing, April O n l y one o f the replicate sets o f leaf-microbial systems exhibited acetylene-reduction rates significantly greater than the acetylene-free control ( A N O V A , P < 0.001): unrinsed leaves f r o m low on living shoots, soaked in seawater ( 1 0 n m o l C 2 H 4 . g -~ organic m a s s . day-~). A s h content for these leaves averaged 2 8 % .

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o

o

u

eat

~,0-

m -o

50o

T

? tD

E -~0-

I

I

;ire 2

Sire 3

Site 4

i

Figure 2. Rates o f acetylene reduction to ethylene per g organic mass (mean + I SD; n = 5; 18 h incubation) for u n r i n s e d , dead, standing leaves of Spartina alterniflora positioned on or near the s e d i m e n t at three m a r s h sites. Left scale, Site 2 a n d 3; right scale, Site 4. F r o m left to right within each set o f six columns, types o f leaf represented are: (1) collapsed onto sediment, on s h o r t - f o r m shoots; (2) collapsed, intermediate shoots; (3) collapsed, tall shoots; (4) two leaves higher on shoots than the collapsed leaf, not on sediment, on s h o r t - f o r m shoots; (5) same as 4, b u t intermediate shoots; (6) same as 4~ b u t tall shoots. A t Site 4, sparsely distributed, intermediate-height shoots were s u b s t i t u t e d for short shoots. Letters o n c o l u m n s indicate m e a n values that were significantly (P < 0.05) different from one another ( A N O V A + m i n i m u m significant range).

Three types of marsh x three types of shoot, M a y , no rinsing At Sites 2 and 4, the highest mean rates of acetylene reduction were found for microbes of collapsed leaves attached to short or sparsely distributed intermediate-height shoots (about 4-30 Izmol C2H 4 . g - l organic m a s s . day-~) (Figure 2). At Site 3, tall-form leaves had higher rates (0-7-0-8 ~tmol C2H 4 . g-1 organic m a s s . day -1) than leaves on shorter shoots ( < 0 - 0 5 p m o l . d a y - l ) , whether collapsed on the sediment or not (Figure 2). Lowest rates were found for leaves not in contact with the sediment at all sites. Ranges of mean ash contents (%) for types of sample were: short (or sparse-intermediate), 21-59; intermediate (not sparse), 21-58; tall, 49-81; collapsed on sediment~ 28-81; not on sediment, 21-72; Site 2, 21-55; Site 3, 28-63; Site 4, 30-81. Time course L e a f samples f r o m both Sites 2 and 4 showed linearly increasing rates (0-9-2-1 l~mol C 2 H 4 " g - l organic m a s s . day-I of acetylene reduction during enclosure and incubation (Figure 3). The rates measured at 21 h of incubation were x 1"7 and × 3.2 the y-intercept (0-h incubation) rates (0-7-1.2 lxmol C2H 4 . g-1 organic m a s s . day-l). Mean percent ash for these samples was 59 (Site 2) and 78 (Site 4).

Patterns of nitrogenase activity

133

2.5

2.0

~

~o

o

g

o. . . . . . . - - 9 " "

U

0-5

I 5

0

I 10

1 15

I 20

Incubation time (hi

Figure 3. Rates of acetylene reduction to ethylene per g organic mass for dead standing leaves (no rinsing) of Spartina alternifloraas determined at different points during a 21 h incubation. Open circles, Site 2 (y = 0.04x + 1.23, r 2= 0.97, n = 12); closed circles, Site 4 (y =0.07x+0.66, r2 = 0.99, n = 12).

TABLE 2. Rates of acetylene reduction (t~mol C2H. • g- ~organic mass. day -~) to ethylene (mean+ 1 SD, n = 5 ; 18 h incubation) for dead standing, tall-form leaves of Spartina alterniflora. Mean percent ash is given for each type of sample as an indicator of clay-film coverage: Abbreviations: C = leaf collapsed onto sediment; 2 - U P = s e c o n d leaf up on shoot from collapsed leaf; 4-UP = fourth leaf up on shoot from collapsed leaf. Half of the leaves of each type were rinsed to partially remove the surface clay film Rinsed

Not rinsed

C

2-UP

4-UP

C

2-UP

4-UP

Rate (±SD) qb Ash

0.2 (0.1) 50

0.9 (0.5) 34

0.5 (0.4) 24

1.1 (1.0) 71

0.5 (0.5) 56

0.3 (0.2) 35

Rate (±SD) %Ash

0-7 (0.5) 48

1-7 (0'5) 39

Site4 ~ 2-6 (2.1) 29

12-0 (9.8) 78

4-8 (2.1) 68

4-0 (1.0) 61

Site 2~

~No significant differences among mean rates for Site 2 (ANOVA, P > 0'05); significant differences detected between rinsed and unrinsed rates for C and 2-UP at Site 4.

Effect of rinsing, June F o r l e a f s y s t e m s f r o m S i t e 2, t h e r e w a s n o s i g n i f i c a n t e f f e c t o f r i n s i n g o n a c e t y l e n e reduction rates, although ash contents were reduced by an average of 33% by rinsing ( T a b l e 2). R a t e s r a n g e d f r o m 0 - 2 - 1 - 1 pmaol C 2 H 4 . g - i o r g a n i c m a s s . d a y - 1 a t S i t e 2. A t S i t e 4, r i n s i n g r e d u c e d a s h c o n t e n t s b y a n a v e r a g e o f 45 % , a n d a c e t y l e n e - r e d u c t i o n r a t e s

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TABLE3. Mean (n = 5) rates of acetylene reduction (nmol C2H4 . g- ~organic mass. day- t, or nmol C~I-I4 . g- ~clay. day "~~)to ethylene (20 h incubation) for tall-form standing green leaves ofSpartina alterniflora (G), tall-form standing dead leaves (D), or for films of clay sediment on glass-fiber filters (C). Rates are shown in increasing order from Ieft to right for data from Sites 1 (1) and 4 (4). Bars connect means which were not significantly different (ANOVA + minimum significant range, P > 0.05)

G- 1 4-4 G- 1 30-7 (0-08 g)

G-4 22-2

Rates per g organic mass C-4~ 214.6

D- 1 476.5

D-4 1372.5

Rates per g clay (mean clay per sample) C-4~ G-4 D-4 36.8 142.8 1916.8 (0.05 g) (0. I0 g) (0-41 g)

D- 1 1919.2 (0.13 g)

aRates for clay on filters (0.03 g) at Site 1 were not significantly different from acetylenefree control.

w e r e also significantly r e d u c e d ( b y 6 5 - 9 4 % ) for t h e two categories o f leaves w i t h t h e largest ash c o n t e n t s (an i n d e x o f c o v e r a g e b y clay film) ( T a b l e 2). Rates at Site 4 r a n g e d f r o m 4 - 1 2 ~tmol C2H 4 . g - l o r g a n i c m a s s . d a y - ~ for u n r i n s e d leaves.

Wholly dead shoots, July D e a d stems w i t h sheaths r e m a i n i n g f r o m Site 4 h a d t h e h i g h e s t a s s o c i a t e d rates o f acetylene r e d u c t i o n (868 n m o l C2H 4 . g-1 o r g a n i c m a s s . d a y -~ [23% ash]) for t h e J u l y s a m p l i n g s , b e i n g statistically i n d i s t i n g u i s h a b l e f r o m stems w i t h o u t sheaths at Site 4 (450 n m o l . d a y - ~ [36% ash]) ( A N O V A + m i n i m u m significant range, n = 5, P > 0.05). A l l o t h e r rates ( n m o l C2H 4 . g - l o r g a n i c m a s s . d a y - ~) for stems a n d for leaves r e m a i n i n g o n d e a d stems at Sites 2 ( i n t e r m e d i a t e - h e i g h t f o r m ) a n d 4 (tall f o r m ) w e r e l o w e r b u t n o t statistically significantly different f r o m o n e a n o t h e r (Site 2, stems w i t h o u t sheaths: 6.4 [22% ash]; Site 2, stems w i t h sheaths: 146 [24% ash]; Site 4, leaves at an average o f 49 c m a b o v e s e d i m e n t : 303 [48% ash]; Site 4, leaves at an average o f 128 c m a b o v e s e d i m e n t : 16 [20% ash]). T h e r e was n o significant difference ( A N O V A , P > 0 - 0 5 ) b e t w e e n rates o f a c e t y l e n e r e d u c t i o n for i n c u b a t i o n in l i g h t vs. i n c u b a t i o n in d a r k n e s s .

Clay + organic base G r e e n leaves w i t h a clay film a n d clay films alone e x h i b i t e d m u c h lower rates o f a c e t y l e n e r e d u c t i o n t h a n d i d d e a d leaves w i t h a clay film (0-215 vs. 4 7 7 - 1 3 7 3 n m o l C2H 4 . g-~ o r g a n i c m a s s . d a y -~) ( T a b l e 3). W h e n c a l c u l a t e d p e r g clay, a c e t y l e n e - r e d u c t i o n rates s h o w e d a similar d i s t r i b u t i o n a m o n g s a m p l e t y p e s ( T a b l e 3). A g r a m o f clay on a d e a d leaf l e d to a b o u t 1.9 ~tmol e t h y l e n e p r o d u c t i o n , d a y - ~, as o p p o s e d to 3 1 - 1 4 3 n m o l for 1 g clay on g r e e n leaves or 0 - 3 7 n m o l for 1 g clay on glass-fiber filters. T h i s c o n t r a s t h e l d even for g r e e n leaves a n d d e a d leaves t h a t h a d a p p r o x i m a t e l y t h e s a m e clay-film c o n t e n t ( G - 4 a n d D - l , T a b l e 3).

Clay content overview R a t e s o f a c e t y l e n e r e d u c t i o n a s s o c i a t e d w i t h s t a n d i n g - d e a d leaves rose r a p i d l y as clay c o n t e n t i n c r e a s e d : b y a factor o f a b o u t 2 b e t w e e n 10% a n d 2 0 % ash; b y a f a c t o r o f a b o u t 5 b e t w e e n 2 0 % a n d 4 0 % ash; b y a factor o f a b o u t 25 b e t w e e n 20 a n d 6 0 % ash ( F i g u r e 4).

Patterns of nitrogenase activity

135

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Figure 4. Mean rates of acetylene reduction to ethylene per g organicmass (logarithmic plot) vs. percent ash (linear plot). Data are for all samplings of unrinsed, standing-dead leaves so~lkedin seawater. Equation for plotted line: log~0y = 0-04x+ 0-62; y = rate of acetylene reduction; x = percent ash; r2= 0.,11 (P < 0-01). All rates plotted were adjusted for incubation effects (Figure 3) to give rates expected prior to sample enclosure.

Clay (ash) content explained about 41% of the variation in logarithm of the acetylenereduction rate (coefficient of variation, r2).

Discussion For dead leaves of rinsed, smooth cordgrass decaying in the attached position (18 cm above the sediment, ash content up to about 15% of dry mass, October-January), we found acetylene-reduction rates of less than 50 nmol C2H 4 . g-~ organic mass. day -~ (Figure 1), similar to many rates reported for terrestrial and freshwater litter (Table 4). However, when we examined unrinsed leaves at advanced stages of decay, bearing heavier clay contents (°/o ash >40), at times between May and August, we found acetylenereduction rates that were commonly > 0.5 lamol C2H 4 . g-~ organic mass. day-~ (Figures 2 and 4; Table 2). These are rates akin to those measured for decaying mangrove leaves (Table 4), and on a par with rates reported for microecosystems manipulated to favour nitrogen fixation in decaying litter (Table 4). Rates found for clay-covered cordgrass leaves and for mangrove leaves approach those found experimentally for nitrogen fixation fuelled by pure carbohydrate polymers or simple sugars (25-60 lamol c 2 n 4 . g-1 organic mass. day-1 (Table 4). For comparison, the theoretical maximum yield of C2H 4 via nitrogenase fuelled by 1 g glucose is about 27 mmol (Harris, 1982).

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TABLE 4. Maximum mean rates (nmol C2H ~ . d a y ~) o f acetylene reduction to ethylene reported for decaying material o f standing smooth cordgrass (Spartina alterniflora) and some other vascular plants. Rates are either per g organic mass (go -t) or per g dry mass (g0-t). Only the first entry below is corrected ( - 2 - 5 ) for effects of enclosure on rates Source

n m o l . day- ~(h incubation)

SMOOTHCORDGRASS(Spartina aherniflora) Figure 4, this paper 13 279 go- ~ (0) July stem expt., this paper 868 go-J (16) G r e e n & E d m i s t e n , 1974 ° 294 gd -t (?) Hanson, 1977 130 ga -t (8) P a t r i q u i n & M c C l u n g , 1978 715 go ~ (35) Casselman, 1979 567 g~ ~ (5) H a i n e s & H a n s o n , 1979 1912 ga-t (15) T h o m s o n & Webb, 1984

Hilletal,, 1990 OTHER CORDGRASS( Spartina maritima) Talbotetal., 1988 RED MANGROVE(Rhizophora mangle) Gotto & Taylor, 1976 Newell et al., 1981

1730 go- ~ 2400 go-~

(2) (3)

831 gd S (24) 10 272 gd-~

(5)

6480 go-t

(2)

AUSTRALIANWHITE MANGROVE(Avicennia marina) V a n d e r V a l k & A t t i w i l l , 1984 11 256 go-~ Hicks & Silvester, 1985 984 go-~ DECAYINGSEAGRASS(Thalassia testudinum) Caponeetal., 1979 5323 gd 'l SEAGRASSRHIZOMES(Zostera marina) Kenworthyetal., 1987 1666 go -~ POND CYPRESS(Taxodium distichum) D i e r b e r g & B r e z o n i k , 1981 886 go-j SUGARMAPLE(Acer saccharum) Howarth & Fisher, 1976 126 go- 3 JAPANESECEDAR(Cryptomeria japonica) N i o h & H a r u t a , 1986 537 gd -~ FORESTLITTER (seventeen species o f trees) Silvester, 1989 365 go- ~ BIRCH (Betula pendula) Nohrstedt, 1988 221 go-m EUCALYPTUS(E. marginata) O ' C o n n e l l & G r o v e , 1987 117 go-~ FORESTLITTV.R(hardwood, pine, mixed-wood sites) Hendrickson, 1990 10 go-~ WOODYRESIDtm(cedar and hemlock) Jurgensen et al., 1987 7 ga- ~ ENGINEEREDWHEATSTRAW(designed microcosms) Hill & Patriquin, 1988 13 368 gd-~ Halsall & Gibson, 1989 36 925 "ga-~ R o p e r & Smith, 1991 3300 ga -~ XYLAN(extracted from oats) Roper & Smith, 1991 24 760 go- J

Comment

Highest corrected leaf value Highest stem-only value Stems, some fallen Whole shoot; based on dead fraction Leaves (some living?) (lag in rate?) Stems ( + leaves?) Shoots heated and ground3 reinoculated Stems (lag in rate?) Stems ( + leaves?); long presubmergence Stems (some living?); lag observed Leaves, aerobic, submerged, 2 weeks decay Leaves, aerobic, submerged, 2 weeks decay

(3) (10)

Leaves, in air, 3 weeks decay Leaves, in air, ? weeks decay

(4)

Dead submerged leaves plus epiphytes; light dependent

(2)

Naturally occurring dead, anaerobic

(3)

Leaf litter, submerged

(2)

Leaf discs, submerged

(24)

Leaves from litter pots

(6)

Leaf and twig material, moistened

(24)

Leaf litter

(24)

Leaf litter, moistened

(24)

L, F and H horizon of forest floor

(24)

Non-surface wood; argon atmosphere

(2) (24) (24) b

Multispecies N-fixing inoculation

(24) b

Preadapted wheat-soil inoculum

Azospirillum + Cellulomonas Preadapted inoculum; ground straw

"Values reported as ltmol, day ~; it is presumed here that this was a calculation error of x 103, since even glucose-supported rates are ~<60 ~ m o l . g - ~. day - ~ (Roper & Smith, 1991). bMontmorillonite clay added.

Patterns of nitrogenase activity

137

Newell et al. (1989) discovered that net change in nitrogen content of naturally decaying, standing, leaves of smooth cordgrass was strongly correlated (r 2 = 0.86) with ash content (post-rinsing) of leaves. T h e y speculated that nitrogen fixation by cyanobacteria in the clay contributed to the positive changes in nitrogen content of the leaves (see Carpenter et al., 1978; Aziz & Nedwell, 1986). In the present study, we have found a highly significant relationship between indicated nitrogen fixation (acetylene reduction) and clay (ash) content of unrinsed, standing, decaying leaves (Figure 4). In fact, as the ash content of unrinsed leaves increased, acetylene-reduction rates increased logarithmically. Our experimental work with clay manipulations (Tables 2 and 3) showed that dead leaves supported acetylene-reduction rates greater than 0.51amol C2H 4 . g-1 organic mass. day-~ with ash contents as low as 24~o, but that the presence of higher clay content (up to 78% ash) enhanced rates (by up to 17 times) (Table 2). Also, when clay was present on non-decaying substrata (green leaves or glass-fiber supports), it had much smaller (e.g., x 52 smaller at Site 4, filters vs. dead leaves) acetylene -reduction rates per unit clay mass for the non-decaying supports. Roper and Smith (1991) also found a strong positive effect of presence of clay on acetylene reduction, for wheat-field microbes with glucose (slope of 13-5 for regression of nitrogenase activity on percent clay in soil; r2= 0"94), and implied that reduced oxygen flow could be one explanation for this phenomenon (see Hill et al., 1990; Paerl, 1990). Nevertheless, since clay content explained only 41% of the variation in acetylene reduction on decaying cordgrass leaves (Figure 4), and rates of acetylene reduction on leaves were highly variable over marsh and leaf types (Figure 2), there are clearly other factors at work. Our higher rates ( > 0"5 ~tmol C2H 4 . g-1 organic mass. day-1) for acetylene reduction associated with leaves of smooth cordgrass were not observed until after April. T h e positive relationship between warmer months and saltmarsh nitrogen fixation is well documented (e.g. Hansori, 1977; Wolfenden & Jones, 1987; Hill et al., 1990). Temperature is clearly at least a partial explanation for this (e.g., Whiting & Morris, 1986). However, we ran all of our incubations at a similar temperature range (25-30 °C, except tagged leaves at 20 °C). Another characteristic of the warmer months that may be a contributing factor is that larger quantities of clay are resuspended for potential deposition on cordgrass shoots during this period (Newell et al., 1988). Clay-organic floc densities in the upper Duplin (salt-marsh) River are on average 3-7 times greater during the summer than the winter. Clay deposition on leaves may be more frequent and persistent in the warmer months. Note that the ash contents of our lowest (ca. 10 cm above the sediment) unrinsed leaves in April ranged only up to 28% (cf. Figure 4). Our finding that the site of acetylene reduction was principally on/in the decaying leaf, not in the clay (Table 3) indicates that cyanobacteria (located primarily in the clay film: Newell et al., 1989) were not the predominant N 2 fixers of decaying cordgrass. An alternative potential source of the high cordgrass-shoot nitrogenase activity (Figure 4) is a synergism between shoot-decomposing fungi and diazotrophic heterotrophic bacteria. Standing, naturally decaying cordgrass leaves (prior to heaviest clay accumulation) harbour a microflora that is dominated, with respect to both microbial standing crop and productivity, by an ascomycetous fungus, Phaeosphaeria spartinicola Leuchtmann (Newell et al., 1989; Leuchtmann & Newell, 1991; Newell & Fallon, 1991). Newell et al. (1989) observed that when the clay (ash) content of rinsed leaves rose to 35% of total mass (after 12 weeks of decay), the bacterial mass increased to 7% of total microbial mass (from <~1%). In contrast, the tagged leaves used in this study (Figure 1), which had not acquired a heavy clay load at the 12-week point (ash content of rinsed leaves < 1 5 ° ) , contained a

138

S . Y . Newell et al.

fungal mass that was > 9 9 % o f the total m i c r o b i a l crop at 12 weeks (Newell et al., u n p u b l . ) . Hill a n d P a t r i q u i n (1990) p r e s e n t e d evidence for the p r e s e n c e o f ftmgal/ bacterial (Helicomyces/Azospiriltum) n i t r o g e n - f i x i n g systems i n d e c o m p o s i n g litter of vascular plants (see T a b l e 4), a n d p r o p o s e d that these m a y be i m p o r t a n t i n w a r m climates. P e r h a p s a Phaeosphaeria/Azospirillum p a r t n e r s h i p develops as s m o o t h cordgrass leaves m o v e into the phase of heavy clay coverage. A l t e r n a t i v e l y , fungal activity m a y decline as clay accumulates, a n d be replaced by a lignocellulolytic/diazotrophic bacterial guild ( B e n n e r et al., 1984; Halsall & G i b s o n , 1989; P o r t e r et al., 1989).

Acknowledgements T h i s research was s u p p o r t e d b y N a t i o n a l Science F o u n d a t i o n grants B S R - 8 6 0 4 6 5 3 a n d O C E - 8 6 0 0 2 9 3 , a n d by the Sapelo I s l a n d Research F o u n d a t i o n . O u r thanks to D o n n a Poppell (word processing), Eileen H e d i c k (graphics), a n d H a n s Paerl a n d C a r o l y n C u r r i n (critical review). U G M I c o n t r i b u t i o n n u m b e r 695.

References Aziz, S. A. Abd. & Nedwell, D. B. 1986 The nitrogen cycle of an east coast, U.K. saltmarsh: II. Nitrogen fixation, nitrification, denitrification, tidal exchange. Estuarine, Coastal and Shelf Science 22, 689-704. Bermer, R., Newell, S. Y., Maccubbin, A. E. & Hodson, R. E. 1984 Relative contributions of bacteria and fungi to rates of degradation of lignocellulosic detritus in salt-marsh sediments. Applied & Environmental Microbiology 48, 36-40. Capone, D. G. 1988 Benthic nitrogen fixation. In Nitrogen Cycling in Coastal Marine Environments (Blackburn, T. H. & Sorensen, J., eds). Wiley, New York, pp. 85-123. Capone, D. G., Penhale, P., Oremland, R. & Taylor, B. 1979 Relationship between productivity and N 2 (C2H2) fixation in a Thalassia testudinum community. Limnology & Oceanography 24, 117-125. Carpenter, E. J., Van Raalte, C. D. & Valiela, I. 1978Nitrogen fixation by algae in a Massachusetts salt marsh. Limnology & Oceanography 23, 318-327. Casselman, M. E. 1979 Biological nitrogen fixation in a Louisiana Spartina alterniflora salt marsh. MSc thesis, Louisiana State University, Baton Rouge. Casselman, M. E., Patrick, W. H. & DeLaune, R. D. 1981 Nitrogen fixation in a Gulf coast salt marsh. Soil Science Society of America Journal 45, 51-56. Dierberg, F. E. & Brezonik, P. L. 1981 Nitrogen fixation (acetylene reduction) associated with decaying leavesof pond cypress (Taxodiumdistichum var. nutans) in a natural and a sewage-enriched cypress dome. Applied & Environmental Microbiology 41, 1413-I 418. Frey, R. W. & Basan, P. B. 1985 Coastal salt marshes. In Coastal Sedimentary Environments (Davis, R. A., ed.). Springer-Verlag, New York, pp. 225-30I. Giller, K. E. 1987 Use and abuse of the acetylene reduction assay for measurement of' associative' nitrogen fixation. Soil Biology & Biochemistry 19, 783-784. Gotto, J. W. & Taylor, B. F. 1976N 2fixation associated with decaying leavesof the red mangrove (Rhizophora mangle). Applied & Environmental Microbiology 31,781-783. Green, F. & Edmisten, J. 1974 Seasonaliry of nitrogen fixation in Gulf coast salt marshes. In Phenology and Seasonality Modeling (Leith, H., ed.). Springer-Verlag, New York, pp. 113-126. Haines, E. B. & Hanson, R. B. 1979 Experimental degradation of detritus made from the salt marsh plants Spartina alterniflora Loisel., Salicornia virginica L., and Juncus roemerianus Scheele. Journal of Experimental Marine Biology & Ecology 40, 27--40. Halsall, D. M. & Gibson, A. H. 1989 Nitrogenase activity by diazotrophs grown on a range of agricultural plant residues. Soil Biology & Biochemistry 21, 1037-1043. Hanson, R. B. 1977 Nitrogen fixation (acetylene reduction) in a salt marsh amended with sewage sludge and organic carbon and nitrogen compounds. Applied & Environmental Microbiology 33, 846-852. Hanson, R. B. 1983 Nitrogen fixation activity (acetylene reduction) in the rhizosphere of salt marsh angiosperms, Georgia, U.S.A. Botanica Marina 26, 49-59. Hardy, R. W. F., Holsten, R. D., Jackson, E. K. & Burns, R. C. 1968 The acetylene-ethylene assay for N z fixation: Laboratory and field evaluation. Plant Physiology 43, 1185-1207. Harris, R. F. 1982 Energetics of nitrogen transformations. InNitrogen in Agricultural Soils (Stevenson, F. J., ed.). Soil Science Society of America, Madison, pp. 833-879. Havelka, U. D., Boyle, M. G. & Hardy, R. W. F. 1982 Biologicalnitrogen fixation. In Nitrogen in Agricultural Soils (Stevenson, F. J., ed.). Soil Science of America, Madison, pp. 365-422.

Patterns of nitrogenase activity

139

Hendrickson, O. Q. 1990 Asymbiotic nitrogen fixation and soil metabolism in three Ontario forests. Soil Biology & Biochemistry 22, 967-971. Hicks, B. J. & Silvester, W. B. 1985 Nitrogen fixation associated with the New Zealand mangrove (Avicennia marina [Forsk.] Vierh. var. resinifera [Forst. f.] Bakh.) Applied & Environmental Microbiology 49, 955-959.

Hill, N. M. & Patriquin~ D. G. 1988 Induction of aerobic nitrogen fixation and enhancement of wheat straw decomposition by inoculation with a crude culture from sugarcane litter. SoilBiology & Biochemistry 20, 613-618. Hill, N. M. & Patriquin, D. G. 1990 Evidence for the involvement of AzospiriUum brasilense and Helicomyces roseusin the aerobic nitrogen-fixing/cellulolytic system from sugarcane litter. Soil Biology & Biochemistry 22, 313-319. Hill, N. M.~ Patriquin, D. G. & Sircom, K. 1990 Increased oxygen consumption at warmer temperatures favours aerobic nitrogen fixation in plant litters. Soil Biology & Biochemistry 22, 321-325. Hopkinson, C. S. & Schubauer, J. P. 1984 Static and dynamic aspects of nitrogen cycling in the salt marsh graminoid Spartina atterniflora. Ecology 65, 961-969. Howarth, R. W. & Fisher, S. G. 1976 Carbon, nitrogen and phosphorus dynamics during leaf decay in nutrient-enriched stream microecosystems. Freshwater Biology 6~ 221-228. Howarth, R. W., Marino, R., Lane, J. & Cole, J. J. 1988 Nitrogen fixation in freshwater, estuarine, and marine ecosystems. 1. Rates and importance. Limnology & Oceanography 33, 669-687. Jurgenson, M. F., Larsen, M. J., Graham, R. T. & Harvey, A. E. 1987 Nitrogen fixation in woody residue of northern Rocky Mountain conifer forests. Canadian Journal of Forestry Research 17, t 283-1288. Kenworthy, W. J., Currin, C., Smith, G. & Thayer, G. 1987 T h e abundance, biomass and acetylene reduction activity of bacteria associated with decomposing rhizomes of two seagrasses, Zostera marina and Thalassia testudinum. Aquatic Botany 27, 97-119. Letzsch, W. S. & Frey, R. W. 1980 Deposition and erosion in a Holocene salt marsh, Sapelo Island, Georgia. Journal of Sedhnentary Petrology 50, 529-542. Leuchtmann, A. & Newell, S. Y. 1991 Phaeosphaeria spartinicola, a new species on Spartina. Mycotaxon 41, 1-7. Newell, S. Y. & Fallon, R. D. 1991 Toward a method for measuring fungat instantaneous growth rates in field samples. Ecology 72, 1547-1559. Newell, S. Y., Arsuffi, T. L., Kemp, P. F. & Scott, L. A. 1991 Water potential of standing-dead shoots of an intertidal grass. Oecologia 85, 321-326. Newell, S. Y., Fallon, R. D. & Miller, J. D. 1989 Decomposition and microbial dynamics for standing, naturally positioned leaves.of a salt-marsh grass, Spartina alterniflora. Marine Biology 101, 471-481. Newell, S. Y., Fallon, R. D., Sherr, B. F. & Sherr, E. B. 1988 Mesoscale temporal variation in bacterial standing crop, percent active cells, productivity and output in a salt-marsh tidal river. Verhandlungen Internationate Vereinigung Limnologie 23, 1839-1845. Newell, S. Y., Fell, J. W., Master, I. M., Miller, C. & Walter, M. A. 1981 Acute impact of an organophosphorus insecticide on microbes and small invertebrates of a mangrove estuary. Archives of Environmental Contamination & Toxicology 10, 427--435. Nioh, I. & Haruta, Y. 1986 Estimation of the amount of asymbiotically fixed nitrogen in the leaf litter of Japanese cedar (Cryptomeria japonica). Journal of the Japanese Forestry Society 68, 314-319. Nohrstedt, H.-0. 1988 Nitrogen fixation (C2H2-reduction) in birch litter. Scandinavian Journal of Forest Research 3, 17-23. O'Connell, A. M. & Grove, T. S. 1987 Seasonal variation in C.,H, reduction (Nz-fixation) in the litter layer of eucalypt forests of southwestern Australia. Soil Biology & Biochemistry 19, 135--I 42. Paerl, H. W. 1990 Physiological ecology and regulation of N., fixation in natural waters. Advances in Microbial Ecology 11,305-344. Patriquin, D. G. 1978 Factors affecting nitrogenase activity (acetylene-reducing activity) associated with excised roots of the emergent halophyte Spartina alterniflora Loisel. Aquatic Botany 4, 193-210. Patriquin, D. G. & McClung, C. R. 1978 Nitrogen accretion, and the nature and possible significance of N., fixation (acetylene reduction) in a Nova Scotian Spartina alterniflora stand. Marine Biology 47,227-242. Pomeroy, L. R. & Wiegert, R. G. (eds). 1981 The Ecology of a Salt Marsh. Springer-Verlag, New York, 271 pp. Porter, D., Newell, S. Y. & Lingle, W. L. 1989 Tunneling bacteria in decaying leaves of a seagrass. Aquatic Botany 35, 395-401. Roper, M. M. 1983 Field measurements of nitrogenase activity in soils amended with wheat straw. Australian Journal of Agricultural Research 34, 725-739. Roper, M. M. & Smith, N. A. 1991 Straw decomposition and nitrogenase activity (C2H 2 reduction) by free-living microorganisms from soil: effects of pH and clay content. Soil Biology & Biochemistry 23, 275-283. Silvester, W. B. 1989 Molybdenum limitation of asymbiotic nitrogen fixation in forests of Pacific Northwest America. Soil Biology & Biochemistry 21,283-289. Sokal, R. R. & Rohlf, F. J. 1981 Biometry (2ndEdn). W. H. Freeman~ San Francisco, 859 pp.

140

S . Y . Nemell et al.

Talbot, M. M. B., Small, J. G. C. & Bate, G. C. 1988 Spatial patterns of N fixation (acetylene reduction) along environmental gradients in a warm-temperate estuary. Journal afExperimemal Marine Biology Ecology 117, 255-270. Thomson, A. D. & Webb, K. L. 1984 T h e effect of chronic oil pollution on salt-marsh nitrogen fixation (acetylene reduction). Estuaries 7, 2-11. Van Der Valk, A. G. & Attiwill, P. M. 1984 Acetylene reduction in an Avicennia marina community in southern Australia. Australian Journal of Botany 32, 157-164. Wadsworth, J. 1980 Geomorphology and hydrography of the Duplin River estuarine system. PhD thesis, University of Georgia, Athens. Whiting, G. J. & Morris, J. T. 1986 Nitrogen fixation (C2H 2 reduction) in a salt marsh: its relationship to temperature and an evaluation of an in situ chamber technique. Soil Biology ~ Biochemistry lg, 515-521. Whiting, G. J., McKellar, H. N., Spurrier, J. D. & Wolaver, T. G. 1989 Nitrogen exchange between a portion of vegetated salt marsh and the adjoining creek. Limnology d¢ Oceanography 34, 463--473. Wolfenden, J. & Jones, K. 1987 Seasonal variation of in situ nitrogen fixation (C~H 2 reduction) in an expanding marsh of Spartina anglica. Journal of Ecology 75, 1011-1021.