Diel patterns of nitrogenase activity associated with macrophytes in a Eutrophic Lake

Aquatic Botany, 28 (1987) 341-352

341

Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

D I E L P A T T E R N S OF N I T R O G E N A S E A C T I V I T Y A S S O C I A T E D W I T H M A C R O P H Y T E S IN A E U T R O P H I C L A K E

JULIE L. CORKRAN and CONRAD E. WICKSTROM

Department of Biological Sciences, Kent State University, Kent, OH 44242 ( U.S.A:) (Accepted for publication 5 March 1987)

ABSTRACT Corkran, J.L. and Wickstrom, C.E., 1987. Diel patterns of nitrogenase activity associated with macrophytes in a eutrophic lake. Aquat. Bot., 28: 341-352. Nitrogenase activity (NA) (acetylene reduction) occurring in the phyllosphere of macrophyte-epiphyte complexes was investigatedin situ,from June to September 1985, in East Twin Lake, Ohio, U.S.A. The epiphytic floraof every macrophyte species examined contained active diazotrophs capable of reducing acetylene at rates of up to 41 682 n M g-1 h-1 during midday incubations. The general diel nitrogenase activity (NA) pattern shared by Heteranthera dubia (Jacq.) MacM., Najas guadalupensis (Spreng.) Morong and Lemna minor L. was unimodal and reproducible throughout the summer: ethylene evolution was low in the morning, peaked around midday (11.00-14.00 h), then declined to a nocturnal minimum. Peaks in N A were generally coincident with lightintensitymaxima. However, rates of ethylene evolution by the Myriophyllurespicatum L. epiphytes studied had low N A which exhibitedan atypicaldielpattern.Epiphyte nocturnal N A ranged from 5 to 46% of a day's totalactivity.Dark (foil-wrapped) bottleN A was constant on a dielbasis and was not dependent on preceding lighthistory.

INTRODUCTION

Various diel patterns of planktonic nitrogen fixation in lakes may be observed. Single peaks in daily activity occur most often around midday ( Stewart et al., 1967; Rusness and Burris, 1970; Granhall and Lundgren, 1971; Ganf and Home, 1975 ), but may be demonstrated during the morning (Vanderhoef et al., 1975) and afternoon (Paerl, 1979; Kellar and Paerl, 1980; Levine and Lewis, 1984) hours as well. Twin peaks have also been reported (Stewart et al., 1971; Peterson et al., 1977). Patterns of planktonic nitrogen fixation may result from diel migrations of cyanobacteria within the water column (Vanderhoef et al., 1975; Levine and Lewis, 1984) or may be a function of physiological responses to light-induced changes in the intracellular or the extracellular milieu (Paerl and Kellar, 1978; Paerl, 1979). The sessile nature of epiphytic diazotrophs precludes responding to the diel 0304-3770/87/$03.50

© 1987 Elsevier Science Publishers B.V.

342 light cycle by altering their physical location; however, their physiological response may additionally reflect gas exchange and exudate cycles of the macrophyte host. Microzone oxygenation affects nitrogenase activity (NA) of planktonic cyanobacteria ( Paerl and Kellar, 1978), while the operation of bacterial nitrogenase activity in the rhizosphere of terrestrial plants is related to exudate release (Balandreau et al., 1974). In situ investigations of phyllosphere-associated nitrogenase activity in lacustrine systems are few (Duong, 1972; Finke and Seeley, 1978; Kostyaev, 1984) and address seasonal and species-level aspects of epiphyte NA. In the single report of diel nitrogenase activity associated with freshwater macrophyte-epiphyte complexes, an afternoon maximum was observed for Myriophyllum spicatum L. in August (Finke and Seeley, 1978). The questions of diel pattern variability among different macrophyte species and of seasonal changes in diel activity remain to be examined. In the present study, macrophytes from the littoral zone of a eutrophic lake were surveyed for acetylene reduction, and diel patterns of NA associated with one floating and three submersed macrophytes were examined. The results demonstrated the universality of macrophyte-associated nitrogen fixation in this lake and indicated that temporal and host-specific differences in the diel activity patterns exist during a portion of the macrophyte growing season. METHODS Littoral zone macrophytes were collected at depths of < 1 m from two sites in East Twin Lake, Portage Co., Ohio, U.S.A. East Twin Lake, a glacial dimictic lake with a maximum depth of 12 m, is eutrophic and the littoral zone occupies ca. 25% of the lake's surface area ( Cooke et al., 1973). Epiphyte nitrogenase activity (NA) associated with the macrophytes was measured using the acetylene reduction assay (Stewart et al., 1967; Stewart, 1968). Incubations were conducted at 5-7-week intervals from June to September 1985 to encompass early-, middle- and late-summer growth periods. Paired survey and diel studies were performed: surveys preceded the corresponding diel studies by 2-11 days. This permitted identification and selection of the two or three abundant macrophyte species with high activities for the diel experiments. Serum bottles (30 ml) containing 10 ml filtered (GF/C) littoral water and macrophyte tissue ( stems and leaves) were prepared for each species collected. Earlier results indicated that above-sediment macrophyte biomass supported most of the nitrogenase activity ( Corkran and Wickstrom, 1985 ) ; thus, roots were not tested. Macrophyte samples were collected with care to minimize epiphyte disturbance, and both apical and older plant portions were used in each sample incubation. Lemna minor L. samples were incubated intact; the other macrophytes were sectioned into 10-mm lengths for incubation. Throughout the summer, each duckweed sample also contained Spirodela

343

polyrhiza (L.) Schleid.; however, since the latter species comprised < 5% of the collection at any one time, these samples are referred to as Lemna. The bottles were stoppered with serum caps, and equal volumes of the atmosphere were exchanged with field-generated acetylene to obtain an atmosphere containing 20% acetylene. During the surveys, samples were incubated around midday under ambient solar radiation in a clear plastic container for 3-4 h. Temperatures similar to those measured in the littoral zone were maintained by changing the container water regularly. For the 24-h experiments, duplicate light and dark bottles were prepared as described above for each of the selected macrophytes. Incubation under in situ light and temperatures was facilitated by suspension at near-surface depths (ca. 5 cm). The bottles were attached by rubber bands to the ends of 20-cm wooden dowels projecting from a styrofoam ring anchored in an unshaded portion of the littoral zone. Light intensities were measured with a quantum sensor and light meter (Lambda Instruments Corp., Model LI-185A). Eight consecutive 3-h incubations provided a diel profile of nitrogenase activity associated with each of the macrophyte species. The incubation periods were designated Periods 1-8: diel studies were initiated at the beginning of Period 1 {11.00-14.00 h) and terminated at the end of Period 8 (08.00-11.00 h) the following day. At the end of each 3-h period, new incubations were initiated with freshly prepared plant material, and the preceding incubations were terminated by sampling the gas phase of each with a sterile 1-ml plastic syringe (Becton-Dickinson). The syringe needles were embedded in rubber stoppers to prevent gas loss during transport to the laboratory for analysis (Horne and Carmiggelt, 1975). Gas samples were processed at 50°C by FID gas chromatography (Perkin-Elmer Sigma 4) on a Porapak R column (Waters Associates) usually within 3 h of collection. Ethylene was quantified according to Wickstrom (1980). Sample activities were corrected for ethylene contamination of the acetylene substrate in gas purity control incubations that were processed along with the samples. Endogenous ethylene production was never detected in control incubations of macrophyte-epiphyte complex without acetylene gas. Rates of ethylene evolution were expressed per gram ash-free dry biomass (AFDB) of macrophyte-epiphyte complex ( n M g-1 h-1 ). The contents of each serum bottle were placed into a tared aluminum weighing pan, taken to dryness at 60 °C for 18 h in a laboratory oven (Grieve Corp., Model LR270C), allowed to equilibrate in a desiccator for 2 h at room temperature and weighed. Dried samples were ashed at 500 ° C in a furnace ( Sybron Thermolyne 1400) for 1 h, equilibrated and weighed. The sample AFDB was calculated as the difference between the dry and ash weights. In addition to the light-bottle incubations, bottles darkened with aluminum foil and containing the selected macrophytes were incubated each period. Thus, each macrophyte was subjected to four treatments: (1) unwrapped {light)

344 TABLE I Nitrogenase activity associated with East Twin Lake macrophytes during summer surveys in 1985. Data represent rates of ethylene evolution per unit ash-free dry biomass of macrophyte-epiphyte complex (nM g-1 h-l) Macrophytespecies

Chara vulgaris L. Ceratophyllum demersum L. Heteranthera dubia (Jacq.) MacM. Lemna minor L. Myriophyllum spicatum L. Najasguadalupensis (Spreng.) Morong Nuphar advena Ait. Nymphaea odorata Ait. Potamogeton crispus L.

Survey date 6 June

18 July

23 August

NE 1 4.62 NE 213.052 46.832 NE NE 3.49 1.57

NE 55.59 70.942 188.012 42.25 38.192 5.69 4.88 0.00

4071.35 5522.24 18 741.412 5947.642 NE 20 848.992 700.08 1150.15 NE

IMacrophyte not evident (NE) at the sample siteson thisdate. 2Species selectedfor subsequent dielstudy.

bottles incubated in the daytime or (2) evening and (3) wrapped (dark) bottles incubated in the daytime or (4) evening. To determine whether these treatments were significantly different from one another, a one-way analysis of variance was performed for each diel data set. Daylight hours were defined as Periods 1 and 2 and evening hours as Periods 5 and 6; this eliminated transition periods encompassing conditions of illumination and darkness. Duncan multiple range analyses ( Steel and Torrie, 1980) were performed on data sets with significant F-ratios to identify the significantly different means. RESULTS

The NA of the m a c r o p h y t e - e p i p h y t e complexes present at the study sites on the three survey dates are presented in Table I. All macrophytes surveyed supported diazotrophic epiphytes throughout the study period, except for Potamogeton crispus in July. Activities ranged from a low of 1.57 for P. crispus in J u n e to a high of 20 848.99 n M g-1 h-1 ethylene evolved per unit A F D B for Najas guadalupensis in August. Light intensities averaged 1669 and 1119/~E m -2 s -1 during the July and August surveys, respectively; intensities were not measured during the J u n e survey. Six macrophytes which had been previously tested were examined in August; each of these species possessed its m a x i m u m survey activity at the later test date (Table I ). A total of four macrophytes were selected for diel studies (Table I ). L. minor was examined each diel study period (Fig. 1 ). Heteranthera dubia and N. guadalupensis (Fig. 2 ) were not p r e s e n t in J u n e b u t had highest activities the other

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months, while Myriophyllum spicatum (Fig. 3 ) was not present at the sample sites in August. Nitrogenase activity associated with three of the macrophytes tracked the diel light curve with maximum rates of ethylene evolution occurring near solar noon (Figs. 1 and 2 ). L. minor, N. guadalupensis ( except July) and H. dubia diel activities were highest during Period 1 (11.00-14.00 h ) in each trial (Table II ). In July, the mean activity of N. guadalupensis (Fig. 2 ) was highest during Period 8 (08.00-11.00 h); however, Periods 1 and 8 mean activities (7921.1 and 8228.8 nM g-1 h-l, respectively) were not significantly different ( t = 0.310; df-- 2). After attaining their nitrogenase maxima around noon, the activities associated with these three macrophytes progressively declined until reaching their low values during Periods 4-7 ( 20.00-08.00 h). The nocturnal minima of

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these species ranged from 19.05 (L. minor, June) to 930.93 nM g-1 h-] (L. minor, September). Excluding L. minor in September, which had a diel minimum that represented 35% its maximum, the mean nocturnal minimum activity for these three species was 5% their diurnal maximum. No relationship TABLE

II

Minimum and maximum diel nitrogenase activities for the macrophytes tested. Data represent rates of ethylene evolution per unit ash-free dry biomass of macrophyte-epiphyte complex (nM g-l h-] ) Date

Activity level]

H. dubia

L. minor

10June

Minimum Maximum

NT 2 NT

20 July

Minimum Maximum

169.84 (6) 55.59 (5) 12 692.87 (1) 6437.01 (1)

NT NT

500.09 (5) 8228.80 (8)

5 September Minimum Maximum

306.53 (4) 930.93 (5) 8778.58 (1) 2639.45 (1)

NT NT

501.11 (7) 41682.48 (1)

19.05 (6) ~ 240.77 (1)

M. spicatum N. guadalupens~s 2.88 (5) 17.99 (6)

NT NT

1Each value represents the mean of duplicate light (unwrapped) bottle incubations. 2Macrophyte not tested (NT) on this date. 3Parenthetical values indicate the diel period of that activity level.

348

between light intensity and levels of diurnal NA was demonstrated statistically using correlation analysis. Significant F-ratios were obtained for L. minor, H. dubia and N. guadalupensis on all study dates when the four treatments were compared by analysis of variance. Light bottles incubated during the daytime (Periods 1 and 2) yielded significantly higher nitrogenase activities than the other combinations (Duncan multiple range test). Daylight/dark bottle, evening/light bottle and evening/dark bottle activities were not statistically different from one another. There was no evidence of differences among the treatment means during the diel study of M. spicatum (F= 3.25; df-- 3.12 ). The diel pattern of associative NA for this macrophyte was atypical (Fig. 3). Although light intensity peaked during Period 1, maximum and minimum NA occurred during successive nocturnal periods (Table II). Acetylene reduction occurred throughout the diel period with total nocturnal NA representing from 5% (H. dubia in July) to 28% (L. minor in September) of the day's total activity per gram AFDB. Nocturnal NA averaged 12% of total daytime activity in the diel studies of L. minor, N. guadalupensis and H. dubia. Nocturnal NA associated with M. spicatum accounted for 46% of the diel activity associated with this macrophyte on the June study date. Phase-contrast microscope examinations (100 and 250 × ) of intact macrophyte-epiphyte complexes and epiphytes loosened by shaking revealed heterocystous cyanobacteria. Although no systematic attempt was made to quantify the nitrogen-fixing species, Gloeotrichia and Anabaena were observed in samples throughout the summer from most of the macrophytes studied. No heterocystous species were observed in samples of M. spicatum. DISCUSSION

Diazotrophic epiphytes were ubiquitous on East Twin Lake macrophytes throughout the summer. Earlier laboratory and in situ investigations have documented phyllosphere-associated NA for some of the macrophytes examined in this report: M. spicatum (Smith and Ornes, 1982; Kostyaev, 1984), Chara vulgaris ( Finke and Seeley, 1978), Ceratophyllum demersum, Lemna, Nuphar, Nymphaea and Potamogeton spp. (Duong, 1972; Finke and Seeley, 1978; Zuberer, 1982; Kostyaev, 1984). The present study represents the first report of NA associated with Heteranthera dubia and Najas guadalupensis. These species supported the greatest activities during the study. No NA was detected in association with P. crispus in July of this study; however, a survey conducted in July 1984 demonstrated that P. crispus, as well as the other macrophytes examined, supported populations of active diazotrophs (Corkran and Wickstrom, 1985). Published reports show Potamogeton-associated NA varies by species as well as the month of study (Finke and Seeley, 1978; Lipschultz et al., 1979; Smith and Ornes, 1982 ). Kostyaev (1984)

349

found that no single species of Potamogeton, including P. crispus, supported NA throughout the summer. The epiphytic, diel NA patterns for L. minor, H. dubia, and N. guadalupensis (the primary study organisms) were similar and exhibited midday maxima and evening minima. We found this activity pattern repeatedly during the summer for these macrophyte species. This is basically the same pattern as that described for planktonic nitrogen fixation in freshwater lakes ( Stewart et al., 1967; Rusness and Burris, 1970; Granhall and Lundgren, 1971; Ganf and Horne, 1975) and for a macrophyte (Finke and Seeley, 1978). Correlations between light intensities and diurnal NA were not significant for the primary macrophytes. This may have resulted from seasonal variability in epiphyte composition and thickness of the epiphyte cover, or simply been due to unrecorded light intensity variations. Although light saturation of epiphyte NA may occur in situ (Wickstrom, 1984), the data here are not conclusive. Nocturnal NA has been demonstrated in a variety of freshwater situations and was noted here as well. The macrophyte-associated NA between sunset and sunrise in East Twin Lake averaged 12% of daily total activity for the primary study organisms. This value is low in comparison to 33% of the day's total nitrogen fixation in a stream (Home, 1975), and ca. 18% by epilithic microflora in an oligotrophic lake (Lundgren, 1978, as estimated from Fig. 1, p. 55). In contrast, M. spicatum nocturnal NA represented 46% of the total diel activity in the June study. Jones {1977) reported that dark fixation by subtropical grassland algal mats responded to light history; this residual activity was greater when the preceding light intensity was high and progressively decreased during the night. In contrast, the NA of our incubations with the primary macrophytes fell rapidly to levels independent of prior light intensity when darkened (Treatment 3 ). Additionally, the levels of ethylene evolved in the absence of light (Treatments 2, 3 and 4) during any period of our diel incubations did not vary significantly. Considering these data and the fact that diurnal activity tracked light intensities, it appears that most of the epiphyte NA associated with three of the East Twin Lake macrophytes was light-dependent, i.e. cyanobacterial. The diel pattern of M. spicatum-associated NA reported here differs from that of the other macrophytes examined: it did not possess a diurnal peak and the NA of light- and dark-bottle incubations were not significantly different. Therefore, it seems likely that the diazotrophic flora of this M. spicatum was primarily composed of light-independent bacterial nitrogen-fixers. These organisms, rather than residual cyanobacterial activity, may also account for the low levels of acetylene reduction experienced in the dark-bottle incubations of the other macrophytes as well. Finke and Seeley (1978) found that the NA maximum for M. spicatum was generally coincident with the diurnal light intensity peak, but they also found the cyanobacterium Gloeotrichia prevalent. The seasonal succession of algal epiphytes in temperate lakes typically

350 involves initial diatom dominance followed by filamentous green algae in midsummer, cyanobacteria in late summer, and culminates in re-establishment of diatom dominance during macrophyte senescence (Stockner and Armstrong, 1971; Cattaneo and Kalff, 1978). Gloeotrichia, the genus generally found to dominate the cyanobacterial component of periphyton assemblages, was observed in our samples and has been shown to reach m a x i m u m biomass in late s u m m e r (Cattaneo and Kalff, 1978). The trend of increasing NA associated with East Twin Lake macrophytes over the s u m m e r probably reflects the seasonal increase of epiphytic nitrogen-fixing cyanobacteria. Kostyaev (1984) demonstrated that macrophyte-associated NA was closely tied to the density of Gloeotrichia colonization. Light-dependent ethylene evolution was also documented by Duong (1972) for an association of cyanobacteria with duckweeds. The L. minor from East Twin Lake evolved ethylene at rates comparable to those obtained by Kostyaev (1984). Duckweed nitrogen fixation may be important to the nitrogen economy of temperate lakes as it consistently possessed epiphyte NA and because populations of this macrophyte typically are one of the first to appear following loss of ice cover and are among the last to collapse in autumn. The NA of our M. spicatum were lower than previously reported. T h e maximum activity obtained in this study represents less than 1% of the average M. spicatum diurnal peak activity obtained by Finke and Seeley (1978). Kostyaev (1984) found that M. spicatum supported the highest in situ fixation rates among 28 species of macrophytes surveyed. Since Eurasian water milfoil is so widely dispersed, the nature and contribution of its diazotrophic epiphytes to the nitrogen budgets of lacustrine systems warrant further study. The current literature and the present study suggest that epiphyte nitrogen fixation on macrophytes occurs with few exceptions in the littoral zones of freshwater lakes. In the majority of instances, attached cyanobacteria account for most of the phyllosphere activity. Although rates of NA vary with light intensity, season and the specific macrophyte-epiphyte complex, the general pattern of a single midday peak is reproducible among different macrophyte species and throughout most of the summer. ACKNOWLEDGMENTS The authors gratefully t h a n k Drs. Michael A. Rogers and Samuel J. Mazzer of Kent State University for translation of Russian literature and aid in macrophyte identification, respectively. Permission from the Twin Lakes Association to conduct the study in East Twin Lake is appreciated. REFERENCES

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352 Stewart, W.D.P., Mague, T., Fitzgerald, G.P. and Burris, R.H., 1971. Nitrogenase activity in Wisconsin lakes of differing degrees of eutrophication. New Phytol., 70: 497-509. Stockner, J.G. and Armstrong, F.A.J., 1971. Periphyton of the experimental lakes area, northwestern Ontario. J. Fish. Res. Board Can., 28: 215-229. Vanderhoef, L.N., Leibson, P.J., Musil, R.J., Huang, C.-Y., Fiehweg, R.E., Williams, J.W., Wackwitz, D.L. and Mason, K.T., 1975. Diurnal variation in algal acetylene reduction (nitrogen fixation) in situ. Plant Physiol., 55: 273-276. Wickstrom, C.E., 1980. Distribution and physiological determinants of blue-green algal nitrogen fixation along a thermogradient. J. Phycol., 16: 436-443. Wickstrom, C.E., 1984. Depression of Mastigocladus laminosus ( Cyanophyta ) nitrogenase activity under normal sunlight intensities. J. Phycol., 20: 137-141. Zuberer, D.A., 1982. Nitrogen fixation (acetylene reduction) associated with duckweed (Lemnaceae) mats. Appl. Environ. Microbiol., 43: 823-828.