Nitrogen fixation by naturally occurring duckweed ...

Nitrogen fixation by naturally occurring duckweed-cyanobacterial associations1 TRANPHUOC DUONG~ AND JAMES M. TIEDJE'

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Michigan State University on 07/11/11 For personal use only.

Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI, U.S.A. 48824 Accepted January 7, 1985 DUONG,T. P., and J. M. TIEDJE.1985. Nitrogen fixation by naturally occurring duckweed-cyanobacterial associations. Can. J. Microbiol. 31: 327-330. Nitrogen fixation as measured by acetylene conversion to ethylene was found to be common for duckweed blooms on ponds, lakes, and streams in Michigan. Twenty-six of 29 sites with duckweed sampled over a 2-year period showed acetylene reduction activity (ARA). These activities corresponded to N inputs of 3.7-7.5 kg N. ha-' per annual cycle for typical blooms but dense Lemna trisulca blooms could be 10 times greater. The ARA was stimulated five- to six-fold by light, was not removed when plants were shaken in water, and was usually associated with the leaves and not the roots. Colonies of heterocyst-bearing cyanobacteria of the genera Nostoc, Gloeotrichia, Anabaena, Calothrix, and Cylindrospermum were observed attached to the lower epidermis or in the reproductive pockets of leaves of Spirodela and Lemna plants but not on Wolfla plants. The ARA correlated reasonably well with the density of cyanobacterial colonies observed. The duckweed appeared to provide a more favorable environment for the cyanobacteria which should result in enhanced nitrogen inputs to aquatic and sediment environments harboring duckweed. The N2 fixation was not sufficient, however, to meet all the nitrogen needs of the duckweed bloom. DUONG,T. P., et J. M. TIEDJE.1985. Nitrogen fixation by naturally occurring duckweed-cyanobacterial associations. Can. J. Microbiol. 31: 327-330. La fixation de l'azote, mesurte par la conversion de I'acCtylkne en Cthylkne, semble commune a la reproduction intensive de la lentille d'eau (Lemna spp.) dans les ttangs, les lacs et les cours d'eau du Michigan. Vingt-six sur 29 sites CchantillonnCs au cours d'une pkriode de 2 ans ont present6 une activitt de reduction dlacCtylene par les lentilles (ARA). Ces activitks ont correspondu une consommation d'azote de 3,7-7,5 kg N-ha-' par cycle annuel pour les dCveloppements typiques, mais dans le cas des dCveloppements denses de Lemna trisulca, les activitCs pouvaient Ctre de 10 fois supkrieures. L'ARA a CtC stimulte par la lumikre, de cinq a six fois; elle n'a pas CtC supprimCe lorsque les plantes furent soumises a agitation et elle fut habituellement associte aux feuilles et non aux racines. Les colonies de cyanobactkries porteuses d'hCtCrocystes, des genres Nostoc, Gloeotrichia, Anabaena, Calothrix, et Cylindrospermum, ont CtC vues attachCes 21 I'Cpiderme infkrieur ou dans les poches reproductrices des feuilles de Spirodela et des plantes de Lemna, mais non des plantes de Wolfla. L'ARA a corrClC relativement bien avec la densite des colonies cyanobactCriennes observCes. Les lentilles d'eau ont paru foumir un environnement plus favorable pour les cyanobacteries, ce qui devrait se traduire par un apport d'azote supkrieur aux milieux ~Cdimentaireset aquatiques comportant des lentilles d'eau. La fixation de l'azote n'est toutefois pas suffisante pour rencontrer tous les besons en azote des lentilles d'eau en dCveloppement intensif. [Traduit par le joumal]

Introduction Duckweeds, members of the family Lemnaceae, are the smallest aquatic flowering plants. They are found floating on the surface of many ponds, shallow pools, and slow-moving streams as well as in some rice paddies. Their distribution is world wide (Daubs 1965). Rao (1953) reported that thick duckweed communities presumably modified the physical and chemical properties of the water underneath favoring the development of blue-green algae over the other algae, although any direct effect of duckweeds on the growth of these blue-green algae was not elucidated. Bottomley (1919) reported that exudates of nitrogen-fixing organisms and other bacteria promoted growth of Lemna. Coler and Gunner (1969) reported that the rhizosphere of Lemna minor was colonized by microorganisms. Studies by T. P. Duong (1972. Ph.D. thesis, Michigan State University, East Lansing), Finke and Seeley (1978), and Zuberer (1982) showed that duckweed blooms contain attached N,-fixing organisms. In this study we report on the quantity of acetylene reduction, the temporal and geographic distribution of the activity, and the species composition of the 'Published as joumal article No. 11443 of the Michigan Agricultural Experiment Station. 'Current address: Tropical Biological Nitrogen Research Center, University of Can Tho, Hau Giang, The Socialist Republic of Viet Nam. 3Author to whom reprint requests should be addressed.

duckweed-algal association. This paper summarizes the findings of T. P. Duong (Ph.D. thesis) as well as additional follow-up studies.

Materials and methods Duckweeds were identified using Daub's Monograph ofLemnaceae (1965). For the acetylene reduction assay, water containing duckweeds was collected and passed through a flour sifter with a metal screen of 1.5 mm square openings. The sifter retained Lemna and Spirodela. The filtrate, which contained Wolfla, was passed through a second modified sifter that was equipped with nylon netting of 35-pm pore size (Nitex, Tobler, Emst Traber, Inc., NY) which retained all Wolffia.Water passing this second filter was called the last filtrate. Since it was too time consuming to separate Lemna from Spirodela, they were assayed together for acetylene reduction. Lemna and Spirodela or Wolfla were removed from the sifter and transferred to 26-mL serum bottles. The last filtrate was used to bring the total volume in each bottle to 16 mL. Triplicate samples of the last filtrate, Wolfla and the mixture of Lemna and (or) Spirodela, were incubated with 1 mL (10%) acetylene for 1 h. The bottles were incubated at the water surface at the site of collection to mimic natural conditions. The reduction was stopped by injecting 0.5 mL of 2% HgCl, into the bottles. Following incubation and ethylene analysis the plants were removed, dried briefly by pressing them gently between paper toweling, and the wet weight was determined. Ten plants selected at random were saved for microscopic examination. One bottle from each category which had HgC12 added at the beginning of the incubation served as a control. Also as a control, duckweed samples were incubated for 1 h without acetylene to observe any plant production of

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TABLE1. Effect of plant part and light on in situ acetylene reduction by plants of Lemna and Spirodela from Wintergreen Lake Conditions

C2H4 formed (nmol-g wet wt.-'. h-')

Whole plants (light) Excised roots (light) Leaves only (light) Shaken water (light) Water (light) Whole plants (dark)

46.8 4.2 92.2 2.1 0.0 10.1

ethylene. Ethylene was quantified by flame-ionization gas chromatography (Varian 600-D) using a 1.O m x 3 mm Porapak N column. The ethylene data reported are means of three replicates. To determine the general occurrence of the Nn-fixing capacity of duckweed, samples were collected from surface waters along a 75mile route (I mile = 1.61 krn) between Kalamazoo and Lansing in southwest Michigan. The sites included lakes, large and small ponds, farm ponds, shallow intermittent ponds, and rivers; these environments are representative of duckweed habitats in the area. In the 1st year (1972), six sites were assayed each Monday from June 13 through September 11. In the 2nd year (1973) more sites were sampled, and they were assayed at I- to 2-week intervals from June 28 through August 7. The incubations were generally in mid-afternoon. Axenic cultures of Lemna perpusilla Torrey and Lemna gibba Linaeus obtained from J. A. D. Zeevaart (Plant Biology Laboratory, Michigan State University) were also tested for acetylene reduction. They were grown in Hunter's medium with or without combined nitrogen. The carbon source was either 1% sucrose (w/v) or 0.1% NaHCOs. They were grown in a temperature-controlled growth chamber at 20-22OC. The acetylene assay was performed with 5- or 7-day-old cultures. Fresh duckweeds were examined under a Leitz stereomicroscopeat lOOX magnification to locate and enumerate algal colonies. For morphologic studies and identification of algal species, the colonized leaves were dissected and the components examined by phase-contrast microscopy. In certain cases leaves were pressed to release the algal colonies from duckweed.

Results Duckweed samples collected from surface waters were found to reduce acetylene to ethylene. This reduction was not catalyzed by water surrounding the duckweed (Table I), by Wolfla, by axenic Lemna, or by field samples of Lemna and Spirodela treated with HgClz prior to incubation with acetylene. Also, duckweed samples did not produce ethylene when incubation without acetylene. As shown in Table 1, most of the acetylene reduction activity (ARA) was associated with the leaves (fronds) and not with the excised roots. The agent responsible for the reduction was firmly attached to the duckweed since minimal activity was found in the water fraction after shaking the plants in water for 5 min (Table 1). Data in Table 1 also show that ethylene production was light dependent since ARA in the light was five times greater than that of samples incubated in the dark. Microscopic examination of active duckweed samples revealed microcolonies of heterocystous cyanobacteria (Fig. I). The cyanobacteria were found attached to the lower epidermis of older leaves, inside the reproductive pockets, or in a few cases, attached to roots of mature Lemna or Spirodela plants. The colonies and immature plants in the pockets could be freed by squeezing the leaf with a dissecting needle. The colonies associated with the lower epidermis could be easily picked free from the host with dissecting needles.

RG.1. Cyanobacterial colony separated from lower epidermis of Lemna. The heterocysts are evident (arrows). Bar = 10 Fm. The majority of the algae observed were identified as Nostoc punctiforme, Gloeotrichiapisum, Anabaena sp., and Calothrix sp. with Cylindrosperum rnajus observed in one case (Table 2). Nostoc punctiforme was generally found in the pockets of Lemna minor and L. trisulca while G . pisum and Anabaena sp. were found on the underside of the leaves and on roots of L. trisulca and L. minor which had sunk below the surface. Lemna trisulca, which was generally more predominate under the layer of L. minor, showed greater ARA than L . minor (Table 2). Both Lemna and Spirodela had ARA capability and associated cyanobacteria while Wolfla had neither. The relative abundance of cyanobacterial colonies associated with duckweed corresponded reasonably well with the magnitude of the ethylene production rate (Tables 2 and 3). The apparent absence of cyanobacteria in a few cases where ARA was observed was probably due to the small subsample examined (10 plants) and the difficulty of exhaustively examining the plants although ARA by nonphototrophs cannot be ruled out. Acetylene reduction associated with duckweed communities occurred in samples from all six sites assayed the 1st year (Table 2) and in samples from 20 to 23 sites assayed in the 2nd year (Table 3). As shown in Table 2 lower quantities of ethylene were produced in June, while much higher quantities were generally produced in July, August, and September. The lower values in the ranges presented in Table 3 were usually from early in the season. Acetylene reduction was always highest in mature blooms and often with plants that had begun to yellow which was when cyanobacterial colonization was most abundant. The relative magnitude of acetylene reduction at the same locations was similar for the 2 years.Values for the Lawrence Rd. Pond and Wintergreen Lake were high, while those for the Potter Park Lagoon were low. Values for the 2nd year are generally higher than for the 1st year because of a more selective sampling for L. nisulca plants which had a higher activity. The effect of light and temperature on ARA by natural duckweed samples is indicated in Table 4. The colder, darker day on August 9 resulted in a much lower activity than occurred the previous and subsequent weeks. Covering the assay bottles with foil caused an approximately sixfold reduction in ARA (Table 4).

DUONG AND TIEDJE

TABLE 2. Occurrence of acetylene-reducing activity by the duckweed-cyanobacterial association in several lakes and ponds during year 1 Source


Wintergreen Lake

Potter Park Lagoon Potter Park Mill Pond Farm Pond Lawrence Rd. Pond Clear Lake

Ethylene produced (nmol .g-' h-I)"

Month

Relative abundance of cyanobacterial coloniesb

Plant speciesc

June July August September July -August July August September July -August June June

"Data are averages obtained from duplicate weekly samplings for the indicated time period. b + , No colonies, a few filaments; + + , small colonies, many plants without colonies; + + + , small colonies, more mature plants with colonies; + + + + , small and large colonies, colonies easily seen on many plants. 'S, Spirodeln; L. Lemna; W, Wolfla.

TABLE 3. Occurrence of acetylene-reducing capability associated with duckweed-algae associations obtained from a variety of surface waters in Michigan. Samples were collected weekly or biweekly from June 28 through August 7 of year 2 Ethylene produced (nmol .g wet wt.-' hK1)

-

Plant"

Source

Mill Pond Hickory Rd. Pond I Hickory Rd. Pond I1 Wintergreen Lake Wintergreen Pond I Wintergreen Pond II Osborne Pond 60 11412, Pond Lacey Rd, 2161, Pond Creek, Lacey Rd. Pond Church Pond, Lacey Butler Rd., S Pond Butler Rd., L Pond Outlet Pond Dowling, M-66 Pond Dowling, 3285 Pond Fawley's Pond I Fawley's Pond II Red Cedar River Potter Park Lagoon ~p

Range

Average

Gloeotrichia Few Gloeotrichia, Nostoc Nostoc None Present Few Few None None Cylindrospermum Few Few None None Fewc None None Few Few Few None None

Lawrence Road Pond Long Lake Pond Clear Lake

-

Algaeb

-

"Lm, L e m minor; ~ Lt, Lemna frisulca; Sp, Spirodela; W, Wolfla puncfam. bDensities of N,-fixing cyanobacteria were qualitatively examined on 10 plants per site. The species were identified in some of the most active samples; they were Gloeofrichia pisum, Nosroc puncfiforme, and Cylindrospermum majus. 'Sample contained a large quantity of planktonic algae.

The nitrate content of the water surrounding the samples was often not detectable (detection limit of 0.2 ppm NO, N) and was never more than 0.6 ppm NO, N. Axenic cultures of L. perpusilla and L. gibba grown in a nitrogen-free medium turned yellowish within 10 days and

eventually died. Upon introduction of a growing unialgal culture of Aphanizomenon, the chlorotic Lemna recovered within 1 week and reproduced normally for several months. However, when field samples of duckweed with high ARA were placed in a nitrogen-free medium and incubated in growth chambers,

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TABLE 4. Environmental effects on acetylene reduction associated with the duckweed-cyanobacterial association Date

Condition, water temperature

Ethylene production (nmol g ' .h- ')

July 31 August 7 August 14 August 21

Full sun, 26°C Cloudy, rainy, 24°C Partly cloud, 25°C Aluminium foil covered (dark) Not covered (natural light) Aluminium foil covered (dark) Not covered (natural light)

112.2 11.2 54.6 15.8 91.0 25.0 156.4


August 28

-

the plants gradually yellowed and ethylene production declined until, at 2 weeks, the majority of the plants were yellow or bleached and no ARA remained. In the same medium with added nitrogen, the plants remained green and grew readily.

Discussion The presence of heterocyst-bearing cyanobacteria and the reduction of acetylene to ethylene strongly suggests that the duckweed-cyanobacteria association has the capacity to fix nitrogen. This finding is consistent with that of Zuberer (1982) who found acetylene reduction by duckweed mats in Texas and Florida. Furthermore the data show that the N2fixation capability rests entirely with the algae and not with the host plant nor the surrounding water. In other studies other nitrogen-fixing organisms, e.g., Rhodopseudomonas (Finke and Seeley 1978) and Klebsiella (Zuberer 1982), were also found on similar plants but their contribution to N2 fixation appears to be minor compared with that by the cyanobacteria. The most common algae observed in these studies were species of Gloeotrichia, Anabaena, Calothrix, and Nostoc; the first three were also found by Finke and Seely (1978) on other freshwater macrophytes. Using the theoretical ratio of 3 :2 (ethylene produced :nitrogen produced), an average bloom density of 4 g wet weight (wt.).lO cm-', and common values of 100-200 nmol ethylene g wet wt.-' h-' , the quantities of nitrogen fixed would be 3.7 -7.5 g nitrogen. ha-' h-' . Assuming these values were a reasonable average for 10 h per day over 100 days, the duckweed-algal association would have contributed 3.7-7.5 kg nitrogen aha-' year-'. While this estimate is reasonable for a single layer of L . minor plants growing on the water surface, it is too low for L. trisulca plants which may extend in masses 30 cm below the surface. In the latter case, reasonable estimates of nitrogen fixed per surface area ranged up to 10 times greater than the above. Thus, it appears that over a period of years the duckweed-cyanobacterial association can be a major source of nitrogen to surface waters and sediments. This source is probably more important to shallow ponds or swampy areas than to larger lakes. The widespread occurrence of the nitrogen-fixing capability in natural duckweed populations from a variety of habitats and over 2 years suggests that it is a rather common phenomenon. The relationship between the duckweed and the cyanobacteria is probably not best described as symbiotic since both organisms are known to succeed separately and because the association could not maintain itself in a nitrogen-free medium.

.

.

Commensalism is probably more descriptive of this association since the cyanobacterial epiphytes appear to benefit by using the duckweed for physical support, protection against direct sunlight, and as a source of carbohydrates and growth factors. The inhibition of N2 fixation in planktonic algae by direct sunlight was also shown in parallel studies at the same sites (T. P. Duong, Ph.D. thesis). Zuberer (1982) found higher fixation in thicker mats, analogous to our experience with L. trisulca, further supporting the importance of the environment provided by the duckweed. Also, in support of the beneficial shading effect, cyanobacteria were never found on the upper surface of the leaves. They were, however, using light as their primary energy source since covering the bottles with foil greatly reduced ethylene production. Cyanobacteria are known to utilize some organic materials (Fay 1976), thus duckweed excretions could provide organic compounds stimulatory to cyanobacterial growth. The fact that both-higher ~~anobact-erial populations and N2 fixation activity were associated with mature and deteriorating blooms suggests that the cyanobacteria may have been responding to a greater quantity of excretions and lytic products. The host duckweed in natural samples did not receive adequate nitrogen from the epiphytic cyanobacteria in a nitrogenfree medium. Finke and Seeley (1978) and Zuberer (1982) estimated that N2 fixation would supply only 30-50 and 15-20%, respectively, of the nitrogen of host macrophytes. However, we demonstrated in a laboratory study using axenic Lemna and Aphanizomenon, that N2 fixation could supply the total nitrogen needs of the plants. In this case, however, the cyanobacterial population density was much higher than found for the associated cyanobacteria in nature.Thus, it is possible that the host plant may receive a portion but not all of its nitrogen from excretions or lysis of the associated cyanobacteria. This could provide a slight competitive advantage for duckweeds in nitrogen-limited environments.

Acknowledgements The authors thank M. Cichowski for help with sampling and by the ethylene analyses. The work was supported in Office of Water Resources Research, United States Department of Interior. BOTTOMLEY, W. B. 1919. The effect of nitrogen fixing organisms and nucleic acid derivatives on plant growth. Proc. R. Soc. London, Ser. B, 91: 83-95. COLER,R. A., and H. B. GUNNER. 1969. The rhizosphere of an aquatic plant (Lemna minor). Can. J. Microbiol. 15: 964-966. DAUBS, E. H. 1965. A monograph of Lemnaceae. 111. Biology Monograph No. 34. The University of Illinois Press, Urbana, IL. FAY, P. 1976. Factors influencing dark nitrogen fixation in a blue-green alga. Appl. Environ. Microbiol. 31: 376-379. FINKE,L. R., and H. W. SEELEY, JR. 1978. Nitrogen fixation (acetylene reduction) by epiphytes of freshwater macrophytes. Appl. Environ. Microbiol. 36: 129- 138. RAO,C. B. 1953. On the distribution of algae in a group of six small ponds. J. Ecol. 41: 62-71. ZUBERER, D. A. 1982. Nitrogen fixation (acetylenereduction) associated with duckweed (Lemnaceae)mats. Appl. Environ. Microbiol. 43: 823-828.