Verh. Internat. Verein. Limnol. 2009, vol. 30, Part 6, p.

941944, Stuttgart, April 2009

by E. Schweizerbartsche Verlagsbuchhandlung 2009

Sandy shore benthic N2-fixation in Lake Malawi, Africa

Mangaliso J. Gondwe, Stephanie J. Guildford and Robert E. Hecky

Introduction
Epipelic N2-fixation in Lake Malawi (930"S to 1425"S) has been shown to be a very important local source of nitrogen (N) for epipelic algal growth, which supports highly diverse and populated endemic cichlid species (Higgins et al. 2001). The rocky habitat, however, covers only about 30 % of the lakes littoral area, while the remainder is almost entirely covered by sand (Ribbink et al. 1983). Despite sand covering a larger expanse of the littoral benthic zone with an almost entirely different biota from that of the rocky zone, N2-fixation in the lakes sand community has never been studied before. This is important particularly because algal growth in the lake has generally been perceived as N limited, which may become critical with the continued threat of eutrophication by increasing human populations and the associated land-use changes in the lakes catchment area (Guildford et al. 2003). Hecky et al. (2003) showed that phosphorus (P) loading to the lake is increasing more rapidly than N loading, so that the epilimnion may become more N limited, thereby causing harmful N2-fixing cyanobacterial species to proliferate in the lake. We report results of a study conducted on 2 sandy beaches in the more productive southern part of Lake Malawi. Study objectives were to (1) estimate N2-fixation rates on sandy sediments by using the acetylene reduction technique, and (2) evaluate the N input from epipsammic N2-fixation to the lakes epilimnion.
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25 cm long), which were incubated at various light levels for N2-fixation analysis using the acetylene reduction technique. Surface sediment samples were also analyzed for chlorophyll concentration, identification and enumeration of dominant algal cells, and Anabaena filaments and heterocysts.

Results
Anabaena filaments and heterocysts were rare in core samples collected from stations A and B and totally absent in core samples from station C at Senga Bay and from all stations along the SEA transect. All the stations were dominated by diatoms, particularly Nitszchia spp. and Navicula spp. (Gondwe 2004). Acetylene reduction was detected in all core samples, regardless of Anabaena filament presence. We detected N2-fixation in samples in the dark as well as in the light (Table 1). The observed N2-fixation has been reported here as dark and total N2fixation where total N2-fixation is the sum of dark and light N2-fixation in the samples. Spatial heterogeneity of both dark and total N2-fixation rates were observed along both transects. Along the Senga Bay transect, dark and total N2-fixation rates for 1999 were significantly higher (p = 0.009 and p < 0.001 respectively) at station C than at station A (Table 1). A similar trend was observed in 2002 along both transects; both dark and total N2-fixation rates increased with depth (Table 1). Both dark and total N2fixation rates were higher at the SEA than at Senga Bay (Table 1). Total N2-fixation rates were particularly higher (p = 0.001) at the SEA than at Senga Bay, while dark rates were only marginally higher (p = 0.119; Table 1). The mean total and dark N2-fixation rates observed in Lake Malawi were 38 g N m2 h1 (= 0.38 mg N m2 day1 during daylight and 32 g N m2 h1 (= 0.45 mg N m2 day1 during night), respectively, giving a total of 0.83 mg N m2 day1 (Table 1). Assuming a sand expanse of 1050 km2 for the whole lake, a total epipsammic N2fixation of 873 kg N day1 can be estimated for the lake. We also estimated the N requirement for primary produc0368-0770/09/0941 $ 1.00
by E. Schweizerbartsche Verlagsbuchhandlung 2009

Key words: acetylene reduction technique, epipelic algal growth, epipsammic N2-fixation, Lake Malawi

Methods
The study was conducted at Senga Bay and Mpwepwe beaches in the southern part of Lake Malawi (see Gondwe et al. 2008) between May and December 1999 and October and December 2002. Mpwepwe in the southeast arm (SEA) of the lake was sampled in October and December of 2002 only. At both study sites, nearshore-offshore transects were used (Table 1). Sand-sediment cores were taken using SCUBA and placed in dark and clear plastic glass cylinders (10 cm i.d.;

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Verh. Internat. Verein. Limnol. 30

Table 1. Mean chlorophyll concentrations, total and dark epipsamic N2-fixation rates (minimum, maximum and mean) along the Senga Bay and SEA transects. SD = Standard deviation. Note * = MayDec 1999, ** = SepDec 2002 and *** = Oct and Dec 2002. Transect/ Station Senga Bay * St A St C Senga Bay** St A St B St C SEA *** St D St E St F Mean Depth (m) 2 10 2 6.5 10 2 5 10 Chla Min Max Mean SD (mg m2) (Total N2-fixed g N m2 h1) 11.39 14.12 42.21 42.02 38.30 30.50 29.76 1.54 32.20 7.04 10.96 18.49 17.48 44.65 32.35 70.69 167.53 27.13 61.98 81.43 56.09 60.00 71.70 17.63 62.91 16.30 30.74 38.79 38.41 51.69 45.38 37.73 19.05 38.05 7.76 19.27 20.68 15.61 6.38 17.74 Min Max Mean SD (Dark N2-fixed g N m2 h1) 0.34 10.14 4.96 6.85 48.47 12.42 41.82 31.01 12.45 63.91 15.51 71.75 64.42 35.67 44.33 98.67 6.09 34.63 9.97 23.54 56.05 24.04 43.07 62.00 32.42 4.88 22.65 4.22 24.49 6.59 13.29 1.77 34.15

tion of epipsammic algal cells in the lake by using Redfields C:N ratio of 5.7:1 (by weight) and a mean epipsammic net CO2 fixation rate of 587 288 mg C m2 day1 from Bootsma & Hecky (2001). The mean N demand was estimated at 104 mg N m2 day1. Using a standard F-ratio value of 0.1 (Charpy-Roubaud et al. 2001), new production in epipsammic algal cells in Lake Malawi was estimated at 10.4 mg N m2 day1, with epipsammic N2-fixers contributing only about 8 %.
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Discussion
Sandy habitats are usually presumed less productive than rocky habits and therefore attract less research attention, including N2-fixation, than the rocky areas. In contrast, algal biomass as chlorophyll concentration in Lake Malawi is higher in sandy (1120 mg Chl m2) than on rocky (416 mg Chl m2) habitats (Gondwe 2004). However, epipsammic N2-fixation rates observed in this study were much lower than epilithic N2-fixation rates reported by Higgins (1999) and Higgins et al. (2001). The mean epipsammic N2-fixation rates in this study ranged from 1663 g N m2 h1 (Table 1), while epilithic N2-fixation rates ranged from 1082585 g N m2 h1 (Higgins et al. 2001). Higgins et al. (2001) observed highest rates at shallower depths (2 m depth) because N2-fixation was strongly light dependent. The N2-fixation was probably due to heterocystous Calothrix species, which were dominant at all sites and depths studied (Higgins et al. 2001). In contrast, we observed highest epipsammic N2fixation rates at deepest depths studied (10 m) along both transects. The scarcity of heterocystic Anabaena fila-

ments at our study sites agrees with findings by Haberyan & Mhone (1991) who, likewise, did not observe Anabaena filaments on sandy sediments at Cape Maclear (1400S, 3450E) in the southwest arm of the lake. As a result, nitrogenase activity measured in this study was not as light-dependent as observed in the pelagic algal community (Gondwe et al. 2008) or on rocky surfaces studied by Higgins et al. (2001). Both dark and light N2fixation rates were variable and of similar magnitudes at all stations along both transects (Table 1). The absence of filamentous cyanobacteria and heterocysts in some core samples studied shows that N2fixation was probably by bacteria rather than by filamentous cyanobacterial species. Some of the N2-fixing bacteria were probably photosynthetic (eubacteria) because in some cases light stimulation of N2-fixation was observed. Charpy-Roubaud et al. (2001) also observed nitrogenase activity in a sand community devoid of cyanobacteria filaments in Tikehau lagoon, French Polynesia. They attributed the nitrogenase activity to small, unidentified eubacteria observed in sand sediments they studied, which may be the same case in Lake Malawis sandy sediments. In addition, dark N2fixation rates in the Tikehau sediments contributed a high proportion of total N fixation, as in our Lake Malawi study. The observed trend of increasing epipsammic N2-fixation rates with water depth may be partly explained by the changing sediment structure with water depth. Sandy beaches in Lake Malawi have compacted fine grains nearshore and loose and coarse grains a little offshore (Fryer 1957). The compacted sediments nearshore limited the diffusion of acetylene molecules to surface

M.J. Gondwe et al., Sandy shore benthic N2-fixation

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reduction sites only, which may have had higher oxygen levels compared to sediments below. We suspect high oxygen levels further reduced the performance of nitrogenase complexes on surface sediments, particularly because the suspected N2-fixers were not heterocystic from microscopic observations. In contrast, acetylene diffused to more reduction sites deeper in coarse and loose sand grains at deeper stations, resulting in higher N2-fixation rates. Spatial dynamics of epipsammic N2-fixation rates in Lake Malawi could have been caused by a non-uniform spatial distribution of total organic matter (TOM) and N2-fixing organisms in the lake. A steady increase in TOM with water depth in Lake Malawi has previously been reported by Buat et al. (2002). Our study showed similar results, indicated by increasing benthic chlorophyll concentrations with water depth, particularly along the Senga Bay transect (Table 1). According to Capone (1988), the TOM provides a source of energy for certain heterotrophic bacteria to fix N2; hence, sites with higher TOM would show higher N2-fixation rates than sites with lower TOM. We did not study the spatial distribution of bacterial N2-fixers in the current study because we had anticipated that light-dependent heterocystic cyanobacteria would dominate N2-fixation both in the planktonic and benthic environments in the lake. This study was done concurrently with a planktonic N2-fixation study (Gondwe et al. 2008). Comparison of areal rates from the 2 studies shows that planktonic daily areal N2-fixation rate (501 g N m2 day1) was lower than the daily rate of 831 g N m2 day1 in the sand community; therefore, epipsammic N2-fixation provides a more concentrated source of new N, which could be sustaining epipsammic algal productivity in the lake. However, on a whole-lake scale, the contribution of
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planktonic N2-fixation to the total epilimnetic N budget was considerably higher at 13 mmol N m2 yr1 because of its enormous area (30 800 km2) compared to the small sandy littoral area (1050 km2). Higgins et al. (2001) obtained mean epilithic areal N2-fixation rates that ranged from 1082585 g N m2 h1 and an annual rate of 1.92 105 kg N y1. In the current study, the mean epipsammic N2-fixation rates ranged from 1663 g N m2 h1 (Table 1) and an annual rate of 3.18 105 kg N y1. Annual inputs of N to the epilimnion from various sources in Lake Malawi (Table 2) show that N2-fixation in the lake contributes <5 % of the total N input to the epilimnion, which suggests that N2-fixation may be a less important source of N to the epilimnion compared with atmospheric deposition, riverine, and upwelling sources. However, Guildford et al. (2000) have argued that the total N input from sources in Table 2 may be only <15 % of the epilimnetic algal N demand of 4000 mol N m2 day1. According to Guildford et al. (2000), water column nutrient regeneration processes (grazing, excretion, and mineralization) may be the most important factors that maintain high rates of primary productivity in the lake while algal biomas remains low.

Acknowledgements
This project was funded by the SADC/GEF Lake Malawi Biodiversity Conservation Project and by the Natural Sciences and Engineering Research Council of Canada (NSERC) through an individual research grant to Dr. Stephanie J. Guildford. We sincerely thank the Department of Fisheries Malawi, Dr. Harvey Bootsma (University of Wisconsin, Milwaukee) and Dr. Ralph E. Smith and Dr. Kistern Mller (University of Waterloo, Canada) for their wonderful contributions to the project.

Table 2. Annual inputs of combined N to the epilimnion of Lake Malawi. Table has been modified from Higgins (1999). The epilithic N input of 0.49 mmol N m2 yr1 has been recalculated from Higgins (1999) estimate of 1.92 105 kg N yr1. Source Atmospheric Riverine Upwelling Planktonic N2-fix Epipsammic N2-fix Epilithic N2-fix Other Total N Input mmol N m2 yr1 139 88199 23 13 0.74 0.49 ? 370481 N Input ( % of total) 2938 1854 56 34 0.150.20 0.100.13 100 Source Bootsma & Hecky (1999) Bootsma & Hecky (1999) Bootsma & Hecky (1999) Gondwe et al. (2007) this study Higgins (1999)

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Verh. Internat. Verein. Limnol. 30 Gondwe, M.J., S.J. Guildford & R.E. Hecky. 2008. Planktonic nitrogen fixation in Lake Malawi/Nyasa. Hydrobiologia. 596: 251267. Guildford, S.J., H.A. Bootsma, E.J. Fee, R.E. Hecky & G. Patterson. 2000. Phytoplankton nutrient status and mean water column irradiance in Lake Malawi and Superior. Aquat. Ecosyst. Health Manag. 3: 3545. Guildford, S.J., R.E. Hecky, W.D. Taylor, R. Mugidde & H.A. Bootsma. 2003. Nutrient enrichment experiments in tropical Great Lakes Malawi/Nyasa and Victoria. J. Gt. Lakes Res. 29: 89106. Haberyan, K.A. & O.K. Mhone. 1991. Algal communities near Cape Maclear, southern Lake Malawi, Africa. Hydrobiologia. 215: 175188. Hecky, R.E., H.A. Bootsma & M. Kingdon. 2003. Impact of land use on sediment and nutrient yields of Lake Malawi/Nyasa (Africa). J. Gt. Lakes Res. 29: 139158. Higgins, S.N. 1999. Epilithic nitrogen fixation in the rocky littoral zones of Lake Malawi, Africa. M.S. thesis, Univ. of Waterloo, Ontario, Canada. Higgins, S.N., R.E. Hecky & W.D. Taylor. 2001. Epilithic nitrogen fixation in the rocky littoral zones of Lake Malawi, Africa. Limnol. Oceanogr. 46: 976982. Ribbink, A.J., B.A. Marsh, A.C. Marsh, A.C. Ribbink & B.J. Sharp. 1983. A preliminary survey of the cichlid fishes of rocky habitats in Lake Malawi. S. Afr. J. Zool. 18: 149 310.

References
Bootsma, H.A. & R.E. Hecky. 1999. Nutrient cycling in Lake Malawi/Nyasa, p. 215241. In H.A. Bootsma & R.E. Hecky [eds.], Water Quality Report: Lake Malawi/ Nyasa Biodiversity Conservation Project. Buat, P., P.S. Ramlal & S.J. Guildford. 2002. The relationship between organic matter, invertebrates and bacteria in the sediments of Lake Malawi. Aquat. Ecosyst. Health Manag. 5: 307313. Capone, D.G. 1988. Benthic nitrogen fixation, p. 85123. In T.H. Blackburn and J. Sorensen [eds.], Nitrogen cycling in coastal marine environments. John Wiley & Sons. Charpy-Roubaud, C., L. Charpy & A.W.D. Larkum. 2001. Atmospheric dinitrogen fixation by benthic communities of Tikehau Lagoon (Tuamotu Archipelago, French Polynesia) and its contribution to benthic primary production. Mar. Biol. 139: 991997. Fryer, G. 1957. The trophic inter-relationships and ecology of some littoral communities of Lake Nyasa with special reference to the fishes, and a discussion of the evolution of a group of rock-f requenting Cichlidae. Northern RodesiaNyasaland Joint Fisheries Research Organisation. Gondwe, M.J. 2004. Planktonic and benthic N2-fixation in the sandy shores of Lake Malawi, Central Africa. M.S. thesis, Univ. of Waterloo, Ontario, Canada.

Authors addresses: M.J. Gondwe, C/O Malawi Fisheries Research Institute, Department of Fisheries, P.O. Box 27, Monkey Bay, Malawi. E-mail: gondwem2005@yahoo.com S.J. Guildford, R.E. Hecky, Department of Biology, University of Waterloo, Waterloo, Ontario, Canada, N2L 3G1. E-mail: sguildfo @scimail.uwaterloo.ca
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