APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1976, p. 942-948 Copyright © 1976 American Society for Microbiology
Vol. 31, No. 6 Printed in U.S.A .
Influence of Sewage Discharge on Nitrogen Fixation and Nitrogen Flux from Coral Reefs in Kaneohe Bay, Hawaii ROGER B. HANSON'* AND K. GUNDERSEN Department of Microbiology, University of Hawaii, Honolulu, Hawaii 96822 Received for publication 15 December 1975
Nitrogen fixation was investigated in Kaneohe Bay, Oahu, Hawaii, a subtropical eutrophic estuary, by using the acetylene reduction technique on algal samples. No active, planktonic, N2-fixing blue-green algae or bacteria were observed. However, Calothrix and Nostoc capable of fixing N., were cultured from navigational buoys and dead coral heads. Nitrogen fixation associated with these structures was greater in the middle sector than in the south and north sectors of the estuary. Experiments demonstrated that the fixation was photosynthetically dependent. Examination of the data showed that there was no significant correlation between rates of nitrogen fixation and concentration of combined nitrogen compounds in the Bay water. Fixation was significantly correlated to the inorganic N/P (atomic) ratio in the south and middle sectors but not in the north sector. The nutrient data indicate there was a flux of combined nitrogen, but not phosphate, from the reef flats.
Nitrogen fixation by the planktonic microorganisms has been reported for open ocean (8, 15) and bay waters (25). However, most marine nitrogen-fixing, blue-green algae are found in the intertidal zone (1), along rocky shores and in brackish water (20). Johannes et al. (13) located within the coral reef complex a variety of blue-green algal communities that were able to fix nitrogen. From available data, biological fixation of nitrogen may be very important to coral reef communities, provided other essential nutrients, such as phosphate, are not limiting (28, 30). Coral reef communities generally exist in environments that do not have wide fluctuations in either physical or chemical parameters (16). Thus, some of these communities could have narrow stress tolerances. Johannes (12), in fact, presented evidence that some are quite susceptible to various forms of pollution. The coral reefs in Kaneohe Bay have been exposed over the last 2 decades to sewage discharge, siltation from heavy urbanization along the shore, and nutrient-rich runoff, all of which have contributed to the killing of the coral communities in several areas of the Bay (4, 18). The destroyed reefs, still standing erect, have subsequently been colonized by an entirely new population of marine organisms (6, 12, 18), including nitrogen-fixing algae. Kaneohe Bay is a subtropical estuary on the I Present address: University of Georgia, Marine Institute, Sapelo Island, Ga. 31327.
northeast coast of Oahu, Hawaii (Fig. 1), located at 21028' N, 157°48' W. Its area and volume are, at mean sea level, 4.60 x 107 m2 and 2.56 x 10 m3, respectively (5). On the basis of water circulation, topography, and nutrient concentration, the Bay can be divided into three sectors: south, middle, and north sectors (Fig. 1). The south sector receives the effluents from the Kaneohe Municipal Sewage Treatment Plant, with a daily discharge of 12,000 m:1 *of secondary-treated sewage, and the Kaneohe Marine Corps Air Station sewage treatment plant, which discharges 3,000 m:1 of secondarytreated sewage per day. In addition to the sewage, the south sector receives, during periods of rain, considerable amounts of silted freshwater through streams and through runoff from the urban and agricultural coastlands. The middle sector opens directly to the ocean through the South Channel and over the barrier reef and is the least influenced by terrestrial runoff. The largest portion of oceanic water entering the middle sector passes over the barrier reef and turns north. The north sector is the most open to the ocean but is considerably influenced by freshwater runoff from streams that empty into the sector. Due to the water movements in the south and middle sectors, water is continuously leaving the north sector through the North Channel, even during incoming tides. In this paper, the influence of the nutrient gradient on the nitrogen-fixing population and the flux of combined nitrogen from the reefs in Kaneohe Bay will be described. 942
VOL. 31, 1976
N2 FIXATION AND N FLUX FROM REEFS
943
FIG. 1. Kaneohe Bay and its location on the northeast coast of Oahu (insert): sewage plant 1 (Kaneohe Municipal) and sewage plant 2 (Kaneohe Marine Corps Air Station) with lines to outfall area; basic current patterns -incoming tides (solid arrow), outgoing tides (broken arrow); major navigational channels, North and South Channels at the respective ends of the extensive outer barrier reef with Kapapa Island; location of the thirteen algal stations (navigational buoys, 1-8, and coral reefs, A-E) in the south, middle, and north sectors of Kaneohe Bay. Coral reefs are outlined by heavy irregular lines.
MATERIALS AND METHODS Sampling and assay of in situ nitrogen fixation. In situ rates of nitrogen fixation were determined by the acetylene reduction method (9). Thirteen stations were selected for this study, representing three different environments in terms of productivity and nutrients (Fig. 1). Algal material encrusting navigational buoys and coral fragments, 5 to 10 mm in size, which were chipped from the surface of dead coral heads (Porites compressa), were suspended in untreated bay water (5 ml). Acetylene reduction experiments were run in triplicate in 38-ml serum bottles sealed with rubber serum stoppers. A 5-ml portion of acetylene (Matheson Gas Co.) was injected into the bottles with a hypodermic syringe, and the excess gas pressure was released through another needle. Final partial pressure of acetylene in the reaction vessel was ca. 0.13 atm. The samples were usually collected and incubated in a water bath
held at sea level at 25 to 26 C between the hours of 0900 and 1300. Samples were not agitated except by the boat motion. After 2 h, gas samples were transferred from the serum bottles into 4-ml Vacutainers using a double-end needle. The gasses (0.5 ml) in the Vacutainers were separated on a stainless-steel Porapak R column (5 feet by 0.125 inch [ca. 152.4 by 0.32 cm]) at 60 C. The ethylene concentration was determined on an Aerograph gas chromatograph, model A-90-P, fitted with a Beckman hydrogen flame ionization detector. Conversion factor. The amount of nitrogen fixed was calculated from the amount of ethylene formed using a ratio of 3:1 to convert ethylene produced to nitrogen fixed (9). An experiment was undertaken to compare the acetylene method and the '5N method, using a dead coral head. Dried coral fragments were pulverized, and 100-g samples were dissolved in 10% HF, centrifuged, and neutralized (pH
944
HANSON AND GUNDERSEN
7.0). The dissolved organic nitrogen was digested in a micro-Kjeldahl, and the NH4 was converted to N2 with alkaline potassium bromide. The isotopic abundance ('5N/'4N ratio) was determined on a modified CEC mass spectrometer, type 21-103C. We found a ratio of 3.3:1. Chlorophyll a (22) and dry weight (100 C for 24 h) were used as a measure of biomass (algal material and coral fragments, respectively), and nitrogen fixation was expressed in terms of amount of nitrogen fixed/unit of biomass per unit of time. Controls containing 1 ml of 50% trichloroacetic acid or 2 ml of saturated NH4Cl solution were run with each acetylene reduction assay. At extremely low nitrogen fixation rates, corrections were made for a small amount of ethylene that was present as a contaminant in the acetylene. Nutrient analysis. Bay water samples were taken in polyethylene bottles at a depth of 0.5 m and analyzed for nitrate, nitrite, and phosphate by the procedures described by Strickland and Parsons (24). Ammonium concentration was determined by the phenolhypochlorite method of Sol6rzano (19), modified by heating the reaction mixture for 10 min at 60 C and cooling at room temperature for 10 min before measuring the extinction spectrophotometrically at 640 nm. Isolation of nitrogen-fixing blue-green algae. The selective enrichment procedures used to isolate nitrogen-fixing blue-green algae were basically those of Allen and Stanier (2). The medium was prepared with open ocean surface water, low in combined nitrogen and phosphorus, supplemented with trace metals and vitamins (3). Planktonic nitrogen fixation. Surface (0.5 m) and subsurface (2 m) water samples, as well as horizontal (0.5 and 2 m) plankton tows (28-,um mesh net), were collected in the middle and south sectors of Kaneohe Bay on several occasions during April and May, 1971. Attempts were also made to determine nitrogen fixation by planktonic organisms by filtering 4.3 liters of Bay water through nitrogen-free acetate and glass filters (pore size, 0.5 ,im; 250-mm Hg vacuum) and resuspending the organisms in 5 ml of filtered Bay water. Enrichment cultures were prepared using 50% artificial seawater for nitrogenfixing blue-green algae and a variety of nitrogenfixing bacteria (2, 3; R. B. Hanson, Ph.D. thesis, Univ. of Hawaii, Honolulu, 1974). Water samples, plankton catch, resuspended organisms, and enrichment cultures were assayed for acetylene reduction.
RESULTS Planktonic nitrogen fixation. Nitrogen fixation was not detected in any of the plankton catches or water samples. Organisms in the water samples concentrated by filtration again gave negative results. On one occasion, a nitrogen fixer belonging to the genus Azotobacter, which demonstrated acetylene reduction, was cultured from the Bay water. All other cultures were negative for acetylene reduction. Nitrogen fixation by algal material from buoys. Nitrogen fixation was detected in the
APPL. ENVIRON. MICROBIOL.
algal material encrusting navigational buoys and in dead coral heads. This nitrogen-fixing activity was highest in the middle sector, compared with the south and north sectors. Stations 5 and C in the middle sector were selected for preliminary studies to determine optimal assay conditions. Algal samples showed a linear rate of fixation (acetylene to ethylene) with time up to 4 h, after which the rate decreased. An enzyme saturation experiment was done to determine the level of acetylene required for maximum production of ethylene by the nitrogenase system. A partial pressure of 0. 13 atm of acetylene was required for maximum production of ethylene. Under the experimental conditions used in these assays, reduction was found to be proportional to the amount of biomass (chlorophyll a and dry weight) in the assay. The rate of acetylene reduction in dark bottles was only 5% of that in light bottles. The precision of the assay procedure for buoy and dead coral material was examined statistically using four sets of algal material, each in triplicate. A mean rate of reduction of 258 nmol of ethylene formed/mg of chlorophyll a per h, with a coefficient of variation of 0.08, for the buoy material and a mean rate of 633 nmol of ethylene formed/ g (dry weight) per h, with a coefficient of variation of 0.27, for the dead coral material was calculated. Nitrogen fixation associated with algal material on buoys in relation to plant nutrients. The rates of nitrogen fixation associated with algal material encrusting buoys and the levels of three plant nutrients in the surrounding water at eight stations (1 through 8) are shown in Fig. 2. Mean fixation rates for stations 1 through 4 in the south sector for June and July, 1971, ranged from 0.11 to 0.60 gg of N/mg of chlorophyll a per h where the plant nutrients were most abundant. The mean rates of fixation at stations 5 through 7 in the middle sector ranged from 1.64 to 1.92 ,tg of N/mg of chlorophyll a per h where the plant nutrients were diluted with ocean water. In the north sector at station 8, the mean rate of 0.28 ,ug of N/mg of chlorophyll a per h was similar to that in the south sector, but the ammonia and phosphate concentrations were lower. The data were examined statistically for correlation between the rates of nitrogen fixation and concentration of phosphate, nitrate, ammonium, and the inorganic N/P ratios (NO3 + NH4+/PO4-3). No correlation was found between the fixation rates and the inorganic nitrogen compounds. However, there was an inverse correlation (P = 0.05; r = -0.92) between nitrogen fixation and the phosphate concentration for all stations ex-
VOL. 31, 1976
N2 FIXATION AND N FLUX FROM REEFS
I mitregeg
I smmsuium-U
tiiatis.
S mitrate-N
ipbsspkate-P
I2.0
1.6
1.6
II
Il
-
,2 E
945
11.2 3 0~~~~
0.8
10.8
'.4
i
0
.,i
i
r 0.4
0.4
0 Ii
I*-. m
mm
1
2
i
3
I_
I
*'IL,= IU 6 7 5 II.
.T
-
Eh
4
Statisis
FIG. 2. Rate of nitrogen fixation in the algal material from buoys and the concentration of some dissolved nutrients in the water (0.5 m) at each station through the estuary. Samples were collected between 0900 and 1200 h and were incubated for 2 h.
cept station 8. The atomic ratio of inorganic nitrogen to phosphorus ranged from 1.6:1 to 2.9:1 in the south sector and 3.9:1 to 6.6:1 in the middle sector and was 4.8:1 in the north sector. A positive correlation (P = 0.05; r = 0.70) between the rates of nitrogen fixation and the NI
I
uitreguo
I
fixat
ion
itrate-N
taummelii
-N
*phesphatg-P
1.2
12.!5
10.C
I
1.0
P ratio was calculated. 0.8 a Nitrogen fixation on coral reefs. In the sum7.555 mer of 1972 the activity of nitrogen-fixing popuI lations on dead coral heads at five stations (A It I through E) in the estuary was determined (Fig. 5.~ 5.0 3). A mean fixation rate of 4.8 ug of NIg per h a: (range, 2.8 to 7.1 A.tg of N/g per h) was found at station A. At station B, in the south sector, the a 2.5 mean rate of 6.1 ,tg of N/g per h (range, 4.1 to 0.2 I 7.5 ,tg of NIg per h) was measured. In the a middle sector, at station C, the mean rate of Wb fixation, 12.5 jig of NIg per h (range, 10.3 to c E 0 Stati INs 15.8 ,ug of NIg per h), was consistently several times higher than that in the south sector. The FIG. 3. Relationship between rates of nitrogen fixation by dead coral fragments and the concentrations mean fixation rate at station D was 2.4 ug of NI of three plant nutrients in the overlying reef waters at g per h (range, 2.1 to 3.0 ,g of N/g per h). The stations A through E in Kaneohe Bay. mean fixation rate at station E in the north sector was 0.9 jig of N/g per h (range, 0.5 to 1.9 ,ug of NIg per h), even lower than the rates in pling in all three sectors. Since the nitrogen concentrations in the reef water did not agree the sewage-influenced south sector. Nutrient concentrations in reef flat water. with the values found in the channel water, As was the case with the phosphate concentra- another set of experiments was done to detertion in the water surrounding the navigational mine the nitrogen levels in the two habitats at buoys, the phosphate in the reef flat water the same time. The concentrations were essenshowed decreasing concentrations from the tially as reported in Fig. 2 and 3 for the two south towards the north sector. The concentra- bodies of water. The rates of fixation associated tions of ammonium and nitrate, on the other with dead coral heads were compared statistihand, remained fairly constant in the water cally to the concentrations of the nutrients, and this time fixation was not significantly correover the reef flats during the periods of sam-
I
A
B
0.6
0.4
946
HANSON AND GUNDERSEN
lated to the concentrations of phosphate, ammonium, and nitrate or the N/P ratios in the reef's overlying water. Nitrogen-fixing blue-green algae isolated from algal material. Since the reduction of acetylene was primarily light dependent, bluegreen algae were suspected of being the major nitrogen fixers. Two types of nitrogen-fixing blue-green algae were isolated from the dead coral surfaces and algal material from buoys, using a nitrogen-free enrichment medium. They were identified as belonging to the genera Calothrix and Nostoc, and both exhibited acetylene reduction in culture.
DISCUSSION A search for nitrogen-fixing activity in the free water of Kaneohe Bay was unsuccessful, except under one cultural condition. The Azotobacter cultured from the water was unable to sustain growth in the growth medium prepared with 100% Bay water. Even though planktonic nitrogen-fixing microorganisms as well as planktonic symbionts may exist in Kaneohe Bay, they were undetected by the enrichment culture techniques and the acetylene reduction method. It is concluded that active planktonic nitrogen-fixing organisms are either rare or non-existent in the water of Kaneohe Bay. In contrast, N2 fixation was found on dead coral and floating structures. The blue-green algae Calothrix and Nostoc were found to be predominant in the material from buoys and dead coral heads. There is little, if any, high-energy surf in the lagoon, except on the barrier reef, to wash these organisms from structures and into the water. Heterotrophic fixation in reef flat sediment will be reported elsewhere. It is generally considered that nitrogen fixation by blue-green algae is suppressed by organic and inorganic nitrogen compounds (7, 14). However, Horne and Fogg (10), Stewart et al. (23), and Vanderhoef et al. (26, 27) found a more rapid reduction of acetylene in eutrophic lakes than in oligotrophic lakes. In Kaneohe Bay, it was found that the fixation was higher in the middle sector than in the south and north sectors. It was suspected that nitrogen fixation was inhibited by the nitrogen-rich water in the south and north sectors. However, when the data were analyzed statistically, there was no apparent negative correlation between nitrogen fixation by the coral and buoy material and the nitrate and ammonium concentrations, separately or together, in the estuary. In addition, the data in Fig. 3 illustrate that the concentration of inorganic nitrogen in reef water does not control blue-green algal nitrogen fixation on
APPL. ENVIRON. MICROBIOL.
the reef flats (cf. station C). Horne and Fogg (10) also reported that an inverse correlation between nitrogen fixation and the levels of inorganic combined nitrogen was not found in Lake Windermere, England. The inverse correlation found between nitrogen fixation and phosphate concentration in the main channel is probably not related to phosphate inhibition in Kaneohe Bay, since it was later determined that there was no significant inhibition or stimulation of acetylene reduction in the buoy material at station 5 when it was further enriched with phosphate (Hanson, Ph.D. thesis). The inorganic N/P ratios in the Bay water were also compared to the rates of N2 fixation to determine if fixation activity was related to or regulated by this ratio. The data (Fig. 2) indicated a positive relationship, whereas there was no relationship between fixation and N/P in coral reef water. The reason for this is still not understood. To our knowledge, there have been no reports on the relationship between periphyton nitrogen fixation and inorganic N/P ratios in coastal and estuarine waters. Recently, however, Vanderhoef et al. (27) have shown that N, fixation was related to some extent to the P/N ratio in lake waters, in contrast to what was found in Kaneohe Bay. There have been reports of nitrogen flux, which was attributed to nitrogen fixation on coral reefs in the Marshall Islands (13, 28). It was also discovered that there was a flux of inorganic nitrogen from reef flats in Kaneohe Bay. The high ammonium-to-nitrate concentration in the water directly over the coral community compared to the low ammonium-to-nitrate concentration in the main channel (middle sector) suggests that nitrogen is being released from the reef. The source of this nitrogen is due, in part, to the high rate of fixation in the community (Fig. 3). Although some of the fixed nitrogen is recycled within the community, nitrogen-fixing algae are known to release newly synthesized nitrogen products (Hanson, Ph.D. thesis). The dissolved organic nitrogen excreted is either assimilated or deaminated by associated microorganisms to ammonium, of which some is oxidized to nitrate. Nitrite in the water was almost undetectable (less than 0.08 ,ugatom/liter), indicating that nitrosofication and nitrification are closely coupled in the benthic community (28, 29). The low nitrogen content in the channel water, negligible terrestrial influence, and high fixation rates in the coral reefs at station C, high ammonium and nitrate content in the reef water, and the decreasing concentration of phosphate in both habitats indicate that there is a nitrogen flux from the coral reefs in the middle sector.
N2 FIXATION AND N FLUX FROM REEFS
VOL. 31, 1976
Unlike ammonium and nitrate, phosphate in the reef flat water decreased with distance from the phosphate-enriched south sector. This suggests that the source of phosphate is primarily the sewage discharge (cf. 4). Since there was no export of phosphate from the reef flats, one is led to believe that the phosphate is probably imported into and recycled within the benthic community. Johannes et al. (13) also reported that no flux of phosphate occurred from the reef community into the overlying water at Eniwetok and concluded that there must be an unusually efficient recycling of the phosphate within the benthic community. The nitrogen cycle is extremely complex, and most of the processes within the cycle are not defined for Kaneohe Bay. The inputs of nitrogen into the estuary are sewage discharge, stream runoff, ocean, atmospheric fallout, and biological nitrogen fixation. The outputs are relatively few: ocean, sedimentation, and denitrification. Based on a preliminary nitrogen budget for Kaneohe Bay (Hanson, Ph.D. thesis), it was estimated that the nitrogen flux from the reefs balanced the sewage input. In contrast, benthic nitrogen fixation represented approximately 10% of the sewage discharge (Hanson and Gunderson, submitted for publication). Nitrogen fixation in the south sector of Kaneohe Bay is probably not influenced by the concentration of combined nitrogen in the water, as evident from the fixation on the reefs and the inorganic nitrogen concentrations in the water over the reefs in the middle sector. There has been considerable evidence that organic and inorganic compounds in sewage, industrial wastes, and runoff from domestic and agricultural land inhibit nitrogen fixation by blue-green algae (11, 21, 23, 31). Heavy metals, herbicides and insecticides, hydrocarbons, and various other toxic materials are potential inhibitors of the process and are no doubt widely distributed in Kaneohe Bay. LITERATURE CITED 1. Allen, M. B. 1963. Nitrogen-fixing organisms in the sea, p. 85-92. In C. H. Oppenheimer (ed.), Symposium on marine microbiology. Charles C Thomas, Springfield, Ill. 2. Allen, M. M., and R. Y. Stanier. 1968. Selective isolation of blue-green algae from water and soil. J. Gen. Microbiol. 51:203-209. 3. Antia, J. J., and R. Kalmakoff. 1965. Growth rates and cell yields from axenic mass cultures of fourteen species of marine phytoplankton. Fish. Res. Board Can. Manuscript Rep. Ser. No. 203, Ottawa. 4. Banner, A. H., and J. H. Bailey. 1970. Effects of urban pollution upon a coral reef system. Hawaii Inst. Mar. Biol. Tech. Rep. No. 25, Kaneohe. 5. Bathen, K. H. 1968. A descriptive study of the physical oceanography of Kaneohe Bay, Oahu, Hawaii. Ha-
947
waii Inst. Mar. Biol. Tech. Rep. No. 19, Kaneohe. 6. DiSalvo, L. H., and K. Gundersen. 1971. Regenerative functions and microbial ecology of coral reefs. I. Assays for microbial population. Can. J. Microbiol. 17:1081-1089. 7. Fogg, G. E., and W. D. P. Stewart. 1965. Nitrogen fixation in blue-green algae. Sci. Prog. 53:191-201. 8. Goering, J. J., R. C. Dugdale, and D. W. Menzel. 1966. Estimates in situ rates of nitrogen uptake by Trichodesmium sp. in the tropical Atlantic Ocean. Limnol. Oceanogr. 11:614-620. 9. Hardy, R. W. F., R. C. Bums, and R. D. Holsten. 1974. Application of the acetylene-ethylene assay for measurement of nitrogen fixation. Soil Biol. Biochem. 5:47-81. 10. Home, A. J., and G. E. Fogg. 1970. Nitrogen fixation in some English lakes. Proc. R. Soc. London Ser. B 175:351-366. 11. Horne, A. J., and C. R. Goldman. 1974. Suppression of nitrogen fixation by blue-green algae in a eutrophic lake with trace additions of copper. Science 183:409411. 12. Johannes, R. E. 1973. Coral reefs and pollution, p. 364375. In Mario Ruivo (ed.), Marine pollution and sea life. Unipub, Inc., New York. 13. Johannes, R. E., et al. 1972. The metabolism of some coral reef communities: a team study of nutrients and energy flux at Eniwetok. Bioscience 22:541-543. 14. Jurgensen, M. F. 1973. Relationship between nonsymbiotic nitrogen fixation and soil nutrient status-a review. J. Soil Sci. 24:512-522. 15. Mague, T. H., N. M. Weare, and 0. Holm-Hansen. 1974. Nitrogen fixation in the North Pacific Ocean. Mar. Biol. 24:109-119. 16. Odum, E. P. 1971. Fundamentals of ecology. W. B. Saunders Co., Philadelphia. 17. Schollhorn, R., and R. H. Burris. 1967. Acetylene as a competitive inhibitor of nitrogen fixation. Proc. Natl. Acad. Sci. U.S.A. 58:213-216. 18. Smith, S. V., K. E. Chave, and T. 0. Kam. 1973. Atlas of Kaneohe Bay: a reef ecosystem under stress. University of Hawaii Sea Grant Program (TR-72-01), University of Hawaii Press, Honolulu. 19. Sol6rzano, L. 1969. Determination of ammonia in natural waters by the phenylhypochlorite method. Limnol. Oceanogr. 14:799-801. 20. Stewart, W. D. P. 1967. Nitrogen turnover in marine and brackish habitats. II. Use of '5N in measuring nitrogen fixation in the field. Ann. Bot. 31:395-407. 21. Stewart, W. D. P. 1972. Algal metabolism and water pollution in the Tay Region. Proc. R. Soc. London Ser. B 71:209-224. 22. Stewart, W. D. P., G. P. Fitzgerald, and R. H. Bums. 1968. Acetylene reduction by nitrogen fixing bluegreen algae. Arch. Mikrobiol. 62:336-348. 23. Stewart, W. D. P., T. Mague, G. P. Fitzgerald, and R. H. Burris. 1971. Nitrogenase activity in Wisconsin lakes of differing degrees of eutrophication. New Phytol. 70:497-509. 24. Strickland, J. D. H., and T. R. Parsons. 1972. A practical handbook of seawater analysis. Bull. Fish. Res. Board Can. Bull. No. 167, Ottawa. 25. Sugahara, I., T. Sawada, and A. Kawai. 1971. Microbiolgical studies on nitrogen fixation in aquatic environments. VI. On the in situ nitrogen fixation in water region. Bull. Jpn. Soc. Sci. Fish. 37:1093-1099. 26. Vanderhoef, L. N., B. Dana, D. Emerich, and R. H. Burris. 1972. Acetylene reduction in relation to levels of phosphate and fixed nitrogen in Green Bay. New Phytol. 71:1097-1105. 27. Vanderhoef, L. N., C. Huang, R. Musil, and J. Williams. 1974. Nitrogen fixation (acetylene reduction) by phytoplankton in Green Bay, Lake Michigan, in
948
HANSON AND GUNDERSEN relation to nutrient concentrations. Limnol. Ocean-
19:119-125. 28. Webb, K. L., W. D. DuPaul, W. Wiebe, W. Sottile, and R. E. Johannes. 1975. Enewetak (Eniwetok) Atoll: aspects of the nitrogen cycle on a coral reef. Limnol. Oceanogr. 20:198-210. 29. Webb, K. L., and W. J. Wiebe. 1975. Nitrification on a ogr.
APPL. ENVIRON. MICROBIOL. coral reef. Can. J. Microbiol. 21:1427-1431. Wiebe, W. J., R. E. Johannes, and K. L. Webb. 1975. Nitrogen fixation in a coral reef community. Science 188:257-259. 31. Wyatt, J. T., G. C. Lawley, and R. D. Barnes. 1971. Blue-green algal responses to some organic-nitrogen substrates. Naturwissenschaft 58:570-571. 30.
Vol. 31, No. 6 Printed in U.S.A .
Influence of Sewage Discharge on Nitrogen Fixation and Nitrogen Flux from Coral Reefs in Kaneohe Bay, Hawaii ROGER B. HANSON'* AND K. GUNDERSEN Department of Microbiology, University of Hawaii, Honolulu, Hawaii 96822 Received for publication 15 December 1975
Nitrogen fixation was investigated in Kaneohe Bay, Oahu, Hawaii, a subtropical eutrophic estuary, by using the acetylene reduction technique on algal samples. No active, planktonic, N2-fixing blue-green algae or bacteria were observed. However, Calothrix and Nostoc capable of fixing N., were cultured from navigational buoys and dead coral heads. Nitrogen fixation associated with these structures was greater in the middle sector than in the south and north sectors of the estuary. Experiments demonstrated that the fixation was photosynthetically dependent. Examination of the data showed that there was no significant correlation between rates of nitrogen fixation and concentration of combined nitrogen compounds in the Bay water. Fixation was significantly correlated to the inorganic N/P (atomic) ratio in the south and middle sectors but not in the north sector. The nutrient data indicate there was a flux of combined nitrogen, but not phosphate, from the reef flats.
Nitrogen fixation by the planktonic microorganisms has been reported for open ocean (8, 15) and bay waters (25). However, most marine nitrogen-fixing, blue-green algae are found in the intertidal zone (1), along rocky shores and in brackish water (20). Johannes et al. (13) located within the coral reef complex a variety of blue-green algal communities that were able to fix nitrogen. From available data, biological fixation of nitrogen may be very important to coral reef communities, provided other essential nutrients, such as phosphate, are not limiting (28, 30). Coral reef communities generally exist in environments that do not have wide fluctuations in either physical or chemical parameters (16). Thus, some of these communities could have narrow stress tolerances. Johannes (12), in fact, presented evidence that some are quite susceptible to various forms of pollution. The coral reefs in Kaneohe Bay have been exposed over the last 2 decades to sewage discharge, siltation from heavy urbanization along the shore, and nutrient-rich runoff, all of which have contributed to the killing of the coral communities in several areas of the Bay (4, 18). The destroyed reefs, still standing erect, have subsequently been colonized by an entirely new population of marine organisms (6, 12, 18), including nitrogen-fixing algae. Kaneohe Bay is a subtropical estuary on the I Present address: University of Georgia, Marine Institute, Sapelo Island, Ga. 31327.
northeast coast of Oahu, Hawaii (Fig. 1), located at 21028' N, 157°48' W. Its area and volume are, at mean sea level, 4.60 x 107 m2 and 2.56 x 10 m3, respectively (5). On the basis of water circulation, topography, and nutrient concentration, the Bay can be divided into three sectors: south, middle, and north sectors (Fig. 1). The south sector receives the effluents from the Kaneohe Municipal Sewage Treatment Plant, with a daily discharge of 12,000 m:1 *of secondary-treated sewage, and the Kaneohe Marine Corps Air Station sewage treatment plant, which discharges 3,000 m:1 of secondarytreated sewage per day. In addition to the sewage, the south sector receives, during periods of rain, considerable amounts of silted freshwater through streams and through runoff from the urban and agricultural coastlands. The middle sector opens directly to the ocean through the South Channel and over the barrier reef and is the least influenced by terrestrial runoff. The largest portion of oceanic water entering the middle sector passes over the barrier reef and turns north. The north sector is the most open to the ocean but is considerably influenced by freshwater runoff from streams that empty into the sector. Due to the water movements in the south and middle sectors, water is continuously leaving the north sector through the North Channel, even during incoming tides. In this paper, the influence of the nutrient gradient on the nitrogen-fixing population and the flux of combined nitrogen from the reefs in Kaneohe Bay will be described. 942
VOL. 31, 1976
N2 FIXATION AND N FLUX FROM REEFS
943
FIG. 1. Kaneohe Bay and its location on the northeast coast of Oahu (insert): sewage plant 1 (Kaneohe Municipal) and sewage plant 2 (Kaneohe Marine Corps Air Station) with lines to outfall area; basic current patterns -incoming tides (solid arrow), outgoing tides (broken arrow); major navigational channels, North and South Channels at the respective ends of the extensive outer barrier reef with Kapapa Island; location of the thirteen algal stations (navigational buoys, 1-8, and coral reefs, A-E) in the south, middle, and north sectors of Kaneohe Bay. Coral reefs are outlined by heavy irregular lines.
MATERIALS AND METHODS Sampling and assay of in situ nitrogen fixation. In situ rates of nitrogen fixation were determined by the acetylene reduction method (9). Thirteen stations were selected for this study, representing three different environments in terms of productivity and nutrients (Fig. 1). Algal material encrusting navigational buoys and coral fragments, 5 to 10 mm in size, which were chipped from the surface of dead coral heads (Porites compressa), were suspended in untreated bay water (5 ml). Acetylene reduction experiments were run in triplicate in 38-ml serum bottles sealed with rubber serum stoppers. A 5-ml portion of acetylene (Matheson Gas Co.) was injected into the bottles with a hypodermic syringe, and the excess gas pressure was released through another needle. Final partial pressure of acetylene in the reaction vessel was ca. 0.13 atm. The samples were usually collected and incubated in a water bath
held at sea level at 25 to 26 C between the hours of 0900 and 1300. Samples were not agitated except by the boat motion. After 2 h, gas samples were transferred from the serum bottles into 4-ml Vacutainers using a double-end needle. The gasses (0.5 ml) in the Vacutainers were separated on a stainless-steel Porapak R column (5 feet by 0.125 inch [ca. 152.4 by 0.32 cm]) at 60 C. The ethylene concentration was determined on an Aerograph gas chromatograph, model A-90-P, fitted with a Beckman hydrogen flame ionization detector. Conversion factor. The amount of nitrogen fixed was calculated from the amount of ethylene formed using a ratio of 3:1 to convert ethylene produced to nitrogen fixed (9). An experiment was undertaken to compare the acetylene method and the '5N method, using a dead coral head. Dried coral fragments were pulverized, and 100-g samples were dissolved in 10% HF, centrifuged, and neutralized (pH
944
HANSON AND GUNDERSEN
7.0). The dissolved organic nitrogen was digested in a micro-Kjeldahl, and the NH4 was converted to N2 with alkaline potassium bromide. The isotopic abundance ('5N/'4N ratio) was determined on a modified CEC mass spectrometer, type 21-103C. We found a ratio of 3.3:1. Chlorophyll a (22) and dry weight (100 C for 24 h) were used as a measure of biomass (algal material and coral fragments, respectively), and nitrogen fixation was expressed in terms of amount of nitrogen fixed/unit of biomass per unit of time. Controls containing 1 ml of 50% trichloroacetic acid or 2 ml of saturated NH4Cl solution were run with each acetylene reduction assay. At extremely low nitrogen fixation rates, corrections were made for a small amount of ethylene that was present as a contaminant in the acetylene. Nutrient analysis. Bay water samples were taken in polyethylene bottles at a depth of 0.5 m and analyzed for nitrate, nitrite, and phosphate by the procedures described by Strickland and Parsons (24). Ammonium concentration was determined by the phenolhypochlorite method of Sol6rzano (19), modified by heating the reaction mixture for 10 min at 60 C and cooling at room temperature for 10 min before measuring the extinction spectrophotometrically at 640 nm. Isolation of nitrogen-fixing blue-green algae. The selective enrichment procedures used to isolate nitrogen-fixing blue-green algae were basically those of Allen and Stanier (2). The medium was prepared with open ocean surface water, low in combined nitrogen and phosphorus, supplemented with trace metals and vitamins (3). Planktonic nitrogen fixation. Surface (0.5 m) and subsurface (2 m) water samples, as well as horizontal (0.5 and 2 m) plankton tows (28-,um mesh net), were collected in the middle and south sectors of Kaneohe Bay on several occasions during April and May, 1971. Attempts were also made to determine nitrogen fixation by planktonic organisms by filtering 4.3 liters of Bay water through nitrogen-free acetate and glass filters (pore size, 0.5 ,im; 250-mm Hg vacuum) and resuspending the organisms in 5 ml of filtered Bay water. Enrichment cultures were prepared using 50% artificial seawater for nitrogenfixing blue-green algae and a variety of nitrogenfixing bacteria (2, 3; R. B. Hanson, Ph.D. thesis, Univ. of Hawaii, Honolulu, 1974). Water samples, plankton catch, resuspended organisms, and enrichment cultures were assayed for acetylene reduction.
RESULTS Planktonic nitrogen fixation. Nitrogen fixation was not detected in any of the plankton catches or water samples. Organisms in the water samples concentrated by filtration again gave negative results. On one occasion, a nitrogen fixer belonging to the genus Azotobacter, which demonstrated acetylene reduction, was cultured from the Bay water. All other cultures were negative for acetylene reduction. Nitrogen fixation by algal material from buoys. Nitrogen fixation was detected in the
APPL. ENVIRON. MICROBIOL.
algal material encrusting navigational buoys and in dead coral heads. This nitrogen-fixing activity was highest in the middle sector, compared with the south and north sectors. Stations 5 and C in the middle sector were selected for preliminary studies to determine optimal assay conditions. Algal samples showed a linear rate of fixation (acetylene to ethylene) with time up to 4 h, after which the rate decreased. An enzyme saturation experiment was done to determine the level of acetylene required for maximum production of ethylene by the nitrogenase system. A partial pressure of 0. 13 atm of acetylene was required for maximum production of ethylene. Under the experimental conditions used in these assays, reduction was found to be proportional to the amount of biomass (chlorophyll a and dry weight) in the assay. The rate of acetylene reduction in dark bottles was only 5% of that in light bottles. The precision of the assay procedure for buoy and dead coral material was examined statistically using four sets of algal material, each in triplicate. A mean rate of reduction of 258 nmol of ethylene formed/mg of chlorophyll a per h, with a coefficient of variation of 0.08, for the buoy material and a mean rate of 633 nmol of ethylene formed/ g (dry weight) per h, with a coefficient of variation of 0.27, for the dead coral material was calculated. Nitrogen fixation associated with algal material on buoys in relation to plant nutrients. The rates of nitrogen fixation associated with algal material encrusting buoys and the levels of three plant nutrients in the surrounding water at eight stations (1 through 8) are shown in Fig. 2. Mean fixation rates for stations 1 through 4 in the south sector for June and July, 1971, ranged from 0.11 to 0.60 gg of N/mg of chlorophyll a per h where the plant nutrients were most abundant. The mean rates of fixation at stations 5 through 7 in the middle sector ranged from 1.64 to 1.92 ,tg of N/mg of chlorophyll a per h where the plant nutrients were diluted with ocean water. In the north sector at station 8, the mean rate of 0.28 ,ug of N/mg of chlorophyll a per h was similar to that in the south sector, but the ammonia and phosphate concentrations were lower. The data were examined statistically for correlation between the rates of nitrogen fixation and concentration of phosphate, nitrate, ammonium, and the inorganic N/P ratios (NO3 + NH4+/PO4-3). No correlation was found between the fixation rates and the inorganic nitrogen compounds. However, there was an inverse correlation (P = 0.05; r = -0.92) between nitrogen fixation and the phosphate concentration for all stations ex-
VOL. 31, 1976
N2 FIXATION AND N FLUX FROM REEFS
I mitregeg
I smmsuium-U
tiiatis.
S mitrate-N
ipbsspkate-P
I2.0
1.6
1.6
II
Il
-
,2 E
945
11.2 3 0~~~~
0.8
10.8
'.4
i
0
.,i
i
r 0.4
0.4
0 Ii
I*-. m
mm
1
2
i
3
I_
I
*'IL,= IU 6 7 5 II.
.T
-
Eh
4
Statisis
FIG. 2. Rate of nitrogen fixation in the algal material from buoys and the concentration of some dissolved nutrients in the water (0.5 m) at each station through the estuary. Samples were collected between 0900 and 1200 h and were incubated for 2 h.
cept station 8. The atomic ratio of inorganic nitrogen to phosphorus ranged from 1.6:1 to 2.9:1 in the south sector and 3.9:1 to 6.6:1 in the middle sector and was 4.8:1 in the north sector. A positive correlation (P = 0.05; r = 0.70) between the rates of nitrogen fixation and the NI
I
uitreguo
I
fixat
ion
itrate-N
taummelii
-N
*phesphatg-P
1.2
12.!5
10.C
I
1.0
P ratio was calculated. 0.8 a Nitrogen fixation on coral reefs. In the sum7.555 mer of 1972 the activity of nitrogen-fixing popuI lations on dead coral heads at five stations (A It I through E) in the estuary was determined (Fig. 5.~ 5.0 3). A mean fixation rate of 4.8 ug of NIg per h a: (range, 2.8 to 7.1 A.tg of N/g per h) was found at station A. At station B, in the south sector, the a 2.5 mean rate of 6.1 ,tg of N/g per h (range, 4.1 to 0.2 I 7.5 ,tg of NIg per h) was measured. In the a middle sector, at station C, the mean rate of Wb fixation, 12.5 jig of NIg per h (range, 10.3 to c E 0 Stati INs 15.8 ,ug of NIg per h), was consistently several times higher than that in the south sector. The FIG. 3. Relationship between rates of nitrogen fixation by dead coral fragments and the concentrations mean fixation rate at station D was 2.4 ug of NI of three plant nutrients in the overlying reef waters at g per h (range, 2.1 to 3.0 ,g of N/g per h). The stations A through E in Kaneohe Bay. mean fixation rate at station E in the north sector was 0.9 jig of N/g per h (range, 0.5 to 1.9 ,ug of NIg per h), even lower than the rates in pling in all three sectors. Since the nitrogen concentrations in the reef water did not agree the sewage-influenced south sector. Nutrient concentrations in reef flat water. with the values found in the channel water, As was the case with the phosphate concentra- another set of experiments was done to detertion in the water surrounding the navigational mine the nitrogen levels in the two habitats at buoys, the phosphate in the reef flat water the same time. The concentrations were essenshowed decreasing concentrations from the tially as reported in Fig. 2 and 3 for the two south towards the north sector. The concentra- bodies of water. The rates of fixation associated tions of ammonium and nitrate, on the other with dead coral heads were compared statistihand, remained fairly constant in the water cally to the concentrations of the nutrients, and this time fixation was not significantly correover the reef flats during the periods of sam-
I
A
B
0.6
0.4
946
HANSON AND GUNDERSEN
lated to the concentrations of phosphate, ammonium, and nitrate or the N/P ratios in the reef's overlying water. Nitrogen-fixing blue-green algae isolated from algal material. Since the reduction of acetylene was primarily light dependent, bluegreen algae were suspected of being the major nitrogen fixers. Two types of nitrogen-fixing blue-green algae were isolated from the dead coral surfaces and algal material from buoys, using a nitrogen-free enrichment medium. They were identified as belonging to the genera Calothrix and Nostoc, and both exhibited acetylene reduction in culture.
DISCUSSION A search for nitrogen-fixing activity in the free water of Kaneohe Bay was unsuccessful, except under one cultural condition. The Azotobacter cultured from the water was unable to sustain growth in the growth medium prepared with 100% Bay water. Even though planktonic nitrogen-fixing microorganisms as well as planktonic symbionts may exist in Kaneohe Bay, they were undetected by the enrichment culture techniques and the acetylene reduction method. It is concluded that active planktonic nitrogen-fixing organisms are either rare or non-existent in the water of Kaneohe Bay. In contrast, N2 fixation was found on dead coral and floating structures. The blue-green algae Calothrix and Nostoc were found to be predominant in the material from buoys and dead coral heads. There is little, if any, high-energy surf in the lagoon, except on the barrier reef, to wash these organisms from structures and into the water. Heterotrophic fixation in reef flat sediment will be reported elsewhere. It is generally considered that nitrogen fixation by blue-green algae is suppressed by organic and inorganic nitrogen compounds (7, 14). However, Horne and Fogg (10), Stewart et al. (23), and Vanderhoef et al. (26, 27) found a more rapid reduction of acetylene in eutrophic lakes than in oligotrophic lakes. In Kaneohe Bay, it was found that the fixation was higher in the middle sector than in the south and north sectors. It was suspected that nitrogen fixation was inhibited by the nitrogen-rich water in the south and north sectors. However, when the data were analyzed statistically, there was no apparent negative correlation between nitrogen fixation by the coral and buoy material and the nitrate and ammonium concentrations, separately or together, in the estuary. In addition, the data in Fig. 3 illustrate that the concentration of inorganic nitrogen in reef water does not control blue-green algal nitrogen fixation on
APPL. ENVIRON. MICROBIOL.
the reef flats (cf. station C). Horne and Fogg (10) also reported that an inverse correlation between nitrogen fixation and the levels of inorganic combined nitrogen was not found in Lake Windermere, England. The inverse correlation found between nitrogen fixation and phosphate concentration in the main channel is probably not related to phosphate inhibition in Kaneohe Bay, since it was later determined that there was no significant inhibition or stimulation of acetylene reduction in the buoy material at station 5 when it was further enriched with phosphate (Hanson, Ph.D. thesis). The inorganic N/P ratios in the Bay water were also compared to the rates of N2 fixation to determine if fixation activity was related to or regulated by this ratio. The data (Fig. 2) indicated a positive relationship, whereas there was no relationship between fixation and N/P in coral reef water. The reason for this is still not understood. To our knowledge, there have been no reports on the relationship between periphyton nitrogen fixation and inorganic N/P ratios in coastal and estuarine waters. Recently, however, Vanderhoef et al. (27) have shown that N, fixation was related to some extent to the P/N ratio in lake waters, in contrast to what was found in Kaneohe Bay. There have been reports of nitrogen flux, which was attributed to nitrogen fixation on coral reefs in the Marshall Islands (13, 28). It was also discovered that there was a flux of inorganic nitrogen from reef flats in Kaneohe Bay. The high ammonium-to-nitrate concentration in the water directly over the coral community compared to the low ammonium-to-nitrate concentration in the main channel (middle sector) suggests that nitrogen is being released from the reef. The source of this nitrogen is due, in part, to the high rate of fixation in the community (Fig. 3). Although some of the fixed nitrogen is recycled within the community, nitrogen-fixing algae are known to release newly synthesized nitrogen products (Hanson, Ph.D. thesis). The dissolved organic nitrogen excreted is either assimilated or deaminated by associated microorganisms to ammonium, of which some is oxidized to nitrate. Nitrite in the water was almost undetectable (less than 0.08 ,ugatom/liter), indicating that nitrosofication and nitrification are closely coupled in the benthic community (28, 29). The low nitrogen content in the channel water, negligible terrestrial influence, and high fixation rates in the coral reefs at station C, high ammonium and nitrate content in the reef water, and the decreasing concentration of phosphate in both habitats indicate that there is a nitrogen flux from the coral reefs in the middle sector.
N2 FIXATION AND N FLUX FROM REEFS
VOL. 31, 1976
Unlike ammonium and nitrate, phosphate in the reef flat water decreased with distance from the phosphate-enriched south sector. This suggests that the source of phosphate is primarily the sewage discharge (cf. 4). Since there was no export of phosphate from the reef flats, one is led to believe that the phosphate is probably imported into and recycled within the benthic community. Johannes et al. (13) also reported that no flux of phosphate occurred from the reef community into the overlying water at Eniwetok and concluded that there must be an unusually efficient recycling of the phosphate within the benthic community. The nitrogen cycle is extremely complex, and most of the processes within the cycle are not defined for Kaneohe Bay. The inputs of nitrogen into the estuary are sewage discharge, stream runoff, ocean, atmospheric fallout, and biological nitrogen fixation. The outputs are relatively few: ocean, sedimentation, and denitrification. Based on a preliminary nitrogen budget for Kaneohe Bay (Hanson, Ph.D. thesis), it was estimated that the nitrogen flux from the reefs balanced the sewage input. In contrast, benthic nitrogen fixation represented approximately 10% of the sewage discharge (Hanson and Gunderson, submitted for publication). Nitrogen fixation in the south sector of Kaneohe Bay is probably not influenced by the concentration of combined nitrogen in the water, as evident from the fixation on the reefs and the inorganic nitrogen concentrations in the water over the reefs in the middle sector. There has been considerable evidence that organic and inorganic compounds in sewage, industrial wastes, and runoff from domestic and agricultural land inhibit nitrogen fixation by blue-green algae (11, 21, 23, 31). Heavy metals, herbicides and insecticides, hydrocarbons, and various other toxic materials are potential inhibitors of the process and are no doubt widely distributed in Kaneohe Bay. LITERATURE CITED 1. Allen, M. B. 1963. Nitrogen-fixing organisms in the sea, p. 85-92. In C. H. Oppenheimer (ed.), Symposium on marine microbiology. Charles C Thomas, Springfield, Ill. 2. Allen, M. M., and R. Y. Stanier. 1968. Selective isolation of blue-green algae from water and soil. J. Gen. Microbiol. 51:203-209. 3. Antia, J. J., and R. Kalmakoff. 1965. Growth rates and cell yields from axenic mass cultures of fourteen species of marine phytoplankton. Fish. Res. Board Can. Manuscript Rep. Ser. No. 203, Ottawa. 4. Banner, A. H., and J. H. Bailey. 1970. Effects of urban pollution upon a coral reef system. Hawaii Inst. Mar. Biol. Tech. Rep. No. 25, Kaneohe. 5. Bathen, K. H. 1968. A descriptive study of the physical oceanography of Kaneohe Bay, Oahu, Hawaii. Ha-
947
waii Inst. Mar. Biol. Tech. Rep. No. 19, Kaneohe. 6. DiSalvo, L. H., and K. Gundersen. 1971. Regenerative functions and microbial ecology of coral reefs. I. Assays for microbial population. Can. J. Microbiol. 17:1081-1089. 7. Fogg, G. E., and W. D. P. Stewart. 1965. Nitrogen fixation in blue-green algae. Sci. Prog. 53:191-201. 8. Goering, J. J., R. C. Dugdale, and D. W. Menzel. 1966. Estimates in situ rates of nitrogen uptake by Trichodesmium sp. in the tropical Atlantic Ocean. Limnol. Oceanogr. 11:614-620. 9. Hardy, R. W. F., R. C. Bums, and R. D. Holsten. 1974. Application of the acetylene-ethylene assay for measurement of nitrogen fixation. Soil Biol. Biochem. 5:47-81. 10. Home, A. J., and G. E. Fogg. 1970. Nitrogen fixation in some English lakes. Proc. R. Soc. London Ser. B 175:351-366. 11. Horne, A. J., and C. R. Goldman. 1974. Suppression of nitrogen fixation by blue-green algae in a eutrophic lake with trace additions of copper. Science 183:409411. 12. Johannes, R. E. 1973. Coral reefs and pollution, p. 364375. In Mario Ruivo (ed.), Marine pollution and sea life. Unipub, Inc., New York. 13. Johannes, R. E., et al. 1972. The metabolism of some coral reef communities: a team study of nutrients and energy flux at Eniwetok. Bioscience 22:541-543. 14. Jurgensen, M. F. 1973. Relationship between nonsymbiotic nitrogen fixation and soil nutrient status-a review. J. Soil Sci. 24:512-522. 15. Mague, T. H., N. M. Weare, and 0. Holm-Hansen. 1974. Nitrogen fixation in the North Pacific Ocean. Mar. Biol. 24:109-119. 16. Odum, E. P. 1971. Fundamentals of ecology. W. B. Saunders Co., Philadelphia. 17. Schollhorn, R., and R. H. Burris. 1967. Acetylene as a competitive inhibitor of nitrogen fixation. Proc. Natl. Acad. Sci. U.S.A. 58:213-216. 18. Smith, S. V., K. E. Chave, and T. 0. Kam. 1973. Atlas of Kaneohe Bay: a reef ecosystem under stress. University of Hawaii Sea Grant Program (TR-72-01), University of Hawaii Press, Honolulu. 19. Sol6rzano, L. 1969. Determination of ammonia in natural waters by the phenylhypochlorite method. Limnol. Oceanogr. 14:799-801. 20. Stewart, W. D. P. 1967. Nitrogen turnover in marine and brackish habitats. II. Use of '5N in measuring nitrogen fixation in the field. Ann. Bot. 31:395-407. 21. Stewart, W. D. P. 1972. Algal metabolism and water pollution in the Tay Region. Proc. R. Soc. London Ser. B 71:209-224. 22. Stewart, W. D. P., G. P. Fitzgerald, and R. H. Bums. 1968. Acetylene reduction by nitrogen fixing bluegreen algae. Arch. Mikrobiol. 62:336-348. 23. Stewart, W. D. P., T. Mague, G. P. Fitzgerald, and R. H. Burris. 1971. Nitrogenase activity in Wisconsin lakes of differing degrees of eutrophication. New Phytol. 70:497-509. 24. Strickland, J. D. H., and T. R. Parsons. 1972. A practical handbook of seawater analysis. Bull. Fish. Res. Board Can. Bull. No. 167, Ottawa. 25. Sugahara, I., T. Sawada, and A. Kawai. 1971. Microbiolgical studies on nitrogen fixation in aquatic environments. VI. On the in situ nitrogen fixation in water region. Bull. Jpn. Soc. Sci. Fish. 37:1093-1099. 26. Vanderhoef, L. N., B. Dana, D. Emerich, and R. H. Burris. 1972. Acetylene reduction in relation to levels of phosphate and fixed nitrogen in Green Bay. New Phytol. 71:1097-1105. 27. Vanderhoef, L. N., C. Huang, R. Musil, and J. Williams. 1974. Nitrogen fixation (acetylene reduction) by phytoplankton in Green Bay, Lake Michigan, in
948
HANSON AND GUNDERSEN relation to nutrient concentrations. Limnol. Ocean-
19:119-125. 28. Webb, K. L., W. D. DuPaul, W. Wiebe, W. Sottile, and R. E. Johannes. 1975. Enewetak (Eniwetok) Atoll: aspects of the nitrogen cycle on a coral reef. Limnol. Oceanogr. 20:198-210. 29. Webb, K. L., and W. J. Wiebe. 1975. Nitrification on a ogr.
APPL. ENVIRON. MICROBIOL. coral reef. Can. J. Microbiol. 21:1427-1431. Wiebe, W. J., R. E. Johannes, and K. L. Webb. 1975. Nitrogen fixation in a coral reef community. Science 188:257-259. 31. Wyatt, J. T., G. C. Lawley, and R. D. Barnes. 1971. Blue-green algal responses to some organic-nitrogen substrates. Naturwissenschaft 58:570-571. 30.
