Denitrification Associated with Periphyton Communities - Applied and ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1981, p. 745-748 0099-2240/81/100745-04$02.00/0

Vol. 42, No. 4

Denitrification Associated with Periphyton Communities FRANK J. TRISKA AND RONALD S. OREMLAND* Water Resources Division, U.S. Geological Survey, Menlo Park, California 94025

Received 4 May 1981/Accepted 1 July 1981

Scrapings of decomposing Cladophora sp. mats (periphyton) covering stream bed rocks produced N20 when incubated under N2 plus 15% C2H2. Denitrification (N20 formation) was enhanced by N03 and was inhibited by autoclaving, Hg2e, and 02. No N20 was formed in the absence of C2H2 (air or N2 atmosphere). Chloramphenicol did not block N20 formation, indicating that the enzymes were constitutive. In field experiments, incubation of periphyton scrapings in the light inhibited denitrification because of algal photosynthetic 02 production. The diurnal periphyton-associated denitrification rate was estimated to be 45.8 ,mol of N20 M2 -day-', as determined by averaging light, aerobic plus dark, and anaerobic rates over a 24-h period. Most studies of denitrification have centered (when our experiments were conducted), the either on agricultural soils or freshwater and filamentous mat undergoes senescence characmarine sediments. Streams, however, have re- terized by dense epiphytic growth of diatoms, ceived much less attention, and studies have bacteria, and accumulation of fine organic parfocused on material balance of nitrate as a con- ticles which coat the filament surface. The fine sequence of solute transport and stream bed particulates, when examined microscopically, denitrification (8, 10, 11). Direct measurements were found to contain a dense and diversified of stream bed denitrification by the acetylene bacterial flora. No attempt was made to quantify blockage technique (1, 13) however, are lacking, bacterial cell density. and with this in mind, we initiated such work in We composited algal tissue into a single samstreams. During the course of these studies, we ple by scraping 20 to 30 large cobbles found observed dense patches of apparently decom- along the margins of a pool (volume of algal posing periphyton (Cladophora sp. mats) on material scraped 700 cm3). The sample was rock surfaces in slowly flowing stream pools. We immediately returned to the laboratory, mixed theorized that this decaying material would be by hand, and dispensed by 10-ml portions into rich in substrates for bacterial heterotrophy yet Erlenmeyer flasks (50 ml) which contained 10 low in oxygen owing to limited permneability and ml of stream water (volume of gas phase = 36 microbial respiration. We now report that these ml). The flasks were sealed with serum caps and, communities support denitrification. with the exception of aerobic controls, were next San Francisquito Creek is a small suburban flushed with N2 for 4 min (flow _ 75 ml/min). drainage about 30 miles south of San Francisco. NaNO3 and HgCl2 were added by syringe injecThe stream drains woodlands, pastures, and a tion (1 ml) from stock solutions at the concengolf course. The experimental site was a per- trations indicated. Chloramphenicol was inmanently flowing reach immediately down- jected as a slurry at about twice its solubility to stream from a golf course and upstream of resi- maintain saturation and to insure penetration dential development. There was no known into the algal material (final concentration _ 4 source of industrial pollution upstream of the mg/ml). Unless otherwise noted, all flasks were experimental site. The experiments were con- injected with C2H2 (initial pressure of C2H2 = ducted during September and October, 1980. On 0.15 atm [14.8 kPa]). The flasks were run in 1 October 1980, the stream had the following duplicate or triplicate as indicated. Immediately concentrations of dissolved inorganic ions: ni- after addition of C2H2, the flasks were placed on trate, 30 umol (423 ug of NO3--N -liter-'); nitrite, a rotary shaker (150 rpm at 200C in the dark) 0.43 /mol (6 jig of N02--N-liter'1); ammonium, for a preincubation period of 2 to 3 min (to 3.4 pmol (48 ,g of NH4+-N-liter-1); and ortho- facilitate gas exchange) before the initial sampling. A longer preincubation period, although phosphate, 1.0 ,mol (32 ,ug of P04-1-P liter-l). The periphyton community consisted of a recommended (6), was not feasible owing to the Cladophora sp. mat which developed over the rapid production of N20 observed upon C2H2 stream bed rocks. During summer low flows addition. After zero-time sampling, the flasks -

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were continuously shaken for the duration of the experiment. At the conclusion of the experiments, the flask contents were dried at 50°C, weighed, and combusted (at 500°C for 4 h) to determine ash-free dry weights, which were used, where indicated, to normalize N20 production per flask. The mean ash-free dry weight per flask was 0.2448 g, with a standard deviation of 0.0583 (42 samples). In field experiments, algal material was collected as outlined above and added in 10-ml portions to prescription bottles (250 ml) containing 80 ml of stream water. Flasks were sealed with serum caps either under air or N2 (flushed for 4 min with N2). All flasks received C2H2 (initial pressure of C2H2 = 0.15 atm) and were incubated in the stream (temperature = 13°C) without shaking. To test the effects of photosynthetic 02 production on denitrification, we incubated the flasks either in the light or in the dark. Light bottles were placed in clear plastic bags, anchored to the stream bottom (0.2 m depth), and exposed to ambient sunlight in a shallow pool. Dark bottles were simultaneously incubated in black plastic bags. Triplicate flasks were run of each experimental variable (light plus air, light plus N2, dark plus air, and dark plus N2) for a total of 12 flasks. Gas samples were taken just before incubation and after 1.5, 3.0, and 6.5 h of incubation. Gas samples (2.5 ml) were withdrawn from the flasks by separate glass syringes and injected into 2-ml Vacutainers to yield a final pressure of 1.25 atm (123.4 kPa). Samples were immediately returned to the laboratory and analyzed as the experiment proceeded. The last sample (taken after 6.5 h of incubation) was taken 1 h after the onset of darkness. Periphyton biomass per unit area of rock surface was estimated by scraping areas (16 cm2) from cobbles found along the pool margins. Five such samples were taken and processed as described previously to determine ash-free dry weights per square meter. To obtain an estimate of denitrification rates occurring in the stream, we converted the rates obtained in the bottles to dry-weight grams, and these were multiplied by the ash-free dry weight per square meter of periphyton. We made corrections for residual N20 in the solution by estimating a Bunsen coefficient of 0.81 at 13°C (12) and applying the coefficient to the equations of Flett et al. (6). N20 and hydrocarbons were analyzed by '3Nielectron capture detector gas chromatography and flame ionization detector gas chromatography (5); these methods made use of 100- and 250-p1 injection volumes, respectively. The 'Nielectron capture detector gas chromatograph

was modified to contain a column (25 by 0.31 cm) of magnesium perchlorate (to absorb water) in line before the Porapak Q column, and a fourway switching value was attached in series after the column to prevent C2H2 from reaching the detector (4). Flasks incubated under N2 plus C2H2 immediately produced N20 (Fig. 1), and production leveled off after 1 h. Exponential production of N20 was reestablished with injection of nitrate, thereby indicating that N20 formation (under these experimental conditions) was nitrate limnited. Controls under air (no C2H2) or inhibited with 1% (wt/vol) Hg+2 did not produce significant N20 (Fig. 1). In addition, no N20.formation was observed from heat-sterilized samples (autoclaved at 15 lb/in2 for 20 min; cooled and incubated under N2 plus C2H2) or samples incubated under N2 without C2H2 (data not shown). We conclude, therefore, that the observed N20 formation was due to denitrification and was not accomplished aerobic nitrifiers (2, 7). Flasks incubated under air plus C2H2 pro-

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FIG. 1. N20 formation by replicate samples of scraped periphyton mats. Samples incubated with air (V, V), air plus C2H2 (0, *), N2 plus C22 plus 1% (wt/vol) HgCl2 (A, A), and N2 plus C2H2 (0, 0). At 4 h (arrow), one of the N2plus C2H2 flasks (0) received IO pmol of NaNO3 by syringe injection (1 ml). Samples incubated under N2 (without C2H2) or heat sterilized and incubated under N2 plus C2H2 did not produce N20 (data not shown).

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duced about 20 times less N20 than did flasks incubated under N2 plus C2H2 (Fig. 1). However, unshaken flasks incubated under air plus C2H2 produced between two and three times as much N20 as did shaken flasks incubated under air plus C2H2 (data not shown). Ethylene levels remained low during these experiments ( dark + air > light + N2 > light + air. These results indicate that denitrification was inhibited by photosynthetic 02 production. An analogous effect was observed on methanogenesis in shallow, tropical marine sediments (9). Thus, in natural lotic environments, denitrification associated with periphyton mats may be a significant nitrogen sink during periods of darkness but is likely to be inhibited during daylight as a consequence of algal photosynthetic oxygen production. An estimation of diurnal denitrification rates can be made by combining the dark + N2 and the light + air flasks used in the field experiment (Fig. 2A and B). Extrapolation to an hourly rate yielded 74.2 ± 6.8 nmol of N20g-'h-1 and 13.1 ± 2.7 nmol of N20 * g-1 h-1 for the dark + N2 and light + air flasks, respectively. When corrected for N20 solubility (6, 12), these rates became 102 ± 9.3 (dark + N2 flask) and 17.2 ± 3.5 (light + air flask). The density of the periphyton mat was 32.04 + 4.76 g.m2 (± standard deviation). Thus, the mean denitrification rates extrapolated to 3.27 (dark + N2 flask) and 0.55 (light + air flask) ,umol of N20m2.h-1. If we assume steady production over 12 h for each condition (39.2 and 6.6 ,mol of N20-m-1-12 h-1, respectively), then the combined daily periphyton mat denitrification was 45.8 ,umol of N20 * day-'. This estimate, however, does not reflect in situ conditions, since the material assayed was scraped from rock surfaces and thereby disrupted. We are currently assaying undisturbed rock surfaces to better assess the contribution of periphyton-associated denitrification to the nitrogen cycle of streams. ACKNOWLEDGMENTS We thank M. Betlach, K. Slack, M. Firestone, and C. Carlson for helpful discussion and comments on the manuscript, M. Betlach for technical advice and assistance, and V. Kennedy for support and interest.

LITERATURE CITED Sherr, and W. J. Payne. 1976. Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus. Appl. Environ. Microbiol. 31:504-508. 2. Bremmer, J. M., and A. M. Blackmer. 1978. Nitrous oxide: emission from soils during nitrification of fertilizer nitrogen. Science 199:295-296. 3. Brock, T. D. 1961. Chloramphenicol. Bacteriol. Rev. 25: 1. Balderston, W. L., B.

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FIG. 2. Denitrification by scrapedperiphyton samples incubated in situ with C2H2. (A) Aerobic bottles incubated in the light (A) and dark (0). (B) Anaerobic (NV2) bottles incubated in the light (0) and dark (A). Points represent the mean + 1 standard deviation of three bottles.

32-48. 4. Chan, Y., and R. Knowles. 1979. Measurements of denitrification in two freshwater sediments by an in situ

acetylene inhibition method. Appl. Environ. Microbiol. 37:1067-1072. 5. Ctilbertson, C. C., A. J. B. Zehnder, and R. S. Oremland. 1981. Anaerobic oxidation of acetylene by estuarine sediments and enrichment cultures. Appl. Environ.

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Microbiol. 41:396-403. 6. Flett, R. J., R. D. Hamilton, and N. E. R. Campbell. 1976. Aquatic acetylene-reduction techniques: solutions to several problems. Can. J. Microbiol. 22:43-51. 7. Goreau, T. J., W. A. Kaplan, S. C. Wofsy, M. B. McElroy, F. W. Valois, and S. W. Watson. 1980. Production of N02- and N20 by nitrifying bacteria at reduced concentrations of oxygen. Appl. Environ. Microbiol. 40:526-532. 8. Hill, A. R. 1979. Denitrification in the nitrogen budget of a river ecosystem. Nature (London) 281:291-292. 9. Oremland, R. S. 1975. Methane production in shallowwater, tropical marine sediments. Appl. Microbiol. 30: 602-608.

APPL. ENVIRON. MICROBIOL. 10. Sain, P., J. B. Robinson, W. N. Stammers, N. K. Kaushik, and H. R. Whiteley. 1977. A laboratory study on the role of stream sediment in nitrogen loss from water. J. Environ. Qual. 6:274-278. 11. Thomson, G. D. 1979. A model for nitrate-nitrogen transport and denitrification in the river Thames. Water Res. 13:855-863. 12. Weiss, R. F., and B. A. Price. 1980. Nitrous oxide solubility in water and seawater. Marine Chem. 8:347359. 13. Yoshinari, T., R. Hynes, and R. Knowles. 1977. Acetylene inhibition of nitrous oxide reduction and measurement of denitrification and nitrogen fixation in soil. Soil Biol. Biochem. 9:177-183.