Denitrification and Assimilatory Nitrate Reduction in - Applied and ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1983, P. 1118-1124

Vol. 46, No. 5

0099-2240/83/111118-07$02.00/0 Copyright © 1983, American Society for Microbiology

Denitrification and Assimilatory Nitrate Reduction in Aquaspirillum magnetotacticum DENNIS A. BAZYLINSKI AND R. P. BLAKEMORE* Department of Microbiology, University of New Hampshire, Durham, New Hampshire 03824

Received 13 April 1983/Accepted 15 August 1983

Aquaspirillum magnetotacticum MS-1 grew microaerobically but not anaerobically with N03- or NH4' as the sole nitrogen source. Nevertheless, cell yields varied directly with N03- concentration under microaerobic conditions. Products of N03- reduction included NH4', N20, NO, and N2. N02- and NH2OH, each toxic to cells at 0.2 mM, were not detected as products of cells growing on NO3-. NO3- reduction to NH4' was completely repressed by the addition of 2 mM NH4' to the growth medium, whereas NO3- reduction to N20 or to N2 was not. C2H2 completely inhibited N20 reduction to N2 by growing cells. These results indicate that A. magnetotacticum is a microaerophilic denitrifier that is versatile in its nitrogen metabolism, concomitantly reducing NO3 by assimilatory and dissimilatory means. This bacterium appears to be the first described denitrifier with an absolute requirement for 02. The process of N03 reduction appears well adapted for avoiding accumulation of several nitrogenous intermediates that are toxic to cells.

Motile bacteria whose principal swimming di- poorly understood. Within this genus, A. iterrections are influenced by magnetic fields, in- sonii and A. psychrophilum also reduce N03 cluding the geomagnetic field, are common in beyond the NO2 stage, but only the latter sediments of diverse aquatic habitats (4, 27). species forms visible gas (15, 20). N20 is the Cells of the bipolarly flagellated, obligate mi- terminal product of N03 reduction in A. itercroaerophile, Aquaspirillum magnetotacticum sonii (7). A. fasciculus, A. gracile, and A. poly(5, 14, 26), synthesize magnetosomes (intracellu- morphum appear to reduce NO3 to N02 only lar, enveloped, iron-rich crystals) consisting of (16, 20). A. dispar (ATCC 27510 and 27650) was magnetite (Fe3O4). Magnetosomes impart to found to grow anaerobically with NO3, reduceach cell a permanent magnetic dipole moment ing it beyond the NO2 stage (21). (3, 13). Cells synthesizing Fe3O4 from soluble Cells of A. magnetotacticum grow microaero(chelated) iron accumulate the hydrous ferric bically with N03 or NH4' as a sole N source. oxide, ferrihydrite (R. B. Frankel, G. C. Pa- N03 is reduced, forming NH3 and nitrous paefthymiou, R. P. Blakemore, and W. O'Brien, oxide (N20) but no detectable N02 (5, 11); Biochim. Biophys. Acta, in press). Thus, bacte- Bazylinski and Blakemore, Abstr. Annu. Meet. rial magnetite synthesis appears to parallel the Am. Soc. Microbiol. 1982, I 53, p. 103). Thus, process of magnetite biomineralization in chi- this bacterium appears to assimilate products of tons (class Mollusca), involving iron reduction N03 reduction while denitrifying. and dehydration of a ferrihydrite precursor (23). True denitrifiers typically reduce 90% or more Iron reduction by nitrate reductase has been of the available N oxide (NO3- or N02) to N suggested for soil microorganisms (29, 30). gas and couple this reduction to electron transMoreover, Sorensen (35) obtained evidence that port phosphorylation (6, 7). Certain non-denitrioxidized iron may replace N03 as a terminal fying N03 reducers, including strains of Escheelectron acceptor in microorganisms found in richia coli, produce N20 in amounts less than surface sediments. 30% of the N oxide (6; J. M. Tiedje, personal This study was undertaken to clarify the bio- communication). Because the gaseous products chemistry of NO3 reduction in A. magnetotac- of N03 reduction in Aquaspirillum species ticum as a prelude to establishing whether en- have not been quantified, the role of these zymes of NO3 reduction are involved in its organisms in denitrification is still unclear. ability to synthesize magnetite through iron re- Moreover, some non-denitrifying bacteria production under microaerobic conditions. duce N20 during NO3 reduction to NH4' (6, N03- reduction in Aquaspirillum species is 33, 34). Thus, another goal of this study was to 1118

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N03- REDUCTION IN A. MAGNETOTACTICUM

establish whether A. magnetotacticum can be considered a denitrifier by currently accepted criteria despite its absolute requirement for 02. MATERIALS AND METHODS Bacteria and growth conditions. The organism used in this study was A. magnetotacticum MS-1. Magnetotactic cells of this strain and those of a non-magnetotactic variant (see below) were routinely cultured in a growth medium containing the following (grams per liter): tartaric acid, 0.75; KH2PO4, 0.69; NaNO3, 0.17; and sodium thioglycolate, 0.06. To each liter of this medium were added 2.0 ml of 10 mM ferric quinate (5), 10 ml of vitamin mixture (36), 5 ml of mineral solution (36), and 0.1 ml of 1% (wt/vol) aqueous resazurin. The mineral solution was modified by the addition of 0.4 g of Na2MoO4 * 2H20 per liter. Ammonium ion was added to the medium as required, either as (NH4)2SO4 or NH4Cl, as indicated. NaNO2 or NH2OH * HCI was added to the medium as indicated. The pH of the medium was adjusted to 6.75 with NaOH added before sterilization. Experiments were carried out with cells cultured microaerobically at 30°C in stoppered 160-ml serum vials each containing 60 ml of culture medium. 02-free N2 or He was bubbled through the medium (ca. 500 ml/min) for 15 min at room temperature before each vial was sealed. The headspace gas of each was then replaced with either N2 or He after the vials were repeatedly evacuated with the use of a vacuum manifold, as described previously by Balch et al. (2). After being autoclaved, the medium was anaerobic (colorless). Just before inoculation, sterile air or 02 was added to the bottles to obtain an initial headspace concentration of 0.2 to 1.0% (vol/vol) 02 (200 to 1,000 Pa 02)- Cells were also mass cultured in 10- to 15-liter batch cultures, as previously described (5). Estimation of cell yield. Cell numbers were determined by direct cell counts with a Petroff-Hausser cellcounting chamber. Dry cell weights were determined by filtering culture samples through 0.2-,um polycarbonate filters (Nuclepore Corp., Pleasanton, Calif.), which were then dried to constant weight at 60°C. Cell magnetism. Cultures were assessed for their magnetism by microscopically noting the fraction of cells, living or dead, that reversed direction when a small magnetic stirring bar 5 to 10 cm away from the microscope stage was rotated 180° from its initial position. Occasionally, cells were negatively stained with 0.5% uranyl acetate (wt/vol; pH 4.2) and examined by electron microscopy for the presence of magnetosomes.

Use of acetylene to block N20 reduction. We used established methods to inhibit N20 reduction with acetylene (C2H2) (12, 38). C2H2 was generated from distilled water and CaC2 (granular; Fisher Scientific Co., Pittsburgh, Pa.). All cultures grown with C2H2 were incubated on a shaker at 30°C. Chemical analyses. N03- was determined with a Beckman SelectIon 2000 ion analyzer (Beckman Instruments, Inc., Irvine, Calif.). N02 was analyzed with sulfanilamide-N-1-naphthylethylene-diamine dihydrochloride (1). NH4' was determined by the reductive amination of a-ketoglutarate (Sigma Technical Bulletin no. 170-UV, Sigma Chemical Co., St. Louis, Mo.). Bound and free NH2OH was assayed by the

1119

Csaky procedure (10) and by the method of Magee and Bums (25). N2O, NO, and 02 were measured by gas chromatography on a Varian series 2400 gas chromatograph equipped with a 63Ni electron capture detector (Varian Instruments, Walnut Creek, Calif.). Two Porapak Q columns (3 mm by 1.8 m) were arranged in a series, the meshes being 80/100 and 60/80, respectively. 02-free N2 at a flow rate of 25 ml/min was the carrier gas. The operating temperatures were as follows: detector, 300°C; column oven, 55C; injector, 70°C. Under these conditions, H2, He, 02, NO, C02, N20, C2H2, and water were separated. N2 was determined with a Perkin-Elmer model 3920A gas chromatograph equipped with a thermal conductivity detector. A molecular sieve 5A column (60/80 mesh; 3 mm by 1.8 m) was used for the stationary phase. 02-free helium at a flow rate of 30 ml/min was the carrier gas. The bridge current was 225 mA, and the operating temperatures were as follows: detector, 130°C; injector, 120°C; column oven, 40°C. Peak areas were determined with a Hewlett-Packard model 3390A computing integrator. For each analysis, standard curves were prepared with pure gases (Scott Environmental Technology, Inc.). Samples of the culture headspace gas were removed with a gas-tight syringe (series A-2; Precision Scientific Co.) that had been previously flushed at least three times with 02-free N2 or He; the samples were then immediately injected into the gas chromatograph. To determine the total concentration of a gaseous product, we calculated the amount present in the solution by using Henry's Law and published values of solubility coefficients (22). Cells grown to late-exponential phase (10 liters; 8 x 10' cells per ml) were harvested by continuous flow centrifugation in a CEPA-model LE (New Brunswick Scientific Co., Edison, N.J.) electrically driven centrifuge equipped with water cooling. Cells were washed several times with 50 mM potassium phosphate buffer (pH 6.90) by centrifugation (11,000 x g for 15 min at 5°C) and dried to constant weight in vacuo over CaSO4 at 110°C. Dried cells were analyzed for total protein, amino acids, and their elemental composition. For amino acid analysis, samples of whole cells were hydrolyzed with HCl and treated with 10.74 mM aqueous Na2 EDTA to remove iron. Amino acids and intracellular NH4' were determined by using a singlecolumn acid-hydrolysate methodology (Spinco Application Note AN-001, 4/77; Beckman Instruments, Spinco Division) with a Beckman model 118CL amino acid analyzer equipped with a Varian model CDS111C peak integrator. The analyzer-integrator system was calibrated with a Beckman standard reference mixture. Total cell protein was determined by the method of Lowry et al. (24), with bovine serum albumin as the standard. Cell elemental composition was determined with a Perkin-Elmer model 240B elemental analyzer with acetanilide as the standard.

RESULTS Effect of nitrogenous compounds on growth and magnetite synthesis. NH4+ and N03- are used as sole sources of nitrogen by A. magnetotacticum

APPL. ENVIRON. MICROBIOL.

BAZYLINSKI AND BLAKEMORE

1120

N20

Na NO2H20H-HCI

24

steadily disappeared throughout growth. N03-, on the other hand, was utilized most extensively after 40 h. The accumulation of N20 appeared to correlate with the extent of NO3 utilization. At about 40 h, the cell growth rate increased from a culture doubling time of about 40 h to a doubling time of 16 h. Traces of N02 or NO or both were occasionally detected during growth of A. magnetotacticum, but neither of these accumulated in significant amounts. Of note, cells actively using N03 also continued to consume 02-

1 1\ I~~~~~~~

72 96 120 144 Hours FIG. 1. Growth response of A. magnetotacticum to added nitrogen compounds. At 48 h, cultures previously grown without a source of fixed nitrogen were provided with NH4Cl, (NH4)2SO4, NaNO3, N20, NaNO2, or NH2OH HCI, each at a final concentration of 2 mM N. The controls received an equal volume of anaerobic growth medium minus an N source. Symbols and bars represent means and standard deviations, respectively, obtained with triplicate cultures. 0

,umols added to the headspace of shaking cultures) had no detectable effect on the growth of cells in the absence of a combined nitrogen source (Fig. 1). Although cultures grown with NH4+ or NO3 each contained magnetotactic cells, those grown with NH4' frequently contained a higher proportion of nonmagnetotactic cells than those grown with N03. Cultures grown with NO3 under microaerobic conditions frequently showed a biphasic growth pattern (Fig. 1 and 2). Figure 3 shows the utilization of N03 and 02 as well as the production of N02 , NQ, and N20 by growing cells. 02

NOj

10

48

(26) (Fig. 1). After an initial lag period, the growth rate observed with N03 was much higher than that with NH4' (Fig. 1). A lag period was not observed with either (NH4)2SO4 or NH4C1. Higher cell yields were obtained with NaNO3- (1.2 x 108 cells per ml) than with (NH4)2SO4 (2.9 x 107 cells per ml) or NH4Cl (2.1

X 107 cells per ml). Cell lysis became apparent 72 h after the addition of N03- or NH4' to the culture medium (Fig. 1). The effect of known intermediates of NO3 reduction by other bacteria was determined. Free NH2OH or N02 (2 mM) were toxic to cells and produced lysis (Fig. 1). When added to growth medium containing no fixed nitrogen source, each compound exhibited toxicity even at a concentration of 0.2 mM (data not shown). However, N02 at less than 1 mM was not toxic for cells actively growing on N03 N20 (120 .

E 7

10

NH

-) o6

0

24

48

72

96

120

144

Hours FIG. 2. Effect of 02 on the growth of A. magnetotacticum with or without NH4+ or N03 . To limit the introduction of 02, 1% inocula (vol/vol) were from cultures grown until 02 had completely disappeared. Cells used as inocula came from culture medium similar to that used in the experiment. Data points and bars represent means and standard deviations, respectively, obtained with triplicate cultures. Symbols: 0, N03 (2 mM), microaerobic conditions (initial PO2, 0.2 kPa); A, N03 (2 mM), anaerobic conditions (resazurin colorless); O, NH4' (2 mM), microaerobic conditions (initial PO,, 0.2 kPa); 0, NH4+ (2 mM), anaerobic conditions (resazurin colorless).

VOL . 46, 1983


091

E

a)

0

24

48

72

96

20

Hours

FIG. 3. Growth of A. magnetotacticum with were grown in a 2-liter serum-stoppered culture vessel containing 1 liter of growth medium with 2 mM N03 and 2 mM NH4'. The inoculum (1%) came from a culture grown in similar medium. 0, Cells per milliliter.

NO3-. Cells

Effect of oxygen on growth of A. magnetotactiIn confirmation of previous results (5), cells did not grow anaerobically (resazurin colorless) with either NO3 or NH4' as the sole nitrogen source (Fig. 2). Under anaerobic conditions, cells eventually became nonmotile, an effect that was reversible for at least several hours. Cells retained their magnetism under anaerobic conditions. Effect of C2H2 on growth and N20 reduction. At a concentration of 0.1 atm (10 kPa), C2H2 inhibited growth and resulted in aberrant nonmotile and coccoid cells. C2H2 at a final headspace concentration of 0.01 atm (1 kPa) did not cum.

1121

adversely affect cell growth or morphology but completely inhibited N20 reduction (Table 1). With NH4' present, together with C2H2 (assimilatory N03 reduction to NH4' repressed), 96.4% of the N supplied as N03 was recovered as N20 (Table 1). Products and mass balance of N03 reduction. Cells growing microaerobically reduced N03 to NH4', N20, and N2 (Tables 1 and 2). Only trace amounts of NO were ever detected. Free (or bound) NH2OH and NO2- were never detected in growing cultures supplied with NO3or NH4' as a nitrogen source. Cells grown with NH4' did not produce NO, N2O, or N2When NO3 was the sole N source and the acetylene block was used, 80% of the N supplied as NO3- was recovered as N20 (Table 1). The remainder was recovered in cell material. Growing cells supplied with NH4' and N03in the presence of C2H2 produced N2O stoichiometrically equivalent to the amount of N03 utilized (Table 1). In the absence of C2H2, and with N03 initially at 0.66 mM or less, N2O accumulated transiently (data not shown). At the end of growth under these conditions, the amount of N2 detected corresponded to the amount of N03 consumed, and no N20 or N03 remained. When the initial NO3 concentration was raised to 2 mM, N2O accumulated through the end of the growth period, and some N03 remained in the culture medium (Fig. 3). Chemical analysis of whole cells. Whole cells consisted of (percentage of dry weight + 0.1): nitrogen, 10.1; carbon, 48.2; and hydrogen, 7.1. Cells harvested in exponential growth consisted of 59.4 0.7% protein. Amino acid analyses of cells grown with N03 were determined (Table 2). The composition of cells grown on NH4' was similar (data not shown). Cells accumulated a large amount of NH4' intracellularly, particularly when grown with NO3 (Table 2). Intracellular NH4' accounted for 87% of the total NH3 detected in cultures grown with NO3. Effect of NO3 on final cell yield. Final cell yields in cultures with levels of 2 mM NH4' ±

TABLE 1. Recovery of N20, NH4', and N2 by cells grown on NO3Products (to N03- N recovered as ):a N20 (%) Culture medium supplement NH4+

N2

NH4+ (2 mM) + NH4+ (2 mM) +

NO3- (0.66 mM) N03- (0.66 mM) N03- (0.66 mM) + C2H2

+

C2H2

96.4

+ 4.1 OC + 2.1

0

101.9

+

1.3

NAb NA

+ 0 3.2 ±.od 80.0 a Values represent means and standard deviations obtained with triplicate cultures and have been corrected for amounts detected in inoculum. b NA, Not applicable. c N20 appeared transiently, as described in the text. d Includes intra- and extracellular NH3. In this experiment, cell N accounted for 19.4 + 2.8% of N03- N. Excreted N was estimated at 2.3%. Total recovery of N03- N, 101.7 + 4.9%.

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TABLE 2. Amino acid composition of whole cells of A. magnetotacticum grown microaerobically with N03 (2 mM) as the sole N source Amino acid

nmol/mg of cell (dry wt)

Alanine ......................

Glycine ...................... Aspartic acid ................. Leucine ..................... Glutamic acid ................ Valine .......................

Lysine ...................... Threonine ................... Serine .......................

Arginine .....................

Isoleucine ................... Proline ......................

Phenylalanine ................ Methionine ..................

Tyrosine ..................... Histidine ....................

% Recovery of proteina..........

560.4 475.9 421.9 405.7 403.7 337.5 291.3 247.8 236.9 216.6 209.8 198.9 177.0 112.9 101.1 97.6

93.9%

Ammonia .................... 1,761.2 a Protein was 59.4% of the cell dry weight.

sufficient to repress assimilatory N03- reduction (Table 1) were higher with larger amounts of N03 (Fig. 4). A substantial change in cell mass occurred when N03 was raised from 1 to 10 mM. No corresponding increase in cell numbers over this range of N03 values was detected. Cells grown with 10 mM N03 or more were abnormally long (20 to 50 ,um) and poorly motile. At lower NO3 concentrations, cultures contained cells that were smaller (2 to 10 ,um), actively motile, and magnetotactic. Cell growth was inhibited at NO3 levels above 40 mM. DISCUSSION A. magnetotacticum MS-1 cells synthesize all of their required nitrogenous compounds de novo from NH4+ or N03 ions. Because they grow with N03 as a sole nitrogen source, thereby producing NH4+, this organism is capable of assimilatory NO3 reduction. This capability is widespread among bacteria and fungi (31) but apparently not among members of the genus Aquaspirillum. A. itersonii and A. delicatum are the only other members known to grow with N03 as the sole nitrogen source (16). Of course, the inability of some species to grow with N03 may reflect requirements for peptides or other constitutents of complex media used in culturing them. Although it is uncertain whether free NH2OH is produced during bacterial assimilatory N03 reduction (18, 31, 32, 37), NO2 has definitely been observed as a free intermediate in other species (19, 31). We did not detect either of these compounds during the growth of A. magnetotacticum with N03 nor ,

did they support growth of this organism. In fact, at concentrations similar to those used by others in culturing bacteria (37), each was toxic, producing cell lysis. Thus, the eight-electron transfer occurring during NO3 reduction to NH3 in A. magnetotacticum may occur without the production of free intermediates. This possibility is supported by data suggesting that a sixelectron transfer was involved in reducing NO2 to NH3 in E. coli (18), Achromobacter fischeri (32), and Veillonella alcalescens (37). A similar process in A. magnetotacticum might preclude the accumulation of NO2 or possibly other toxic intermediates of assimilatory NO3 reduction. Assimilatory reduction of NO3- to NH3 was repressed by 2 mM NH4' in the culture medium, as evidenced by the complete conversion of nitrogen supplied as N03 to an N gas (N20 in the presence of C2H2; N2 in its absence) under these conditions. Moreover, neither N20 nor N2 was detected when N03 was omitted. This is consistent with the well-recognized repression of assimilatory NO3- reductase by NH4' and by other reduced nitrogenous compounds (31). These findings also suggest that a dissimilatory pathway of N03 reduction to NH4', recently found to be of significance in the production of NH4' in soils (9, 34) and in the bovine rumen (17), is not present in cells of A. magnetotacticum MS-1. 90

80

108~~~~~~~~~~~~7 loB~~~~~~~~~~G

,8.

.

106

,

o~~~~~.4

0

N 0O (mM)

FIG. 4. Effect of initial N03- concentration on final cell yields of A. magnetotacticum. Cells were grown microaerobically (initial Po29 1 kPa) in 500-,ml batch cultures. Final cell yields are reported as direct cell counts (0) and dry cell weights (0). Symbols and bars represent means and standard deviations, respectively, of triplicate analysis.

VOL. 46, 1983


The similar amino acid composition of cells grown with either NH4' or N03 suggests a similar mechanism of NH3 assimilation by each cell type. Production of free N02, which characterizes dissimilatory NO3 reduction by many organisms (19, 31), was never apparent during denitrification by A. magnetotacticum. Thus, cells of strain MS-1 possess an efficient means of reducing toxic NO2. It seems likely that the rate of N02 reduction may be higher than the rate of N03 reduction. Cells of A. magnetotacticum produced only trace amounts of NO and accumulated N20 while reducing NO3. These appeared as transient intermediates and were subsequently reduced to N2. With sufficient NH4' present to repress assimilatory NO3 reduction, increased concentrations of N03 resulted in increased final growth yields. This suggests that NO3 reduction under microaerobic conditions is coupled to energy conservation in this organism. True denitrifiers typically reduce 90% or more of the N oxide to N gas and couple this reduction to electron transport phosphorylation (6, 7). By these criteria, our data confirm that A. magnetotacticum is indeed a denitrifier. Of the N supplied to cells as N03 alone, 80%o was recovered as N gas. The remainder was recovered in cell material and excreted nitrogenous products, including NH4'. Thus, under conditions in which NO3 is the sole N source, cells of A. magnetotacticum concomitantly carry out denitrification and assimilatory N03 reduction to NH4+. Cell growth with NO3 in batch culture is biphasic. The onset of rapid cell growth appeared to correlate with the onset of N20 production from N03 . The data also suggest that NO3 dissimilation commenced when the dissolved 02 reached ca. 4.1 ,umol/liter. Denitrification is generally associated with anoxic conditions because 02 not only inhibits denitrifying enzyme activity but represses synthesis of new denitrifying enzymes as well (19). However, some organisms tolerate limited quantities of 02 while denitrifying (31). Cells of A. magnetotacticum are obligately microaerophilic and do not grow anaerobically, even with N03 (5). Moreover, they consume 02 while denitrifying. Thus, this bacterium appears to be the first described denitrifier which actually requires rather than tolerates 02. This may reflect a specific requirement for 02 as a substrate for oxygenases participating in cell biosynthesis (e.g., heme or lipid synthesis). However, we have been unable to relieve this 02 requirement by growing cells in complex media or by adding hemin. We lack evidence that 02 iS specifically required for N03 reduction. Moreover, our data do not enable us to determine whether,

1123

under microaerobic conditions, respiration involving 02 and N03 as terminal electron acceptors occurs simultaneously. Cultures grown microaerobically with NH4' or N03 as the sole nitrogen source contain some non-magnetotactic cells. However, we have frequently observed that cultures grown with NH4', in contrast to those grown with N03 , contain a larger proportion of cells that are not magnetotactic and do not contain magnetosomes. This is consistent with the possible involvement of NO3 reducing enzymes in magnetite synthesis. Dissimilatory NO3 reductase is an induced enzyme in most bacteria which synthesize it (31) and therefore would not be synthesized by cells growing with NH4' as the sole N source unless a suitable inducer (perhaps even Fe+3) was present. However, it is not yet known whether any of the enzymes involved in denitrification in A. magnetotacticum can reduce ferric iron. Alternatively, the formation of bacterial magnetite might result from the oxidation of ferrous hydroxide [Fe(OH)2] coupled with a reduction of N03- or N20. This reaction can occur nonbiologically at pH 8 (8, 28), although it has not yet been shown to occur enzymatically. Our results, which confirm and extend those of Escalante-Semerena et al. (11), clearly establish that A. magnetotacticum is a microaerophilic denitrifier. The possibility that denitrification is a characteristic shared by other magnetotactic bacteria is an interesting one. Knowledge of this process in strain MS-1 can be expected to lead to more information concerning the ecological niche of these interesting organisms. We recently showed that growing cells of A. magnetotacticum reduce C2H2 microaerobically (Bazylinski and Blakemore, Curr. Microbiol., in press). Thus, in addition to its capacity for assimilatory and dissimilatory N03 reduction, this species also fixes atmospheric N2. Its versatility with respect to nitrogen metabolism may play a significant role in magnetite synthesis and can be expected to favor its survival in microaerobic aquatic habitats. ACKNOWLEDGMENTS We gratefully acknowledge valuable communications, comments, and encouragement from J. M. Tiedje and P. Cornell of Michigan State University and R. B. Frankel of the Massachusetts Institute of Technology. We are grateful to N. Blakemore and A. Geshnizgani for valuable technical assistance. Amino acid and elemental analyses were performed through the Instrumentation Center of the University of New

Hampshire. This work was supported by National Science Foundation grants PCM 79-22224 and PCM 82-15900 and Office of Naval Research contract N0014-80-C-0029. LITERATURE CITED 1. American Public Health Association. 1980. Standard methods for the examination of water and wastewater, 15th

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