This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cooper, D. C.
Right arrow Articles by Coby, A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cooper, D. C.
Right arrow Articles by Coby, A. J.
Agricola
Right arrow Articles by Cooper, D. C.
Right arrow Articles by Coby, A. J.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, June 2003, p. 3517-3525, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3517-3525.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Chemical and Biological Interactions during Nitrate and Goethite Reduction by Shewanella putrefaciens 200

D. Craig Cooper,1* Flynn W. Picardal,1 Arndt Schimmelmann,2 and Aaron J. Coby1

Environmental Science Research Center, School of Public and Environmental Affairs,1 Department of Geological Sciences, Indiana University, Bloomington, Indiana 47405-14052

Received 15 August 2002/ Accepted 5 March 2003


arrow
ABSTRACT
 
Although previous research has demonstrated that NO3- inhibits microbial Fe(III) reduction in laboratory cultures and natural sediments, the mechanisms of this inhibition have not been fully studied in an environmentally relevant medium that utilizes solid-phase, iron oxide minerals as a Fe(III) source. To study the dynamics of Fe and NO3- biogeochemistry when ferric (hydr)oxides are used as the Fe(III) source, Shewanella putrefaciens 200 was incubated under anoxic conditions in a low-ionic-strength, artificial groundwater medium with various amounts of NO3- and synthetic, high-surface-area goethite. Results showed that the presence of NO3- inhibited microbial goethite reduction more severely than it inhibited microbial reduction of the aqueous or microcrystalline sources of Fe(III) used in other studies. More interestingly, the presence of goethite also resulted in a twofold decrease in the rate of NO3- reduction, a 10-fold decrease in the rate of NO2- reduction, and a 20-fold increase in the amounts of N2O produced. Nitrogen stable isotope experiments that utilized {delta}15N values of N2O to distinguish between chemical and biological reduction of NO2- revealed that the N2O produced during NO2- or NO3- reduction in the presence of goethite was primarily of abiotic origin. These results indicate that concomitant microbial Fe(III) and NO3- reduction produces NO2- and Fe(II), which then abiotically react to reduce NO2- to N2O with the subsequent oxidation of Fe(II) to Fe(III).


arrow
INTRODUCTION
 
In recent years, dissimilatory reduction of ferric (hydr)oxide minerals has been documented for a large number of microorganisms in a wide range of environments (31, 37) and has become recognized as an important constituent of the global carbon and iron cycles (31, 32). This process has a profound effect on groundwater geochemistry and places a key control on the fate of contaminant metals (12, 13, 19, 30, 60) and organic compounds (24, 42) in anoxic groundwater. As a consequence, processes that affect the rate and extent of microbial iron reduction in natural systems are of great interest. NO3- is a common groundwater contaminant in the United States (38) and may interfere with bioremediation schemes that seek to use microbial iron reduction to engender reductive immobilization of metal contaminants.

Shewanella putrefaciens 200 is a facultative anaerobe capable of utilizing O2, NO3-, NO2-, Fe(III), Mn(IV), and a number of other compounds as terminal electron acceptors for carbon metabolism (14, 40, 41). Obuekwe and Westlake (40, 41) reported that NO3-, NO2-, and a number of other terminal electron acceptors can inhibit reduction of Fe(III)-phosphate by S. putrefaciens 200, and subsequent work (1, 51) has demonstrated that the presence of NO3- can inhibit microbial Fe2+ production during sediment incubations. Additional studies with S. putrefaciens and other bacteria (14) have also observed that NO3- or NO2- inhibit microbial iron reduction. Studies with S. putrefaciens 200 suggest that this inhibition may result from kinetically favorable transport of electrons to NO3- or NO2-, a mechanism suggested by Arnold et al. to explain inhibition of Fe(III) reduction by oxygen (3-5, 14). Although the respiratory electron transport chain of S. putrefaciens is known to include a variety of b- and c-type cytochromes and quinones (36, 42, 55), the sequential arrangement of electron carriers has not been elucidated, and terminal NO3-, NO2-, or Fe(III) reductases have not been fully purified. While the NO3- and NO2- reductases for S. putrefaciens MR-1 have been partially purified by Krause and Nealson (27), MR-1 is a denitrifier, whereas the S. putrefaciens 200 used in our studies primarily reduces NO3- and NO2- to ammonia (Cooper, Coby, and Picardal, unpublished data). It is therefore likely that strains 200 and MR-1 have substantial enzymatic differences.

Although illuminating, previous studies provide only limited insight into the dynamics of interactions between dissimilatory iron and NO2- or NO3- reduction (NOx- reduction) when solid-phase, ferric (hydr)oxide minerals (e.g., ferrihydrite, goethite, lepidocrocite) are the Fe(III) source. The chemistry of ferric (hydr)oxide minerals (FeOOH) is fundamentally different from that of aqueous or microcrystalline Fe(III) used by others in the previous studies. Not only does the FeOOH provide a textured surface that sorbs metal cations, but Fe2+ adsorption to a FeOOH surface forms highly reactive ferrous-ferric surface complexes that have distinctive chemical properties that are often similar to those of green rust (16, 20, 22, 35, 52). Biogeochemical interactions in systems containing NO3- and solid-phase Fe(III) therefore clearly may be different than those previously described in systems using aqueous Fe(III). The objective of this study is to determine the nature of the biological and geochemical interactions between microbial ferric (hydr)oxide and NO3- reduction using S. putrefaciens 200 as a model microorganism capable of reducing both Fe(III) and NO3- and synthetic goethite as a model ferric (hydr)oxide mineral.


arrow
MATERIALS AND METHODS
 
Bacterial strain and growth conditions.
S. putrefaciens is a gram-negative motile rod with an obligate respiratory metabolism (49). The strain used in these experiments, S. putrefaciens 200, was originally isolated from a Canadian oil pipeline by Obuekwe (39). The culture was maintained on a solid medium of nutrient agar containing 5 g of yeast extract/liter (Difco, Detroit, Mich.), as previously described (42). Liquid cultures were grown in a 2.5-liter Bioflow 3000 fermentor (New Brunswick Scientific Company, Edison, N.J.) in a medium that consisted (per liter) of 2.0 g of Na2SO4, 0.5 g of K2HPO4, 1.0 g of NH4Cl, 0.198 g of CaCl2 · 2H2O, 0.1 g of MgSO4 · 7H2O, 19.35 mg of FeCl3 · 6H2O, 0.5 g of yeast extract, and 3 ml of 60% (wt/vol) aqueous sodium lactate. Following an initial period of aerobic growth, anaerobic reductase activity was induced by reducing the airflow to approximately 100 ml min-1 and maintaining the cells under suboxic ([aqueous O2 {O2(aq)}] < 2.5 µmol/liter) conditions for a period of 12 h. It has been previously demonstrated that this culture technique induces anaerobic reduction systems for a wide range of terminal electron acceptors, including nitrate, nitrite, and Fe(III) (14). Cells were subsequently harvested by centrifugation and resuspended to a target optical density (A600, ~1.2) in low-ionic-strength, artificial groundwater medium (AGW) (12).

A nitrate-reducing enrichment culture was developed from nitrate-contaminated sandy sediments obtained from a Uranium Mill Tailings Remedial Action (UMTRA) site in Shiprock, New Mexico, in October 1999 during sediment collection by the U.S. Department of Energy. Sediments were collected with a backhoe, placed in whirl-pack bags, flushed with argon, placed in sealed argon-flushed bottles, and stored at 4°C aphotically for 1 month prior to use. Enrichment of nitrate-reducing cultures was done with a sulfate-free AGW medium with 10 mM lactate as the electron donor and 2.5 mM nitrate as the electron acceptor. The sulfate-free medium was created by substituting chloride salts for sulfate salts in the AGW medium previously described (12). To establish enrichments, sterile sulfate-free AGW medium was degassed with nitrogen and dispensed (50 ml) into 120-ml serum bottles in an anaerobic chamber (Coy Laboratory Products, Grass Lake, Mich.). After the addition of approximately 10 g of sediment, the bottles were crimp sealed with butyl rubber stoppers and statically incubated in the dark at room temperature. Transfers (10% volume) were done every 7 to 10 days into identical medium in anoxic serum bottles or tubes. After eight transfers, an enrichment culture was developed that was capable of complete utilization of 2.5 mM nitrate in 1 to 3 days. Subsequent experiments revealed that the enrichment culture was also capable of slow reduction of solid-phase Fe(III) oxides.

Fe(III) reduction and NO3- reduction experiments.
The AGW medium and goethite slurries used in these experiments have been described previously (12). When required, 1.0 ml of sterile NO3- or NO2- stock solutions of NaNO3 and NaNO2 (respectively) dissolved in Milli-Q water were added to the AGW medium (goethite free) or AGW + goethite slurry (NOx- + goethite). Although the general methodology employed in the Fe(III) and NO3- reduction experiments has been previously described (12), a summary of the procedure is presented here. Experiments were initiated by inoculating slurries with 2 ml of S. putrefaciens (or UMTRA enrichment culture) suspension under anoxic conditions (ca. 2 x 106 cells ml-1). For each experiment, a series of 150-ml (nominal volume) glass serum bottles were crimp sealed with butyl rubber stoppers and incubated horizontally on a shaker table at room temperature (95% N2-5% H2 headspace). Initial samples (t0) were taken prior to inoculation, and subsequent samples were taken at regular intervals thereafter. During sampling, bottles were transferred to the anoxic chamber, shaken vigorously to resuspend any settled solids, and then immediately sampled with a 3-ml syringe (18-gauge needle). One 1.5-ml aliquot was extracted for 2 h in 2 M KCl for total ammonium-nitrogen (NH3-N) measurements. The second 1.5-ml aliquot of slurry was placed into a separate microcentrifuge tube, and particles were separated by centrifugation (25 min at 14,900 x g). The supernatant was immediately sampled for pH, Fe2+(aq), NH3-N(aq), NO3-, and NO2-. Aqueous Fe2+ was immediately analyzed via a modification of the ferrozine method (26, 53). Samples for NH3-N, NO3-, and NO2- were diluted 2x to 5x in Milli-Q water, frozen, and stored for later analysis. NO3- and NO2- were analyzed via ion chromatography (Dionex Series 4500i; column type, IonPac AS17 [4 by 250 mm]; eluent, 50 mM NaOH and Milli-Q H2O gradient), and aqueous NH3-N was analyzed colorimetrically via the phenate method (21). Extraneous supernatant was then discarded, and the pellet was resuspended and extracted for 2 h in 0.5 N HCl. Both slurries (KCl and HCl) were separated via centrifugation (10 min at 14,900 x g). The HCl supernatant was immediately analyzed for bound Fe2+ via the ferrozine method, and the KCl supernatant was diluted 2x in Milli-Q water, frozen, and stored for later analysis via the phenate method. Total 0.5 N HCl-extractable Fe2+ (Fe2+-HCl) was defined as the sum of aqueous and bound Fe2+, and the precision of the phenate analysis was monitored by comparing total and aqueous NH3-N in the goethite-free bottles.

For N2O analyses, 25.0 µl of headspace gas was withdrawn with a Hamilton gas-tight syringe and analyzed via gas chromatography with a Hewlett Packard 5890 Series II gas chromatograph equipped with a GS-Q megabore capillary column (conditions: oven temperature, 75°C; detector temperature, 275°C; pressure, 130 kPa) and electron capture detector. The instrument was calibrated with both laboratory and commercial N2O standards (Scotty I analyzed gases), and gas concentration was defined as the average of data for three injections. The concentration of aqueous N2O was calculated from temperature and headspace N2O by Henry's law (59), and total N2O was defined as the sum of headspace and aqueous N2O. For N2 analyses, 3.0-ml aliquots of headspace were withdrawn with a syringe and immediately injected into a Shimadzu model 14A gas chromatograph equipped with a CR501 Chromatopac, 1.0-ml sample loop, 3.0 m MS-5A 60/80, and 1.0 m porapak R 80/100 columns (2.5 min run time; isothermal at 75°C) and thermal conductivity detector. To guard against contamination from atmospheric N2, the syringe was slowly compressed during injection, and excess volume acted as a purge for the sample loop. Check standards and He blanks were also analyzed at regular intervals. The instrument was calibrated with laboratory N2 standards, and gas concentration was defined as the average of data for two injections. With the sole exception of the stable isotope studies (described below), the exact same methodology was used for all experiments with S. putrefaciens 200 and the UMTRA enrichment cultures. All experiments were conducted with the necessary controls.

Stable isotope experiments.
With noted exceptions, stable isotope experiments were conducted as described previously. Experiments were conducted under a He atmosphere in specially fabricated Pyrex media bottles (~500 ml) equipped with a vacuum stopcock and crimp-sealed sampling port. After preparation of the experimental medium (100 ml) and replacement of the headspace with He, the bottles were allowed to equilibrate for 48 h, and t0 samples were taken from all bottles prior to inoculation (biological and combined experiments) or NO2- addition (chemical experiments). Microbially reduced goethite (MrG) for the chemical experiment was harvested from previous NO3--free experiments, sterilized via pasteurization (25 min at 95°C), and washed three times with sterile, anoxic, lactate-free AGW medium prior use in isotope experiments.

Isotopic analytical requirements dictated that the entire ~400-ml headspace be collected. Thus, four bottles (two inoculated and two uninoculated) were continuously monitored for aqueous parameters and headspace N2O, while six isotope bottles were continuously monitored for headspace N2O but sampled for aqueous parameters only at the initial and final times. Two isotope bottles were sacrificed at each sampling point, and procedural standards were randomly extracted to monitor the overall precision of the extraction process.

The isotope extraction was conducted in two stages. During the first stage, aqueous slurries were frozen immediately after wet chemical sampling was complete (2 to 4 h; standard commercial freezer) and the headspace-N2O was subsequently removed and stored for isotope analysis. The headspace separation was achieved by cryogenically trapping the condensable gasses (N2O, CO2, H2O) in a liquid-N2 cold trap and slowly removing the incondensable gas (vacuum pumping) while cryogenically trapping any remaining headspace condensable gas. Next, the condensable gas was warmed and cryogenically transferred to a vacuum-tight storage vessel containing a few grains of degassed coconut charcoal. During the second stage, the condensable gas in the storage vessel was further purified on a more advanced vacuum line by cryogenically separating water and traces of incondensable gas from N2O and CO2. The purified N2O and CO2 was then cryogenically transferred to a glass ampoule containing excess granular Cu0 (ca. 2 g), sealed under vacuum, heated overnight at 500°C to reduce the N2O to N2, and stored for subsequent determination of {delta}15N values. Since the biological experiments did not produce the requisite 5 µmol total N2O needed for accurate {delta}15N analysis, 5 µmol of standard N2O was injected into the sampling port of the isotope bottle during the initial trapping of condensable gasses.

The values for {delta}15N of N2O ({delta}15N-N2O) for the biological experiment are based on dilution calculations utilizing the {delta}15N-N2O value for the standard, the {delta}15N-N2O value for a mixture of the sample and standard, and the mass ratio of sample to standard N2O. The N2O standard for {delta}15N-N2O analyses was a 2.0-liter aliquot of medical-grade N2O gas (UN1070, Air Products Corp.) stored at 3,000 kPa to prevent fractionation effects from N2O liquid-gas phase changes at high pressure. Two different aliquots of standard gas were needed for these studies, and these standards had {delta}15N-N2O values of 0.02 ± 0.17{per thousand} (n = 9) (biological experiments) and -2.12 ± 0.40{per thousand} (n = 9) (chemical and combined experiments). The fractionation effect associated with the previously described method for extracting headspace N2O in equilibrium with AGW medium was calculated to be -2.8 ± 1.0{per thousand}. All reported {delta}15N-N2O values account for this extraction bias. {delta}15N values of elemental nitrogen samples were determined off-line, manually, in dual-inlet mode with a Finnigan MAT 252 mass spectrometer with a cryogenic trap at the inlet. We used the ammonium sulfate nitrogen stable isotope standards NBS-N-1 and NBS-N-2 as internal standards for calibration and report our results relative to air nitrogen (air nitrogen {delta}15N {equiv} 0{per thousand}), with a mass-spectrometric precision of ± 0.2{per thousand}.


arrow
RESULTS
 
NO3- inhibition of goethite reduction.
Our batch experiments with goethite as the Fe(III) source in the absence of NO3- (data not shown) yielded results similar to those of previous researchers who have investigated microbial reduction of iron (hydr)oxide minerals. Fe2+ production rates are initially rapid and then slow with time, possibly as a result of passivation of the cell and iron oxide surfaces by adsorbed Fe(II) (44, 45). It is important to note that approximately 50% of the microbially produced Fe2+ is associated with the mineral or cellular surface (precipitated, adsorbed, or a reactive surface complex). Data in Fig. 1 illustrate the effect of NO3- on the rate and extent of Fe2+ production during goethite reduction by S. putrefaciens 200. All slurries reduced NO3- (data not shown), but only the slurries containing initial concentrations of 0.0, 1.0, and 2.5 mM NO3- produced Fe2+ at concentrations significantly above those in the uninoculated controls. The presence of 1.0 mM NO3- resulted in a 50% reduction in the initial rate (~28 h) of Fe2+ production (ca. 2x slower) and prevented significant Fe2+ production for a period of 8 h. The presence of 2.5 mM initial NO3- prevented significant Fe2+ production for over 200 h, and no Fe2+ production was observed in the 5 mM NO3- treatment. No measurable effect on the ultimate extent of iron reduction was observed for the 1 mM NO3- treatment.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 1. Effect of NO3- on Fe(II) production (sum of aqueous and bound Fe) via dissimilatory reduction of synthetic goethite by S. putrefaciens 200. Data from experiments using no NO3- (•), 1.0 mM initial NO3- ({circ}), 2.5 mM initial NO3- ({square}), 5.0 mM initial NO3- ({triangleup}), and uninoculated controls ({blacktriangledown}) are plotted versus time. Solid lines connect the points for experiments with no NO3- and 1 mM NO3-, and dotted lines connect all other points. Data represent average values of three replicates, and the error bars reflect standard deviation. Note the split axes.

Subsequent experiments were conducted with goethite plus one concentration of NO3- (2.5 mM initial NO3-). This decision was made because the large sampling matrix prevented experimentation at multiple NO3- concentrations, and 2.5 mM represented a good midpoint between the 0.0 mM NO3- and 5 mM NO3- end members. These experiments show typical patterns of NO2- and N2O production by S. putrefaciens 200 during NO3- reduction in the absence (Fig. 2a) and presence (Fig. 2b and c) of goethite and reveal that a very small amount of surface-associated Fe2+ (~0.1 mM) is produced while NO3- is still present even though Fe2+ production rates are minimal until after the NO2- supply has been exhausted (Fig. 2b and c). More than 70% of the added NO3- was recovered as NO3-, NO2-, N2, N2O, or NH3-N in all experiments with NO3- and goethite (data not shown). In these experiments, the proportion of nitrogen recovered was initially 100% and then decreased as experiments progressed and cell growth occurred. Since the modest amount of cell growth could not account for the unrecovered N, it is likely that products or complexes were formed that were not included in the N species quantified. Ammonia was the dominant end product of NOx- reduction in our experiments, and only minimal amounts of N2 were produced (Fig. 3). Since the Fe2+-Fe3+ redox transition (EH0 {cong} 0.8 V) is dominant in our system, this observation is consistent with a report by Samuelsson that ammonia is the dominant end product (and that N2O is a minor by-product) of NO3- reduction by a different strain of S. putrefaciens at redox potentials greater than or equal to ~0 mV (46).



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 2. NO3- reduction by S. putrefaciens 200 in the presence and absence of goethite. (a) Experiment with goethite absent, showing data for NO3- ({circ}), NO2- (•), and N2O ({square}). (b) Experiment with goethite present, showing data for NO3- ({circ}) and NO2- (•). (c) Experiment with goethite present, showing data for N2O ({blacktriangledown}) and Fe(II)-HCl ({square}). Data represent average values of three replicates, and the error bars reflect standard deviation. Note the split axes and the dual y axes in Fig. 2a.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 3. NH3-N (circles) and N2 (squares) production during 2.5 mM NO3- reduction by S. putrefaciens 200 in the presence (solid symbols) or absence (open symbols) of goethite. Whereas NO3- and biogenic NO2- were completely consumed in bottles lacking goethite, a mean NO2- concentration of 1.0 mM remained in bottles containing goethite at the conclusion of the experiment. Data represent average values of three replicates, and the error bars reflect standard deviation. Note the split y axis.

Goethite inhibition of NO3-and NO2- reduction.
Results from NO3- reduction experiments conducted with 2.5 mM NO3- in the absence of goethite are summarized in Fig. 2a. Most NO2- was reduced within 48 h, but low levels persisted through the first 100 h. Observed trends in mass balance were statistically indistinguishable from experiments conducted in the presence of goethite, and trends in NH3-N and N2-N product distribution were similar as well. Examination of these data and comparison with results summarized in Fig. 2 reveal three important observations, namely (i) significantly more N2O was produced when NO3- was reduced in the presence of goethite (Fig. 2c) than in the absence of goethite (Fig. 2a); (ii) in both systems, N2O production was not observed until after all NO3- had been consumed (Fig. 2a to c); and (iii) the rates of NO3- and NO2- reduction were notably higher in the absence of goethite (Fig. 2a) than in the presence of goethite (Fig. 2b).

Table 1 provides summary data showing that the presence of goethite can inhibit the rate of dissimilatory NO3- and NO2- reduction by S. putrefaciens. This table summarizes results from experiments that compare the initial rates of Fe(III) and NOx- reduction across various experimental matrices. The presence of NO3- resulted in an ~20-fold decrease in the rate of microbial goethite reduction, and the presence of goethite resulted in an ~10-fold decrease in the rate of NO2- reduction and an ~2-fold decrease in the rate of NO3- reduction. In addition, data presented in Fig. 4 indicate that the presence of goethite has a marked affect on the observed degree of N2O production in slurries containing 2.5 mM NO3-. Indeed, the N2O produced in slurries containing goethite and 2.5 mM NO3- was even substantially higher than in incubations containing 5.0 mM NO3- lacking goethite.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Comparison of initial rates of NO3- reduction, NO2- reduction, and Fe2+ production across various experimental matrices



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 4. Effect of goethite on N2O production rate with 5 mM initial NO3- without goethite ({square}), 2.5 mM initial NO3- without goethite ({circ}), and 2.5 mM initial NO3- with goethite (•). Data represent average values of three replicates, and the error bars reflect standard deviation. Note the split x and y axes.

In order to determine if the observed mutual inhibition was due to a process unique to S. putrefaciens 200, similar experiments were also conducted with enrichment cultures. Data from these experiments (Fig. 5) indicate that the presence of goethite can also inhibit dissimilatory reduction of NO3- and NO2- by a natural enrichment culture under conditions that are analogous to those used in experiments with S. putrefaciens. These cultures produced 200 µM HCl-soluble Fe(II) and virtually no N2O over the course of the experiments (data not shown). Although these experiments yielded different trends in NO3- reduction by-products, the data clearly demonstrate that inhibition of microbial NO3- and NO2- reduction by goethite is not limited to S. putrefaciens 200. Thus, this phenomenon may be important in a wide variety of sedimentary systems.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 5. Effect of goethite on NO3- reduction by UMTRA enrichment culture with NO3- without goethite ({circ}), NO2- without goethite ({square}), NO3- with goethite (•), and NO2- with goethite ({blacksquare}). All experiments had 2.5 mM initial NO3-. Data represent average values of three replicates, and the error bars reflect standard deviation. When not shown, error bars are smaller than the symbol size.

Stable isotope studies ({delta}15N-N2O).
Data presented in Fig. 6 display the relationship between extent of reaction (percentage of available NO2- reduced) versus the {delta}15N-N2O produced by the reduction of NO2- by (i) S. putrefaciens in the absence of goethite (microbial experiment), (ii) sterile microbially reduced goethite in the absence of S. putrefaciens (chemical experiment), and (iii) NO3- reduction by S. putrefaciens in the presence of goethite (combined experiment). This comparison and subsequent calculations assume that all N2O is produced via NO2- reduction and that significant N2O and ammonia production does not begin until all NO3- has been converted to NO2-. This assumption is supported by experimental results (Fig. 2 to 4) and allows us to assume no fractionation between NO3- and NO2- in the combined experiment.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 6. {delta}15N values of N2O for microbial ({circ}), chemical (•), and combined ({diamond}) experiments plotted versus extent of reaction.

Based on this assumption, the combined data have been normalized to account for the difference in the {delta}15N of the initial NO3- (+6.8 ± 0.2{per thousand}, combined experiment) and NO2- (2.0 ± 0.2{per thousand}, chemical and microbial experiments) in order to express all data on the same basis (NO2- reduction). The clear separation between the {delta}15N-N2OMICROBIAL (ca. +45 ± 27{per thousand}) and the {delta}15N-N2OCHEMICAL (ca. -25 ± 8{per thousand}) indicates that the stable isotopic signature of nitrogen in N2O ({delta}15N-N2O) can be used in our experiments to discriminate between N2O of microbial origin and N2O of chemical origin. Because we wanted to limit our arguments to cases where the NO2- concentration is at least 10x greater than the N2O concentration, we have considered only {delta}15N-N2OMICROBIAL data points where at least 50 µmol NO2-/liter remains (98% of original NO2-). Even if all of the N2OMICROBIAL data points are considered, however, the new value of {delta}15N- N2OMICROBIAL (ca. +31 ± 29{per thousand}) is still notably different from that of the {delta}15N-N2OCHEMICAL (ca. -25 ± 8{per thousand}). The large degree of error in the values for {delta}15N- N2OMICROBIAL arises from the large standard dilutions needed to obtain the 5 µmol of N2O needed for isotopic analysis (microbial experiments typically yielded less than 0.3 µmol N2O). The observed value for the NO2--normalized {delta}15N-N2OCOMBINED (ca. -28 ± 5{per thousand}) coincides closely with the observed value for {delta}15N-N2OCHEMICAL and therefore indicates that more than 95% of the N2O produced during the combined reaction is of chemical origin.

Although the {delta}15N-N2OMICROBIAL data presented in Fig. 6 are enriched in 15N with respect to the initial NO2- pool in all cases, the extent of enrichment varied substantially in different replicate experiments when >95% of the available NO2- had been consumed. Although the cause for this variation at the conclusion of the experiments is not clear, the variation does not materially affect our conclusion that the N2O from the combined experiment is primarily of chemical origin.


arrow
DISCUSSION
 
NO3- inhibition of goethite reduction.
It has been previously reported that inhibition of iron (hydr)oxide reduction by NO3- also inhibited changes in the speciation of oxide-sorbed metals (12), and previous researchers have also reported NO3- inhibition of iron reduction by S. putrefaciens species. DiChristina (14) reported that 15 mM initial NO3- inhibited ferric chloride reduction by 46 to 92% and inhibited ferric citrate reduction by 2 to 22%. Similarly, Obuekwe and Westlake (40, 41) reported that the presence of 1 mM NO3- resulted in an ~50% decrease in the rate of soluble Fe3+-phosphate (2% solution) reduction by a Pseudomonas sp. (later identified as S. putrefaciens 200). More recent experiments by Lee et al. with S. putrefaciens DK-1 also showed significant inhibition of 10 mM Fe(III) citrate reduction in the presence of 10 mM NO3- (28). These authors observed no inhibition until 25 h, after which Fe(II) production ceased and concentrations decreased by more than 30% over the course of their 95-h experiment. It is difficult to directly to compare data for extent of iron reduction due to differences in medium composition. Nevertheless, a comparison of reaction rates indicates that NO3- clearly inhibited the microbial reduction of crystalline iron (hydr)oxide minerals more strongly than it inhibits reduction of ferric citrate or the microcrystalline sources produced when ferric chloride is added to a circumneutral buffered medium.

Data in Fig. 1 also indicate that instead of simply slowing the rate of Fe2+ production, the initial presence of 1 mM NO3- appears to prevent any significant accumulation of Fe2+ for the first two sampling periods. This observation would, on the surface, seem contrary to the results of DiChristina (14) and Obuekwe and Westlake (40, 41). DiChristina's studies of competitive usage of NO3- and Fe(III) by S. putrefaciens 200 suggested only partial inhibition of Fe(II) production by 15 mM NO3-. His studies were done with either ferric citrate or ferric chloride as the Fe(III) source. Ferric citrate is an aqueous complex, whereas an acidic solution of ferric chloride would immediately precipitate a ferric oxyhydroxides mineral at circumneutral pH. Thus, the experiments with ferric chloride were, in effect, done with an amorphous or microcrystalline Fe(III) oxyhydroxides, while the experiments with ferric citrate were done with a true aqueous Fe(III) source. Interestingly, his work showed that NO3- inhibition of Fe(II) production by microaerobically grown cells was 3- to 23-fold greater in the presence of this solid-phase Fe(III) (FeCl3) than in the presence of Fe(III) citrate. Obuekwe's work, which also showed simultaneous NO3- reduction and Fe(II) production, utilized Fe(III) phosphate in which the Fe(III) is typically chelated by additional citrate. It would therefore appear that significant Fe(II) production occurs simultaneously with NO3- reduction when the Fe(III) is supplied in a chelated form. When Fe(III) is presented in the form of a solid-phase oxyhydroxide that would allow sorption of biogenic Fe(II), inhibition of Fe(II) production by NO3- is more substantial.

With respect to the N speciation and N balance in our system, our results are comparable to those of other researchers who have also reported incomplete recovery of N species during nitrate reduction to ammonia. Samuelsson and Rönner (47) reported that only 25 to 75% of NO3- added to isolates from the Baltic Sea was recovered as ammonia. In experiments with a different strain of S. putrefaciens (formerly Pseudomonas putrefaciens), Sammuelsson was unable to account for 6 to 91% of the NO3- consumed. In those experiments, only NH4+, N2O, and NO2- were measured, and the missing N was attributed to N2 or some other unmeasured N species (46). Using S. putrefaciens strain MR-1, Krause and Nealson, however, found little or no ammonia production and suggested that the carbon/NO3- ratio may also play a role in determining whether S. putrefaciens produces gaseous compounds (N2 or N2O) or ammonia during dissimilatory NO3- reduction (27).

It is generally accepted that under most conditions, microorganisms can generate more energy from dissimilatory NO3- reduction than from dissimilatory Fe(III) reduction. Consequently, NO3- inhibition of microbial iron (hydr)oxide reduction may result from differences in the rate of electron transport to NOx- and Fe(III) that have evolved to allow preferential use of the most favorable oxidant (1, 14, 41, 50, 51). Alternate explanations, however, are also possible. Since a small amount of Fe(II) was produced in our goethite-containing bottles as NO3- reduction proceeded (Fig. 2c), NO3-, NO2-, and Fe2+ were simultaneously present in the same system. Under suitable conditions, Fe2+ can chemically reduce both NO3- to NO2- (11) and NO2- to N2O and/or ammonia (10, 40, 57). Thus, concomitant reduction of NO3- and Fe(III) may create an apparent inhibition of microbial iron reduction by oxidizing Fe2+ to Fe(III) via chemical NOx- reduction. Results from previous studies testing this possibility are somewhat contradictory but do reveal two general themes. First, the chemical rate of NO2- reduction via oxidation of aqueous Fe2+ (produced via the microbial reduction of ferric chloride or ferric citrate) is too slow to account for a significant degree of NOx- inhibition of microbial Fe(III) reduction (14). Second, the chemical rate of NO2- reduction via oxidation of surface-associated Fe2+ [produced via the microbial reduction of iron (hydr)oxide minerals] is notably faster than the rate of NO2- reduction by aqueous Fe2+ (57). This finding that Fe2+-Fe(III) moieties (e.g., Fe2+ adsorbed to FeOOH minerals, green complexes, and/or ferrous precipitates) can reduce NO2- more rapidly than aqueous Fe2+ is supported by previous geochemical research into the catalytic effect of these moieties on the rate of NOx- reduction to N2O and/or ammonium (22, 23, 52). This information, coupled with the observation that goethite reduction in the presence of NO3- produced notably more N2O than NO3- reduction alone (Fig. 4), suggests that concomitant NO3- and goethite reduction in our systems does result in chemical reoxidation of Fe2+ coupled to NO2- reduction to N2O. However, the extremely small amount of N2O produced by this reaction indicates that most NO2- in our system is reduced enzymatically.

Goethite inhibition of NO3- and NO2- reduction.
While our work agrees with previous studies with S. putrefaciens 200 which indicate that the presence of NO3- will substantially inhibit microbial Fe(III) reduction (14), the observation that goethite inhibits NO3- and NO2- reduction is unprecedented and differs from the results of DiChristina (14) and Lee et al. (28), who reported that the presence of Fe(III)-chloride and/or Fe(III)-citrate had no notable effect on the observed rates of NO3- and NO2- reduction by S. putrefaciens. This disparity likely arises from significant differences in the nature of the Fe(III) source used in these experiments and the time scale of the experiments described by DiChristina (minutes to hours) and by the present work (hours to weeks). In addition, the observation that natural enrichment cultures can also show goethite inhibition of NO3- reduction (Fig. 5) indicates that this process may be important in natural systems with high iron content and low flow rates and thus merits significant further investigation.

Several mechanisms can potentially explain the goethite inhibition of NOx- reduction in both S. putrefaciens 200 and the UMTRA enrichment culture.

(i) The presence of goethite particles might have a toxic effect. Direct goethite toxicity is probably not a factor, since goethite is not known to be toxic, especially to microorganisms capable of dissimilatory iron reduction. To examine the prospect that goethite particles could have abraded the cells and either increased cell mortality or made cells more susceptible to other toxic effects, cells were incubated (5 days at 30°C with no carbon substrate and in the same experimental medium) in the presence and absence of goethite and a series of other sediments. In all cases, cells incubated in the presence of a solid surface yielded a higher final cell number than cells incubated in the absence of a solid surface (data not shown). Thus, cell abrasion probably does not contribute significantly to the observed goethite inhibition of microbial NOx- reduction.

(ii) If all NOx--reducing cells are coated by a dense layer of goethite crystals, such a coating might reduce the diffusive flux of NOx- to the cell surface. There is currently no evidence, however, that such dense coatings are formed or that they would limit NOx- diffusion to the cell surface.

(iii) If rates of electron transport to NOx- and Fe(III) are similar, goethite inhibition of NOx- reduction could result from simple kinetic competition between the two electron acceptors, a mechanism previously suggested to explain inhibition of Fe(III) reduction by oxygen (4). However, given the very small amount of Fe(III) reduction observed in the presence of NOx-, the extent of inhibition shown in Table 1 probably cannot be explained via a competitive mechanism alone.

(iv) Prolonged exposure to elevated concentrations of NO2- may be toxic (2). This mechanism, however, would not explain why NO2- reduction proceeded much more slowly in cultures containing goethite than in cultures absent goethite. Oxygen uptake studies on cells that had been cultured aerobically in the presence of NO2- indicated that NO2- showed little toxicity at concentrations up to 5 mmol/liter (data not shown). When combined with the observation that the NO2- reduction rate in the absence of goethite and presence of high (~2 mM) NO2- concentrations (Fig. 2a) was markedly greater than the NO2- reduction rate in the presence of goethite and similar NO2- concentrations (Fig. 2b), these experiments indicate that NO2- toxicity could not explain the observed goethite inhibition of NOx- reduction.

(v) Chemical oxidation of biogenic Fe2+ by NO2- results in the nucleation of ferric (hydr)oxide minerals on the surface of a cell, forming coatings which impede transport of NOx- into the cell. Such a hypothetical mechanism would proceed via three steps. Firstly, S. putrefaciens concomitantly reduces NO3- and, to a lesser extent, goethite, producing NO2- and small amounts of aqueous Fe2+. Some of the microbially produced Fe2+ then sorbs to the cellular surface, forming a reactive complex that can catalyze the oxidation of Fe2+ to Fe(III) via the chemical reduction of NO2- to N2O. Most of the NO2- produced is reduced biologically to ammonia and/or N2, with N2O as a minor by-product. Lastly, the Fe(III) precipitates as an iron oxyhydroxide mineral. This mineral can nucleate on the cell surface and thereby inhibit transport of NO3- and NO2- to NOx- reductases inside the cell. Since chemical NO2- reduction via Fe2+ oxidation is known to produce N2O (22, 23, 52, 57), this mechanism is supported by the increased N2O production observed to occur during concomitant NOx- and goethite reduction (Fig. 4). In addition, the data presented in Fig. 6 provide compelling evidence that N2O is being produced via chemical NO2- reduction via Fe2+ oxidation. An inhibitory mechanism involving the presence of Fe oxyhydroxide coatings on the cell surface is supported by the recent work of Liu et al. (29), who observed the formation of Fe mineral coatings on S. putrefaciens after exposure of cells to Fe2+. These coatings coincided with a lag phase during which reduction of Fe(III) citrate was inhibited. The mineral coatings disappeared as cells recovered from the lag phase, and the authors suggested that the cells had developed a mechanism for coating removal. Formation and subsequent removal of such coatings in our experiments would explain the long lag period seen in Fig. 2b and c (between ~50 and 300 h), during which very little NO2- or Fe(III) was reduced.

At the current time, however, a definitive mechanism of NOx- reduction inhibition by goethite cannot be completely substantiated by the data generated in these experiments. Since all cultures in our experiments were grown identically prior to resuspension in slurries containing or lacking goethite, levels of nitrate or nitrite reductase were initially induced equally in all reactors, regardless of whether goethite was present or absent.

{delta}15N2O studies of interactions between microbial NOx- and goethite reduction.
We are not aware of any reported values for the isotopic fractionation between NO2- and N2O for the chemical reduction of NO2- by Fe2+. However, it is possible to compare our values for biological NO2- reduction to N2O with values reported in the literature. Because the actual {delta}15N-N2O value is somewhat dependent on the {delta}15N of the starting NOx- species, these comparisons were made on the basis of the difference in {delta}15N between reactant and product ({Delta}15N = [{delta}15N-NOx- species] - [{delta}15N-N2O]). The key difference between our microbial studies and previously reported {Delta}15N values for N2O formation during denitrification and NH4+ oxidation (7, 33, 34, 56, 58) is that we report a negative {Delta}15N value (enrichment in 15N with respect to the source), whereas previous studies reported positive {Delta}15N values (depletion in 15N with respect to the source). Considering these reports, it should be noted that the N2O produced during denitrification is produced as an intermediate or final product, whereas the N2O produced during nitrate reduction to ammonia is thought to be produced as a minor side reaction (6, 54). As these processes are quite different, it is not surprising that our {Delta}15N values display a trend not reported in the literature. The production of 15N-enriched N2O during reduction of NOx- by S. putrefaciens 200 can easily be explained if (i) active biological NO2- reduction to ammonia by S. putrefaciens favors 14N over 15N and creates a pool of 15N-enriched NO2- and (ii) the N2O-producing side reaction utilizes this remnant pool of 15N-enriched NO2- to produce N2O that is depleted in 15N relative to the remnant pool but that is still enriched in 15N relative to the original NO2-.


arrow
Conclusion
 
This work has demonstrated not only that NO3- inhibits microbial reduction of crystalline Fe3+ sources more strongly than that of aqueous Fe3+ sources but also that the presence of crystalline Fe3+ (e.g., goethite) inhibits microbial NO3- and NO2- reduction. Increased N2O production in the presence of goethite and stable isotopic evidence both indicate that abiotic NO2- reduction to N2O coupled to Fe2+ oxidation is occurring concomitantly with microbial NOx- and goethite reduction.

The implications of this process can be dramatic in a number of ways. Most relevantly, it provides for a series of geochemical reactions that can potentially inhibit a microbiological process by nucleating minerals on an active cellular surface. While novel, the idea that the formation of minerals on a cell membrane can inhibit intracellular processes is not without precedent. Both gram-negative and gram-positive bacterial cell membranes are documented to be ideal sites for mineral nucleation (8, 9, 15, 17, 18, 25, 48), and previous researchers have demonstrated that the production of Fe2+ and ferrous minerals may be responsible for the cell passivation commonly observed during batch mode iron reduction experiments (29, 43). Further studies of the metabolic and environmental implications of abiotic Fe2+ oxidation occurring concomitantly with microbial goethite and NOx- reduction are needed.


arrow
ACKNOWLEDGMENTS
 
We acknowledge funding by the Natural and Accelerated Bioremediation Research (NABIR) program, Biological and Environmental Research (BER), U.S. Department of Energy (grant no. DE-FG02-97ER62482).

We also thank Phil Long for assistance in collection of sediments from the DOE UMTRA site in Shiprock, N.Mex., Eric Roden for invaluable discussions, and the anonymous reviewers for useful suggestions for improving the manuscript.


arrow
FOOTNOTES
 
* Corresponding author. Present address: Geosciences Research, IF-IRC MS 2107, Idaho National Engineering and Environmental Laboratory, P.O. Box 1625, Idaho Falls, ID 83404-2107. Phone: (208) 526-5395. Fax: (208) 526-0875. E-mail: coopdc{at}inel.gov. Back


arrow
REFERENCES
 
    1
  1. Achtnich, C. A., F. Bak, and R. Conrad. 1995. Competition for electron donors among nitrate reducers, ferric iron reducers, sulfate reducers, and methanogens in anoxic paddy soil. Biol. Fertil. Soils 19:65-72.
    2. 2
  2. Almeida, J. S., S. M. Julio, M. A. M. Reis, and M. J. T. Carrondo. 1995. Nitrite inhibition of denitrification by Pseudomonas fluorescens. Biotechnol. Bioeng. 46:194-201.[CrossRef]
    3. 3
  3. Arnold, R. G., T. J. DiChristina, and M. R. Hoffmann. 1986. Inhibitor studies of dissimilative Fe(III) reduction by Pseudomonas sp. strain 200 ("Pseudomonas ferrireductans"). Appl. Environ. Microbiol. 52:281-289.[Abstract/Free Full Text]
    4. 4
  4. Arnold, R. G., M. R. Hoffman, T. J. DiChristina, and F. W. Picardal. 1990. Regulation of dissimilatory Fe(III) reduction activity in Shewanella putrefaciens. Appl. Environ. Microbiol. 56:2811-2817.[Abstract/Free Full Text]
    5. 5
  5. Arnold, R. G., T. M. Olson, and M. R. Hoffman. 1986. Kinetics and mechanism of dissimilative Fe(III) reduction by Pseudomonas sp. 200. Biotechnol. Bioeng. 28:1657-1671.[CrossRef]
    6. 6
  6. Averill, B. A., and J. M. Tiedje. 1982. The chemical mechanism of microbial denitrification. FEBS Lett. 138:8-12.[CrossRef][Medline]
    7. 7
  7. Barford, C. C., J. P. Montoya, M. A. Altabet, and R. Mitchell. 1999. Steady-state nitrogen isotope effects of N2 and N2O production in Paracoccus denitrificans. Appl. Environ. Microbiol. 65:989-994.[Abstract/Free Full Text]
    8. 8
  8. Beveridge, T. J., and W. S. Fyfe. 1985. Metal fixation by bacterial cell walls. Can. J. Earth Sci. 22:1893-1898.
    9. 9
  9. Beveridge, T. J., J. D. Meloche, W. S. Fyfe, and R. G. E. Murray. 1983. Diagenesis of metals chemically complexed to bacteria: laboratory formation of metal phosphates, sulfides, and organic condensates in artificial sediments. Appl. Environ. Microbiol. 45:1094-1108.[Abstract/Free Full Text]
    10. 10
  10. Brons, H. J., W. R. Hagen, and A. J. B. Zehnder. 1991. Ferrous iron dependent nitric oxide production in nitrate reducing cultures of Escherichia coli. Arch. Microbiol. 155:341-347.[Medline]
    11. 11
  11. Buresh, R. J., and J. T. Moraghan. 1976. Chemical reduction of nitrate by ferrous iron. J. Environ. Qual. 5:320-325.[Abstract/Free Full Text]
    12. 12
  12. Cooper, D. C., F. Picardal, J. Rivera, and C. Talbot. 2000. Zinc immobilization and magnetite formation via ferric oxide reduction by Shewanella putrefaciens200. Environ. Sci. Technol. 34:100-106.
    13. 13
  13. Cummings, D. E., F. Caccavo, S. Fendorf, and R. F. Rosenzweig. 1999. Arsenic mobilization by the dissimilatory Fe(III)-reducing bacterium Shewanella alga BrY. Environ. Sci. Technol. 33:723-729.[CrossRef]
    14. 14
  14. DiChristina, T. J. 1992. Effects of nitrate and nitrite on dissimilatory iron reduction by Shewanella putrefaciens200. J. Bacteriol. 174:1891-1896.[Abstract/Free Full Text]
    15. 15
  15. Douglas, S., and T. J. Beveridge. 1998. Mineral formation by bacteria in natural microbial communities. FEMS Microbiol. Ecol. 26:79-88.[CrossRef]
    16. 16
  16. Erbs, M., H. C. B. Hansen, and C. E. Olsen. 1999. Reductive dechlorination of carbon tetrachloride using iron(II) iron(III) hydroxide sulfate (green rust). Environ. Sci. Technol. 33:307-311.
    17. 17
  17. Fein, J. B., C. J. Daughney, N. Yee, and T. A. Davis. 1997. A chemical equilibrium model for metal adsorption onto bacterial surfaces. Geochim. Cosmochim. Acta 61:3319-3328.
    18. 18
  18. Fortin, D., F. G. Ferris, and T. J. Beveridge. 1997. Surface-mediated mineral development by bacteria., p. 161-181. In J. F. Banfied and K. H. Nealson (ed.), Geomicrobiology, vol. 35. Mineralogical Society of America, Washington, D.C.
    19. 19
  19. Fredrickson, J. K., J. M. Zachara, D. W. Kennedy, M. C. Duff, Y. A. Gorby, S.-M. Li, and K. M. Krupka. 2000. Reduction of U(VI) in goethite({alpha}-FeOOH) suspensions by a dissimilatory metal-reducing bacterium. Geochim. Cosmochim. Acta 64:3085-3098.[CrossRef]
    20. 20
  20. Génin, J.-M. R., G. Bourrié, F. Trolard, M. Abdelmoula, A. Jaffrezic, P. Refait, V. Maitre, B. Humbert, and A. Herbillon. 1998. Thermodynamic equilibria in aqueous suspensions of synthetic and natural Fe(II)-Fe(III) green rusts: occurrences of the mineral in hydromorphic soils. Environ. Sci. Technol. 32:1058-1068.[CrossRef]
    21. 21
  21. Greenberg, A. E., L. S. Clesceri, and A. D. Eaton (ed.). 1992. Standard methods for the examination of water and wastewater, 18th ed. American Public Health Association, Washington D.C.
    22. 22
  22. Hansen, H. C. B., O. K. Borgaard, and J. Sørensen. 1994. Evaluation of the free energy of formation of Fe(II)-Fe(III) hydroxide-sulfate (green rust) and its reduction of nitrite. Geochim. Cosmochim. Acta 58:2599-2608.
    23. 23
  23. Hansen, H. C. B., C. B. Koch, H. Nancke-Krogh, O. K. Borggaard, and J. Sørensen. 1996. Abiotic nitrate reduction to ammonium: key role of green rust. Environ. Sci. Technol. 30:2053-2056.[CrossRef]
    24. 24
  24. Kim, S., and F. W. Picardal. 1999. Enhanced anaerobic biotransformation of carbon tetrachloride in the presence of reduced iron oxides. Environ. Toxicol. Chem. 18:2142-2150.[CrossRef]
    25. 25
  25. Konhauser, K. O., W. S. Fyfe, F. G. Ferris, and T. J. Beveridge. 1993. Metal sorption and mineral precipitation by bacteria in two Amazonian river systems: Rio Solimões and Rio Negro, Brazil. Geology 21:1103-1106.[Abstract/Free Full Text]
    26. 26
  26. Kostka, J. E., and G. W. Luther. 1994. Partitioning and speciation of solid phase iron in saltmarsh sediments. Geochim. Cosmochim. Acta 58:1701-1710.
    27. 27
  27. Krause, B., and K. H. Nealson. 1997. Physiology and enzymology involved in denitrification by Shewanella putrefaciens. Appl. Environ. Microbiol. 63:2613-2618.[Abstract]
    28. 28
  28. Lee, I.-G., S.-J. Kim, and T.-Y. Ahn. 2000. Inhibitory effect of nitrate on Fe(III) and humic acid reduction in Shewanella putrefactions DK-1. J. Microbiol. 38:180-182.
    29. 29
  29. Liu, C., J. M. Zachara, Y. A. Gorby, J. E. Szecsody, and C. F. Brown. 2001. Microbial reduction of Fe(III) and sorption/precipitation of Fe(II) on Shewanella putrefaciens strain CN32. Environ. Sci. Technol. 35:1385-1393.[Medline]
    30. 30
  30. Lloyd, J. R., P. Yong, and L. E. Macaskie. 2000. Biological reduction and removal of Np(V) by two microorganisms. Environ. Sci. Technol. 34:1297-1301.[CrossRef]
    31. 31
  31. Lovely, D. R. 1991. Dissimilatory Fe(III) and Mn(IV) reduction. Microbiol. Rev. 55:259-287.
    32. 32
  32. Lovely, D. R., and E. J. P. Phillips. 1986. Organic matter mineralization with reduction of ferric iron in anaerobic sediments. Appl. Environ. Microbiol. 51:683-689.[Abstract/Free Full Text]
    33. 33
  33. Mandernack, K. W., T. Rahn, C. Kinney, and M. Wahlen. 2000. The biogeochemical controls of the {delta}15N and {delta}18O of N2O produced in landfill cover soils. J. Geophys. Res. 105:709-720.
    34. 34
  34. Mariotti, A., J. C. Germon, and A. Leclerc. 1982. Nitrogen isotope fractionation associated with the NO2-->N2O step of denitrification in soils. Can. J. Soil Sci. 62:227-241.
    35. 35
  35. Misawa, T., K. Hashimoto, and S. Shimodaira. 1974. The mechanism of formation of iron oxide and oxyhydroxides in aqueous solutions at room temperature. Corr. Sci. 14:131-149.[CrossRef]
    36. 36
  36. Myers, C. R., and J. M. Myers. 1993. Role of menaquinone in the reduction of fumarate, nitrate, iron(III), and manganese(IV) by Shewanella putrefaciens MR-1. FEMS Microbiol. Lett. 114:215-222.[CrossRef]
    37. 37
  37. Myers, C. R., and K. H. Nealson. 1990. Iron mineralization by bacteria: metabolic coupling of iron reduction to cell metabolism in Alteromonas Putrefaciens strain MR-1, p. 131-149. In R. B. Frankel and R. P. Blakemore (ed.), Iron biominerals. Plenum Press, New York, N.Y.
    38. 38
  38. Nolan, B. T., and J. D. Stoner. 2000. Nutrients in groundwaters of the conterminous United States, 1992-1995. Environ. Sci. Technol. 34:1156-1165.[CrossRef]
    39. 39
  39. Obuekwe, C. O. 1980. Microbial corrosion of a crude oil pipeline. Ph.D thesis, University of Alberta, Edmonton, Alberta, Canada.
    40. 40
  40. Obuekwe, C. O., and D. W. S. Westlake. 1981. Effect of nitrate on reduction of ferric iron by a bacterium isolated from crude oil. Can. J. Microbiol. 27:692-697.[Medline]
    41. 41
  41. Obuekwe, C. O., and D. W. S. Westlake. 1982. Effect of reducible compounds (potential electron acceptors) on reduction of ferric iron by Pseudomonas species. Microbiol. Lett. 19:57-62.
    42. 42
  42. Picardal, F. W., R. G. Arnold, H. Couch, A. M. Little, and M. E. Smith. 1993. Involvement of cytochromes in the anaerobic biotransformation of tetrachloromethane by Shewanella putrefaciens200. Appl. Environ. Microbiol. 59:3763-3770.[Abstract/Free Full Text]
    43. 43
  43. Roden, E. E., and M. M. Urrutia. 1999. Ferrous iron removal promotes microbial reduction of crystalline iron(III) oxides. Environ. Sci. Technol. 33:1847-1853.[CrossRef]
    44. 44
  44. Roden, E. E., M. M. Urrutia, and C. J. Mann. 2000. Bacterial reductive dissolution of crystalline Fe(III) oxide in continuous-flow column reactors. Appl. Environ. Microbiol. 66:1062-1065.[Abstract/Free Full Text]
    45. 45
  45. Roden, E. E., and J. M. Zachara. 1996. Microbial reduction of crystalline iron(III) oxides: influence of oxide surface area and potential for cell growth. Environ. Sci. Technol. 30:1618-1628.[CrossRef]
    46. 46
  46. Samuelsson, M.-O. 1985. Dissimilatory nitrate reduction to nitrite, nitrous oxide, and ammonium by Pseudomonas putrefaciens. Appl. Environ. Microbiol. 50:812-815.[Abstract/Free Full Text]
    47. 47
  47. Samuelsson, M.-O., and U. Rönner. 1982. Ammonium production by dissimilatory nitrate reducers isolated from Baltic Sea water, as indicated by 15N study. Appl. Environ. Microbiol. 44:1241-1243.[Abstract/Free Full Text]
    48. 48
  48. Schultze-Lam, S., D. Fortin, and T. J. Beveridge. 1996. Mineralization of bacterial surfaces. Chem. Geol. 132:171-181.
    49. 49
  49. Semple, K. M., and D. W. S. Westlake. 1987. Characterization of iron-reducing Alteromonas putrefaciens strains from oil-field fluids. Can. J. Microbiol. 33:366-371.
    50. 50
  50. Sørensen, J. 1987. Nitrate reduction in marine sediment: Pathways and interactions with iron and sulfur cycling. Geomicrobiol. J. 5:401-421.
    51. 51
  51. Sørensen, J. 1982. Reduction of ferric iron in anaerobic, marine sediment and interaction with reduction of nitrate and sulfate. Appl. Environ. Microbiol. 43:319-324.[Abstract/Free Full Text]
    52. 52
  52. Sørensen, J., and L. Thorling. 1991. Stimulation by lepidocrocite ({gamma}-FeOOH) of Fe(II)-dependent nitrite reduction. Geochim. Cosmoschim. Acta 55:1289-1294.
    53. 53
  53. Stookey, L. L. 1970. Ferrozine—a new spectrophotometric reagent for iron. Anal. Chem. 42:779-781.[CrossRef]
    54. 54
  54. Stouthamer, A. H. 1988. Dissimilatory reduction of oxidized nitrogen compounds, p. 245-303. In A. J. B. Zehnder (ed.), Biology of anaerobic microorganisms. John Wiley & Sons, New York, N.Y.
    55. 55
  55. Tsapin, A., K. Nealson, T. Meyers, M. A. Cusanovich, J. Van Beuumen, L. D. Crosby, B. A. Feinberg, and C. Zhang. 1996. Purification and properties of a low-redox-potential tetraheme cytochrome c3 from Shewanella putrefaciens. J. Bacteriol. 178:6386-6388.[Abstract/Free Full Text]
    56. 56
  56. Ueda, S., N. Ogura, and E. Wada. 1991. Nitrogen stable isotope ratio of groundwater N2O. Geophys. Res. Lett. 18:1449-1452.
    57. 57
  57. Weber, K. A., F. W. Picardal, and E. E. Roden. 2001. Microbially-catalyzed nitrate-dependent oxidation of biogenic solid-phase Fe(II) compounds. Environ. Sci. Technol. 35:1644-1650.[Medline]
    58. 58
  58. Webster, E. A., and D. W. Hopkins. 1996. Nitrogen and oxygen isotope ratios of nitrous oxide emitted from soil and produced by nitrifying and denitrifying bacteria. Biol. Fertil. Soils 22:326-330.[CrossRef]
    59. 59
  59. Wilhelm, E., R. Battino, and R. J. Wilcock. 1977. Low-pressure solubility of gases in liquid water. Chem. Rev. 77:219-262.[CrossRef]
    60. 60
  60. Zachara, J. M., J. K. Fredrickson, S. C. Smith, and P. L. Gassman. 2001. Solubilization of Fe(III) oxide-bound trace metals by a dissimilatory Fe(III) reducing bacterium. Geochim. Cosmochim. Acta 65:75-93.

Applied and Environmental Microbiology, June 2003, p. 3517-3525, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3517-3525.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Rakshit, S., Matocha, C. J., Coyne, M. S. (2008). Nitrite Reduction by Siderite. Soil Sci. 72: 1070-1077 [Abstract] [Full Text]  
  • Matocha, C. J., Coyne, M. S. (2007). Short-term Response of Soil Iron to Nitrate Addition. Soil Sci. 71: 108-117 [Abstract] [Full Text]  
  • Coby, A. J., Picardal, F. W. (2005). Inhibition of NO3- and NO2- Reduction by Microbial Fe(III) Reduction: Evidence of a Reaction between NO2- and Cell Surface-Bound Fe2+. Appl. Environ. Microbiol. 71: 5267-5274 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cooper, D. C.
Right arrow Articles by Coby, A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cooper, D. C.
Right arrow Articles by Coby, A. J.
Agricola
Right arrow Articles by Cooper, D. C.
Right arrow Articles by Coby, A. J.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»