INTRODUCTION

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Autotrophic ammonia-oxidizing bacteria (AOB) have been considered ideal microbial indicators of perturbations caused by pollutants in natural environments (20, 54, 58). At least three reasons for this can be formulated: (i) AOB perform a vital bottleneck role in N cycling in many natural environments because of their unique ability to oxidize NH₄⁺ to NO₂ (1, 45, 51); (ii) AOB are generally sensitive to pollutants and often require a long time for recovery (20, 25, 33, 54); and (iii) there is a simple and cheap method for measuring NH₄⁺ oxidation activities in environmental samples (25). In addition, the AOB constitute a very promising model group for studies of microbial diversity and activity, since the methods used for studying these bacteria have been drastically improved recently (9). Given the impressive development of methods, it is likely that AOB will continue to be considered attractive indicators of environmental perturbations in the years to come.

Inhibitory effects of various compounds on AOB in various environments have been extensively reported. However, due to the difficulties involved in cultivation of AOB, there is still a general lack of data concerning the effects of toxic chemicals on the general physiology of these bacteria. Previous studies have dealt mostly with specific nitrification inhibitors targeting the NH₄⁺ monooxygenase enzyme (5, 42), and very little is known about other targets of toxicants in AOB cells.

Xenobiotic surfactants comprise a very important group of potentially toxic compounds that are believed to be harmful due to disruption of the function and structure of bacterial membranes (11, 13, 14, 43, 64). The linear alkylbenzene sulfonates (LAS) constitute the quantitatively most important group of synthetic surfactants used today, and the use of these compounds will likely increase in the years to come (12). Concern has been raised about possible toxic effects of LAS on susceptible biota in agricultural soils receiving high loads of these anionic surfactants from recycled sewage sludge or contaminated irrigation water (38, 61). Indeed, recent research has demonstrated that autotrophic NH₄⁺ oxidation in agricultural soil may be affected at LAS concentrations occasionally encountered in soils after application of municipal sewage sludge (19, 60, 61; L. Elsgaard, S. O. Petersen, and K. Debosz, submitted for publication). Here we describe a detailed study of the effects of LAS on four strains of AOB belonging to different phylogenetic clusters in the  subgroup of the class Proteobacteria. Surfactant-induced toxic effects on various essential physiological parameters, such as metabolic activity, specific growth rate, and microcolony formation, were investigated. Our aims were to obtain toxicological information for each of these parameters and to compare the overall physiological status of nonstressed and LAS-stressed cultures of ammonia-oxidizing cells.

	MATERIALS AND METHODS

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Bacterial strains. Three soil isolates, Nitrosomonas europaea NCIMB 11850 (63), Nitrosospira multiformis (formerly Nitrosolobus multiformis) NCIMB 11849 (59), and Nitrosospira sp. strain AV (41), and one marine isolate, "Nitrosococcus mobilis" NC2, of AOB belonging to the  subclass of the Proteobacteria were used. Based on its 16S ribosomal DNA sequence, "N. mobilis" should be reclassified as a member of the genus Nitrosomonas (27), and below we refer to the "N. mobilis" strain as a Nitrosomonas strain. The marine strain and the soil isolates were kindly supplied by H.-P. Koops (University of Hamburg, Hamburg, Germany) and J. I. Prosser (University of Aberdeen, Aberdeen, United Kingdom), respectively.

Culture conditions. An autotrophic growth medium modified from the medium of MacDonald and Spokes (40) was used for all experiments. This medium contained (per liter of membrane-filtered water) 0.5 g of (NH₄)₂SO₄, 0.2 g of KH₂PO₄, 20 mg of CaCl₂ · 2H₂O, 40 mg of MgSO₄ · 7H₂O, 3.8 mg of FeNaEDTA, 1 µg of phenol red, 4.77 g of HEPES, and 1 ml of a trace element solution (16). The pH of the medium was adjusted to 7.5 with 10 M NaOH, and the medium was autoclaved at 121°C for 20 min. For all growth experiments with suspended or filter-grown cells (see below), sterile filtered NaHCO₃ was added as a carbon source to a final concentration of 1.5 mM. The purity of the strains used was routinely tested on tryptic soy agar plates and by epifluorescense microscopy (Zeiss Axioscope or Axioplan). Only AOB cultures that produced no visible colonies after 5 days of incubation on tryptic soy agar plates were used for experiments. Prior to individual experiments, an LAS was added to each experimental flask or petri dish from a stock solution in which methanol was the solvent. The methanol was subsequently allowed to evaporate in a sterile hood (Holten LaminAir, Allerød, Denmark) before fixed volumes of medium were added to each container. The LAS used was a C₁₂ homologue [4-(2-dodecyl)benzenesulfonic acid, sodium salt; molecular mass, 348 g mol¹; purity, >97%; impurities consisted mainly of sodium salts)] that was synthesized and supplied by Risø National Laboratory (Roskilde, Denmark). All other chemicals were analytical grade and were obtained from commercial suppliers. Acid-washed glassware was used for all experiments.

Growth rates in liquid batch cultures. Growth experiments were carried out in triplicate in sterile 120-ml serum vials containing 20 ml of newly inoculated growth medium (0.1%[vol/vol] from an early-stationary-phase culture) and different concentrations of LAS. The vials were capped with Teflon-coated rubber septa and shaken to resolubilize the surfactant added. Incubation was performed at 30°C in the dark on a rotary shaker (200 rpm), and subsamples were taken at regular intervals by means of a sterile syringe. To measure NO₂ production, NO₂ samples were frozen (18°C) and examined within a few days. Cell counts were determined directly by using acridine orange-stained black polycarbonate membrane filters (pore size, 0.2 µm; diameter, 25 mm; Poretics Products, Livermore, Calif.) as described previously (28). Specific growth rates and generation times were calculated from log-transformed growth curves based on the accumulation of NO₂, since this approach has been shown to give more precise estimates of specific growth rates than direct cell counting in batch cultures of AOB (52). When cultures were challenged with surfactant, a slow initial exponential growth phase was sometimes followed by a faster exponential growth phase; in these cases the steepest part of the log-transformed growth curve was used to determine the specific growth rate. Cultures showing no or only weak NO₂ accumulation (<200 µM) were monitored for 3 months to confirm that they were negative for growth.

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Microcolony formation by early-stationary-phase cells exposed to LAS. The viability of AOB was determined by using a modified version of the microcolony technique previously used as a viability index for AOB in our laboratory (28, 63). The assay measures the proportion of cells that are able to initiate growth and form microcolonies consisting of four or more cells when they are incubated on a membrane filter floating on autotrophic growth medium. However, instead of using naked membrane filters as in the original protocol (28), the cell-inoculated filters were mounted on a circular glass coverslip coated with a thin layer of silicone oil as described previously (29). The modification was essential because surfactant-containing medium disrupted microcolony formation on naked filters. Briefly, early-stationary-phase cells from the four AOB cultures were filtered onto white polycarbonate membrane filters (pore size, 0.2 µm; diameter, 25 mm; Poretics Products). The filters were then transferred to the surface of 10 ml of sterile filtered autotrophic growth medium without (NH₄)₂SO₄ by using a sterile petri dish with blotting paper at the bottom. The membrane filters were then mounted with the bacterial side towards a silicone oil-coated glass coverslip. The mounted filters were finally transferred (filter side down) to the surface of sterile growth medium in a glass petri dish divided into four wells (each containing 4 ml of medium with the same LAS concentration; i.e., there were four replicates). Optimal incubation times were determined for each strain by determining the time necessary to maximize microcolony formation for counting; the strain-specific optimal incubation times (between 5 and 10 days) were subsequently used for all the LAS exposure studies. Incubation was performed in moist chambers at 30°C in the dark. Incubation was terminated by acridine orange staining and subsequent mounting of the filters on microscope slides as described elsewhere (63). It was important to wipe off any drops of LAS-containing medium from the filters and to rinse the filters with water in order to prevent nonspecific staining of the filters exposed to LAS. The filters were inspected with an epifluorescense microscope (Zeiss Axioscope). Finally, NO₂ levels were measured (see below) in the filter-containing wells at the end of incubation.

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NH₄⁺ oxidation and CO₂ fixation rates in early-stationary-phase cells exposed to LAS. The effects of LAS on NH₄⁺ oxidation and CO₂ fixation rates were determined with early-stationary-phase cultures of all four AOB. Cells were grown in 500-ml batch cultures (1,500-ml batch cultures for Nitrosospira sp. strain AV) and harvested when the NO₂ level had increased to 5 to 6 mM. The cultures were added to 300-ml centrifuge bottles and centrifuged (Beckman J2-21 M/E; 9,000 × g, 20°C, 30 min). The pellets were resuspended in NH₄⁺-free growth medium by using 25% (strain AV) or 50% (other strains) of the original culture volume.

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Adaptability of N. europaea after exposure to LAS. To investigate the short-term adaptability of AOB to LAS, the physiological stress parameter tests described above were repeated with N. europaea cells which had been cultured for about 20 generations in medium containing a sublethal concentration of LAS. The N. europaea strain used for these experiments was first inoculated (0.1%, vol/vol) into fresh medium containing 10 mg of LAS liter¹. After a second transfer to LAS-containing medium, the cells (in the early stationary phase) were used for activity, growth, and microcolony formation assays as described above, except that the NH₄⁺ oxidation experiments were performed in medium with a high HCO₃ concentration (1 mM).

Analytical techniques. LAS contents were measured by reverse-phase high-performance liquid chromatography by using a 25-cm Nucleosil 100-5 C₁₈ column (Macherey-Nagel, Düren, Germany) and UV detection (Dionex spectral array detector; Dionex, Sunnyvale, Calif.) as described previously (50). NO₂ contents were measured spectrophotometrically by using a plate reader (EL312e; Bio-Tek Instruments, Winooski, Vt.) as described previously (63). The radioactivity associated with radiolabelled AOB cells was determined by liquid scintillation counting by using 10 ml of Ultima Gold XR (Packard) as the scintillation cocktail. Samples were stored for 24 h in the dark to reduce quenching and then counted with a Packard 1600 TR liquid scintillation counter.

Estimation of key toxicological parameters. The lowest-observed-effect concentrations and highest-no-effect concentrations were determined by Dunnett's t test by using an SAS analysis of variance procedure (57; Elsgaard et al., submitted), and 50% effective concentrations were estimated by nonlinear regression of nontransformed data as described by Nyholm et al. (44).

	RESULTS

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Effects of LAS on growth in liquid cultures. It was important for data interpretation if LAS could be degraded by the AOB strains. When N. europaea and "N. mobilis" were cultivated in the presence of a sublethal LAS concentration, 3 mg liter¹, all of the LAS was recovered in the fully grown cultures. In contrast, the two Nitrosospira strains removed significant amounts of LAS from the medium, as only 88% (N. multiformis) and 58% (Nitrosospira sp. strain AV) of the added LAS remained in the fully grown cultures. Removal of LAS by adsorption to the AOB cells was negligible, however, because the LAS levels measured in the liquid phase were similar in flasks with and without added cells (data not shown).

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Representative growth curves for the four AOB strains investigated grown in liquid medium containing different LAS concentrations. (A) N. europaea; (B) "N. mobilis"; (C) N. multiformis; (D) Nitrosospira sp. strain AV. Different symbols indicate different concentrations of LAS (in milligrams per liter). d, day.

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View larger version (11K): [in this window] [in a new window]   FIG. 2.   Influence of LAS on the specific growth rates of the four AOB strains investigated. d1, day1; l1, liter1.

Effects of LAS on microcolony formation. Early-stationary-phase cells of all four AOB strains were used to test the toxic effect of LAS on cell viability, as measured by the ability of cells to form microcolonies on a membrane filter surface. It was previously suggested that this assay provides a useful viability index for AOB in cultures (28), and we have used it to study specific physiological responses to osmotic stress in N. europaea (63). Formation of colonies containing four or more cells, corresponding to at least two cell divisions on the filters, was taken to indicate that AOB cells were viable by the standard protocol (28). In the present study, however, we supplemented the standard protocol for enumeration of viable cells in N. europaea and "N. mobilis" cultures by also recording the formation of microcolonies containing 16 more cells, corresponding to at least four cell divisions on the filters.

View larger version (12K): [in this window] [in a new window]   FIG. 3.   Influence of LAS on microcolony formation (MCFU) (i.e., the percentage of membrane-immobilized cells forming microcolonies). Symbols: , microcolonies consisting of four or more cells; , microcolonies consisting of 16 or more cells. l1, liter1.

Effects of LAS on NH₄⁺ oxidation and CO₂ fixation rates. Early-stationary-phase cells of all four AOB strains were used to test the acute toxic effects of LAS on NH₄⁺ oxidation and CO₂ fixation activities in short-term experiments performed with resting cells from stationary-phase cultures. Figure 4 shows that both NH₄⁺ oxidation and CO₂ fixation rates progressively decreased as a function of LAS amendment, but N. europaea and "N. mobilis" were generally less susceptible than the two Nitrosospira strains. In general, LAS inhibited both NH₄⁺ oxidation and CO₂ fixation rates to the same degree, but an exception was observed with N. europaea, where NH₄⁺ oxidation was markedly more sensitive than CO₂ fixation in the 3- to 18-mg liter¹ range. Repeating the experiments resulted in almost perfect reproduction of the dose-response curves, indicating that the apparent differences in the shapes of the dose-response curves for the NH₄⁺ oxidation and CO₂ fixation rates in N. europaea were real.

View larger version (22K): [in this window] [in a new window]   FIG. 4.   Influence of LAS on CO2 fixation (A to D) and NH4+ oxidation (E to H) rates (relative numbers) in the four AOB strains investigated. The absolute rates of NH4+ oxidation are given in Table 1. l1, liter1.

Estimation of physiological and toxicological test parameters. Table 1 shows that the generation times, specific cell yields, and specific NH₄⁺ oxidation activities varied within a factor of 4 for the four AOB strains. In contrast, the percentages of cells forming microcolonies varied by more than 2 orders of magnitude. Stationary-phase cells of the two Nitrosomonas strains (including "N. mobilis") clearly exhibited a much better ability to form microcolonies than cells of the two Nitrosospira strains.

Effect of LAS preexposure in N. europaea. Although spontaneously inhibited by LAS treatment, growth occasionally resumed in some AOB cultures after long lag periods, and the subsequent growth rates were similar to those of the untreated controls. This is most clearly shown in Fig. 1 for the N. europaea and "N. mobilis" cultures grown at an LAS concentration slightly above the lowest observed effect concentration. Based on these observations, we examined whether physiological parameters in N. europaea, such as specific metabolic activity (NH₄⁺ oxidation rate), lag period prior to growth, specific growth rate, and microcolony formation, were reversibly or irreversibly affected by preexposure to LAS. We compared cultures by using untreated inocula (controls) and inocula which had been preexposed for approximately 20 cell generations (two culture transfers) to a sublethal LAS level, 10 mg liter¹. The comparison failed to reveal any difference in the lengths of the lag phases or the subsequent growth rates (data not shown). We did observe, however, that when the length of exposure to LAS was extended by repeated culturing (tested up to approximately 100 cell generations) in the presence of 10 mg of LAS liter¹, cultures frequently stopped growing, while this was never the case during routine cultivation of N. europaea without LAS (data not shown). The observations indicated that long-term exposure to LAS, even at a sublethal level (10 mg liter¹), eventually led to compromised cell functions that resulted in irreversible cessation of growth.

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View larger version (17K): [in this window] [in a new window]   FIG. 5.   Effect of preexposure for approximately 20 generations to a sublethal LAS level (10 mg liter1) on microcolony formation (A) and the cell-specific NH4+ oxidation rate (B) of N. europaea. Symbols: , nontreated control cells; , preexposed inoculant cells. MCFU, microcolony formation; l1, liter1.

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	DISCUSSION

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Physiological characteristics of the AOB strains. The generation times, specific cell yields, and NH₄⁺ oxidation rates of the four strains investigated (Table 1) were generally within the ranges of values reported previously for pure cultures of AOB (7, 51), although each strain grew quite rapidly in the present study. In earlier reports (3, 47-49) generation times of 11.6 to 15.4 h were recorded for N. europaea NCIMB 11850, while the same strain grew with generation times of only 7.8 to 9.6 h under similar conditions in our laboratory (63) (Table 1). Likewise, longer generation times have been reported for N. multiformis (18 h) (59), Nitrosospira sp. strain AV (21 to 40 h) (6, 7), and "N. mobilis" NC2 (12 to 13 h) (36) compared to those reported in this study (Table 1). This comparison supports the hypothesis that AOB strains may adapt to higher growth rates when they are cultured repeatedly in laboratory media, as suggested previously (2, 48).

LAS interactions with the AOB strains. The two Nitrosomonas strains did not degrade LAS, while N. multiformis and Nitrosospira sp. strain AV removed 12 and 42% of the LAS added, respectively. Our study thus represents the first report of LAS removal by autotrophic AOB. We found no evidence that physical or chemical processes (adhesion, volatilization, or precipitation) explain the observed removal of LAS, and we therefore suggest that the two Nitrosospira strains have a limited capacity for LAS degradation. It has recently been shown that some methanotrophic bacteria are able to cometabolize LAS by means of a methane monooxygenase (31, 32), which functionally resembles the ammonia monooxygenase of AOB. Thus, our finding was not totally unexpected, but it was surprising that only the two Nitrosospira strains were able to remove LAS. Attempts to promote degradation of LAS by N. europaea by using higher LAS concentrations (6, 12, or 18 mg liter¹) were all unsuccessful (data not shown). The biodegradation data did not affect interpretation of our LAS toxicity experiments, however, since constant or almost constant LAS levels would have been present throughout the incubation periods. Only in the long-term experiments performed with Nitrosospira sp. strain AV may significant LAS degradation have obscured interpretation of the levels of toxicity. Degradation products of LAS are less toxic than the parent compound (46), and the toxicity of LAS might thus have been slightly underestimated in these experiments. Finally, the observed variability among AOB strains in terms of degradation potential strongly suggests that use of N. europaea as a model organism for studies of the bioremediation potential of AOB (17, 18, 30) should be complemented with studies of Nitrosospira strains.

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