Response of Nitrobacter spp. Ribosomal Gene and Transcript

Dec 29, 2007 - transition from a minimum value at nitrite starvation (∼0.07) to a maximum value with excess nitrite present (∼4), and 5 h were req...
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Environ. Sci. Technol. 2008, 42, 901–907

Response of Nitrobacter spp. Ribosomal Gene and Transcript Abundance Following Nitrite Starvation and Exposure to Mechanistically Distinct Inhibitors S H A W N A . H A W K I N S , * ,†,§ K E V I N G . R O B I N S O N , ‡,§ A L I C E C . L A Y T O N , §,| A N D G A R Y S . S A Y L E R §,| Department of Biosystems Engineering and Soil Science, Department of Civil and Environmental Engineering, The Center for Environmental Biotechnology, and Department of Microbiology, The University of Tennessee, Knoxville, Tennessee 37996

Received June 29, 2007. Revised manuscript received November 5, 2007. Accepted November 12, 2007.

The Nitrobacter spp. rRNA gene (rDNA) and relative rRNA transcript abundance (rRNAt/rDNA ratio) were evaluated in response to sudden changes in the nitrite oxidation rate. The rDNA abundance poorly indicated sudden transitions in the rate, whereas the relative rRNAt abundance usually varied quickly and significantly. In response to changes in nitrite concentration, 8 h were required for the rRNAt/rDNA ratio to transition from a minimum value at nitrite starvation (∼0.07) to a maximum value with excess nitrite present (∼4), and 5 h were required for this metric to return to the minimum value after nitrite starvation re-ensued. Generally, the relative rRNAt abundance dropped significantly after 4.5 h of exposure to three different inhibitors. A sharp decline in the rRNAt/rDNA ratio occurred during exposure to 3,5-DCP (from 4 down to 0.2) even as the fractional inhibition level remained low (0.8). Interestingly, when the pH was suddenly changed to 4.5, inhibiting nitrite oxidation completely, the rRNAt/rDNA metric did not decline suggesting that rRNAt processing was inhibited. This effect was not observed during severe inhibition with 3,5-DCP and azide. Overall, the findings indicate the relative rRNAt abundance can be used to closely track in situ Nitrobacter spp. activity and in most instances will reveal inhibition events with the potential to impact treatment performance in reactors where Nitrobacter spp. are dominant.

Introduction Nitrite-oxidizing bacteria (NOB) occupy a nexus of interests in wastewater treatment, with some processes requiring high

nitrite-oxidizing activity, while others seek to limit NOB activity. More specifically, nitrite oxidation rates should be continuously high to promote nitrification (the oxidation of ammonia to nitrite and subsequently nitrate) and denitrification (the use of nitrate as a terminal electron acceptor). The widely practiced nitrification process removes the oxygen demand and toxicity effects of ammonia on receiving streams and controls the design of single-sludge carbon/ammonia oxidation reactors (1). Denitrification removes the nitrate produced during nitrification that contributes to eutrophication. Conversely, NOB activity is inhibited to promote the use of nitrite as a terminal electron acceptor during anaerobic ammonia oxidation (anammox) (2) and denitritification (3). These processes remove nitrogen more efficiently than nitrification, followed by denitrification, with respect to oxygen utilization. Each of the nitrogen (N)-conversion processes described is prone to instability caused or witnessed by changes in the activity of aerobic nitrite oxidizers. An in situ indicator that could be used to identify shifts in NOB activity before treatment efficiency significantly deteriorates would enhance process reliability through implementation of corrective actions prior to system failure (e.g., influent diversion, extending or shortening the reactor solids retention time, performing bioaugmentation, or changing reactor operating conditions to better promote or eliminate NOB activity). There are a variety of ways in which nitrite-oxidizing activity could be monitored. However, it is clear that simply tracking N-conversion intermediates would be insufficient because simultaneous oxidation and reduction of nitrite can occur during denitritification and anammox. Cell abundance could be monitored, for example, with the ribosomal ribonucleic acid gene (rDNA), but rDNA abundance does not correlate well with nitrite-oxidizing activity as a result of the low growth rate and yield of NOB and because the discriminatory power of current molecular assays such as real time PCR is relatively low (4, 5). Bacterial activity is better measured by enumeration of the mature ribosomal ribonucleic acid (rRNA) or the RNA transcripts for key catalytic enzymes; however, these parameters do not respond quickly to declining activity in the ammonia-oxidizing bacteria (AOB) that perform the first stage of nitrification (6, 7). The rRNA transcript (rRNAt), an intermediate in the process of generating mature rRNA, provides excellent sensitivity for monitoring activity in both fast and slow growing bacteria (8, 9). Recently, the relative rRNAt abundance or rRNAt/rDNA activity metric was shown to correlate well with nitrite oxidizing activity in Nitrobacter spp. (5). Although promising, this previous study did not assess how quickly changes in the relative transcript abundance reflected transitions in the nitrite oxidation rate. In addition, the study did not address the fact that declining N-treatment efficiency may follow exposure to sundry unidentified inhibitors transiently present in wastewater. The current study presents batch nitrite oxidation experiments that assess how quickly the Nitrobacter spp. rDNA and relative rRNAt abundance reflect variations in the nitrite oxidation rate caused by changing substrate availability and exposure to mechanistically distinct inhibitors.

Experimental Procedures * Corresponding author e-mail: [email protected]; phone: 865974-7722; fax: 865-974-4514. † Department of Biosystems Engineering and Soil Science. ‡ Department of Civil and Environmental Engineering. § The Center for Environmental Biotechnology. | Department of Microbiology. 10.1021/es0716002 CCC: $40.75

Published on Web 12/29/2007

 2008 American Chemical Society

Bench-Scale Nitrification Reactor. Experiments were conducted using mixed liquor collected from a 10 L fill and draw, complete mix, bench-scale nitrification reactor (BSNR). The reactor continuously received 1 L/day of influent containing 54 mM (NH4)2SO4 (1500 mg N/L), 1.5 mM K2HPO4, 1.5 mM VOL. 42, NO. 3, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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KH2PO4, 0.75 mM MgSO4, 0.20 mM CaCl2, 16.6 µM EDTA, 9.9 µM FeSO4, and 0.5 µM CuSO4. The reactor pH, dissolved oxygen (DO), and temperature were maintained at 7.2 ( 0.1, g3 mg/L, and 30 ( 1 °C, respectively. The dominant NOB in the BSNR were Nitrobacter spp. (5). Nucleic Acid Extraction. The protocols for DNA and RNA sample collection, preservation, and extraction have been previously described (5). Briefly, DNA was extracted using the FastDNA kit (Q-BIOgene; Carlsbad, CA), diluted 1:5 in 10 mM Tris-HCl (pH 8.0), and frozen at -80 °C until analyzed by real-time PCR. RNA was extracted using the RNeasy Mini Kit (Qiagen; Valencia, CA) with lysozyme lysis and on-column DNase digestion, diluted 1:5 in 1 mM sodium citrate, and frozen at -80 °C until analyzed by real-time RT-PCR. Typically three, but a minimum of two, replicate samples were extracted. Real-Time PCR and Real-Time RT-PCR. The real-time PCR detection system for Nitrobacter rDNA and rRNAt targeted a genera-specific 16S to 23S rDNA intergenic spacer region and consisted of the primers NITISRf (5′-CCATTCACTATCTCCAGGTC-3′) and NITISRr (5′-TGATTAGAAAGACCAGCTTGC-3′) and the probe NITISRp [5′-(6-carboxyfluorscein)TCGAACCGATAGCGAGGCGG-(carboxytetramethlyrhodamine)3′] (5). Nitrobacter spp. rDNA abundance was quantified in 1:5 dilutions of the sample DNA extracts using a real-time PCR reaction containing: 12.5 µL of QuantiTect Probe PCR master mix (Qiagen; Valencia, CA), 5.125 µL of nuclease free water, 400 nM concentrations of each primer (1µL), a 150 nM concentration of probe (0.375 µL), and 5 µL of template. The rDNA assay temperature protocol was as follows: 50 °C for 2 min, Taq activation at 95 °C for 15 min, and 40 cycles with melting at 94 °C for 15 s and annealing/extension at 61 °C for 1 min. Nitrobacter spp. rRNAt abundance was quantified in 1:5 dilutions of the sample RNA extracts by real-time RT-PCR using the following reaction mix prepared on ice: 12.5 µL of QuantiTect Probe RT-PCR master mix (Qiagen; Valencia, CA), 4.875 µL of RNase-free water, 400 nM concentration of each primer (1µL), a 150 nM concentration of probe (0.375 µL), 0.25 µL of RT enzyme mix, and 5 µL of template. The rRNAt assay temperature protocol was as follows: RT reaction at 50 °C for 30 min, Taq activation at 95 °C for 15 min and 40 cycles with melting at 94 °C for 15 s and annealing/extension at 61 °C for 1 min. Both assays were performed on a MJ DNA Engine Opticon thermocycler in triplicate using the average fluorescence for cycles 3–7 for baseline subtraction, a fluorescence threshold of 0.005, and external standard curves (5). Nitrobacter rDNA and rRNAt Abundance during Transitional Starvation Conditions. The range and rate of change of Nitrobacter rDNA and rRNAt and the rRNAt/rDNA ratio were investigated in a batch nitrite oxidation experiment using a BI-2000 electrolytic respirometer (Bioscience; Bethlehem, PA). Six 1 L vessels were prepared containing 400 mL of BSNR mixed liquor and 500 mL of deionized water. Samples were collected from one vessel after more than nine hours of nitrite starvation to establish baseline levels of Nitrobacter spp. rDNA and rRNAt. Immediately thereafter, 70 mL of a 1000 mg NO2--N/L solution was spiked into all six vessels, which were sequentially sampled to monitor the transition from and back to nitrite starvation. A nitrite sample (0.45 µm filtered) along with samples for DNA (3 replicates) and RNA (2 replicates) extractions were collected during each sample event. Nitrite samples were analyzed with a DX 500 (Dionex; Sunnyvale, CA) ion chromatograph. Inhibition Experiments. A BI-2000 electrolytic respirometer was used to monitor batch inhibition assays. Nitrite was added to several vessels (100 mg/L) containing dilute BSNR mixed liquor and nitrite oxidation (oxygen consumption) was monitored over 4.5 h without the inhibitor present. This period of uninhibited activity was quantified with a linear 902

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regression fit (OUR1) of the final 4 h of cumulative oxygen uptake and time data. An inhibitor was then added to the vessels at different concentrations. After a 30 min acclimation period, inhibited NOB activity was quantified with a linear regression fit of 4 h of cumulative oxygen uptake and time data (OUR2). Fractional inhibition values were calculated as (OUR1 - OUR2)/OUR1. The overall approach to the inhibition study was to first establish a dose–response relationship for each compound investigated using the assay described above. Two final experiments were then conducted for each inhibitor to assess the effect on Nitrobacter rDNA and rRNAt across a range of inhibitor concentrations and fractional inhibition levels. For the final two experiments, five test vessels were used; one vessel served as an uninhibited control, while the remaining four received different inhibitor doses. DNA and RNA samples (3 replicates each) were collected from one vessel immediately before the inhibitor was added and from all five vessels 4.5 h later. Inhibitors. Three inhibitors that likely impact Nitrobacter spp. in mechanistically distinct modes were investigated: 3,5-dichlorophenol (DCP), azide, and hydrogen ion (H+ acid spike) (10). Briefly, 3,5-DCP is a hydrophobic ionogenic compound that inhibits bacterial cells via uncoupling. The mechanism likely involves a protonophoric shuttle system whereby phenoxide and phenol forms of 3,5-DCP repeatedly transport protons from the periplasm to the cytoplasm thereby dissipating the proton motive force (11, 12). Azide may inhibit Nitrobacter spp. by two separate mechanisms. First, it is well-known that azide selectively inhibits the mitochondrial terminal cytochrome oxidase by binding heme aa3 and thereby preventing enzyme turnover (12). In Nitrobacter spp., the terminal cytochrome oxidase is also type aa3 (13); therefore, azide could inhibit electron transport. Azide also inhibits dissimilatory nitrate reductase via a molybdenum cofactor (14). In both Nitrobacter winogradskyi and Nitrobacter hamburgensis, the nitrite oxidoreductase enzymes use a molybdenum cofactor (15, 16); therefore azide could directly inhibit nitrite oxidation. The mechanisms involved when bacteria succumb to low pH are not precisely known and may vary among phylogenetic groups. However, low pH likely produces a drop in the cytoplasmic pH that causes systemic, nonspecific enzyme inhibition and cell death via membrane damage (17, 18).

Results Nitrobacter rDNA and rRNAt Abundance during Transitional Starvation Conditions. A composite respirogram was created by averaging oxygen consumption data from six vessels containing dilute BSNR mixed liquor (Figure 1A). The oxygen uptake rate (OUR) was approximately 0.02 mg/L/h during a 9 h time period preceding the nitrite spike, confirming that the NOB were in a state of starvation. Following nitrite addition, the vessel OURs increased to 7.0 ( 0.4 mg/L/h for 10.4 ( 0.7 h. The OUR in all vessels decreased between approximately 19 and 21 h, so that during the final two hours of the experiment the vessel OURs were only 0.1 ( 0.2 mg/L/h. The return to a very low OUR indicated the added nitrite had been oxidized to nitrate. Linear regression fits of the composite OUR and nitrite data (Figure 1A) yielded a stoichiometry for nitrite oxidation (1.18 mg O2/mg NO2--N) near the theoretical value (1.14 mg O2/mg NO2--N), which confirmed that oxygen consumption accurately reflected nitrite oxidizing activity in the vessels. Molecular data from the experiment are presented in Figure 1B. A linear regression fit of the Nitrobacter rDNA data confirmed the ribosomal gene abundance changed relatively little over the course of the experiment (6.2 ( 1.1 × 109 copies/L; range ) 4.5–8.2 × 109 copies/L). In all cases, the rDNA gene abundance in samples collected from the

FIGURE 1. Cumulative oxygen consumption and nitrite concentration (A) and Nitrobacter spp. rDNA and rRNAt abundance and the rRNAt/rDNA ratio (B) in six vessels containing dilute BSNR mixed liquor transitioned from and back to nitrite starvation. Oxygen consumption is the average ( standard deviation of the six vessels; nitrite is the average ( standard deviation of three replicate analyses of a sample taken from each vessel sequentially. Nitrobacter rDNA and rRNAt data are the median ( minimum/maximum assay mean for replicate extracts of samples collected from each vessel sequentially (for each sample event, 3 replicate samples were collected for DNA extraction and 2 replicate samples were collected for RNA extraction from a single vessel). The rRNAt/rDNA ratio was computed with the average rDNA and rRNAt concentrations; error bars are the interquartile range of all possible ratios computed with the rDNA and rRNAt concentrations for replicate DNA and RNA extracts. same vessel at different times was not significantly different (Tukey HSD analysis; p > 0.05). This result and the small difference between the maximum and minimum average rDNA gene abundance ( NOB > heterotrophs) has been observed for other inhibitors (22). For azide, the inhibition level gradually increased with dose, and complete inhibition (fractional inhibition >0.95) was not obtained as with 3,5-DCP (Figure 2B). The fractional inhibition was low ( 5.7) but declined approximately 1 order of magnitude if the fractional inhibition level exceeded 0.85 (∼1.9 × 109 copies/L; pH ) 5.2–5.3). Surprisingly, the transcript abundance in completely inhibited vessels (∼1.5 ( 0.7 × 1010 copies/L; pH ) 4.5) was not significantly different from that measured in the uninhibited control vessels (Tukey HSD analysis; p > 0.05). For the inhibition experiments presented in Figure 3, the vessel Nitrobacter spp. population levels varied significantly (ANOVA; p < 0.05) even though identical procedures were used to prepare the vessels for each experiment and the reactor was operated with the strictest uniformity over time. Normalization of rRNAt to rDNA (rRNAt/rDNA ratio) allowed the variation in rRNAt abundance following only 4.5 h of exposure to the different inhibitors to be more accurately evaluated (Figure 4). The ratio metric clearly decreased as the azide and 3,5-DCP concentrations were increased. However, the most severely inhibited acid-dosed vessels displayed an rRNAt/rDNA metric similar to the uninhibited

FIGURE 4. Compiled rRNAt/rDNA ratio data gathered in inhibition experiments with 3,5-DCP (A), azide (B), and H+ (C). The rRNAt/rDNA ratio was computed with the average rDNA and rRNAt concentration; error bars are the interquartile range of all possible ratios computed with the rDNA and rRNAt concentrations for three replicate DNA and RNA extracts. Fractional inhibition results that correspond to the rRNAt/rDNA data are plotted as filled circles. In each plot, a solid line displays the previously established dose–response relationship (Figure 2). The rRNAt/rDNA data are fit with dashed lines based on a point to point (A and C) or linear regression (B) basis. controls (Figure 4C). The apparent lower (∼0.07) and upper limits (∼2–4) of the metric value observed in Figure 1 were reinforced in Figure 4. This suggests that if a “normal” range of Nitrobacter spp. rRNAt/rDNA metric values could be established for an operating reactor (presumably between the upper and lower metric limits), it would aid in distinguishing an inhibiting condition in the reactor, identified as an unusually low rRNAt/rDNA ratio value.

Discussion Changes in NOB activity should reflect a drop in nitrification, denitritification, or anammox efficiency during biological wastewater treatment, so efforts to improve the design and operational reliability of these processes may be aided by an in situ measure of nitrite-oxidizing activity. For nitrification, where high NOB activity is required, this postulate should hold true whether the drop in treatment efficiency is caused by AOB inhibition, in which case the NOB would be starved of nitrite, or resulted from direct NOB inhibition. Conversely, for denitritification and anammox, the NOB activity level must remain low or the efficiency of these processes is reduced (2, 3). In this investigation, the response time and magnitude of changes in Nitrobacter spp. rDNA and relative rRNAt abundance were evaluated after a starved nitrifierenriched biomass was spiked with nitrite. Nitrobacter rDNA abundance poorly reflected the ensuing changes in the nitrite VOL. 42, NO. 3, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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oxidation rate but the rRNAt/rDNA ratio varied quickly and significantly. During starvation, the Nitrobacter rRNAt/rDNA metric was at a minimum value (∼0.07), required 8 h to transition to the upper limit (∼4) with excess nitrite present, but only 5 h of starvation to return back to the lower limiting value. Since rapid transitions in the nitrite oxidation rate were easily detectable in just a few hours with this metric, in situ changes in Nitrobacter spp. activity in an operating reactor may be discernible well before a significant decline in treatment performance occurs. These results contrast with amoA transcript abundance, which remained at elevated levels long after ammonia had been removed from solution (6). This may indicate that transcript levels for key catalytic enzymes used by lithotrophic bacteria remain abundant to ensure rapid growth when substrates become available. However, this has not been verified for the nitrite oxidoreductase enzymes used by NOB. For the slow-growing, lithotrophic NOB, the relative rRNAt abundance may better reflect the nitrite oxidation rate because this would affect the cell energy charge and, in turn, the concentration of the rRNAt initiating nucleotide that may regulate rDNA transcription (28). The uncoupler 3,-5 DCP produced significant declines in relative rRNAt abundance even when the nitrite-oxidizing rate was unaffected. Such a differential inhibitory effect was also measured in Pseudomonas denitrificans, for which active transport was strongly inhibited by the uncoupler carbonyl cyanide m-chlorophenylhydrazone, while the denitrification rate remained unaffected (29). Another study showed that 100 µM 2,4-DNP did not affect nitrite oxidation in N. winogradskyi but completely inhibited the formation of NADH (30), which is required for Nitrobacter spp. growth (31). Thus, it appears that the growth prospect for Nitrobacter decreased upon exposure to 3,5-DCP, although the ability to oxidize nitrite was not significantly impacted. Such a scenario illustrates the value of monitoring the rDNA abundance because nitrification may not be initially affected after the relative rRNAt abundance decreases; however, treatment performance would likely decrease as the NOB population level dropped over time because of growth inhibition. In fact, a slow decrease in rDNA abundance and treatment performance accompanied by low relative rRNAt abundance may suggest growth inhibition and thus the identity of an otherwise unknown inhibiting compound. The azide- and acid-inhibition results emphasize that the characteristics of the rRNAt response is inhibitor/mechanism dependent. Unlike 3,5-DCP, azide and low pH produced low relative rRNAt abundance only when the inhibition levels were relatively high. However, transcript abundance in the most severely acid inhibited vessels was not significantly different than in uninhibited control vessels. This may indicate that the low pH eliminated the ability of Nitrobacter spp. to process existing ribosomal transcripts. The antibiotic chloroamphenicol, which prevents rRNAt processing, inhibited both Escherichia coli and Mycobacterium tuberculosis growth under a variety of conditions, resulting in a high rRNAt abundance (8, 32). Chloroamphenicol similarly affected Acinetobacter calcoaceticus (33), and it was suggested that unusually high rRNAt levels during exposure of this organism to municipal wastewater samples indicated inhibitors were present that prevented ribosome synthesis (34). Perhaps most revealing, researchers linked the slow growth of E. coli in mouse intestinal content to inhibition of rRNAt processing, and both chloroamphenicol-treated cells and those grown in mouse intestinal contents gave ring-shaped fluorescent signals during in situ rRNAt hybridization (35). These results indicated that rRNA transcripts were stuck in inhibited membrane bound endonucleases. Likewise, the low pH environment studied in this investigation may have inhibited endonuclease activity and thus processing of existing rRNAt. 906

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This again illustrates the value of measuring rDNA abundance because under this inhibition scenario nitrification treatment performance would decline along with the NOB population level, while the relative rRNAt abundance remained high. The overall implication of this study is that the relative rRNAt abundance can be used to closely track in situ Nitrobacter spp. activity, thereby providing a metric to potentially improve design and implementation of N-conversions during wastewater treatment. For example, the relative rRNAt abundance could be used to develop better operating schemes to promote long-term inhibition of nitrite oxidation for improved nitrogen removal efficiency via denitritification (36). The results also reveal the utility of independent examination of reactor treatment performance data in the context of long-term NOB population trends (rDNA abundance), particularly in light of recent research that demonstrates significant Nitrospira and Nitrobacter spp. population variations during nitrite oxidation inhibition (20). Together, the response pattern of treatment performance, nitrogen intermediates, rDNA, and rRNAt/rDNA data during inhibition events could conceivably narrow the search for some inhibiting compounds. These findings remain to be confirmed for Nitrospira spp., which are the dominant NOB in many wastewater treatment reactors (37).

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