Article

pubs.acs.org/est

Impact of Heavy Metals on Transcriptional and Physiological Activity

of Nitrifying Bacteria
Vikram Kapoor, Xuan Li, Michael Elk, Kartik Chandran, Christopher A. Impellitteri,
and Jorge W. Santo Domingo*,

U.S. Environmental Protection Agency, Oce of Research and Development, Cincinnati, Ohio 45268, United States
Pegasus Technical Services, Inc., Cincinnati, Ohio 45268, United States

Department of Earth and Environmental Engineering, Columbia University, New York, New York 10027, United States

S Supporting Information
*

ABSTRACT: Heavy metals can inhibit nitrication, a key

process for nitrogen removal in wastewater treatment. The
transcriptional responses of amoA, hao, nirK, and norB were
measured in conjunction with specic oxygen uptake rate
(sOUR) for nitrifying enrichment cultures exposed to dierent
metals (Ni(II), Zn(II), Cd(II), and Pb(II)). There was
signicant decrease in sOUR with increasing concentrations
for Ni(II) (0.033 mg/L), Zn(II) (0.110 mg/L), and Cd(II)
(0.031 mg/L) (p < 0.05). However, no considerable changes
in sOUR were observed with Pb(II) (1100 mg/L), except at
a dosage of 1000 mg/L causing 84% inhibition. Based on RTqPCR data, the transcript levels of amoA and hao decreased
when exposed to Ni(II) dosages. Slight up-regulation of amoA, hao, and nirK (0.51.5-fold) occurred after exposure to 0.33
mg/L Zn(II), although their expression decreased for 10 mg/L Zn(II). With the exception of 1000 mg/L Pb(II), stimulation of
all genes occurred on Cd(II) and Pb(II) exposure. While overall the results show that RNA-based function-specic assays can be
used as potential surrogates for measuring nitrication activity, the degree of inhibition inferred from sOUR and gene
transcription is dierent. We suggest that variations in transcription of functional genes may supplement sOUR based assays as
early warning indicators of upsets in nitrication.

INTRODUCTION

sentinel genes may serve as early warning indicators of

nitrication inhibition in WWTPs.
Inhibition of nitrication by heavy metals has traditionally
been studied using physiological responses, such as those based
on ammonia-dependent specic oxygen uptake rate
(sOUR).11,12 A wide range of nitrifying bacteria inhibition
rates (i.e., sOUR) has been reported in the presence of specic
metals, variability that may be explained by a number of factors.
For example, the reported values for concentration of Cu that
can cause a 50% decrease in nitrication activity range from 61
to 173 mg/L.1315 Few studies have attempted to understand
this variability by characterizing the physiological, transcriptional, and proteomic responses of nitrifying bacteria in
response to nitrication inhibition by heavy metals.5,7,8 Most
of these studies have used pure cultures (i.e., Nitrosomonas
europaea), and therefore limited data is available on how
complex nitrifying microbial communities (such as those from
activated sludge) respond to the presence of various metals.

Nitrifying bacteria play a critical role in biological nitrogen

removal (BNR) in wastewater treatment plants (WWTPs) by
oxidizing ammonia to nitrate. Heavy metals, such as Ni(II),
Zn(II), Cd(II), and Pb(II), that enter WWTPs through
industrial discharges can impact nitrication performance.1,2
The oxidation of ammonia to nitrite by ammonia-oxidizing
bacteria (AOB) is often considered the most sensitive step in
nitrication.3 This biochemical step is linked to ammonia
monooxygenase (amoA) and hydroxylamine oxidoreductase
(hao) activity.4 As a result, the transcripts of these two genes
have been suggested as targets to study nitrication inhibition
due to their sensitivity to environmental perturbations.58 For
instance, exposure of Nitrosomonas europaea cells to Cd(II)
caused a decrease in amoA expression, along with the reduction
in specic ammonia oxidation activity.7 However, exposure to
Zn(II) caused up-regulation of amoA, while there were no
signicant changes in the expression of hao.8 The change in
expression of the genes coding for autotrophic nitrite reduction
to nitric oxide (nirK) and nitric oxide reduction to nitrous oxide
(norB) has also been linked to imbalances in the metabolism of
AOB.9,10 From a treatment performance perspective, such
2015 American Chemical Society

Received:
Revised:
Accepted:
Published:
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June 5, 2015
October 21, 2015
October 26, 2015
October 26, 2015
DOI: 10.1021/acs.est.5b02748
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Dosing Pump (Model BL 7916, Hanna Instruments, Ann

Arbor, MI). Filtered laboratory air was provided at 1 L/min to
ensure adequate aeration. Mixing was provided by magnetic
stirring at 200 rpm. Reactor performance was monitored via
total chemical oxygen demand (tCOD) of the mixed liquor, as
well as euent NH4+-N concentration. Once steady state was
achieved within the bioreactor (euent NH4+-N concentration
less than 5 mg/L), samples (100 mL) were withdrawn from the
reactor and used in batch studies.
Batch Experiments. The inhibition studies were performed
in batch vessels with samples from the nitrifying bioreactor with
and without (control) exposure to Ni(II), Zn(II), Cd(II), or
Pb(II). The ranges of concentrations of metals used in the
batch assays were 0.033 mg/L for Ni(II), 0.110 mg/L for
Zn(II), 0.031 mg/L for Cd(II), and 11000 mg/L for Pb(II).
These concentrations were chosen based on previously
reported inhibition thresholds,11,17,18 and since many metalcontaminated wastewaters typically had concentrations below
or within these ranges,1,2,21,22 they likely represented worst-case
scenario for municipal WWTPs. Nickel chloride, zinc chloride,
cadmium chloride, and lead chloride (ACS grade certied,
Fisher Scientic, Fair Lawn, NJ) were used to prepare stock
solutions for batch studies. All reagents and their dilutions were
prepared using autoclaved Milli-Q water.
Briey, samples (50 mL) were collected from the continuous
stirred-tank reactor and diluted with deionized autoclaved water
(50 mL). MOPS (3-(N-morpholino)propanesulfonic acid) was
used as a buer (20 mM nal concentration).17 Metals were
then added to the biomass suspensions, and the pH was
adjusted to 7.5. The mixtures were placed on a magnetic stirrer
and constantly mixed at 100 rpm and 25 1 C for 12 h to
simulate prolonged exposure to metals. Biomass samples (1
mL) were collected from both the control and treatment vessels
before and after metal exposure and centrifuged at 21000 g
for 5 min. The supernatant was removed, and the pellets were
stored at 80 C for nucleic acid extraction. Considering the 12
h exposure time, batch experiments for a particular
concentration for a given metal were performed in duplicates
on the same day. All experiments for a given metal were
conducted within a week. Controls were performed for each
individual batch experiment and consisted of samples in which
metals were not added during the tested period.
Oxygen Uptake Measurement. Ammonia-dependent
oxidation rates (i.e., sOUR) were determined using respirometry as previously described.17 At the end of the 12 h exposure
period, biomass was transferred to water-jacketed glass vessels
and aerated with pure oxygen followed by injection of a
substrate aliquot (20 mg NH4+-N L1). A decrease in the
dissolved oxygen (DO) level in the vessel due to ammonia
oxidation was measured in real-time via a DO probe (YSI
5300A, Yellow Springs Instruments, OH) and continuously
recorded using a computer interface.23 The inhibition of
nitrication activity was expressed as the dierence between the
measured sOUR with and without metal exposure. The
respirograms were used to express inhibition by measuring
the dierences between the maximum specic oxygen uptake
rates in the absence (sOURcontrol) and presence (sOURsample) of
metal exposure as follows:

Moreover, there has been minimal data on the correspondence

between results obtained from biochemical and molecular
assays for the same samples of nitrifying populations in
response to heavy metal inhibition.7,8,16 To address this
research gap, we examined the physiological and transcriptional
responses of nitrifying enrichment culture upon exposure to
several heavy metals, specically, Ni, Zn, Cd, and Pb.
It has been demonstrated in previous studies that the degree
of nitrication inhibition is generally dependent on the
concentration of metal.17,18 Furthermore, it has been suggested
that inhibition may be related to metal complexation with
intracellular functional groups, such as sulfhydryl (SH), which
may disrupt protein structure and function.19 Active multivalent
metal cations may also displace essential metals from their
metabolic sites and inhibit function of various enzymes (e.g.,
Cu2+ with Zn2+ in amoA).20 However, very little is known about
the relationship between functional gene expression and metal
interactions in nitrifying bacteria. Heavy metals are known to
cause oxidative stress in bacteria,19 and, therefore, the changes
in transcript levels of nitrication genes encoding proteins
associated with oxidative stress might be putative indicators of
inhibition. The oxidation of ammonia to hydroxylamine
(NH2OH) and NH2OH oxidation to NO2 are carried out
by amoA and hao, respectively. In addition, autotrophic
denitrication may be favored during nitrication inhibition
resulting in transcription of NO2 reductase and nitric oxide
(NO) reductase genes (such as nirK and norB, respectively).
Since the expression of these respiratory genes can correlate
with the activity of enzymes thereby encoded, it is reasonable to
assume that exposure of nitrifying bacteria to heavy metals can
induce stress that would impact the transcription of genes
coding for these enzymes. Thus, the primary objectives of this
study were to examine the impact of heavy metals on the
physiological and genetic activities of nitrifying enrichments.
Specically, we examined the transcriptional responses of
nitrifying enrichments upon long-term exposure (12 h) to
Ni(II), Zn(II), Cd(II), and Pb(II). The transcription of amoA,
hao, nirK, and norB, four key functional genes involved in redox
N transformations in AOB, was measured in response to metal
inhibition using reverse transcriptase-quantitative PCR (RTqPCR) assays. The 12 h exposure period was chosen since
short-term assays (15 min to 1 h) may not adequately reect
the response observed in wastewater systems subject to
prolonged toxic exposure.11 An important assumption in our
study is that changes in RNA transcription levels reect
sensitive cellular responses to changing environmental
conditions and could be precursors of more detrimental
whole cell impacts. We compared the transcriptional responses
to ammonia-dependent sOUR measurements for the same set
of samples to evaluate the ecacy of using functional-gene
transcript levels as indicators of nitrication inhibition in cells
exposed to heavy metals.

MATERIALS AND METHODS

Nitrifying Bioreactor. Nitrifying enrichment cultures were
cultivated in a lab-scale bioreactor (V = 10 L) as described
earlier18 operated at solids retention time (SRT) of 35 d and
hydraulic retention time (HRT) of 1 d. The reactor was fed an
inorganic medium devoid of organic carbon, with ammonium
(500 mg of NH4-N/L, (NH4)2SO4) as the sole energy source
with essential macro- and micronutrients. The pH of the
reactor was automatically maintained at 7.5 0.1 with 1 M
sodium bicarbonate using a pH Controller and Chemical

% inhibition = (sOUR control sOUR sample)/sOUR control 100%

(1)

RT-qPCR. The transcriptional levels of amoA, hao, nirK, and

norB in the tested samples were determined using RT-qPCR
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assays. RNA and DNA were extracted from the stored cell
pellets using AllPrep DNA/RNA Mini Kit (Qiagen GmbH,
Hilden, Germany) as described previously.24 cDNA was
synthesized from the puried total RNA extracts using the
Superscript III, following the manufacturers instructions (Life
Technologies, San Francisco, CA). RT-qPCR was performed
on the QuantStudio 6 Flex Real-Time PCR system (Life
Technologies) with Power SYBR Green Master Mix (Applied
Biosystems, Foster City, CA) using published primers (Table
S1). AOB relative abundance was determined by qPCR using
AOB 16S rRNA gene primers (Table S1). The transcript levels
of each functional gene were normalized against AOB 16S
rRNA gene copies as quantied by qPCR since 16S rRNA gene
copies were relatively stable in the batch cultures exposed to
metals (data not shown). Additional details regarding RNA and
DNA extraction and qPCR assays are provided in the
Supporting Information. The relative fold change in gene
transcription was dened as log2 (T/C), where T is the gene
expression level in the testing sample (presence of metal), and
C is the gene expression level in the control sample (absence of
metal).
Bacterial 16S rRNA Gene Sequencing. The bacterial
composition of nitrifying bioreactor was evaluated via highthroughput sequencing of 16S rRNA gene libraries generated
with selected DNA extracts and barcoded primers 515F/
806R.25 PCR reactions were performed in 25 L volumes using
the Ex Taq kit (Takara) with 200 nM each of the forward and
reverse primer and 2 L of template DNA (210 ng/L).
Cycling conditions involved an initial 5 min denaturing step at
94 C, followed by 30 cycles of 45 s at 94 C, 60 s at 50 C, and
90 s at 72 C, and a nal elongation step of 10 min at 72 C.
Prior to multiplexed sequencing, PCR products were visualized
on an agarose gel to conrm product sizes. Sequencing of the
pooled library was performed on an Illumina Miseq benchtop
sequencer using pair-end 250 bp kits at the Cincinnati
Childrens Hospital DNA Core facility. Sequence reads were
processed using MOTHUR v1.25.126 as described earlier.27
Details of sequence processing are provided in the Supporting
Information.
Statistical Analyses. The results for sOUR based
inhibition were expressed as relative percent inhibition. The
results for RT-qPCR based inhibition were represented as fold
change (in log2 scale) in relative gene expression. All
experiments were performed as experimental duplicates. The
Students t-test method was used for the statistical analyses with
p 0.05 for the statistical signicance of sOUR and RT-qPCR
results. Correlation coecients were determined between the
16S rRNA gene sequencing data sets based on relative
abundance of taxonomic groups. All analyses were performed
using Microsoft Excel (2013).

Figure 1. Bioreactor performance as measured by qPCR derived

relative abundance of AOB 16S rRNA gene copies and reactor tCOD
concentrations.

Figure 2. Relative gene transcript concentration of amoA during

steady-state operation of the reactor as characterized by >99%
ammonia removal.

amoA was consistent with ammonia oxidation in the bioreactor

(Figure 2). Similar proles for AOB 16S rRNA gene copies and
amoA gene transcripts have been observed earlier for nitrifying
cultures cultivated in a lab-scale bioreactor having similar
conguration as the current study.9
The bacterial community composition of the nitrifying
bioreactor was also examined to conrm the presence and
determine diversity of nitrifying bacteria in the enrichment
cultures. More than 200,000 16S rRNA gene sequences were
generated from nitrifying biomass DNA extracts obtained on
days 5, 10, 15, 25, and 30 of bioreactor operation after
attainment of steady state. These libraries obtained on multiple
sampling dates displayed high degrees of similarity, based on a
comparison of taxonomic groups identied and their
abundance proportions (correlation coecients, r > 0.9)
indicating that the microbial composition of the bioreactor
was similar over the experimental period. Analyses of these
sequences indicated that most AOB in the bioreactor belonged
to the family Nitrosomonadaceae under the class Betaproteobacteria (>60% of total sequences) (Figure 3). Remaining
sequences included members related to the groups Bacteroidetes, alphaproteobacteria, and gammaproteobacteria. While
an additional 10 dierent classes were also identied, they
represented less than 1% each. Previous analyses of next
generation sequencing data of nitrifying enrichments established from wastewater inoculum also observed that Nitrosomonas-like populations constituted the major group in such
enrichments.9,28
sOUR-Based Inhibition. The inhibition of nitrication
activity based on the sOUR method was plotted against the
total metal dosage for each concentration (Figure 4). The
specic ammonium oxidation rate decreased as the applied

RESULTS AND DISCUSSION

Reactor Performance and Population Dynamics. The
reactor achieved steady state within a month and maintained
stable operation during the course of the study, characterized
by >99% ammonia removal. During steady-state nitrication,
the average euent NH4+-N concentration was 1.77 0.41
mg/L (n = 49), and average tCOD was 1923 130 mg/L (n =
49). The background levels of AOB 16S rRNA gene copies and
amoA gene transcripts in the bioreactor were measured for
multiple dates to observe AOB temporal dynamics. The relative
abundance of AOB varied between 105 to 106 16S rRNA gene
copies/mL (Figure 1). The relative mRNA concentration for
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between the measured and predicted inhibition using Solver

(MS Excel, 2013). The predicted inhibition based on the metal
concentration was calculated as follows:
% inhibition = 100/(1 + K i /I ))

(2)

Based on the empirical estimates of Ki the degree of

inhibitory eect of metals toward ammonium oxidation was Cd
> Ni > Zn > Pb (Ki values for Cd, Ni, and Zn were estimated as
0.76 mg/L, 1.9 mg/L, and 17.29 mg/L, respectively, based on
noncompetitive inhibition model). Interestingly, with the
exception of Pb, the inhibitory character of the metals tested
in our study corresponded well with their sulde complexation
potential series, which follows Pb > Cd > Ni > Zn (stability
constants for metal sulde complexes, log K, 25 C: 27.5,
27.0, 26.6, 24.7 for Pb, Cd, Ni, and Zn, respectively).29 In
another study,18 the molar inhibitory eect toward ammonium
oxidation followed: Cu = Zn > Cd > Ni; however, their results
did not correspond well with the metal-sulde complexation
potential series.
The decrease in respiration rates as a response to increasing
metal concentrations has previously been observed for
nitrifying bacteria batch cultures.17,18 In our study, we observed
that ammonia-dependent oxygen uptake rates were signicantly
inhibited after a 12-h exposure to Ni(II), Zn(II), or Cd(II). In
contrast, cells exposed to Pb(II) did not show signicant
changes in sOUR for Pb(II) concentrations up to 100 mg/L.
Nitrication was only severely inhibited when Pb(II) levels
reached 1000 mg/L. This is consistent with previous reports on
lack of inhibition of nitrication at Pb concentration as high as
40 mg/L.30 It has been suggested that nitrication inhibition in

Figure 3. Bacterial community composition of the nitrifying bioreactor

based on 16S rRNA gene sequencing of nitrifying biomass DNA
extracts obtained on multiple dates (n = 5).

metal dose to nitrifying biomass increased for both Ni(II) and

Cd(II) exposure. For Zn(II) exposure, nitrication inhibition
increased with metal concentration up to 3 mg/L Zn(II), after
which activity did not increase upon exposure to 10 mg/L
Zn(II). There was no change observed when the nitrifying
biomass was exposed to Pb(II) up to 100 mg/L; however, there
was an 84% decrease in ammonia oxidation upon exposure to
1000 mg/L Pb(II) (Figure 4). An empirical noncompetitive
inhibition model was used to estimate the half-inhibition
coecient, Ki, by minimizing the sum of squared errors

Figure 4. Comparison of ammonia oxidation inhibition measured by sOUR as a function of total metal concentration after a 12-h exposure of (a)
Ni(II) (0.033 mg/L), (b) Zn(II) (0.110 mg/L), (c) Cd(II) (0.031 mg/L), and (d) Pb(II) (11000 mg/L). Metal eect was measured in two
independent experiments for each concentration (experimental duplicates).
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batch assays with exposure time of up to 12 h continuously
increases for Ni, Zn, and Cd, probably due to slow
internalization kinetics,11,18 whereas Pb has been shown to be
less inhibitory than Ni, Zn, and Cd.12
Several studies have used respirometry to assess the
inhibitory concentrations of heavy metals impacting biological
wastewater treatment. Although ammonia or nitrite may be
used as nitrogen substrate to measure oxygen uptake rates by
AOB or NOB, respectively, we used ammonia-dependent
sOUR assays since ammonia oxidation, being the rst step of
nitrication, is often the rate limiting step and more prone to
environmental perturbations.3,31 These methods are considered
by many as useful tools for evaluating the eects of organic and
inorganic compounds on nitrication. However, a wide range of
inhibitory levels has been reported using these methods. For
example, using municipal wastewater Juliastuti et al.32 observed
complete nitrication inhibition at a Zn(II) concentration of
1.2 mg/L. In another study, more than 3 mg/L of Zn(II) was
required to attain greater than 90% inhibition.11 Previously,
Cenci and Morozzi33 reported 50% inhibition for Zn2+
concentration of 16 mg/L, which is most close to the value
of Ki (i.e., 17.29 mg/L) observed in this study. The dierences
in exposure time is a likely explanation, although there may be
other experimental dierences (e.g., biomass used, nutritional
growth conditions) that could have contributed to the range of
concentrations reported as inhibitory. Thus, alternative
methods are required to truly assess nitrication inhibition in
WWTPs. A 50% decrease in nitrifying activity has been
observed with Cd concentration of 8.3 mg/L15 and 13 mg/L14
when using nitrifying enrichment culture and activated sludge,
respectively. On the other hand, the concentration of Cu at
which 50% inhibition occurred for nitrifying enrichments (173
mg/L)15 was at least an order of magnitude higher than that for
mixed liquor (18 mg/L).14 The inhibitory values reported for
Cu vary up to more than 3-fold using activated sludge,14,34
suggesting that inhibition rates may dier even when the same
type of biomass is used in inhibition studies.
Transcriptional Responses to Heavy Metals. The amoA
transcript levels decreased after exposure to Ni(II), with
greatest reduction after the cells were exposed to 3 mg/L
Ni(II) (Figure 5). The transcript levels of hao decreased with
increasing Ni(II) dosages; however, no signicant change was
observed with 3 mg/L Ni(II) (p > 0.05). The levels of nirK and
norB decreased after exposure to 1 mg/L and 3 mg/L Ni(II),
respectively. Increased transcription of amoA, hao, and nirK
ranging from 0.5- to 1.5-fold occurred after exposure to 0.3, 1,
and 3 mg/L Zn(II). Although the changes in the transcript
levels of amoA and hao with Zn(II) exposure were not
signicant (p > 0.05), amoA and hao transcripts decreased by
0.5-fold when exposed to 10 mg/L Zn(II). Stimulation of norB
expression was observed at a Ni(II) dose of 0.3 mg/L for batch
nitrifying cultures. There were signicant changes in the levels
of all four genes when exposed to Cd(II) (p < 0.05). The
transcription of amoA was considerably increased at 0.3 mg/L
Cd(II), while hao was increased at 1 mg/L Cd(II). Although
amoA transcripts were increased upon exposure to all
concentrations of Cd(II), the linear correlation of amoA
transcripts with Cd(II) concentrations was not statistically
signicant (p > 0.05). The transcript levels of amoA, hao, nirK,
and norB increased by more than half-fold in cells exposed to
100 mg/L Pb(II), but amoA decreased by 2-fold and hao
decreased by 1-fold when exposed to 1000 mg/L Pb(II).

Figure 5. Relative fold change (in log2 scale) in the transcript levels of
amoA, hao, nirK, and norB measured by RT-qPCR as a function of
metal concentration after 12-h exposure of (a) Ni(II) (0.033 mg/L),
(b) Zn(II) (0.110 mg/L), (c) Cd(II) (0.031 mg/L), and (d)
Pb(II) (11000 mg/L). Each bar graph represents an average of
experimental duplicates.

Based on 16S rRNA gene sequencing, most AOB detected in

this study were closely related to the Nitrosomonas group.
When Nitrosomonas-like functional genes were measured, on
average, we observed that amoA transcript levels decreased
when exposed to Ni(II), but such a decrease was not as high as
compared to the relative decrease in activity as suggested by
sOUR data. A more marked discrepancy between molecular
data and respirometry was observed for cells exposed to Cd(II)
in which amoA transcript levels increased, while there was a
reduction in sOUR. For Zn(II) and Pb(II), amoA expression
was inhibited at 10 and 1000 mg/L, respectively (Figure 5).
The results suggest that the intracellular RNA content of these
functional genes may not strictly correlate with the activity of
enzymes under heavy metal exposure. Similar results have been
shown in previous studies.7,16 The changes in the RNA content
of a cell reect sensitive cellular responses and have been used
to estimate the relative growth rates and are an indicator of the
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physiological state of the organism.35,36 However, the lack of
correlation between 16S rRNA abundance and physiological
activity in AOB under stress conditions has been observed
previously;7,37 therefore, monitoring changes in the transcript
levels of a functional genes such as amoA and hao has been
suggested by a few.7,10 Other sentinel genes that are identied
to be sensitive to heavy metal inhibition include genes encoding
for mercury resistance proteins (merTPCADE)5,38 and genes
involved in DNA replication and recombination (such as
tnpAR).5 The results presented here assist in understanding the
interplay between physiological and transcriptional responses of
genes involved in nitrication for mixed populations under
environmental perturbations.
The current study focused on acute toxicity (<24 h
exposure) of heavy metals and did not account for chronic
toxicity. The chronic eects of heavy metal inhibition to
nitriers may be underappreciated, considering the everincreasing industrialization and large-scale mining over recent
years and corresponding elevated levels of metals in wastewaters.21,22 Metals may be frequently or even continuously
present in activated sludge systems, causing long-term impact
on microorganisms. In some cases, bacterial communities may
develop resistance to heavy metal inhibition and recover their
activities upon long-term exposure to inhibitors.39,40 For
instance, Yeung et al.41 evaluated inhibition and adaptation to
nickel using nitrifying biomass established from nitrifying
activated sludge of a full-scale wastewater treatment plant. They
were able to select for nickel-resistant nitrifying cultures
obtained by long-term batch incubations of decaying activated
sludge with high levels of added inhibitor. After incubating
activated sludge with dierent concentrations of Ni, they
observed that nitrifying communities were not impacted with 1
mg/L of Ni and showed considerably low levels of nitrication
inhibition at 5 and 10 mg/L Ni. Incubation with 50 mg/L Ni
resulted in signicant inhibition as reected by decreased amoA
transcript abundance. By contrast, we observed signicant
down-regulation of amoA transcript levels with 3 mg/L of
added Ni(II). The results indicate that, although Ni is
inhibitory at concentrations above 1 mg/L, there is
considerable variation in reported threshold values due to
dierences in experimental congurations and biomass source
and adaptation to metals. We used nitrifying enrichment
cultures, while the aforementioned study used nitrifying
activated sludge from the aeration basin of a full-scale
wastewater treatment plant as the source of nitrifying biomass
for batch incubations. When the tested biomass or sludge is
from dierent sources, or is harvested at varying growth
conditions, the nitrifying process could be dierent since the
physiological state of the cells may impact the uptake of
inhibitory compounds including metals.11,42 In another study,
the inhibitory eect of zinc, copper, nickel, and lead on pure
cultures was compared with activated sludge.43 It was observed
that pure cultures were more sensitive to heavy metal inhibition
than nitrifying sludge samples, suggesting that pure culture
studies may overestimate true inhibition of a given contaminant. Furthermore, the dierences between the chemical and
biological properties of nitrifying samples used (pure culture
versus activated sludge) represent yet another source of
variation in the observed eects of heavy metal inhibition.
The transcript levels of amoA, hao, nirK, and norB were
slightly up-regulated (less than 1-fold) at Zn(II) concentration
of 3 mg/L, while a decrease in the transcript levels of all the
functional genes was observed for a Zn(II) concentration of 10

mg/L. The overall changes in the transcript levels of functional

genes were not signicant when exposed to Zn(II) (p > 0.05).
In a series of studies using nitrifying bacteria pure cultures, the
amoA and hao expression were monitored in response to ZnCl2
additions.8,20 The transcriptional responses of Nitrosococcus
mobiliz, a halophilic nitrier, to 0.06 and 0.6 mg/L Zn revealed
that amoA and hao expression levels were maintained or slightly
up-regulated during ZnCl2 additions.20 For N. europaea, a
chemolithoautotrophic nitrier, signicant up-regulation of
amoA occurred after addition of 30 and 90 M ZnCl2 (1.96
and 5.88 mg/L Zn(II), respectively), while the expression of
hao was not signicantly inhibited.8 Additionally, both studies
suggested that Zn is capable of competing with Cu and
replacing it in the metal active site in the AMO enzyme, thereby
inhibiting nitrication. Similar results for amoA were obtained
in our study with the exception of hao which was not
signicantly up-regulated in the previous study.
In our study, the impact of Pb exposure, as inferred by sOUR
and amoA gene expression, was not statistically dierent for
Pb(II) doses 100 mg/L Pb (p > 0.05). At 1000 mg/L Pb(II),
nitrifying enrichments were signicantly inhibited (p < 0.05)
based on both sOUR and amoA transcript levels. The addition
of Cd(II) resulted in decreased sOUR; however, there was an
increase in the transcript levels of amoA, hao, nirK, and norB,
which is compatible with studies using N. europaea pure
cultures.16 The relative expression of amoA increased in
continuously cultured N. europaea cells with each pulse addition
of Cd and reached a maximum of 7.6-fold up-regulation after
exposure to 60 M Cd (6.7 mg/L Cd).16 The overexpression
of nirK in N. europaea cells exposed to Cd has been previously
reported.5 The up-regulation of amoA observed in the previous
study16 and in our study upon Cd exposure suggests that the
nitrifying populations may be able to recover from Cd
inhibition through the de novo synthesis of AMO enzymes.
The preferential synthesis of AMO and HAO mRNAs during
energy-limiting conditions has been observed in a previous
study, where levels of new AMO and HAO mRNA and de novo
AMO enzyme activity correlated with increasing ammonium
concentrations.44
The inuence of heavy metals (e.g., Zn, Ni, Pb, Cd) on
nitrifying bacteria at the physiological and transcriptional level
is of interest because these metals and their complexes can
potentially inhibit nitrication activity by disrupting proteins
once they are transported across bacterial cell membranes and
interact with protein functional groups.19,45 The complexation
of metals with the sulfhydryl functional group has been
suggested earlier, and the correspondence between inhibition
coecients and metal sulde stability constants observed in this
study further demonstrates the inhibitory character of the
metals. Moreover, active multivalent metal cations may replace
essential metals from their metabolic sites and inhibit function
of various enzymes. For instance, heavy metals such as Zn have
been shown to replace active metal binding cores in
enzymes;8,20 Cu2+ and Fe3+ are two redox active multivalent
cations present in the active site of AMO. Another possible
inhibition mechanism is that the binding of metal cation at a
nonactive site in the enzyme may alter the structure and
function of the enzyme. There are limited studies describing the
uptake of Cd and Pb by nitrifying bacteria at the molecular
level. Nonetheless, they are known to cause nitrication
inhibition in pure cultures of AOB and also in activated sludge
obtained from WWTPs. In general, it should be considered that
heavy metal inhibition to the activated sludge may be
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most of these studies used pure cultures of N. europaea or

nitrifying enrichment cultures. By not capturing the diversity of
wastewater nitrication systems, the results obtained from pure
culture studies may not always reect the dierent metal
tolerance levels of the various nitrifying populations that coexist
in these systems.49,50 While this may in part explain the range in
the reported inhibition values, we must consider that microbialmetal interactions are expected to occur with many other
species (i.e., non-nitriers) present in any given WWTP. How
the latter interactions impact nitriers is poorly understood.
Consequently, future studies will benet from examining
nitrication inhibition in wastewater using a more holistic
(i.e., total community) system approach employing a
combination of emerging technologies (metagenomics and
metatranscriptomics).

inuenced by changes in pH, temperature, DO, presence of

suspended particles and/or other metals, SRT, and HRT.18,46,47
The aforementioned factors may impact the metal bioavailability and thus strongly alter the observed metal toxicity.
Future studies examining the relationship between metal
partitioning and microbial toxicity are needed to elucidate the
exact mechanism of heavy metal-based nitrication inhibition.
Here we applied RNA-based RT-qPCR assays to measure
transcript level responses of several functional genes in
nitrifying enrichment exposed to dierent concentrations of
heavy metals. The application of RNA-based function specic
assays to measure microbial nitrication activity was also
employed in previous studies.9,10 While overall the results of
these studies show that the RNA-based approach may not be a
viable indicator of inhibition of nitrication by heavy metals,
our results further support that measuring transcript levels may
not be directly correlated to responses at the enzyme activity
level. Nonetheless, dierences in the relative expression of
some of these functional genes have been shown to correlate
with sOUR rates during the imposition of metal inhibition.7 In
wastewater treatment systems prone to acute exposure, changes
in gene transcription levels may be more dramatic and could
supplement sOUR based assays as early warning indicators to
prevent excessive nitrication inhibition. Periodic shocks of
heavy metals (or other nitrication inhibitors) at loads much
higher than baseline levels are quite common, especially in
primarily domestic wastewater treatment plants. As most plants
may be simultaneously exposed to a mixture of metals,
understanding antagonistic or synergistic scenarios will be
even more complex and will require a better understanding of
mechanistic behavior at the molecular/genetic level.
The applicability of sOUR as a sensitive indicator of
inhibition has been demonstrated earlier6,7 and in this study.
However, it does not allow for discrimination between activities
of multiple AOB in mixed populations. Additionally, the
metabolic response in a given system to the presence of
inhibitory compounds may be largely dependent on characteristics of the bacterial community. Therefore, it is useful to
understand how the metabolic pathways (such as transcription
of functional genes) of the nitrifying populations can be
inuenced upon exposure to heavy metals. Another advantage
of using the expression of key functional genes as a measure of
nitrication activity is that this approach could in some cases
predict impending process upsets or recovery as shown in
AOB cultures10 and anaerobic ammonia oxidation reactors
previously.48 Along this, a critical research need is the ability to
identify and quantify inhibition in dierent steps that involve
autotrophic ammonia or nitrite oxidation. Such distinctions are
needed when faced with selective inhibitors of nitrication
steps. For instance, while certain heavy metals could inhibit
ammonia to hydroxylamine oxidation (by displacing essential
cations in the active site of amoA), they may not have an impact
on hydroxylamine oxidation or nitrite reduction. Such
inferences can only be made by targeting functional genes
rather than structural genes (such as the 16S rRNA gene).
Accordingly, specic biomarkers to quantify the expression of
key functional genes involved in nitrication are needed, and
additional studies are required to implement these molecular
approaches in a WWTP scenario.
Heavy metal inhibition of nitrifying bacteria has been widely
measured in terms of specic ammonia oxidation rates11,17,18
and transcription of several specic genes,8,16,20 as well as by
studying whole-genome transcriptional changes.5 However,

ASSOCIATED CONTENT

S Supporting Information
*

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.est.5b02748.
Table of the qPCR primers and probes used in this study
and additional details regarding RNA and DNA
extraction, qPCR assays, and next-generation sequencing
processing (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: santodomingo.jorge@epa.gov.
Notes

The authors declare no competing nancial interest.

ACKNOWLEDGMENTS
We are grateful for comments provided by Michael Elovitz. The
views expressed in this article are those of the authors and do
not necessarily represent the views or policies of the U.S.
Environmental Protection Agency. This research was supported
in part by an appointment to the Postdoctoral Research
Program at the U.S. Environmental Protection Agency (EPA),
Oce of Research and Development, Cincinnati, OH,
administered by the Oak Ridge Institute for Science and
Education through an Interagency agreement between the U.S.
Department of Energy and the U.S. Environmental Protection
Agency.

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