Journal of Water and Environment Technology, Vol.13, No.

1, 2015

Enrichment of Nitrifiers and the Influence of

Antimicrobials on Nitrification during Batch
Experiment

Jingru DU1), Chao TANG2), Weikang QI1), Yu QIN1), Yu-You LI3)

1) Dept. of Environ. Science, Graduate School of Environ. Studies, Tohoku Univ., 6-6-06 Aoba,
Aramaki, Aoba-ku, Sendai 980-8579, Japan
2) Gansu Academy of Environmental Sciences, Yanerwan Road 225, Lanzhou 730000, China
3) Dept. of Civil and Environ. Engineering, Tohoku Univ., 6-6-06 Aoba, Aramaki, Aoba-ku,
Sendai 980-0845, Japan

ABSTRACT
The enrichment of nitrifying bacteria was realized and sludge yield was calculated for the batch
experiment period. The influence of four kinds of antimicrobials on ammonia-oxidizing bacteria
(AOB) and nitrite-oxidizing bacteria (NOB) was investigated using batch experiments. The
specific ammonia-oxidizing activity (SAA) was 0.44 gN/gVSS/d and the specific
nitrite-oxidizing activity (SNA) was 0.64 gN/gVSS/d. Below 400 mg/L antimicrobials,
Cefalexin (CLX) almost has no adverse effect on nitrification and the highest inhibition caused
by Sulfamethoxazole (SMX) was 15.4% and 23.9% to SAA and SNA respectively. Azithromycin
(AZI) exhibited the most intense inhibitory effect. The relative activity of AOB and NOB were
39.0% and 29.8% respectively when AZI was 400 mg/L. Due to the complexation of
Tetracycline (TC) with Ca2+ and Mg2+, biomass showed obvious reduction with the foam on
water surface. With the addition of 400 mg/L TC, only 76.9%, 13.7% and 8.1% of sludge
remained when the substrate contained calcium or magnesium ions or both of them respectively.

Keywords: antimicrobials, inhibition, nitrification

INTRODUCTION
Antimicrobials have been detected widely in the final effluents of sewage treatment
plants (STPs) around the world. Conventional treatment may remove a proportion of
these compounds, but there is a potential for residues of antimicrobials to be released in
treated effluent into the aquatic environment. The occurrence and biological impacts of
pharmaceutically active compounds in the environment could cause an emerging issue.
Antimicrobial compounds have the potential to promote antibiotic resistance in
pathogens, changing the community structure/diversity of native bacteria. The
antimicrobials included in this study are Cefalexin (CLX), Azithromycin (AZI),
Sulfamethoxazole (SMX), and Tetracycline (TC), which belong to β-lactams,
Macrolides, Sulfonamides and Tetracyclines respectively – four different classifications
of antimicrobials. Their broad use to treat bacterial diseases and infections for both
humans and animals attracts more attention recently (Kolpin et al., 2002). In general,
the concentrations of antimicrobials were in the low range in surface waters,
groundwater, seawater and municipal wastewater, but in the higher µg/L or mg/L range
in hospital and pharmaceuticals industry effluent (Xu et al., 2007). Fifty nine
pharmaceuticals had been detected in the effluent of drug industries and the
concentration of 3 antimicrobials was on the order of mg/L (Larsson et al., 2007). Thus
the antimicrobials concentrations used in this research (between 20 and 400 mg/L) are
on the basis of pharmaceutical industry effluents (Larsson et al., 2007). Moreover,

Address correspondence to Yu-You LI, Department of Civil and Environmental Engineering, Tohoku
University, Email: yyli@epl1.civil.tohoku.ac.jp
Received May 23, 2014, Accepted September 10, 2014.
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 Journal of Water and Environment Technology, Vol.13, No.1, 2015

higher concentrations have been previously used for similar assays in other studies and
could adapt the antibiotic concentration variations in the wastewater influence (Amin et
al., 2006; Chelliapan et al., 2006).

Nitrification, the oxidation of reduced forms of nitrogen to nitrite and nitrate, is an

essential link in the nitrogen cycle of natural, industrial and agriculture systems.
However, microbial nitrification is recognized as being difficult to maintain in practical
wastewater treatment plants (WWTPs) owing to the lower reaction rate, yield, and the
sensitivity of nitrifying bacteria to physical, chemical, and environmental disturbances.
There are two steps in a conventional nitrification process. First, ammonia is oxidized to
nitrite by ammonia-oxidizing bacteria (AOB). Second, nitrite is oxidized to nitrate by
nitrite-oxidizing bacteria (NOB).

There are several reports about the effect of antimicrobials on activated sludge process,
which include nitrification process (Louvet et al., 2011; Tugce Katipoglu-Yazan, 2013).
Some of them have found significant inhibition to ammonification, nitritation and
nitration at different concentrations of antimicrobials. However, as a kind of autotrophic
bacteria, nitrifier constitutes only a minority of microbial population in the activated
sludge system. As well as most of the research on this field focuses on single
classification of antimicrobials (Nakata et al., 2005; Perez et al., 2005). After the
previous cultivation of nitrifier, this study increased the nitrogen loading rate (NLR)
step by step and did the batch experiment under high NLR. The influence of four
antimicrobials was investigated. The specific ammonium-oxidizing activity (SAA) of
AOB and specific nitrite-oxidizing activity (SNA) of NOB were calculated via batch
experiments at different concentrations of antimicrobials. The aim of this paper was to
evaluate the effect of antimicrobials on nitrification and indicate the potential risk of
pharmaceutical wastewater treatment process.

MATERIALS AND METHODS

Experimental set up
Figure 1 shows the experimental apparatus for the enrichment of nitrifying bacteria. A
10 L aeration tank was used for nitrifying, in which folded plate sediment zone was also
included. The seed sludge was taken from the Sendai municipal sewage treatment plant
(oxidation ditch) and the initial MLSS (mixed liquor suspended solids) concentration
was 2,430 mg/L. Liquid temperature inside the reactor was maintained at (30 ± 1.5)°C
with a heating rod. During all aeration reaction stages, DO (dissolved oxygen) was
controlled within 2 – 3 mg/L. This experiment was conducted at different NLR (from
0.05 gN/L/d to 0.4 gN/L/d) with influent NH4+–N concentration of 50 mg/L. The
composition of the synthetic wastewater used in this experiment was described in Table
1.

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 Journal of Water and Environment Technology, Vol.13, No.1, 2015

2 4 5

1 6 7

Fig. 1 - Schematic diagram of the nitrification reactor.

(1. substrate tank; 2. air pump; 3. peristaltic pump;
4. heating rod; 5. thermometer; 6. sludge outlet;
7. aeration tank).

Table 1 - Composition of the synthetic wastewater.

Concentration in
Substrate Concentration [g/L] Microelement the microelement
solution [g/L]
NH4Cl 0.191 Na2-EDTA·2H2O 8.304
NaHCO3 0.800 FeSO4·7H2O 5.000
KH2PO4 0.081 ZnSO4·7H2O 0.215
Na2HPO4·12H2O 0.214 CoCl2·6H2O 0.120
Na2SO4 0.213 MnCl2·4H2O 0.495
CaCl2·2H2O 0.036 CuSO4·5H2O 0.125
MgCl2·6H2O 0.051 (NH4)6Mo7O24·4H2O 0.089
NiCl2·6H2O 0.095
Microelement 1 [mL/L] Na2SeO3 0.078
H3BO3 0.007

Analytical methods
The pH, DO, NH4+-N, MLSS, MLVSS (mixed liquor volatile suspended solids) and
alkalinity were measured according to the Japan Standard Testing Method for
Wastewater (APHA-AWWA-WEF, 1998). Nitrite and nitrate were measured with
capillary electrophoresis (Agilent technologies 7100, CA, USA). Before the analysis of
NH4+-N, NO2–-N and NO3–-N, samples were filtered through 0.45 µm cellulose acetate
membrane.

Batch experiments
The batch experiments followed the methods from a previous article (Third et al., 2001).
Nitrifying activity for AOB and NOB were respectively measured under completely
aerobic conditions (initial DO ≈ 7.5 mg/L). Sludge was taken from the reactor and

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 Journal of Water and Environment Technology, Vol.13, No.1, 2015

incubated in a 500 mL beaker at 30˚C in shaking water bath (Yamato BT200, Japan)
and sample interval was half an hour. The experiment for each antimicrobial was
repeated at least 3 times and the standard deviation is shown as bars in the figures.

With the same nitrogen loading rate, for AOB test, the substrate was the same with the
synthetic wastewater while for NOB test 246 mg/L NaNO2 (50 mg/L NO2–-N) was
utilized as the nitrite source. Antimicrobials were spiked into the beakers respectively in
order to investigate their effect at different concentrations (0 – 400 mg/L). In this way
the SAA and SNA at different antimicrobial concentrations were obtained by the curve
slope of the ammonia reduction rate and nitrite production rate based on the following
equations:

SAA = ∆ [NH4+-N] / (MLVSS × h) (1)

SNA = ∆ [NO2–-N] / (MLVSS × h) (2)

The relative activity was expressed as % and the inhibition rate caused by
antimicrobials were obtained from the following equations:

Relative activity [%] = Inhibited activity (with the addition of antimicrobials)

/ Initial activity (without antimicrobials) × 100 (3)
Inhibition rate [%] = 100 – Relative activity [%] (4)

Statistics
The experimental data was analyzed by SPSS and the data was expressed as the mean
standard error (S.E.) The data evaluation was done using Duncan’s post-hoc test in the
analysis of variance (ANOVA). Indeed, p < 0.01 was regarded as statistically and
extremely significant while p < 0.05 was regarded as the rejection of statistically
significant. For all experiments, the related SAA and SNA were set as 100% in the
groups without antimicrobials. The statistical difference among three antimicrobials was
obtained by comparing their effect on activity at each concentration.

RESULTS AND DISCUSSION

Setup experiment
In order to enrich the nitrifier, the reactor run without carbon source and the data of
continuous operation are shown in Fig. 2. At first, the activated sludge taken from the
oxidation ditch performed low ammonia removal efficiency (around 19%) at 0.05
gN/L/d. About 24 days later, the removal efficiency of NH4+-N began to increase and
approached to 95%. No accumulation of nitrite was found during this period and it
completely transferred to nitrate. This steady stage was sustained for 40 d and then the
NLR increased to 0.1 gN/L/d by decreasing HRT (hydraulic retention time) to 12 h. The
ammonia removal and nitrate production efficiency appeared to no obvious variation.
After being stable at this stage, NLR was increased to 0.2 and 0.4 gN/L/d respectively.
The AOB and NOB in this reactor adapted to operate under higher NLR so as to obtain
higher microbial activity than the bacteria in common activated sludge system
(Alimahmoodi et al., 2013). This reactor successfully realized the operation of activated
sludge under high NLR and was helpful to the research of nitrification and
denitrification.

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 Journal of Water and Environment Technology, Vol.13, No.1, 2015

The alteration of HRT led to different growth rates of MLVSS at each stage. In the
beginning of this continuous operation, the heterotrophic bacteria cannot survive due to
the lack of organic substance and was under the endogenous metabolism stage (Okabe
et al., 2005). The microbe growth rate was lower than its death rate so that the
concentration of MLVSS showed 32% decrease at the first 35 d. Then, nitrifying
bacteria started to occupy dominant population and had rapid proliferation with
sufficient nitrogen source. From day 51, the growth rate of microbe became higher than
the death rate and the concentration of MLVSS began to increase under the cultivated
condition. However, the shortest HRT resulted in part of the sludge discharged with the
effluent and that the MLVSS reduced from 1,590 mg/L to 1,380 mg/L at the third stage.
After approaching NLR to 0.4 gN/L/d, the reactor no longer reduced HRT again and
kept certain increase of MLVSS. The yield of nitrifying activated sludge was 0.06
kg-MLVSS/kg-NH4+-N according to calculation. This value supplied the available
frequency of the batch experiment for the purpose of maintaining a constant MLVSS
concentration in the reactor. According to this enrichment and purification of the
nitrification bacteria, the original activated sludge could operate without carbon source
under high NLR. This is the preparation step for the batch experiment in order to keep
the stabilization of microbe and its activity (Chen et al., 1988).

1 2 3 4
60
NLR [Ng/L/d] MLVSS [mg/L] NO3--N [mg/L] NH4+-N [mg/L]

40

20 Influent
Effluent
0
60

40

20
NO3-N
0
3,000

2,000

1,000
MLVSS
0
0.6

0.4

0.2
NLR
0
0 20 40 60 80 100 120 140 160 180 200
Time [d]
Fig. 2 - Concentration of ammonia, nitrate and MLVSS with different NLR.

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Effect of CLX on nitrification

In order to evaluate the acute impact of toxicant on the biodegradation of substrate, the
assessment of the microbial activity of the biomass in batch experiments is suggested.
In this study, before the analysis of antimicrobial influence on nitrification, the initial
SAA and SNA were measured. The microbial activity of AOB and NOB were around
0.44 and 0.64 gN/(gVSS·d) without antimicrobials, respectively. This demonstrated that
NOB showed faster reaction rate than AOB which was consistent with a previous
research (Schramm, 2003). The value of reaction rate can explain no accumulation of
nitrite in the reactor – once nitrite was generated from ammonia, it could be transferred
to nitrate immediately. The measurement of nitrification activity aimed at estimating the
nitrifying capacity of the inoculated population at the time of sampling and supplied the
reference to the decision of NLR.

It could be anticipated that different activities of nitrifiers, rely on their sensitivity

toward CLX. The effect of CLX at different concentrations on SAA and SNA according
to the metabolic activity is shown in Fig. 3. Evidently, the effect of CLX was no strict
dose-dependent for AOB but had limited inhibition for NOB, which was in conformity
with the results obtained from the statistical analysis (concentration as factor: pAOB >
0.05; pNOB < 0.05). When the concentration of CLX was below 400 mg/L, no significant
adverse effect was observed on SAA, while 10.6% inhibition was found in NOB below
this concentration. Based on the research of American Water Works Association
(AWWA), most but not all nitrifier species have a Gram-negative multilayered cell wall.
However, the marine and some other strains of NOB may have additional cell wall
layers just like Gram-positive bacteria (AWWA, 2006). Thus, the experimental results
had great agreement with the characteristic of CLX as a first-generation cephalosporin:
it was effective against a wide range of Gram-positive and a limited range of
Gram-negative bacteria. The mechanism of CLX with no obvious interference to
nitrifiers suggested that its target position (bacterial cell wall) had apparent difference
between Gram-positive and Gram-negative (Waxman and Strominger, 1983). Among
these four antimicrobials used in this study CLX was the slightest inhibitor and it can be
easily degraded by microorganisms owing to its opening of the strained β-lactam ring.
This structure could be destroyed by β-lactamase, which is a kind of widespread
enzyme in bacteria (Lamm et al., 2009). The reduction of its concentration could be
another reasonable explanation for the resistance of nitrification bacteria to CLX.

SAA Relative activity SNA Relative activity

0.7 0.7
100 100
0.6 0.6
Relative activity [%]
SAA [gN/gVSS/d]

SNA [gN/gVSS/d]

Relative activity [%]

0.5 80 0.5 80

0.4 60 0.4 60
0.3 0.3
40 40
0.2 0.2
20 20
0.1 0.1

0.0 0 0.0 0
0 20 50 100 200 400 0 20 50 100 200 400
Cefalexin concentration [mg/L] Cefalexin concentration [mg/L]

Fig. 3 - Relative SAA and SNA at different CLX concentrations.

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 Journal of Water and Environment Technology, Vol.13, No.1, 2015

Effect of AZI on nitrification

As shown in Fig. 4, the inhibition of AZI on AOB and NOB was evaluated by activity
measurement. There was remarkable inhibitory effect of AZI on nitrifying bacteria
(concentration as factor: pAOB < 0.01; pNOB < 0.01). Both for AOB and NOB, a dose–
effect was found. With the rising concentration of AZI, the microbial activity decreased
substantially during the whole concentration range. This result indicated that just as a
kind of toxicant, the addition of AZI had exerted several pressures on nitrification,
which reduced the activity of microbes (Babich and Stotzky, 1985). At the
concentration of 400 mg/L the relative SAA and SNA were 39.1% and 20.2%,
respectively. Thus, the EC50 of AZI for AOB and NOB is supposed to be about 198
mg/L and 71.3 mg/L, respectively. Compared with NOB, AZI exerted around 20%
higher inhibition to AOB and this was due to the different tolerance between these two
kinds of nitrifier.

Alighardashi et al. (2009) investigated the influence of Erythromycin (ERY) on

nitrification which is a parent compound of AZI. The NH4+-N removal efficiency of
activated sludge nitrifiers decreased half at a concentration of 267 mg/L in batch tests.
In another literature, wastewater spiked with 10 mg/L ERY inhibited the specific
NH4+-N evolution rate by 41% (Louvet et al., 2010). As a kind of antimicrobial drug
which inhibited the protein synthesis and upgrade production from ERY, AZI was more
powerful to reduce the bacterial activity (Amsden, 1996). The major target microbe of
AZI and ERY was Gram-positive but most nitrifiers were Gram-negative and AZI still
exhibited significant adverse effect on nitrification. The results of previous and this
research suggested that the pharmaceutical spectra of antimicrobials could not apply to
the microorganism in the wastewater treatment processes directly. Thus, the influence
of antimicrobials on biological treatment processes need more strict research and could
not infer generally from the conclusion of medical purposes.

SAA Inhibition rate SNA Inhibition rate

0.7 100 0.7
100
0.6 0.6
80
SAA [gN/gVSS/d]

80
SNA [gN/gVSS/d]
Inhibition rate [%]

0.5 0.5
Inhibition rate [%]

0.4 60 0.4 60
0.3 40 0.3
40
0.2 0.2
20 20
0.1 0.1

0.0 0 0.0 0
0 20 50 100 200 400 0 20 50 100 200 400
Azithromycin concentration [mg/L] Azithromycin concentration [mg/L]

Fig. 4 - Inhibition rate of AOB and NOB at different AZI concentrations.

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 Journal of Water and Environment Technology, Vol.13, No.1, 2015

Effect of SMX on nitrification

The influence of SMX on nitrification is shown in Fig. 5. Unlike CLX, it was almost
resisted by nitrifiers and AZI caused the sharp reduction of microbial activity. The
addition of SMX yielded an evident, but limited influence on the activity of nitrification.
The relative activity of AOB and NOB were 0.37 and 0.49 gN/gVSS/d at the SMX
concentration of 400 mg/L, respectively (concentration as factor: pAOB < 0.01; pNOB <
0.01). The experimental result also indicated that the decrease of ammonia reduction
and nitrate production efficiency gradually increased with the exposure concentration of
SMX. In order to distinguish the effect of different antimicrobials, the kinds of
antimicrobials were used as factor in the statistical calculation, pAOB and pNOB approach
to 0 and considered that the difference of antimicrobial effect was statistically and
extremely significant. According to the post-hoc test, this distinction was reflected by
the different inhibitory levels of each antimicrobial at the same concentration. As a kind
of sulfa drug, SMX derived from its antagonistic competition with p-aminobenzoic acid
for dihydropteroate synthase enzyme, which is necessary for bacterial folic acid
synthesis (Aga, 2007). A previous study reported an average decrease in NO2–
production rates of 49% compared to the control by groundwater bacteria (Underwood
et al., 2011). The inconformity of the SMX inhibition might rely on the different
experimental conditions and called further research.

On the other hand, the stronger adverse effect on NOB than AOB indicated the different
sensitivity to SMX between AOB and NOB. It was confirmed that NOB was more
sensitive than AOB to heavy metal, toxicant, organic compounds and other substances
in previous studies (Cervantes, 2009; Mota et al., 2005). The inhibition to NOB was
higher than to AOB that was corresponding with the results of CLX and AZI. We could
consider that AOB was more tolerant to toxicant and hostile condition.

SAA Inhibition rate SNA Inhibition rate

0.7 100 0.7
100
0.6 0.6
80
80
SNA [gN/gVSS/d]
Inhibition rate [%]
SAA [gN/gVSS/d]

0.5 0.5
Inhibition rate [%]

0.4 60 0.4 60
0.3 40 0.3
40
0.2 0.2
20 20
0.1 0.1

0.0 0 0.0 0
0 20 50 100 200 400 0 20 50 100 200 400
Sulfamethoxazole concentration [mg/L] Sulfamethoxazole concentration [mg/L]

Fig. 5 - Inhibition rate of AOB and NOB at different SMX concentrations.

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 Journal of Water and Environment Technology, Vol.13, No.1, 2015

Effect of TC on nitrification
In the addition of TC, foam appeared in the reaction beaker and led to obvious reduction
of biomass as shown in Fig. 6a. Thus, the activities of AOB and NOB in the presence of
TC could not be measured. The results indicated that TC could react with several
substrates under the aeration condition. In order to find which substrate could lead to
this phenomenon, several substrates were tested separately in the presence of TC. Every
experiment used one substrate added into TC solution and there were 8 kinds of
substrates shown in Table 1 (including microelement as one kind). The addition of most
substrates did not produce the foam except Ca2+ and Mg2+. The presence of Ca2+ or
Mg2+ in the TC solution led to a similar phenomenon with the initial experiment.
Previous research also reported this result and a possible explanation was TC could
chelate certain divalent cations and form stable complexes (Lambs et al., 1988). In order
to distinguish the effect of Ca2+ or Mg2+ or both of them, the residual biomass was
detected in different concentrations of TC at the end of the batch experiment. As shown
in Fig. 6b, MLVSS had no remarkable change during the whole reaction process
without TC. However, foam was obviously observed immediately on the liquid surface
after adding TC. In the addition of 20 mg/L TC, the MLVSS exhibited 6.3% and 45.0%
reduction with Ca2+ and Mg2+, respectively, while 55.3% reduction appeared in the
presence of both Ca2+ and Mg2+. When the concentration of TC was 400 mg/L, 76.9%
and 13.7% biomass remained in the reaction tank for Ca2+ and Mg2+, respectively, while
only 8.1% MLVSS left in the beaker with Ca2+ and Mg2+ together. These results were in
general agreement with those previously reported that the complexes formed with
calcium were slightly weaker than those formed with magnesium (Jogun and Stezowski,
1976; Martin, 1979). On the other hand, several papers reported SAA and SNA were
3.3% and 25.7% inhibited at TC concentration of 10 mg/L in a long-term exposure
experiment (Shi et al., 2013). From another study, TC did not show an inhibitory effect
on ammonium oxidation and nitrification when TC was 10 µg/L (Islam, 2009). These
results indicated a markedly toxic effect of TC on AOB and NOB at higher
concentration. This inhibition of TC was stronger for NOB and its complexation with
calcium and magnesium ions could enhance the risk of the pharmaceutical wastewater
treatment under aerobic condition.

CaCl2 MgCl2 MgCl2+CaCl2

a 2,000
b
1,600
MLVSS [mg/L]

1,200

800

400

0
0 100 200 300 400
Tetracycline concentration [mg/L]

Fig. 6 - Biomass reduction in the presence of TC.

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 Journal of Water and Environment Technology, Vol.13, No.1, 2015

CONCLUSIONS
Below the NLR of 0.4 gN/L/d, the enrichment of nitrifying bacteria based on activated
sludge was successful by reducing HRT. According to the sludge yield, the reactor
could operate with constant biomass in the case of taking sludge out for repeated batch
experiment. Among the tested kinds of antimicrobials, CLX has no obvious adverse
effect on nitrifier, while SMX shows slight inhibition to the nitrification process. To the
nitrifiers AZI is the strongest inhibitor, but the relative activity of nitrifying bacteria
cannot be detected in the presence of TC. Foam appeared soon after the addition of TC
which caused biomass reduction and the experiment could not be continued. Compared
with AOB, NOB is more sensitive to antimicrobials, so it may lead to the accumulation
of nitrite during the nitrification treatment of antimicrobials wastewater.

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