オゾン処理による下水中のアンピシリン耐性菌と細胞内遺伝子の除去効率に及ぼす温度の影響
Effect of Temperature on the Removal Efficiency of Ampicillin Antibiotic
Resistant Bacteria and Intracellular Genes in Wastewater by Ozonation○ Jiaxing Wang, Kyoung-Soo Park, Fumitake Nishimura* : Kyoto University

Abstract

The emergence and spread of antibiotic resistance have become a growing concern for human health.
Domestic wastewater treatment plants, on the other hand, have been identified as key hotspots forthe spread of antibiotic resistance in the environment. The capacity of conventional wastewater
treatment to control and eliminate these micropollutants, particularly antibiotic resistance genes(ARGs), is limited. Here, the current study investigated the effect of temperature on the removal
efficiency of ampicillin antibiotic resistant bacteria and intracellular genes by ozonation treatment
in order to check the concrete impacts of an operating factors from the engineering point of views.
Meanwhile, the variation of intracellular ARGs was determined as antibiotic resistant bacteria (ARB)
are removed during treatment. In this study, E. coli DH5𝛼 competent cells carrying the ampicillin
resistant gene blaTEM were employed for ozonation removal experiment, in which the secondary
effluent of wastewater was prepared as the matrix. Then the ARB and ARGs were quantified using
a culture-based method, DNA extraction, and qPCR. As a result, it shows temperature has no
significant effect on the removal efficiency of ARB and ARGs in wastewater by ozonation with ozone
CT. However, for ozone injection time, the removal efficiency of ARGs is higher under lower
temperature.
Keywords: Wastewater Treatment, Antibiotic resistance, Ozonation
1. Introduction

Antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs) are becoming moreprevalent in the environment as a result of their diversity and capacity to transfer across genera1).
Urban wastewater treatment plants have been identified as significant hotspots for the spread ofantibiotic resistance in the environment over the last decade2). As a result, the prevalence of ARBand ARGs in wastewater treatment plants (WWTPs) has recently attracted increased attention.
Biological reactors in activated sludge systems, in particular, are regarded to be one of the mostimportant compartments for containing both ARB and ARGs2). Traditionally, before discharging into
the environment, pathogens and fecal contamination in treated wastewater effluent can becontrolled by disinfection methods such as chlorination, which is the most common treatment
applied. However, in terms of recent studies, the capacity of chlorination to remove ARB and ARGs
is restricted3). Despite the fact that it efficiently reduces ARB, complete DNA remains may persistand transfer ARGs to downstream bacteria via transformation or transduction3,4). In this case,ozonation has been widely used and researched considering its effective inactivation of ARB andelimination of ARGs from wastewater effluent. A previous study mentioned that the ozone depletion
rate varies with the increasing temperature5). However, it is still not clear how they affect theremoval efficiency of ARGs.
2. Materials and Methods

2.1 Bacterial preparation and culture
E. coli DH5𝛼 competent cells were selected to be transformed by the plasmid pUC19 carrying
the ampicillin resistant gene blaTEM. The formed ampicillin resistant bacteria at the concentrationof 108 CFU/mL can be used for the ozonation removal experiment further. The decrease in thenumber of viable E. coli colonies following ozonation was measured using a culture-based technique.
Disinfected (for all the collection times) and nondisinfected samples containing E. coli DH5𝛼 (pUC19)
were serially diluted with phosphate buffer to produce between 30 and 300 colonies on each XM-G
agar medium plate containing ampicillin at the concentration of 32 µg/mL (minimum inhibitory
concentration). After 18h-24h of incubation at 37 ℃, the colonies on the plates were counted. For
each sample, duplicate plates were prepared, and the result was averaged.

2.2 Wastewater samples
All experiments were performed using actual wastewater effluent samples collected from thesecondary clarifier of an WWTP located in Japan. This WWTP, serving a population equivalent of
10,000, has an average flow of 88400 m3/day and employs conventional activated sludge as secondary
treatment. In order to eliminate the impact of the bacteria existing in the wastewater originally,
1.5L secondary effluent sample for each experiment went through 0.45 µm pre-filtration. For eachcomparable set of experiments, the wastewater sample should be originated from the same sample.
The qualitative parameters (pH, conductivity, TOC) of the secondary-treated wastewater samples
used during the experimental period were determined via routine analysis. The pH of wastewater
sample was adjusted to 7 for each experiment. 10 mL ampicillin-resistant E. coli transformed before
was performed centrifugation at 2000 × g for 20 minutes so as to remove most of the medium
components. The concentrate was added to 1.5 L of 0.45 µm filtered secondary effluent water toadjust the concentration of ampicillin-resistant E. coli in the sample to approximately 105 CFU/mL.

2.3 Ozone treatment experiment set-up and procedure
This study carried out the ozone treatment experiment using the cylindrical semi batch reactor.
The capacity of the reaction tank is 2.0 L. Ozone gas is generated from ozone generator and flows
into the reaction tank. The ozone gas concentration entering the reaction tank is measured by an
input ozone gas monitor, and the flow rate of the input ozone gas is measured using a flowmeter.
The ozone gas concentration expelled from the reaction tank is determined by an output ozone gasmonitor. The temperature of the reaction tank can be kept at a certain temperature during thereaction period using a constant temperature tank. The water sample in the reaction tank is stirred
by magnetic stirrer.
The temperature was set at 10, 20 and 30 ℃. The input ozone gas concentration was maintained
at 1.0 g/m3 approximately. The acquired samples were transferred into 10 mL tubes containing 0.1
mL 1% Na2S2O3 solution and 50 mL glass tubes including 5mL indigo solution in order to remove
any residual oxidants. The ozone concentration in the liquid phase was measured by the indigo
colorimetric method. The experiment was performed in triplicate and average values are quoted asresults.

2.4 DNA extraction and qPCR
DNA was extracted using the Fast DNATM Spin Kit for soil following the protocol of themanufacturer. Single DNA extraction was performed for each sample. The concentration and qualityof the extracted DNA were measured by spectrophotometry. The extracted DNA samples will bestored at −20 °C until quantification analysis.
Antibiotic resistant genes in ozone-treated water samples were quantified by Real-time PCR
using TaqMan probes. The genes examined to this study is plasmid pUC19 with the ampicillin
resistance gene blaTEM. For the quantification of pUC19, standard DNA for calibration curve wasprepared. As the standard DNA, pUC19 extracted by NucleoBond Xtra Midi Plus from theampicillin-resistant Escherichia coli culture solution was used. Quantification of standard DNA
processed using a Qubit 2.0 Fluorometer, and was converted to concentration (copies/µL). The DNA
for preparing the calibration curve was prepared to 102 to 106 copies/µL by 10-fold serial dilution,and each dilution was subjected to the determination operation in triplicate.
3. Results and Discussion

Figure 1 and 2 show the increasing removal rate of ARGs and ARB as the CT value increased
under different temperature. As can be seen, the removal rate of ARGs and ARB with each CT value
under 10°C, 20°C, 30°C intermingles together and there is no significant difference under different
temperature, implying that temperature has no significant impact on the removal rate of ARB andARGs with ozone CT value. Generally, the inactivation rate constant (k) for ARB and ARGs by ozone
inactivation will be calculated using the first-order Chick−Watson expression ln (Ct/C0) = −kCT.
According to previous study6), the inactivation rate constant k of ARGs can approach 102 L/(mg・min)approximately in clean systems. However, based on our results using real wastewater effluent, theinactivation rate constant k of 20°C is 4.12 L/(mg・min). The discrepancy between the clean systemand real wastewater effluent indicates that inactivation of antibiotic resistance genes in real
wastewater effluent is relatively ineffective.
Figure 3 and 4 show the increasing removal rate of ARGs and ARB as the time increased under
different temperature. In contrast, the removal rate of ARGs shows a significant increase with thedecreasing temperature, which can be attributed to 1) Given that ozone decomposition rate increases
with the increasing temperature5), the ozone concentration would be lower under higher

5.0

5.0

4.0

4.0

3.0

ARGs

2.0

10°C20°C
30°C

1.0

Log10(C0/Ct)

Log10(C0/Ct)

temperature 2) Considering the dissolvability of gas, dissolved ozone concentration increases a lot

3.0ARB

2.010°C
20°C
30°C

1.0
0.0

0.0
0.0

2.0
CT value (mg・min/L)

4.0

Figure 1: removal rate of ARGs with CT value

0.0

1.0
2.0
3.0
CT value (mg・min/L)

4.0

Figure 2: removal rate of ARB with CT value

under the lower temperature. Combined with these two factors, the dissolved ozone concentration
increased with lower temperature simultaneously, which facilitated the reaction further. In figure
4, the removal rate of ARB shows a tiny increase as the temperature is lower as well.
10°C
30°C

5.020°C

ARGs

5.0

4.0

4.0

3.0

3.0

2.0

2.0

1.0

1.0

0.0

10°C
30°C

20°C

ARB

0.0
0.0

10.0
20.0
30.0
Ozone Injection Time (min)

40.0

0.0

Figure 3: removal rate of ARGs with Time

10.0
20.0
30.0
Ozone Injection Time (min)40.0

Figure 4: removal rate of ARB with Time

4. Conclusion

1)

Temperature has no significant effect on the removal efficiency of ampicillin antibiotic resistantbacteria (ARB) and intracellular genes (ARGs) in secondary effluent of wastewater (pH7.0, TOC
6.9 mg/L) by ozonation with ozone CT value. However, for ozone injection time, the removal
efficiency of ARGs is higher under lower temperature.

2)

1 mg・min/L and 0.2 mg・min/L of CT may be required to inactivate ARGs and ARB sufficiently(2-log reduction) in wastewater under 20°C and 1.0 g/m3 ozone gas concentration.

3)

It is suggested that ozonation can be applied to wastewater treatment plants relatively more
efficiently in cold regions in order to achieve better removal of ARGs and get the most out ofozonation.

Acknowledgement
This research was partially supported by the JST-Mirai Program, and the River Fund of The
River Foundation, Japan.
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