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Invest Clin 60(5): 1371-1387, 2019

Isolation of a Highly Efficient Sulfamethoxazole-degrading Bacteria and Its Degradation Properties

Ying Yue

College of Life and Science and Technology, Harbin Normal University, Harbin 150025, Heilongjiang, China

Jihua Wang* College of Life and Science and Technology, Harbin Normal University, Harbin 150025, Heilongjiang, China

*Corresponding author(E-mail: wangjihua333@hotmail.com)

Qi Zeng College of Life and Science and Technology, Harbin Normal University, Harbin 150025, Heilongjiang, China

Xu Liu

College of Life and Science and Technology, Harbin Normal University, Harbin 150025, Heilongjiang, China

Ning Zhang Environmental Engineering, Tsinghua University, Beijing 100084, China

Miao Li

Environmental Engineering, Tsinghua University, Beijing100084, China

Xiang Liu Environmental Engineering, Tsinghua University, Beijing 100084, China

Abstract With respect to antibiotic-contaminated sites, sulfamethoxazole (SMX) is considered at the highest risk level and a substance for priority control. In order to deepen the application of microbial remediation for the degradation of SMX, SMX-degrading bacteria were enriched and fortified from contaminated samples through co-metabolism. Strain 2-T was isolated and identified as Pseduochrobactrum sp. In terms of the SMX degradation, this strain was highly efficient when compared to the other isolated strains, exhibiting degradation efficiency up to 92%. Results show that the optimal degradation conditions of strain 2-T are at 25°C and pH 7.0, with a 10% bacterial inoculum and a low initial SMX concentration. Under the optimal conditions, the initial SMX concentration was within 1-30 mg/L and the degradation process conformed to first-order kinetics. The SMX concentration was affected by the bacterial biomass and degradation time. Furthermore, SMX degradation was in accordance with the Monod equation. The activation energy of the SMX degradation reaction by strain 2-T at different temperatures was lower than 44.89 kJ/mol. This study provides a basis for use of the SMX-degrading strain 2-T for bioremediation of SMX-contaminated wastewater. Keywords: Sewage Treatment, Sulfamethoxazole, Degrading Bacteria, Kinetic Equations, Monod Equation

Aislamiento de Bacterias Altamente Eficientes que Degradan el

Sulfametoxazol y Sus Propiedades de Degradación Resumen Por lo que se refiere a los lugares contaminados con antibióticos, se considera que el sulfonamidazole (SMX) es la sustancia que presenta el mayor nivel de riesgo y UN control prioritario. Con el fin de profundizar aún más la aplicación de la técnica de reparación microbiana en la degradación de SMX, se han enriquecido y reforzado las bacterias de degradación SMX a partir de muestras contaminadas por cometabolismo. Isolación de la cepa 2-t, identificada como pseudocrobacillus sp. En cuanto a la degradación de SMX, la cepa es más eficiente en degradación que otras cepas aisladas, con una eficiencia de degradación del 92%. Los resultados indicaron que las condicionesóptimas de degradación de las cepas 2-T eran 25ºC, pH=7.0, una cobertura de 10%de las

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bacterias y una baja concentración inicial de smx. En las mejores condiciones,la concentración inicial de smx oscila entre 1 y 30 mg/L y el proceso de degradación se ajusta a la cinética primaria.Las concentraciones de SMX se ven afectadas por la biomasa bacteriana y el tiempo de degradación.Además,la degradación del SMX se ajusta a la ecuación de Mond.Las cepas bacterianas 2-T degradan el SMX y su activación puede ser inferior a 44,89 kJ/mol a diferentes temperaturas.El estudio sirve de base para la biorehabilitación de las aguas residuales contaminadas por el smx mediante cepas de bacterias degradadas por el SMX 2-T. Palabras clave: Tratamiento de Aguas Residuales, Sulfametoxazol, Bacterias Degradantes, Ecuaciones Cinéticas, Ecuación de Monod

1. Introduction

At present, China is seriously short of water resources, and groundwater is an important source of water supply for many cities in China, especially in the north where water is scarce all the year round. Therefore, wastewater recycling and groundwater recharge have become one of the best way to alleviate water shortage. However, sewage treatment plant of tail water discharge and reuse in a variety of ways (such as irrigation, infiltration of surface water, artificial reinjection, etc.) infiltration recharge of groundwater, which includes the changes in plants could also migrated to the groundwater pollution in accumulating, lead to the risk of groundwater contamination, including antibiotics is a class of emerging contaminants in highly attention. Therefore, the reuse of municipal sewage (i.e., reclaimed water) after deep treatment has become a potential source of pollution, leading to more and more antibiotics entering the environment from sewage and reclaimed water into the contaminated groundwater. China has been the world's largest antibiotic producer and consumer, producing approximately 210,000 tons of antibiotic raw materials each year [1, 2]. Studies have shown that about 75-90% of the antibiotics used for medical or animal farming are excreted into the municipal sewage system as parent compounds or metabolites. Most of these compounds enter the sewage treatment plant along with the wastewater via the sewage collection system and remain in the reclaimed water, eventually entering the environment. The real harm of antibiotic abused and environmental pollution lied in the intensification of bacterial antibiotic resistance [3]. The sewage treatment plant acts as a receiving water body and an important point source for antibiotic emission into the environment. Considering the current status of antibiotic residues in sewage, optimizing the existing treatment processes, or developing new methods, and improving the removal of antibiotics in wastewater treatment plants has become a hot topic in environmental research. The removal of many antibiotics by sewage treatment plants is not obvious, resulting in large amounts of residual drugs entering the natural environment, potentially causing harm. Carballa tested the content of antibiotics and other drugs in a sewage treatment plant in Galicia, Canada, and found that concentrations were between ng·L-1 and μg·L-1. Furthermore, the activated sludge method could only remove a portion of the substances [4].

Sulfonamides (SAs) are a class of synthetic antibacterial drugs based on p-aminobenzene sulfonamide that are widely used globally. They have a wide variety of antibacterial spectrums and their curative effects are stable. They were easy to use, inexpensive and storable for long periods [5]. They were one of the most widely used antibiotics in the medical field (prevention), livestock industry (treatment of livestock diseases), aquaculture and agriculture (growth catalysts) [6-9]. China's SAs produced 5,000 tons in the 1980s. In 2003, production exceeded 20,000 tons and increased thereafter [10]. The widespread use of SAs, as well as their structural stability and difficulty to degrade, have attracted the attention of scientists. Sulfamethoxazole (SMX), within the SA class, is frequently and universally detected in the environment, especially in water, due to its wide application. The detection concentration ranged from ng/L to μg/L [11-13]. Therefore, it is necessary to remove SMX from wastewater.

Refractory organic pollutants, such as antibiotics, can be removed from wastewater using physical, chemical and biological methods. Among these, biological methods were primarily used to degrade organic pollutants because of their low investment, good effect, complete mineralization and lack of secondary pollution [14-16]. Antibiotics degraded by microorganisms may be converted into cellular components or eventually transformed into non-toxic inorganic or organic small molecules, thereby achieving a harmless antibiotic treatment process, in which resistant bacteria play the most important role. The drug can directly destroy and modify the antibiotic to inactivate it. At present, there are many reports on SA biodegradation under natural conditions (soil, water, etc.) and the artificial screening of antibiotic-degrading bacteria has attracted increasing attention from scholars. Lin et al studied aerobic SMX degradation in soil and found that SMX adsorption of 40 μg·kg-1 was weak and biodegradation was significant [17]. Gao et al. investigated the removal efficiency of 15 antibiotics from a sewage treatment plant in Michigan, USA, and explored the migration process of drugs using the mass balance method [18]. It is believed that removal of several of the studied antibiotics was negligible and that biotransformation and degradation were the main mechanisms. Bioaccumulation can enrich SMX-degrading microorganisms and further enhance the removal of SMX. At present, most studies have

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focused on SMX degradation by non-acclimated activated sludge. Deng's latest article , they studied the aerobic microorganisms degrading sulfonamides, including the classification, degradation pathway, degradation characteristics and degradation functional genes of the degradation bacteria, while there were few studies on the removal of SMX by domestic activated sludge [19].

Hence, the objectives of this paper were to: 1) screen for antibiotic-degrading bacteria that can effectively degrade SMX in a previously studied sewage treatment plant; 2) enhance the degradation efficiency of the SMX-degrading bacteria through different co-metabolism acclimation cycles; and 3) identify the properties of the strain and characterize SMX degradation under different influencing factors. The kinetic degradation characteristics of the bacteriostatic strain 2-T were investigated and used to understand the feasibility of the strain as a biofortification culture. Additionally, the Monod equation was used to further analyze the degradation characteristics of strain 2-T. Characterization of the SMX degradation kinetics by domesticated activated sludge is very useful for designing biological treatment units, while providing a basis for antibiotic application and operation in microbial enhanced remediation in wastewater treatment plants. This study has great significance in alleviating water resources crisis, preventing groundwater pollution and enhancing the prevention and control of sulfamethoxazole in groundwater. It also brings significant social and environmental benefits while ensuring the safe reuse of water resources. 2. Experimental

2.1. Study Area and Sampling

Research was carried out at the sewage treatment plant in the Shijiazhuang, China. The community lies at approximately latitude 38°0′44″N and longitude 114°41′46″E. The sewage treatment plant is mainly responsible for the production of wastewater from many large pharmaceutical companies in the vicinity, most of which are among the largest antibiotic producers in China. A large amount of recalcitrant antibiotics remain in the treated sewage, which is discharged into the environment as reclaimed water, subsequently causing contamination of the groundwater. In order to solve this problem, wastewater from the sewage treatment plant was collected for experimentation. All glassware used for sampling was washed with deionized water, methanol, and ethanol and high-purity water three times. Next, it was placed in a muffle furnace and baked at 450°C for 5 h. Can not be used for high temperature burning of the vessel after washing three times and then placed in a blast hot dryer to dry; glass fiber filter before use was also baked in the muffle furnace 450°C for 5 h. The sample was stored at 4°C and the pre-treatment required for subsequent experiments was completed within 24 h. 2.2. Enrichment and Detection of Antibiotics

In this experiment, solid phase extraction (SPE) was used to enrich antibiotics of the water samples. The specific operations were as follows:

(1) Water sample pretreatment A water sample (1 L) was taken for extraction filtration and Na2EDTA was added to complex the cations

present in the sample. The mixture was filtered through a 0.7 μm glass fiber filter and the pH was adjusted to 3 with HCl.

(2) HLB cartridge activation Before use, 5 mL of methanol (chromatographically pure) and 15 mL of high-purity water were used to

rinse the HLB cartridge and the small column packing was activated. (3) Water sample filtration with the HLB cartridge The pretreated sample was extracted using an SPE device. The flow rate of the sample was controlled

within a range of 2-4 min/L. Following filtration, the Na2EDTA residue in the small column was washed twice with 10 mL of ultrapure water. Finally, the column was placed in a freeze dryer to remove residual moisture and then stored dry.

(4) Antibiotic elution and concentration The antibiotics in the cartridge were eluted with 2 mL, 2 mL and 2 mL of methanol in sequence. The eluate

was nitrogen-blown, reconstituted with 200 μL of methanol and mixed with a vortex mixer. The resulting solution was passed through a 0.22 μm membrane to be enriched and extracted. The antibiotics were stored at -20°C for testing.

The antibiotic concentration was determined with the Ultra High Liquid Chromatography Tandem Mass Spectrometer SOE-083(Waters, USA) equipped with an American Thermo Scientific C18 liquid chromatography column (150mm × 4.6mm, 5μm). The detection wavelength was 270 nm; the injection volume was 20 μL; the flow rate was 0.6 mL·min-1; and the column temperature was 30°C. In order to achieve better separation, a dual mobile phase was used during the measurement process. The mobile phase consisted of solution A (an ultrapure aqueous solution of formic acid at pH 3) and solution B (methanol) at an A : B ratio of 65% : 35%.

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Analysis of antibiotics in the sample: The presence of the following antibiotics was determined: (1) 7 kinds of sulfonamides: sulfamethoxazole (SMX), sulfathiazole (STZ), sulfamethazine (SMR),

sulfamethazine (SMN), sulfamethoxazole (SML), sulfadiazine (SDZ) and sulfadiazine (SDM) (2) 5 kinds of fluoroquinolones: norfloxacin (NOR), ofloxacin (OFL), enrofloxacin (ENR), lomefloxacin

(LOM) and ciprofloxacin (CIP) (3) 4 kinds of tetracyclines: tetracycline (TCN), oxytetracycline (OTC), chlortetracycline (CTC) and

doxycycline (DOX) (4) 4 kinds of macrolides: azithromycin (AZN), roxithromycin (ROX), clarithromycin (CLA) and

erythromycin (ERY)

2.3. Domestication of Sulfamethoxazole Degrading Bacteria

According to the concentration of antibiotics detected in the environment, SMX was selected as the standard. SMX (purity >99.5%) was purchased from Shanghai SiYu Chemical Technology Co. LTD. The molecular structure of SMX is shown in Figure 1. And the physical and chemical properties are shown in Table 1.

Domestication of SMX-degrading bacteria was performed following the methods of Wang [20], with modifications corresponding to our specific experiments. The experimental SMX acclimation process was performed over 3 cycles, with different types of carbon sources used in each cycle. The collected sewage sample was centrifuged at 8000 rpm for 5 min and the supernatant was removed. The sample was rinsed three times by adding phosphate buffer (pH 7.0) to remove organic compounds left from the acclimation process. The rinsed sample (20 mL) was resuspended under sterile conditions in 180 mL of synthetic wastewater. The synthetic wastewater, which was necessary for the growth of the microorganisms, consisted of inorganic salts and trace elements (1 ml/L; Table 2). Next, SMX and auxiliary carbon sources (yeast) were added. The sodium acetate and succinic acid were brought to a final concentration of 1 mg/L and 1 g/L in medium. After incubating at 30°C on a 150 rpm/min shaker in the dark for 10 d, the mixture was centrifuged at 5000 rpm for 15 min and washed three times with phosphate buffer. Connect 1% of the bacterial solution to the same medium, adjust the concentration of SMX and sodium acetate was adjusted according to a certain gradient. The gradually increasing concentration gradient of SMX was 1, 5, 10, 20, 40 mg/L. The concentration of the auxiliary carbon source was gradually decreased at 1, 0.5, 0.3, 0.1, 0.01 g/L. The mixed cultures obtained by centrifugation following the first acclimation stage are referred to as MX-1. The culture obtained by acclimatization of MX-1 was inoculated into the same synthetic wastewater prepared above with an inoculation amount of 1%. The auxiliary carbon source of the medium was simplified to sodium acetate and succinic acid, while the subsequent domestication method was the same as the first cycle. The resulting mixed cultures are referred to as MX-2. The third domestication cycle continued to further simplify the auxiliary carbon source to sodium acetate. The domestication method was the same as the first cycle, resulting in the MX-3 domesticated mixed cultures.

Figure 1. Sulfamethoxazole molecular structural formula

Table 1. SMX physical and chemical properties

Compound Abbreviation CAS Molecular formula

Molecular weight

Solubility LogKow PKa

Sulfamethoxazole

SMX 723-46-

6 C10H11N3O3S 253.28 4.00×102 0.91 1.6/5.7

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Table 2. Synthetic wastewater Culture medium Composition and conditions

Inorganic salt medium (g·L-1)

Na2CO3 0.1,Na2HPO4·12H2O,KH2PO4 0.34,(NH4)2SO4 0.24,MgSO4·12H2O,CaCl2 0.004 g/L, trace element solution 1mL/L, pH=7.0。

Trace elements (g·L-1)

FeCl2·4H2O 1.5,CoCl2·6H2O 0.19,MnSO4·7H2O 0.1,ZnCl2 0.07,NiCl2·4H2O 0.024,Na2MoO4·2H2O 0.024,MnCl2·4H2O 0.006,CuCl2·2H2O 0.002。

2.4. Isolation, Identification and Comparison of the Degradation Ability of Sulfamethoxazole-degrading Bacteria

The mixed MX-3 culture cells were centrifuged at 5000 rpm for 5 min and then were washed three times with PBS. The culture was suspended in 100 mL of synthetic wastewater. After gradient dilution, the solution was uniformly coated on a solid medium containing 50 mg/L SMX. Cells were cultured at 30°C for 72 hours at a constant temperature until the colonies grew. Colonies of different shapes were picked and used to repeat the purification experiment until pure colonies were obtained. The strain was inoculated on a medium containing SMX and cultured in a constant temperature incubator at 30°C for 72 h. After a single colony was obtained, the morphology was observed. Microbial physiological and biochemical experiments were further carried out to identify the different bacteria.

Strains with strong degradation ability were selected for molecular identification by Beijing Saimo Lily Biotechnology Co., Ltd., as follows:

(1) Genomic DNA extraction: the lysozyme-SDS-phenol/chloroform method was used to extract genomic DNA from the bacteria.

(2) PCR amplification (Table 3) (3) PCR thermal cycle: pre-denaturation at 94°C for 5 min; 30 cycles at 94°C for 30 s (denaturation), 58°C

for 30 s (annealing) and 72°C for 1.5 min (elongation); and a final extension at 72°C for 10 min. Reactions were then held at 4°C.

(4) Construction of phylogenetic tree: BLAST alignment was performed using NCBI based on the 16S rDNA sequencing results of the obtained strain.

2.5. High-Throughput Sequencing Was Used To Analyze the Diversity of Cultures at Different Domestication Stages

The FastDNA Spin Kit (MP) was used to isolate the total DNA of microorganisms from different domestication cycles according to the instructions. Primers 515F (5'-gtgccagcmgccgcggtaa-3') and 8106R (5'-ggactachvgggtwtctaat-3') were used for PCR amplification of the V4 region of the 16S rRNA gene. Miseq or Hiseq was used for sequencing.

2.6. Factors Affecting the Degradation Performance of the SMX-Degrading Strain 2-T

During the process of degrading the SMX substrate, in addition to the adaptive change of SMX toxicity, the change of external factors had a great influence on the SMX degradation. Temperature and pH had a strong influence on the activity of the biological enzymes, which directly affected the activity of the microorganisms. In addition, inoculated-pathogen quantities and the initial concentration of the substrate had an effect on bacterial SMX degradation. In this study, the biodegradable bacteria 2-t was domesticated and sorted, and sulfamethoxazole was used as the only carbon source to conduct relevant experimental research and treatment on these factors (Table.4), and the kinetic fitting was carried out to further analyze the ability of biodegradable bacteria 2-t to remove SMX.

Table 3. 30μL PCR reaction system

Reactants 30μL 10×PCR Buffer 3μl dNTP(10mM) 2μl Primer27F(10μM) 1μl Primer1492R(10μM) 1μl Genomic DNA 100ng rTaq (5U/μl) 0.3μl ddH2O add to 30μl

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Table 4. Different treatment groups Treatment Experiment treatment

SMX initial concentration

Inorganic salt synthesis medium + Degrading bacteria 2-T 10%(5ml/50ml)+SMX(1, 5, 10, 15, 20, 30mg/mL), pH=7.0, 30°C

Inoculated pathogen quantities

Inorganic salt synthesis medium + SMX(1mg/L) + Degrading bacteria 2-T(1%, 5%, 10%), pH=7.0, 30°C

pH Inorganic salt synthesis medium + SMX(1mg/L) + Degrading bacteria

2-T(10%), pH(5.0, 7.0, 10), 30°C

Temperature Inorganic salt synthesis medium + SMX(1mg/L) + Degrading bacteria

2-T(10%), pH=7.0, T(10°C, 15°C, 20°C, 25°C, 30°C, 35°C) 3. Results and Discussion

3.1. Antibiotic Levels in the Contaminated Area and the Ecological Risks

Among 21 detected antibiotics in the collected environmental samples, the SMX content was the highest at a concentration as high as 542.47 ng/L (Figure 2). As proposed by Verlicchi et al., considering the environmental and ecological risks of detected antibiotics, according to the value of Hazard quotient (HQ), three levels of antibiotic ecological risk are classified, including low risk (HQ<0.1), medium risk (0.1<HQ<1) and high risk (HQ>1). In this study, the maximum concentration of antibiotic detection (MECs) was used instead of the predicted environmental concentration. The LC50 or EC50 values can be queried using EPI software. The physical and chemical properties of the detected antibiotics are shown in Table 5. The potential risks of antibiotics in sewage water were evaluated by considering the five antibiotics with concentrations >100 ng/L. The HQs are shown in Figure 3. The ecological risks of antibiotics detected in sewage are high, of which SMX has the highest potential risk; thus, it is the priority control risk substance. Furthermore, when SMX and TMP are simultaneously present, TMP will enhance the toxicity of SMX. Therefore, it is necessary to effectively deal with the high potential risk substance SMX.

Figure 2. (a) The level of antibiotics in the sample. (b) Major antibiotic HQ in the sample.

Table 5. Physicochemical properties of antibiotics detected

Compound CAS# Water-soluble Log Kow PNEC

ERY 114-07-8 733.90mg/L 3.06 2.00×10-2

OFL 82419-36-1 361.37mg/L 0.35 1.6×10-2

ROX 80214-83-1 837.07mg/L 2.75 7.1

SMX 723-46-6 253.27mg/L 0.89 2.7×10-2

CIP 85721-33-1 427.0 mg/L 0.4 6.33×10-2

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3.2. Analysis on the Diversity of Microflora Structure and Composition in Different Domestication Stages

Table 6. Alpha diversity index in each domestication stage

Sample Richness Chao1 Shannon Simpson Dominance Equitability

MX-0 548.0 548.2 4.79 0.116 0.884 0.526

MX-1 174.0 175.9 4.16 0.110 0.890 0.559

MX-2 161.0 165.1 3.19 0.271 0.729 0.435

MX-3 163.0 163.9 4.44 0.0844 0.916 0.604

Samples from undomesticated samples (MX-0) to domesticated samples at each stage (MX-1, MX-2,

MX-3) were obtained 106288, 82937, 72360 and 81461 sequences (as shown in Fig3.), and OUTs cluster analysis was performed at the same sequencing depth. After clustering, 548, 174, 161 and 163 OTUs were generated in each domestication stage. Table 6 shows that the abundance distribution of OTUs in samples at each stage is absolutely uniform. The change in the number of MX-3 OTUs compared with MX-2 is not obvious, which indicates that the strain tolerance increases with the acclimation tolerance, which helps to enhance the strain degradation ability.

According to Figure 3(b), the addition of SMX in different domestication stages subtly changes the structural characteristics at the phylum level, especially the community structure of the dominant flora. In each domestication stage, the most dominant flora was Proteobacteria, with an average abundance of 49.8%-90.1%. Zhang previous research also shows that the deformation under pressure from the choice of antibiotic bacteria door is the most advantage bacterium group [21].

Figure 3. (a) OTUs cluster analysis of each sample (b) Community structure composition of door

classification level in different domestication stages

According to Figure 4(a), the co-metabolism domestication of SMX significantly changed the abundance and composition of species. Concentration gradient to high concentration of SMX inhibited the growth of smx-sensitive strains in the bacterial community. However, high concentration of SMX also enriched and enhanced the propagation of some functional bacterial communities with degradability. As shown in Figure 4(b), under the pressure of SMX concentration, some genera were transformed into dominant ones, and some were inhibited to become non-dominant ones. In this study, the composition of the major bacteria for co-metabolism domestication was significantly different from that of other researchers [21-24]. The difference in the composition of the community at the level of genus classification may be different from previous studies, which may be due to the influence of factors such as the species concentration of auxiliary carbon source added in the domestication process, the concentration of antibiotics and other experimental influencing conditions.

With the increasing of SMX stress concentration and domestication time, the flora in the domestication system may need to constantly adjust the composition structure of its own flora to adapt to the living environment. In the domestication system, microorganisms that can utilize SMX as carbon source and energy source or can utilize other microorganisms to degrade intermediate products as carbon source are retained, the microbiota with weak resistance to SMX or unable to use SMX as carbon source and energy source were eliminated.

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Figure 4. (a) Community structure composition of different domestication stages (b) Community structure of

different domestication stages 3.3. Selection of SMX Dominant Degrading Bacteria

After 3 cycles of co-metabolism and domestication using SMX as the sole carbon source, the MX-3 degrading bacteria were isolated by agar plate separation. Seven strains that could grow under a high concentration of SMX, namely strains 1-2, 2-1, 2-2, 3-1, 2-T, 3-T and 4-T, were obtained by separation and screening. Bioacclimation can strengthen the related microorganisms capable of degrading organic pollutants and further enhance the removal ability of the pollutants.

When using SMX as the sole carbon source, the absorbance of the 7 strains gradually increased with time (Figure 5(a)), indicating that each strain can grow and reproduce under SMX pressure. Each strain entered the logarithmic growth phase within 24 h of culture and their growth patterns were basically the same; however, their adaptation periods were different.

As shown in Figure 4b, all 7 strains can degrade SMX to a certain extent. Strains 2-T and 1-2 exhibited better degradation effects with degradation rates reaching 69% and 62%, respectively, while strain 4-J exhibited the worst removal of SMX at only 26% within 10 days (Table 7). The other 4 strains (2-1, 2-2, 3-1 and 3-T) had SMX removal rates of 47%, 54%, 51% and 48%, respectively. The difference in the removal ability of each strain was related to the strain species. Jiang et al. screened the psychrophilic SMX-degrading strain HA-4 from activated sludge samples, the SMX degradation rate was 34.3% within 192 h. After domestication, the degradation rate under the optimal growth conditions was 74% [25]. This indicates that the metabolism and domestication performed during this experiment enhanced the degradation ability of the strain.

According to Figure 5(b), SMX degradation occurs at the beginning of the experiment. Compared with Figure 4(a), there was no corresponding relationship between the SMX degradation ability of the strain and its growth and reproduction. Thus, the proliferation ability of the degrading bacteria does not represent the removal effect. It has also been shown that strains can grow with SMX as the sole carbon source. Nguyen studies have confirmed that biodegradation of SMX is not caused by co-metabolism and that the growth of degrading bacteria does not prove SMX removal [26].

In order to understand the SMX degradation characteristics of each strain, first-order kinetic fitting was carried out according to the experimental SMX degradation data obtained from the 7 strains. In order to understand the degradation characteristics of SMX by each strain, plot lnS-t (Figure 5c). The fitting parameters and kinetic equations are shown in Table 8. The first-order kinetic fitting results show that the linear correlation coefficient R2 of the kinetic equation was greater than 0.9, indicating that the SMX removal process of each strain agrees with the first-order kinetic equation. The rate constant and half-life indicate that the removal ability of each strain was ordered as: 2-T >1-2>2-1>2-2>3-T>4-J. Comparison of the biodegradation rates showed that the degradation ability of strain 2-T was also the highest. In summary, strain 2-T is the best strain for degrading SMX. Therefore, strain 2-T was used as the experimental strain due to its high-efficiency degradation ability.

The genomic DNA of strain 2-T was extracted and then the 16S rDNA was amplified using universal primers. Figure 6(a) shows that the 16S rDNA fragment was 1500 bp. NCBI blast alignment (Genbank login number: FJ983141.1) was conducted according to the sequencing results. The phylogenetic tree (Figure 6(b)) was constructed according to the Neighbor-Joining method using MEGA software. According to the phylogenetic tree, the strain 2-T belongs to the genus Pseudoochrobactrum sp., which has the highest similarity and homology to the genus C. pallidum. In the current study, a series of strains that degrade SMX were isolated, such as Bacillus subtilis, Pseudomonas aeruginosa, Rhodococcus erythropolis and Brevundimonas sp. SMXB12 [27], Bacillus licheniformis BR3 and Pseudomonas hydrophila HA-4 [28, 29]. However, there are few reports on the degradation ability of strain 2-T that was isolated and screened in this study.

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Table 7. The ability of the strain to degrade SMX

Microorganisms Degradation rate (%)

1-2 0.62±0.05

2-1 0.47±0.05

2-2 0.54±0.05

3-1 0.51±0.06

2-T 0.69±0.07

3-T 0.48±0.06

4-J 0.26±0.05

Table 8. Kinetic reaction equations for SMX removal by different microorganisms

Microorganisms First-order reaction kinetic

equation R2 K/d-1 Half-period /d

1-2 lnS=-0.0808x + 1.7063 0.956 0.0808 8.58

2-1 lnS= -0.0751x + 2.0757 0.905 0.0751 9.23

2-2 lnS= -0.0749x + 1.9036 0.901 0.0749 9.25

3-1 lnS= -0.062x + 1.71050 0.918 0.0620 11.18

2-T lnS= -0.1229x + 1.6993 0.964 0.1229 5.63

3-T lnS= -0.0687x + 1.6334 0.937 0.0687 10.09

4-J lnS= -0.0271x + 1.7756 0.929 0.0271 25.57

Figure 5. (a) Growth curve of different bacteria. (b) Degradation of SMX by 7 strains of microorganisms (c)

Kinetics fitting of 7 strains of microbial SMX degradation

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Figure 6. (a) PCR amplification product agarose gel electrophoresis detection results (b) Phylogenetic tree

3.4. Degradation Characteristics of the SMX-degrading Strain 2-T

① Effect of initial SMX concentration SMX degradation curves for strain 2-T cultured with different initial concentrations of SMX are shown in

Figure 7(a). For this experiment, a 10% (5 mL/50 mL) 2-T suspension was cultured at 30°C and 150 r/min, with an initial pH of 7.0. It can be seen from Figure 7(a) that the concentration of SMX decreased with time, indicating that strain 2-T can perform the SMX removal reaction at different initial concentrations of SMX. As the initial concentration of SMX increased, the SMX removal rate decreased from 90.2% to 75.8%.

According to the experimental data of SMX degradation at different initial concentrations, first-order kinetic fitting was performed (Figure 7b), and the fitting parameters and kinetic equation were shown in Table 9.

Table 9. Kinetic equation of 2-T degradation of SMX under different initial concentrations

Initial concentrations(mg/L)

First-order reaction kinetic equation

R2 K/d-1 Half-period /d

1.015 lnS=-0.13114x-0.04373 0.971 0.13114 5.28

5.038 lnS=-0.12981x+1.42737 0.919 0.12981 5.33

9.97 lnS=-0.12484x+2.03583 0.95 0.12484 5.55

14.877 lnS=-0.09989x+2.49379 0.914 0.09989 6.94

18.963 lnS=-0.082x+2.7018 0.93 0

0.08200 8.45

29.77 lnS=-0.07173x+3.01707 0.9 0.07173 9.66

Figure 7. (a) Effect of different initial concentrations on 2-T degradation of SMX (b) Kinetic fit of 2-T degradation SMX under different initial concentration conditions

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Figure 8. (a) Effect of different bacterial biomass on 2-T degradation of SMX (b) Kinetic fit of 2-T

degradation SMX under different biomass conditions

② Effect of bacterial biomass SMX degradation curves for strain 2-T cultured with different bacterial suspensions are shown in Figure

8(a). For this experiment, the bacteria were cultured at 30°C and 150 r/min, with an initial pH of 7.0 and an initial SMX concentration of 1 mg/L.

The biomass of the degrading bacteria is an important factor affecting the degradation of SMX. As the inoculum of the experimental bacterial suspension increased, the degradation rate of SMX was faster Figure 8(a). This is because as the bacterial inoculum amount increased, the biomass in the culture solution increased. In turn, the bacterial surface binding sites and the sites where the degradation reactions occur increased within the same solution volume; therefore, SMX removal increased during the same time. In addition, the rate significantly accelerated, indicating an increase in the inoculum of the bacterial suspension and shortening the reaction process for removing SMX. For the experimental data with different inoculums, the kinetic fitting results are shown in Figure 8(b); and the fitting parameters and kinetic equations are shown in Table 10. Furthermore, the linear coefficients (R2) were all above 0.92. The rate constant and half-life indicate that the half-life of SMX degraded as the inoculum size increases.

Table 10. Kinetic equation of 2-T degradation of SMX under different biomass conditions

Biomass First-order reaction kinetic equation R2 K/d-1 Half-period/d

1% lnS=-0.0657x - 0.0774 0.917 0.0657 10.55

5% lnS=-0.0853x - 0.1374 0.950 0.0853 8.120

10% lnS=-0.1751x + 0.1578 0.979 0.1751 3.960

③ Effect of pH SMX degradation curves for strain 2-T cultured with different initial pH values are shown in Figure 13. For

this experiment, a 10% (5 mL/50 mL) 2-T suspension was cultured at 30°C and 150 r/min, with an initial SMX concentration of 1 mg/L.

As shown in Figure 9(a) the SMX concentration decreased with time under different pH conditions, indicating that strain 2-T can degrade SMX in the range of pH 5-10. However, with increasing pH, the removal effect of SMX first increased and then decreased. At pH 7.0, the SMX biodegradation effect of strain 2-T was the best, with a removal efficiency of 90.2%. In contrast, when the pH was 10, the removal efficiency was 81.7%, and when the pH was 5, SMX was only slightly biodegraded. The pH has a significant effect on the removal of SMX by the strain. The acidic conditions had an inhibitory effect on the SMX conversion and removal by the degrading bacteria, while the alkaline conditions had a slight inhibitory effect. Zheng has shown that peracid or overbase affects the transport and absorption of nutrients by microorganisms [29]. The pH of the culture solution mainly affected the ionic state of SMX, as well as the physiological characteristics, solubility and degradative enzyme activity of the microorganisms in the experiment. SMX has two pKa values. At pH < 1.6, SMX is in the form of a cation; at pH > 5.7, SMX is in an anionic form; when the pH is between 1.6 and 5.7, SMX is in a neutral form. In advanced oxidation process (AOPs), anionic forms are more susceptible to

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degradation than the other two forms [25]. According to previous studies, the SMX degradation ability of SMX-degrading bacteria under neutral conditions is better than that under acidic conditions [30]. The results of this study are consistent with these findings. Under acidic conditions (pH=5.0), the SMX degradation effect is not significant; however, under alkaline conditions, the SMX degradation rate by strain 2-T is low but it can degrade SMX. The change of pH affects the degradation of SMX by affecting the growth of the degrading bacteria; thus, SMX degradation is also closely related to the biomass of strain 2-T. In addition, the ability to remove SMX is affected by changing the physiological characteristics of the strain, which affects the biomass [31]. In this study, the optimum pH of strain 2-T was 7.0.

Figure 9. (a) Effect of different initial pH on 2-T degradation of SMX (b) Kinetic fit of 2-T degradation SMX

under different pH conditions The kinetic fit was performed according to the experimental data of SMX degradation under different

initial pH conditions. Figure 9(b) shows the kinetic fitting parameters and kinetic equations. The linear correlation coefficient R2 was above 0.93 (Table 11), indicating that the SMX degradation reaction under different initial pH conditions is consistent with first-order kinetics. According to the calculated rate constant and half-life, the SMX degradation ability of the strain is significantly affected by the change of initial pH. Compared with alkaline conditions, an acidic environment is more detrimental to SMX degradation by strain 2-T, indicating that this strain is suitable for a neutral alkaline reaction environment.

Table 11. Kinetic equation of 2-T degradation of SMX under different pH conditions

pH First-order reaction kinetic equation R2 K/d-1 Half-period /d

5 lnS=-0.0107x+0.0012 0.926 0.0107 64.77

7 lnS=-0.1465x-0.0430 0.982 0.1465 4.73

10 lnS=-0.1137x+0.2156 0.978 0.1137 6.09

④ Effect of temperature SMX degradation curves for strain 2-T cultured at different temperatures are shown in Figure 10a. For this

experiment, a 10% (5 mL/50 mL) 2-T suspension was cultured at 150 r/min, with an initial pH of 7.0 and an initial SMX concentration of 1 mg/L.

Temperature has a great influence on microbial activity. The concentration of SMX decreased over time at all experimental temperatures (Figure 10a), indicating that strain 2-T can degrade SMX within the tested temperature range (10-35°C). However, the degradation effect of SMX first increased and then decreased with increasing temperature. At 25°C, the biodegradation effect of strain 2-T was the most obvious indicating that the microbial activity was highest at this temperature. The change of temperature has a significant effect on the degradation of SMX. After day 16, the degradation rate of SMX at 10°C was 50.3%, whereas at 25°C it was 92%. The degradation curves show that the removal effect of SMX was weak at lower temperatures (10-20°C) and stronger at 25-35°C. Temperature has an effect on the activity of SMX-degrading bacteria and the solubility of SMX. Generally, the solubility of SMX increases with increasing temperature; however, there is a specific optimum temperature for microbial activity. A previous study shows that the optimal temperature for SMX degradation by Acinetobacter sp. is 25°C, whereas it is 10°C for Pseudomonas psychrophlia HA-4 [25]. In fact, changes in temperature affect the activity of the microorganisms, mainly with respect to the expression of related genes. Wu found that ammonia monooxygenase gene abundance is higher at 25°C than at 4°C [32]. Gui found that temperature has an effect on the expression of denitrifying genes (napA, nirS, conorB, nosZ), which in turn affects the removal of organic pollutants [33]. Within the experimental temperature range (5-35°C), Pseudomonas psychrophlia HA-4 reduces SMX, but the removal efficiency is significantly different [29]. Our data shows that the strain 2-T can degrade SMX between 10-35°C, and that the degradation ability is most significant at 25°C. Therefore, the optimum temperature for SMX degradation by strain 2-T is 25°C.

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Table 12. Kinetic equation of 2-T degradation of SMX under different temperature conditions

Temperature (°C)

First-order reaction kinetic equation R2 K/d-1 Half-period /d

10 lnS=-0.0445x-0.00547 0.949 0.045 15.4

15 lnS=-0.0616x-0.1174 0.912 0.082 8.45

20 lnS=-0.0952x+0.00639 0.958 0.0952 7.28

25 lnS=-0.1509x-0.1986 0.92 0.1519 4.56

30 lnS=-0.1361x+0.1482 0.961 0.132 5.25

35 lnS=-0.1303x+0.1632 0.97 0.1293 5.36

As shown in Figure 10b, the kinetic fit was performed according to the experimental SMX degradation data

under different temperature conditions. The kinetic fitting parameters and kinetic equations are shown in Table 12. The first-order kinetic fitting results show that the linear correlation coefficient R2 of the kinetic equation is above 0.91 (Table 12), indicating that the degradation reaction of SMX at different temperatures is consistent with first-order kinetics. Comparing the degradation rate constants and half-lives shows that the effect of pH on SMX degradation by strain 2-T in this study is greater than the effect of temperature (Table 12). The SMX degradation ability of strain 2-T first increased and then weakened with increasing temperature, and the SMX degradation rate was highest at 25°C.

According to long-term experimental results, the optimal conditions for the degradation of SMX by strain 2-T are: initial concentration at low concentration level, high biomass, 25℃ and neutral environment. Under the optimal conditions, the SMX degradation rate by strain 2-T reached 92% within 16 days. In the previous research results (Table 13), 2-T was studied less. The temperature and initial SMX concentration are the main factors affecting the degradation rate. Different strains have different degradation effects on SMX, and may also be due to different genus. The results highlight the potential application of strain 2-T to remove SMX from sewage through biological amplification. Adaptability of the bacteria to the toxicity of the metabolites during the degradation process is different. Thus, this process needs to be further studied.

Table 13. Microbial ability to degrade SMX

Microorganisms Conditions Degradation efficiency

Rhodococcus rhodochrous [34]

26 °C, 150rpm,31.6mg/L SMX,36d 20%

Pseudomonas psychrophila HA-4 [25]

10 °C, 150rpm,100mg/L SMX,192h 34.3%

Bacillus subtilis [35]

26 °C, 150rpm, 6mg/L SMX 48h 2.8%

Phanerochaete chrysosporium [27]

Room temperature, 150 rpm, 10mg/LSMX , 10d 100%

Shewanella oneidensis MR-1 [36]

30°C, 10 mg/L, pH=7.0–8.0 59.88%

Ochrobactrum [37] 30°C, 150rpm, pH=7.0, 5mg/L SMX, 10d 45.2%

Achromobacter denitrificans strain [26]

30°C, 150rpm, 0.5mg/L SMX,16h 60%

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Figure 10. (a) Effect of different temperature on 2-T degradation of SMX (b) Kinetic fit of 2-T degradation

SMX under different temperature conditions 3.5. SMX Degradation t Thermodynamics of Strain 2-T

The activation energy (Ea) of the SMX reaction process was obtained using the Arrhenius formula as Eq. (1) according to the fitting results of the SMX kinetic equation for strain 2-T under different temperature conditions.

ln(k) = −���

��� + ��� (1)

Where k is the rate constant, s-1; R is the atmospheric constant, 8.314 J (mol·K); T is the thermodynamic temperature, K; and A is the pre-exponential factor.

The thermodynamic fitting results of strain 2-T under different temperature conditions had a linear correlation coefficient R2>0.9 (Figure 11), indicating that lnk and 1/T have a good linear relationship within the temperature range of 10-30°C. The parameters of the Arrhenius equation can be obtained by fitting the slope of the line with thermodynamics, Ea=44.89 kJ/mol. According to previous studies, the activation energy of biochemical reactions is lower when the reaction activation energy is between 10-420 kJ/mol [38]. Therefore, the SMX degradation reaction of strain 2-T in this study can easily occur. From the economic point of view, it can be reused to the sewage treatment plant to repair the pollution of sulfamethoxazole.

Figure 11. Degradation bacteria 2-T degrades the activation energy of SMX 3.6. Monod Equation for SMX Degradation by Strain 2-T

In 1942, Monod, the founder of modern cell growth kinetics, proposed the Monod equation, which is used to describe the relationship between the specific growth rate of a cell and the concentration of a limiting substrate when an organism uses organic compounds as the sole carbon source. It can be expressed by the following Eq. (2):

μ = μmax�

���� (2)

Where μ is the growth ratio of the cell (min-1); μmax is the maximum specific growth rate (min-1); S is the concentration of the limiting substrate (mg/L); and Ks is the half saturation constant (mg/L). The Monod equation is usually simplified linearly. The inverse solution is obtained Eq. (3):

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�

�=

�

����+

��

����· (

�

�) (3)

Regression analysis by 1/μ versus 1/S is plotted in Figure 12a. A straight line is obtained with a slope of Ks/μmax and a 1/μ intercept on the vertical axis. The values of μmax and Ks can be calculated using the plot.

According to the previous experiment, the μ values of the different initial concentrations were calculated as μ-S curves. The growth rate ratio of strain 2-T increases with the concentration of SMX (Figure 12b), thereby increasing the driving force of microbial degradation due to the increased SMX concentration.

As per the fitting results of the Monod equation, 1/μ has a good linear relationship with 1/S. Growth of the strain during the SMX degradation by strain 2-T conforms to the Monod equation (R2=0.989) through the vertical axis. The intercept and slope of the Monod equation show that Ks is 63.364 mg/L and μmax is 0.285 min-1. The Monod kinetic equation for SMX degradation by strain 2-T is Eq. (4):

μ = 0.285�

��.����� (4)

In summary, the Monod model better simulates the SMX degradation process of strain 2-T. The degradation rate of SMX in the degradation process is related to the substrate concentration and the cell density of the strain. The concentration of SMX is affected by the cell density and degradation time of the strain. Therefore, there is a dynamic in the degradation of SMX and the strain itself. Balance. In the 1960s and 1970s, Lawrence confirmed that the Monod equation is fully applicable to sewage biological treatment studies. The activated sludge purification process is divided into the initial adsorption removal and microbial metabolism. They have shown that the reduction of sulfonamide concentrations is due to biodegradation by bacteria rather than simple biosorption or accumulation. According to the results of kinetics analysis, it is inferred that SMX is mainly decomposed and utilized by a series of biochemical reactions as a source of carbon and nitrogen for self-growth, but further analysis of degradation products is needed to clarify the degradation pathway and molecular mechanism of SMX by strains.

Figure 12. (a) Fitting of Monod Kinetic Equation for Degrading Bacterium 2-T Degrading SMX (b) μ-S

diagram of degrading bacteria 2-T degrading SMX 4. Conclusions

In this study, the analysis of sample determination showed that: a) SMX was the substance with the highest pollution level at the site. SMX has become a ubiquitous refractory pollutant due to its wide application, which highlights its potential risks in the ecological environment and human health. As such, this hazard must be removed from the wastewater. Due to the low abundance of microorganisms, the low bioavailability of SMX and the residence time of sewage within the biological treatment units, SMX treatment by sewage treatment plants is not satisfactory. b) Cyclic co-metabolic gradient concentration enrichment and domestication method was used to enrich and obtain MX-3, which had the ability to degrade SMX efficiently. In the process of domestication, bacteria constantly adjust their community structure to adapt to the living environment. c) Seven strains of bacteria with SMX as the sole carbon source were isolated from MX-3. According to the results of growth status and degradation ability of culture, strain 2-T was determined as the experimental degradation bacteria in this study. The strain 2-T was identified by 16S rDNA as Pseduochrobactrum sp. d) According to the experiments of different influencing factors, the optimum degradation conditions of 2-T were identified as temperature 25 C, pH=7.0, higher strain biomass and lower initial concentration of SMX. Under the optimum conditions, the degradation rate of SMX was 92%, and the effect of pH on 2-T degradation of SMX was the most obvious. e) According to the thermodynamic fitting results of 2-T degradation of SMX at different temperatures, the Ea of 2-T degradation of SMX was lower (Ea=44.89 kJ/mol). Therefore, the degradation reaction of SMX by the degrading bacteria 2-T in this study is easy to occur. From an economic point of view, it

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can be reused to sewage treatment plants to remedy the pollution caused by sulfamethoxazole. f) In the experiment of SMX degradation by 2-T, the growth of the strain in the process of SMX degradation by 2-T conformed to the Monod equation (R2=0.989), which proved that the degradation bacteria could be a highly efficient degradation bacteria with SMX as the sole carbon source. The Monod kinetic equation of SMX

degradation by degrading bacteria 2-T was as follows: μ = 0.285�

��.����� . In order to promote the application

of microbial SMX degradation, and to improve the safety of water resources reuse in the future. Future researches should focus on the biodegradability and toxicity of SMX degradation intermediates because some intermediates may be more stable and more toxic than SMX. Acknowledgment

This research was supported by the National Natural Science Foundation of China (51378287), the Program for Distribution characteristics and transmission mechanism of antibiotic resistance genes in the process of reclaimed water infiltration into groundwater. This work was supported by Integration and Demonstration of Urban Water Pollution Process Control and Comprehensive Improvement of Water Environment of National Major Science and Technology Program-Water Body Pollution Control and Remediation (2017ZX07202002-06).

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