Optimized Operational Parameters for the Biodegradation of the

β-lactam Antibiotics and Intermediates in Treating High-strength

Organic Wastewater Treatment

Qian-yi Wang

, Zhao-Bo Chen

, Dong-Xue Hu

, Yu-Bo Cui

, Li-qiang Yu

Li-Xue Liu

, Hui Ge

, Xue-Jun Zou

and Ze-Hao Li

College of Environment and Resources, Dalian Minzu University, 18 Liaohe West Road, Dalian 116600, China

616476984@qq.com, gehui@dlnu.edu.cn, zouxuejun@dlnu.edu.cn, 1293420933@qq.com

Keywords: Biodegradation, High-Strength Organic Wastewater, β-lactam Antibiotics, Target Contaminants.

Abstract: Many attentions have been devoted to the fate of antibiotics such as β-antibiotics in the soil and water,

especially in the developing countries. The problems of their fate in the environment have to be clearly

identified to prevent any environmental pollution. Therefore, it’s necessary to research the biodegradation

law of β-lactam antibiotics and intermediates if the antibiotics entered the sewage treatment plants (SATS),

and to estimate the removals of SATs. Ceftriaxone sodium, cefoperazone sodium, ampicillin sodium,

amoxicillin, 6-APA and 7-ACA, typical β-antibiotics, were chosen as the analytes. Typical biologic

treatment active sludges, which has been widely used in the developing countries, was transformed in

experimental at laboratory-sized in this study to measure the β-lactam antibiotics and intermediates’

removal efficiency during the biologic treatment on different carbon sources, temperature, dissolved oxygen

and pH value conditions. When using glucose as plus carbon source, the effect of β-lactam antibiotics and

intermediate was best, and the optimum mass ratio of glucose and analytes was 1:8. Considering the

treatment effect and the processing cost factors, then gained a relatively appropriate condition: controlling

temperature at the range from 25 to 35 ℃, pH value in neutral and dissolved oxygen at 2.5 mg/L.

1 INTRODUCTION

In recent years, there are many reports confirmed

that different concentration level of drugs was found

in surface water, groundwater, drinking water,

sludge, soil, biological body and the others kinds of

environmental medium (Magda 2011, Halling-

Sùren, 1998). And its influences to environment and

human health has caused the international social

attention. Antibiotics was harmless to human cells,

however, while the antibiotics abusing in the

developing countries is common, which will produce

terrible consequences, that build the pathogenic

bacteria resistant ability to antibiotics. At the same

https://orcid.org/0000-0001-7719-1878

https://orcid.org/0000-0003-1878-6139

https://orcid.org/0000-0003-4247-0302

https://orcid.org/0000-0003-3563-1956

https://orcid.org/0000-0003-4281-4259

https://orcid.org/0000-0003-0046-7631

time, the antibiotic chemical structure is so

complicated with strong bacteriostatic and

sterilization effect, belongs to the difficult

biodegradable material. Beta-lactam

antibioticcontain typical beta lactam ring structure,

occupy an important position in antibiotic use. Beta-

lactam antibiotic’s mechanism of action is to inhibit

synthesis of cell wall, it has bactericidal broad-

spectrum and very good antibacterial, a good

curative effect and hypoallergenic with low adverse

(Buynak 2016, Calamari 2003, Bel 2009).

So, antibiotics once polluted the environment,

will leave a long-term serious influence on the

environmental micro-ecological. Antibiotic

substances enter municipal sewage and sewage

treatment plants (STPs). If they are not eliminated

during sewage treatment, they are emitted into

surface water and may reach drinking water (Edward

Turos, 2007). As a consequence, the occurrence and

fate of pharmaceutically active compounds in the

natural environment has been recognized as one of

Wang, Q., Chen, Z., Hu, D., Cui, Y., Yu, L., Liu, L., Ge, H., Zou, X. and Li, Z.

Optimized Operational Parameters for the Biodegradation of the -lactam Antibiotics and Intermediates in Treating High-strength Organic Wastewater Treatment.

DOI: 10.5220/0011388700003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 1213-1220

ISBN: 978-989-758-595-1

1213

the emerging issues in environmental chemistry.

Knowledge of pharmaceuticals in the environment is

little.

Emad researched that UV/ZnO photocatalysis

can be used for amoxicillin, ampicillin and

cloxacillin degradation in aqueous solution (James

2010). Besides, they also examined degradation of

the antibiotics amoxicillin, ampicillin and cloxacillin

in aqueous solution by the photo-Fenton process.

Using the application of sonolysis for the

degradation of fluoroquinolone antibiotic

ciprofloxacin (Kümmerer 2013), the pH effect on

the degradation rate is studied and changes in

biodegradability.

Besides chemical and photochemical degradation

(Kummerer 2003), the biodegradability of antibiotic

released into the environment is an important aspect

of their partial or complete decomposition by

microorganisms (Chaudhuri 2009), which is the

major progress in the sewage treatment plants. Thus,

it is important to study on the biodegradation of

antibiotic substances in the active sludges. As

antibiotics are considered to have an adverse effect

on microorganisms (Brosillon 2013).

Using the Closed Bottle test (CBT) (Emad 2010),

the biodegradation of some clinically important

antibiotic drugs was investigated as the first step of

an environmental risk assessment. The CBT was

performed according to test guidelines in the dark

room temperature (20±1℃) was described elsewhere

in detail. The standard test period of the CBT is 28

days. The CBT conditions are low bacterial density

and low concentration of organic carbon i.e. test

compound. As a result, the antibiotics tested were

not biodegraded in the CBT. The results indicate that

the various antibiotics were active against different

groups of bacteria present in wastewater.

Hybrid processes (Reddy 2006) involving a

physical-chemical and pre-treatment like

photocatalysis coupled to a biological treatment

have been considered for antibiotics’ removal. Using

photocatalysis as pre-treatment as prior to a

biological treatment, an irradiation time of 2h ensure

a significant residual organic content for the

biological treatment. A decrease of the residual

amount of antibiotics contained in the irradiated

solution s was recorded, which can be related to an

inherent biodegradation since these residual

concentrations were below their inhibitory

thresholds, 18 and 9 mg/L for TC and TYL.

So, the biodegradation of the beta-lactam

antibiotics in the environment, especially in the

SATs, is important to the antibitics problems. This

study, therefore, investigated the biodegradability of

some typical beta-lactam antibiotics in the actives on

lab-scales: ceftriaxone sodium, cefoperazone

sodium, ampicillin sodium, amoxicillin, 6-APA and

7-ACA to observe the existing conventional biologic

treatments ability to remove the antibiotics.

2 EXPERIMENTAL

2.1 Materials

This context selected four beta lactam antibiotics

(ceftriaxone sodium, cefoperazone sodium,

ampicillin sodium, amoxicillin) and two

intermediates (6 - APA ,7 - ACA) as the research

object, with a purity of > 99 % determined by HPLC

according to the Chinese Pharmacopoeia 2005 (ChP

2005). But methanol (MeoH), formic acid, disodium

hydrogen phosphate (Na

HPO

), potassium

dihydrogen phosphate (KH

) (purchased from

Tianjin Chemical Reagent Co., China) were of

analytical-reagent grade. The water used was

domestic sewage. They were filtered by 0.45 µm

filter membrane before experiment. Pencil core

aerator was used to control the DO of the system,

and electric-heated thermostatic water bath was used

to control the temperature.

Figure 1: Schematic view of the active sludges. (a)

thermostatic bath; (b) air pump; (c) tank; (d) pencil core

aerator; (e) thermostatic devices; (f) pH, DO and detector;

(g) electrode.

2.2 Characterization

This context selected four beta lactam antibiotics

(ceftriaxone sodium, cefoperazone sodium,

ampicillin sodium, amoxicillin) and two

intermediates (6 - APA 7 - ACA) as the research

object. The experimentally characterization for the

research object are summarized in Table 1. An

Agilent Technologies 1200 HPLC system with an

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

1214

UV detector and an Eclipse XDB-C18 column (150

mm × 4.6 mm×5 µm) were employed to study the

degradation process (Yahiat 2011).

2.3 Degradation Experiments

Activated sludge was collected from municipal

wastewater treatment. The basic culture medium

contains the following mineral supplementation:

HPO

(34g/L), CaCl

(18g/L), MgSO

(25g/L).

Firstly, biodegradation in different carbon source

(regarded as co-metabolism substance) including

glucose, sugar, starch and was investigated at 298 K

and almost neutral pH with the initial respective

concentration of 80 mg/L, and the check test was not

added in the culture medium. Selected glucose as the

carbon source used in co-metabolism action and was

investigated at 298K and almost neutral pH and

maintianed DO at 2.5 mg/L, tested biodegradation in

series of glucose concentration/respective target

contaminants concentration (1:1, 1:3, 1:5, 1:8, 1:10

and 1:15) system.

In the glucose concentration/respective target

concentration contaminants (1:8) system, the

influence of the pH on the degradation of target

contaminants was determined at 5, 7, 9 and with the

initial concentration of 400 mg/L, with glucose (50

mg/L) and primary nitrogen source ammonium (32.5

mg/L NH

Cl) at 298 K and DO at 2.5 mg/L, each

condition was aerated for 20h, deposited for 1h per

day and run for 20 days. The temperature effect on

the degradation of target contaminants was

determined at 288, 298 and 308K with the initial

concentration of 400 mg/L under pH around 7 and

DO around 2.5 mg/L, and also each condition was

aerated for 20h, deposited for 1h per day and run for

20 days. Finally the DO effect on the degradation

was determined at 1, 2.5 and 4 mg/L with initial

concentration of 400 mg/L.

Table 1: Overview of study β-lactam antibiotics.

Optimized Operational Parameters for the Biodegradation of the -lactam Antibiotics and Intermediates in Treating High-strength Organic

Wastewater Treatment

1215

3 RESULT AND DISCUSSION

3.1 Degradation of Target

Contaminants in Different Carbon

Sources System

Fig.2 demonstrates the temporal evolution of the

concentration during the temporal evolution of in

different carbon sources solvent systems. It shows

that, on the basis of the degradation in the out-added

carbon sources, the introduction of different carbon

sources increased the degradation rate of the target

contaminants, the possible reason is that the out-

added carbon sources were oxidative decomposed

by microorganisms, which used the release of large

amounts of energy in decomposition, for the

synthesis of their own cellular material, so that the

microorganism bloomed, and also letted key enzyme

of the β-lactam antibiotics and intermediates’s

decomposition keep active, and it was conducive to

the further degradation (Heberer 2002). And this

process is called Co-Metabolism (Smital 2004).

Among the three out-added carbon sources, glucose

has the monosaccharide structure, and sucrose and

starch has disaccharides and polysaccharides

structure, so their molecular structure is more

complex compared with glucose, the reactions

required more kinds of enzymes involved, not as

directly and quickly as glucose. Obviously all the

target contaminants had the best removal efficiency

in carbon sources system of glucose.

Figure 2. Different target contaminants’ removal

efficiency in different carbon system.

3.2 Degradation of Target

Contaminants under Different Cgc:

Ctc (Gc: Glucose Concentration,

Tc: Respective Target

Contaminants Concentration)

Fig.3 presents removal of target contaminants under

different Cgc: Ctc. Under the constant target

contaminants concentration condition, between 1:15

and 1:8 the co-substance increased with the increase

of removal efficiency of all the target contaminants,

and it had a maximum value of all with Cgc at 1/8 of

Ctc. When Cgc: Ctc was above 1:8, the removal

efficiency decreased with the increase of the co-

substance glucose concentration, excessive growth

of substance would appear. Therefore, refractory

organic matters were disadvantageous for contesting

enzymes, resulting in the decreased removal

efficiency.

Figure 3: Removal of target contaminants under different

Cgc:Ctc.

3.3 pH-Dependent Degradation in the

Glucose System

The concentration of target contaminants was

monitored during the degradation under different pH

condition ranging from 5 to 9, as shown in Fig.3. It

shows the process of the active sludges cultivated

under different pH with time series. And the average

removal efficiency and effluent concentration of all

target contaminants was shown in Fig.4. From Fig.4

and Fig.5, it can see that under acidic conditions,

degradation is relatively low. And with pH

increased, ceftriaxone sodium and ampicillin

sodium’s degradation efficiency increased with a

little margin compared with pH=7, the removal

efficiency of 6-APA almost constant, but removal

efficiency of cefoperazone sodium, amoxicillin and

7-APA decreased. Considered the appropriate pH of

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

1216

water treatment plants’ flows is ranging from 6.5 to

8.5. Fig.3 or Fig.4 showed that when under pH was

around 7 (neuter), removal efficiency of all the

target contaminants was better considered the

relative conditions. Adjusting the pH around 7 for

degradation will be helpful to improve the removal

efficiency.

Figure 4: Different contaminants’ average removal efficiency and average effluent concentration under different pH.

Figure 5: The effluent concentrations and removal efficiency of the different β-lactam antibiotics and intermediates during

the 20 days. (a) 7-APA; (b) 6-APA; (c) ceftriaxone sodium; (d) cefoperazone sodium; (e) ampicillin sodium; (f)

amoxicillin.

Optimized Operational Parameters for the Biodegradation of the -lactam Antibiotics and Intermediates in Treating High-strength Organic

Wastewater Treatment

1217

3.4 Temperature Effect on the

Degradation of β-lactam Antibiotics

and Intermediates

Temperature has a remarkable effect on reaction

according to the biodegradation theory. To

determine the effect of temperature on target

contaminants degradation, the average effluent

concentrations and removal efficiency was

monitored at 288, 298 and 308K, as shown in Fig.6.

The curve lines and histograms in Fig.5 shows the

consequent changes of effluent concentrations and

removal efficiency in sludge domestication under

different temperature. Generally, temperature had a

strong effect on degradation, especially under 288K

and 298K. It is found that the increase in

temperature dramatically accelerates the degradation

efficiency, and participated in aerobic

biodegradation process microorganism are classified

to mesophile microorganism, its optimum growth

temperature ranges from 293 to 310K. It also shows

that when under 298k or under 308k, the active

sludges has similar effluent concentrations and

removal efficiency. Considered the energy-

consumed, temperature should be controlled under

298K.

Figure 6: Temperature effect on the effluent concentrations and removal efficiency of the different β-lactam antibiotics and

intermediates during the 20 days. (a) 6-APA; (b) 7-APA; (c) amoxicillin; (d) ampicillin Sodium; (e) cefoperazone sodium;

(f) ceftriaxone sodium.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

1218

3.5 DO Effect on the Degradation of

β-lactam Antibiotics and

Intermediates

Dissolved oxygen is an important element

affecting microbial metabolism. The

microorganisms involved in wastewater treatment

are mainly based in aerobic respiration. The DO in

the reactor must be guaranteed in order to maintain

normal aerobic respiration and physiological

metabolism of microorganisms in the active sludges.

So the low DO concentration has a seriously

negative effects, even worse, it decrease the

performance of the treatment reactor. While the high

DO contents would not only increase the operation

costs and reduce the feasibility of the treatment

process, but also increase decomposition of

pollutants, causing a lack of nutrients for

microorganisms, loosely structures of active sludges

and poor performance. Average removal efficiency

and effluent concentration of the target contaminants

during the 20 days’ active sludges domestication are

show in Fig.6. It shows that with the DO increased,

the degradation efficiency increased. There have a

little improvement between DO under 2.5 mg/L and

under 4.0 mg/L, while removal efficiency of 7-APA

decreased when DO contents increased. So

considered the more operation costs under a higher

DO contents, controlling the DO contents under 2.5

mg/L is more rational.

Figure 7: Different contaminants’ average removal

efficiency and average effluent concentration under

different DO (points represented concentration, columns

represented removal efficiency).

4 CONCLUSIONS

The degradation of the target contaminants in

different systems was determined using HPLC. Out-

added carbon sources can accelerate the degradation

and out-added-glucose system has higher removal

efficiency than sucrose and starch as the carbon

sources. And when the initial glucose concentration

the initial target contaminants concentration was

under 1:8, it has the best removal efficiency.

Investigating the effects of the pH on the

degradation of target contaminants in the glucose

system revealed that the degradation of 6-APA,

amoxicillin and ceftriaxone sodium accelerated

generally with the increasing pH, but which was a

little improvement compared with under pH=7. So

when pH around 7, all the target contaminants had a

rational removal efficiency.

The effect of temperature on the degradation of

target contaminants is strong. On the basis of the

experimental results, when temperature increased

from 288K to 298K, the degradation accelerated a

lot. While under 298k or 308K, the degradation was

similar. So main active microorganism were

mesophile.

DO also has a strong effect on the degradation.

Experiments results shows that with the DO contents

increased, the degradation increased. But there was a

small improvement between 2.5 mg/L and 4 mg/L.

Considered the operation costing, keep DO under

2.5 mg/L is more practical.

REFERENCES

Bel, E. D. Dewulf, J. De, B. (2009). Influence of pH on

the sonolysis of ciprofloxacin: Biodegradability,

ecotoxicity and antibiotic activity of its degradation

products. Chemosp. 77, 291–295.

Brosillon, S. (2013). Guideline for Testing of Chemicals

(301D). Closed Bottle Test. 54, 134-156. Buynak, D.

(2016). Understanding the longevity of the β-lactam

antibiotics and of antibiotic/β-lactamase inhibitor

combinations. Biochem Pharmacol. 71(30), 930–940.

Calamari,. D. Zuccato, E. Castiglioni,.S. Bagnati,.R.

Fanelli, R. (2003). Strategic survey of therapeutic

drugs in the rivers Po and Lambro in northern Italy.

Environ. Sci. Technol. 37, 1241–1248.

Chaudhuri, M. (2019). Degradation of the antibiotics

amoxicillin, ampicillin and cloxacillin in aqueous

solution by the photo-Fenton process. J Hazar Mater.

30, 1476–1481.

Edward Turos, G. Greenhalgh, K. (2007). Penicillin-

bound polyacrylate nanoparticle: Restoring the activity

of β-lactam antibiotics against MSRA. Bioorganic &

Medicinal Chemistry Letters. 17(12), 3468-3472.

Emad, S. (2010). Degradation of amoxicillin, ampicillin

and cloxacillin antibiotics in aqueous solution by the

UV/ZnO photocatalytic process. J Hazar Mater. 12,

445–449.

Halling-Sùren sen, B. NorsNielsen, S. Lanzky, P.F.

Ingerslev, Holten, F. Utzhùft, L. Jùrgensen, H.C.

(1998). Occurrence fate and effects of pharmaceutical

substances in the environment - a review. Chemosp.

36, 357-393.

Optimized Operational Parameters for the Biodegradation of the -lactam Antibiotics and Intermediates in Treating High-strength Organic

Wastewater Treatment

1219

Heberer, T. (2002). Occurrence, fate, and removal of

pharmaceutical residues in the aquatic environment: a

review of recent research data. Toxicol. Lett. 131, 5–

17. James, H. (2010). Fate of amoxicillin in mixed-

culture bioreactors and its effects on microbial growth

and resistance to silver ions. Sci. Technol. 44, 1827-

1832.

Kummerer, K. Henninger, A. (2003). Promoting resistance

by the emission of antibiotics from hospitals and

households into effluent, Clin. Microbiol. Infect. 9,

1203–1214.

Kümmerer, K. Ahmad, A. Sundermann, V. (2103).

Biodegradability of some antibiotics, elimination of

the genotoxicity and aection of wastewater bacteria in

a simple test. Chemosp. 11(14), 15-23.

Magda, A. Mona, A. Pharma, J. Ramadan, A. (2011).

Validation of an HPLC-UV method for the

determination of ceftriaxone sodium residues on

stainless steel surface of pharmaceutical

manufacturing equipments. Bio Analysis. 55, 247-252.

Reddy, S.K. (2006). Effects of Drugs: The International

Encyclopedia of adverse. Fifteenth Edition. 7, 478–

502.

Smital, T. Luckenbach, T. Sauerborn, R. (2004). Emerging

contaminants-pesticides, PPCPs, microbial

degradation products and natural substances as

inhibitors of multixenobiotic defense in aquatic

organisms. Mutation Res. 552 (1-2), 101-117.

Yahiat, S. Fourcade, F. Amrane, A. (2011). Removal of

antibiotics by an integrated process coupling

photocatalysis and biological treatment -Case of

tetracycline and tylosin. International Biodeterioration

& Biodegradation. 65, 997-1003.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

1220