Microbial degradation and transformation of the

antibiotic sulfamethoxazole under aerobic and anaerobic

conditions

vorgelegt von

M. Sc.

Wei-Ying Ouyang

Promotionsausschuss:

Vorsitzender: Prof. Dr. Juri Rappsilber

Gutachter: Prof. Dr. Lorenz Adrian

Gutachter: Prof. Dr. Peter Neubauer

Gutachter: PD Dr. Hans-Hermann Richnow

Tag der wissenschaftlichen Aussprache: 04. Juni 2021

“Microbial degradation and transformation of the antibiotic sulfamethoxazole under

aerobic and anaerobic conditions”

This research was performed at the Helmholtz Centre for Environmental Research-UFZ,

Leipzig, Germany between November, 2015 and December, 2019 under the supervision of

Prof. Dr. Lorenz Adrian and PD Dr. Hans-Hermann Richnow. I herewith declare that the

results of this dissertation were my own research and I also certify that all sentences were

written by myself.

Signature Date

Acknowledgement

I

group of Prof. Dr. Lorenz Adrian at the Department of Isotope Biogeochemistry, Helmholtz

Centre for Environmental Research Leipzig (UFZ). The research project was funded by the

Chinese Scholarship Council, and was also supported by Institute of Urban Environment,

Chinese Academy of Sciences.

my dissertation. Thanks to Prof. Dr. Peter Neubauer for the review of my dissertation and Prof.

Dr. Juri Rappsilber to be the chairperson of the dissertation committee.

I want to thank my colleagues Dr. Rohit Budhraja, Dr. Chang Ding and Dr. Katja Seidel for

the working time we spent together, sharing beauty of science and life. Thank Dr. Camelia

Algora, Dr. Alba Trueba-Santiso, Dr. Clara Reino-Sanchez and other group members for their

creative discussion during the relaxing lunch time. Thanks to Benjamin Scheer together with

Danny Frauenstein and Felicitas Ehme for their technical support in the lab work, and

organization of many unforgettable group activities. I thank my students Jimmy Köpke,

Susiddharthak Chakraborty and Joana Kühnert for their productive work and interesting

feedback on my supervision.

Abstract

II

functions. The broad occurrence of antibiotic residues contributes to the development and

propagation of antibiotic resistance genes, causing a significant reduction of antibiotics

available for treatments of infectious disease. Sulfamethoxazole (SMX) is one of the most

frequently detected antibiotics in the environment, especially enriched in manure, sludge, and

farm-impacted soil. Microbial degradation is the major sink of SMX in both natural and

engineered systems. Aerobic SMX-degraders have been enriched and isolated, whereas

identification of in-situ degrading populations is still challenging. Besides, field studies

revealed the contribution of anaerobic processes to SMX mitigation, while knowledge about

the anaerobic SMX-transforming bacteria is limited and no pure strain was described until now.

In addition, a robust assessment method is needed to evaluate attenuation of SMX in complex

matrices. In this study, we aimed to identify in-situ aerobic SMX-degraders, characterize

anaerobic SMX-transforming cultures, and develop assessment techniques to evaluate removal

of SMX in the environment.

We investigated aerobic SMX-degrading bacteria in soil microcosms by culture-independent

Intrasporangiaceae, Nocardioidaceae, and the order Solirubrobacterales, which was

consistent with the results of DNA-SIP. The 13C abundance of 60 to 80% in several

taxonomically relevant proteins indicated that Intrasporangiaceae directly acquired carbon

from 13C6-labeled SMX, acting as primary SMX-degraders in the soil.

Furthermore, we examined how microbial anaerobic transformation contributes to removal of

SMX. We enriched SMX-transforming mixed cultures from sediment of a constructed wetland

and digester sludge from a wastewater treatment plant. Transformation of SMX was observed

in both sulfate-reducing and methanogenic cultures, whereas nitrate-reducing cultures showed

no SMX transformation. In sulfate-reducing cultures, up to 90% of an initial SMX

concentration of 100-250 µM was removed within 6 weeks of incubation. Our results

Abstract

III

vulgaris Hildenborough for its capacity to transform SMX and observed the same

transformation products at similar rates. We then investigated the physiology of the mixed

SMX-transforming cultures, and found that the mixed cultures did not grow on SMX.

Transformation of SMX by D. vulgaris Hildenborough was identified as a cometabolic process.

Addition of electron donor can promote transformation of SMX by D. vulgaris Hildenborough.

Intact-cell activity test revealed that anaerobic transformation of SMX was an enzymatic

reduction process, and SMX did not induce expression of specific transformation related

proteins. Besides, in the intact-cell activity test, exponential phase cells showed higher activity

towards SMX transformation compared with stationary phase cells.

Significant carbon and hydrogen isotope fractionations (𝜀C=-5.8±0.7‰, 𝜀H=-33.8±9.2‰)

the fact further verifies the robustness of CSIA to differentiate reaction mechanisms. Good

correlation was observed between change of carbon isotopes and change of hydrogen isotopes.

The distinctive lambda value (Λbulk

SMX can be applied in field study to assess attenuation of SMX.

In sum, our work revealed that Intrasporangiaceae as non-cultivable bacteria played key roles

in aerobic degradation of SMX in pig-farm impacted soil, and sulfate-reducing bacteria

especially D. vulgaris transformed and detoxified SMX under anaerobic conditions.

Processes for removal of SMX can be differentiated by the robust CSIA method. This study

extended the understanding of microbial degradation and transformation of SMX under aerobic