Anaerobic degradation of anionic surfactants by denitrifying bacteria Ana Paulo Thesis committee Promotor Prof. Dr A.J.M. Stams Personal chair at the Laboratory of Microbiology Wageningen University Co-promotor Prof. Dr P. A. García-Encina Professor at the Department of Chemical Engineering and Environmental Technology University of Valladolid, Spain Dr C.M. Plugge Assistant Professor at the Laboratory of Microbiology Wageningen University Other members Prof. Dr B. Philipp, University of Münster, Germany Dr F.P. van der Zee, Veolia Water Technologies / Biothane Systems International B.V., Delft, The Netherlands Prof. Dr H. H. M. Rijnaarts, Wageningen University Prof. Dr M. M. Alves, University of Minho, Portugal This research was conducted under the auspices of the Graduate School for Socio-Economic and Natural Sciences of the Environment (SENSE) Anaerobic degradation of anionic surfactants by denitrifying bacteria Ana Paulo Thesis submitted in fulfillment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr M. J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Wednesday 15th of October 2014 at 11 a.m. in the Aula. Ana Paulo Anaerobic degradation of anionic surfactants by denitrifying bacteria, 148 pages. PhD thesis, Wageningen University, Wageningen, NL (2014) With references, with summaries in Dutch and English ISBN 978-94-6257-124-2 TABLE OF CONTENTS SUMMARY 1 CHAPTER I GENERAL INTRODUCTION 3 CHAPTER II ANAEROBIC DEGRADATION OF SODIUM DODECYL SULFATE (SDS) BY DENITRIFYING BACTERIA 25 CHAPTER III BACTERIAL DIVERSITY IN ANOXIC AND AEROBIC ENRICHMENTS DEGRADING SODIUM LAURYL ETHER SULFATE (SLES) 43 CHAPTER IV DEGRADATION OF SODIUM LAURYL ETHER SULFATE (SLES) BY FACULTATIVE ANAEROBIC BACTERIA 65 CHAPTER V BEHAVIOR OF SURFACTANT-DEGRADING BACTERIA AT INCREASING ANIONIC SURFACTANT CONCENTRATIONS IN DENITRIFYING CONDITIONS 77 CHAPTER VI DOMAINOME ANALYSIS OF PSEUDOMONAS NITROREDUCENS DSM 14399T 99 CHAPTER VII GENERAL DISCUSSION 137 LIST OF REFERENCES 145 SAMENVATTING 161 ABOUT THE AUTHOR 163 LIST OF PUBLICATIONS 164 ACKNOWLEDGMENTS 165 OVERVIEW OF TRAINING ACTIVITIES SUMMARY Surfactants are produced and used in the formulation of many different commercial products. After use, these compounds end up in wastewater treatment plants (WWTPs) or in the environment. Although many surfactants can be degraded in aerobic conditions, anaerobic conditions are also common in Nature and in WWTPs. For achieving nutrients removal from wastewater, biological removal of nitrogen and phosphorus can be performed in a WWTP using the anaerobic-anoxic-aerobic (A2/O) concept. Using the A2/O process sequence, surfactants can be degraded anaerobically before reaching the aerobic compartment. In the anoxic compartment, facultative anaerobic bacteria can degrade surfactants by using nitrate/nitrite as electron acceptor. However, not much is known about surfactant-degrading denitrifying bacteria. In this thesis, Pseudomonas stutzeri strain SN1 and Pseudomonas nitroreducens strain SN2 were isolated from activated sludge of a WWTP with the A2/O process, using the anionic surfactant sodium dodecyl sulfate (SDS) as sole carbon and energy source. Both strains were able to completely degrade SDS coupled to nitrate reduction to dinitrogen gas (Chapter II). In the A2/O process, the diversity of bacterial communities involved in the degradation of surfactants may differ between anoxic and oxic compartments, where two different electron acceptors are involved. Surfactants can directly affect the biological activity of microorganisms present in WWTPs and disturb the treatment efficiency. In this way, increased concentrations of surfactants may give rise to a different bacterial diversity selection in anaerobic, anoxic and oxic conditions. The degradation of the anionic surfactant sodium lauryl ether sulfate (SLES) in aerobic conditions is known, but not in denitrifying conditions. In this thesis, the bacterial diversity of enrichments cultures able to degrade different concentrations of SLES in anoxic and aerobic conditions was determined. Aeromonas hydrophila strain S7, Pseudomonas stutzeri strain S8 and Pseudomonas nitroreducens strain S11 were isolated from anoxic enrichments. Comamonas testosteroni strain S13 and Acinetobacter sp. S15 were isolated from aerobic enrichments (Chapter III). SLES initial degradation steps by pure bacterial cultures were previously investigated, but much is still unknown about how the cleavage of ether bonds from chemical compounds is catalyzed by bacterial enzymes. Aeromonas hydrophila strain S7, Pseudomonas stutzeri strain S8 and Pseudomonas nitroreducens strain S11 are able to use SLES in anoxic conditions coupled to nitrate reduction (Chapter III). SLES degradation in anoxic conditions was compared between the three strains. P. nitroreducens strain S11 was found to be the best SLES degrader in anoxic conditions and also to be an excellent aerobic SLES degrader (Chapter IV). Sulfatases and ether cleaving enzymes were probably used by P. nitroreducens strain S11 in both conditions, although differences between SLES 1 degradation in aerobic and anoxic conditions indicated that ether cleavage and following SLES complete degradation is faster under aerobic conditions. Although surfactants can be toxic to microorganisms, surfactant-degrading bacteria are known to be resistant to high surfactants concentration, in aerobic conditions. This was not previously investigated using surfactant-degrading denitrifying bacteria. Surfactant- resistant bacteria, with the ability to couple surfactant degradation to nitrate reduction, can be very useful for degrading the surfactants arriving to the anoxic compartments of a WWTP at high concentration. In this thesis, high concentrations of SDS and SLES were used to investigate the effect of these on SDS/SLES-degrading bacteria (P. stutzeri strain SN1, P. nitroreducens strain SN2, P. stutzeri strain S8 and P. nitroreducens strain S11), under anoxic conditions (Chapter V). P. stutzeri strain SN1 was inhibited by increasing SDS and SLES concentrations, after degrading a certain amount of the surfactants. Overall, P. nitroreducens strains showed to be more resistant to high surfactant concentrations compared to P. stutzeri strains. Nevertheless, high concentrations of SDS and SLES did not inhibit growth and nitrate reduction ability of any of the tested Pseudomonas sp.. Protein domains represent the evolutionary conserved autonomously folding functional building blocks of the proteins. Prediction of protein domains from genomes can be used for species classification and validation of known physiological abilities. P. nitroreducens are facultative anaerobic bacteria from the P. aeruginosa group, which can degrade complex compounds. P. nitroreducens DSM 14399T shares with P. nitroreducens strain SN2 the ability for SDS degradation in anoxic conditions. For increasing the insight into P. nitroreducens DSM 14399T phylogenetic classification and physiological properties (e.g. SDS degradation) its genome was sequenced, annotated and compared to other Pseudomonas spp. genomes. This was performed by comparing functional profiles, based on protein domains presence or absence, with physiological data (Chapter VI). Functional profile comparison confirmed P. nitroreducens classification. Protein domain analysis and genes annotation validated SDS degradation by P. nitroreducens DSM 14399T. This study showed that protein domains prediction and functional profiles comparison can be used for studying and comparing different Pseudomonas species at the physiological level. 2 CHAPTER I GENERAL INTRODUCTION 3 Surfactants can be found in industrial and domestic wastewater and may negatively affect biological treatment processes. Wastewater treatment plants (WWTPs) have often an anaerobic-anoxic-oxic (A2/O) regime, used for organic matter and nutrients removal from the wastewater. Therefore, it is important to get insight into the biodegradation and toxicity of surfactants in the anaerobic and anoxic compartments. This thesis describes research on the degradation of anionic surfactants in the anoxic compartment of WWTPs. 1. SURFACTANTS Surfactants constitute a diverse group of chemicals designed mainly for cleaning and solubilization purposes. These compounds can be used in personal care products and pesticide formulations, among other applications (Lara-Martin et al. 2008). The name surfactant results from the shortening of the term surface active agent due to the ability of these compounds to change surface properties (Holmberg et al. 2002). A surfactant is characterized by its tendency to adsorb to surfaces and interfaces (Holmberg et al. 2002). Soaps are the first known and oldest surfactants used for cleaning purposes. These are composed of sodium or potassium salts of fatty acids produced from the hydrolysis of fats in a chemical process called saponification (Stalmans et al. 2007) (Figure 1). Figure 1 Saponification reaction where fats are hydrolyzed to produce soap (sodium salt of fatty acid) and glycerol. The first synthetic soap was made in Germany in 1916, due to shortage of natural materials. After this moment, synthetic soaps commonly referred to as detergents, have undergone many improvements. Detergents and soaps are used for removing dirt particles, and suspend them to be rinsed away with water (Stalmans et al. 2007). Commercial detergents are composed of 10 to 30 % of surfactants