Anaerobic degradation of anionic surfactants by denitrifying bacteria

172 Pages·2014·3.75 MB·English

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
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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.

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CHAPTER I

GENERAL INTRODUCTION

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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 (often referred to as
the active components), larger proportions of builders used for softening the water by
removing cations from it (e.g. sodium tripolyphosphate and/or other suitable chelates)
and a number of other ingredients in smaller amounts (Swisher 1987).
Surfactants started to be produced after the chemical revolution that occurred in the
detergent industry, around 1950, when more economical and more efficient chemical
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