Degradation of benzene and other aromatic hydrocarbons by anaerobic bacteria

138 Pages·2008·3.91 MB·English

Degradation of benzene and
other aromatic hydrocarbons
by anaerobic bacteria

Sander A.B. Weelink
Promotoren
Prof. dr. ir. A.J.M. Stams
Persoonlijk hoogleraar bij het Laboratorium voor Microbiologie
Wageningen Universiteit

Promotiecommissie
Dr. ir. H.H.M. Rijnaarts
TNO Bouw en Ondergrond/Deltares, Utrecht

Prof. dr. P.C. de Ruiter
Wageningen Universiteit

Prof. dr. D. Springael
Katholieke Universiteit Leuven, België

Prof. dr. H.J. Laanbroek
Nederlands Instituut voor Ecologie (NIOO-KNAW), Nieuwersluis

Dit onderzoek is uitgevoerd binnen de onderzoekschool WIMEK/SENSE
Degradation of benzene and
other aromatic hydrocarbons
by anaerobic bacteria

Sander A.B. Weelink

Proefschrift
ter verkrijging van de graad van doctor
op gezag van de rector magnificus
van Wageningen Universiteit,
Prof. dr. M.J. Kropff,
in het openbaar te verdedigen
op dinsdag 18 november 2008
des namiddags te vier uur in de Aula
Degradation of benzene and other aromatic hydrocarbons by anaerobic bacteria
Sander A.B. Weelink

PhD thesis Wageningen University, Wageningen, The Netherlands (2008)
128 pages, with summary in Dutch

ISBN 978-90-8585-239-1
Voor Pa en Ma

Abstract

Accidental spills, industrial discharges and gasoline leakage from underground storage tanks have
resulted in serious pollution of the environment with monoaromatic hydrocarbons, such as benzene,
toluene, ethylbenzene and xylene (so-called BTEX). High concentrations of BTEX have been detected
in soils, sediments and groundwater. The mobility and toxicity of the BTEX compounds are of major
concern. In situ bioremediation of BTEX by using naturally occurring microorganisms or introduced
microorganisms is a very attractive option. BTEX compounds are known to be transformed (or
degraded) by microorganisms under aerobic and anaerobic conditions. As BTEX compounds are often
present in the anaerobic zones of the environment, anaerobic bioremediation is an attractive
remediation technique. The bottleneck in the application of anaerobic techniques is the lack of
knowledge about the anaerobic biodegradation of benzene. In particular, little is known about the
bacteria involved in anaerobic benzene degradation and the anaerobic benzene degradation pathway
has still not been elucidated. The aim of the research presented in this thesis was to gain more insight
in the degradation of benzene and other aromatic hydrocarbons by anaerobic bacteria. In particular,
the physiology and phylogeny of the bacteria responsible for the degradation were studied and the
results have been presented in this thesis.
Anaerobic benzene and toluene degradation was studied with different electron acceptors in batch
experiments inoculated with material from an aquifer polluted by BTEX-containing landfill leachate
(Banisveld landfill near Boxtel, The Netherlands). Benzene was not degraded during one year of
incubation. Toluene degradation, on the other hand, was observed with nitrate, MnO and Fe(III)NTA
2
as electron acceptors. After further enrichment and several isolation attempts, a novel
betaproteobacterial bacterium, strain G5G6, was obtained in pure culture. Strain G5G6 is able to grow
with toluene as the sole electron donor and carbon source, and amorphous and soluble Fe(III)-
species, nitrate and MnO as electron acceptors. Strain G5G6 has several other interesting
2
physiological and phylogenetic characteristics, which will be subject of future research. Strain G5G6
represents a novel species in a novel genus for which we propose the name Georgfuchsia toluolica.
In general, aerobic degradation of BTEX is a faster process than anaerobic BTEX degradation.
However, for a number of reasons application of oxygen-dependent processes in the subsurface are
technically and financially often not appealing. Therefore, an alternative bioremediation strategy would
be to introduce oxygen in an alternative way, e.g. by in situ production. Chlorate reduction is a way to
produce molecular oxygen in situ under anaerobic conditions. The formation of oxygen during chlorate
reduction may result in rapid oxidation of compounds which are slowly degraded under anaerobic
conditions; an example of such a compound is benzene. Therefore, benzene degradation coupled to
the reduction of chlorate (ClO -) was studied in this thesis. With mixed material from a wastewater
3
treatment plant and soil samples obtained from a location contaminated with benzene, a benzene-
degrading chlorate-reducing stable enrichment culture was obtained. This stable enrichment consisted
of about five different bacterial species. Cross feeding involving interspecies oxygen transfer is a likely
mechanism in this enrichment. One of these species, strain BC, was obtained in pure culture.
Phylogenetic analysis showed that strain BC is a Alicycliphilus denitrificans strain. Strain BC is able to
degrade benzene in conjunction with chlorate reduction. Oxygenase genes putatively encoding the
enzymes performing the initial steps in aerobic degradation of benzene, were detected in strain BC.
This demonstrates the existence of aerobic benzene bacterial biodegradation pathways under
essentially anaerobic conditions. Thus, aerobic pathways can be employed under conditions where no
external oxygen is supplied.
The new insights into toluene degradation under anaerobic conditions and benzene degradation
coupled to chlorate reduction, as described in this thesis, can be applied for the improvement or
development of in situ bioremediation strategies for BTEX contamination.

Table of contents

Chapter 1 General introduction 1

Chapter 2 Anaerobic benzene and toluene biodegradation studies with different
electron acceptors in contaminated aquifer microcosms 27

Chapter 3 Anaerobic degradation of aromatic hydrocarbons with Fe(III), Mn(IV) or nitrate
by the Betaproteobacterium, Georgfuchsia toluolica, gen. nov., sp. nov. 41

Chapter 4 Physiological and phylogenetic characterization of a stable
benzene-degrading, chlorate-reducing microbial community 59

Chapter 5 Isolation and characterization of Alicycliphilus denitrificans strain BC, which
grows on benzene with chlorate as the electron acceptor 73

Chapter 6 Physiological characterization of two Alicycliphilus denitrificans strains 91

Chapter 7 General discussion and summary 95

Nederlandse samenvatting 106

References 110

List of publications 122

Curriculum vitae 123

Dankwoord 124

WIMEK/SENSE certificate 126