Table Of ContentMicrobial community analysis in sludge of
anaerobic wastewater treatment systems
Integrated culture-dependent and culture-independent approaches
Kees Roest
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Promotoren: Prof. Dr. W. M. de Vos
Hoogleraar Microbiologie
Wageningen Universiteit
Prof. Dr. Ir. A. J. M. Stams
Persoonlijk Hoogleraar bij het Laboratorium voor
Microbiologie, Wageningen Universiteit
Co-promotoren: Dr. A. D. L. Akkermans†
Universitair Hoofddocent, Laboratorium voor Microbiologie
Wageningen Universiteit
Dr. H. Smidt
Universitair Docent, Laboratorium voor Microbiologie
Wageningen Universiteit
Leden van de Prof. Dr. M. Alves
Promotiecommissie: Universidade do Minho, Portugal
Dr. G. Muyzer
Technische Universiteit Delft
Prof. Dr. H. J. Laanbroek
Nederlands Instituut voor Oecologisch Onderzoek,
Centrum voor Limnologie,
Nieuwersluis
Prof. Dr. Ir. J. B. van Lier
Wageningen Universiteit
Dit onderzoek is uitgevoerd binnen de onderzoekschool SENSE
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Microbial community analysis in sludge of
anaerobic wastewater treatment systems
Integrated culture-dependent and culture-independent approaches
Kees Roest
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 woensdag 12 december 2007
des namiddags te half twee in de Aula.
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Kees Roest - Microbial community analysis in sludge of anaerobic wastewater
treatment systems: Integrated culture-dependent and culture-independent approaches -
2007
Ph.D. thesis Wageningen University, Wageningen, The Netherlands
ISBN 978-90-8504-839-8
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Abstract
The need for clean water is increasing and anaerobic wastewater treatment can be
used as a cost-effective solution for purification of organically polluted industrial
waste streams. This thesis presents results from microbiological investigations of
several full-scale and lab-scale anaerobic wastewater treatments systems. Anaerobic
wastewater treatment has gained popularity and is now one of the key technologies in
environmental biotechnology. However, knowledge of the microbial community
structure – function relationships is limited. A combination of cultivation-dependent
and cultivation-independent techniques can be used to improve this knowledge. In this
thesis, batch serial dilution incubations from a methanol-fed lab-scale thermophilic
(55(cid:176) C) methanogenic bioreactor indicated that syntrophic interspecies hydrogen
transfer-dependent methanol conversion is at least equally important as direct
methanogenesis in this lab-scale reactor. A direct methanol-utilizing
Methanomethylovorans hollandica-related strain was detected up to a 108-fold
dilution, while Thermodesulfovibrio relatives and Methanothermobacter
thermoautotrophicus strains were found till 109–fold dilutions in the presence of
H /CO . Microbial diversity was further evaluated in two expanded granular sludge
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bed reactors fed with increasing oleic acid loading rates. The archaeal community in
the reactor inoculated with granular sludge stayed quite stable and active, whereas the
relative abundance of Methanosaeta-like organisms gradually decreased in the reactor
inoculated with suspended sludge when oleate loads were increased to 8 kg of
chemical oxygen demand m-3 day-1. Desulfomicrobium and Methanobacterium were
found to dominate the start-up of a full-scale synthesis gas fed gas-lift reactor treating
metal and sulphate rich wastewater. Most Probable Number (MPN) counts confirmed
that heterotrophic sulphate reducing bacteria (SRB) were dominant (1011-1012 cells/g
VSS) compared to homoacetogens (105-106 cells/g VSS) and methanogens (108-
109cells/g VSS). Methanogens can still persist in sulphate-reducing bioreactors with
short sludge retention time, since competition for hydrogen is determined by Monod
kinetics and not by hydrogen threshold values. The microbial community in a full-
scale upflow anaerobic sludge blanket reactor treating paper mill wastewater operated
at 37(cid:176) C was relatively stable over a period of 3 years as indicated by a high similarity
(>75%) of denaturing gradient gel electrophoresis profiles of 16S ribosomal RNA
gene fragments. Batch incubations at different temperatures resulted in microbial
community changes. While the archaeal community composition differed
significantly between incubations at 45 and 55(cid:176) C, the bacterial composition changed
between 37 and 45(cid:176) C. Overall the bacterial community was dominated by Firmicutes
(68% of the clones) and Delta-Proteobacteria (17% of the clones). A sequential
degradation of first butyrate and then propionate at 37(cid:176) C was linked to strong
presence of Syntrophomonas sp. and Desulfobulbus propionicus, respectively. MPN
series allowed estimating the number of micro-organisms per ml sludge that could use
propionate without sulphate (109), propionate and sulphate (105), butyrate without
sulphate (108), butyrate with sulphate (105), glucose (109) and H /CO (1010). Archaea
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were mainly dominated by Methanosaeta, but also Crenarchaeota-relatives were
identified. Bacterial clone sequences were related to a variety of different known
species, with expected functions in anaerobic digestion like fermentative bacteria,
syntrophic short chain fatty acids oxidisers and SRB. However, about 80% of the
clones was similar to sequences in the database without close cultured relatives, but
many of these appeared to be present in anaerobic environments. It is important to
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improve knowledge of these unknown micro-organisms and fast accurate monitoring
and identification could be instrumental in realising this. Therefore, a pilot macro-
array was developed and tested. It appeared that combining probes generated by PCR
amplification of the V1 and V6 variable regions of the 16S rRNA gene provided
accurate differentiation of closely related organisms. The integrated application of
molecular and cultivation dependent analyses of microbiota structure and function of
a broad variety of anaerobic wastewater treatment systems described in this thesis has
been used to improve insight of the ecophysiology in such reactors. Some general
commonalities of anaerobic systems have been found, but also system-specific
characteristics. This provides potential identification of general and system-specific
indicator populations, allowing improved diagnostics and reactor predictability.
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Content
Abstract 5
Preface and thesis outline 8
Chapter 1 Microbial ecophysiology of anaerobic wastewater treatment
systems – State of the art and approaches towards improved
understanding of structure-function relationships 11
Chapter 2 Changes in anaerobic microbial community from a paper mill
wastewater treatment reactor after incubation with different
substrates and at different temperatures. 63
Chapter 3 Enrichment and detection of micro-organisms involved in direct
and indirect methanogenesis from methanol in an anaerobic
thermophilic bioreactor 79
Chapter 4 Molecular monitoring of microbial diversity in expanded
granular sludge bed (EGSB) reactors treating oleic acid 89
Chapter 5 Occurrence of methanogenesis during start-up of a full-scale
synthesis gas-fed reactor treating sulfate and metal-rich wastewater 101
Chapter 6 Community analysis of a full-scale anaerobic bioreactor treating
paper mill wastewater 113
Chapter 7 Detection of the microbial diversity in anaerobic wastewater
treatment sludge 129
Summary and concluding remarks 145
Acknowledgements / dankwoord 153
About the author 156
Educational activities 159
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Preface and thesis outline
The research presented in this thesis was financed by the Technology Foundation,
STW, applied science division of NWO, the Netherlands, and entitled: ‘Molecular
monitoring of microbial diversity in anaerobic wastewater treatment systems’.
Anaerobic wastewater treatment is a commonly applied technology for the
purification of industrial wastewater with high organic matter content. Relatively
small, high-rate reactors with biomass retention are applied for an efficient conversion
of organic compounds into methane and carbon dioxide. The Upflow Anaerobic
Sludge Blanket (UASB) reactor is the most common anaerobic reactor type, but new,
more efficient reactors with biomass retention have been developed. The microbial
composition of anaerobic bioreactors is rather complex. Besides conventional culture-
dependent microbiological methods culture-independent molecular biological
methods, mostly targeting 16S ribosomal RNA and the encoding gene, can be used
nowadays to investigate the microbial composition of anaerobic bioreactors. The
research described in this thesis mainly focused on fatty acid-degrading communities
which form the core of all methanogenic bioreactors. Syntrophic micro-organisms
rely on other organisms, such as sulphate reducing bacteria (SRB) or methanogens,
for the removal of their metabolic products. These SRB and methanogens benefit
from the products produced by syntrophs, which results in a delicate balance and
fierce competition. Improved knowledge of these organisms and their interactions is
important for wastewater treatment reactor stability and control.
Chapter 1 gives an overview of current knowledge of the ecophysiology of micro-
organisms in anaerobic wastewater treatment systems. A short description of different
applications of anaerobic wastewater treatments is given, followed by a description of
tools and techniques to study micro-organisms in such ecosystems, including culture-
dependent and especially molecular approaches that can be used to provide insight
into microbial community structure – function relationships that govern wastewater
treatment processes. Advantages and disadvantages of the various approaches are
discussed. A summary of known cultivated key micro-organisms in anaerobic
wastewater treatment plants is given, followed by a detailed description of published
molecular inventories of the microbial diversity in such systems. Furthermore,
chapter 1 provides an outlook on emerging integrated approaches, stressing the need
for concerted applications of innovative culture-dependent as well as culture-
independent techniques, pivotal to achieve a better understanding and predictability of
anaerobic wastewater treatment processes and reactor performance.
Culture-dependent and culture-independent techniques have been applied to study the
bacterial and archaeal population dynamics in anaerobic sludge, incubated at different
temperatures with different feed mixtures, as described in chapter 2. The biochemical
results of the incubations were compared with the microbial community shifts using
Denaturing Gradient Gel Electrophoresis (DGGE) fingerprinting and subsequent
cloning and sequencing analysis (324 bacterial clones analysed). With this approach
the identity of microbes could be linked to their function in the mixed communities.
In chapter 3 micro-organisms linked to direct and indirect methane formation from
methanol in a lab-scale thermophilic (55(cid:176) C) bioreactor were investigated. Reactor
sludge was disrupted and serial dilutions were incubated in specific growth media
containing methanol and possible intermediates of methanol degradation as substrates.
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Dominant micro-organisms in the different dilutions were identified by 16S rRNA-
gene sequence analysis. Furthermore, dilution PCR was used to determine the relative
abundance of Archaea and Bacteria in all investigated samples.
In other lab-scale reactors the influence of granular and suspended sludge as inoculum
was investigated. Bacterial and archaeal diversity was evaluated in two expanded
granular sludge bed reactors fed with increasing oleic acid loading rates up to 8 kg of
chemical oxygen demand m-3 day-1 as described in chapter 4. During operation, the
sludge in both reactors was segregated in two layers: a bottom settled one and a top
floating one. Both layers in the two reactors were microbiologically characterised.
The start-up of a full-scale synthesis gas fed gas-lift reactor treating metal and
sulphate rich wastewater was investigated and presented in chapter 5. Most Probable
Number (MPN) counts were used to determine the abundance of heterotrophic SRB,
homoacetogens and methanogens. Furthermore, 16S rRNA gene analysis was applied
to identify the dominant microbial groups, and an explanation for the co-existence of
SRB and methanogens was given.
To get insight into the microbial community of a full-scale upflow anaerobic sludge
blanket reactor treating paper mill wastewater, conventional microbiological methods
were combined with 16S rRNA gene analyses, as described in chapter 6. Particular
attention was paid to micro-organisms able to degrade propionate or butyrate in the
presence or absence of sulphate. Serial enrichment dilutions allowed estimating the
number of micro-organisms per ml sludge and quantitative RNA dot-blot
hybridisation revealed the ratio Archaea/Bacteria. The microbial community
composition was further characterised by 16S rRNA-gene-targeted DGGE
fingerprinting, and via cloning and sequencing of the most dominant amplicons from
the bacterial and archaeal patterns.
Chapter 7 describes the integrated application of more extensive bio-molecular and
cultivation-based experiments to further improve the knowledge on complex
microbial community structure - function relationships in the full-scale anaerobic
paper-mill wastewater treatment system addressed in previous chapters 2 and 6. MPN
series allowed estimating the number of micro-organisms per ml sludge that could use
propionate with and without sulphate, butyrate with and without sulphate, glucose and
H /CO . Cloning of 16S rRNA genes, screening of clones with amplified ribosomal
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DNA restriction analysis and partial sequencing revealed the expected high species
richness. From this clone library a set of 121 abundant and not closely related clones
was nearly completely sequenced. This information was used to develop and test a
pilot macro-array to provide the means for high throughput microbial community
analysis tools. Such tools could prove useful for microbial diagnostics in
environmental biotechnology towards an improved ability to predict functionality and
failure of anaerobic reactor systems.
Finally, the results obtained in the different chapters are discussed. In general, a
combination of culture-dependent and culture-independent microbiological techniques
has been used to get insight of the ecophysiology of anaerobic bioreactors. Since these
bioreactors were mainly methanogenic, expected methanogenic Archaea such as
Methanosarcinales (mainly Methanosaeta) and Methanobacteriales, with known
functions were detected, but also members of the Crenarchaeota have been found.
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Further research is needed to elucidate the functional role of these Crenarchaeota in
anaerobic wastewater treatment. Besides expected fermenting bacteria, syntrophic
short chain fatty acids oxidisers and SRB, many bacterial phylotypes have been
detected with unknown function. Advanced molecular techniques have been applied
for rapid monitoring and identification of the microbes present. Still, integrated
multidisciplinary research approaches are needed to understand and control useful
(and less-wanted) microbes in anaerobic environments, such as engineered
wastewater treatment systems.
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Description:Universitair Hoofddocent, Laboratorium voor Microbiologie the reactor inoculated with granular sludge stayed quite stable and active, whereas the . number of micro-organisms per ml sludge and quantitative RNA dot-blot.
