PhD Thesis - Aida Azrina Binti Azmi

Transcription

CHARACTERIZATION AND PROFILING OF MICROBIAL
COMMUNITY DIVERSITY ASSOCIATED WITH ANAEROBIC
SULFUR OXIDATION IN AN UPFLOW ANAEROBIC SLUDGE
BLANKET REACTOR TREATING MUNICIPAL SEWAGE
(
B
)A
AIDA AZRINA BINTI AZMI
DOCTOR OF ENGINEERING
NAGAOKA UNIVERSITY OF TECHNOLOGY
2015
(
CHARACTERIZATION AND PROFILING OF MICROBIAL COMMUNITY
DIVERSITY ASSOCIATED WITH ANAEROBIC SULFUR OXIDATION IN
AN UPFLOW ANAEROBIC SLUDGE BLANKET REACTOR TREATING
MUNICIPAL SEWAGE
(
B
(
)A
By
AIDA AZRINA BINTI AZMI
A Dissertation Submitted to the Graduate School of Engineering, Nagaoka
University of Technology, in Partial Fulfillment of the Requirements for the
Degree of DOCTOR OF ENGINEERING
(Energy and Environmental Engineering)
February 2015
!
i!
This thesis was submitted to the Graduate School of Engineering, Nagaoka University
of Technology and has been accepted as fulfillment of the requirement for the degree
of Doctor of Engineering (Energy and Environmental Engineering). The members of
the Supervisory Committee were as follows:
Takashi YAMAGUCHI, Dr.Eng.
Professor
Department of Civil and Environmental Engineering
Nagaoka University of Technology
(Chairman)
Masashi HATAMOTO, Dr.Eng.
Assistant Professor
Department of Civil and Environmental Engineering
Nagaoka University of Technology
(Member)
!
ii!
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS
v
CHAPTER
1
INTRODUCTION
1
2
LITERATURE REVIEW
4
2.1
Microbial Communities in Sewage Treatment
4
2.2
Sulfur Cycle Bacteria
7
2.3
Anaerobic Sulfide/Sulfur Oxidation
25
2.4
Bioelectrochemical Reactor
27
3
References
31
REPRODUCTION OF ANAEROBIC SULFUR
41
OXIDATION REACTION BY USING POTENTIAL
CONTROL DEVICE AND ANALYSIS OF MICROBIAL
COMMUNITIES INVOLVED IN THE REACTION
Abstract
41
3.1
Introduction
42
3.2
Materials and Methods
43
3.3
Results and Discussion
47
3.4
Conclusion
55
References
!
56
iii!
4
MOLECULAR CHARACTERIZATION OF ANAEROBIC
58
SULFUR-OXIDIZING MICROBIAL COMMUNITIES IN
UPFLOW ANAEROBIC SLUDGE BLANKET REACTOR
TREATING MUNICIPAL SEWAGE
5
Abstract
58
4.1
Introduction
59
4.2
60
4.3
63
4.4
Conclusion
73
References
74
DIVERSITY PROFILE OF MICROBES ASSOCIATED
80
WITH ANAEROBIC SULFUR OXIDATION IN AN
UPFLOW ANAEROBIC SLUDGE BLANKET REACTOR
TREATING MUNICIPAL SEWAGE
6
Abstract
80
5.1
Introduction
81
5.2
82
5.3
86
5.4
Conclusion
99
References
100
SUMMARY
108
LIST OF PUBLICATIONS
!
111
iv!
ACKNOWLEDGEMENTS
First of all, I am grateful to Almighty Allah for empowering me with His
guidance and granted me all these graces throughout my life to achieve my missions
in life and to fulfill this PhD study.
I would like to express my sincere and deepest gratitude to my advisor,
Professor Takashi Yamaguchi, for accepting me as his student and letting me
experience the research of wastewater treatment, for his caring, patience, motivation,
and also for providing a good environment for me to do research as well as financially
supported my research. I would like to thank Dr. Masashi Hatamoto, for his guidance,
enthusiasm, immense knowledge and insightful comments, helping me in developing
my background in this field, have faith in me and patiently corrected my writing.
Without their guidance and persistent help, these research and dissertation would not
have been possible. I could not imagine having a better advisor and mentor for my
PhD study.
I would like to thank the staff members for being nice and friendly and also
for their help. I would also like to thank my fellow lab members for their help and
stimulating discussion and also for the fun we had in the last few years.
Last but not least, I am extremely grateful to my parents for their prayers,
supporting and encouraging me, and standing by me through the good times and bad.
May Allah bless and grants them a long and healthy life.
!
v!
CHAPTER 1
INTRODUCTION
Wastewater treatment systems are designed to achieve improvements in the
quality of the wastewater besides providing low cost sanitation and environmental
protection. To date, there are varieties of treatment technology used for the handling
of various types of industrial and municipal wastewaters such as filter bioreactor,
fluidized bed reactor, sequencing batch reactor (SBR), expanded granular sludge bed
(EGSB) reactor and upflow anaerobic sludge blanket (UASB) reactor. Among all
these treatment technologies, UASB is one of the most popular and widely applied
technologies in many countries such as the developing countries because of its high
efficiency and cost effective sewage treatment process. In these series of wastewater
treatment studies, a system consisting an UASB reactor together with a down-flow
hanging sponge (DHS) reactor as a post-treatment was developed for municipal
sewage treatment under low temperature conditions. An aerobic post-treatment
system, DHS, was chose because of the sufficient and effective process performance
in treating the effluent from the UASB reactor, besides providing a low investment,
operation and maintenance costs.
Initially, these series of wastewater treatment studies was conducted to
investigate the sulfate reduction reaction that contributes to the degradation of organic
matters in the UASB reactor under low temperature conditions. However, an
interesting phenomenon was observed while this investigation was being conducted
where anaerobic sulfur oxidation was occurred in the UASB reactor without the
presence of electron acceptors such as oxygen, nitrate, nitrite and oxidized-irons. In
!
1!
the previous studies, the occurrence of sulfur oxidation reaction was observed with
the presence of these electron acceptors. This discovery has not been reported yet
previously and seems to be a new finding. Therefore, as the main objective of this
study, the probable main factors that are responsible in actuating the anaerobic sulfur
oxidation reaction to occur in the UASB reactor was investigated.
To achieve this main objective, information regarding this matter was gathered
and also several steps were set and conducted as in chapters as stated below:
CHAPTER 2 explored the literature that is relevant to understanding the global cycle
of sulfur and its microbes. Several studies on sulfur cycle microbes under various
conditions were discussed and their characteristics were reviewed. Besides that,
bioelectrochemical reactor (BER) approaches to studying fundamental microbial
interaction and its mechanisms were also discussed.
CHAPTER 3 aimed to reproduce the anaerobic sulfur oxidation reaction by
controlling the oxidation-reduction potential (ORP) and other parameters of the
cultured sludge in the UASB reactor under low temperature. Additionally, the
microbial community structure in the sludge, before and after culture, was
investigated to estimate the microbial communities involved in the reaction.
CHAPTER 4 aimed to investigate the microbial communities existing in the UASB
reactor during the anaerobic sulfur oxidation period by molecular analysis of the 16S
rRNA gene.
!
2!
CHAPTER 5 aimed to analyze comprehensively the abundance, distribution,
characteristics and phylogenetic diversity of microbes involved in anaerobic sulfur
oxidation in the UASB reactor for twenty-four months of operation period by next
generation sequencing analysis. On top of that, the influence of environmental
conditions on the microbial community structure and diversity was observed.
Finally, the summary and conclusion of this study were stated in CHAPTER
6 which all the chapters were summarized and the results of each chapter were
compared. The findings from this study were then concluded.
The framework of the research is shown below in Fig. 1.1.
Introduction
Description of the problem addressed and purpose of the research as well as steps
to achieve the main objective discussed.
CHAPTER 1
Literature Review
Literature that is relevant to understanding the global cycle of sulfur and its
microbes reviewed.
CHAPTER 2
ChaChapter!
Study 1
Anaerobic sulfur oxidation
reaction reproduced and
microbial
communities
involved in the reaction
analyzed.
CHAPTER 3
Study 2
Microbial
communities
existing in the UASB
reactor during anaerobic
sulfur oxidation period
investigated.
Study 3
Diversity of microbes
exists in the UASB reactor
over a period of 2 years
analyzed.
CHAPTER 4
CHAPTER 5
Summary
Summary and conclusion of the research stated.
CHAPTER 6
Fig. 1.1 Research framework.
!
3!
CHAPTER 2
LITERATURE REVIEW
2.1
Microbial Communities in Sewage Treatment
Microorganisms are important and represent the core component in the
treatment of wastewater. They play an essential role in converting the organic wastes
to more stable less polluting substances resulting in water purification and energy
production. Many studies were conducted on the analysis of microbial communities in
various types of wastewater and reactors until now (Table 2.1), and from time to time,
many new findings or microbes are discovered. These analyses and the new
discoveries will help to increase the microbial biodiversity data of the wastewater
which will lead to a better and effective performance of the reactor in treating the
wastewater.
According to these studies of microbial communities analysis under different
conditions, the most frequently retrieved 16S rRNA gene were those affiliated with
the phylum Proteobacteria, followed by phyla Chloroflexi, Bacteroidetes, Firmicutes
and Actinobacteria (Table 2.1). Despite having these analyses conducted and
retrieving the microbial biodiversity data, there are still a large number of
uncharacterized microbes exist in the environment. Furthermore, according to
Achtman and Wagner (2008), more than 85 novel bacterial phyla have been
discovered and not even one representative isolate have been cultured nor a single
genome sequence have been recovered from most of these phyla since 1987 (Fig.
2.1). The population structures as well as the cohesive forces of the immeasurable
!
4!
majority microbes on this planet are also not yet known. Therefore, the development
of novel concepts for improving the microbial communities analysis is necessary to
have a thorough knowledge on the ecology of the microbes exist in the wastewater
treatment plants and reactors as well as on the earth.
Fig. 2.1 Numbers of phyla and genomic sequences among microbes since 1987. Each
schematic tree shows the number of known phyla for each indicated year. Vertical
line represents each phylum. Numbers above each horizontal bar indicate the numbers
of phyla that they span. (Achtman and Wagner, 2008)
!
5!
Nitrifying/denitrifying
industrial plant
Nitrifying sequencing
biofilm batch reactor
Enhanced biological
phosphorous removal
sequencing batch
reactor + sodium
acetate
Enhanced biological
phosphorous removal
continuous flow
reactor
Sugar processing
wastewater - upflow
anaerobic sludge
bioreactor
Vinegar processing
wastewater - upflow
anaerobic sludge
bioreactor
Clear-liquor
processing wastewater
- upflow anaerobic
sludge bioreactor
Amino-acid
processing wastewater
- upflow anaerobic
sludge bioreactor
Relative frequency of bacterial 16S rRNA gene (%)
No.of clones
No. of OTUs
Chao1
Coverage (%)
Proteobacteria
Alphaproteobacteria
Betaproteobacteria
Gammaaproteobacteria
Deltaproteobacteria
Epsilonproteobacteria
Chloroflexi
Bacteroidetes
Firmicutes
Actinobacteria
Planctomycetes
Nitrospira
Deferribacteres
Spirochaetes
Chlorobi
Acidobacteria
Verrucomicrobia
Fusobacteria
Others
High-load aeration
basin municipal plant
Table 2.1 Summary of bacterial 16S rRNA-based diversity surveys of wastewater treatment plants and reactors.
62
25
32
77
94
53
83
64
96
33
42
78
97
69
144
48
51
30
59
51
84
43
84
68
104
42
87
76
103
46
107
74
117
65
124
67
3
52
18
26
31
5
51
22
4
1
13
33
8
3
2
4
5
1
4
13
3
16
8
8
4
4
6
6
1
7
2
15
2
16
2
10
1
1
12
2
1
8
37
8
18
1
32
11
8
13
22
20
21
11
9
4
2
5
32
10
5
9
7
33
2
11
1
2
14
1
1
3
6
2
1
5
3
5
3
1
3
3
2
2
2
23
14
2
3
11
10
1
1
10
4
(Adapted from Wagner and Loy, 2002; Narihiro et al., 2009)
!
6!
2.2
Sulfur Cycle Bacteria
The role of microorganisms is crucial in the global cycle of various elements
such as sulfur, nitrogen, carbon and phosphorus. In the sulfur cycle, sulfate reducing
bacteria (SRB) and sulfide oxidizing bacteria (SOB) in particular, are extremely
important from the domestic, industrial and environmental perspectives. Their
activities under carefully controlled conditions could resolve and improve some
processing and environmental problems. For example, the contribution of SRB
together with the SOB in the treatment of acid mine drainage can overcome the severe
environmental problem occurred in the mining industry (Tang et al., 2009).
The first study of the sulfur cycle microbes was initiated at the end of the 19th
century by the famous microbiologist Winogradsky and Beijerinck (Lens et al.,
2002). To date, there were many studies done on sulfur cycle microbes under different
conditions, such as in wastewater treatment reactors, soil, oil and mining, hypersaline
and hydrothermal fields, which were summarized in Table 2.2. The studies were
mostly doing isolation, quantification, characterization and investigation on the
phylogenetic relationships of the microbes in general. However, the knowledge on the
sulfur cycle microbes, in terms of the mechanism, physiology as well as the
interactions among themselves and other microbial communities and also with the
environment, is still inadequate and not fully known.
!
7!
Table 2.2 Summary of several studies on sulfur cycle microbes under different conditions.
Sulfur Cycle
Microbial Study
Purpose
Analysis Method
Brief Outcome
Reference
Activated sludge,
anaerobic digesters,
wastewater biofilms
and water treatment
plants
Isolation and characterization
of an organism which is
capable of both facultatively
autotrophic and facultatively
anaerobic growth
Isolation, physiological
and biochemical analyses
• The new isolate is significantly different compared to 2
similar species, Thiobacillus A2 and Paracoccus
denitrificans.
• Generic name of Thiosphaera was given in view of its
ability to oxidize reduced sulfur compounds and because it
is a chain-forming coccus rather than a rod.
• Species name pantotropha was given in recognition of its
wide range of possible substrates.
Robertson and
Kuenen, 1983
Elucidation of phylogenetic
relationships of strain THI
011T and related bacteria, and
comparison of the
physiological characteristics
of these organisms
Physiological and
biochemical analyses,
HPLC and 16S rRNA gene
sequence analysis
Paracoccus thiocyanatus sp. nov., a new species of
thiocyanate-utilizing facultative chemolithotroph isolated
from activated sludge was proposed.
Katayama et al.,
1995
Investigation of phylogenetic
relationships of filamentous
sulfur bacteria, Thiothrix spp.
and Eikelboom type 021N
bacteria, isolated from
wastewater treatment plants
Isolation and 16S rRNA
gene sequence analysis
It is proposed that Eikelboom type 021N bacteria should be
accommodated within the genus Thiothrix as a new species,
Thiothrix eikelboomii sp. nov., and 3 further new Thiothrix
species were described, Thiothrix unzii sp. nov., Thiothrix
fructosivorans sp. nov. and Thiothrix defluvii sp. nov.
Howarth et al., 1999
!
!
!
8!
Table 2.2 (Continued).!
Sulfur Cycle
Microbial Study
Crop lands, orchards
and garden soil
Purpose
Analysis Method
Brief Outcome
Reference
Isolation and partial
characterization and in situ
detection of a novel aerobic
chemolithoautotrophic sulfuroxidizing bacterium inhabiting
wastewater biofilms
analysis, FISH analysis
and 16S rRNA gene
sequence analysis
Strain SO07 was one of the important sulfur-oxidizing
populations involved in the sulfur cycle occurring in the
wastewater biofilm and was primarily responsible for the
oxidation of hydrogen sulfide and elemental sulfur to sulfate
under oxic conditions.
Ito et al., 2004
Identification of
microorganisms present in the
scum layer of the settler
compartment of UASB
reactors and evaluate their role
in biological oxidation of
sulfides
Isolation, cultivation,
microscopic analysis and
chemical analysis
• Microbes similar to Beggiatoa sp., Thiotrix sp. and species
of cyanobacteria were identified, and most of them shown
to be capable of carrying out sulfur oxidation.
• Biological oxidation of sulfide tests using scum and cultures
of the cyanobacteria Phormidium and Pseudoanabaena
showed a significant decrease in sulfide concentrations,
suggesting that the microbes present in the scum layer can
play a role in the minimization of odor emissions.
Garcia et al., 2012
Elucidation of thiosulfateoxidizing bacteria from an
Italian rice field soil
Restriction fragment
length polymorphism
(RFLP) and sequencing
7 phylogenetic groups within the α- and β-subclass of
Proteobacteria were isolated:
Graff and Stubner,
2003
• Xanthobacter sp. and Bosea sp. related strains (already
described as sulfur-oxidizers in rice field soil)
• Mesorhizobium loti, Hydrogenophaga sp., Delftia sp.,
Pandoraea sp. and Achromobacter sp. related strains
(represented new sulfur-oxidizers in rice field soil)
!
9!
Sulfur Cycle
Microbial Study
Petroleum and
mining industries
!
Purpose
Analysis Method
Brief Outcome
Reference
Diversity study of sulfur
chemolithotrophs of αProteobacteria from Indian
soils
and biochemical analyses,
and 16S rRNA gene
sequence analysis
5 facultative sulfur chemolithotrophs were isolated:
Deb et al., 2004
Isolation and characterization
of chemolithotrophic strains
from temperate orchard soil in
India
and 16S rRNA gene
sequence analysis
Tetrathiobacter kashmirensis gen. nov., sp. nov., a novel
mesophilic, neutrophilic, tetrathionate-oxidizing, facultatively
chemolithotrophic β-Proteobacteria was isolated from soil and
proposed.
Ghosh et al., 2005
Selectively enumerate and
determine the relative
abundances of planktonic
nitrate-reducing bacteria
(NRB) and SRB in some
western Canadian oil field
waters
Chemical analysis, and
bacterial enumeration and
MPN culture analyses
• Microbial numbers in the produced waters near or at the
wellheads very low but increased in the aboveground
facilities.
• No thiosulfate-oxidizing NRB were detected in the oil field
waters but other types of NRB were detected.
• Each of the oil fields contained NRB, which might be
stimulated by nitrate amendment to control sulfide
production by SRB.
Eckford and
Fedorack, 2002
Sulfate and heavy metals
removal by SRB in a benchscale upflow anaerobic packed
bed reactor
Chemical analysis
SRB activity increased the water pH from ~4.5 to 7.0, and
enhanced the removal of sulfate and metals.
Jong and Parry,
2003
•
•
•
•
Paracoccus versutus related strains (2 strains)
Paracoccus alcaliphilus related strain
Pseudaminobacter salicylatoxidans related strain
Unclassified chemolithoautotrophic arsenite oxidizing strain
10!
Sulfur Cycle
Microbial Study
Hypersaline and/or
hyperalkaline
!
Purpose
Analysis Method
Brief Outcome
Reference
Quantification of acid mine
drainage microbial
communities and
understanding the importance
of SRB in attenuating acid
mine drainage
Chemical analysis,
microorganisms
quantification by sybr
green I direct count and
CARD-FISH, and
scanning electron
microscopy (SEM)
analysis
• Members of sulfate-reducing genera Syntrophobacter,
Desulfosporosinus and Desulfurella were dominant in
sediment with pH 4.2-6.0, reducing redox potential -210
mV to 50 mV and a lower metals and iron solubility.
• Suggesting that the attenuation of acid mine drainage
characteristics is biologically driven by sulfate reducers and
the consequent precipitation of metals and iron as sulfides.
Sánchez-Andrea et
al., 2012
Thiocyanate utilization study
by microbes under highly
alkaline conditions
electron microscopy and
16S rRNA gene sequence
analysis
• Active thiocyanate biodegradation may occur under highly
alkaline conditions.
• Thiocyanate can be used by heterotrophic and autotrophic
alkaliphilic bacteria either as a nitrogen source or as an
electron donor and energy source.
Sorokin et al., 2001
Phototrophic community
study found in Lake
Khilganta, an alkaline saline
lake
Physicochemical analysis,
bacteria enumeration and
electron microscopy
• Cyanobacteria is abundant in upper zone.
• Purple bacteria is developed in lower zones.
• Ectothiorhodospira sp. are dominant among the
anoxyphotobacteria and predominantly develops when pH
increased to 10.4.
• Purple bacteria of genera Allochromatium, Thiocapsa and
Rhodovulum are also present.
• Main role in sulfide oxidation belongs to phototrophic
anoxyphotobacteria and cyanobacteria.
Kompantseva et al.,
2005
11!
Sulfur Cycle
Microbial Study
Metal-rich marine
geothermal fields,
craters of volcanoes,
volcanic hot springs,
solfataras,
subseafloor habitats
and hydrothermal
vent fields
Purpose
Analysis Method
Brief Outcome
Reference
Diversity analysis of
halophilic SOB inhabiting
various types of hypersaline
environment
electron microscopy and
analysis
• Unexpectedly high culturable diversity of
chemolithoautotrophic SOB in hypersaline habitats, ranging
from moderate to extreme halophiles and including aerobic,
facultatively anaerobic and thiocyanate-utilizing
phenotypes.
• Halophilic SOB found belong to 6 different phylotypes
within the γ-proteobacteria, 4 of which were not previously
known.
Sorokin et al., 2006
Describing a group of
halotolerant marine metalmobilizing rod-shaped
thiobacilli
light and electron
microscopy, and DNADNA hybridization
Thiobacillus prosperus sp. nov., a new species of halotolerant
metal-mobilizing bacterium isolated from a marine
geothermal field was proposed.
Huber and Stetter,
1989
Bacterial diversity analysis in
subseafloor fluids from a
diffuse flow hydrothermal
vent shortly after volcanic
eruption created the site in
1998, 1999 and 2000
Geochemical analysis and
analysis
• Bacterial diversity is high in diffuse fluids, and that it
changes with the post-eruptive evolution of vent fluid
chemistry and temperature.
• Species richness increase with time within the εproteobacteria, the dominant phylotype found to be unique
to the subseafloor environment.
Huber et al., 2003
!
!
!
12!
Sulfur Cycle
Microbial Study
!
Purpose
Analysis Method
Brief Outcome
Reference
Enzymatic and genetic
characterization of carbon and
energy metabolisms by deepsea hydrothermal
chemolithoautotrophic isolates
of ε-proteobacteria
Isolation, enzyme assays,
radioisotope labeling,
zymogram, PCR
amplification and
sequencing of genes for
rTCA, Calvin-Benson and
soluble hydrogenase
• ε-proteobacteria use rTCA cycle for carbon assimilation.
• Sulfur-oxidizing ε-proteobacteria showed enzyme activity
of a potential sulfite: acceptor oxidoreductase for a direct
oxidation pathway to sulfate but no activity of AMPdependent adenosine 5’-phosphate sulfate reductase for
indirect oxidation pathway.
• No thiosulfate-oxidizing enzymes activity detected.
Takai et al., 2005
Elucidation of genetic and
biochemical aspects of εproteobacterial energy
metabolism, and
understanding the
ecophysiological roles of these
bacteria
electron microscopy, fatty
acid analysis, DNA-DNA
hybridization and 16S
rRNA gene sequence
analysis
Sulfurimonas paralvinellae sp. nov., a novel mesophilic,
hydrogen- and sulfur-oxidizing chemolithoautotroph within
the ε-proteobacteria isolated from a deep-sea hydrothermal
vent polychaete nest was proposed.
Takai et al., 2006
Isolation, characterization and
ecological analysis of coldactive, chemolithotrophic,
SOB from perennially icecovered Lake Fryxell
Water analysis,
microscopy, MPN analysis
and SSU rRNA gene
sequence analysis
• Isolated 3 strains SOB resembling Thiobacillus thioparus.
• Psychrotolerant Antarctic isolates showed an adaptation to
cold temperatures, thus should be active in the nearly
freezing waters of the lake.
• Sulfur-oxidizing chemolithotroph population peaks
precisely at the oxycline, although viable cells exist well
into the anoxic, sulfidic waters of the lake.
• SOB described likely play a key role in biogeochemical
cycling of carbon and sulfur in Lake Fryxell.
Sattley and
Madigan, 2006
13!
Sulfur Cycle
Microbial Study
!
Purpose
Analysis Method
Brief Outcome
Reference
Determination of
biogeochemical processes and
key organisms relevant for
primary production in surface
sediments of an ultramafic
hydrothermal vent field
Biochemical analysis,
CARD-FISH and 16S
rRNA gene sequence
analysis
In conductively heated surface sediments microbial sulfur
cycling is the driving force for bacterial biomass production
although ultramafic-hosted systems are characterized by fluids
with high levels of dissolved methane and hydrogen.
Schauer et al., 2011
Clarification of sulfur cyclerelated microbial community
structures, as well as microbial
linkages with the cycles of
other elements such as carbon
and nitrogen in hydrothermal
chimneys on the Southwest
Indian Ridge
Physicochemical analyses,
pyrosequencing of
metagenomes and
bioinformatics analyses
• Few dominant bacteria, particularly sulfate-reducing δproteobacteria, participated in the microbial sulfur cycle.
• Contained highly abundant genes related to sulfur oxidation
and reduction.
• Several carbon metabolic pathways, in particular the
Clavin-Benson-Bassham pathway and the reductive
tricarboxylic acid cycles for CO2 fixation, were identified in
sulfur-oxidizing autotrophic bacteria.
• Highly abundant genes related to the oxidation of shortchain alkanes were grouped with SRB, suggesting an
important role for short-chain alkanes in sulfur cycle.
• SOB were associated with enrichment for genes involved in
the denitrification pathway, while SRB displayed
enrichment for genes responsible for hydrogen utilization.
Cao et al., 2014
14!
Generally, there are various states of oxidation of sulfur in the sulfur cycle.
These three oxidation states are the most significant in nature which is (i) -2 (sulfide
and reduced organic sulfur) (ii) 0 (elemental sulfur), and (iii) +6 (sulfate). These states
can be transformed both chemically and biologically. Furthermore, the sulfur cycle is
closely related to other element cycles such as carbon and nitrogen cycles (Muyzer
and Stams, 2008). As shown in Fig. 2.2, sulfur cycle consists of reduction and
oxidation reaction. In reduction reaction, sulfate act as an electron acceptor which is
used by a wide range of microorganisms and is converted to sulfide. As for the
oxidation reaction, reduced sulfur compounds such as sulfide act as an electron donor
used by phototrophic and chemolithothropic bacteria and is converted to elemental
sulfur or sulfate (Robertson and Kuenen, 2006; Tang et al., 2009).
Fig. 2.2 Schematic representation of the main sulfur cycle process.
(Meulepas et al., 2010)
!
15!
2.2.1
Sulfate-reducing bacteria (SRB)
A direct anaerobic reduction of sulfate, elemental sulfur and thiosulfate to
sulfide is done by SRB. Besides that, sulfide can also be produced by anaerobic
microorganisms through breakdown of proteins to amino acids and further
degradation of amino acids to sulfide. SRB comprise a diverse obligate anaerobes
group. This group grows in the anoxic environments with the presence of organic
materials and sulfate where the organic compounds or hydrogen is used as electron
donor by SRB in sulfate reduction to sulfide. Mostly, the electron donor and the
carbon source are the same compound, but when an electron donor used is H2, CO2 or
organic compounds such as acetate is needed as the carbon source (Postgate, 1984;
Tang et al., 2009).
To date, more than 220 species of 60 genera of SRB were known. The largest
group of SRB (more than 25 genera) is Deltaproteobacteria (such as order
Desulfobacterales, Desulfovibrionales and Syntrophobacterales), followed by
Firmicutes
(such
as
genera
Desufotomaculum,
Desulfosporomusa
and
Desulfosporosinus), Nitrospira (such as genus Thermodesulfovibrio) and two phyla
represented by Thermodesulfobacteria and Thermodesulfobium (Barton and Fauque,
2009). Besides that, Archaeoglobus spp. of phylum Euryarchaeota is the sulfatereducer group of the domain archaea (Woese et al., 1990).
Currently, SRB can be divided into two main groups, with regard to the
metabolic function, which are complete oxidizers and incomplete oxidizers. Complete
oxidizers are acetate oxidizers which are capable to oxidize the organic compound to
carbon dioxide. Incomplete oxidizers are non-acetate oxidizers which oxidize the
organic compound incompletely to acetate and CO2 (Muyzer and Stams, 2008). Table
!
16!
2.3 shows some of the bacterial and also archaeal species that belong to these two
groups. The other group of obligate anaerobes, sulfur-reducing bacteria, which can
reduce sulfur to sulfide but unable to reduce sulfate to sulfide consist of genera such
as Desulfuromonas, Desulfurella, Sulfurospirrilium and Campylobacter (Tang et al.,
2009).
Table 2.3 Summary of some complete oxidizers and incomplete oxidizers SRB and
archaea (Tang et al., 2009).
Complete Oxidizers
Incomplete Oxidizers
(Acetate oxidizers)
(Non-acetate oxidizers)
Desulfobacter
Desulfovibrio
Desulfobacterium
Desulfomicrobium
Desulfococcus
Desulfobotulus
Desulfonema
Desulfofustis
Desulfosarcina
Desulfotomaculum
Desulfoarculus
Desulfomonile
Desulfoacinum
Desulfobacula
Desulforhabdus
Archaeoglobus
Desulfomonile
Desulfobulbus
Desulfotomaculum acetoxidans
Desulforhopalus
Desulfotomaculum sapomandens
Thermodesulfobacterium
Desulfovibrio baarsii
!
17!
Electron donors
Recently, many studies have done and extended the number of electron
donors. More than one hundred compounds are potential electron donors for SRB
including sugars such as fructose and glucose, amino acids such as glycine, serine and
alanine, monocarboxylic acids such as formate, acetate, propionate, butyrate, lactate
and pyruvate, dicarboxylic acids such as fumarate, succinate and malate, alcohols
such as methanol, ethanol, 1-propanol, 2-propanol and 1-butanol, and aromatic
compounds such as benzoate and phenol (Rabus et al., 2006; Barton and Fauque,
2009).
Electron acceptors
Most species of SRB can utilize thiosulfate and sulfite as electron acceptors
besides sulfate. Some studies reported that some SRB species belonging to
Desulfohalobium, Desulfofustis, Desulforomusa and Desulfospirs are to grow with
elemental sulfur (Rabus et al., 2006). Some SRB species can also utilize
organosulfonates as electron acceptors (Lie et al., 1996; Laue et al., 1997). According
to Tang et al. (2009), some SRB species also utilized other non sulfur-containing
electron acceptors such as nitrate and nitrite (Mitchell et al., 1986; Moura et al.,
1997), ferric iron (Lovley et al., 1993; Bale et al., 1997), arsenate, chromate and
uranium (Newman et al., 1997; Macy et al., 2000; Lovley and Phillips, 1994).
!
18!
Environmental conditions
SRB can also thrive in a range of different environmental conditions where
they can be found in many natural and engineered environments with the presence of
sulfate (Muyzer and Stams, 2008). They have been found in environments with
extreme pH values. Willow and Cohen (2003) reported that SRB grow in the
environments with pH values in the range 5-9. Furthermore, according to Neculita et
al. (2007), pH values outside the range 5-9 usually results in reduced activity. In
anaerobic reactor, the sulfate reducers grow optimally at pH values in the range 6.98.5 and tolerated pH values as high as 10 (Visser et al., 1996; Tang et al., 2009). SRB
comprise both mesophilic and thermophilic strains. Their growth and sulfate
reduction kinetics being affected significantly by temperature (Stetter et al., 1993; van
Houten et al., 1997; Weijma et al., 2000). SRB are mostly living independently.
However, some of them are present in consortia with other microbes such as
methanotrophic archaea or together with SOB (Muyzer and Stams, 2008).
The characteristics of SRB mentioned above were summarized in Table 2.4.
2.2.2
Sulfur-oxidizing bacteria (SOB)
The biological removal of sulfide can be divided into two ways which are
direct and indirect ways. In direct way, photoautotrophic or chemolithotrophic SOB
use sulfide as an electron donor and converted it to sulfur or sulfate. Photoautotrophic
SOB use CO2 as the terminal electron acceptor. Chemolithotrophic SOB use O2 (for
aerobic species) or nitrate and nitrite (for anaerobic species) as the terminal electron
!
19!
acceptors. In indirect way, the reduced sulfur compound oxidation is performed
chemically by ferric iron as the oxidizing agent and the ferric iron is regenerated by
iron oxidizing bacteria for further use (Pagella and De Faveri, 2000; Tang et al.,
2009).
Photoautotrophic oxidation of sulfide is an anaerobic process performed by
green sulfur bacteria such as Chlorobium, and purple sulfur bacteria such as
Allochromatium by utilizing H2S as an electron donor for CO2 reduction in a
photosynthetic reaction. Most of the purple sulfur bacteria store the produced
elemental sulfur as globules within the cell and further oxidation of sulfur results in
formation and release of sulfate from the cells (Madigan and Martinko, 2006; Tang et
al., 2009). Purple sulfur bacteria comprise several genera such as Chromatium,
Thioalkalicoccus,
Thiorhodococcus,
Thiospirillum, Thiodictyon and
Thiocapsa,
Thiopedia.
The
Thiocystis,
genera
Thiococcus,
Ectothiorhodospira,
Thiorhodospira and Halorhodospira are of special interest because the sulfur
produced resides outside the cell (Madigan and Martinko, 2006). Additionally,
lithoautotrophic growth in the absence of light has been reported for some of the
purple bacteria such as Allochromatium vinosum and Thiocapsa roseopersicina
(Friedrich et al., 2001). Green sulfur bacteria comprise genera such as Chlorobium,
Prosthecochloris, Pelodictyon, Ancalochloris and Chloroherpeton, which use H2S as
an electron donor to oxidize it first to elemental sulfur and then to sulfate. The sulfur
produced by these bacteria resides outside the cell. Green sulfur bacteria are able to
grow and function at low light intensities because of the presence of the chlorosomes
(Madigan and Martinko, 2006).
Chemolithotrophic SOB, which are also known as colorless sulfur bacteria,
are diverse in term of morphological, physiological and ecological properties. They
!
20!
are able to grow chemolithotrophically on reduced inorganic sulfur compounds such
as sulfide, sulfur and thiosulfate. In some cases, they are able to grow on organic
sulfur compounds such as methanethiol, dimethylsulfide and dimethyldisulfide
(Robertson and Kuenen, 2006; Madigan and Martinko, 2006). Sulfide oxidation
involves the transfer of six electrons from sulfide to the cell electron transport system
and then to the terminal electron acceptor, which is primarily oxygen as many of the
species are aerobic, to produce sulfite. Nonetheless, some species can grow
anaerobically using nitrate and nitrite as the terminal electron acceptor. There are two
pathways for the oxidation of sulfite to sulfate. In the first pathway, the electrons from
sulfite are transferred directly to cytochrome c by sulfite oxidase with concomitant
formation of adenosine triphosphate (ATP) as a result of electron transport and proton
motive force. The second pathway is a reversal of adenosine phosphosulfate reductase
activity where one high energy phosphate bond is produced by converting adenosine
monophosphate (AMP) to adenosine diphosphate (ADP). When thiosulfate is used as
electron donor, it is split into elemental sulfur and sulfite which then oxidized to
sulfate (Madigan and Martinko, 2006; Tang et al., 2009). The colorless sulfur bacteria
comprise many genera such as Thiobacillus, Acidithiobacillus, Achromatium,
Beggiatoa, Thiothrix, Thioplaca, Thiomicrospira, Thiosphaera and Thermothrix to
name a few (Tang et al., 2009).
Electron donors
The colorless sulfide-oxidizers are categorized into four groups, in term of
energy and carbon source, as stated below (Tang et al., 2009):
!
21!
(i)
Obligate chemolithotrophs which need an inorganic source for
energy and use CO2 as carbon source. The species belong to this
category are some species of Thiobacillus, at least one species of
Sulfolobus and all of the known species of Thiomicrospira.
(ii)
Facultative
chemolithotrophs
which
can
grow
either
(a)
chemolithoautotrophically with CO2 and an inorganic energy
source, (b) heterotrophically with complex organic compounds as
carbon and energy source, or (c) mixotrophically using both
pathways simultaneously. The species belong to this category are
Thiobacilli, Thiosphaera pantotropha, Paracoccus denitrificans
and some species of Beggiatoa.
(iii)
Chemolithoheterotrophs which are characterized by the ability to
generate energy from reduced sulfur compounds oxidation while
being unable to fix CO2. The species belong to this category are a
few species of Thiobacillus and some species of Beggiatoa.
(iv)
Chemoorganoheterotrophs
which
oxidize
reduced
sulfur
compounds without deriving energy from them. This reaction is
used as a means for detoxifying the metabolically produced
hydrogen peroxide. The species belong to this category are such as
Thiobacterium, Thiothrix and some species of Beggiatoa.
Electron acceptors
Generally, oxygen is used as a universal electron acceptor by the colorless
sulfide-oxidizers, but the degree of aerobiosis varies among species. During sulfur
!
22!
compounds oxidation, the electrons produced are transferred to dissolved oxygen and
O2 is reduced to H2O. Under anaerobic conditions, various colorless sulfur-oxidizers
grow differently and the use of nitrate or nitrite as terminal electron acceptors are one
of the best known pathways. Sulfide oxidation under denitrifying conditions could
lead to formation of sulfur, sulfate and nitrite or nitrogen. The use of ferric iron and
molybdate as electron acceptor has been reported under microaerobic conditions
(Tang et al., 2009).
Environmental conditions
As mentioned by Tang et al. (2009), colorless sulfur-oxidizers are diverse as
far as the growth pH and temperature are concerned. There are studies reported that
these bacteria grow at pH values in the range 1-9 and temperatures ranging from 4 to
90°C. The optimal pH varies among the species of SOB and the outcome of
competition for common substrate in the mixed cultures is dictated mainly by pH.
The characteristics of SOB mentioned above were summarized in Table 2.4.
!
23!
Table 2.4 Summary of characteristics of SRB and SOB.
Characteristic
SRB
Electron donor
•
•
•
•
•
•
Sugars
e.g. fructose & glucose
Amino acids
e.g. glycine, serine &
alanine
Monocarboxylic acids
e.g. formate, acetate,
propionate, butyrate,
lactate & pyruvate
Dicarboxylic acids
e.g. fumarate, succinate &
malate
Alcohols
e.g. methanol, ethanol,
1-propanol, 2-propanol &
1-butanol
Aromatic compounds
e.g. benzoate & phenol
•
•
•
•
•
•
•
•
Hydrogen sulfide
Sulfide
Elemental sulfur
Thiosulfate
Sulfite
Ferrous iron
Hydrogen
Carbon monoxide
Sulfate
Thiosulfate
Sulfite
Elemental sulfur
Organosulfonates
Nitrate
Nitrite
Ferric iron
Arsenate
Chromate
Uranium
•
•
•
•
•
Oxygen
Nitrate
Nitrite
Ferric iron
Molybdate
Electron acceptor
•
•
•
•
•
•
•
•
•
•
•
Environmental
condition
• pH values in range 5-10
• Mesophilic and
thermophilic
!
SOB
• pH values in range 1-9
• Temperature ranging
from 4 to 90°C
24!
2.3
Anaerobic Sulfide/Sulfur Oxidation
Anaerobic sulfide/sulfur oxidation, as mentioned earlier, can occur under two
reactions either photoautotrophic or chemolithotrophic sulfide/sulfate oxidation
reaction. Under photoautotrophic oxidation of sulfide/sulfate, the bacteria utilize H2S
as an electron donor for CO2 reduction in a photosynthetic reaction referred to as the
van Niel reaction as shown below (Madigan and Martinko, 2006; Tang et al., 2009):
Light
2H2S + CO2
2S0 + CH2O + H2O,
∆G0 = 75.36 kJ mol-1
(1)
Under chemolithotrophic sulfide/sulfate oxidation, the bacteria use nitrate or nitrite as
terminal electron acceptors. Oxidation of sulfide under denitrifying conditions could
lead to formation of sulfur, sulfate and nitrite or nitrogen based on the following
reactions below (Cardoso et al., 2006):
S2- + 1.6NO3– + 1.6H+
SO42- + 0.8N2 + 0.8H2O,
∆G0 = -743.9 kJ/reaction
S2- + 0.4NO3– + 2.4H+
(2)
S0 + 0.2N2 + 1.2H2O,
S2- + 4NO3–
SO42- + 4NO2–,
!
(3)
(4)
25!
S2- + NO3– + 2H+
S0 + NO2– + H2O,
(5)
Sulfide conversion to sulfate coupled to complete denitrification (Eq. (2)) consumes
four times more nitrate when compared with conversion to sulfur (Eq. (3)). Under
complete sulfide oxidation to sulfate, complete denitrification to nitrogen (Eq. (2))
decreases the amount of required nitrate by a factor of 2.5 when compared with
incomplete denitrification to nitrite (Eq. (4)) (Cardoso et al., 2006; Tang et al., 2009).
Sulfur and thiosulfate oxidation under denitrification can be described by the
following reactions below:
S0 + 1.2NO3– + 0.4H2O
SO42- + 0.6N2 + 0.8H+,
S2O32- + 1.6NO3– + 0.2H2O
(6)
2SO42- + 0.8N2 + 0.4H+,
(7)
Besides that, bacterial disproportionation of partially oxidized sulfur
compounds such as elemental sulfur, thiosulfate and sulfite is also quantitatively
important and ecologically relevant process in the sulfur cycle (Bak and Cypionka,
1987; Cypionka et al., 1998; Poser et al., 2013). The sulfur compounds serve
simultaneously as both electron donors and electron acceptors, and transformed into
oxidized and reduced species during the disproportionation process (Poser et al.,
2013). Under standard conditions, the disproportionation of elemental sulfur to sulfate
and sulfide is an endergonic process (Eq. (8)), whereas disproportionation of
!
26!
thiosulfate or sulfite to sulfate and sulfide (Eq. (9) and (10)) or to sulfur and sulfate
(Eq. (11) and (12)) is an exergonic process (Bak and Cypionka, 1987).
4S0 + 4H2O
2SO42- + 3HS- + 5H+,
∆G0 = 10.2 kJ mol-1 (per sulfur)
S2O32- + H2O
SO42- + HS- + H+,
∆G0 = -21.9 kJ mol-1
4SO32- + H+
(9)
3SO42- + HS-,
∆G0 = -58.9 kJ mol-1 (per sulfite)
3S2O32- + 2H+
(11)
S0 + 2SO42- + H2O,
∆G0 = -62.3 kJ mol-1 (per sulfite)
2.4
(10)
4S0 + 2SO42- + H2O,
∆G0 = -35.5 kJ mol-1 (per thiosulfate)
3SO32- + 2H+
(8)
(12)
Bioelectrochemical Reactor (BER)
Bioelectrochemical reactors (BERs) are widely used nowadays in research as
a platform for studying fundamental microbial interaction and have real application
on fields like wastewater treatment, energy production and storage as well as
resources production recycling and recovery. The most practically applied is in
wastewater treatment such as domestic, industrial and agricultural wastewater.
!
27!
Although laboratory experiments have shown BER can work, implementing the fullscale of bioelectrochemical wastewater treatment is still insufficient and inefficient
because certain microbiological, technological and economic challenges need to be
resolved which have not been encountered previously in other wastewater treatment
system (Rozendal et al., 2008). According to Rozendal et al. (2008) again, more
studies are required with real wastewaters which will help to develop strategies for
improving the degradation of complex materials and controlling the microbial
reactions occurring in the system. Furthermore, new designs are also required to
minimize potential losses and optimize performance for full-scale BERs. Importantly,
the capital costs of BERs have to be reduced so that the field of large-scale
wastewater treatment can be targeted.
BER technology, biological research in particular, is changing very quickly
and it could be two to five years before first generation technologies are available
commercially. Furthermore, nothing controversial with this technology has been
identified to this point and commercial uptake is expected to be driven by cost and
reliability (European Commission, 2013).
BER mechanisms
BER is an electrochemical system in which electrochemically active
microorganisms catalyse the anode and/or the cathode reaction (Rozendal et al.,
2008). BERs have been used for culturing organisms, influencing metabolite
production and biotransformation of a wide array of compounds (Thrash and Coates,
2008). As shown in Fig. 2.3, there are two routes of stimulation in a BER where in
one route is the cathodic reduction of either a mediator or part of the bacterial electron
!
28!
transport chain which serves as the energy source for the bacteria. The bacteria
transfer the electrons to terminal reductases via their electron transport chain, which
then reduce an oxidized substrate, for example, a contaminant such as nitrate or
uranium. In other route, the BER system provides a continuous supply of a suitable
electron acceptor either through direct anodic oxidation of a terminal reductase or
through indirect anodic oxidation of a soluble mediator that is used by the
microorganism as a suitable electron acceptor to oxidize various reduced substrates
such as ammonia or ferrous iron (Thrash and Coates, 2008).
Fig. 2.3 Schematic diagram of electrical stimulation of microorganisms.
(Thrash and Coates, 2008)
!
29!
The main objective of BER is the electron transfer between a microorganism
and a working electrode. The electron transfer between a working electrode and a
bacterial cell can occur in three ways, either directly at the electrode surface,
indirectly mediated by a soluble electron shuttling agent or by the electrolysis of
water (Fig. 2.4). Direct electron transfer at the electrode surface have been
demonstrated by Gregory et al. (2004) with the direct cathodic transfer of electrons to
a bacterial cell. The pure cultures of Geobacter metallireducens and G.
sulfurreducens were directly utilizing the electrons from the surface of graphite
electrodes for reducing nitrate and fumarate, respectively. For indirectly mediated by
a soluble electron shuttling agent, the microorganisms can selectively oxidize or
reduce the electroactive substrates such as quinones, phenazines and humic
substances, without consuming them and leave them free for recycling at an electrode,
which means that the electrons are shuttled between microbes and electrodes. These
electroactive substrates can be used as electron donors and/or acceptors by bacteria in
a nondegradative manner (Thrash and Coates, 2008). In cathodic electrolysis of water
where hydrogen is produced can be oxidized by microorganisms coupled to the
reduction of many substrates, including nitrate. A wide variety of microorganisms use
hydrogen as an electron donor, and thus the electrolytic production of hydrogen is an
effective strategy for stimulating metabolism and growth of those organisms
(Schlegel and Lafferty, 1965; Ohmura et al., 2002; Thrash and Coates, 2008).
!
30!
Fig. 2.4 Mechanisms of electron transfer.
(Thrash and Coates, 2008)
References
Achtman, M. and Wagner, M. (2008). Microbial diversity and the genetic nature of
microbial species. Nature Reviews Microbiology, 6: 431-440.
Bak, F. and Cypionka, H. (1987). A novel type of energy metabolism involving
fermentation of inorganic sulfur compounds. Nature, 326: 891-892.
Bale, S.J., Goodman, K., Rochelle, P.A., Marchesi, J.R., Fry, J.C., Weightman, A.J.
and Parkes, R.J. (1997). Desulfovibrio profundus sp. nov., a novel barophilic
sulfate reducing bacterium from deep sediment layers in the Japan Sea.
International Journal of Systematic Bacteriology, 47: 515-521.
!
31!
Barton, L.L. and Fauque, G.D. (2009). Biochemistry, physiology and biotechnology
of sulfate-reducing bacteria. Advances in Applied Microbiology, 68: 41-98.
Cao, H., Wang, Y., Lee, O.O., Zeng, X., Shao, Z. and Qian, P-Y. (2014). Microbial
sulfur cycle in two hydrothermal chimneys on the Southwest Indian Ridge.
mBio, 5: 1-11.
Cardoso, R.B., Sierra-Alvarez, R., Rowlette, P., Flores, E.R., Gomez, J. and Field,
J.A. (2006). Sulfide oxidation under chemolithoautotrophic denitrifying
conditions. Biotechnology and Bioengineering, 95: 1148-1157.
Cypionka, H., Smock, A.M. and Böttcher, M.E. (1998). A combined pathway of
sulfur compound disproportionation in Desulfovibrio desulfuricans. FEMS
Microbiology Letters, 166: 181-186.
Dannenberg, S., Kroder, M., Dilling, W. and Cypionka, H. (1992). Oxidation of H2,
organic compounds and inorganic sulfur compounds coupled to reduction of
O2 or nitrate by sulfate-reducing bacteria. Archives of Microbiology, 158: 9399.
Deb, C., Stackebrandt, E., Pradella, S., Saha, A. and Roy, P. (2004). Phylogenetically
diverse new sulfur chemolithotrophs of alpha-proteobacteria isolated from
Indian soils. Current Microbiology, 48: 452-458.
Dilling, W. and Cypionka, H. (1990). Aerobic respiration in sulfate-reducing bacteria.
FEMS Microbiology Letters, 71: 123-128.
Eckford, R.E. and Fedorack, P.M. (2002). Planktonic nitrate-reducing bacteria and
sulfate-reducing bacteria in some western Canadian oil field waters. Journal of
Industrial Microbiology and Biotechnology, 29: 83-92.
!
32!
European Commission (2013). Bioelectrochemical systems: wastewater treatment,
bioenergy and valuable chemicals delivered by bacteria. Science for
Environment Policy, issue 5: 1-12.
Friedrich, C.G., Rother, D., Bardischewsky, F., Quentmeier, A. and Fischer, J. (2001).
Oxidation of reduced inorganic sulfur compounds by bacteria: emergence of a
common mechanism? Applied and Environmental Microbiology, 67: 28732882.
Garcia, G. P., Souza, C. L., Glória, M., Silva, S. Q. and Chernicharo, C. A. L. (2012).
Biological oxidation of sulphides by microorganisms present in the scum layer
of UASB reactors treating domestic wastewater. Water Science and
Technology, 66: 1871-1878.
Ghosh, W., Bagchi, A., Mandal, S., Dam, B. and Roy, P. (2005). Tetrathiobacter
kashmirensis gen. nov., sp. nov., a novel mesophilic, neutrophilic,
tetrathionate-oxidizing, facultatively chemolithotrophic betaproteobacterium
isolated from soil from a temperate orchard in Jammu and Kashmir, India.
International Journal of Systematic and Evolutionary Microbiology, 55: 17791787.
Graff, A. and Stubner, S. (2003). Isolation and molecular characterization of
thiosulfate-oxidizing bacteria from an Italian rice field soil. Systematic and
Applied Microbiology, 26: 445-452.
Gregory, K.B., Bond, D.R. and Lovley, D.R. (2004). Graphite electrodes as electron
donors for anaerobic respiration. Environmental Microbiology, 6: 596-604.
Howarth, R., Unz, R.F., Seviour, E.M., Seviour, R.J., Blackall, L.L., Pickup, R.W.,
Jones, J.G., Yaguchi, J. and Head, I.M. (1999). Phylogenetic relationships of
filamentous sulfur bacteria (Thiothrix spp. and Eikelboom type 021N bacteria)
!
33!
isolated from wastewatertreatment plants and description of Thiothrix
eikelboomii sp. nov., Thiothrix unzii sp. nov., Thiothrix fructosivorans sp. nov.
and Thiothrix defluvii sp. nov. International Journal of Systematic and
Evolutionary Microbiology, 49: 1817-1827.
Huber, H. and Stetter, K.O. (1989). Thiobacillus prosperus sp. nov., represents a new
group of halotolerant metal-mobilizing bacteria isolated from a marine
geothermal field. Archives of Microbiology, 151: 479-485.
Huber, J., Butterfield, D.A. and Baross, J.A. (2003). Bacterial diversity in a
subseafloor habitat following a deep-sea volcanic eruption. FEMS
Microbiology Ecology, 43: 393-409.
Ito, T., Sugita, K. and Okabe, S. (2004). Isolation, characterization and in situ
detection of a novel chemolithoautotrophic sulfur-oxidizing bacterium in
wastewater biofilms growing under microaerophilic conditions. Applied and
Environmental Microbiology, 70: 3122-3129.
Jong, T. and Parry, D.L. (2003). Removal of sulfate and heavy metals by sulfate
reducing bacteria in short-term bench scale upflow anaerobic packed bed
reactor runs. Water Research, 37: 3379-3389.
Katayama, Y., Hiraishi, A. and Kuraishi, H. (1995). Paracoccus thiocyanatus sp.
nov., a new species of thiocyanate-utilizing facultative chemolithotroph, and
transfer of Thiobacillus versutus to the genus Paracoccus as Paracoccus
versutus comb. nov. with emendation of the genus. Microbiology, 141: 14691477.
Kompantseva, E.I., Sorokin, D., Gorlenko, V.M. and Namsaraev, B.B. (2005). The
phototrophic community found in Lake Khilganta (an alkaline saline lake
located in the southeastern Transbaikal region). Mikrobiologiia, 74: 410-419.
!
34!
Laue, H., Denger, K. and Cook, A.M. (1997). Taurine reduction in anaerobic
respiration of Bilophila wadsworthia RZATAU. Applied and Environmental
Microbiology, 63: 2016-2021.
Lens, P., Vallero, M., Esposito, G. and Zandvoort, M. (2002). Perspectives of sulfate
reducing
bioreactors
in
environmental
biotechnology.
Reviews
in
Environmental Science and Biotechnology, 1: 311-325.
Lie, T.J., Pitta, T., Leadbetter, E.R., Godchaux III, W. and Leadbetter, J.R. (1996).
Sulfonates: novel electron acceptors in anaerobic respiration. Archives of
Lovley, D.R. and Phillips, E.J.P. (1994). Reduction of chromate by Desulfovibrio
vulgaris and its c3 cytochrome. Applied and Environmental Microbiology, 60:
726-728.
Lovley, D.R., Roden, E.E., Phillips, E.J.P. and Woodward, J.C. (1993). Enzymatic
iron and uranium reduction by sulfate-reducing bacteria. Marine Geology,
113: 41-53.
Macy, J.M., Santini, J.M., Pauling, B.V., O’Neill, A.H. and Sly, L.I. (2000). Two new
arsenate/sulfate-reducing bacteria: mechanism of arsenate reduction. Archives
of Microbiology, 173: 49-57.
Madigan, M. T. and Martinko, J. M. (2006). Brock Biology of Microorganisms, 11th
edition, Prentice Hall, Upper Saddle River, NJ.
Meulepas, R.J.W., Stams, A.J.M. and Lens, P.N.L. (2010). Biotechnological aspects
of sulfate reduction with methane as electron donor. Reviews in Environmental
Science and Biotechnology, 9: 59-78.
!
35!
Mitchell, G.J., Jones, J.G. and Cole J.A. (1986). Distribution and regulation of nitrate
and nitrite reduction by Desulfovibrio and Desulfotomaculum species.
Archives of Microbiology, 144: 35-140.
Moura, I., Bursakov, S., Costa, C. and Moura, J.J.G. (1997). Nitrate and nitrite
utilization in sulfate-reducing bacteria. Anaerobe, 3: 279-290.
Muyzer, G. and Stams, A.J.M. (2008). The ecology and biotechnology of sulphatereducing bacteria. Nature Reviews Microbiology, 6: 441-454.
Narihiro, T., Terada, T., Kikuchi, K., Iguchi, A., Ikeda, M., Yamauchi, T., Shiraishi,
K., Kamagata, Y., Nakamura, K. and Sekiguchi, Y. (2009). Comparative
analysis of bacterial and archaeal communities in methanogenic sludge
granules from upflow anaerobic sludge blanket reactors treating various foodprocessing, high-strength organic wastewaters. Microbes and Environments,
24: 88-96.
Neculita, C.M., Zagury, G.J. and Bussiere, B. (2007). Passive treatment of acid mine
drainage in bioreactors using sulfate-reducing bacteria: critical review and
research needs. Journal of Environmental Quality, 36: 1-16.
Newman, D.K., Kennedy, E.K., Coates, J.D., Ahmann, D., Ellis, D.J., Lovley, D.R.
and Morel, F.M.M. (1997). Dissimilatory arsenate and sulfate reduction in
Desulfotomaculum auripigmentum sp. nov. Archives of Microbiology, 168:
380-388.
Ohmura, N., Matsumoto, N., Sasaki, K. and Saiki, H. (2002). Electrochemical
regeneration of Fe(III) to support growth on anaerobic iron respiration.
Applied and Environmental Microbiology, 68: 405-407.
Pagella, C. and De Faveri, D.M. (2000). H2S gas treatment by iron bioprocess.
Chemical Engineering Science, 55: 2185-2194.
!
36!
Poser, A., Lohmayer, R., Vogt, C., Knoeller, K., Planer-Friedrich, B., Sorokin, D.,
Richnow, H.-H. and Finster, K. (2013). Disproportionation of elemental sulfur
by haloalkaliphilic bacteria from soda lakes. Extremophiles, 17: 1003-1012.
Postgate, J. R. (1984). The sulphate reducing bacteria, 2nd edition, University Press,
Cambridge.
Rabus, R., Hansen, T. A. and Widdel, F. (2006). Dissimilatory sulfate- and sulfurreducing. In M. Dworkin, S. Falkow, E. Rosenberg, K.H. Schleifer, E.
Stackebrandt (Eds.), The Prokaryotes, vol. 2, 3rd ed., Springer, New York, p.
659-768.
Robertson, L.A. and Kuenen, J.G. (1983). Thiosphaera pantotropha gen. nov. sp. nov.,
a facultatively anaerobic, facultatively autotrophic sulphur bacterium. Journal
of General Microbiology, 129: 2847-2855.
Robertson, L.A. and Kuenen, J.G. (2006). The colorless sulphur bacteria. In M.
Dworkin, S. Falkow, E. Rosenberg, K.H. Schleifer, E. Stackebrandt (Eds.),
The Prokaryotes, vol. 2, 3rd ed., Springer, New York, p. 985-1011.
Rozendal, R.A., Hamelers, H.V.M., Rabaey, K., Keller, J. and Buisman, C.J.N.
(2008). Towards practical implementation of bioelectrochemical wastewater
treatment. Trends in Biotechnology, 26: 450-459.
Sánchez-Andrea, I., Knittel, K., Amann, R., Amils, R. and Sanz, J.L. (2012).
Quantification of Tinto River sediment microbial communities: importance of
sulfate-reducing bacteria and their role in attenuating acid mine drainage.
Sattley, W.M. and Madigan, M.T. (2006). Isolation, characterization, and ecology of
cold-active, chemolithotrophic, sulfur-oxidizing bacteria from perennially ice-
!
37!
covered Lake Fryxell, Antarctica. Applied and Environmental Microbiology,
72: 5562-5568.
Schauer, R., RØy, H., Augustin, N., Gennerich, H.H., Peters, M., Wenzhoefer, F.,
Amann, R. and Meyerdierks, A. (2011). Bacterial sulfur cycling shapes
microbial communities in surface sediments of an ultramafic hydrothermal
vent field. Environmental Microbiology, 13: 2633-2648.
Schlegel, H.G. and Lafferty, R. (1965). Growth of “Knallgas” bacteria
(Hydrogenomonas) using direct electrolysis of the culture medium. Nature,
205: 308-309.
Sorokin, D.Y., Tourova, T.P., Lysenko, A.M. and Kuenen, J.G. (2001). Microbial
thiocyanate utilization under highly alkaline conditions. Applied and
Sorokin, D.Y., Tourova, T.P., Lysenko, A.M. and Muyzer, G. (2006). Diversity of
culturable halophilic sulfur-oxidizing bacteria in hypersaline habitats.
Stetter, K.O., Huber, R., Blöchl, E., Kurr, M., Eden, R.D., Fielder, M., Cash, H. and
Vance, I. (1993). Hyperthermophilic archaea are thriving in the deep north sea
and Alaskan oil reservoirs. Nature, 365: 743-745.
Takai, K., Campbell, B.J., Cary, S.C., Suzuki, M., Oida, H., Nunoura, T., Hirayama,
H., Nakagawa, S., Suzuki, Y., Inagaki, F. and Horikoshi, K. (2005).
Enzymatic and Genetic Characterization of Carbon and Energy Metabolisms
by
Deep-Sea
Hydrothermal
Chemolithoautotrophic
Isolates
of
Epsilonproteobacteria. Applied and Environmental Microbiology, 71: 73107320.
!
38!
Takai, K., Suzuki, M., Nakagawa, S., Miyazaki, M., Suzuki, Y., Inagaki, F. and
Horikoshi, K. (2006). Sulfurimonas paravinellae sp. nov., a novel mesophilic,
hydrogen-
and
sulfur-oxidizing
chemolithoautotroph
within
the
Epsilonproteobacteria isolated from a deep-sea hydrothermal vent polychaete
nest, reclassification of Thiomicrospira denitrificans as Sulfurimonas
denitrificans comb. nov. and emended description of the genus Sulfurimonas.
International Journal of Systematic and Evolutionary Microbiology, 56: 17251733.
Tang, K., Baskaran, V. and Nemati, M. (2009). Bacteria of the sulphur cycle: An
overview of microbiology, biokinetics and their role in petroleum and mining
industries. Biochemical Engineering Journal, 44: 73-94.
Thrash, J.C. and Coates, J.D. (2008). Review: direct and indirect electrical stimulation
of microbial metabolism. Environmental Science and Technology, 42: 39213931.
van Houten, R.T., Yun, S.Y. and Lettinga, G. (1997). Thermophilic sulphate and
sulphite reduction in lab-scale gas-lift reactors using H2 and CO2 as energy
and carbon source. Biotechnology and Bioengineering, 55: 807-814.
Visser, A., Hulshoff Pol, L.W. and Lettinga, G. (1996). Competition of methanogenic
and sulfidogenic bacteria. Water Science and Technology, 33: 99-110.
Wagner, M. and Loy, A. (2002). Bacterial community composition and function in
sewage treatment systems. Current Opinion in Biotechnology, 13: 218-227.
Weijma, J., Stams, A.J.M., Hulshoff Pol, L.W. and Lettinga, G. (2000). Thermophilic
sulphate reduction and methanogenesis with methanol in a high rate anaerobic
reactor. Biotechnology and Bioengineering, 67: 354-363.
!
39!
Willow, M.A. and Cohen, R.R.H. (2003). pH, dissolved oxygen and adsorption
effects on metal removal in anaerobic bioreactors. Journal of Environmental
Quality, 32: 1212-1221.
Woese, C.R., Kandler, O. and Wheelis, M.L. (1990). Towards a natural system of
organisms: Proposal for the domains Archaea, Bacteria, and Eucarya.
Proceedings of the National Academy of Sciences of the United States of
America, 87: 4576-4579.
!
40!
CHAPTER 3
REPRODUCTION OF ANAEROBIC SULFUR OXIDATION REACTION BY
USING POTENTIAL CONTROL DEVICE AND ANALYSIS OF MICROBIAL
COMMUNITIES INVOLVED IN THE REACTION
Abstract
A phenomenon where sulfide is oxidized to sulfate without the presence of electron
acceptors was observed in the up-flow anaerobic sludge blanket (UASB) reactor used
for treatment of municipal sewage. In the previous study, the anaerobic oxidation of
sulfur (AOS) is known to occur in relatively high oxidation-reduction potential (ORP)
under anaerobic condition and low temperature environment. However, the
information and knowledge regarding this phenomenon is still limited. Therefore, this
study aimed to culture the sludge in the UASB reactor under low temperature by
controlling the ORP to reproduce the AOS. Additionally, the microbial community
structure in the sludge, before and after culture, was investigated to estimate the
microbial communities involved in the reaction. A two chamber-type potential control
device operated at 15℃ was used in this study with the working electrode potential
electrochemically regulated to
200 mV using potentiostat. Stirring condition and
presence of methane production inhibitor were also monitored when AOS is
reproduced. As a result, the sulfate concentration in the medium increased in the ORP
control system but decreased in the non-ORP control system. In the stirring system,
the sulfate concentration decreased in the early stage but later the sulfur oxidized.
There were no significant changes in the sulfate concentration of the 2-bromo
!
41!
ethanesulfonic acid system which was added with methane production inhibitors.
These results showed that by controlling the ORP, the occurrence of AOS and the
microbial community structure was able to confirm. The cloning analysis result for
the
ORP
control
system
where
sulfur
oxidation
occurred
shows
that
Desulfovibrionaceae and Syntrophobacteraceae belong to the phylum Proteobacteria
had a higher proportion. Therefore, it can be presumed that these microbial
communities also probably involved in the AOS.
3.1
Introduction
Nowadays, anaerobic treatment technology has turned into a popular treatment
technology for a various types of industrial and municipal wastewater. Up-flow
anaerobic sludge blanket (UASB) reactor has becoming one of the most widest
application due to its advantages of low cost, energy saving and low sludge
production (Lettinga et al., 1980). In our series of municipal sewage treatment
studies, a UASB reactor with sulfur-redox reaction under low-temperature conditions
was used (Takahashi et al., 2011). In the UASB reactor, an interesting phenomenon
had occurred where sulfide is oxidized to sulfate without the presence of electron
acceptors. As the need for more detail observation, bioelectrochemical reactors
(BERs) were used to regenerate the anaerobic oxidation of sulfur (AOS) by
controlling the oxidation-reduction potential (ORP). Currently, research using BERs
are growing vastly. BERs have been applied for water treatment, energy production
and storage, resources production recycling and recovery studies (Logan and Regan,
2006; Rozendal et al., 2008). The BERs flexibleness offers almost infinite range of
solutions for metabolic stimulation and downstream application. (Thrash and Coates,
!
42!
2008). Therefore, the aim of this study was to investigate the probable main factors
that caused this reaction to occur by controlling the ORP and other parameters.
Additionally, the microbial community structures in the UASB and BERs were also
been monitored to check the probability of these microbes contribution in this
reaction.
3.2
Feed material
Anaerobic granular sludge in the UASB reactor with total volume of 1178 L,
column diameter of 0.56 m, reactor height of 4.7 m, located at the municipal sewage
treatment plant in Nagaoka, Niigata, Japan was used as a seed sludge. This UASB is
supplied with sewage that is stored in a temporary tank of 2 m3 capacity and the
hydraulic retention time (HRT) was set to 6 h. The UASB is operated around 9-28 ℃
with no pH adjustment and chemical inputs such as SS flocculant. In this experiment,
the sludge taken from the second port (total 11 ports) which is located 0.50 m from
the bottom of the UASB was used.
BERs
Four sets of BER were utilized in this study. Each BER was assembled with
two glass chambers (350 mL capacity) with glass tubing and a pinch-clump assembly,
and was separated into two units by a proton exchange membrane (Nafion membrane,
0.125 mm thick) (Fig. 3.1). A carbon electrode (100×10×5 mm) served as the
supporting material in each unit, and in order to control the potential of the working
electrode, an Ag/AgCl reference electrode was inserted into the working unit. Each
!
43!
electrode was connected to a potentiostat (PS-08, Tohogiken, Japan). The potential of
the working electrode was maintained at
200 mV (vs. Ag/AgCl) in each BER. All
voltages reported in this paper are with respect to the Ag/AgCl reference electrode
(type: saturated KCl). The working volume in each unit was 350 mL. The medium
used in the counter electrode unit was 300 mL of 100 mM NaCl.
Potentiostat
RE
WE
PEM
CE
Fig. 3.1 Schematic diagram of BER. RE, reference electrode; WE, working electrode;
CE, counter electrode; PEM, proton exchange membrane.
!
44!
BER operation
The synthetic medium composition used in the BER culture chamber was as
follow: KH2PO4, 140 mg/L; NH4Cl, 540 mg/L; MgSO4 · 7H2O, 307 mg/L
(40 mg-S/L); CaCl2 · 2H2O, 150 mg/L; NaHCO3, 4200 mg/L; Trace Element (FeCl2 ·
4H2O, 199 mg/L; CoCl2, 130 mg/L; MnCl2 · 4H2O, 198 mg/L; ZnCl2, 136 mg/L;
H3Bo, 36.2 mg/L; NiCl2, 13 mg/L; AlCl3, 13.3 mg/L; Na2MoO4 · 2H2O2, 4.2 mg/L;
CuCl2 · 2H2O, 17 mg/L), 1 ml/L; Se/W (Na2SeO3, 1.7 mg/L, Na2WO4 · H2O,
3.3 mg/L), 1 ml/L. The pH of the medium was adjusted to about 6.8-7.3. 80 mg-S/L
of sodium sulfate and 2 mM of anthraquinone-2,6-disulfonate (AQDS) was added in
the medium as a substrate and electron mediator, respectively. The UASB sludge was
centrifuged and the supernatant was discarded. Then, the sludge was washed with
phosphate buffered saline (PBS). This centrifugation and washing step was repeated
for a couple of time. 18-20 g of remained sludge was taken and added into the culture
chamber. After that, the chamber was purged with nitrogen gas and sealed to maintain
the anaerobic condition in the chamber. The electrodes were then connected to the
potentiostat placed in each chamber and placed the culture device (the whole chamber
units) in the incubator where the light is blocked. The initial temperature of the
operation was set at 15°C. In this experiment, one standard system and three control
systems were operated. The standard system is a reference system where the ORP is
controlled. Meanwhile, the three control systems were: i) non-ORP control system, to
observe the state of reaction due to the diffusion, ii) stirring system, to monitor the
impact of the stirring parameter, and iii) BES (2-bromo ethanedisulfonic acid) system,
to investigate the effect of this methanogenic inhibitor towards the reproduction of
sulfur oxidation reaction. Final concentration of 5 mM of BES was added to the
system.
!
45!
High performance liquid chromatography (HPLC) analysis
Approximately 2 ml of samples from each systems were collected and filtered
through 0.2 µm membrane filter (Advantec) into a tube. The filtered samples were
then subjected to HPLC for sulfate concentration measurement.
16S rRNA gene clone library and phylogenetic analysis
Genomic DNA was isolated from the sludge sample and subjected to PCR
amplification of 16S rRNA gene using Premix Ex Taq Kit (Takara Bio, Shiga, Japan).
A set of bacteria-specific primer, EUB338f and 907r, was used for PCR amplification.
The PCR products were purified and then cloned using TOPO PCR cloning kit
(Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The
positive clones were selected and sent to Dragon Genomics Center (TAKARA BIO,
Yokkaichi, Japan) for sequencing. The raw sequencing data of the 16S rRNA gene
were analyzed for possible chimeras using the Bellerophon program (http://compbio.anu.edu.au/bellerophon/bellerophon.pl). The sequences with 97% similarity were
then grouped into operational taxonomic units (OTUs) using FastGroup II program
(http://biome.sdsu.edu/fastgroup/index.htm).
Representative
sequences
were
phylogenetically classified using the Classifier tool from the Ribosomal Database
Project (RDP) (http://rdp.cme.msu.edu/) and the ARB program.
Terminal-restriction fragment length polymorphism (T-RFLP) analysis
PCR was performed by using fluorescently labeled 907r and non-labeled
EUB338f primers. HhaI and TaqI were selected to digest the PCR products of
bacteria and archaea 16S rRNA gene purified with QIAquick PCR purification kit
(Qiagen, Hilden, Germany), respectively and were run through capillary sequencer.
!
46!
Multidimensional scanning (MDS) analysis
The T-RFLP profile patterns were analyzed by multidimensional scaling
(MDS) analysis. The presence and absence of T-RFLP peaks and their intensity over
all profiles were recorded in a binary matrix by using the Bray-Curtis coefficient,
which was then analyzed with the program SPSS 9.0 J for Windows (SPSS Japan)
using a stress value of <0.1. The MDS map showed most similar communities are
grouped closer together.
3.3
The regeneration of AOS reaction in the BERs had been confirmed by
measuring the sulfate concentrations in the medium. The sulfate concentrations were
compared between the ORP control system, non-ORP control system, stirring system
and BES system. As shown in Fig. 3.2, the sulfate concentration in the ORP control
system increased after 13 days of operation which shows that AOS reaction was
occurred. However, the sulfate concentration in the non-ORP control system
decreased where 20.1 mg/L of sulfate had reduced. This shows that sulfur reduction
had occurred, not AOS. This is probably due to the nitrogen purging inside the
chamber which caused the ORP to decrease and thus the AOS did not occur. With
these results, it can be concluded that by controlling the ORP, AOS occurred. For
stirring system, initially the sulfate concentration decreased which shows that sulfur
reduction had occurred. However, the AOS reaction had occurred in the latter part of
the operation where the sulfate concentration suddenly increased. This is probably
because when stirring, the entire sulfate is equally scattered in the medium and
consumed by the sulfur reducing bacteria which cause the reduction of sulfur to
!
47!
occur. However, a lot of sulfide is produced during this reaction and latter being
consumed by sulfur oxidizing bacteria which then caused the sulfur oxidation to
happen. Therefore, it can be presumed that the stirring effect can be related to the
AOS reaction. Meanwhile, in the BES system where BES was added to inhibit the
methanogenic activity, there were no changes in the sulfate concentration observed
during the whole period of the operation. This shows that the inhibition of the
methanogenic activity gave negative effect on the production of the AOS reaction. In
addition, there is a high possibility of the coexistence of sulfur oxidizing bacteria and
Sulfate conc. (mg-S/L)
methane-producing bacteria in producing AOS reaction.
ORP control system
Non-ORP control system
Stirring system
BES system
Time (day)
Fig. 3.2 Sulfate concentration measured in the BERs.
!
!
!
48!
In addition to the analyses, the microbial community structures in the UASB
reactor and BERs were characterized and identified through microbial 16S rRNA
gene analyses. The microbial 16S rRNA gene analyses conducted were cloning and
T-RFLP. For the cloning analysis, the samples analyzed were seed sludge directly
took from the UASB reactor and cultured samples from the ORP control system
chamber where the AOS reaction occurred in both units. In addition, cultured samples
from the non-ORP control system chamber where the AOS reaction did not occur was
also analyzed to compare the microbial community structures between the AOS
reaction occurred and non-occurred units. From the 16S rRNA cloning analysis
shown in Table 3.1, a diversity of bacteria species was present in the UASB reactor
and BERs. Phylum Proteobacteria, Bacteroidetes and Caldiserica were the major
bacterial groups observed in all reactors. δ-Proteobacteria under phylum
Proteobacteria was found to be the most abundant group of bacteria with more than
90% of total clones in all samples whereas phylum Bacteroidetes was the major
bacteria group found in seed sludge (Sample 1) and non-ORP control system (Sample
2) samples. The major clones of phylum Bacteroidetes were most belongs to
Bacteroidia class. Bacteroidia class bacteria presents in the gastrointestinal system of
mammals which allows it to be abundant in the feces (Krieg et al., 2010). Thus, the
presence of these bacteria in the UASB sludge, which is a treated municipal sewage,
is common. Meanwhile, phylum Caldiserica, which was also abundant in all samples,
is a bacterium that is observed with the reduction of sulfur compounds such as
thiosulfate, sulfite and elemental sulfur (Mori et al., 2009). In ORP control system
sample (Sample 3), where AOS reaction occurred, δ-Proteobacteria, WCHB1-03,
Spirochaetes and OP8_1 were slightly increased which seems these bacteria were
probably contribute to the AOS reaction occurrence. However, it cannot be confirmed
!
49!
yet. As we look more into the δ-Proteobacteria class of phylum Proteobacteria which
was the most abundant group of bacteria as shown in Table 3.2, all the bacteria family
are
sulfur
reducing
bacteria
groups
(Garrity
et al.,
2005)
except
for
Syntrophorhabdaceae which decompose aromatic compounds (Qiu et al., 2008) and
Geobacteraceae, an iron reducing bacteria (Holmes et al., 2007). In seed sludge
sample, Syntrophaceae and Syntrophobacteraceae family were the major groups
identified with 60% of total clones. Meanwhile, in non-ORP control system sample,
Desulfobulbaceae and Desulfomicrobiaceae family were the major groups identified
in the sample. In ORP-control system sample where the AOS reaction occurred, all
the
mentioned
bacteria
groups
were
extremely
decreased
except
for
Syntrophobacteraceae and Desulfovibrionaceae family which were significantly
increased with 80% of total clones identified in the sample. However,
Syntrophobacteraceae and Desulfovibrionaceae family are known as sulfur reducing
bacteria (Garrity et al., 2005) and there are no studies mentioned that these bacteria
are sulfur-oxidizing bacteria and related to the AOS reaction.
!
50!
Table 3.1 Overview of phylogenetic affiliations and number of bacteria clones
obtained from the BER chamber samples where AOS reaction occurred.
Sample 1, seed sludge sample; Sample 2, non-ORP control system sample;
Sample 3, ORP control system sample.
!
51!
Table 3.2 Phylogenetic affiliations and number of bacteria clones in each family
specifically for class δ-Proteobacteria obtained from the BER chamber samples
where AOS reaction occurred.
No of clones (％ of total clones)
Bacte ria
Others
Total -Proteobacteria
Sample1
Sample2
Sample3
8 (26.7)
6 (18.2)
15 (41.7)
2 (6.7)
-
1 (2.8)
10 (33.3)
4 (12.1)
2 (5.6)
3 (10.0)
-
-
3 (10.0)
4 (12.1)
15 (41.7)
1 (3.3)
10 (30.3)
-
3 (10.0)
8 (24.2)
2 (5.6)
-
1 (3.0)
-
-
-
1 (2.8)
30 (100)
33 (100)
36 (100)
Sample 1, seed sludge sample; Sample 2, non-ORP control system sample;
Sample 3, ORP control system sample.
The T-RFLP analysis results presented in Fig. 3.3 revealed that the major TRFs were 60, 164, 194, 197, 231, 345 and 592 base pair (bp). Peak 197 bp is the
dominant peak in all samples. In seed sludge sample, all peaks were low except 197
bp peak. Peak 164 bp in non-ORP control system sample was a bit high compared to
seed sludge sample whereas the rest had no changes. Thus, the microbial communities
of both samples are similar. Meanwhile, in ORP control system sample, 60 and 194
bp peaks were higher compared to the other samples. Hence, these two peaks, which
probably represent two different or same species, may have contributed in the AOS
reaction as this reaction occurred in the ORP control system. As mentioned earlier,
both stirring system and ORP control system had AOS reaction occurred. However,
compared to the ORP control system sample, peak 60 and 164 bp decreased
!
52!
meanwhile peak 231 bp increased. From this result, it seems that the microbial
community structures in both systems were different and thus it can be believed that
the microbial community structure may change by stirring process. The T-RFLP
profile from BES added system shows a similar pattern with the ORP control system
profile even though the AOS reaction did not occur in the BES added system. This
indicates that the addition of BES in the sample had no impact towards the bacterial
community presence in the sample even though the amount of BES added was enough
for complete suppression of methanogenic activity. Hence, methane producing
archaea activity may be important for the occurrence of AOS reaction.
Fluorescence intensity (-)
197
60
60
60
164
164
0
231
197
231
60
164
60
194 197
164
231
100
592
(a)
345
592
(b)
345
592
(c)
592
(d)
592
(e)
197
194 197
164
231
194
345
200
345
345
300
400
500
600
700
T-RFs size (bp)
Fig. 3.3 T-RFLP profiles of bacterial 16S rRNA gene from different BER with HhaI
digestion. (a) Seed sludge sample, (b) Non-ORP control system sample, (c) ORP
control system sample, (d) Stirring system sample, (e) BES added system sample.
!
53!
2.0
Sample4
1.0
Demension2
Sample2
0.0
Sample3
Sample5
-1.0
Sample1
-2.0
-2.0
-1.0
0.0
1.0
2.0
Demension1
Fig. 3.4 MDS plot of the T-RFLP data from each BER chambers. Sample 1, seed
sludge sample; Sample 2, non-ORP control system sample; Sample 3, ORP control
system sample; Sample 4, stirring system sample; Sample 5, BES added system
sample.
The two-dimensional plot of MDS scores for the T-RFLP profiles of bacteria
in the UASB reactor and BERs were shown in Fig. 3.4. The ratios of the peak height
of the T-RF detected were statistically calculated in order to determine the differences
in the microbial community structure in each BER. Most notably, the MDS analysis
showed that the samples were well separated between non-ORP control group
(Sample 1 and Sample 2) and ORP control group (Sample 3, Sample 4 and Sample 5).
Sample 1 (seed sludge) and Sample 2 (non-ORP control system), which were located
in the positive region of Dimension 1, were seems to be the closest in distance
compared to the other samples as both samples were not controlled by ORP.
!
54!
However, there is a bit distance between them due to the microbial community
differences as there was AOS reaction occurred in Sample 1 but not in Sample 2
which is consistent with the results of controlling the ORP and other parameters
mentioned earlier. On the other hand, Sample 4 (stirring system) was located a bit far
from the clustered samples (Sample 3 and Sample 5). This shows that there is a bit
differences in the microbial community structure due to the influence of the stirring
cultivation process. The proximity distance of Sample 3 (ORP control system) and
Sample 5 (BES system) indicates that they are closely related and proved that the
addition of BES in the sample did not affect the bacterial community structure of the
sample.
3.4
Conclusion
In this study, the BERs was successfully operated and confirmed that AOS
reaction occurred by controlling the ORP. The microbial community structures
present in the reactors were also successfully identified where Syntrophobacteraceae
and Desulfovibrionaceae family were the most abundant bacteria groups found in the
AOS reaction sample. In addtition, methanogenic activity may be important for the
occurrence of AOS reaction. However, more analyses are still needed for further
characterization and identification of the microbial community structures in the AOS
reaction sample. This is to have a better confirmation on which microbes that really
contribute to the AOS reaction occurrence.
!
55!
References
Garrity, G.M., Brenner, D.J., Krieg, N.R. and Staley, J.T. (eds.) (2005). Bergey's
Manual of Systematic Bacteriology, Volume Two: The Proteobacteria, Part
C: The Alpha-, Beta-, Delta-, and Epsilonproteobacteria. New York, New
York: Springer. ISBN 978-0-387-24145-6.
Holmes, D.E., O’Neil, R.A., Vrionis, H.A., N’Guessan, L.A., Ortiz-Bernad, I.,
Larrahondo, M.J., Adams, L.A., Ward, J.A., Nicoll, J.S., Nevin, K.P., Chavan,
M.A., Johnson, J.P., Long, P.E. and Lovley, D.R. (2007). Subsurface clade of
Geobacteraceae that predominates in a diversity of Fe (III)-reducing
subsurface environments. The ISME Journal, 1: 663-677.
Krieg, N.R., Ludwig, W., Whitman, W.B., Hedlund, B.P., Paster, B.J., Staley, J.T.,
Ward, N., Brown, D. and Parte, A. (Eds.) (2010). Bergey's Manual of
Systematic Bacteriology, Volume 4: The Bacteroidetes, Spirochaetes,
Tenericutes
(Mollicutes),
Dictyoglomi,
Acidobacteria,
Gemmatimonadetes,
Fibrobacteres,
Lentisphaerae,
Fusobacteria,
Verrucomicrobia,
Chlamydiae, and Planctomycetes. New York, New York: Springer. ISBN 9780-387-68572-4.
Lettinga, G., van Velsen, A.F.M., Hobma, S.W., de Zeeuw, W. and Klapwijk, A.
(1980). Use of the upflow sludge blanket (USB) reactor concept for biological
wastewater treatment, especially for anaerobic treatment. Biotechnology and
Bioengineering, 22: 699–734.
Logan, B.E. and Regan, J.M. (2006). Electricity-producing bacteria communities in
microbial fuel cells. Trends in Microbiology, 14: 512-518.
!
56!
Mori, K., Yamaguchi, K., Sakiyama, Y., Urabe, T. and Suzuki, K. (2009).
Caldisericum exile gen. nov., an anaerobic, thermophilic, filamentous
bacterium of a novel bacterial phylum, Caldiserica phyl. nov., originally
called the candidate phylum OP5, and description of Caldisericaceae fam.
nov., Caldisericales ord. nov. and Caldisericia classis nov. International
Journal of Systematic and Evolutionary Microbiology, 59: 2894-2898.
Qiu, Y-L., Hanada, S., Ohashi, A., Harada, H., Kamagata, Y. and Sekiguchi, Y.
(2008). Syntrophorhabdus aromaticivorans gen. nov., sp. nov., the First
Cultured Anaerobe Capable of Degrading Phenol to Acetate in Obligate
Syntrophic Associations with a Hydrogenotrophic Methanogen. Applied and
Rozendal, R.A., Hamelers, H.V.M., Rabaey, K., Keller, J. and Buisman, C.J.N.
(2008). Towards practical implementation of bioelectrochemical wastewater
treatment. Trends in Biotechnology, 26: 450-459.
Takahashi, M., Yamaguchi, T., Kuramoto, Y., Nagano, A., Shimozaki, S., Sumino,
H., Araki, N., Yamazaki, S., Kawakami, S. and Harada, H. (2011).
Performance of a pilot-scale sewage treatment: An up-flow anaerobic sludge
blanket (UASB) and a down-flow hanging sponge (DHS) reactors combined
system by sulfur-redox reaction process under low-temperature conditions.
Bioresource Technology, 102: 753-757.
Thrash, J.C. and Coates, J.D. (2008). Review: Direct and Indirect Electrical
Stimulation of Microbial Metabolism. Environmental Science & Technology,
42: 3921-3931.
!
57!
CHAPTER 4
MOLECULAR CHARACTERIZATION OF ANAEROBIC SULFUROXIDIZING MICROBIAL COMMUNITIES IN UPFLOW ANAEROBIC
SLUDGE BLANKET REACTOR TREATING MUNICIPAL SEWAGE
Abstract
A novel wastewater treatment system consisting of an up-flow anaerobic sludge
blanket (UASB) reactor and a down-flow hanging sponge (DHS) reactor with sulfurredox reaction was developed for treatment of municipal sewage under lowtemperature conditions. In the UASB reactor, a novel phenomenon of anaerobic sulfur
oxidation occurred in the absence of oxygen, nitrite and nitrate as electron acceptors.
The microorganisms involved in anaerobic sulfur oxidation have not been elucidated.
Therefore, in this study, we studied the microbial communities existing in the UASB
reactor that probably enhanced anaerobic sulfur oxidation. Sludge samples collected
from the UASB reactor before and after sulfur oxidation were used for cloning and
terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S
rRNA genes of the bacterial and archaeal domains. The microbial community
structures of bacteria and archaea indicated that the genus Smithella and uncultured
bacteria within the phylum Caldiserica were the dominant bacteria groups.
Methanosaeta spp. was the dominant group of the domain archaea. The T-RFLP
analysis, which was consistent with the cloning results, also yielded characteristic
fingerprints for bacterial communities, whereas the archaeal community structure
yielded stable microbial community. From these results, it can be presumed that these
!
58!
major bacteria groups, genus Smithella and uncultured bacteria within the phylum
Caldiserica, probably play an important role in sulfur oxidation in UASB reactors.
4.1
Introduction
Up-flow anaerobic sludge blanket (UASB) technology has been widely used for
sewage treatment especially in warm regions because of its high treatment efficiency
and cost efficacy (Sato et al., 2007). However, the efficiency of the anaerobic
methanogenic process tends to decrease at low temperature because of the suspension
methanogenic activity (Uemura and Harada, 2000; Yamaguchi et al., 2006). Thus, an
aerobic post-treatment system, which has also been reported to achieve high chemical
oxygen demand (COD) removal at short hydraulic retention time (HRT) and has
proven to be an effective method for various types of wastewater treatment (Chan et
al., 2009), is necessary. In these series of studies for treatment of municipal sewage
under low-temperature conditions, a wastewater treatment system consisting of a
UASB and down-flow hanging sponge (DHS) reactor with a sulfur-redox reaction
was developed. Previous studies showed that the sulfur-redox UASB-DHS system is
applicable for low-strength wastewater treatment under low-temperature conditions
such as below 10ºC with over 90% biochemical oxygen demand (BOD) removal
(Yamaguchi et al., 2006; Takahashi et al., 2011a).
In these series of wastewater treatment studies, an interesting phenomenon
occurred in the UASB reactor tank where anaerobic sulfur oxidation reaction was
initiated without the presence of electron acceptors such as oxygen, nitrate, nitrite and
oxidized-irons. It was presumed that this sulfur oxidation reaction was mediated
biologically by the microorganisms that actuate the reaction. Sulfur-oxidizing
!
59!
microbes catalyze a central step in the global sulfur cycle (Loy et al., 2009). These
microbes share the ability to oxidize reduced sulfur compounds such as sulfide,
elemental sulfur, polythionates and thiosulfate to sulfate as a final oxidation product
(Tang et al., 2009). Many studies have evaluated the characteristics of the sulfuroxidizing microbial community (Dunn et al., 1999; Kletzin, 2008; Shao et al., 2010).
However, information regarding sulfur oxidation under anaerobic conditions and the
actual diversity and distribution pattern of the microorganisms is still inadequate. In
contrast
to
well-characterized
laboratory-cultured
microorganisms,
the
microorganisms in the reactor could have unknown behavior or contain novel or notyet-cultured species (Overmann and van Gemerden, 2000). Therefore, the aim of this
study was to analyze and identify the bacteria and archaea groups that contribute to
the contrasting performance and microbial community characteristics during
anaerobic sulfur oxidation by molecular analysis of the 16S rRNA gene.
4.2
Reactor operation, sample collection and water quality analysis
A 1,178 L UASB reactor with a height of 4.7 m was set up at the municipal
sewage treatment plant in Nagaoka, Niigata, Japan without an open settling
compartment where no light enters (Fig. 4.1). The system was fed with raw sewage
that was supplemented with 150 mg-S/L sodium sulfate. The system was operated at
ambient temperature and the HRT was set to 8 h. The sludge samples were collected
from each port of the UASB reactor on day 91, before sodium sulfate was added, and
on days 111, 167 and 335, after the addition of sodium sulfate. The collected samples
were then kept in a container containing ice during delivery to the laboratory and
!
60!
stored at -20°C for microbial analysis. The temperature, pH, oxidation-reduction
potential (ORP) and dissolved oxygen (DO) were measured on site by portable
devices. Sulfate was analyzed using high-performance liquid chromatography
(HPLC) (Shimadzu LC 20-ADsp) equipped with a Shin-pack IC-A3 column after
filtration using 0.2 µm membrane filter. The mobile phase used consist of 3.2 mM
bis-tris, 8 mM p-hydroxybenzoic acid and 50 mM boric acid at a flow rate of 1.0
ml/min. Sodium sulfate was used as standard. The COD was analyzed using a HACH
water quality analyzer (HACH DR2500). Sulfide concentration was measured by
iodometric titration method based on standard methods published by the Japan
Sewage Works Association (1997).
Fig. 4.1 Schematic diagram of the UASB reactor.
!
61!
DNA extraction, polymerase chain reaction (PCR), cloning and sequencing
Genomic DNA was isolated from approximately 500 mg of sludge sample
using a FastDNA® SPIN Kit for Soil (MP Biomedicals, LLC, USA) according to the
manufacturer’s protocol. The DNA concentration was determined by using a
NanoDrop® Spectrophotometer ND-1000 (Thermo Fisher Scientific, USA). PCR
amplification of the 16S rRNA gene from extracted DNA was conducted using
Premix Ex Taq Kit (Takara Bio, Shiga, Japan). A set of bacteria-specific primers,
EUB338f and UNIV1500r, and a set of archaea-specific primers, ARC109f and
Univ1500r, were used for PCR amplification. Amplification of the 16S rRNA gene
was performed in a final volume of 20 µl containing 20 ng of extracted DNA under
the following conditions: initial denaturation step of 94°C for 2 min, followed by 35
cycles of denaturation at 94°C for 30 s, annealing at 50°C for 1 min, and extension at
72°C for 1 min. The final cycle was followed by final extension at 72°C for 4 min.
The PCR products were purified according to the protocol provided in the QIAquick
PCR purification kit (Qiagen, Hilden, Germany) and then cloned using a TOPO PCR
cloning kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s
protocol. The positive clones were selected and sent to Dragon Genomics Center
(Takara Bio, Yokkaichi, Japan) for sequencing. The raw sequencing data of the 16S
rRNA gene were analyzed for possible chimerism using the Bellerophon program
(http://comp-bio.anu.edu.au/bellerophon/bellerophon.pl).
Sequences
with
97%
similarity were then grouped into operational taxonomic units (OTUs) using the
FastGroup II program (http://biome.sdsu.edu/fastgroup/index.htm) and classified
using
the
Classifier
tool
from
the
Ribosomal
Database
Project
(RDP)
(http://rdp.cme.msu.edu/).
!
62!
Phylogenetic analysis
Phylogenetic analyses were conducted using the 16S rRNA gene sequence.
The 16S rRNA gene sequences were aligned with an ARB data set using ARB
software and the resulting alignment was re-corrected manually considering primary
and secondary structures. A phylogenetic tree was constructed using the neighborjoining method and a bootstrap analysis of 1,000 replicate data sets was conducted
using the ARB software for tree topology confidence estimation.
Terminal-restriction fragment length polymorphism (T-RFLP) analysis
PCR was performed by using fluorescently labeled primers 907r and
ARC912r for bacteria and archaea, respectively. HaeIII and HhaI were selected to
digest the PCR products of bacteria, whereas TaqI was selected to digest the PCR
products of archaea 16S rRNA gene purified with the QIAquick PCR purification kit
(Qiagen, Hilden, Germany). The digested PCR products were then run through a
CEG-2000XL capillary sequencer (Beckman Coulter, Fullerton, CA) as described
previously by Hatamoto et al. (2007). In silico T-RF prediction was performed using
TRiFLe as described by Junier et al. (2008) and the predicted T-RFs were correlated
with the cloning results.
4.3
Reactor performance
Over one year of operation, the COD, BOD, sulfate and sulfide concentration of
the process performance were recorded (Fig. 4.2). The average total COD
concentration of the UASB effluent was 162.4 mg/L with a removal rate of 48.1%.
!
63!
The total BOD concentration of the UASB effluent was 67.4 mg/L with a 57.2%
removal rate. This reactor performance is common in UASB reactors treating
domestic sewage under ambient conditions (Takahashi et al., 2011a; Sumino et al.,
2007). Before addition of sodium sulfate, the average UASB influent sulfate
concentration was 7 mg-S/L and the effluent sulfide concentration was 29 mg-S/L.
Sodium sulfate was added to the influent sewage for the reproduction of the anaerobic
sulfur oxidation reaction because based on our previous analysis, anaerobic sulfur
oxidation had occurred when approximately 40-150 mg-S/L sodium sulfate was
added (Sumino et al., 2003). After addition of sodium sulfate, the average UASB
influent sulfate concentration was 54 mg-S/L and the effluent sulfide concentration
was 29 mg-S/L. The occurrence of the sulfur oxidation reaction in the UASB reactor
can be determined through the changes in the sulfate and sulfide concentrations
during the operation. Our results (Fig. 4.2) suggest that the sewage temperature affect
the concentrations of sulfate and sulfide in the UASB reactor. The level of oxidized
sulfur as indicated by the increase in sulfate concentration was inversely proportional
to the temperature after addition of sodium sulfate, where sulfide was oxidized to
sulfate during the cold season (average of 13.1°C) but was not oxidized during the hot
season (average of 23.3°C).
This finding is supported by the sulfate and sulfide concentrations in the UASB
reactor on days 91, 111, 167 and 335 (Fig. 4.3). Over the 91 days of operation, which
was before the addition of sodium sulfate, the temperature was 23.2°C and sulfur
oxidation did not occur. On day 111, which was 13 days after sodium sulfate addition,
the temperature slightly decreased to 18.7°C but sulfur oxidation still did not occur,
which indicates that the microbial community had not changed. Sulfur oxidation
occurred after 167 days of operation where the temperature dropped gradually and
!
64!
sulfate concentration increased, which means the microbial community had changed.
However, the sulfur oxidation stopped after day 335 when the temperature started to
increase (23.4°C). When sulfur oxidation occurred, the ORP was always less than
-300 mV, and the DO, nitrate and nitrite were not detected, which shows that
anaerobic conditions were maintained. Additionally, lower sewage temperature seems
to be important for the occurrence of the anaerobic sulfur oxidation reaction.
Fig. 4.2 COD, sulfate and sulfide concentrations in the UASB reactor.
!
65!
Fig. 4.3 UASB profiles of sulfide and sulfate concentrations along the reactor height.
Table 4.1 Phylogenetic affiliation and numbers of bacterial 16S rRNA gene clones in
the UASB sludge.
!
66!
Table 4.2 Phylogenetic affiliation and numbers of archaeal 16S rRNA gene clones in
the UASB sludge.
Phylogenetic analysis of bacterial and archaeal microbial communities
The microbial community present in the UASB reactor was successfully
characterized through microbial 16S rRNA gene analyses. Samples from days 91, 111
and 167 were used for cloning analysis to monitor changes in microbial community
structure between sulfur oxidation and non-sulfur oxidation conditions. The 16S
rRNA cloning analysis results shown in Table 4.1 and 4.2 indicate that bacteria and
archaea species were present in the reactor. Among the bacteria species, the major
bacterial groups in the UASB reactor were phylum Proteobacteria comprising an
average of 39% of the total clones, followed by the phyla Firmicutes (18%),
Bacteroidetes (16%) and Caldiserica (12%). The acetate-utilizing methanogen
Methanosaeta spp. was the dominant group of the domain archaea at all times. The
microbial structure was almost the same as that of other UASB reactors treating
sewage except for phylum Caldiserica (Takahashi et al., 2011b). However, at the
genus level, genus Smithella of phylum Proteobacteria was the major bacterial group
!
67!
observed in the UASB reactor comprising an average of 19% of the total clones,
followed by uncultured bacteria of phylum Caldiserica (12%). Within phylum
Proteobacteria, which was the most abundant group of bacteria, various genera were
present and most of the bacteria are sulfur-reducing bacteria (SRB) including the most
dominant phylotype, genus Smithella (Garrity et al., 2005).
Mori et al. (2008, 2009) discovered a new bacteria strain that represented a new
genus and novel species named Caldisericum exile within the phylum Caldiserica.
This strain was previously known as uncultured phylum OP5 and obtained from
anaerobic environments. Anaerobic growth of this bacterium was observed with the
reduction of sulfur compounds such as thiosulfate, sulfite and elemental sulfur but
without the presence of sulfate, fumarate, nitrate, nitrite and oxygen as electron
acceptors. Furthermore, some of the OP5 clones were retrieved from relatively sulfurrich environments. Based on the description of Mori et al., the uncultured bacteria of
phylum Caldiserica found in this study were not closely related and had low sequence
similarity (the sequence similarity of the 16S rRNA gene was approximately 83%) to
any other known bacteria in recognized phyla (shown in Fig. 4.4). Therefore, the
uncultured bacteria of phylum Caldiserica present in this UASB reactor were
probably involved in the sulfur cycle and may thus have participated in the sulfur
oxidation and/or sulfate reduction reactions in the UASB reactor.
!
68!
Fig. 4.4 Phylogenetic relationships of bacterial 16S rRNA gene sequences constructed
using neighbor-joining method. The sequences determined in this study are shown in
bold.
Other dominant bacterial species were members of the genus Smithella but these
phylotypes were phylogenetically related to S. propionica (the sequence similarity of
the 16S rRNA gene was approximately 97%). S. propionica only grows in anaerobic
conditions and is unable to use sulfate as an electron acceptor (Liu et al., 1999); it is
also able to grow on propionate in syntrophic association with methanogens. S.
propionica oxidizes propionate to acetate, carbon dioxide and hydrogen, particularly
acetate. Under this condition where acetate is extensively produced, Methanosaeta
spp., which have high affinity for acetate (Smith and Ingram-Smith, 2007), will
present a higher growth rate and higher yield, as observed in this study.
As mentioned above, most of the bacteria groups found in the UASB reactor
were SRB and no signs of sulfur-oxidizing bacteria (SOB) were present. Previous
!
69!
reports indicated that members of genera such as Acidianus (Friedrich, 1998),
Bacillus (Aragno, 1992), Beggiatoa (Strohl, 1989), Paracoccus, Pseudomonas and
Thiobacillus (Friedrich and Mitrenga, 1981) in aerobic conditions; Allochromatium
(formerly known as Chromatium) (Imhoff, 1998), Chlorobium, Rhodobacter,
Rhodopseudomonas and Rhodovulum (Brune, 1989) in anaerobic conditions; and the
purple sulfur bacteria Thiocapsa roseopersicina (Kondratieva et al., 1976) and
Allochromatium vinosum (Kämpf and Pfennig, 1986) in addition to the purple nonsulfur bacteria Rhodovulum sulfidophilum (Hiraishi and Umeda, 1994), Rhodocyclus
genatinosus and Rhodopseudomonas acidophila (Kondratieva, 1989; Siefert and
Pfennig, 1979) which grows in the dark, are frequently observed SOB (Friedrich et
al., 2001). However, none of these bacteria groups existed in the UASB reactor
during the entire operating period. Therefore, it is likely that the SRB present in the
UASB reactor had another function as oxidizing bacteria in sulfur oxidation reaction
in an unknown manner.
One possibility of anaerobic sulfur oxidation is the reverse reaction of anaerobic
oxidation of methane (AOM) with sulfate reduction reaction. Actually, AOM
consortia could catalyze the reverse direction of AOM with sulfate (Holler et al.,
2011). A consortium consisting of anaerobic methanotrophic (ANME) archaea and
SRB mediates the AOM with sulfate (Knittel et al., 2005). In the UASB reactor,
ANME-related archaea was not detected, however, methanogenic archaea and SRB
were detected. Thus, the SRB present in the UASB reactor may play a key role in
anaerobic sulfur oxidation reaction. Nevertheless, further research is needed to
elucidate the phenomena.
!
70!
Fig. 4.5 T-RFLP profile of bacterial (HaeIII and HhaI) and archaeal (TaqI) 16S rRNA
gene from UASB sludge on different sampling days. A, Before sodium sulfate
addition (Day 91); B, After sodium sulfate addition (Day 111); C, Sulfur oxidation
occurrence (Day 167); D, Sulfur oxidation end (Day 335).
!
71!
Succession of microbial community
The T-RFLP profile presented in Fig. 4.5 confirms the trend detected in the
clone libraries. The T-RFLP analysis of bacteria showed some changes before and
after the addition of sulfate with characteristic fingerprints for bacterial communities
obtained using both HaeIII and HhaI restriction enzymes (REs). The archaeal
community structure demonstrated stable microbial community until the end of the
oxidation process using TaqI RE where the predominant T-RF corresponds to
Methanosaeta spp. HaeIII and HhaI were used for fingerprinting of bacterial
communities because each RE has its own limitation in deriving certain terminal
restriction fragment sizes that can be measured within a measurable size range
(Engebretson and Moyer, 2003). As shown in Fig. 4.5, when using HaeIII, T-RF sizes
lower than 50 bp, where the simulated peak determined by TRiFLe for uncultured
bacteria of phylum Caldiserica (~33 bp) is located, could not be measured, whereas
when using HhaI, T-RF sizes higher than 500 bp, where the simulated peak for
Smithella (~561 bp) is located, could not be measured.
The T-RFLP profiles showed consistent results with the cloning analysis
where the major peaks observed corresponded to the genus Smithella (~185 bp) and
uncultured bacteria of phylum Caldiserica (~197 bp) in the HaeIII and HhaI T-RF
profiles, respectively. Several peaks showed changes in their profiles during the
oxidation reaction. During the oxidation period, the height of some of the peaks in the
HaeIII profile such as the peaks at 183 and 185 bp increased, and these peaks were
identified as representing the genera Desulfarculus and Smithella, respectively. When
the oxidation ended, some peaks such as those at 181 bp (representing genus
Desulfovibrio) and 183 bp disappeared, and the height of some of the peaks such as
the peaks at 71 bp (representing genus Bacteroides) and 185 bp decreased. In the
!
72!
HhaI profile, the height of the peaks at 68, 163, 165 and 197 bp increased during the
oxidation period but then decreased when the oxidation ended. These peaks
represented the genera Desulfobulbus, Desulfitibacter and Desulfarculus, and
uncultured bacteria of phylum Caldiserica, respectively. The peaks that disappeared
or showed reduced magnitude when the oxidation ended thus probably represented
the bacteria groups contributing to the sulfur oxidation reaction.
4.4
Conclusion
In summary, these findings allowed us to identify the microbial community
structures of the bacterial and archaeal groups present in the UASB reactor. The
bacterial community presented high diversity, as determined by the 16S rRNA gene
analysis, whereas the archaeal community had relatively low and simple diversity. It
can therefore be concluded that (i) uncultured bacteria within the phylum Caldiserica
and genus Smithella probably play an important role in sulfur oxidation, (ii) some
SRB probably have a dual function and play a role in sulfur oxidation, and/or (iii)
missing peaks and peaks with the decreased height during the non-oxidation condition
probably represent bacteria involved in sulfur oxidation in the UASB reactor.
However, further analyses are necessary for a deeper understanding of microbial
diversity and its contribution to the anaerobic sulfur oxidation reaction in the UASB
reactor.
!
73!
References
Aragno, M. (1992). Aerobic, chemolithoautotrophic, thermophilic bacteria. In
Kristjansson, J.K. (ed.), Thermophilic Bacteria, CRC Press Inc., Boca Raton
U.S.A.. p. 77-103.
Brune, D.C. (1989). Sulfur oxidation by phototrophic bacteria. Biochimica et
Biophysica Acta, 975: 189-221.
Chan, Y.J., Chong, M.F., Law, C.L. and Hassell, D.G. (2009). A review on anaerobicaerobic treatment of industrial and municipal wastewater. Chemical
Engineering Journal, 155: 1-18.
Dunn, J.P., Stenger Jr., H.G. and Wachs, I.E. (1999). Oxidation of sulfur dioxide over
supported vanadia catalysts: molecular structure - reactivity relationships and
reaction kinetics. Catalysis Today, 51: 301-318.
Engebretson, J.J. and Moyer, C.L. (2003). Fidelity of select restriction endonucleases
in determining microbial diversity by terminal-restriction fragment length
polymorphism. Applied and Environmental Microbiology, 69: 4823-4829.
Friedrich, C.G. and Mitrenga, G. (1981). Oxidation of thiosulfate by Paracoccus
denitrificans and other hydrogen bacteria. FEMS Microbiology Letters, 10:
209-212.
Oxidation of reduced inorganic sulfur compounds by bacteria: emergence of a
Friedrich, C.G. (1998). Physiology and genetics of sulfur-oxidizing bacteria.
Advances in Microbial Physiology, 39: 235-289.
!
74!
Garrity, G.M., Brenner, D.J., Krieg, N.R. and Staley, J.T. (eds.) (2005). Bergey's
Manual of Systematic Bacteriology, Volume Two: The Proteobacteria, Part C:
The Alpha-, Beta-, Delta-, and Epsilonproteobacteria. New York, New York:
Springer.
Hatamoto, M., Imachi, H., Yashiro, Y., Ohashi, A. and Harada, H. (2007). Diversity
of anaerobic microorganisms involved in long-chain fatty acid degradation in
methanogenic sludges as revealed by RNA-based stable isotope probing.
Hiraishi, A. and Umeda, Y. (1994). Intragenic structure of the genus Rhodobacter:
transfer of Rhodobacter sulfidophilus and related marine species to the genus
Rhodovulum gen. nov. International Journal of Systematic Bacteriology, 44:
15-23.
Holler, T., Wegener, G., Niemann, H., Deusner, C., Ferdelman, T.G., Boetius, A.,
Brunner, B. and Widdel, F. (2011). Carbon and sulfur back flux during
anaerobic microbial oxidation of methane and coupled sulfate reduction.
America, 108: E1484-E1490.
Imhoff, J.F., Süling, J. and Petri, R. (1998). Phylogenetic relationship and taxonomic
reclassification of Chromatium species and related purple sulfur bacteria.
International Journal of Systematic Bacteriology, 48: 1029-1043.
Japan Sewage Works Association (1997). Japanese standard testing methods for
sewage, Japan Sewage Works Association, Tokyo, Japan. (in Japanese)
Junier, P., Junier, T. and Witzel, K.-P. (2008). TRiFLe, a program for in silico
terminal restriction fragment length polymorphism analysis with user-defined
sequence sets. Applied and Environmental Microbiology, 74: 6452-6456.
!
75!
Kämpf, C. and Pfennig, N. (1986). Chemoautotrophic growth of Thiocystis violacea,
Chromatium gracile and C. vinosum in the dark at various O2-concentrations.
Journal of Basic Microbiology, 26: 517-531.
Kletzin, A. (2008). Oxidation of sulfur and inorganic sulfur compounds in Acidianus
ambivalens. In Dahl, C. & Friedrich, C.G., (ed.), Microbial Sulfur
Metabolism, Springer Verlag, Berlin, p. 184–201.
Knittel, K., Lösekann, T., Boetius, A., Kort, R. and Amann, R. (2005). Diversity and
Distribution of Methanotrophic Archaea at Cold Seeps. Applied and
Kondratieva, E.N., Zhukov, V.G., Ivanovsky, R.N., Petushkova, Yu.P. and Monosov,
E.Z. (1976). The capacity of phototrophic sulfur bacterium Thiocapsa
roseopersicina for chemosynthesis. Archives of Microbiology, 108: 287-292.
Kondratieva, E.N. (1989). Chemolithotrophy of phototrophic bacteria. In Schlegel,
H.G. and Bowien, B. (ed.), Autotrophic bacteria, Springer, Berlin, and Science
Tech Publishers, Madison, WI, p. 283-287.
Liu, Y., Balkwill, D.L., Aldrich, H.C., Drake, G.R. and Boone, D.R. (1999).
Characterization of the anaerobic propionate-degrading syntrophs Smithella
propionica gen. nov., sp. nov. and Syntrophobacter wolinii. International
Journal of Systematic Bacteriology, 49: 545-556.
Loy, A., Duller, S., Baranyi, C., Mußmann, M., Ott, J., Sharon, I., Béjà, O., Paslier,
D.L., Dahl, C. and Wagner, M. (2009). Reverse dissimilatory sulfite reductase
as phylogenetic marker for a subgroup of sulfur-oxidizing prokaryotes.
Mori, K., Sunamura, M., Yanagawa, K., Ishibashi, J.-i., Miyoshi, Y., Iino, T., Suzuki,
K.-i. and Urabe, T. (2008). First cultivation and ecological investigation of a
!
76!
bacterium affiliated with the candidate phylum OP5 in hot springs. Applied
and Environmental Microbiology, 74: 6223-6229.
Mori, K., Yamaguchi, K., Sakiyama, Y., Urabe, T. and Suzuki, K. (2009).
Caldisericum exile gen. nov., an anaerobic, thermophilic, filamentous
bacterium of a novel bacterial phylum, Caldiserica phyl. nov., originally
called the candidate phylum OP5, and description of Caldisericaceae fam.
nov., Caldisericales ord. nov. and Caldisericia classis nov. International
Overmann, J. and van Gemerden, H. (2000). Microbial interactions involving sulfur
bacteria: implications for the ecology and evolution of bacterial communities.
FEMS Microbiology Reviews, 24: 591-599.
Sato, N., Okubo, T., Onodera, T., Lalit, K.A., Ohashi, A. and Harada, H. (2007).
Economic evaluation of sewage treatment processes in India. Journal of
Environmental Management, 84: 447-460.
Shao, M.-F., Zhang, T. and Fang, H.H.-P. (2010). Sulfur-driven autotrophic
denitrification: diversity, biochemistry, and engineering applications. Applied
Microbiology and Biotechnology, 88: 1027-1042.
Siefert, E. and Pfennig, N. (1979). Chemoautotrophic growth of Rhodopseudomonas
species with hydrogen and chemotrophic utilization of methanol and formate.
Smith, K.S. and Ingram-Smith, C. (2007). Methanosaeta, the forgotten methanogen?
Trends in Microbiology, 15: 150-155.
Strohl, W.R. (1989). Beggiatoales. In Stanley, J.T., Bryant, M.P., Pfennig, N. and
Holt, J.G. (ed.), Bergey's Manual of Systematic Bacteriology, Volume Three,
Williams & Wilkins, Baltimore, p 2089-2106.
!
77!
Sumino, H., Takahashi, M., Yamaguchi, T., Abe, K., Araki, N., Yamazaki, S.,
Shimozaki, S., Nagano, A. and Nishio, N. (2007). Feasibility study of a pilotscale sewage treatment system combining an up-flow anaerobic sludge blanket
(UASB) and an aerated fixed bed (AFB) reactor at ambient temperature.
Sumino, H., Yamaguchi, T., Tanikawa, D., Okazaki, Y., Araki, N., Kawakami, S.,
Yamazaki, S. and Harada, H. (2003). Sewage treatment by sulfur redox cycle
action in a system consisting of a UASB pre-reactor and a aerobic postreactor. Environmental Engineering Research, 40: 431-440. (in Japanese)
Takahashi, M., Ohya, A., Kawakami, S., Yoneyama, Y., Onodera, T., Syutsubo, K.,
Yamazaki, S., Araki, N., Ohashi, A., Harada, H. and Yamaguchi, T. (2011b).
Evaluation of treatment characteristics and sludge properties in a UASB
reactor treating municipal sewage at ambient temperature. International
Journal of Environmental Research, 5: 821-826.
Takahashi, M., Yamaguchi, T., Kuramoto, Y., Nagano, A., Shimozaki, S., Sumino, H.,
Araki, N., Yamazaki, S., Kawakami, S. and Harada, H. (2011a). Performance
of a pilot-scale sewage treatment: An up-flow anaerobic sludge blanket
(UASB) and a down-flow hanging sponge (DHS) reactors combined system
by sulfur-redox reaction process under low temperature conditions.
Tang, K., Baskaran, V. and Nemati, M. (2009). Bacteria of the sulphur cycle: An
Uemura, S. and Harada, H. (2000). Treatment of sewage by a UASB reactor under
moderate to low temperature conditions. Bioresource Technology, 72: 275-
!
78!
282.
Yamaguchi, T., Bungo, Y., Takahashi, M., Sumino, H., Nagano, A., Araki, N., Imai,
T., Yamazaki, S. and Harada, H. (2006). Low strength wastewater treatment
under low temperature conditions by a novel sulfur redox action process.
Water Science and Technology, 53: 99–105.
!
79!
CHAPTER 5
DIVERSITY PROFILE OF MICROBES ASSOCIATED WITH ANAEROBIC
SULFUR OXIDATION IN AN UPFLOW ANAEROBIC SLUDGE BLANKET
REACTOR TREATING MUNICIPAL SEWAGE
Abstract
An analysis of the diversity of microbes involved in anaerobic sulfur oxidation in an
upflow anaerobic sludge blanket (UASB) reactor used for treating municipal sewage
under low-temperature conditions was conducted. Anaerobic sulfur oxidation
occurred in the absence of oxygen, nitrite and nitrate as electron acceptors; yet,
reactor performance parameters demonstrated that anaerobic conditions were
maintained. In order to gain insights into the underlying basis of anaerobic sulfur
oxidation, the microbial diversity exist in the UASB sludge were analyzed
comprehensively to determine their identities and contribution to sulfur oxidation.
The sludge samples were collected from the UASB reactor over a period of 2 years
and used for bacterial 16S rRNA gene-based terminal restriction fragment length
polymorphism (T-RFLP) and next-generation sequencing analyses. Both T-RFLP and
sequencing results showed that microbial community patterns changed markedly from
day 537 onwards. Bacteria belonging to the genus Desulforhabdus within the phylum
Proteobacteria and uncultured bacteria within the phylum Fusobacteria were the
major groups observed during the period of anaerobic sulfur oxidation. Their
abundance was shown to correlate with temperature, suggesting that these bacterial
groups are perhaps involved with anaerobic sulfur oxidation in UASB reactors.
!
80!
5.1
Introduction
Microorganisms are diverse and complex life forms. They play varied roles in
the cycles of elements such as sulfur, nitrogen, carbon and iron, and have an
important environmental impact. In the sulfur cycle, sulfur-oxidizing and –reducing
bacteria play various crucial roles in different anaerobic environments; they also
represent a key element in biological wastewater treatment plants (Tang et al., 2009).
Sulfur-oxidizing bacteria (SOB), in particular, are the main microorganisms
contributing to the bioremediation of the sulfide-rich wastewater generated by many
industries such as the petroleum, mining, textile dyeing, pulp and paper, food
processing and sulfate-containing wastewater treatment industries, as well as by
tanneries (Janssen et al., 1999; Tang et al., 2009). SOB also play a role in wastewater
treatment that involves anaerobic sulfur oxidation in upflow anaerobic sludge blanket
(UASB) reactors that are used for the treatment of municipal sewage (Ono et al.,
2011).
In general, SOB oxidize hydrogen sulfide, sulfur, sulfite, thiosulfate and
polythionates such as tri-, tetra- and pentathionate to sulfate as the major oxidation
product under acidic, neutral or alkaline environments (Rohwerder and Sand, 2007).
Oxidation of these reduced sulfur compounds is mainly associated with the
chemolithotrophic
and
photoautotrophic
bacteria
(Friedrich
et al.,
2005).
Chemolithotrophic bacteria, which are also referred to as colorless sulfur bacteria, use
oxygen or oxidized-iron under aerobic conditions or nitrate and nitrite under
anaerobic conditions as the terminal electron acceptors. As with photoautotrophic
bacteria, carbon dioxide is used as the terminal electron acceptor by green and purple
sulfur bacteria under anaerobic conditions (Friedrich et al., 2001; Tang et al., 2009).
!
81!
Many studies have reported the structures and activities of microbial
communities present in the anaerobic bioreactors that are used for treating various
types of wastewater under different parameters (Casserly and Erijman, 2003; Buzzini
et al., 2006; Briones et al., 2009; Narihiro et al., 2009; Li et al., 2011; Khemkhao et
al., 2012). However, the knowledge and understanding of the mechanism of anaerobic
sulfur oxidation as well as the interactions between microbial communities and
environmental components remains incomplete and undefined in some areas. There
are significant differences in physiology among the many bacterial species albeit they
share certain structural, genetic and metabolic characteristics.
Therefore, in this study, an analysis of the abundance, distribution,
characteristics and phylogenetic diversity of microorganisms exist in the UASB
reactor over a period of 2 years was conducted to gain greater insights into microbial
biodiversity and its role in anaerobic sulfur oxidation. This will also eventually help
in supplementing novel and additional information to the present microbial
biodiversity data.
5.2
Reactor operation, sample collection and water quality analysis
A closed settling compartment UASB reactor with a total volume of 1,178 L
and a height of 4.7 m was operated at ambient temperature. The hydraulic retention
time of the system was set to 8 h. Additional detail on the UASB reactor has been
described in a previous report (Takahashi et al., 2011). The system was fed with raw
sewage that was added with 150 mg-S/L sodium sulfate initially and after that
maintained to 50 mg-S/L sodium sulfate throughout the operation. The system was set
!
82!
up at the municipal sewage treatment plant in Nagaoka, Niigata, Japan. Before and
after the addition of sodium sulfate, sludge samples were collected from port 5 of the
UASB reactor, which is located 1.278 m from the bottom of the reactor, over a period
of 2 years of operation and kept in a container containing ice during delivery to the
laboratory.
The
collected
samples
were
then
immediately
stored
at
-20°C until required for microbial analysis. Portable devices were used to measure the
temperature and pH by using pH meter (TOA DKK HM-20P), oxidation-reduction
potential (ORP) by using ORP meter (TOA DKK RM-20P) and dissolved oxygen
(DO) by using DO meter (YSI 58-115V) on site. A high-performance liquid
chromatography (HPLC) system (Shimadzu LC 20-ADsp) was used to analyze
sulfate, nitrate and nitrite contents, whereas a HACH water quality analyzer (HACH
DR2500) was used for chemical oxygen demand (COD) and iron analysis. Sulfide
analysis was conducted according to the standard methods published by the Japan
Sewage Works Association (1997) which was briefly described as follows: hydrogen
sulfide gas, which was produced after the addition of sulfuric acid into the sample,
was absorbed into the zinc acetate solution and produced zinc sulfide. Iodine solution
and hydrochloric acid were then added into the solution containing zinc sulfide.
Starch was also added into the solution as an indicator, which produced a color
change from blue to transparent after titrated with sodium thiosulfate to measure the
sulfide content.
DNA extraction, polymerase chain reaction (PCR) and terminal-restriction
fragment length polymorphism (T-RFLP) analysis
Genomic DNA isolation from the sludge samples was performed using a
FastDNA SPIN Kit for Soil (MP Biomedicals, LLC, Carlsbad, CA, USA) according
!
83!
to the manufacturer’s protocol. The DNA concentration was determined using a
NanoDrop Spectrophotometer ND-1000 (Thermo Fisher Scientific, Waltham, MA,
USA). PCR was performed by using a set of bacteria-specific primers, EUB338f and
fluorescently labeled primers 907r (Hatamoto et al., 2007a) under the following
conditions: an initial denaturation step of 94°C for 2 min, followed by 35 cycles of
denaturation at 94°C for 30 s, annealing at 50°C for 1 min, and extension at 72°C for
1 min. The final cycle was followed by final extension at 72°C for 4 min. HhaI
restriction enzyme was used to digest the PCR products of bacteria 16S rRNA gene
fragments that had been purified with a QIAquick PCR purification kit (Qiagen,
Hilden, Germany) and were then analyzed through a CEG-2000XL capillary
sequencer (Beckman Coulter, Fullerton, CA, USA) as described previously by
Hatamoto et al. (2007b). The TRiFLe program was used to perform an in silico
terminal restriction fragment (T-RF) prediction as described by Junier et al. (2008)
and the predicted T-RFs were then correlated with the sequencing results.
Next-generation sequencing and data analysis
PCR amplification of 16S rRNA gene from extracted DNA for sequencing
was carried out according to Caporaso et al. (2011) by using the universal primers
515F and 806R. Reactions were held at 94°C for 3 min as an initial denaturation step,
with amplification proceeding for 35 cycles at 94°C for 45 s, 50°C for 60 s, and 72°C
for 90 s. A final extension of 10 min at 72°C was added to ensure complete
amplification. The PCR products were purified according to the protocol provided in
the QIAquick PCR purification kit (Qiagen, Hilden, Germany) and were then
analyzed through MiSeq sequencer (Illumina San Diego, CA, USA) targeting the V4
region of the bacterial 16S rRNA gene (Caporaso et al., 2011).
!
84!
The sequencing data of the 16S rRNA gene were analyzed using Quantitative
Insights Into Microbial Ecology (QIIME) software for microbial community analysis
(Caporaso et al., 2010). The 100 base reads were cut with quality scores of two or
more consecutive base calls below 1e-5. A minimum length of 75 bases was required
for inclusion in the analysis and any reads that contained N character were discarded.
The operational taxonomic unit (OTU) picking was performed by clustering the
sequence at 97% identity using UCLUST (Edgar, 2010) and blast against the SILVA
database (Quast et al., 2013). The BLAST matches were chosen based on the E-value
(maximum value is 1e-10), the percent sequence identity of the alignment between a
BLAST match and the read which must be greater than or equal to the OTU selection
threshold (0.97 here, corresponding to species-like OTUs), and the match that
achieves the longest alignment to the read. Chimeric sequences were identified with
ChimeraSlayer (Haas et al., 2011). Alpha diversity was determined using the
calculation of observed species, Chao1, Phylogenetic diversity (PD), Shannon index,
Simpson index and sampling intensity (coverage) at each sampling depth. Weighted
UniFrac, a quantitative measure of beta diversity, was used to perform principal
coordinate analysis (PCoA) to determine the similarities between samples (Lozupone
et al., 2007).
Sequence data accession number
The nucleotide sequence data was deposited to the DDBJ Sequence Read
Archive under accession number DRA002293.
!
85!
5.3
Performance of the UASB reactor
As shown in Fig. 5.1, changes in sewage temperature influenced the
concentrations of reduced sulfur (sulfide) and oxidized sulfur (sulfate) throughout the
2 years of operation of the reactor. After addition of sodium sulfate, the level of
oxidized sulfur increased during low temperature periods but decreased during high
temperature periods. This suggested that lower sewage temperature is essential for
stimulating anaerobic sulfur oxidation reaction to be occurred. This can be further
seen in the UASB profiles during anaerobic sulfur reduction and oxidation occurrence
period shown in Fig. 5.2. The UASB profiles showed that during high temperature
(Fig. 5.2A), a high sulfate concentration of the UASB influent decreased significantly
at the bottom part of the reactor and from then a low sulfate concentration was
observed until the top part of the reactor, whereas a low sulfide concentration
increased significantly at the bottom part and maintained a high concentration until
the top part of the reactor which indicates that anaerobic sulfur reduction reaction had
occurred. During low temperature (Fig. 5.2B), a high sulfate concentration of the
UASB influent was reduced to sulfide at the bottom part until the middle part of the
reactor, but at the middle part of the reactor, the concentration of sulfide decreased
whereas the sulfate concentration increased which indicates that anaerobic sulfur
oxidation reaction had occurred.
The average total BOD concentration of the UASB effluent was 85.1 mg/L
with a removal rate of 46.7%, whereas the average total COD concentration of the
UASB effluent was 176.4 mg/L with a 45.5% removal rate. The average pH and ORP
during the operational period was 7.0 and -281 mV, respectively. The average total
!
86!
iron (Fe), ferrous iron (Fe2+) and ferric iron (Fe3+) concentrations of the UASB
influent were 0.6 mg/L, 0.4 mg/L and 0.2 mg/L, respectively. These iron
concentration results were consistent with the facts that municipal wastewater
typically contains metals in small amounts and the concentration level of particularly
iron is typically 0.4-1.5 mg/L (Henze et al., 1997; Hvitved-Jacobsen, 2002; Nielsen et
al., 2005). Fumarate, which is the salt and esters of fumaric acid, biodegrades faster in
more polluted waters, thus it does not have a lasting effect in sewage and therefore,
the measurement of fumarate concentration of the UASB influent was unnecessary
(Howard, 1997). More details on the performance of the UASB reactor are shown in
Table 5.1. From the data observed, the total BOD removal rate showed a significant
reduction during low temperature periods which was consistent with the results
reported by Singh and Viraraghavan (2003). Besides that, these data indicated a stable
system and were typical for the operation of such a reactor maintained under
anaerobic conditions, where no indications of DO, nitrate or nitrite were identified.
This suggested that, contrary to earlier report (Ono et al., 2011), the sulfur oxidation
reaction in this study occurred in the absence of any electron acceptors.
!
87!
30
50
20
25
10
0
Temperature ( )
Sulfur (mg-S/L)
Sodium sulfate added
75
0
0
100
200
300
400
500
600
700
800
Time (days)
Fig. 5.1 Time course of the reduced ( ) and oxidized ( ) sulfur concentration and
influent sewage temperature (–) of the UASB reactor. Reduced and oxidized sulfur
concentrations were calculated from the UASB profile results.
Fig. 5.2 UASB profiles of sulfide and sulfate. A – non-occurrence of anaerobic sulfur
oxidation; B – occurrence of anaerobic sulfur oxidation.
!
88!
Table 5.1 Operational conditions and performance of the UASB reactor.
Influent
temperature
(°C)
Influent
pH
91
23.5
6.7
111
18.2
167
Sample
(day)
Influent
ORP
(mV)
Sulfide
concentration
(mg-S/L)
Sulfate
concentration
(mg-S/L)
Influent nitrate
concentration
(mg-N/L)
Influent nitrite
concentration
(mg-N/L)
Influent
ammonium
concentration
(mg-N/L)
DO
(mg/L)
BODtotal
removal
rate
(%)
CODtotal
removal
rate
(%)
Influent
Effluent
Influent
Effluent
-258
5.5
9.6
9.2
1.1
0.0
0.0
32.6
0.1
93
57
6.9
-224
0.0
19.5
142.4
129.9
0.0
0.0
14.8
0.1
79
54
11.6
6.8
-306
0.0
19.0
92.4
75.4
0.0
0.0
23.2
0.5
37
27
214
10.9
7.0
-245
0.0
27.2
83.6
44.6
0.0
0.0
28.4
0.5
31
39
255
12.6
7.3
-258
0.0
32.8
69.1
71.1
0.0
0.0
33.2
0.3
39
31
284
16.0
7.0
-211
2.2
27.7
41.4
8.6
0.0
0.0
43.5
0.2
64
32
335
22.9
7.2
-279
4.0
43.0
51.0
1.8
0.0
0.0
29.2
0.2
51
48
363
26.4
6.8
-278
5.7
39.1
45.0
2.5
0.0
0.0
32.4
0.2
63
58
379
25.4
6.7
-275
11.3
41.2
47.0
4.2
0.1
0.0
26.5
0.2
80
78
406
26.2
6.8
-242
4.0
43.1
37.0
2.4
0.2
0.0
27.4
0.2
84
77
421
26.0
6.8
-369
3.4
23.6
23.0
1.4
0.2
0.0
29.1
0.2
78
53
453
21.6
7.0
-228
3.1
17.5
38.0
3.3
0.1
0.0
41.2
0.2
68
52
537
11.7
7.7
-203
1.7
13.5
52.0
43.7
0.3
0.0
16.0
0.5
18
42
634
14.2
7.4
-206
2.4
31.7
33.8
12.8
0.1
0.0
13.2
0.0
41
53
699
22.1
6.6
-212
1.3
27.7
12.1
12.8
0.3
0.0
32.5
0.4
52
40
747
26.0
6.9
-321
9.1
32.0
45.8
4.1
0.1
0.0
33.0
0.1
66
57
!
89!
Microbial communities structure and diversity
In this study, a total of 259,912 sequence reads were generated from 16
samples collected from the UASB reactor during 2 years of operation (Table 5.2).
Approximately 11,000-23,000 sequence reads per sample were analyzed. According
to the Chao1 estimator, the estimated species increased on average by approximately
5- to 7-fold in the samples. So, the coverage values were relatively low (58-71%
coverage), which indicates an underestimation of species richness due to the high
microbial diversity that exists in the sludge samples. These results were consistent
with previous studies of various wastewater treatment analyses in which low coverage
values were reported (Narihiro et al., 2009; Chaganti et al., 2012). PD, Shannon and
Simpson diversity indexes, which were applied to compare the microbial diversity
among the samples, also exhibit a high richness and evenness in microbial diversity
existed across all sludge samples. These results were consistent with a previous study
in which high microbial diversity was observed in sewage sludge samples (Narihiro et
al., 2009; Chaganti et al., 2012; Sundberg et al., 2013).
PCoA plot clustering of phylogenetic diversity (weighted UniFrac distances)
of the sludge samples is shown in Fig. 5.3. PCoA plots showed a distinct overall
bacterial community composition in each anaerobic sulfur reduction and oxidation
periods. The bacterial communities of day 167 from the oxidation period showed
similarities between the other bacterial communities from the reduction periods,
where they were clustered together and seemed to exhibit slight changes in microbial
activity, which is probably because it was a start-up period. The bacterial
communities of day 214 and 255 from the oxidation period were located a bit distant
and clustered together which indicated that their microbial communities had change
during anaerobic sulfur oxidation. Day 284, which was clustered together with day
!
90!
214 and 255, showed that their microbial communities had changed after the
oxidation had ended. After the oxidation had totally ended, the bacterial communities
of day 335-453 were shifted back to the previous cluster indicating that their
microbial communities had changed to it was before. Day 537 formed a cluster on its
own, which indicated that the bacterial communities on that day differed from the
other clusters and that a sudden change had occurred in the microbial communities
during anaerobic sulfur oxidation. The microbial communities of day 634-747 showed
an obvious shift where they formed a distinct cluster indicating that they were
different from the other clusters and that their microbial communities had changed
completely after the oxidation ended. The marked differences between the anaerobic
sulfur oxidation and reduction period show that the phylogenetic diversity of the
sludge samples is related to microbial communities which is, in turn, influenced by
the temperature and probably by the surrounding environment.
Fig. 5.3 PCoA analysis of UASB sludge samples with weighted UniFrac.
– oxidation period sludge samples; X – reduction period sludge samples.
!
91!
Table 5.2 Diversity indices of the UASB sludge samples.
Sample
(day)
Anaerobic sulfur
Number of
reduction/oxidation sequence
Number of
OTU
Chao1
Coverage
(%)
PD*
Shannon*
Simpson*
91
Reduction
14343
5823
36628
65
258.94
9.16
0.97
111
Reduction
15096
6007
30853
66
245.38
8.97
0.96
167
Oxidation
17752
8156
47198
60
298.38
9.97
0.98
214
Oxidation
21972
10254
63745
60
312.42
10.34
0.99
255
Oxidation
16233
7719
43872
59
294.50
10.21
0.99
284
Reduction
11836
5717
34783
58
289.77
10.11
0.99
335
Reduction
16213
7444
44072
60
291.35
9.93
0.98
363
Reduction
13716
5207
29837
68
235.85
8.91
0.96
379
Reduction
13756
5191
29366
68
233.32
8.69
0.95
406
Reduction
15017
5647
31792
68
241.72
8.97
0.97
421
Reduction
15512
5317
29618
71
219.46
8.48
0.95
453
Reduction
15590
5874
30759
68
231.66
8.74
0.96
537
Oxidation
14001
5988
37038
63
284.03
9.18
0.96
634
Reduction
17354
7740
43674
62
283.90
9.91
0.99
699
Reduction
22886
9205
46527
66
262.72
9.55
0.98
747
Reduction
18635
7690
40045
65
266.06
9.38
0.97
Abbreviation: OTU - Operational taxonomic unit; PD - Phylogenetic diversity
*Calculations determined at 0.03 dissimilarity based on 10,000 reads.
!
92!
Bacterial distribution in UASB sludge
T-RFLP and next generation sequencing analyses of the bacterial 16S rRNA
gene were performed to characterize and identify the microbial communities exist in
the UASB reactor. Both analyses were used to increase the reliability of the results
and to minimize biases produced by both methods. The distribution of the bacterial
communities in the sludge samples, before and after the sodium sulfate was added as
well as during anaerobic sulfur oxidation and non-oxidation, was determined. The TRFLP results shown in Fig. 5.4 yielded a large number of T-RFs in all 16 samples and
there were 11 dominant T-RFs observed. However, during anaerobic sulfur oxidation,
a sudden increase in a 348 bp T-RF was observed. Among the dominant T-RFs, only
the 60, 163, 194, 197, 231 and 348 bp T-RFs appeared in all samples. The 163, 194,
197 and 231 bp T-RFs were simulated by TRiFLe as genera Desulfitibacter,
Singulisphaera, uncultured bacteria of the phylum Caldiserica and genus
Blastopirellula, respectively. As for 60 and 348 bp T-RFs, each of it had two
simulated bacteria species, which were genera Prosthecochloris and Treponema for
60 bp T-RF, and Desulfomicrobium and Desulfovibrio for 348 bp T-RF. Some of the
T-RFs represented more than one species, as different species can produce the same
T-RF length when using a particular enzyme due to the probability of non-unique
restriction enzyme cutting sites and their variation across species (Junier et al., 2008;
Schütte et al., 2008). In addition, the T-RFs predicted in silico and those measured in
vivo mostly differed by a few base pairs (Winderl et al., 2008), thus the identity of the
bacteria in the community cannot be analyzed directly. Since it is not possible to
quantify the contribution of each of the species to the T-RF accurately, sequencing
analysis was conducted as a means of identity confirmation and precise quantification
of representation of bacteria in the community.
!
93!
The sequencing results shown in Fig. 5.5 showed virtually the same patterns
as the T-RFLP results, particularly from day 537 onwards where both of the results
showed distinctive changes in the microbial community patterns. During the initial
anaerobic sulfur oxidation (days 167, 214 and 255), the microbial community patterns
only changed slightly as compared to the other non-oxidation days such as a slight
increase in bacteria belonging to the phyla Proteobacteria, Firmicutes and
Bacteroidetes, and a slight decrease in bacteria belonging to the phyla Caldiserica and
Chloroflexi. However, during the second anaerobic sulfur oxidation period (days
537), a marked difference in the microbial community patterns was observed
compared to that seen during the initial oxidation period. Representatives of the phyla
Caldiserica and Chloroflexi greatly decreased in number, whereas those of the
phylum Proteobacteria only slightly increased compared to the earlier period.
Previous study (Aida et al., 2014) showed that uncultured bacteria of phylum
Caldiserica was the major bacteria group observed in the UASB reactor, which was
consistent with the results observed in this study. But in both initial and second
anaerobic sulfur oxidation period, the number of representatives of the phylum
Caldiserica decreased. One possible reason is the difference of primers used in both
studies, but a quantitative method such as real-time PCR was needed to clarify the
phenomena. Interestingly, bacteria belonging to the phylum Fusobacteria
demonstrated a sudden and marked increase in the second compared to the first
oxidation period. Thus, it may be possible that the phylum Fusobacteria can only be
detected during anaerobic sulfur oxidation periods. The microbial community patterns
also began to show some changes after day 537, similar to the PCoA analysis (Fig.
5.3), which was probably due to the adaptation of the microbial communities to the
environment.
!
94!
75
50
25
0
91
111
167 214 255 284 335 363 379 406 421 453 537 634 699 747
Detection rate of 16S rRNA gene at phylum level (%)
Relative abundance of T-RFs (%)
100
100
75
50
25
0
91
111
167 214 255 284 335 363 379 406 421 453 537 634 699 747
Time (days)
Time (days)
60 bp
231 bp
68 bp
344 bp
163 bp
348 bp
166 bp
563 bp
194 bp
592 bp
197 bp
Others
Proteobacteria
Bacteroidetes
Acidobacteria
Caldiserica
Spirochaetes
Chlorobi
Firmicutes
Synergistetes
Fusobacteria
Chloroflexi
Planctomycetes
Others
Fig. 5.4 Distribution of different phylogenetic bacteria communities
Fig. 5.5 Bacterial community structures of UASB sludge samples at
as determined by T-RFLP profiles after HhaI digestion of the 16S
the phylum level.!
rRNA gene.!
Overall, the major bacterial groups monitored in the UASB reactor were found
to belong to the phylum Proteobacteria which dominated the UASB sludge samples
(27.3±10.4% of the total taxa), followed by the phyla Caldiserica (17.8±8.9%),
Firmicutes (11.3±4.1%), Chloroflexi (11.5±6.1%), Bacteroidetes (8.0±4.4%) and
Spirochaetes (6.5±4.0%). Meanwhile, bacteria belonging to the other phyla
constituted less than 3% of the total taxa. These bacterial groups were those generally
detected in wastewater treatment plants and reactors, particularly representing the
phylum Proteobacteria which has been described as the most abundant bacterial
group present in reactors (Wagner and Loy, 2002; Diaz et al., 2006; Narihiro et al.,
2009;
Yang
et
al.,
2011).
Within
the
phylum
Proteobacteria,
class
Deltaproteobacteria was the major group accounting for 78.7±16.7% of the total taxa,
followed
by
classes
Gammaproteobacteria
Alphaproteobacteria
(3.2±6.5%
and
(13.6±9.3%),
2.6±3.0%,
Beta-
and
respectively),
and
Epsilonproteobacteria (1.0±1.7%), which is similar to the microbial community
structure previously detected in various types of UASB sludge (Narihiro et al., 2009).
The major groups within class Deltaproteobacteria were found to be genus
Desulfovibrio, followed by the genera Desulforhabdus and Smithella. However, no
previous studies have mentioned that these bacterial groups are SOB or involved in
sulfur oxidation.
The reverse reaction of the anaerobic oxidation of methane (AOM) with
sulfate reduction seems to be the possibility of anaerobic sulfur oxidation to occur.
Holler et al. (2011) demonstrated that the reverse direction of AOM with sulfate
could be catalyzed by AOM consortia. These consortia, which mediate AOM with
sulfate, are composed of anaerobic methanotrophic (ANME) archaea and SRB
(Knittel et al., 2005). Although ANME-related archaea were not detected in the
!
96!
UASB reactor, methanogenic archaea and SRB were detected, as reported previously
by Aida et al. (2014). This suggested that SRB that are present in the UASB reactor
might act as the main players in the anaerobic sulfur oxidation.
Bacteria belonging to the phylum Fusobacteria, are obligate anaerobic nonspore-forming gram-negative bacilli. Initially, these bacteria were associated with the
human mouth and gastrointestinal tract (Robrish et al., 1991; Bennett and Eley,
1993). Later, these bacteria were also isolated from anoxic sediment and sludge
(Schink, 1984; Brune and Schink, 1992), anaerobic mud (Janssen and Liesack, 1995;
Watson et al., 2000), and cold deep-marine sediment (Zhao et al., 2009), which
produce H2 and acetate as major fermentation products under different conditions
(e.g. variations in carbon source and temperature). Some of these bacteria are
psychrotrophic bacteria with an optimal growth temperature of 18.5°C, while some
are mesophilic bacteria with an optimal growth temperature of 28-37°C. Therefore,
the uncultured bacteria of the phylum Fusobacteria found in this study, which could
be detected during anaerobic sulfur oxidation, are probably not related to any other
known bacteria in this phylum and are likely to play a role in anaerobic sulfur
oxidation. Nevertheless, further studies are needed to elucidate the function of these
uncultured bacteria.
!
97!
Influence of environmental conditions on microbial community structure and
diversity
Environmental conditions strongly influence the structure and diversity of
microbial communities which are related to the types of reactions that can occur under
these conditions. Temperature acclimation, in particular, may alter the microbial
community activity, structure and diversity (Sekiguchi et al., 2002; Levén et al.,
2007). In this study, temperature appeared to influence the microbial activity and
diversity in the UASB sludge which then stimulated the sulfur redox reaction to take
place. As mentioned earlier, anaerobic sulfur oxidation occurred at low temperatures
and the major bacterial groups observed during the period of oxidation belong to the
genera Desulfovibrio, Desulforhabdus, Smithella and uncultured bacteria of the
phylum Fusobacteria. Thus, a correlation was observed between the abundance of
members of these four genera and the temperature in the reactor. The results in
Fig. 5.6 show that members of the genus Desulforhabdus and uncultured bacteria of
the phylum Fusobacteria were highly abundant when the temperature was low and
vice versa. In contrast, the abundance of members of the genera Desulfovibrio and
Smithella was not related to temperature. This suggested that bacteria belonging to the
genus Desulforhabdus and uncultured bacteria of the phylum Fusobacteria probably
related to anaerobic sulfur oxidation. Although genus Desulforhabdus is SRB,
perhaps these bacteria have a dual function and also play a role in anaerobic sulfur
oxidation in the UASB reactor. However, further validation methods such as stable
isotope probing and microautoradiography-fluorescent in situ hybridization methods
(Dumont and Murrell, 2005; Ito et al., 2012) are needed to gain more detailed insights
on the physiological properties of these bacteria.
!
98!
Fig. 5.6 Significant relationships between relatively abundant bacterial groups ( )
present in the UASB sludge samples and sewage temperature (–) of the UASB
reactor. A - Desulfovibrio; B - Desulforhabdus; C - Smithella; D – uncultured bacteria
of phylum Fusobacteria.
5.4
Conclusion
In summary, this microbial diversity analysis yielded a comprehensive
overview of the abundance, distribution, characteristics and phylogenetic diversity of
microbes exist in the UASB reactor. A highly diverse bacterial community was
present where the genera Desulfovibrio, Desulforhabdus and Smithella of the phylum
Proteobacteria as well as uncultured bacteria of the phylum Fusobacteria were the
major bacterial groups observed during the period of anaerobic sulfur oxidation.
However, only the genus Desulforhabdus and uncultured bacteria of the phylum
Fusobacteria were influenced by the temperature which suggested that these two
bacterial groups are likely to be related to the sulfur cycle and to play a role in
anaerobic sulfur oxidation. After all, it still remains an uncertainty with which
!
99!
mechanism or pathway that these bacteria oxidize sulfur to sulfate. Further analyses
are required to unravel all these issues and also provide a clearer and better
understanding on the basis of anaerobic sulfur oxidation in the UASB reactor
thoroughly.
References
Aida, A.A., Hatamoto, M., Yamamoto, M., Ono, S., Nakamura, A., Takahashi, M.
and Yamaguchi, T. (2014). Molecular characterization of anaerobic sulfuroxidizing microbial communities in up-flow anaerobic sludge blanket reactor
treating municipal sewage. Journal of Bioscience and Bioengineering, 118:
540-545.
Bennett, K.W. and Eley, A. (1993). Fusobacteria: new taxonomy and related diseases.
Journal of Medical Microbiology, 39: 246-254.
Briones, A.M., Daugherty, B.J., Angenent, L.T., Rausch, K., Tumbleson, M. and
Raskin, L. (2009). Characterization of microbial trophic structures of two
anaerobic bioreactors processing sulfate-rich waste streams. Water Research,
43: 4451-4460.
Brune, A. and Schink, B. (1992). Anaerobic degradation of hydroaromatic
compounds by newly isolated fermenting bacteria. Archives of Microbiology,
158: 320-327.
Buzzini, A.P., Sakamoto, I.K., Varesche, M.B. and Pires, E.C. (2006). Evaluation of
the microbial diversity in an UASB reactor treating wastewater from an
unbleached pulp plant. Process Biochemistry, 41: 168-176.
Caporaso, J.G., Kuczynski, J., J. Stombaugh, Bittinger, K., Bushman, F.D., Costello,
!
100!
E.K., Fierer, N., Peña, A.G., Goodrich, J.K., Gordon, J.I., Huttley, G.A.,
Kelley,
S.T., Knights, D., Koenig, J.E., Ley, R.E., Lozupone, C.A.,
McDonald, D., Muegge, B.D., Pirrung, M., Reeder, J., Sevinsky, J.R.,
Turnbaugh, P.J., Walters, W.A., Widmann, J., Yatsunenko, T., Zaneveld, J.
and Knight, R. (2010). QIIME allows analysis of high-throughput community
sequencing data. Nature Methods, 7: 335-336.
Caporaso, J.G., Lauber, C.L., Walters, W.A., Berg-Lyons, D., Lozupone, C.A.,
Turnbaugh, P.J., Fierer, N. and Knight, R. (2011). Global patterns of 16S
rRNA diversity at a depth of millions of sequences per sample. Proceedings of
the National Academy of Sciences of the United States of America, 108
(Supplement 1): 4516-4522.
Casserly, C. and Erijman, L. (2003). Molecular monitoring of microbial diversity in
an UASB reactor. International Biodeterioration and Biodegradation, 52: 712.
Chaganti, S.R., Lalman, J.A. and Heath, D.D. (20129. 16S rRNA gene based analysis
of the microbial diversity and hydrogen production in three mixed anaerobic
cultures. International Journal of Hydrogen Energy, 37: 9002-9017.
Diaz, E.E., Stams, A.J.M., Amils, R. and Sanz, J.L. (2006). Phenotypic properties and
microbial diversity of methanogenic granules from a full-scale upflow
anaerobic sludge bed reactor treating brewery wastewater. Applied and
Dumont, M.G. and Murrell, J.C. (2005). Stable isotope probing - linking microbial
identity to function. Nature Reviews Microbiology, 3: 499-504.
Edgar, R.C. (2010). Search and clustering orders of magnitude faster than BLAST.
Bioinformatics, 26: 2460-2461.
!
101!
Friedrich, C.G., Bardischewsky, F., Rother, D., Quentmeier, A. and Fischer, J. (2005).
Prokaryotic sulfur oxidation. Current Opinion in Microbiology, 8: 253-259.
Oxidation of reduced inorganic sulfur compounds by bacteria: Emergence of a
Hatamoto, M., Imachi, H., Ohashi, A. and Harada, H. (2007a). Identification and
cultivation of anaerobic, syntrophic long-chain fatty acid-degrading microbes
from mesophilic and thermophilic methanogenic sludges. Applied and
Hatamoto, M., Imachi, H., Yashiro, Y., Ohashi, A. and Harada, H. (2007b). Diversity
of anaerobic microorganisms involved in long-chain fatty acid degradation in
methanogenic sludges as revealed by RNA-based stable isotope probing.
Haas, B.J., Gevers, D., Earl, A.M., Feldgarden, M., Ward, D.V., Giannoukos, G.,
Ciulla, D., Tabbaa, D., Highlander, S.K., Sodergren, E., Methe´, B., DeSantis,
T.Z., The Human Microbiome Consortium, Petrosino, J.F., Knight, R. and
Birren, B.W. (2011). Chimeric 16S rRNA sequence formation and detection in
Sanger and 454-pyrosequenced PCR amplicons. Genome Research, 21: 494504.
Henze, M., Harremoës, P., la Cour Jansen, J. and Arvin, E. (1997). Wastewater
treatment: Biological and chemical processes, 2nd edition, Springer, Berlin.
Holler, T., Wegener, G., Niemann, H., Deusner, C., Ferdelman, T.G., Boetius, A.,
Brunner, B. and Widdel, F. (2011). Carbon and sulfur back flux during
anaerobic microbial oxidation of methane and coupled sulfate reduction.
!
102!
America, 108: E1484-E1490.
Howard, P.H. (1997). Handbook of environmental fate and exposure data for organic
chemicals, volume 5, CRC Press, Boca Raton, Florida.
Hvitved-Jacobsen, T. (2002). Sewer processes – Microbial and chemical process
engineering of sewer networks, 1st edition, CRC Press, Boca Raton, Florida.
Ito, T., Yoshiguchi, K., Ariesyady, H.D. and Okabe, S. (2012). Identification and
quantification of key microbial trophic groups of methanogenic glucose
degradation in an anaerobic digester sludge. Bioresource Technology, 123:
599-607.
Janssen, A.J.H., Lettinga, G. and de Keizer, A. (1999). Removal of hydrogen sulphide
from wastewater and waste gases by biological conversion to elemental
sulphur Colloidal and interfacial aspects of biologically produced sulphur
particles. Colloids and Surfaces A: Physicochemical and Engineering Aspects,
151: 389-397.
Janssen, P.H. and Liesack, W. (1995). Succinate decarboxylation by Propionigenium
maris sp. nov., a new anaerobic bacterium from an estuarine sediment.
Japan Sewage Works Association (1997). Japanese standard testing methods for
sewage. Japan Sewage Works Association (in Japanese).
Junier, P., Junier, T. and Witzel, K.-P. (2008). TRiFLe, a program for in silico
terminal restriction fragment length polymorphism analysis with user-defined
sequence sets. Applied and Environmental Microbiology, 74: 6452-6456.
Khemkhao, M., Nuntakumjorn, B., Techkarnjanaruk, S. and Phalakornkule, C.
(2012). UASB performance and microbial adaptation during a transition from
!
103!
mesophilic to thermophilic treatment of palm oil mill effluent. Journal of
Environmental Management, 103: 74-82.
Knittel, K., Lösekann, T., Boetius, A., Kort, R. and Amann, R. (2005). Diversity and
Distribution of Methanotrophic Archaea at Cold Seeps. Applied and
Levén, L., Eriksson, A.R.B. and Schnürer, A. (2007). Effect of process temperature
on bacterial and archaeal communities in two methanogenic bioreactors
treating organic household waste. FEMS Microbiology Ecology, 59: 683-693.
Li, J., Wang, J., Luan, Z., Deng, Y. and Chen, L. (2011). Evaluation of performance
and microbial community in a two-stage UASB reactor pretreating acrylic
fiber manufacturing wastewater. Bioresource Technology, 102: 5709-5716.
Lozupone, C.A., Hamady, M., Kelley, S.T. and Knight, R. (2007). Quantitative and
Qualitative β Diversity Measures Lead to Different Insights into Factors That
Structure Microbial Communities. Applied and Environmental Microbiology,
73: 1576–1585.
Narihiro, T., Terada, T., Kikuchi, K., Iguchi, A., Ikeda, M., Yamauchi, T., Shiraishi,
K., Kamagata, Y., Nakamura, K. and Sekiguchi, Y. (2009). Comparative
analysis of bacterial and archaeal communities in methanogenic sludge
granules from upflow anaerobic sludge blanket reactors treating various foodprocessing, high-strength organic wastewaters. Microbes and Environments,
24: 88-96.
Nielsen, A.H., Lens, P., Vollertsen, J. and Hvitved-Jacobsen, T. (2005). Sulfide-iron
interactions in domestic wastewater from a gravity sewer. Water Research, 39:
2747-2755.
Ono, S., Takahashi, M., Hatamoto, M., Kawakami, S., Yamaguchi, T., Yamazaki, S.,
!
104!
Araki, N. and Harada, H. (2011). Performance of a pilot-scale sewage
treatment by UASB and DHS reactor combined system enhancing sulfatereducing reaction. The 4th IWA-ASPIRE Conference & Exhibition (IWAASPIRE 2011), p.307, No. 14-22-7, Tokyo, Japan.
Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., Peplies, J. and
Glöckner, F.O. (2013). The SILVA ribosomal RNA gene database project:
improved data processing and web-based tools. Nucleic Acids Research,
41(D1): D590-D596.
Robrish, S.A., Oliver, C. and Thompson, J. (1991). Sugar metabolism by
fusobacteria: regulation of transport, phosphorylation, and polymer formation
by Fusobacterium mortiferum ATCC 25557. Infection and Immunity, 59:
4547-4554.
Rohwerder, T. and Sand, W. (2007). Oxidation of inorganic sulfur compounds in
acidophilic prokaryotes. Engineering in Life Sciences, 7: 301-309.
Schink, B. (1984). Fermentation of tartrate enantiomers by anaerobic bacteria, and
description of two new species of strict anaerobes, Ruminococcus pasteurii
and Ilyobacter tartaricus. Archives of Microbiology, 139: 409-414.
Schütte, U.M.E., Abdo, Z., Bent, S.J., Shyu, C., Williams, C.J., Pierson, J.D. and
Forney, L.J. (2008). Advances in the use of terminal restriction fragment
length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize
microbial communities. Applied Microbiology and Biotechnology, 80: 365380.
Sekiguchi, Y., Kamagata, Y., Ohashi, A. and Harada, H. (2002). Molecular and
conventional analyses of microbial diversity in mesophilic and thermophilic
!
105!
upflow anaerobic sludge blanket granular sludges. Water Science and
Technology, 45: 19-25.
Singh, K.S. and Viraraghavan, T. (2003). Impact of temperature on performance,
microbiological, and hydrodynamic aspects of UASB reactors treating
municipal wastewater. Water Science and Technology, 48: 211-217.
Sundberg, C., Al-Soud, W.A., Larsson, M., Alm, E., Yekta, S.S., Svensson, B.H.,
Sørensen, S.J. and Karlsson, A. (2013). 454 pyrosequencing analyses of
bacterial and archaeal richness in 21 full-scale biogas digesters. FEMS
Microbiology Ecology, 85: 612-626.
Takahashi, M., Ohya, A., Kawakami, S., Yoneyama, Y., Onodera, T., Syutsubo, K.,
Yamazaki, S., Araki, N., Ohashi, A., Harada, H. and Yamaguchi, T. (2011).
Evaluation of treatment characteristics and sludge properties in a UASB
reactor treating municipal sewage at ambient temperature. International
Journal of Environmental Research, 5: 821-826.
Tang, K., Baskaran, V. and Nemati, M. (2009). Bacteria of the sulfur cycle: An
Wagner, M. and Loy, A. (2002). Bacterial community composition and function in
sewage treatment systems. Current Opinion in Biotechnology, 13: 218-227.
Watson, J., Matsui, G.Y., Leaphart, A., Wiegel, J., Rainey, F.A. and Lovell, C.R.
(2000). Reductively debrominating strains of Propionigenium maris from
burrows of bromophenol-producing marine infauna. International Journal of
Systematic and Evolutionary Microbiology, 50: 1035-1042.
Winderl, C., Anneser, B., Griebler, C., Meckenstock, R.U. and Lueders, T. (2008).
Depth-resolved quantification of anaerobic toluene degraders and aquifer
!
106!
microbial community patterns in distinct redox zones of a tar oil contaminant
plume. Applied and Environmental Microbiology, 74: 792-801.
Yang, C., Zhang, W., Liu, R., Li, Q., Li, B., Wang, S., Song, C., Qiao, C. and
Mulchandani, A. (2011). Phylogenetic diversity and metabolic potential of
activated sludge microbial communities in full-scale wastewater treatment
plants. Environmental Science and Technology, 45: 7408-7415.
Zhao, J.-S., Manno, D. and Hawari, J. (2009). Psychrilyobacter atlanticus gen. nov.,
sp. nov., a marine member of the phylum Fusobacteria that produces H2 and
degrades nitramine explosives under low temperature conditions. International
!
!
107!
CHAPTER 6
SUMMARY
The anaerobic sulfur oxidation reaction occurred in the upflow anaerobic
sludge blanket (UASB) reactor treating municipal sewage is an interesting
phenomenon where it occurred without the presence of electron acceptors. This
phenomenon seems to be a new discovery where there were no studies reported
previously regarding this phenomenon and the knowledge on this matter is still
lacking and unclear. Therefore, several analyses were conducted to identify the
factors that made this anaerobic sulfur oxidation to occur.
In this study, anaerobic sulfur oxidation was regenerated by using
bioelectrochemical reactor (BER) under several conditions, which were oxidationreduction potential (ORP) control, non-ORP control, stirring and BES (2-bromo
ethanedisulfonic acid) added condition, to investigate the effect of these parameters
on the regeneration of anaerobic sulfur oxidation. Under the ORP control condition,
the anaerobic sulfur oxidation was regenerated, whereas other conditions failed to
regenerate the anaerobic sulfur oxidation reaction. The microbial community
structures in the ORP control BER system, where the anaerobic sulfur oxidation were
regenerated, were analyzed using cloning and terminal restriction fragment length
polymorphism (T-RFLP) analyses. The results showed that phylum Proteobacteria
was the major group observed where Syntrophobacteraceae and Desulfovibrionaceae
family of sub-phylum δ-Proteobacteria were the most abundant bacteria groups
found in the anaerobic sulfur oxidation reaction samples with 80% of total clones.
These bacteria groups are known as sulfur reducing bacteria.
!
108!
The microbial community structures in the UASB reactor were also been
analyzed and identified using cloning and T-RFLP analyses. The archaeal community
had just relatively low and simple diversity which acetate-utilizing methanogen
Methanosaeta spp. was the dominant group of the domain archaea at all times.
Whereas, the bacterial community presented high diversity which genus Smithella of
phylum Proteobacteria was the major bacterial group observed in the UASB reactor
comprising an average of 19% of total clones, followed by uncultured bacteria of
phylum Caldiserica (12%). The bacterial communities observed in the UASB reactor
and in the anaerobic sulfur oxidation regenerated BER system showed a similar result
where phylum Proteobacteria was the most abundant bacteria group found in the
anaerobic sulfur oxidation samples.
To gain greater insights into microbial biodiversity and its role in anaerobic
sulfur oxidation, an extensive analysis of the abundance, distribution, characteristics
and phylogenetic diversity of microorganisms exist in the UASB reactor was
conducted by next generation sequencing analysis. Consistent with the results
mentioned above, phylum Proteobacteria was the most abundant bacteria group
observed during the period of anaerobic sulfur oxidation comprising an average of
27.3% of total clones which genera Smithella as well as Desulfovibrio and
Desulforhabdus were the major bacterial groups present. Besides phylum
Proteobacteria, uncultured bacteria of the phylum Fusobacteria was also the major
bacteria group observed during the period of anaerobic sulfur oxidation, where it
demonstrated a sudden and marked increase in the second compared to the first
oxidation period. Representatives of the phyla Caldiserica, which were the major
bacterial group observed earlier, decreased gradually in number in both initial and
second anaerobic sulfur oxidation period. However, only the genus Desulforhabdus
!
109!
and uncultured bacteria of the phylum Fusobacteria were influenced by the
temperature which suggested that these two bacterial groups are likely to be related to
the sulfur cycle and to play a role in anaerobic sulfur oxidation.
From these observations, it can be suggested that this anaerobic sulfur
oxidation reaction was mediated biologically by the microorganisms, and it can
therefore be concluded that (i) genus Desulforhabdus, which is SRB, could have a
dual function and can also act as SOB, and/or (ii) uncultured bacteria of the phylum
Fusobacteria probably related and play an important role in anaerobic sulfur
oxidation. However, further analyses are required to elucidate the pathways that are
utilized by these bacteria to oxidize sulfur to sulfate and to gain greater insight into
the basis of anaerobic sulfur oxidation in the UASB reactor. In addition, some other
SRB probably have a dual function and play a role in anaerobic sulfur oxidation as
well. These findings will be a novel discovery in this field of study and will give a
great impact on the development of sulfide removing process of domestic and
industrial wastewater treatment.
!
110!
LIST OF PUBLICATIONS
1. Aida, A.A., Hatamoto, M., Yamamoto, M., Ono, S., Nakamura, A., Takahashi,
M. and Yamaguchi, T. (2014). Molecular characterization of anaerobic sulfuroxidizing microbial communities in up-flow anaerobic sludge blanket reactor
treating municipal sewage. Journal of Bioscience and Bioengineering, 118:
540-545.
2. Aida, A.A., Kuroda, K., Yamamoto, M., Nakamura, A., Hatamoto, M. and
Yamaguchi, T. (2015). Diversity profile of microbes associated with anaerobic
sulfur oxidation in an upflow anaerobic sludge blanket reactor treating
municipal sewage. Microbes and Environments (In press).
!
111!

PhD Thesis - Aida Azrina Binti Azmi

Transcription

Similar documents

Sir M. Visvesvaraya senior scientist`s state award for science and

Nutra-JOINT Brochure

Bio-Mite Waste Water Treatment Plants

UNWANTED HOUSEGUESTS

PDF (Author`s version) - OATAO (Open Archive Toulouse Archive

Kinetico 2060f Sulfur Guard System Non

Epulopiscium fishelsoni - Academic lab pages

Petro-Marine