Research Collection
Doctoral Thesis
Population dynamics and kinetics of nitrifying bacteria in
membrane and conventional activated sludge plants
Author(s):
Manser, Reto
Publication Date:
2005
Permanent Link:
https://doi.org/10.3929/ethz-a-005083976
Rights / License:
In Copyright - Non-Commercial Use Permitted
This page was generated automatically upon download from the ETH Zurich Research Collection. For more
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ETH Library
DISS
ETH NO
16217
POPULATION DYNAMICS AND KINETICS OF NITRIFYING
BACTERIA IN MEMBRANE AND CONVENTIONAL ACTIVATED
SLUDGE PLANTS
A dissertation submitted to the
SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH
for the
degree
of
Doctor of Technical Sciences
presented by
RETO MANSER
Dipl Ing
ETH
born 24 03 1972
citizen of
accepted
on
the recommendation of
Prof Dr Willi
Prof Dr Hansruedi
Prof Dr
Appenzell
Gujer, examiner
Siegnst, co-examiner
Jurg Keller,
2005
co-examiner
Summary
Summary
Introduction
the last
During
decades, municipal
effects of ammonia in
increasingly
comes
water reuse
have
biological
due to
prices
of MBR will
new
significantly
single
households
plants
wastewater
comparison
have attracted in¬
the conventional activated
sludge process
secondary settling tank, need
to CAS are
still
a
mostly higher
costs
drawback of MBR, beside
for the investor.
Nonetheless, decreasing membrane
and lessened energy
consumption by optimized operation
coming years. In addition, tightened
dealing
with
Finally,
new
substances e.g. endocrine
MBR
systems for the
disrupters
treatment
may
con¬
of the wastewater
apartment houses with subsequent recycling of the treated
were
conventional activated
water are
put into operation in past years, adapting the experiences from
sludge plants.
ing,
which leads to
tors
of wastewater treatment
a
At the
available.
already commercially
First full-scale
or
to
municipal
advance their attraction in the
tribute to the extension of MBR.
areas.
Conversely, space be¬
same time, pressure for
the effluent free of solids. However,
technology
and the
discharge regulations
from
carbon to
regions.
Compared
in
growing competition
treatment.
due to the omission of the
provide
consumption
a
in arid
biological
for the treatment of
footprint
tanks and
of
uncertainty
only
of coastal waters and to prevent adverse
in urban
particularly
shortage
(MBRs)
lower
a
and elevated energy
the
factor
attention in recent years.
(CAS) they
smaller
filtration step after the
rises due to water
Membrane bioreactors
creasing
a
limiting
a
from
waters. As a consequence,
necessitate
additionally
eutrophication
developed
the required volume for the bio¬
receiving
risen. Furthermore, stringent regulations for sensitive receiving waters
processes has
logical
has
wastewater treatment
nutrient removal in order to reduce the
On the other
hand, long-term experiences
certain caution and reluctance of the
plants (WWTPs)
designing engineers
in terms of MBR
are
still miss¬
and the opera¬
technology.
Aim of thesis
The aim of this work
vated
was
sludge properties.
to
investigate
This included the
sion of kinetic information. The
edge
and
placed
in
on
experience
the influence of the membrane
new
study
insights
from CAS systems
can
of
population dynamics
should
help
to
as
well
on
as
the acti¬
the
provi¬
understand how the broad knowl¬
be transferred to MBR systems.
nitrification and the involved microbial
municipal
separation
populations
because of their
Emphasis
was
important
role
wastewater treatment.
l
Summary
Methodology
A conventional and two membrane bioreactor
They
two years.
were
fed with
municipal
pilot plants
showing
wastewater
variation. The CAS and the MBRs treated the wastewater
2
(0.56 m3
d"1) population equivalents, respectively.
hollow fibre modules from Zenon
submerged
and
the
a
characteristic pore size of 0.1
sludge
was
age
kept
at 20
The
um.
days.
were
in
parallel
typical
a
corresponding
The MBRs
(ZW-10)
plants
were run
with
operated
with
than
more
diurnal
hydraulic
m3
d"1) and
(18
to 60
equipped
were
surface
a
for
with four
of 0.93
area
m2
each
and
pre-denitrification
The startup of the MBR and CAS
done without in¬
was
oculation.
The
nitrifying population
(FISH).
was
Different batch tests
Fluorescent In Situ
quantitatively analyzed using
performed
were
to
information
gain
on
Hybridization
the kinetics of nitrifica¬
tion.
Quantification of nitrifying bacteria
Quantification of bacteria using Fluorescence
and
scanning microscopy (CLSM)
availability
of
image analysis
expensive microscope. Therefore,
an
teria in activated
quantification
trifying
was
In Situ
sludge using
FISH and
of the biovolume is based
only
image analysis.
nitrifying
minutes, compared
The method
proved
bacteria in activated
to
rapid
manual
on
about
developed.
the time needed for
hour for the
quantification
of
analysis
bac¬
quantify nitrifying
was
the
requires
of the aggregates formed
Accordingly,
one
be suitable for the
to
method to
counting
and
consuming
epifluorescence microscopy
bacteria and determination of their size.
5 to 15
a
confocal laser
Hybridization (FISH),
is very time
one
The
ni¬
by
sample
with CLSM and
population dynamics
of
sludge.
Population dynamics
The identification of the
differences between the CAS and the MBR for both
nitrite-oxidizing
bacteria
(NOB). Accordingly,
nitrification rates. Confocal laser
nitrifying
bacteria
were
located
a
possible
ammonia-oxidizing
mostly
of AOB
influence of the
high
by
NOB
were
were
larger
nitrifying community composition
Plants
rich
nor
generally
in the CAS
shear forces induced
within the MBR. It is concluded that the membrane
the nitrification
only
(AOB)
same
by
separation
were
overgrown
of
nitrifying
by
by
smaller than those formed
compared
the
coarse
to
the MBR in¬
bubble aeration
itself does affect neither the
performance.
-
community composition
and
maximum
treating ammonium-rich wastewater e.g. separate treatment of digester liquids
nitrifiers, which could be used for bioaugmentation. However, such reactors harbor
ferent
minor
showed that the aggregates formed
in the inner part of the floes and
built up
bacteria
both systems exhibited the
scanning microscopy
heterotrophic bacteria. Aggregates
by AOB. Furthermore, aggregates
dicating
FISH revealed
nitrifying community composition using
bacteria that is
poorly adapted
to
-
a
en¬
dif¬
the conditions
h
Summary
prevailing
in WWTP. It
conditions and
rapidly
Hence, the enriched
was
shown that the enriched nitrifiers
were
not
able to
adapt
washed out after addition to the CAS and MBR
were
community composition
is crucial for
and failure of
success
to
those
pilot plants.
bioaugmen¬
tation.
Kinetics
The half-saturation constants for substrate
significantly
stants
between nitritation and nitratation. On the other
for oxygen of the CAS
were
AOB and NOB. The differences
prevailing
at
attributed to
in the conventional system.
anoxic conditions is
in
significantly higher
were
mass
hand, the half-saturation
comparison
negligible
can
be
the measured loss of
sludge
model
no.
3
diverge. However,
were
detected.
endogenous respiration concept
(ASM3).
floes
MBR consisted of
for both AOB and NOB. No
of AOB and NOB did not
only partly explained by
large
neglected.
sludge
vated
con¬
the MBR for both
transfer effects within the
between membrane and conventional activated
the
to
By contrast, the sludge from the
very small floes where diffusion resistance
Decay
low in both MBR and CAS and did not differ
were
significant differences
Likewise, aerobic decay
autotrophic activity
incorporated
as
Unlike NOB, AOB showed
a
was
e.g. in the acti¬
pronounced decay
of their
enzymes.
Conclusions
Existing
models for activated
netic parameters for oxygen
quence, MBRs
can
be
sludge
affinity
operated
at
processes
of
nitrifying
pre-denitrification.
The extension of activated
sludge
mass
also be
applied
to MBRs.
bacteria have to be
low oxygen concentrations
tion energy and to enhance
of
can
(1
go2
m"3)
adapted.
However, ki¬
As
in order to
a
conse¬
save aera¬
models with enzyme kinetics of AOB and the consideration
transfer effects within the floes may
improve
our
understanding
of nitrogen
dynamics
in wastewater treatment.
in
Zusammenfassung
Einleitung
Während der letzten Jahrzehnte entwickelten sich kommunale
(ARA)
der ausschliesslichen Elimination
von
Nährstoffelimination.
Damit
konnte
reduziert werden und andererseits
werden. In der
um
den
Folge
Faktor
Zudem
5-10.
des
Wiederverwendung
zunehmende
(MBR)
zur
deutlich
Energieverbrauch
-
fallende
immer noch
-
nachteilig
Membranpreise
Energieverbrauch
durch
Membran-Kleinanlagen
für das
optimierten
Hauptsächlich
Abwassers
von
neue
Verfahren
Bemessung
Allerdings
gingen
stützte
fehlen
Vorsicht und
zu
einer
Reinigung
und
werden können. Weiter
höhere Kosten und ein höherer
aus.
zunehmenden
neuen
Trotzdem
Technologie für den
ist anzunehmen, dass
Wettbewerbs
zu
und
Verbreitung
anschliessender
sinkender
mehr Attraktivität in
Einleitbedingungen
weiteren
und die Becken
und der
Umgang
führen.
MBR
von
Wiederverwendung
des
einzelnen Mehrfamilienhäusern sind bereits kommerziell erhältlich.
In den letzten Jahren
der
gebaut
einigen
konventionellen
zu
Betrieb dem Membranverfahren
dürften
zur
Wasserknappheit.
im
neben der Unsicherheit einer
aufgrund
Mikroverunreinigungen
mit
wird
der Druck
kommunalen Abwassers kommen seit
den kommenden Jahren verhelfen werden. Verschärfte
mit
Regionen
Stufe
empfindliche
knapper. Gleichzeitig steigt
MBR
MBR einen keimfreien Ablauf.
wirken sich
für
benötigen
Vergleich
weniger Platz, da die Nachklärung entfällt
zu.
vorgebeugt
biologischen
biologischen Reinigung. Umgekehrt
Abwassers in
Behandlung
Küstengewässer
Ammoniak
Einleitbedingen
strenge
der höheren Belebtschlammkonzentrationen kleiner
garantieren
Investor
gereinigten
Bedeutung
Belebtschlammanlagen
aufgrund
verlangen
eine Filtrationsstufe nach der
Membranbioreaktoren
Jahren
von
mit
Anlagen
zu
der
Eutrophierung
nachteiligen Auswirkungen
in städtischen Gebieten zunehmend
Platzangebot
für eine
die
erhöhten sich die erforderlichen Beckenvolumina der
Oberflächengewässer
das
Stoffe weiter
organischer
einerseits
Abwasserreinigungsanlagen
erste MBR zur
man
kommunalen
sich auf die breite
Erfahrung
langfristige praktische Erfahrungen
Zurückhaltung
bei den
Abwasserreinigung
von
in Betrieb. Bei
konventionellen
mit MBR. Dies führt
planenden Ingenieuren
und Betreibern
von
zu
Anlagen.
gewisser
ARA.
Ziel der Arbeit
Das Ziel dieser Arbeit
war
die
Untersuchung
des Einflusses des Membranverfahrens auf den
Belebtschlamm. Dies beinhaltete das Studium der
von
kinetischer Information.
Verständnis
Anlagen
Die
gewonnenen
Populationsdynamik
Erkenntnisse
sollten
führen, wie das breite Wissen und die langjährige Erfahrung
auf MBR
übertragen
die Nitrifikation und die
kommunalen
werden können. Die
Untersuchung
beteiligten Mikroorganismen aufgrund
und die
Erarbeitung
zu
einem
besseren
von
konventionellen
beschränkte sich dabei auf
derer zentraler Rolle in der
Abwasserreinigung.
IV
Zusammenfassung
Vorgehen
Eine konventionelle und zwei MBR
wurden im Parallelbetrieb während zwei
Pilotanlagen
Jahren betrieben. Kommunales Abwasser mit einem
Die konventionelle und die
respektive
mit
(0.56 m3
2
Membrananlagen
je
Oberfläche
wurden mit
m2
0.93
von
von
vorgeschalteter
Pilotanlagen
Jedes Modul
(ZW-10).
Zenon
von
waren
hat
0.1
Anlagen
Quantifizierung
der
Situ
Hybridization).
Verschiedene
fand ohne externe
Nitrifikantenpopulation erfolgte
Batchexperimente
Animpfung
mit Hilfe
wurden
von
(18 m3
60
von
spezifische
Die
um.
d"1)
ausgerüstet
eine
Denitrifikationszone und bei einem Schlammalter
Die
zu
Die MBR
und eine charakteristische Porenweite
betrieben. Die Inbetriebnahme der
der Nitrifikanten
behandelten das Abwasser
d"1) Einwohnergleichwerten.
vier Hohlfasermodulen
diente als Zulauf.
typischen Tagesgang
von
Anlagen
20
Tagen
statt.
FISH
durchgeführt,
(Fluorescent
In
die Kinetik
um
bestimmen.
Resultate
Quantifizierung
Die
nitrifizierender Bakterien
Quantifizierung
Bakterien mittels FISH, confocal laser
von
und
scanning microscope (CLSM)
ist zeitintensiv und setzt ein teures
digitaler Bildauswertung
wurde eine Methode entwickelt,
zuverlässig
CLSM
manuelles
zu
Zählen
und
Bestimmen
Die
Quantifizierung
der
Grösse
der
Vorgehen (ca.
Stunde) lediglich
des Biovolumens
erfolgt
Nitrifikanten-Cluster
Der Zeitaufwand pro Probe
eine
Deshalb
voraus.
die Nitrifikanten im Belebtschlamm schnell und ohne
quantifizieren.
Epifluoreszenz-Mikroskop.
beschriebenen
um
Mikroskop
beträgt
im
unter
dem
zum
oben
Vergleich
5-15 Minuten. Die
durch
Populationsdynamik
der Nitrifikanten in Belebtschlamm konnte mit der entwickelten Methode
erfolgreich verfolgt
werden.
Populationsdynamik
Die
Bestimmung
keine
der
Zusammensetzung der Nitrifikantenpopulationen mittels
Unterschiede
nennenswerten
den
bezüglich
(NOB)
Dementsprechend
auch die maximalen Nitrifikationsraten in beiden
Untersuchungen
mit dem CLSM
Innern der Flocken
der konventionellen
(AOB)
oxidierenden Bakterien
waren
zwischen
Ammonium-
zeigten,
da sie
sind,
angesiedelt
waren
von
den schnell
Zusammensetzung
Anlagen
zur
Faulwasser
der
geschlossen werden,
Nitrifikantenpopulationen
Behandlung
-
waren
die AOB-Cluster der konventionellen
Aus den Resultaten kann
reichern
und
Nitrit-
den MBR.
Systemen gleich.
grösstenteils im
wachsenden, heterotrophen
noch die
an,
die
allgemein
Anlage
dass die
ammoniumreichen Abwassers
Nitrifikanten
Anlage
und
dass die Nitrifikanten-Cluster
Bakterien überwachsen werden. Die Cluster der NOB
AOB. Ferner
FISH erbrachte
kleiner als
kleiner als
jene
Membranseparierung
jene
der
der MBR.
weder die
Nitrifikationsleistung beeinflusst.
-
z.
grundsätzlich
B.
separate Behandlung
für
die
von
Unterstützung der
v
Zusammenfassung
in ARA
Nitrifikationsleistung
weisen
Allerdings
solche
Reaktoren
wurde
Es
in
Umweltbedingungen
ARA
konventionelle und MBR
der
dass
gezeigt,
eine
die schlecht
Nitrifikantenpopulationen auf,
sind.
werden könnten
eingesetzt
sich
die
Pilotanlagen
andere
an
die
so
angereicherten
konnten
anpassen
(auch Bioanreicherung genannt).
deutlich
sofort
angereicherten Nitrifikantenpopulationen
Nitrifikanten
nach
folglich
ausgewaschen
ist
in ARA angepasst
Umweltbedingungen
und
der
Zusammensetzung
der
wurden. Die
daher entscheidend
nicht
an
die
in
die
Zugabe
Zusammensetzung
für den
Erfolg
der
B i oanrei cherung.
Kinetik
In beiden
Systemen
Andererseits
höher
die Monodkonstanten für Sauerstoff im konventionellen
waren
für
wurden tiefe Monodkonstanten für Ammonium und Nitrit
und
AOB
Massentransportlimitierung
System. Umgekehrt
in
anoxischen
unter
für AOB
vernachlässigbar
aeroben Zerfall
von
den
Unterschiede
nur
aus
System
deutlich
zurückzuführen
im
kleinen
auf
konventionellen
Flocken, worin der
spielt.
Bedingungen
und NOB.
im
ist
Keine
AOB und NOB wurden
Abnahme der Aktivität
sind
Belebtschlammflocken
grossen
besteht der Belebtschlamm in den MBR
Diffusionswiderstand keine Rolle
Zerfall
Die
NOB.
gefunden.
teilweise mit dem
Gegensatz
signifikanten
gefunden. Allerdings
Sludge
AOB ein Teil ihrer
Enzyme bei knappem Substratangebot ab.
3)
aeroben
Bedingungen
zwischen
dem
konnte die gemessene
Konzept der endogenen Atmung (inkorporiert
z.B. im Activated
Model No.
zu
Unterschiede
erklärt werden. Im Unterschied
zu
den NOB bauen
Schlussfolgerungen
Die
verfügbaren
müssen
Belebtschlammmodelle können auch für MBR verwendet werden.
die kinetischen Parameter für die
werden. Dadurch können MBR bei tiefen Sauerstoffkonzentrationen
werden,
um
einerseits
Denitrifikation
Die
zu
Erweiterung
Berücksichtigung
Belüftungsenergie
Allerdings
Sauerstoffaffinität der Nitrifikanten angepasst
zu
sparen
und
(1
andererseits
g02
m"3)
die
vorgeschaltete
betrieben
steigern.
der Belebtschlammmodelle mit
von
Verständnis für die
Enzymkinetik
Massentransportlimitierung
Stickstoffdynamik
in der
der AOB
zusammen
in den Flocken kann dazu
Abwasserreinigung
zu
mit der
beitragen,
unser
verbessern.
vi
Acknowledgements
Many people have contributed
to
this PhD thesis with their work and advices.
First of all I would like to thank my advisors. Hansruedi
me
during
Siegrist encouraged
many valuable discussions. His enthusiasm and close relation to
in the field of wastewater treatment
this research work in
a
flexible way
with his time whenever I needed
the thread of this work.
was
having
time for
help, provided
Jürg Keller
was
Willi
inspiring.
me
willing
to
Gujer
gave
practicing
the
me
practical problems
the
piano.
and motivated
possibility
He
with lots of ideas while
was
do
very free
keeping
co-examine my thesis and
to
in mind
produced
new
ideas.
I
am
very
plants
grateful
and
all who
Haag. Chapter
3
work
are
Finally,
diploma
also due to the
was an
important prerequisite
providing
an
essentially
Lorena
based
operation and supervision
Jaramillo, Kathrin Muche
on
the
experimental
of the
pilot
and Justine
data that Kathrin
thesis.
laboratory
I wish to thank my
fields and for
were
of this thesis is
Muche collected for her
Thanks
for the
responsible
gathered experimental data, namely
to
staff led
for the
colleagues
enjoyable
and
by
success
for
Jack
of the
Eugster. Their
accurate
and reliable
experiments.
encouraging stimulating
interesting atmosphere
discussions in various
in- and outside the
Eawag.
vu
Contents
i
Summary
iv
Zusammenfassung
vii
Acknowledgements
ix
Abbreviations
Introduction
1
Rapid
Method to
Chapter
1:
A
Chapter
2:
Membrane
Quantify
Bioreactor
Population Dynamics
Chapter
3:
Bioaugmentation
Chapter
4:
Consequences
Chapter
5:
Decay
vs.
Nitrifiers in Activated
Conventional
Sludge
Activated
Sludge
7
System
of Nitrifiers
20
of Nitrifying Bacteria
of Mass Transfer Effects
Processes of Nitrifying Bacteria
-
31
on
the Kinetics of Nitrifiers
47
62
Conclusions
75
References
78
Index
86
Curriculum vitae
90
Vlll
Abbreviations
Nitrogen compounds
NH3;
NH4+;
NH4-N
Ammonia; Ammonium; Ammonium-nitrogen
NH2OH
Hydroxylamine
N02"; NO2-N
Nitrite; Nitrite-nitrogen
N03"; NO3-N
Nitrate; Nitrate-nitrogen
N2
Nitrogen
Kj-N
Kj eldahl-nitrogen (sum
gas, elemental
nitrogen
of ammonium and
organic nitrogen)
Bacteria
bacteria
AOB
Ammonia-oxidizing
AMO
Ammonia monooxygenase
HAO
Hydroxylamine
N.
Nitrosomonas
NOB
Nitrite-oxidizing
NO2-OR
Nitrite oxidoreductase
HET;H
Heterotrophic
oxidoreductase
bacteria
bacteria
Others
ASM3
Activated
ATU
Allylthiourea
ATP
Adenosine
CAS
Conventional activated
CLSM
Confocal laser
COD
Chemical oxygen demand
CV
C oeffi ci ent of vari ati on
DOC
Dissolved
sludge
model
no.
3
triphosphate
sludge plant
scanning microscopy
organic
carbon
IX
FISH
Fluorescent in situ
H202
Hydrogen peroxide
HCl
Hydrogen
chloride
HRT
Hydraulic
retention time
MBR
Membrane bioreactor
NaHC03
Sodium
bicarbonate, sodium hydrogen carbonate
NaOH
Sodium
hydroxide
ORP
Oxidation-reduction
OUR
Oxygen
P042";
PO4-P
utilization
hybridization
potential
(uptake)
rate
Phosphate; Phosphate-phosphorus
rRNA
Ribosomal ribonucleic acid
SBR
Sequencing batch
SF
Safety
SRT; SA
Sludge
TSS
Total
WWTP
Wastewater treatment
reactor
factor
retention
suspended
time; Sludge
age
solids
plant
Kinetic parameters
b
Endogenous respiration
Mmax
Maximum
D
Diffusion coefficient
K
Half-saturation constant of Monod kinetics
e
Temperature coefficient
r
Rate
growth
rate
rate
Introduction
Introduction
Application
of membranes in wastewater treatment
While membrane
it is
applied
tion to
in
technology
municipal
only
wastewater treatment
sedimentation in the activated
replace
(Stephenson, 2000).
membrane
is state of the art in e.g.
Yamamoto et al.
submerged
order to prevent
into
fouling
an
drinking
sludge
of the membrane.
since
decades,
since recent years. Membrane ultrafiltra¬
sludge
(1989) reported
activated
water treatment
process
first
reactor. A
was
experiences
with
a
hollow-fibre
low permeate flux had to be
of this unit
Operation
first described in 1969
was
very similar to
kept
in
today's
membrane bioreactors
(MBRs). In the mean time, a lot of research was done particularly on
the fouling issue (e.g. Fane et al, 2000; Field et al, 1995; Judd, 2004; Nagaoka et al., 1998;
Roorda and van der Graaf, 2000; Stephenson, 2000; Wintgens etal, 2003). Furthermore, first
full-scale
plants
significant
pal
cost
were
put into operation (Figure 1). The further technical developments and
reductions have
sharply
Compared
wastewater treatment.
increased the interest in MBR
technology
with the traditional tubular membranes
applied
other hand,
past, hollow-fibre and plate membranes require much less energy. On the
brane
have
prices
decreased in past years
sharply
within the next five years is assumed
(Engelhardt
membranes with pore sizes between 0.04
um
(Figure 2).
and
and 0.4
50%
commercially
used.
CO
W
600'000
<u
100
o
o
»
c
re
re
400'000
a>
c
re
200'000
n
E
1995
2000
Year of
Figure
2005
and
vs.
Figure
up of
a
cake
layer
on
MBR with the conventional activated
include
an
2000
2005
2010
2
Development
of membrane costs.
conventional system
retain bacteria and the vast
building
1995
Year
1 Full-scale MBRs worldwide.
completely
1990
2010
operation
Membrane bioreactor
esses
by
800'000
'5
MBR
mem¬
1000
"E
rooo'ooo
re
>
A further reduction
in the
Rothe, 2001). Today, mostly synthetic
urn are
1'200'000
c
for munici¬
majority
the membrane.
sludge
process
of viruses due to the flocculation proc¬
Therefore,
(CAS)
a
correct
comparison
in terms of effluent
additional filtration and disinfection step after sedimentation
quality
of the
must
(Figure 3).
2
Introduction
permeate
nitrification
denitrification
pump
-®—— effluent
excess
mechanically
treated
sludge
internal recirculation
wastewater
denitrification
sedimentation filtration
nitrification
disinfection
"a
o
o
°
_
effluent
return
Figure
Comparison
3
providing
As it
of
similar effluent
can
sludge
sludge
excess
(MBR) with the conventional activated sludge process (CAS)
membrane bioreactor
a
quality.
be seen, MBR
are
particularly
suited if
stringent regulations (e.g. germfree effluent)
have to be met and limited space is available. Other fields of
wastewater treatment
plants (WWTP)
taurants, apartment houses), which
with
be
can
highly dynamic
application
load
small
(e.g.
steadily operated requiring
are
decentralized
settlements,
minimal
res¬
ef¬
monitoring
fort.
from the omission of the
Apart
can
be
operated
at
membrane
leading
reached at
a
constant
smaller
the
kgxss
15
their
are
and
is
reduced
dramatically
sludge production
was
of the
not
WWTP and is
existing
hand, mostly higher
still
the
hamper
compensate by
spread
sludge
MBR
costs
at
they
form
a
of
(Günder, 1999;
sludge
and little
Van
an
efficiency
Further
be
mi-
and membrane
advantages
of
barrier and
originally expected decreased
der Roest et al, 2002; Wagner
a
flexible and
architectonic
point
experience compared
to
into account
phased
exten¬
of view.
conventional WWTP
secondary settling
However, taking
can
concentrations
highly germ-selective
is suited for
in terms of
MBR.
a
ages
the
by
slowly degradable
high
very
volume index. The
of MBR. The omission of the
far for the costs of
degradation
because aeration
technology
interesting
On the other
retention of solids
(summarized by Wedi, 2004).
verified
Rosenwinkel, 2000). Finally,
sion of
MBR
their tolerance to shock loads because
operation independently
excess
of
operation
complete
Alternatively, higher sludge
may favor the
m"3) is not economically advantageous,
performance
MBR
volume.
biological
volume, which
cropollutants. However,
(>
concentrations due to the
higher sludge
to a
filtration and disinfection step, MBR
secondary settling tank,
tank itself does not
savings
for filtration
possible simplifications of sludge stabilization, investment costs are
similar for both systems or even lower for large plants (Engelhardt and Rothe, 2001). None¬
theless, operation costs of MBR are still higher in comparison to CAS. They are dominated by
the amortization of the membranes, about whose lifetime in municipal wastewater treatment
and disinfection units and
can
only
were
be
speculated due
assumed
to
(Wedi, 2004).
lack of full-scale
A
specific
energy
experience. Hitherto,
consumption
lifetimes of 5 to 10 years
of 1 kWh
m"3
wastewater
treated
3
Introduction
was
reported (Stein, 2003;
Main energy
Wedi et
of MBRs
consumers
al, 2004) compared
about 0.3-0.5 kWh
to
m"3
for CAS.
the aeration systems, which represent about 60% of the
are
total energy demand. Thereof accounts about 70% for the cross-flow aeration of the
mem¬
branes and about 30% for the fine bubble aeration.
In
conclusion,
MBR
still possesses
technology
optimization potential
with
regard
to
material
and process
to
engineering aspects. Increasing competence and sales volumes probably will lead
membrane costs. Furthermore, the coming EU directive concerning the quality of
lower
bathing
requires
waters
reduction of the germs
Nitrification
the
monitoring
Without
nitrification, municipal
into the
receiving
waters. On
the surface waters and
vated
sequential
on
WWTP would
the
one
the other
discharge large
hand, ammonium
bacteria
able to
directly
high
hand, ammonium dissociates
(nitratation).
are
amounts
causes
to
fish
steps: oxidation of ammonia to nitrite
dation of nitrite to nitrate
lithoautotrophic
and targets
a
significant
process within wastewater treatment
pH associated with algae growth, which is toxic
sists of two
are
microbiological parameters
being discharged.
important
-
of
involved.
In each
Hitherto,
oxygen
consumption
ammonia
populations.
(nitritation)
reaction, distinct
no
to
(NH4+)
of ammonium
(NH3)
at
Nitrification
and
in
ele¬
con¬
subsequent
oxi¬
groups of aerobic chemo-
autotrophic organisms
are
known which
oxidize ammonia to nitrate.
Nitritation
The overall reaction of ammonia to nitrite is
same
with
are
hydroxylamine (NH2OH)
(HAO)
NH3
+
to
produce
02 +2H+
NH2OH
+
AMO
+2e-
HAO
H20
respiratory
0.5O2 +2H+
two-stage process, but is catalyzed by the
chain
+2e~
as an
intermediate
(AMO)
(Eq.
as
the substrate
1 and
(Suzuki
Eq. 2). The
for ammonia oxidation and
et
al, 1974)
enzymes involved
hydroxylamine
oxidoreduc¬
nitrite.
>NH2OH+H20
>N02~ +5H+
The electrons released in the
the
a
Ammonia rather than ammonium is used
organism.
ammonia monooxygenase
tase
again
(Eq. 3)
+
4e~
hydroxylamine
oxidation
and for CO2 assimilation
are
required
for ammonia
1-3
1
Eq.
2
oxidation,
(Wood, 1986).
Eq.
>H20
Combining equations
Eq.
yields
3
the overall formula for the oxidation of ammonia to nitrite
(nitritation):
NH3+1.502
>NOf +H+ +H20
This reaction is
isms.
Although
Eq.
catalyzed by gram-negative mostly obligate chemolithoautotrophic
Nitrosomonas europaea is the model ammonia-oxidizer
according
4
organ¬
to text-
4
Introduction
books and
tant in WWTP
analysis
other
previous studies,
ammonia-oxidizing bacteria (AOB)
(reviewed by Wagner
Fluorescent In Situ
al, 2002).
et
to
seem
be
impor¬
more
Hybridization (FISH)
mobilis, members
of various WWTP revealed that members related to Nitrosococcus
of the Nitrosomonas marina
quently present (Purkhold
et
lineage and Nitrosomonas europaea/eutvopha were most fre¬
al, 2000). Today it is generally accepted that nitrosomonads (in¬
and not nitrosospiras
cluding Nitrosococcus mobilis)
Nitrosolobus
sion,
andNitrosovibrio)
WWTP harbor
underestimated
ing
WWTP
present in
diversity
dominated
responsible
of AOB of the
a
single
numbers.
(Daims
which
Nitrosospira,
species (Juretschko
co-existing
enormously
was
FISH results indicate that
ammonia-oxidizer
et
the genera
Betaproteobacteria,
harbor at least five different
plants
significant
by
(encompassing
for ammonia oxidation in WWTP. In conclu¬
previously. Interestingly, quantitative
are
whereas other
a
are
et
populations,
AOB
nitrify¬
some
al, 1998),
which
are
al, 2001b).
Nitratation
Nitrite is further oxidized to nitrate
oxygen atom in
trons
released
by
the enzyme nitrite oxidoreductase
the nitrate molecule is derived from water
are
used in the electron transport chain
according
(Eq. 3)
and
to
partly
and the
(NO2-OR)
Eq.
5. The two elec¬
also for C02 assimila¬
tion.
N°2~OR
N02~ +H20
Traditionally,
important
Nitrobacter
Eq.
+2e~
belonging
nitrite-oxidizer in WWTP
detected in
of
>NOf +2H+
Alphaproteobacteria was considered as
(Henze et al, 2002). However, no Nitrobacter
to
the
from nine different WWTP
samples
(Wagner
et
al, 1996), whereas the
in
novel, yet uncultured Nitrospira-\ike nitrite-oxidizing bacteria (NOB)
5
the most
could be
occurrence
nitrifying
WWTP
could be demonstrated
et
al, 2001; Okabe
Nitrospira
et
have been
(Burrell et al, 1998; Daims et al, 2001a; Daims et al, 2000; Gieseke
al, 1999). Today, four different phylogenetic lineages within the genus
recognized (Daims
dominance of Nitrospira-\ike bacteria
affinity
to
nitrite and oxygen
et
over
al, 2001a).
postulated
that the pre¬
Nitrobacter in most WWTP is due to their
(reviewed by Wagner
An overview of the distribution and
It has been
ecology
of
et
higher
al, 2002).
nitrifying
bacteria is
given
in
Koops and
Pommerening-Roser (2001).
The well-known overall nitrification process summarizes
NH3+202
Goals and
The
yields Eq.
6:
Eq.
6
questions
underlying goal
to
3-5 and
>NOf +H+ +H20
of this
effect of the membrane
help
equations
project
separation
understand how the broad
ferred to MBR systems. The
was
on
to
obtain
a more
the activated
knowledge
and
following questions
fundamental
sludge properties.
experience
understanding
The
new
about the
insights
from CAS systems
can
should
be trans¬
should be addressed:
5
Introduction
How
bacteria
nitrifying
can
How does the membrane
reasonably
quantified
influence the
separation
How does the membrane
be
in activated
sludge?
of nitrifying bacteria?
populations
influence the floe structure? What
separation
are
the
conse¬
quences for the kinetics of nitrification?
Does the
of
decay
bacteria differ from that of
nitrite-oxidizing
bacteria? What is the effect of different oxidation-reduction
How is the
competition
among different groups of
tation? Are MBR systems
activated
existing
Can
Which
adaptations
advantageous
sludge
models
extensions
or
in this
are
ammonia-oxidizing
potentials
decay?
nitrifying bacteria,
on
e.g.
bioaugmen¬
regard?
reliably
be
applied
membrane bioreactors?
to
necessary?
Outline of this thesis
The
study
interest.
of
population dynamics
Chapter
1
presents
reliably quantified
with
bridization
and
(FISH)
a
a
procedure
chapter
performance
of the
parallel, tracking
placed
was
SRT
on
using
ing
bacteria in activated
nitrifying
the
on
developed
months of
a
the evolution of ammonia- and
separation
is often
enriched
involving
using
separation
the method
subsequent eight
without the need of
ated
the
quantify
organisms
sludge
be
can
on
Fluorescence In Situ
of
nitrifying
of
Hy¬
populations
in
chapter
MBR
are
bacteria is de¬
1. The startup behavior and the
compared
nitrite-oxidizing
with
CAS
a
bacteria.
running
in
Special emphasis
of the influence of the membrane from other factors e.g.
composition.
Bioaugmentation
tion
how
method to
epifluorescence microscopy.
the strict
influent
or
2
adequate
an
reasonable effort. The method is based
The influence of the membrane
scribed in
presumes
the
suggested
enlarging
as
a
concept
to
the aeration tanks. In
nitrifying sludge
investigation
improve
chapter
the nitrification
3 the
potential
from the separate treatment of
of the co-existence and
of
performance
bioaugmenta¬
digester liquids
is evalu¬
among different
competition
nitrify¬
groups.
4 demonstrates the
Chapter
Based
on
impact
of the membrane
floe size distribution measurements and batch
transfer limitation within the activated
modeling
Decay
as
Major
play
Therefore,
chapter
well
of nitrification
processes
MBRs.
separation
a
are
an
sludge
were
experiments,
the
sludge
structure.
the effects of
mass
studied and their consequences for
highlighted.
important
role with
closer look at the
5. Differences between MBRs and
are
briefly
increasing sludge
ages
characteristically
for
presented
in
CAS, ammonia-and nitrite-oxidizing bacteria
as
decay
the effect of the oxidation-reduction
results of this thesis
floes
on
kinetics of
potential
were
nitrifying
bacteria is
investigated.
summarized in the conclusions.
6
CHAPTER 1
A
Rapid
Method to
Quantify
Nitrifiers in
Activated
Reto
Sludge
Manser, Kathrin Muche, Willi Gujer, Hansruedi Siegrist
Water Research
39(8),
1585-1593
2005
A
Rapid
Method to
Activated
Nitrifiers in
Quantify
Sludge
Manser, Kathrin Muche, Willi Gujer, Hansruedi Siegrist
Reto
Abstract
Quantification of bacteria using Fluorescence
scanning microscopy (CLSM)
availability
of
image analysis is
expensive microscope. Therefore,
an
bacteria in activated
The
quantification
evaluated
as a
microscopic
10-15
trite-oxidizing
function of the number of
fields for
bacteria per
the time needed for
ingly,
hour for the
FISH and
very time
method to
rapid
a
manual
counting
quantification
sample
one
were
sample
and 6-8
requires
the
was
developed.
of the aggregates formed
uncertainty
fields. It
analyzed microscopic
ammonia-oxidizing bacteria
confocal laser
and
quantify nitrifying
epifluorescence microscopy
on
of the method
was
microscopic
found that
fields for ni¬
optimal regarding effort and accuracy. Accord¬
only 5 to 15 minutes, compared to about one
was
with CLSM and
image analysis.
As
also allows for the measurement of extended time series with
parison
consuming
bacteria and determination of their size. The overall
by nitrifying
was
sludge using
of the biovolume is based
Hybridization (FISH),
In Situ
and
of the determined biovolume and the measured
a
activity
a
consequence, this method
reasonable effort. The
showed
an
explicit
com¬
correla¬
tion.
sludge; FISH; Nitrifying bacteria; Quantification; Epifluorescence
Activated
Keywords:
microscopy
1. Introduction
Increasing
demands
ticated treatment
moval to
plants
higher sludge
removal
can
wastewater treatment in
plants.
with
The extension of the
scumming
and
e.g.
crobiology
questions
as
et
and
structure
larger
and
more
sophis¬
steps from only COD
As
et
a
consequence,
Stable
are
only inadequately
re¬
still to be
complex
al., 2002; Wagner
et
approaches
applications
done with confocal laser
bacteria have to be
the FISH
of
engineering
in molecular mi¬
level of
insight
ecosystems like activated
sludge
al., 1995).
a new
This molecular
vistas for the solution of the above mentioned issues. However,
applying
potential
explored.
addressed with traditional
New technical
operating
biological phosphorus
failure and the
Hybridization (FISH) permit
and function of
al., 2002; Wagner
new
performance
light microscopy.
remain unanswered when
1. For most
complex ecosystems.
bulking sludge
increasing.
Furthermore, nitrification
Fluorescence In Situ
population
(Juretschko
more
enhance nitrification
respirometry
such
tool opens up
treatment
are
Until now, these issues could be
into the
biological
often not be maintained.
bioaugmentation to
methods,
the last decades led to
nitrification, denitrification and biological phosphorus removal implied
retention times and
due to
problems
on
a
biological
couple
of
technique.
quantified. Previously, quantification
scanning microscopy (CLSM)
followed
was
mainly
by image analysis (Bouchez
8
1. A
et
al, 2001; Daims
and
threshold
uses a
biovolume of the
detected
by
al, 2001c; Kuehn
et
signal intensity
of thick
quality
Firstly,
tioned
(Wagner
of
analysis
et
a
directly
signal
geneous
cells with low
cles
the
of
an
sludge
bacteria,
is needed. The im¬
expensive microscope
floes
conventional
by
unless the
mi¬
epifluorescence
samples
are
physically
sec¬
al., 1998). Secondly, the technique is time consuming, which makes the
at the
microscope
intensities may
and therefore low
activity
showing high signal
intensities
are
setting
when
or
hamper
feasible.
hardly
the threshold
procedure using image analysis,
done either
the biovolume
on
al., 1990). However, several constraints
subsequent image analysis
time series tedious and
long
Amann et
availability
like activated
samples
croscopy does not allow
the
sludge
discriminate between target and non-target cells. The
to
general probe (e.g. EUB338;
a
al, 1998). This method focuses
activated
in
bacteria of interest is then referred to the biovolume of all
specific
have to be considered.
age
et
rapid method to quantify nitrifiers
is still
analyzing
the threshold
subjective
a
the
images.
setting (Daims
signal intensity
not
the semi-automatic
Thirdly, despite
task which has to be
Problems with inhomoet
al, 2001c). Hence,
excluded. Moreover, debris
are
omitted and would have to be deleted
parti¬
manually
in
image analysis step.
2. Other methods
assign
images (Eikelboom
ence
cently
enhanced
and
can
Jenkins et
Buijsen, 1983;
van
FISH and
using
differences
only large
the abundance of filamentous bacteria to
categories
based
on
al., 1993). This technique
refer¬
was re¬
epifluorescence microscopy (Hug et al., 2005). However,
filaments, nitrifiers mainly appear as
sludge floes (Wagner et al., 1995). The size of these aggre¬
be detected. In contrast to
dense aggregates within activated
gates is crucial regarding the abundance. Therefore, the quantification of nitrifiers based
reference
images
quantification by
is not
flow cytometry
3. Uncertainties of the
must
possible.
a
are
quantification
be determined in order to
the results involves
For the
higher
not
of
counting
single
cells
or
feasible.
with
identify
time
reason, manual
same
on
regard
to
the main
demand,
the
the
sampling,
sources
of
the method and the operator
error.
Since
uncertainty analysis
a
higher
allows the
accuracy of
optimization
of
work to meet the individual criteria.
4. It has to be clarified whether
(measured
new
as
of the
intensity
quantification
methods
their
also
activity,
has still to be
during periods
does not
stringently
5. It is not
spira fraction
a
quantify
While
proportional
to
activity (Wagner
to link
e.g.
as
as
cell numbers
were
activity
determined,
or
biovolume. The consideration of the
nitrifiers
seem
to
keep
their rRNA content
al., 1995). Consequently, the signal intensity
activity.
these numbers with measured parameters such
ammonia oxidation
quantified by
numbers, biovolume
the rRNA content of the cells and therefore
explored. However,
et
cell
traditionally
the abundance
reflect the actual metabolism
clear, how
activity (measured
finding
with low
should
probe signal).
measure
which is
probe signal intensity,
one
rates).
Hall et al.
as
COD, TSS
(2003) compared
or
the Nitro-
FISH and CLSM to measured nitrate
clear correlation between the biovolume and the rate.
production rates without
Thus, the link to the above
mentioned parameters has still to be evaluated.
9
1. A
The
of this
goal
in activated
have
study
sludge
was
like activated
of
use
with manual
methods have been
producible
of
development
without the
experience
some
the
rapid method to quantify nitrifiers
an
a
method to
rapid
expensive
sludge
bacteria
many researchers
might
quantification using epifluorescence microscopy,
properly developed
no re¬
Especially in engineering systems
nitrifying bacteria, a reasonably fast
far.
so
processes with low enrichment of
sludge
activated
quantify nitrifying
Although
CLSM.
in
and reliable
quantification method is needed to follow population dynamics, e.g. during bioaugmentation studies. Furthermore, the quantitative microbiological information has to be
linked to measured
activity
in order to evaluate its
for
potential
modeling
purposes.
2. Materials and Methods
Activated
reactor
used for the
sludge samples
and
(MBR)
MBR and
conventional activated
a
(Manser
wastewater
et
sequencing
a
uncertainty analysis
al, 2004). Activity
batch
pilot
the SBR for the separate treatment of
(WWTP)
of Biilach
vated
sludge
m"3
above 10 gN
trite)
taken
were
done
measurements were
m3)
volume 2
digester liquid
membrane bio¬
domestic
using sludge
inoculated with
from the
sludge
the wastewater treatment
at
from
plant
activity (Batch experiments)
the MBR. A batch reactor
and
a
(Switzerland).
2.1 Measurement of the
A) Sludge from
taken from
sludge (CAS) pilot plant treating
(SBR,
reactor
were
continuously
and 4 g02
regularly
for the
liter)
1
was
inoculated with diluted acti¬
aerated. Ammonium and oxygen concentrations
m"3, respectively.
measured
simultaneously
(Volume
by
(conversion of ammonia to ni¬
(Massone et al., 1998). Samples were
Nitritation rates
titration with NaOH
analysis
always
were
with FISH. The
experiment
was
performed
pH
at
7.5
and 20°C.
B) Sludge from
try (Drtil
trations
et
al., 1993)
were
taneously
the SBR. The nitritation rate
always
for the
in
a
above 100 gN
analysis
2.2 Fluorescence In Situ
Samples
were
(35 kHz,
prior
hybridization
to
five
m"3
(Volume
once a
liter).
day by
means
of respirome-
Ammonium and oxygen
concen¬
m"3, respectively. Samples were taken
and 4 g02
were
performed
at
pH
simul¬
7.5 and 19°C.
Hybridization (FISH)
paraformaldehyde
minutes)
was
applied
in order to break up
fluorescently
7.5
with FISH. All measurements
fixed in 4%
fication
formed with
batch reactor
measured
was
labeled
as
to
described
the fixed
large
by
Amann et al
samples
floes. In situ
(1990).
Ultrasoni-
from the CAS and the SBR
hybridizations
of cells
were
per¬
(Cy3), rRNA-targeted oligonucleotide probes according
to
Manz et al
(1992) (Table 1). Oligonucleotide probes were obtained from Microsynth (Balgach, Switzerland) and Thermo Hybaid (Interactiva Division, Ulm, Germany).
10
1. A
Table 1
and
Oligonucleotide probes
rapid method to quantify nitrifiers
hybridization conditions applied
in this
Reference
Ntspa662a
Genus
Daims et
NEUa
Most
Nitrospira
halophilic
sludge
study
Target organisms
Probe
activated
in
and halotolerant ammonia oxidizers in the
al., 2001a
Wagneretal.,
1995
beta-subclass of Proteobacteria
Nmll
Many members of the
Nitrosomonas
communis
Nmo218
Many members of the
Nitrosomonas
ohgotropha lineage
lineage
PommereningRoser
Gieseke et
All known ammonia oxidizers in the beta-subclass except N.
Nso 1225
al.,
et
al.,
1996
2001
Mobarry étal.,
1996
mobilis
a
used with unlabeled
2.2
indicated in the reference
with
samples hybridized
United
Canterbury,
was
done
using
technik AG,
lOOOx
an
oligonucleotide probes
Kingdom) prior
Olympus
to
microscopic
was
used
For
quantification
an
embedded in Citifluor
were
observation.
microscope, equipped
BX50
Tübingen, Germany). Microscopic
magnification.
distance
fields
with
(diameter:
ocular with
an
(Citifluor,
Epifluorescence microscopy
a
filter HQ-CY3
200
urn)
were
(Analysen¬
of 10
implemented grid
with
analyzed
urn
equi¬
(Figure 1).
Quantification
Microscopic
fields
were
the slide varied from
on
as
Microscopy
All
2.3
competitor
each
microscopic
field
randomly chosen by the operator. Since the thickness of the sample
field to field, only one focus plane instead of the complete depth of
was
analyzed. Positively
and the average diameter of every aggregate
1). Accordingly,
dirt
could be
particles
was
easily
labeled aggregates
were
determined with the aid of the
excluded and
a
large
counted
manually
range of
grid (Figure
in¬
probe signal
tensities could be taken into account.
The biovolume instead of the bioarea
measurements
and to estimate
the aggregates, which
slide
was
found to be
was
single
true
was
used to allow
cell rates. It
for to the vast
negligible
was
counted and
assigned
a
diameter of 1
and 0.7
urn
single
As reference system, the
the
counting
blurry
cells
area
density
compression
occupied by
signal
of the
within the floes
was
a
negligible
of every
sludge
-
was
considered
as
cells
were
using
on
sludge
extent to
floe
microscopic
estimated
was
of
the
the ruler
approximately
oxidizing
taken in order to estimate their
the activated
c
Single
for ammonia- and nitrite
were
contributed to
of the aggregates, the coverage
auto-fluorescence
the biomass
only
urn
activity
assuming spherical shape
for aggregates with diameters below 20 urn,
teria, respectively. Digital images of single cells
diameter. However,
the abundance to
of aggregates. The
majority
the fine focus wheel to estimate their vertical dimension.
on
as
linking
calculated
bac¬
mean
the abundance.
chosen. In addition to
field
by
the floes
(Figure 1).
part of the overall
-
visible
The variation of
uncertainty.
11
1. A
The abundance
(as
biovolume per floe
rapid method to quantify nitrifiers
cross-section)
was
calculated
using
in activated
the
sludge
for¬
following
mula:
bvfloc=-^^[um3mmfloc"2]
C
with:
A
'
bvfioc
field
bvfieid
c
=
biovolume per
=
=
area
reference system
plied
field
of microscopic field
[urn3 field"1]
was
chosen, since the
[mmfloc2 mmfieid"2]
[mmfieid2 field"1]
the dimension of the abundance
glance,
ducibility
thermore,
microscopic
coverage of microscopic field with floes
Afield
At first
[urn3 mmfioc"2]
biovolume per floe cross-section
=
as
volume per
area
may be
purpose of this method is to
of the results rather than absolute numbers based
on a
the correlation of the biovolume with measured
This
confusing.
provide
a
good
repro¬
certain reference system. Fur¬
activity
is
independent
of the ap¬
reference system.
containing
hybridized
Figure
with
a
Principle
1
sludge samples
specific probe
activated
Digital image from
epifluorescence microscopy
(bar 20 |xm)
Microscopic field (d 200 \m)
10 |xm,
grid (equidistance
implemented in ocular)
=
with
and nomenclature of
=
=
quantification procedure
2.4.
Uncertainty analysis
The
uncertainty analysis provides quantitative
data about the accuracy and
reliability
of the
method, which is crucial for its application. The overall uncertainty of the quantification
method
was
activated
sample
determined
by analyzing
sludge sample (MBR).
of the
complete
45 to 55 fields for each
However, the values
distribution. Since the
probe (FISH), using
gained (bvfioc) represent only
complete
the
a
same
random
distribution is not known and cannot
be
assigned from a priori knowledge, the nonparametric bootstrap method was applied (Efiron
and Tibshirani, 1993). This statistical method allows estimating an empirical distribution
without the assumption of normality. Basically, for an entity of N values, a bootstrap sample b
is obtained
by randomly sampling
uncertainty
as a
every
probe (N
standard
error
N times with
function of the number of
=
45 to
55)
were
replacement.
analyzed fields, B
generated. Subsequently,
sen and coefficient of variation CVn
were
In order to evaluate the overall
=
lO'OOO
bootstrap samples
the estimated
calculated
value
p,n,
following
for¬
expected
using
the
for
mulas:
12
1. A
rapid method to quantify nitrifiers
in
activated
sludge
.8=10 000
2>nb
Û
=
2>V,
—^1
I
A
i
fi=10 000
sen
=J^ty
with:
n
2^nb-Mn)
Hn
microscopic
1.. .B: number of bootstrap
=
bvlb
p,n
=
sen
expected value
expected
estimated standard
=
CVn
estimated
estimated
=
fields
sample
biovolume per floe cross-section of field i and
=
An b
=^
CV
1.. .N: number of
=
b
h=—
of bootstrap
value when
error
when
coefficient of variation when
=
b
bootstrap sample
b
sample
analyzing n fields
analyzing
analyzing
fields
n
n
fields
3. Results
The result of
affected
and
interest, namely the estimation of nitrifier biovolume
several
by
sources
of
uncertainty. They
by
the measurement
sludge,
is
procedure
stem from:
mainly
1.
the
2.
the FISH
3.
the natural
spatial variability
4.
systematic
errors
missing representativeness
The first two
of the
grab sample
protocol (fixation, hybridization)
sources
taken from
were
caused
are
in the activated
of the aggregates and their
subjective
estimation
among different operators
will be excluded in the further
completely
mixed
pilot
reactors
considerations, because the samples used
and the FISH
protocol
was
standardized.
However, the representativeness of samples from full scale plants should be examined by
tracer
experiments,
tection of
which allow for the assessment of the
stirred
insufficiently
zones.
The latter two
hydraulics
sources are
of the
plant
and the de¬
assessed in sections 3.1 and
3.2.
3.1 Natural
spatial variability
probe
For every
between 60 and 600 aggregates
average cell diameter of
and
bers
Nmll)
was
1-1.2
(~1 aggregate
tected
by
of the aggregates and their
ammonia-oxidizing
urn.
per
Nmo218 made up the
major
The diameter of the aggregates
oxidizing
bacteria
bacteria
While cells detected
microscopic field)
(NOB: probe Ntspa662)
by
enumerated
were
same
operator. The
present in low
num¬
the NEU
16
the
NEU
probe
were
heterogeneously distributed,
(~4 aggregates
up to
by
estimation
(AOB: probes Nsol225, Nmo218,
and rather
fraction
ranged
were
subjective
um.
generally
per
microscopic field)
In contrast,
smaller
(up
cells de¬
of AOB.
aggregates of nitriteto 8
urn). However,
the
13
1. A
cell diameter of NOB
8
detected
as
by probe Ntspa662
aggregates per microscopic field
The observed differences of the
frequency
their
spatial
enumerated
more
homogeneous spatial
some
this effect.
pronounced
which
on
also smaller
activated
(0.5-0.8 urn).
sludge
About
the average.
(Figure 2).
All groups of AOB showed
a
pattern reflects the transformation from diameters to bio-
asymmetric
symmetric pattern,
was
in
distribution and size of the aggregates influenced the
(spherical aggregates). Moreover,
average value further
rather
were
distribution of the measured abundances
wide distribution. The
volume
rapid method to quantify nitrifiers
can
be
fields with abundances far
Despite
explained by
distribution
compared
this
the
to
transformation,
general
higher
than the
NOB showed
a
smaller aggregate size and
the AOB.
DNmll
Nso1225
NEU
D Nmo218
„o
»^
s
,0
"T
<o
Û
fl
'v
<V
abundance
Figure
45)
Histograms
2
were
analyzed
Ä
J^
5
"5
,o
<o
D
Ao
"D
A
(103 mm3 mn>|OC"2)
<o
abundance
\
probe using
the
same
activated
&
(103 mm3 mmf|OC"2)
of the series used for the estimation of the uncertainties. 55
for each
Q>
Ntspa662
microscopic
sludge sample (MBR).
fields
(Ntspa662:
AOB: Nsol225, Nmo218,
Nmll, NEU; NOB: Ntspa662.
The overall
fields
was
racy of the
to
be
uncertainty
estimated
of the
using
the
mean
bootstrap
quantification, Figure
analyzed.
As it could be
method
as a
function of the number of
(Figure 3). Depending
on
the
microscopic
required
accu¬
3 allows the determination of the number of fields that need
expected
ferences between the detected groups,
teria. As
abundance
from the distributions
especially
(Figure 2),
between ammonia- and
there
are
large
nitrite-oxidizing
dif¬
bac¬
fields the estimated CV is 0.33 for the Nsol225
illustration,
microscopic
this
value
is
however,
probe,
only 0.10 for the Ntspa662 probe, which reflects the pronounced
an
difference between the
at 10
spatial variability
of AOB and NOB.
14
1. A
rapid method to quantify nitrifiers
in
activated
sludge
--NEU
—
Nso-1225
Nmo218(Op A)
—
-
—
-
-
Nmo218(Op B)
-
Nmll
—
^^^Ntspa662
0
5
10
15
20
25
number of
Figure
Estimation of the overall
3
cient of variation. Probe Nmo218
3.2
Systematic
Two main
and assessed.
of
uncertainty
was
analysed
systematic
Systematic
35
microscopic
errors
of the
twice
among different
errors
sources
30
40
two
abundance
Firstly,
Therefore,
five operators
significant
for each
due to the
subjectivity
of the operator
mean
B)
as
=
coeffi¬
described in section 3.2.
were
abundance stem either from the
10
microscopic
(Figure 4).
or
from the
(nonparametric
subjective
identified
subjective
choice of mi¬
were
which
hampers
significantly higher
(stained
found
(p
=
probe Nmo218)
of
showed
0.98). However,
the CV
(average: 0.22).
quantified microscopic
systematic
lower
variance)
analysis
field varied between 0.01 and 0.57
the identification of
with
or
this method for the first
using
were
version of one-way
differences among the operators
quantified microscopic
high variance,
fields
Three of them
sequence, the differences between the operators for each
a
method. CV
operators (Op.
the number and size of the counted aggregates could be
analyzed by
no
bootstrap
fields.
time. A Kruskal-Wallis test
that
the
by
operators
deviations of the
among different operators.
were
55
A and
estimation of the number and the size of the aggregates
croscopic
50
fields
mean
by
45
As
a con¬
field also had
effects.
123456789
Operator
A
Operator
B
D
Operator
C
D
Operator
D
D
Operator
E
10
microscopic field
Figure
4
Results from the
analysis
of
systematic
errors
due to the estimation of the number and size of the ag¬
gregates (Probe Nmo218).
Secondly, systematic
errors
may not be
chosen.
randomly
among operators
Therefore,
are
11 wells
introduced because the
(stained
with
microscopic
probe Nmo218),
fields
encompass-
15
1. A
ing
microscopic
6
erator A in
in both
fields
the first
runs
each,
and
run
were
twice. The
analyzed
B in the second
by Operator
in order to prevent
rapid method to quantify nitrifiers
run.
Operator
tween
the two persons
deviations for
single
(p
mixing systematic
wells
(Figure 5).
influence the result due to the
A hit
not
microscopic
contain
fields
the
this
yielded
18
urn)
found for the two series
reason are
by Op¬
all fields
in well 11
15.3+1.8 and 13.5+1.8
x
pronounced
aggregates, which
large
(Figure 5),
calculation
differences be¬
involved
of the
example,
103 urn3 mmjoc'2
for
Operator
by Opera¬
value
average
strongly
while
the fields chosen
over
Operator
mean
the
A and
abundance
(Figure 3).
D
1
sludge
rank test indicates that
B, respectively. An almost identical shape of the overall uncertainty of the
was
chosen
quantified
significant
of the aggregates. For
The
aggregate.
A
high spatial variability
The main
spherical shape
large aggregate (diameter
did
B
tor
66
one
signed
fields did not lead to
microscopic
0.31). However,
=
were
activated
effects due to the choice of the fields and
due to the estimation of number and size of the aggregates. A
the effect of the choice of the
fields
analyzed
in
23456789
10
Operator
A
Operator
B
11
well
Figure
scopic
5
Results from the
fields
3.3 Time demand for
Depending
croscopic
analysis
of
systematic
errors
due to the choice of the
quantification
the abundance of the aggregates, the time demand for the
on
field
ranged
from 20 seconds
(probe NEU)
average for operators familiar with the FISH
to 90
technique
and
3.4 Link between abundance of nitrifiers and measured
In activated
sludge models,
By contrast, quantitative
biomass concentrations
FISH is
a
direct
measure
are
seconds
into inhibtion of bacteria and
competition
of AOB
population
(conversion
estimated
by measuring
their
activity.
of the biomass concentration. The combina¬
examined. The
sludge
activity provides
from the MBR
pilot plant
oligotropha lineage (probe Nmo218),
sludge
of ammonia to
the
activity
eutropha/europaea lineage (probe NEU)
in the
on
sludges
members of the Nitrosomonas
nitritation rates
(probe Ntspa662)
mi¬
insights
dominated
sentatives of the AOB
one
new
populations
members of the Nitrosomonas
of
among different bacteria. Two activated
with different
were
analysis
epifluorescence microscopy.
tion of the information about biomass concentrations and their
by
fields. 6 micro¬
microscopic
analyzed per well (Probe Nmo218).
were
from the SBR. The
nitrite)
were
was
whereas
the main repre¬
comparison
of measured
with the biovolume of AOB revealed
a
lin-
16
1. A
relationship
ear
r
0.94).
=
A
for both the MBR
linear
weighted
0.02 for the MBR and
the
assumptions
of 10-30
per cell
can
fields
(Mueller
one
(depending
al., 1966),
corresponding
values for the
for the
not
intercept (p
urn3 and 1 ul
cell is 0.5-1
=
sludge
plant (Figure 6B,
slope (p
0.37 and
of fixed
=
0.008 and
0.34).
sample
Under
consists
the
plant, respectively. Assuming
the
and the SBR
0.96)
=
activated
in
sludge concentration), specific nitritation rates
yielded 1-7 x 10"3 and 3-18 x 10"3 pmol cell"1 h"1 for
on
be estimated. The calculation
MBR and the SBR
r
regression yielded significant
SBR, respectively) but
the MBR and the SBR
et
(Figure 6A,
that the biovolume of
microscopic
rapid method to quantify nitrifiers
values
are
a
biomass
1600kgSsm"3
kgN kgss"1 d"1 for the
of
density
0.3-2.0 and 0.8-5.0
plant, respectively.
<f~100
250
800
nitritation rate
Figure
plant.
Relationship
6
12
microscopic
fields
croscopic
one
standard
were
(mgNgcoD1 d"1)
nitritation rate
(mgNgcoD1 d"1)
A) Sludge from the
between measured nitritation rate and abundance of AOB.
fields
were
analyzed
analyzed
for each
sample (probe Nsol225). B) Sludge
for each
sample (sum
is
nowadays
of
Nmo218 and
probes NEU,
MBR
pilot
from the SBR. 14 mi¬
Nmll). Error bars indicate
error.
4. Discussion
the FISH
Although
technique
plants,
wastewater treatment
samples.
as
On the other
substrate
This
its benefits for
fast and reliable methods to
hand,
study
hand,
consumption
focuses
on
the
rates
the
often
quantify
quantitative
applied
of interest for
are
and their appearance in
need of
use
nitrifying population,
for
The
an
new
expensive
quantification
method
dance of nitrifiers in activated
ability
studies. Their
large
as
one
number of
modeling.
are
generally
an
vulnerable to
important step
perturbations
low abundance in activated
facilitate their
quantification
and
sludge
without the
subsequent image analysis.
was
successfully applied
sludge. Although
of the aggregates and their
limited. On the
needed to process
because nitrification is
densely packed aggregates
CLSM with
are
such
data must be linked to traditional parameters such
which allows its
bioaugmentation
engineering systems
practical applications
bacteria
within the treatment of wastewater. Moreover, nitrifiers
are
to
subjective
reproducibly
determine the abun¬
uncertainties due to the natural
estimation
results revealed that the main part of the overall
to
were
uncertainty
not
separated
in the
arises from the
spatial
vari¬
analysis,
the
spatial variability
17
1. A
of the aggregates within the activated
among different operators, albeit the
to
subjectivity. Systematic
size of the aggregates
Due to the
microscopic
mean
sludge.
15
Therefore,
the
on
values
-
optimum
probe
and
probe.
of AOB, many
independently
the
analyzed sample,
spatial variability
much lower
Quantification by
These accuracies
plants (Manser
required
low
were
when
compared
et
were
prone
fields
probe
only present
were
approximately
image analysis typically
A
higher
tests in
modeling
batch
takes about
are
one
also
a
sample
Consequently,
(Figure 3).
microscopic
the
The
fields per
higher
time de¬
needed per
sample.
hour per
population dynamics
involving
accuracy
these results for
10 minutes
accu¬
from 35 to 22% de¬
from 13 to 11%. Due to the
uncertainty
result,
in very low numbers in
of 6 to 8
optimum
an
ob¬
found to be 10 to
was
time demand of 5 to 15 minutes per
to a
of NOB led to
its
only slightly improves
uncertainty
to
Below 10
increase of the accuracy of the
large
shown to be sufficient to track
al, 2004).
applied.
many fields without any stained bacteria.
field for NOB,
activity
are
analyzed
than for the other groups of AOB examined
higher
CLSM and
using
to
the NEU
CVs of the overall
microscopic
a
method
quantification
CVs of the overall
corresponds
targeted by
spatial variability
sample involving
mand per
of the
microscopic
probe involving
there
was
fields must be
microscopic
between effort and accuracy of the result
used. This
Since AOB
deviations
significant
several tasks which
implies
sludge
in the choice of microscopic fields.
nor
fields per
microscopic
pending
method
fields every additional field leads to
an
did not show
analysis
activated
in
found neither in the estimation of the number and the
whereas every additional field above 10
racy.
The
presented
errors were
high spatial variability
tain accurate
rapid method to quantify nitrifiers
sample.
in activated
sludge
time demand would be
higher
purposes. However, the time demand is still
experiments.
The clear correlation between the abundance and the nitritation rate of AOB suggests that the
biovolume is
a
reasonable
reference system
of
state
also at
periods
and starvation would
x
The choice of the
possibly
for
al., 1999).
reflects
Nitrosospira
in
activity (Wagner
et
to
the
a
(Koops
and
known to
keep
population
by
the floe
carried out at
composition.
While
in the
substrate, members of the
growth
SBR)
cell"1
pmol
in
found
in
(various
members
exhibit
high
Nitrosomonas
rates
a
their ribosome
activity. The
h"1
(MBR)
of
ranging from
0.95 x 10"3 pmol
the
growth
rates
high affinity
to
et
sludges
Nitrosomonas
and
oligotropha lineage (prevailing
a
and
Prosser, 1989; Schramm
maximum
combined with
esti¬
literature,
nitritation rates between the two examined
specific
as
al., 1995), alternating stages of growth
incubated biofilm culture
have lower maximum
MBR)
are
were
for pure cultures of Nitrosomonas europaea to
pha/europaea lineage (prevailing
affinity
covered
alter the correlation between abundance and
The difference of the
their
area
results. However, all measurements
cell divisions. Since nitrifiers
with low
10"3 pmol cell"1 h"1
x
cell"1 h"1
When
activity.
l-7xl0"3
nitritation
of
rates
specific
10"3 pmol cell"1 h"1 (SBR) agree with values
mated
11-23
for
gives reproducible
growth supporting
content
3-18
measure
eutroa
low
in the
the substrate
Pommerening-Roser, 2001; Prosser, 1989).
applying
probe targeting
this
method,
a
preliminary analysis
AOB and about 30
microscopic
of about 50
microscopic
fields for every
fields for every
probe targeting
NOB of
a
18
1. A
typical sample
lows
on
required
standard
ple
recommended. The
the number of
defining
the
are
error
of the
although
This
only
has
study
scope.
cells
there
are
fields
dealt with uncertainties
the
result,
neities, which
are
caused
of the overall
uncertainty
the method cannot be
break up
during
sample depending
is then
sample
sludge
expressed
as
the
sampling
is
be
applied
to
quantification
performed
within the whole FISH pro¬
uncertainties due to
of the activated
have
sludge containing
only
inhomoge-
tanks. The estimation
sludge
of the
requires homogeneity
activated
a
the micro¬
at
hybridization steps probably
prone to
more
by incomplete mixing
in this method
the
encompassing
to
in
resulting
cells.
containing targeted
However, several preliminary steps have
on
per
for bacteria with low abundance
can occur
cedure. While small modifications of the fixation and
minor influence
activated
described above al¬
as
analyzed
of the result for every
uncertainty
targeted
median of zero,
fields needed to be
in
Mean values rather than the median must be used because multi¬
mean.
fields without any
subsequent uncertainty analysis
microscopic
accuracy. The
rapid method to quantify nitrifiers
very
samples. Furthermore,
dense floes, which do not
the treatment with ultrasonic.
5. Conclusions
The
presented
method allows fast and reliable
without the need of
applicable
for all
an
expensive
users
large spatial variability
bust in terms of the
confocal laser
of FISH. The overall
of the
subjective
quantification
sludge
scanning microscope (CLSM). Moreover, it is
uncertainty
involving
a
sludge,
10-15
choice of the
microscopic
compared
sequence, this method is
trification
microscopic
or
to
helpful
a
inhibition of
about
fields
one
hour
sample
using
bacteria which
well
by nitrifying
are
imply
bacteria facilitates the
is far
than
quicker
Furthermore, the
made
a
activity
application
of FISH for
tests in batch
measurement
of the abundance
can
shown to be
ro¬
the
found to be
subjective
exam¬
optimal regarding
As
a con¬
bioaugmentation strategies
the
of
analysis
be done at
a
large
activity
the
and could therefore
esti¬
fields for NOB
image analysis.
modeling. Moreover,
experiments
the
bacteria. For the
The demonstrated correlation between the biovolume and the measured
ing
was
as
by
microscopic
CLSM and
tool for the evaluation of
nitrifying
as
fields for AOB and 6-8
time demand of about 5-15 minutes per
effort and accuracy,
of the method is dominated
bacteria. However, the method
nitrifying
mation of the size and number of the aggregates formed
ined activated
of nitrifiers in activated
of ni¬
time series.
of the
presented
nitrify¬
method
partly replace
later stage. This
them.
study
has
step towards the implementation of molecular biological techniques into engineering
fields like
modeling
wastewater treatment
plants.
Acknowledgements
The authors would like to thank
Helge Daebel,
Marc Neumann and
Swiss Federal Institute for Environmental Science and
Christoph
Technology (EAWAG)
Ort of the
for their sup¬
port in the statistical evaluation.
19
CHAPTER 2
Membrane Bioreactor
Activated
vs.
Conventional
Sludge System Population
Dynamics of Nitrifiers
-
Reto
Manser, Willi Gujer, Hansruedi Siegrist
Presented at the 4
IWA World
Congress
Marrakech, September
2004
Membrane Bioreactor
Activated
Sludge System
Dynamics
of Nitrifiers
Reto
Conventional
vs.
Population
-
Manser, Willi Gujer and Hansruedi Siegrist
Abstract
Although
membrane bioreactors have attracted
search has been undertaken
subsequent eight
Both
pilot plant.
same
ing
months of
plants
two reactors for both
ingly,
were
membrane bioreactor with
a
in
operated
hybridization (FISH)
ammonia-oxidizing
both systems exhibited the
nor
on
separation
the nitrification
performance
conventional activated
sludge
revealed
only
bacteria and
were
of
sludge
age and treated the
us¬
minor differences between the
nitrite-oxidizing
bacteria. Accord¬
bacteria
by nitrifying
overgrown
re¬
the microbial
maximum nitrification rates. Confocal laser
in the inner part of the floes and
concluded that the membrane
composition
separation
nitrifying community composition
showed that the aggregates formed
ning microscopy
mostly
same
a
at the same
parallel
domestic wastewater. The identification of the
fluorescent in situ
attention in recent years, little
This paper compares the startup behaviour and the
community composition.
the
increasing
the influence of the membrane
on
by heterotrophic
itself does affect neither the
performance. However, impacts
were
scan¬
located
bacteria. It is
nitrifying community
kinetic parameters
on
are
emphasized.
Keywords: CLSM; Cluster;
Conventional activated
sludge system; FISH;
Membrane biore¬
actor; Nitrifying bacteria; Population dynamics
1. Introduction
Membrane bioreactors
creasing
attention in recent years.
(CAS) they
smaller
into
for the treatment of
(MBR)
have
a
biological
operation
lower
footprint
tanks and
Compared
wastewater
have attracted in¬
the conventional activated
due to the omission of the
adapting
the
sludge
process
secondary settling tank,
the effluent free of solids. First full-scale
provide
in past years,
to
municipal
plants
need
were
put
from conventional activated
sludge
models from the conventional activated
sludge
experiences
plants.
However, the
correct
application
process for MBR systems
and kinetics of activated
son
of the two systems
withdrawn,
factors
with
(Cicek
requires
sludge
were
synthetic
et
of
or
existing
additional information about the
in MBR. The few
special
al, 1999; Luxmy
al
(2000)
found
al.
(2002)
showed that the membrane
significantly
investigations
carried out at different
industrial
et
sludge
feed, fully
ages
done
et
so
or even
aerobic
al, 2000; Witzig
population composition
or a
far
on
the
sludge
combination of these
al, 2002). While Luxmy
different bacterial communities in the two systems,
sludge population
compari¬
without any
was more
Witzig
substrate-limited than
et
et
con-
21
2. MBR
ventional activated
sludge
membrane
on
separation
the nitrification
tings
were
operated
were
the
previous research,
In contrast to
these differences
sludge. However,
age, since the MBR
on
the
in
without any
of this
goal
Therefore
study
parallel.
The
sludge
age
was
Population dynamics ofnitrifiers
-
level
with diurnal variation
provided
of the
and
with the
set¬
pilot plant
in both
impact
(nitrifying bacteria)
same
in order to
plants
separate the influence of the membrane from the sludge age. The
wastewater
the
investigate
to
CAS
equal
high
withdrawn.
was
a
due to the
probably only
are
at genus
MBR and
a
CAS
sludge
community composition
performance.
operated
vs.
strictly
of domestic
treatment
real conditions.
2. Material and Methods
2.1 Pilot
The
plants
pilot plants (Figure 1)
draulic
variation.
60(18m3d"1)
The
fed with
were
and
CAS
the
municipal
treated
MBR
equipped with 4 submerged hollow fibre modules from
0.93 m2 each and a characteristic pore size of 0.1 urn.
tently operated according
bic tanks
the
to
deteriorating
specifications
the membrane
gen concentration
was
sludge
systems due
TSS in
higher
age
(SA)
MBR).
Zenon
hy¬
corresponding
to
with
of the manufacturer. A fine
kept
was
(ZW-10)
at 20
The
MBR
surface
a
Coarse bubble aeration
in the MBR. Both
temporarily higher
denitrification and the
to
wastewater
performance. Oxygen
m"3. Due to the coarse
controlled between 2.5 and 3 g
was
the
diurnal
typical
a
2(0.56m3d_1) population equivalents, respectively.
and
tained hairs from
showing
wastewater
area
of 2
mm re¬
concentration in the
bubble
plants
days (aerobic
SA
aeration,
was
The startup of both MBR and CAS
10
was
aero¬
the oxy¬
with pre-
operated
were
of
intermit¬
was
screen
was
days
in both
done without
inoculation.
Screen 6
mm
Permeate
Waste
Screen 2
water
'o
mm
Effluent
o
o
do
Anoxic
(7
Primary
clarifier
Figure
Setup
1
of pilot
Aerobic
(7
5
plants (left:
of cells
probes according
The
probes
T
|
used
SeSecondary
claclarifier
conventional activated
Hybridization (FISH)
were
were
performed
to Manz et al
are
i
Aerobic tank with
submerged
membrane
modules
(0
14
m3)
Anoxic
m3)
Microsynth (Balgach, Switzerland)
many).
do
o
o
from the CAS and the MBR
hybridizations
cleotide
m3)
,
-%-
2.2 Fluorescent In Situ
Samples
5
(5 m3)
o
-%
and
21
sludge plant, right:
and
m3)
&membrane
bioreactor)
microscopy
by Amann et al (1990). In situ
fluorescently labeled, rRNA-targeted oligonu¬
fixed
with
(0
(9 m3)
as
described
(1992). Oligonucleotide probes were obtained
Thermo Hybaid (Interactiva Division, Ulm,
from
Ger¬
listed in Table 1.
22
2. MBR
Table 1
Oligonucleotide probes
and
Probe
Target organisms
EUB338
Most Bacteria
CAS
vs.
hybridization conditions applied
Population dynamics ofnitrifiers
-
in this
study
Reference
Amaim et
and other Bacteria not detected
by
al.,
1990
Daims et
al,
1999
Daims et
al,
1999
EUB3 38-11
Planctomycetales
EUB338-III
Verrucomicrobiales and other Bacteria not detected
Nit3a
Nitrobacter spp.
Wagner
Ntspa662a
Genus
Daims et
al., 2001a
Ntspa712a
Phylum Nitrospira
Daims et
al., 2001a
NEUa
Most
EUB338
by
EUB338
Nitrospira
halophilic
and halotolerant ammonia oxidizers in the beta-
Wagner
et
et
al,
al,
1996
1995
subclass of Proteobacteria
a
Mobarrye/a/.,
Nsv443
Nitrosospira
NmV
Nitrosococcus mobilis
Nmll
Many members of the Nitrosomonas
communis
Nmo218
Many members of the
oligotrophia lineage
Nsol225
All known ammonia oxidizers in the beta-subclass except N. mobilis
used with unlabeled
All
competitor
samples hybridized
Canterbury,
time series
croscope,
United
were
spp.
as
with
equipped
Nitrosomonas
were
lineage
oligonucleotide probes
Giesekee/a/.,2001
Mobarrye/a/.,
standard
observation.
microscopic
to
embedded in Citifluor
were
For every
Samples
epifluorescence microscopy
with filters HQ-CY3 and HQ-FLUOS
(2005c).
PommereningRoser
étal, 1996
of
sample
and
(both
bacteria
nitrifying
probe,
12
on an
from
for the
Olympus
chosen
randomly
of
BX50 mi¬
Analysentechnik AG,
carried out
was
(Citifluor,
analysis
according
microscopic
to
fields
analyzed.
Images for the spatial analysis of the floes
recorded
were
using
a
TCS NT confocal laser
scanning microscope (Leica Microsystems, Bensheim, Germany) equipped
laser
1996
indicated in the reference
Tübingen, Germany). Quantification
Manser et al
PommereningRoser
étal, 1996
lineage
Kingdom) prior
analyzed by
1996
(450
to 514
nm)
constructions of floes
2.3 Batch
and two Helium-Neon lasers
were
experiments
generated
and
Maximum nitrification rates
volume
as
described
and set to 7.5 in all
by
from stacked thin
analytical
were
and 633
optical
one
Argon
Three-dimensional
nm).
re¬
sections.
measurements
by respirometry in batch reactors with 7.5 liter
(e.g. Cech et al, 1985). The pH value was controlled
measured
many authors
experiments using
17°C between the first and last
(543
with
HCl and NaOH.
experiment.
Temperature decreased from
Initial concentrations of ammonia
were
23 to
between 6
and8gNH4-Ntn"3.
The nitrification
one
capacity
of the biofilm
module five weeks after chemical
filled with tap water and
equipped
on
the membrane surface
cleaning
with
an
and
immersing
air diffuser.
was
assessed
it into
Oxygen
a
by taking
cylindrical
concentration
was
out
vessel
about
23
2. MBR
8 go2
rn"3.
The initial concentration of ammonia
stock solution.
of the bulk medium
Samples
vs.
CAS
-
Population dynamics ofnitrifiers
set to 10 gNH4-N
was
rn"3 by adding
regularly analyzed
were
ammonia
for nitrate to calculate
the nitrification rate.
The wastewater
P042"
N02", total N,
P042",
N02",
composition
uors were
were
analyzed
analyzed
for TSS, total COD, soluble COD,
and total P. The effluents of both
soluble COD
composite samples
was
taken two to three times
twice
performed according
to
and DOC,
(only MBR)
a
a
plants
were
analyzed
and TSS
alkalinity
week. Grab
samples
for
NH4+, NO3",
NH4+, N03",
(only CAS).
from the mixed
week for TSS, total COD, total N and total P. All
Standard Methods
(American
Public Health
24h-
analyses
Association, 1992/
liq¬
were
3. Results and Discussion
3.1 Performance of the MBR and CAS
Table 2 summarizes the effectiveness of the MBR and the CAS system in the treatment of
domestic wastewater. Excellent effluent
90% of the total COD
was
94%. The
matter.
Both
higher
plants
the CAS exhibited
were
value of the MBR is
exhibited
a more
possible
reasons
complete
pronounced
for the
proximately
tent
of
mostly
due to the
variation and
on
the increased air
liquor properties
compared
supply
due to the
to the CAS
(data
not
organics, nitrogen
particulate
Further nitrification
coarse
as
bubble aeration
Furthermore, the
shown) probably
efficiency
of
reduced the
nitrogen
respectively, assuming
was
similar
ap¬
con¬
in the CAS effluent.
are
of solids from the treated wastewater, the total
Equal
of the MBR
retention of
slightly higher.
of the MBR and the CAS system
and
efficiency
stable nitrite oxidation in the MBR.
in the permeate
the recirculation flow rate.
of
were
67%) and 74% for the CAS and MBR system,
separation
complete
Approximately
nitrification. Nitrite concentrations in the effluent of
centration in the aerobic tank of the MBR system is
tent
obtained in both systems.
transfer effects within the floes. Removal
organic nitrogen
The mixed
direct
mass
or
more
smaller floe size in the MBR
influence of
was
removed in the CAS, whereas the removal
within the pores of the membrane
are
quality
higher
listed in Table 3. Due to the
suspended
solids
than in the anoxic
values for both activated
sludges
were
(TSS)
con¬
tank, depending
found for the
con¬
phosphorus.
24
2. MBR
Summary
Table 2
steady-state period
of the
performance
of five months
TSS
gm
Total COD
gm-3
Soluble COD
gm"3
0
pilot plants. Average
-
Population dynamics ofnitrifiers
values and standard deviations
DOC
gem"3
NH4+
Influent
97
CAS effluent
over a
31
+
MBR effluent
8.0 + 2.9
32±4a
326 +102
n.d.c
19+6
n.d.c
8 + 1
n.d.c
gNm"3
16 ±5
0.16 + 0.14
0.16 + 0.09
N03"
gNm"3
0.8 + 0.6
6.4 + 2.3
5.0 + 1.3
N02
gNm"3
0.5 + 0.4
0.21+0.17
0.17 + 0.05
Total N
gNm"3
31+6
Total soluble N
gNm"3
n.d.c
9.5+2.2
n.d.c
P042"
gpm"3
1.8 + 0.6
1.6 + 1.1
1.1+0.7
Total P
gpm"3
4.0+1.0
1.7 ±
148
+
66
mol m";
Alkalinity
b
of the two
CAS
shown.
Unit
Parameter
a
are
vs.
1.2b
n.d.c
7.3 + 1.2
(~ 3*DOC) and COD
Calculated
as sum
of soluble COD
Calculated
as sum
of phosphate and P content of TSS
content of TSS
not determined
Table 3
state
Mixed
period
liquor properties
of five months
are
of the two
Unit
Parameter
over a
steady-
MBR
3650 ± 844
4730 ± 573
(aerobic)
2980 ± 473
(anoxic)
gcOD gTSS
1.09+0.15
1.09 + 0.06
ip
gp gcOD
0.022+0.005
0.022 + 0.004
In
gN gcOD
0.065+0.011
0.069 + 0.008
ICOD
Nitrifying population
3.2
The evolution of the
(Figure 2).
nated
by
The
were
of
a
similar pattern for both CAS and MBR
ammonia-oxidizing
bacteria
(AOB)
oligotropha lineage (Nmo218),
is domi¬
which made
80%> of all detected AOB. The smaller fraction consisted of members of the
europaea/eutropha (NEU)
and the Nitrosomonas communis
lineage (Nmll).
present throughout the whole period and accounted for 5%> and 15%> of all AOB,
respectively.
Members of the iV.
europaea/eutropha-\ike
and
bacteria shows
heterogeneous population
approximately
Both
nitrifying
members related to the Nitrosomonas
Nitrosomonas
iV.
values and standard deviations
CAS
gm"3
TSS
up
pilot plants. Average
shown.
advantageous
high
substrate
Since ammonia concentrations
plants (WWTPs) treating
for TV.
show
and iV. communis-\ike AOB and possess
Pommerening-Roser, 2001).
water treatment
oligotropha lineage
oligotropha-\ike
are
domestic wastewater, the
AOB.
affinity compared
urease
to
activity (Koops
generally
prevailing
low in waste¬
conditions
are
However, the predominance of iV. oligotropha-
25
2. MBR
like AOB is
contrasting previous investigations
Nitrosomonas
somonas
marina cluster
Unfortunately,
operated
europaea/eutropha lineage,
at
it is not
low
of WWTPs, where members related to the
detected
probably provides
niches for iV.
were
of FISH to
a raw
fering signals
microscope.
after 150
composition
enhanced the
possibly
2001b).
No
signals
days
of
were
coccus
detected after
generally
are
of
operation.
hybridizations
Nitrosospira
only sporadically
with
Nsol225 confirmed that
no
more
with
group
nor
plants.
inter¬
signifi¬
community
but
performance,
redundancy (Daims
NmV and
probes
the
application
causing
diverse
com-
during
increased
lineage
The
varia¬
and iV.
The
fraction of dirt
WWTPs
high
NEU
al,
et
Nsv443, indicat¬
members related to Nitroso¬
This result is consistent with the
hypothesis
for ammonia oxidation in WWTPs and that ni-
in these systems
trosospiras
cant
days
responsible
occur
probe
high
sewer.
did not influence the nitrification
mobilis could accumulate in the
nitrosomonads
inoculation from the
of the process due to functional
that neither members related to the
that
by probe
Members of the iV. communis
operation
stability
by
on
AOB. The
europaea/eutropha-\ike
failed because of the
in both MBR and CAS after circa 150
cantly
ing
sample
wastewater
under the
also caused
was
al, 2002).
et
carried out
specified
munis-Wkt AOB to occupy. But the low number of AOB detected
investigated period possibly
and the Nitro¬
lineage
(reviewed by Wagner
ages, which would favour the above
tion of the influent load
Population dynamics ofnitrifiers
whether most of these studies
reported
sludge
-
the Nitrosococcus mobilis
frequently
most
were
CAS
vs.
(Purkhold
et
al, 2000). Hybridization
further hitherto known AOB
were
present in signifi¬
number.
Nitrite-oxidizing
bacteria
(NOB) belonged
and CAS, but could be detected
Since
no
supposed
only
to
the genus
after about 120
members related to the genus Nitrobacter
that either unknown
rRNA content of the cells
was
nitrite-oxidizing
days
of
(NIT3)
bacteria
in both MBR
Nitrospira (Ntspa662)
complete
found in any
were
were
nitrification
samples,
present in the first
below the detection limit for FISH.
(Figure 2).
period
it is
or
the
Alternatively, hybridization
with
probe Ntspa712 targeting the phylum Nitrospira did not reveal the presence of further
NOB (data not shown). Thus, the low nitrite concentrations in the reactors favor the adapta¬
tion of K-strategists
(high affinity
to
the
substrate, low maximum growth rates)
like NOB, while Nitrobacter'-like NOB
strategist
with low
affinity
to
nitrite
are
(Schramm
postulated
et
to
be
a
as
Nitrospira-
relatively fast-growing
r-
al, 1999).
26
2. MBR
a)
_
vs.
CAS
-
Population dynamics ofnitrifiers
60
AIIAOB(Nso1225)
D
.2
at
ë I
(0
O N
20
to
N oligotrophia lineage
(Nmo218)
i
40
lineage
(Nmll)
europaea/eautropha
lineage (NEU)
A N
i
i-A-i—$—*-
0
b)
communis
-
O
<f- 60
a)
-
I
=
E
ra
40
Nitrospira
(Ntspa662)
Genus
1
i20i
E
(0
tO
O
£-
0
•
••••••
30
c)
--
D
E
ANH4
--
a0
z
D)
liM,
6000
4000
£
Q
a
•
O
¥ 2000
50
100
150
time
200
»
•
N03
ON02
DCOD
250
(d)
AIIAOB(Nso1225)
N oligotrophia lineage
(Nmo218)
O N
communis
lineage
(Nmll)
europaea/eutropha
lineage (NEU)
A N
e)
<f~ 60
S
|
=
"E
E
(0
40
H
»
|20
Nitrospira
(Ntspa662)
Genus
to
o
~
<-•—•-
0
30
a
20
a
-
*
E
z
D)
10
n£P
ru
djd
a*A
Ai
^
CL10
n^^n
u
rn
^
n
D
D
DSb ^
DÖ=n-,
D
D
6000
,
D
D
4000
rn
Q
q$S]CP
o
o
D
*
+
iL «»%••
••.•
Bum
100
150
time
2
Time series of ammonia-
(a-c)
a
ANH4
ON02
•
N03
DCOD
0
50
Figure
2000
«
0
the CAS
'£
and MBR
(d-f).
250
(d)
(a, d) and nitrite-oxidizing bacteria (b, e) and concentration profiles (c, f) in
Error bars indicate
trations refer to the effluent of the
200
plants,
one
standard deviation.
Ammonia, nitrate and nitrite
COD concentration to the activated
concen¬
sludge.
27
2. MBR
MBR
CAS
vs.
-
Population dynamics ofnitrifiers
MBR
CAS
DCAS
80
NOB
AOB
60
1
-
40
jj (1
20
0
CO
O
CM
*
cd
V
V
V
CO
O
CM
cluster size
Figure
3
Cluster
(probe Ntspa662).
five months.
the
largest
cells
were
The
cluster size
possibly
the
gates
Nitrospira
12
was
lead to the
was
tected
urn.
located
large
over a
steady-state period
of
groups
the aggregates formed
(Figure 3).
Daims et al
by
(2001a)
clusters in conventional full-scale activated
Similarly
development
availability. Although
mostly
urn were
to
present in the CAS system,
the floe
size, the high shear forces
of smaller aggregates. As
(Nmo218)
the floes
confocal laser
were
were
a
located in clusters
were
sludge
consequence,
larger
than 10
rather
floe is crucial in terms of
compressed
scanning microscopy
in the inner part of the floes
EUB338mix
slow-growing
nitrifiers in
Generally
distribution of the nitrifiers within the activated
only by probe
grew the
(nm)
35% in the CAS system.
hybridization procedure,
were
counted for each distribution
ammonia-oxidizing
15% of all detected AOB in the MBR
substrate and oxygen
to
were
grew in dense clusters.
preferably
cluster size in the MBR
spatial
CD
While AOB clusters up to diameters of 18
urn, whereas it
co
i
excluded.
smaller than those of
within the MBR
only
CD
CD
(jim)
found similar average diameters of
sludge plants.
co
i
Between 350 and 1400 cluster
Single
were
CD
(=aggregate) size distribution of ammonia- (probe Nmo218) and nitrite-oxidizing bacteria
Both AOB and NOB
NOB
|^
rfl^
(Figure 4).
considered to be
on
showed that the aggre¬
Most of the bacteria de¬
heterotrophic bacteria,
nitrifiers. This observation suggests
a
the slide due
potential
which
over¬
oxygen limitation of
floes.
28
2. MBR
Figure
of the
Analysis
4
shows AOB detected
detected
spatial
distribution of AOB within
scanning microscope (CLSM).
focal laser
by
an
CAS
vs.
activated
Population dynamics ofnitrifiers
-
sludge
The distance between the horizontal
both the Nmo218
probe
and EUB338mix
probe.
floe from the CAS
layers
is 2
um.
plant by
con¬
The white color
The grey color shows other bacteria
EUB338mix.
only by
3.3 Nitrification rates
Both the CAS and the MBR system exhibited
age values of 33+5 and 36+5 mgN
First,
gcoD-1 d"1
at 20
led to the
However, the smaller floe size in the MBR
by
similar maximum nitrification rate with
aver¬
°C, respectively (Figure 5).
unlimited conditions with respect to substrate and oxygen
at
nitrifying community composition
ated
a
same
nitrification
compared
performance
the CAS
to
the location of the nitrifiers in the inner part of the
concentrations, the
(data
not
same
in both systems.
shown),
floes, possibly lead
to
accentu¬
smaller
mass
transfer effects at limited conditions in MBR systems.
Secondly, knowing
(SF)
ri
in terms of
the maximum nitrification rates and
overloading
can
be estimated
applying
nitrogen balance,
the
following
the
safety
factor
formula:
Nit, max
T7
overloading
with:
*
ALNlt
ALNlt
j
=
max
=
ave
maximum ammonium load that
The calculation
yielded
suming typical
maximum
constants
factors
(0.2 d"1
was
be nitrified
at 20
(gN
growth
rates
°C; Siegrist
et
(1 d"1
at 20
al, 1999),
d"1)
(CAS: 349, MBR: 11) (gN
2.7 and 2.6 for the CAS and the
a
d"1)
MBR, respectively. Furthermore,
°C; Gujer
et
as¬
al, 1999) and aerobic decay
good agreement
with the calculated
safety
found.
Since the membrane surface
capacity
can
average ammonium load that is nitrified
of
these
provides large
biofilms
was
areas
for
investigated.
potential
biofilm
Surprisingly,
growth,
the
the nitrification
measured
rate
was
29
2. MBR
15
2.5
±
mgNm"2 d"1,
3000 mgN
formance
4000 mgN
m"2 d"1
is
since
negligible
m"2 d"1 in respect
it is
far
°C; Boiler et
at 15
is
module). Thus,
backwash
which
to
supposed
the
lower
than
al, 1990).
CAS
vs.
for
of
growth
1
(8
that the shear stress of the
the
(2000-
Its contribution to the overall nitrification per¬
the membrane surface
procedure efficiently prevent
biofilms
typical nitrifying
nitrification
average
Population dynamics ofnitrifiers
-
of the
rate
h"1 m"2
average
was
stable biofilm
on
about
load per
hydraulic
bubble aeration and the
coarse
a
reactor
cyclic
the membrane
sur¬
face.
o
-a
•
CAS
OMBR
150
180
210
5
indicate
Overall maximum nitrification rates. Values
one
270
(d)
time
Figure
240
given
are
corrected for temperature at 20 °C. Error bars
standard deviation.
4. Conclusions
The
impact
of the membrane
formance of the process
ing
the
same
wastewater. It
the membrane
tion. The
bility
was
prove the
although
nitrifying community composition
and
heterogeneity
to
seem
age. It is
separation
be
of the AOB
hypothesized
activated
on
pilot
in
and the per¬
parallel
treat¬
that
nitrifying community composi¬
population
and its associated sta¬
other factors like wastewater
that this
finding
can
also be
composi¬
assigned
to
other
sludge population.
does neither enhance the nitrification
provide
a
or
sludge
performance
nor
im¬
wash-out.
large
surface
contribution to the overall nitrification rate
Consequently, existing
MBR and CAS
experiments
dependent
safety against overloading
the membranes
a
itself does not influence the
groups within the activated
the membrane
the
is concluded from the
composition
sludge
on
investigated by running
separation
of the process
tion and
separation
models
can
are
be
area
for
potential
biofilm
growth,
its
neglected.
valid also for MBR systems in terms of
the model structure, but the characterization of the kinetics needs further research. The
smaller floe size in the MBR
ers
in the inner part of the
compared to
floes, possibly
the CAS, accentuated
by
lead to smaller
transfer effects in MBR sys¬
mass
the location of the nitrifi¬
tems.
30
CHAPTER 3
Bioaugmentation
Reto
of Nitrifying Bacteria
Manser, Kathrin Muche, Willi Gujer, Hansruedi Siegrist
Manuscript
in preparation
of Nitrifying Bacteria
Bioaugmentation
Reto
Manser, Kathrin Muche, Willi Gujer, Hansruedi Siegrist
Abstract
Bioaugmentation
is often
without the need of
separate
highly
the
of
potential
uids. It
riched
enlarging
treatment of
enriched
suggested
Nitrosomonas
a
By
means
oligotropha
were
and
days.
Nitrospira)
digester liquids
municipal
Activated
Keywords:
a
studies,
indicate that the enriched
formance of
As
detected
as
low substrate
a
This
by
affinity
were
contributing
the effect of
digester liq¬
present in the
pilot plants (dominated by
fluorescent in situ
and
en¬
to the ac¬
not able to
were
hybridization
adapt
to low
fully nitrifying pi¬
to the conversion of ammonia and nitrite.
continuously adding sludge
to wastewater treatment
nitrifying sludge
study investigated
Nitrobacter) compared
consequence, the nitrifiers added to the
washed out without
of simulation
treatment of
nitrifying
bacteria
conventional and two membrane bioreactor
substrate levels within
plants
of
populations
sludge digestion provides
seeding.
from the separate treatment of
Nitrosomonas europaea and
(FISH). The former exhibited
lot
associated with centralized
bioaugmentation using sludge
of
sludge
improve the nitrification performance
Furthermore, the increased application of
to
which could be used for
nitrifying sludge,
sludge (dominated by
tivated
the aeration tanks.
digester liquids
found that different
was
concept
as a
plants
from the separate
The results
investigated.
was
is not suitable to enhance the nitrification per¬
wastewater treatment
plants.
Membrane
sludge; Bioaugmentation; FISH; Kinetics;
Bioreactor;
Modeling; Nitrification; Population dynamics
1. Introduction
Historically, only
relatively
COD
removed in wastewater treatment
was
small tank volumes.
N-removal have to be met.
trophic bacteria,
Nowadays,
bacteria have low
Nitrifying
which results in
stricter effluent
large
addition, the separate
treatment
of
in terms of
growth
rates in
comparison
to
hetero¬
volumes of aeration tanks to guarantee all-season nitri¬
fication. Denitrification needs often to be achieved too,
In
plants (WWTPs), involving
regulations especially
leading
to even
larger
tank volumes.
indeed decreases the overall ni¬
sludge digester liquids
trogen load, but increases the diurnal variation of the N-Kj (total Kjeldahl nitrogen) load,
which augments the
Bioaugmentation
out
the need of
was
introduced
enlarging the
tion is carried out in
a
are
dewatering. The
trifiers, whereas
high
typically
main
sludge
as a
retention time
concept
improve
to
(SRT).
the nitrification
aeration tanks. Accumulation of
nitrifying
separate reactor either outside of the activated
Oleszkiewicz, 2004; Plaza
Both systems
aerobic
required
et
al, 2001)
within the return
seeded with ammonium-rich
advantage
sludge
nitrifying
concentrations,
bacteria
e.g. the
sludge
line
seems
to
stream
(Salem
liquids originating
SHARON®
with¬
bacteria used for addi¬
of the latter system is the enrichment of
different type of
influent ammonium
or
performance
et
(Head
and
al, 2003).
e.g. from
sludge
already adapted
ni¬
be present in processes with
process
(Logemann
et
al, 1998).
32
Bioaugmentation ofnitrifying
3.
However, it is
to
not
clear whether nitrifiers grown at
low substrate concentrations
(MBR)
reactors
plants (CAS,
exhibit
a
Manser et
prevailing
different
goal
of this
study
brane
was
to
investigate
the
potential
bridization
on
(FISH)
the
for
enriched nitri¬
using
This included the
of the
analysis
of their kinetics and examination of the
Furthermore, the potential positive effect of
bioaugmentation performance
applied
was
of bioaugmentation
digester liquid.
measurement
among the different groups.
separation
addition,
conventional activated
to
settleability.
autotrophic community compositions,
competition
adapt
can
membrane bio¬
sludge
compared
sludge
al, 2005a) and completely retain bacteria, which prevents loss of
bacteria from the separate treatment of
fying
municipal
WWTPs. In
structure
seeded bacteria due to insufficient
The
in
ammonium concentrations
high
bacteria
was
explored.
tracking population dynamics.
mem¬
Fluorescent in situ
Batch
hy¬
experiments provided
kinetic data.
2. Materials and Methods
2.1 Pilot
plants
A CAS and two identical MBR
treating municipal
wastewater
pilot plants (Manser
following
the MBRs treated the wastewater
typical
a
corresponding
al, 2004)
et
diurnal
to 60
in
operated
were
parallel
variation. The CAS and
hydraulic
m3
d"1) and
(18
(0.56 m3
2
d"1) popula¬
equivalents, respectively. Oxygen concentration in the aerobic tanks was controlled be¬
tween 2.5 and 3 g m"3. Due to the coarse bubble aeration inducing a cross-flow at the mem¬
tion
brane
operated
were
days
A
surface, the
oxygen concentration
with
pre-denitrification
for the CAS and MBR 1 and 40
sequencing
batch reactor
the separate treatment of
synthetic
set to 7.5+0.1.
sludge
volume 3
at
m3) was
inoculated with
aerated. The
operated
over
2.2 Batch
this
sludge
from the SBR for
(Switzerland),
pH value
was
fed with
controlled
Community compositions
a
using
period
did not
experiments
constants for
substrate
(ammonia, nitrite).
batch reactors with 7.5 liter volume for at least two hours
that all
biodegradable
matters were
tively. Oxygen
concentration
limitations. AOB
regularly
were
was
inhibited
until all substrate
Activated
prior
using
HCl
10 g
was
adding
or
NaOH in all
controlled between 3 and 4 g
by
to
sludge
consumed. The temperature and
trolled and set to 15.0+0.2°C and 7.50+0.05
taken
at 20
period.
A) Half-saturation
sure
plants
kept
twice for about two weeks. Each
started with inoculum from the WWTP of Biilach.
change
(SRT)
was
.
the WWTP of Biilach
continuously
was
in the MBRs. Both
retention time
for the MBR 2
days
The SBR
temporarily higher
and the
digester liquid
ammonia solution and
NaC03 and
was
(SBR,
was
m"3 allylthiourea (Ks
m"3
was
aerated in
the substrate to
pH
value
en¬
was con¬
experiments,
respec¬
in order to prevent oxygen
for nitrite
only). Samples
consumed. Substrate concentrations of the
samples
were
were
33
Bioaugmentation ofnitrifying
3.
measured
according
Standard Methods
to
oxygen utilization rate
tionally,
maximum nitrification
in
al., 1993)
et
trolled and set to 7.50+0.05 in all
(15
Public Health
used for the estimation of the K values.
were
batch reactor
a
Association, 1992/ Addi¬
for the determination of the
experiments
Nitritation and nitratation rates
B) Maximum nitrification activity.
respirometry (Drtil
data from
(OUR)
activity (B)
(American
bacteria
experiments.
(Volume
7.5
were
liter).
measured
The
by
pH value
means
was con¬
The temperature in the batch reactors
the
of
kept
was
Added ammonium and nitrite concentrations
17°C) during
experiments.
m"3/20 gNo2-N m"3, 10 gNH4-N m"3/4 gNo2-N m"3 and 5 gNH4-N m"3/2 gNo2-N m"3
for sludge mixtures; 100 gNH4-N m"3/20 gN02-N rn"3 for the bioaugmentation experiment (Chap¬
ter 2.3); 500 gNH4-N m"3/75 gN02-N rn"3 for the decay experiment (C).
constant
or
100 gNH4-N
were:
A batch reactor
C) Autotrophic decay.
reactor
and
perature
continuously
was
kept
aerated. The
at 15°C. At
from the batch reactor and
tion rates
were
measured
(130 liter)
pH value
intervals of
placed
in
a
one
secondary
sludge
from the SBR
pilot
controlled and set to 7.5+0.1. The tem¬
was
day,
filled with
was
7.5 liter of activated
sludge
was
withdrawn
batch reactor, where nitritation and nitrata¬
by respirometry. Nitrite-oxidizing
bacteria
are
probably
inhibited
by
the elevated nitrate concentrations. Batch
nitrate concentration from 60 to 300 gN
However,
a
centrations
2.3
inhibition
constant
only slightly
can
increased
experiments revealed that a sudden increase of the
m"3 reduced the maximum nitratation rates by 50%.
be assumed
during
the
during
the
experiment,
since the nitrate
con¬
experiment.
Bioaugmentation experiment
Large
of SBR
sludge were once-only
m3
(0.05
each). The spiking of the
amounts
and MBRs
instantaneously
was
were
Amann et al
from the CAS
cells
were
CAS took about
hour,
(2.7
m3)
whereas the MBRs
Hybridization (FISH)
taken at maximum OUR and fixed in 4%
(1990).
prior
one
seeded.
2.4 Fluorescence In Situ
Samples
added to the anoxic tanks of the CAS
Ultrasonification
to
performed
hybridization
with
(35 kHz,
five
paraformaldehyde
minutes)
in order to break up
large
was
applied
floes. In situ
as
to
described
fixed
by
samples
hybridizations
fluorescently labeled, rRNA-targeted oligonucleotide probes
of
ac¬
to Manz et al
(1992) (Table 1). Oligonucleotide probes were obtained from Microsynth (Balgach, Switzerland) and Thermo Hybaid (Interactiva Division, Ulm, Germany).
cording
All
samples hybridized
Canterbury,
was
done
(both
was
United
on an
from
and
oligonucleotide probes
Kingdom) prior
Olympus
BX50
to
microscopic
were
embedded in Citifluor
observation.
microscope, equipped
(Citifluor,
Epifluorescence microscopy
with filters HQ-CY3 and HQ-FLUOS
Analysentechnik AG, Tübingen, Germany). Quantification of nitrifying bacteria
carried out
sample
with
according
to Manser et al
(2005c).
14
microscopic
fields
were
analyzed
per
probe.
34
Bioaugmentation ofnitrifying
3.
Table 1
Oligonucleotide probes
and
hybridization conditions applied
in this
NEUa
Most
halophilic
study.
Reference
Target organisms
Probe
bacteria
and halotolerant ammonia oxidizers in the
Wagner
al.,
et
1995
beta-subclass of Proteobacteria
a
Nmll
Many members of the Nitrosomonas communis lineage
PommereningRoser et al.,
Nmo218
Many members of the Nitrosomonas oligotropha lineage
Giesekeetal.,2001
Nit3a
Nitrobacter spp.
Wagneretal.,
Ntspa662a
Genus
Nitrospira
Daims et al, 2001a
used with unlabeled
competitor
1996
1996
indicated in the reference
as
Modeling
2.5
representation of the investigated processes, the activated sludge model No. 3
(ASM3, Gujer et al, 1999) was extended with two-step nitrification to enable separate model¬
For
a
correct
ing
of ammonia and nitrite oxidation. Denitrification of nitrite and from nitrate to nitrite
not
included, because this study
AOB and NOB
dix).
exhibiting
focused
on
nitrification.
distinct kinetic parameters
Kinetic data of the SBR
and MBRs to the second
was
were
assigned
to one
Furthermore,
introduced
were
(XAoBr, XNoBr),
two
was
populations
(Table 3,
see
of
appen¬
kinetic data of the CAS
population (XAoBk, XNOBk)-
3. Results and Discussion
of
Community composition
3.1
The
analysis
of the
ammonia-oxidizing
clear difference between the SBR
nicipal wastewater, Figure 1).
Nitrosomonas
bacteria
nitrifying
(fed
bacteria
with
(AOB) community composition
digester liquid)
The CAS and MBRs
oligotropha lineage (Probe Nmo218,
detected AOB. These bacteria show
therefore well
a
were
high
bars),
affinity
in
pilot plants (fed
by
and
Pommerening-Roser, 2001; Prosser, 1989). By contrast, in the SBR
the Nitrosomonas
europaea/eutropha (Probe NEU,
group is characterized
tolerance
prevailing
(Bollmann
mu¬
which made up 70-80% of all
and low maximum
are
to
with
a
members related to the
and
adapted
conditions
dominated
white
substrate
and the
exhibited
growth
rates
completely nitrifying plants (Koops
black
bars)
were
reactor
most
members of
abundant. This
by a low substrate affinity, high maximum growth rates and a high salt
al, 2002; Koops and Pommerening-Roser, 2001), which is advanta¬
et
geous in environments with elevated ammonium concentrations. The third group made up ap¬
proximately
20% in all reactors and consisted of members of the Nitrosomonas communis
lineage (Probe Nmll,
oligotropha-like
and iV.
grey
bars).
Their
europaea-like
phylogenetic
characteristics
are
between the iV.
AOB.
35
bacteria
Bioaugmentation ofnitrifying
3.
100%
m
o
D N
N
H
oligotrophia
communis
N europaea
SBR
Figure
Population compositions
1
of each group of AOB
Nitrite-oxidizing
MBRs
belonged
(n
=
of AOB before
(NOB)
the genus
substrate and low maximum
and
high
Nitrospira (Probe Ntspa662). They
growth
rates
maximum
growth
rates
-
(Schramm
words, the
maximum
SBR
was
growth rates;
Andrews and
modeling
requires
(iVax-b)
are
outcompeted by
was
laid
(Umax)
on
exhibiting
a
low
present. Unfortunately, problems
of further
to
the
in the SBR
affinity
the sub¬
to
with the
hy¬
samples.
by r-strategists (low affinity to the substrate and high
Harris, 1986), whereas K-strategists (high affinity to
were
most
abundant in the CAS and the MBRs.
of the co-existence of different bacteria
detailed
knowledge
about the kinetic parameters. In
(KNH4
and KN02 of
crucial, because they determine whether
the better
adapted
populations using
particular,
Monod-kinetics)
a
of AOB and NOB.
of the latter
impairing
Half-saturation constants
(KNH4
Half-saturation constants of SBR
monium and nitrite
and
sludge
were
the half-
growth
growth
or
Therefore, the main focus
adversely
rates
(data
not
growth
rates
influenced the
shown).
KN02)
sludge
were
(Figure 2). Additionally,
CAS and MBR
inhibition effects
the estimation of the
same
certain bacterium is washed out
groups under certain conditions.
Unfortunately, product
the
and the net
the determination of the half-saturation constants and the maximum
measurement
SBR,
-
quantification
growth rates)
saturation constants for the substrate
rate
high affinity
a
parameters
The reliable
substrate
have
dominated
the substrate and low maximum
3.2 Kinetic
standard deviation
one
al, 1999). Conversely,
et
(Probe NIT3)
were
bridizations of probe NIT3 did not allow the
In other
Error bars indicate ±
bioaugmentation.
showed similar differences. The NOB in the CAS and
members related to the genus Nitrobacter
strate
MBR1 MBR2
3).
bacteria
to
CAS
estimated from concentration
OUR data from different batch
used for the estimation of the K values
profiles
of
experiments
am¬
with
(Figure 3).
36
Bioaugmentation ofnitrifying
3.
01
Figure
0 05
02
(d)
time
(d)
time
(d)
time
\a)
of SBR
profiles
Lines represent best fit to the extended ASM3
sludge.
SBR
1500
0 1
time
2 Measuredd concentration
bacteria
CAS
800
1
-|
-i
1
AOB
0
400
*
o
o
300
200
(W^
-
o oo ooo
a.
3
o
100
o
0 00
0 05
time
Figure
3
0 10
-
NOB
-
0 15
0 05
(d)
time
Measured and modeled OUR used for estimation of Knh4 and KN02 values. Lines represent best fit to
the extended ASM3.
visible in the
Upper diagrams: Nitratation due
to
nitrite
produced
lags
behind nitritation
(particularly
CAS).
The estimation of the half-saturation constants of SBR
AOB and NOB
ues
01
(d)
(Table 2), compared
from the OUR data
files for all examined
erably possibly
correspond
to
sludge yielded high
values for both
low values in the CAS and MBR1. The obtained val¬
well with the values estimated from the concentration pro¬
sludges. Although
due to inhibition effects
the KNH4 and KN02 values in the SBR vary consid¬
(Chapter 2.2),
SBR exceed those obtained from the CAS and MBR1
it
can
by
This kinetic observation is in agreement with the detected
be stated that the values in the
one
to two
orders of
magnitude.
community compositions (Chapter
3.1).
37
Bioaugmentation ofnitrifying
3.
Table 2 Estimated Knh4 and KN02 from the measurement of the concentration
(OUR).
rates
Given
respectively. A)
are
standard
estimated from concentration
profiles, B)
SBR
KNH4(gNm"3)
KN02(gNm"3)
9.9 ±0.5
9.2± 1.1
14.1 ±0.6
2.9 ±0.3
6.0 ±0.3
15.2 ±2.3
mean
estimated from OUR
and the oxygen utilization
Km«
and
mean
values,
MBR1
fem"3)
KN02
(gNm"3)
KNH4
(gNm"3)
i)
0.28
±0.20°
0.13
±0.05^
0.14 ±0.07
single
profiles.
CAS
KN02 (gN
0.17
m"3)
±0.06^
A)
0.11 ±0.01
0.21 ±0.02
4.2 ±3.0
)
profiles
of the estimations and standard deviations for
errors
bacteria
0.1
0.21 ±0.05
B)
0.24 ±0.02
value of three measurements
(Manser
et
al, 2005a)
Autotrophic decay
The net
growth
bacterium
rate
grow in
can
about the
knowledge
defined
-
an
of the SBR
4).
The
Qnob
values
significantly
a
converted
to
bacteria
minus
decay
is washed out at
-
a
bioaugmentation
The estimation of the
determines whether
given
Therefore,
SRT.
is needed in order to
endogenous respiration
lower value for the AOB than for the NOB
rates
(Figure
0.105°C_1,
(temperature correction factors: 9aob
d"1
d"1
and 0.31
for AOB and NOB, respectively. In
0.22
20°C
are
a
=
to
r-strategists)
patterns
or
rate
endogenous respiration rates estimated from activated sludge, which was
by K-strategists (Manser et al, 2005b), the same values here (sludge dominated by
comparison
dominated
sludge system
augmented plants.
sludge yielded
growth
of the nitrifiers used for
0.062°C"1, Jenkins, 1969)
=
maximum
activated
decay
model their fate in the
as
are
about 50%
higher. Interestingly,
the AOB fractions showed distinct
decay
(Figure 4, middle). While the abundance of the TV. europaea-like
(black) sharply decreased, iV. communis-like bacteria (grey) were less reduced and iV.
as
analyzed by
oligotropha-like
FISH
bacteria
(white)
strategists) probably outcompeted
microbial
decay
composition
were
even
able to grow
slightly.
the other groups for the released
and could therefore maintain its presence,
leading
The latter group
nitrogen originating
to a
shift in the
(Kfrom
community
of AOB.
~
300
N europaea
AN
communis
o N
oligotrophia
200
100
-
4
time
Figure
FISH
4
2
(d)
Decay of AOB (left) and NOB (right) from SBR sludge.
analysis
of AOB. Error bars indicate
one
standard deviation
4
time
(n
T
=
=
15°C, pH
7.5. Middle:
6
(d)
Quantitative
14).
38
3.
3.3 Batch
Batch
experiments
with
with
experiments
sludge
sludge
model parameters: XAoBr,
(SBR,
mixtures
mixtures
reflects the measured
reasonably
activity
XNOBr)
carried out in order to test whether the model
were
associated with the co-existence of
and
conditions with low substrate concentrations.
sludge
At
high
were
substrate
sludge correspond
are
not
not
limited
impair
the
by
rapidly adapt
Therefore, maximum nitrification
concentrations, the measured
well to the calculated
the substrate
sum
(Figure 5).
slower-growing K-strategists
rates
rates
to
of
of different mixtures of SBR and CAS
of the individual rates, since both
The
fast-growing r-strategists
populations
from the SBR did
from the CAS.
R
AOB
a
can
determined at different substrate levels.
2500
—~
r-strategists
K-strategists (CAS, MBRs, model parameters:
XAOBk, XNoBk)- On the other hand, it is uncertain, whether r-strategists
mixtures
bacteria
Bioaugmentation ofnitrifying
D measured AOB
2000
U calculated AOB
'E
1500
CM
1000
m calculated NOB
|
X
500
E
measured NOB
j
O
O)
z.
0
0%
10%
30%
% of SBR
Figure
(NH4:
5
100%
mJ, N02:
bars indicate ±
one
20 gN
are
mJ).
0% of SBR
equal
or
rates
sludge
means
slightly
lower than the
corresponding
sludge yielded
strategists (CAS, MBR) possibly
high substrate concentrations,
In contrast to
poor
sludge
100%
at
substrate concentrations
high
sludge.
activity
due to
significant
competitors
sludge
sum
T
17°C, pH
:
=
7.5. Error
the
at low substrate concen¬
of the individual rates
similar results
(data
r-strategists (SBR)
r-strategists only
substrate limitation.
lower substrate concentrations in the short term
are
70%
sludge
100% of CAS
of mixtures of SBR and CAS
Mixtures of SBR and MBR1
maximum
50%
% of SBR
sludge
interfere with the
to
30%
standard deviation.
Likewise, the measured
6).
10%
Measured and calculated OUR of mixtures of CAS and SBR
100 gN
trations
50%
at
(Figure
The K-
low substrate levels.
reach 30-50%) of their
Obviously, they
(hours, Figure 6).
shown).
not
As
are
a
not
able to
adapt
consequence,
they
in WWTPs with full nitrification associated with low ammonium and
nitrite concentrations.
Nonetheless, the addition of
nitrification
performance;
SBR
if the
sludge
to
the CAS and MBRs may contribute to the overall
indigenous nitrifying
(Figure 10). However, only partial
nitrification
can
bacteria
be achieved
are
washed out in winter
depending
on
the amount of
added nitrifiers.
39
Bioaugmentation ofnitrifying
3.
NH4:10gNm^,NO2:4gNm^
800
bacteria
NH4:5gNm^,N02:2gNm^
800
measured AOB
M
calculated AOB
U
calculated NOB
measured NOB
CAS
Figure
SBR
CAS
+
SBR
CAS
Finally,
the
ments at
=
15°C, pH
dynamic
=
7.5. Error bars indicate ±
behavior of the
sludge
one
mixture
experiments (Table 2), indicating
lations
according
activity
of both
bution of the
to
r-
+
SBR
at low substrate
standard deviation.
was
low substrate concentrations could be well
from individual
CAS
(~ 50%) and CAS (~ 50%) sludge
Measured and calculated OUR of mixtures of SBR
6
concentrations. T
SBR
a
examined
reproduced
parallel
(Figure 7).
The
with the parameters found
substrate
uptake
of the two popu¬
their individual kinetic characteristics. The model shows that
and
K-strategists
r-strategists
close to their maximum
to
was
activity
the
although
beginning of the experiment, the contri¬
quickly declined, whereas the K-strategists were
equal
the overall OUR
measure¬
at
the
until almost all substrate
was
consumed.
measured OUR
calculated overall OUR
AOBk
AOBr
NOBk
NOBr
(CAS) KNH4
(SBR) KNH4
(CAS) KN02
(SBR) KN02
=
=
=
=
0 1 gN
6 gN
0 2 gN
4 gN
m"3
m"3
rrï3
rrï3
NH4
NO,
0 04
time
Figure
0 08
0 02
time
(d)
0 04
0 06
(d)
7 Measured and modeled OUR of mixtures of CAS and SBR
at low substrate concentrations.
Bioaugmentation
3.4
The influence of the addition of nitrifiers from the SBR
on
sludge
the
seen
community composition
that the maximum nitritation rates
in batch
day
experiments sharply
14. The observed rise
on
the nitritation
of AOB in the CAS and MBRs is shown in
as
determined at
increased in all
agreed
non-limiting
pilot plants
performance
Figure
8. It
and
can
be
substrate concentrations
after the addition of SBR
sludge
on
well with the added amount of AOB. However, the aug-
40
3.
mented
with
and 30
large
days (MBR2). Although
uncertainties due to the
able information
washed out
versely,
by
decreased and reached the initial value after about 20
activity exponentially
1)
MBR
was
obtained. It
simultaneously
the AOB
the
quantification
caused
high spatial variability
was
of the AOB
found that the added AOB
in the
alter
the
during
r-strategists (AOBr)
within the
decay
and the dilution rates. The
period
were
of the
3.3). Otherwise,
not
able to
al, 2000)
amount
experiment, equally
nor
et
performance.
Con¬
enriched
plants.
The modeled
the de¬
pilot plants. Hence,
the different
bacteria. A
the amount of
organization
exhibited the
sludge
larger
nitrifying
are
the batch
prevailing
experiment
in the
pilot plants
for the short-term
(Chap¬
inadequately protected against prédation (Bouchez
of AOB and NOB
same
as
a
sharper
decrease of the added
dense aggregates
behavior. Neither the
structure in MBRs
SRT
only delays
bacteria is
consequently
(Manser
only
et
complete
(Manser
al, 2005a)
retention of
seem
to
the wash-out of the added AOB
directly dependent
of the modeled nitritation rates
gen load and
to
the conditions
the measured data suggests
et
al,
al, 1995) possibly prevent autotrophic bacteria from major prédation.
pilot plants
nitrifying
to
observed since it would have led to
of nitrifiers. The
All three
terns
were
the observed decrease of the iV. europaea should have been decelerated.
was not
2004; Wagner
cells
europaea)
iV.
inactivated due to the low substrate
good agreement with
adapt
The fact that accumulated bacteria may be
et
valu¬
sludge,
only slightly
in all
(CAS,
associated
of the modeled maximum nitritation rates and the biomass concentrations of XAoBr
reflect
that the added AOB
ter
were
experiment
were
concentrations and did not contribute to the nitrification in the
only
the added
(mostly
pilot plants (iV. oligotropha)
significantly
nitritation rates show that the
crease
by
partly
was
with the reduction of the maximum nitritation
dominating
the addition and did not
bacteria
Bioaugmentation ofnitrifying
reflect the
identical for all three
on
the nitrified
play
a
(MBR 2).
nitrogen load,
temporal variability
single
role for
Since
the pat¬
of the influent nitro¬
pilot plants.
41
Bioaugmentation ofnitrifying
3.
CAS
(SRT
Max. nitritation rates
600
=
MBR 1
20d)
(SRT
=
MBR 2
20d)
(SRT
=
bacteria
40d)
(batch experiments)
-
<
~
600
fe
-
W
D
~400
X.
-
U.T^c^-^
o
1200
-
z
0
-
Nitritation rates in aerobic reactor
300
200
„
-
-
!
all AOB
4l00H
AOBk
AOBr
Effluent concentration of
1
,-0
8
NH4
1
4
,-0
8
H
£06
|0 4
|0 4-
-
I
I
OT02
O
O
-
OT02
o
0
0 +0-020
40
time
Figure
8
20
Measured and modelled data of AOB for
sludge
was
added
on
day
the CAS and MBRs is shown in
tion rates
as
determined at
increased
according
to
Figure
9.
Analogous
the added amount of NOB in all
analysis
hybridizations
of the
Accordingly,
were
the nitratation
only
equally
performance
in
nitritation, the maximum nitrata¬
experiments sharply
pilot plants, subsequently quickly
(CAS,
of the
MBR
1)
and 30
probe targeting
days (MBR2).
Nitrobacter
de¬
Un¬
(r-strategist)
Nonetheless it is assumed that the
attributed to the wash-out of the added NOB
because the model suggests that their
the added NOB
the
community composition.
decline of the maximum nitratation rates is
(r-strategists),
to
on
substrate concentrations in batch
creased and reached the initial value after about 20
fortunately, problems
± one stan¬
14.
non-limiting
with the
60
(d)
Error bars indicate
bioaugmentation experiment.
The influence of the addition of nitrifiers from the SBR
did not allow the
40
time
(d)
dard deviation. SBR
ble.
-
activity
in the aerated tanks
to the AOB not able to
adapt
was
negligi¬
to low substrate
concentrations.
42
3.
(SRT
CAS
Max. nitritation rates
=
20d)
Bioaugmentation ofnitrifying
(SRT=20d)
MBR1
bacteria
MBR2(SRT=40d)
(batch experiments)
150
250
250
200
200
Nitratation rates in aerobic reactor
100
J" 150
a
1
1100
3
I)
f
50
-
0
Effluent concentration of NO2
20
40
time
Figure
9
of
potential
of
sludge
was
added
digester liquids
was
tion.
simulations
aerobic SRT
This
plant
system is
4d,
would
enriched
higher
HRT
=
(2003)
performed assuming
6h, influent ammonium
completely nitrify
days.
namic calculations
nificantly change
pressed
separate
± one stan¬
in
The results of
assuming
the results
in percent of the total
treatment
(data
not
It exhibits however
required
simulations reveal that the maximum addition
daily
depicted
amount
input N-Kj
(30%
almost 50% due to
of the
growth
T
a
=
CAS:
10°C.
only partial
in
Figure
10.
Dy¬
did not
sig¬
of added nitrifiers is
ex¬
municipal
nitrifying
no or
of
for all-season nitrification is
are
wastewater
which is returned in the
and used for the enrichment of
required
is most
for nitrifica¬
=
calculations
The
shown).
input N-Kj load,
by
bioaugmentation
the
diurnal variation of
ammonium is limited up to around 30% of the
monium load in the effluent
showed that
following plant layout
25 gN m"3 and
concentration
summer season.
steady-state
typical
a
from the separate treatment
somewhat below the minimal SRT
operated
were
nitrifying sludge
nitrification in winter season, because the minimal SRT
than four
(d)
Error bars indicate
bioaugmentation experiment.
elucidated. Salem et al
a
=
40
time
14.
bioaugmentation using
beneficial when
Therefore,
day
on
20
(d)
for wastewater treatment
Significance
The
40
time
Measured and modeled data of NOB for
dard deviation. SBR
3.5
20
(d)
digester liquid
bacteria. The internal
load
(Salem
et
input N-Kj load)
of the added
to
the
feeding
of
al, 2003).
The
reduces the
am¬
r-strategists (AOBr)
in
43
3.
the CAS. The
formance.
indigenous
By contrast, the bioaugmentation leads
fluent, because the added
quence, nitrite oxidation is
r-strategists (NOBr)
mentation dose.
are
of
abundant
takes
digester liquids
enriched bacteria
rates
place
therefore
are
is not
or even
Nonetheless, The NOBk
growth
are
compared
at
adapted
K-strategists (NOBk), although
poorly adapted
a
°
to
AOBr
produced
ni¬
the AOBr. Moreover, the separate treat¬
warm
influent. The
the low temperatures in the basins. Hence,
to
performance
negative impact
is
possibly overestimated,
on
nitrification rates
since
a
(Abeysinghe
NOB
-
*
*
*
*
m
*
oxidize the
completely
bioaug¬
-
30-
10
the added
Oleszkiewicz, 2004).
S?ng
a conse¬
exceed the former at the maximum
AOB
E 40
low nitrite levels. As
to
elevated temperatures due to the
sudden decrease in temperature may have
etal,
elevated nitrite concentrations in the ef¬
able to
not
the simulated enhancement of the nitrification
2002; Head and
to
attributed to the
mainly
equally
trite because of its lower
ment
population
NOB
bacteria
does not contribute to the nitritation per¬
population (AOBk)
AOB
Bioaugmentation ofnitrifying
*
-
AOBk
*
0-
o
S10
0%
10%
20%
30%
0%
addition of nitrifiers
(%
Figure
T
=
the
10
Simulation
10°C. As
an
of feeded
study
example,
digester liquid.
for
input N-Kj load)
Results of
(%
bioaugmentation assuming
10% addition of nitrifiers
steady-state
10%
20%
30%
addition of nitrifiers
means
calculations
are
CAS
of feeded
input N-Kj load)
plant layout
that 10% of the total
with SRT
input N-Kj
=
4d,
HRT
=
6h,
load is returned in
shown.
44
3.
Bioaugmentation ofnitrifying
bacteria
4. Conclusions
of
Bioaugmentation
using
enriched
a
conventional activated
sludge
and two membrane bioreactor
from the separate treatment of
nitrifying sludge
digester liquids
pilot plants
was
studied. It
is concluded that:
1.
from the separate treatment of ammonium-rich
Sludge
harbors
different
a
than activated
exhibits
with
sludge
are
same
able to
are
age and their
sludge
municipal
not
are
levels, they
sedimentation in the
the
in
dominating
low substrate
the
municipal
adapt
to
tion. The
use
community composition
of enriched
not
necessarily
the
one
the
europaea,
Nitrobacter)
K-strategists (iV. oligotropha,
Ni¬
in the WWTPs. At
prevailing
K-strategists
and washed out
depending
of added bacteria in the effluent due to poor
negligible
is crucial for
nitrifying sludge
since the CAS and the MBRs showed
suited for the
long-term
On the other
nitrify.
nitrifying
hand,
bacteria if the seeded
diurnal variation of the influent
the
required
biological
tanks of
increasing
indigenous population
its
plant
N-Kj
a
and failure of
in the effluent may
potential
does
performance.
if the seeded
occur
is limited due to the poor
sludge
is
On
plant
of the
adaptation
already partially nitrify. Therefore, heightened
load due to the separate treatment of
WWTP. Process
bioaugmenta¬
digester liquids
enhancement of the nitrification
aerobic SRT cannot be
in the return
success
from the separate treatment of
hand, elevated nitrite concentrations
does not
The former
plants (WWTPs).
r-strategists (iV.
the conditions
rates. Loss
clarifier is
secondary
oxidizing bacteria
WWTPs due to their very low saturation constants.
outcompeted by
decay
sludge digesting)
behavior.
3. The enriched
the
wastewater treatment
saturation constants for ammonia and nitrite.
high
2. The added nitrifiers
on
from
from
of both ammonia- and nitrite
substrate concentrations that favor
high
trospira)
community composition
liquids (e.g.
compensated by adding
engineering
stream
enriched
solutions which
(Salem
et
digester liquids
directly
al, 2003)
sludge
into
enrich the
avoid these draw¬
backs.
The
recently developed
bination with batch
municipal
experiments
WWTPs. As
duction of control
and oxygen
method to
an
quantify nitrifying
should be further
example,
on
the
applied
it would allow to
strategies involving
availability
bacteria
for
(Manser
et
in
com¬
tracking population dynamics
investigate
in
the influence of the intro¬
different environmental conditions
nitrifying
al, 2005c)
regarding
substrate
bacteria.
45
3.
Bioaugmentation ofnitrifying
bacteria
Appendix
Table 3 Kinetic parameters of
autotrophic
bacteria at 20°C. Bold values
are
used for simulations.
Symbol
Characterization
Units
|aA0Br
max.
growth
rate of
XA0Br
0.6-1.1-1.6
d1
Prosser,
|aA0Bk
max-
growth
rate of
XA0Bk
0.8
d1
Bollmanne/a/.,2002
|aN0Br
max.
growth
rate of
XN0Br
0.6-1.0-1.4
d1
Prosser,
|aN0Bk
max.
growth
rate of
XN0Bk
0.75
d1
Value
Reference
1989
1989
measurements
own
(un¬
published)
bAOBnaer
aerobic
endogenous respiration rate
of XA0Br
0.22
d1
this
bAOBbaer
aerobic
endogenous respiration rate
of XA0Bk
0.15
d1
Manser et
0.31
d1
this
0.2
d1
Manser et
al, 2005b
al, 2005b
bNOBnaer
aerobic
bNOBbaer
aerobic
bAOBnanox
anoxic
endogenous respiration rate
of XAoBr
0.01
d1
Manser et
al, 2005b
bAOBk,anox
anoxic
endogenous respiration rate
of XA0Bk
0.01
d1
Manser et
al, 2005b
bNOBnanox
anoxic
endogenous respiration rate
of XN0Br
0.01
d1
Manser et
al, 2005b
bNOBk,anox
anoxic
endogenous respiration rate
of XN0Bk
0.01
d1
Manser et
al, 2005b
KMHAOBr
saturation constant for ammonium of XAoBr
6-14
gNm"3
endogenous respiration rate
endogenous respiration
of XN0Br
study
rate of
XN0Bk
this
study
study, Stehre/a/.,
1995
KNH4,AOBk
saturation constant for ammonium of XA0Bk
0.15
gNm"3
Manser et
KN02,NOBr
saturation constant for nitrite of XN0Br
1-4-9
gNm"3
this
KN02 NOBk
saturation constant for nitrite of XN0Bk
0.25
gNm"3
Manser et
XAoBr:
Ammonia-oxidizing bacteria (r-strategists)
from SBR reactor
XN0Br:
Nitrite-oxidizing bacteria (r-strategists)
bacteria
from SBR reactor
XAoBk:
Ammonia-oxidizing
XAoBk:
Nitrite-oxidizing bacteria (K-strategists)
(K-strategists)
from CAS
from CAS
or
or
treating
al, 2005a
study, Prosser,
al, 2005a
ammonium-rich wastewater
treating ammonium-rich
wastewater
treating municipal
wastewater
MBR
MBR
1989
treating municipal
wastewater
46
CHAPTER 4
Consequences
on
of Mass Transfer Effects
the Kinetics of Nitrifiers
Reto
Manser, Willi Gujer, Hansruedi Siegrist
Water Research
39(19),
4633-4642
2005
of
Consequences
mass
transfer effects
on
the kinetics of nitrifiers
Reto
Manser, Willi Gujer, Hansruedi Siegrist
Abstract
The influence of membrane
evaluated
was
by running
sludge (CAS) plant
treated the
in
separation
a
and
transfer effects
mass
membrane bioreactor
parallel.
Both
pilot plants
on
(MBR) and
were
operated
a
the kinetics of nitrifiers
conventional activated
at the same
sludge
age and
domestic wastewater. The half-saturation constants for the substrate
same
low in both MBR and CAS and did not differ
significantly
were
between the two processes
0.17±0.06 gN m"3 and
(Knh4= 0.13±0.05 gN m"3 and 0.14±0.10 gN m"3 and KN02
m"3
for the MBR and CAS respectively). However, the half-saturation con¬
0.28±0.20 gN
=
stants for oxygen exhibited a
monia-oxidizing (AOB)
Ko,aob
and
0.18±0.04
=
0.47±0.04 g02
major
difference between the two processes for both the
=
m"3 (substrate only N02) for the
values of the CAS
were
attributed to
mass
MBR and CAS
sludge
floes for which the diffusion resistance
be
of
quences for the
Keywords:
eling;
mass
of MBRs
sludge;
higher K0
large
prevailing
from the MBR consisted of very small
neglected.
transfer effects in activated
operation
Activated
can
The
floes
respectively.
transfer effects within the
in the conventional system. In contrast, the
implementation
am¬
nitrite-oxidizing (NOB) bacteria. The experiments yielded
0.79±0.08 g02 m"3 as well as K0,nob
0.13±0.06 and
and
On the basis of these
results, the
models is discussed and
sludge
conse¬
highlighted.
are
Floe size
distribution; Kinetics; Membrane Bioreactor; Mod¬
Nitrification
1. Introduction
Membrane bioreactors
wastewater
when
Due to their low
limited
or
strict
new
However, the
operation
correct
on
application
provide
have to be met. The first full-scale
by adapting
the
experience gained
from
plants
conven¬
of nitrifiers when
conducted
(2004)
comparing
as
an
so
far
the
modeling
MBR with
the
are
on
did not find
yield
a
a
significantly
CAS. As
coefficient
in both processes. On the other
oxygen and substrate used for
sludge
additional information about the kinetics of activated
kinetic parameters, such
equal
models from the conventional activated
sludge properties were
performance (e.g. Judd, 2004; Shimizu et al., 1997)
kinetic data. Manser et al
be
quality
existing
investigations
not
to
of
requires
with respect to the membrane
expected
effluent
in recent years
out
nity-associated
plants are planned or existing ones redeveloped.
effluent, MBRs are particularly suitable if space is
sludge plants (CAS).
in MBRs. Most
composition
considered for the treatment of domestic
increasingly
and solid-free
requirements
process for MBR systems
sludge
are
wastewater treatment
footprint
have been put into
tional activated
(MBR)
or
a
different
that
commu¬
growth rate,
hand, the half-saturation
typically lumped parameters
and do
community
consequence,
maximum
carried
constants
are
are
for
additionally
48
Consequences of mass transfer effects
4.
influenced
by potential
transfer effects within the activated
mass
found small floe sizes in MBRs
et
with CAS systems
compared
Cicek et
(e.g.
ofnitrifiers
floes. Various authors
sludge
al., 1997), which would also support the supply of the complete
the kinetics
on
al., 1999; Zhang
MBR floes with oxygen at
low concentrations.
This
study investigates
the
impact
kinetics of nitrifiers. An MBR and
parallel (Manser
of membrane
a
Measurement of the
size distribution
particle
sion resistance is taken into account and its
and
with the
pilot plant
CAS
al., 2004). Half-saturation
et
separation
data
for
implication
settings
same
obtained
constants were
provided
transfer effects
mass
the
on
sludge
the
operated
were
batch
by
on
in
experiments.
structure.
The diffu¬
is discussed.
modeling
2. Materials and Methods
2.1 Pilot
plants
A CAS and
MBR
an
tic wastewater
wastewater
pilot plant (Manser
following
typical
a
to 60
corresponding
al., 2004)
et
diurnal
(18
m3d"1)
were
hydraulic
and 2
Because
a coarse
pre-denitrification
2.2 Batch
temporarily higher
was
and the
sludge
retention time
constants for
batch reactors with
that all
a
substrate
biodegradable
matter was
The oxygen concentration
were
was
larly
until the substrate
were
measured
was
according
experiments
B) Half-saturation
were
to
entirely
2000).
kept
at 20
were
surface,
operated
with
days.
were
to
adding
using
HCl and NaOH in all
m"3
was
aerated in
the substrate to
pH value
were con¬
experiments
respec¬
in order to prevent
(American
Public Health
samples
Association, 1992/
carried out for the calculation of each value.
Activated
prior
to
sludge
adding
consumed. The temperature and
inhibited
was
rates at
pH
g
value
were
ensure
m"3).
as
that all biode¬
Nitritation and nitratation
previously
Neither NOB
a
controlled and set to
various oxygen concentrations
respectively,
by allylthiourea (10
aerated in batch reactors with
the substrate to
experiments respectively.
titration with NaOH and H202
AOB
prior
sludge
consumed. The substrate concentrations of the
constants for oxygen.
matter was
Activated
10 g
heterotrophic endogenous respiration
by
was
plants
consumed. The temperature and
Standard Methods
20.0+0.1°C and 7.50+0.01 in all
ured
(SRT)
m"3.
m"3 allylthiourea (Ks for nitrite only). Initial
by
were between 5 and 8 gN m"3. Samples were taken regu¬
volume of 1 liter for at least two hours
as
in the MBR. Both
controlled between 3 and 4 g
inhibited
concentrations of the added substrate
gradable
cross-flow at the membrane
volume of 7.5 liters for at least two hours
oxygen limitation. AOB
Three
a
respec¬
controlled between 2.5 and 3 g
(ammonia, nitrite).
trolled and set to 15.0±0.2°C and 7.50+0.05
tively.
to treat domes¬
experiments
A) Half-saturation
ensure
was
bubble aeration is needed to induce
the oxygen concentration
parallel
m3d"1) population equivalents
(0.56
tively. The oxygen concentration in the aerobic tanks
in
operated
variation. The CAS and MBR treated
described
nor
as
were
(Ficara
heterotrophic
well
meas¬
et
al.,
bacte-
49
Consequences of mass transfer effects
4.
ria
rates, the activated
for two
inhibited in any
chemically
were
sludge
was
experiment.
all oxygen utilization rate
fed with wastewater
(OUR).
Four
ofnitrifiers
Prior to the measurement of the nitratation
containing
in order to increase the difference between
days
the kinetics
on
experiments
a
greater nitrite concentration
endogenous respiration
were
and the
over¬
carried out for the calculation of
each value.
2.3 Floe size distribution
The floe size distribution
tersizer X
based
was
are
the
on
principle
formed with
Olympus
an
activated
sludge
2.4 Floe
density
of laser ensemble
dense
light scattering. However,
particles. Therefore, the floe number
in order to obtain
manually
once
regularly over a period of three months using a MasLtd., Worcestershire, United Kingdom). The measurement
Instruments
inherently single,
not
mined
(Malvern
measured
was
1X50 inverse
from the CAS
Samples (80 ml)
of activated
utes at 1000 rpm.
The
comparison
were
The
measurement.
microscope using
sludge
microscopy
was
per¬
100 u.1 of diluted
magnification.
40x
floes
deter¬
analyzed.
were
sludge
pellet was
a
activated
and sizes
then
from the CAS and MBR
and the
weighed
density
were
centrifuged
for five min¬
of the floe calculated
according
to:
Mpellet Ppellet
'
with:
MLSS
Mpeiiet
Ppeiiet
Mixed
=
Pellet
weight (g)
Pellet
density (~
=
=
liquor suspended
1 g
solids
(kgTSs
m"3)
ml"1)
2.5 Diffusion model
The diffusion model
floes,
was
slightly
proposed by
were
distributed
presented by
homogeneously
teria
were
MBR
to
was
These
Monod terms
as
are
as
by applying
The
following,
K, apparent half-saturation
was
rates
(Manser
et
constants
in water at 20 °C
were con¬
al., 2004). The floe size
uptake
rates
of the bac¬
half-saturation constants measured in the
applied
monia and nitrite
respectively (Milazzo, 1963;
including
were
by
bac¬
heterotrophic
can
be
neglected
intrinsic half-saturation constants of the bacteria
sion coefficients
of the
estimated
floe, whereas the nitrifiers
intrinsic values because the diffusion resistance
the small floes. In the
cated
(1998).
subdivided into 12 classes. The oxygen and substrate
expressed
sludge.
growth
spherical shape
boundary layer
Nicolella et al
within the whole
centrated in the inner part due to their low
distribution
that assumed
(1992),
modified. The thickness of the external
the Sherwood number correlation
teria
Beccari et al
2.2,
mass
transfer effects
1.7 and 1.7
Schwarzenbach
x
10"9 m2 s"1
as
are
due
indi¬
K'. The diffu¬
for oxygen,
am¬
etal, 2003).
50
4.
The model
was
USA) equipped
Consequences of mass transfer effects
solved
numerically using
with
non-stiff ordinary differential
a
MATLAB
(v7.0,
on
the kinetics
The Math Works Inc,
solver
equation
ofnitrifiers
Natick, Ma,
(ODE45).
3. Results and discussion
3.1 Floe size distribution
Large differences between the
CAS and the MBR
found. The floes from the MBR
were
were
about ten times smaller than those from the CAS and confirm the results of other authors
(Cicek
al, 1999; Huang
et
et
al., 2001; Zhang
et
al., 1997). Since there is
tleable floes in MBR systems, the bacteria have
floes. The
hibit the
is
probably
relative
of
comparison
be made
checked
more
floes. The
large
caused
sonably
by
selection for set-
inducement to build up
coarse
aeration
during
the observed
by using
critically.
A
the
measurement
same
comparison
of the median values
technique,
of the automatic laser
a
While
a
can rea¬
absolute values have to be
scattering
However, the distribution obtained
(Table 1).
sludge
method with manual
from the CAS revealed
grab sample
(Table 1)
period (fall).
of the floe size distribution between the MBR and CAS
large
also in¬
possibly
of the floe size distribution
temporal variability
the temperature decrease
determination of floe number and size of
ment
physical
shear stresses in MBR systems due to the
higher
growth
no
no
good
manually
agree¬
much
was
narrower.
Volumetric
Table 1
quantiles
of the floe size distribution of activated
per part: Mean values and standard deviations
Lower
one
sample
from the CAS. A total of 230 floes
10% -quantile
CAS
(|j.m)
73+21
MBR
Laser
12
scattering
Manual
a
(temporal variability only)
from the CAS and the MBR.
over a
period
of three months
(n
Up¬
=
14).
part: Comparison of the automatic laser scattering method with manual determination of floe number and
size for
As
sludge
CAS
counting
CAS
+
3
enumerated and their
50% -quantile
307
+
72
35
+
9
(\aa)
mean
diameter estimated.
90%.-quantile
90 + 23
408
1038
262
419
629
display,
the distribution of the
(|j.m)
733 + 115
112
consequence of the volumetric
normal
were
density
functions is
log-
(Figure 1).
51
Consequences of mass transfer effects
4.
100
-
the kinetics
ofnitrifiers
_,
-80
>,
o
|
60
-
on
£
-40
LL
Ë
-20
5
0
0
500
1000
Floe Size
Figure
Volumetric floe size distributions of
1
density
The floe
density expressed
as
gxss
rnfloc"3
fer effects within the floes because it is
is available.
pending
ties
on
were
et al.
Bishop
(1995)
found for biofilms with
solid retention time
9
=
days)
days).
20
on
was
Liss et al.
compact and physically
of the activated
suspension
regard
the volumetric
to
to mass trans¬
Unfor¬
activity.
direct measurement
no
technique
rn"3 de¬
sludge.
gTss"1, corresponding to
a
The
floe
the
the data from the literature vary
discussed in
Chapter
pellet comprised
higher
SRTs
(4
(16
and
pilot plants (SRT
10-14 Vol% and
This
corre¬
for the CAS and MBR
sludge
sludge respectively.
floes,
a
of 20 gTss
rough
The
floes at
in the lower range of these
floe
Volume Index
density
considerably.
were
mfloc"3
of the
a
sludge
from the examined
study
sludges,
words, the applied techniques only allow
the CAS floe
examined the influence of the
(2001)
expected
Sludge
to
stable than those at lower SRTs
more
counting
corresponding
urn
for the CAS and MBR
the data from manual
calculated for the CAS
about 50 ml
quently
thickness of 130-240
a
similar floe densities of 25-35 and 27-42 gTSs
was
In other
difficult, because
with
found densities of biofilms from 30 up to 120 gTss
study (Table 1).
centrifugation
respectively. Using
mfloc"3
directly proportional
However, the densities determined in this
14-22 Vol% of the total
to
months)
of three
investigated period
important parameter
the floe structure and concluded that
were more
After
findings.
sponds
an
Rather dense floes should therefore be
days).
the
the thickness of the biofilm and the location within the biofilm. Maximum densi¬
diameters measured in this
and 20
(urn)
light scattering.
is
its determination turns out to be
tunately,
2000
grab samples (within
from CAS and MBR measured with laser ensemble
3.2 Floe
1500
density
(SVI)
of
only
of the CAS
sludge
rnfloc"3.
estimation of the floe
sensitivity
18 gTss
density
of this parameter is
and
conse¬
3.3.
3.3 Kinetics
Different bacteria
populations
have
inherently
distinct
ecophysical properties.
The
analysis
of
community composition revealed the same populations of nitrifying bacteria in both MBR
and CAS (Manser et al., 2004). Consequently, equal kinetic properties can be expected. How¬
the
ever,
way
mass
transfer effects
(Figure 2). Oxygen
additionally
influence the kinetics of the nitrifiers in
and ammonia concentrations decrease
across
the
floe,
a
different
whereas nitrite
52
Consequences of mass transfer effects
4.
is
mainly produced
consequence, the
within the floe
availability
the AOB and
by
of nitrite is unaffected
consumed
immediately
diffusion. In
by
the kinetics
on
the NOB. As
by
addition,
ofnitrifiers
the outer
a
purely
reduces the oxygen available for nitrification.
heterotrophic layer already
Laminar
Concentrat'0n
î
y
Boundary
Layer Ï
HETonly
HET, AOB,
NOB
Figure
2
Schematic 2D slice of
a
spherical
activated
floe with radius R and
sludge
a
purely heterotrophic
outer
layer
A diffusion model allows the effect of the
mass
transfer
on
the oxygen and substrate utiliza¬
tion rates to be described. However, several uncertainties affect the model output.
firstly
from the
uniform
uncertainty
of the model structure, e.g. the
of the floes. But uncertainties
density
of the parameters. Since these two
the
uncertainty
of the parameters
the
uncertainty
of the parameter of floe
of the actual floe
edge
tween
density
of
sources
can
partly
are
assumptions
also introduced
uncertainty
are
by
of
is
but also for the
responsible
variability
not
only
of the floe
and
knowledge
other,
of each
reflect that of the model structure. As
density
stem
spherical shape
the limited
independent
not
They
an
example,
for the limited knowl¬
within and be¬
density
the floes.
A local
sensitivity analysis
revealed that the parameter with the
to
the
floe diameter results in
a
Ko' value that is about 20-40% higher (Figure 3D, G). Furthermore, the floe diameter is
a
model output is the floe size. An increase of 100
rather theoretical
true.
since the
value,
Therefore, the uncertainty
tainty
assumption
Apart
et
considerably.
al
(1995)
(Dfioc/Dwater)
were
the modeled
Assuming
(Bishop
K0,aob'
the outer
increased
heterotrophic layer (Figure 3F). Although
slightly
most
by
leading
to
only
small
reality
the
uncer¬
readily
sensitive to the model out¬
et
al., 1995;
were
were
found to be
Matson and
Characklis,
kgxss rnfloc"3
in biofilms.
strongly dependent
purely heterotrophic layer
about 20%
compared
to a
to
on
mass
over
the
be 20%
floe without
the diffusion coefficient of ammonia is
smaller than that of oxygen, the utilization rate of ammonia is
than that of oxygen,
to
not
measured and the data from
found floe densities from 30 up to 120
within the biofilm.
spatial position
radius,
significantly
The diffusion coefficients within the floe
However, both the diffusion coefficient and the floe density
of the floe
of the floes is in
size, the floe density and the reduction
these parameters cannot be
between 30% and 60% of those in pure water
1976). Bishop
mean
spherical shape
from the floe
of the diffusion coefficients within the floe
put (Figure 3). Unfortunately,
a
the
in the absolute floe size contributes
of the calculated K0' values.
the literature vary
of
urn
in
highest sensitivity
a
even
three times lower
transfer effects due to the
stoichiometry
53
4.
(Figure 3 A-C).
In
Consequences of mass transfer effects
addition, heterotrophic activity
on
the kinetics
ofnitrifiers
in the form of ammonia turnover
can
be
ne¬
glected.
The results
clearly
role in activated
reveal that the
sludge floes,
mass
but is
transfer limitation due to diffusion
negligible
for floes with
the K' values of MBR
D, G). Consequently,
(Figure
of the bacteria, because more than
3 A,
smaller than 100
urn
a
sludge
Floe
heterotrophic layer (C, F, I).
thickness of the
rA0B
=
H ET
5
40
Inob
:
(Df/Dw)
10
(A-C) and
and floe size
oxygen
(A, D, G),
Default parameter values used
=
=
20
size
=
(D-I)
floe
were:
as a
function of the reduc¬
density (B, E, H)
floe
density
=
60
and thick¬
kgC0D nifioc-3,
400 |am. Calculation of rates for
130 g02
kgcoD-1 d"1,
rN0B
=
35 g02
Knh4:
kgcoD-1 d"1,
kgcoD-1 d"1 (= endogenous respiration). Calculation of rates for K0,nob (NOB slightly enriched):
90 g02 kgcoD-1 d"1 (= endogenous respiration).
g02 kgcoD-1 d"1, rHET
:
45
=
Measurement of the
degradation
half-saturation constants
a
15
% of radius
heterotrophic layer 10% of floe radius and floe
gMM.N kgcoD-1 d"1. Calculation of rates for K0,Aob: i"aob
Half-saturation constants for the substrate
no
layer
100 go2
=
rHET
35
consisted of floes
sludge
density
Calculated half-saturation constants for substrates
tion of the diffusion coefficient within the floe
of the
urn
reflect the intrinsic K values
k9coD nifioc"
ness
significant
(Table 1).
20
3
a
diameter smaller than 100
90% of the floe volume in the
Floe size
Figure
plays
significant
difference
yielded
was
(Ks)
of the substrates
(Figure 4)
and
subsequent
low values for both AOB and NOB
estimation of the
(Table 2). Furthermore,
found between the CAS and the MBR. Mass transfer
plays only
minor role here because the conversion rate of ammonia is lower than that of oxygen and
heterotrophic activity
with respect to ammonia and nitrite
However, the KNob' values of the CAS varied within
a
can
be
broad range
neglected (Figure 3A-C).
over
time. This variation
54
Consequences of mass transfer effects
4.
may be
values also correlate with
influence of diffusion resistance within
0
20
40
60
Time
Figure
Examples
4
represent the best fit
It should be
effects
can
floes
(Table 2)
100
0
profiles
and therefore
imply
the
(Figure 3 A).
20
40
60
Time
that nitrite is
equal Ks'
prevailing composition
80
100
(min)
used for the estimation of Knh A0B and
normally produced
the
within the floes where
experimental setup
values of both CAS and MBR
did not exclude
can
Kno,nob-
Lines
ecophysical properties.
potential
transfer
diffusion
be
surprising
More
mass
explained by the same
(Manser et al., 2004), which
largely
of nitrifier communities in both systems
exhibited similar
probably
80
floe sizes
(min)
neglected. However,
resistance. The
ofnitrifiers
to the Monod model.
emphasized
be
higher
large
of measured concentration
(chi2)
the kinetics
uncertainties associated with the estimation of such low values. But
explained by
higher KNob'
on
were
the low values of
mostly higher values were reported in previous studies (Bollmann
al, 2002; Prosser, 1989; Schramm et al, 1999). The analysis of the community composi¬
both AOB and NOB, since
et
tion revealed that the AOB
lineage,
are
whereas NOB
regarded
Schramm et
as
were
belonged
al., 1999). Stehr
study (Table 2).
to even
We
et
al
m"3
Nitrospira (Manser
high
assume
study (Table 2). Finally,
steady-state
the
which
other AOB
(FISH).
slightly
Nitrospira,
conditions of
(Gieseke
were
oligotropha
Both groups
et
al., 2001;
m"3 for
0.42-1.05 gN
exceed the values estimated
pilot plants
present which
Schramm et al
dynamic
al., 2004).
Knh,aob values of
that either the AOB in the
or
et
substrate affinities
found
(1995)
for members of the genus
does not reflect the
members of the Nitrosomonas
oligotropha lineage,
lution of the detection method used
in this
with
lower ammonia concentrations
of 0.14 gN
by
the genus
typical iC-strategists
members of'the Nitrosomonas
in this
dominated
to
(1999)
were
are
obtained
which is confirmed
by
able to
beyond
a
the
KNo,nob
adapt
reso¬
value
the values found
measurement
of the ammonium concentrations
continuously
fed systems and may have led to
very low values.
55
4.
Consequences of mass transfer effects
Table 2 Estimated intrinsic Ks values for MBR and apparent
enors
from parameter estimation
experiments
were
distributed
given. 50%-quantiles
period
over a
Ks' for CAS of AOB and NOB
are
listed. The
of six months.
Floe size
Rate AOB
(gNkgcoDd_1)
Kno,nob
(gN m-3)
Floe size
Rate NOB
50%-q. (|am)
(gN kgcoD d"1)
0.10
+
0.06
73
31 + 1.5
0.12 + 0.02
73
28 + 0.9
0.15
+
0.03
79
32+0.9
0.22 + 0.04
73
35 + 1.2
0.12 + 0.04
78
38 + 1.1
0.15+0.02
78
29 + 0.8
Knh,aob
CAS
ofnitrifiers
at 15°C. Standard
of the measured floe size distributions
50%-q. (|am)
Knh,aob
(gN m-3)
MBR
are
the kinetics
on
Kno,nob
0.07 ± 0.02
315
41 + 1.0
0.50 + 0.03
593
46 + 0.9
0.20 ± 0.06
529
42 + 1.5
0.22 + 0.04
406
27 + 0.8
0.16 + 0.03
418
39+0.8
0.11+0.02
184
31 + 1.0
Half-saturation constants for oxygen
(Ko)
Table 3 summarizes the half-saturation constants obtained for oxygen with AOB, NOB and
heterotrophic
bacteria of the MBR. Since
Monod model
was
However, kinetic data from
possess
a
values for NOB than AOB
previous
Nitrobacter, which is regarded
Conversely,
higher affinity
NOB
to oxygen
are
as a
based
respiration
chemically
were
sumed oxygen. Since
nitratation rate
inhibited
Table 3
was
of
(consumption
consistent with
et
bacteria
each)
during
H2O2),
to
the genus
with low
simple
Nitrospira
(Belser,
also
are
nor
MBR
to
corre¬
al, 2001;
simultaneously
con¬
influences the measurement of the
the half-saturation constant of
no
thought
heterotrophic endogenous
nitritation and therefore
sludge.
to
to oxygen
1979; Gieseke et
heterotrophic
determined. Its estimation
of MBR
belonging
affinity
al., 1999). The K0 values of the
(= apparent Ko' values,
(endogenous respiration)
diffusion
bacteria
very low
value,
resistance) for AOB, NOB and het¬
(four experi¬
Mean values and standard deviations
at 20 °C.
Ko,AOB (g02
Ol
)
Ko,NOB (§02
0.18 + 0.04
In contrast to the
111
)
0.13+0.06
Ko,HET,endo (g02
(Figure 3D, G).
The
application
a
high
floe
density (80 kgcoD
)
must
take
mass
trans¬
of the diffusion model to measured
data of the CAS showed that the observed values could well be
(Figure 5). However,
HI
0.05+0.02
MBR, the estimation of the K0' values for larger floes
fer effects into account
tance
with NOB
produced a
previous findings (Belser, 1979; Wiesmann, 1994).
was
Estimated intrinsic K0 values
erotrophic
experiments
heterotrophic endogenous respiration
during endogenous respiration
which
the
neglected,
(Belser, 1979; Prosser, 1989).
typical r-strategist
belonging
(Schramm
on
It should be considered that neither nitratation
Prosser, 1989).
ments
studies
to values found in the literature for pure cultures
spond
be
Interestingly, the K0 (= K0')
significantly different, which contrasts with
found not to be
were
previous findings stating higher K0
and the substrate.
can
used for the estimation of the parameters.
values for AOB and NOB
the genus
transfer effects
mass
rnfloc"3)
explained by
and low
diffusion resis¬
D0,f/D0,w
ratio
(0.3)
56
Consequences of mass transfer effects
4.
had to be assumed in order to
by
S VI) and the
(low
et al., 1995;
applied
Matson and
the floes from the CAS
Although
parameter values
are
were
purely
very compact
still in the range of the
Characklis, 1976), other factors such
published data (Bishop
larger floe size, different
as
of the nitrifiers between the CAS and MBR systems
ecophysical properties
ofnitrifiers
the differences between the CAS and the MBR
explain
transfer effects within the floe.
mass
the kinetics
on
or mass
transfer
effects within the dense aggregates of nitrifiers may have contributed to the measured dissimi¬
larities. In
particular,
the
lead to dissolved oxygen
found
(2004)
ment
(as
of the overall oxygen
compared
uptake
sludge
rate
(as
bacteria
the outer
addition,
from CAS than from MBR. The
the H2O2
consumption rate)
confirmed that the AOB
heterotrophic
K0,het (Table 3),
to
in
consumption rate)
concentrations than
around the aggregates. In
(DO) gradients
larger aggregates
the NaOH
distribution of AOB and NOB in the floes may
non-homogenous
were more
(Figure 5C). Apart
Manser et al
parallel
measure¬
and the nitritation rate
inhibited at low oxygen
from the
higher K0,aob
value
enhanced this ef¬
purely heterotrophic layer probably
fect.
measured overall OUR
A
measured NaOH
utilization rate
2
Figure
(g02
Examples
5
2
4
DO
rn3)
radius, D0,f/D0,w
(measured
as
modeled nitritation rate
2
4
m"3)
DO
(g02
m"3)
(OUR) as a function of the bulk oxygen
(B) and without (A) inhibition of AOB. Substrate concentrations were non-
Estimated parameters: floe
=
(g02
modeled overall OUR
—
of measured and calculated oxygen utilization rates
concentration for the CAS with
limiting.
4
DO
——
0.3.
density
Comparison
=
80
kgC0D nifioc-3,
of nitritation rate
H202 utilization rate) of CAS (C). For the
thickness of the
(measured
as
heterotrophic layer
NaOH utilization
rates used for the
calculations,
rate)
see
the
10% of floe
=
with overall OUR
of
legend
Fig.
3.
Accordingly,
the estimation of the apparent half-saturation constants for the CAS from the
experimental
data led to
be considered that the
influences the
strongly
Ko,aob'
value
riod, which
(>250
not
ml
(0.50
was
gTss"1)-
g02
significantly higher
temporal variability
mass
rn"3)
values
compared
to
the MBR
(Table 4).
of the conventional activated
It should
sludge properties
transfer effects and therefore the actual K0' values. A lower
was
found with conventional activated
associated with smaller floes
In contrast, the
(50%-quantile:
sludge properties
197
sludge
urn)
and
of the MBR did not vary
from
a
a
high
second pe¬
SVI value
significantly (data
shown).
57
Consequences of mass transfer effects
4.
Estimated apparent K0' values for
Table 4
CAS
sludge
sity
80
=
at 20 °C.
kgcoD nifioc"3,
Standard
thickness of the
Ko,aob' (go2
heterotrophic layer
0.79 + 0.08
l)
Without AOB
=
Ko,nob' (go2
)
m
are
Ko,het' (go2
)
m
0.47+0.04
The microbial
(Figure 6).
of NOB
activity
the maximum
of AOB leads to
activity
duction of NOB
activity
tribution of nitrifiers).
due to
high
activity
AOB
activity
could
cannot
of the nitrifiers. The presence of
yet explain the
outer
an
K0,aob' during high
AOB
activity
of ele¬
dis¬
would
heterotrophic layer
due to the
(Table 3). Conversely,
the K0' val¬
by strongly increasing
temperature due to
diminishing
occurrence
obviously
enhance
decreasing temperature,
substantially
reduced micro¬
activity (Figure 6D).
DO control for CAS could be
Consequently,
would
be
certainly
possible
high nitrogen loading
of COD
sumption
achieving
optimized
the DO to low levels
drop
to
whereas the DO should be increased to
of
only slightly raises the K0,aob',
the K0,nob'- Nonetheless, this re¬
diffusion resistance increases with
(Figure 6C). Although
Ko' values become smaller with
der
within the floe and therefore
(assuming homogenous spatial
inhibit nitrification
noticeably
and heterotro¬
of NOB
30% increase of
a
is also lower than
K0,nob'
heterotrophic activity
bial
)
(Figure 6B)
AOB
lower intrinsic K0 value of the NOB used for the calculations
this effect
0.3.
function of the active proc¬
as a
consumption
vated nitrite concentration in the effluent of CAS systems
ues
m
=
0.16 + 0.02
(Figure 6A),
influences the oxygen
(Figure 6C)
also alters the diffusion resistance. While the
slightly
radius, D0,f/D0,w
activity
bacteria
phic
ofnitrifiers
Model parameters used: floe den¬
given.
10% of floe
The diffusion model allows the K0' values to be calculated
esses
the kinetics
AOB, NOB and heterotrophic bacteria (endogenous respiration) of
of parameter estimation
enors
on
more
the basis of these
during
low
findings.
higher
available for
aeration costs, limit the aerobic
save
denitrification)
It
nitrogen loading periods,
levels to counteract the diffusion limitation
situations. This would
(thus leaving
on
and
improve
the
un¬
con¬
possibility
simultaneous nitrification and denitrification.
08
08
35
B
07
m— 06
07
-
y
AOB
m—2 5
^_NOB
......
S 05
S 05
layer
-
04
03
g"
-
S|mo2
=
0 9n
..
SN02
=
0 05 gN
SNo2
=
0 3 gN
SN02
=
-
02
.
2 gN
m
nT3
nT3
J
"
04
"
03
.
02
nT3
0 1
NOB with HET
layer
^
3
S
J
30
AOB with HET
E
E
"g
-
-
m"3
Simh4
=
0 9n
SNH4
=
0 05 gN
Snh4
=
0 3 gN
Snh4
=
2 gN
nT3
m"3
nT3
0 1
00
05
10
15
Snh4(gN
Figure
6
m
20
25
00
05
)
10
15
Sno2 (gN
m
20
)
25
100
200
300
OURHET(go2
Calculated half-saturation constants for oxygen of the CAS
400
kgcoD-1 d"1)
depending
on
=
=
100
HET
of
15
20
25
the active processes at 20°C
layer
(except D). Endogenous respiration
g02 kgC0D d"1,
0.15 gNm"3, KNO2
KNH4
0.15gN m"3 (Table 2). Substrate levels indicate steady
Sno2 0 gN m"3 means that NOB are not active.
=
10
Temperature (°C)
10%
of radius
state
is
assumed.
concentrations, e.g.
=
58
Consequences of mass transfer effects
4.
The
an
for
Implications
3.4
important
prohibitive
values in CAS systems
basis of these
depend
the
results,
models
sludge
be reflected in
reasonably
tance can
activated
extending
ofnitrifiers
nitrification. Mass transfer effects
modeling
role in CAS but not in MBR systems. Since
for
the kinetics
of nitrification
modeling
results have consequences for
reported
on
by
computation
model,
diffusion
a
half-saturation constants.
dynamic
the diffusion resis¬
Accordingly,
the active processes and the temperature
on
the K0'
(Figure 6).
On the
line-fit formulas for the calculation of the K0' values
following
play
times still appear to be
were
derived:
AOB2CPC
~~
AOB20°Cmin
rxO
Ss
^+a
aHET
„
~~
^"O NOB 2CPC
+
mm
9HET
rXNH4
^S
,
^0
ix
r\(-i
"\r
_
_
—
vO
t
T
,xO
e
2CPC
The Ko' values
IX
rXN02 NOB
°NH4
O
,
°N02
"^
^NH4
^NH4 AOB
^S+^S
ix
Sno2
n
q
"^
AOB
„
+aAOB2
,0
i
^aNOB«"»
0
.
°S
'
,x
Snh4
n
O
,
"^
^S
K-ONOB20°C
|
^aAOB1 "AUDI
0
.
JX
,,
-K
rxO
„
K
+
^NH4
036(20-T)
are
divided into
a
and
constant
fraction. The constant
process-dependent
a
be estimated from the results shown in
3
0.60 gQ2 m
(Ko,AOB,20°c,mm'
both including heterotrophic endogenous respiration of
0.47 g02
Ko,NOB,2o°c,min'
100 go2kgcoD1d"1). Similarly, the contributions of nitritation (aAOB,i -0.1 go2 m"3, a
0.2 go2 rn"3, Figure 6A-B) and nitratation (aNOB- 0.1 g02 rn"3, Figure 6A) can also be
aob,2
found. The OUR due to heterotrophic growth was correlated with the availability of readily
fractions
can
-3
rn"
=
Figure
6
2 gcoD
rn"3, Figure 6C).
=
,
~
biodegradable
substrate Ss
the nitrite concentration
justed
to
the temperature
The formulas
were
on
1999)
MBR with two
a
consequently
set
point
of So
=
rn"3,
were
The results indicate that
typical
values
independently
Ks'
~
neglected. Finally,
was
Figure
a common
activated
=
age
a
one
are
typical
dynamics
mean
rn"3 (Figure 7).
of the system may lead to
a
performance (Figure 7).
diminished microbial
activity
is
partly
at
high
as
were
Figure
7.
autotrophic K0'
small diurnal variation of
application
large
an
by
(CAS only)
aerated with
shown in
increases the
relatively
a
al,
CAS
oxygen concentra¬
In contrast, the
The loss of
lessened
tank
et
K0' values (K=const) yield similar efflu¬
errors.
Ko' values from CAS systems to MBR systems results in
nitrification
Both reactors
negligible
setup of
common
domestic influent
and leads to
(ASM3, Gujer
secondary settling
days.
set to 10
was
consequence,
1 go2
model
sludge
heterotrophic growth only marginally
a
the K0' values must be ad¬
in the nitrification process. A
mixed reactors and
sludge
The influence of
6D.
since diffusion effects
domestic COD
concentrations at So
in
to
rn"3,
carried out with
the calculated K0' values. As
ent
K0,nob
completely
1 go2
0.8 g02
sensitivity
chosen. The
tions. All simulations
values for
the
incorporated
and
was
~
according
in order to test their
a
(aHET
a
As
of constant K0'
example,
significant
performance during
the transfer of
underestimation of the
cold
seasons
owing
to
the reduced diffusion resistance.
59
Consequences of mass transfer effects
4.
CAS: K0'
=
f(t)
K0'
vs.
1 2
=
on
the kinetics
const
-
SNH4, Kq1
Influent
D COD load
ofnitrifiers
NH4 load
f(t)
=
SN02, Kq1
=
Snh4, Ko'
=
const
=
const
sno2, K0'
f(t)
Ko aob', K0'=f(t)
Konob', K0'=fi(t)
Ko aob', K0'
const
=
Konob', K0'= const
Snh4, CAS
SNH4l
MBR
Ko aob', CAS
Kqaob', MBR
02
04
06
Time
Figure
02
(d)
7 Influent load and calculated effluent concentrations of
and 10 °C. K0
const
=
corresponds
These considerations
to mean
based
are
on
values, dependent
the
assumption
an
on
composition
aeration in order to enhance
different
mass
or
06
(d)
aerobic CAS and MBR
validated in real systems
on
its
1 g02
:
m"3)
at 20
significantly
over
un¬
time due to fluctua¬
temperature. Moreover, control strategies (e.g. reduced
nitrogen elimination)
compositions,
(S0
that the floe structure remains constant
may alter the floe structure associated with
transfer effects. Differences between WWTPs
different influent
Of
temperature only.
der various conditions. However, floe sizes may vary
tions of the influent
04
Time
loads
or
likely
the
proposed
operation. Therefore,
are
improved ability
to
reproduce
because of
to occur
model should be
ammonia and nitrite
dynamics.
4. Conclusions
In this paper,
we
have demonstrated the consequences of
of nitrification. A conventional activated
pilot plant
constants
were
operated
in
parallel
for both substrate and oxygen
were
It is concluded that diffusion resistance is
separation
leads to small floe sizes.
sludge (CAS)
to treat
the
same
mass
and
a
transfer effects
Consequently,
the kinetics
(MBR)
domestic wastewater. Half-saturation
estimated from batch
negligible
on
membrane bioreactor
experiments.
for MBR systems because the membrane
the measured
(apparent)
half-saturation
constants in MBRs
reflect the low intrinsic K values of the bacteria. MBR systems may there¬
fore be
low oxygen concentrations
operated
Additionally,
at
the internal
recycle
from the membrane compartment
(1
go2
rn"3),
which would
from the aerobic to the anoxic
(high
DO due to necessary cross-flow
minimize the oxygen transfer from the aerobic to the anoxic
effects
play
a
key
role within the
zone
save
large
floes
prevailing
zone.
aeration energy.
should not be directed
aeration)
In contrast,
in order to
mass
transfer
in the CAS. The apparent half-
60
4.
saturation constants for oxygen
Consequences of mass transfer effects
are
significantly higher
on
in CAS because
the kinetics
include both the
they
intrinsic K values of the bacteria and the effect of the diffusion resistance.
multaneous nitrification and denitrification
within the
ters
large
such
WWTPs.
mined
as
floes. However, the
floe size and floe
Apparent
individually.
implementation
depend strongly
which may vary
thus
are
Accordingly,
be achieved in CAS due to the DO
transfer effects
density,
KBOB' values
The
mass
can
ofnitrifiers
on
gradients
variable parame¬
significantly
between different
system-dependent parameters
and must be deter¬
of these
findings
in activated
sludge
models is
step towards the understanding of ammonia and nitrite dynamics in CAS, particularly
oxygen concentrations.
results for
common
Temperature-dependent
CAS with
probably requires dynamic
Accordingly,
typical
K0' values
diurnal
the
on
on
seem
a
to
highly
yield
first
at
low
reasonable
variable influent
proposed
model extension.
other kinetic parameters such
provide
a
the active processes.
future research is needed in order to validate the
should also be examined in order to
modeling
static K0' values
variation, whereas
depending
However, the impact of membrane separation
stants
si¬
a more
as
decay
comprehensive parameter
con¬
set
for
of MBR.
61
CHAPTER 5
Decay
Processes of Nitrifying Bacteria
Reto
Manser, Willi Gujer, Hansruedi Siegrist
Submitted to Water Research
Processes of
Decay
Reto
Bacteria
Nitrifying
Manser, Willi Gujer, Hansruedi Siegrist
Abstract
The extension of wastewater treatment
increased
found
technologies
new
about
knowledge
were
carried out in order to
separation
bacteria. It
was
found that
decay
nitrite-oxidizing
bacteria. No
activated
were
sludge
bacteria did not
model
bacteria showed
significance
Keywords:
the
a
no.
3
findings
Activated
pro¬
the effect of membrane
negligible
nitrite-oxidizing
for both ammonia- and
differences between membrane and conventional
the measured loss of
autotrophic activity
endogenous respiration concept
incorporated e.g. in the
Unlike
(ASM3).
nitrite-oxidizing bacteria, ammonia-oxidizing
the
as
of their enzymes. Based
pronounced decay
of these
higher
increasingly important.
of ammonia- and
decay
at
Consequently,
ages.
study
potential (ORP)
operated
are
Likewise, aerobic decay of ammonia- and nitrite-oxidizing
significantly diverge. However,
only partly explained by
sludge
on
at anoxic conditions is
significant
detected.
sludge
processes and kinetics becomes
decay
and different ORP conditions
activated
like membrane bioreactors
concentrations associated with elevated
sludge
Therefore, batch experiments
was
COD to nutrient removal leads to
only
ages and exposes bacteria to different oxidation-reduction
sludge
conditions. Moreover,
activated
from
plants
on
on an
extended
ASM3, the
wastewater treatment is discussed.
sludge; Decay; Enzyme kinetics;
Membrane
Bioreactor; Modeling;
Nitrification
1. Introduction
Autotrophic decay
rates
fore
and
ance
on
the
design
against
wash-out
the extension of the
have
significant impact
a
analysis
or
the nitrification
of wastewater treatment
overload
biological
on
directly depend
treatment
on
steps from
performance
plants. Safety
of the
and there¬
plant perform¬
Furthermore,
these kinetic parameters.
only
cation, denitrification and biological phosphorus removal
COD removal to
plants
with nitrifi¬
exposes bacteria to different oxida¬
tion-reduction
potential (ORP) conditions, which appear to influence the decay rates (Lee and
Oleszkiewicz, 2003; Siegrist et al, 1999). Additionally, membrane bioreactors are operated at
higher
activated
sludge
concentrations associated with elevated
State of the art models consider loss of biomass
mass
concentration
biological activity
respiration
1999).
current
reduce
or
ages.
proportional to the bio¬
(e.g. lysis, endogenous respiration). Looking into more details, loss of
can
lysis
sludge
as a
single
be the result of mechanisms like:
process
prédation by protozoa, endogenous
due to adverse environmental conditions
In the maintenance process
-
as
another concept
-
(Van
Loosdrecht and Henze,
external substrate is used to
keep
the
biological activity, but the amount of bacteria is not altered. Prédation and lysis both
cell number, biomass and activity, whereas endogenous respiration
from a microbi¬
ological point
-
of view
-
only
leads to
a
loss of
activity
and
slightly
reduces biomass.
Finally,
63
Decay processes ofnitrifying
5.
degradation
enzyme
loss of
causes a
activity,
which is
rapidly
bacteria
restored if substrate becomes
available.
However, it is
process. In
clear, whether these mechanisms
not
studies
addition, previous
nitrification
systems combining
Possible differences between
ria
decay products
on
of nitrate
was
The
of this
goal
better
a
(summarized
process
bacteria
are
on
from
heterotrophic
bacteria
product
or
decay
and
only
in activated
Martinage
(AOB)
of nitrite
comprehension
and
sludge
Paul, 2000).
nitrite-oxidizing
bacte¬
dynamics. Eventually,
growth
of
one
from
nitrifying
bac¬
inhibition due to accumulation
taken into account.
study
the
was
investigation
of decay processes for both AOB and NOB at dif¬
ferent ORP conditions and for different systems
Loss of
in
it does not become clear whether simultaneous
investigations
many
teria
merely provide
one
ammonia-oxidizing
may contribute to
(NOB)
as
described with
reasonably
are
kinetic data
activity
measured in batch
was
discussed and consequences for
(conventional
and membrane
bioreactor).
experiments by respirometry. Underlying
of activated
modeling
processes
sludge systems presented.
2. Materials and Methods
2.1 Activated
sludge samples
Samples
taken from
reactor
were
conventional activated
a
(MBR) pilot plant (Manser
mestic wastewater
following
treated the wastewater
a
et
al, 2004),
typical
corresponding
sludge system (CAS)
which
were
operated
and
in
a
membrane bio¬
parallel treating
do¬
diurnal
to 60
hydraulic variation. The CAS and the MBR
(18 m3 d"1) and 2 (0.56 m3 d"1) population equiva¬
lents, respectively. Oxygen concentration in the aerobic tanks was controlled between 2.5 and
3 g m"3. Due to the coarse bubble aeration inducing a cross-flow at the membrane surface, the
oxygen concentration
pre-denitrification
was
and the
2.2 Batch
experiments
Activated
sludge samples
(decay
under aerobic
of both for
once a
day
plant
one
of 7-10
in
placed
a
(SRT)
was
stirred
enabling
stirred
with 33% anoxic volume
phenomenon
seven
(permeate)
minutes.
and
placed
sludge
(decay
at
a
anoxic
For the anoxic
on
was
were
with
was
continuously
conditions)
experiment,
or a
nitrate
Furthermore, the
a
combination
(NaN03)
reactor was
pool.
aerated
was
aerated
An
experiment
typical
wastewater
conducted in order to
decay (Lee
and
investigate a possible ef¬
Oleszkiewicz, 2003). At intervals of
withdrawn from the batch reactor and
secondary
operated
days.
(16 hours) phases simulating
was
Subsequently,
in
at 20
the nitrifiers to restore their ATP
and aerated
(8h)
kept
plants
batch reactor that either
days (Figure 1).
liter of activated
3000 rpm for
MBR
retention time
conditions), only
fect of the feast-famine
day,
were
for five minutes
alternating
treatment
one
sludge
added in order to prevent anaerobic conditions.
manually
with
period
a
in the MBR. Both
temporarily higher
the
pellet
was
resuspended
centrifuged
at
with effluent of the
batch reactor. The main purpose of the
centrifuga-
64
Decay processes ofnitrifying
5.
tion step
low nitrate
provide
to
was
measurements, because
20%) reduction of the nitratation rate
utilization rate
gNm"3)
6-8
(OUR; Figure
and ammonia
and nitritation rates
B)
pH
controlled
was
1
(data
experiment) concentrations for the
only 50 gN m"3 was found to lead to a
1:
C).
were
determining
Oxygen
maximum nitratation
to
carried out at
were
the base oxygen
with nitrite
successively spiked
experiments
HCl and NaHCÛ3.
After
m"3), leading
10-18 gN
All
shown).
not
A), samples
:
(NH4C1,
(Figure
using
for anoxic
(ammonia
nitrate concentration of
a
bacteria
concentration
pH
(NaNÛ2,
(Figure
7.5 and 20.0°C.
controlled between 3
was
m"3.
and 4 gQ2
©
N02 NH4
Centnfugation
700
-I
Effluent MBR
O
600
500
ocfed-
-
--
Pellet
400
P°)
"^
T =20dfl.3°C
T =20dfl.l°C
JOD,
jjj
200
°
100
Figure
pH
V
nitratation rate, C
=
scribed
for batch
were
samples
°
o o
=
I
4
3
-
2
--
60
from the CAS
zations of cells
were
120
time
A
(min)
base oxygen utilization rate
(heterotrophic rate),
Hybridization (FISH)
Ultrasonification
(1990).
prior
hybridization
to
performed
with
(35 kHz,
five
paraformaldehyde
minutes)
in order to break up
large
was
applied
floes. In situ
as
to
de¬
fixed
hybridi¬
fluorescently labeled, rRNA-targeted oligonucleotide
to Manz et al
(1992) (Table 1). Oligonucleotide probes were obtained
Microsynth (Balgach, Switzerland) and Thermo Hybaid (Interactiva Division, Ulm,
probes according
5
-
I
experiments.
6
o o o o o
taken at maximum OUR of AOB and fixed in 4%
Amann et al
by
\
°°o-
ooooo
nitritation rate.
max.
2.3 Fluorescence In Situ
Samples
®
-
0
Experimental setup
1
max.
0
=7.5dfl.l
V =50 I
=
300
=7.5dfl.l
H-
B
S
7
-
--
pH
1:
from
Ger¬
many).
All
samples hybridized
Canterbury,
was
done
(both
was
United
on an
from
Kingdom) prior
Olympus
BX50
according
NEUa
to
microscopic
were
embedded in Citifluor
observation.
microscope, equipped
to Manser et al
Oligonucleotide probes
Probe
oligonucleotide probes
and
Epifluorescence microscopy
of
halophilic
nitrifying
bacteria
(2005c).
hybridization conditions applied
in this
study.
Reference
Target organisms
Most
(Citifluor,
with filters HQ-CY3 and HQ-FLUOS
Analysentechnik AG, Tübingen, Germany). Quantification
carried out
Table 1
with
and halotolerant ammonia oxidizers in the
Wagner
et
al.,
1995
beta-subclass of Proteobacteria
a
Nmll
Many members of the
Nitrosomonas
communis
Nmo218
Many members of the
Nitrosomonas
oligotropha lineage
used with unlabeled
competitor
as
lineage
PommereningRoser et al.,
1996
Giesekeetal.,2001
indicated in the reference
65
Decay processes ofnitrifying
5.
bacteria
3. Results and Discussion
3.1
Decay experiments
The activated
sludge
model
no.
trification to enable separate
tion of
heterotrophic
(OUR)
rate
due to
also
probably
heterotrophic
ure
(OURaob)
was
et
al, 1999)
not
included. Loss of
lumped
of protozoa
associated with loss of bac¬
activity
Endogenous respira¬
process, which accounts for the oxygen utilization
decay products, growth
on
extended with two-step ni¬
in the ASM3.
(endogenous respiration)
a
was
of ammonia and nitrite oxidation. Denitrification of
on
slowly degradable
very
Loosdrecht and Henze,
(Van
In the
1999).
COD and
following,
OUR represents the base OUR measured before the addition of substrate
(OURnob)
after addition of nitrite
after addition of ammonia and
sludge
CAS
growth
respiration
dynamics
enzyme
process
bacteria is
nitratation rates
A),
1:
as one
(ASM3, Gujer
modeling
nitrite and from nitrate to nitrite
teria is modeled
3
are
discussed in
are
complete
chapter
shown, experiments with
3.2.
MBR
enzyme
(Figure
1:
B)
synthesis (Figure
data of the
Only graphical
sludge yielded
(Fig¬
and nitritation rates
1:
C).
Effects of
experiments
with
similar trends.
Aerobic conditions
Most activated
models
sludge
summarize nitrification
(e.g. ASM3)
as one
process. As
sequence, nitrite concentrations cannot be modeled and kinetics of AOB and NOB
into
one
parameter
problems
in the
receiving
step nitrification is
NOB. Our
uid
water
due to its
required involving
previous
suggested
However, elevated effluent concentrations of nitrite may
set.
toxicity.
activity
a
are
lumped
occur
causing
model extension with two-
the separate determination of the kinetics of AOB and
research with activated
that the
In that case,
a con¬
from the separate treatment of
sludge
of NOB may decline faster
compared
to AOB
digester liq¬
(unpublished
data).
_—
o
800
200
600
150
6000
co
'E
O)
\
CM
100
O
5
O)
m
J
Q
o
o
g
z
a.
200
4000
CM
O
400
150
50
2000
3
o
Figure
2 Results of batch
sent best fit
applying
the
experiment for
adapted
The results of the aerobic
activity
ing
the
CAS
sludge
under aerobic conditions at 20°C and
7.5. Lines repre¬
ASM3.
experiment
for
sludge
from the CAS
is reduced to about 60%> of the initial value after 7
experiment, large
pH
amounts
of
organic nitrogen
are
days
are
shown in
Figure
2. The
for both AOB and NOB. Dur¬
released due to the
endogenous
res-
66
piration
of
heterotrophic bacteria, hydrolyzed
Since this
trate.
focuses
study
process in the ASM3
Therefore,
duced
and the
(Table 2)
ammonium and
the
nitrifying bacteria,
the observed
nitrogen
nitrified to ni¬
immediately
heterotrophic endogenous respiration
in order to better fit the accumulation of nitrate.
slightly adapted
was
considering
term
a
on
to
bacteria
Decay processes ofnitrifying
5.
of the nitrate accumulation
lag phase
of bacteria
content
assumed to be 10%>
was
intro¬
was
referring
to
COD.
Adaptation
Table 2
of ASM3. XH
=
bacteria.
heterotrophic
fiag
=
fit parameter, t
=
simulation
time, to
=
initial
time.
Process
Aerobic
Despite
Process rate
endogenous respiration
the fact that nitrate accumulation and
the released
is well
nitrogen
the first two
activity during
model processes.
lying
rameter
b
of XH
reproduced by
of the
days
Thus,
data
the
at t
to
=
0
seems
No differences between AOB and NOB
sludge
from the separate treatment of
confirming
endogenous respiration
(Gujer et al, 1999; Koch
et
,
,,
ah
lag +Ct-to)
of
autotrophic
loss of
sharp
bacteria
on
autotrophic
be consistent with the under¬
not to
rates
in
Paul,
were
(summarized
found.
resulted in
a
with
measurements
higher
50%
in
with
for NOB
rate
The aerobic
modeling
heterotrophic
previous
from the MBR
sludge
and
Martinage
By contrast,
(unpublished data).
agreed
al, 2000). Experiments
I
the
well with values used for
rates
•
7
autotrophic endogenous respiration
digester liquids
observation
previous
our
'
p
omitted for the estimation of the pa¬
were
literature data for one-step nitrification
2000).
than AOB
;r~
K0+^0
adapted ASM3,
listed in Table 3. The values of the estimated
correspond
~
subsequent growth
experiment
points
'
H.aer
equation
studies
yielded
simi¬
lar results.
Table 3 Estimated aerobic
endogenous respiration
rates
AOB
ning
ing
the
CAS
0.15IL0.020
O.lSiO.Ol0
0.28IL0.050
MBR
0.14±0.01
0.14±0.01
0.23±0.03
value from two
of AOB
This
readily
a
finding
sludge samples
percentages
are
No shift within the
dormant state
rather
were
sharp
(below
autotrophic
taken from
reduction of
population
indicates that after two
days
detection limit for
accessible bacteria in the
20%) percent of the overall
adapted
ASM3.
in the
begin¬
experiments
FISH confirmed the
experiment (Figure 3).
has either fallen into
Since
using
the
heterotrophic
bHET.aerobic (d )
experiment.
Prédation of
using
bNOB.aerobic (d )
quantification
of the
NOB
at 20°C
bAOB.aerobic (d )
mean
The
(with standard errors)
beginning
pilot plants running
the
organization
of AOB
was
significant
FISH)
may be
bacteria would have to
unlikely. Moreover,
a
activity
an
amount
of AOB
grazed by protozoa.
explanation, although
belong
close to
or
observed dur¬
to
a
this group
steady state,
of AOB and NOB
as
10-
(Figure 2).
such
high
dense aggre-
67
Decay processes ofnitrifying
5.
gates (Manser
from
et
al, 2004; Wagner
et
al, 1995)
bacteria
additionally prevent autotrophic
may
bacteria
major prédation.
20
—
A N
oligotrophia
ON
communis
• N europaea
Sum AOB
2
4
6
time
Figure
3
(d)
in the aerobic
Quantification of ammonia-oxidizing bacteria (AOB)
experiment using
FISH.
Anoxic conditions
5000
150
120Ï
4000
a
100
^
*\
CO
-
'E
90
3000
CM
O
S
O)
-
A
A
Q
60
O
heterotrophic
COD
60
\
2000
40
O
30
A
o
80
*
*
s\
1000
X
ÙN03
-
0
4
Figure
4
Results of batch
sent best fit
applying
(d)
activity
time
for CAS
sludge
negligible
experiment
for
sludge
rates
of
autotrophic
al, 1999) stated only
from the CAS
over a
bacteria
sludge (Table 4). By contrast, previous
period
were
studies
(Lee
are
of
d"1
0.02
and
the
missing
pool)
tions.
Likewise,
a
of short aerobic
significant
very low
periods (enabling
decrease of the
7.5. Lines repre¬
the OURhet data would
Figure
4. Loss of
lower for either CAS
Oleszkiewicz, 2003; Siegrist
compared
decay products
to
or
et
aerobic
in the batch
the nitrifiers to restore their ATP
heterotrophic endogenous respiration
OUR. The estimation of the anoxic
on
or
autotrophic activity
measured nitrate concentrations. However, the model
bacteria based
pH
week. Estimated anoxic
30-50%) reduction of these parameters under anoxic
may have led to
erotrophic
shown in
one
conditions. Inhibition effects associated with the accumulation of
reactors or
(d)
under anoxic conditions at 20°C and
for both AOB and NOB
endogenous respiration
MBR
6
the ASM3.
The results of the anoxic
is
4
experiment
20
0
6
time
°
also at anoxic condi¬
rates were
clearly diverges
estimated from the
from the measured het¬
endogenous respiration rates
yield 0.16 d"1 and 0.13 d"1 for the
of
heterotrophic
CAS and
MBR,
68
5.
respectively.
Other processes like
decline of the
pend
heterotrophic
the estimated
on
Decay processes ofnitrifying
of protozoa may have contributed to the observed
decay
Furthermore, the modeled heterotrophic
OUR.
bacteria
endogenous respiration
rates
from the aerobic
OUR
experiments.
mainly
de¬
While OUR
due to
heterotrophic endogenous respiration and prédation is reasonably lumped into one pa¬
rameter under aerobic conditions, this simplification may not reliably reproduce measured
OUR under anoxic conditions.
Table 4 Estimated anoxic
endogenous respiration
rates
(with standard errors)
AOB
:
NOB
(d"1)
CAS
0.015±0.004
MBR
0.01±0.003
anoxic-aerobic conditions
Bacteria
exposed
are
removal. Since
ent
nitrifying
0.001
0.033±0.002
0.064±0.002
ORP conditions in wastewater treatment
alternating
to
bacteria
are
obligate aerobes, they
their metabolisms may
occur
under anoxic conditions
requirements during the aerobic period,
Liu, 1999; Chen etal, 2001).
_—
800
r
also known
200
as
plants
with nutri¬
unable to store
are
their substrate under anoxic conditions due to the lack of oxygen. As
age to
ASM3.
bHET.anoxic (d )
0.02±0.009
Alternating
using the
heterotrophic
(d"1)
<
at 20°C
a
leading
feast-famine
result,
to
utilize
or
stress and dam¬
increased substrate
phenomenon (Chen
and
5000
'a
—«
600
'a
NOB
-
-
150
o
4000
.-~
c,
F
D)
CM
AOB
DC
400
4x
3
o
g
o
Q
z
o
DC
heterotrophic
200
50
3000
z
S
2000
O
z
1000
0
5
Results of batch
was
omitted for better
The results of the
in
Figure
4x
(d)
experiment
7.5. Lines represent best fit
pH
-
6
time
Figure
CO
co
o
ë
4
E
S
-100-
for CAS
applying
the
sludge
adapted
under
ASM3.
alternating
Activity
alternating
anoxic-aerobic
5. Likewise to the aerobic
measured decrease of
(Figure 5).
experiment
experiment,
While
autotrophic activity
The estimated aerobic
shown in Table 5,
(0.01 d"1).
of AOB and NOB
during
anoxic
periods
readability.
assuming
mean
for
sludge
from the CAS
the ASM3 had to be
der to fit the accumulation of nitrate. In contrast to the
ASM3
anoxic-aerobic conditions at 20°C and
could be
completely
reasonably
endogenous respiration
endogenous respiration
of AOB and
shown
adapted (Table 2)
aerobic
modeled
in
experiment,
using
the
heterotrophic
or¬
the
adapted
rates of AOB and NOB
anoxic rates estimated from the anoxic
rates
are
are
experiment
bacteria from the
69
aerobic
experiment (Table 3)
and anoxic
were
endogenous respiration
confirmed, aerobic endogenous respiration
rates
of
of NOB
rates
bacteria had to be increased to better
heterotrophic
fit the measured data. The results suggest that NOB
bacteria
Decay processes ofnitrifying
5.
are more
stressed under
alternating
an¬
oxic-aerobic conditions than AOB. Increased
oxic
endogenous respiration
survive short anoxic
Estimated
Table 5
20°C
rates
of
periods.
endogenous respiration
using the adapted
grazing may be responsible for the elevated an¬
heterotrophic bacteria, because protozoa are known to
rates
(with standard errors) from the anoxic-aerobic experiment
at
ASM3.
AOB
NOB
heterotrophic
bAOB.aerobic (d )
bAOB.anoxic (d )
bNOB.aerobic (d )
bNOB.anoxic (d )
bHET.aerobic (d )
bnET.anoxic (d )
CAS
0.15±0.01
0.01^
0.22±0.01
0.01^
0.27±0.02
0.06±0.004
MBR
0.13±0.01
0.01^
0.18±0.01
0.01^
0.23±0.02
O.lOiO.01
value derived from anoxic
mean
In
conclusion,
sludge
were
activity
significant
no
found
indicating
differences between conventional and membrane activated
the
same
processes
in the two systems. The feast-famine
microbial
ments
experiment
activity
as
previously reported (Lee
a
of
microbiological point
for the loss of
in
phenomenon resulting
and Oleszkiewicz, 2003)
suggest that environmental conditions
However, from
being responsible
autotrophic
reduced decrease of
was
it is
likely
experi¬
not. Our
crucial for the loss of microbial
are
view,
a
activity.
that most bacteria do not die
-
unless
and
they are exposed to e.g. toxic compounds -, they rather become dormant (Kaprelyants
Kell, 1996). Our observations fit the hypothesis that prédation plays a major role in acti¬
vated
sludge systems (Van
and anoxic
Loosdrecht and Henze,
heterotrophic endogenous respiration
the rates under aerobic conditions. In
since protozoa
1999),
rates are
significantly
are
obligate
reduced
compared
addition, the decline of the heterotrophic
consistent with the decrease of the denitrification
however uncertain how protozoa affect
aerobic
to
OUR is not
under anoxic conditions. It remains
activity
nitrifiers, because the latter preferably
grow in dense
aggregates. The negligible loss of autotrophic activity under anoxic conditions is possibly
rather due to the inhibited metabolism of the
3.2
obligate
aerobic
nitrifying
bacteria.
Enzyme dynamics
Enzyme
processes
(activation, synthesis, degradation, inhibition)
therefore cannot account for the loss of
experiments. Nonetheless,
Enzyme
enzyme
autotrophic activity
dynamics
can
activation is the fastest mechanism to
tivated within
minutes, depending
on
play
a
regulate
the presence of
in the
role for
a
an
are
beginning
plants
process and
with
can
inhibitor. In
found constant activation times of about 10 minutes for nitritation
comparably
of the aerobic
fluctuating
be
our
fast and
stopped
loads.
or reac¬
measurements we
(Figure 6A).
The subse¬
quent enzyme synthesis (ammonia monooxygenase AMO and hydroxylamine oxidoreductase
HAO)
took between 1 and 2
2004; SayavedraSoto
et
hours, which is
al, 1996). By
OUR. It is concluded that NOB do not
in agreement with literature data
contrast, NOB
or
immediately
only marginally degrade
(Aoi
et
al,
reached their maximum
their enzymes
regardless
of
70
Decay processes ofnitrifying
5.
the environmental conditions. The enzyme saturation for every measurement
as
ratio of the OUR after the activation time and the maximum OUR after
Cell
synthesis.
growth during
tion. As shown in
Figure 6B,
ammonia due to
saturation.
1996),
Similarly,
half-life times
conditions, although
AOB
the fact that
(Figure 6C). Despite
conditions,
complete
neglected
to
enzyme
for this calcula¬
about 60%> of their maxi¬
decline. The continuous release of
the AOB to
keep
a
the order of hours
were
reported (SayavedraSoto etal,
causes
on
able to
were
synthesis
aerobically degraded
degradation
enzyme
of environmental
are
was
calculated
minimal level of the enzyme
could still detect one-third of the enzymes 24h after the start
(2004)
Surprisingly,
of enzyme
following only marginal
a
heterotrophic decay
whereas Aoi et al
of starvation.
phase
enzymes
level after 6 hours with
mum
nia
the
was
bacteria
enhanced at aerobic than at anoxic
was more
continuously nitrify
degradation
anoxic conditions
small amounts of released
of enzymes
seem
to
basically
prevent
a
takes
ammo¬
place regardless
significant
decrease of the
enzyme saturation in AOB.
1000
1 0
m
~
800
o
<
08
ra
06
I
04
^zAaa^
a
**£
|
°
600
I
enzyme
ai
cell
activation
200
°Q-
synthesis
'
400
A
growth
10m in
aerobic
anoxic-aerobic
anoxic
C
ai
00
30
60
time
Figure
6
periments (C).
(AOB)
time
enzyme
Data from
was
24
synthesis (AOB)
calculated
as
from Wild et al
for
one
measurement
in order to test its
in wastewater treatment systems
of enzymes
6
(d)
(A). Short-term degrada¬
(AOB) during decay
ex¬
ratio of the OUR after the activation time and the maxi¬
Lines represent best fit
with conventional activated
(1995)
4
time
(B). Long-term degradation
synthesis.
experiments
1£
(h)
using
sludge
(with already implemented two-step nitrification)
adapted
dynamics
at aerobic conditions
complete
(Table 6).
The ASM3
netics
12
150
The enzyme saturation
OUR after
kinetics
120
(min)
Illustration of enzyme activation and
tion of enzymes
mum
90
00
the ASM3 extended with enzyme
are
was
shown.
extended with enzyme ki¬
sensitivity
on
ammonia and nitrite
(Table 6).
71
Stoichiometric matrix and process rate
Table 6
ration
equations (adapted
from Wild et
Esat
Process rate
Enzyme
=
satu¬
(p)
s
Enzyme synthesis of
s
^O
JNH
u
'
'
synth
Xaob
„
„
„
^MiAOB ~t~°NH
Aerobic enzyme
of XAob
decay
S°
k
-r^
o
rate
nitritation enzymes. Esat is
„
to
+ÖNH
processes allow
new
(Figure 6C),
treatment
(sample
plants.
shown in
depicted
Figure 6A)
The obtained values at 20°C
ksynth
=
the aerobic
which however should not
Data
Figure
are as
kdecay
,
'v-
O
+öO
*)
siA>
the
ALK, AOB
-y-
degree
+ ö
-p
'^AOB'ßsat
O
,
"
ALK
of cell saturation with
of nitritation enzymes is
decay
the measured values
decay experiment
play
a
role in
by
the
negligible.
(Figure 6). Only
is underestimated
practical operation
by
the
of wastewater
6B and several data sets from batch
used to estimate the unknown parameters
were
d"1
30±4
in
—F
the actual total amount of enzymes divided
reasonably reproducing
long-term degradation during
model
^O.AOB
multiplied by
actual biomass of XAob- It is assumed that anoxic
The two
JALK
'„
„
of XAob has to be
proportional
^fi
ci
J0
'
"NRAOB
ASM3, the growth
'
+^0
JNH
H-m,AOB
JALK
sat
^
^O.AOB
s
_i_CT^
_i_C
"t"°0
^ALK.AOB "t"°ALK
F
.
decay
^O.AOB
o
Growth of Xaob
the
al, 1995). Esat
(-).
Process
In
bacteria
Decay processes ofnitrifying
5.
experiments
ksynth
and
kdecay-
follows:
=
d"1
3.0±0.3
The maximum enzyme saturation is lower than 1 due to the continuous
decay
of the enzymes.
With the estimated parameters, the maximum achievable enzyme saturation is 0.9 at 20°C.
The temperature
3.3
Significance
A simulation
equal
to
the
growth
of AOB.
for wastewater treatment
study
autotrophic decay
configurations
is assumed to be
dependency
was
carried out in order to test the
and the enzyme kinetics
based
on
ASM3 default. Anoxic
Model B:
Anoxic
Model C:
Including
decay
=
0. No
autotrophic decay
autotrophic decay
=
at
were
applied:
50%> of aerobic
decay.
anoxic conditions.
enzyme kinetics. Model B extended with enzyme kinetics for AOB
Maximum
growth
l/ESat,max= 0.88 (Figure 8)
growth rate.
A CAS with three
completely
chosen to be 12h.
Temperature
ammonia load for
the nitrification process. Three different model
the ASM3 with two-step nitrification
Model A:
(Table 6).
on
of the reduction of anoxic
sensitivity
municipal
rate
(nm,AOB)
was
increased
in order to exclude the effect of
mixed reactors
was
of AOB
was
assumed. The
set to 15°C. A
wastewater was
used
typical
hydraulic
an
by
factor
overall reduced
retention time
was
diurnal influent variation of the
(Figure 7).
72
Decay processes ofnitrifying
5.
The
ure
of the model
sensitivity
predictions
investigated
was
two common
on
bacteria
plant layouts (Fig¬
aerobic and with 33% anoxic volume. The dissolved oxygen concentration in the
7): fully
aerobic reactors
was
fixed at 2.5 g02
m"3,
whereas the aerobic
sludge
age
was
6d in both
set to
layouts.
Influent variation
Mod
Figure
A ASM3 default
Mod
B
anoxic
»Mod
C
mcl
decay
=
0
enzyme kinetics
02
04
06
time
(d)
04
08
time
effluent for
an
impact
diurnal variation in the influent.
typical
on
the ammonia concentrations in the
Neglecting
enzyme
underestimation of the ammonia concentrations for
fully
However, the differences
compensated by
the maximum
performance
(without
the
growth
not
to a
ration is
and could be
large
of AOB. Pre-denitrification
rate
aerobic and
obviously
dynamics
partly
e.g.
a
leads to
anoxic
an
plants.
10%> increase of
decreases the nitrification
in Model A due to loss of nitrifiers within the anoxic reactor, whereas Model B
anoxic
fully
are
autotrophic decay) predicts equal
aerobic
plant layout. Similarly,
the enzyme saturation of AOB
ing
(d)
Simulated concentrations of ammonia and nitrite in the effluent of the third reactors.
7
The results reveal that enzyme kinetics has
to
06
always
above 75%
peak
even
at
aerobic
in the effluent
fully
degradation of enzymes assumed)
(Figure 7). Nonetheless, the enzyme
characteristics.
Interestingly,
results in almost identical ammonia effluent concentrations
default ASM3
(Model A)
concentrations
are
barely
the combined effect of reduced anoxic
for the
plant
influenced
by
lead¬
satu¬
aerobic conditions for the chosen domestic influent
ics
(Model C)
compared
anoxic conditions diminish the diurnal variation of
(Figure 8, only
lower ammonia
slightly
ammonia effluent concentrations
with
decay
and enzyme kinet¬
compared
pre-denitrification. By contrast,
neither anoxic
decay
nor
enzyme
to
the
effluent nitrite
dynamics
of AOB.
73
Decay processes ofnitrifying
5.
X
09
bacteria
-
TO
CA
LU
I
08-
fully
aerobic
33%
anoxic
N
S
06
—i
02
\
i
\
04
06
08
time
Figure
8 Diurnal variation of enzyme saturation
1
(d)
Esat of AOB (Model C).
4. Conclusions
Batch
pilot plant
teria. It
was
cept
as
performed
were
1. Loss of
in order to
autotrophic activity
incorporated
activated
activity
treatment
may
possibly
play
plants
phic decay
rates
3. Ammonia- and
Likewise,
over
a
decay
no
were
other mechanisms
several
a
of ammonia- and
days.
nitrite-oxidizing
the
were
same
Therefore, decay
model
membrane bioreactor
nitrite-oxidizing
bac¬
the
endogenous respiration
con¬
role.
(ASM3). Other processes
Furthermore, it must be considered
no.
3
analyzed
that
in batch
bacteria incubated at anoxic conditions
This must be considered when
modeling
wastewater
Otherwise, nitrification performance will be underes¬
alternating
anoxic-aerobic conditions
phenomenon
was
not
bacteria exhibited similar
the autotro¬
on
detected.
endogenous respiration
rates.
found between the conventional and the membrane activated
processes
being responsible
for loss of
activity
parameters estimated from conventional systems
in the two sys¬
can
also be ap¬
membrane bioreactors.
nitrite-oxidizing bacteria, ammonia-oxidizing
of their enzymes. The extension of activated
monia-oxidizing
bacteria may lead to
wastewater treatment
a
sludge
better
bacteria showed
a
pronounced decay
models with enzyme kinetics for
understanding
of ammonia
in activated
am¬
dynamics
in
plants.
Further research is needed to elucidate the processes involved in the loss of
ity
like
occur.
nitrite-oxidizing
due to the feast-famine
differences
sludge
significant
with anoxic volume.
sludge suggesting
4. Unlike
conventional and
taken from continuous flow systems and
timated. An additional influence of
to
a
only partly explained by
was
of both ammonia- and
did not decrease
plied
the
study
e.g. in the activated
sludge samples
systems, where
tems.
from
sludge
found that:
prédation by protozoa
2. The
activated
experiments using
sludge systems.
In
particular,
in situ measurement of the
labeled nitrifiers in continuous flow systems would avoid the
change
autotrophic
decay
to
activ¬
of e.g. radio¬
batch conditions.
74
Conclusions
Conclusions
The
facilitates
microscopy
cence
ing
method based
developed
bacteria in activated
ferent
fluorescence in situ
on
a
sludge.
comprehensive
A
groups is available
nitrifying
hybridization (FISH)
reliable and fast identification and
allowing
for
of
set
quantification
analysis
of the
nity composition. Although FISH and epifluorescence microscopy is still
for research, operators of large wastewater treatment plants may apply
monitoring
mized based
on
The membrane
a
dramatic
ages
does not influence the
the floe structure.
on
expected
are
model
play
parameters. However,
small floe size
compared
centrations in order to
Likewise, decay is
not
the oxidation-reduction
was
only marginally
diminished their
strategies optimizing
a
tool
this
commu¬
mostly
used
technique
for
wash-out of the nitrifiers
and
could be
foaming
opti¬
non-elevated
sludge
by
potential.
a
and
MBRs
negligible
be
exhibiting
to CAS
other processes such
implicitly
are
can
at
compared
conversions,
sludge systems
Consequently,
as mass
considered in
lumped
in MBRs due to their
operated
at
low oxygen
con¬
improve pre-denitrification.
the membrane
separation.
It however
strongly depends
on
The
reduced under
As
operated
MBRs
bacterial groups
aeration energy and to
affected
activity.
same
transfer limitation is
mass
to CAS.
save
or
nitrifying
dif¬
but has
the microbial
role in activated
a
nitrify¬
nitrifying community composition
Therefore,
harbor the
to
equal physiological properties. Apart
transfer effects
against sludge bulking
measures
of
of filamentous bacteria.
analysis
separation
impact
(10-30d)
mitigation
or
the
nitrification failures due to inhibition
Thus,
purposes.
could be detected
epifluores¬
oligonucleotide probes targeting
detailed
a
and
activity of both ammonia- and nitrite-oxidizing bacteria
anoxic conditions, whereas aerobic conditions similarly
consequence, WWTPs extensions of anoxic volumes
aerated volumes enhance nitrification
capacity
or
in terms of
control
remaining
nitrifiers rather than reduce it.
The enriched
WWTPs
community composition is crucial for success and failure of bioaugmentation.
harbor nitrifiers, which are well adapted to low substrate concentrations (mostly Ni¬
trosomonas
oligotropha
gester liquids lead
to a
ties to the substrates
is not able to
sludge
term
adapt
and
Nitrospira),
nitrifying community
(mostly
to
bacteria due to the membrane
structure.
high
stants
maximum
models used for CAS
can
Nitrobacter).
is not
play
reliably
a
influent
have to be
variation, dynamic (depending
mimicking
mass
transfer effects may
be
adapted.
on
applied
rapidly degrade
their enzymes unlike
enriched
suited for the
complete
long-
retention of
to MBRs in terms
mass
For CAS
the active
improve
model of ammonia oxidation must include enzyme
but low affini¬
role in this context.
However, kinetic parameters implicitly considering
-
rates
di¬
Since the latter group
necessarily
For this reason, the
performance.
does not
growth
sludge
days, bioaugmentation using
digester liquids
separation
half-saturation constants for oxygen
a
high
low substrate levels within
from the separate treatment of
sludge
with
Nitrosomonas europaea and
enhancement of the nitrification
Activated
whereas ammonium-rich influent like
the
transfer effects
exhibiting large
processes)
of model
-
such
floes and
half-saturation
of the model. A
as
con¬
detailed
quality
kinetics, since ammonia-oxidizing bacteria
nitrite-oxidizing
more
bacteria.
76
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84
Index
Index
abundance, 9, 12, 16, 17,
activated
aerobic
sludge
model
conditions,
digester liquids, 10, 44, 45,
39
no.
3, 36, 59,
66
ammonia monooxygenase, 4, 70
ammonia-oxidizing bacteria, 4, 13, 26, 36,
64
anoxic
liquids,
conditions,
ATP, 64,
33
68
68
biovolume, 9, 11,
activation,
enzyme
degradation,
enzyme
kinetics,
enzyme
synthesis, 66,
70
71
71
70
sludge production,
feast-famine
18
filamentous
12
4
29
coefficient of variation, 12
co-existence, 37,
phenomenon,
bacteria,
FISH, 5, 8, 36,
13
CLSM, 8, 17, 24,
density, 11, 52,
floe
size, 25,
floe size
69
9
56
53
distribution,
51
2
growth rate, 30, 37, 49, 51, 72,
60
conventional activated
sludge plant, 10, 22,
73
half-saturation constants, 37, 55, 56, 57, 58
Half-saturation constants, 37
64
heterotrophic layer, 53,
costs, 3
cross-flow
3
67
floe
fouling,
40
strategies,
34, 49,
enzyme
excess
chemolithoautotrophic,
control
energy, 3
35,65
44
biofilm, 24, 31,54
diameter,
67
epifluorescence microscopy, 9, 11, 16, 24,
5
bioaugmentation, 33,
cell
73
enzymes, 4
Betaproteobacteria,
bootstrap,
quality, 25, 49, 59, 66,
endogenous respiration, 30, 39, 56, 63, 66,
66
aggregates, 9, 13, 29, 42, 68, 70
ammonium-rich
effluent
67
aeration,
4
hollow-fibre, 2,
23
4
58
decay, 39, 63,
69
hydroxylamine,
decentralized,
3
hydroxylamine oxidoreductase, 4,
diffusion
coefficient,
diffusion
model, 51, 53,
image analysis,
54
58
70
8
inhibition, 35, 37, 68,
70
86
kinetic parameters, 47, 49
partial nitrification, 40,
K-strategists, 27, 37, 39,
pilot plants,
linear
regression,
loss of
lysis,
activity,
66
pore
effects, 53, 56,
58
Monod
73
quantification, 11, 14, 18, 24,
63
receiving water,
removal
15
4
efficiency,
25
respirometry, 8, 10, 24, 35,
25
model, 37,
nitratation, 5, 43,
rRNA, 9,
56
safety factor,
nitrification, 4, 42, 45, 63,
56
30
sensitivity analysis, 53, 59,
66
nitritation rates, 42
single cells,
nitrite-oxidizing bacteria, 5, 13, 27, 37,
64
sludge
71
batch reactor, 10, 34
sequencing
66
65
27
r-strategists, 27, 37, 40,
66
nitratation rates, 43
nitritation, 4, 41,
42
reference system, 11
3
microscopic field, 11, 12,
liquor,
67
protozoa, 69, 70
bioreactor, 2, 10, 22, 34, 49,
micropollutants,
22
23
pre-denitrification,
63
maximum nitrification rate, 30, 40
mixed
size, 2,
prédation, 42, 63,
transfer
membrane
23
population dynamics, 18,
17
63
maintenance,
mass
40
44
11
23,
age,
34
Nitrobacter, 5, 27, 37, 43,
56
sludge structure, 42, 51,
Nitrosococcus
mobilis, 5,
27
sludge
Nitrosomonas
communis, 26,
volume
index, 3,
60
57
spatial distribution, 14, 29, 54,
36
58
Nitrosomonas europaea, 4, 16, 18, 26, 36
spatial variability, 13, 14, 16, 18, 42,
Nitrosomonas
marina, 5,
specific
Nitrosomonas
oligotropha, 16, 18, 26, 36,
27
55
27
Nitrospira, 5, 27, 37,
oxidation-reduction
55
5
potential, 63,
of the art, 2
systematic
errors, 15
temperature correction factors, 39
oligonucleotide probes, 10, 23, 36,
oxidoreductase,
startup, 23
state
Nitrosospira, 5, 18,
nitritation rates, 18
65
69
temporal variability,
threshold,
9
57
53
Index
time
demand,
titration,
16
10
ultrafiltration,
uncertainty, 12, 13, 19,
urease
2
ultrasonification, 10
activity,
viruses,
2
26
53
Curriculum vitae
Curriculum vitae
2001
-
2005
Dissertation at the Swiss Federal Institute of
ence
1998
1992
-
-
-
Technology (Eawag),
2000
Teaching assistance,
1998
M.S. in Environmental
of
1995
and
1996
Technology
Internships
at
Aquatic
Sci¬
Duebendorf
ETH Zurich
Zurich
engineering
at
the Swiss Federal Institute
(ETHZ)
Ecosens, Brutisellen and Office for the
en¬
vironment, Glarus
school in Wattwil
1987 -1992
High
March 24, 1972
Born in
Männedorf, Switzerland
90