A
+
=
*
* 1 u u
u
X
AOB
Y
AOB
b
AOB
* x
Integrating microbial data and process
models
Good fit between theoretical
predictions and measurement
Betaproteobacterial AOB
responsible for observed
nitrification
Deviations from theoretical
predictions suggest
Novel AOB
Failing nitrification
Model not universal
X
AOB
/X
v
predicted (%)
X
A
O
B
/
X
v
m
e
a
s
u
r
e
d
(
%
)
Regression
95% CI
8
4
0
0 1 2 3 4 5 6 7
y = 0.94x 1.42
Integrating microbial data and process
models
Coskunur et al., AEM, 2005
Data useful for resource management?
Can high biomass
plants be made to work
harder?
Can such data permit
more intelligent balance
between process
performance and
process cost?
4
5
6
7
8
9
0.01 0.1 1 10 100
l
o
g
A
O
B
c
e
l
l
s
/
m
l
Cell-specific ammonia
oxidation rate fmol/cell/h
Coskunur et al., AEM, 2005
Cell Specific Ammonia Oxidation Rate (fmol/cell/hr)
L
o
g
A
O
B
c
e
l
l
s
/
m
l
10.0 1.0 0.1
1.0E+09
1.0E+08
1.0E+07
Stability
Failing
Irregular
Stable
23
22
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
Quantifying AOB in relation to failure
Pickering et al., submitted Environ. Sci. Tchnol. (under review)
Pickering, 2008
Dissolved
oxygen
(mg l
-1
)
Ammoniacal
nitrogen
(mg l
-1
)
1. Influent
2. Anoxic zone
3. Mechanical aeration
4. Mechanical aeration
5. Diffuse aeration
6. Diffuse aeration
7. Diffuse aeration
8. Secondary effluent
9. Return Activated Sludge
Sutton-in-Ashfield
Toton
Nitrosomonas europaea NCIMB 11850
T
1. Influent
2. Anoxic zone
3. Mechanical aeration
4. Mechanical aeration
5. Diffuse aeration
6. Diffuse aeration
7. Diffuse aeration
8. Secondary effluent
9. Return Activated Sludge
Sutton-in-Ashfield
Toton
Nitrosomonas europaea NCIMB 11850
T
Wanlip
DNA
Wanlip
RNA
Presence versus Activity
Milner et al., 2008, Water Research
1. Influent
2. Anoxic zone
3. Mechanical aeration
4. Mechanical aeration
5. Diffuse aeration
6. Diffuse aeration
7. Diffuse aeration
8. Secondary effluent
9. Return Activated Sludge
Sutton-in-Ashfield
Toton
Nitrosomonas europaea NCIMB 11850
T
1. Influent
2. Anoxic zone
3. Mechanical aeration
4. Mechanical aeration
5. Diffuse aeration
6. Diffuse aeration
7. Diffuse aeration
8. Secondary effluent
9. Return Activated Sludge
Sutton-in-Ashfield
Toton
Nitrosomonas europaea NCIMB 11850
T
Wanlip
DNA
Wanlip
RNA
Presence versus Activity
AOB
abundance
( 10
7
ml
-1
)
Ammoniacal
nitrogen
(mg l
-1
)
Nitrosomonas europaea
-like organism
Milner et al., 2008 Water Research
Are AOB mixotrophic?
Y = (M
t
M
t0
)/ (S
t
S
t0
)
Observed yield
Assumptions: All ammonia is consumed by AOB with little used for maintenance,
no heterotrophic assimilation
26 mg mg
-1
ammonia removed
0.03 - 0.34 mg mg
-1
ammonia removed
Can we make the biomass work harder?
Bellucci et al., 2011 Appl. Environ. Microbiol.
43 37 31 25 19 13 7 1
100
80
60
40
20
0
Time (days)
C
o
n
c
e
n
t
r
a
t
i
o
n
a
s
N
(
m
g
/
L
)
RH4
43 37 31 25 19 13 7 1
100
80
60
40
20
0
Time (days)
C
o
n
c
e
n
t
r
a
t
i
o
n
a
s
N
(
m
g
/
L
)
RH3
43 37 31 25 19 13 7 1
100
80
60
40
20
0
Time (days)
C
o
n
c
e
n
t
r
a
t
i
o
n
a
s
N
(
m
g
/
L
)
RL2
43 37 31 25 19 13 7 1
100
80
60
40
20
0
Time (days)
C
o
n
c
e
n
t
r
a
t
i
o
n
a
s
N
(
m
g
/
L
)
RL1
0.25 mg l
-1
0.5 mg l
-1
3.4 mg l
-1
3.0 mg l
-1
NH
3
NO
2
-
NO
3
-
RHs RLs
2.5
2.0
1.5
1.0
0.5
0.0
Treatments
Y
i
e
l
d
(
V
S
S
a
o
b
/
N
H
4
+
-
N
r
e
m
o
v
e
d
)
All biological communities are characterised by a species abundance
distribution
Area under the curve is the total number of taxa, S
T
i.e. total (alpha)
diversity or species richness
0
0 5 10 15 20 25
Log2 Bacterial abundance (arbitrary units)
N
u
m
b
e
r
o
f
s
p
e
c
i
e
s
(
S
)
S
T
Intermediate abundance
Rare
species
Common
species
Most diversity is undetected
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
0 5 10 15 20 25
log2 species abundance (arbitrary units)
N
u
m
b
e
r
o
f
s
p
e
c
i
e
s
p
r
e
s
e
n
t
a
t
a
g
i
v
e
n
a
b
u
n
d
a
n
c
e
Even cloning and sequencing efforts will only sample the
far right hand end of the species curve
Most diversity is undetected
Next generation sequencing
Generates orders of magnitude greater sequencing depth (i.e. number
of sequences) than conventional Sanger-sequencing of clones
Clone-sequencing: 100 clones reads is a big library takes
approx. 2 days from PCR
Parallel sequencing: up to 1,000,000 reads takes hours to days
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
0 5 10 15 20 25
log2 species abundance (arbitrary units)
N
u
m
b
e
r
o
f
s
p
e
c
i
e
s
p
r
e
s
e
n
t
a
t
a
g
i
v
e
n
a
b
u
n
d
a
n
c
e
An explosion of data
Key tools and theories still
being developed
Genbank growth now
exceeding Moores law
Economist 2009
Next generation sequencing
Next generation sequencing
ABI SOLiD (~50 100 bp)
Illumina Solexa and Hi-Seq genome analyser
(~75 150 bp)
Roche 454 pyrosequencing (~400 1000 bp)
Ion Torrent (~100 200 bp)
Pyrosequencing
PCR using fusion primers (so-called tags)
Generates PCR amplicons with A and B adaptor tags
One fragment binds to one bead
Emulsified in oil to give microreactors with reagents
for PCR (emPCR)
Multiple copies made of single fragment
Initially each bead has a single DNA molecule attached
Pyrosequencing
Sequencing by synthesis as nucleotides are flowed across plate in turn
Incorporated bases emit light with intensity proportional to homopolymer
length n
Pyrosequencing
Flowgrams are translated into sequences (by rounding to integers)
Can cause noise which can be removed (Quince et al., 2009, Nature Methods)
Pyrosequencing
Beware of sequencing error!
Quince et al., Nat.Meths 2009, BMC Bioinf. 2011
Species diversity in activated sludge
30,000 sequences
from UK AS plants
Sequencing noise
removed
1000s of species in AS
plants!
Just to sequence 90%
of diversity in 0.25 ml
requires
2-8 MILLION
sequences
0
1000
2000
3000
4000
5000
6000
7000
8000
Lognormal Inverse
Gaussian
Derby Wanlip
N
u
m
b
e
r
o
f
S
p
e
c
i
e
s
Davenport et al., unpublished
Thanks!