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Engineering Biology through

Molecular Microbial Ecology


Russell Davenport
Global challenges
Zimmerman et al., 2008 ES&T
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9
5
0

2
0
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Life expectancy
Y
e
a
r
s

Year
20
40
30
50
60
70
Riley, 2005, Pop Dev Rev
Growing population,
urbanisation, wealth,
industrialisation, and
consumption
Global challenges
UN Millenium Development Goals 2.6 billion without improved sanitation
WHO/Unicef, 2010
Global challenges
Rockstrm et al., 2009 Nature
Anthropocene

Ecological
footprint has been
exceeded

Affect air, land and
water

21
st
Century of the
environment or
biology
Living beyond our planetary boundaries
Double burden and emerging hazards
Lvovsky, 2001
World Bank, 2003
Traditional hazards: water-borne diseases from inadequate water supply & sanitation

Modern hazards: exposure to agro-industrial chemicals
Dual jeopardy risks to health
Emerging hazards chemical exposure
Personal care and domestic
hygiene products, pesticides,
pharmaceuticals and plastics
300 Mt per annum in water courses
Present in picogram (10
-12
) nanogram (10
-9
) per litre level
143,000 registered chemicals in Europe
< 1% of chemicals assessed for toxicity/health risks
Emerging hazards chemical exposure
Personal care and domestic
hygiene products, pesticides,
pharmaceuticals and plastics
Mitigation:
Manufacturing source chemical regulation (e.g. REACH)
Engineered intervention of emissions wastewater regulation
(e.g. Integrated Pollution Prevention & Control; IPPC and
Water Framework Directive; WFD)
Environmental engineering
Provide appropriate water and
wastewater treatment
governance and technologies


Protection of human health and
the environment through the
regulation of chemicals
World Bank, 1992
Empiricism and opportunism
1910
1891
1914
..practices do not represent the zenith
of scientific treatment, nor are they
the product of a logical
and rational and design process.
Feachem et al., 1983
- 4 M tonnes total CO
2
(0.5% total UK) emissions
- 1.5% of UK electricity
Economic and Environmental Costs
Organic carbon, N, P
Waste sludge
(WAS)
Return Activated Sludge
(RAS)
CO
2
emissions
CO
2
emissions
Treated
effluent
to sludge
treatment
O
2

Empirical wastewater treatment
Sometimes result in failure
Unpredictable and inexplicable
Situation bound
Proximity to failure unknown
Adequate theories and models
Aggregate behaviour
Poorly calibrated
Over design
Over aeration

Why theory? How difficult can it be?
Stars in the our galaxy : Stars in the universe :
Measures of complexity
Bacteria in a STW : Bacteria in the world :
10
30

10
21

10
18

10
11

Causey Arch, Built 1725
Severan Bridge, Turkey ~200 AD
The limits of empiricism
Roman design: unchanged since 179 BC
The Forth railway bridge:1885
Rationally designed 1882
Classical structural theory and new materials
Theory allows to transcend experience
A new era
New molecular based tools based on
evolutionary principles

Novel (ecological) theories
Evolutionary tree of life
Woeses tree (1977) based on rRNA gene sequence
comparisons
Tree dominated by microbial forms
Revolution in microbial ecology
The basic molecular tool box
Clone, screen &
sequence
DNA extraction
Amplification
Community analysis
RNA sequence determination
Fingerprint gel
Sample
Fix cells
Fluorescence in situ
Hybridisation (FISH)
Probe/primer design
Abundance Diversity
Quantitative real-time PCR
Measure diversity and abundance

Microbial ecology
Understanding the problems
0
10
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30
40
50
60
70
Time
B
a
c
t
e
r
i
a
l

N
u
m
b
e
r
s
Identification and quantification is central in
achieving population dynamics
- Empirical monitoring (reactive management)
- (Ecological) theory (predictive management)
Challenges facing biological treatment
Nitrification Foaming
Phosphorus removal
Bulking Denitrification
Removal of micropollutants including Endocrine
Disrupting Compounds (EDC)
Nutrient removal Solids separation
BOD removal
Nitrification
3 step process involving 2 distinct groups of bacteria
Ammonia oxidation (autotrophic AOB)
NH
3
+ O
2
+ 2H
+
+ 2e
-
NH
2
OH + H
2
O
Nitrite oxidation (nitrite-oxidizing bacteria)
NH
2
OH + H
2
O NO
2
-
+ 5H
+
+ 4e
-

NO
2
-
+ H
2
O NO
3
-
+ 2H
+
+ 2e
-

Rate limiting step
NH
4

NO
2
-

NO
3
-

N
2

N- Fixation
Quantitative analysis of AOB
AOB occur in microcolonies
in activated sludge flocs

Can contain thousands of
individual cells
R
2
= 0.8704
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10
Microcolony radius (microns)
Cell No.
4/3 H
3
R
2
= 0.87
n = 80
Quantitative analysis of AOB
On the basis of this theoretical model the proportion of AOB
to total biomass can be predicted from ammonia removal




Values from cultured AOB are used to provide yield and
growth rate
Xv
Ammonia
x bnit
Ynit x
Xv
Xnit
(

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
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20
19
18
17
16
15
14
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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
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d
a
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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!

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