Bacterioplankton communities:
single-cell characteristics and
physiological structure
Paul del Giorgio
Université du Québec à Montrèal
Why study aquatic bacteria?
• They are responsible for much of organic matter
and nutrient transformation and mineralization
• Bacteria are responsible for much of the aerobic
respiration and all of anaerobic respiration in
aquatic systems
• Aquatic bacteria are one of the largest living
reservoirs of carbon, P, N, Fe and other materials
• Aquatic bacteria represent the largest surface in
oceans and lakes
• Bacterial biomass may be a significant food
resource in aquatic food webs
• Some bacteria pose sanitary or environmental
problems
Ecosystem processes
Carbon cycling
Gas exchange
Bacterial community structure
Bacterial processes:
Production
Respiration
Nutrient cycling
Resource supply: the nature and amount
of organic matter and nutrients
Trophic interactions:
Grazing (predation)
Viral mortality
Competition
What is community structure at
the microbial level?
•
•
•
•
•
•
Bacterial biomass
Bacterial cell size and morphology
Attached versus free-living cells
The distribution of cells with different functions
Taxonomic (phylogenetic) composition
The distribution of cells with different growth and
metabolic rates
From Cole et al. (1988)
BP mgC m-3 d -1
100
10
1
10
100
NPP mgC m-3 d -1
1000
Bacterial response to changes in resources and conditions
?
Δ Environment
Δ bacterial
community
metabolism
Bacterial response to changes in resources and conditions
?
Δ Environment
Δ bacterial
community
metabolism
Bacterial response to changes in resources and conditions
?
Δ Environment
Changes in abundance
Δ bacterial
community
metabolism
Ducklow 1999
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Bacterial response to changes in resources and conditions
?
Δ Environment
Changes in abundance
Δ bacterial
community
metabolism
Changes in composition
of bacterial community
Bacterial response to changes in resources and conditions
?
Δ Environment
Changes in composition of bacterial community
Changes in abundance
Δ bacterial
community
metabolism
Changes in composition
of bacterial community
Bacterial response to changes in resources and conditions
?
Δ Environment
Changes in composition of bacterial community
Δ bacterial
community
metabolism
?
Changes in abundance
Changes in composition
of bacterial community
Bacterioplankton black box
Large viruses
or
small bacteria
CH
4
Aerobic
or
quimioautotrophs
or
fermenters
h 
With or w/o
external
structure
O2
O2
O2
Attached
or
free-living
Small
or
large
Phototrophic
or
heterotrophic
z
z
z
z
Active
or
dormant
Alive
or
dead
Caja
negra del picoplancton
Bacterioplankton
black box
Starvation, dormancy, slow
growth
• Dormancy, starvation-survival, slow
growth, and inactivity are often used
interchangeably to denote low levels of
cellular activity in marine bacteria, but these
terms are not synonyms and refer to
different states
Spec substrate consumption
Microbial bioenergetics: maintenance
versus growth
}
}
Death
Dormancy
e (µ)
m (µ)
}
me (µ = 0.0 h-1)
Growth rate µ (h-1)
Starvation survival
• Under conditions of extreme substrate and energy deprivation, marine
bacteria may undergo a “starvation” response
• The starvation response is regulated by specific genes and involves cell
miniaturization, and profound changes in macromolecular composition,
with the synthesis of specialized protective proteins
• Prolonged starvation may lead to cell “dormancy”, which is a state of
complete metabolic arrest that allows long-term survival under
unfavorable conditions. Cells in a dormant state are still more resistant to
other environmental stresses
• There are costs and benefits associated to entering dormancy as opposed
to maintaining a slow level of metabolic activity and growth as a response
to low substrate availability
• Resource patchiness and temporal variability play a major role in shaping
the survival strategies of marine bacteria, whether it is slow growth,
starvation response or dormancy
The distribution of cells into different
physiological categories is termed the
“physiological structure” of bacterioplankton
• Within a bacterial community there is a continuum of activity, from
dead to highly active cells
• The categories used to describe the physiological structure are
operational and depend on the methods used
• The physiological structure is related, albeit in complex ways, to the
size structure of the community, as well as to the phylogenetic
structure, i.e. the distribution of cells into operational taxonomical
units
• The physiological structure is dynamic, i.e. the proportions of cells in
various physiological states may vary at short time scales and small
spatial scales
Nyström et al. 1992
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
The starvation sequence
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Joux and Le Baron 2000
The reality of our disciplne:
• Thomas Brock's classic microbial ecology
text (Brock 1966) is prefaced by a quote
attributed as a graduate student motto. The
motto simply states, 'microbial ecology is
microbial physiology under the worst
possible conditions'.
“If I could do it all over again, I would be a microbial
ecologist. Ten billion bacteria live in a gram of soil... They
represent thousands of species, almost none of each are
known to science”
Wilson, E.O. 1994. Naturalist. Island Press
Approaches to measuring single-cell properties
Abundance:
Nucleic acid staining
(SYTO13)
Phylogenetic composition:
Fluorescence In Situ Hybridization
(FISH)
Physiological state
Highly active cells
(CTC, HDNA)
DNA
ETS
3H
3H
Metabolism
C respiration (O2 consumption)
C production (3H-thym incorporation)
Bacterial Growth Efficiency (BGE)
Ribosomes
Physiological state:
Physiological state:
Altered membrane
(BackLight)
Depolarized cells
(DiBac)
Some approaches used to assess bacterial
characteristics in situ that are culture
independent
• Microautoradiography to assess uptake of
radiolabeled organic compounds
• RNA (and other macromolecular) contents
• Vital stains as indices of cell metabolism
(Fluorescein, Calcein, INT, CTC)
• Stains that reflect membrane polarization and
integrity (PI, Oxonol, SYTOX, TOPRO)
• Structural integrity under TEM
Heissenberger et al.
1996
Examples of cell
and capsule
structure observed
by TEM in
bacterioplankton
samples
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Zweifel & Hagström (1995)
Site
Baltic Sea, NB1
Baltic Sea, SR5
Baltic Sea, US5b
North Sea, Skagerrak-1
North Sea, Skagerrak-2
Mediterranean, Point B
BT (106)
2.5 - 3.2
0.7 - 1.2
0.6 - 2.7
1.1 - 1.4
0.2 - 0.8
0.5
NuCC (%)
4-6
17 - 27
12 - 27
2-5
4 - 32
20
MPN (%)
0.1 - 0.3
7 - 14
6 - 15
0.5 - 0.6
0.2 - 0.8
16
Marie et al. 1997
Marine picoplankton
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Cytometric enumeration of in situ
aquatic bacteria using green nucleic acid
stains
Cytometric detection of dead or injured
bacteria in situ using exclusion nucleic
acid stains
Cytometric detection of in situ bacteria
with depolarized membranes using the
Oxonol DiBAC
Cytometric detection of in situ actively
respiring bacteria using CTC
In situ hybridation visualized with epifluorescence
microscopy
RNA probing of bacterioplankton using
epifluorescence and cytometry
Figs. 1 y 2 from Heissenberger et al. (1996)
Intact cells
Damaged cells
Empty cells
% of bacterial community
60
50
40
30
20
10
0
1
2
3
4
Station in a gradient
5
6
From Hoppe (1976)
Autoradiography
3H-AA
3H-thymidine
3H-aspartic
14C-glucose
CFU
0
20
40
60
Percentage of total cells
80
100
Smith and del Giorgio 2003
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Bouvier et al. 2007
Bouvier et al. 2007
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Bouver et al. 2007
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Lebaron et al. 2001
River and coastal samples
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Longnecker et al. 2006
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
del Giorgio and
Gasol in press
All bacteria
Motile
Membrane+
FISH-EUB+
HNA
INT+
NucC
MAR+
Capsule
Direct viable count
Esterase activity
CTC+
0
20
40
60
80
% (average, SE, 25 and 75% quartiles and range)
100
Søndergaard and Danielsen 2001
The highly active “CTC” fraction is
seasonally much more dynamic than the
total bacterial abundance in lakes
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Enzyme
probes
esterases
Respiration
probes
+ - - Membrane polarization
- + +- + + Membrane
-permeability
ETSchain
DNA
Ribosomes
rRNA probes
NA probes
DNA Duplication
*
***
*
*
*
* Metabolism:
organic substrate incorporation
CO2, O 2 exchange...
The universe of DAPI-positive particles
High
activity
Medium
activity
Low activity
Dormancy
Death
Lysis No BT
TEM
PI (damage)
Dibac (depolarization)
CTC
Microautoradiography
DNA content
The regulation of the physiological structure of
bacterioplankton communities has three main components
• Environmental factors that influence the
individual level of metabolic activity and cell
integrity and damage, such as substrate and
nutrient availability, UV and temperature
• Physical and biological factors that influence the
persistence and loss of the various physiological
fractions, such as selective grazing and viral
infection, and selective degradation
• Intrinsic phylogenetic characteristics that
modulate the response of different bacterial strains
to the above factors
Example: Bacterial succession along the
transition between fresh and salt waters
• Does bacterial composition change abruptly
along a salinity gradient in an estuary?
• Is the compositional succession
accompanied by changes in the
physiological structure of the community
along this salinity gradient?
Bacterial composition
Upper
Fresh
Middle Salt
Lower Upper
FreshMiddle Lower
Salt
50
March
May
July
Sept
BETA
25
ALPHA
0
50
25
0
0
20 40 60 80 100 0
20 40 60 80 100
Distance downriver, Km
BP
12
Salinity (‰)
10
2.5
T urbidity
Maximum
8 Km
2
8
1.5
6
4
1
2
0.5
0
Bacterial production (µg C l
Salinity
-1
15
10
5
0
0
River sites----------Estuarine sites
h -1)
-2
20
Salinity
%CTC
12
T urbidity
Maximum
8 Km
15
8
6
10
4
2
5
0
-2
20
15
10
5
0
0
River sites----------Estuarine sites
%CTC + cells
Salinity (‰)
10
20
%Dibac +
12
40
10
35
30
8
25
6
20
4
15
2
10
0
-2
20
5
15
10
5
0
0
River sites----------Estuarine sites
% DiBac + cells
Salinity (‰)
Salinity
Salinity
%Dead
12
25
10
Salinity (‰)
8
6
15
4
10
2
5
0
-2
20
15
10
5
0
0
River sites----------Estuarine sites
%Dead cells (PI+)
20
%EUB +
12
Salinity (‰)
10
90
T urbidity
Maximum
8 Km
80
70
8
60
6
50
4
40
2
30
0
-2
20
20
15
10
5
0
10
River sites----------Estuarine sites
% cells detected with EUB probe
Salinity
Environmental stress influences the
physiological structure of bacterioplankton
• What about biological interactions, such as
grazing and viral infection
Viles and Sieracki 1992
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Fukuda et al. 1998
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Gonzalez et al. 1990
Flagellate and ciliate grazing is
strongly size-dependent.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
This had strong implications on
the influence of bacterial
structure on food web
interactions within the microbial
loop
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Hahn and Höfle 1999
Grazing influences the size
distribution within
individual bacterial taxa.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Great morphological
plasticity in bacteria
Gasol et al. (1995)
50
Total
Percent
40
Dapi +
30
Active
20
10
0
Relative grazing
efficiency
120
96
González et al. 1990
72
48
24
Chrzanowski & Simek. 1990
0
0.01
0.1
Size (µm3)
1
CTC +
Selective grazing of live and active cells by protists
In situ dyalysis bag experiments in the Mediterranean Sea to follow
the dynamics of active and inactive cells in the presence and
absence of protistan grazing showed selective grazing and
significant cell inactivation
-0.43
Black box approach
0.87
1.09
0.08
-0.77
Using single-cell
measurements
0.69
A
I
0.06
0.81
0.24
0.44
0.86
-0.19
-0.19
del Giorgio et al. 1996
A
I
Lake Microcosm Experiments:
(with David Bird, Rox Maranger and Yves Prairie, UQÀM)
• Water samples were filtered through 0.8 µm
(to remove grazers), or unfiltered
• Water samples were incubated in dialysis
bags in situ in Lac Cromwell (Québec)
• Three UV/light treatments
• We followed thee abundance of highly
active cells (CTC+) and injured/dead cells
(TOPRO+)
How do environmental and
biological factors interact to
shape the physiological structure
of bacterioplankton?
Experimental design
Unfiltered water
Filtered water (0.8 µm)
Lake surface
Surface
Plexiglass
5 cm depth
Deep
1.5 m depth
Reducing protozoan grazers resulted in higher
proportions of CTC+ cells. The grazing effect may
be related to size-selective removal
40
Proportion of active cells
35
No Grazers
30
25
Grazers
Grazers
20
15
10
5
0
Deep Filt
Deep Unfilt
Lake
There was an interaction between grazing and light
(or UV) that affected the proportion of CTC+ cells.
40
No grazers
Percent active bacteria
35
30
25
No grazers
No grazers
20
Grazers
15
10
5
0
Surface Filt Plexi Filt
Deep Filt
Lake
Percent dead / injured bacteria
The proportion of cells that took up the exclusion
strain TOPRO increased with UV exposure
15
PAR
80% UVA
70% UVB
PAR
30% UVA
0% UVB
PAR
0% UVB
0% UBA
10
5
0
Surface Filt Plexi Filt
Deep Filt
W
Lake
Maranger et al. 2001
% TOPRO+ and CTC+ cells
There is an inverse pattern of CTC+ and
TOPRO+ cells in relation to UV/light
exposure
30
25
CTC%
TOP%
20
15
10
5
0
SURF
S+P
DEEP
Treatment
LAKE
Some conclusions regarding the
link between grazing and
bacterioplankton activity (I)
• Grazing and UV radiation both affect the
physiological structure of bacterioplankton
• Grazing is highly selective and preferentially
removes active cells
• Active cells appear to be on average larger than
less active or dormant cells
• Grazing selectivity may be based on size
Some general ecological patterns
in microbial (II)
• In aquatic microbial communities, small size and
low activity represent a refuge against predation
and perhaps viral infection
• Large cells must find alternative refuges:
attachment, parasitism, chemical defenses
• In other types of communities it is often the the
small and the weak that are selectively removed
• General allometric rules, i.e. size versus specific
activity, do not necessarily apply to aquatic
microbial communities
Are there links between singlecell activity and the phylogenetic
affiliation of bacterial cells?
Single-cell analyses that link composition with
activity and function
• Hibridization (FISH) and in situ reverse trabscription (ISRT)
16S rRNA & mRNA
Chen et al. 1997
• In situ hibridization and microautoradiography (MAR-FISH)
16S rRNA & 3H-TdR
Lee et al. 1999
Cottrell & Kirchman 2000
• Activity probes, cytometry cell sorting and molecular analyses
CTC, FACS, DGGE
Bernard et al. 2000
Zubkov et al. 2001
• In situ hybridization and DNA synthesis
16S rRNA & BrdU
Pernthaler et al 2002
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Zubkov et al. 2001
Celtic Sea
Does the active fraction (CTC+) have the same composition
than the inactive fraction ?
Bernard et al. 2000
Urbach et al. 1999
Used BromodeoxyUridine
(BrdU), an analog of thymidine,
to detect growing cells
Cells incorporating BrdU can be
detected using
immunofluorescence
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Linking growth to phylogeny: BrdUincorporation
•
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Hamasaki et al. 2004
Found that the BrdUincorporating (growing)
communities were
substantially different
from the total
communities
• This suggests that the
numerically dominant
groups are not
necessarily those that
are the most active
Hamasaki et al. 2007
Cottrel and Kirchman 2003
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Showed that the
contribution of the
major groups to Tdr
and Leu assimilation
varied greatly along a
salinity gradient
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Also showed that
some groups
contribute
disproportionately to
total bacterial activity
Leucine
Mixed aminoacids
Glucose
Protein
Thymidine
Bacterial subgroup
alfaproteobacteria
Roseobacter
SAR11
Bacteroidetes
gammaproteobacteria
0
20
40
60
80
% incorporating the substrate
del Giorgio and Gasol in press
What is the link between single-cell activity
and phylogenetic affiliation?
•
MAR-FISH analysis analyses show that in most cases there is a mixture of
cells that are active and inactive in substrate uptake within any given bacterial
group, suggesting that the level of single-cell activity is not intrinsic but rather
that members of the same group may express very different levels of activity
depending on their microenvironment and of their immediate history
•
This scenario would further suggest that resource microheterogeneity may play
a key role in determining the distribution of activity within bacterial
assemblages
•
Alternatively, the heterogeneity of single-cell activity detected within broad
phylogenetic groups may indicate that within these groups there is a wide
range of genetic diversity, that is expressed as a wide range in metabolic
responses of different cells to the same set of environmental conditions
•
This establishes two extreme scenarios, i.e. the physiological structure entirely
due to environmental heterogeneity, microscale patchiness and temporal
variability, versus physiological heterogeneity due entirely to
genetic/phenotypic diversity. Where along this gradient lie natural
bacterioplankton assemblages is still a matter of study
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Azam 1998
Microscale variability in coastal bacterial community structure
Seymour et al. 2004
Total BA
% HDNA
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
LDNA
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
D1 group
Some general ecological
conclusions from these examples:
• There are intense bacterioplankton phylogenetic
successions along environmental gradients,
associated to physiological stress and possibly cell
mortality
• Predation is a major structuring factor in microbial
communities, but predator-prey interactions may
be distinct in microbial systems
• Some general ecological notions, such as
allometric relationships, refuge and succession
theory, may not effectively describe the microbial
world
Ducklow 2001
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Variability in specific BP (BP / BA) and BR (BR / BA) in marine waters
del Giorgio and Gasol in press
Variability in abundance of microbial components
Region
HNLC Equator
Parameter
Mean
S.D.
C.V.
HBACT
PRO
SYN
PEUK
716,000
126,000
18%
145,000
38,000
26%
9,800
3,400
35%
6,300
1,800
28%
172,000
72,000
42%
2,300
2,600
113%
870
450
51%
183,000
45,000
25%
1,700
1,100
65%
720
360
50%
Western Equator Mean
S.D.
C.V.
HOT
Mean
S.D.
C.V.
444,000
119,000
27%
Landry and Kirchman 2002
We know that there is an upper limit
to bacterial growth rate, but how
slowly can a bacterial cell grow?
• There are thermodynamic constraints that
determine both the upper and lower limits
of cell growth
• Slow growth still requires the operation of
tranport systems, the maintenance of cell
membranes, and the turnover of proteins
and nucleic acids.
Spec substrate consumption
Microbial bioenergetics: maintenance
versus growth
}
}
Death
Dormancy
e (µ)
m (µ)
}
me (µ = 0.0 h-1)
Growth rate µ (h-1)
del Giorgio and Gasol in press
B
Homogeneous
All cells have equal growth
rates and growth yield
P
R
Bulk BGE
Heterogeneous
Low growth,
Low yield
cells
P
R
High growth,
High yield
cells
P
Bulk BGE
R
del Giorgio and Gasol in press
What about other components of the microbial
food web: The coupling between protist
predators and their bacterial prey
• There is evidence that protist grazing may
profoundly affect the physiological (and
taxonomic) structure of bacterioplankton
• But does the distribution of single cell
activity affect protist activity?
Bacteria
High-DNA
Low-DNA
Heterotrofic flagellates
HNF abundance (cells ml -1)
A
1.2 107
1 107
1.5 104
8 106
1 104
6 106
4 106
5000
2 106
0
0
20
40
60
80
100
0
120
Bacterial abundance (cells ml -1)
2 104
Percent High-DNA or CTC+ cells
100
10
%High-DNA
%CTC+
10
100
1000
104
105
Heterotrophic flagellates (ml -1)
h -1)
-1
Total enzyme activity (nmol l
b-GAM
Beta-glucosaminidase activity
10
TEA = 0.005 x HNF
r2 = 0.18
0.52
1
0.1
0.01
0.001
10
100
1000
104
105
Heterotrofic flagellates (cells ml
-1
)
-1 -1
Specific enzyme (nmol HNF h )
SE = 5.84 x CTC 1.68
r2 = 0.94
0.001
0.0001
10-5
10-6
1
10
%CTC+ bacteria
100
Feedback at the population level
Protist biomass
Protist grazing
Feedback at the
cellular level
Bacterial
Biomass/
production
?
Protist single
cell activity
?
Bacterioplankton
structure
Some patterns concerning protistbacteria interactions:
• Microbial predators can respond to prey
fluctuations at the population level, like predators
in other types of systems
• But microbial predators can also respond at the
level of cellular metabolism
• This response is much faster and allows microbial
predator-prey systems to be more tightly coupled
than any other system
• This tight coupling provides overall stability to the
ecosystem
300 l-1
Zooplankton
2000 l-1
Ciliates
Microphyto100 ml-1
Flagellates
Nanophyto1000 ml-1
107 ml-1
?
Picoplancton
103 ml-1
?
?
An important aspect of the functioning of
bacterial communities is social behavior
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Ducklow 2001
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
14
Size does matter!
Smaller organisms have higher surface area (SA) to
volume (V) ratios. Consider a spherical microbe:
SA= 4r2
V= 4/3 r3
So, SA:V = 4r2/4/3 r3 ~ 1/r
That is, as organisms get bigger, SA:V gets smaller
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Approaches
Bacterial physiological parameters
intact membrane
Potentiel Membrane
Intégrité Membrane
PI (Live/Dead Baclight)
damaged membrane
intact membrane
DiOC6(3)
ADN
damaged membrane
DiBAC4(3)
Respiration
ETS
Content: SYTO13
ADN
3
Turn
Turnover:
over:HH3Thymidine
Thymidineuptake
uptake
Protéines
ATP
Protein
Turn over: H3 Leucine uptake
CTC
Firefly Luciferase
Bioluminescence
enzyme
Enzymatic
Activity
Luciferin
Substrate
Biolog
-Bulk metabolism
-Single cell activity