microbial ecology vl 7 marine habitats: past, current and future … · fatty acids, that were...
Post on 23-Jun-2020
6 Views
Preview:
TRANSCRIPT
1
Microbial ecology
VL 7 Marine habitats:
Past, current and future research
Bert Engelen engelen@icbm.de
www.icbm.de/pmbio
Marine Microbiology
View on the Pacific ocean
App. 71% of the Earth´s surfaceis covered by oceans
Mean depth. app. 4000 meters
All oceans are connected
General circulation patterns: Equatorial- and coastal currents(upwelling, outwelling)
Coastal zones are governed by tides
The sea is salty: app. 2.7% NaCl and 0,8% other ions (SO4
--, Mg+, Ca, K)
Dissolved nutrients are limited (Oligotrophy)
Ecological features of the ocean:
© Bert Engelen
2
Zonation of the ocean
verändert nach E. P. Odum, 1983
Where can we find marine microorganisms?
Global
Higher numbers in shallow areas due to:
Terrigenic impact
Ratio productive vs. konsumptive volume
Inflow of limiting factors (Fe, N, P) from sediments
Vertical
Maxima in:
The euphotic Zone (dependant on site < 50 m, < 200 m)
At interphases (halocline, thermocline, chemokline, sediment surface)
Small scale
Free living vs. particulate (Detritus, marine snow)
3
Distribution of chlorophyll in the ocean
Distribution of chlorophyll in the ocean
4
Shelf vs. Abyss
Primary production
Secundary production
Sediment deposition
Photic zone
Transfer of POC from the photic zone into the sediment
Open ocean Coastal zone
Deposition at the seafloor
[g C m-2 a-1]30 120
3 30Exportproduction
0,3 8Bruttodeposition
Nettodeposition 0,01 1
Tekt
onik
, Ver
witt
erun
g
Sedimenting POC
Deg
rada
tion,
resp
iratio
n
5
The Namibian Upwelling Area
Transect
Dep
th[m
bsf]
Total cell counts [cm-3]
0
1
2
3
4
5
6
12802 water depth 79012803 water depth 1940
12806 water depth 13012807 water depth 300
12808 water depth 3800
104 105 106 107 108 109 1010
MUCGC
6
High organic load supports growth of giants
Beggiatoa filaments
Thiomargarita namibiensis
7
Phase contrast SybrGreen staining
Thiomargarita namibiensis and Beggiatoa
Phages in the food web
8
Viruses
viralloop
What is the role of bacteria in the marine foodweb?
Consumers(animals)
Destruents(bacteria)
Producer(plants)
too simple?!
bacterialloopDetritus
Grazer(Ciliates, Flagellates)
Mesozooplankton
Detritus Detritus
Detritus
Light
Phytoplankton Herbivores Carnivores
Disolved- and particulate organic substances
(DOM, POM)
Bacterioplankton
Nutrients(P, N, Fe)
modified after Cypionka, 1999
Size classes of microbes in SeawaterMesoplankton 0.2 - 20 mm
Microplankton 20 - 200 µm
Nanoplankton 2 - 20 µm
Picoplankton 0.2 - 2 µm
Femtoplankton 0.02 - 0.2 µm
BacterioplanktonBacteria size: 0.03 - 0.4 µm
The most important groups:
PhytoplanktonDiatoms DinoflagellatesMicroflagellatesCyanobacteria
ZooplanktonHeterotrophic nanoflagellatesCopepodes
What are the dominating organisms the ecosystem?
9
How many microbes are there?
Depth (m) (Golfstream and Sargasso sea)0 - 50 6050 - 100 40100 - 200 25200 - 500 15500 - 1000 52000 - 5000 <1
Phytoplankton volumes in cm3/1000 m3
What is limiting their number?Absence of important growth factors or lightLoss via grazing, lysis or sedimentation
Viruses10 - 100 * NBacteria
Bacterial numbers/mlEstuaries 106- 107
Schelf arear 1- 3 * 106
Open ocean 104 -106
Usually: Filtration techniques to determine viral abundance
Problem: Direct counting in sediments difficult due to small size and particle background
Solution: Growth experiments on isolates via induction of prophages
How to Analyze Marine Phages ?
10
Viral Infections as Controlling Factorsfor the Deep Biosphere ?
100 nm
Photo: Phages from Rhodobacter capsulatus strain E32ODP Site 1230, sediment depth: 268 mbsf
Lytic and Lysogenic Life Modes
Viral particles
10µm
Release of cell compoundsShown: free DNA
Prophages in ~70% of all bacterial genomes detected(Canchaya et al. 2003)
11
addition of mitomycin C
incubation
washing steps
OD
600
0
0.5
1
1.5
2
0 5 10 15 20 25
Time [h]
Control
Mitomycin C
addition of mitomycin C
incubation
washing steps
OD
600
0
0.5
1
1.5
2
0 5 10 15 20 25
Time [h]
Control
Mitomycin C
Phage Induction Experiments
DNA damage via the antibiotics "Mitomycin C" induces the assembly of phages
Control Mitomycin C
Selection of Deep-Biosphere Isolates
13 out of 162 isolates from ODP Leg 201 tested
Rhizobium radiobactermost frequently isolated (40 strains)
Same amount from the Eastern Mediterranean(Süß et al., 2004)
Batzke et al., 2007
12
P. glucanolyticus P073ODP Site 1225, 198 mbsf
0 5 10 15 20
Time [h]
0
1
0.5
0 5 10 15 20
Time [h]
0
1
0.5
0
1
0.5
OD
OD
600
V. diazotrophicus P082ODP Site 1230, 1 mbsf
0
1
0.5
0
1
0.5
Time [h]
0 5 10 15 200
1
0.5V. diazotrophicus R6ODP Site 1230, 320 mbsf
Time [h]
0 5 10 15 200
1
0.5
0
1
0.5
ControlControl
Mitomycin C
Induction of Prophages in SelectedDeep-Biosphere Isolates
R. radiobacter P007ODP Site 1225, 198 mbsf
OD
600
0
1
2
OD
600
0
1
2
Rhv. sulfidophilum P122AODP Site 1231, 43 mbsf
0
0.3
0.6
20 30 40 50 60
Time [h]
OD
600
Rhb. capsulatus E32ODP Site 1230, 268 mbsf
20 30 40 50 60Time [h]
0
0.3
0.6
20 30 40 50 60
Time [h]
OD
600
20 30 40 50 60Time [h]
Control
Mitomycin C
ControlControl
Induction of Prophages in SlowGrowing Deep-Biosphere Isolates
13
Selection of Deep-Biosphere Isolates
6 out of 13 isolates from ODP Leg 201 positive
All phages nominated for sequencing by the Gordon & Betty Moore Foundation
Batzke et al., 2007
Open Questions to be Answered
What is the extent of viral infections within the deep biosphere?deep-biosphere specific viruses ? inter- and intra-species diversity ?
Are representative isolates from deep sediment-layers infected by the same viruses as their relatives from other habitats ?
correlation of phage/host biogeography ?
What can viral infections tell us about the physiological state of indigenous microorganisms?
prophages inducible from starving host cells ?
How important is the viral shunt for the deep biosphere ?essential nutrients for deep-biosphere populations ?
14
Anoxic basins and paleoclimate
15
The principle of the estuarine circulation in the Baltic
Saltwater is pressed via autum storms into the BalticSubduction of fresh and brackish water bodies
(aus Rheinheimer 1995)
Inflow of saltwater into the Gotland basin=> visible by exchanging geochemical profiles of deep anoxic waters
before after
16
0 10 20 30 40 50H S2
(µmol liter -1 )
Oxygen (ml liter -1 )
Temperature (°C)
Salinity (%)
0
50
100
150
200
250
Dep
th(m
)
Temperature
Oxygen
H 2S
0 0.5 1 1.5 2
Bacterial abundance
(10 6 ml -1 )
Bacterial Production
(pmol Leu L -1 h -1 )
Salinity
RNA concentration
( µg l -1 )
Production
AbundanceDNA
RNA
0 0.5 1 1.5 2
DNA concentration
( µg l -1 )
0 5 10 15 0 1 2 3 0 0.5 1 1.5 2
Sept.17th-18th. 1998
Fingerprinting bacterial communities of the Gotland deep
G7
G9G11G8
Ajax
LL12T4
G4
G6G5
G2Sr5G1
T6T7
T5
G10
T1T2
G3
Station Teilideep samples
( 90-140m)
Surface(5-30m)
anoxic(138-225m)
T3 oxic(80-120m)
Winter water
40 60 80 10020
Similarity (%)
138m
175m200m150m
5m
90m
80m
120m110m
30m
5m
125m140m
105m
225m
5m30m
50m
80m
5m
5m
Gotland deep
TGGE fingerprints of microbial communities within the central Baltic
Gotland deep TeiliSurface
5 2255 30 50 80 110120138 150175200 5 30 80 90 1051251405 5 Depth [m]
+
-32°C
44°C
Δ T 12°C
Gra
dien
t
StSt
Winter waterSurface Oxic zone
Anoxic zoneSurface Oxic zone
17
Eastern Mediterranean:Paleo climate and current situation
Theoretical background:
Querschnitt durch das MittelmeerDeep water formation
18
Climate change led to Sapropel formationvia differences in circulation patterns
⇒ Warm climate
⇒ Higher inflow of fresh water
⇒ Rise of the halocline
⇒ Reverse current patterns
⇒ Enhanced primary production
⇒ Formation of anoxic basins
Sapropel formation
19
Top Bottom
Gravity core with Sapropels S1, S3, S4 und S5 (85 cm)
Meteor cruise 51/3
11.11.2001 – 10.12.2001
20
15 352520 3010
40
30
35
45
583
562563
567
569571
573576577
Longitude (°E)
Latit
ude
(°N
)
592599
575
Malta
Istanbul
Sampling sites
0 1 2 3 4 5 6 7
Dep
th (c
m)
0
100200
300
400
Total cell count (107 cm3)0 1 2
S1
S3
S4
S5
Station 567-1
/ / / / / /
ATP concentrations ( ) und TCC ( ) at the sediment surface, in Sapropels und Corg-lead intermediate layers
Microbial abundances (Sediment)
21
MUC / GC
8 mbsfSEDIMENT
MUCOXICseafloor
ANOXICseafloor
EemianSapropel
CTD
MUC
ANOXIC
OXIC
INTERFACE
WATER
Two depthprofiles
Two transectsMUC / GC
8 mbsfSEDIMENT
MUCOXICseafloor
ANOXICseafloor
EemianSapropel
CTD
MUC
ANOXIC
OXIC
INTERFACE
WATER
Two depthprofiles
Two transects
The Black Sea
The four model organisms
Roseobacter species
Rhizobium andPhotobacterium species
Chloroflexi
Hypotheses:
enter sediments only by sedimentationare buried, may survive for some timewill not thrive in deeper and older layers
are able to adapt and grow in the deep subsurfacemight be opportunistic subsurface bacteria
true deep-biosphere bacteria
22
• How far is the subsurface dominated by surface bacteria that survived?Which role have groups hat are specifically adapted, and are more relevant for geochemical processes?
• Are anoxic conditions sufficient to promote growth of our model organisms? What is the influence of the oxic-anoxic transition on their abundance?Effects of physicochemical settings (pressure, sedimentation rates, TOC)?
• Are model groups specific for different habitats?What are the differences and specific adaptations of sediment inhabitants? Or, are the same types present everywhere?
Questions
Work schedule
• Distribution of model organisms from different compartments of the water column and the sediments
CARD-FISH Quantitative PCR
• Tracing the model organismsin enrichment cultures
Group specific PCR
• New enrichments for Chloroflexi Unusual culture media:e.g. halogenated compounds
• Molecular identification and characterization of accompanied communities
• Molecular and physiological characterization of novel ecotypes of the model organisms
• Detailed analysis of the Eemian sapropel
Isolation of model organismsPhylogeny below species level (ERIC)Adaptation to: pressure, anoxia, TOC
DGGE of enrichmentsIsolation of interesting strains
High resolution screening
23
Time frame
Year 1 Year 2 Year 3Adaptation of the detection systems to environmental samples PhD thesis
Specific detection of the model organisms in the environment PhD thesis
Molecular and physiological characterization of novel isolates PhD thesis
Isolation of model organisms and other community members PhD thesis
Detailed analysis of the Eemian sapropel 2. Master thesis
Attempts to isolate subsurface members of the Chloroflexi 1. Master thesis
Screening of enrichment cultures for the model organisms 2 Research projects
Identification of enriched accompanied microbial communities 1 Research project
The oceanic crustand pressure adaptations
24
Pressure
... 1 bar pressure rise per 10 m; at 1000 m, pressure is a 100 times higher
Bacteria do not have a Schwimmblase.
Are bacteria pressure sensitive?
Experiment: Bring a balloon to a water depth of 1000 m
? ... or with water
?... filled with air °O (1 %)
O O (almost 100 %)
However...
high pressures have an influence on:- Boiling point and viscosity of water
- Membrane fluidity
- Stability of certain biomolecules
Barophilic microorganisms are adapted to high pressures e.g. higher amount of unsaturated fatty acids within their membrane, or modifed enzymes
25
Fatty acids, that were found in bacterial lipids
saturated
iso-branching
ante iso-branchingunsaturated
alicyclic
Glycerol diether
Diglycerol tetraether
Rule of thumb: The higher the pressure,the more fluent is the membrane.High content of unsaturated fatty acids.
Fluids from the ocean crust support life in the deep biosphereBert Engelen, Katja Ziegelmüller, Jörn Logemann and Heribert Cypionka
ICBM, University of Oldenburg, Germany
26
Extension: largest aquifer on earth, amount equivalent to ice coverage
Annual flow: as big as the fluvial input into the oceans
Hydrothermal fluids in the upper basaltic crust
Motor: thermal, tides, seismic and tectonic events,topography of the upper crust
Speciality: oxidised compounds SO42-, NO3
- (O2) still present
potential electron acceptorsfor microbial respiration
Crustal fluids might fuel the deep biospherein marine sediments from belowHypothesis
SeafloorOrganic matter
Anaerobicmicrobial
degradation
SO42- reduction
NO3- reduction
Mn(IV) reduction
AOMMethanogenesis
Fe(III) reduction
Diffusion
O2 respiration
O2, NO3-, Mn(IV), Fe(III), SO4
2-
Bottom seawater
Ocean crustDiffusion
NO3-, Mn(IV), Fe(III), SO4
2-
Volatiles (H2, CO2, CH4)Crustal fluids
Fe(III) reduction
SO42- reductionAOM
Mn(IV) reduction
NO3- reduction
‚Upside-down‘ redox profile
(DeLong 2004)
Is microbial life in deep subsurface sedimentsstimulated by fluids from the ocean crust?
27
http
://w
ww
.ngd
c.no
aa.g
ov
Crustal fluids might fuel deep-biosphere populations on a global scale
IODP Exp. 301 to theEastern flank of theJuan de Fuca Ridge
Site 1301PacificPlate
Juan de FucaPlateJu
an d
eFu
caRi
dge
Leg 168
PacificPlate
Juan de FucaPlateJu
an d
eFu
caRi
dge
Leg 168
Vancouver Island
U 1301
Dep
th[m
bsf]
500
0
250Sediment
Grizzly Bareoutcrop
Baby Bare Mama BareSeafloor
2650 mWater column
~3.5 Ma Ocean crust
IODPSite U1301
Intense microbiological studies* Huber et al., 2006** Cowen et al., 2003; Nakagawa et al., 2006
* ODPSite 1026**
Diffusion
Diffusion
electron donors & acceptors
Fluid circulation at IODP Site U1301
52 kmSouth North14 km
SeawaterRecharge
~2°C
Alteration~60°C
Discharge Discharge
~20°C
28
Sulfate reduction rates(pmol·cm-3·d-1)
AOM rates(pmol·cm-3·d-1)
0 1 2 3 4 5 23 7600
SRR
AOM rates
0 1 2 3 4 5 6 7
SG IAODC
4 5 6 7 8 9
Log10 cell counts(cm-3)
4 5 6 7 8 9
ArchaeaBacteria
Log10 total cell counts(cm-3)
Dep
th[m
bsf]
0
25
100
175
200
225
250
275
50
75
125
150
300
Pore water sulfate(mM)
Methane(mmol·kg-1)
0 10 20 30
CH4
SO42-
0 2 4 6
Seafloor
Oceanic crust
Sediment
Depth profiles at IODP Site U1301
Dep
th[m
bsf]
0
25
100
175
200
225
250
275
50
75
125
150
300
MUF-P (mmol·cm-3·d-1)
Phosphate(µmol·kg-1)
Phosphatase activity
Site 1026 Site 1301
600 20 40
0 1 2 3 4 5
Engelen et al., 2008
Identified sulfate-reducing bacteria
Dep
th[m
bsf]
0
25
100
175
200
225
250
275
50
75
125
150
300
Pore watersulfate (mM)
Methane(mmol·kg-1)
0 10 20 30
0 2 4 6
Seafloor
Oceanic crust
Sediment Delta-ProteobacteriaDesulfovibrio aespoeensis*Desulfotignum balticumDesulfovibrio indonesiensis
Firstly isolated from thehard-rock laboratory(deep groundwater)
(Motamedi & Pedersen et al., IJSB 1998)
Desulfovibrio indonesiensisDesulfotignum balticum
3 strains 240-260 mbsf260 mbsf
FirmicutesDesulfotomaculum sp.
Desulfosporosinus sp.
Desulfosporosinus sp.
1.3 mbsf
4 strains 1-30 mbsf
Delta-ProteobacteriaDesulfovibrio aespoeensis
+1.3 mbsf + 2 strains 240 and 260 mbsf
29
Observations on ourmoderate thermophilic sulfate-reducing isolates
Isolation conditions: 20°C, 0.1 MPa
Growth range: 10 - 48 °C
In-situ conditions: 56-61°C, ~30 MPa
Question?
Will our strains grow at in-situ temperatureswhen we let them grow under in-situ pressure?
30
20 µm
D. indonesiensis 250-260 mbsf, lactate medium 45°C
Measuring of growth?
0.1 MPa 40 MPa
Pressure and temperature effect ongrowth of D. indonesiensis strains
Shift in temperature range: 0.1 MPa = 10 - 48 °C200 MPa = 15 - 52 °C
At 20°C: piezotolerant behaviour
Pressure: Decelerates growth (stress!)
20 °C
At 45°C: piezophilic behaviour
Pressure: Stimulates growth
45 °C
top related