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