Benthic bacteria in the German Bight: Characterising community structure and influencing environmental factors

(1)

‚Living along gradients‘

Doctoral defense Rebecca Störmer Hamburg, 18.01.2013 ¹

Benthic bacteria in the German Bight:

Characterising community structure and influencing environmental factors

or

(2)

Introduction Methods Results

Spatiotemporal variations of benthic bacterial communities in the German Bight

Impact of ocean dumping on benthic bacterial communities Conclusions

Future perspectives

Outline

(3)

Motivation: Ocean dumping

• deposition of waste at sea

• waste includes:

– liquid wastes (dilute acid, sludge) – construction waste

– dredged material (harbours, rivers)

3

geo.de

Introduction

climateforce.net presstv.ir fr.wikipedia.org

www.greenpeace.org

Threat for ecosystems

(4)

Motivation: Ocean dumping

4

Introduction

climateforce.net presstv.ir fr.wikipedia.org

www.greenpeace.org

Overview of the number and distribution of dumpsites within the OSPAR area

OSPAR 2009b

(5)

5

since 2005: ~ 6 mio m³

3 m high rising containing mainly sand

Introduction

fr.wikipedia.org

HPA Report 2010

sediment origin dumping site

(6)

6

Introduction

Motivation

• monitoring focuses on macrozoobenthic communities and flatfishes

•

“Progress on investigating biological responses to the disposal of dredged material has been slow in OSPAR and more effort is needed for a wider and more systematic application of bioassays in the testing of dredged sediments.“ (OSPAR 2009b)

• bacterial community structure affected by – physical disturbance (Findlay et al 1990) – heavy metal and oil contamination

(Gremion et al 2004, dos Santos et al 2011)

www.bgisequence.com

(7)

7

Introduction

Motivation

• benthic bacterial communities:

– largest variety of metabolic types

– heterotrophic, phototrophic and lithotrophic bacteria – highly abundant: 10⁸ - 10¹¹ cells per ml

www.bgisequence.com

Problem: sublittoral benthic bacterial communities uncharacterised in the German Bight

Are bacterial community analyses a useful supplement for monitoring programs at dumping sites?

(8)

Introduction

German Bight

• hydrographic regime pre- dominantely influenced by discharges of Elbe and Weser River

• most euthrophied region in the North Sea

• shallow part of the North Sea with max. dephts ~ 50 m

• Helgoland

Elbe Weser

Jade

(9)

Sediments in the German Bight

• Grain size fractions

– Clay particles < 4 µm – Silt particles 4 - 63 µm

– Fine sand particles 63 – 200 µm

– Medium sand particles 200 – 630 µm – Coarse sand particles 630 – 2000 µm – Gravel particles 2000 – 6000 µm

• Proportion of each grain size fraction determines the classification into sandy or muddy sediments

9

Introduction

(10)

Sediments in the German Bight

Introduction

www.bfn.de

(11)

noaa.gov

11

Introduction

Spatial gradients Temporal gradients

Jan Feb Mar Apr May Jun Oct Dec

Temperature (°C)

0 2 4 6 8 10 12 14 16 18

Environmental gradients

(12)

Characterising sublittoral benthic bacterial communities

I: Spatiotemporal variations influencing benthic bacterial communities in the German Bight

II: Impact of ocean dumping on benthic bacterial communities a) Community structure

b) Community function

Research aims

(13)

• Bacterial community analysis – ARISA fingerprinting

– 16S pyrosequencing – functional gene arrays

• Environmental data (additional data provided by Prof. Karen Wiltshire and HPA)

– CHN analysis

– particle size analysis

• Statistical analysis

– univariate and multivariate statistics – Geostatistics

13

Methods

Data analysis

(14)

Characterising sublittoral benthic bacterial communities

Research aims

(15)

Results

• monthly sampling from September 2010 to August 2011

• three replicates for bacterial community analysis (ARISA)

• environmental data includes:

– temperature – salinity

– chl a – CHN

– particle size of the sediments

15

I: Spatiotemporal variations influencing benthic bacterial communities

offshore transect nearshore transects

(16)

Results I: Spatiotemporal variations influencing benthic bacterial communities

Transect I Offshore Transect II Nearshore

P8 II P8 III P8 IV P8 V P8 VI

Fine sand (%)

0 20 40 60 80 100

Elbe II E3 Elbe IIIElbe IV Elbe V Elbe VI

Fine sand (%)

0 20 40 60 80 100

Stable environmental conditions offshore vs

variable environmental conditions nearshore

(17)

17

(18)

Results

Fingerprinting via Automated Ribosomal Intergenic Spacer Analysis (ARISA)

Ribosomal Operon of Bacteria

ITS

16S 23S

Infrared labeled tag

Conversion of community profiles into numeric data

Community profiles

(19)

Variable Pseudo-F P

Proportion of

variance Variable Pseudo-F P

Proportion of variance

Coarse gravel 0 1 0.000 Temperature 47,382 0.0001 0.114

Medium gravel 0 1 0.000 Fine sand 30,272 0.0001 0.069

Fine gravel 0 1 0.000 Chlorophyll a 20,515 0.0016 0.045

Coarse sand 15,685 0.0425 0.041 Salinity 16,793 0.0132 0.036

Medium sand 2,362 0.0021 0.060 Hydrogen 13,296 0.1106 0.029

Fine sand 26,317 0.0007 0.066 Coarse silt 11,434 0.2703 0.024

Coarse silt 26,109 0.0012 0.066 Medium sand 12,879 0.1348 0.027

Medium silt 25,044 0.001 0.063 Coarse sand 10,608 0.3847 0.022

Fine silt 23,395 0.0024 0.060 Fine silt 0.92303 0.5777 0.020

Clay 22,599 0.0035 0.058 Medium silt 12,812 0.1628 0.027

Temperature 47,382 0.0001 0.114 Clay 10,797 0.3552 0.023

Salinity 24,043 0.0007 0.061 Nitrogen 0.77095 0.7846 0.016

Nitrogen 16,197 0.0468 0.042 TOC 0.67199 0.8882 0.014

TOC 17,895 0.0219 0.046

Hydrogen 19,452 0.0106 0.050 Chlorophyll a 38,587 0.0001 0.094

19

Results

I: Spatiotemporal variations influencing benthic bacterial communities Distance based multivariate multiple regression model (DISTLM)

p < 0.01

Marginal test Sequential test

(20)

Results

-40 -20 0 20 40 20

-40 -20 0 20 40

d b R D A 2 ( 1 9 . 9 % o f f i t t e d , 9 . 3 % o f t o t a l v a r i a t i o n )

Resemblance: S7 Jaccard

Site

Elbe II E3 Elbe III Elbe IV Elbe VI Elbe V

Temperature

Fine sand

Chl a

Salinity Hydrogen Coarse silt

Medium sand

Coarse sand Fine silt Medium silt

Clay Nitrogen

Carbon

Near estuary community

-40 -20 0 20 40

d b R D A 2 ( 1 9 . 9 % o f f i t t e d , 9 . 3 % o f t o t a l v a r i a t i o n )

Resemblance: S7 Jaccard

Month

April

December February January

June March May October

Temperature

Fine sand

Chl a

Salinity Hydrogen Coarse silt

Medium sand

Coarse sand Fine silt

Medium silt Clay

Nitrogen

Carbon

Early summer to autumn community

Winter to

spring community

Spatial

Nearshore bacterial communities exhibit strong spatiotemporal variations

Marine community

marine estuarine

distance-based Redundancy analysis

Temporal

(21)

Results

21

Elbe II E3 Elbe III

Elbe IV Elbe V

Elbe VI

ARISA OTUs

0 20 40 60 80

January February

Ma rch

April Ma

y June

October Decemb

er

ARISA OTUs

0 20 40 60 80

Spatial Temporal

* * p < 0.05

Diversity of bacterial communities in nearshore habitats affected by temporal variations Distribution of operational taxonomic units (OTUs)

OTUs defined according to ITS fragment lenghts

(22)

Main findings

• amplitude of environmental gradients determines the bacterial community structure

• temporal variations affect bacterial community structure and diversity

(23)

Characterising sublittoral benthic bacterial communities

23

Research aims

(24)

Results II: Impact of ocean dumping on benthic bacterial communities

(25)

Results

25

2 months

9 months 2 months 4 months

08/08 - 10/08 08/09 12/09 – 02/10 04/10 08/10

II: Impact of ocean dumping on benthic bacterial communities Timeline sampling campaigns

Dumping campaigns Sampling campaigns

(26)

Results

• sampling campaigns in August 2009 and April and August 2010

• each campaign comprises 125 sampling sites

• three replicates for bacterial community analysis (ARISA)

II: Impact of ocean dumping on benthic bacterial communities

Grain size fractions Sum Hexachlorocyclohexane (HCH)

< 20µm alphaHCH

20-63µm betaHCH

63-100µm gammaHCH

100-200µm deltaHCH

200-630µm

630-1000µm Sum Dichlorodiphenyldichloroethane (DDT) and metabolites

1000-2000µm ppDDE

opDDD

S, N, P, C ppDDD

TOC (C) opDDT

nitrogen (N) ppDDT

sulphur (S)

phosphor (P) Sum Organotin Compounds

monobutyltin (MBT)

Hydrocarbons dibutyltin (DBT)

tributyltin (TBT) Sum Polycyclic Aromatic Hydrocarbons (PAH) tetrabutyltin naphthaline

fluorene Heavy Metals

phenanthrene arsenic

anthracene lead

fluoranthene cadmium

pyrene chrome

benz(a)anthracene copper

chrysene nickel

benzo(b)fluoranthene mercury

benzo(k)fluoranthene zinc

benzo(a)pyrene dibenz(ah)anthracene benzo(ghi)perylene indeno(1.2.3cd)pyrene

Sum Chlorinated Diphenyls (PCB) PCB28

PCB52 PCB101 PCB118 PCB138 PCB153 PCB180

Contextual data

(27)

Results

27

Grain size fractions Sum Hexachlorocyclohexane (HCH)

< 20µm alphaHCH

20-63µm betaHCH

63-100µm gammaHCH

100-200µm deltaHCH

200-630µm

630-1000µm Sum Dichlorodiphenyldichloroethane (DDT) and metabolites

1000-2000µm ppDDE

opDDD

S, N, P, C ppDDD

TOC (C) opDDT

nitrogen (N) ppDDT

sulphur (S)

phosphor (P) Sum Organotin Compounds

monobutyltin (MBT)

Hydrocarbons dibutyltin (DBT)

tributyltin (TBT) Sum Polycyclic Aromatic Hydrocarbons (PAH) tetrabutyltin naphthaline

fluorene Heavy Metals

phenanthrene arsenic

anthracene lead

fluoranthene cadmium

pyrene chrome

benz(a)anthracene copper

chrysene nickel

benzo(b)fluoranthene mercury

benzo(k)fluoranthene zinc

benzo(a)pyrene dibenz(ah)anthracene benzo(ghi)perylene indeno(1.2.3cd)pyrene

Sum Chlorinated Diphenyls (PCB) PCB28

PCB52 PCB101 PCB118 PCB138 PCB153 PCB180

Contextual data

(28)

Results

-0.8 0.8 28

-0.60.8

20 – 63 µm TOC 100 – 200 µm

200 – 630 µm 630 - 1000 µm

1000 – 2000 µm

nitrogen sulfur

phosphor hydrocarbons Sum PAH

PCBSum Sum HCH

Sum DDX

Sum organotin compounds

cadmium

chrome copper

mercury

II: Impact of ocean dumping on benthic bacterial communities Biplot Redundancy analysis August 2009

Dumping site Surrounding Transects

Reference

10.1 % of variance

7 % of variance

Bacterial communities at the dumping site significantly affected by fine sand (100 – 200 µm) and organic pollutants

(29)

Results

29

1.5km (surrounding1) dumping centre

3km_2 (surrounding 5) 3km_1 (surrounding 4)

2km_ (surrounding 3)

2km_1 (surrounding 2)

reference 1 reference 2

• based on significant differences (analysis of similarities) in the communitity structure nine representative samples were selected

• via pyrosequencing the V1-V5 region of the 16S rDNA was sequenced

(30)

Elbe

dum ping

centre sur

rounding 1

sur

rounding 2

sur

rounding 3

sur

rounding 4

sur

rounding 5

referenc e 1

referenc e 2

No. 454 OTUs

0 500 1000 1500 2000 2500

Results

30

Singletons (n=1) were removed prior to the analysis OTUs = sequence similarity > 97 %

Distribution of operational taxonomic units (OTUs)

lowest diversity was observed in Elbe and at dumping centre

(31)

Results

31

Elbe

dum ping

centre sur

rounding 1

sur rounding

2

3

4

5

referenc e 1

referenc e 2

No. sequences

0 100 200 300 400 500

Flavobacteriaceae

Flavobacteriales unclassified

singletons (n=1) were removed prior to the analysis

Flavobacteriales

considerable higher abundance of Flavobacteriaceae at the dumping centre indication for organic pollution?

(32)

Results

Elbe

dum ping

centre sur

rounding 1

sur

rounding 2

sur

rounding 3

sur

rounding 4

sur

rounding 5

referenc e 1 referenc

e 2

No. sequences

0 200 400 600 800 1000 1200 1400

Alcaligenaceae

Betaproteobacteria unclassified Burkholderiales unclassified Burkholderiales_incertae_sedis Comamonadaceae

Hydrogenophilaceae Methylophilaceae Rhodocyclaceae

Betaproteobacteria

typical freshwater groups still detectable nine months after dumping activity

(33)

Results

33

Elbe

dum ping

centre sur

rounding 1

2

3

4

5

referenc e 1

referenc e 2

No. sequences

0 500 1000 1500 2000 2500 3000

3500 Cystobacteraceae

Deltaproteobacteria unclassified Desulfobacteraceae

Desulfobacterales unclassified Desulfobulbaceae

Desulfuromonadaceae

Desulfuromonadales unclassified Geobacteraceae

Myxococcales unclassified Polyangiaceae

Syntrophaceae

Syntrophobacteraceae

Syntrophobacterales unclassified Syntrophorhabdaceae

II: Impact of ocean dumping on benthic bacterial communities Deltaproteobacteria

considerable higher numbers of Desulfuromonadaceaea and lower numbers of Desulfurobacteraceae

indication for organic pollution?

(34)

Characterising sublittoral benthic bacterial communities

Research aims

(35)

Results

35

1.5km (surrounding1) dumping centre

3km_2 (surrounding 5) 3km_1 (surrounding 4)

2km_ (surrounding 3)

2km_1 (surrounding 2)

reference 1 reference 2

(36)

Results

• GeoChip 4.2 (functional gene array) (He et al 2007, Lu et al 2012)

• contains 103 666 probes encoding for functional genes involved in biogeochemical key processes

• genes are categorised according to these processes e.g. sulphur cycling, heavy metal resistance, organic remediation

GeoChip analysis

(37)

Results

37

Elb e a

Elb e b

Elbe c

dumping centre a dumping center b

dumping centre c 1.5 km a

1.5 km b 1.5 km c

2 km_1 a 2 km_1 b

2 km_1 c 2 km_2 a

2 km_2 b 2 km_2 c

3 km_1 a 3 km_1 b

3 km_1 c 3 km_2 a

3 km_2 b 3 km_2 c

referenc e_1 a referenc

e_1 b referenc

e_1 c referenc

e_2 a referenc

e_2 b referenc

e_2 c

% of total genes

0 2 4 6 8 10 12 14 16 18 20

Distribution of functional genes of the gene category „organic remediation“

p < 0.05

*

significantly lower functional diversity at the dumping centre as compared with the reference sites

(38)

Results

a c b d 38

I II

III

genes

Hierachical clustering based on Euclidean distance for the gene category „organic remediation“

Elbe

Dumping site

Reference

samples

(39)

Results

Main findings

• similar results for all gene categories

• no accumulation of genes involved in pollution related processes at the dumping centre detectable

• significant lower functional diversity at the dumping centre

• differences among detected gene groups based on phylogenetic background

39

(40)

Conclusions

• most pronounced environmental gradients affect the bacterial community structure significantly

• bacterial community structure at the dumping site was significantly influenced by the dumping activity

– lower alpha and functional diversity

– mix-community containing fresh water and adapted marine bacteria

• bacterial community analysis represent a useful supplement for monitoring programs

• But: further elaboration is needed!!

Mainconclusions

(41)

Future perspectives

• deepening the knowledge about benthic bacterial communities in the German Bight

– identifiying community composition and function

„Metagenomic approaches“

– simultaneous investigation of pelagic and benthic bacteria

„Benthopelagic coupling“

• adaptation of monitoring conditions and experimental set up for the inclusion of bacterial community analysis

– controlled experiments focusing on the impact of relevant pollutants on the bacterial communities

– identifying indicator organisms

– inclusion of physicochemical parameters such as pH, oxygen penetration, bioavailability of pollutants

41

(42)

Acknowledgement

This research was only possible with the help of all of you!!!

Many thanks to:

Dr. Antje Wichels and Dr. Gunnar Gerdts My committee:

Prof. Dr. Wolfgang Streit, PD Dr. Andreas Pommerening, Prof. Dr. Friedrich Buchholz and Jun. Prof. Dr. Mirjam Perner

Hamburg Port Authority (Dr. Maja Karrasch und Rolf Lüschow) MLUR

NLWKN BafG

POLMAR graduate school Prof. Dr. Karen Wiltshire Dr. Jörg Peplies

Dr. Christian Hass

Julia Haafke and Bettina Oppermann Kristine Carstens and Sylvia Peters Mathis van Ahnen

all my colleagues and friends at the BAH