Bacterial Diversity in Oligotrophic Marine Sediments

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Bacterial Diversity in Oligotrophic Marine
Sediments

• Ian Hewson, B.Sc (Hons)
• Fuhrman Lab

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www.micrographia.com
What is the bacterial diversity in oligotrophic
marine sediments? What controls this diversity?
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Outline of Presentation

• Introduction to sediment bacteria
• Oligotrophic Sediment Bacterial Diversity
• Nutrient Availability
• Water Motion
• Vertical Distribution / Anoxia
• Grazing
• Virus Infection
• Summary of Factors Shaping Bacterial Communities

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Outline of Presentation

• Introduction to sediment bacteria
• Oligotrophic Sediment Bacterial Diversity
• Nutrient Availability
• Water Motion
• Vertical Distribution / Anoxia
• Grazing
• Virus Infection
• Summary of Factors Shaping Bacterial Communities

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Sediment Bacterial Communities
Bacterial abundance in sediments high 107 - 109
Cells cm-3 (105 - 107 Cells cm-3 in water
column) Productive up to 2.1 d-1 in muddy
sediments (Camman et al. 91) Biogeochemically
important Denitrification almost exclusively
benthic
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Sediment Bacterial Communities
Component mg C dm-3 Total Bacteria
2.0 28 Viruses
0.1 1.4 Protists 1.6
22 DOC 3.6 49
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Bacterial Fingerprinting

• Allows rapid analysis of bacterial communities
  from small samples
• Sensitive (detection threshold 0.09 of
  community)
• Two methods targeting different groups
• Terminal Restriction Fragment Length Polymorphism
  (TRFLP)
• gtgt 16S rDNA
• gtgt Functional Genes (nifH, amoA, nirS, ureC,
  alkA)
• Based upon sequence heterogeneity
• Automated rRNA Intergenic Spacer Analysis
• gtgt16S 23S rDNA ITS length heterogeneity

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Comparison of fingerprinting vs sequencing
Fingerprinting Sequencing
Allows entire community to be analysed in one
sample Quantitative (with biases) Not positive
ID but theoretical (database) Low Cost (4-8 per
community)
Entire community may require large number of
clones Not-Quantitative (subject to same PCR
biases as fingerprinting) More positive ID but
still theoretical (GenBank database) High Cost
(100s per community)
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Automated rRNA Intergenic Spacer Analysis (ARISA)

• DNA extracted from mixed community from
  sediment core
• PCR performed with one tagged primer
• Forward universal, reverse eubacterial

Each peak represents an operational taxonomic
unit
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Interpretation of ARISA Data

• Diversity - Simpsons Index (D) - 1/?pi2
• Each OTU considered one species (but may be more
  than one)
• Integrated area under each peak relative
  abundance in community

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Outline of Presentation

• Introduction to sediment bacteria
• Oligotrophic Sediment Bacterial Diversity
• Nutrient Availability
• Water Motion
• Vertical Distribution / Anoxia
• Grazing
• Virus Infection
• Summary of Factors Shaping Bacterial Communities

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Characteristics of oligotrophic sediments

• Oligotrophic
• Nutrient Coral Reef (µM) Moreton Bay
  (µM)
• NH4 6 8 50
• NOx 10 10
• PO43- 3 5-6
• (Rasheed et al. 2002) (Dennison
  Abal, 1999)
• Bioclastic Carbonate Sediments
• Coral rubble
• Algal fragments (I.e. Halimeda spp. Fragments,
  crustose algae)
• Foraminifera
• Modal Grain Size large - 125 - 250 mm

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Outline of Presentation

• Introduction to sediment bacteria
• Oligotrophic Sediment Bacterial Diversity
• Nutrient Availability
• Water Motion
• Vertical Distribution / Anoxia
• Grazing
• Virus Infection
• Summary of Factors Shaping Bacterial Communities

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Arsnicker Key, Florida
USA
July 2001
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Nutrient Addition Experiment

• Sediment and water in enclosed in 50 cm diameter
  polycarbonate collars
• Area in sheltered, shallow water
• Nutrients (10 mM NH4 and SiO4 and 1 mM PO43-)
  added daily
• 3 X Control (no addition)
• 2 X NH4 SiO4
• 2 X PO43- SiO4
• 2 X NH4 PO43-
• Samples _at_ 0,7 and 14 d for bacterial DNA,
  abundance

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Microbial Abundance

• All amendments result in lower bacterial
  abundance

2.0 x 108 1.0 x 108 0
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ARISA

• Few containment artifacts
• Few changes upon nutrient amendment
• Unclear from electropherograms

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Bacterial Diversity

• Nutrient addition stimulated diversity

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Outline of Presentation

• Introduction to sediment bacteria
• Oligotrophic Sediment Bacterial Diversity
• Nutrient Availability
• Water Motion
• Vertical Distribution / Anoxia
• Grazing
• Virus Infection
• Summary of Factors Shaping Bacterial Communities

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Australia
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Water Motion Transect

• January 2003
• 200m Transect across southern Heron Reef Flat
• (Windward Side)
• Strong spatial gradient in water motion
• Heterogeneity in microalgal abundance

Water Motion
Reef Flat
Beach
Reef Crest
Gutter
200m
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Gutter
Reef Flat
Reef Flat
Reef Crest
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ARISA
Gutter
Reef Flat
Distance from Island
Relative Fluorescence
Reef Crest
0
0 140 340 540
740 940
ITS Length (bp)
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Water Motion

• No clear trend between re-working (due to water
  motion) and diversity / OTU richness

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Outline of Presentation

• Introduction to sediment bacteria
• Oligotrophic Sediment Bacterial Diversity
• Nutrient Availability
• Water Motion
• Vertical Distribution / Anoxia
• Grazing
• Virus Infection
• Summary of Factors Shaping Bacterial Communities

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Vertical Heterogeneity

• Sediment Core withdrawn from one location
• Sectioned into 1 cm depth horizons
• Subsamples extracted for DNA

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Vertical Heterogeneity
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• Diversity greatest near surface
• Decreased diversity below oxic/anoxic boundary
• Habitat Overlap?

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Outline of Presentation

• Introduction to sediment bacteria
• Oligotrophic Sediment Bacterial Diversity
• Nutrient Availability
• Water Motion
• Vertical Distribution / Anoxia
• Grazing
• Virus Infection
• Summary of Factors Shaping Bacterial Communities

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Australia
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Metazoan Grazing

• Grazing by Holothuria atra extensive on reef
• Approx 10-40 of reef grazed d-1
• (Moriarty et al. 1985)
• Samples of sediment in front of holothurians
• Samples of fecal pellets

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Before
Before
After
After

• Species Richness Before 132 16, After 85
  40
• Simpsons Index Before 52 8, After 25 1

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Pre-Grazed 1
Fecal Pellet 1
Fecal Pellet 2
Transect 3
Transect 2
Transect 4
Transect 5
Transect 6
Transect 7
Transect 8
Transect 1
Pre-Grazed 2
0 - 1 cm
1 - 2 cm
2 - 3 cm
3 - 4 cm
4 - 5 cm
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
Similarity (Jaccard Index)
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Outline of Presentation

• Introduction to sediment bacteria
• Oligotrophic Sediment Bacterial Diversity
• Nutrient Availability
• Water Motion
• Vertical Distribution / Anoxia
• Grazing
• Virus Infection
• Summary of Factors Shaping Bacterial Communities

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Experimental Design

Florida Bay
USA
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Virus Concentrate Preparation

• Virus- Free Seawater prepared by passing 1-2
  Liters of seawater through a 0.02 mm filter
• 2 L flocculent layer collected
• Virus-free seawater added to sediments
• Viruses and Bacteria eluted from sediments by
  mechanical agitation (80 viruses , 50
  bacteria)
• (Hewson Fuhrman, in press)
• Eluate settled for 10 minutes prior to processing

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Incubation and Innoculation

• gt30,000 Da concentrate prepared
• Half of concentrate heat-killed
• Concentrate added to sediment samples (50 ml) in
  whirlpaks
• Incubated at in situ temperature and light

20 ml
50 ml
100 ml
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Florida Bay

• Strong effect of live virus addition compared to
  killed controls
• Significant containment artifacts
• Incubations not screened to remove grazers
• Difficult to homogenize

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Florida Bay
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Clustering Analysis
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Outline of Presentation

• Introduction to sediment bacteria
• Oligotrophic Sediment Bacterial Diversity
• Nutrient Availability
• Water Motion
• Vertical Distribution / Anoxia
• Grazing
• Virus Infection
• Summary of Factors Shaping Bacterial Communities

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Summary of Factors Affecting Oligotrophic
Sediment Bacterial Communities

• Strong positive effects of nutrient availability
  ?S 65 ?D 31
• Strong positive effects of virus infection ?S
  52 ? D 13
• Strong negative effects of sediment depth ?S
  -103 ?D -35
• Strong negative effects of metazoan grazing ?S
  -47 ?D -27
• Moderate effects of Water motion / sediment
  re-working ?S -17 ?D 5

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Acknowledgements
The Fuhrman Lab Mike Schwalbach Josh
Steele Xiaolin Liang Ximena Hernandez John
Griffith Lusine Sargsyan Heron Island Research
Station Dave Logan Collette Bagnato Ted Upton
USF Gabriel Vargo Gary Hitchcock Jennifer
Jurado Merrie Beth Neely Dani Ault Keys Marine
Lab, Layton, FL Great Barrier Reef Marine Park
Authority Queensland State Government
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Species Richness 111 18 Simpsons Index 43
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• Spatially heterogeneous over small scales (cm)
• Diversity / OTU richness homogeneous

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16S rDNA TRFLP