Scott Gifford, Oscar Sosa, Dan Repeta, Ed DeLong

Methylotrophs Dominate Dilution-to-Extinction Cultures Enriched with Marine Dissolved Organic Matter

Particulate carbon

Dissolved Organic Carbon (DOC)

750 GT C

10-20 GT C

660 GT C

CO2

CO2

Release Mechanisms

LysisGrazingLeakage

Dissolved Organic Carbon (DOC)

660 GT C

labile semi-labile refractory

?

lability

hr -days 1,000 – 2000 years

HMW

Dissolved Organic Carbon (DOC)

DOC

Chemical Characterization

Dan Repeta

Rene Boiteau

Biology

Oscar Sosa

Ed DeLong

What is the chemical composition of DOC?

Which microorganisms consume DOC?

What are the genes involved in DOC degradation?

www.nelha.org

Karl et al 1998

HMW DOM collection

Tangential flow filtration with 1kDa filter

Ultrafiltration to obtain DOM

Courtesy Dan Repeta

Chemical Characterization

Isolates

transfer transfer106 cells ml-1

5-10 cells ml-1

incubate

[DOM]FALC

ON

1

B

C

D

E

F

2 3 4 5 6 7 8A

inoculated well

control well

Dilution to extinction culturing

Dil-to-Ext with a DOM twist

DOM

North Pacific : NEHLA

InoculantNorth Atlantic: Nahant Bay

BostonNahant

Bay

noDOM 4X DOM 10X DOM total

wells inoculated 840 840 840 2520 controls 54 54 54 162

growth >105 inoculated 1 40 52 93 controls 0 0 0 0

50 to 100 100 to 500 500 to 1000 >10000

5

10

15

20

25

30

35

40

no DOM

4X DOM

10X DOM

cells per ml

wells

<105 105 5 X 105 106 or >

Screen wells for growth with flowcytometry

side scatter

log green fluorescence

Flow cytometry

100x DOM

20x DOM

5x DOM

no DOM

days

cells per ml

Isolates have a distinct growth response to DOM

Isolate Phylogeny (small subunit rDNA)

93 Isolates

88 obtain 16S rDNA

10 Gammaproteobacteria

5 Alteromonads3 SAR922 Vibrios

78 Betaproteobacteria

78 unclassified Methylophilales_ otu_2801_

88% of isolates are methylotrophs

Isolate growth on methanol….

cells per ml

days

No MeOH 150 µM methanol

3

2

1

cells per ml X 106

No MeOH 150 µM methanol

days

Isolates can degrade C1 compounds

McCarren et al 2010: Gamma: Alteromonads Gamma: Methylophaga

Current Study: Gamma: Alteromonads Beta: Methylophilales

DOM

transcripts

Same TaxaSame Functional Group

microcosm

Alteromonads

Methylotrophs

CH3 CH3

HMW DOC

TranscriptomicsGenomics

Draft genomes for 73 isolates

Substrate experiments

Purified DOMC1 compoundsComplex Carbs. porphyrin, xylan pectin, etc.,

Current work…..

Isolates have a distinct growth response to DOM

Conclusions

Dilution cultures enriched with DOM produced more isolates

Hypothesize methylotrophs may have ability to use C1 compounds from DOM polymer

Isolate collection is dominated by methylotrophs

Acknowledgements

Jamie BeckerRene BoiteauChon Martinez

Tsultrim PaldenJohn EppleySara Lincoln