1. Scott Gifford, Oscar Sosa, Dan Repeta, Ed DeLong Methylotrophs Dominate Dilution-to-Extinction Cultures Enriched with Marine Dissolved Organic Matter

2. Particulate carbon Dissolved Organic Carbon (DOC) 750 GT C 10-20 GT C 660 GT C CO 2 Release Mechanisms Lysis Grazing Leakage

3. Dissolved Organic Carbon (DOC) 660 GT C labilesemi-labilerefractory ? lability hr -days 1,000 – 2000 years HMW

4. 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?

5. www.nelha.org Karl et al 1998 HMW DOM collection Tangential flow filtration with 1kDa filter

6. Ultrafiltration to obtain DOM Courtesy Dan Repeta Chemical Characterization Isolates

7. transfer 10 6 cells ml -1 5-10 cells ml -1 incubate [DOM] FALCON 1 B C D E F 2345678 A inoculated well control well Dilution to extinction culturing

8. Dil-to-Ext with a DOM twist DOM North Pacific : NEHLA Inoculant North Atlantic: Nahant Bay Boston Nahant Bay

9. noDOM4X DOM10X DOMtotal wells inoculated 840 2520 controls 54 162 growth >10 5 inoculated 1405293 controls 0000 <10 5 10 5 5 X 10 5 10 6 or > Screen wells for growth with flowcytometry side scatter log green fluorescence Flow cytometry

10. 100x DOM 20x DOM 5x DOM no DOM days cells per ml Isolates have a distinct growth response to DOM

11. Isolate Phylogeny (small subunit rDNA) 93 Isolates 88 obtain 16S rDNA 10 Gammaproteobacteria 5 Alteromonads 3 SAR92 2 Vibrios 78 Betaproteobacteria 78 unclassified Methylophilales_ otu_2801_ 88% of isolates are methylotrophs

12. Isolate growth on methanol…. cells per ml days No MeOH 150 µM methanol 3 2 1 cells per ml X 10 6 No MeOH 150 µM methanol days Isolates can degrade C1 compounds

13. McCarren et al 2010:Gamma: AlteromonadsGamma: Methylophaga Current Study:Gamma: AlteromonadsBeta: Methylophilales DOM transcripts Same Taxa Same Functional Group microcosm

14. Alteromonads Methylotrophs CH 3 3 HMW DOC

15. Transcriptomics Genomics Draft genomes for 73 isolates Substrate experiments Purified DOM C1 compounds Complex Carbs. porphyrin, xylan pectin, etc., Current work…..

16. 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 Becker Rene Boiteau Chon Martinez Tsultrim Palden John Eppley Sara Lincoln