Anaerobic degradation of gaseous alkanes by sulfate-reducing bacteria from marine gas and oil cold seeps

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1 Anaerobic degradation of gaseous alkanes by sulfate-reducing bacteria from marine gas and oil cold seeps ISMOS 2015 Florin Musat Helmholtz entre for Environmental Research, Leipzig (Max Planck Institute for Marine Microbiology, Bremen)

2 Propane and butane - abundant in the environment Natural gas (gas seeps, hydrates) crude oil (oil seeps) Oil and gas seeps, natural gas hydrates Suess, 2010 Gulf of Mexico structure II gas hydrates Etiope and iccioli, 2010 Ethane 11% Propane 32% Methane 44% Pentane 0.5% Isobutane 9.5% Butane 3% Sulfate Reduction Rates >> Anaerobic Oxidation of Methane Rates Sassen et al., 2004 Orcutt et al., 2004

3 Anaerobic degradation of alkanes AOM Strain HxN1 Strain OcN1 D glaeba alkanexedens Lake D glaeba alkanexedens ALD Strain TD3 Strain Hxd3 Strain HdN1 Strain AK-01 Sediment, enrichment and isolation Hydrate Ridge Gas seeps, hydrates Guaymas Basin rude oil seeps Gulf of Mexico Gas seeps MacDonald, I.R. Monterey Bay Aquarium Research Institute

4 Hydrocarbon range Degrade only 3 and 4 Guaymas Basin rude oil seeps But28-GuB (butane) Strain BuS5 10 m Hydrate Ridge Gas seeps, hydrates (butane) No isolate Gulf of Mexico Gas seeps (propane) But12-GMe (butane) No isolate No isolate

5 Microbial communities of the enrichment cultures Aggregates 5 mm Desulfosarcina/Desulfococcus Sequence-specific probe Group-specific probe Abundance

6 Microbial communities of the enrichment cultures Aggregates 5 mm Sequence-specific probe Group-specific probe Abundance

7 Do the dominant cells degrade the hydrocarbons? substrate ARD-FISH F-containing tyramides -propane -butane H2S produced (mm) H 2 S But12-GMe F Nano-scale-Secondary Ion Mass Spectrometry nanosims Time (d) enrichment Primary ion beam (O -, s + ) hit the sample / 12 (%) But12-GMe Atoms / polyatomic fragments released from the upper layers (1-2 nm) some are ionized => secondary ions Time (d)

8 Do the dominant cells degrade the hydrocarbons? nanosims -propane -butane N (Biomass) 19 F (Identity) / 12 (Activity) H2S produced (mm) H 2 S But12-GMe Time (d) ARD-FISH F-containing tyramides 1.70 enrichment 1.60 / 12 (%) But12-GMe Time (d)

9 Do the dominant cells degrade the hydrocarbons? nanosims -propane -butane N (Biomass) 19 F (Identity) / 12 (Activity) H2S produced (mm) H 2 S But12-GMe Time (d) 1.70 enrichment But12-GMe 1.60 / 12 (%) But12-GMe Time (d) Jaekel et al., ISME J 20

10 Activation of propane and butane Extraction and analysis of metabolites by G-MS Butane activation at subterminal OO OO OO OO Propane activation at terminal and subterminal OO OO OO OO OO OO OO OO Kniemeyer, Musat et al., Nature 2007

11 Activation of propane at the terminal atom: significant or just side reaction? Strain BuS5 incubated with position-specific D-labeled substrates D 2 D 3 D 3 D 2 D 2 D 3 D 3 onsistent activation of propane at the primary (terminal) atoms Jaekel et al., EMI 2014

12 Physiology of strain BuS5 genome and proteome analysis True specialists, able to utilize a very limited substrate range (propane & butane)? Hints at other possible substrates Ecological niche Support for the proposed degradation pathway (based on methylalkylsuccinates and fatty acids) Identification of activating genes (mas/ass), possible use as functional markers Strain BuS5, other substrates tested, no growth: Hydrocarbons methane, ethane, pentane, hexane, 2-methylbutane, 2-methylpentane, 3- methylpentane, propylene, 1-butylene Hydrogen Sugars glucose, fructose Alcohols, ketones methanol, ethanol, 1-propanol, 2- propanol, 1-butanol, 2-butanol, 2- butanone, acetone Acids formate, acetate, propionate, butyrate, valerate, capronate, 2- methylbutyrate, 2-methylpropionate, 3-methylvalerate, 4-methylvalerate, succinate, fumarate, malate, pyruvate, lactate, benzoate

13 Physiology of strain BuS5 genome and proteome analysis Genome Genome sequence summary DNA scaffolds 217 DNA total number of bases 3,600,571 DNA coding number of bases 3,081,150 Genes total number 3636 Protein coding genes 3561 RNA (rrna, trna) genes 75 5S rrna 1 16S rrna 1 23S rrna 1 Genes with function prediction 2549 Genes without function prediction 1012 JGI Genome sequencing project S Sievert, WHOI 2D gel electrophoresis R Rabus, Uni Oldenburg Shotgun proteome analysis, Orbitrap L-MS L Adrian, UFZ Leipzig Proteome Shotgun, Orbitrap L-MS Sequest Mascot 634 proteins Focus alkane activation & -oxidation terminal oxidation potential utilization of other substrates energy metabolism 500 proteins

14 onclusions Strain HxN1 Strain OcN1 Strain HdN1 AOM D glaeba alkanexedens Lake D glaeba alkanexedens ALD Strain Hxd3 Strain AK-01 Phylogeny luster SA degraders Metabolites Environment Isotope fractionation MasD (function)

15 Acknowledgements MPI, Microbiology Ulrike Jaekel Olaf Kniemeyer Olav Grundmann Ramona Appel Friedrich Widdel MPI, Biogeochemistry Niculina Musat Birgit Adam Tomas Vagner Daniela Franzke Marcel Kuypers UFZ Leipzig Hans-Hermann Richnow arsten Vogt Anko Fischer Lorenz Adrian Benjamin Sheer Sediment samples Antje Boetius Samantha Joye Uni Oldenburg Ralf Rabus WHOI Stefan Sievert Financial support DFG SPP19 Max Planck Society UFZ Leipzig JGI