OCCURRENCE OF ANAEROBIC N-DRIVEN METHANE OXIDIZING BACTERIA BELONGING TO NC10 PHYLUM IN

EUROPEAN LAKE SEDIMENTS.

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Presentation outline:

Introduction: 1. Methane? 2. Methane production 3. Methane consumption

This research: 1. Aims2. M & M3. Results and Conclusions4. Remarks and Suggestions 5. Acknowledgement

Introduction: 1- Methane?

• Contribution to global warming 25x > CO2

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Last updated on 9 July 2010 by John Cook.

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From Abby Anderegg and Natalie Cac Sarah Hestekin

Introduction: 2- methane production

• ca 70% is biogenic (aerobic and anaerobic methanogens) • ca 25% is from other sources (mining, combustion of fossil

fuels, burning biomass, and aerobic degradation of plant matter by UV light exposure in soil as well)

Introduction:

2- biogenic methane consumption:

- Aerobic 1 γ-proteobacteria type I 2 α-proteobacteria “ II 3 verrucomicrobia “ III

- Anaerobic 1 S-DMO 2 Fe/Mn-DMO 3 N-DMO

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What is known about N-DMO briefly:

N-DM Oxidizers spotted in ≠ ecosystems (wetlands, rice fields, peat bogs, minerotrophic peatland, wastewater treatment plants, lake sediment);

3CH4 + 8NO2- + 8H+ → 3CO2 + 4N2 + 10H2O ΔG˚ = -928 Kj/mol CH4

N-DMOs are affiliated to a new phylum the NC10 and there are not yet isolated pure cultures available;

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This research:

1- Aims

• Contribute to understanding ecology of N-DMO

• Expand knowledge on the occurrence of N-DMO in lake sediments

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2- M & M

• DNA extracts (Xμls) from lake sediments (ca 115) see Tab1; • PCR approaches for detection and quantification

• Cloning, sequencing, and phylogenetic inferences

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Lake

Sample position Sample name

Country Depth

CH4 conc surf 1

CH4 conc surf 2

CH4 conc tophyp

CH4 conc bottom

CH4 conc +10

CH4 conc bottom - surf

O2 conc surf

O2 conc tophyp

O2 conc bottom

O2 conc surf - bottom T surf

T bottom

CH4 diff flux

CH4 tot flux

BUR deep BUR deep CH 30,0 0,76 1,23 0,41 241,26 434,75 240,03 9,92 0,08 0,05 9,87 21,4 4,5 1,22 1,14

BUR

intermediate

BUR intermediate CH 10,0 0,90NA NA NA 17,52NA NA NA NA NA 21,4NA 1,18 1,11

BURshallow BUR shallow CH 2,5 1,05NA NA NA 75,54NA NA NA NA NA 21,4NA 1,01 9,09

ERS deep ERS deep SE 5,0 2,37 3,33NA 6,59 19,75 3,26 8,20NA 7,00 1,20 16,9 15,2 2,08 1,97

ERS

intermediate

ERS intermediate SE 3,0NA NA NA NA 1,96NA NA NA NA NA 16,9NA 1,18 1,14

ERSshallow ERS shallow SE 2,0 0,58NA NA NA 1,40NA NA NA NA NA 16,9NA 0,28 0,43

GER deep GER deep CH 9,3 3,30 3,52 191,78 798,23 1139,39 794,71 10,94 0,09 0,05 10,89 23 9,1NA 11,64

GER

intermediate

GER intermediate CH 6,0 2,99NA NA NA 319,20 NA NA NA NA NA 23NA NA 11,42

GERshallow GER shallow CH 3,6 3,08NA NA NA 5,45NA NA NA NA NA 23NA 3,52 8,57

GLI deep GLI deep SE 14,0 0,10 0,12 0,05 0,04 0,10 -0,08 9,26 7,87 7,41 1,85 16,5 6,5 0,19 0,17

GLI

intermediate

GLI intermediate SE 8,3NA NA NA NA 0,05NA NA NA NA NA 16,5NA 0,12 0,11

GLIshallow GLI shallow SE 4,2 0,18NA NA NA 0,16NA NA NA NA NA 16,5NA 0,12 0,11

GRI deep GRI deep SE 12,2 0,24 0,22NA 0,14 0,39 -0,08 8,82NA 1,85 6,97 16,5 5,4 0,14 0,13

GRI

intermediate

GRI intermediate SE 7,4NA NA NA NA 0,99NA NA NA NA NA 16,5NA 0,10 0,10

GRIshallow GRI shallow SE 3,1 0,24NA NA NA 0,31NA NA NA NA NA 16,5NA 0,05 0,06

HAR deep HAR deep SE 6,2 0,30 0,29NA 1,17 3,16 0,88 9,49NA 5,87 3,62 14,9 13,9NA 4,26

HAR

intermediate

HAR intermediate SE 3,3NA NA NA NA 0,70NA NA NA NA NA 14,9NA NA 2,66

HARshallow HAR shallow SE 2,9 0,28NA NA NA 0,51NA NA NA NA NA 14,9NA 0,16 0,29

HAS deep HAS deep CH 5,5 0,21 18,70NA 12,92 1199,47 -5,78 6,60NA 0,27 6,33 22,7 20,2NA 26,19

HAS

intermediate

HAS intermediate CH 3,7 14,31NA NA NA 5,90NA NA NA NA NA 22,7NA 11,43 16,30

HASshallow HAS shallow CH 2,5 6,48NA NA NA 9,73NA NA NA NA NA 22,7NA 5,60 7,45

HIJK deep HIJK deep NL 1,2 0,79 0,65NA 0,83 0,92 0,18 5,95NA 5,58 0,37 20,8 20,7NA 28,04

HIJKshallow HIJK shallow NL 1,0 0,59NA NA NA 0,55NA NA NA NA NA 20,8NA NA 11,84

HIN deep HIN deep CH 11,0 2,63 2,98 0,92 14,27 1238,58 11,29 10,83 1,38 0,13 10,70 14,3 6,8 1,64 1,58

HIN

intermediate

HIN intermediate CH 7,2 2,76NA NA NA 9,92NA NA NA NA NA 14,3NA 2,03 1,95

HINshallow HIN shallow CH 4,3 3,14NA NA NA 27,64NA NA NA NA NA 14,3NA 3,95 7,17

HUT deep HUT deep CH 15,4 0,66 1,13 0,51 636,25 888,87 635,12 10,91 0,51 0,08 10,83 19,6 6,6 0,48 1,38

Tab 1. An extract from the list of the samples from lakes sediments with the description of a number of samples ‘ features.

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Example of the sampling site

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2- M & M • PCR approaches: TG-, N-, HS-, and direct PCR; with N-DMO specific primers targeting pmoA and 16S rDNA genes;

• Cloning, sequencing, and phylogenetic inferences: PCR products cloned by using the vector pGEM-T Easy, phylogenetic inferences by MEGA4 and 5.

Sample position

Country-Sample code pmoA

16S rRNAqP1r/f

16S rRNAqP2r/f

8f/1043r

193f/1043r

deep CH-BUR-d ± ± + + +

intermediate BUR-i ± + + + +

shallow BUR-s - - - - -

deep SE-GLI d + + + + +

intermediate GLI i + + + + +

shallow GLI s - - - - ±

deep FI-SYR d + + + + +

intermediate SYR i - - - - -

shallow SYR s - - - - -

deep FI-VAL d - - - - -

intermediate VAL i - - - - -

shallow VAL s + + + + +

Tab 2. Positive PCR product from screening functional gene pmoA and phyloge-netic gene 16S rDNA;

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Tab 3. Summary of the main features of the lake sediments samples that gave positive PCR product.

Name Long name CountryMax

depth pHCH4

tot fluxCH4

diff flux

O2 conc surf -

bottom

(m)(mmol/m2/

day)(mmol/m2/

day) (mmol/m2/day)BUR Burgäschisee CH 30,0 8,60BUR-d deep 1,14 1,22 9,87BUR-i intermediate 1,11 1,18 NABUR-s shallow 9,09 1,01 NAGLI Glimmingen SE 15,3 6,9GLI-d 14 0,17 0,19 1,85GLI-i 8,3 0,11 0,12 NAGLI-s 4,2 0,11 0,12 NASYR Syrjänalunen FI 8,5 6,1SYR-d 9,4 1,05 1,05 9,92SYR-i 6 0,67 0,67 NASYR-s 1,3 0,64 0,64 NAVAL Valkea-Kotinen FI 6,5 5,9VAL-d 5,5 0,04 0,04 8,90VAL-i 3,4 0,05 0,05 NAVAL-s 1,9 0,17 0,17 NA

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Sequences of pmoA PCR products from one lake sediment;

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inferences Sequences of pmoA PCR products from two lake sediments;

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PCR-products indicating presence of NC10-like bacteria were found in 6 samples (out of 115) of the lake sediments; of which two originated from the same lake at different depth;

Among the lake sediments that gave positive PCR product could not be extrapolated any features-pattern;

Phylogenetic inferences showed that all cloned pmoA-PCR products belonged to the NC10-like bacteria phylum; the sequences originating from the two lakes did not form any separated clade.

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4- Ongoing work:

Q-PCR by pmoA and 16S primers;

16S rDNA PCR products cloning/sequencing /phylogenetic inferences

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5- Personal remarks and suggestions

Project limitations:1.Sampling site (.....);2.Availability of replicates;3.Sampling time data points;4.Absence of funding.

5- Personal suggestions:1.Repeat sampling on

the sites where PCR product were obtained;2.Provide replicates for both sampling and time data points; 3.Provide funding.

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6- Acknowledgement:

NIOO-ME

Dr. P. Bodelier for hosting and providing all research facilities besides the research project.

ECO-nsultancy P.J.M. Middeldorp for financial support

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