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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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(M. B
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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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(M. B
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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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