1. OCCURRENCE OF ANAEROBIC N-DRIVEN
METHANE OXIDIZING BACTERIA BELONGING TO
NC10 PHYLUM IN EUROPEAN LAKE SEDIMENTS.

2. Presentation outline:
Introduction:
1. Methane?
2. Methane production
3. Methane consumption
This research:
1. Aims
2. M & M
3. Results and Conclusions
4. Remarks and Suggestions
5. Acknowledgement

3.  Introduction:
1- Methane?
• Contribution to global warming 25x > CO2
Last updated on 9 July 2010 by John Cook.

4. 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)

5.  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

6.  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;

7. This research:
1- Aims
•Contribute to understanding ecology of N-DMO
•Expand knowledge on the occurrence of N-DMO
in lake sediments

8. 2- M & M
• DNA extracts (Xμls) from lake sediments (ca 115)
see Tab1;
• PCR approaches for detection and quantification
• Cloning, sequencing, and phylogenetic inferences

9. Lake
Samp
le
positi
on Sample name Country Depth CH4 conc surf 1 CH4 conc surf 2
CH4 conc
tophyp
CH4 conc
bottom CH4 conc +10CH4 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
inter
media
te BUR intermediateCH 10,0 0,90NA NA NA 17,52NA NA NA NA NA 21,4NA 1,18 1,11
BUR
shallo
w 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
inter
media
te ERS intermediateSE 3,0NA NA NA NA 1,96NA NA NA NA NA 16,9NA 1,18 1,14
ERS
shallo
w 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
inter
media
te
GER
intermediate CH 6,0 2,99NA NA NA 319,20NA NA NA NA NA 23NA NA 11,42
GER
shallo
w 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
inter
media
te GLI intermediate SE 8,3NA NA NA NA 0,05NA NA NA NA NA 16,5NA 0,12 0,11
GLI
shallo
w 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
inter
media
te GRI intermediate SE 7,4NA NA NA NA 0,99NA NA NA NA NA 16,5NA 0,10 0,10
GRI
shallo
w 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
inter
media
te HAR intermediateSE 3,3NA NA NA NA 0,70NA NA NA NA NA 14,9NA NA 2,66
HAR
shallo
w 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
inter
media
te HAS intermediateCH 3,7 14,31NA NA NA 5,90NA NA NA NA NA 22,7NA 11,43 16,30
HAS
shallo
w 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
HIJK
shallo
w 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
inter
media
te HIN intermediate CH 7,2 2,76NA NA NA 9,92NA NA NA NA NA 14,3NA 2,03 1,95
HIN
shallo
w 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.

10. Example of the sampling site

11. 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.

12. Sample
position
Country-
Sample code
pmoA
16S rRNA
qP1r/f
16S rRNA
qP2r/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;
3- Results: PCR screening

13. Tab 3. Summary of the main features of the lake sediments samples that gave positive
PCR product.
Name Long name Country
Max
depth pH
CH4
tot flux
CH4
diff flux
O2 conc
surf - bottom
(m) (mmol/m2/day) (mmol/m2/day) (mmol/m2/day)
BUR Burgäschisee CH 30,0 8,60
BUR-d deep 1,14 1,22 9,87
BUR-i intermediate 1,11 1,18 NA
BUR-s shallow 9,09 1,01 NA
GLI Glimmingen SE 15,3 6,9
GLI-d 14 0,17 0,19 1,85
GLI-i 8,3 0,11 0,12 NA
GLI-s 4,2 0,11 0,12 NA
SYR Syrjänalunen FI 8,5 6,1
SYR-d 9,4 1,05 1,05 9,92
SYR-i 6 0,67 0,67 NA
SYR-s 1,3 0,64 0,64 NA
VAL Valkea-Kotinen FI 6,5 5,9
VAL-d 5,5 0,04 0,04 8,90
VAL-i 3,4 0,05 0,05 NA
VAL-s 1,9 0,17 0,17 NA

14. 3- Results: Phylogenetic inferences
Sequences of pmoA PCR products from one lake sediment;

15.  3- Results: Phylogenetic inferences
Sequences of pmoA PCR products
from two lake sediments;

16. 3- Results and Conclusions:
 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.

17. 4- Ongoing work:
 Q-PCR by pmoA and 16S primers;
 16S rDNA PCR products cloning/sequencing
/phylogenetic inferences

18. 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.

19. 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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