1. Maria Briglia, M.T. Vaello, M. Meima, and P.L.E. Bodelier
Distribution and diversity of anaerobic nitrite-driven
methane oxidizing NC10 phylum bacteria in European lakes
THE DEPARTMENT OF MICROBIAL ECOLOGY
Wageningen, The Netherlands
www.nioo.knaw.nl – m.briglia@nioo.knaw.nl
Nederlands Instituut
voor Ecologie
Het NIOO is een instituut van de Koninklijke
Nederlandse Akademie van Wetenschappen
Introduction
The novel phylum NC10 comprehends so far only bacteria that are able to oxidise methane (CH4) in the absence of oxygen, as described in the equation below
(Raghoebarsing et Al., 2006-Nature, 440:136-141).
3CH4 + 8NO2
- + 8H+ → 3CO2 + 4N2 + 10H2O ΔG˚ = -928 Kj/mol CH4
The emissions of methane from freshwater lakes account for 2 to 10% of the total emission of the greenhouse gas methane (Bastviken et Al., 2004-Global Biogeochem.
Cy., 18:1-12). To better understand the controls and variability in these emissions more information on the distribution, quantification, and diversity of microbes involved
in methane cycling is necessary. About the ecological and biogeochemical role of the NC10 bacteria freshwater lakes almost nothing is known. In this we screened a large
range of European lakes in order to provide more information on the occurrence and biodiversity of NC10 bacteria in relation to a set of measured environmental
variables.
Name Max depth pH
CH4
tot flux
CH4
diff flux
O2 conc
surf - bottom
(m) (mmol/m2/day) (mmol/m2/day) (mmol/m2/day)
BUR 30,0 8,60
BUR-d 30,0 1,14 1,22 9,87
BUR-i 10,0 1,11 1,18 NA
BUR-s 2,5 9,09 1,01 NA
GLI 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 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 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
Tab 2. Positive PCR product from screening functional gene pmoA and phylogenetic gene 16S rDNA;
Materials and Methods
The samples screened in this investigation originated from 32 different European freshwater lakes (Fig.1). Sediment cores were collected at three different depths (shallow, intermediate, and deep)
in the lake. The surface 0-2cm layer of each sediment core sampled were used for further analyses. In Table 1 only the characteristics of the lakes that gave positive results for NC10 are described.
Four PCR approaches were needed for detection of NC10 like bacteria: TG-, Nested-, HS-, and direct PCR. The functional gene pmoA and the phylogenetic gene16S rRNA were used as target. The
used PCR primers sets for both targets were specific for NC10-like bacteria (Luesken et Al., AEM-2011, 92:845-854; Baoli Zhu et Al., AEM-2012, 78:8735-87420. Phylogenetic inference was
performed by using MEGA5.
Results
• From all the lakes that were screened, only four sediment samples (two of Finland) were positive for NC10, of which two were from the same lake (Sweden).
• The functional gene pmoA as well as the 16S rRNA for NC10 were found at in 02-cm surface layer retrieved in the lake at different water-depths (see Tab2.).
• The functional gene pmoA for aerobic gamma and alpha proteobacterial MOB- (type 1a,1b, and II) was detected next to NC10 but not in all samples (see Tab2.).
• Phylogenetic inferences confirmed that the isolated clones from NC10 pmoA PCR products belonged to the NC10 phylum (see Fig1.).
Further work
Quantification of the positive samples by Q-PCR is in progress; therefore it is not possible to provide any estimation of cell density.
Tab 1. Description of the characteristics of the lakes where the screening for functional
gene pmoA and phylogenetic gene 16S rRNA revealed the presence of NC10 –like bacteria
Lake
(Country/Sample
code)
Sample
description
PCR-targets
pmoA 16S rRNA
NC10 *MOB-1a MOB-1b MOB-II qP1r/f qP2r/f 8f/1043r 193f/1043r
CH / BUR-d deep ± + - - ± + + +
“ -i intermediate ±
+ +
- + + + +
“ -s shallow - - - - - - - -
SE / GLI-d deep + + + + + + + +
“ -i intermediate + + + + + + + +
“ -s shallow - + + + - - - ±
FI / SYR-d deep + + + + + + + +
“ -i intermediate - + + + - - - -
“ -s shallow - + + - - - - -
FI / VAL-d deep - + + + - - - -
“ -i intermediate - + + + - - - -
“ -s shallow + + + - + + + +
*=MOB 1a ,1b, and II cell density was between 6,7x106 and 5x109 as determined by Q-PCR.
Conclusions
•These results show that NC10 occurs in the top layer of sediments of only a small sub-set of the large range of lakes sampled.
•Considering the presence of aerobic proteobacterial MOB the wide-spread absence or low abundance of NC10 hints at low nitrate/nitrite availability.
•The presence of proeobacterial MOB apparently does not exclude the presence of NC10-like bacteria.
Fig2. Phylogenetic tree of the NC10 bacterial pmoA sequences obtained from Lakes SYR
and VAL sediments. The tree was constructed using neighbor-joining and bootstrap
analysis of 1,000 replicates. All sequences found belong to NC10 phylum.
Fig1. Geographical description of the location of the lakes which were used for this study.
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