Planktonic Archaea in Lake Kivu
Marc Llirós
1, F Darchambeau
2, A Plasencia
1, F Gich
1, B Leporcq
2, AV Borges
3, B Delille
3, P Isumbisho
4, EO Casamayor
5, CM Borrego
1& JP Descy
21Institute of Aquatic Ecology, University of Girona, Campus de Montilivi, E-17071 Girona (Spain); 2, Laboratory of Freshwater Ecology-URBO, Facultés Universitaires Notre-Dame de la Paix, University of Namur, B-5000 Namur, Belgium; 3, Chemical
Oceanography Unit, Institut of Physique, B-4000 Liège, Belgium;4, Unité d’Enseignement et de Recherche en Hydrobiologie Appliquée, Institut Supérieur Pédagogique, BP 854-Bukavu, DR Congo; 5Group of Limnology, Centre d'Estudis Avançats de
Blanes-CSIC, Accés Cala Sant Francesc, E-17300 Blanes (Spain)
Study Site
Background information
Blanes-CSIC, Accés Cala Sant Francesc, E-17300 Blanes (Spain)
marc.lliros@udg.es
Lake Kivu (2ºS, 29ºE) is a deep meromictic and oligotrophic high altitude (1,463m a.s.l.) tropical lake. Kivu has a permanent thermal- and haline stratification that produces a wide oxic mixolimnion and a deep anoxic
monimolimnion (from 60 to 489 m depth) which is very rich in both CO2 and CH4. Although most of this CH4
The use of culture independent techniques to aquatic microbial ecology has showed that archaea are ubiquitous and abundant in prokaryotic planktonic assemblages from both freshwater and marine environments. In some freshwater lakes,
mesophilic Crenarchaeota have been found to constitute between 1 and 10% of the total planktonic community. Recently, monimolimnion (from 60 to 489 m depth) which is very rich in both CO2 and CH4. Although most of this CH4
comes from the reduction of magmatic CO2, degradation of organic matter in anoxic conditions by active
methanogenic Archaea counts for one third of CH4 production.
mesophilic Crenarchaeota have been found to constitute between 1 and 10% of the total planktonic community. Recently, the discovery that some mesophilic Crenarchaeota can act as chemolithoautotrophs has broadened our view of archaeal metabolism and has posed questions on their impact on biogeochemical cycles.
Experimental desing
Experimental desing
Water samples
• K-1 to K-4 sampling stations (see figure for details)• 1L to 500 mL water filtrated through 5.0 and 0.22 µm pore size filters • 60ºC, 160 V, 16h
Water samples
• 1L to 500 mL water filtrated through 5.0 and 0.22 µm pore size filters
DNA extraction
• CTAB+β-mercaptoethanol+PVP • Centricon purificationDGGE analysis
• 60ºC, 160 V, 16h • 20% to 80% denaturing gradient • SybrGold® staining • SequencingDNA extraction
• Centricon purification• Only 0.22 µm filters analysed
• Sequencing
• ARB-software analysis (in progress) • Phylogenetic trees (in progress)
16S rDNA extraction
• Universal PCR: UA21f/U958r
• Archaea nested-PCR: ARC344f-GC/ARC915r
• Crenarchaeota nested-PCR: CREN337f-GC/ARC915r
Phylogenetic analysis
• Crenarchaeota nested-PCR: CREN337f-GC/ARC915r
Results
Results
Bitacora of Lake Kivu (March-April 2007)
A sampling campaing was carried out during March-April 2007 in Lake Kivu. Four
stations (labelled K1 to K4, see map) were sampled during the campaing. Thermal and
chemical stratification of the water column with oxic and anoxic water layers were clearly defined in all stations. Water samples were collected every 10 meters along the
K-1 (Kibuye) K-2 (Ishungu) K-3 (RD Congo) K-4 (Bukavu)
K -1 1 0 m n A R C K -1 4 0 m n A R C K -1 5 0 m n A R C K -1 1 0 0 m n A R C K -2 1 0 m n A R C K -2 4 0 m n A R C K -2 5 0 m n A R C K -2 9 0 m n A R C K -3 1 0 m n A R C K -3 4 0 m n A R C K -3 5 0 m n A R C K -3 8 5 m n A R C K -4 1 0 m n A R C K -4 4 0 m n A R C K -4 5 0 m n A R C K -4 8 5 m n A R C K -1 1 0 0 m d A R C K -2 5 0 m d A R C K -2 9 0 m d A R C K -3 8 5 m d A R C K -4 1 0 m d A R C M a rk e r M a rk e r M a rk e r M a rk e r M a rk e r
clearly defined in all stations. Water samples were collected every 10 meters along the vertical profile of each station and they were used for molecular fingerprinting analysis.
K K K K K K K K K K K K K K K K K K K K K Ma rk e r M a rk e r M a rk e r M a rk e r M a rk e r 1 1 1 1 1 K1-Kibuye D O (m g L-1) 0 2 4 6 8 10 12 pH 6,5 7,0 7,5 8,0 8,5 9,0 9,5 K3-D R C ongo DO sat (% ) DO (m g L-1) 0 2 4 6 8 10 12 pH 6,5 7,0 7,5 8,0 8,5 9,0 9,5 K1 1 1 1 1 1 2 1 1 3 4 3 4 1 1 1 1 1 1 1 5 1 5 5 1 0 10 20 30 40 DO sat (% ) 0 20 40 60 80 100 120 D e tp h ( m ) 0 10 20 30 DO sat (% ) 0 20 40 60 80 100 120 K1 (Kibuye) K3 (Goma) 1 2 2 2 4 2 2 2 D e tp h ( m ) 40 50 60 70 80 90 D e tp h ( m ) 40 50 60 70 80
Code Type Clone Environment
1 Crenarchaeota MP104-SW-a5 (DQ088780) Deep fracture water (crustal biome) 2 Crenarchaeota VIARC-2 (UCR240005) Freshwater lake
3 Crenarchaeota NRP-N (AB243805) Rice paddy soil (anaerobic) 4 Crenarchaeota GA55 (AB046184) leachate from landfill site
5 Crenarchaeota PET1-19 (AY278101) Soil, grassland,Crenarchaeota group 1.1b 1 Euryarchaeota ASC34 (AB161335) Petroleum cont soil
Tem perature (ºC) 22 23 24 25 26 100 Conductivity (µS cm-1) 1000 1200 1400 1600 1800 2000 2200 C hl (m g L-1) 0 5 10 15 20 25 Tem perature (ºC) 22 23 24 25 26 80 C onductivity (µS cm-1) 1000 1200 1400 1600 1800 2000 2200 Chl (m g L-1) 0 5 10 15 20 25
1 Euryarchaeota ASC34 (AB161335) Petroleum cont soil
2 Euryarchaeota Methanosaeta sp. Clone A1 (MSP133791) Methane formation from crude oil 3 Euryarchaeota ESYB47/EJ_CO2 (AB119603/AY454667) Estuarine sediment
4 Euryarchaeota MB-01 (AY175387) Methanogen peatland 5 Euryarchaeota pTN-19 (AB182768) Rice paddy soil
1 Unknown
Negative images of SybrGold®-stained DGGE fingerprints of 16S rRNA gene
C hl (m g L-1) K2-Ishungu Bay D O (m g L-1) pH 6,5 7,0 7,5 8,0 8,5 9,0 9,5 Lake Kivu K 4-Bukavu bay DO (m g L-1) 0 2 4 6 8 10 12 pH 6,5 7,0 7,5 8,0 8,5 9,0 9,5 K2 (Ishungu)
Negative images of SybrGold®-stained DGGE fingerprints of 16S rRNA gene
amplicons from Archaea (above) and Crenarchaeota (below) planktonic community
thriving in Lake Kivu. Archaea and Crenarchaeota nested-PCR products loaded onto 20%-80%
DGGE gels gave clear and distinct band profiles. Some of the obtained bands were common in the different samples analysed while some others were depth specific. We are currently trying to optimize PCR-DGGE
0 10 20 30 DO sat (% ) 0 20 40 60 80 100 120 0 2 4 6 8 10 12 0 10 20 D O sat (% ) 0 10 20 30 40 50 60 70 80 90 100 110 120 0 2 4 6 8 10 12 K4 (Bukavu)
(Ishungu) samples analysed while some others were depth specific. We are currently trying to optimize PCR-DGGE performance to obtain more clear fingerprints.
K-1 (Kibuye) K-2 (Ishungu) K-3 (Congo) K-4 (Bukavu)
D e tp h ( m ) 30 40 50 60 70 80 D e tp h ( m ) 30 40 50 60 70 Temperature DO sat Conductivity
K-1 (Kibuye) K-2 (Ishungu) K-3 (Congo) K-4 (Bukavu)
K -1 1 0 m n C R E N K -1 4 0 m n C R E N K -1 5 0 m n C R E N K -1 1 0 0 m n C R E N K -2 1 0 m n C R E N K -2 4 0 m n C R E N K -2 5 0 m n C R E N K -2 9 0 m n C R E N K -3 1 0 m n C R E N K -3 4 0 m n C R E N K -3 5 0 m n C R E N K -3 8 5 m n C R E N K -4 1 0 m n C R E N K -4 4 0 m n C R E N K -4 5 0 m n C R E N K -4 8 5 m n C R E N M a rk e r M a rk e r M a rk e r Tem perature (ºC) 22 23 24 25 26 90 100 Conductivity (µS cm-1) 1000 1200 1400 1600 1800 2000 2200 0 5 10 15 20 25 T em perature (ºC) 22 23 24 25 26 80 Conductivity (µS cm-1) 1000 1200 1400 1600 1800 2000 2200 0 5 10 15 20 25 Chlorophyll DO DO sat pH K K K K K K K K K K K K K K K K M a rk e r M a rk e r M a rk e r 1 1 2 1 1 1 1 1 C hl (m g L-1) 0 5 10 15 20 25 Chl (m g L-1) 0 5 10 15 20 25
Preliminary conclusions
2 1 1 1 1 1 1 1 1 1 1 K-1 (Kibuye) 0 20 K-2 (Ishungu bay) 0 20
Planktonic archaea seems to be widely distributed in Lake Kivu.
The main archaeal phylotypes recovered were mainly assigned to:
1 1 D e p th ( m ) 40 60 80 100 D e p th ( m ) 40 60 80 100
Code Type Clone Environment
1 Crenarchaeota MP104-SW-a5 (DQ088780) Deep fracture water (crustal biome) 2 Crenarchaeota VIARC-2 (UCR240005) Freshwater lake
1 Unknown
The main archaeal phylotypes recovered were mainly assigned to:
•
Euryarchaeota: mainly methanogenic uncultured clones (as
expected according to lake chemistry).
•
Crenarchaeota: mainly related to different non-thermophilic
% of bands 0 20 40 60 80 100 % of bands 0 20 40 60 80 100 K-3 (Goma) 0 20 40 K-4 (Bukavu bay) 0 20 40
•
Crenarchaeota: mainly related to different non-thermophilic
clones from anoxic environments.
0 20 40 60 80 100 D e p th ( m ) 40 60 80 100 0 20 40 60 80 100 D e p th ( m ) 40 60 80 100 % Crenarchaeota % Euryarchaeota % Unknown
Affiliation (in %) of 16S rRNA gene sequences retrieved from DGGE fingerprints of
Lake Kivu samples.
ACKNOWLEDGEMENTS: This work was founded through ECOSYKI project from the belgium government & REN 2003-08333-C02-01-GLO project from the spanish Ministerio de Educación y Ciencia (MCYT). M. Ll. is supported by PhD student fellowship (BES-2004-5127)
from the MCYT associated to the project referred above. We would like to thank all co-workers for tremendous efforts during sampling campaign.
% of bands
0 20 40 60 80 100
% of bands
