226
13.5 Community Composition of Bacteria
Associated to TEP
We used combined protocols to observe TEP and the compo-
sition of attached and free-living bacterial cells. The bacterial
community composition was analysed with fl uorescent in situ
hybridization (FISH) using group-specifi c rRNA oligonucle-
otidic probes according to Moter and Göbel ( 2000 ). This
method was coupled with a coloration using Alcian Blue to
visualize TEP. Detailed methods can be found in Lemarchand
et al. ( 2006 ). The following oligonucleotidic probes were
used to analyse bacterial community composition: EUB338
for Eubacteria , ALF1b and BET42a for the alpha- and beta-
subclasses of Proteobacteria , and CF319a for the Cytophaga-
fl avobacterium group (now renamed Bacteroidetes , group
Sphingobacteria-Flavobacteria ). Probes were labelled with
the indocarbocyanine fl uorescent dye Cy3 (MWG-Biotech).
Our results indicated that bacteria associated with TEP con-
stituted 2.6 % of the total DAPI- stained bacteria and the pro-
portion of Eubacteria is different between the free-living and
attached fractions. The contribution of Eubacteria to particle-
associated and DAPI-stainable bacteria averaged 75 % while
their contribution to free-living bacteria accounted for only
50 %. As noted previously in different studies in both marine
and freshwater environments (Brachvogel et al. 2001 ;
Bockelmann et al. 2000 ; Brümmer et al. 2000 ; Selje and
Simon 2003 ) we also found high proportions of
Betaproteobacteria and Cytophaga-Flavobacteria in parti-
cle-associated bacteria compared with free-living cells (Table
13.2 ). In contrast, the contribution of Alphaproteobacteria to
particle-associated bacteria was lower than their contribution
to free-living cells. Thus, our results clearly indicate that the
composition of particle- associated bacteria is different from
that of free-living bacteria highlighting the signifi cant role of
TEP in driving microbial diversity.13.6 Activity of Free-Living Bacteria
and Particle-Associated BacteriaPotential ectoenzyme activities by bacterial extracellular
enzymes were measured in fractionated water samples incu-
bated at 20 °C in the dark for 2 or 6 h according to the proto-
col of Hoppe ( 1983 ). The 0.2–1.2 μm fraction contained
free-fl oating bacteria while the fractions 1.2–5 μm and > 5
μm contained bacteria associated to detrital organic particles.
The α-glucosidase and leucine aminopeptidase activities
were estimated on duplicate fi lters for each size fraction.
Most activity was associated with the 1.2–5 μm fraction and
glycolytic activities were three times lower than proteolytic
activities. Values of K m for leucine uptake were on average
higher in the 1.2–5 μm fraction than in the 0.2–1.2 μm frac-
tion and the > 5 μm fraction. The K m values of α-glucosidase
activity were lower than the K m of leucine aminopeptidase
activity. Glucose uptake affi nity had the lowest values in the
0.2–1.2 μm fraction. Cell-specifi c hydrolysis rates in the
attached bacterial fraction (1.2–5 μm and > 5 μm fractions)
were signifi cantly higher than those in the free-living bacte-Table 13.2 Comparison (one-way ANOVA) between free and attached fractions of the various bacterial groups expressed as mean abundances or
as percentages of DAPI or EUB338 -stained bacteria in Lake Pavin
Free Attached P
Total DAPI cells (nb.ml −1 ) 3.8 ± 1.4 × 10^6 9.9 ± 3.6 × 10^4 <0.0001
EUB 338 (cell.ml −1 ) 1.9 ± 0.4 × 10^6 7.5 ± 0.9 × 10^4 0.043
EUB338/DAPI (%) 49.4 ± 11.8 76.6 ± 9.3 0.043
ALF 1b (cell.ml −1 ) 4.2 ± 1.2 × 10^5 1.1 ± 0.2 × 10^4 <0.0001
ALF1b/EUB338 (%) 21.9 ± 6.4 15.3 ± 2.1 <0.0001
BET42a (cell.ml −1 ) 5.5 ± 0.9 × 10^5 3.4 ± 0.4 × 10^4 <0.0001
BET42a/EUB338 (%) 29.1 ± 4.9 45.7 ± 6.1 <0.0001
CF319a (cell.ml −1 ) 4.7 ± 0.6 × 10^5 2.4 ± 0.4 × 10^4 0.008
CF319a/EUB338 (%) 24.7 ± 3.1 32.2 ± 5.3 0.008
Mean values were calculated from data of different dates and depths
Modifi ed from Lemarchand et al. ( 2006 )
Table 13.1 Average values for bacterial colonization of TEP in Lake Pavin during spring 2000
Size interval Bacteria per particle Bacteria per unit area (N per μm^2 ) Number of attached bacteria (× 10^3 ml −1 )
2–5 μm 3.05 0.32 0.04
5–10 μm 6.18 0.21 0.81
10–15 μm 10.47 0.17 1.21
15–20 μm 16.28 0.12 0.90
Modifi ed from Carrias et al. ( 2002 )
J.-F. Carrias et al.