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Figure 10 gives a tentative overview of the distri-
bution of clone sequences among the biofilm and the
core layers. It is obvious that the biofilm is com-
posed mostly of cyanobacterial OTUs (operational
taxonomic units), discriminating the presence
of other taxa, possibly caused by PCR bias (von
Wintzingerode et al. 1997) and by the insufficient
number of clones analysed. Indeed, though several
hundred clones were analysed from each layer, rare-
faction curves (unpublished) indicated that none of
the seven clone libraries were sufficiently sampled
to reach diversity saturation.
While cyanobacterial are still present in large
numbers in layer 1, they are insignificant in deeper
layers. Proteobacteria emerge as a major taxon in
layer 1 and dominate the deeper layers. Other taxa
appear in low (2 - 12%) but rather constant per-
centage in layers 3 through 6, i.e. Bacteriodetes,
Acidobacteria, Planctomycetes, and Actinobacteria.
OTUs of different candidate phyla especially
emerge through layers 1 to 6 but were not found in
each layer.
common to both studies. Acidobacteria are major
contributors to diversity in all 4 tufa sites, ranging
between 7.3 and 12.8% in the Taiwanese samples
and 10.4% in the WB sample. The percentage
values are also high for Alphaproteobacteria (6.0 -
12.9% versus 19.5% for the WB tufa) and Beta-
proteobacteria (4.5 - 8.8% versus 9.5%). On the
other hand, Gammaproteobacteria constitute a
lower part of the WB community (5% versus 9.6 -
15.9%), while the presence of Firmicutes of the
WB tufa (6.6%) only matches that of the Eternal
Spring Shrine tufa sample (6.4%). No Firmicutes
were identified in the other two Taiwanese
samples. Phylum affiliation of clones between the
geographically different freshwater samples may
be higher in case the high portion of the unassigned
clone sequences (.30%) of the Taiwanese studies
will be more thoroughly analysed.
Extracellular polymeric substances
As in other mineralizing biofilm systems, such as
marine and lacustrine cyanobacterial microbialites
(e.g. Trichet & D´farge 1995; D´farge et al. 1996;
Arp et al. 1998; Kawaguchi & Decho 2002;
Gautret et al. 2004; Decho et al. 2005), extracellular
polymeric substances (EPS) are considered to play a
crucial role by providing mineral nucleation sites in
tufa-forming biofilms of karstwater streams (Pente-
cost 1985). Microbiologically produced EPS are
involved in calcium carbonate precipitation by pro-
viding diffusion-limited microenvironments that
create alkalinity gradients in response to metabolic
processes, and by attracting and binding of cal-
cium ions to negatively charged sites. This would
result in an inhibition of precipitation (Kawaguchi
& Decho 2002). However, EPS is under constant
modification through physico-chemical alteration
(e.g. by UV radiation, pH, free radicals) and/or
microbial degradation (e.g. hydrolysis, decarboxy-
lation) (for review see Dupraz & Visscher 2005).
In the present study, biofilm structure and spatial
distribution of bacteria and phototrophic organisms
as well as extracellular polymeric substances (EPS)
within tufa-forming biofilms were investigated in
the fully hydrated state using multi-channel con-
focal laser scanning microscopy (CLSM). Analysis
of tufa-forming biofilms by CLSM revealed that
extracellular polymeric substances can be divided
into three major structural domains. Lectin-binding
analysis firstly allowed the detection of EPS glyco-
conjugates (i.e. polysaccharides, including those
ones covalently linked to proteins and/or lipids)
which were clearly associated with phototrophic
organisms (Fig. 11a, d, g). Secondly, network-like
EPS glycoconjugates were detectable as extended
sheet-like structures (Fig. 11b, e, h). Thirdly,
glycoconjugates were found as more diffuse and
Molecular assessment and interpretation. It is not
surprising that the distribution of sequences of
clones and isolates differ significantly, inasmuch
the isolation did not attempt to recover phototrophic
(e.g. Chloroflexi), autotrophic, lithotrophic and
anaerobic organisms. The swarming capacities of
many fast growing flavobacteria and colonies with
slimy appearance also hindered the growth of
slowly growing heterotrophic cultures (e.g. Acido-
bacteria, Planctomycetes, Verrucomicrobia, Gem-
matimonadetes) and the inability of members of
candidate phyla to grow on any of the artificial
growth media provided is well known. The fact
that Firmicutes, e.g. Bacillus and Paenibacillus,
are present among the isolates while almost absent
in the clone libraries (Fig. 10) can be attributed to
the spore stage in which these organisms may rest
in the tufa core matrix. Spores are known to resist
most of the less harsh DNA isolation techniques,
applied to avoid shearing of DNA which would
support the formation of chimeric PCR structures.
Those sequences clustering with candidate phyla
or representing novel candidate phyla will be fully
sequenced in order to better analyse the presence
of chimeric structure.
Comparison with the results of Ng et al. (2006)
on non-cyanobacterial taxa from three freshwater
tufa environments in Taiwan is hardly possible. In
contrast to this study, not individual layers but 3
homogenized core samples were pooled and ana-
lysed. For comparison we therefore present the
Westerh ¨fer rivulet (WB) data as if they were ana-
lysed from a pooled sample as well. Though the
number of phyla listed by Ng et al. (2006) is much
lower, comparison is possible with a few phyla
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