Geology Reference
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to calculate ion activities and PCO 2 of the water samples as
well as saturation state with respect to calcite. Hydroche-
mical model calculations have been carried out using the
same program. Contrary to an incorrect statement
in Altermann et al. (2006: 156), the model calculations
take into account the formation of ion pairs and the pH
control of HCO 3 2 :CO 22 ratios. Total carbon, nitrogen
and sulfur determination on tufa carbonate samples was
conducted with a EuroEA CNS-analyser (Hekatech), and
organic and inorganic carbon content was analysed with
a Multiphase Carbon Determinator LECO RC - 412.
SSU rDNA cloning and sequence analyses of diatoms
and cyanobacteria: In brief, SSU rDNAs were amplified
using PCR primers that preferentially bind to rDNAs
of cyanobacteria (Wilmotte et al. 1993) or diatoms
(N. Brinkmann, unpubl.) from DNA directly extracted of
biofilm samples, cloned and sequenced. Phylogenetic
trees were generated using all cyanobacterial 16S and
diatom (raphid and araphid pennate) 18S rDNA sequences
as currently (May 2009) available from public databases
within the ARB database programme (Ludwig et al.
2004; www.arb-home.de). To this the newly determined
sequences were added using the parsimony algorithm
tool in ARB. An alignment of full length sequences
extracted from ARB was then subjected to phylogenetic
analyses using the Maximum Likelihood method. The phy-
logenies were obtained with the programme Treefinder
(Jobb 2008). Confidence values for the obtained groups
(edge support) were inferred from expected-likelihood
weights (Strimmer & Rambaut 2002) applied to local
rearrangements of tree topology as provided in Treefinder.
Only values at or above 87% were recorded and support
at
analysed by the RDP Classifier program (http:// rdp.cme.
msu.edu/classifier/classifier.jsp).
Laser-scanning microscopy and lectin-binding: Due to
the occurrence of different pigments in cyanobacteria
(chlorophyll a, phycocyanin and phycoerythrin) and
algae (chlorophyll a), the specific autofluorescence of
various phototrophic organisms was recorded in two
different channels. Signals from phycocyanin and phyco-
erythrin were excited at 561 nm and detected in the
range of 585 - 625 nm. Autofluorescence of chlorophyll a
(chla) was detected in a second channel (excitation
633 nm; emission 650-800 nm) (Neu et al. 2004). Bacterial
cell distribution and biomass were determined after stain-
ing with nucleic acid-specific fluorochrome SYTO 9
(Molecular Probes, final concentration 1 mgml 21 ). Glyco-
conjugate distribution within biofilms was measured by
using fluorescence lectin-binding analysis (FLBA) (Neu
et al. 2001). A screening with 76 commercially available
lectins revealed that approximately 30 lectins with differ-
ent specificities were suitable for the detection of EPS
within tufa-forming biofilms of both creeks investigated.
Detailed information of methodological procedures and
results are given in Zippel et al. (submitted). Calcein
(Sigma) was used for the detection of calcium carbonate
present in tufa-forming biofilms. Tufa pieces were
stained with the calcein solution (10 mgl 21 ) for 12 h at
room temperature (Moran 2000).
Methods for embedding and sectioning of tufa biofilm
samples have been described in Arp et al. (2001b, 2003)
and Shiraishi et al. (2008c). For microelectrode measure-
ments and flux calculations see Bissett et al. (2008a, b)
and Shiraishi et al. (2008a, b). Mass balance calculations
of Ca loss via photosynthetic biofilms versus Ca loss
from bulk water system have been calculated as follows:
(1) The annual Ca loss via biofilms [mol year 21 ] is the
annual average flux of PS-induced CaCO 3 deposition
(1.85 - 2.86 10 26 mol m 22 s 21 ), multiplied by the
biofilm surface area recorded by field mapping (94 m 2 )
and time (60 60 12 365 seconds per year): 2.7 -
4.2 10 3 mol year 21 ; (2) The total annual Ca loss
from creek waters [mol year 21 ] is the decrease in Ca
concentration during the course of the stream
(0.35 mmol L 21 ), multiplied by the water flow rate at
site WB 5 (c. 2.0 L s 21 ) and time (60 60 24 365
seconds per year): 2.2 10 4 mol year 21 . As a result, the
annual Ca loss via biofilms is only 12 - 19% of the
total annual Ca loss from stream waters.
internal
branch
lengths
(edges)
is
indicated
by
a
filled circle.
Non-phototrophic prokaryotes: In order to determine
whether the composition of a tufa biofilm differs from
that of the rivulet water to which it is exposed, a core
sample was drilled from tufa deposited at a downstream
site (23.05.2006). The core, 4 cm Ø and 5 cm deep, was
obtained using a modified Stihl motorsaw equiped with a
coring device. The core was immediately rinsed with
sterile 0.7% NaCl solution, transported to the laboratory
on ice, and then frozen at 280 8C. The tufa core sample
was separated into several layers (CL, core layers) of
c. 4 - 5 mm thickness, each of them corresponding to an
annual laminae couple of the tufa stromatolite; thus the
core represents the CaCO 3 deposition of about 10 years.
The assessment of the taxonomic status of isolates from
the biofilm and tufa layers followed isolation on R2A
medium. Partial 16S rRNA gene sequence analysis
(c. 500 bp) were analysed by BLAST (Altschul et al. 1997)
which allows a rough affiliation of sequences to homolo-
gous sequences of described taxa which are deposited in
public databases. For the identification and biodiversity
assessment of uncultivated prokaryotes, DNA was isolated
from the biofilm and from several 4 - 5 mm layers of
the tufa. Following sequence analysis and exclusion of chi-
meric sequences (RDP) 2.351 partial sequences were
References
A LI , Z., C OUSIN , S., F R ¨ HLING , A., S CHUMANN , P.,
Y ANG ,J.&S TACKEBRANDT , E. 2009. Description
of Flavobacterium rivuli, sp. nov., Flavobacterium
subsaxonicum sp. nov., Flavobacterium swingsii sp.
nov., and Flavobacterium reichenbachii sp. nov., iso-
lated from a hard water rivulet. International Journal
of
Systematic
and
Evolutionary
Microbiology, 59,
2610 - 2617.
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