Geology Reference
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10 mL CaCl
2
and 1.5 mL NaHCO
3
solution. Blank
experiments without polysaccharides were prepared
by adding agarose beads into the mixture containing
10 mL CaCl
2
and 1.5 mL NaHCO
3
solution. Initial
saturation index in respect to calcium carbonate
(SI) is 1.96.
Each day for 5 days, vials were gently agitated
and samples were extracted with a sterile syringe
and filtered through 0.2 mm polycarbonate filters,
washed with NH
3
solution (pH ΒΌ 8), air-dried and
filters were stored in a desiccator. Dry material
was then deposited onto the SEM stub with carbon
tabs. The carbon layer underneath the particles
allowed us to analyse the uncoated specimens. The
morphology of the precipitates was characterized
by scanning electron microscopy (SEM, Philips
XL30, LaB6 filament) and the elementary compo-
sition of the crystals was determined qualitatively
with an EDAX EDS detector.
Functional groups revealed by infrared
spectroscopy
FTIR spectra (Fig. 4) revealed extensive homology
between the samples and indicated the presence of
the same functional groups mentioned in previous
studies (Comte et al. 2006c; Beech & Tapper
1999). Absorption bands have been assigned to the
different functional groups of the skeleton, that is,
ether, carboxylic, carboxylate or sulphate groups.
All of the samples analyzed in this study were
characterized by a broad band above 3000 cm
21
and intense absorptions of around 1650 and
1050 cm
21
(Fig. 4). Characteristic absorption
peaks of around 3500-3200 cm
21
reflect the
stretching of the N-H bond of amino groups
present in the polymers. This N-H stretching peak
lies in a spectrum region occupied by a broad and
strong band (3500-3000 cm
21
), which may be
due to hydroxyl groups that are hydrogen bonded
to various degrees. The weak peak at 2850 cm
21
indicates the presence of saturated carbohydrates
in samples of PCC strains. The C-H stretching
bands between 2800-3000 cm
21
were poorly
resolved and their intensities were weak.
The corresponding CH
2
deformationmodes were
located in the region 1430-1400 cm
21
. Protein
related bands, the nCvO of amide I was present at
1650 cm
21
and the region in the spectrum of poly-
saccharides. However, the presence of N-acetyl
groups may also be manifested by the absorbance
band in this range (Beech & Tapper 1999).
Vibrations due to the carbohydrate backbone
were common in all spectra. Strong complex
absorptions, centred between 1060-1080 cm
21
for
the exopolymers, are ascribed to complex vibrations
of the carbohydrate skeleton, ring structures, includ-
ing bending, stretching and coupling between these
modes (Beech & Tapper 1999). The absorption
peaks between the 1000-1200 cm
21
regions ascer-
tained the presence of gluuronic and mannuronic
acids, the main carboxylic building blocks of algi-
nate (Kazy et al. 2002). Absorption bands at a
region of 1350 cm
21
, assigned to the nCZOof
carboxylic acids, suggested that the exopolymer
were acidic. The significant differences between
the spectra of PCC 7942 and Syn Red are observed
in the sugar/sugar phosphate region at around
950 cm
21
.
The complex absorptions at the c. 2920 cm
21
region are ascribed to the asymmetric stretching of
nCZH bond of ZCH
2
groups combined with that
of the CH
3
groups. The corresponding symmetric
stretching of the same bond was found at the
c. 2850 cm
21
region (Beech & Tapper 1999). The
observed peaks in the spectra at the 1400 to
1450 cm
21
region are characteristic for the presence
of carboxyl groups (Kazy et al. 2008).
Extraction protocol
The content and composition of EPS require eluci-
dation to clarify their role in various geochemical
processes. However, the first step in the studies,
the extraction protocols of extracellular polymers
is a matter of debates. Comte et al. (2006a)
noted that applied chemical reagents could con-
taminate collected EPS. Further study by Comte
et al. (2007) revealed that applied chemical reac-
tants could affect the high-pressure size exclusion
chromatography fingerprint of EPS whereas phys-
ical extraction methods only affect correspond-
ing molecular weight distributions. Additionally,
the authors noted that physical means (such as cen-
trifugation) were either inefficient for extraction or
could induce significant cell lysis (e.g. heating) and
contaminate the EPS. Recently, extracellular
polymeric substances were extracted from aerobic
granules using seven extraction methods (Adav &
Lee 2008). Aerobic granules are compact bio-
aggregates with a compact interior structure.
Ultrasound followed by the chemical reagents for-
mamide and NaOH outperformed other methods in
extracting EPS from aerobic granules of compact
interior. The collected EPS revealed no contami-
nation by intracellular substances and consisted
mainly of proteins, polysaccharides, humic sub-
stances and lipids. We just started the work on
the role of extracellular polymeric substances of
picocyanobacteria strains in geochemical pro-
cesses. The work is now in progress to compare
this other extraction protocol in terms of quantities
and qualities of extracellular polymeric substances
(Comte et al. 2006b). More research is needed to
determine the most effective protocol for extra-
cellular polymers collections.
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