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Extraction of Extracellular Polymeric Substances

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by

Hannah Thomas

on 2 October 2014

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Transcript of Extraction of Extracellular Polymeric Substances

Extraction of Extracellular Polymeric Substances from Extreme Acidic Microbial Biofilms
Extracellular Polymeric Substances
EPS are a complex mixture of:
- Proteins
- Neutral hexoses
- Acid polysaccharides
- Lipids
- DNA
- Humic acid substances

Essential for the development of a biofilm matrix

Attract attention because of their potential to remove toxic heavy metals from water.


Extreme Acidic Environments
Unusually high levels of heavy metals

Rio Tinto river in Spain maintains a constant low pH (0.9-2.5)

Buffered by ferric iron and have high concentrations of heavy metals

Low pH could affect the charge of the functional groups of the EPS changing their metal binding capabilities
Aim of the Study
To evaluate:
the influence of low internal biofilm pH in the heavy metal sorption of the EPS

their heavy metal extraction effectiveness

To compare:
Five different EPS extraction methods
Sample Collection

Three eukaryotic biofilms taken from the bottom surface of the river (name RT LZ and ST)

Sterile plastic spatula

Sub-samples taken for:
- EPS analysis
- Identification of eukaryotic components

Water samples filtered: 0.45um membrane

Concentrations of 10 recoverable metals were measured
Extraction of Colloidal and Capsular Exopolymers
Carbohydrates and proteins that are loosely bound
Tightly bound compounds
Freeze-Drying
Chemical Characterisation of EPS
Protein and humic acid contents Lowry Method

Neutral hexose sugars Dubois Method

DNA content Burton Method

Metal content Guibaud Method

G6PDH activity Trinity Biotech Kit



Results from biochemical analysis were statistically compared by analysis of variance using Statistica V.6.0
Fraction
Method
Results: Community Description
Mixture of microorgansims dominated by one or two species

RT biofilm: 80% Euglena mutabilis
15% Pinnularia sp.

LZ biofilm: 95% Chlorophyta Chlorella sp.

ST biofilm: 85% Filamentous green algae
10% Unicellular red algae

The remaining species in all biofilms were amoebas and flagellates
Results: Different Extraction Techniques
Higher EPS with NaCl + Crown Ether
Lowest EPS with MilliQ water
Colloidal/Capsular ratios similar in 3 biofilms except for MilliQ
EPS Chemical Characterisation
G6PDH Activity:
NaCl: highest amount of activity
No significant differences between other methods

Hexose Sugar Concentrations:
MilliQ: lowest concentrations
No significant differences between other methods

DNA Content:
NaCl + MilliQ: 5x more DNA than others
No significant differences between other methods

Humic acid:
no significant differences

Heavy metal extraction:
EDTA: highest yield
No significant differences between other methods
All primarily composed
of hexose sugars
Humic acid and
heavy metals - second main components
Protein + DNA
lowest values in
composition
Conclusions
High levels of heavy metals in the colloidal fraction support a significant protective role of EPS against their toxic effect

Heavy metal composition in EPS greatly resembled the composition of the surrounding water

No single extraction method can extract all the potential EPS components with the same efficacy

Combined extractions are needed for more precise characterisation
Any Questions
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