Ecology of Acidovorax in activated sludge

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CHAPTER 2:Materials and Methods

General buffers and solutions

The water used throughout this study was sterile, micro-filtered and deionised by a Millipore purification system. Unless stated otherwise, solutions were sterilised by autoclaving at 121 °C and 15 psi for 20 minutes. Heat sensitive solutions were sterilised by membrane filtration using a 0.2 µm membrane (Sartorius AG, Gottingen, Germany).

Microbiological methods
Unless stated otherwise, bacterial culture and enumeration were carried out according to methods described in Standard Methods (Eaton, 2005). All centrifugation was performed in an Eppendorf 5810, a Sorvall® RC-5B or a Biofuge Stratos (Thermo).

Bacterial strains and culture conditions

A number of different bacterial strains were used in this research. The strains and their required growth media are shown in Table 2.2. Specific mutant variants of the strains mentioned below are described in each separate chapter.

Bacterial growth conditions

Unless specified, A. temperans CB2 cultures were grown at 28 oC either on R2A agar (Difco) or in R2A broth (Difco) with shaking at 120 rpm. Overnight broth cultures were obtained by inoculating a single colony isolate into a 125 ml conical flask containing 20 ml of broth followed by shaking incubation at 120 rpm for 16 – 18 hours.For attachment experiments overnight cultures of A. temperans CB2 Hpos were diluted 1:100 in R2A broth. From the resulting culture 100 µl was used to inoculate 4 ml of fresh R2A broth in 20 ml universal bottles. These cultures were incubated with shaking for up to seven days. To investigate the influence of cell attachment to glass wool, approximately 15 mg of 10 – 30 mm long glass wool fibres were added to half of the universal bottles (GW+) and sterilised by autoclaving prior to inoculation.

Optical density measurements

For optical density measurements, 1 ml bacterial culture was transferred to a plastic cuvette (Biolab) and the absorbance was measured at 600 nm (OD600) using a Helios Beta spectrophotometer (Thermo Scientific). Dilutions were prepared to maintain the linear relationship between cell densities and OD600 measurements. Cell density was estimated by direct plate count enumeration of viable cells (Eaton et al., 2005).

Glycerol stock preparation

Glycerol stocks were made by freezing 1 ml of overnight grown culture in a final glycerol concentration of 15% at -80˚C.

 Media

All media were sterilised by autoclaving at 15 psi for 20 min. Solid media was made from liquid media with the addition of 1.5% (w/v) of granulated agar (Invitrogen).

ABSTRACT
ACKNOWLEDGEMENTS 
TABLE OF CONTENTS 
ABBREVIATIONS 
LIST OF FIGURES 
LIST OF TABLES 
1 GENERAL INTRODUCTION
1.1 The biofilm mode of life 
1.2 Wastewater treatment
1.2.1 Activated sludge
1.2.1.1 Microbial flocs
1.2.1.2 Cell-cell interactions
1.2.1.3 Surface attachment
1.3 Acidovorax temperans 
1.3.1 Ecology of Acidovorax in activated sludge
1.3.1.1 A. temperans phenotype variation
1.3.1.2 Genome sequence
1.4 Exopolysaccharides (EPS) 
1.4.1 Introduction
1.4.2 Gram-negative cell wall
1.4.2.1 Lipopolyssacharide
1.4.2.2 Lipid A
1.4.2.3 Core oligosaccharide
1.4.2.4 The O-specific polysaccharide (EPS)
1.4.3 Generally accepted functions of bacterial EPS
1.4.3.1 Cell-cell recognition, aggregation and structural function
1.4.3.2 Viral infection/antibiotics
1.4.3.3 Nutrition
1.4.4 Commercial polysaccharides
1.4.5 EPS of closely related proteobacteria
1.4.5.1 Pseudomonas
1.4.5.2 Rhizobium
1.4.5.3 Betaproteobacteria
1.4.5.4 Ralstonia
1.4.5.5 Burkholderiales
1.4.5.6 Leptothrix
1.4.5.7 Comamonadaceae
1.4.5.8 Summary of EPS compositional studies
1.4.6 Carbohydrate analysis
1.4 Research Objectives 
2 MATERIALS AND METHODS 
2.1 General buffers and solutions 
2.2 Microbiological methods 
2.2.1 Bacterial strains and culture conditions
2.2.2 Bacterial growth conditions
2.2.3 Optical density measurements
2.2.4 Glycerol stock preparation
2.2.5 Media
2.2.6 Gram stain
2.2.7 Biofilm assay
2.3 General nucleic acids techniques 
2.3.1 Agarose gel electrophoresis
2.3.2 Preparation of plasmid DNA
2.3.3 Nucleic acid quantification
2.3.4 RNA isolation
2.3.5 Genome sequence and random mutagenesis
2.4 Microscope analysis 
2.4.1 Light microscopy
2.4.2 Fluorescent microscopy
2.4.3 Electron microscopy
2.4.3.1 Preparation TEM grids
2.4.3.2 Preparation of bacterial cells for TEM viewing
2.4.3.3 Embedding of cells in resin
2.4.3.4 Immuno-labeling of thin sections
2.4.3.5 Cryo SEM
2.5 Protein visualisation and quantification 
3 ANALYSIS OF EXTRACELLULAR POLYSACCHARIDES
PRODUCED BY Acidovorax temperans CB2 
3.1 Introduction 
3.1.1 EPS in activated sludge
3.2 Aim 
3.3 Materials and Methods 
3.3.1 EPS Production, harvest and purification
3.3.1.1 Bacterial growth
3.3.1.2 EPS harvest
3.3.1.3 Total carbohydrate assay
3.3.1.4 Amino- and N-acetyl assay
3.3.1.5 Rheological test
3.3.2 Analysis of monosaccharide composition
3.3.2.1 Acid hydrolysis of EPS
3.3.2.2 Thin layer chromatography on monosaccharides
3.3.2.3 Conversion of monosaccharides to alditol acetates
3.3.2.4 Gas chromatography (GC) of alditol acetates
3.3.2.5 Gas chromatography mass spectrometry (GC-MS) of
alditol acetates
3.3.2.6 Uronic acid assay
3.3.2.7 Methylation
3.4 Results 
3.4.1 A. temperans CB2 growth characteristics
3.4.2 Light microscopy
3.4.3 Transmission electron microscopy (TEM)
3.4.4 EPS isolation, purification and recovery
3.4.4.1 Cell growth
3.4.4.2 EPS isolated and purification
3.4.4.3 EPS recovery
3.4.4.4 EPS purification
3.4.5 Thin layer chromatography of hydrolysed EPS
3.4.6 Gas chromatography (GC)
3.4.6.1 Monosaccharide composition
3.4.6.2 Analysis of the carbohydrate composition of the media
3.4.7 Combined gas chromatography-mass spectrometry (GC-MS)
3.4.8 Linkage analysis
3.4.9 Identification of candidate genes involved in the formation of
nucleotide sugars
3.4.9.1 Glycosyl transferases
3.5 Discussion 
3.5.1 Microscopy
3.5.2 EPS isolation and properties
3.5.3 EPS composition
3.5.4 EPS structure determination
3.5.5 EPS related genomic analysis
3.5.6 Phase variation
4 THE OCCURRENCE AND FUNCTION OF EXTRACELLULAR DNA PRODUCED BY Acidovorax temperans CB2 
4.1 Introduction 
4.1.1 Genetic structure of Type IV pili
4.1.2 Acidovorax motility phenotypes
4.2 Aim 
4.3 Materials and methods 
4.3.1 Culture conditions
4.3.2 Cell attachment assays
4.3.3 MALDI-ToF protein analysis of DNase I
4.4 Results 
4.4.1 DNase effect of biofilm formation in microtiter plates
4.4.2 DNase activity
4.4.3 DNase purity
4.4.4 Influence of enzymes on cell viability
4.4.5 Attachment to glass wool
4.4.6 Disruption of attached cells with DNase
4.4.7 Effect of DNase on attached cells
4.4.8 Mechanism of eDNA attachment
4.4.9 Role of electrostatic interactions in attachment
4.4.10 Relationship of eDNA to cell density and viability
4.4.10.1 Growth curve
4.4.11. Visualisation of attachment structures
4.4.11.1 Light microscopy
4.4.11.2 Fluorescent microscopy
4.4.11.2.1 Visualisation of eDNA
4.4.11.3 Transmission electron microscopy
4.4.11.3.1 Antibody labeling
4.4.11.4 Cryo SEM visualisation of the attachment matrix
4.4.11.4.1 DNase treatment
4.5 Discussion 
4.5.1 Use of DNase to identify eDNA-mediated attachment
4.5.2 Role of eDNA in A. temperans CB2 Biofilm formation
4.5.3 Attachment to glass wool
4.5.3.1 Addition of DNase
4.5.4 Concentration and source of extracellular DNA
4.5.5 Mechanism of attachment
4.5.6 Microscopic analysis of attachment networks
5 CONCLUDING DISCUSSION
5.1 Introduction 
5.2 Characterisation of extracellular polysaccharides 
5.3 EPS composition and properties 
5.4 The occurrence and function of extracellular DNA 
5.5 Prevention of attachment 
5.6 DNA versus EPS 
5.7 Final conclusion 
REFERENCES

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Extracellular polymers of Acidovorax temperans

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