Molecular analysis

The following sections detail the molecular analysis aspect of the current study, which were undertaken at the Cardiff School of Bioscience, Cardiff University. The biofilms incubated within the current study were evaluated and compared by 16S ribosomal ribonucleic acid (rRNA) gene polymerase chain Reaction-denaturing gradient gel electrophoresis (PCR-DGGE), DNA and EPS quantification.

3.7.1 EPS and DNA extraction protocol

The protocol used within the current study for EPS and DNA extraction was modified from Brown and Lester (1980) as described by Zhang et al. (1999). Zhang et al. (1999) found that the protein and carbohydrate fractions extracted using the method outlined by Brown and Lester (1980) compared well to other commonly used extraction protocols. The modified method is outlined herein.

To the cotton bud (with collected biofilm) contained within a 1.5 ml non-stick tube 1.0 ml of sterile phosphate buffer saline (PBS) was added and mechanically shaken (Lab Line, Multi-Wrist Shaker) for 10 minutes. The cotton bud and PBS was then sonicated in a ultrasonic water bath (Grant XB2) for 30 s at approximately 60 Hz; and then centrifuged (Eppendorf 5424) for 10 minutes at 6000 g. The supernatant was then removed and placed in a sterile 2.0 ml (non-stick) centrifuge tube. This stage was referred to as the wash step.

The bud was then re-extracted, by adding 1.0 ml of 2% ethylenediaminetetraacetic acid (EDTA) in PBS. The solution was then sonicated for 30 s and incubated for 3 h at 4 ⁰C. After the incubation period the supernatant was then removed and added to the wash step supernatant. The combined supernatant was then used for EPS evaluation.

The remaining cellular material and cotton bud were then further centrifuged for 5 minutes at 14000 g. The cell pellet which formed was then used for DNA extraction and subsequent bacterial community analysis.

The supernatant and pellet obtained from this process were defined as the primary EPS and DNA extract. The whole EPS extraction process was then repeated on the extracted cotton bud and the resultant supernatant and pellet were defined as the secondary extract.

The efficiency of the employed biofilm removal technique was an important consideration within the current study. Cotton swabs have been found to be more effective than polyester and Rayon swabs at removing substrate from surfaces (Rose et al. 2004). Moreover, it is widely suggested that when using cotton swabs multiple passes of the same area are undertaken (Rose et al. 2004; Assere et al. 2008). Both of these criteria were met within the current study. ESEM images taken before and after swabbing indicated the effectiveness of this studies removal technique on a microscopic level (see Figure 3.29). The ESEM images presented in Figure 3.29 were captured as part of a different aspect of the current study although, the removal procedure was the same as outlined within the current section.

Figure 3.29 ESEM images showing a surface at 200x magnification: a) before incubation, b) after incubation and c) after incubation and swabbing (sample size: 0.5x0.5mm²).

3.7.2 Total carbohydrate and protein assays

A typical EPS matrix will contain a wide variety of extracellular constituents, including polysaccharides (i.e. carbohydrates), proteins, nucleic acids and lipids. Carbohydrates and proteins are generally the largest constituent of the matrix, representing over 50% of the overall EPS fraction (Horan and Eccles 1986; Jahn and Nielsen 1998; Wingender et al. 2001;

a) Before incubation b) After incubation c) After incubation and swabbing

Tsuneda et al. 2003; Flemming and Wingender 2010; Andersson et al. 2011). In particular, Tsuneda et al. (2003) found that proteins and polysaccharides can potentially account for 75-90% of a biofilm’s overall EPS. Furthermore, carbohydrates and proteins have been found to contribute to several essential biofilm properties, namely mechanical stability and cohesion (Pratt and Kolter 1999; Wloka et al. 2004; Simoes et al. 2007; Celmer et al. 2008, Flemming and Wingender 2010, Ahimou et al. 2010). Consequently, only the carbohydrate and protein fractions were analysed within the current study.

3.7.2.1 Total extracellular carbohydrate concentration

The total carbohydrate concentration in the EPS for the respective biofilms was determined using a standard phenol-sulphuric acid based assay kit (Sigma MAK104). Glucose (2.0 mg/ml solution) was used as the calibration standard in the range 0-20 µg/50µl (i.e. 0-400 µg/ml).

The procedure used to determine the total carbohydrate concentration was modified from the manufacturer's specification and is documented herein.

Firstly the required glucose standards were prepared. Glucose (2mg/ml solution) of concentrations 0, 2, 4, 6, 8 and 10µl was added to a 1.5ml (non-stick) centrifuge tube. The total volume of the solution was brought up to 50 µl using sterile nuclease-free molecular-grade water. The EPS sample of volume of 50 µl was then added to a 1.5 ml centrifuge tube.

Concentrated sulphuric acid of volume of 150 µl was added to each of the centrifuge tubes.

The solutions were then incubated for 15 minutes at 90 ⁰C in a heating block (Techne Dri-Block-3). The samples were protected from natural light during the incubation period. After the incubation period 30 µl of phenol-based developer was added to each of the centrifuge tubes. The solutions were then left for 5 minutes before being transferred to 1.5 ml cuvettes (Bio-one 613101). The volume of the solutions was made up to 1.0 ml, by using sterile nuclease-free molecular-grade water. The absorbance was then measured using a Spectrophotometer (Jenway 6300), using a wavelength was set to 490 nm.

A preliminary analysis of the unused cotton buds indicated that they contained a considerable amount of carbohydrate (see Appendix A.8, Table A.5). In particular, repeatability tests indicated that the cotton buds had carbohydrate concentration of 279.45 ± 40.98 µg/ml (Based on 7 repeats). Carbohydrate within the bud was thought to have derived from the cellulose within the cotton. The significant carbohydrate concentration within the cotton bud would have had a considerable impact on the established extracellular carbohydrate concentrations.

Consequently, 279.45 µg/ml was subtracted from all carbohydrate measurements.

Nevertheless, the documented measurements should be used with caution.

The levels of protein from the unused cotton swabs was below the detection limit in all assessed cases and thus, negligible.

3.7.2.2 Total extracellular protein concentration

The total concentration of protein in the EPS was measured using the standard Bradford assay (Sigma B6916), with bovine serum albumin (BSA) as the calibration standard in the range 0-20 µg/ml. The procedure used to determine the total protein concentration was outlined by Bradford (1976). The absorbance was measured at 595nm using a Spectrophotometer (Jenway 6300).

Typical standard curves for protein and carbohydrate are presented in Appendix A.8 in Figure A.13. It should be noted that all standard curves used within the current study had R² values of at least 0.95.

3.7.3 DNA and community analysis 3.7.3.1 DNA extraction and purification

The total community genomic DNA was extracted from the biofilm samples using a standard DNA isolation kit (Next-Tec X150). The procedure was per the manufacturer’s specification with the exception that after adding 90.0 μl of Buffer, 10.0 μl of Lysozyme and 20.0 μl of RNase A the sample was mechanically shaken (Lab Line, Multi-Wrist Shaker) for 5 minutes.

In addition, in the final stage all of the extracted DNA was passed through the filter column resulting in a total volume of 200 µl. The DNA extracts were then stored at -80ºC until required for Polymerase Chain Reaction (PCR) amplification. DNA extractions from unused cotton buds were also carried out and analysed, as a negative control.

3.7.3.2 PCR conditions

To minimise potential contamination issues, the PCR was carried out under aseptic conditions using autoclaved and/or UV-treated instruments and sterile nuclease-free molecular-grade water. The amplification of Bacterial 16S rRNA gene sequences were amplified by nested PCR using primer combination 357F-GC-518R (Webster et al. 2003). All PCR reactions were performed within a DNA Engine Dyad Thermal Cycler (MJ Research). PCR conditions were as described by Muyzser et al. (1993) and Webster et al. (2003).

Sterile nuclease-free molecular-grade water and Acetobacterium sp. Ac1 DNA was used as a negative and positive control, respectively in all sets of PCR reactions.

The reaction mixtures were held at 95°C for 5 minutes followed by 10 cycles of 94°C for 30 seconds, 55 °C for 30 s and 72°C for 60 s plus 20 cycles of 94°C for 30 s, 52°C for 30 s and 72°C for 60 s, with an extension step of 5 minutes at 72 °C. A typical set of PCR products

are present in Figure 3.30 demonstrating that that 16S rRNA gene PCR products were approximately 200 bp.

Figure 3.30 A typical Polymerase Chain Reaction (PCR) product.

The bacterial diversity within the biofilm and water samples was determined carrying out Denaturing Gradient Gel Electrophoresis (DGGE) on the nested PCR products (Schäfer and Muyzer 2001; Webster et al. 2002; 2006).

3.7.3.3 DGGE analysis

The PCR products were separated using a DCode Universal Mutation Detection System (Bio-Rad Laboratories) and 1.0 mm thick (16.0 x 16.0 cm² glass plate) 8.0% (w/v) polyacrylamide gels (Acrykogek 2.6 solution, acrylamide: N,N’-methylenebisacrylamide; 37:1; BDH Laboratory Supplies) with denaturant gradient between 30.0% and 60.0% (Webster et al.

2003). The polyacrylame gels were prepared with a 1 x Tris-Acetate EDTA (TAE) buffer (pH 8; 40mM Tris base, 20.0mM acetic acis, 1.0mM EDTA), using a 50.0 ml volume Gradient Mixer (Fisher Scientific). The electrophoresis was performed at 60 ºC and 200 Volts for 5 h (with an initial 10 minutes at 80 Volts). The polyacrylame gels were stained with SYBRgold nucleic acid gel stain (Molecular Probes) for 30.0 minutes and viewed under UV.

A Gene Genius Bio Imaging System (Syngene) was used to capture images of the Gel.

Distinguishable DGGE bands were excised from the gel and washed in sterile nuclease-free molecular-grade water for 10 minutes. The bands were then air-dried, crushed and re-amplified by PCR for Sanger sequencing as described by O'Sullivan et al. (2008). The sequencing within the current study was undertaken by Eurofins Genomics GmBH.

DNA Ladder

(Bioline hyperladder I)

DNA Samples Control Samples (bud)

+ and – PCR controls

3.7.3.4 Total DNA quantification

The total DNA concentration was measured within the biofilm and bulk water samples using a fluorescent dye assay kit (Quant-iT PicoGreen dsDNA) and a multimode microplate reader (Tecan Infinite M200 Pro). Samples and standards were prepared on a 96 microplate (OptiPlate-96F, black) as per the manufacturer’s specification. The pre-defined PicoGreen programme (Magellon 7.1) was used to calibrate the microplate reader. Critically, the absorbance was measured at 485 nm. A typical standard curve for the DNA concentration is presented in Appendix A.8 in Figure A.14 (R² = 0.99).

A strong linear relationship has been reported within the literature between DNA concentration and the total acridine orange direct cell concentration (McCoy and Olson 1985). The recommendations outlined by McCoy and Olson (1985) were used within the current study to estimate the total cell concentration within biofilm and bulk water samples.