3.3.1
Sampling
Unless otherwise stated all reagents were purchased from Merck Ltd, (Manukau City, New Zealand). The samples in this study were supplied from a primary treated dairy wastewater system of a dairy milk powder plant. Wastewater is collected from all areas of the manufacturing plant, treated using a DAF tank before irrigating onto pasture.
Six different samples, two biofilm and four wastewater samples were analysed in this study. One biofilm sample consisted of a frozen stock sample of the extreme biofilm that initiated this investigation. One wastewater sample collected after the DAF tank (DAF sample) was
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analysed using Next Generation Sequencing (NGS) to determine the complete microbial profile entering the wastewater system. The second biofilm sample (fresh biofilm), collected by scraping the inside of a wastewater storage silo with a clean 250ml sample pot and the remaining three wastewater samples (collected from different irrigator nozzles) were analysed for the culturable fraction due to the interest in their growth and biofilm forming ability. All wastewater samples were collected by opening a valve and left running for 30s to clean out stagnant wastewater before collecting fresh wastewater in a clean, sterile 250ml sample pot. Samples were transported chilled to the laboratory for isolation (a journey of about 24 h).
3.3.2
Next Generation Sequencing (NGS) and analysis
DNA from the DAF sample was extracted using a PrestoTM Mini gDNA Bacteria Kit. After extraction, the quality of the DNA was assessed using a 2% agarose EGel and the concentration was measured using a Colibri Spectrometer, (Berthold Detection Systems, Germany). DNA from the extreme biofilm was extracted utilising the same method, however, the quality of the DNA was not good enough for further analysis. The DAF sample DNA was sent to New Zealand Genomics Ltd. (NZGL; Massey Genome Service at Massey University, Palmerston North) where sequencing was carried out using an Illumina MiSeq 2× 250 base PE run.
The resulting sequences were analysed using the fastq-mcf tool algorithm from the ea- utils suite of tools (v. 1.1.2-621; ("ea-utils," 2016)) to remove any sequencing adapters from the read files. The sequences files were checked to ensure they had the same numbers of sequences, and a fastq to fasta converter script was used to generate fasta files. Each of the two fastq files were then sequentially mapped to a local copy of the NCBI nr database using the DIAMOND blastx algorithm (v. 0.7.9;(Buchfink et al., 2015)). This copy of the nr database had been previously indexed using DIAMOND. The reads were then converted into the DIAMOND equivalent of tabular BLAST+ output (format 6). These mapping results, and the fasta sequences were then used as input for MEGAN (v. 5.11.3;(Huson et al., 2007)) using default parameters to enable taxonomic viewing of the sequences.
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3.3.3
Isolation of culturable bacteria
Standard agar plate techniques were used in the isolation of the bacteria from the two- biofilm samples and three wastewater irrigator samples. The 14-streak method was performed on using the selective agars; Pseudomonas, MacConkey, and nutrient. These were then incubated at 10°C, 30°C and, 55°C for 48 h. Individual colonies were then immediately re-streaked onto the same agar to ensure pure single isolates were obtained. The pure isolates were then incubated in 20 ml of Tryptic soy broth (TSB) for 24 h. In total 23 isolates were obtained.
3.3.4
16S ribosomal RNA sequencing
To identify the unknown isolates, universal primers 27F (5’ AGA GTT TGA TCC TGG CTC AG) and U1492R (5’ TAC GGC TAC CTT GTT ACG ACT) (Edwards et al., 1989) targeting the 16S ribosomal RNA gene, were used for PCR to amplify an approximately 800 bp fragment for sequencing. The template for this PCR was prepared from a loop inoculation of each isolate, added to 20 ml of TSB and incubated at 30°C overnight. DNA was extracted crudely by heating the TSB culture to 80°C and holding for 10 min then cooling to room temperature.
The PCR mix was made using 25µl DNA/RNA free water, 20µl Mastermix (5 Prime MasterMix100 Runs GmbH, Germany) consisting of Taq polymerase, dNTPs and magnesium chloride, 1µl of each primer (10µmol) and 4μl of heat shocked (>80°C for 10 min) culture.
3.3.5
Extracellular polymeric substance (EPS) analysis
The extreme biofilm sample was washed and freeze-dried as described in Lanham (2012). PHA analysis was done using gas chromatography (Prominence, Shimadzu) equipped with an FID detector and fitted with a DB-5MS Ultra Inert (30 m length, 0.250 m diameter, and 0.25 µm film) column (Agilent Technology, USA) as described in Oehmen, et al. (2005).
Microscopy images were taken of mixed isolate biofilm samples smeared onto a glass slide. Cultures were fixed onto slide by applying heat then stained with 3% w/v Sudan Black in 70% Ethanol for 10 min and then counter stained with safranin for 10s. The sample was then observed on a bright field microscope (Mesquita et al., 2015; Wei et al.,2011).
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3.3.6
Microtiter plate biofilm assay
A microtiter plate assay (Oh et al., 2007) was used to determine the ability of the isolated bacteria to form biofilms. The microtiter plate assay is an important tool in assessing the potential for biofilm formation. The high through put capability of the test allows for the testing of multiple strains of bacteria under varying conditions (O’Toole, 2011). This method has been used for bacteria isolated from different environments such as dental (Yoshida & Kuramitsu, 2002), mussel production facilities (Nowak et al., 2017), pork meat processing (Wang et al., 2017) and dairy manufacture (Zain et al. 2016). For isolates in dairy manufacture, the assay has been widely used to screen for biofilm formation, even though the test cannot replicate the flow experienced in the manufacturing plant. This method was chosen as it allows a rapid assessment of initial biofilm formation, and is a test that measures biomass resulting from bacterial colonisation of the well surfaces (Azeredo et al.,2017). The microtiter plate assay is a convenient method to quickly assess the effect of various nutrients and ion levels on biofilm formation and was therefore chosen for the present study.
Three wells of a sterile 96 well tissue culture plate (Falcon, In Vitro Technologies NZ PTY LTD) were inoculated with 20 µl bacterial suspension in 230 µl TSB. Negative control wells contained 250 µl TSB only. The TSB has a higher nutrient content than dairy wastewater, however, the use of actual dairy wastewater is inappropriate due to the highly variable nature of dairy manufacturing plant wastewater. This variable nature would introduce an uncontrolled variable to the test and was therefore not used in these trials.
The plates were then covered and incubated overnight at 30°C. Each well was then emptied and washed 3 times with 250 μl sterile distilled water to remove any non-attached bacteria to the plastic. The wells were then filled with 250 μl methanol to fix the biofilm to the plastic for 15 min then emptied and air dried. The wells were stained with 0.5% Crystal Violet dye and left for 5 min. The stain was removed from the plates and the wells rinsed with running distilled water and air-dried. After the plates were dried, 250 μl of 33% glacial acetic acid was used to solubilize the dye and optical density readings were taken at 570 nm using an automatic
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96 well plate reader (BMG Labtech Spectrostar microplate reader, Bio-Tek Instruments, INC, Winooski, VT, USA).
To compare the ability of the different isolates to form biofilms, criteria consisting of four separate categories were adapted from Stepanović et al. (2000). The cut off optical density (ODC) was considered three standard deviations above the mean control OD. Strains were classified as per in Table 3-1:
Table 3-1: Biofilm formation criteria