A further experiment was conducted to evaluate if the 0.5% somase interfered

with the measurement o f cellular ATP. A sample o f Pseudomonas fluorescens PI 7 was

split in tw o and one set dosed with 0.5% somase. Total and free ATP measurements were taken and the light emitted due to the cellular ATP evaluated. The tw o samples yielded values with no significant difference as shown in Table 3.5.

Table 3.5. Results o f test to determine the effect o f 0.5% somase on the calculation o f cellular ATP

Light emitted (mV) per 100 |.il sample due to cellular ATP

Average Standard Deviation

25.472 1.54Î

somase 23.678 1.638

3.1.4.9 An experiment was then undertaken to see the effect the addition of somase has on the value of biofilm ATP derived from samples taken from the experimental pipe rig. Eight sections of the polyethylene pipe rig were extracted from the rig after 20 days exposure. The sections were sampled and sonicated for 15 mins. The removed biomass was divided into two sub-samples and one set analysed using the somase protocol and the other set without somase addition. Table 3.6 summarises the results.

Table 3.6. Summary of the biofilm ATP derived from ATP analysis with and without somase.

Cellular ATP (ne/dm^)

Section 0.5% Somase No Somase

1 7.52 3.61 2 3.73 7.16 3 4.57 18.65 4 8.00 17.14 5 5.59 18.28 6 3.55 25.80 7 11.37 10.04 8 8.78 11.37 Average 6.64 14.01 Standard Deviation 2.75 7.24 (St. Dev./Ave) % 41 52

3.1.4.10 The table shows that the levels of free ATP have a significant influence over the determination of cellular ATP. The level of biofilm from samples dosed with 0.5% somase are on average lower than the sample without somase. The somase samples are more consistent than the other set of results and may therefore be considered more

accurate. The standard deviation of the samples dosed with somase is 41% of the average where as the no-somase samples had a standard deviation of 52% of the average.

3.1.4.11 The level of free ATP in the biofilm was seen to vary with time. This will affect the gain in accuracy by using Somase. It was stated in section 3.1.4,2 that the level of free ATP varied between 7 and 98% of the total ATP for the polyethylene pipe rig. This data looked at more closely may provide an insight into biofilm behaviour. Figure 3.4 shows the variation of free ATP as a percentage of the total ATP and the total ATP for one of the pipe rig streams over time. It can be seen that the proportion of free ATP steadily increases with time to a maximum around 30 days. From this point the proportion of free ATP reduces until approximately 45 days after which the free ATP begins to rise again. During this time the total ATP steadily increases as more microcolonies are formed. This is consistent with the biofilm increasing in thickness, producing a substrate limited dead base layer (at 30 days) resulting in sloughing after this time. When the biofilm has rejuvenated (45 days) subsequent increases in biofilm free ATP occur as the cycle begins again.

3.1.4.12 After 45 days the free ATP reduces to only 50% of the total. The biofilm appears not to be completely rejuvenated but appears to retain the dead under-section of the biofilm despite the sloughing action. This may appear to be the case as the graph represents the net behaviour of the biofilm. The action of the biofilm is better explained with reference to the individual microconsortia making up the film. If one assumes that the biofilm consists of many discrete microcolonies on the surface then the biofilm magnitude will be a summation of these colonies. It may also be assumed that a microcolony will produce a dead layer at its base. The dead layer will contain large amounts of free ATP and where the layer is sufficiently thick the biofilm will slough from the surface. The thickness of this layer may be related to the ratio of the free to total ATP. Figure 3.5 shows a theoretical model for the sloughing of microcolonies. The individual microcolonies increase in the ratio of free to total ATP and when this reaches a certain level the colony sloughs. The biofilm may be represented by the sum of the microcolonies as shown by the dotted line. It can be seen that this line reaches a

maximum initially and then fluctuates about a mean as sloughing incidents occur. The frequency of the oscillations and the magnitude of the peaks and troughs will depend on the rate of sloughing, the rate of microcolony formation and the critical ratio of free to total ATP for sloughing. This theory can be seen to match the actual results on figure 3.4. Figure 3.7 shows that the water distribution system biofilms studied consist of many microcolonies. It may also be suggested that with time the % of total ATP that is free will level as the biofilm consists of many colonies of different ages. If one increases the number of microcolonies on figure 3.5 to infinity the net biofilm line would plateau (figure 3.6). Initially the variation of free ATP with time in biofilms may be exaggerated due to the sparse coverage of the pipe surface. Further research could bear out this hypothesis.

3.1.4.13 A limitation of the analysis of ATP for biofilm measurement is that the amount of ATP is not consistent between cells. ATP may vary by species, cell age and metabolic activity. This may be acceptable for measuring biofilm of a mixed population where an average value can be given. It may not be possible however to compare biofilm values taken in the summer with those taken in the winter due to the difference in metabolic activity. The winter samples may appear to be lower than the summer samples because the amount of ATP per cell will be reduced although the amount of cells may not. Table 3,7 shows the ATP per culturable cell during autumn and winter periods of the pipe rig experiment.

Tables 3.7. Comparison of ratio of ATP per culturable cell for polyethylene pipe rig biofilm during autumn and winter periods. No disinfection.

AUTUMN WINTER

Exposure period Oct - Dec 1994 Jan - Mar 1995

Temp (Ave. & Range) °C 12.75 10.5-16.5 8.75 7.0-9.75

Plate count analysis Ave. Biofilm Density

(cfu/dm^)

69.9 X 10^ 9 2 x lO f

ATP technique (ng ATP/dm^)

69.22 12.8

ATP per culturable cell (ng ATP/cfu)

3 .1.4.13 The autumn value can be seen to be very clo se to the value o f 10'^^ g A TP/cell

FIGURE 3.1. EFFECT OF SONICATION ON CULTURABILITY OF BACTERIA 120 -1

I

8

SONICATION TIME (Mins)

FIGURE 3.2. EFFECT OF SONICATION ON ATP STABILITY

110 105 -- ? 100 95 - 90 - 85 - 40 35 30 25 20 15 10 5 0 ' Bidistilled-sonicated ■Bidistilled sonicated

FIGURE 3.3. RATE OF HYDROLYSIS OF ATP WITH INCREASING