Challenges with feed

The CT07 feed had several challenges that had to be understood and overcome before the feed could be used for experimentation. The main question was how to grow CT07 repeatedly to the same concentration and to then be sure the concentrations were relatively constant. A general understanding of the growth patterns of CT07 was therefore necessary. Microbiological plating and optical density (OD) had to be investigated to determine quick methods of determining CT07 concentrations. Growth curves were repeated to ensure the repeatability of feed preparation. Finally, a number of controls were put in place that limited variability in CT07 concentrations.

To determine the general growth pattern of CT07, a full growth curve was done over 24 hours. Three bacteria cultures were grown from different stock cultures and full dilutions and plating were done every 2 hours to create the full 24-hour growth curve with standard deviations. The plates were left to grow for 48 hours at room temperature. The average bacterial concentrations can be seen for the three cultures grown on Figure 15, the standard deviation is so small that it cannot be seen.

Figure 15: 24-hour growth curve for CT07

The lag phase is the first 4 hours, the exponential growth phase is the next 12 hours and the stationary phase is after 16 hours. The stationary phase was identified as the phase to be used for experiments, because it ensures repeatability as bacterial concentrations are then relatively constant. The stationary phase also ensures the feed is usable for a few hours as it remain constant, therefore it simplifies experimental control.

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As an alternative method to determine bacterial concentrations, absorbance was investigated and compared to bacterial concentrations calculated through plating. A wavelength of 600 nm was used for the absorbance, i.e. OD600, and the absorbance was measured every 2 hours for 24 hours with

three samples just as the bacterial concentration growth curve was done. The OD600 versus time curve

can be seen for the full 24-hour growth on Figure 16.

Figure 16: OD600 for 24-hour growth curve

The growth phases are recognisable, with a lag phase the first four hours, exponential growth the next ten hours and a decrease in OD600 observed after 16 hours. A difference between the bacterial

concentration and OD600 curves can best be seen when they are plotted against each other, Figure 17.

Figure 17: OD600 vs bacterial concentration

The OD600 relationship is linear compared to the bacterial concentration for the first 14 hours, with an

R-squared value of 0.9902, but thereafter there is no linear relationship. The OD600 increases until it

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concentration is increasing. The OD600 will therefore not be a sufficient measurement on its own to

determine an estimated bacterial concentration during the stationary phase.

The variability of bacterial growth is not noticeable on the full growth curve with the log axis, Figure 15. The three cultures were grown at the same time from different stock cultures, but showed repeatable results. The repeatability had to be investigated for different days. Three different cultures were grown on three different days, but only the growth curve for the stationary phase was investigated, i.e. bacterial concentrations and OD600 was measured for 16-hours, 18-hours and 20-

hours. Figure 18 represents all the data of the six cultures grown for the stationary phase, 3 cultures grown on the same day and 3 cultures grown on separate days. 16-hours seem to be the start of the stationary phase, with concentrations still increasing to the 18-hour mark. From 18-hours the bacterial concentrations are more than 0.5 x109 _{cfu/mL and less than 2.0 x10}9 _{cfu/mL. A bacterial concentration}

of between 0.5 x109 _{cfu/mL and 2.0 x10}9 _{cfu/mL was accepted as a relative repeatable concentration}

for CT07 grown for 20 hours.

Figure 18: Box and whisker diagram for stationary growth phase for all growth data recorded

Coliform bacterial contamination can vary from 0 cfu/mL to 106 _{cfu/mL, depending on the water}

source (Ackermann 2010). It is therefore difficult to quantitatively define efficient disinfection. It was decided to create a worst-case scenario for the batch treatment process. The Plankenberg River, outside Stellenbosch, has bacterial concentrations of up to 106 _{cfu/ml, which is high for a natural water}

source (Ackermann 2010). To test the robustness of the disinfection procedure a bacterial concentration of 107 _{cfu/ml was chosen as a feed concentration. The bacterial cultures grown in TSB}

were diluted by 1:100 to achieve a concentration between 0.5 x107 _{and 2.0 x10}7 _{cfu/ml. The treatment}

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to have been determined as successful in disinfection. This means a 5-log reduction was required by the disinfection process to pass the bacterial deactivation requirements.

The physical, chemical, and microbiological characteristics of tap water feed were expected to vary slightly. However, it was assumed that the characteristics would still be within a range that would not influence the disinfection. The slight variability in the feed was chosen to determine the practical application of the treatment on water from natural sources. Tap water was expected to have a low chlorine residual and small amounts of nutrients, minerals, and salts. The chlorine residual and any microbiological content was expected to be removed when the water was sterilised in the autoclave. The nutrients, minerals and salts were of value as they would support the survival of the CT07 and prevent it from dying due to a lack of nutrients.

A quick bacterial concentration indicator was difficult to develop. The OD600 would have worked for

low concentrations that fall within the exponential growth phase, but at the stationary phase there was no relationship. Figure 19 shows two OD600 graphs compared for 24-hour growth curves on

different days.

Figure 19: Comparing OD600 curves for cultures grown on different days

The curves have the same shape and pattern, while a slight delay for the 8 February curve can be observed. The OD600 did not drop to below 0.600 again within the 24-hour period. An OD600 above

0.600 can therefore be an indication that the bacterial culture had reached its stationary phase. The growth curves showed repeatability when comparing culture age and concentration and this could function as an indicator of expected bacterial concentrations. The OD value is very quick to get from the spectrometer, and therefore useful in combination with the age of the culture which will give an indication of growth phase and concentration. A bacterial culture that is older than 16 hours with an

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OD600 of above 0.600 was, most probably, in the stationary phase at a concentration of about 109

cfu/ml.