Incubation phase

Biofilms were incubated within the pilot-scale pipeline under representative, albeit artificial conditions. This provided greater variable control of the biofilms conditioning, which was beneficial from a scientific and safety standpoint. Variable control was essential within the current study for the purpose of establishing the impact of discrete ecological factors on

biofilm frictional development. Furthermore, continuous biofilm monitoring without disruption could also be achieve by incubating the biofilms within a laboratory environment.

Most biofouling investigations documented within the literature have incubated biofilms under field conditions and then evaluated them in the laboratory (Schultz 1998; Schultz and Swain 1999; Schultz 2000; Barton 2006; Lambert et al. 2008; 2009; Andrewartha et al. 2008, Andrewartha 2010; Perkins et al. 2013; 2014; Walker et al. 2013). Sufficient variable control and continuous monitoring are limited or precluded by such an approach, although, the resultant biofilm will be extremely representative of the natural ecology. Moreover, the transportation of a biofilm could lead to irreversible damage.

The principal ecological factor under review within the current study was flow hydrodynamics. Several assays were carried out within the pipeline facility in order to evaluate the impact of flow hydrodynamics on biofilm frictional behaviour over time. In particular, biofilms were incubated with synthetic wastewater at three different steady state flow regimes, namely 𝑈̅ = 0.60 m/s, 𝑈̅ = 0.75 m/s, and 𝑈 ̅ =1.00 m/s. For the purpose of repeatability, all other significant ecological factors, such as temperature and nutrient content were controlled and remained reasonably constant within each of the discrete flow assays. A summary of the key ecological factors within the facility during the three flow assays is presented in Table 3.4.

Table 3.4 Average environmental and operational parameters within the ReD = 5.98x10⁴, ReD = 7.82x10⁴, and ReD = 1.00x10⁴ assays. sufficient nutrient supply (see Table 3.4) was deemed adequate for the biofilms to reach a state of equilibrium in terms of their frictional resistance (Picologlou et al. 1980; Lambert et al. 2008; 2009; Andrewartha 2010).

The average freestream velocities investigated within the current study were of particular industrial relevance. Generally, as a rule of thumb, the minimum 𝑈̅ required for a pipe to be self-cleansing is 0.60 m/s (Fair and Geyer 1954). Typically, within the UK and US

wastewater pipelines are designed to achieve an average freestream velocity of 0.60 and 0.75 m/s, respectively. Field measurements documented by Lauchlan et al. (2005), who investigated wastewater pumping mains within the UK, illustrated the prevalence of such design criteria within actual systems. In particular, Lauchlan et al. (2005) found that the majority of the assessed UK systems operated at average freestream velocities between 0.6-1.0 m/s, with most of them operating at the upper limit of this range.

The flow assays will be referred to herein in terms of ReD, i.e. the 0.60 m/s, 0.75 m/s and 1.00 m/s assays will be referred to as the ReD = 5.98x10⁴, ReD = 7.82x10⁴, and ReD = 1.00x10⁴ assays, respectively.

During the incubation phase a complete set of PG and mean-velocity traverses were taken at least three times a day, with the exception of the ReD = 7.82x10⁴ assay were only PG data was collected. A total of 60 PG and velocity (if applicable) profiles were taken during each incubation phase.

The specific details of the pipe’s ecology during each of the respective incubations are outlined herein.

3.8.2.1 Incubation temperature

The fluid temperature within the pipeline for all three assays was controlled using the external cooling system outlined in Section 3.2.2. The average fluid temperature recorded during the incubation phase of the three flow assays was 21.5 ± 0.9 °C, as shown by Figure 3.31a. The fluid temperatures recorded during the incubation phase of each of the flow assays are shown within Figure 3.31a, which presents the average daily values, recorded using the universal temperature probes (outlined in Section 3.3.5).

Naturally, the temperature within typical wastewater systems is highly variable and seasonal dependant. As a result, it can range from 5°C < T < 22°C (Hoes et al. 2009; Cipolla and Maglionico 2014). Cipolla and Maglionico (2014) reported that the temperature within a sewer system in Bologna, Italy was approximately: 10°C < T < 14°C in the winter; 14°C < T

< 18°C in the spring/autumn and 18°C < T < 22°C in the summer. Consequently, in terms of temperature, all three flow assays were accurate for real sewer systems and provided the maximum representative levels of microbial activity (i.e. summer conditions).

The comprehensive temperature control ensured that µ and ρ remained reasonably constant within the respective flow assays. The maximum variation in µ and ρ recorded within the each of the assays was ±2.5% and ±0.02%, respectively.

Figure 3.31 a) Water temperature and b) Reynolds Numbers recorded during the biofilm incubation phase of the ReD = 5.98x10⁴, ReD = 7.82x10⁴ and ReD = 1.00x10⁵ assays.

3.8.2.2 Incubation Reynolds Numbers

The hydrodynamic conditions within each of the flow assays was regulated, through a combination of pump and temperature control. In order to satisfy the required steady state conditions within the respective assays, the flow rate was periodically adjusted using the system’s gate valve. As a result of the flow rate regulation, the increase in frictional resistance caused by the biofilm manifested itself in terms of an increase in headloss and pump power requirements. Figure 3.31b illustrates the daily average ReD recorded within each of the flow assays during the incubation phase. The maximum variation in ReD recorded was ±3%, which indicated that the flow conditions within the respective assays was reasonably constant and homogenous.

3.8.2.3 Hydraulic retention time (HRT)

To ensure a system is well mixed with negligible stagnation periods, the general rule of thumb is to maintain an internal system HRT (= V/Q) of less than a few minutes (Stoodley and Warwood 2003; Teodósio et al. 2010). This criteria was desirable as it would have fostered greater microbial development upon the surface as opposed to within the water column (Eisnor and Gagnon 2003; Stoodley and Warwood 2003; Teodósio et al. 2010). The internal system HRT used within each of the three flow assays was in the range of 73.1s < HRT <

74.3s (as shown in Appendix A.9 in Table A.6, which shows the internal HRT for each of the components of the pipeline facility) and was controlled by adjusting and regulating the

recirculation tank’s volume. The volume regulation also maintained steady state conditions within the three flow assays. Consequently, the wastewater within the three flow assays was well-mixed and of a constant volume.

3.8.2.4 Nutrient content – Average daily water concentrations

During the incubation phase, the wastewater’s COD, TN and TP concentrations were monitored and regulated daily, in order to ensure the respective parameters remained reasonably constant and satisfied the target concentration criteria. Water samples (20-50 mm) were taken and evaluated from the recirculating tank every 24 h. The results of this evaluation were then used to determine whether the system required diluting or further concentrating, and to what degree. If adjustments were required, a secondary water sample was taken and evaluated; for the purpose of establishing whether the re-adjusted wastewater met the required target criteria. If this was not the case, then the whole process was repeated. The concentrations of NH4+, NO3-, Fe, Mn and Cl were also measured during each of the incubation phases.

Table 3.5 presents the average chemical parameters recorded during the incubation phase of each of the flow assays at both pre- and post- adjustment time intervals. Figure 3.32 presents the daily average concentrations of COD, TN and TP recorded during the incubation phase of the three flow assays.

The Cl concentration of the local water source was found to be extremely high (< 0.2 mg/l) and as a consequence, it was extensively monitored. It should be noted, that the source water for the pipeline facility was local to the School of Engineering and it differed from the local drinking water. The Cl concentration within the source water was neutralised using sodium thiosulfate. As a result, the concentration of Cl within the synthetic wastewater remained below the detection limit (i.e. < 0.02 mg/l) during each of the flow assays (see Table 3.5).

The COD concentration during each of the incubation phases required daily adjustment, typically through further concentrating. On average the COD concentrations within the respective assays reduced by 34% per day. Daily re-concentrating with case-specific amounts of macro-nutrients, namely Peptone and meat extract compensated for this reduction. The concentrations of TP and TN within the three flow assays required less extensive adjustment.

Typically, the respective parameters only required minor adjustments 2-3 times throughout each of the incubation phases. It can be seen from Table 3.5 and Figure 3.32 that as a result of the extensive daily monitoring, the concentrations of COD, TN and TP during the incubation phases of the flows assays remained reasonably constant.

Table 3.5 Average chemical parameters recorded during the incubation phases of the ReD = 5.98x10⁴, ReD = 7.82x10⁴ and ReD = 1.00x10⁵ assays (for both pre- and post- concentration

adjustment time intervals).

Parameter

ReD = 5.98 x 10⁴ ReD = 7.82 x 10⁴ ReD = 1.00 x 10⁵ (mg/l)

n (mg/l)

n (mg/l)

Av. σ Av. σ Av. σ n

Pre Adjustment COD 371.8 111.0 20 347.7 101.1 20 357.9 108.7 20

TN 49.8 0.7 10 51.3 3.1 8 50.6 4.8 11

TP 10.6 0.97 20 9.9 0.6 20 10.7 1.1 20 pH 8.09 0.13 20 7.96 0.18 20 7.80 0.26 20

Post Adjustment

COD 536.4 40.5 20 545.6 21.2 20 548.1 23.4 20 TOC 238.2 16.1 10 251.2 9.5 20 241.6 12.2 9

DOC 211.5 14.3 10 - - - 190.6 9.6 9

TN 49.5 0.6 2 50.3 0.4 2 51.2 0.9 11

TP 12.1 1.20 2 10.8 0.8 4 11.0 0.8 3

pH 8.09 0.13 20 7.96 0.18 20 7.80 0.26 20 NH4+ 0.41 0.23 6 0.25 0.23 4 0.75 0.02 4 NO3- 0.50 0.28 6 0.33 0.28 4 1.09 0.11 4 Fe 0.11 0.05 6 0.10 0.07 4 0.15 0.09 5 Mn 0.13 0.06 6 0.30 0.20 4 0.39 0.10 5 Cl 0.01 0.01 6 0.00 0.01 4 0.00 0.01 5

* Derived from COD using Equation 3.25

Figure 3.32 Concentrations of a) COD b) TP and c)TN recorded during the incubation phase of the ReD = 5.98x10⁴, ReD = 7.82x10⁴ and ReD = 1.00x10⁵ assays, post concentrations adjustments, with

the exception of TN, which represent the pre concentration adjustment values.

200.0