Factors affecting biofilm development, specifically the materials of the pipe, were investigated. Two laboratory scale bioreactor systems were constructed to study biofilm formations: a pipe loop bioreactor with continuous flow at 10.1 liters per minute (LPM), and a tank bioreactor under stagnant conditions with a minimal flow of 0.0095 LPM. The continuous flow bioreactors were constructed using cross-linked polyethylene (PEX), copper, and galvanized steel pipes. The tank bioreactors consisted of glass chambers containing coupons made from the pipe materials, as well as glass microscope slides. Municipality tap water was used in the experimentation, with no nutrients added. Legionella pneumophila was spiked into all the pipe loop bioreactors, and only in one tank bioreactor. Detection of heterotrophic bacteria, coliforms and Legionella using tryptic soy agar (TSA), Brilliance, and buffered yeast charcoal extract (BYCE), respectively. Over ten weeks, biofilms were developed on PEX, copper, and steel, in the pipe loop bioreactors and the tank bioreactors. Heterotrophic bacteria were detected in all systems; however, no coliforms were detected, and Legionella pneumophila was only detected on a coupon in the copper pipe loop bioreactor, as measured by bacterial concentration on test materials. In the tank bioreactors, biofilms developed the most rapidly on PEX, followed by galvanized steel, and finally copper. Out of the four materials, copper had the lowest bacterial growth, which can be ascribed to the bactericidal impact of copper ions on the bacterial cells attaching to the copper surface. After biofilm aging, higher bacterial colonization on copper and accumulation of dead bacterial layer on the surface may act as a protective barrier against copper ions. Bacterial densities in the biofilm reached a high concentration of 1.40 x 105 CFU/cm2 on the PEX pipe loop bioreactor, and 1.05 x 104 CFU/cm2in the PEX coupon in the tank bioreactors. Comparing the turbulent conditions in the pipe loop bioreactors to the stagnant conditions in the tank bioreactor, showed that biofilms formed more rapidly under stagnant conditions, but in larger quantities under turbulent conditions.

The impact of physical/chemical properties of gray water on microbial inactivation in gray water using chlorine was investigated through creating artificial gray water in lab, varying specific components, and then measuring microbial inactivation. Gray water was made through taking autoclaved nanopure water, and increasing the concentration of surfacants, the turbidity, the concentration of organic content, and spiking E. coli grown in tryptic soy broth (TSB); chlorine was introduced using Clorox Disinfecting Bleach2. Bacteria was detected using tryptic soy agar (TSA), and E. coli was specifically detected using the selective media, brilliance. The log inactivation of bacteria detected using TSA was shown to be inversely related to the turbidity of the solution. Complete inactivation of E. coli concentrations between 104-105 CFU/100 ml in gray water with turbidities between 10-100 NTU, 0.1-0.5 mg/L of humic acid, and 0.1 ml of Dawn Ultra, was shown to occur, as detected by brilliance, at chlorine concentrations of 1-2 mg/L within 30 seconds. These result in concentration time (CT) values between 0.5-1 mg/L·min. Under the same gray water conditions, and an E. coli concentration of 104 CFU/100 ml and a chlorine concentration of 0.01 mg/L, complete inactivation was shown to occur in all trials within two minutes. These result in CT values ranging from 0.005 to 0.02. The turbidity and humic acid concentration were shown to be inversely related to the log inactivation and directly related to the CT value. This study shows that chlorination is a valid method of treatment of gray water for certain irrigation reuses.