1. ) biofilm bacterial community structure. Biofilm bacterial community response to an experimental discolouration event in a pilot- scale water distribution system Cindy J. Smith 1 *, Rebecca L. Sharpe 1 and Joby B. Boxall 1 1 Pennine Water Group, Department of Civil and Structural Engineering, Sir Frederick Mappin Building, Mappin Street, University of Sheffield, Sheffield, S1 3JD, UK. *Discipline of Microbiology, School of Natural Science, NUI Galway, Ireland. * E-mail: cindy.smith@nuigalway.ie 1.Discolouration of potable water, due to fine insoluble particles, is a major cause for customer contacts to water companies. Within water distribution systems (WDS) pipe walls are sites for biofilm development and the accumulation of particulate material (Fig. 1). 2.The stability and amount of material accumulated is known to be influenced by the maximum shear stress exerted by the daily flow profile but the processes and mechanisms explicitly involved are poorly understood. Mobilisation of material into the bulk water occurs when shear stress exceed the conditioning values. The effect of shear stress on the development and mobilisation of biofilm bacterial communities unknown. 3.The internationally unique temperature controlled pipe-rig test facility at the University of Sheffield (Fig. 2) can be used to bridge the gap between field and bench scale studies by simulating field conditions in a controlled laboratory setting to determine the underpinning factors that contribute to discolouration and the role biofilm microorganisms play.. Fig. 1. Pipe wall biofilm development. Biofilm develops, trapping microorganisms and fine particles such as Mn and Fe. Increased shear stress above the daily conditioning shear results in mobilisation of material from the pipe wall and discolouration of drinking water. Fig. 2. The temperature controlled pipe loop test facility at the University of Sheffield. Excerpt the Pennine Water Group coupon (Deines et al., 2010), 52 coupons are inserted along the length of each loop to facilitate examination of the pipewall biofilm. AIM: To examine the response of WDS bacterial communities to an experimental discolouration event using the internationally unique pipe-loop test facility at the University of Sheffield (Fig. 2). OBJECTIVES: 1: To test the hypothesis that biofilm stability and bacterial community composition is influenced by the maximum conditioning shear stress of the water flowing through it. 2: Determine the affect of incremental shear stress flushing steps on biofilm bacterial community structure conditioned at three different shear stresses. Shear stress (N/m²) coupon removal 0.44 0.96 1.1 1.65 2.2 0.66 Fig. 3: Schematic of mobilisation experiment. Incremental shear stress applied to each loop after 28 days biofilm development. SUMMARY 1: Loop 1, conditioned during the 28 day growth phase at the lowest shear stress (0.11 N/m2) resulted in the greatest amount of material associated with the pipe-wall is released into the bulk water as evidenced by the highest turbidity, Mn and DAPI cell counts of the three loops (Fig. 1). The increase in DAPI count cell numbers indicates that biological material is released into the bulk water during a discolouration event. 2: Neither the diverse bacterial community structure nor 16S rRNA gene copy numbers on the pipe-wall of the three loops varied significantly between loops or after the flushing event. In conclusion, pipe-wall material was mobilisied into the bulk water during the experimental discolouration event, the amount mobilised was influenced by the daily conditioning shear. Neither the conditioning shear stress or the incremental shear stress applied during the flushing altered the pipe-wall bacterial community structure. RESULTS References: Deines et al., 2010 Appl. Microbiol.Biotechnol. 87: 749 -746; Smith et al., 2005 FEMS Microbiol. Ecol. 54: 375-380; Smith et al., 2006 Environ. Microbiol. 8: 804 -815. Results 3: Quantitative changes in 16S rRNA gene copy numbers from pipe wall pre- and post-flushing. Loop 1 Loop 2 Loop 3 4m 9m 52 coupons per loop Increasing shear stress INTRODUCTION Methods 28 day build up Table 1: Conditioning steady state shear stress conditions pipe wall biofilms were developed at for 28 days at 8°C. After 28 days, 5 coupons were taken from each loop, and bacterial community composition analyzed by T-RFLP. Mobilisation 3 coupons were removed from each loop at start and end of mobilisation event. Bacterial community diversity was examined by T-RFLP and 16S rRNA gene clone library analysis. Gene copy numbers were quantified by Q-PCR. Turbidity, Mn concentration and DAPI cell counts were measured in the water after each incremental increase in shear stress. Effect of conditioning shear stress on - i) Mobilisation of material from pipe wall 2D Stress: 0.08 ANOSIM analysis showed that conditioning shear stress had no effect on pipe wall biofilm community structure (R = 0.095, P = 0.2). Loop 1 Loop 2 Loop 3 Green line indicates 50% community similarity, based on Bray-Curtis index. Most material was mobilised from the loop conditioned at the lowest shear stress. Effect of conditioning shear stress on - ii) Pipe wall bacterial community structure Shear stress (N/m 2 ) Turbidity (NTU)

2. Fig. 5: (A-C) MDS analysis of T-RFLP data from loop 1, 2 & 3 respectively and (D) combined T-RFLP data from all 3 loops before & after flushing. ANOSIM analysis did not show any statistical difference in community structure before and after the flushing event for any loop. Results 2: Pipe-wall bacterial community structure pre- and post- flushing. Results 1: Mobilistation of pipe wall material into bulk water during flushing event Fig. 6: 16S rRNA gene copy numbers per mm 2 of pipe wall, quantified pre- and post-flushing for each loop. No statistical difference in gene copy numbers pre- and post- flushing was observed for any of the three loops (P < 0.05). Turbidity Fig. 4: Normalised increase in A) tubidity, B) manganese and C) DAPI cell counts in the bulk water after each flushing step for loop 1, 2 and 3. 2D Stress: 0.06 A. 2D Stress: 0.07 B. 2D Stress: 0.05 C. Flavobacterium sp. AKB-2008-JO41 L3B_ 12 (B1) L1B_ 17 (B1, A2) L3B _33 (B1) Uncultured bacterium caohai-58 L3B _30 (B1) Mycobacterium chelonae strain MC3 L2B_31 (B2) uncultured Hyphomicrobium sp. clone A5 L2B_24 (B1) L1B_3 (B4) Rhizobium etli, isolate PhyCEm-90 uncultured Rhizobium sp. clone 1P-2-L24 L2B_13 (B2) L3B_ 9 (B2, A2) L3A_ 23 (A1) L3B_37 (B2) Caulobacter vibrioides strain 212 L1A_ 8 (A2) uncultured bacterium clone GL_DU15 Bradyrhizobium sp. Is-D308 L3B_44 (B1) L2B_39 (B1) L3A _16 (A1) uncultured alpha proteobacterium AKB-2008-TA3A L2B_15 (B1) L3B_2 (B2) L2B_6 (B1, A1) L3B_40 (B1) Erythromicrobium ramosum strain DSM 8510 L1A_5 (A1) Sphingomonas sp. V1-2 L2b_30 (B1) L2B_25 (B1, A2) uncultured bacterium clone LW18m-1-60 L3B_4 (B1) uncultured bacterium clone 6S_2a02 L1A_31 (A1) L1B_29 (B2)_ Sphingomonas sp. Y57 L3B_21 (B1) L2B_20 (B1) L1A_38 (A1) L1A_18 (A1) Delftia acidovorans strain REG131 L1A 39 (A1) uncultured bacterium FATNRMAR041 L3B_18 (B1) L2B_28 (B1, A1) L1A_22 (A14) Acidovorax facilis strain 228 L1A_11 (A1) uncultured bacterium clone I4-40 Polaromonas sp. BAC41 L1A_32 (A1) L3B_5 (B1, A1) uncultured bacterium clone 1C227268 L2B_16 (B2, A2) L1A_26 (A1) uncultured Methylibium sp.. L2B_38 (B1) L3B_16 (B3) L2B_18 (B2) uncultured bacterium A0640 L2 _B1 (B1) L1B_26 (A1) L2A35 (A1) L3B_38 (B1, A1) Cupriavidus basilensis Uncultured clone F3Boct.43 L2_B8 (B2) L2B_32 (B1) L1B_ 25 (B1, A1) Azospira sp. IHB B 2277 L3B_31 (B1, A1) Dechlorosoma suillum L2B_17 (B3) L1B _19 (B2) Pseudoxanthomonas japonensis L3B_8 (B3) L2A_6 (A6) Uncultured bacterium clone K46 L3B_17 (B4) L1A_1(A1) L2B_33 (B1, A1) Stenotrophomonas maltophilia R551-3 Uncultured bacterium clone lp93-1c02.w2k Uncultured bacterium Nera_L6-C1-T7 L3A_41 (A1) Legionella fallonii strain LLAP10 Nevskia ramosa strain OL1 Uncultured bacterium HOClCi73 DWDS L2_A21 (A5) L3B_45 (B2, A2) L1A_6 (B2) L1A _17 (A1) L3B_15 (B1) uncultured gamma proteobacteria 7.1.14_S L2A_3 (B1, A1) L1B_9 (B13,A4) L1B_1 (B11, A5) L2B_2 (B7, A3) L3B_1 (B14, A18) Pseudomonas trivialis Cf2F Pseudomonas fluorescens ATCC 17556 uncultured bacterium clone 50p7_2515 E. coli U00096 0.05 γ-proteobacteria β-proteobacteria α- proteobacteria Flavobacteria Actinobacteria Holophage