) biofilm bacterial community structure.

Biofilm bacterial community response to an experimental discolouration event in a pilot- scale water distribution system

Cindy J. Smith1*, Rebecca L. Sharpe1 and Joby B. Boxall1

1Pennine 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 .

Biofilm microbial cells

pipe wall

EPSwater flow

Fe & MnBiofilm microbial cells

pipe wall

EPSwater flow

Fe & Mn

. 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.

SUMMARY1: 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

INTRODUCTION

Methods28 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 3Green 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/m2)

Turb

idity

(NTU

)

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 mm2 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).

0

2.0106

4.0106

6.0106 P =0.2139 P =0.9863 P =0.7351

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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.

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