Potable water pipe-wall biofilm bacterial community response to conditioning shear stress
and a hydraulic disturbance in a full-scale pipe loop facility.
C. J. Smith*, R. Sharpe** I. Douterelo** and J. B. Boxall**
*School of Natural Sciences, Microbiology, NUI Galway, University Road, Galway, Ireland.
**Pennine Water Group, Department of Civil and Structural Engineering, Sir Frederick Mappin Building, Mappin Street, University of Sheffield, Sheffield,
S1 3JD, UK.
E-mail: cindy.smith@nuigalway.ie
water flow
Biofilm
microbial
cells
EPS
The Problem: Discolouration of potable water, due to fine insoluble particles, is a major cause for
Fe & Mn
customer contacts to water companies. Within Water Distribution Systems (WDS) pipe walls are sites for
biofilm development and the accumulation of particulate material. 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
pipe wall
water occurs when shear stress exceeds the conditioning values (Husband et al 2008).
The Hypothesis: Biofilms play an important role in understanding causes and consequences of material
layer at the pipe wall and hence in discolouration.
The Objective: To examine the effect of shear stress on biofilm bacteria community development and the
.
Increasing Shear Stress
subsequent response of WDS biofilm bacterial communities to increased hydraulic conditions in a full scale
Figure 1: Pipe wall biofilm formation and drinking water discoloration with
increasing shear stress above daily conditioning shear.
WDS test facility.
Experimental Set-up & Methodology
2.2
1.65
Shear stress (N/m²)
• 3 x 200m pipe-loop test facility
1.1
coupon removal
• Biofilm material layers were accumulated for 28 days at
0.96
0.66
8°C under three different steady state boundary shear
stresses – 0.11, 0.22 and 0.44 (N/m²).
0.44
• After 28 days, each loop was individually flushed
according to figure 3. Each flushing step was conducted for
Figure 3: Schematic of incremental shear stress applied
three turnovers of water.
to each loop. Star indicates coupon removal.
• Turbidity and DAPI cell counts were measured in the bulk
water after three turnovers.
• Coupons were taken before and after the flushing event to
analyze the bacterial community on the pipe wall.
The effect of the mobilization event on pipe-wall biofilm
community structure
A: Loop 1
C: Loop 3
2D Stress: 0.07
D: Q-PCR of biofilm
Shear stress (N/m2)
loop 1, 2 & 3 pipe wall after 28 days at 8°C.
water after each incremental increase in shear stress for
loops 1, 2 & 3.
ANOSIM analysis showed that conditioning
shear stress had no effect on pipe wall biofilm
community structure (R = 0.095, P = 0.2).
The most material was mobilized from loop 1,
conditioned at the lowest daily shear, as
evidenced by the increase in cell numbers and
turbidity in the drinking water.
Summary
• Conditioning shear stresses did not affect the bacterial community structure of a 28-day-old biofilm.
• Conditioning shear stress did affect mobilization of material into the bulk water - more material was
mobilized by the lowest conditioning shear stress than the highest.
• Pipe wall biofilm community structure and 16S rRNA gene copy numbers were not altered by the
mobilization event
2.010 6
Coupon Pre-flush
Low
Medium
st
e
L3
Po
Pr
L3
Po
e
L2
Po
L1
Pr
Coupon Post-flush
L1
Resemblance: S17 Bray Curtis similarity
st
0
Transform: Square root
Figure 5: A) DAPI cell counts and B) turbidity in drinking
P =0.7351
P =0.9863
4.010 6
e
Figure 4: MDS analysis of T-RFLP data from
P =0.2139
6.010 6
16S rRNA genes mm 2
Green line indicates 50% community similarity
based on Bray-Curtis similarity index.
B : Loop 2
Turbidity (NTU)
Loop 1 0.11 N/m²
Loop 2 0.22 N/m²
Loop 3 0.44 N/m²
Resemblance: S17 Bray Curtis similarity
Transform: Square root
Transform: Square root Resemblance: S17 Bray Curtis similarity
B: Turbidity (NTU) after each incremental
increase in shear
2D Stress: 0.07
2D Stress: 0.08
Pr
A: DAPI cell counts in drinking water after each
incremental increase in shear
2D Stress: 0.08
sequencing.
L2
Conditioning shear stress and
mobilization of pipe-wall material
Conditioning shear stress and biofilm
community structure
gene amplified for T-RFLP, Q-PCR and gene
st
Figure 2: The temperature controlled pipe loop test facility. Insert the Pennine
Water Group coupon (Deines et al., 2010), 52 coupons are inserted along the
length of each loop to facilitate examination of the pipe-wall biofilm.
• DNA was extracted from coupon and the 16S rRNA
High
Similarity (%) 50
Figure 6: (A-C) MDS analysis of T-RFLP data from loop 1, 2 & 3 before &
after mobilization. ANOSIM analysis showed no difference in community
structure before and after the mobilization event for any loop. (D) 16S
rRNA gene copy numbers mm2 of pipe-wall before and after mobilization.
No statistical difference in gene copy numbers was observed.
Significance
• Results are moving us closer to an understanding of the link between
daily conditioning shear, biofilm formation and discoloration.
• Flushing alone will not remove bacteria from WDS pipe-wall.