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Investigating Bacterial Community Response During Discolouration …/file/C… · ·...
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Investigating Bacterial Community Response During Discolouration Events in an Experimental Water Distribution System Cindy J. Smith*, Rebecca L. Sharpe and Joby B. Boxall Pennine Water Group, Department of Civil and Structural Engineering, Sir Frederick Mappin Building, Mappin Street, University of Sheffield, Sheffield, S1 3JD, UK. *E‐mail: [email protected] INTRODUCTION Discolouration of potable water, due to fine insoluble particles, is a major cause for customer contacts to water companies. Within water distribution systems (WDS) particulate material accumulates on pipe walls. The stability and amount of material within these layers is known to be influenced by the maximum shear stress exerted by daily flow profile but the processes and mechanisms explicitly involved are poorly understood. Mobilisation of material into the bulk water occurs when the shear stress exceeds the conditioning values (Fig. 1). Whilst discolouration is predominantly an aesthetic issue association with microbiological content has been made. Biofilm microbiological loads are known to be significant within WDS. It seems likely that microbial biofilms may be important in understanding causes and consequences of discolouration. 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 EPS water flow Fe & Mn . Biofilm microbial cells pipe wall EPS water flow Fe & Mn . Fig. 1. Increased shear stress (right to left) above the daily conditioning shear resulting in discolouration of drinking water as material associated with the pipe wall is mobilised. Loop 1 Loop 2 Loop 3 4m 9m 52 coupons per loop 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 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. METHODS Pipe wall material was developed in each loop for 28 days at 8°C using the following constant shear stress; Loop 1 low ‐ 0.11 N/m 2 , Loop 2 medium ‐ 0.22 N/m 2 & Loop 3 high – 0.44 N/m 2 . After 28 days, each loop was individually flushed according to figure 3. Each flushing step was conducted for three turnovers of water. Turbidity changes in the bulk water were continuously measured and water samples were collected for DAPI and Mn analysis after one turnover. 5 coupons were Shear stress (N/m²) coupon removal 0.44 0.96 1.1 1.65 2.2 0.66 Fig. 3: Schematic of incremental shear stress applied to each loop. Star indicates coupon removal. taken from each loop before and after the flushing event. Biofilm was removed from the coupons using a nylon brush as described by Deines et al., 2010, filtered and DNA was extracted. T‐RFLP was conducted using F63‐FAM & R518 primer set, followed by individual digest with CfoI and AluI. T‐RFLP profiles were aligned using T‐Align (Smith et al., 2005) and subsequently analysed using a Bray‐Curtis similarity matrix in PRIMER‐6. Q‐PCR was conducted using the primer set F1369 & R1492 with TaqMan probe 1389 according to Smith et al., 2006. 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 Coupon Post‐flush Similarity (%) 50 60 Transform: Square root Resemblance: S17 Bray Curtis similarity 2D Stress: 0.08 A: Loop 1 Transform: Square root Resemblance: S17 Bray Curtis similarity 2D Stress: 0.07 B : Loop 2 Transform: Square root Resemblance: S17 Bray Curtis similarity 2D Stress: 0.07 C: Loop 3 Transform: Square root Resemblance: S17 Bray Curtis similarity Loop 1 2 3 B B B B B A A A A B B B B B A A A A B B B B A A A A A 2D Stress: 0.16 Sample B = Pre‐flush A = Post‐flush D: All Loops Coupon Pre‐flush 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. 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 3: Quantitative changes in 16S rRNA gene copy numbers from pipe wall 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). L1 Pre L1 Post L2 Pre L2 Post L3 Pre L3 Post 0 2.0× 10 6 4.0× 10 6 6.0× 10 6 P =0.2139 P =0.9863 P =0.7351 Low Medium High 16S rRNA genes mm 2 B. Manganese A. 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. C. DAPI Cell counts Shear stress (N/m 2 ) Turbidity (NTU) Shear stress (N/m 2 ) Shear stress (N/m 2 ) Mn (μg/l) Cells/ml
Transcript of Investigating Bacterial Community Response During Discolouration …/file/C… · ·...
Investigating Bacterial Community Response During Discolouration Events in an Experimental Water Distribution SystemCindy J. Smith*, Rebecca L. Sharpe and Joby B. Boxall
Pennine Water Group, Department of Civil and Structural Engineering, Sir Frederick Mappin Building, Mappin Street, University of Sheffield, Sheffield, S1 3JD, UK.*E‐mail: [email protected]
INTRODUCTION
Discolouration of potable water, due to fine insoluble particles, is a major cause for customer contacts to water companies. Within water distribution systems (WDS) particulate material accumulates on pipe walls. The stability and amount of material within these layers is known to be influenced by the maximum shear stress exerted by daily flow profile but the processes and mechanisms explicitly involved are poorly understood. Mobilisation of material into the bulk water occurs when the shear stress exceeds the conditioning values (Fig. 1). Whilst discolouration is predominantly an aesthetic issue association with microbiological content has been made. Biofilm microbiological loads are known to be significant within WDS. It seems likely that microbial biofilms may be important in understanding causes and consequences of discolouration. 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 & Mn
.
Biofilm microbial cells
pipe wall
EPSwater flow
Fe & Mn
.Fig. 1. Increased shear stress (right to left) above the daily conditioning shear resulting in discolouration of drinking water as material associated with the pipe wall is mobilised.
Loop 1
Loop 2
Loop 3
4m
9m
52 couponsper loop
Fig. 2. The temperature controlled pipe loop test facility at the University of Sheffield. Excerpt the PennineWater 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 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.
METHODS
Pipe wall material was developed in each loop for 28 days at 8°C using the following constant shear stress; Loop 1 low ‐ 0.11 N/m2, Loop 2 medium ‐ 0.22 N/m2 & Loop 3 high – 0.44 N/m2. After 28 days, each loop was individually flushed according to figure 3. Each flushing step was conducted for three turnovers of water. Turbidity changes in the bulk water were continuously measured and water samples were collected for DAPI and Mn analysis after one turnover. 5 coupons were
Shear stress (N/m²)
coupon removal
0.44
0.96
1.1
1.65
2.2
0.66
Fig. 3: Schematic of incremental shear stress applied to each loop. Star indicates coupon removal.
taken from each loop before and after the flushing event. Biofilm was removed from the coupons using a nylon brush as described by Deines et al., 2010, filtered and DNA was extracted. T‐RFLP was conducted using F63‐FAM & R518 primer set, followed by individual digest with CfoI and AluI. T‐RFLP profiles were aligned using T‐Align (Smith et al., 2005) and subsequently analysed using a Bray‐Curtis similarity matrix in PRIMER‐6. Q‐PCR was conducted using the primer set F1369 & R1492 with TaqManprobe 1389 according to Smith et al., 2006.
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
Coupon Post‐flushSimilarity (%) 5060
Transform: Square root Resemblance: S17 Bray Curtis similarity
2D Stress: 0.08
A: Loop 1
Transform: Square root Resemblance: S17 Bray Curtis similarity
2D Stress: 0.07
B : Loop 2
Transform: Square root Resemblance: S17 Bray Curtis similarity
2D Stress: 0.07C: Loop 3
Transform: Square rootResemblance: S17 Bray Curtis similarity
Loop 1 2 3
B
B
B
B
B
A
A
AA
B
B
B
B
BA
A
AA
B
B
B
B
A
A
A
A
A 2D Stress: 0.16
Sample B = Pre‐flushA = Post‐flush
D: All Loops
Coupon Pre‐flush
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.
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 3: Quantitative changes in 16S rRNA gene copy numbers from pipe wall 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).
L1 Pre
L1 Post
L2 Pre
L2 Post
L3 Pre
L3 Post
0
2.0×106
4.0×106
6.0×106 P =0.2139 P =0.9863 P =0.7351
Low Medium High
16S
rR
NA
gen
es m
m2
B. Manganese
A. 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.
C. DAPI Cell counts
Shear stress (N/m2)
Turbidity (NTU
)
Shear stress (N/m2)
Shear stress (N/m2)
Mn(µg/l)
Cells/m
l
