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Characterisation of the physical composition and microbial community structure of biofilms within a model full-scale drinking water distribution system.

Fish KE, Collins R, Green NH, Sharpe RL, Douterelo I, Osborn AM, Boxall JB - PLoS ONE (2015)

Bottom Line: The volume of EPS was 4.9 times greater than that of the cells within biofilms, with carbohydrates present as the dominant component.Additionally, the greatest proportion of EPS was located above that of the cells.Moreover, biofilms from different positions were similar with respect to community structure and the quantity, composition and three-dimensional distribution of cells and EPS, indicating that active colonisation of the pipe wall is an important driver in material accumulation within the DWDS.

View Article: PubMed Central - PubMed

Affiliation: Pennine Water Group, Department of Civil and Structural Engineering, The University of Sheffield, Sheffield, United Kingdom; NERC Biomolecular Analysis Facility, Department of Animal and Plant Sciences, Western Bank, Sheffield, United Kingdom.

ABSTRACT
Within drinking water distribution systems (DWDS), microorganisms form multi-species biofilms on internal pipe surfaces. A matrix of extracellular polymeric substances (EPS) is produced by the attached community and provides structure and stability for the biofilm. If the EPS adhesive strength deteriorates or is overcome by external shear forces, biofilm is mobilised into the water potentially leading to degradation of water quality. However, little is known about the EPS within DWDS biofilms or how this is influenced by community composition or environmental parameters, because of the complications in obtaining biofilm samples and the difficulties in analysing EPS. Additionally, although biofilms may contain various microbial groups, research commonly focuses solely upon bacteria. This research applies an EPS analysis method based upon fluorescent confocal laser scanning microscopy (CLSM) in combination with digital image analysis (DIA), to concurrently characterize cells and EPS (carbohydrates and proteins) within drinking water biofilms from a full-scale DWDS experimental pipe loop facility with representative hydraulic conditions. Application of the EPS analysis method, alongside DNA fingerprinting of bacterial, archaeal and fungal communities, was demonstrated for biofilms sampled from different positions around the pipeline, after 28 days growth within the DWDS experimental facility. The volume of EPS was 4.9 times greater than that of the cells within biofilms, with carbohydrates present as the dominant component. Additionally, the greatest proportion of EPS was located above that of the cells. Fungi and archaea were established as important components of the biofilm community, although bacteria were more diverse. Moreover, biofilms from different positions were similar with respect to community structure and the quantity, composition and three-dimensional distribution of cells and EPS, indicating that active colonisation of the pipe wall is an important driver in material accumulation within the DWDS.

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An example of the arrangement, volume and distribution of cells, carbohydrates and proteins of drinking water biofilms.A) 3D projection of example Day 0 and Day 28 biofilms, plotting region shown is 420 μm × 420 μm × 30.6 μm and 420 μm × 420 μm × 94.4 μm, respectively; B) Volume (log) of biofilm components at Day 28, relative to the thresholds used in digital image analysis, each data point (n = 25) represents a FOV, box and whisker plots show the range, interquartile range and median—indicated by the solid black line; C) Day 28 area distribution plot, each line (n = 25) indicates a FOV, note the different x-axis scales between components. Area coverage fraction refers to the proportion of each XY image of the Z-stack covered by the particular component, blue dashed line at “0” indicates the cell peak location; peak location is the aligned slice number at which the maximum area fraction occurs. Area fractions for carbohydrates and proteins are plotted relative to cells (see Materials and Methods for details).
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pone.0115824.g004: An example of the arrangement, volume and distribution of cells, carbohydrates and proteins of drinking water biofilms.A) 3D projection of example Day 0 and Day 28 biofilms, plotting region shown is 420 μm × 420 μm × 30.6 μm and 420 μm × 420 μm × 94.4 μm, respectively; B) Volume (log) of biofilm components at Day 28, relative to the thresholds used in digital image analysis, each data point (n = 25) represents a FOV, box and whisker plots show the range, interquartile range and median—indicated by the solid black line; C) Day 28 area distribution plot, each line (n = 25) indicates a FOV, note the different x-axis scales between components. Area coverage fraction refers to the proportion of each XY image of the Z-stack covered by the particular component, blue dashed line at “0” indicates the cell peak location; peak location is the aligned slice number at which the maximum area fraction occurs. Area fractions for carbohydrates and proteins are plotted relative to cells (see Materials and Methods for details).

Mentions: Visualisation and Characterisation of DWDS Biofilms after 28 Days of Growth. After 28 days of growth, biofilms were heterogenic in their coverage and morphology (e.g. Fig. 4A). A FOV generally contained all three stained components but no complete co-localisation was observed; the extent of carbohydrate coverage in contrast to that of proteins was illustrated, along with areas where either only cells or EPS were present. The median total biofilm volume at Day 28 was 252325 μm3 (per 420 μm2 FOV), of which carbohydrates were the dominant component (Fig. 4; Table 3) occurring at a significantly greater volume than either cells (W = 179.0, p = 0.0090), or proteins (W = 40.0, p<0.0001). Proteins occurred at a significantly lower volume than the cells (W = 543.0, p<0.0001) and were consistently the least abundant biofilm component (Fig. 4; Table 3). The range in volume of each of the components (Table 3) indicates the substantial heterogeneity in biofilm coverage and supports the choice of analysing a greater number of sample replicates.


Characterisation of the physical composition and microbial community structure of biofilms within a model full-scale drinking water distribution system.

Fish KE, Collins R, Green NH, Sharpe RL, Douterelo I, Osborn AM, Boxall JB - PLoS ONE (2015)

An example of the arrangement, volume and distribution of cells, carbohydrates and proteins of drinking water biofilms.A) 3D projection of example Day 0 and Day 28 biofilms, plotting region shown is 420 μm × 420 μm × 30.6 μm and 420 μm × 420 μm × 94.4 μm, respectively; B) Volume (log) of biofilm components at Day 28, relative to the thresholds used in digital image analysis, each data point (n = 25) represents a FOV, box and whisker plots show the range, interquartile range and median—indicated by the solid black line; C) Day 28 area distribution plot, each line (n = 25) indicates a FOV, note the different x-axis scales between components. Area coverage fraction refers to the proportion of each XY image of the Z-stack covered by the particular component, blue dashed line at “0” indicates the cell peak location; peak location is the aligned slice number at which the maximum area fraction occurs. Area fractions for carbohydrates and proteins are plotted relative to cells (see Materials and Methods for details).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4338064&req=5

pone.0115824.g004: An example of the arrangement, volume and distribution of cells, carbohydrates and proteins of drinking water biofilms.A) 3D projection of example Day 0 and Day 28 biofilms, plotting region shown is 420 μm × 420 μm × 30.6 μm and 420 μm × 420 μm × 94.4 μm, respectively; B) Volume (log) of biofilm components at Day 28, relative to the thresholds used in digital image analysis, each data point (n = 25) represents a FOV, box and whisker plots show the range, interquartile range and median—indicated by the solid black line; C) Day 28 area distribution plot, each line (n = 25) indicates a FOV, note the different x-axis scales between components. Area coverage fraction refers to the proportion of each XY image of the Z-stack covered by the particular component, blue dashed line at “0” indicates the cell peak location; peak location is the aligned slice number at which the maximum area fraction occurs. Area fractions for carbohydrates and proteins are plotted relative to cells (see Materials and Methods for details).
Mentions: Visualisation and Characterisation of DWDS Biofilms after 28 Days of Growth. After 28 days of growth, biofilms were heterogenic in their coverage and morphology (e.g. Fig. 4A). A FOV generally contained all three stained components but no complete co-localisation was observed; the extent of carbohydrate coverage in contrast to that of proteins was illustrated, along with areas where either only cells or EPS were present. The median total biofilm volume at Day 28 was 252325 μm3 (per 420 μm2 FOV), of which carbohydrates were the dominant component (Fig. 4; Table 3) occurring at a significantly greater volume than either cells (W = 179.0, p = 0.0090), or proteins (W = 40.0, p<0.0001). Proteins occurred at a significantly lower volume than the cells (W = 543.0, p<0.0001) and were consistently the least abundant biofilm component (Fig. 4; Table 3). The range in volume of each of the components (Table 3) indicates the substantial heterogeneity in biofilm coverage and supports the choice of analysing a greater number of sample replicates.

Bottom Line: The volume of EPS was 4.9 times greater than that of the cells within biofilms, with carbohydrates present as the dominant component.Additionally, the greatest proportion of EPS was located above that of the cells.Moreover, biofilms from different positions were similar with respect to community structure and the quantity, composition and three-dimensional distribution of cells and EPS, indicating that active colonisation of the pipe wall is an important driver in material accumulation within the DWDS.

View Article: PubMed Central - PubMed

Affiliation: Pennine Water Group, Department of Civil and Structural Engineering, The University of Sheffield, Sheffield, United Kingdom; NERC Biomolecular Analysis Facility, Department of Animal and Plant Sciences, Western Bank, Sheffield, United Kingdom.

ABSTRACT
Within drinking water distribution systems (DWDS), microorganisms form multi-species biofilms on internal pipe surfaces. A matrix of extracellular polymeric substances (EPS) is produced by the attached community and provides structure and stability for the biofilm. If the EPS adhesive strength deteriorates or is overcome by external shear forces, biofilm is mobilised into the water potentially leading to degradation of water quality. However, little is known about the EPS within DWDS biofilms or how this is influenced by community composition or environmental parameters, because of the complications in obtaining biofilm samples and the difficulties in analysing EPS. Additionally, although biofilms may contain various microbial groups, research commonly focuses solely upon bacteria. This research applies an EPS analysis method based upon fluorescent confocal laser scanning microscopy (CLSM) in combination with digital image analysis (DIA), to concurrently characterize cells and EPS (carbohydrates and proteins) within drinking water biofilms from a full-scale DWDS experimental pipe loop facility with representative hydraulic conditions. Application of the EPS analysis method, alongside DNA fingerprinting of bacterial, archaeal and fungal communities, was demonstrated for biofilms sampled from different positions around the pipeline, after 28 days growth within the DWDS experimental facility. The volume of EPS was 4.9 times greater than that of the cells within biofilms, with carbohydrates present as the dominant component. Additionally, the greatest proportion of EPS was located above that of the cells. Fungi and archaea were established as important components of the biofilm community, although bacteria were more diverse. Moreover, biofilms from different positions were similar with respect to community structure and the quantity, composition and three-dimensional distribution of cells and EPS, indicating that active colonisation of the pipe wall is an important driver in material accumulation within the DWDS.

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