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Exploration of flow cytometry for rapid detection of changes in the microbial communities of drinking water after washing of slow sand filters

Pullerits, Kristjan LU (2016) KMB820 20152
Applied Microbiology
Biotechnology
Abstract
Microbial water quality is often assessed with heterotrophic plate counts (HPC) which are not trustworthy and time consuming, and flow cytometry (FCM) with DNA staining by SYBR® Green I (SG) has been suggested as a suitable tool for analyzing the bacterial community in drinking water. This study investigated the existing staining protocol and then applied this protocol to monitoring of slow sand filter function.

After testing the published protocol with different staining temperatures and times, it was found to be a robust and reliable method for FCM using 15 minutes of staining time with 37°C incubation temperature. The protocol was used successfully to investigate: lake water contamination into tap water; differentiate stagnant from... (More)
Microbial water quality is often assessed with heterotrophic plate counts (HPC) which are not trustworthy and time consuming, and flow cytometry (FCM) with DNA staining by SYBR® Green I (SG) has been suggested as a suitable tool for analyzing the bacterial community in drinking water. This study investigated the existing staining protocol and then applied this protocol to monitoring of slow sand filter function.

After testing the published protocol with different staining temperatures and times, it was found to be a robust and reliable method for FCM using 15 minutes of staining time with 37°C incubation temperature. The protocol was used successfully to investigate: lake water contamination into tap water; differentiate stagnant from flowing water; and discriminate bottled water from tap water. Natural organic matter can interact with SG, and humic acid added to water samples quenched the fluorescence signals and affected the bacterial counts as well as the fingerprint.

Two new slow sand filters were built in winter 2015. One filter contained completely new sand (SSF-new) and one contained new sand and washed sand from older sand filters (SSF-mix). These were compared to an old established slow sand filter (SSF-est). The effect on the bacterial community and traditional water quality parameters due to washing of these three slow sand filters was studied using FCM with bacterial staining, HPC, coliform and Escherichia coli data. Coliforms and E. coli counts were detected in the outgoing water from SSF-new and SSF-mix whereas SSF-est removed coliforms and E. coli. However, SSF-est had the least total cell reduction when counts in the incoming water were compared to the outgoing water suggesting that this well-functioning slow sand filter can select which bacteria are present in the outgoing water. SSF-est also had the most stable biofilm, with fluorescent fingerprints remaining stable throughout the sampling period, while shifting the microbial community to more cells with low nucleic acid (LNA) content and overall a reduced percentage of high nucleic acid (HNA) bacteria. The community shift to more LNA bacteria in the outgoing water was to some extent seen in SSF-mix but not in SSF-new. All sand filters seeded the water with more percentage intact cells. (Less)
Popular Abstract
Water is the fundamental factor for human survival and safe drinking water is essential for the wellbeing of people. Still, almost 1 billion people around the globe lack access to secure drinking water. Examples of pathogen contamination of the drinking water in Sweden include outbreaks of Cryptosporidium in Östersund 2010, that affected 27 000 people, and Norovirus in Lilla Edet where 2 400 individuals got sick. Also in Lund 2013, the citizens were urged to boil their water due to increased levels of coliform bacteria. Temperature change due to global warming could affect the source waters used for drinking water production by raising the nutrient levels in the water. This nutrient boost might stimulate microbial growth and also cause... (More)
Water is the fundamental factor for human survival and safe drinking water is essential for the wellbeing of people. Still, almost 1 billion people around the globe lack access to secure drinking water. Examples of pathogen contamination of the drinking water in Sweden include outbreaks of Cryptosporidium in Östersund 2010, that affected 27 000 people, and Norovirus in Lilla Edet where 2 400 individuals got sick. Also in Lund 2013, the citizens were urged to boil their water due to increased levels of coliform bacteria. Temperature change due to global warming could affect the source waters used for drinking water production by raising the nutrient levels in the water. This nutrient boost might stimulate microbial growth and also cause higher risk for pathogen contamination in the future.

To protect drinking water from microbial contamination, there are numerous drinking water treatment methods involving physical, chemical and biological barriers. Ringsjöverket, which provides drinking water to Helsingborg, Landskrona and Lund, among other cities, uses source water from Lake Bolmen. Chemicals are added to the water that interact with contaminants and form floccules that settle in the bottom of sedimentation basins. The water is then filtered through rapid sand filters to remove the last floccules. Afterwards, big basins filled with sand, called slow sand filters are used. Here microorganisms are living attached to sand and are able to degrade organic matter and other contaminants. The last step before the water goes out to the distribution system is to add a small amount of chlorine to inactivate microbes.

To make sure that the water is safe to consume, the microbial water quality is tested with a method involving cultivation of microbes called heterotrophic plate counts (HPCs). Not all drinking water bacteria are cultivable, in fact, less than 0.01% of all bacteria in drinking water can be detected by HPCs and drinking water contains thousands of bacteria per mL, with most of these being harmless for humans. Specific “indicator” organisms are also often targeted, for example Escherichia coli and coliforms. These cultivation based methods take days to acquire the results.

In this thesis, a method called flow cytometry (FCM) using DNA staining was investigated. This method has recently been introduced as a faster and more sensitive method to examine microbes in drinking water. FCM is a rapid method compared to cultivation based methods and results are obtained within hours. The DNA of the bacteria in a water sample is stained so they will emit light and can be counted. Water was sampled at Ringsjöverket in August 2015, before and after three slow sand filters, to compare two which were newly built (March 2015) with an old established slow sand filter. All samples were examined using HPC, E. coli, coliform plate counts and FCM data. This was done to both gain more knowledge of how slow sand filters work, and to compare FCM to HPCs, E. coli and coliform data. FCM with DNA staining gave fast and reliable data for investigation of drinking water bacteria whereas HPC data were difficult to interpret. All data, both FCM and traditional parameters also showed that newly built slow sand filters using completely new sand have a different, and poorer, performance than if sand filters are built using a mix of mature and new sand. In addition, neither of the newly built sandfilters were as effective at cleaning the water in terms of coliforms and E. coli as an old established slow sand filter, suggesting that it requires more than 5 months for the new slow sand filters, and their water-cleaning biofilms, to become a stable functioning biofilter. (Less)
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author
Pullerits, Kristjan LU
supervisor
organization
course
KMB820 20152
year
type
H2 - Master's Degree (Two Years)
subject
keywords
biofilm, slow sand filter, microbial communities, drinking water, flow cytometry, applied microbiology, teknisk mikrobiologi
language
English
id
8894154
date added to LUP
2016-11-14 15:37:24
date last changed
2016-11-14 15:37:24
@misc{8894154,
  abstract     = {Microbial water quality is often assessed with heterotrophic plate counts (HPC) which are not trustworthy and time consuming, and flow cytometry (FCM) with DNA staining by SYBR® Green I (SG) has been suggested as a suitable tool for analyzing the bacterial community in drinking water. This study investigated the existing staining protocol and then applied this protocol to monitoring of slow sand filter function.

After testing the published protocol with different staining temperatures and times, it was found to be a robust and reliable method for FCM using 15 minutes of staining time with 37°C incubation temperature. The protocol was used successfully to investigate: lake water contamination into tap water; differentiate stagnant from flowing water; and discriminate bottled water from tap water. Natural organic matter can interact with SG, and humic acid added to water samples quenched the fluorescence signals and affected the bacterial counts as well as the fingerprint.

Two new slow sand filters were built in winter 2015. One filter contained completely new sand (SSF-new) and one contained new sand and washed sand from older sand filters (SSF-mix). These were compared to an old established slow sand filter (SSF-est). The effect on the bacterial community and traditional water quality parameters due to washing of these three slow sand filters was studied using FCM with bacterial staining, HPC, coliform and Escherichia coli data. Coliforms and E. coli counts were detected in the outgoing water from SSF-new and SSF-mix whereas SSF-est removed coliforms and E. coli. However, SSF-est had the least total cell reduction when counts in the incoming water were compared to the outgoing water suggesting that this well-functioning slow sand filter can select which bacteria are present in the outgoing water. SSF-est also had the most stable biofilm, with fluorescent fingerprints remaining stable throughout the sampling period, while shifting the microbial community to more cells with low nucleic acid (LNA) content and overall a reduced percentage of high nucleic acid (HNA) bacteria. The community shift to more LNA bacteria in the outgoing water was to some extent seen in SSF-mix but not in SSF-new. All sand filters seeded the water with more percentage intact cells.},
  author       = {Pullerits, Kristjan},
  keyword      = {biofilm,slow sand filter,microbial communities,drinking water,flow cytometry,applied microbiology,teknisk mikrobiologi},
  language     = {eng},
  note         = {Student Paper},
  title        = {Exploration of flow cytometry for rapid detection of changes in the microbial communities of drinking water after washing of slow sand filters},
  year         = {2016},
}