Extracellular DNA in Monochloraminated Drinking Water and Its

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Extracellular DNA in Monochloraminated Drinking Water and its Influence on DNA-based Profiling of Microbial Community Bairoliya Sakcham, Amit Kumar, and Bin Cao Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.9b00185 • Publication Date (Web): 21 Apr 2019 Downloaded from http://pubs.acs.org on April 22, 2019

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Environmental Science & Technology Letters

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Extracellular DNA in Monochloraminated Drinking Water and its Influence on DNA-

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based Profiling of Microbial Community

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Bairoliya Sakcham1,2, Amit Kumar1,3, Bin Cao1,3*

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1

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University, Singapore 637551

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2

Interdisciplinary Graduate School, Nanyang Technological University, Singapore 637553

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School of Civil and Environmental Engineering, Nanyang Technological University, Singapore

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639798

Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological

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*Corresponding author: Dr. Bin Cao, School of Civil and Environmental Engineering, Nanyang

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Technological University, 50 Nanyang Ave, N1-01C-69, Singapore 639798; e-mail:

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[email protected]; Tel: (+65) 6790 5277; Fax: (+65) 6791 0676.

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1 ACS Paragon Plus Environment


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Abstract

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The interaction between biofilms and disinfectant in drinking water distribution systems

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(DWDS) as well as the role of this interaction in the formation of disinfection byproducts

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(DBPs) has been extensively studied in recent years. In contrast, lysis of cells and/or release of

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intracellular biomolecules from inactivated/damaged cells and their fate and implications are an

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overlooked aspect in DWDS. In particular, DNA, once released into DWDS, may persist in

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water as extracellular DNA (eDNA). In this study, we report for the first time that the total DNA

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extracted from monochloraminated drinking water contains a high fraction of eDNA. Drinking

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water samples were obtained from Locations 1 (~20-year-old pipeline) and 2 (~7-year-old

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pipeline). At Location 1, 85-386 ng of eDNA was found per litre of sampled water, which

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accounted for 52 ±12 % of total DNA, while at Location 2, 33-58 ng of eDNA was found per

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litre of sampled water, accounting for 42±8% of the total DNA. We further showed that the

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removal of eDNA reduced α-diversity, increased community evenness, and changed relative

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abundance of detected taxa. Our findings lead to future research questions about the source, fate

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and implications eDNA in DWDS.


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INTRODUCTION

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Drinking water distribution systems (DWDS) house a huge diversity of microorganisms in the

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form of planktonic cells in the bulk water and sessile microbial communities, i.e., biofilms, on

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the inner pipe surfaces. Majority of the microorganisms (~90-98%) are found attached to the

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inner surfaces of the distribution pipelines in the form of biofilms1. Biofilms are believed to

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provide more favorable conditions for the sustenance of the microorganisms as they can protect

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the residing organisms from the harsh conditions in the bulk water (e.g., low nutrient content,

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dynamic flow patterns and/or presence of disinfectant residuals)2. Cells along with the biofilm

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matrix and loose mineral deposits can make their way into the bulk water stream due to natural

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dispersion of biofilms or changes in operational parameters (e.g., flow velocity, nutrient loading,

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disinfection residual) causing biofilm detachment3.

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To manage biofilms in DWDS, a secondary disinfectant monochloramine (MCA) is often used3.

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MCA can inactivate cells in bulk water4-5, reducing biofilm formation. It can also effectively

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penetrate biofilms and inactivate cells within the biofilms6-8. Various studies have found that the

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interaction between the disinfectant and the organic matter in the form of DWDS biofilms

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contributes greatly to the formation of disinfection by-products (DBPs), a group of emerging

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contaminants potentially genotoxic and carcinogenic9. An overlooked aspect of the cell-

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disinfectant interaction in the DWDS is the possible lysis of cells and release of intracellular

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biomolecules into the drinking water. In particular, DNA, once released from the cells and

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biofilms in DWDS, may persist in water as extracellular DNA (eDNA) because DNA reacts

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slowly and moderately with MCA (kMCA =10-1 to 101 M-1 s-1) and chlorine (kchlorine =101 to 103

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M-1 s-1), respectively10. DNA has been shown to be stable in various environmental conditions

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and has been observed to persist and accumulate for long periods in freshwaters 11, marine

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environments 12 and soils 13, engineered water systems14-15, especially in the presence of natural

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organic matters 16. Although the occurrence and fate of eDNA in many natural environments

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including soils and surface waters have been investigated, there have been no studies examining

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the presence of eDNA in DWDS17. In addition, many studies characterized DWDS microbial

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communities using DNA-based profiling methods18, however, eDNA has not been taken into

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consideration. Whether and how eDNA influences DNA-based profiling of DWDS microbial

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communities still remains a question. 3 ACS Paragon Plus Environment


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In this study, we report for the first time that the total DNA extracted from monochloraminated

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drinking water contains a high fraction of eDNA and the presence of eDNA obscures the DNA-

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based estimation of microbial diversity and community profiling. Our results suggest that the

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occurrence, fate and implications of eDNA, an overlooked component in drinking water, should

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be carefully examined in future studies.

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MATERIALS AND METHODS

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Sampling of Drinking Water. Drinking water samples were obtained from two different

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locations, Location 1 (~20-year-old pipeline) and Location 2 (~7-year-old pipeline), both

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distributing water treated with MCA as a secondary disinfectant. Glass fiber membrane (25 mm

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in diameter, 0.4 µm nominal pore size, Grade GB-140; Advantec) packed in polypropylene in-

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line filter holders (Sterlitech Corporation) were used to filter the drinking water directly from the

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taps. The glass filter membranes have been shown to be capable of effectively retaining DNA19

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and have been applied to extract environmental DNA from water samples20. The initial flow rate

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for filtration ranged between 200-215 mL/min and a total of ~15 L of water was filtered through

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each membrane. For each location, 4 samples were collected in parallel using a flow splitter from

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the same tap (Figure S1 in the Supporting Information (SI)). The filter samples were then

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subjected to various treatments and analyses (Figure S2) which are described below in details.

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The SEM imaging methods and representative SEM images (Figure S3) can be found in SI.

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Water quality parameters have been provided in Table S1.

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DNase Treatment. After filtration, the membranes were taken out and divided into two equal

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parts and one part was randomly assigned to DNase treatment (refer to SI for details). DNase

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treatment has been previously shown to remove DNA only from membrane-compromised cells

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without affecting the intact cells21. The samples were incubated with DNase (DNase-

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Treated)/MilliQ water (Control) at 37 °C for 1 h and then washed twice with 0.1 M Phosphate

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Buffered Saline (PBS) before proceeding for the DNA extraction. The effectiveness of the

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DNase treatment was evaluated using a cell suspension (OD600 ~0.1) of Pseudomonas

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aeruginosa (a bacterial strain isolated from DWDS) spiked with known amounts (2, 3 or 4 µg) of

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genomic DNA.


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DNA Staining and Confocal Microscopy Imaging. A small portion (~2.5 × 2.5 mm) from the

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center of each half was randomly assigned as a control or for DNase treatment. After DNase

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treatment, samples were rinsed with sterile MilliQ water and stained using TOTO-1 (2 µM in

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DMSO, excitation/emission spectrum of 488/488-640 nm) and counter stained with SYTO-60

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(10 µM in DMSO, excitation/emission spectrum of 633/644-664 nm). The effectiveness of the

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combination of dyes was evaluated using a cell suspension of P. aeruginosa imaged before and

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after treatment with DNase to remove eDNA (refer to SI for details).

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DNA Extraction and Quantification. DNA extraction was carried out using a modified

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cetyltrimethylammonium bromide (CTAB) –Proteinase K method (refer to SI for details). DNA

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was quantified using Quant-iT ™ dsDNA High-Sensitivity Assay Kit from Invitrogen. The

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percentage of eDNA in the extracted samples was estimated based on the difference between the

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amount of DNA extracted from the filter samples with and without DNase treatment.

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qPCR, PCR Amplification and Sequencing. The abundance of eDNA was estimated using

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qPCR based on the difference between the 16S rRNA gene copy numbers from the filter samples

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with and without DNase treatment. Fungal contribution to eDNA was also assessed by

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quantifying the 18S rRNA gene. PCR amplification was carried out in duplicate in a 20 µL

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reaction volume using universal 16S rRNA gene primers. The amplicons were purified using

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AMPure XP beads (Beckman Coulter, Brea, 138 CA) and quantified using Quant-iT™ dsDNA

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High-Sensitivity Assay Kit. Following library preparation and quality control, pair ended

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sequencing (300 × 300 bp) was carried out on an Illumina MiSeq platform. Experimental details

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can be found in SI.

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Sequencing Data Processing and Analysis. The sequences were processed using the DADA2

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pipeline v1.8 in R v3.5.122. The resulting ASV (Amplicon Sequence Variants) table was filtered

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for reads which were unassigned at Kingdom level or assigned as the Eukaryotic and Chloroplast

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sequences. Only the prokaryotic sequences were kept for further analysis. ASV table is just a

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higher resolution version of a traditional OTU table22. The ASV table obtained from the DADA2

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pipeline was further analyzed in R (v3.5.1) using the “phyloseq”23 and “vegan”24 packages. The

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resulting plots were made using the package “ggplot2”25. All analysis was conducted on data-sets

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rarefied to the minimum number of sequences obtained for a sample. More details can be found 5 ACS Paragon Plus Environment


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in the SI. Sequencing results have been deposited to the Sequence Read Archive (SRA) under

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Biosample number SAMN10667470- SAMN10667485 and Bioproject number PRJNA512437.

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RESULTS AND DISCUSSION

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Significant Fraction of eDNA in Total DNA Extracted from Drinking Water. Representative

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CLSM images of the filter samples with and without DNase treatment stained with TOTO-1

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(green fluorescence) and SYTO 60 (red fluorescence) are shown in Figure S4. TOTO-1 is

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impermeable to intact cells and selectively stains eDNA and DNA in membrane-compromised

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cells, while SYTO 60 stains intracellular DNA in intact cells as well26. Using a cell suspension

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spiked with DNA, we have shown that these stains work appropriately in terms of cell

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permeability/impermeability (Figure S5) and the DNase treatment effectively removed eDNA,

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while it did not substantially reduce cell viability (>95% after the DNase treatment for all tested

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conditions) (Figure S6). Treatment with DNase greatly reduced the TOTO-1 signal in the

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images, suggesting a high abundance of eDNA in the samples. Further quantitative analyses

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showed a 2-4-fold reduction in the biovolume of eDNA in the treated samples while the

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biovolume for the intact cells remains unchanged (Figure S7A). In addition, greater proportion of

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the green fluorescence signal colocalizes with the red fluorescence signal (Figure S7B),

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indicating the presence of partially permeabilized cells, which is not surprising given the

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monochloramine treatment of the collected water27. The used disinfectant monochloramine

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might be a possible driver of DNA release in the DWDS (Figure S8).

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Pairwise comparison was made for the DNA extracted from the control and the DNase-treated

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halves from each filter sample and the amount of eDNA was estimated assuming that all eDNA

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was removed by DNase treatment. The proportion of eDNA varied in different samples. At

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Location 1, 85-386 ng of eDNA was found per liter of sampled water, which accounted for 52

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±12 % of total DNA, while at Location 2, 33-58 ng of eDNA was found per liter of sampled

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water, accounting for 42±8% of the total DNA (Figure 1A). These proportions for eDNA are

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comparable with what have been reported in surface and lake water samples28. The proportion of

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eDNA was also estimated using qPCR based on the total copy number of the 16S rRNA gene.

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The qPCR-based estimation of eDNA facilitates the comparison of our data with relevant

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literature in other systems such as soils and sediments13, 28 because most eDNA studies used 6 ACS Paragon Plus Environment

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marker genes such as the 16S rRNA gene to estimate the proportion of eDNA in the samples. It

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also helps to confirm that by analysing the bacterial community through the 16S rRNA gene

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amplicon sequencing approach, we are covering the contribution of a major proportion of the

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eDNA. The qPCR results showed an eDNA fraction of 48±12% and 29±11% at Locations 1 and

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2, respectively (Figure 1B). Single factor Analysis of Variance (ANOVA) showed no significant

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difference between the estimated proportion of eDNA obtained through extraction and that

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through 16S rRNA gene-based qPCR (p = 0.66 and 0.13 for Location 1 and 2, respectively),

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suggesting that majority of the eDNA in the system is of bacterial and archaeal origin and is

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expected to contribute to the analysis of the microbial community through the 16S rRNA gene

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amplicon sequencing approach. For verification, fungal contribution to eDNA was also assessed

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by quantifying the 18S rRNA gene (Table S2). Our results show that in 1 L water sample, a copy

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number of about 103 – 104 were detected for the 18S rRNA gene, which is substantially lower

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than the 16S rRNA genes (~108). Thus, taking 18S rRNA gene copy numbers into account does

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not change the calculated proportion of eDNA based on the quantitative results using qPCR.

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Figure 1. Percentage of eDNA found in the samples based on (A) DNA extraction (n = 4) and

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(B) total 16S rRNA gene copies quantified in the control and treated halves of the filters (n = 4).

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Locations 1 and 2 refer to systems which are ~20- and ~7-year-old, respectively. S1, S2, S3 and

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S4 are samples from different filters (annotated by different colors) collected in parallel from one

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tap at each location. 8 ACS Paragon Plus Environment

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Influence of eDNA on Estimation of α- and β-diversity. The α-diversity indices for samples

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with and without DNase treatment are shown in Figure 2A1 and 2B1 for Locations 1 and 2,

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respectively. The Chao1 index and Abundance-based Coverage Estimator (ACE) are estimators

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of richness, taking into consideration rare organisms that may have been missed due to under

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sampling29, while the observed richness is the actual number of ASVs detected in the samples.

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Both Chao1 and ACE have values comparable with the observed richness (Location 1, p = 1.0,

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0.99; Location 2, p = 0.99, 0.99 for Chao1 and ACE, respectively), confirming that the sampling

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was adequate. The DNase treatment, i.e., removal of eDNA, significantly reduced the Chao1 and

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ACE indices for samples from Location 2 (p 0” indicates more detection in the

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control samples; “FC

Extracellular DNA in Monochloraminated Drinking Water and Its

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