Response of simulated drinking water biofilm mechanical and structural properties to long-term disinfectant exposure. Yun Shen, Conghui Huang, Guillermo L ...
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Response of simulated drinking water biofilm mechanical and structural properties to long-term disinfectant exposure Yun Shen, Conghui Huang, Guillermo L Monroy, Dao Janjaroen, Nicolas Derlon, Jie Lin, Rosa M. EspinosaMarzal, Eberhard Morgenroth, Stephen A. Boppart, Nicholas J. Ashbolt, Wen-Tso Liu, and Thanh H. Nguyen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b04653 • Publication Date (Web): 12 Jan 2016 Downloaded from http://pubs.acs.org on January 13, 2016
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Response of simulated drinking water biofilm mechanical and structural properties
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to long-term disinfectant exposure
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Yun Shen1, Conghui Huang1, Guillermo L. Monroy2, Dao Janjaroen1, Nicolas Derlon4,
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Jie Lin1, Rosa Espinosa-Marzal1, Eberhard Morgenroth4,5, Stephen A. Boppart 2,3,
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Nicholas J. Ashbolt 6, Wen-Tso Liu1, Thanh H. Nguyen1
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Champaign, 2Department of Bioengineering, University of Illinois at Urbana-Champaign,
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Department of Civil and Environmental Engineering, University of Illinois at Urbana-
Department of Electrical and Computer Engineering, University of Illinois at Urbana-
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Champaign, 4Eawag: Swiss Federal Institute of Aquatic Science and Technology, 8600
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Dübendorf, Switzerland, 5ETH Zürich, Institute of Environmental Engineering, 8093
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Zürich, Switzerland, 6School of Public Health, University of Alberta, AB T6G 2G7
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Canada.
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Abstract
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Mechanical and structural properties of biofilms influence the accumulation and release
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of pathogens in drinking water distribution systems (DWDS). Thus, understanding how
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long-term residual disinfectants exposure affects biofilm mechanical and structural
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properties is a necessary aspect for pathogen risk assessment and control. In this study,
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elastic modulus and structure of groundwater biofilms was monitored by atomic force
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microscopy (AFM) and optical coherence tomography (OCT) during three months of
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exposure to monochloramine or free chlorine. After the first month of disinfectant
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exposure, the mean stiffness of monochloramine or free chlorine treated biofilms was 4 to
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9 times higher than those before treatment. Meanwhile, the biofilm thickness decreased
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from 120±8 m to 93±6 -107±11 m. The increased surface stiffness and decreased biofilm
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thickness within the first month of disinfectant exposure was presumably due to the
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consumption of biomass. However, by the second to third month during disinfectant
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exposure, the biofilm mean stiffness showed 2 to 4-fold decrease while the biofilm
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thickness increased to 110±7 -129±8 m, suggesting that the biofilms adapted to
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disinfectant exposure. After three months of the disinfectant exposure process, the
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disinfected biofilms showed 2-5 times higher mean stiffness (as determined by AFM) and
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6-13 fold higher ratios of protein over polysaccharide, as determined by differential
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staining and confocal laser scanning microscopy (CLSM), than the non-disinfected
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groundwater biofilms. However, the disinfected biofilms and non-disinfected biofilms
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showed statistically similar thickness (t-test, p>0.05), suggesting that long-term
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disinfection may not significantly remove net biomass. This study showed how biofilm
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mechanical and structural properties vary in response to a complex DWDS environment,
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which will contribute to further research on the risk assessment and control of biofilm-
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associated-pathogens in DWDS.
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Introduction
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Biofilms in drinking water distribution systems (DWDS) can facilitate pathogen
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persistence and transmission1 by harboring pathogens2, supplying nutrients,3-7 and
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protecting pathogens from disinfection.8, 9 It is further reported that biofilms can capture or
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accumulate planktonic pathogens and then release these pathogens via the detached biofilm
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materials.1 This process (biofilms accumulating and releasing pathogens) can be highly
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influenced by biofilm structural and mechanical properties. For example, biofilm
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roughness was observed to control pathogen accumulation to biofilms by increasing the
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interception of pathogens with biofilms.10-13 Biofilm elasticity and cohesiveness are shown
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to be essential for detachment of biofilms and biofilm-associated pathogens.14-16 Therefore,
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comprehensive understanding of the mechanical and structural properties for drinking
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water biofilms will provide information to predict, assess, and aid in controlling the risk of
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pathogens associated with DWDS biofilms.
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A disinfectant residual is required in most drinking waters by the U.S. Environmental
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Protection Agency (EPA). Of particular interest here is that disinfectant residuals may
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influence the biofilm mechanical and structural properties through biomass loss and change
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in biofilm chemical composition. Thinner and rougher Pseudomonas aeruginosa biofilms
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were observed after a relatively short term (1-6 days) of continuous exposure to free
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chlorine stream.17 The cohesiveness of multi-species drinking water biofilms did not
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significantly change after 60 mins of exposure to quiescent free chlorine solution. 18 Longer
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disinfectant exposure (8 weeks) was also reported to lead to a reduction in groundwater
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biofilm thickness.19 However, it is unknown how longer term (i.e., normal operational)
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disinfectant exposure may influence mechanical and structural properties other than
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thickness. In addition to disinfectant exposure, hydrodynamic shear stress is known to
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influence biofilm mechanical and structural properties. 18,
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developed under high shear stress up to 10 Pa were shown to be cohesively stronger.15, 21
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Reduction of biofilm thickness was observed under a continuous exposure to shear stress
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up to 0.9 Pa.21, 22 During disinfectant exposure, shear can accelerate biofilm-disinfectant
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reaction by enhancing mass transfer of disinfectant into the biofilms, 26 presumably leading
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to significant biofilm property variation. However, the combined effect of disinfectant
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For example, biofilms
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exposure and shear stress on properties of biofilm grown under low nutrient conditions
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over a longer time appears to be unreported.
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To fill these research gaps, mechanical and structural properties of simulated drinking
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water biofilms were monitored during three months of disinfectant exposures.
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Monochloramine and free chlorine are the two most commonly used disinfectants in
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DWDS and were separately used to treat groundwater-grown biofilms. Both shear and
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quiescent conditions were explored during disinfectant exposure to simulate dynamic and
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stagnant zones in DWDS. In this study, we measured biofilm elastic modulus with atomic
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force microscopy (AFM) and biofilm structure (thickness and roughness) with optical
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coherence tomography (OCT) to determine the role of disinfectant exposure, shear
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conditions, and exposure duration time on biofilm mechanical and structural properties.
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The results of this study show how biofilm mechanical and structural properties vary in
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response to a complex DWDS environment and contribute to further research on the risk
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assessment and control of biofilm-associated-pathogens in DWDS.
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Materials and Methods
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Biofilm preparation and disinfectant exposure assay
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A groundwater source for drinking water in Urbana-Champaign, IL was selected for
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growing biofilms on PVC coupons (RD 128-PVC, BioSurface Technologies Corporation,
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Bozeman, MT) in CDC reactors (CBR 90-2, BioSurface Technologies Corporation,
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Bozeman, MT), as described previously.11, 12 This groundwater source mainly contained
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1.6 mM Ca2+, 1.2 mM Mg2+, and 1.0 mM Na+. Continuous stirring at 125 RPM in the CDC
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reactors created a shear condition with a Reynolds (Re) number of 2384. The groundwater
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biofilms were developed in CDC reactors for one year and then distributed in six reactors
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for the subsequent biofilm disinfectant exposure.
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In the biofilm disinfection step, these one-year-old biofilms were exposed to either free
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chlorine or monochloramine for 3 months. Specifically, groundwater containing 4 mg
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Cl2/L of monochloramine or free chlorine were continuously introduced to the biofilm
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reactors in either a stirred or non-stirred condition (Figure 1). Reactors 1 and 4 were
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continuously exposed to groundwater containing freshly prepared monochloramine, while 4 ACS Paragon Plus Environment
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Reactors 2 and 5 were similarly exposed to freshly prepared free chlorine. Reactors 3 and
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6 were exposed only to disinfectant-free groundwater and were used as controls. Reactors
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1-3 were stirred at 125 RPM to simulate pipe flow conditions, while Reactors 4-6 were
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unstirred to simulate stagnant conditions. Both stirred and non-stirred conditions were used
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because the shear stress caused by stirring could influence the biofilm mechanical and
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structural properties. 18, 20-25 In a real DWDS, both quiescent and shear conditions are likely
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to occur and affect biofilm structure. The six reactors were operated three months at 4 mg
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Cl2/L of total disinfectant, which is the maximal residual disinfectant concentration in
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DWDS required by the EPA. The feed disinfectant solutions were prepared and replenished
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every other day.
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Biofilm elastic modulus determination by AFM indentation test
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AFM probe preparation
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The indentation measurements were conducted with a silica sphere (with a diameter of 20
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m) glued to a tipless cantilever (calibrated with a normal spring constant of 0.6-1.2 N/m,
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Mikromasch, Lady's Island, SC). Similar to previous studies using spherical probes ranging
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in size from 10m to 50m to detect mechanical properties of polymers, cells, and
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biofilm,
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