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Sustainability Engineering and Green Chemistry
Membrane distillation biofouling: Impact of feed water temperature on biofilm characteristics and membrane performance Anne Bogler, and Edo Bar-Zeev Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02744 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 7, 2018
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Membrane distillation biofouling: Impact of feed water temperature
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on biofilm characteristics and membrane performance
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Anne Bogler* and Edo Bar-Zeev*
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Zuckerberg Institute for Water Research, The Jacob Blaustein Institutes for Desert
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Research, Ben-Gurion University of the Negev, Sede Boker, 84990, Israel
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* Corresponding Author: Dr. Edo Bar-Zeev;
[email protected], Anne Bogler;
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[email protected] 8
Key words
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Membrane distillation, feed water temperature, wetting, biofouling, biofilm characteristics
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ABSTRACT
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Membrane distillation (MD) is a temperature driven membrane separation technology
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that holds great potential for decentralized and sustainable wastewater treatment
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systems. Yet, similarly to all membrane based systems, microbial fouling (biofouling)
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might be a critical hurdle for MD wastewater treatment applications. In this study we
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determined the impact of increasing feed water temperatures (47 °C, 55 °C and 65 °C)
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on biofilm growth and MD performance via dynamic biofouling experiments with
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Anoxybacillus sp. as a model bacterium. Our results indicated that cell growth was
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reduced at 47 °C, resulting in moderate distillate water flux decline (30%). Differently,
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extensive growth of Anoxybacillus sp. at feed water temperature of 55 °C caused severe
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distillate water flux decline (78%). Additionally, biofouling induced membrane wetting,
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which facilitated the passage of bacteria cells and endospores through the membrane
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structure into the distillate. Although bacteria growth was impaired at feed water
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temperature of 65 °C, excessive production of EPS (compared to bacterial abundance)
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crippled membrane separation due to severe pore wetting. These results underline the
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importance of optimized operating conditions and development of anti-biofouling and
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anti-wetting membranes for successful implementation of MD in wastewater treatment
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with high biofouling propensity.
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GRAPHICAL ABSTRACT
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INTRODUCTION
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Membrane distillation (MD) is a promising technology for sustainable fresh water
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production, due to the possibility of utilizing waste heat or low-grade solar and
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geothermal heat for separation of high quality fresh water from contaminated feed
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solutions.1–3 Distillate water flux is driven by a water vapor pressure gradient across a
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water-excluding microporous hydrophobic membrane.4,5 Air-filled membrane pores only
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allow the passage of vapors that evaporate from the heated feed water and condense 2 ACS Paragon Plus Environment
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into the cold distillate. Therefore, all non-volatile contaminants are rejected at the vapor-
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liquid interface.1,4 MD operates at ambient hydraulic pressure, which reduces system
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complexity and facilitates the application of MD in decentralized wastewater treatment
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systems.2,3,6 Such MD systems may be powered by an unlimited supply of solar energy,
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thus provide remote communities that are not connected to the national water and
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energy grids with potable water and reduce critical water scarcity.
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Similarly to all membrane systems, treatment of contaminated feed water via MD
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induces the challenge of fouling and especially microbial fouling (herein termed
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biofouling).3,7,8 Biofouling develops as bacteria and organic material, ubiquitous in natural
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and engineered systems, form biofilms on membrane and spacer surfaces.9–11 Biofilm
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formation follows a phased paradigm, in which individual cells or planktonic bacterial
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clusters (protobiofilms) adhere to an organic conditioning film.12 Following irreversible
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attachment, bacteria and protobiofilms develop into a multi-layered biofilm structure,
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comprising live and dead cells encased in a protective matrix of self-produced
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extracellular polymeric substances (EPS).11,13 Despite substantial research on
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membrane biofouling, very little is known about biofilm formation and community
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composition in MD systems.14,15 In addition to factors that influence biofilm formation in
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other membrane systems, the elevated feed water temperature is unique to MD.8,14,16
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Feed water temperature of MD systems can range between 40 °C to 80 °C,3,17 leading to
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a shift in bacterial community composition due to thermally induced succession, after
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which thermophilic and heat resistant bacteria will constitute the majority of the bacteria
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population.14,15,18,19
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MD biofilm can reduce the vapor pressure driving force by hindering mass and heat
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transport, consequently enhancing temperature and salt concentration at the membrane
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surface.15,20,21 The hydrophilic nanoporous structure of the biofilm can further affect the 3 ACS Paragon Plus Environment
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driving force via water vapor pressure depression.21,22 Concomitantly, depending on the
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biofilm properties (i.e., thickness, density, organic composition, etc.), hydraulic
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resistance can also impair water transfer through the biofilm.21,23 Another fouling effect,
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that is unique to MD, is membrane wetting, which refers to the penetration of
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contaminated feed water through the pore structure into the distillate.15,24,25 Among other
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mechanisms, wetting can be caused by a reduction of membrane surface hydrophobicity
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via attachment of surface active compounds in the feed water.15,20,24 Some surface
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active compounds, termed biosurfactants, can be actively secreted to the feed water as
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amphiphilic EPS components by bacteria.15,25
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In this study we identified the effects of feed water temperature on MD biofouling with a
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monoculture of a thermophilic Anoxybacillus sp. at three feed water temperatures (47
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°C, 55 °C and 65 °C) in a direct contact MD system containing feed and distillate
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spacers. System performance was analyzed in real time over three days via distillate
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water flux and distillate water quality measurements. In addition, biofilm properties were
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characterized at the end of each experiment. Our results highlight that selection of the
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appropriate feed water temperature is crucial for the success of MD application in
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wastewater treatment.
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MATERIALS AND METHODS
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Bench-scale MD system. A custom-made bench-scale direct contact MD system with a
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transparent polycarbonate cross flow cell was used for dynamic biofouling experiments
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(Figures S1 and S2). Feed and distillate temperature were controlled by a recirculating
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heater (Polyscience) and chiller (KNF Lab). Flow conditions were held constant using
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two gear pumps with Masterflex® pump heads (Cole-Parmer). Distillate water flux was
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monitored by weighing the distillate solution on a digital scale (Phoenix instrument)
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interfaced with a data acquisition system. Additionally, values of feed (Sensorex) and 4 ACS Paragon Plus Environment
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distillate conductivity (Eutech Instruments, Thermo Scientific) as well as temperature
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(DS18B20 Digital Temp Sensor, Adafruit) in the reservoirs and at in-/outlets of the cross
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flow cell were collected every minute via a microprocessor (Arduino UNO). Further
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details and experimental parameters are available in the Supporting Information.
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Hydrophobic Durapore® membranes with nominal pore size 0.45 µm (HVHP, Merck
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Millipore, Ireland) were used as MD membranes. The active membrane area in the cross
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flow cell was 6.5 x 3.5 cm2 and spacers (46 mils = 1.2 mm, Conwed®, USA) determined
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channel height on feed and distillate side. The spacer had a diamond shaped pattern
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comprised of slightly hydrophobic (contact angel of ~94°) polypropylene filaments,
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molded together in two layers. Filaments were oriented at a 45° angle towards the flow
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direction (Figure S1, insert).
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Bacteria strain and feed water medium. Thermophilic, Gram-positive, spore forming
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Anoxybacillus sp. was isolated from effluent of an operational wastewater membrane
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bioreactor (MBR). An MD biofouling experiment (see details below) was carried out with
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effluent from the MBR for one day at a feed water temperature of 55 °C. Bacteria were
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removed from the biofouled MD membrane with mild sonication and plated on Luria–
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Bertani broth agar (LB, Becton, Dickinson and Company). Single colonies were isolated,
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grown overnight (~12h) in LB and plated again. Isolation process was repeated three
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times before genomic DNA was extracted from sub-samples using a phenol chloroform
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approach.14 Amplification of the V1–V3 regions of the bacterial 16S rRNA was achieved
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with a 28F and 519R primer pair 26 and the bacterium strain was identified by sanger-
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sequencing (Hy-labs, Israel), with high confidence (99% similarity to the Silva database)
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as an Anoxybacillus sp. (and most likely Anoxybacillus gonensis).
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Artificial sterile wastewater medium was prepared fresh for each experiment according
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to Bar-Zeev et al. 2014,27 comprising 8.0 mM NaCl, 0.15 mM MgSO4, 0.50 mM NaHCO3,
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0.20 mM CaCl2, 0.20 mM KH2PO4, 0.40 mM NH4Cl, and 0.6 mM Na3C6H5O7 (sodium
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citrate). Each salt was dissolved in 60 mL double distilled water (DDW), individually
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autoclaved and then added to sterile DDW (10 L). The resulting feed water medium had
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a conductivity of 1 mS cm-1 and pH 7.
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Biofouling procedure. Prior to each experiment, the MD system was thoroughly
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cleaned in three steps by passing 10% bleach, Ethylenediaminetetraacetic acid solution
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(EDTA, 5 mM) at pH 9 and 90% ethanol through the system for 30 min each. Between
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steps, the system was rinsed twice for 5 min with DDW. Finally, the system was
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thoroughly rinsed five times with DDW to remove any traces of the cleaning agents
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before the membrane and spacers were inserted into the cross flow cell.
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Initially, baseline (control) experiments were carried out for three days with artificial
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sterile wastewater (10 L) as feed water and without bacterial inoculation to determine the
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distillate water flux and salt passage at the different temperatures. Distillate temperature
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was adjusted to reach the same initial distillate water flux of 23 ± 2 L m-2 h-1 for all three
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feed water temperatures (further details on the operating parameters are provided in
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Table S1). Every 24 h, collected distillate water was removed from the distillate
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reservoir.
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For each biofouling experiment a fresh and concentrated bacterial suspension was
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prepared by inoculating 300 mL of LB broth with Anoxybacillus sp. and letting it grow
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overnight to a mid-exponential stage (OD600 of ~0.5). Supernatant was removed and
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replaced with sterile artificial wastewater after short centrifugation (4000 rpm for 20 min).
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The feed reservoir was inoculated with the bacteria culture (~7x107 cells mL-1) once the
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initial distillate water flux was stable at 23 ± 2 L m-2 h-1. The feed water was constantly
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aerated by a small air pump and sampled every 24 h to monitor pH and bacterial
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concentration. At the same time, glucose (Sigma-Aldrich) was supplemented as an
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additional carbon source to maintain a constant concentration of ~0.55 mM.
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Real time observation with optical coherence tomography. OCT is an approach that
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has recently gained importance for in situ non-invasive fouling observation in membrane
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systems 28–30 and was used in this study to characterize biofilm formation and membrane
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wetting. The scan lens (Thorlabs, LSM03-BB) fitted on a spectral domain Optical
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Coherence Tomograph (Ganymede-II, Thorlabs, Germany) was placed above the cross
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flow cell to allow observation through the viewing window of the top cell half. Cross
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sectional scans were taken every 24 h between spacer filament crossings and along
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filaments with a resolution of 2 µm/pixel. Sensitivity of image acquisition was set to
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medium at an A-scan rate of 15 kHz with averaging over six A-scans.
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Biofilm characterization. After three days of biofouling membrane and feed spacer
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were removed from the cross flow cell, avoiding separation or shifting, and dissected into
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1 cm2 coupons for subsequent analysis.27,31
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Confocal laser scanning microscopy (CLSM). Imaging was carried out directly at the end
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of each experiment. SYTO® 9, propidium iodide (LIVE/DEAD BacLight™, molecular
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probes) and concanavalin A (Alexa Fluor® 633, molecular probes) were used to stain
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membrane and spacer coupons for identification of live cells, dead cells and extracellular
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polysaccharides as part of biofilm EPS. Stained coupons were mounted in an
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unconfined biofilm characterization chamber 27 for image acquisition with an inverted
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CLSM (LSM 510 META, Carl Zeiss, Inc.). Random Z stacks (7 to 14) with slice thickness
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of 2 µm were captured for each membrane coupon using an LD C-Apochromat 40x
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magnification objective. Images were analyzed to obtain biovolume and biofilm thickness
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of the three biofilm components. For spacer samples, a Plan-Neofluar 10x magnification
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objective was used to capture CLSM images with 50 µm slice thickness at and between
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each filament crossing. The resulting eight adjacent images were combined into one
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image showing one square spacer element. Further details can be found in the
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Supporting Information.
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Bacterial abundance (BA). At the end of each experiment membrane and spacer
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subsamples were re-suspended in 1 mL of sterile wastewater with 2.5 mM EDTA. Cells
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were removed from samples by short (6 min) bath sonication and were diluted with
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sterile wastewater. Dilutions were stained according to the manual with LIVE/DEAD™
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BacLight™ Bacterial Viability and Counting Kit (molecular probes). BA in the distillate
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was measured by staining with SYBR® Green II RNA gel stain (molecular probes) for
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total bacteria count. All stained samples were measured at a flow speed of 25 µL min-1
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using an Attune NxT flow cytometer (life technologies). Representative flow-cytometry
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plots that exhibit the distinction between live and dead cells as well as total bacteria
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count can be found in the Supporting Information (Figure S3).
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Total organic carbon (TOC). Measurements were obtained from membrane and spacer
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samples placed in acid-cleaned glass vials with 7 mL DDW and 40 µL 2 M HCl. Probe
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sonication (Sonicator Model CL-188, QSONICA) for 3 min (six 30 s cycles) was required
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to disperse cells from membrane and spacer surface. A TOC analyzer (Multi N/C,
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Analytik-Jena, Germany) measured TOC using a five point calibration procedure.
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Biofilm density. Organic carbon density was calculated by dividing TOC per area by total
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biovolume per area obtained from the biofilm components in CLSM and is given in fg
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µm-3. Additionally, cell density was calculated by dividing BA per area by total biovolume
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per area, given in cells µm-3.
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Transmission (TEM) and scanning (SEM) electron microscopy. Samples were fixed,
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stained and imaged according to Bar-Zeev et al. 2015 32. Briefly, membrane and spacer
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coupons were fixed at 4 °C overnight with 2.5% glutaraldehyde fixation buffer (0.1 M
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Cacodylate, 0.1% CaCl2, pH 7.2). Staining of bacterial cells was done with osmium
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tetroxide followed by sequential dehydration steps. For TEM, membrane samples were
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then infiltrated with epoxy resin at room temperature and cut into 70 nm slices before
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imaging with an FEI Tecnai T12 G2 TWIN transmission electron microscope operating at
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120 kV. For SEM, membrane samples after dehydration were freeze-fractured and then
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both membrane and spacer samples were sputter coated with gold and imaged with an
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JSM-7400F (JEOL) ultrahigh resolution cold FEG-SEM at an acceleration voltage of 3
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KeV. Further details are available in the Supporting Information.
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Statistical analysis. Distillate water flux values collected every minute were aggregated
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into 4 h rolling averages. Averages were normalized to the last hour before inoculation,
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as well as to the corresponding hour of two averaged baseline experiments (Figure S4).
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Distillate salinity was averaged in the same manner, but normalized to the initial salinity
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of the distillate, equaling DDW. TOC and BA were displayed as means with
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corresponding standard deviations, obtained from three or four biologically different
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experiments. Biofilm parameters were normalized to the area of membrane or spacer
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coupon, measured with ImageJ software. The spacer area was measured as the area
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covered and not the surface area of the spacer filaments, which the supplier estimated
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to be 0.98 m2 m-2. Statistical analysis was conducted in Excel with the XLSTAT Add-In
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using ANOVA pairwise comparison at a significance level of p
