Scalable and Sustainable Total Pathogen Removal Filter Paper for

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Scalable and Sustainable Total Pathogen Removal Filter Paper for Point of Use Drinking Water Purification in Bangladesh Olof Gustafsson, Levon Manukyan, Simon Johan Gustafsson, Gopi Krishna Tummala, Sharmin Zaman, Anowara Begum, Md. Almujaddade Alfasane, Khondkar Siddique-e-Rabbani, and Albert Mihranyan ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.9b03905 • Publication Date (Web): 06 Aug 2019 Downloaded from pubs.acs.org on August 13, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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ACS Sustainable Chemistry & Engineering

Scalable and Sustainable Total Pathogen Removal Filter Paper for Point of Use Drinking Water Purification in Bangladesh Olof Gustafsson‡,1 Levon Manukyan‡,1 Simon Gustafsson,1 Gopi Krishna Tummala,1 Sharmin Zaman2, Anowara Begum3, Md. Almujaddade Alfasane,4 Khondkar Siddique-eRabbani,5 Albert Mihranyan1* 1Nanotechnology

and Functional Materials, Department of Engineering Sciences,

Regementsvägen 1, Box 534, Uppsala University, 751 21 Uppsala, Sweden 2Center

for Advanced Research in Sciences (CARS), University of Dhaka, Dhaka 1000, Bangladesh

3Department

of Microbiology, Science Complex, University of Dhaka, Dhaka 1000, Bangladesh

4Department

of Botany, Curzon Hall Campus, University of Dhaka, Dhaka 1000, Bangladesh

ACS Paragon Plus Environment

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ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

5Department

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of Biomedical Physics and Technology, Curzon Hall Building, University of Dhaka, Dhaka 1000, Bangladesh

*Corresponding author: [email protected]


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KEYWORDS Pithophora algae cellulose, Cladophora algae cellulose, water-borne infections, viruses, filtration

ABSTRACT

This article describes for the first time the full cycle of development from raw material cultivation to real-life application of a truly sustainable and scalable filter paper material intended for point of use drinking water purification in Bangladesh. The filter paper, featuring tailored pathogen removal properties, is produced from nanocellulose extracted from Pithophora green macroalgae, growing locally in Bangladesh, a new unexploited resource that can address a global problem. We demonstrate that the Pithophora cellulose filter paper can be used as a total pathogen barrier to remove all types of infectious viruses and bacteria from water. The performance of the filter is validated using surrogate latex nanobeads, in vitro model viruses, and real-life water samples collected from Turag river and Dhanmondi lake in Dhaka, Bangladesh. Access to clean drinking water is a persistent problem in Bangladesh, affecting tens of millions of people every day. Mortality rate due to water-borne diarreal infections, including viral infections, among susceptible population groups, especially among children under age of 5, is still very high. The proposed solution can dramatically improve the quality of lives for millions of people in the entire South East Asian region including and beyond the borders of Bangladesh.


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INTRODUCTION The water crisis in Bangladesh is a serious societal problem that affects both rural and urban areas. Bangladesh is a country with a population of over 162 million people, where 43% of the population survives on less than 1.25 USD per day (UN 2010 data).1 The projected growth rates suggest that the population of Bangladesh will reach 200-225 million people mark by 2050.1 Today, about 20 million people lack access to safe water and 63 million people lack access to improved sanitation.1 Both rural and urban regions of Bangladesh have extremely high densities of population. The cities of Dhaka and Chittagong with average density of population in slums on the order 205,000 people/km2 are the only cities with extensive piped water and sewage system.2 Besides, the water is available at most a few hours per day.3 All of the piped water get contaminated on the supply route and needs to be boiled for consumption. In 2008, the residents of Dhaka city required 2.1 billion L water/day.2 While the recommended amount of water is around 200 L/day/person (for all purposes), slum-dwellers may manage with less than 10 L/day.2 With Dhaka population growing over 300,000 persons/year,2 access to clean water is critical for normal and sustainable life. Water-borne diarrheal diseases constitute a major health problem in Bangladesh, especially among children under the age of 5 years, and may account for up to 11% of all deaths in the country.4 Densely populated urban areas constitute a major hotspot for spreading diarrheal diseases, and Bangladesh is among the most risky zones for epidemic


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in the world.5 While there are many different water-borne pathogens, viruses have the greatest infectivity among them all and exhibit the longest survival potential in the environment.6 Water-borne viruses, e.g. rotavirus, adenovirus, norovirus, and hepatitis A/E viruses, are difficult to clear from water due to their small size and high resistivity.7 In the past, several studies focused particularly on the occurrence of water-borne viral infections in Bangladesh. High quantities of rotavirus and adenovirus were detected in both surface- and ground-water.8-9 The viral occurrence frequency (rotavirus and adenovirus) and mean log concentration exceeded those of the bacterial pathogens, being at least ten-fold higher in some cases.9 It was reported that overall proportion of rotavirus outbreaks in Bangladesh in children under the age of 5 years has increased from about 25% in 1993–97 to 42% in 2008–12, respectively.10 International Centre for Diarrhoeal Disease Research Bangladesh (icddr,b) reported that Vibrio cholera and rotavirus constitute about 30-40% of all diarrheal cases in the country.11 Rotavirus is one of the most persistent and infectious water-borne pathogens (ID50 is ~6 viral units), and it is also featured with high excretion rate with stool, i.e. >1010–1012/mL.5 Major viral outbreaks caused by water-borne pathogens other than rotavirus have also been reported. In particular, hepatitis E virus outbreak, i.e. acute jaundice, was recorded in Rajshashi City in northern Bangladesh in 2010, causing high mortality among pregnant women and neonates.12-13 The source of the epidemic was associated with drinking


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municipally supplied water, likely to be caused by sewage contamination.12 Another common water-borne diarrheal viral pathogen in Bangladesh is norovirus.14 It should, however, be noted that the challenges of pathogen safety of drinking water are common not only for Bangladesh but also many other countries in the world. In order to fight rotavirus infection, WHO recommends state-level vaccination programs. Although vaccines against rotavirus (RotaTeq® or Rotarix®) have been highly effective in highincome countries, they are considerably less potent in low-income countries.15 The reasons for the discrepancy may include malnutrition, zinc deficiency, avitaminosis, gut microbiota, co-infections, functional deficiency of infants immunity, and environmental enteropathy in children from low-income countries.15 Overall, vaccines are expensive and they are designed to fight a specific virus, whereas prevention of water-borne diseases should preferably be pluripotent, protecting against all types of known and potentially emerging pathogens. To prevent the spread of water-borne infections, affordable point-of-use (POU) water treatment strategies that can protect against all kinds of water-borne pathogens are needed. Pooi and Ng recently reviewed available technology for low-cost POU water treatments, highlighting benefits and shortcomings of each method.16 Collecting rainwater and/or disinfection by boiling as well as using sunlight are some of the common practices for reducing the incidence of water-borne infectious diseases. Boiling is highly energy consuming and can be unaffordable due to high fuel costs for the poorest people.


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If wood is used for heating, it can lead to deforestation. Furthermore, boiling is a high risk of scalding.16 Sunlight has been explored for disinfection, by filling transparent plastic bags or bottles with water and exposing it extensively to solar rays.16 However, efficiency is highly dependent on the treatment time and weather conditions, since it may require up to 48 h of exposure on a cloudy day for disinfection. Also, prolonged exposure to sunlight may lead to building up toxic degradation products from plastic.16 At University of Dhaka, solar water pasteurization technique has been tested that uses a solar light absorbing (black) base, water in plastic (transparent polythene or polypropylene) bags, and extra transparent covers. The latter configuration allows achieving the ‘Green House effect’ and raising the water temperature to more than 70 0C in about 2 hours of exposure to clear sun.17-19 A synergy of heat and UV destroys hazardous pathogens in water. However, toxic degradation of the plastic bags remains an issue here as well. A few years ago a new type of virus removal filter paper was described for biotechnology applications that is capable of removal of various types of viruses, including the smallest and most resistant to disinfection viruses, by straightforward dead-end filtration.20-29 This filter paper is produced from nanocellulose extracted from filamentous green Cladophora sp. algae.30 Recently, researchers at Uppsala University have shown that the filter paper produced from Cladophora algae cellulose could also be used for water purification applications.31 This article explores the possibility of making water purification filter paper


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from cellulose extracted from algae species that are endemic in Bangladesh and therefore can be locally cultivated and processed into advanced filters. In many parts of Bangladesh, especially in shallow natural water pools, grows a kind of green macroalgae, viz. Pithophora. Pithophora algae have long, branched filaments made up of numerous cells, often referred to by local people as “Shewla” [শেওলা]. Pithophora algae are very robust and show unusually high survival potential at varying environmental conditions.32-33 It has previously been reported that Pithophora algae are growing especially fast in nitrate and phosphate rich environments.34 Thus, it is possible to cultivate and harvest large quantities of Pithophora in Bangladesh. The applications of Pithophora algae have so far been limited. It has been used for environmental remediation,35-39 Cu(II) and Pb(II)40 sorption or as a livestock feed additive enriched with microelements.41 Extract from Pithophora oedogonia was also used as a reducing agent to produce Au42-43 or Ag nanoparticles.44 Here, we report for the first time the properties of a water purification filter paper produced from cellulose extracted from Pithophora green macroalgae, which was cultivated locally at the University of Dhaka, Bangladesh, in order to demonstrate its huge potential in making advanced filters for drinking water purification.

EXPERIMENTAL SECTION Materials and methods


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Reagent and materials other than cellulose Specialty reagents such as bis(ethylenediamine)copper(II) hydroxide solution (442305, lot number BCBW3059), waste water –simulated matrix (MMW001, lot number LRAB5412) and 30 nm fluorescent latex beads (carboxylate-modified polystyrene) (L5155, lot number 012K1566V) were obtained from Sigma Aldrich (currently Merck). Common chemical reagents, such as sodium hydroxide (pellets), hydrochloric acid (37% w/v), hydrogen peroxide (30% w/v), sodium chloride were of reagent grade and were purchased from Sigma Aldrich (currently Merck). Cultivation of algae Field experiment was carried out in several concrete tanks at the Botanical garden of Department of Botany, Curzon Hall campus, University of Dhaka. Phosphate-rich organic fertilizer in the form of 2.3 mm powder granules (Bone Meal, 2 kg, Corbel Int Ltd., Dhaka, Bangladesh) was used. The fertilizer contained (% wt): total P: 8% min, total N: 3% min, moisture: 8% max. The algae were grown in a water tank filled with soil (25 kg) gathered from the surrounding garden and the phosphate-rich fertilizer. The fertilizer was added initially and then after every 7 days. The dimensions of each tank and water medium taken were as follows: LxBxD 1.56 m x 0.76 m x 0.31 m; water height 0.19 m; volume of water used 225 L. Bleaching of Algae Cellulose.


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A four stage bleaching sequence was used to purify Pithophora green algae. The process has washing steps with deionized water between the bleaching stages and after the final bleaching stage to remove the extracted constituents from algal mass. First stage consists of an alkaline extraction step where 3 % wt. algae was cooked in 0.1 M NaOH at 0.83 bar in a pressure vessel for 120 minutes. Second stage consists of an acid treatment step where 3 % wt. algae was cooked in 0.3 M HCl at 0.83 bar in a pressure vessel for 120 minutes. Third stage is an alkaline peroxide bleaching step where 3 % wt. algae was cooked in 0.1 M NaOH and 2 % wt. H2O2 at 0.83 bar in a pressure vessel for 120 minutes. Final bleaching stage is an ethanol extraction step at room temperature where washed cellulose obtained after third bleaching stage was dispersed in ethanol to obtain a consistency of 10 % wt. and the suspension was allowed to stay at room temperature for 48 hrs. Cellulose recovered after final washing step was freeze-dried and stored at room temperature. Intrinsic Viscosity and Degree of Polymerization The intrinsic viscosity of the Pithophora cellulose was measured according to the ASTM standard

test

method

D

1795-96

by

dissolving

the

cellulose

powder

in

bis(ethylenediamine)copper(II) hydroxide solution.45 An Ubbelohde viscometer (Paragon Scientific Ltd, UK) was used, and five repeated measurements were performed for the algae cellulose and the solvent at 25 °C. The performed experiments were recorded with a video camera and the outflow time was determined from the recordings. Intrinsic


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viscosity was calculated according to the standard D 1795-96 and the degree of polymerization was obtained by multiplying the intrinsic viscosity by a factor of 190.45 XRD The degree of crystallinity (CI) was determined using Equation 1, where the intensity of the crystalline peak at 22.8° was used as the total intensity, Itot, and the intensity at the minimum between the crystalline peaks at 16.7 and 22.8° was used as the intensity of the amorphous cellulose, Iam. 𝐶𝐼 = 100 ∙

(

𝐼𝑡𝑜𝑡 ― 𝐼𝑎𝑚 𝐼𝑡𝑜𝑡

)

(1)

Zeta potential A 0.1 wt % Pithophora cellulose dispersion in water was prepared using high-shear ultrasonication (750 W; 20 kHz; 13 mm probe; Vibra-cell; Sonics, USA) with 30 s pulsing at 70% amplitude for 20 min. Eight mL of the cellulose dispersion was diluted to 0.02 wt % using 32 mL of 10 mM NaCl solution. One mL of the diluted dispersion was added to a quartz glass cell with electrodes, and the zeta potential was measured using a Malven Nano ZS DLS instrument. Preparation of Filter Paper A 0.2 wt % dispersion of Pithophora cellulose was prepared. The dispersion was then run three times in succession through a 200 µm sized chamber and then once through a 100 µm sized chamber at 1800 bar, using an LM20 Microfluidizer. Filter papers of 26 μm thicknesses was then prepared by adjusting the solids content of dispersion. The resulting


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nanocellulose dispersions were then drained over a nylon filter membrane (Durapore, 0.65 µm DVPP, Merck Millipore) fitted in a funnel using vacuum. The resulting wet cellulose mass was then dried at 80 °C using a hot-press (Carver, USA). Nitrogen Gas Sorption Porometry Pore size distribution evaluation of the nanocellulose filter papers through the BarretJoyner-Halenda (BJH) method6 was performed using the desorption branch of the isotherm. This was done using an ASAP 2020 (Micrometrics, USA) instrument. The filter sample was degassed at 90 °C in vacuum for four hours and the following analysis of nitrogen sorption was then carried out at 77 K using liquid nitrogen as a coolant. Total porosity The total porosity of the filter was calculated from the ratio between the bulk and true density as follows : (2) where ε% is the total porosity, ρbulk is the bulk density, calculated from the dimesnions of the filter disc (47 mm in diameter; 26 μm thickness) and ρtrue true density (1.64 g/cm3 for crystalline algae cellulose).

Dynamic Light Scattering Particle size distribution of the suspended solids in the SWW was analyzed through dynamic light scattering (DLS) using a Malvern Mastersizer 3000. Refractive index of the


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dispersant was set to that of water, i.e. 1.33, and the refractive index of the particles was set to that of diatomite, i.e. 1.4346. Particle geometry was set as non-spherical and Mie scattering47 was used as the scattering model. Scanning Electron Microscopy Post-filtration imaging of the nanocellulose filter papers were performed using a scanning electron microscope (LEO1550, Zeiss, Germany). The filters were sputtered prior to scanning electron microscopy (SEM) analysis with Au/Pt at 2 kV, 25 mA for 35 seconds to avoid charging of the material. SEM pictures were retrieved at acceleration voltages of 1.00 kV. Filtration of Surrogate Latex Particles in SWW Filtration of 30 nm fluorescent latex particles in SWW was carried out using an Advantech KST 47 filter holder. The nanocellulose filter papers were fitted with a Munktell General Purpose Filter Paper as a mechanical support in the cell and the filters were then pre-wetted with deionized water. Feed dispersions were prepared by diluting 2 mL of imitated waste water – simulated matrix and 80 µL of 30 nm fluorescent latex particles with deionized water up to a total volume of 200 mL. Filtrations were carried out at overhead pressures 1 bar and 3 bar. The permeate solution was collected in fractions of 40 mL each and the real flux was monitored using a scale (Mettler Toledo, MS1602TS) registering the change in weight of the collected permeate solution over time. The absorption of the collected permeate was analyzed at 500 nm using a Shimadzu UV-


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1650PC spectrophotometer. The fluorescence of the collected permeate fractions was measured between 450-580 nm, with an excitation wavelength of 264 nm, using a TECAN M200 spectrophotometer. The area under the curve (AUC) was calculated for the fluorescence peak, after subtraction of background emission from water, by integration of the measured wavelengths. The particle removal rate is described by the logarithmic reduction value (LRV) and is calculated using Equation 3. 𝐴𝑈𝐶𝑓𝑒𝑒𝑑

(3)

𝐿𝑅𝑉 = 𝑙𝑜𝑔10𝐴𝑈𝐶𝑝𝑒𝑟𝑚𝑒𝑎𝑡𝑒

AUCfeed is the area under the curve for the feed solution and AUCpermeate is the area under the curve for the permeate solution. Microbiological materials Bacteriophages ΦΧ174 (13706-B1), MS2 (15597-B1), and PR772 (BAA-769-B1), and host bacteria Escherichia coli (Migula) Castellani and Chalmers C (13706), C-3000 (15597), and K12 J53-1(R15) (BAA-769) strains were purchased from the American Type Culture Collection (ATCC). NZCYM broth (N3643), maltose (M5885) and sodium chloride (S5886) were purchased from Sigma-Aldrich. Tryptone (Oxoid) (LP0042) and yeast extract (Oxoid) (LP0021) were purchased from Thermo Fisher Scientific. Agar (214530) was purchased from BD. Bacteriophage propagation The PR772 bacteriophage was propagated in E. coli strain K12 J53-1(R15) [HER 1221] host bacteria. Host bacteria strain was diluted 1:200 with Luria-Bertani (1% tryptone, 0.5%


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yeast extract, and 1% NaCl in deionized water) broth (LB), and cultured by incubating at 37°C for 3 hours in a shaking incubator (INCU-Line® ILS 4) with agitation at 220 rpm. Bacteriophage was propagated by inoculation of host bacteria at exponential growth phase. The incubation continued at 37°C for 7 hours with an agitation speed at 150 rpm. Bacteriophages were harvested by centrifugation at 5000 g for 10 min, and the suspension was collected and stored at 4°C. The small-size ΦΧ174 bacteriophage was propagated by inoculation of E. coli strain C host bacteria at exponential growth phase in LB broth for 5 hours at 37°C with an agitation (150 rpm). Bacteriophage was harvested by centrifugation at 5000 g for 10 min, and the suspension was collected and stored at 4°C. The MS2 bacteriophage was propagated in E. coli strain C-3000 host bacteria. Briefly, host bacteria strain was diluted 1:200 with NZCYM (2.2% NZCYM, and 0.2% maltose in deionized water), broth and cultured by incubating at 37°C for 3 hours in a shaking incubator (INCU-Line® ILS 4) with agitation at 220 rpm. Bacteriophage was propagated by inoculation of host bacteria at exponential growth phase. The incubation continued at 37°C for 7 hours with an agitation speed at 150 rpm. Bacteriophages were harvested by centrifugation at 5000 g for 10 min, and the suspension was collected and stored at 4°C. The enumeration of the bacteriophage was performed by double-layer agar plate assay and expressed as plaque-forming units (PFU)/mL. For each bacteriophage suspension a series of tenfold serial dilutions were prepared in LB broth. Diluted bacteriophage was


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mixed with the host bacteria at exponential growth phase and molten soft agar and poured on a Petri dish with the layer of hard agar. PFU was calculated using the Equation 4: N (PFU mL ) = log10(VP ∙ DF)

(4),

log10

where PFU is plaque forming units, N is number of plaques, VP is volume of phage added in mL, and DF is dilution factor. Feed suspensions were prepared by dilution of SWW-simulated matrix (1:10) in 200 mL of deionized water and were spiked with appropriate bacteriophage stock dispersion to obtain the titer of ca. 106 PFU/mL. Filtrations were carried out in an Advantec KST-47 filter holder at three different overhead pressures, i.e., 1, 3 or 5 bar. To substitute the air in the pores of the filters with the water, the filters were pre-filtered at 4 bar overhead pressure with 20 mL of deionized water. The flux during filtration was monitored using precision balance (Mettler Toledo MS1602TS) by registering the permeate samples weight changes over the time. Permeate samples were collected in 40 mL fractions and bacteriophage removal rate is expressed by log reduction values (LRV): PFU (PFU mL )𝑓𝑒𝑒𝑑 ― log10( mL )𝑝𝑒𝑟𝑚𝑒𝑎𝑡𝑒

𝐿𝑅𝑉 = log10

(5),

where LRV is logarithmic reduction value and PFU is plaque forming unit. Bacteriophage enumeration was performed in duplicates with the limit of detection, i.e., ≤0.7 PFU/mL, which refers to ≤5 bacteriophages per mL. Microbiological analysis


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The following media were used for microbiological analysis of real-life water samples collected in Dhaka, Bangladesh: Tryptic Soy agar (TSA), MacConkey agar, Modified Faecal Coliform agar (MFC), Thiosulphate-citrate-bile salt-sucrose agar (TCBS), and SalmonellaShigella agar (SS). All media components were supplied by BD Difco Oxford, (UK). Water samples were aseptically collected in sterilized sampling bottles from 0.5 m depth at Dhanmondi lake and Turag river. Temperature, pH and TDS were measured. Collected sample was transported to laboratory within 2 hours in a cool box. The filter paper, support filter paper and rubber gasket were UV radiated prior to use. Autoclaved filter holder was filled with 150 mL of water sample. Overhead pressure of 5 bar was applied to drive filtration. After a while, filtered water was collected dropwise in a previously sterilized conical flask. For analysis, 0.1 mL of the sample (from control and treated water sample) was spread onto respective culture media, i.e. TSA, MacConkey, MFC, TCBS, and SS agar. Ten-fold dilution was done only for total aerobic bacterial count. The inoculated Petri dishes were incubated at 37 °C and 45 °C temperature for 18-24 hours. MFC plates were incubated at 45 °C for fecal coliform. After 24 hours incubation, CFU/mL were counted. The limit of detection was ≤ 10 cfu/mL for total aerobic bacteria, total coliform, E. coli, Salmonella, Shigella, and Vibrio sp.

RESULTS AND DISCUSSION Pithophora algae growth


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Figure 1 shows Pithophora algae, cultivated in concrete pits at University of Dhaka. With addition of a phosphor-rich nutrient, rapid algae growth was achieved. In particular, the fresh weight of algae reached 3.45 kg/m2 within 18 days, doubling in weight every 3 days. The algae were then used to extract cellulose, which was further processed into paper sheets at Uppsala University, Sweden. Table 1 summarizes the physical-chemical properties of Pithophora cellulose. The crystallinity index of Pithophora cellulose was high, i.e. 96.6%, which is comparable to that of cellulose extracted from Valonia and Cladophora algae.48 Further, the intrinsic viscosity of the Pithophora cellulose was 11.2 dL/g, which corresponds to a cellulose degree of polymerization of 2120, measured according to the ASTM standard.49 For comparison, microcrystalline cellulose produced by mineral acid hydrolysis of wood pulp under similar conditions has a crystallinity degree of only 80% and a degree of polymerization of less than 350.50 The high degree of crystallinity and high degree of polymerization are characteristic properties of Cladophora-type celluloses.30 Removal of surrogate latex nanoparticles from simulated waste water The following studies were performed at Uppsala University, Sweden, to investigate the feasibility of using Pithophora cellulose filter paper for water purification. The filtration properties of the produced filter papers were evaluated using simulated waste-water (SWW) matrix spiked with excessive amount of surrogate latex nanoparticles at 1011/mL concentration. The source of the turbidity-contributing solids in the SWW was diatomite


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and formazin. As reported earlier, the majority of the colloidal particle size distribution in SWW has a peak value of 880 nm, and no particles ≤ 590 nm and ≥ 31.1 µm could be detected.31 In order to mimic virus contamination conditions, the SWW was further spiked with surrogate 30 nm latex beads, having a particle size comparable to that of the smallsize viruses. It should be noted that the latex nanobeads were functionalized with fluorescent groups, to be able to monitor the efficiency of their removal, as described previously.20, 31 Figure 2 shows the observed fluxes through Pithophora filter paper with SWW. For initial characterization, the filtration of the SWW solutions, spiked with surrogate latex nanobeads, was performed at 2 different pressures, i.e. 1 and 3 bar. No significant fouling was observed in the samples under the experimental conditions for entire load volume, i.e. 140 L/m2. The observed flux values were 25 and 75 L/m2/h for 1 and 3 bar, respectively. It should be noted that the observed fluxes of SWW in Pithophora filter paper were nearly 2-fold slower than those reported earlier for filter papers produced from Cladophora cellulose at corresponding filter thickness, i.e. 60 and 150 L/m2/h at 1 and 3 bar, respectively.31 The latter suggests that Pithophora filter paper has denser structure, since the modal pore size values were around 17 nm in both cases. Figure 3 shows the scanning electron microscopy images of Pithophora filter paper before and after latex nanobead-spiked SWW filtration. The observed structure of the Pithophora filter paper prior to filtration was largely similar to that previously reported for


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Cladophora cellulose.21-25,

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In particular, randomly oriented individual cellulose

nanofibers and their bundles were visible. After filtration, a deposit of solids was observed with spherical nanometer-sized particles embedded in a layer of another material, covering the surface of the filter paper. The nanofibrous texture of Pithophora filter paper was not visible after filtration of SWW, as it was covered by sediment. Figure 4 shows the photographs of SWW liquid before and after filtration. A clearly visible decrease in the turbidity of permeate, as compared to feed, was noticed after filtration. Table 2 summarizes the transmittance values (at λ=500 nm wavelength) before and after filtration. The transmittance of the feed solution increased from about 95% to 100% after filtration. Furthermore, The fluorescence signal due to latex nanobeads in the permeate was below the limit of detection for entire load volume, suggesting consistent ≥2 LRV efficiency at both applied pressures. The promising results on removal of surrogate 30 nm fluorescently-labeled nanobeads from SWW motivated continued studies using model viruses as presented below. Removal of model viruses from simulated waste water Figure 5 illustrates the mechanism of virus removal by the filter paper. For virus removal filtration the most critical properties of the viruses include their particle size and pI values. The size of most viruses varies between 20 and 200 nm, while pI values range between 3.5 and 7.0. PR772 coliphage (70 nm; pI 4.0) was used as a large size model virus. MS2 and ΦX174 coliphages were used as the worst-case small-size virus models, i.e. 27 and 28


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nm, respectively. The pI values for MS2 and ΦX174 phages were 3.9 and 6.6, respectively.52 For comparison, the sizes for most common water-borne infective viruses are 30 nm for hepatitis E virus;53 30-40 nm for norovirus;54 70 nm for rotavirus,55 and 100 nm for human adenovirus.56 Figure 6 shows the results of the SWW filtrations spiked with three model viruses. The filtration of SWW spiked liquids was first performed at 1 and 3 bar, as in the case of latex nanobeads above, and then additionally at 5 bar. The filtration at 5 bar was beneficial to increase both the throughput and virus removal capacity as it will be discussed below. No flux decline was observed under the experimental conditions. The average flux values for SWW filtrations through Pithophora filter paper were 22, 82 and 115 L/m2/h at 1, 3, and 5 bar, respectively. It is seen from Figure 5 that under experimental conditions virus removal was sufficiently high to enable the application of filter in POU water purification, since ≥4 LRV was observed in all tests for all types of model viruses. The removal of large-size model viruses, i.e. 70 nm PR772 phage, was especially high, and ≥6 LRV was detected irrespectively of the applied pressure. For small-size model viruses, the virus removal capacity was higher at increasing pressure. No detectable model virus particles were detected in the permeate for 28 nm ΦX174 phage at 3 and 5 bar for all fractions, whereas at 1 bar 1 PFU/mL virus was detected in the undiluted permeate fractions above 90 L/m2. The lower virus removal capacity could be related to Brownian motion as observed earlier.26 It should be noted that the virus


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clearance at 1 and 3 bar observed for Pithophora cellulose filter paper in this study were slightly better than those reported for Cladophora cellulose filter paper reported earlier, which could be related to the more compact structure of Pithophora filter paper.31 Filtration at 5 bar provided highest LRVs with respect to removal of MS2 phage. Combined with high flux values, filtration at 5 bar appears to provide very high level of protection (LRV >5) even under the worst-case experimental conditions. It should be noted that the removal of the small-size MS2 phages was more challenging than that of ΦX174 phage, which could be related to the pI differences between the phages. The pH of SWW during experiments was 6 which is around the pI of ΦX174 phage, suggesting that this phage may at least partly aggregate under the experimental conditions. On the other hand, MS2 phages (pI 3.9) are negatively charged at this pH, and therefore the probability of aggregation is low. In all, the results of the model virus removal studies showed excellent model small-size virus clearance capacity, motivating continued studies using real-life samples, as discussed below. Filtration of real-life water samples from Dhanmondi lake and Turag river, Dhaka, Bangladesh In order to further study the feasibility of using Pithophora filter paper for drinking water purification, the filters were tested using real-life water samples collected in Dhaka, Bangladesh, at two different locations, i.e. Dhanmondi lake and Turag river, during summer of 2018. Figure 7 shows the sites of collection of water samples. The collected


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water samples were very turbid but turned completely transparent following filtration at 5 bar, as shown in Figure 7. The pH and total solids contents at the site of sampling were 7.3±0.03 and 85±4 mg/mL as well as 7.3±0.04 and 101±7 mg/mL in Turag river and Dhanmondi lake, respectively. The microbiological analysis of the collected water samples showed varying levels of bioburden at both locations, including detectable levels of pathogenic bacteria, such as Vibrio, Salmonella and Shigella. Figure 8 shows the typical images for agar plates to detect various microbes in the real-life samples collected from Turag river before and after filtration. Table 3 and 4 summarize the results of the microbiological tests at both locations. It is seen from these tables that all microbes that were detectable in the feed were absent in permeate, even after enrichment procedure. This is the first time the Pithophora filter paper is shown to completely remove real-life infectious pathogens from real-life water samples. Following the microbiological analysis, the collected samples were additionally analyzed for presence of human viruses using PCR method at Uppsala University Hospital Clinical Virology division. In particular, the samples from Dhanmondi lake and Turag river were monitored for the presence of a typical water-borne virus, i.e. human adenovirus. Human adenovirus was detected in the pre-treatment samples from Turag river but not from the Dhanmondi lake. Table 5 shows the results of PCR analysis for adenovirus from water samples collected from Turag river. In the Turag river samples, the detected number of copies per mL varied


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between 1 412 and 22 203 copies. Following the filtration, no human adenovirus DNA was detected in 3 out of 4 samples, whereas in 1 sample it was significantly reduced. It should be noted that the PCR method quantifies the number of nucleic acid copies in the sample and does not necessarily reflect the number of infectious particles despite its high sensitivity.57 Thus, the presented results confirm the high virus removal capacity of Pithophora nanocellulose filter paper also in real-life water samples. Filter sizing considerations for practical applications In order to enable the practical applications of the filter paper, filter sizing should be considered. In particular, the filter should be operated under such conditions that it maximizes the water throughput and favors the highest level of pathogen safety with minimal footprint at POU. Therefore, the minimum surface area necessary for filtering a specific water volume needs to be calculated. For the sake of analysis, we assumed that an average person requires 2-3 L of drinking water per day, depending on the climate. Then, the sizing calculations were performed for filtering 3 L (i.e. Vb) of water in less than half an hour (i.e. tb=25 min). According to equation 6, the sizing is dependent on two parameters, i.e. the maximum volume that can be processed before complete fouling occurs (Vmax) and the initial flow rate (J0). According to Equation 7, if α > 10, it means that the filtration is limited mainly by the initial flux (J0) and not by the filter’s volumetric throughput capacity (Vmax), and if α < 0.01 the filtration is limited by the flux. Because no


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filter fouling was observed during the tests with SWW, we assume that the filter paper is not limited by its throughput capacity, and thus the extrapolated Vmax is ≥103 L/m2.

(6)

(7)

In this study, 47 mm in diameter flat disc sheets were used. Under the experimental conditions, the rate of flow was between 0.04 to 0.2 L/h depending on the applied pressure. It is concluded that for practical applications 47 mm filters cannot provide sufficient and reasonable throughput, and the surface area of the filter paper, therefore, needs to be increased. For a flat sheet disc filter, increasing the filter diameter from 47 to 300 mm, i.e. 6-fold increase, will scale the surface area by a factor of 40, i.e. from 0.00174 to 0.07065 m2, which in turn will result in a dramatic increase in the volumetric throughput. Table 6 shows the minimum surface area required to process 3 L of water in 25 min using 300 mm in diameter flat filter sheets at different overhead pressures. The calculated α was >10 for all pressures, indicating that the flux is the rate-limiting factor. The calculations suggest that a single flat disc sheet of 300 mm in diameter size operated at 5 bar would be sufficient to process 3L of water in ca. 25 min, producing water of the highest microbiological safety. For comparison, if operated at 1 bar, almost 4-4.5 times more filter area will be needed. In a low-income setting, such as that in Bangladesh, the operation of a single sheet 300 mm in diameter filter paper at 3-5 bar is fully reasonable


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to produce enough drinking water per person per day and could dramatically improve the population’s quality of life, ultimately saving thousands of lives. Future, studies should focus on integrated solutions to remove other types of water contaminants such as heavy metals, pesticides, and organic molecules.

CONCLUSION In this article, for the first time, we show the potential of processing locally growing green Pithophora macroalgae to produce total pathogen barrier filter paper for POU drinking water purification. In particular, the produced filter paper is shown to efficiently remove all types of pathogens, including bacteria and viruses. The performance of the filter paper was sequentially verified with surrogate latex nanoparticles, worst-case model viruses and real-life bioburden from water samples collected in Bangladesh. It was shown that by using 300 mm flat sheet Pithophora filter paper it would be possible to produce 3L of drinking water of highest microbiological safety within 25 min when operated at moderate overhead pressures. The presented filter paper could alleviate a severe societal problem of access to safe drinking water in Bangladesh and other regions with high density of population and poor access to drinking water.

AUTHOR INFORMATION ‡These authors contributed equally to first authorship


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* Corresponding author: [email protected]

ACKNOWLEDGEMENTS The authors thank Dr. C. Öhrmalm at Clinical Virology, Uppsala Academic Hospital, for conducting the human adenovirus PCR analysis.

CONFLICT OF INTEREST The corresponding author (Albert Mihranyan) is the inventor behind IP related to virus removal filter paper.

FUNDING This work was mainly funded by the Swedish Research Council’s Development Research program (Ventenskapsrådet Utvecklingsforskning No. 2016-05715). Part of the salaries for Uppsala co-authors (AM, GKT, SG) was covered by the Knut and Alice Wallenberg Foundation as a part of Wallenberg Academy Fellow grant (KAW 2013.0190).

ABBREVIATIONS ASTM - American Society for Testing and Materials ATCC- American Type Culture Collection


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AUC- Area Under Curve BJH- Barret-Joyner-Halenda method DLS- Dynamic Light Scattering DNA- Deoxyribonucleic Acid ID50 - Median Infectious Dose LB- Luria-Bertani broth LRV- Log10 Removal Value MFC- Modified Faecal Coliform agar PCR - Polymerase Chain Reaction PFU- Plaque Forming Unit POU- Point-of-Use SS- Salmonella-Shigella agar SWW- Simulated Waste Water TCBS- Thiosulphate-citrate-bile salt-sucrose agar TSA- Tryptic Soy Agar


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46. Ciullo, P. A., Diatomite. In Industrial Minerals and Their Uses: A Handbook and Formulary, Noyes Publications: Westwood, N.J, 1996. 47. Mie, G., Beiträge zur Optik trüber Medien, speziell kolloidaler Metallösungen. Ann. Phys. 1908, 330, 377-445. 48. Mihranyan, A., Cellulose from Cladophorales Green Algae: From Environmental Problem to High‐Tech Composite Materials. J. Appl. Polym. Sci. 2011, 119 (4), 2449-2460. DOI: 10.1002/app.32959. 49. ASTM D1795-13, Standard Test Method for Intrinsic Viscosity of Cellulose. ASTM International: West Conshohocken, PA, 2013. 50. Cellulose, Microcrystalline. In European Pharmacopenia 7.0, 2009; pp 1634-1637. 51. Metreveli, G.; Wågberg, L.; Emmoth, E.; Belák, S.; Strømme, M.; Mihranyan, A., A Size-Exclusion Nanocellulose Filter Paper for Virus Removal. Adv. Healthcare Mater. 2014, 3 (10), 1546-1550. DOI: 10.1002/adhm.201300641. 52. Michen, B.; Graule, T., Isoelectric points of viruses. J Appl Microbiol 2010, 109 (2), 388-97. DOI: 10.1111/j.1365-2672.2010.04663.x. 53. Ahmad, I.; Holla, R. P.; Jameel, S., Molecular virology of hepatitis E virus. Virus Res 2011, 161 (1), 47-58. DOI: 10.1016/j.virusres.2011.02.011.


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54. Robilotti, E.; Deresinski, S.; Pinsky, B. A., Norovirus. Clin Microbiol Rev 2015, 28 (1), 134-164. DOI: 10.1128/CMR.00075-14. 55. Group, W. H. O. S. W., Rotavirus and other viral diarrhoeas: WHO scientific working group. Bull World Health Organ 1980, 58 (2), 183-198. 56. Kennedy, M. A.; Parks, R. J., Adenovirus virion stability and the viral genome: size matters. Mol Ther 2009, 17 (10), 1664-1666. DOI: 10.1038/mt.2009.202. 57. Rodriguez, R. A.; Pepper, I. L.; Gerba, C. P., Application of PCR-based methods to assess the infectivity of enteric viruses in environmental samples. Appl Environ Microbiol 2009, 75 (2), 297-307. DOI: 10.1128/aem.01150-08.


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Figure 1. Cultivation of Pithophora algae at University of Dhaka, Bangladesh, extracted algae cellulose and filter paper sheets made from it at Uppsala University, Sweden.


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Figure 2. Observed fluxes at overhead pressures of 1 and 3 bar for filtrations of 30 nm latex nanobeads from SWW through Pithophora filter paper.


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Figure 3. SEM image of the Pithophora cellulose filter paper before (left panel) and after 30 nm latex beads-spiked SWW filtration (right panel).


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Figure 4. Effect of filtration of SWW spiked with surrogate 30 nm latex particles through Pithophora cellulose filter papers: before (left image) and after (right image).


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Figure 5. Illustration of the size-exclusion mechanism of pathogen removal by Pithophora cellulose filter paper.


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Figure 6. Log10 reduction values (LRV) for filtration of SWW spiked with ΦX174, MS2 and PR772 bacteriophages through Pithophora nanocellulose filter papers at 1, 3, and 5 bar. The horizontal dashed line represents desirable level of virus clearance for POU water purification, i.e. LRV=4 (99.99%). Arrows in the graph indicate that virus titer was below the detection limit.


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(A)

(B)

(C)


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Figure 7. Images of locations where water was sampled at (A) Dhanmondi lake and (B) Turag river, including a water sample before and after filtration (C).

Total aerobic bacteria (before-after)

Faecal coliform (before-after)

Total coliform/E. coli (before-after)

Salmonella-Shigella (before-after)

Vibrio (before-after)


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Figure 8. Detectable CFU before and after filtration in the Turag river. All images represent samples before enrichment procedure.


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Table 1. Physico-chemical characteristics of Pithophora cellulose filter paper.

Property

Unit

Pore size mode [nm]

17

Total porosity [%]

31.5±2.5

Specific surface area [m2/g] 62±3 Crystallinity index [%]

96.6

Intrinsic viscosity [dL/g]

11.2

Degree of polymerization

2120

Zeta-potential [mV] at pH -8.3±1.0 7.4


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Table 2. Measured transmittance for filtrations of 30 nm latex particles in SWW through nanocellulose filter papers. TSS was 0.251 mg/L. T = 100% corresponds to the transmittance of pure water. ΔP [bar]

Tfeed500nm [%]

Tpermeate500nm [%]

LRV30 nm latex

1

94.9 ±1.2

100 ±0

≥2

3

95.0 ±1.2

100 ±0

≥2

LRV=log10 removal value of 30 nm latex nanoparticles based on fluorescence intensity


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Table 3. Dhanmondi lake, n=4. Bacterial count, CFU per mL Pre-treatment range

Post-treatment range no enrichment

with enrichment

Total aerobic bacteria

1.12∙103 - 4.96∙104

ND

Absent

Total coliform

7.70∙102 - 7.12∙103

ND

Absent

Faecal coliform

7.00∙101 - 1.3∙102

ND

Absent

E. coli

1.00∙101 - 1.10∙102

ND

Absent

Vibrio

ND; positive after enrichment

ND

Absent

Salmonella


ND

Absent

Shigella

ND - 1∙101

ND

Absent


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Table 4. Turag river, n=5. Bacterial count, CFU per mL Pre-treatment range

Post-treatment range no enrichment

with enrichment

Total aerobic bacteria

2.22∙104 - 5.64∙104

ND

Absent

Total coliform

2.44∙103 - 7.76∙103

ND

Absent

Faecal coliform

1.90∙102 – 5.00∙102

ND

Absent

E. coli

8.00∙101 - 5.90∙102

ND

Absent

Vibrio


ND

Absent

Salmonella

1.00∙101 – 7.00∙101

ND

Absent

Shigella

ND-8.00∙101

ND

Absent


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Table 5. qPCR detection of adenovirus from Turag river, n=4. #

Number of copies per mL Pre-treatment

Post-treatment

1

2 032

ND

2

3 166

92

3

1 412

ND

4

22 203

ND


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Table 6. Scaling of 26 μm filters (300 mm in diameter) to process 3 L of water in 25 min at varying overhead pressures. Pressure, bar

J0, L/m2/h

α

Amin, m2

Scaling factor*

1

23

104

0.32

4.4

3

80

30

0.09

1.3

5

115

21

0.06

0.9

*Based on Equation 1 and data from flux using SWW spiked with MS2 *Scaling factor is Amin/A300mm or the number of 300 mm flat disc filters necessary to process fixed Vb at tb.


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TOC image

SYNOPSIS Filter paper for water purification is shown, capable of removing all types of infectious pathogens, which is produced from sustainable, locally growing seaweed in Bangladesh.


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Scalable and Sustainable Total Pathogen Removal Filter Paper for

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