Abstract Effective point-of-use devices for providing safe drinking water are urgently needed to reduce the global burden of waterborne disease. Here we show that plant xylem from the sapwood of coniferous trees – a readily available, inexpensive, biodegradable, and disposable material – can remove bacteria from water by simple pressure-driven filtration. Approximately 3 cm3 of sapwood can filter water at the rate of several liters per day, sufficient to meet the clean drinking water needs of one person. The results demonstrate the potential of plant xylem to address the need for pathogen-free drinking water in developing countries and resource-limited settings.

Citation: Boutilier MSH, Lee J, Chambers V, Venkatesh V, Karnik R (2014) Water Filtration Using Plant Xylem. PLoS ONE 9(2): e89934. https://doi.org/10.1371/journal.pone.0089934 Editor: Zhi Zhou, National University of Singapore, Singapore Received: October 17, 2013; Accepted: January 23, 2014; Published: February 26, 2014 Copyright: © 2014 Boutilier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the James H. Ferry, Jr. Fund for Innovation in Research Education award to R.K. administered by the Massachusetts Institute of Technology. SEM imaging was performed at the Harvard Center for Nanoscale Systems, a member of the National Nanotechnology Infrastructure Network (NNIN), which is supported by the National Science Foundation under NSF award no. ECS-0335765. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction The scarcity of clean and safe drinking water is one of the major causes of human mortality in the developing world. Potable or drinking water is defined as having acceptable quality in terms of its physical, chemical, and bacteriological parameters so that it can be safely used for drinking and cooking [1]. Among the water pollutants, the most deadly ones are of biological origin: infectious diseases caused by pathogenic bacteria, viruses, protozoa, or parasites are the most common and widespread health risk associated with drinking water [1], [2]. The most common water-borne pathogens are bacteria (e.g. Escherichia coli, Salmonella typhi, Vibrio cholerae), viruses (e.g. adenoviruses, enteroviruses, hepatitis, rotavirus), and protozoa (e.g. giardia) [1]. These pathogens cause child mortality and also contribute to malnutrition and stunted growth of children. The World Health Organization reports [3] that 1.6 million people die every year from diarrheal diseases attributable to lack of access to safe drinking water and basic sanitation. 90% of these are children under the age of 5, mostly in developing countries. Multiple barriers including prevention of contamination, sanitation, and disinfection are necessary to effectively prevent the spread of waterborne diseases [1]. However, if only one barrier is possible, it has to be disinfection unless evidence exists that chemical contaminants are more harmful than the risk from ingestion of microbial pathogens [1]. Furthermore, controlling water quality at the point-of-use is often most effective due to the issues of microbial regrowth, byproducts of disinfectants, pipeline corrosion, and contamination in the distribution system [2], [4]. Common technologies for water disinfection include chlorination, filtration, UV-disinfection, pasteurization or boiling, and ozone treatment [1], [2], [5]. Chlorine treatment is effective on a large scale, but becomes expensive for smaller towns and villages. Boiling is an effective method to disinfect water; however, the amount of fuel required to disinfect water by boiling is several times more than what a typical family will use for cooking [1]. UV-disinfection is a promising point-of-use technology available [1], yet it does require access to electricity and some maintenance of the UV lamp, or sufficient sunlight. While small and inexpensive filtration devices can potentially address the issue of point-of-use disinfection, an ideal technology does not currently exist. Inexpensive household carbon-based filters are not effective at removing pathogens and can be used only when the water is already biologically safe [1]. Sand filters that can remove pathogens require large area and knowledge of how to maintain them [1], while membrane filters capable of removing pathogens [2], [4] suffer from high costs, fouling, and often require pumping power due to low flow rates [6] that prevents their wide implementation in developing countries. In this context, new approaches that can improve upon current technologies are urgently needed. Specifically, membrane materials that are inexpensive, readily available, disposable, and effective at pathogen removal could greatly impact our ability to provide safe drinking water to the global population. If we look to nature for inspiration, we find that a potential solution exists in the form of plant xylem – a porous material that conducts fluid in plants [7]. Plants have evolved specialized xylem tissues to conduct sap from their roots to their shoots. Xylem has evolved under the competing pressures of offering minimal resistance to the ascent of sap while maintaining small nanoscale pores to prevent cavitation. The size distribution of these pores – typically a few nanometers to a maximum of around 500 nm, depending on the plant species [8] – also happens to be ideal for filtering out pathogens, which raises the interesting question of whether plant xylem can be used to make inexpensive water filtration devices. Although scientists have extensively studied plant xylem and the ascent of sap, use of plant xylem in the context of water filtration remains to be explored. Measurements of sap flow in plants suggest that flow rates in the range of several liters per hour may be feasible with less than 10 cm-sized filters, using only gravitational pressure to drive the flow [7]. We therefore investigated whether plant xylem could be used to create water filtration devices. First, we reason which type of plant xylem tissue is most suitable for filtration. We then construct a simple water filter from plant xylem and study the resulting flow rates and filtration characteristics. Finally, we show that the xylem filter can effectively remove bacteria from water and discuss directions for further development of these filters.

Materials and Methods Materials Branches were excised from white pine growing on private property in Massachusetts, USA, with permission of the owner and placed in water. The pine was identified as pinus strobus based on the 5-fold grouping of its needles, the average needle length of 4.5 inches, and the cone shape. Deionized water (Millipore) was used throughout the experiments unless specified otherwise. Red pigment (pigment-based carmine drawing ink, Higgins Inks) was dissolved in deionized water. Nile-red coated 20 nm fluorescent polystyrene nanospheres were obtained from Life Technologies. Inactivated, Alexa 488 fluorescent dye labeled Escherichia coli were obtained from Life Technologies. Wood sections were inserted into the end of 3/8 inch internal diameter PVC tubing, sealed with 5 Minute Epoxy, secured with hose clamps, and allowed to cure for ten minutes before conducting flow rate experiments. Construction of the Xylem Filter 1 inch-long sections were cut from a branch with approximately 1 cm diameter. The bark and cambium were peeled off, and the piece was mounted at the end of a tube and sealed with epoxy. The filters were flushed with 10 mL of deionized water before experiments. Care was taken to avoid drying of the filter. Filtration and Flow Rate Experiments Approximately 5 mL of deionized water or solution was placed in the tube. Pressure was supplied using a nitrogen tank with a pressure regulator. For filtration experiments, 5 psi (34.5 kPa) pressure was used. The filtrate was collected in glass vials. For dye filtration, size distribution of the pigments was measured for the input solution and the filtrate. Higgins pigment-based carmine drawing ink, diluted ∼1000× in deionized water, was used as the input dye solution. For bacteria filtration, the feed solution was prepared by mixing 0.08 mg of inactivated Escherichia coli in 20 mL of deionized water (∼1.6×107 mL−1) after which the solution was sonicated for 1 min. The concentration of bacteria was measured in the feed solution and filtrate by enumeration with a hemacytometer (inCyto C-chip) mounted on a Nikon TE2000-U inverted epifluorescence microscope. Before measurement of concentration and filtration experiments, the feed solution was sonicated for 1 min and vigorously mixed. Imaging Xylem structure was visualized in a scanning electron microscope (SEM, Zeiss Supra55VP). Samples were coated with gold of 5 nm thickness before imaging. To visualize bacteria filtration, 5 mL of solution at a bacteria concentration of ∼1.6×107 mL−1 was flowed into the filter. The filter was then cut longitudinally with a sharp blade. One side of the sample was imaged using a Nikon TE2000-U inverted epifluorescence microscope and the other was coated with gold and imaged with the SEM. Optical images were acquired of the cross section of a filter following filtration of 5 mL of the dye at a dilution of ∼100×. Particle Sizing Dynamic light scattering measurements of particle size distributions were performed using a Malvern Zetasizer Nano-ZS.

Discussion Wood has been investigated recently as a potential filtration material [13], showing moderate improvement of turbidity. While we showed filtration using freshly cut xylem, we found that the flow rate dropped irreversibly by over a factor of 100 if the xylem was dried, even when the xylem was flushed with water before drying. We also examined flow through commercially available kiln-dried wood samples cut to similar dimensions. Wood samples that exhibited filtration showed two orders of magnitude smaller flow rates than in the fresh xylem filter, while those that had high flow rates did not exhibit much filtration effect and seemed to have ruptured tracheids and membranes when observed under SEM. Wetting with ethanol or vacuuming to remove air did not significantly increase the flow rate in the wood samples that exhibited the filtration effect, suggesting that the pit membranes may have a tendency to become clogged during drying. These results are consistent with literature showing that the pit membranes can become irreversibly aspirated against the cell wall, blocking the flow [14]. In fact, the pit membranes in the SEM images (Figure 1d,e and Figure 4d,e), which were acquired after drying the samples, appear to be stuck to the walls. Regardless, our results demonstrate that excellent rejection (>99.9%) of bacteria is possible using the pit membranes of fresh plant xylem, and also provide insight into the mechanism of filtration as well as guidelines for selection of the xylem material. Peter-Varbanets et al. [2] have outlined the key requirements for point-of-use devices for water disinfection: a) performance (ability to effectively remove pathogens), b) ease of use (no time-consuming maintenance or operation steps), c) sustainability (produced locally with limited use of chemicals and non-renewable energy), and d) social acceptability. Meeting all of these requirements has proved to be challenging, but point-of-use methods that have been successfully used for low-cost water treatment in developing countries include free-chlorine/solar disinfections, combined coagulant-chlorine disinfection, and biosand/ceramic filtrations [5]. While chlorine is a very effective biocide, its reaction with organic matter can produce carcinogenic by-products [15] and some waterborne pathogens such as Cryptosporidium parvum and Mycobacterium avium are resistant to the chlorine [16]. Solar disinfection based on ultraviolet irradiation can effectively inactivate C. parvum, but this requires low turbidity of source water [17] and is not effective for control of viruses [16]. Filtration based on biosand and ceramic filters is also effective at removing pathogens, but the effectiveness against viruses is low or unknown [18]. Coagulation combined with chlorine disinfection removes or inactivates viruses and pathogens effectively. However, necessity of an additional filtration step and relatively high cost are potential barriers for practical use [18]. Among these methods, a review on field studies by Sobsey et al. [5] suggested that biosand and ceramic filtration are the most effective methods in practice, because once the apparatus is installed, the effort for use and dosage is significantly reduced and therefore promotes persistent use compared to disinfection approaches. Although membrane-based filtration is the most widely used for water treatment in industrialized nations and the cost of membranes has significantly decreased, membranes are still unaffordable to poor communities in the developing world [2]. Ultrafiltration systems run by hydrostatic pressure [19] and some recently invented point-of-use devices using ultrafiltration membranes may provide water to developing regions at reasonable cost [2]. However, membranes still require specialized chemicals and processes for manufacture, and need cleaning or replacement. Xylem filter technology could be an attractive option for low-cost and highly efficient point-of-use water treatment by filtration, overcoming some of the challenges associated with conventional membranes. Xylem filters could provide the advantage of reduced human effort compared to existing point-of-use water treatment options, requiring only simple periodic filter replacement. In addition, the pressures of 1–5 psi used here are easily achievable using a gravitational pressure head of 0.7–3.5 m, implying that no pumps are necessary for filtration. The measured flow rates of about 0.05 mL/s using only ∼1 cm2 filter area correspond to a flow rate of over 4 L/d, sufficient to meet the drinking water requirements of one person [20]. This is comparable to chlorination and biosand filtration, which have the highest production rates of prevalent point-of-use water treatment methods, and far exceeds typical production rates for solar disinfection. Xylem filters could potentially be produced locally and inexpensively, and disposed of easily owing to their biodegradability. The high flow rates and low cost would certainly help address the issues of maintenance and replacement. For example, 200 filters of 10 cm2 area and 0.5 cm thickness could be packaged into a volume of about 1 L, which will be inexpensive and last a few years even with weekly replacement. Furthermore, as suggested by the dye filtration experiment, xylem filters should be able to significantly reduce water turbidity, enhancing the aesthetic qualities of the drinking water, which is hardly achieved by chlorination and solar disinfection. Wood is an easily available material. While use of fresh xylem does not preclude its use as a filter material, dried membranes have definite practical advantages. Therefore, the process of wood drying and its influence on xylem conductivity needs further study. In particular, processes that yield intact yet permeable xylem tissues that can be stored long-term will be helpful for improving the supply chain if these filters are to be widely adopted. In addition, flow through xylem of different plants needs to be studied to identify locally available sources of xylem, which will truly enable construction of filters from locally available materials. In the present study, we report results using xylem derived from only one species. These xylem filters could not filter out 20 nm nanoparticles, which is a size comparable to that of the smallest viruses. It will be interesting to explore whether there exist any coniferous species that have pit membranes with smaller pore sizes that can filter out viruses, or whether conifer xylem can be impregnated with particles such as carbon black to improve rejection of viruses. In their absence, angiosperms with short vessels that yield practical filter lengths may be the best alternative due to their smaller pit membrane pore sizes [8]. Further exploration of xylem tissues from different plants with an engineering perspective is needed to construct xylem filters that can effectively reject viruses, exhibit improved flow rates, or that are amenable to easy storage. It is also conceivable that plants could be selected or developed for enhanced filtration characteristics, as has been the norm in agriculture for enhancement of many desirable characteristics including resistance to pests, flavor, or productivity.

Conclusions Plant xylem is a porous material with membranes comprising nanoscale pores. We have reasoned that xylem from the sapwood of coniferous trees is suitable for disinfection by filtration of water. The hierarchical arrangement of the membranes in the xylem tissue effectively amplifies the available membrane area for filtration, providing high flow rates. Xylem filters were prepared by simply removing the bark of pine tree branches and inserting the xylem tissue into a tube. Pigment filtration experiments revealed a size cutoff of about 100 nm, with most of the filtration occurring within the first 2–3 mm of the xylem filter. The xylem filter could effectively filter out bacteria from water with rejection exceeding 99.9%. Pit membranes were identified as the functional unit where actual filtration of the bacteria occurred. Flow rates of about 4 L/d were obtained through ∼1 cm2 filter areas at applied pressures of about 5 psi, which is sufficient to meet the drinking water needs of one person. The simple construction of xylem filters, combined with their fabrication from an inexpensive, biodegradable, and disposable material suggests that further research and development of xylem filters could potentially lead to their widespread use and greatly reduce the incidence of waterborne infectious disease in the world.

Acknowledgments The authors thank Yukiko Oka for assistance with preparation of illustrations and Sunandini Chopra for help with dynamic light scattering measurements.

Author Contributions Conceived and designed the experiments: MSHB JL VV VC RK. Performed the experiments: MSHB JL VV VC. Analyzed the data: MSHB JL RK. Contributed reagents/materials/analysis tools: VC. Wrote the paper: MSHB JL RK.