WO2020181167A1 - Living filtration membrane - Google Patents

Abstract

The present disclosure provides methods and systems for treating water comprising passing water through a living, self-healing cellulose membrane to obtain treated water.

Description

LIVING FILTRATION MEMBRANE

CROSS-REFERENCE TO RELATED APPLICATIONS

10001] This application claims the benefit of U.S. Provisional Application No. 62/814,596, filed March 6, 2019 and U.S. Provisional Application No. 62/880,397, filed July 30, 2019, the contents of each of which are incorporated herein by reference.

GOVERNMENT RIGHTS

[0002] This invention was made with government support under Grant No. 1828523 awarded by the National Science Foundation and Cooperative Agreement Number W911NF- 15-2-0020 awarded by the Army Research Laboratory. The government has certain rights in the invention.

FIELD

[0003] The present disclosure provides methods and systems to treat water with a living, self-healing cellulose membrane.

BACKGROUND

[0004] Access to sufficient amounts of clean water is a persistent global problem. In 2015, the United Nations identified as a Sustainable Development Goal providing access to clean veater for all by the year 2030, as two thirds of the world’s population (about 3.6 billion people) experience water scarcity for at least one month of the year. This number could increase to 4.8-5.7 billion by 2050. Current w¾ter use coupled with global population increases, makes the development of easy to use, chemicaliy-bemgn, and inexpensive water treatment technologies a challenge.

[0005 j To combat water scarcity and degrading water quality', many are turning to advanced engineering solutions for water treatment. Micro- and ultra- filtration membranes can be used in water treatment to remove pathogens, for example, protozoa like Giardia and Cryptosporidium , bacteria like E. coii, and viruses, without reiving on complex water chemistry. Membranes also have a lower footprint, making them more desirable in urban and decentralized locations. In industry, micro- and ultra- filtration membranes are used to filter and/or concentrate milk, fruit juice, and beer, and they are also used in biomedical equipment. Currently, most membrane installations are in the micro- and ultra- filtration range, and most of those installations are used by industry, with the membrane market valuation expected to reach $11.95 billion by 2021.

SUMMARY

[0006] Disclosed herein are methods of treating water, tire method comprising the steps of: passing water through a living, self-healing cellulose membrane; and obtaining treated water, wherein the membrane rejects at least about 80% of particles having a size of at least about 30 nm.

10007} Also disclosed herein is a system for treating water comprising: a water input line for receiving non-treated water and at least one living, self-healing cellulose membrane which is used to convert the non-treated water into treated water, wherein the membrane rejects at least about 80% of particles having a size of at least about 30 nm.

[0008] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The patent or application file contains at least one drawing executed in color.

Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0010] FIGS. 1.4-lF show the characteristics of a living filtration membrane (LFM). FIG. 1A is a digital photo of an LFM on a gloved hand. FIG. IB and FIG. 1C are scanning electron micrograph (SEM) images of an LFM showing coated cellulose fibers (LFM prepared with lyophilization and gold coating, FIG. IB) and bacteria embedded in fibers (bleached LFM prepared with post-critical point drying and gold coating, FIG. 1 C). FIG. ID is Fourier Transform Infrared (FTIR) spectra for LFM and a cellulose nanofiber membrane. FIG. IE is a graph of pure water flux as a function of transmembrane pressure for a pristine LFM. FIG. IF is LFM selectivity measured with gold and polypropylene nanoparticles. Prior to permeability and selectivity measurements, membranes were compacted at 3.1 bar for 1 h in a dead-end filtration cell. Membrane thickness was 1.3 ± 0.2 mm, and each coupon had a diameter of 25 mm. Where applicable, data is presented as the average with the error bars denoting the standard deviation.

|60l l] FIGS. 2A-2F show that the microorganisms within LFMs impart a self-healing property. FIGS. 2A-2C show normalized self-healing permeability for 4 mm long surface incision slit (FIG. 2A), for three 450 pm diameter holes (FIG. 2B) and for one 2 mm hole (FIG. 2C). For each test, permeability was measured before and immediately after damage. Membranes were placed in a growth solution to heal for a period of 1-17 days. Ail graphs are normalized to pristine membrane intrinsic permeability. FIG. 2D and FIG. 2E are confocal images of LFM damaged using a 450 pm tapered needle immediately following damage (FIG. 2D) or after 14 days of healing (FIG. 2E): side views of LFM are shown the bottom portion of image. FIG. 2F is an image of a damaged LFM with 2 m hole secured in the 10 mL dead-end filtration cell.

[00121 FIGS. 3A-3D show the effect of LFM permeability (FIG. 3A and FIG. 3B) and LFM selectively (FIG. 3C and FIG. 3D) as a function of storage environment: acidic media without a carbon source (FIG. 3A and FIG. 3C) or deionized water (FIG. 3B and FIG. 3D).

[0013] FIGS. 4A-4C show a point of use application. FIG. 4A and 4B are images of one possible operational setup for gravity LFM filtration. FIG. 3C shows the production rate for gravity LFM filtration setups.

[0014] FIGS. 4A-4C show a point of use application FIG 4A and 4B are images of one possible operational setup for gravity LFM filtration. FIG. 3C shows the production rate for gravity LFM filtration setups.

[0015] FIG. 5 is a graph of normalized flux for living fi ltration membranes (LFM 1 and LFM 2) and mixed cellulose ester membranes (MCE 1 and MCE 2) when processing drinking water pre-treated with coagulation and flocculation.

DETAILED DESCRIPTION

[0016 j The present disclosure provides methods of treating water, the methods comprise passing water through a living, self-healing cellulose membrane to obtain treated water. In some aspects, the water is wastewater.

[0017] Living filtration membranes (LFMs) were fabricated on lab-scale from a mixture of deionized water, black tea, sucrose, acetic acid (5%), and a starter culture of bacteria and yeast (SCOBY) and characterized for water filtration, structural, and self-healing properties. Pristine LFM permeability and size cutoff was about 135 L-ufMr'-har ¹ and about 30 mn, respectively. However, the LFMs disclosed herein experienced no change to intrinsic permeability and selectivity when stored outside of synthetic growth conditions for about 10 days. Self-healing tests resulted in return to 175-180% of the original flux in a period of about 4 to about 17 days, based on the type of applied damage, following incubation of LFMs in a growth solution at about 25 °C Successful lab-scale gravity filtration with the LFMs demonstrated ease of use and world-wide accessibility, especially in places lacking reliable drinking water.

[0018] Section headings as used m this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.

1. Definitions

[0019 j The terms“comprise(s),”“include(s),”“having,”“has,”“can,”“contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,”“and” and“the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments“comprising,”“consisting of’ and“consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0920 j For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0021] Unless otherwise defined herein, scientific and technical temis used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, environmental and chemical engineering, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular

[0022 j “Living, self-healing cellulose membrane,”“living filtration membrane,” and “LFM” are used interchangeably herein to refer to membranes comprising a microbial cellulose matrix with an associated or intertwined microbial community. The microbial community is living and responsible for regeneration of the cellulose matrix which allows the membrane to self-heal following damage or rupture. [00231 “Flat sheet,” as used herein refers to flat membrane structures having a separating layer present at the surface.

[0024] ‘Wastewater,” as used herein, refers to any used water from any combination of domestic, industrial, commercial or agricultural activities, surface runoff or stonnwater, and any sewer inflow or sewer infiltration. In some embodiments, the wastewater is domestic or municipal sewage or blackwater, winch is contaminated with fecal matter, or greywater, which is wastewater without fecal contamination.

[0025] Preferred methods and materials are described below , although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

2, Methods for Treating Water

[0026] Tie present disclosure provides methods for treating water. The methods comprise passing water through a living, self-healing cellulose membrane and obtaining treated water.

[0027] The methods may be applied to any source of water that needs purification or treatment for removal of contaminants. In some embodiments, the water is wastewater

a) Membrane properties

[0028] A membrane is essentially a semi-permeable barrier that allows some components of a solution to pass through while rejecting others. One basis for rejection of a component by a membrane is due to size. Particles too large to pass through the pores created by the matrix of the membrane will be rejected.

[0029] The living, self-healing cellulose membrane may reject at least 80% of particles having a size of at least about 30 lira In some embodiments, the membrane may reject at least 80% of particles having a size of at least about 30 nm, at least about 31 nm. at least about 32 nm, at least about 33 nm, at least about 34 nm, at least about 35 nm, at least about 36 nm, at least about 37 nm, at least about 38 nm, at least about 39 nm, at least about 40 nm, at least about 41 nm, at least about 42 nm, at least about 43 nm, at least about 44 nm, at least about 45 nm, at least about 46 nm, at least about 47 nm, at least about 48 nm, at least about 49 nm, at least about 50 nm, at least about 52 nm, at least about 55 nm, at least about 58 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nni . or at least about 100 nm.

|00301 In some embodiments, the membrane may reject at least 85% of particles having a size of at least about 30 nm, at least about 31 nm, at least about 32 nm, at least about 33 nm, at least about 34 nm, at least about 35 nm, at least about 36 nm, at least about 37 nm, at least about 38 nm, at least about 39 nm, at least about 40 nm, at least about 41 nm, at least about 42 nm, at least about 43 nm, at least about 44 nm, at least about 45 nm, at least about 46 nm, at least about 47 nm, at least about 48 nm, at least about 49 nm, at least about 50 nm, at least about 52 nm, at least about 55 nm, at least about 58 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm .

10031] In some embodiments, the membrane may reject at least 90% of particles having a size of at least about 30 nm, at least about 31 nm, at least about 32 nm, at least about 33 mn, at least about 34 nm, at least about 35 nm, at least about 36 nm, at least about 37 nm, at least about 38 nm, at least about 39 nm, at least about 40 nm, at least about 41 nm, at least about 42 nm, at least about 43 nm, at least about 44 nm, at least about 45 nm, at least about 46 nm, at least about 47 nm, at least about 48 nm, at least about 49 nm, at least about 50 nm, at least about 52 nm, at least about 55 nm, at least about 58 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 mn, at least about 90 nm, at least about 95 nm, or at least about 100 nm.

[0032] In some embodiments, the membrane may reject at least 95% of particles having a size of at least about 30 nm, at least about 31 nm, at least about 32 nm, at least about 33 nm, at least about 34 nm, at least about 35 nm, at least about 36 nm, at least about 37 nm, at least about 38 nm, at least about 39 nm, at least about 40 mn, at least about 41 mn, at least about 42 nm, at least about 43 nm, at least about 44 nm, at least about 45 nm, at least about 46 nm, at least about 47 nm, at least about 48 mn, at least about 49 nm, at least about 50 nm, at least about 52 nm, at least about 55 nm, at least about 58 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 mn, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm.

[0033] Flux is the flow of a solution through a filter. The ability to maintain a reasonably high flux is essential in tire membrane separation/filtration process. Low flux can result in long filtration times or require large filter assemblies, resulting in increased cost and large hold-up volumes retained in the modules and associated filter system equipment. The living, self-healing cellulose membrane may have a flux of at least about 100 L/m²/hr (LMH) at 2 bar.

[0034J In some embodiments, the flux is at least about 125 L/m²/hr (LMH), at least about 150 L/m²/hr (LMH), at least about 175 L/m²/hr (LMH), at least about 200 L/m²/hr (LMH), at least about 225 L/m²/far (LMH), at least about 250 L/nr/hr (LMH), at least about 300 L/mblir (LMH), at least about 350 L/nr/hr (LMH), 400 at least about L/m²/hr (LMH), at least about 450 L/nr/hr (LMH), or at least about 500 L/m²/hr (LMH) at 2 bar. In some embodiments, the flux is between about 100 L/m²/hr (LMH) and about 500 L/m/hr(LMH) at 1-3 bar. In certain embodiments, the flux is between about 100 L/m²/hr(LMH) and about 300 L/m²/hr(LMH) at 1-3 bar.

f0035J Similar to flux, permeability is a measure of the solution passing through a filter at a given applied force. Essentially, the permeability or specific flux, measures the amount of force necessary to produce a given flow through a membrane. The permeability can be used as a measure of membrane becoming fouled or being compromised by a tear, puncture, or rupture. Tire living, self-healing cellulose membrane may have a permeability of at least 50 Lmfii^bar¹.

[0036] In some embodiments, the permeability is at least about 55 Lm²h^!bar¹, at least about 60 Lmffli^bar¹, at least about 65 Lm^h^bar¹, at least about 70 LufT bar¹, at least about 75 Lm^h^bar¹, at least about 80 Lm^h^bar¹, at least about 85 Luffli^har¹, at least about 90 Lm²h^!bar¹, at least about 95 Lnwirbar¹, at least about 100 Lm^h^bar¹, at least about 105 LufT bar¹, at least about 110 LufT bar¹, at least about 115 Lm²h^_1bar ^l, at least about 120 Lm^b^bar¹, at least about 125 Lm^h^bar¹, at least about 130 Lirffb^bar¹, at least about 135 Lm^h^bar¹, at least about 140 Lmdisbar¹, at least about 145 Lm^h^bar¹, or at least about 150 LmTrbar¹.

[0037] In some embodiments, the permeability is less than about 300 Lm²h^lbar^_1, less than about 250 Lm^h^bar¹, less than about 200 Lm^h^bar¹, less than about 175 Lmfltybar , less than about 150 Lm²h^!bar¹, less than about 145 Lm^h^bar¹, less than about 140 Lm ²h¹bar^!, less than about 135 Lmfltybar^’1, less than about 130 Lnr ltybar¹, less than about 125 Lm^h^bar¹, less than about 120 Lmfiv'bar¹, less than about 115 Lmflr'bar¹, less than about 110 Lm^h^bar¹, less than about 105 Lm²h^_1bar % less than about 100 Lmfii^bar¹, less than about 95 Lm^h^bar¹, less than about 90 Lm^h^bar¹, less than about 85 Lm^h^bar¹, less than about 80 Lm²h^!bar less than about 75 Lm²h^!bar less than about 70 Lm²h^!bar less than about 65 Lmfir'bar¹, or less than about 60 Lm²h^!bar\ [ϋ©38| The membrane may take on a variety of configurations, shapes, and sizes based on the end-use application. In some embodiments the membrane is a flat sheet.

|00391 The thickness of the membrane may be varied by known methods to achieve the desired permeability and flux for the anticipated application. Tire membrane may have a thickness of at least about 0.1 mm. In some embodiments, the thickness is at least about 0.2 mm, at least about 0.3 mm, at least about 0.4 mm, at least about 0.5mm, at least about 0.6 mm, at least about 0.7 mm, at least about 0.8 mm, at least about 0.9 mm, at least about 1.0 mm, at least about 1.25 mm, at least about 1.5 mm, at least about 1.75mm, at least about 2.0 mm, at least about 2.5 mm, at least about 3.0 mm, at least about 3.5 mm, at least about 3.75 mm, at least about 4.0 mm, at least about 4.5 mm, at least about 4.75 mm, or at least about 5 mm .

[0040] In some embodiment, the thickness of the membrane is less than about 10 mm, less than about 7.5 mm, less than about 5 mm, less than about 4.5 mm, less than about 4.0 mm, less than about 3.5 mm, less than about 3.0, less than about 2.5 mm, less than about 2.0 mm, less than about 1 5mm, less than about 1.0 mm, or less than about 0.5 mm

[0041 j The shape and size of the membrane will be chosen based on the end use application. Common filter shapes include, but are not limited to, a circle, an o val, a square, and a rectangle. The membrane may be a circle and have a diameter at least about 0.50 cm. In some embodiments, the diameter is at least about 0.75 cm, at least about 1.0 cm, at least about 1.5 cm, at least about 2 cm, at least about 2.5 cm, at least about 3.0 cm, at least about 3.5 cm, at least about 4.0 cm, at least about 4.5 cm, at least about 5.0 cm, at least about 7.5 cm, at least about 10 cm, at least about 1 meter, at least about 5 meters, or at least about 10 meters. In some embodiments, the diameter may be up to about 10 meters. The membrane may be a square or rectangle and have side length up to about 10 meters.

b) Living, Self-Healing Cellulose Membrane

[0042] The living, self-healing cellulose membranes, or living filtration membranes (LFMs) described herein comprise microbial cellulose and an associated microbial community. The membranes have innate antifouling and self-healing properties. Antifouling is due to the high surface hydrophi!icity of the cellulose and the presence of microorganisms on the membrane, which may repel other microorganisms. The living microorganisms are also responsible for self-healing due to microbial generation of cellulose after damage.

[0043 J The living, self-healing cellulose membrane may be derived from one or more cellulose producing microorganisms. Any microorganism capable of producing cellulose may be suitable for the methods disclosed herein. For example, bacteria from the genera

Aerobacter, Acetobacler , Achromobacter, Agrobacterium, Alacaligenes, Azotobaster, Pseudomonas, Rhizobium, and Sarcina are all capable synthesizing cellulose. In some embodiments, the microorganisms comprise a symbiotic cul ture of bacteria and yeast (SCOBY) from kombucha tea. The species comprising a SCO BY generally

include Acetobacler bacterial species, as well as various Saccharomyces species or other yeasts. In some embodiments, the microorganisms comprise Acetobacler, Rhizobium, Agrobacterium, Aerobacter, Salmonella, Escherichia, Zygosaccharomyces rouxii, Candida sp. , or combinations thereof.

10044} Living, self-healing cellulose membranes may be fabricated using water, organics, nutrients, sucrose or other carbon source, acetic acid, and a microbial culture. The membrane may be fabricated using water streams which are high in organics and nutrients, including, for example, municipal wastewater, environmental waste streams or waste streams from the food industry. In one embodiment, the living, self-healing cellulose membrane is made by a method comprising combining boiling water, tea and a carbon source to form a tea mixture; steeping the tea mixture; adding acetic acid and the one or more cellulose producing microorganisms and yeast to form a culture; and incubating the culture.

[0045} The carbon source may he any source of carbon amenable to uptake and breakdown by the microbial organisms comprising the membrane. In some embodiments, the carbon source comprises sucrose, fructose, glucose, maltose, or a combination thereof. The carbon source may be a natural product, for example, honey or agave nectar, or purified, such as pure sucrose or glucose. The carbon source may be provided in any form including, but not limited to, powders, granules, syrups or solution.

[0046} The tea may include green tea, white tea, black tea or a combination thereof. In some embodiments, the tea is black tea, including, but not limited to, Oolong, Pekoe, Ceylon, Assam and Darjeeling. In certain embodiments, the black tea is a combination of Pekoe and Ceylon.

10047} The tea mixture may be steeped for varying amounts of time depending on the scale of the process. In general, the tea mixture needs to cool to between about IS to about 30 °C (e.g. about 25 °C) before proceeding to the next step.

[0048} Acetic acid is added to adjust the pH to less than 5 and the one or more cellulose producing microorganisms are added to form the culture. In some embodiments, the acetic acid is added to a pH of between 3.5 and 5, or between 3 5 and 4. [0049J The final culture may he incubated as long as necessary until a uniform membrane of desired thickness and diameter is obtained. In general, the incubation is carried out at temperatures which promote growth and cellulose production of the microorganisms. In some embodiments the culture is incubated at about 25°C. A constant incubation temperature results in consistent and uniform membrane characteristics. In some embodiments, the incubation lasts about 7 to about 10 days.

[01)50] The method may further comprise treating the membranes with a treatment solution, including, but not limited to, sodium hydroxide, hydrogen peroxide, and sodium hypochlorite, that remove microorganisms and excess organic matter. With low

concentrations of treatment solutions, inert cellulose membranes with similar permeability and selectivity characteristics can be fabricated and used like conventional polymeric membrane. These membranes may be useful for specific applications, including filtration in medical devices, in which the presence of microorganisms should be avoided. Higher concentration of the treatment solutions can change the porous structure of the membrane, such that the treatment results in a new inert cellulose membrane with higher penneability and lower selectivity or, upon fusing of fibers, lower permeability and higher selectivity. Treated membranes may be restored by reintroducing microorganisms and giving them a food source to create LFMs with different filtration properties.

3. System for Treating Water

[0051 ] The present disclosure provides systems for treating water. The systems for treating water comprise a water input line for receiving non-treated water and at least one living, self-healing cellulose membrane which is used to convert the non-treated water into treated water, wherein the membrane rejects at least 80% of particles having a size of at least 30 nm.

10052 J The system may be applied to any source of water that needs purification or treatment for removal of contaminants. In some embodiments, the water is wastewater. In some embodiments, the water is potable water

a) Membrane properties

0053 j The living, self-healing cellulose membrane may reject at least 80% of particles having a size of at least about 30 nm In some embodiments, the membrane may reject at least 80% of particles having a size of at least about 30 nm, at least about 31 nm, at least about 32 nm, at least about 33 nm, at least about 34 nm, at least about 35 nm, at least about 36 nm, at least about 37 nm, at least about 38 nm, at least about 39 nni . at least about 40 nm, at least about 41 nm, at least about 42 nm, at least about 43 nm, at least about 44 nm, at least about 45 nm, at least about 46 ran, at least about 47 nm, at least about 48 nm, at least about 49 nm, at least about 50 nm, at least about 52 nm, at least about 55 nm, at least about 58 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm

[0054] In some embodiments, the membrane may reject at least 85% of particles having a size of at least about 30 nm, at least about 31 nm, at least about 32 nm, at least about 33 nm, at least about 34 nm, at least about 35 nm, at least about 36 nm, at least about 37 mn, at least about 38 mn, at least about 39 nm, at least about 40 nm, at least about 41 nm, at least about 42 nm, at least about 43 nm, at least about 44 nm, at least about 45 nm, at least about 46 nm, at least about 47 nm, at least about 48 nm, at least about 49 mn, at least about 50 mn, at least about 52 nm, at least about 55 nm, at least about 58 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 mn, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm.

[0055] In some embodiments, the membrane may reject at least 90% of particles having a size of at least about 30 mn, at least about 31 nm, at least about 32 nm, at least about 33 n , at least about 34 nm, at least about 35 nm, at least about 36 nm, at least about 37 mn, at least about 38 nm, at least about 39 nm, at least about 40 nm, at least about 41 nm, at least about 42 nm, at least about 43 nm, at least about 44 nm, at least about 45 mn, at least about 46 nm, at least about 47 nm, at least about 48 nm, at least about 49 nm, at least about 50 nm, at least about 52 nm, at least about 55 nm, at least about 58 urn, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm.

|0056] In some embodiments, the membrane may reject at least 95% of particles having a size of at least about 30 nm, at least about 31 nm, at least about 32 nm, at least about 33 mn, at least about 34 mn, at least about 35 nm, at least about 36 nm, at least about 37 nm, at least about 38 nm, at least about 39 nm, at least about 40 nm, at least about 41 nm, at least about 42 nm, at least about 43 urn, at least about 44 nm, at least about 45 nm, at least about 46 nm, at least about 47 nm, at least about 48 nm, at least about 49 nm, at least about 50 nm, at least about 52 mn, at least about 55 nm, at least about 58 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 mu, at least about 90 nm, at least about 95 nm, or at least about 100 nm.

|00571 The living, self-healing cellulose membrane may have a flux of at least about 100 L/m²/hr (LMH) at 2 bar. In some embodiments, the flux is at least about 125 L/m²/hr (LMH), at least about 150 L/m²/hr (LMH), at least about 175 L/m²/hr (LMH), at least about 200 L/nr/hr (LMH), at least about 225 L/m²/hr(LMH), at least about 250 L/m²/hr (LMH), at least about 300 L/m²/hr (LMH), at least about 350 L/m²/hr (LMH), at least about 400 L/m²7hr (LMH), at least about 450 L/m²/hr (LMH), or at least about 500 L/m²/hr (LMH) at 2 bar. In some embodiments, the flux is between about 100 L/rrb/hr (LMH) and about 500 L/nr/hr (LMH) at 1-3 bar. In certain embodiments, the flux is between about 100 L/nr/hr (LMH) and about 300 L/m²/hr(LMH) at 1-3 bar.

10058J The living, self-healing cellulose membrane may have a permeability of at least about 50 Lm²h^!bar¹. In some embodiments, the permeability is at least about 55 Lm²h'bar ', at least about 60 Lnrlr'bar¹, at least about 65 Lm^h^bar¹, at least about 70 Lm^h^bar', at least about 75 LmHr^bar¹, at least about 80 Lmflv'bar¹, at least about 85 Lm^h^bar¹, at least about 90 Lm²h^!bar at least about 95 LmfVbar¹, at least about 100 LnfVbar¹, at least about 105 Lmflr'bar¹, at least about 110 Lnrir'bar¹, at least about 115 Lm²h^lbar^!, at least about 120 Lnrlr'bar¹, at least about 125 Lnrif'bar¹, at least about 130 LnfTfTrar', at least about 135 Lm^h^bar ', at least about 140 Lmrh^bar ¹, at least about 145 Lmrh^bar¹, or at least about 150 Lm²h'bar '.

[0059| In some embodiments, the permeability is less than about 300 Lmrir'bar¹, less than about 250 Lmfir'bar¹, less than about 200 Lmflr'bar¹, less than about 175 Lm²h^!bar ', less than about 150 Lm²h^!bar less than about 145 Lmflr'bar¹, less than about 140 Lm flf'bar¹, less than about 135 Lm²h^!bar¹, less than about 130 Lm^h^bar¹, less than 125 about Lm^h^bar¹, less than about 120 Lnrlr'bar¹, less than about 115 Lrnrh/ 'bar¹, less than about 110 Lnfiflr^bar¹, less than about 105 Lm^h^bar¹, less than about 100 Lmrh^bar ', less than about 95 Lnrh^bar¹, less than about 90 Lnrlr'bar¹, less than about 85 Lm²h 'bar', less than about 80 Lm²h^lbar', less than about 75 Lm²h^lbar', less than about 70 Lm ²h'bar', less than about 65 Lm²h'bar', or less than about 60 Lm²h^!bar¹.

10660 j Hie membrane may take on a variety of configurations, shapes, and sizes based on the end-use application. In some embodiments the membrane is a flat sheet.

[0061 ) The thickness of the membrane may be varied by known methods to achi eve the desired permeability and flux for the anticipated application. Tire membrane may have a thickness of at least about 0.1 mm. In some embodiments, the thickness is at least about 0.2 mm, at least about 0.3 mm, at least about 0.4 mm, at least about 0.5mm, at least about 0.6 mm, at least about 0.7 mm, at least about 0.8 mm, at least about 0.9 mm, at least about 1.0 mm, at least about 1 .25 mm, at least about 1.5 mm, at least about 1 75mm, at least about 2.0 mm, at least about 2.5 mm, at least about 3.0 mm, at least about 3.5 mm, at least about 3.75 mm, at least about 4.0 mm, at least about 4.5 mm, at least about 4.75 mm, or at least about 5 mm.

[0062] In some embodiments, the thickness of the membrane is less than about 10 mm, less than about 7.5 mm, less than about 5 mm, less than about 4.5 mm, less than about 4.0 mm, less than about 3.5 mm, less than about 3.0, less than about 2.5 mm, less than about 2.0 mm, less than about 1.5 mm, less than about 1.0 mm, or less than about 0.5 mm.

[0063] The shape and size of the membrane will be chosen based on the end use application. Common filter shapes include, but are not limited to, a circle, an oval, a square, and a rectangle. The membrane may be a circle and have a diameter at least about 0.50 cm. In some embodiments, the diameter is at least about 0.75 cm, at least about 1.0 cm, at least about 1.5 cm, at least about 2 cm, at least about 2.5 cm, at least about 3.0 cm, at least about 3.5 cm, at least about 4.0 cm, at least about 4.5 cm, at least about 5.0 cm, at least about 7.5 cm, at least about 10 cm, at least about 1 meter, at least about 5 meters, or at least about 10 meters. In some embodiments, the diameter may be up to about 10 meters. Hie membrane may be a square or rectangle and have side length up to about 10 meters

b) Living, Self-Healing Cellulose Membrane

[0064] The living, self-healing cellulose membranes comprise microbial cellulose and an associated microbial community.

[0065] The living, self-healing cellulose membrane may be derived from one or more cellulose producing microorganisms. Any microorganism capable of producing cellulose may be suitable for the methods disclosed herein. For example, bacteria from the genera

Aerobacter, Acetobacter, Achromobacter, Agrobacterium, Alacaligenes, Azotobaster, Pseudomonas, Rhizobium, and Sarcina are all capable synthesizing cellulose. In some embodiments, the microorganisms comprise a symbiotic culture of bacteria and yeast (SCOBY) from kombucha tea. The species comprising a SCOBY generally

include Acetobacter bacterial species, as well as various Saccharomyces species or other yeasts. In some embodiments, the microorganisms comprise Acetobacter, Rhizobium, Agrobacterium, Aerobacter, Salmonella, Escherichia Zygosaccharomyces rouxii, Candida sp. , or combinations thereof.

|00661 Living, self-healing cellulose membranes may be fabricated using water, organics, nutrients, sucrose or other carbon source, acetic acid, and a microbial culture. The membrane may be fabricated using water streams which are high in organics and nutrients, including, for example, municipal wastewater, environmental waste streams or waste streams from the food industry. In one embodiment, the living, self-healing cellulose membrane is made by a method comprising combining boiling water, tea and a carbon source to form a tea mixture; steeping the tea mixture; adding acetic acid and the one or more cellulose producing microorganisms and yeast to form a culture; and incubating the culture

f 60671 The carbon source may be any source of carbon amenable to uptake and breakdown by the microbial organisms comprising the membrane. In some embodiments, the carbon source comprises sucrose, fructose, glucose, maltose, or a combination thereof. The carbon source may be a natural product, for example, honey or agave nectar, or purified, such as pure sucrose or glucose. The carbon source may be provided in any form including, but not limited to, powders, granules, syrups or solution.

100681 The tea may include green tea, white tea, black tea or a combination thereof. In some embodiments, the tea is black tea, including, but not limited to, Oolong, Pekoe, Ceylon, Assam and Darjeeling. In certain embodiments, the black tea is a combination of Pekoe and Ceylon.

10669] The tea mixture may be steeped for varying amounts of time depending on the scale of the process. In general, the tea mixture needs to cool to between about 13 to about 30 °C (e.g., about 25 °C) before proceeding to the next step.

[0070 j Acetic acid is added to adjust the pH to less than 5. and the one or more cellulose producing microorganisms are added to form the culture. In some embodiments, the acetic acid is added to a pH of between 3.5 and 5, or between 3 5 and 4.

[0671] The final culture may be incubated as long as necessary until a uniform membrane of desired thickness and diameter is obtained. In general, the incubation is carried out at temperatures which promote growth and cellulose production of the microorganisms. In some embodiments the culture is incubated at about 25°C. A constant incubation temperature results in consistent and uniform membrane characteristics. In some embodiments, the incubation lasts about 7 to about 10 days. [00721 The method may further comprise treating the membranes with a treatment solution, including, but not limited to, sodium hydroxide, hydrogen peroxide, and sodium hypochlorite, that remove microorganisms and excess organic matter. With low

concentrations of treatment solutions, inert cellulose membranes with similar permeability and selectivity characteristics can be fabricated and used like conventional polymeric membrane. These membranes may be useful for specific applications, including filtration in medical devices, in which the presence of microorganism s should be avoided. Higher concentration of the treatment solutions can change the porous structure of the membrane, such that the treatment results in a new' inert cellulose membrane with higher permeability and lower selectivity or, upon fusing of fibers, lower permeability and higher selectivity. Treated membranes may be restored by reintroducing microorganisms and giving them a food source to create LFMs with different filtration properties.

4, Examples

Materials and Methods

[0073] Membrane growth and LFM thickness. A culture of symbiotic bacteria and yeast (Kombucha, 20 g, Cultures for Health) w¾s added to sterile black tea made by boiling 700 mL deionized water (DI) and steeping 4.6 g generic mix of pekoe black teas for 1 hour. This culture was supplemented with sucrose (85 g, generic, granulated) and distilled white vinegar (200 mL, 5% acetic acid, generic). Mixtures were covered with paper towels, secured with rubber bands, and placed in an incubator at 25 °C for 10 days. After a 10-day growth period, the membranes were used within 2 days.

[0074] Prior to any experiments, LFM thicknesses were quantified by placing a portion of each membrane sample on a clean microscope slide and measuring the thickness using calipers (United States Plastic Corp, Stainless Steel Caliper) in three different sample regions to obtain an average thickness. Duong the course of experimentation, three different media were used: a growth media with the same composition listed above to fabricate an LFM, an acidic media without a carbon source (growth media minus sucrose), and DI.

[0075] Permeability and selectivity. Permeability and selectivity tests were performed in a dead-end stirred cell with a 24.5 mm sample diameter (Amicon 8101, Millipore co.). The cell was pressurized using a compressed air tank while flux was determined by monitoring the change of permeate mass with time with a balance connected to a computer. Prior to any flux or selectivity measurements, membrane samples were compacted for 1 hour at 3.1 bar using deionized (DI) water as the feed solution and increasing the pressure slowly by 5 bar per minute. Each feed solution was filtered through separate membrane samples. Permeability was measured for 20 min at 4 different applied pressures (0.7, 1.4, 2.1, and 3.1 bar).

Selectivity tests were carried out at 1.4 bar using polymer microspheres solutions (3, 0 2, and 0.1 pm; Polyscience, Inc.) or gold nanopartide solutions (5, 10, and 20 nm; NN-Labs). Confirmation of particle diameters for PolySciences Poly Beads used in selectivity testing were run on a Malvern Zetasizer.

[0076] Electrospinning of Cellulose Nanofibers. Cellulose acetate nanofiber mats were electrospun using a modification of a previous method ( K. A. Rieger et al. , RSC Adv. 6, 24438-24445 (2016)), and regenerated into pure cellulose nanofiber mats. Briefly, solutions consisting of 15 wt% cellulose acetate in acetone were mixed for 24 hours at 20 rpm using an Arma-Rotator A-l apparatus (Elmeco Engineering, Rockville, MD). A cellulose acetate solution was loaded into a 5 mL Luer-Lock tip syringe capped with a Precision Glide 18- gauge needle (Becton, Dickinson & Company, Franklin Lakes, NJ) after which the syringe was secured to an infusion syringe pump (Cole Parmer, Vernon Hills, IL) Alligator clips were used to connect the electrode of a high-voltage supply (Gamma High Voltage Research, Ormond Beach, FL) to the needle and the electrode of a copper plate (152.4 mm ^c 152.4 mm x 3.2 mm, McMaster-Carr, Robbinsville, NJ). The copper plate was wrapped in aluminum foil and held at a fixed separation distance of 10 cm. A constant feed rate of 3 mL Irl and an applied voltage of 25 kV were used to electrospin the cellulose acetate solutions. The assembled electrospinning apparatus was housed in a custom-built environmental chamber equipped with a desiccant unit (Drierite, Xenia, OH) that maintained the temperature at 22 ±

1 °C and the relative humidity at 55%. To generate nanofiber layers with a consistent bulk thickness, cellulose acetate was electrospun for 1 hour. After being peeled off the collector plate, the cellulose acetate nanofiber layer was sandwiched between Teflon sheets (3.2 mm ^c 101 6 mm x 152.4 mm, McMaster-Carr) and placed in a furnace for 1 hr at 208°C. To generate cellulose nanofibers, the heat-treated cellulose acetate nanofibers were submerged in a 0.1 M sodium hydroxide/ethanol solution (4: 1 v/v) for 14 hours before being washed three times with DT water.

[0077] Storage and self-healing. Permeability and selectivity tests were performed on

LFMs stored in two different media: 1.1% acetic acid and black tea media without a carbon source, and deionized water. Membranes in both media were stored in the incubator at 25 °C.

Both permeability and selectivity tests were performed with pristine LFMs, and membrane samples were stored in the two media for 5, 10, and 15 days. Three different membrane samples were tested for each storage solution and storage time.

|007Sj Three different self-healing experiments were performed. In tire first self-healing test a 2 mm diameter hole was placed in the center of the sample using a needle. To prevent further tearing of the membrane, the sample was secured in the base of the dead-end cell during puncture and healing. Permeate flux was measured for 10 min at an applied pressure of 1.4 bar immediately after the puncture and after 22, 78 and 100 hours in growth solution. The second self-healing test was conducted by placing three pinholes in the membrane (45Qpm, standard patchwork pin) and conducting the same set of storage permeability tests that were conducted during the first self-healing test. The third self-healing test conducted on the membrane began by measuring LFM intrinsic permeability and followed by placing a 3mm incision using a sterile scalpel in the membrane surface. The same set of storage permeability tests were conducted on the membrane.

|0 79| Chemical and microscopic characterization. Pristine membrane samples were lyophilized prior to Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) characterizations. Specifically, samples were allowed to freeze slowly for two days in a sterile 50 mL falcon tube using a freeze dryer (Labconco FreeZone 2.5 set at - 46°C and 2.5E-4 bar). Membrane samples were then analyzed using FTIR spectroscopy (Nicolet iS5, iD5 with ATR attachment) and scanning electron microscopy (Tescan Mira3). 3D images of membrane samples were also obtained through confocal laser scanning microscopy. Confocal images were captured using a Leica SP8 laser scanning confocal microscope equipped with a Plan-Apochromat 10 ^c /0.4 numerical aperture objective.

Calcafluor White was excited with a 405 nm laser, and an emission window of 569-61 1 nm was used.

Example 1

Growing Living Filtration Membranes (LFMs)

[0080] Polymeric membrane fabrication commonly requires a large amount of harmful solvents, as well as other high-purity chemicals. To combat drawbacks associated with traditional polymeric membrane implementation, many have looked to biological systems for inspiration, creating several classes of biomimetic membranes. Inside the human body, biological membranes, e.g., kidneys and eye lenses, make use of the phospholipid bilayer to sieve contaminants. Aquaporin-incorporated membranes have been extensively studied for their potential ability to provide sustainable desalination, but experience difficulties in scale- up. Polymeric membranes fabricated with carbon nanotubes (CNT) are sturdy with potentially ultra-high membrane flux. But, the use of CNTs in membranes may lead to CNT release into the environment and other biological systems, leading to CNT toxicity in living organisms and subsequent consequences in other biological organisms.

10081] In an attempt to eliminate the use of toxic ingredients, while also exploiting the propensity of living ceil membranes as filters, LFMs were grown from ingredients found in even very modest grocery stores, along with a culture that may be obtained from an array of sources LFMs were fabricated from a mixture of 4.4 mg-L ¹ dried black tea leaves, 5 vol% acetic acid (i.e., distilled white vinegar), 94.4 mg-L ¹ sucrose, and 22: 100 starter culture by mass (Cultures for Health). After these ingredients were combined, the mixtures fermented for 7-10 days at 25 °C until the LFMs reached a thickness of 1-1.5 mm. LFMs were harvested from the top of the fermented mixture.

10082] LFMs were fabricated from several classes of bacteria, including Acetobacter, Rhizobium, Agrobacterium, Aerobacter, Salmonella , and Escherichia and yeast including: Zygosaccharomyces rouxii and Candida sp. that make up the culture used to ferment kombucha. When a culture is grown under stirred conditions, bacterial cellulose forms balls; however, when a stagnant culture is grown, the cellulose forms a layer at the liquid-air interface. Longer growth times led to thicker layers and associated iowur filtration membrane water permeability. Hie membranes described here used thinner layers that incorporated both bacterial cellulose and the microorganisms that synthesize, maintain, and repair the cellulose.

[0083] Microorganisms in the starter culture feed on the sucrose to synthesize cellulose pellicles into a floating cellulose network. This matrix of cellulose formed a thin layer on top of the fermented mixture. As inoculation time increased, the LFM grew in thickness (as long as the LFM carbon food source is not removed from the broth) forming a zoogleai mat often referred to as a tea-mushroom, tea fungus, or any of over 80+ wOrldwide names for the compound. LFM growth may be slowed by adding a lesser amount of starter culture prior to inoculation leading to changes in network morphology.

Example 2

LFM Composition & Characterization

[0084 j FTIR spectra w'ere collected using a Nicolet iS5, iD5, with ATR attachment for the

LFM and cellulose nanofibers synthesized by electrospinning. As a long-chain carbohydrate used by living organisms for energy purposes, cellulose (CeHioOs)» is the most abundant organic polymer on Earth; it is a building block in vegetable tissue cell walls and is secreted by bacteria to form biofilms. Cellulose acetate was electrospim and regenerated into a pure cellulose membrane comprised of a random network of nanofibers that had a continuous and cylindrical morphology with an average diameter of 0.9 ± 0.5 pin. By the absence of a peak at 1750 cm % the FTIR spectra indicated that the acetate groups of cellulose acetate were replaced with hydroxyl groups and pure cellulose nanofiber mats were successfully fabricated. These pure cellulose nanofiber mats can act as a chemical and morphological control to the LFM membranes. Characteristic peaks around 1020 and 1046 cm ¹ for both LFM and the cellulose electrospun membranes (FIG. ID) were indicative of C-C, C-OH, C-H ring and side group vibrations, respectively, confirming that the basis of the LFM was also cellulose.

(8085J The morphology of the LFM cellulose matrix was vi sualized using SEM. The fibrous structure of the samples was preserved by mounting lyophiiized (Labconco FreeZone 2.5) or critical point dried (CPD) samples (Autosamdri-931 CPD) that were sputter-coated with gold (Denton- Vacuum, Model: Desk-1) and imaged via SEM (Tescan MiraS). FIG. IB, obtained from a lyophiiized LFM, suggested that biopolymers encase the cellulose strands, forming a non-uniform porous network. FIG. 1 C, obtained from a bleach-treated CPD membrane, revealed fibers with a small average diameter, 38 nm ± 11.4 nm. Pure cellulose fibers were exposed and intertwined with bacteria. The SEM images were consistent with earlier work on bacterial cellulose crystalline structure. Cellulose mats that undergo treatments (usually alkali) show decreased swelling with a more distinct fiber network, with fiber diameters in the range of 5-70 nm.

[0086] Die hydrophilicity of the LFMs was determined using air bubble-under-water contact angle measurements. Their contact angle was found to be 63.1 ° ± 5.1° consistent with hydrophilic cellulose-based materials literature ranging from 60° to 80°.

Example 3

LFM Properties

10087] The LFM structure as a non- woven mat of cellulose fibers lends to its use as a selective water filtration membrane. LFMs were tested for filtration properties using a bench- scale laboratory setup. Permeability tests were conducted with a 10 mL dead-end filtration cell (Amicon 8101, Millipore Co.) connected to an external 800 mL reservoir (Amicon). Pure water permeability was tested after a compaction period of one hour, using pristine membranes, taken from the top of a fermented brew after a 10-day growth period. Flux across the LFM was monitored until stabilization for the applied pressures of 0 70, 1.4, 2.1, and 3.1 bar. Average permeability across LFM was 89 9 L m ¾ ^!bar ¹ (FIG IE) Depending on applied pressure, traditional polymeric ultrafiltration membrane permeability can reach to 1000 L m ²h^_1.

[0088] LFM selectivity was tested using the same 10 ml, dead-end cell at an applied pressure of 1 38 bar. Feed solutions consisted of deionized water and a concentration of particles of known diameter. Rejection was calculated by measuring absorbance of feed and permeate solutions at 350 and 500 nm wavelength for polypropylene and gold beads, respectively using UV-Vis spectroscopy (Agilent Technologies, Cary 60 UV-Vis). Results were plotted and pristine LFM 90% particle diameter cutoff was interpolated at 45 nm (FIG. IF). Therefore, the LFM cutoff would remove bacteria and protozoa and would reject both Zika and Hepatitis C viruses (both are 50 nm) to some extent. Traditional polymeric UF membranes, on the other hand, remove contaminants in the range of 1-30 nm.

Example 4

LFM Self-Healing

[0089] Self-healing materials have the innate ability to propagate an autonomous mobile phase, occurring in a damaged unit. Intrinsic self-healing materials may still require external stimuli, typically thermal. Extrinsic self-healing can be: 1) capsule-based via the

incorporation of microcapsules or hydrogels, 2) a vascul ar network that is capillary-based with the incorporation of hollow' glass fibers, stainless steel wires, or 3) a microvascular foam that creates pipelines within the polymer composite matrix, acting as post-healing reinforcement. Self-healing polymers include classes of formaldehydes, epoxies, acrylic acids, and polyelectrolytes, among others. Traditional self-healing repairs could be detrimental to water filtration properties due to the specific surface structure required to control the desired permeability and selectivity

[0090] Three self-healing tests were conducted using 10 mL or 50 ml, dead-end filtration cells (Amicon, Milhpore) The first test involved placing a 4 mm incision into the membrane using a sterile scalpel (FIG. 2A). The second test involved placing 3 puncture holes in the membrane with a 450 pm tapered needle (FIG. 2B), according to the method of Getachew, et al (B. A. Getachew, S. R Kim, I H Kim, Environ. Sci. Technoi. (2017),

doi: lQ.1021/acs.est.6h04574.), who tested self-healing abilities of microcapsule-embedded membranes. The third test involved placing a 2 mm diameter hole in the membrane using a large tapered needle (FIG. 2C and FIG. 2D). All tests started by measuring pristine membrane intrinsic permeability, followed with intentional damage to the membrane, and continued by placing the membrane in growth solution and incubator set at 25 °C to inoculate self-healing.

[0091] For test one, permeability immediately following damage increased by 33%, and then returned within 10% of pristine membrane intrinsic permeability following 10 days of self-healing. For test two, permeability immediately following damage increased by 277% of pristine membrane permeability and returned within 43% of the pristine membrane permeability after 17 days of self-healing. For test three, post-puncture permeability' increased by 1874% immediately after damage, and returned within 357% of pristine membrane permeability after 4 days. Though complete flux return was not fully achieved in short-term self-healing tests, the trend indicates with appropriate time, pristine membrane permeability will again return to the LFM. All damaged membranes regained selectivity after the conclusion of each self-healing experiment. Tims, when damaged, the membranes were able to self-heal, regain flux, and regain selectivity after being damaged by up to a 2 mm diameter puncture.

[0092] When the LFM is placed in growth solution with a sucrose food source, the microorganisms inside the LFM can synthesize new' cellulose pellicles, performing the self- healing process. This process is similar to ceil membrane repair in tire human body wiiere new cells help slough away the old, dead, or damaged cells and assist in the growth of new cells. Using confocal microscopy, the growth of a new cellulose layer atop the damaged layer was confirmed (FIG. 2E). The area of LFM subjected to damage (from a 450 pm puncture) has new cellulose growth on top of the membrane. The newly healed portion is thinner than the surrounding membrane.

Example 5

LFM Stability

[0093] To grow the microfibril cellulose network and employ the LFM as a water filtration membrane, the microorganisms in the starter culture need and a carbon source to synthesize cellulose pellicles and an acidic environment pFf 2.5-3.5 to protect from infection. During filtration, the LFM is taken away from the food source and acidic environment that it needs to continue synthesis; once outside of the growth solution, the cellulose generation begins to slow as the microorganisms become stressed.

[0094] To monitor LFM stability outside of its growth environment, pristine LFMs were stored in both DI water and an acidic media without a carbon source at 2.5 °C. LFM selectivity and permeability were tested after different lengths of time outside of the growih environment after 5, 10, and 13 days. Permeability and selectivity data for storage time in both solutions is shown in FIGS. 3A-3D. In DI and acidic media, average flux measured for fresh membranes and membranes stored for 5 and 10 days in Dl water was 284 ± 12 L m^_2h^_l at a pressure of 0.78 bar. For 15 days storage in DI water, the flux increased to 393 L m^-2h^-1.

Example 6

LFM Applications

|01)95] Increasing ease of access to clean drinking water is a UN Sustainable Development Goal, and a described above a method for producing an ultrafiltration membrane that removes suspended particles with size similar or smaller than protozoa, bacteria, and larger viruses using materials commonly available at a grocery store or market was developed. Not only can the membranes described herein produce purified drinking water, the membranes can be employed using common household items, such as a pour-over coffee maker without any additional equipment. FIGS. 4A and 4B show how LFMs can easily withstand placement in a coffee filtration device with several inches of head. With this setup, 300 mL of clean water was obtained after 8 hours without any pressure. Notably, the flowrate could be increased by applying gravity pressure using an elevated water tank and an in-line filter. FIG. 4C show's possible output operational capabilities based on size of filter and height of head. Die average drinking water requirement for a 4-person family (16 L) is marked with the black dotted line. The required drinking water may be achieved by modifying the membrane area or the amount of pressure on the membrane.

10096] As a form of water treatment, the LFM may have lower membrane installation costs thanks to higher intrinsic permeability at lower trans-membrane pressures compared to commercially available UF membranes. Additionally, LFMs may have krwer maintenance and replacement costs thanks to self-healing and potential antifouling properties as well as an inexpensive and safe method of membrane fabrication, as all materials needed to fabri cate membranes are common household items. LFM applications may be in various forms of water treatment. Due to the ability to remove organic mater, bacteria, and microorganisms, they can be employed in wastewater treatment, ultrafiltration, and/or as a pretreatment for RO. In sum, LFMs have the potential to bring accessible water treatment to anyone, anywhere. Example 7

LFM Foisting

|00971 Folding is the accumulation of unwanted material on the surface or within the pores of a membrane such that performance is compromised. Flux decline with time due to fouling can be one of the most serious shortcomings of microfiltration and ultrafiltration membranes, and for water filtration, can severely affect the quality of the water produced. 10098] Relative antifouling properties were determined for LFMs with a comparison to commercial nitrocellulose membranes. The LFM and nitrocellulose membranes, each having an approximate 50 kDa molecular weight cut-off, were cut to size and compressed in a 15 mL dead-end filtration cell at lOpsi for 1 minutes, 20 psi for 1 minute, 30 psi for 1 minute and 45 psi for 1 hour. Raw basin creek reservoir water (1.5 L) was mixed with an aluminum chlorohydrate (ALCH) coagulant (11.2 mL) using a radial mixer for 30 seconds with high agitation and an additional 5 minutes a low agitation until flocculation was observed. A peristaltic pump was used to put the top -90% of flocculated water mixture into the testing reservoir and to prime the filtration cell. The filtration cell is gradually brought up to testing pressure (10 psi for 15 minutes, 20 psi for 15 minutes and 30 psi for 15 minutes) before a 45 psi hold for the duration of the antifouling test. After 7 hours, the cell was depressurized and normalized flux was calculated using mass data acquired over time.

10099) The flux data (FIG. 5) indicated that living filtration membranes had a 40% decrease in flux compared to a 95% reduction in flux for a commercial mixed cellulose ester membrane. A smaller decrease in flux is atributed to the LFM’s anti-fouling property. Both membranes had a similar size cutoff and similar starting fluxes.

[0190] Additional flux measurements are taken at various pressures. Immediately after the fouling tests, the fouling layer is treated with calcoflouro white (cellulose and chitin) and/or a live-dead (microorganism) stain for analysis of the fouling layer under a con focal microscope and quantification of bacteria on the surface of the membranes. Tire water chemistry, membrane surface charge, and surface roughness are used to further analyze the fouled membrane. DNA extraction and analysis are used for both the membrane and the fouling layers.

[0191 j It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.

2.3 [01 21 Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the disclosure, may be made without departing from the spirit and scope thereof.

Claims

CLAIMS What is claimed is:

1. A method of treating water, the method comprising the steps of:

a. passing water through a living, self-healing cellulose membrane; and b. obtaining treated water,

wherein the membrane rejects at least about 80% of particles having a size of at least about 30 nm.

2. The method of claim 1, wherein the water is wastewater or potable water.

3. The method of claims 1 or 2, wherein the membrane rejects at least about 80% of particles having a size of at least about 48 nm.

4. The method of claims 1-3, wherein the membrane rejects at least about 85% of particles having a size of at least about 30 nm.

5. The method of claims 1-4, wherein the membrane rejects at least about 90% of particles having a size of at least about 30 nm.

6. The method of any one of claims 1-5, wherein the membrane has a flux of at least about 100 L/nr/hr (LMH) at 2 bar.

7. The method of any one of claims 1-6, wherein the membrane has a permeability of at least about 50 Lm Trhar ¹.

8. The method of any one of claims 1-7, wherein the membrane has a permeability of at least about 135 Lrrwh har ¹

9. The method of any one of claims 1-8, wherein the membrane is a flat sheet.

10. The method of claim 9, wherein the membrane has a thickness of at least about 0.1mm.

11. The method of any one of claims 1-10, wherein the membrane has a diameter of at least about 0.50 cm.

12. The method of any one of claims 1-1 1 , wherein the membrane is derived from one or more cellulose producing microorganisms.

13. The method of claim 12, wherein the cellulose producing microorganisms comprise a symbiotic culture of bacteria and yeast from kombucha tea.

14. The method of claim 13, wherein the cellulose producing microorganisms comprise Acetobacter, Rhizobium. Agrobacterium, Aerobacter. Salmonella, Escherichia,

Zygosaccharomyces rouxii, Candida sp , or combinations thereof.

15. The method of any of claims 1 -14, wherein the living, self-healing cellulose membrane is made by a method comprising:

combining boiling water, tea, and a carbon source to form a tea mixture;

steeping the tea mixture;

adding acetic acid and the one or more cellulose producing microorganisms to form a culture; and

incubating the culture.

16. The method of claim 15, wherein the carbon source comprises sucrose, fructose, glucose, maltose, or a combination thereof

17. A system for treating water comprising: a water input line for receiving non-treated water and at least one living, self-healing cellulose membrane which is used to convert the non-treated water into treated water, wherein the membrane rejects at least about 80% of particles having a size of at least about 30 am.

18. The system of claim 17, wherein the water is wastewater or potable water.

19. The system of claims 17 or 18, wherein the membrane rejects at least about 80% of particles having a size of at least about 48 nm.

20. The system of any one of claims 17-19, wherein the membrane rejects at least about 85% of particles having a size of at least about 30 nm.

21. The system of any one of claims 17-20, wherein the membrane rejects at least about 90% of particles having a size of at least about 30 nm.

22. The system of any one of claims 17-21, wherein the membrane has a flux of at least about 100 L/m²/hr (LMH) at 2 bar.

23. The system of any one of claims 17-22, wherein the membrane has a permeability of at least about 50 Lm^h^bar ¹.

24. The system of any one of claims 17-23, wherein the membrane has a permeability of at least about 135 Lm^h^bar ¹.

25. The system of any one of claims 17-24, wherein the membrane is a flat sheet

26. The system of claim 25, wherein die membrane has a thickness of at least about 0.1mm.

27. The system of any one of claims 17-26, wherein the membrane has a diameter of at least about 0.50 cm.

28. The system of any one of claims 17-27, wherein the water is wastewater or potable water.

29. The system of any one of claims 17-28, wherein the membrane is derived from one or more cellulose producing microorganisms.

30. The system of claim 29, wherein the cellulose producing microorganisms comprise a symbiotic culture of bacteria and yeast from kombucha tea

31. The system of claim 29, wherein the cellulose producing microorganisms comprise Acetobacter, Rhizobium, Agrobacterium. Aerobacter, Salmonella, Escherichia,

Zygosaccharomyces rouxii. Candida sp. , or combinations thereof.

32. The system of any of claims 17-31, wherein the living, self-healing cellulose membrane is made by a method comprising:

combining boiling water, tea, and a carbon source to form a tea mixture;

steeping the tea mixture;

adding acetic acid and the one or more cellulose producing microorganisms to form a culture; and

incubating the culture.

2.7

33. The system of claim 32, wherein the carbon source comprises sucrose, fructose, glucose, maltose, or a combination thereof.