)

(
1

(51) International Patent Classification: DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN,
B01D 61/02 (2006.01) 11) 61/58 (2006.01) HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP,
B 11) 61/04 (2006.01) KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME,
MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ,
(21) International Application Number:
OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA,
PCT/US2020/021327
SC, SD, SE, SG, SK, SL, ST, SV, SY, TH, TJ, TM, TN, TR,
(22) International Filing Date: TT, TZ, UA, UG, US, UZ, VC, VN, WS, ZA, ZM, ZW.
06 March 2020 (06.03.2020)
(84) Designated States (unless otherwise indicated, for every
(25) Filing Language: English kind of regional protection available) . ARIPO (BW, GH,
GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ,
(26) Publication Language: English
UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
(30) Priority Data: TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
62/814,596 06 March 2019 (06.03.2019) US EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
62/880,397 30 July 2019 (30.07.2019) US MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,
TR), OAPI (BF, BJ, CF, CG, Cl, CM, GA, GN, GQ, GW,
(71) Applicants: MONTANA TECHNOLOGICAL KM, ML, MR, NE, SN, TD, TG).
UNIVERSITY [US/US]; c/o Bev Hartline VC Research,
1300 W . Park, Butte, Montana 59701 (US). UNIVERSI¬
Published:
TY OF MASSACHUSETTS AMHERST [US/US]; 240 — with international search report (Art. 21(3))
Thatcher Rd., Amherst, Massachusetts 01003-9364 (US).
POLITECNICO DI TORINO [IT/IT]; DIATI - Depart¬
ment of Environment, Land and Infrastructure Engineering,
Corso Duca degli Abruzzi 24, 10129 Torino (IT).
(72) Inventors; and
(71) Applicants: ZODROW, Katherine R. [US/US]; c/o Mon¬
tana Technological University, 1300 W . Park, Butte,
Montana 59701 (US). EGGENSPERGER, Christina G.
[US/US]; c/o Montana Technological University, 1300 W .
Park, Butte, Montana 59701 (US). GIAGNORIO, Mat-
tia [IT/IT]; c/o Politecnico di Torino, Corso Duca degli
Abruzzi 24, 10129 Torino (IT). HOLLAND, Marcus C.
[US/US]; c/o Montana Technological University, 1300 W .
Park, Butte, Montana 59701 (US). DOBOSZ, Kerianne
M. [US/US]; c/o University of Massachusetts Amherst,
240 Thatcher Rd., Amherst, Massachusetts 01003-9364
(US). SCHIFFMAN, Jessica D. [US/US]; c/o Universi¬
ty of Massachusetts Amherst, 240 Thatcher Rd., Amherst,
Massachusetts 01003-9364 (US). TIRAFERRI, Alberto
[IT/IT] ; c/o Politecnico di Torino, Corso Duca degli Abruzzi
24, 10129 Torino (IT). BECHTEL, Carson [US/US]; c/o
Montana Technological University, 1300 W . Park, Butte,
Montana 59701 (US). JIANG, Daqian [CN/US]; c/o Mon¬
tana Technological University, 1300 W . Park, Butte, Mon¬
tana 59701 (US).
(74) Agent: MUELLER, Lisa V.; Casimir Jones, S.C., 2275
Deming Way, Suite #3 10, Middleton, Wisconsin 53562
(US).
(81) Designated States (unless otherwise indicated, for every
kind of national protection available) : AE, AG, AL, AM,
AO, AT, AU, AZ , BA, BB, BG, BH, BN, BR, BW, BY, BZ,
CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO,

(54) Title: LIVING FILTRATION MEMBRANE

(57) 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.
 LIVING FILTRATION MEMBRANE

CROSS-REFERENCE TO RELATED APPLICATIONS

10 ] This application claims the benefit of U.S. Provisional Application No. 62/8 14,596,
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. 828523
awarded by the National Science Foundation and Cooperative Agreement Number W91 1NF-
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
a er 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 $ 1.95 billion by 202 1.

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.
100 7 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 8 % 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 O F THE DRAWINGS

000 ] 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.
[00 ] FIGS. . - F show the characteristics of a living filtration membrane (LFM). FIG.
A 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. 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 s LFM selectivity measured with gold and polypropylene nanoparticles. Prior to
permeability and selectivity measurements, membranes were compacted at 3.1 bar for h in a
dead-end filtration cell. Membrane thickness was 1.3 ± .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.
|60ll] 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 m diameter holes (FIG. 2B) and for one 2 m 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- 7 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 0
mL dead-end filtration cell.
[ 0 2 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).
[0 3] 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.
[00 4] 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.
[ 5] FIG. 5 is a graph of normalized flux for living fi ltration membranes (LFM 1 and
LFM 2) and mixed cellulose ester membranes (MCE and MCE 2) when processing
drinking water pre-treated with coagulation and flocculation.

DETAILED DESCRIPTION

[0 6 The present disclosure provides methods of treating water, the methods comprise
passing water through a living, self-healing cellulose membrane to obtain treated water. I
some aspects, the water is wastewater.
[0 ] 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 ' ar 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 this section and the entire disclosure herein are merely
for organizational purposes and are not intended to be limiting.

1. Definitions

[0019 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.
[0 2 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.
[0 21] 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.
[002 “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] ie 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
[0 2 ] 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.
[002 ] 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 3 1 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 east about 38 n , at least about 39 nm, at least about 40 nm,
at least about 4 1 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 n , at least about 75 n , at least
about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 . or at least about
100 nm.
|00301 some embodiments, the membrane may reject at least 85% of particles having a
size of at least about 30 nm, at least about 3 1 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 3 nm, at least about 39 nm, at least about 40 nm, at least about 4 1 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
6 5 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.
10 In some embodiments, the membrane may reject at least 90% of particles having a
size of at least about 30 nm, at least about 3 nm, at least about 32 nm, at least about 33 mn,
a 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 4 1 n , 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 9 5 nm, or at least about 100 nm.
032 In some embodiments, the membrane may reject at least 95% of particles having a
size of at least about 30 nm, at least about 3 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 3 nm, at least about 39 nm, at least about 40 n, at least about 4 1 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 n , 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, a least about 85 nm,
at least about 90 nm, at least about 95 nm, or at least about 1 0 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/m 2/hr (LMH) at 2
bar.
[ 034 In some embodiments, the flux s at least about 125 L/m /hr (LMH), at least about
150 L/m /hr (LMH), at least about 175 L/m /hr (LMH), at least about 200 L/m 2/hr (LMH), at
least about 225 L/m /far (LMH), at least about 250 L/nr/hr (LMH), at least about 300 L/m r
(LMH), at least about 350 L/nr/hr (LMH), 400 at least about L/m /hr (LMH), at least about
450 L/nr/hr (LMH), or at least about 500 L/m /hr (LMH) at 2 bar. In some embodiments, the
flux is between about 100 L/m /hr (LMH) and about 500 L/m /hr(LMH) at 1-3 bar. In certain
embodiments, the flux is between about 100 L/m /hr(LMH) and about 300 L/m 2/hr(LMH) at
1-3 bar.

03 Similar to flux, permeability s 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. T re living, self-healing cellulose membrane may have a permeability of at least 50
Lm fi bar 1.
[0 36] In some embodiments, the permeability is at least about 55 Lm h bar 1, at least
about 60 L fl bar , at least about 65 Lm^h^bar , at least about 70 L bar 1, at least
about 75 Lm^h^bar 1
, at least about 80 Lm^h^bar , at least about 85 L fl ar 1, at least
about 90 Lm h bar 1, at least about 95 Ln bar 1, at least about 100 Lm^h^bar 1
, at least
about 105 L bar 1, at least about 1 10 L bar 1, at least about 1 5 Lm 2h _ bar l , at least
about 120 Lm ^bar 1, at least about 125 Lm^h^bar 1, at least about 130 L f f bar 1, at least
about 135 Lm ba , at least about 140 Lm ba , at least about 145 Lm^h^bar 1, or at
least about 150 Lm bar 1.
[0037] In some embodiments, the permeability is less than about 300 Lm h l bar _ , less
than about 250 Lm^h^bar , less than about 200 Lm^h^bar 1
, less than about 175 L fltybar
, less than about 150 Lm h bar 1, less than about 145 Lm^h^bar 1
, less than about 140 Lm
h bar , less than about 35 Lm fltybar , less than about 130 Ln ltybar , less than about
’

125 Lm^h^bar 1, less than about 120 Lm fiv'bar 1 , less than about 1 5 L flr'bar 1
, less than
about 110 Lm ba , less than about 105 Lm h _ bar less than about 1 0 Lm fii^bar 1, less
than about 95 Lm^h^bar 1
, ess than about 90 Lm^h^bar 1
, ess than about 85 Lm^h^bar 1
,
less than about 80 Lm h ba less than about 75 Lm h ba less than about 70 Lm h bar
less than about 65 Lm fir'bar 1
, or less than about 60 Lm h bar
[ 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. T re 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.
[0 40] 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 .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. n
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 meter, at least about 5 meters, or at least about 0
meters. In some embodiments, the diameter may be up to about 0 meters. The membrane
may be a square or rectangle and have s de length up to about 0 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 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.
4 } 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. he
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 to about 30
°C (e.g. about 25 °C) before proceeding to the next step.
[004 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 s added to a pH of between 3.5 and 5, or between 3 5 and 4 .
[0 49 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.
[ 5 ] 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 w th 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

[ 05 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 8 % of particles having a size of at least
3 nm.
1 05 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 wa ter
a) Membrane properties
53 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 3 1 nm, at least about 32
nm, at least about 33 nm, at least about 34 n , at least about 35 nm, at least about 36 nm, at
least about 37 nm, at least about 38 nm, at least about 39 . at least about 40 nm, at least
about 4 1 nm, at least about 42 nm, at least about 43 nm, at least about 44 nm, at least about
4 5 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 n , 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 0
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 3 1 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 4 1 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 n , 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 3 1 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 n , at least about 37 mn, at least
about 38 nm, at least about 39 nm, at least about 40 nm, at least about 4 1 nm, at least about
42 nm, at least about 43 nm, at least about 44 nm, at least about 45 mn, a 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
6 5 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.
0 56] 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,
a least about 34 mn, a 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 n , at least about 4 1 n , at least about
42 m, 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 1 0
L/m /hr (LMH) at 2 bar. In some embodiments, the flux is at least about 2 5 L/m /hr (LMH),
at least about 150 L/m 2/hr (LMH), at least about 175 L/m 2/hr (LMH), at least about 200
/n hr (LMH), at least about 225 L/m /hr(LMH), at least about 250 L/m 2 hr (LMH), at least
about 300 L/m /hr (LMH), at least about 350 L/m /hr (LMH), at least about 400 L/m 27hr
(LMH), at least about 450 L/m 2 hr (LMH), or at least about 500 L/m 2/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/m 2/hr(LMH) at 1-3 bar.
1 58 The living, self-healing cellulose membrane may have a permeability of at least
about 50 Lm h bar 1 . In some embodiments, the permeability is at least about 55 Lm h 'bar
' , at least about 60 Lnrlr'bar 1
, at least about 65 Lm^h^bar 1 , at least about 70 Lm^h^bar ' ,
at least about 75 Lm r bar 1 , at least about 80 Lm flv'bar 1, at least about 85 Lm^h^bar , at
least about 90 Lm 2h bar at least about 95 L bar 1, at least about 100 Ln bar 1, at
least about 105 Lm flr'bar 1, at least about 110 L r r 'bar 1, at least about 115 Lm 2h lbar , at
least about 120 L r l 'bar 1 , at least about 25 Lnr 'ba 1 , at least about 0 Ln rar ' , at
least about 35 m h ba ' , at least about 40 Lmr h ba , at least about 4 5 Lmrh^bar ,
or at least about 150 Lm h 'ba ' .
059 In some embodiments, the permeability is less than about 300 L r r 'bar 1, less
than about 250 Lm 'bar 1, less than about 200 Lm 'bar 1, less than about 175 Lm 2h bar
' , less than about 150 Lm 2h bar less than about 145 Lm flr'bar 1 , less than about 140 Lm
flf'bar 1, less than about 135 Lm h bar 1, less than about 130 Lm^h^bar , less than 125
about Lm^h^bar 1 , less than about 120 L r l 'bar 1 , less than about 115 L r 'bar 1, less
than about 1 Lnfiflr^bar 1 , less than about 5 Lm^h^bar 1
, less than about 00 Lmrh^bar
' , less than about 95 Lnrh^bar 1
, less than about 90 Lnrlr'bar 1
, less than about 85 Lm h
'bar ' , less than about 80 Lm h l bar ' , less than about 75 Lm h l bar ' , less than about 70 Lm
h 'bar ' , less than about 65 Lm h 'bar ' , or less than about 60 Lm h bar 1.
10 6 H e 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.
[0 61 ) 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 . 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
m , at least about .25 mm, at least about 1.5 mm, at least about 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 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, ess than about 3.0, less than about 2.5 mm, ess than about 2.0
mm, less than about .5 mm, ess than about 1.0 mm, or ess than about 0.5 mm.
0 63 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 .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 meter, at least about 5 meters, or at least about 0
meters. In some embodiments, the diameter may be up to about 0 meters. e 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.
| 61 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
06 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.
1 6 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.
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 to about 30
°C (e.g., about 2 °C) before proceeding to the next step.
007 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 s added to a pH of between 3.5 and 5, or between 3 5 and 4 .
067 ] 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.
[0072 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
[ 73] Membrane growth a d 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 w th the same composition listed above to fabricate an LFM, an
acidic media without a carbon source (growth media minus sucrose), and DI.
[ 07 ] Permeability and selectivity. Permeability and selectivity tests were performed in a
dead-end stirred cell with a 24.5 m 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 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 -
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 χ 152.4 mm
x 3.2 mm, McMaster-Carr, Robbinsville, NJ). The copperplate was wrapped in aluminum
foil and held at a fixed separation distance of 10 cm. A constant feed rate of 3 mL 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 w th a desiccant unit (Drierite, Xenia, OH) that maintained the temperature at 22 ±
°C and the relative humidity at 55%. To generate nanofiber layers with a consistent bulk
thickness, cellulose acetate was electrospun for hour. After being peeled off the collector
plate, the cellulose acetate nanofiber layer was sandwiched between Teflon sheets (3.2 mm χ

1 6 mm 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 D water.
[0077] Storage and self-healing. Permeability and selectivity tests were performed on
LFMs stored in two different media: 1. 1% acetic ac d 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.
|0 7 j 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 /0.4 numerical aperture objective.
Calcafluor White was excited with a 405 nm laser, and an emission window of 569-61 n
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.
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 vo %
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.
0082] 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 s grown, the cellulose forms a layer at the liquid-air
interface. Longer growth times led to thicker layers and associated ow r filtration membrane
water permeability. e membranes described here used thinner layers that incorporated both
bacterial cellulose and the microorganisms that synthesize, maintain, and repair the cellulose.
008 ] 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 8 + 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
84 j FT R spectra w'ere collected using a Nicolet iS5, iD5, w th ATR attachment for the
LFM and cellulose nanofibers synthesized by electrospinning. As a long-chain carbohydrate
used by living organisms for energy purposes, cellulose (C H i O )» 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 . By the absence of a peak
at 1750 cm the FT R 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 for both
LFM and the cellulose electrospun membranes (FIG. D) 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.
8 85 The morphology of the LFM cellulose matrix was vi sualized using SEM. T e
fibrous structure of the samples was preserved by mounting lyophiiized (Labconco FreeZone
2.5) or critical point dried (CPD) samples (Autosamdri-93 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 anon-uniform porous network. FIG. 1C, obtained from a bleach-treated CPD
membrane, revealed fibers with a small average diameter, 38 nm ± 1.4 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 w th a more distinct fiber network, with
fiber diameters in the range of 5-70 nm.
[0086] D e 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
0087] The LFM structure as a non-woven mat of cellulose fibers lends to ts 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 -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 ba (FIG E) Depending on
applied pressure, traditional polymeric ultrafiltration membrane permeability can reach to
1000 L m h _ 1 .
[0088 LFM selectivity was tested using the same 10 m , 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 5 0 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 n ) 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 selecti vity
[ 09 ] Three self-healing tests were conducted using 10 mL or 50 ml, dead-end filtration
cells (Amicon, Mil pore) The first test involved placing a 4 m 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
a (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 ho e n 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
th e 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 w th 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 w ere
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). h e 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
[ 93 ] 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.
[ 94 ] 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 grow
environment after 5, , and days. Permeability and selectivity data for storage time
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 D water was 284 ± 12 L m _ h _ l
at a pressure of 0.78 bar. For 5 days storage in D water, the flux increased to 393 L m- h- 1.
Example 6
LFM Applications
| 195] 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. D e average
drinking water requirement for a 4-person family ( 6 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.
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
|0 71 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.
1 098 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 minute, 30 psi for 1 minute and 45
psi for hour. Raw basin creek reservoir water (1.5 L) w as mixed with an aluminum
chlorohydrate (ALCH) coagulant ( 1 1.2 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 s gradually brought up to testing
pressure (10 psi for 1 minutes, 20 psi for 15 minutes and 30 psi for 15 minutes) before a 45
ps 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.
0 99 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.
01 0 ] Additional flux measurements are taken at various pressures. Immediately after the
fouling tests, the fouling layer s treated with calcoflouro white (cellulose and chitin) and/or a
live-dead (microorganism) stain for analysis of the fouling layer under a con ocal 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.
[01 1 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
[0 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

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 or 2, wherein the membrane rejects at least about 80% of particles
having a s ze 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 3 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
00 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 5 Lm h ar 1.

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

about 35 Lrr ar

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

0 . The method of claim 9, wherein the membrane has a thickness of at least about 0 . 1mm.

1. 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 , 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 m .

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

19. The system of claims 7 or 8, 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 7-19, 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 7-21, wherein the membrane has a flux of at least
about 100 L/m /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 e 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

3 . 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 17-3 1, 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.
Form PCT/ISA/210 (continuation of first sheet (2)) (July 2019)
Form PCT/ISA/2 10 (second sheet) (July 20 19)