# US 11,440,827 B2 · Method for the preparation of biopolymers

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- Number: US 11,440,827 B2
- Document ID: US11440827B2
- Status: Granted
- Kind: B2
- Application number: 16/051,742
- Filed: 2018-08-01
- Published: 2018-11-29
- Granted: 2022-09-13
- Earliest claimed: 2013-12-30
- Assignees: United Utilities PLC
- Applicants: United Utilities PLC
- Inventors: Minh Son Le
- Web page: https://patentgrep.com/patent/US11440827B2

## CPC

- C02F 11/00: Treatment of sludge; Devices therefor
- C07K 1/14: Extraction; Separation; Purification
- C07K 1/30: by precipitation
- C07K 9/00: Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C02F 1/36: ultrasonic vibrations
- C02F 1/54: using organic material
- C02F 2303/06: Sludge reduction, e.g. by lysis
- C02F 2303/26: Reducing the size of particles, liquid droplets or bubbles, e.g. by crushing, grinding, spraying, creation of microbubbles or nanobubbles
- Y02W 10/40: Valorisation of by-products of wastewater, sewage or sludge processing
- C02F: TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE (separation in general B01D; special arrangements on waterborne vessels of installations for treating water, waste water or sewage, e.g. for producing fresh water, B63J; adding materials to water to prevent corrosion C23F; treating radioactively-contaminated liquids G21F9/04; regeneration of reactants for recirculation into processes, see the relevant places for the processes)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02W: CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT

## Abstract

A method of preparing and separating biopolymers and biopolymer fractions is useful for wastewater treatment applications from sewage sludge. The method comprising the steps of disrupting the bacterial cell walls of bacteria present in the sewage sludge by at least 75% to release the intracellular contents of the bacterial cells and separating the biopolymers from any contaminants present.

## Claims

### Claim 1

A method of removing phosphorus from wastewater comprising:
i) removing extracellular polymeric substances (EPS) from bacterial cells in sewage sludge to provide an EPS-removed sewage sludge;
ii) disrupting bacterial cell walls of the bacterial cells present in the EPS-removed sewage sludge by at least 75% to release intracellular contents of the bacterial cells so as to form a cell lysate;
iii) separating the biopolymers or biopolymer fractions from any contaminants present, wherein step i) is performed before steps ii) and iii);
iv) adding the isolated biopolymers or biopolymer fractions to said wastewater; and
v) allowing said isolated biopolymers or biopolymer fractions to remove said phosphorus from the wastewater.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the method of isolating the biopolymers or biopolymer fractions further comprises the step of:
precipitating the soluble biopolymers from the cell lysate prior to separating the biopolymers from any contaminants present.

### Claim 3 (depends on 2)

The method according to claim 2 wherein the soluble biopolymers precipitated comprise nucleic acids.

### Claim 4 (depends on 1)

The method according to claim 1 wherein the cell lysate is anaerobically digested and peptidoglycan present in the cell lysate is separated and isolated.

### Claim 5 (depends on 1)

The method according to claim 1 wherein the sewage sludge is a biological sludge.

### Claim 6 (depends on 1)

The method according to claim 1 wherein the ruptured bacterial cell walls are ground to disperse or dissolve peptidoglycan present therein.

### Claim 7 (depends on 1)

The method according to claim 1 wherein the extracellular polymeric substances (EPS) are removed using a mechanical means.

### Claim 8 (depends on 1)

The method according to claim 1 wherein the extracellular polymeric substances (EPS) are removed using a chemical means.

### Claim 9 (depends on 1)

The method according to claim 1 wherein the extracellular polymeric substances (EPS) are removed using enzyme treatments.

### Claim 10 (depends on 1)

The method according to claim 1 wherein the bacterial cell walls are disrupted by means of one or more of: ultra-sonication, and/or bead mills, and/or caustic treatments.

### Claim 11 (depends on 1)

The method according to claim 1 wherein peptidoglycan present in the cell lysate is separated and isolated from the cell DNA.

### Claim 12 (depends on 11)

The method according to claim 11 wherein the cell lysate is anaerobically digested.

### Claim 13 (depends on 11)

The method according to claim 11 wherein the cell lysate is filtered.

### Claim 14 (depends on 2)

The method according to claim 2 wherein the biopolymers are treated before, during or post the precipitation step with one or more chemicals.

### Claim 15 (depends on 1)

The method according to claim 1, wherein the extracellular polymeric substances (EPS) are removed by at least 50%.

### Claim 16 (depends on 1)

The method according to claim 1, wherein the extracellular polymeric substances (EPS) are removed by anaerobic digestion.

### Claim 17 (depends on 16)

The method according to claim 16, wherein the extracellular polymeric substances (EPS) are removed by at least 50%.

### Claim 18

A method of preparing and separating biopolymers from sewage sludge; and subsequently using said biopolymers to remove phosphorus from wastewater in a method comprising adding biopolymers to said wastewater, and allowing said biopolymers to remove said phosphorus from the wastewater; wherein the biopolymers are prepared and separated by the following steps:
i) removing extracellular polymeric substances (EPS) from bacterial cells in sewage sludge to provide an EPS-removed sewage sludge;
ii) disrupting the bacterial cell walls of bacteria present in the EPS-removed sewage sludge by at least 75% to release the intracellular contents of the bacterial cells; and
iii) separating the biopolymers from any contaminants present, wherein step i) is performed before step ii), and wherein step (ii) is performed before step iii).

### Claim 19

A method of removing phosphorus from wastewater comprising:
i) removing extracellular polymeric substances (EPS) from bacterial cells in sewage sludge by anaerobic digestion to provide an EPS-removed sewage sludge;
iia) disrupting bacterial cell walls of the bacterial cells present in the EPS-removed sewage sludge by at least 75% to release intracellular contents of the bacterial cells so as to form a cell lysate and solubilising nucleic acids and contaminants present in the cell lysate;
iib) precipitating the nucleic acids onto peptidoglycan debris present in the cell lysate to provide a precipitate comprising a blend of the nucleic acids and the peptidoglycan debris;
iii) separating the biopolymers or biopolymer fractions from any contaminants present, wherein the biopolymers or biopolymer fractions comprise a blend of nucleic acids and peptidoglycan and wherein step i) is performed before steps iia), iib) and iii);
iv) removing the precipitate from the cell lysate using a solid liquid separation technique;
v) adding the removed precipitate to said wastewater; and
vi) allowing the removed precipitate to remove said phosphorus from the wastewater.

### Claim 20

A method of removing phosphorus from wastewater comprising:
i) removing extracellular polymeric substances (EPS) from bacterial cells in sewage sludge to provide an EPS-removed sewage sludge;
ii) disrupting bacterial cell walls of the bacterial cells present in the EPS-removed sewage sludge by at least 75% to release intracellular contents of the bacterial cells so as to form a cell lysate and solubilising nucleic acids and contaminants present in the cell lysate;
iii) removing the nucleic acids, any EPS present, and the contaminants present in the cell lysate by anaerobic digestion; before removing peptidoglycan debris present in the digestate formed by the anaerobic digestion using a solid liquid separation technique to provide biopolymer fractions, wherein step i) is performed before steps ii) and iii);
iv) adding the biopolymer fractions to said wastewater; and
v) allowing the biopolymer fractions to remove said phosphorus from the wastewater.

### Claim 21 (depends on 19)

The method of claim 19, wherein the extracellular polymeric substances (EPS) are removed by at least 50%.

### Claim 22 (depends on 19)

The method of claim 19, wherein the precipitate is a blend of nucleic acids and peptidoglycan with a purity of at least 50%.

### Claim 23 (depends on 21)

The method of claim 21, wherein the precipitate is a blend of nucleic acids and peptidoglycan with a purity of at least 50%.

### Claim 24 (depends on 20)

The method of claim 20, wherein the extracellular polymeric substances (EPS) are removed by at least 50%.

### Claim 25 (depends on 20)

The method of claim 20, wherein the peptidoglycan debris has a purity of at least 50%.

### Claim 26 (depends on 24)

The method of claim 24, wherein the peptidoglycan debris has a purity of at least 50%.