# US 12,600,941 B2 · Method for producing biomass using hydrogen-oxidizing bacteria

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- Number: US 12,600,941 B2
- Document ID: US12600941B2
- Status: Granted
- Kind: B2
- Application number: 17/996,826
- Filed: 2021-04-26
- Published: 2023-06-29
- Granted: 2026-04-14
- Earliest claimed: 2020-04-24
- Assignees: AERBIO AIS (formerly Marsor DK ApS)
- Applicants: Aerbio A/S
- Inventors: Robert Patrick William Mansfield; Bart Pander; Preben Krabben; Peter Nicholas Rowe
- Web page: https://patentgrep.com/patent/US12600941B2

## CPC

- C12N 1/205: Bacterial isolates
- A23J 1/008: {from microorganisms (A23J1/18 takes precedence)}
- A23K 10/12: by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
- A23K 20/147: Polymeric derivatives, e.g. peptides or proteins
- A23K 50/10: for ruminants
- A23K 50/30: for swines
- A23K 50/75: for poultry
- A23K 50/80: for aquatic animals, e.g. fish, crustaceans or molluscs
- C12N 1/20: Bacteria; Culture media therefor
- C12R 2001/01: Bacteria or Actinomycetales {; using bacteria or Actinomycetales}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A23J: PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23K: FODDER
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS

## Abstract

The present invention provides a method for producing a biomass comprising at least 65% protein from hydrogen-oxidising microorganisms using one or more input streams comprising one or more gaseous substrates and a nutrient composition which are controlled and wherein the biomass is produced at a rate of more than 10 g/l/d. The produced and isolated biomass is useful as feed or to provide nutrition to one or more organisms.

## Claims

### Claim 1

A continuous method for producing a biomass comprising at least 65% protein of total biomass by dry weight from hydrogen-oxidizing microorganisms comprising bacteria selected from the genus Cupriavidus sp, using one or more input streams comprising one or more gaseous substrates, comprising hydrogen and oxygen, the method comprising:
contacting the microorganisms in a liquid phase with a nutrient composition comprising nitrogen and phosphorous-comprising compounds and the gaseous substrates, wherein the one or more gaseous substrates comprises carbon dioxide, or wherein the nutrient composition comprises carbon-comprising compounds, and wherein the input stream and nutrient composition are controlled and wherein the biomass is produced at a rate of more than 10 g/l/d, the method further comprising:
i. controlling the input stream comprising adding a molar ratio of hydrogen: oxygen to the liquid phase of from 1:1 to 10:1;
ii. controlling the input stream and nutrient composition comprising maintaining a specific growth rate of the microorganisms of from 1.0 to 8.0 d−1 or from 0.04 to 0.3 h−1;
iii. controlling the input stream comprising maintaining hydrogen at a transfer rate of from 0.03 to 1.2 mol/l/h in the liquid phase or oxygen at a transfer rate of from 0.003 to 0.4 mol/l/h in the liquid phase; and
iv. replacing the liquid phase of the bioreactor where the microorganisms are grown and maintained at a volume of from 4 to 30% per hour; wherein a microbial concentration of at least 10 g/l is maintained in the liquid phase.

### Claim 2 (depends on 1)

The method of claim 1, wherein controlling the input stream comprises adding hydrogen in a concentration of from 70 to 100% (v/v), oxygen in a concentration of from 20 to 100% (v/v) and carbon dioxide in a concentration of from 5 to 100% (v/v) to the liquid phase, individually or as any premixed combination thereof.

### Claim 3 (depends on 1)

The method of claim 1, wherein controlling the input stream and nutrient composition comprises maintaining a concentration of the microorganisms in the liquid phase of the bioreactor, wherein the concentration comprises at least 10 g/l.

### Claim 4 (depends on 1)

The method of claim 1, wherein controlling the input stream and nutrient composition comprises maintaining a concentration of the microorganisms in the liquid phase of the bioreactor, wherein the concentration comprises from 10 g/l to 100 g/l.

### Claim 5 (depends on 1)

The method of claim 1, wherein controlling the input stream comprises adding a molar ratio of hydrogen:oxygen:carbon dioxide of from 3.88 to 51.94:0.85 to 2:0.75 to 2 to the liquid phase.

### Claim 6 (depends on 1)

The method of claim 1, wherein controlling the input stream comprises maintaining a molar ratio of dissolved hydrogen:oxygen:carbon dioxide of from 3.183 to 12.748:0.795 to 4.25:0.75 to 2.0 in the liquid phase.

### Claim 7 (depends on 1)

The method of claim 1, wherein controlling the input stream comprises maintaining a concentration of hydrogen of from 0.5 to 20 mg/l, a concentration of oxygen of from 0.5 to 80 mg/l and a concentration of carbon dioxide of from 20 to 2000 mg/l in the liquid phase at a temperature of from 28 to 45° C. and at a pressure in the gaseous phase of from 100 to 2000 kPa.

### Claim 8 (depends on 1)

The method of claim 1, wherein controlling the input stream comprises maintaining a molar ratio of hydrogen:oxygen gas hold-up in the liquid phase of from 0.5:1 to 7:1 or from 1:1 to 3.5:1.

### Claim 9 (depends on 1)

The method of claim 1, wherein the microorganisms utilize the hydrogen at a rate of from 0.05 to 0.5 mol/g/h, the oxygen at a rate of from 0.04 to 0.18 mol/g/h, and the carbon dioxide at a rate of from 0.03 to 0.13 mol/g/h.

### Claim 10 (depends on 1)

The method of claim 1, wherein the carbon dioxide is derived from the exhaust gas of a production or combustion process.

### Claim 11 (depends on 10)

The method of claim 10, wherein the carbon dioxide is purified and concentrated to a concentration of from 40 to 100% (v/v).

### Claim 12 (depends on 1)

The method of claim 1, wherein the step of contacting the microorganisms in a liquid phase with a nutrient composition comprises adding a nutrient composition which is adjusted to a pH of from 1 to 4 prior to adding, wherein the nutrient composition comprises DSMZ medium for chemolithotrophic growth 81 (H-3) without NaHCO3.

### Claim 13 (depends on 12)

The method of claim 12 wherein, prior to adding, the medium is supplemented with an additional 1.5 g/L ammonium chloride, 3×10−4 g/L NiCl2×6H2O, and 1.5×10−3 g/L ZnSO4×7H2O, and 1.5×10−4 g/L CuCl2×2H2O, and 0.15 g/L Ferric ammonium citrate.

### Claim 14 (depends on 1)

The method of claim 1, wherein the microorganisms comprise bacteria selected from the species Cupriavidus necator.

### Claim 15 (depends on 1)

The method of claim 1 further comprising removing the nutrient composition and isolating the biomass produced by dewatering or drying the biomass such that the biomass comprises a water content of less than 5% by weight.

### Claim 16 (depends on 15)

The method of claim 15 further comprising using the dried or dewatered nutrient composition removed by dewatering or drying the biomass as the nutrient composition for contacting the microorganisms in a liquid phase.